Biochemistry, Genetics and Molecular Biology › Molecular Biology

Metabolism, Diabetes, and Cancer

Works Count: 50279

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This cluster of papers explores the role of AMP-activated protein kinase (AMPK) in regulating cellular metabolism, including its involvement in glucose and lipid metabolism, insulin signaling, mitochondrial homeostasis, and the potential impact on cancer risk. The cluster also delves into the mechanisms of action of metformin, a drug known to activate AMPK and its effects on cellular energy balance and gene expression regulation.

Keywords

AMP-activated Protein Kinase; Metformin; Glucose; Insulin Signaling; Cancer Risk; Cellular Energy; Mitochondrial Homeostasis; Glycogen Metabolism; Regulation of Gene Expression; Tumor Suppression

10-year follow-up of diabetes incidence and weight loss in the Diabetes Prevention Program Outcomes Study

2009-10-30

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Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer's disease – is this type 3 diabetes?

2005-03-03

Eric van Steen , Benjamin M. Terry , Enrique J. Rivera +6 more

The neurodegeneration that occurs in sporadic Alzheimer's disease (AD) is consistently associated with a number of characteristic histopathological, molecular, and biochemical abnormalities, including cell loss, abundant neurofibrillary tangles and dystrophic … The neurodegeneration that occurs in sporadic Alzheimer's disease (AD) is consistently associated with a number of characteristic histopathological, molecular, and biochemical abnormalities, including cell loss, abundant neurofibrillary tangles and dystrophic neurites, amyloid-β deposits, increased activation of pro-death genes and signaling pathways, impaired energy metabolism/mitochondrial function, and evidence of chronic oxidative stress. The general inability to convincingly link these phenomena has resulted in the emergence and propagation of various heavily debated theories that focus on the role of one particular element in the pathogenesis of all other abnormalities. However, the accumulating evidence that reduced glucose utilization and deficient energy metabolism occur early in the course of disease, suggests a role for impaired insulin signaling in the pathogenesis of AD. The present work demonstrates extensive abnormalities in insulin and insulin-like growth factorm typer I and II (IGF-I and IGF-II) signaling mechanisms in brains with AD, and shows that while each of the corresponding growth factors is normally made in central nervous system (CNS) neurons, the expression levels are markedly reduced in AD. These abnormalities were associated with reduced levels of insulin receptor substrate (IRS) mRNA, tau mRNA, IRS-associated phosphotidylinositol 3-kinase, and phospho-Akt (activated), and increased glycogen synthase kinase-3β activity and amyloid precursor protein mRNA expression. The strikingly reduced CNS expression of genes encoding insulin, IGF-I, and IGF-II, as well as the insulin and IGF-I receptors, suggests that AD may represent a neuro-endocrine disorder that resembles, yet is distinct from diabetes mellitus. Therefore, we propose the term, "Type 3 Diabetes" to reflect this newly identified pathogenic mechanism of neurodegeneration.

Metformin: An Old Drug for the Treatment of Diabetes but a New Drug for the Protection of the Endothelium

2015-01-01

Mustafa Kinaan , Hong Ding , Chris R. Triggle

The anti-diabetic and oral hypoglycaemic agent metformin, first used clinically in 1958, is today the first choice or ‘gold standard' drug for the treatment of type 2 diabetes and polycystic … The anti-diabetic and oral hypoglycaemic agent metformin, first used clinically in 1958, is today the first choice or ‘gold standard' drug for the treatment of type 2 diabetes and polycystic ovary disease. Of particular importance for the treatment of diabetes, metformin affords protection against diabetes-induced vascular disease. In addition, retrospective analyses suggest that treatment with metformin provides therapeutic benefits to patients with several forms of cancer. Despite almost 60 years of clinical use, the precise cellular mode(s) of action of metformin remains controversial. A direct or indirect role of adenosine monophosphate (AMP)-activated protein kinase (AMPK), the fuel gauge of the cell, has been inferred in many studies, with evidence that activation of AMPK may result from a mild inhibitory effect of metformin on mitochondrial complex 1, which in turn would raise AMP and activate AMPK. Discrepancies, however, between the concentrations of metformin used in in vitro studies versus therapeutic levels suggest that caution should be applied before extending inferences derived from cell-based studies to therapeutic benefits seen in patients. Conceivably, the effects, or some of them, may be at least partially independent of AMPK and/or mitochondrial respiration and reflect a direct effect of either metformin or a minor and, as yet, unidentified putative metabolite of metformin on a target protein(s)/signalling cascade. In this review, we critically evaluate the data from studies that have investigated the pharmacokinetic properties and the cellular and clinical basis for the oral hypoglycaemic, insulin-sensitising and vascular protective effects of metformin.

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Disruption of IRS-2 causes type 2 diabetes in mice

1998-02-01

Dominic J. Withers , Julio Sanchez Gutierrez , Heather Towery +7 more

AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism

2005-01-01

Barbara B. Kahn , Thierry Alquier , David Carling +1 more

Glycemic Control With Diet, Sulfonylurea, Metformin, or Insulin in Patients With Type 2 Diabetes Mellitus<SUBTITLE>Progressive Requirement for Multiple Therapies (UKPDS 49)</SUBTITLE>

1999-06-02

Robert C. Turner

ContextTreatment with diet alone, insulin, sulfonylurea, or metformin is known to improve glycemia in patients with type 2 diabetes mellitus, but which treatment most frequently attains target fasting plasma glucose … ContextTreatment with diet alone, insulin, sulfonylurea, or metformin is known to improve glycemia in patients with type 2 diabetes mellitus, but which treatment most frequently attains target fasting plasma glucose (FPG) concentration of less than 7.8 mmol/L (140 mg/dL) or glycosylated hemoglobin A1c(HbA1c) below 7% is unknown.ObjectiveTo assess how often each therapy can achieve the glycemic control target levels set by the American Diabetes Association.DesignRandomized controlled trial conducted between 1977 and 1997. Patients were recruited between 1977 and 1991 and were followed up every 3 months for 3, 6, and 9 years after enrollment.SettingOutpatient diabetes clinics in 15 UK hospitals.PatientsA total of 4075 patients newly diagnosed as having type 2 diabetes ranged in age between 25 and 65 years and had a median (interquartile range) FPG concentration of 11.5 (9.0-14.4) mmol/L [207 (162-259) mg/dL], HbA1c levels of 9.1% (7.5%-10.7%), and a mean (SD) body mass index of 29 (6) kg/m2.InterventionsAfter 3 months on a low-fat, high-carbohydrate, high-fiber diet, patients were randomized to therapy with diet alone, insulin, sulfonylurea, or metformin.Main Outcome MeasuresFasting plasma glucose and HbA1c levels, and the proportion of patients who achieved target levels below 7% HbA1c or less than 7.8 mmol/L (140 mg/dL) FPG at 3, 6, or 9 years following diagnosis.ResultsThe proportion of patients who maintained target glycemic levels declined markedly over 9 years of follow-up. After 9 years of monotherapy with diet, insulin, or sulfonylurea, 8%, 42%, and 24%, respectively, achieved FPG levels of less than 7.8 mmol/L (140 mg/dL) and 9%, 28%, and 24% achieved HbA1c levels below 7%. In obese patients randomized to metformin, 18% attained FPG levels of less than 7.8 mmol/L (140 mg/dL) and 13% attained HbA1c levels below 7%. Patients less likely to achieve target levels were younger, more obese, or more hyperglycemic than other patients.ConclusionsEach therapeutic agent, as monotherapy, increased 2- to 3-fold the proportion of patients who attained HbA1c below 7% compared with diet alone. However, the progressive deterioration of diabetes control was such that after 3 years approximately 50% of patients could attain this goal with monotherapy, and by 9 years this declined to approximately 25%. The majority of patients need multiple therapies to attain these glycemic target levels in the longer term.

LKB1 Is the Upstream Kinase in the AMP-Activated Protein Kinase Cascade

2003-11-01

Angela Woods , Stephen R. Johnstone , Kristina Dickerson +7 more

Insulin and insulin resistance:

2004-01-01

James R. Sowers , Edward D. Fröhlich

Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain

2000-06-07

Mark R. Owen , E. Doran , Andrew P. Halestrap

Although metformin is widely used for the treatment of non-insulin-dependent diabetes, its mode of action remains unclear. Here we provide evidence that its primary site of action is through a … Although metformin is widely used for the treatment of non-insulin-dependent diabetes, its mode of action remains unclear. Here we provide evidence that its primary site of action is through a direct inhibition of complex 1 of the respiratory chain. Metformin (50 μM) inhibited mitochondrial oxidation of glutamate+malate in hepatoma cells by 13 and 30% after 24 and 60 h exposure respectively, but succinate oxidation was unaffected. Metformin also caused time-dependent inhibition of complex 1 in isolated mitochondria, whereas in sub-mitochondrial particles inhibition was immediate but required very high metformin concentrations (K0.5, 79 mM). These data are compatible with the slow membrane-potential-driven accumulation of the positively charged drug within the mitochondrial matrix leading to inhibition of complex 1. Metformin inhibition of gluconeogenesis from L-lactate in isolated rat hepatocytes was also time- and concentration-dependent, and accompanied by changes in metabolite levels similar to those induced by other inhibitors of gluconeogenesis acting on complex 1. Freeze-clamped livers from metformin-treated rats exhibited similar changes in metabolite concentrations. We conclude that the drug's pharmacological effects are mediated, at least in part, through a time-dependent, self-limiting inhibition of the respiratory chain that restrains hepatic gluconeogenesis while increasing glucose utilization in peripheral tissues. Lactic acidosis, an occasional side effect, can also be explained in this way.

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The Kinase LKB1 Mediates Glucose Homeostasis in Liver and Therapeutic Effects of Metformin

2005-11-25

Reuben J. Shaw , Katja Lamia , Debbie S. Vasquez +5 more

The Peutz-Jegher syndrome tumor-suppressor gene encodes a protein-threonine kinase, LKB1, which phosphorylates and activates AMPK [adenosine monophosphate (AMP)-activated protein kinase]. The deletion of LKB1 in the liver of adult mice … The Peutz-Jegher syndrome tumor-suppressor gene encodes a protein-threonine kinase, LKB1, which phosphorylates and activates AMPK [adenosine monophosphate (AMP)-activated protein kinase]. The deletion of LKB1 in the liver of adult mice resulted in a nearly complete loss of AMPK activity. Loss of LKB1 function resulted in hyperglycemia with increased gluconeogenic and lipogenic gene expression. In LKB1-deficient livers, TORC2, a transcriptional coactivator of CREB (cAMP response element-binding protein), was dephosphorylated and entered the nucleus, driving the expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), which in turn drives gluconeogenesis. Adenoviral small hairpin RNA (shRNA) for TORC2 reduced PGC-1alpha expression and normalized blood glucose levels in mice with deleted liver LKB1, indicating that TORC2 is a critical target of LKB1/AMPK signals in the regulation of gluconeogenesis. Finally, we show that metformin, one of the most widely prescribed type 2 diabetes therapeutics, requires LKB1 in the liver to lower blood glucose levels.

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AMPK Phosphorylates and Inhibits SREBP Activity to Attenuate Hepatic Steatosis and Atherosclerosis in Diet-Induced Insulin-Resistant Mice

2011-04-01

Li Yu , Shanqin Xu , Maria M. Mihaylova +7 more

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Insulin signalling and the regulation of glucose and lipid metabolism

2001-12-01

Alan R. Saltiel , C. Ronald Kahn

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AMPK: a nutrient and energy sensor that maintains energy homeostasis

2012-03-22

D. Grahame Hardie , Fiona A. Ross , Simon A. Hawley

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Metformin and reduced risk of cancer in diabetic patients

2005-04-22

Josie Evans , Louise A. Donnelly , Alistair Emslie‐Smith +2 more

Voluntary surveillance systems are subject to under-reporting.Figures may underestimate true numbers.Miscategorisation of probable country of infection by presuming the country with the highest prevalence as the likely country of infection … Voluntary surveillance systems are subject to under-reporting.Figures may underestimate true numbers.Miscategorisation of probable country of infection by presuming the country with the highest prevalence as the likely country of infection will underestimate the number of infections acquired in the United Kingdom, particularly among people originating from countries with high prevalence.New HIV diagnoses do not represent new HIV infections, as diagnosis can occur at any point between infection and death, which in the natural course of infection is typically 10-12 years.Furthermore, surveillance reports do not distinguish between partners infected in high prevalence countries while visiting and partners infected before migrating from those countries.The number of people becoming infected with HIV through heterosexual intercourse in the United Kingdom is rising steadily.As the number of heterosexuals living with HIV (diagnosed and undiagnosed) in the United Kingdom grows, the likelihood of heterosexual transmission within the country will increase, particularly among ethnic minorities.The continuing collaboration of those who contribute to the voluntary HIV/AIDS reporting system in England, Wales and Northern Ireland is gratefully acknowledged, as is the help, advice, and support of Kevin Fenton (Health Protection Agency (HPA)), Noel Gill (HPA), Phillip Mortimer (HPA), Linda Lazarus (Department of Health), and Daniel Thomas (Communicable Disease Surveillance Centre Wales), and administrative support provided by Fay Peyman (HPA) and Fateha Begum (HPA).Contributors: VLG followed up reports.SD analysed surveillance data and wrote the first draft.VLG, SD, KS, and BGE were involved in the drafting of subsequent versions.BGE is guarantor

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Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase

2002-10-07

Toshimasa Yamauchi , Junji Kamon , Yasuhiko Minokoshi +7 more

The LKB1–AMPK pathway: metabolism and growth control in tumour suppression

2009-07-24

David B. Shackelford , Reuben J. Shaw

Insulin Resistance and a Diabetes Mellitus-Like Syndrome in Mice Lacking the Protein Kinase Akt2 (PKBβ)

2001-06-01

Han Cho , James Mu , Jason K. Kim +7 more

Glucose homeostasis depends on insulin responsiveness in target tissues, most importantly, muscle and liver. The critical initial steps in insulin action include phosphorylation of scaffolding proteins and activation of phosphatidylinositol … Glucose homeostasis depends on insulin responsiveness in target tissues, most importantly, muscle and liver. The critical initial steps in insulin action include phosphorylation of scaffolding proteins and activation of phosphatidylinositol 3-kinase. These early events lead to activation of the serine-threonine protein kinase Akt, also known as protein kinase B. We show that mice deficient in Akt2 are impaired in the ability of insulin to lower blood glucose because of defects in the action of the hormone on liver and skeletal muscle. These data establish Akt2 as an essential gene in the maintenance of normal glucose homeostasis.

Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein

1991-07-01

Xiao‐Jian Sun , Paul Rothenberg , C. Ronald Kahn +6 more

Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase

2005-07-01

Simon A. Hawley , David Pan , Kirsty J. Mustard +5 more

Oncogenic Kras Maintains Pancreatic Tumors through Regulation of Anabolic Glucose Metabolism

2012-04-01

Haoqiang Ying , Alec C. Kimmelman , Costas A. Lyssiotis +7 more

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Coated Charcoal Immunoassay of Insulin

1965-10-01

Victor Herbert , Kam‐Seng Lau , Chester W. Gottlieb +1 more

Charcoal premixed with dextran of average molecular weight 80,000 almost instantly adsorbs free insulin but rejects antibody-bound insulin. The use of such dextran-coated charcoal makes simpler and more rapid the … Charcoal premixed with dextran of average molecular weight 80,000 almost instantly adsorbs free insulin but rejects antibody-bound insulin. The use of such dextran-coated charcoal makes simpler and more rapid the immunoassay of insulin in biologic fluids, using radioisotope dilution with 131I-insulin and "biopsy" of the insulin pool by antibody to insulin. The procedure here described yields a straight line graph when insulin added is plotted against insulin recovered.

The AMPK signalling pathway coordinates cell growth, autophagy and metabolism

2011-09-01

Maria M. Mihaylova , Reuben J. Shaw

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Glycemic Durability of Rosiglitazone, Metformin, or Glyburide Monotherapy

2006-12-04

Steven E. Kahn , Steven M. Haffner , Mark Heise +7 more

The efficacy of thiazolidinediones, as compared with other oral glucose-lowering medications, in maintaining long-term glycemic control in type 2 diabetes is not known. The efficacy of thiazolidinediones, as compared with other oral glucose-lowering medications, in maintaining long-term glycemic control in type 2 diabetes is not known.

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Insulin and insulin-like growth factor signalling in neoplasia

2008-11-24

Michaël Pollak

Metabolic syndrome: a closer look at the growing epidemic and its associated pathologies

2014-11-18

Sadhbh O’Neill , Lorraine O’Driscoll

Summary Obesity is reaching epidemic proportions with recent worldwide figures estimated at 1.4 billion and rising year‐on‐year. Obesity affects all socioeconomic backgrounds and ethnicities and is a pre‐requisite for metabolic … Summary Obesity is reaching epidemic proportions with recent worldwide figures estimated at 1.4 billion and rising year‐on‐year. Obesity affects all socioeconomic backgrounds and ethnicities and is a pre‐requisite for metabolic syndrome. Metabolic syndrome is a clustering of risk factors, such as central obesity, insulin resistance, dyslipidaemia and hypertension that together culminate in the increased risk of type 2 diabetes mellitus and cardiovascular disease. As these conditions are among the leading causes of deaths worldwide and metabolic syndrome increases the risk of type 2 diabetes mellitus fivefold and cardiovascular disease threefold, it is of critical importance that a precise definition is agreed upon by all interested parties. Also of particular interest is the relationship between metabolic syndrome and cancer. Metabolic syndrome has been associated with a plethora of cancers including breast, pancreatic, colon and liver cancer. Furthermore, each individual risk factor for metabolic syndrome has also an association with cancer. Our review collates internationally generated information on metabolic syndrome, its many definitions and its associations with life‐threatening conditions including type 2 diabetes mellitus, cardiovascular disease and cancer, providing a foundation for future advancements on this topic.

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The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress

2004-02-25

Reuben J. Shaw , Monica Kosmatka , Nabeel Bardeesy +4 more

AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status found in all eukaryotic cells. AMPK is activated by stimuli that increase the cellular AMP/ATP ratio. Essential … AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status found in all eukaryotic cells. AMPK is activated by stimuli that increase the cellular AMP/ATP ratio. Essential to activation of AMPK is its phosphorylation at Thr-172 by an upstream kinase, AMPKK, whose identity in mammalian cells has remained elusive. Here we present biochemical and genetic evidence indicating that the LKB1 serine/threonine kinase, the gene inactivated in the Peutz-Jeghers familial cancer syndrome, is the dominant regulator of AMPK activation in several mammalian cell types. We show that LKB1 directly phosphorylates Thr-172 of AMPKα in vitro and activates its kinase activity. LKB1-deficient murine embryonic fibroblasts show nearly complete loss of Thr-172 phosphorylation and downstream AMPK signaling in response to a variety of stimuli that activate AMPK. Reintroduction of WT, but not kinase-dead, LKB1 into these cells restores AMPK activity. Furthermore, we show that LKB1 plays a biologically significant role in this pathway, because LKB1-deficient cells are hypersensitive to apoptosis induced by energy stress. On the basis of these results, we propose a model to explain the apparent paradox that LKB1 is a tumor suppressor, yet cells lacking LKB1 are resistant to cell transformation by conventional oncogenes and are sensitive to killing in response to agents that elevate AMP. The role of LKB1/AMPK in the survival of a subset of genetically defined tumor cells may provide opportunities for cancer therapeutics.

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Diabetes and Cancer

2010-06-29

Edward L. Giovannucci , David M. Harlan , Michael C. Archer +6 more

Epidemiologic evidence suggests that cancer incidence is associated with diabetes as well as certain diabetes risk factors and diabetes treatments.This consensus statement of experts assembled jointly by the American Diabetes … Epidemiologic evidence suggests that cancer incidence is associated with diabetes as well as certain diabetes risk factors and diabetes treatments.This consensus statement of experts assembled jointly by the American Diabetes Association and the American Cancer Society reviews the state of science concerning 1) the association between diabetes and cancer incidence or prognosis, 2) risk factors common to both diabetes and cancer, 3) possible biologic links between diabetes and cancer risk, and 4) whether diabetes treatments influence risk of cancer or cancer prognosis.In addition, key unanswered questions for future research are posed.

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From the Triumvirate to the Ominous Octet: A New Paradigm for the Treatment of Type 2 Diabetes Mellitus

2009-03-31

Ralph A. DeFronzo

Insulin resistance in muscle and liver and β-cell failure represent the core pathophysiologic defects in type 2 diabetes. It now is recognized that the β-cell failure occurs much earlier and … Insulin resistance in muscle and liver and β-cell failure represent the core pathophysiologic defects in type 2 diabetes. It now is recognized that the β-cell failure occurs much earlier and is more severe than previously thought. Subjects in the upper tertile of impaired glucose tolerance (IGT) are maximally/near-maximally insulin resistant and have lost over 80% of their β-cell function. In addition to the muscle, liver, and β-cell (triumvirate), the fat cell (accelerated lipolysis), gastrointestinal tract (incretin deficiency/resistance), α-cell (hyperglucagonemia), kidney (increased glucose reabsorption), and brain (insulin resistance) all play important roles in the development of glucose intolerance in type 2 diabetic individuals. Collectively, these eight players comprise the ominous octet and dictate that: 1 ) multiple drugs used in combination will be required to correct the multiple pathophysiological defects, 2 ) treatment should be based upon reversal of known pathogenic abnormalities and not simply on reducing the A1C, and 3 ) therapy must be started early to prevent/slow the progressive β-cell failure that already is well established in IGT subjects. A treatment paradigm shift is recommended in which combination therapy is initiated with diet/exercise, metformin (which improves insulin sensitivity and has antiatherogenic effects), a thiazolidinedione (TZD) (which improves insulin sensitivity, preserves β-cell function, and exerts antiatherogenic effects), and exenatide (which preserves β-cell function and promotes weight loss). Sulfonylureas are not recommended because, after an initial improvement in glycemic control, they are associated with a progressive rise in A1C and progressive loss of β-cell function. The natural history of type 2 diabetes has been well described in multiple populations (1–16) (rev. in (17,18). Individuals destined to develop type 2 diabetes inherit a set of genes from their parents that make their tissues resistant to insulin (1,16,19–24). In liver, the insulin resistance is manifested by …

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AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy

2007-08-22

D. Grahame Hardie

AMP-Activated Protein Kinase Induces a p53-Dependent Metabolic Checkpoint

2005-04-01

Russell G. Jones , David R. Plas , Sara Kubek +5 more

PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes

2003-06-13

Vamsi K. Mootha , Cecilia M. Lindgren , Karl‐Fredrik Eriksson +7 more

Acarbose for prevention of type 2 diabetes mellitus: the STOP-NIDDM randomised trial

2002-06-01

Jean‐Louis Chiasson , Robert G. Josse , Ramón Gomis +3 more

Quantitative Insulin Sensitivity Check Index: A Simple, Accurate Method for Assessing Insulin Sensitivity In Humans

2000-07-01

Arie Katz , Sridhar Nambi , Kieren J. Mather +4 more

Insulin resistance plays an important role in the pathophysiology of diabetes and is associated with obesity and other cardiovascular risk factors. The "gold standard" glucose clamp and minimal model analysis … Insulin resistance plays an important role in the pathophysiology of diabetes and is associated with obesity and other cardiovascular risk factors. The "gold standard" glucose clamp and minimal model analysis are two established methods for determining insulin sensitivity in vivo, but neither is easily implemented in large studies. Thus, it is of interest to develop a simple, accurate method for assessing insulin sensitivity that is useful for clinical investigations. We performed both hyperinsulinemic isoglycemic glucose clamp and insulin-modified frequently sampled iv glucose tolerance tests on 28 nonobese, 13 obese, and 15 type 2 diabetic subjects. We obtained correlations between indexes of insulin sensitivity from glucose clamp studies (SI(Clamp)) and minimal model analysis (SI(MM)) that were comparable to previous reports (r = 0.57). We performed a sensitivity analysis on our data and discovered that physiological steady state values [i.e. fasting insulin (I(0)) and glucose (G(0))] contain critical information about insulin sensitivity. We defined a quantitative insulin sensitivity check index (QUICKI = 1/[log(I(0)) + log(G(0))]) that has substantially better correlation with SI(Clamp) (r = 0.78) than the correlation we observed between SI(MM) and SI(Clamp). Moreover, we observed a comparable overall correlation between QUICKI and SI(Clamp) in a totally independent group of 21 obese and 14 nonobese subjects from another institution. We conclude that QUICKI is an index of insulin sensitivity obtained from a fasting blood sample that may be useful for clinical research.

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Diabetes Mellitus, Fasting Glucose, and Risk of Cause-Specific Death

2011-03-03

Sreenivasa Rao Kondapally Seshasai , Stephen Kaptoge , Alexander Thompson +7 more

The extent to which diabetes mellitus or hyperglycemia is related to risk of death from cancer or other nonvascular conditions is uncertain. The extent to which diabetes mellitus or hyperglycemia is related to risk of death from cancer or other nonvascular conditions is uncertain.

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Mechanism by Which Fatty Acids Inhibit Insulin Activation of Insulin Receptor Substrate-1 (IRS-1)-associated Phosphatidylinositol 3-Kinase Activity in Muscle

2002-12-01

Chunli Yu , Yan Chen , Gary W. Cline +7 more

Recent studies have demonstrated that fatty acids induce insulin resistance in skeletal muscle by blocking insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase). To examine the mechanism by … Recent studies have demonstrated that fatty acids induce insulin resistance in skeletal muscle by blocking insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase). To examine the mechanism by which fatty acids mediate this effect, rats were infused with either a lipid emulsion (consisting mostly of 18:2 fatty acids) or glycerol. Intracellular C18:2 CoA increased in a time-dependent fashion, reaching an ∼6-fold elevation by 5 h, whereas there was no change in the concentration of any other fatty acyl-CoAs. Diacylglycerol (DAG) also increased transiently after 3–4 h of lipid infusion. In contrast there was no increase in intracellular ceramide or triglyceride concentrations during the lipid infusion. Increases in intracellular C18:2 CoA and DAG concentration were associated with protein kinase C (PKC)-θ activation and a reduction in both insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1 associated PI3-kinase activity, which were associated with an increase in IRS-1 Ser307 phosphorylation. These data support the hypothesis that an increase in plasma fatty acid concentration results in an increase in intracellular fatty acyl-CoA and DAG concentrations, which results in activation of PKC-θ leading to increased IRS-1 Ser307 phosphorylation. This in turn leads to decreased IRS-1 tyrosine phosphorylation and decreased activation of IRS-1-associated PI3-kinase activity resulting in decreased insulin-stimulated glucose transport activity. Recent studies have demonstrated that fatty acids induce insulin resistance in skeletal muscle by blocking insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase). To examine the mechanism by which fatty acids mediate this effect, rats were infused with either a lipid emulsion (consisting mostly of 18:2 fatty acids) or glycerol. Intracellular C18:2 CoA increased in a time-dependent fashion, reaching an ∼6-fold elevation by 5 h, whereas there was no change in the concentration of any other fatty acyl-CoAs. Diacylglycerol (DAG) also increased transiently after 3–4 h of lipid infusion. In contrast there was no increase in intracellular ceramide or triglyceride concentrations during the lipid infusion. Increases in intracellular C18:2 CoA and DAG concentration were associated with protein kinase C (PKC)-θ activation and a reduction in both insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1 associated PI3-kinase activity, which were associated with an increase in IRS-1 Ser307 phosphorylation. These data support the hypothesis that an increase in plasma fatty acid concentration results in an increase in intracellular fatty acyl-CoA and DAG concentrations, which results in activation of PKC-θ leading to increased IRS-1 Ser307 phosphorylation. This in turn leads to decreased IRS-1 tyrosine phosphorylation and decreased activation of IRS-1-associated PI3-kinase activity resulting in decreased insulin-stimulated glucose transport activity. insulin receptor substrate-1 insulin receptor phosphatidylinositol 3-kinase protein kinase C diacylglycerol long-chain acyl-CoA liquid chromatography tandem mass spectrometry 4-morpholinepropanesulfonic acid tumor necrosis factor α Insulin resistance in skeletal muscle is a major factor in the pathogenesis of type 2 diabetes. Recent studies in animals and humans have demonstrated a strong relationship with increased intramuscular triglyceride content (1Kraegen E.W. Cooney G.J., Ye, J.M. Thompson A.L. Furler S.M. Exp. Clin. Endocrinol. Diabetes. 2001; 109: S189-S201Crossref PubMed Scopus (118) Google Scholar, 2Kim J.K. Gavrilova O. Chen Y. Reitman M.L. Shulman G.I. J. Biol. Chem. 2000; 275: 8456-8460Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 3Kim J.K. Fillmore J.J. Chen Y., Yu, C. Moore I.K. Pypaert M. Lutz E.P. Kako Y. Velez-Carrasco W. Goldberg I.J. Breslow J.L. Shulman G.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7522-7527Crossref PubMed Scopus (598) Google Scholar, 4Pan D. Lillioja S. Kriketos A. Milner M. Baur L. Bogardus C. Jenkins A. Storlien L. Diabetes. 1997; 46: 983-988Crossref PubMed Google Scholar) and intramyocellular triglyceride content as assessed by 1H NMR (5Krssak M. Falk Petersen K. Dresner A. DiPietro L. Vogel S.M. Rothman D.L. Roden M. Shulman G.I. Diabetologia. 1999; 42: 113-116Crossref PubMed Scopus (1037) Google Scholar, 6Perseghin G. Scifo P., De Cobelli F. Pagliato E. Battezzati A. Arcelloni C. Vanzulli A. Testolin G. Pozza G. Del Maschio A. Luzi L. Diabetes. 1999; 48: 1600-1606Crossref PubMed Scopus (761) Google Scholar, 7Jacob S. Machann J. Rett K. Bretchel K. Volk A. Renn W. Maerker E. Matthaei S. Schick F. Claussen C.D. Harring H.U. Diabetes. 1999; 48: 1113-1119Crossref PubMed Scopus (557) Google Scholar). In addition, infusions of lipid emulsions with heparin to acutely raise plasma fatty acid concentrations have also been shown to cause profound insulin resistance in rat and human skeletal muscle within 4–6 h (8Boden G. Chen X. J. Clin. Invest. 1995; 96: 1261-1268Crossref PubMed Scopus (291) Google Scholar, 9Roden M. Price T.B. Perseghin G. Petersen K.F. Rothman D.L. Cline G.W. Shulman G.I. J. Clin. Invest. 1996; 97: 2859-2865Crossref PubMed Scopus (1272) Google Scholar, 10Dresner A. Laurent D. Marcucci M. Griffin M.E. Dufour S. Cline G. Slezak L.A. Andersen D.K. Hundal R.S. Rothman D.L. Petersen K.F. Shulman G.I. J. Clin. Invest. 1999; 103: 253-259Crossref PubMed Scopus (1049) Google Scholar, 11Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Crossref PubMed Scopus (999) Google Scholar). The mechanism by which fatty acids induce insulin resistance in skeletal muscle remains controversial. Randle et al. (12Randle P.J. Garland P.B. Hales C.N. Newsholme E.A. Lancet. 1963; i: 785-789Abstract Scopus (3879) Google Scholar, 13Randle P.J. Newsholme E.A. Garland P.B. Biochem. J. 1964; 93: 652-665Crossref PubMed Scopus (508) Google Scholar) first suggested that fatty acids might induce insulin resistance in skeletal muscle by inhibiting pyruvate dehydrogenase activity, resulting in an increase in intracellular citrate concentration, which would then result in inhibition of phosphofructokinase activity leading to an increase in intracellular glucose-6-phosphate; this in turn would inhibit hexokinase activity, resulting in decreased glucose uptake. More recent 31P/13C NMR studies in humans have revealed a very different mechanism of fatty acid-induced insulin resistance whereby an increase in plasma fatty acid concentration was shown to result in lower intramyocellullar glucose 6-phosphate (9Roden M. Price T.B. Perseghin G. Petersen K.F. Rothman D.L. Cline G.W. Shulman G.I. J. Clin. Invest. 1996; 97: 2859-2865Crossref PubMed Scopus (1272) Google Scholar, 14Roden M. Krssak M. Stingl H. Gruber S. Hofer A. Furnsinn C. Moser E. Waldhausl W. Diabetes. 1999; 48: 358-364Crossref PubMed Scopus (170) Google Scholar) and glucose concentrations (10Dresner A. Laurent D. Marcucci M. Griffin M.E. Dufour S. Cline G. Slezak L.A. Andersen D.K. Hundal R.S. Rothman D.L. Petersen K.F. Shulman G.I. J. Clin. Invest. 1999; 103: 253-259Crossref PubMed Scopus (1049) Google Scholar), suggesting that fatty acids inhibit insulin-stimulated glucose transport activity (10Dresner A. Laurent D. Marcucci M. Griffin M.E. Dufour S. Cline G. Slezak L.A. Andersen D.K. Hundal R.S. Rothman D.L. Petersen K.F. Shulman G.I. J. Clin. Invest. 1999; 103: 253-259Crossref PubMed Scopus (1049) Google Scholar). These changes were associated with reduced insulin-stimulated IRS-11 tyrosine phosphorylation (11Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Crossref PubMed Scopus (999) Google Scholar) and IRS-1-associated phosphatidylinositol 3-kinase (PI3-kinase) activity (10Dresner A. Laurent D. Marcucci M. Griffin M.E. Dufour S. Cline G. Slezak L.A. Andersen D.K. Hundal R.S. Rothman D.L. Petersen K.F. Shulman G.I. J. Clin. Invest. 1999; 103: 253-259Crossref PubMed Scopus (1049) Google Scholar, 11Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Crossref PubMed Scopus (999) Google Scholar) suggesting that fatty acids cause insulin resistance through inhibition of insulin signaling, which we hypothesized might occur through activation of a serine kinase cascade involving PKC-θ (11Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Crossref PubMed Scopus (999) Google Scholar). To explore the possible roles of different intracellular fatty acid metabolites such as fatty acyl-CoA, diacylglycerol (DAG), ceramides, and triglycerides in mediating fatty acid-induced insulin resistance in skeletal muscle, we measured these metabolites at different time intervals during a lipid infusion in relation to insulin stimulation: (i) insulin receptor tyrosine phosphorylation, (ii) IRS-1 tyrosine phosphorylation, and (iii) IRS-1-associated PI3-kinase activity as well as PKC-θ translocation. In a separate group of in vitro soleus muscle studies, we also examined whether fatty acid-induced defects in insulin signaling were coupled to defects in insulin-stimulated glucose uptake across a range of insulin concentrations. LCACoA standards (C16:1, C16:0, C17:0, C18:2, C18:1, and C18:0), diacylglyceride standards, and ceramide standards (C6:0, C16:0, C18:0) were purchased from Sigma.N-Arachidoyl-d-sphingosine andN-lignoceroyl-d-sphingosine were purchased from Avanti Polar Lipids (Arlington, AL). Antibody against IRS-1 was purchased from Upstate Biotechnology (Lake Placid, NY). Antibody against the insulin receptor subunit andZymed phosphotyrosine and rabbit anti-peptide against nPKC-θ were from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-mouse IgG antibodies conjugated to horseradish peroxidase were obtained from Caltag Laboratories (Burlingame, CA). Mouse monoclonal antibody against PKC-θ was from Transduction Laboratories (Lexington, KY). Male Wistar rats (Charles River, Wilmington, MA) weighing between 250 and 300 g (for the time course study) and 50 and 75 g (for insulin dose response) were housed in an environmentally controlled room with a 12-h light/dark cycle and fed with regular rat chow diet. The rats were catheterized in the right jugular vein and carotid artery; the catheters were externalized through an incision in the skin flap behind their head. The rats were allowed to recover from surgery until they reached preoperative weight (∼5–7 days) and were fasted overnight (∼15 h) before the infusion experiment. All procedures were approved by the Yale University Animal Care and Use Committee. The rats were divided randomly into five study groups (6–8 rats/group). The control group was infused with isotonic saline solution for 5 h. The other groups were infused with a 20% triglycerides emulsion (Liposyn II, Abbott Laboratories, North Chicago, IL) (5 ml/kg/h) combined with heparin (6 units/h) for 1, 3 and 5 h. A fifth wash-out group was infused with lipid/heparin for 5 h, which was then discontinued and followed with an isotonic saline infusion for another 3 h. Identical studies were performed for muscle DAG analysis (3–9 rats/group) with the addition of a 4-h lipid/heparin infusion group (n = 4). At the end of the infusions, rats were anesthetized with pentobarbital (50 mg/kg); soleus muscle samples, rapidly dissected and freeze-clamped in situ, were stored at −70 °C for measurement of fat metabolites. Soleus muscle was selected for all studies because it consists of mostly type 1 fiber, which is highly insulin-responsive and best reflects insulin action in human skeletal muscle (10Dresner A. Laurent D. Marcucci M. Griffin M.E. Dufour S. Cline G. Slezak L.A. Andersen D.K. Hundal R.S. Rothman D.L. Petersen K.F. Shulman G.I. J. Clin. Invest. 1999; 103: 253-259Crossref PubMed Scopus (1049) Google Scholar, 11Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Crossref PubMed Scopus (999) Google Scholar). To study the effect of fatty acids on insulin signaling in muscle at the same time points, we performed another set of identical parallel studies in five groups (basal, 1, 3, an 5 h lipid/heparin infusion and 3 h wash-out) under conditions identical to those described above, adding a 20-min hyperinsulinemic euglycemic clamp following the lipid/heparin or saline infusion. In these studies an intravenous bolus (150 milliunits/kg for 45 s, 75 milliunits/kg for another 45 s) of insulin (humulin regular insulin, Eli Lily, Indianapolis, IN) was followed by a constant insulin infusion at 10 milliunits/kg/min, with plasma glucose concentration clamped at 5.5 mm using a variable infusion of glucose (50g/dl) to maintain euglycemia as described previously (11Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Crossref PubMed Scopus (999) Google Scholar). At the end of the clamps, rats were anesthetized with pentobarbital (50 mg/kg). Soleus muscle samples were rapidly dissected, freeze-clamped in situ, and stored at −70 °C for insulin signaling assays. Rats were euthanized with a lethal dose of pentobarbital. LCACoAs were extracted from frozen tissue samples (∼100 mg) and purified using a solid phase extraction method described previously by Deutsch et al. (15Deutsch J. Grange E. Rapoport S.I. Purdon A.D. Anal. Biochem. 1994; 220: 321-323Crossref PubMed Scopus (102) Google Scholar) with minor modifications for desalting. A known amount of heptadecanoyl-CoA was added as an internal standard. OPC columns (Applied Biosystems, Foster City, CA) were used for solid phase extraction. Samples were dissolved in 100 μl of methanol/H2O for LC/MS/MS analysis. DAGs and ceramides were extracted from frozen tissue (∼100 mg) with chloroform/methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene. Prior to the extraction, known amounts of 1,3-dipentadecanoin, triheptadecanoin, and hexanoylsphingosine were added as internal standards. Extracted samples were evaporated to dryness and redissolved in 1 ml of hexane-methylene chloride-ethyl ether (95:5:0.5, v/v/v). DAGs were isolated from triglycerides by use of a diol bonded-phase SPE column (Waters, Inc., Milford, MA) under vacuum, as described previously (16Pacheco Y.M. Perez-Camino M.C. Cert A. Montero E. Ruiz-Gutierrez V. J. Chromatogr. B Biomed. Sci. Appl. 1998; 714: 127-132Crossref PubMed Scopus (17) Google Scholar). Briefly, the SPE column was preconditioned with 4 ml of hexane. The lipid extract was then placed on the column, and triglycerides were eluted with 8 ml of hexane-methylene chloride-ethyl ether (89:10:1, v/v/v). DAGs were eluted with 8 ml of hexane-ethyl acetate (85:15, v/v) into a second set of collection tubes. The solvent was evaporated to dryness under vacuum and redissolved in 0.5 ml of hexane-ethyl acetate (85:15, v/v) for LC/MS/MS analysis. Monitoring for the presence of triheptadecanoin in the DAG fraction assessed the separation of triglycerides from DAGs. A bench-top tandem mass spectrometer, API 3000 (PerkinElmer Life Sciences), interfaced with a TurboIonspray ionization source or atmospheric pressure chemical ionization source was used. Peripherals included a PerkinElmer series 200 micro-pump and an autosampler. LCACoAs were ionized in negative electrospray mode. Doubly charged ions and corresponding product ions were chosen as transition pairs for each CoA species (C16:1, C16:0, C18:2, C18:1, and C18:0) for selective reactions monitoring (SRM) quantitation. Total LCACoAs contents were obtained from the sum of individual species. Methanol/H2O (60/40) was used as continuous flow at 300 μl/min, and 5 μl of sample was injected for analysis. DAGs (derived from C16:1, C16:0, C18:2, C18:1, and C18:0) and ceramides (C16:0, C18:0, C20:0, C22:0, C24:1, C24:0) were ionized in positive atmospheric pressure chemical ionization mode. [M+H-18]+/product ions from corresponding fatty acid moiety were monitored for SRM quantitation for DAGs. [M+H-18]+/264.3 were monitored for ceramide species for quantitation. The same mobile phase was used for LCACoAs at 300 μl/min with 3 μl of sample injected. After a 5-h infusion with glycerol (as control) or lipid/heparin at 85 μl/kg/min, rats were anesthetized with an intravenous injection of sodium pentobarbital (50 mg/kg). Soleus muscles were isolated from the rats and preincubated in oxygenated (95% O2, 5% CO2) Krebs-Henseleit bicarbonated (KHB) buffer containing 2 mm pyruvate, 36 mm mannitol, and 0.1% bovine serum albumin (preincubation buffer) to recover for 30 min at 18 °C. The soleus muscles were then incubated at 29 °C in oxygenated preincubation buffer with various concentration of insulin (0, 50, 1,000, or 10,000 microunits/ml) for 35 min. After incubation, the muscles were rinsed with ice-cold saline and freeze-clamped in liquid nitrogen for analysis of insulin-stimulated IRS-1 tyrosine phosphorylation and insulin-stimulated IRS-1-associated PI3-kinase activity. To measure the insulin-stimulated glucose uptake in the muscle, soleus muscles were preincubated at 29 °C with various concentrations of insulin (0, 50, 1,000, or 10,000 microunits/ml) for 35 min followed by incubation in KHB buffer containing 1 mm [3H]2-deoxyglucose and 39 mm [1-14C]mannitol for an additional 20 min. For IRS-1 serine phosphorylation analysis, after a 5-h lipid infusion, soleus muscles were freeze-clamped in situ and kept in liquid nitrogen until analysis. Muscle samples were ground under liquid nitrogen and homogenized in a ice-cold Hepes buffer, pH 7.4, containing 150 mm NaCl 50 mm β-glycerol phosphate, 2 mm dithiothreitol, 1 mmNaVO4, 2 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 1% Triton-100, 10% glycerol, and 10 μg/ml aprotinin. The homogenates were centrifuged at 20,500 × g for 1 h. Supernatants were collected, and protein concentration was measured with the Bradford protein assay reagent (Bio-Rad). Muscle homogenates (4 mg protein) were immunoprecipitated with 4 μg of anti-IRS-1 antibody for 18 h for IRS-1 tyrosine phosphorylation and PI3-kinase activity assay or with 4 μg of anti-IR antibody for IR tyrosine phosphorylation. Immunoprecipitates were washed three times by brief centrifugation and gentle resuspension in ice-cold homogenization buffer plus 0.1% SDS. Immunoprecipitates were subjected to SDS-PAGE on a 4–12% gradient gel. Proteins were transferred to nitrocellulose membrane using a semidry electro-blotter (Owl Separation System, Portsmouth, NH). The membranes were immunoblotted with anti-phosphotyrosine antibody, and bands were visualized using enhanced chemiluminescence (Amersham Biosciences) and quantified by densitometry (Amersham Biosciences). The membrane was stripped with 100 mm glycine, pH 3.0, and reblotted with anti-IRS-1 antibody to determine the amount of IRS-1 proteins. IRS-1 serine phosphorylation was measured using a site-specific antibody, phospho-Ser307, generated in Dr. Morris White's laboratory (17Aguirre V. Uchida T. Yenush L. Davis R. White M. J. Biol. Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1199) Google Scholar). Immunoprecipitation and Western blotting procedures were the same as for IRS-1 tyrosine phosphorylation. The immunoprecipitates were washed twice with phosphate-buffered saline, twice with 100 mm Tris, pH 7.5, containing 500 mmLiCl2, and twice with 10 mm Tris containing 150 mm NaCl and 1 mm EDTA. 100 μmNa3VO4 was included in all the wash buffers. Kinase reactions were done as described previously (11Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Crossref PubMed Scopus (999) Google Scholar).32P was captured with a storage phosphor-screen, and the screen was scanned with a Storm system. Images were analyzed and quantified using ImageQuant software. 100 mg of soleus muscle was homogenized and extracted in 4× (w/v) ice cold 20 mm MOPS, pH 7.5, 250 mm mannitol, 1.2 mm EGTA, 1 mm dithiothreitol, 2 mmphenylmethylsulfonyl fluoride, leupeptin (200 μg/ml), and 2 mm benzamidine. The homogenate was solubilized by hand for 2 min and centrifuged at 4 °C for 10 min at 100,000 ×g. Separation of cytosol and membrane fraction was done as described previously (18Laybutt D.R. Schmitz-Peiffer C. Saha A.K. Ruderman N.B. Biden T.J. Kraegen E.W. Am. J. Physiol. 1999; 277: E1070-E1076Crossref PubMed Google Scholar). 5 (cytosolic) or 10 μg (particulate) of protein was loaded and subjected to SDS-PAGE (10% gel, 187 V). Proteins separated on the gels were electrophoretically transferred to polyvinylidene difluoride filter membranes (Amersham Biosciences) in 19 mm Tris, pH 8.9, buffer containing 140 mmglycine at 90 V for 90 min. Polyvinylidene difluoride membranes were probed with 0.625 μg/ml anti-PKC-θ antibody (Transduction Laboratories) for 2 h at room temperature, followed by horseradish peroxidase-conjugated goat anti-mouse antibody (1:5,000) for 2 h. PKC isozymes visualized by enhanced chemiluminescence reagents and quantitated by densitometry using a Medical Dynamics Personal Densitometer SI and IP Lab Gel H software (Signal Analytics, Vienna, VA). Individual band densities were adjusted for inter-gel variability using the standard, and the amount of PKCθ in each fraction were calculated according to the total amount of protein in the final volume of supernatant extracted. Plasma fatty acid concentration was determined with an acyl-CoA oxidase-based colorimetric kit (Wako NEFA-C, Wako Pure Chemicals, Osaka, Japan). Tissue triglycerides were extracted by adapting the method described by Storlien et al. (19Storlien L.H. Jenkins A.B. Chisholm D.J. Pascoe W.S. Khouri S. Kraegen E.W. Diabetes. 1991; 40: 280-289Crossref PubMed Scopus (960) Google Scholar), and triglyceride content was measured using a kit from Sigma. Data were expressed as means ± S.E. Analysis of data using analysis-of-variance with one-way post-hoc tests (Fisher's protected least significant difference) was done to determine the differences between control and different time courses of lipid infusion groups at a minimum p < 0.05 threshold. Basal plasma fatty concentration increased rapidly following the lipid/heparin infusion and remained constant until the saline wash-out period during which time it returned to base-line concentration (Fig. 1 A). This increase in plasma fatty acid concentration in the lipid-infused group resulted in increases in both intramuscular LCACoAs and DAG concentration in the soleus muscle compared with the control group (Fig. 1, B andC). Although the LCACoA continued to increase throughout the lipid infusion, the DAGs reached a peak concentration at 3–4 h and then surprisingly decreased to basal concentrations despite continued lipid infusion (Fig. 1 C). In contrast, lipid infusion had no effect on intramyocellullar ceramide content (Fig. 1 D) or muscle triglyceride (Fig. 1 E) content except at the 1-h time point, at which time the concentration decreased compared with base line. The increase in total LCACoA concentration could be accounted for entirely by a selective increase in C18:2 CoA (major fatty acid composition in liposyn II) (3.86 ± 0.46 nmol/g of weight for control group, 9.30* ± 0.87, 16.17** ± 2.37, and 18.89** ± 2.51 nmol/g of weight after a 1-h, 3-h, and 5-h lipid infusion and 7.22 ± 1.22 nmol/g of weight after wash-out period; *, p < 0.05 versus control; **,p < 0.001 versus control; Fig. 2 A). In contrast the transient ∼3–4-fold increase in total DAG content at 3–4 h (0.65 ± 0.14 μmol/g of weight for control group, 1.43 ± 0.51, 2.73 ± 0.83+, 2.54 ± 0.79+, 1.36 ± 0.40, 0.96 ± 0.31 μmol/g of weight for 1-h, 3-h, 4-h, 5-h and wash-out groups, respectively; +, p ≤ 0.006versus control) could be attributed to an increase in virtually all DAG species (Fig. 2 B). These increases in intracellular LCACoA and DAG concentrations were associated with PKC-θ activation, as reflected by a significant reduction in the fraction of PKC-θ in the cytosol and a significant increase in the PKC-θ membrane-associated/cytosol fraction after 5 h of lipid infusion (both p = 0.04 versus control group; Fig. 3). There was also a reduction in total PKC-θ content, which is consistent with previous observations in a high-fat fed rat model that had increased intramuscular lipid accumulation (20Schmitz-Peiffer C. Browne C.L. Oakes N.D. Watkinson A. Chisholm D.J. Kraegen E.W. Biden T.J. Diabetes. 1997; 46: 169-178Crossref PubMed Google Scholar).Figure 2Time course for the concentration profiles of LCACoAs and DAG in soleus muscles during the lipid infusion. A, individual LCACoAs species were quantitated:C16:1, palmitoleoyl-CoA; C16:0, palmitoyl-CoA;C18:2, linoleoyl-CoA; C18:1, oleoyl-CoA; andC18:0, stearoyl-CoA. Values are means ± S.E. for 6–10 experiments. *, p < 0.05 versus control group; **, p < 0.001 versus control group.B, DAG species were abbreviated as two contributing fatty acyl groups. S, stearoyl; O, oleoyl;L, linoleoyl; P, palmitoyl; Po, palmitoleoyl. Values are means ± S.E. for 3–9 experiments. *,p < 0.05 versus control group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Time course for the effects of fatty acids on PKC -θ activity in soleus muscle in vivo. PKC-θ protein levels were determined in the cytosolic and membrane fraction by immunoblotting with PKC-θ specific antibodies. Total PKC-θ levels were calculated from the sum of cytosolic and membrane-associated amounts, and PKC-θ distribution was expressed as the ratio of membrane-associated to cytosolic amounts.W/O, without. Values are means ± S.E. for 6–10 experiments. *, p < 0.05 versus control groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The increase in intracellular fatty acyl-CoA and PKC-θ activation were also associated with a significant impairment in insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI3-kinase activity after 5 h of lipid infusion (Fig. 4). These changes were associated with a 1.6-fold increase (p = 0.002 versus control) in IRS-1 Ser307 phosphorylation following 5 h of lipid infusion (Fig. 5). In contrast lipid infusion did not inhibit insulin-stimulated insulin receptor tyrosine phosphorylation (Fig. 4).Figure 5Effects of fatty acids on IRS-1 Ser307 phosphorylation in soleus muscle in vivo. A, IRS-1 Ser307phosphorylation was detected with a polyclonal antibody raised specifically for phosphorylated Ser307 (upper panel). Nitrocellulose membranes were stripped and reprobed with IRS-1 antibody to ensure equal amount of protein loading (lower panel). IP, immunoprecipitate; INS, insulin; Gly, glycerol; WB, Western blot.B, degree of IRS-1 Ser307 phosphorylation in glycerol- and lipid-infused groups. Values are means ± S.E. from six rats for each group. *, p < 0.05 versusglycerol-infused rats.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Following the 3-h lipid wash-out period, intracellular 18:2 acyl-CoA returned to base-line concentrations, and PKC-θ activity returned to normal (Figs. 1 and 3). In parallel with these results insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI3-kinase activity also returned to normal. To determine whether higher concentrations of insulin could overcome these lipid-induced defects in insulin signaling and action, we also examined insulin-stimulated muscle glucose uptake and insulin signaling across a wide range of insulin concentrations (50, 1,000, and 10,000 microunits/ml) in an in vitro soleus muscle preparation following 5 h of either lipid or glycerol infusion. Consistent with our previous results, 5 h of lipid infusion induced a profound defect in insulin-stimulated glucose uptake, which occurred across all insulin concentrations (Fig. 6). This reduction in insulin-stimulated glucose uptake was paralleled by similar reductions in insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI3-kinase activity across all insulin concentrations, but there was no change in insulin receptor tyrosine phosphorylation (Fig. 7). Taken together these results demonstrates that fatty acids induce a defect in insulin activation of PI3-kinase at the level of IRS-1 tyrosine phosphorylation that cannot be overcome with supraphysiologic concentrations of insulin.Figure 7Insulin dose response for the effects of fatty acids on insulin signaling in soleus muscle in vitro. A, IR tyrosine phosphorylation was detected with phosphotyrosine-specific antibody (upper panel). Nitrocellulose membranes were stripped and reprobed with insulin receptor antibody to ensure equal amount of protein loading (lower panel). The bar graph shows the degree of IR tyrosine phosphorylation in glycerol- or lipid-infused soleus musclein vitro. IP, immunoprecipitate.B, IRS-1 tyrosine phosphorylation was detected with phosphotyrosine-specific antibody (P-tyr, upper panel). Nitrocellulose membranes were stripped and reprobed with IRS-1 antibody to ensure equal amounts of protein loading (lower panel). The bar graph shows the degree of IRS-1 tyrosine phosphorylation in glycerol- or liposyn-infused soleus musclein vitro. WB, Western blot. C, IRS-1-associated PI3-kinase activity was detected by measuring32P incorporation into phosphatidylinositol (PI3P). The bar graph shows the IRS-1-associated PI3-kinase activity in glycerol- or liposyn-infused soleus muscle in vitro. Values are means ± S.E. from eight independent experiments. *, p < 0.05versus glycerol-infused rats.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine the possible roles of fatty acyl-CoA, diacylglycerol, ceramides, and triglycerides in mediating fatty acid induced insulin resistance in skeletal muscle, we assessed the intracellular concentration of these metabolites at different time intervals during a lipid infusion in awake rats. The changes in these fatty acid metabolite concentrations were then compared with changes in insulin-stimulated insulin receptor tyrosine phosphorylation, IRS-1 tyrosine phosphorylation, IRS-1-associated PI3-kinase activity, and PKC-θ translocation. We found that during the lipid infusion intra- myocellullar C18:2 CoA concentration increased by ∼6-fold and that it was the only intracellular fatty acyl-CoA to increase. Because the infused intralipid consisted mostly of C18:2 fatty acids, these data strongly suggest that this intracellular fatty acyl-CoA was derived from the infused lipid. Following the increase in intracellular C18:2 CoA, there was a ∼3-fold increase in intracellular DAG, which peaked at 3–4 h and then surprisingly declined despite persistent elevation in plasma fatty acid concentrations. In contrast to the fatty acyl-CoA, which consisted mostly of C18:2 fatty acids, the increase in DAG consisted of virtually all measured fatty acids. Taken together these data suggest that an increase in intracellular fatty acyl-CoA activates a phospholipase that leads to production of DAG from endogenous lipid sources, which might explain the observed decrease in intramuscular triglyceride content during the first couple of hours of the lipid infusion. In contrast to the increases in intracellular fatty acyl-CoA and DAG, there were no significant increases in intracellular ceramides or triglyceride concentrations during the 5-h lipid infusion, which suggests that these metabolites do not play a major role in mediating fatty acid-induced insulin resistance in skeletal muscle. In parallel with the increases in intracellular fatty acyl-CoA, we observed a ∼30% reduction in insulin activation of IRS-1 tyrosine phosphorylation and an ∼50% reduction in IRS-1-associated PI3-kinase activity after 5 h of lipid infusion, which coincided with activation of PKC-θ. These data might explain the 3–5 h delay for fatty acid-induced insulin resistance in skeletal muscle resulting from an intralipid/heparin infusion (8Boden G. Chen X. J. Clin. Invest. 1995; 96: 1261-1268Crossref PubMed Scopus (291) Google Scholar, 9Roden M. Price T.B. Perseghin G. Petersen K.F. Rothman D.L. Cline G.W. Shulman G.I. J. Clin. Invest. 1996; 97: 2859-2865Crossref PubMed Scopus (1272) Google Scholar). In contrast, the lipid infusion had no effect on insulin receptor tyrosine phosphorylation. Overall these data demonstrate that increases in plasma fatty acid concentration inhibit insulin activation of IRS-1-associated PI3-kinase at the level of IRS-1, possibly though activation of PKC-θ, a known serine kinase. To gain further insights into this mechanism we assessed IRS-1 Ser307 phosphorylation. Previous in vitrostudies by Aguirre et al. (17Aguirre V. Uchida T. Yenush L. Davis R. White M. J. Biol. Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1199) Google Scholar) demonstrated that IRS-1 Ser307 phosphorylation is a critical site in mediating TNFα-induced insulin resistance in Chinese hamster ovary cells. When IRS-1 Ser307 was mutated to IRS-1 Ala307, these cells were protected from TNFα-induced insulin resistance. Indeed, in the present study we found that after 5 h of lipid infusion there was a 1.6-fold increase in IRS-1 Ser307 phosphorylation in soleus muscle, which suggests that fatty acids may mediate insulin resistance through the same common final pathway as TNFα (21Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2251) Google Scholar). To determine whether higher concentrations of insulin could overcome these fatty acid-induced defects in insulin signaling and action, we also examined these parameters in vitro, across a wide range of insulin concentrations, in soleus muscles obtained from rats following 5 h of either lipid or glycerol infusion. Consistent with our current and previous in vivo results, 5 h of lipid infusion induced a profound defect in insulin-stimulated glucose uptake (9Roden M. Price T.B. Perseghin G. Petersen K.F. Rothman D.L. Cline G.W. Shulman G.I. J. Clin. Invest. 1996; 97: 2859-2865Crossref PubMed Scopus (1272) Google Scholar, 10Dresner A. Laurent D. Marcucci M. Griffin M.E. Dufour S. Cline G. Slezak L.A. Andersen D.K. Hundal R.S. Rothman D.L. Petersen K.F. Shulman G.I. J. Clin. Invest. 1999; 103: 253-259Crossref PubMed Scopus (1049) Google Scholar, 11Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Crossref PubMed Scopus (999) Google Scholar), which occurred across all insulin concentrations. This reduction in insulin-stimulated glucose uptake was paralleled by similar reductions in insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI3-kinase activity across all insulin concentrations, but there was no change in insulin-stimulated IR tyrosine phosphorylation. Taken together these results demonstrate that the fatty acid-induced inhibition of insulin-stimulated glucose transport activity in muscle can be explained for the most part by decreased activation of PI3-kinase at the level of IRS-1 tyrosine phosphorylation, which cannot be overcome with supraphysiologic concentrations of insulin. In conclusion, these data provide new insights into the pathogenesis of fat-induced insulin resistance in skeletal muscle and support the hypothesis that an increase in plasma fatty acid concentration results in an increase in intracellular fatty acyl-CoA and DAG concentrations, which then results in activation of PKC-θ leading to increased IRS-1 Ser307 phosphorylation. These changes in turn result in decreased IRS-1 tyrosine phosphorylation and decreased activation of IRS-1-associated PI3-kinase, resulting in decreased insulin-stimulated glucose transport activity. We acknowledge the expert technical assistance of Hyegeong Kim, Lynn Croft, Anthony Romanelli, Taca Higashimori, and Theresa Choi.

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Role of AMP-activated protein kinase in mechanism of metformin action

2001-10-15

Gaochao Zhou , Robert P. Myers , Ying Li +7 more

Metformin is a widely used drug for treatment of type 2 diabetes with no defined cellular mechanism of action. Its glucose-lowering effect results from decreased hepatic glucose production and increased … Metformin is a widely used drug for treatment of type 2 diabetes with no defined cellular mechanism of action. Its glucose-lowering effect results from decreased hepatic glucose production and increased glucose utilization. Metformin’s beneficial effects on circulating lipids have been linked to reduced fatty liver. AMP-activated protein kinase (AMPK) is a major cellular regulator of lipid and glucose metabolism. Here we report that metformin activates AMPK in hepatocytes; as a result, acetyl-CoA carboxylase (ACC) activity is reduced, fatty acid oxidation is induced, and expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin or an adenosine analogue suppresses expression of SREBP-1, a key lipogenic transcription factor. In metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced; activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, we find that AMPK activation is required for metformin’s inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. Activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug; these results also suggest that alternative means of modulating AMPK should be useful for the treatment of metabolic disorders.

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Role of Fatty Acids in the Pathogenesis of Insulin Resistance and NIDDM

1997-01-01

Guenther Boden

Evidence is reviewed that free fatty acids (FFAs) are one important link between obesity and insulin resistance and NIDDM. First, plasma FFA levels are elevated in most obese subjects. Second, … Evidence is reviewed that free fatty acids (FFAs) are one important link between obesity and insulin resistance and NIDDM. First, plasma FFA levels are elevated in most obese subjects. Second, physiological elevations in plasma FFA concentrations inhibit insulin stimulated peripheral glucose uptake in a dose-dependent manner in normal controls and in patients with NIDDM. Two possible mechanisms are identified: 1) a fat-related inhibition of glucose transport or phosphorylation, which appears after 3-4 h of fat infusion, and 2) a decrease in muscle glycogen synthase activity, which appears after 4-6 h of fat infusion. Third, FFAs stimulate insulin secretion in nondiabetic individuals. Some of this insulin is transmitted in the peripheral circulation and is able to compensate for FFA-mediated peripheral insulin resistance. FFA-mediated portal hyperinsulinemia counteracts the stimulation of FFAs on hepatic glucose production (HGP) and thus prevents hepatic glucose overproduction. We speculate that, in obese individuals who are genetically predisposed to develop NIDDM, FFAs will eventually fail to promote insulin secretion. The stimulatory effect of FFAs on HGP would then become unchecked, resulting in hyperglycemia. Hence, continuously elevated levels of plasma FFAs may play a key role in the pathogenesis of NIDDM in predisposed individuals by impairing peripheral glucose utilization and by promoting hepatic glucose overproduction.

Pathogenesis of NIDDM: A Balanced Overview

1992-03-01

Ralph A. DeFronzo , Riccardo C. Bonadonna , Eleuterio Ferrannini

Non-insulin-dependent diabetes mellitus (NIDDM) results from an imbalance between insulin sensitivity and insulin secretion. Both longitudinal and cross-sectional studies have demonstrated that the earliest detectable abnormality in NIDDM is an … Non-insulin-dependent diabetes mellitus (NIDDM) results from an imbalance between insulin sensitivity and insulin secretion. Both longitudinal and cross-sectional studies have demonstrated that the earliest detectable abnormality in NIDDM is an impairment in the body's ability to respond to insulin. Because the pancreas is able to appropriately augment its secretion of insulin to offset the insulin resistance, glucose tolerance remains normal. With time, however, the β-cell fails to maintain its high rate of insulin secretion and the relative insulinopenia (i.e., relative to the degree of insulin resistance) leads to the development of impaired glucose tolerance and eventually overt diabetes mellitus. The cause of pancreatic “exhaustion” remains unknown but may be related to the effect of glucose toxicity in a genetically predisposed β-cell. Information concerning the loss of first-phase insulin secretion, altered pulsatility of insulin release, and enhanced proinsulin-insulin secretory ratio is discussed as it pertains to altered β-cell function in NIDDM. Insulin resistance in NIDDM involves both hepatic and peripheral, muscle, tissues. In the postabsorptive state hepatic glucose output is normal or increased, despite the presence of fasting hyperinsulinemia, whereas the efficiency of tissue glucose uptake is reduced. In response to both endogenously secreted or exogenously administered insulin, hepatic glucose production fails to suppress normally and muscle glucose uptake is diminished. The accelerated rate of hepatic glucose output is due entirely to augmented gluconeogenesis. In muscle many cellular defects in insulin action have been described including impaired insulin-receptor tyrosine kinase activity, diminished glucose transport, and reduced glycogen synthase and pyruvate dehydrogenase. The abnormalities account for disturbances in the two major intracellular pathways of glucose disposal, glycogen synthesis, and glucose oxidation. In the earliest stages of NIDDM, the major defect involves the inability of insulin to promote glucose uptake and storage as glycogen. Other potential mechanisms that have been put forward to explain the insulin resistance, include increased lipid oxidation, altered skeletal muscle capillary density/fiber type/blood flow, impaired insulin transport across the vascular endothelium, increased amylin, calcitonin gene-related peptide levels, and glucose toxicity.

THE AMP-ACTIVATED/SNF1 PROTEIN KINASE SUBFAMILY: Metabolic Sensors of the Eukaryotic Cell?

1998-06-01

D. Grahame Hardie , David Carling , Marian Carlson

Mammalian AMP-activated protein kinase and yeast SNF1 protein kinase are the central components of kinase cascades that are highly conserved between animals, fungi, and plants. The AMP-activated protein kinase cascade … Mammalian AMP-activated protein kinase and yeast SNF1 protein kinase are the central components of kinase cascades that are highly conserved between animals, fungi, and plants. The AMP-activated protein kinase cascade acts as a metabolic sensor or “fuel gauge” that monitors cellular AMP and ATP levels because it is activated by increases in the AMP:ATP ratio. Once activated, the enzyme switches off ATP-consuming anabolic pathways and switches on ATP-producing catabolic pathways, such as fatty acid oxidation. The SNF1 complex in yeast is activated in response to the stress of glucose deprivation. In this case the intracellular signal or signals have not been identified; however, SNF1 activation is associated with depletion of ATP and elevation of AMP. The SNF1 complex acts primarily by inducing expression of genes required for catabolic pathways that generate glucose, probably by triggering phosphorylation of transcription factors. SNF1-related protein kinases in higher plants are likely to be involved in the response of plant cells to environmental and/or nutritional stress.

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AMPK in Health and Disease

2009-07-01

Gregory R. Steinberg , Bruce E. Kemp

The function and survival of all organisms is dependent on the dynamic control of energy metabolism, when energy demand is matched to energy supply. The AMP-activated protein kinase (AMPK) αβγ … The function and survival of all organisms is dependent on the dynamic control of energy metabolism, when energy demand is matched to energy supply. The AMP-activated protein kinase (AMPK) αβγ heterotrimer has emerged as an important integrator of signals that control energy balance through the regulation of multiple biochemical pathways in all eukaryotes. In this review, we begin with the discovery of the AMPK family and discuss the recent structural studies that have revealed the molecular basis for AMP binding to the enzyme's γ subunit. AMPK's regulation involves autoinhibitory features and phosphorylation of both the catalytic α subunit and the β-targeting subunit. We review the role of AMPK at the cellular level through examination of its many substrates and discuss how it controls cellular energy balance. We look at how AMPK integrates stress responses such as exercise as well as nutrient and hormonal signals to control food intake, energy expenditure, and substrate utilization at the whole body level. Lastly, we review the possible role of AMPK in multiple common diseases and the role of the new age of drugs targeting AMPK signaling.

Cellular and molecular mechanisms of metformin: an overview

2011-11-11

Benoı̂t Viollet , Bruno Guigas , Nieves Sanz Garcia +3 more

Considerable efforts have been made since the 1950s to better understand the cellular and molecular mechanisms of action of metformin, a potent antihyperglycaemic agent now recommended as the first-line oral … Considerable efforts have been made since the 1950s to better understand the cellular and molecular mechanisms of action of metformin, a potent antihyperglycaemic agent now recommended as the first-line oral therapy for T2D (Type 2 diabetes). The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory chain complex I. In addition, the resulting decrease in hepatic energy status activates AMPK (AMP-activated protein kinase), a cellular metabolic sensor, providing a generally accepted mechanism for the action of metformin on hepatic gluconeogenesis. The demonstration that respiratory chain complex I, but not AMPK, is the primary target of metformin was recently strengthened by showing that the metabolic effect of the drug is preserved in liver-specific AMPK-deficient mice. Beyond its effect on glucose metabolism, metformin has been reported to restore ovarian function in PCOS (polycystic ovary syndrome), reduce fatty liver, and to lower microvascular and macrovascular complications associated with T2D. Its use has also recently been suggested as an adjuvant treatment for cancer or gestational diabetes and for the prevention in pre-diabetic populations. These emerging new therapeutic areas for metformin will be reviewed together with recent findings from pharmacogenetic studies linking genetic variations to drug response, a promising new step towards personalized medicine in the treatment of T2D.

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Reduction in the Incidence of Type 2 Diabetes with Lifestyle Intervention or Metformin

2002-02-07

William C. Knowler , Elizabeth Barrett‐Connor , Sarah Fowler +4 more

Type 2 diabetes affects approximately 8 percent of adults in the United States. Some risk factors — elevated plasma glucose concentrations in the fasting state and after an oral glucose … Type 2 diabetes affects approximately 8 percent of adults in the United States. Some risk factors — elevated plasma glucose concentrations in the fasting state and after an oral glucose load, overweight, and a sedentary lifestyle — are potentially reversible. We hypothesized that modifying these factors with a lifestyle-intervention program or the administration of metformin would prevent or delay the development of diabetes.

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Complexes between the LKB1 tumor suppressor, STRADα/β and MO25α/β are upstream kinases in the AMP-activated protein kinase cascade

2003-09-24

Simon A. Hawley , Jérôme Boudeau , Jennifer L. Reid +5 more

The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of … The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs) that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRADα/β and MO25α/β. We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRADα and MO25α, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRADα/β and MO25α/β activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRADα or STRADβ and MO25α or MO25β are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1), but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

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Homeostasis model assessment: insulin resistance and ?-cell function from fasting plasma glucose and insulin concentrations in man

1985-07-01

David R. Matthews , J P Hosker , A. Rudenski +3 more

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AMP-activated protein kinase—an energy sensor that regulates all aspects of cell function

2011-09-15

D. Grahame Hardie

AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains cellular energy homeostasis. It arose very early during eukaryotic evolution, and its ancestral role may have been in … AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains cellular energy homeostasis. It arose very early during eukaryotic evolution, and its ancestral role may have been in the response to starvation. Recent work shows that the kinase is activated by increases not only in AMP, but also in ADP. Although best known for its effects on metabolism, AMPK has many other functions, including regulation of mitochondrial biogenesis and disposal, autophagy, cell polarity, and cell growth and proliferation. Both tumor cells and viruses establish mechanisms to down-regulate AMPK, allowing them to escape its restraining influences on growth.

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Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage

2007-10-23

Ranganath Muniyappa , Sihoon Lee , Hui Chen +1 more

Insulin resistance contributes to the pathophysiology of diabetes and is a hallmark of obesity, metabolic syndrome, and many cardiovascular diseases. Therefore, quantifying insulin sensitivity/resistance in humans and animal models is … Insulin resistance contributes to the pathophysiology of diabetes and is a hallmark of obesity, metabolic syndrome, and many cardiovascular diseases. Therefore, quantifying insulin sensitivity/resistance in humans and animal models is of great importance for epidemiological studies, clinical and basic science investigations, and eventual use in clinical practice. Direct and indirect methods of varying complexity are currently employed for these purposes. Some methods rely on steady-state analysis of glucose and insulin, whereas others rely on dynamic testing. Each of these methods has distinct advantages and limitations. Thus, optimal choice and employment of a specific method depends on the nature of the studies being performed. Established direct methods for measuring insulin sensitivity in vivo are relatively complex. The hyperinsulinemic euglycemic glucose clamp and the insulin suppression test directly assess insulin-mediated glucose utilization under steady-state conditions that are both labor and time intensive. A slightly less complex indirect method relies on minimal model analysis of a frequently sampled intravenous glucose tolerance test. Finally, simple surrogate indexes for insulin sensitivity/resistance are available (e.g., QUICKI, HOMA, 1/insulin, Matusda index) that are derived from blood insulin and glucose concentrations under fasting conditions (steady state) or after an oral glucose load (dynamic). In particular, the quantitative insulin sensitivity check index (QUICKI) has been validated extensively against the reference standard glucose clamp method. QUICKI is a simple, robust, accurate, reproducible method that appropriately predicts changes in insulin sensitivity after therapeutic interventions as well as the onset of diabetes. In this Frontiers article, we highlight merits, limitations, and appropriate use of current in vivo measures of insulin sensitivity/resistance.

Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34). UK Prospective Diabetes Study (UKPDS) Group.

1998-09-12

AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance

2017-06-01

Daniel Garcia , Reuben J. Shaw

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The mechanisms of action of metformin

2017-08-03

Graham Rena , D. Grahame Hardie , Ewan R. Pearson

Metformin is a widely-used drug that results in clear benefits in relation to glucose metabolism and diabetes-related complications. The mechanisms underlying these benefits are complex and still not fully understood. … Metformin is a widely-used drug that results in clear benefits in relation to glucose metabolism and diabetes-related complications. The mechanisms underlying these benefits are complex and still not fully understood. Physiologically, metformin has been shown to reduce hepatic glucose production, yet not all of its effects can be explained by this mechanism and there is increasing evidence of a key role for the gut. At the molecular level the findings vary depending on the doses of metformin used and duration of treatment, with clear differences between acute and chronic administration. Metformin has been shown to act via both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms; by inhibition of mitochondrial respiration but also perhaps by inhibition of mitochondrial glycerophosphate dehydrogenase, and a mechanism involving the lysosome. In the last 10 years, we have moved from a simple picture, that metformin improves glycaemia by acting on the liver via AMPK activation, to a much more complex picture reflecting its multiple modes of action. More work is required to truly understand how this drug works in its target population: individuals with type 2 diabetes.

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AMPK: guardian of metabolism and mitochondrial homeostasis

2017-10-04

Sébastien Herzig , Reuben J. Shaw

Current Situation and Problems for Weight Reduction Using GLP-1 Receptor Agonists

2025-06-25

| JOURNAL OF MEDICAL AND CLINICAL STUDIES

A genetic screen reveals a key role for Reg1 in 2-deoxyglucose sensing and yeast AMPK inhibition

2025-06-24

Alberto Ballin , Véronique Albanèse , Samia Miled +5 more | bioRxiv (Cold Spring Harbor Laboratory)

Abstract The yeast Saccharomyces cerevisiae thrives in sugar-rich environments by rapidly consuming glucose and favoring alcoholic fermentation. This metabolic strategy is tightly regulated by glucose signaling pathways, particularly the glucose … Abstract The yeast Saccharomyces cerevisiae thrives in sugar-rich environments by rapidly consuming glucose and favoring alcoholic fermentation. This metabolic strategy is tightly regulated by glucose signaling pathways, particularly the glucose repression pathway, which prevents the expression of genes required for the utilization of alternative carbon source. Central to this regulatory network is the yeast ortholog of the heterotrimeric 5′AMP-activated protein kinase (AMPK), which adjusts gene expression in response to glucose availability. The activity of the yeast AMPK complex is primarily regulated by the phosphorylation state of its catalytic subunit Snf1, a process orchestrated by a balance between upstream kinases and phosphatases. Among the latter, the Protein Phosphatase 1 (PP1) complex Reg1/Glc7 plays a critical role in repressing Snf1 activity under glucose-rich conditions. Despite its importance, the precise mechanism by which glucose availability leads to Snf1 inhibition remains incompletely understood. Evidence suggests that hexokinase 2 (Hxk2) participates in this pathway, potentially coupling the early steps of glucose metabolism to Snf1 signaling. Notably, the toxic glucose analog 2-deoxyglucose (2DG)- which is phosphorylated by Hxk2 but not further metabolized-mimics glucose in its ability to repress Snf1, implicating glucose or 2DG phosphorylation as a key regulatory signal. In this study, we explore the molecular basis of 2DG resistance in yeast through a large-scale genetic screen. The identified mutations confer resistance either by reducing 2DG phosphorylation (e.g., mutations in HXK2 ) or by enhancing constitutive Snf1 activity, via gain-of-function alleles in AMPK subunits or loss-of-function mutations in REG1 and GLC7 . We describe a novel class of REG1 missense mutations, including reg1-W165G , that maintain normal basal Snf1 activity but fail to mediate 2DG-induced Snf1 inhibition, rendering cells effectively immune to 2DG toxicity. These findings position Reg1 as a central mediator in glucose sensing and further suggest that 2DG toxicity primarily results from aberrant Snf1 inactivation rather than a direct metabolic disturbance.

Understanding semaglutide action in the brain

2025-06-24

Sarah Crunkhorn | Nature Reviews Drug Discovery

Metformin in Colorectal Cancer: Epidemiological Evidence, Predictive Biomarkers, and Implications for Prevention and Treatment

2025-06-24

Seokho Myung , Youn Young Park , Man S. Kim | International Journal of Molecular Sciences

Interest in metformin as a potential anticancer agent for colorectal cancer (CRC) has increased. However, compelling epidemiological links and strong preclinical evidence suggest that metformin has variable efficacy in patients … Interest in metformin as a potential anticancer agent for colorectal cancer (CRC) has increased. However, compelling epidemiological links and strong preclinical evidence suggest that metformin has variable efficacy in patients with CRC. This variability highlights the need to identify the patients who are most likely to benefit from effective stratification. We aimed to review the evidence concerning the diverse roles of metformin in CRC prevention and treatment, focusing on identifying and validating the predictive biomarkers essential for selecting patient subgroups that are likely to respond positively. We explored the various molecular pathways through which metformin acts and investigated how these diverse mechanisms might explain the observed differences in patient responses. Epidemiological studies and large meta-analyses have consistently reported reduced CRC incidence and improved survival among patients with diabetes treated with metformin. However, successfully extending these benefits broadly across all patients with CRC or achieving predictable outcomes in advanced disease settings remains a significant challenge. This review consolidates the current knowledge, highlights how different mechanisms interact, critically assesses clinical evidence in light of patient heterogeneity, and advocates for the development and implementation of biomarker-guided personalized therapeutic strategies as key to optimally utilizing the potential of metformin in CRC management. The current challenges and vital future research priorities in this critical area are also outlined.

Metformin slows intestinal glucose absorption in type 2 diabetes, irrespective of the timing of its administration

2025-06-24

Malcolm J. Borg , Cong Xie , Chang Chen +7 more | Diabetes Obesity and Metabolism

Exploring multifaceted roles of metformin in therapeutic applications, mechanistic insights, and innovations in drug delivery systems across biological contexts: a systematic review

2025-06-24

Nor Akmalyati Sulong , Vannajan Sanghiran Lee , Chin Fei Chee +1 more | Drug Delivery and Translational Research

Triglyceride Glucose Index: As A Glycemic Control Indicator

2025-06-24

Muzaffer Katar , Osman Demir | Harran Üniversitesi Tıp Fakültesi Dergisi

Background: Type-2 Diabetes Mellitus (T2DM) continues to be the most common endocrine disease today. Easily accessible, accurate and reproducible markers are needed in addition to the accepted markers to evaluate … Background: Type-2 Diabetes Mellitus (T2DM) continues to be the most common endocrine disease today. Easily accessible, accurate and reproducible markers are needed in addition to the accepted markers to evaluate insulin resistance (IR) and glycemic control. Therefore, our study aimed to evaluate the use of triglyceride glucose index (TyGI) as an indicator for insulin resistance and glycemic control. Materials and Methods: Triglyceride(TG), HbA1c, fasting blood glucose (FBG), and total insulin (TI) values of 953 samples, studied simultaneously in our Faculty of Medicine Hospital Laboratory between March 2023 and August 2023, were retrospectively evaluated. The patients were divided into two groups as good and/or poor glycemic control regarding their HbA1c, and the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) values. Receiver Operating Characteristic (ROC) analysis was performed to assess the ability of TyGI to discriminate between good and/or poor glycemic control for each of HOMA-IR and HbA1c. Statistical significance level was accepted as p&lt;0.05. Multivariate logistic regression analysis was performed as well. Results: A total of 953 patients; with the mean age of 40,83±16,78 participated in the study. According to gender, all parameters except age (p: 0,613) showed significant differences (p&lt;0.001). There were significant differences for FBG, HbA1c, HOMA-IR, TI, TG and TyGI parameters according to cut-off values in all two study groups (p&lt;0.001). TG showed high positive correlation with TyGI (r: 0.796, p&lt;0.001) and moderate positive correlation with FBG (r: 0.616, p&lt;0.001) for both study groups, but low positive correlation with the others. TyGI, had a high selectivity and specificity for HOMA-IR with ≥8,76 cut-off value (AUC:0,72, Se:65%, Sp:70% (p&lt;0.001: 95% CI:0,69-0,75)). In ROC analysis, TyGI had the highest AUC value for HbA1c, and the lowest for HOMA-IR group. The risk of poor glycemic control for HOMA-IR in men is 2.247 times higher than in women. As age increases by one unit, the risk of poor glycemic cont-rol for HOMA-IR increases by 1.045 times. Conclusions: TyGI was significantly raised in incident T2DM patients with poor glycemic control. TyGI can act as s simple and useful markers that have the strong predictive capability to identify insuline re-sistance and anticipate the development of incident T2DM.

A new pathway for neuroprotection against tau hyperphosphorylation via δ-opioid receptor initiated inhibition of CDK5 and AMPK signaling

2025-06-24

Jiahui Li , Yuan Xu , Gianfranco Balboni +1 more | Frontiers in Aging Neuroscience

Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by decreased memory and cognitive impairment. Abnormal tau hyperphosphorylation ultimately forms neurofibrillary tangles, which is one of the most important … Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by decreased memory and cognitive impairment. Abnormal tau hyperphosphorylation ultimately forms neurofibrillary tangles, which is one of the most important pathological features of AD. Since we have previously shown that the δ-opioid receptor (DOR) is neuroprotective in the brain, we asked if DOR plays any role in the control of tauopathy. Methods In the PC12 cell model with okadaic acid-induced tau hyperphosphorylation, cell viability and cytotoxicity were evaluated by using CCK8 assay kit and lactate dehydrogenase cytotoxicity assay kit. The techniques of western blot and immunofluorescence were used to investigate the effect of DOR on tau hyperphosphorylation. Results We found that DOR activation inhibited okadaic acid-induced tau hyperphosphorylation in PC12 cells and attenuated the cell cycle reactivation and apoptosis. The DOR effect was blocked by Naltrindole, a DOR antagonist. Furthermore, the mechanistic studies showed that the DOR displayed its effect by reducing the expression of cyclin-dependent kinase (CDK) 5 and AMP-activated protein kinase (AMPK) in the model of tauopathy. Discussion Our novel findings suggest that DOR signaling may protect neurons from AD injury by inhibiting tau hyperphosphorylation.

Impact of Ghrelin‐Depleting Gastrectomy on Long‐Term Endocrine and Metabolic Health With a Focus on Skeletal Muscle and Bone Mineral Content

2025-06-23

Hiroki Harada , Takuya Goto , Keishi Yamashita +7 more | Annals of Gastroenterological Surgery

ABSTRACT Background Advances in diagnostic and surgical techniques have improved survival rates for gastric cancer patients. However, gastrectomy involving ghrelin‐secreting regions of the upper gastric greater curvature can lead to … ABSTRACT Background Advances in diagnostic and surgical techniques have improved survival rates for gastric cancer patients. However, gastrectomy involving ghrelin‐secreting regions of the upper gastric greater curvature can lead to long‐term endocrine and metabolic disturbances, including reductions in serum ghrelin and insulin‐like growth factor‐1 (IGF‐1), potentially contributing to skeletal muscle and bone mineral loss. Methods This prospective observational study included 35 gastric cancer patients who underwent gastrectomy between 2016 and 2018, with follow‐up for 3–5 years. Patients were categorized into ghrelin‐depleted (total or proximal gastrectomy) and ghrelin‐preserved (distal gastrectomy) groups. Serum desacyl‐ghrelin, IGF‐1, and insulin‐like growth factor‐binding protein‐3 (IGFBP‐3) levels were measured, and skeletal muscle mass and bone mineral content were assessed. Results The ghrelin‐depleted group exhibited significantly lower serum desacyl‐ghrelin (56.9 ± 27.9 vs. 111.2 ± 54.8 fmol/mL, p = 0.0006), skeletal muscle mass (87.7% ± 2.1% vs. 95.1% ± 2.4%, p = 0.0229), and bone mineral content (90.9% ± 13.0% vs. 99.5% ± 6.3%, p = 0.0249). Additionally, IGF‐1 levels showed a significant positive correlation with skeletal muscle mass ( r = 0.53, p = 0.020). While the correlation between IGF‐1 and bone mineral content did not reach statistical significance, a positive trend was observed ( r = 0.44, p = 0.062). Conclusion Gastrectomy involving resection of ghrelin‐rich regions leads to long‐term reductions in serum desacyl‐ghrelin levels, adversely affecting skeletal muscle mass and bone mineral content. These findings highlight the importance of considering the endocrine consequences when selecting surgical procedures.

Retraction Notice to “Insulin-Induced Gene 2 Expression Is Associated with Breast Cancer Metastasis” [Am J Pathol 191 (2021) 385–395]

2025-06-23

Ning Lü , Mei Zhang , Lu Lu +3 more | American Journal Of Pathology

Glucose transporter 1 is essential to maintain brain endothelial cell homeostasis under hyperglycemia condition

2025-06-23

Benjamin Miao , Mohammad Sarif Mohiuddin , Rashu Barua +7 more | AJP Cell Physiology

Diabetic patients are prone to developing cerebrovascular disease (CVD) due to a multitude of factors. Particularly, the hyperglycemic environment is a key contributor to the progression of diabetes-associated complications. However, … Diabetic patients are prone to developing cerebrovascular disease (CVD) due to a multitude of factors. Particularly, the hyperglycemic environment is a key contributor to the progression of diabetes-associated complications. However, there is a dearth of knowledge regarding glucose transporter 1 (GLUT1, also known as SLC2A1)-dependent mechanisms responsible for these adverse effects. Here, we revealed the importance of glucose transporter 1 in preserving brain endothelial cell homeostasis beyond regulating glucose uptake. To elucidate the GLUT1-mediated protective mechanism, we used bulk RNA-seq to analyze transcriptomic alterations under hyperglycemia and GLUT1 deficiency conditions and validated the critical gene changes in cultured human brain endothelial cells and diabetic mouse models. We found that GLUT1 downregulation is linked to increased expression levels of podocalyxin (PODXL) and decreased thioredoxin-interacting protein (TXNIP) within healthy brain endothelial cells incubated with high glucose, demonstrating an anti-stress response mechanism. Interestingly, brain endothelial cells isolated from diabetic mice no longer showed a similar protection mechanism. Instead, the diabetic endothelial cells are characterized by considerably enriched GLUT1 and TXNIP expression under a hyperglycemic state. GLUT1 overexpression recaptures the diabetic features, such as elevated expression of TXNIP and NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome, along with increased IL-1β production and permeability. Our findings of a GLUT1-dependent regulatory mechanism for the endothelium provide a potentially deeper insight into mechanistic shifts that occur due to the diabetic disease state and the pathogenesis of diabetes-associated vascular complications.

Synergistic targeting of cancer cells through simultaneous inhibition of key metabolic enzymes

2025-06-23

Jan Dreute , Julia Stengel , Jonas Becher +7 more | Cell Death and Differentiation

As cancer cell specific rewiring of metabolic networks creates potential therapeutic opportunities, we conducted a synthetic lethal screen utilizing inhibitors of metabolic pathways. Simultaneous administration of (R)-GNE-140 and BMS-986205 (Linrodostat) … As cancer cell specific rewiring of metabolic networks creates potential therapeutic opportunities, we conducted a synthetic lethal screen utilizing inhibitors of metabolic pathways. Simultaneous administration of (R)-GNE-140 and BMS-986205 (Linrodostat) preferentially halted proliferation of ovarian cancer cells, but not of their non-oncogenically transformed progenitor cells. While (R)-GNE-140 inhibits lactate dehydrogenase (LDH)A/B and thus effective glycolysis, BMS-986205, in addition to its known inhibitory activity on Indoleamine 2,3-dioxygenase (IDO1), also restricts oxidative phosphorylation (OXPHOS), as revealed here. BMS-986205, which is being tested in multiple Phase III clinical trials, inhibits the ubiquinone reduction site of respiratory complex I and thus compromises mitochondrial ATP production. The energetic catastrophe caused by simultaneous interference with glycolysis and OXPHOS resulted in either cell death or the induction of senescence in tumor cells, with the latter being eliminated by senolytics. The frequent synergy observed with combined inhibitor treatment was comprehensively confirmed through testing on tumor cell lines from the DepMap panel and on human colorectal cancer organoids. These experiments revealed highly synergistic activity of the compounds in a third of the tested tumor cell lines, correlating with alterations in genes with known roles in metabolic regulation and demonstrating the therapeutic potential of metabolic intervention.

The metformin administration on pancreatic tissue damage in metastatic rat prostate cancer and STZ induced diabetes model

2025-06-21

Pınar Köroğlu , Onur Ertik , Ömür Karabulut Bulan +1 more | Journal of Molecular Histology

Hub biomarkers and their clinical relevance in glycometabolic disorders: A comprehensive bioinformatics and machine learning approach

2025-06-20

Liping Xiang , Bing Zhou , Yunchen Luo +3 more | Chinese Medical Journal

Abstract Background: Gluconeogenesis is a critical metabolic pathway for maintaining glucose homeostasis, and its dysregulation can lead to glycometabolic disorders. This study aimed to identify hub biomarkers of these disorders … Abstract Background: Gluconeogenesis is a critical metabolic pathway for maintaining glucose homeostasis, and its dysregulation can lead to glycometabolic disorders. This study aimed to identify hub biomarkers of these disorders to provide a theoretical foundation for enhancing diagnosis and treatment. Methods: Gene expression profiles from liver tissues of three well-characterized gluconeogenesis mouse models were analyzed to identify commonly differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA), machine learning techniques, and diagnostic tests on transcriptome data from publicly available datasets of type 2 diabetes mellitus (T2DM) patients were employed to assess the clinical relevance of these DEGs. Subsequently, we identified hub biomarkers associated with gluconeogenesis-related glycometabolic disorders, investigated potential correlations with immune cell types, and validated expression using quantitative Polymerase Chain Reaction in the mouse models. Results: Only a few common DEGs were observed in gluconeogenesis-related glycometabolic disorders across different contributing factors. However, these DEGs were consistently associated with cytokine regulation and oxidative stress (OS). Enrichment analysis highlighted significant alterations in terms related to cytokines and OS. Importantly, osteomodulin ( OMD ), apolipoprotein A4 ( APOA4 ), and insulin like growth factor binding protein 6 ( IGFBP6 ) were identified with potential clinical significance in T2DM patients. These genes demonstrated robust diagnostic performance in T2DM cohorts and were positively correlated with resting dendritic cells. Conclusions: Gluconeogenesis-related glycometabolic disorders exhibit considerable heterogeneity, yet changes in cytokine regulation and OS are universally present. OMD , APOA4 , and IGFBP6 may serve as hub biomarkers for gluconeogenesis-related glycometabolic disorders.

Cymbopogon proximus Chiov’s extract improves insulin sensitivity in rats with dexamethasone-induced insulin resistance and underlying mechanisms

2025-06-20

Amany M. Hamed , Noha A. Seif-Eldein , Shymaa A Thabet +7 more | Scientific Reports

Abstract The worldwide prevalence of type 2 diabetes mellitus (T2DM) is increasing swiftly. Cymbopogon proximus ( C. proximus ) is a wild herbaceous plant utilized as a potent remedy in … Abstract The worldwide prevalence of type 2 diabetes mellitus (T2DM) is increasing swiftly. Cymbopogon proximus ( C. proximus ) is a wild herbaceous plant utilized as a potent remedy in Egyptian folk medicine, sometimes referred to as “Halfabar.” This study examined the hypoglycemic, hypolipidemic, and antioxidant properties of the methanolic extract from the aerial parts of C. proximus , as well as its impact on pancreatic tumour necrosis factor-α (TNF-α) and Glucose Transporter-4 (GLUT4) in skeletal muscles within an experimental model of insulin resistance. Additionally, bioactive metabolites in the extract were analyzed via liquid chromatography-mass spectrometry (LC/MS) technology. Insulin resistance was induced by administering 1 mg/kg of dexamethasone to rats over a period of 14 days. The rats received two doses of the extract: a low dose of 100 mg/kg body weight and a high dose of 200 mg/kg body weight, along with the reference drug; Metformin (M) at a dose of 40 mg/kg body weight, supplied once daily by gastric tube for 14 days. The treatment of dexamethasone led to a significant ( P < 0.05) elevation in serum fasting glucose, fasting insulin, HOMA-IR, and pancreatic TNF-α, along with a significant ( P < 0.05) reduction in GLUT4 expression in skeletal muscles. Both extract and reference treatments significantly ( P < 0.05) mitigated these abnormalities. The highest dose of the extract exhibited a significantly ( P < 0.05) greater antioxidant impact, a more pronounced reduction in insulin levels and HOMA-IR, as well as an enhanced rise in GLUT4 expression and insulin sensitivity index compared to the lowest dose and the M. Histopathological and immunohistochemical analyses corroborate the biochemical results. The LC–ESI–MS/MS profiling resulted in the characterization and tentative identification of 95 metabolites’ structures. Identified substances purported to possess anti-diabetic effect include apigenin, luteolin, tricin flavone glycosides, cyanidin, malvidin anthocyanin glycosides, and caffeic acid. These findings suggest that C. proximus can mitigate insulin resistance. Additional clinical trials are necessary to validate these findings and assess the extract’s effectiveness in human insulin resistance.

Diabetes Mellitus and Lung Cancer Screening Outcomes in the National Lung Screening Trial

2025-06-20

Robert P. Young , Ralph Ward , R.J. Scott +1 more | Annals of the American Thoracic Society

Current eligibility criteria for lung cancer (LC) screening are derived from randomized controlled trials and largely based on age and smoking history. However, the individualised benefits of screening are highly … Current eligibility criteria for lung cancer (LC) screening are derived from randomized controlled trials and largely based on age and smoking history. However, the individualised benefits of screening are highly variable and may be mediated by the presence of co-existing comorbid disease, including diabetes mellitus (DM). This study examines in detail screening outcomes for those reporting a prior diagnosis of DM. This was a secondary analysis of 53,452 high-risk subjects from the National Lung Screening Trial (NLST), and compared outcomes following screening with computed tomography (CT) or chest x-ray (CXR) stratified according to DM status. Models of LC mortality were derived after adjustment and LC rate ratios (per 1000 person years), including 95% Confidence Intervals (95% CI), were examined according to screening arm and DM status. Compared to those without DM, DM subjects (N=5,174, 9.7%) had a 2-fold greater baseline cardiovascular comorbidity (p<0.0001), 2-fold greater non-LC mortality (p<0.0001) and greater LC lethality (p=0.02), with more late-stage lung cancer (p=0.03). We found comparable stage shift and surgical rates, favouring those randomised to CT relative to CXR, for both DM and non-DM subgroups. However, we found no reduction in LC mortality for DM subjects favouring CT (2.2% vs 2.1% respectively, Rate ratio per 1000 person years (RR)=1.03, 95%CI 0.71-1.49, p=0.89), contrasting with non-DM subjects (RR=0.83, 95%CI 0.73-0.95, p=0.006)(p for interaction 0.28). In a modified Cox-Proportional Hazard model for dying of lung cancer by screening arm, adjusting for relevant co-variables, DM was associated with a hazard ratio (HR)=1.03, 95%CI=0.71-1.50, p=0.88) compared to non-DM (HR=0.82, 95%CI=0.72-0.94, p=0.003). LC mortality for those randomised to CT was greater for DM vs non-DM subjects (2.2% vs 1.6%, RR=1.35 (95% CI 1.02-1.79, p=0.033) but no different for CXR (2.1% vs 2.0%, RR=1.09 (95% CI 0.82-1.44, p=0.55). The significant reduction in LC mortality favouring CT-based screening found in non-DM subjects was not observed in those reporting DM. While study design (under-powering), collider/confounder effects (bias) and newer treatment modalities remain possible limitations, the findings from this clinical trial data support simulation studies suggesting LC screening outcomes may be attenuated by comorbidity such as DM.

In Vivo PK-PD and Drug–Drug Interaction Study of Dorzagliatin for the Management of PI3Kα Inhibitor-Induced Hyperglycemia

2025-06-19

Guanqin Jin , Kewei Zheng , Shihuang Liu +5 more | Pharmaceuticals

Objectives: The anticancer effects of PI3Kα inhibitors (PI3Ki) are constrained by their hyperglycemic side effects, while the efficacy of conventional hypoglycemic agents, such as insulin, metformin, and SGLT-2 inhibitors, in … Objectives: The anticancer effects of PI3Kα inhibitors (PI3Ki) are constrained by their hyperglycemic side effects, while the efficacy of conventional hypoglycemic agents, such as insulin, metformin, and SGLT-2 inhibitors, in mitigating PI3Ki-induced hyperglycemia remains suboptimal. Dorzagliatin, a novel glucokinase activator, has been approved in China for the management of hyperglycemia, offering a promising alternative. This study aims to investigate the pharmacokinetic properties and potential mechanisms of drug interactions of dorzagliatin in the regulation of PI3K-induced hyperglycemia. Methods: Plasma concentrations of WX390, BYL719, and Dorz in mice were measured using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Pharmacokinetic (PK) parameters and PK/PD models were derived by using Phoenix WinNonlin 8.3.5 software. Blood glucose levels at various time points and tumor volume changes over a four-week period were assessed to explore the interactions when PI3Ki were combined with dorzagliatin. Results: The results indicated that, compared to the Dorz group, the combination groups (Dorz + BYL719, Dorz + WX390) exhibited increases in AUC0→t of dorzagliatin by 41.65% and 20.25%, and in Cmax by 33.48% and 13.32%, respectively. In contrast, co-administration of these PI3Ki with dorzagliatin resulted in minimal increase in their plasma exposure. The combination therapy group (Dorz+BYL719) exhibited superior antitumor efficacy compared to the BYL719 group. Conclusions: Our findings indicate that the drug–drug interactions (DDIs) between dorzagliatin and multiple PI3Ki (including WX390 and BYL719) may partially account for the enhanced antitumor efficacy observed in the combination therapy group compared to PI3Ki monotherapy. This interaction may be explained by the inhibition of P-glycoprotein (P-gp) and the pharmacological mechanism of dorzagliatin regarding the activation of insulin regulation.

Gene Dynamics in Maternal Nutrition: The Role of Chromium in Mitigating Offspring Obesity

2025-06-19

Xiaofeng Li , Tao Wang , Weimin Wang +4 more | Biological Trace Element Research

Glycemic control determines the response to immunotherapy for hepatocellular carcinoma in subjects with diabetes

2025-06-18

Meng‐Wei Lin , Hung‐Tsung Wu , Huang-Pin Chen +6 more | Research Square (Research Square)

<title>Abstract</title> <italic><bold>Aims: </bold></italic>Although diabetes is highly correlated with hepatocellular carcinoma (HCC), it remains unclear how glycemic status affects responses to immunotherapy for HCC. Therefore, this study aimed to determine the … <title>Abstract</title> <italic><bold>Aims: </bold></italic>Although diabetes is highly correlated with hepatocellular carcinoma (HCC), it remains unclear how glycemic status affects responses to immunotherapy for HCC. Therefore, this study aimed to determine the prognostic implications of glycemic status and its impacts on immunotherapy treatment in HCC. <italic><bold>Methods: </bold></italic>This retrospective study included 151 subjects with HCC who were treated with atezolizumab and bevacizumab. Subjects were categorized into three groups, including non-diabetes group (N=83), well-controlled diabetes group (HbA1c<7.0%; N=37), and poorly-controlled diabetes group (HbA1c≥7.0%; N=31). Therapeutic response was evaluated using modified response evaluation criteria in solid tumors. Progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan-Meier method. Prognostic factors were analyzed with Cox proportional hazards models. <italic><bold>Results:</bold></italic><italic> </italic>The poorly-controlled diabetes group exhibited the worst therapeutic response and significantly unfavorable PFS and OS, as compared with well-controlled diabetes and non-diabetes groups. Subgroup analysis showed that PFS and OS for well-controlled diabetes group were comparable to non-diabetes group. Furthermore, the poorly-controlled diabetes was an independent predictor of reduced PFS (HR=3.52, 95% CI:1.98-6.24, P<0.001) and OS (HR=5.16, 95% CI:2.38-11.18, P<0.001). <italic><bold>Conclusions: </bold></italic>Our findings show that poor glycemic control in subjects with diabetes predicts worse survival and immunotherapy treatment response to HCC.

Phenformin, But Not Metformin, Delays Development of T Cell Acute Lymphoblastic Leukemia/Lymphoma via Cell-Autonomous AMPK Activation

2025-06-17

Diana Vara‐Ciruelos , Madhumita Dandapani , Fiona M. Russell +7 more | Cell Reports

Vitamin D3 augments cytotoxic effect of Itraconazole on chronic myelogenous leukemia cell line: a synergic effect on AMPK/AKT signaling pathway

2025-06-16

Moein Tahvili , Vahid Nejati , Yaghub Pazhang | Molecular Biology Reports

Metformin‐Associated Lactic Acidosis During Docetaxel Therapy for Castration‐Resistant Prostate Cancer: A Case Report

2025-06-16

Yoshitsugu Inoue , Aya Niimi , Takuya Kudo +3 more | IJU Case Reports

ABSTRACT Introduction Docetaxel is a key treatment for castration‐resistant prostate cancer and is administered with prednisolone, which increases the risk of steroid‐induced diabetes. Its myelosuppressive effect also increases vulnerability to … ABSTRACT Introduction Docetaxel is a key treatment for castration‐resistant prostate cancer and is administered with prednisolone, which increases the risk of steroid‐induced diabetes. Its myelosuppressive effect also increases vulnerability to febrile neutropenia. Metformin is widely used for glycemic control; however, elderly cancer patients are particularly susceptible to metformin‐associated lactic acidosis, necessitating careful management of sick‐day and febrile neutropenia during chemotherapy. Case Presentation We report a 70‐year‐old male with castration‐resistant prostate cancer and diabetes mellitus who developed febrile neutropenia on Day 5 following docetaxel initiation. He progressed to shock with severe metabolic acidosis on Day 7 and died despite intensive care, including continuous renal replacement therapy. A retrospective review revealed continued metformin use despite prodromal fatigue and loss of appetite, likely due to impaired judgment. Conclusion This case may have involved septic shock, but metformin likely worsened the lactic acidosis. It highlights the need for sick‐day education and monitoring in elderly cancer patients.

Rac1 signaling mediates the protection of apigenin against hepatic lipid accumulation and insulin resistance

2025-06-16

Man‐Chen Hsu , Bei‐Chia Guo , Chia-Hui Chen +3 more | Biomedicine & Pharmacotherapy

Effect of Metformin on vascular endothelial injury in patients with non-small cell lung cancer treated with chemotherapy combined with bevacizumab

2025-06-16

Hongyan Li , Zhenye Pu , Fang Jin | Cancer Chemotherapy and Pharmacology

Activin receptor type IIA/B blockade increases muscle mass and strength, but compromises glycemic control in mice

2025-06-15

Michala Carlsson , Emma Frank , Joan M. Màrmol +7 more | bioRxiv (Cold Spring Harbor Laboratory)

Purpose Blocking the Activin receptor type IIA and B (ActRIIA/IIB) has clinical potential to increase muscle mass and improve glycemic control in obesity, cancer, and aging. However, the impact of … Purpose Blocking the Activin receptor type IIA and B (ActRIIA/IIB) has clinical potential to increase muscle mass and improve glycemic control in obesity, cancer, and aging. However, the impact of blocking ActRIIA/IIB on strength, metabolic regulation and insulin action remains unclear. Methods Here, we investigated the effect of short- (10 mg/kg once, 40h) or long-term (10 mg/kg twice weekly, 21 days) antibody targeting ActRIIA/IIB (αActRIIA/IIBab) in lean and diet-induced obese mice and engineered human muscle tissue. Results Short-term αActRIIA/IIB administration in lean mice increased insulin-stimulated glucose uptake in skeletal muscle by 76-105%. Despite this, αActRIIA/IIB-treated mice exhibited 33% elevated fasting blood glucose and glucose intolerance. Moreover, long-term αActRIIA/IIB treatment increased average muscle mass (20%) and reduced fat mass (-8%) in obese mice but did not change insulin-stimulated glucose uptake in skeletal muscle or adipose tissue, yet induced marked glucose intolerance, and increased hepatic glucose output in response to pyruvate. Concomitantly, long-term αActRIIA/IIBab treatment increased strength (30%) in mouse soleus muscle and prevented activin A-induced loss of tissue strength in engineered human muscle tissue. Surprisingly, long-term αActRIIA/IIBab treatment lowered volitional running (-250%). Conclusion Our findings demonstrate that, in accordance with human studies, ActRIIA/IIB blockade holds promise for increasing muscle mass, strength, and insulin sensitivity. However, contrary to the improved glycemic control in humans, ActRIIA/IIB blockade in mice causes severe glucose intolerance and lowers voluntary physical activity. Our study underscores the complex metabolic and functional consequences of ActRIIA/IIB blockade, and highlight species differences on glycemic control, which warrant further investigation.

Transient AMPK activation by nutrient stress of high fat diet preserves cardiac electrophysiological stability and protects against arrhythmias

2025-06-15

Michael W. Rudokas , Margaret McKay , Xiaohong Wu +7 more | bioRxiv (Cold Spring Harbor Laboratory)

Sudden cardiac death (SCD) is a major complication of obesity, yet it remains unclear whether early metabolic stress, prior to the onset of overt obesity or structural remodeling, can independently … Sudden cardiac death (SCD) is a major complication of obesity, yet it remains unclear whether early metabolic stress, prior to the onset of overt obesity or structural remodeling, can independently promote arrhythmias. In vitro studies suggest that fatty acids can allosterically activate AMP-activated protein kinase (AMPK), a key metabolic sensor known to preserve myocardial viability and mitochondrial function following ischemia-reperfusion (I/R) injury. We hypothesized that AMPK signaling critically modulates the electrophysiological (EP) response to high-fat diet (HFD)-induced metabolic stress. Methods: To test this, wild-type (WT) and AMPK kinase-dead (AMPK-KD) mice were subjected to an 8-week HFD regimen beginning at 4 weeks of age. Controls remained on normal diet (ND) for the same duration. Arrhythmia susceptibility was assessed ex vivo using rapid pacing and I/R challenge protocols. Changes in the EP substrate were defined by high-resolution optical action potential mapping. Underlying mechanisms were probed using western blotting, confocal and transmission electron microscopy. Results: HFD-fed wild-type (WT) hearts did not display increased arrhythmia susceptibility in response to either burst pacing or I/R challenge. On the contrary, they exhibited a paradoxical enhancement in post-ischemic EP recovery compared to ND-fed controls. This improvement was associated with increased phosphorylation of canonical AMPK targets, including acetyl-CoA carboxylase (ACC) and raptor, consistent with the activation of a cardioprotective metabolic program. In sharp contrast, AMPK-deficient (AMPK-KD) hearts demonstrated heightened vulnerability to inducible ventricular tachycardia (VT), irrespective of diet. Conduction slowing emerged as an early EP abnormality in these hearts and served as the initial substrate (or 'first hit') that promoted their increased incidence of non-sustained VT. Notably, this conduction impairment arose in conjunction with an increase (rather than decrease) in Cx43 and Nav1.5 protein expression. Mechanistically, defective conduction in AMPK-KD hearts was linked to impaired autophagic degradation of intercalated disc proteins resulting from reduced phosphorylation of ULK1, a downstream effector of AMPK. Consequently, unphosphorylated Cx43 accumulated at the intercalated disc, likely replacing phosphorylated isoforms (p-Cx43). In addition, AMPK-KD hearts exhibited swollen, fragmented mitochondria and reduced levels of mitochondrial fusion proteins. Upon HFD challenge, this vulnerable mitochondrial substrate generated excessive reactive oxygen species (ROS) coinciding with accelerated repolarization. Together, impaired conduction and ROS-driven action potential shortening promoted VT sustenance in HFD-fed AMPK-deficient hearts. Conclusions: Our findings identify AMPK as a key metabolic regulator that integrates redox balance, mitochondrial integrity, and protein homeostasis to preserve cardiac excitability during early nutrient overload. Loss of AMPK signaling, as occurs with aging and advanced metabolic disease, may therefore represent a pivotal mechanism linking HFD to increased SCD risk.

Frequency of New Onset Diabetes Mellitus in Patients Receiving Treatment for Diffuse Large B Cell Lymphoma

2025-06-13

A. Younas , Ainan Arshad , Muhammad Arslan | Indus journal of bioscience research.

Objectives: To determine the frequency of new-onset diabetes mellitus in patients receiving treatment for diffuse large B-cell lymphoma. Methodology: This study was done at Shaukat Khanum Memorial Cancer Hospital & … Objectives: To determine the frequency of new-onset diabetes mellitus in patients receiving treatment for diffuse large B-cell lymphoma. Methodology: This study was done at Shaukat Khanum Memorial Cancer Hospital & Research Center, Lahore after Six months following approval of the study synopsis from 7th February to 7th May. We enrolled 150 patients aged 18–40 years with newly diagnosed DLBCL were enrolled using non-probability consecutive sampling. Patients with prior diabetes or central nervous system lymphoma were excluded. Glycemic status was assessed post-chemotherapy using fasting blood glucose and HbA1c. Associations with demographic and clinical variables were analyzed. Results: New-onset diabetes mellitus was observed in 32.0% (n = 48) of patients. A significant association was found with family history of diabetes (p = 0.009) whereas age, BMI, gender, smoking, alcohol use, lymphoma stage, or type of therapy had no significant association. Conclusion: The frequency of new-onset diabetes among DLBCL patients undergoing chemotherapy is substantial. Family history of diabetes emerged as a significant risk factor. Routine screening for hyperglycemia should be incorporated into oncology care to ensure early diagnosis and management of diabetes during cancer treatment.

328-OR: Impact of Diabetes and Chemotherapy on Cardiovascular Outcomes in Breast Cancer Survivors—A Population-Based Study

2025-06-13

Amanda Leiter , JENNY J. LIN , Chung Yin Kong +1 more | Diabetes

Introduction and Objective: Older breast cancer (BC) survivors with diabetes mellitus (DM) have worse mortality outcomes than survivors without DM. DM is a major risk factor for cardiovascular disease (CVD), … Introduction and Objective: Older breast cancer (BC) survivors with diabetes mellitus (DM) have worse mortality outcomes than survivors without DM. DM is a major risk factor for cardiovascular disease (CVD), now the leading cause of death in BC survivors. Anthracycline chemotherapy (AC), a widely used BC treatment, also increases CVD risk. We assessed the impact of DM and AC, and their interaction, on specific CVD outcomes in older BC survivors. Methods: We analyzed Surveillance, Epidemiology and End Results cancer data linked to Medicare, a US population-based dataset. We included patients ≥66 years old diagnosed with Stage 2-3 BC between 2008-2017 and excluded patients with preexisting CVD. DM and incident CVD (myocardial infarction [MI], coronary artery disease [CAD], congestive heart failure [CHF]), and AC were ascertained from claims. We used inverse probability treatment weighting with propensity scores to balance baseline sociodemographic characteristics and comorbidities in patients who received AC vs. no AC. Cox regression was used to assess the impact of DM, AC and their interaction on CVD incidence. Results: In 7,182 included BC survivors with median 5 years follow up and median age 72, 1235 (17%) received AC, 1408 had preexisting DM (20%), 332 (4.6%) developed MI, 1289 (18.0%) developed CAD, and 1101 (15.3%) developed CHF. DM increased the risk of MI (hazard ratio [HR] 1.98, 95% confidence interval [CI] 1.53-2.55), CAD (HR 1.63, 95% CI 1.41-1.86), and CHF (HR 1.91, 95% CI 1.65-2.21). AC also increased risk of CAD (HR 1.23, 95% CI 1.12-1.35) and CHF (HR 1.29, 95% CI 1.16-1.44)), but not MI (HR 0.99, 95% CI 0.81-1.23). There were no significant interactions between DM and AC in increasing any CVD event risk. Conclusion: In older BC survivors, both DM and AC significantly increase CVD risk separately, but do not interact to further increase CVD risk. Future research should focus on mitigating CVD risk in BC survivors with DM receiving AC, a particularly high-risk group. Disclosure A. Leiter: None. J.J. Lin: None. C. Kong: None. J. Wisnivesky: Other Relationship; PPD, Banook, American Medical Association, Sanofi, Biontech. Research Support; Axella, Regeneron Pharmaceuticals, Sanofi. Funding NIDDK/NIH (K08DK137022)NCI/NIH (R01CA271604)

1459-P: Impact of Endocrine Adjuvant Therapy for Breast Cancer on Diabetes Risk—A Population-Based Study

2025-06-13

Varshini Srinivasan , Samyuktha Prabu , Carin Y. Smith +5 more | Diabetes

Introduction and Objective: Rapid changes in estrogen levels while undergoing adjuvant endocrine therapy for breast cancer may have an adverse glycemic impact. This study is designed to determine the risk … Introduction and Objective: Rapid changes in estrogen levels while undergoing adjuvant endocrine therapy for breast cancer may have an adverse glycemic impact. This study is designed to determine the risk of diabetes mellitus (DM) in women on endocrine adjuvant therapy for treatment of non-metastatic breast cancer in the Rochester Epidemiology Project (REP) records-linkage system. Methods: A cohort of 9771 women aged 18 and over with a primary breast malignancy diagnosis was defined within the REP resources and screened for eligibility. Index date was defined as the date of breast cancer diagnosis. DM was diagnosed based on ADA criteria, a new DM diagnosis code or start of a new medication for DM. Baseline characteristics were compared between those who did and did not use aromatase inhibitors (AI). Univariate Cox regression models to evaluate the risk of DM, using AI and tamoxifen as time dependent variables, and multivariable models adjusted for age, body mass index (BMI) and baseline fasting glucose were utilized. Results: From our initial cohort, 3882 women met the criteria for inclusion. Women who used AI were older, were more frequently white, had higher BMI, and had more prior glucocorticoid use. 334 women progressed to DM. Univariate models for time from breast cancer diagnosis to onset of DM (or to follow-up if no DM), showed a significantly increased risk with AI [HR 1.38 (1.10-1.73), p 0.006] and a significantly decreased risk with tamoxifen [HR 0.52 (0.37-0.72), p &lt;0.001]. In multivariable models, after adjustment for age, BMI and fasting glucose, AI were no longer significantly associated, but the reduced risk associated with tamoxifen remained. Conclusion: Risk of DM was greater in women on AI for breast cancer, but this increased risk was not significant after adjustment for covariates. Tamoxifen was associated with a lower risk for DM that remained significant after adjustment. Prospective comparative studies are needed to provide more insight into their glycemic effects. Disclosure V. Srinivasan: None. S.B. Prabu: None. C. Smith: None. C. Wood-Wentz: None. K.R. Bailey: None. A. Vella: Research Support; Novo Nordisk A/S, Dexcom, Inc. Advisory Panel; Boehringer-Ingelheim, Rezolute. A.M. Egan: None. K. Muthusamy: None.

285-OR: A Kinase-Mediated Signaling and Glucose Uptake Defect in Human Insulin Resistance

2025-06-13

Nida Haider , Tomer M. Yaron , Jared L. Johnson +3 more | Diabetes

Introduction and Objective: Insulin resistance is a major risk factor in the development of type 2 diabetes (T2D) and metabolic syndrome. Although ~25% of people within the general non-diabetic population … Introduction and Objective: Insulin resistance is a major risk factor in the development of type 2 diabetes (T2D) and metabolic syndrome. Although ~25% of people within the general non-diabetic population are insulin resistant, the primary underlying cause of insulin resistance remains elusive. Methods: In this study, we have used induced pluripotent stem cells (iPSC) derived from non-diabetic humans at both ends of the insulin sensitivity spectrum, i.e., the top 20% of insulin resistance vs. the top 20% of insulin sensitivity differentiated into myoblasts (iMyos) to model insulin resistance in vitro. Results: Global phosphoproteomics analysis of these cells showed a large network of protein phosphorylations linked to differences in insulin sensitivity including 378 up-regulated and 393 down-regulated insulin stimulated phosphosites in I-Res iMyos. To identify drivers of altered phosphorylation in insulin resistance, we used an in silico AI kinome analysis to query which of the 300+ Ser/Thr kinases encoded in the human genome might be responsible for the protein phosphorylation changes. We could identify 16 kinases whose predicted activities were significantly increased in I-Res iMyos, suggesting their potential link to the pathogenesis of insulin resistance. To functionally link these altered kinases to the downstream defect of impaired glucose uptake associated with insulin resistance, we conducted a loss-of-function screen using a CRISPR-based approach to identify candidate kinases, or phosphatases that regulate glucose uptake in I-Res iMyos. Among the 16 kinases predicted to have increased activity in insulin resistance, one kinase, DYRK2, was identified as a potential modulator of insulin resistance by being both increased in predicted activity in I-Res iMyos and associated with rescued glucose uptake in I-Res iMyos by CRISPR-mediated knockdown. Conclusion: In summary, combining a Kinome analysis with CRISPR screening and population genetics reveals DYRK2 as an important upstream regulator of human insulin resistance. Disclosure N. Haider: None. T. Yaron-Barir: Other Relationship; DeStroke. J.L. Johnson: None. L. Cantley: Stock/Shareholder; Larkspur, Volastra, Cell Signaling Technologies. Consultant; Manas. P. Yi: None. C. Kahn: Consultant; Novo Nordisk, Cellarity. Advisory Panel; TIXiMED. Board Member; 1825.

286-OR: Spatial Regulation of Glucose and Lipid Metabolism by Hepatic Insulin Signaling

2025-06-13

Ben He , Kyle D. Copps , Morris F. White +1 more | Diabetes

Introduction and Objective: Hepatic insulin sensitivity is critical for systemic glucose and lipid homeostasis. The liver is spatially organized into zones in which hepatocytes express distinct metabolic enzymes; however, the … Introduction and Objective: Hepatic insulin sensitivity is critical for systemic glucose and lipid homeostasis. The liver is spatially organized into zones in which hepatocytes express distinct metabolic enzymes; however, the functional significance of this zonation to metabolic dysregulation caused by insulin resistance is undetermined. Methods: Here, we used CreER mice to selectively disrupt insulin signaling in periportal (PP) and pericentral (PC) hepatocytes. Results: PP-insulin resistance has been suggested to drive combined hyperglycemia and excess lipogenesis in type 2 diabetes. However, we found that PP-insulin resistance in mice impaired lipogenesis and suppressed high-fat diet (HFD)-induced hepatosteatosis, despite elevating blood glucose and insulin. Conversely, PC-insulin resistance reduced HFD-induced pericentral steatosis while preserving normal glucose homeostasis, in part by shifting glycolytic metabolism from the liver to muscle. Conclusion: These results demonstrate distinct roles of insulin in PP versus PC, and suggest that PC-insulin resistance might be therapeutically useful to combat hepatosteatosis without compromising glucose homeostasis. Disclosure B. He: None. K.D. Copps: None. M.F. White: Board Member; HPRL (Housey Pharmaceutical Research Laboratory). R. Tao: None. Funding NIH grants DK133388

823-P: AT278, a Novel Ultra-fast Highly Concentrated Insulin Aspart (500 IU/ml), Shows Ultra-fast Properties Independent of BMI

2025-06-13

EVA SVEHLIKOVA , Gabriele Fluhr , Bettina Lackner +7 more | Diabetes

Introduction and Objective: High insulin doses are required due to the rise in obesity and insulin resistance, in both type 1 and type 2 diabetes (T2DM). AT278 is a new … Introduction and Objective: High insulin doses are required due to the rise in obesity and insulin resistance, in both type 1 and type 2 diabetes (T2DM). AT278 is a new ultra-rapid formulation of concentrated insulin aspart (500 IU/ml). We compared pharmacodynamics (PD) and pharmacokinetics (PK) of AT278 (500 IU/ml) with insulin aspart (InsAsp, 100 IU/ml) and human insulin (HumIns, 500 IU/ml) in T2DM over a wide range of BMI. Methods: In a randomised, double-blind, crossover, euglycemic clamp study, a single sc injection of 0.5 IU/kg of AT278 was compared to InsAsp, with an additional open comparison to HumIns in 41 adults with T2DM (BMI 25.0 - 38.7 kg/m2). Results: AT278 showed significantly accelerated onset of glucose-lowering effect (AUCGIR,0-60min) compared with InsAsp (TR 2.02) and HumIns (TR 3.91). AT278 showed 5-min earlier onset of appearance and 24 min shorter tEarly50%Cmax compared with InsAsp. PK and PD of HumIns was significantly retarded. Overall insulin exposure and glucose-lowering effect were comparable for all formulations. While BMI had a decelerating effect on the onset PK and PD parameters for InsAsp, AT278 retained all ultra-fast properties regardless of BMI. Conclusion: Concentrated AT278 (500 IU/ml) offers ultra-fast characteristics independent of BMI and thus has the potential to improve quality of life for people with diabetes with high prandial insulin needs. Disclosure E. Svehlikova: None. G. Fluhr: None. B.C. Lackner: None. M. Ratzer: None. D.J. Gerring: None. J. Jezek: None. S.J. Howell: None. L. Zakrzewski: None. S. Murray: Employee; Arecor Ltd. T. Pieber: Consultant; Arecor. Speaker's Bureau; Novo Nordisk A/S. Consultant; Eli Lilly and Company. Advisory Panel; Sanofi. Research Support; AstraZeneca. Advisory Panel; ADOCIA. Speaker's Bureau; Roche Diagnostics.

889-P: Enhancing Glycemic Control through Orlistat Combination Therapy with Pioglitazone and Metformin in Patients with Obesity Type 2 Diabetes—A Multicenter Randomized Controlled Trial

2025-06-13

Jia Liu , Hui You , Xin Wen +3 more | Diabetes

Introduction and Objective: Obesity further impairs blood glucose regulation, so patients with obesity and type 2 diabetes should undergo aggressive weight loss treatment. We aimed to evaluate the efficacy of … Introduction and Objective: Obesity further impairs blood glucose regulation, so patients with obesity and type 2 diabetes should undergo aggressive weight loss treatment. We aimed to evaluate the efficacy of orlistat in patients with obesity and type 2 diabetes treated with pioglitazone-metformin. Methods: In this double-blind, placebo-controlled, 12-week, multicenter trial, patients aged 18-65 years with type 2 diabetes and a BMI &gt; 28 kg/m² on pioglitazone-metformin. They were randomly assigned to receive placebo or 120 mg orlistat three times daily. The primary endpoint was HbA1c change from baseline to week 12. Secondary endpoints included improvements in blood glucose, HOMA-IR, body weight, and abdominal obesity. Results: After 12 weeks, HbA1c improvement in the orlistat group was not significant. However, insulin resistance improved notably, with HOMA-IR decreasing by -15.6% vs -7.86% in the placebo group (P&lt;0.05). Blood glucose levels also improved significantly; fasting blood glucose dropped by -0.73 vs -0.17 mmol/L (P&lt;0.05). This improvement is linked to orlistat’s weight loss effect. At week 12, the orlistat group had significantly lower BMI (30.16 vs 32.70 kg/m², P&lt;0.001) and reduced subcutaneous fat (246.43 vs 307.38 cm²) and visceral fat (120.70 vs 142.46 cm², P&lt;0.05) compared to the placebo group. Conclusion: Orlistat is effective for patients with obesity and type 2 diabetes who are receiving pioglitazone-metformin therapy, it reduces body weight, improves fat distribution, alleviates insulin resistance, and enhances glycemic control. Disclosure J. Liu: None. H. You: None. X. Wen: None. Y. Fang: None. L. Bu: None. S. Qu: None.

Exploring lactylation and cancer biology: insights from pathogenesis to clinical applications

2025-06-13

Zixuan Gou , Qifan Sun , Jiannan Li | Frontiers in Cell and Developmental Biology

As a byproduct of glycolysis, lactate functions as a signaling molecule, a substrate for energy metabolism, and a regulator of the tumor microenvironment (TME). It is involved in various biological … As a byproduct of glycolysis, lactate functions as a signaling molecule, a substrate for energy metabolism, and a regulator of the tumor microenvironment (TME). It is involved in various biological processes, including energy shuttling, tumor growth and invasion, drug resistance, and immune evasion. Lactylation, a recently identified post-translational modification (PTM), acts as a bridge between gene regulation and cellular metabolism, thus playing a crucial role in tumor biology. Similar to other epigenetic modifications, lactylation influences the spatial conformation of chromatin, modulates DNA accessibility, and regulates gene expression. It intricately participates in TME-related processes by orchestrating immune state transitions and enhancing the malignant characteristics of tumors. This review summarizes lactylation-related genes in tumors, the role of lactylation in the TME, the interactions of the genes with other metabolic pathways, and the potential mechanisms underlying tumor progression as well as their clinical implications. Despite its nascent stage, research on the epigenetic regulation of tumor-related genes by lactylation holds promise. In this review, we highlighted unresolved challenges in this field and provided insights that may guide the development of novel targeted therapies for cancer.

847-P: ASC47, a Muscle-Preserving Weight Loss Drug Candidate for Obesity, in Combination with Semaglutide, Demonstrated Superior Weight Loss to Semaglutide Monotherapy in a Preclinical Model

2025-06-13

JINZI JASON WU , Chuyan Wu | Diabetes

Introduction and Objective: ASC47 (selective THR-β agonist) is an adipose-targeted, once-monthly subcutaneously (SQ) injected, muscle-preserving weight loss drug candidate for the treatment of obesity, discovered and developed in-house at Ascletis. … Introduction and Objective: ASC47 (selective THR-β agonist) is an adipose-targeted, once-monthly subcutaneously (SQ) injected, muscle-preserving weight loss drug candidate for the treatment of obesity, discovered and developed in-house at Ascletis. This study in diet-induced obese (DIO) mice demonstrated a powerful synergy in weight loss and muscle preservation when combining ASC47 and semaglutide. Methods: C57BL/6J mice were fed a high-fat diet for 16 weeks and received different treatments for 28 days. Body weight and food intake were recorded daily. Body composition was assessed by EchoMRI weekly. Results: ASC47 low dose combination 1 demonstrated superior weight loss compared to semaglutide monotherapy, showing an average total body weight reduction of 36.2%, a 56.7% greater reduction in body weight compared to semaglutide monotherapy. At the end of treatment, the percentage of total muscle mass over the total body weight of obese mice treated with ASC47 low dose combination treatments (68.8%) were close to healthy non-obese mice (66.0%), indicating healthy weight loss. Semaglutide monotherapy was unable to restore body composition to healthy levels. Conclusion: ASC47 low dose combination treatments were well tolerated in obese mice and produced a significant and healthy WL. Disclosure J. Wu: Employee; Ascletis Pharma (China) Co., Limited. C. Wu: Employee; an affiliate of Ascletis Pharma (China) Co., Limited,. Funding Ascletis Pharma (China) Co., Limited

1636-P: Liraglutide Mitigates Lipid Accumulation, EMT, and Fibrosis in MASLD via AMPK Activation

2025-06-13

Chun-Cheng Liao , Chien‐Ning Huang , Chih‐Li Lin +1 more | Diabetes

Introduction and Objective: Metabolic-associated steatotic liver disease (MASLD) is a prevalent condition associated with obesity and diabetes, frequently progressing to fibrosis and cirrhosis. Effective pharmacological treatments for this condition remain … Introduction and Objective: Metabolic-associated steatotic liver disease (MASLD) is a prevalent condition associated with obesity and diabetes, frequently progressing to fibrosis and cirrhosis. Effective pharmacological treatments for this condition remain scarce. This study aimed to evaluate the potential of liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, to regulate lipid metabolism and inhibit epithelial-mesenchymal transition (EMT), thereby reducing fibrosis through the activation of AMP-activated protein kinase (AMPK). Methods: The study employed human HepG2 hepatocyte cells exposed to high-glucose and high-fatty acid conditions to simulate MASLD, with or without liraglutide treatment. Additionally, male db/db mice fed a high-fat diet (HFD) were administered liraglutide. Key analyses included lipid accumulation assessment using Oil Red-O staining, quantification of reactive oxygen species (ROS), Western blotting, and immunofluorescence for EMT markers. Liver tissues were evaluated for fibrosis and collagen deposition using histological staining and quantitative polymerase chain reaction (qPCR). Results: Liraglutide significantly reduced lipid accumulation and ROS levels, preserved mitochondrial function, and inhibited EMT by restoring E-cadherin expression and downregulating Snail and vimentin in an AMPK-dependent manner. In vivo, liraglutide treatment reduced hepatic lipid deposition, decreased fibrosis markers, and improved liver histology, including reduced collagen accumulation. Conclusion: Liraglutide demonstrates significant potential as a therapeutic agent for MASLD by modulating lipid metabolism, suppressing EMT, and reducing fibrosis. These findings suggest a promising strategy for the management of MASLD and its complications and provide a basis for further clinical investigation into liraglutide’s therapeutic applications. Disclosure C. Liao: None. C. Huang: None. C. Lin: None. S. Tsou: None. Funding National Science and Technology Council, R.O.C. and grant unmber is 113-2314-B-663-001

287-OR: Physiological and Cellular Effects of Ligand and Kinase Independent Action of the Insulin Receptors in Liver

2025-06-13

Anindya Ghosh , Vítor Rosetto Muñoz , Arijeet K. Gattu +4 more | Diabetes

Introduction and Objective: Insulin signals through the insulin receptor (IR) tyrosine kinase to mediate its well-known metabolic and mitogenic functions. Recently, we have identified a second IR signaling pathway in … Introduction and Objective: Insulin signals through the insulin receptor (IR) tyrosine kinase to mediate its well-known metabolic and mitogenic functions. Recently, we have identified a second IR signaling pathway in preadipocytes that is dependent on intracellular domain of IR, but is ligand and tyrosine kinase-independent (LYK-I). Here, we studied this pathway in liver using liver specific IR-KO mice (LIRKO), since liver naturally contains almost no IGF1R. Methods: We have re-expressed either wildtype (WT) or kinase-dead (K1030R) -IR in LIRKO mice using adenoviral over-expression. Results: As expected, infection of LIRKO hepatocytes with the WT-IR, but not the kinase-dead IR, restored classical insulin signaling. In vivo, re-expression of WT-IR in liver of LIRKO mice normalized fed hyperglycemia, glucose tolerance, insulin sensitivity, reduced plasma insulin levels and increased liver weight, compared to mock-infected LIRKO mice. By contrast, somewhat surprisingly, expressing K1030R mutant IR in LIRKO mice worsened these parameters, despite the absence of endogenous IR and minimal IGF1R to inhibit. In isolated LIRKO hepatocytes, expression of either WT-IR or the K1030R mutant increased etoposide induced apoptosis and reduced NFκB expression compared to mock infected cells, similar to LYK-I effects observed in preadipocytes. Cholesterol biosynthesis genes (e.g. Hmgcs1), were upregulated by the expression of either WT-IR or the K1030R mutant, while inflammatory markers (Tnfα, Cxcl10) were downregulated compared to mock infected mice. Mechanistically, using LC-MS/MS proteomics we have shown that both WT-IR and kinase dead IR can interact with several inflammatory mediators, including IFITM2/3, which may partially contribute to apoptosis and inflammatory responses related to LYK-I actions of IR. Conclusion: Thus, LYK-I actions of IR in liver could modulate several cellular actions including cholesterol biosynthesis, inflammation, and apoptosis. Disclosure A. Ghosh: None. V.R. Muñoz: None. A. Gattu: None. Y. Watanabe: None. M. Lino: None. G. Ruiz: None. C. Kahn: Consultant; Novo Nordisk, Cellarity. Advisory Panel; TIXiMED. Board Member; 1825.

70-OR: Risks of Cancers among Novel Subtypes of Type 2 Diabetes—A Longitudinal Analysis from the United States

2025-06-13

Zhongyu Li , Bertha Cecilia Salazar , Jiali Guo +5 more | Diabetes

Introduction and Objective: Type 2 diabetes (T2D) may be associated with higher cancer risk through chronic inflammation, insulin resistance, and obesity. We assessed cancer risks across T2D subtypes. Methods: Newly … Introduction and Objective: Type 2 diabetes (T2D) may be associated with higher cancer risk through chronic inflammation, insulin resistance, and obesity. We assessed cancer risks across T2D subtypes. Methods: Newly diagnosed T2D (n = 727,076; age: 64.4 years [SD:13.3], 52% female) over 2012-2023 from the Epic Cosmos platform were classified into Severe Insulin-Deficient Diabetes (SIDD, 21.6%), Mild Obesity-Related Diabetes (MOD, 23.8%), or Mild Age-Related Diabetes (MARD, 40.9%) using reliable algorithms. Unclassified cases were labeled as Mixed (13.7%). First occurrence of cancers (colorectal, liver, pancreatic, female [breast, endometrial, and ovarian], prostate) within ten years after T2D diagnosis were identified using ICD-10-CM codes. Cox proportional hazards models were used to estimate absolute and relative hazards (HR) by subtype, adjusted for age, sex, and smoking. Results: Cumulative cancers were highest in MOD and Mixed. Patterns for specific cancer risks differed: compared to MOD (Figure), SIDD and MARD had 35-111% higher hazards of liver, pancreatic, and prostate cancers but 10-36% lower hazards of colorectal and female cancers. Conclusion: Novel subtypes exhibit varying risks for different cancers, highlighting opportunities for early screening and tailored prevention strategies. Disclosure Z. Li: None. B. Salazar: None. J. Guo: None. K.O. Sanaka: None. P. Vellanki: Advisory Panel; Eli Lilly and Company. M.K. Ali: Advisory Panel; Eli Lilly and Company. C. Hofmeister: Research Support; AbbVie Inc, Sanofi, Bristol-Myers Squibb Company. J. Varghese: None.

1701-P: Activin E–Blocking Antibody for Treatment of Metabolic Diseases

2025-06-13

C. J. Schwartz , Charity G. Moore , ALEXANDER TAGUCHI +5 more | Diabetes

Introduction and Objective: Activin E, a product of the INHBE gene, has been implicated in body weight regulation and adiposity. A novel antibody that potently blocks Activin E-mediated signaling was … Introduction and Objective: Activin E, a product of the INHBE gene, has been implicated in body weight regulation and adiposity. A novel antibody that potently blocks Activin E-mediated signaling was developed based on the hypothesis that this mechanism could improve metabolic outcomes in a diet-induced obesity (DIO) mouse model. Specifically, the objective was to determine whether anti-Activin E antibody, alone or in combination with semaglutide, could cause weight loss, preserve lean muscle mass, and favorably alter other metabolic parameters. Methods: A potent anti-Activin E antibody was engineered and confirmed to block Activin E signaling in both reporter cell assays and a human adipocyte cell line. Its efficacy was then evaluated in DIO mice as a single agent and in combination with semaglutide. Body weight, food intake, body composition (including lean mass and fat distribution), and metabolic parameters were monitored throughout treatment. Statistical comparisons were performed using appropriate tests (e.g., ANOVA or t-test), with significance at p&lt;0.05. Results: As a monotherapy, anti-Activin E antibody achieved weight loss in DIO mice without substantially altering food intake. When combined with semaglutide further enhancements in weight reduction were achieved compared to either therapy alone. Preliminary data related to muscle mass retention, body fat distribution, and additional metabolic measures was also promising. Conclusion: These findings suggest an anti-Activin E antibody is a promising candidate for treating obesity as both a monotherapy and in combination with incretins such as semaglutide. Further investigations are warranted to confirm its therapeutic potential and safety profile. Disclosure C. Schwartz: Employee; iBio, Inc. C. Moore: None. A. Taguchi: Employee; iBio Inc. H. Zhang: None. T. Phuong: Employee; IBIO INC. T. Hsu: Employee; iBio Inc. M. Dent: Employee; iBio Inc. M.B. Brenner: Employee; iBio Inc.

849-P: Eloralintide (LY3841136), a Selective Amylin Mimetic, Lowered Body Weight with Improved Quality of Weight Loss and GI Tolerability in Rats Compared with Cagrilintide

2025-06-13

Daniel A. Briere , A. Long , Delia Bullock +6 more | Diabetes

Introduction and Objective: Non-selective amylin analogs have shown clinical weight loss benefits, though clinical incidence of nausea and vomiting are high and may require prolonged dosing titrations. Moreover, preclinical studies … Introduction and Objective: Non-selective amylin analogs have shown clinical weight loss benefits, though clinical incidence of nausea and vomiting are high and may require prolonged dosing titrations. Moreover, preclinical studies in rats suggest lean mass preservation versus GLP-1 receptor agonists. Herein, we evaluated weight loss efficacy, GI tolerability, and lean-mass preservation in rats administered eloralintide compared to the non-selective amylin analog, cagrilintide. Methods: Peptides were made using automated peptide synthesizers and evaluated in amylin and calcitonin receptor functional and binding assays. PK and conditioned taste avoidance studies performed using lean rats. Weight loss studies were carried out in lean and DIO rats using QNMR to determine body composition. Results: Eloralintide is an amylin peptide analog modified with a C20 fatty diacid moiety that binds to amylin receptors while retaining selectivity from calcitonin receptors. Eloralintide demonstrated prolonged plasma half-life and reduced clearance compared to cagrilintide. In lean rats, eloralintide matched the food intake reductions of cagrilintide, resulting in a similar degree on body weight change, without the same degree of conditioned taste avoidance. In DIO rats, eloralintide lowered body weight primarily by loss of fat mass and with more potent effects for weight loss efficacy, food intake lowering, and fat mass reductions compared to cagrilintide, with head-to-head studies demonstrating reduced loss of lean mass in comparison to cagrilintide. Conclusion: Eloralintide demonstrated preclinical improvements in efficacy, GI tolerability, and quality of weight loss compared to the non-selective amylin analog cagrilintide. These findings support clinical investigation of eloralintide as a weight loss therapeutic. Disclosure D.A. Briere: Employee; Lilly USA LLC. A. Long: Employee; Eli Lilly and Company. D.M. Bullock: None. L. O'Farrell: None. B. Bowen: Employee; Eli Lilly and Company. K. Lansu: Employee; Eli Lilly and Company. T. Coskun: None. J.S. Moyers: Employee; Eli Lilly and Company. Stock/Shareholder; Eli Lilly and Company. H. Qu: Employee; Eli Lilly and Company.

845-P: MBX 1416, a Selective GLP-1 Antagonist, Elevates and Sustains Blood Glucose in Rats without Change in Body Weight

2025-06-13

KISHORE THALLURI , Michael Hackett , Jessica J. Pellman +5 more | Diabetes

Introduction and Objective: MBX 1416 is a selective, reversible glucagon-like peptide-1 receptor (GLP-1R) antagonist being investigated for treating post-bariatric hypoglycemia. In vivo studies examined pharmacokinetic/pharmacodynamic (PK/PD) properties of MBX 1416. … Introduction and Objective: MBX 1416 is a selective, reversible glucagon-like peptide-1 receptor (GLP-1R) antagonist being investigated for treating post-bariatric hypoglycemia. In vivo studies examined pharmacokinetic/pharmacodynamic (PK/PD) properties of MBX 1416. Methods: MBX 1416 single subcutaneous dose PK was assessed in Sprague-Dawley rats for dose selection. The GLP-1 antagonist mechanism of action was tested in rats by reversing effects of a potent GLP-1 agonist, semaglutide. Differential effects of MBX 1416 on glucose elevation following glucose challenge in DIO mice vs an unmodified GLP-1 antagonist exendin 9-39 (Ex9) were assessed with an intraperitoneal glucose tolerance test (IPGTT) 24 h post dosing to highlight MBX 1416’s lengthy time action. Results: Dose-proportional plasma MBX 1416 exposures were seen with a single dose (Cmax and AUCinf, ≤2-fold across doses). In healthy rats, MBX 1416 completely abrogated anorectic and weight loss activity of semaglutide but when given alone, had no effect on body weight or food intake vs controls. In DIO mice, MBX 1416 led to a greater glucose excursion in an IPGTT that was sustained 24 hours; in contrast, the GLP-1 antagonist Ex9 showed no activity at 24 h. Conclusion: MBX 1416 demonstrated expected PK/PD effects in rats, increasing blood glucose excursions in DIO mice with no change in food intake or body weight vs vehicle in healthy rats. Disclosure K. Thalluri: None. M.J. Hackett: None. J.J. Pellman: None. A. Joice: None. E. Fabbrini: Employee; Janssen Pharmaceuticals, Inc. Stock/Shareholder; Janssen Pharmaceuticals, Inc. Employee; MBX Biosciences. D. Perez-Tilve: Research Support; MBX Biosciences, Novo Nordisk A/S. Other Relationship; BlueWater LLC, Ghrelco LLC. Consultant; Eli Lilly and Company, Ampeptec. R. DiMarchi: Research Support; MBX Biosciences. M.A. Dorato: Stock/Shareholder; Eli Lilly and Company.

2158-LB: Additive Hepatoprotective Effects of DA-1241, a GPR119 Agonist, in Combination with Efruxifermin in a Diet-Induced Obese and Biopsy-Confirmed Mouse Model of MASH

2025-06-13

YUNA CHAE , TAE-HYOUNG KIM , SU JIN LEE +3 more | Diabetes

Introduction and Objective: DA-1241, a novel G protein-coupled receptor 119 agonist, recently completed a Phase 2a clinical trial in presumed metabolic dysfunction-associated steatohepatitis (MASH) patients. This study extended previous findings … Introduction and Objective: DA-1241, a novel G protein-coupled receptor 119 agonist, recently completed a Phase 2a clinical trial in presumed metabolic dysfunction-associated steatohepatitis (MASH) patients. This study extended previous findings on its hepatoprotective synergy with glucagon-like peptide-1-based drugs and evaluated its anti-MASH effects in combination with a fibroblast growth factor 21 analogue. Methods: Male C57BL/6JRj mice were fed a GAN diet for 36 weeks before receiving vehicle, DA-1241 (100 mg/kg, QD, oral), Efruxifermin (EFX; 1 mg/kg, QW, subcutaneous), or their combination for 12 weeks. Results: DA-1241 was weight-neutral, while EFX induced 17% weight loss (p&lt;0.05 vs. vehicle), with no further reduction in combination. Each treatment ameliorated plasma transaminases and liver cholesterol levels, with combination therapy providing greater improvement compared to monotherapies. In combination, 94% of mice had a ≥2-point improvement in non-alcoholic fatty liver disease activity score from baseline, indicating an additive effect. Liver lipid area and steatotic hepatocytes also decreased more with the combination. Liver immunohistochemistry revealed significantly reduced inflammatory (galectin-3) and fibrotic (type 1 collagen and α-SMA) markers, suggesting enhanced effects over monotherapy. mRNA analysis showed marked decreases in inflammatory (Tnfα -58%, Cxcl10 -56%) and fibrotic (Col1a1 -72%) genes, confirming synergy. Notably, hedgehog-interacting protein, a suppressor of hepatic stellate cell activation, was upregulated more (+321%) in the combination than in each alone. These findings suggest that combination therapy benefits liver pathology in MASH. Conclusion: For the first time, we suggest a beneficial combination effect of DA-1241 and EFX in MASH treatment, highlighting the therapeutic potential of combining GPR119 agonists and FGF21 analogues, likely by further inhibiting steatosis, fibrosis, and inflammation. Disclosure Y. Chae: None. T. Kim: None. S. Lee: None. I. Jung: None. M. Kim: None. H. Kim: Employee; Dong-A ST Co., Ltd., MetaVia Inc.

2152-LB: Prediabetes Phenotypes Defined by Glucose-Stimulated Insulin Response Using Hyperglycemic Clamp in Healthy Young People

2025-06-13

VIDHYA NATARAJAN , BHANU PRAKASH REDDY ATTUNURU , Deepa Shankar +7 more | Diabetes

Introduction and Objective: Prediabetes, often classified by static glucose measures, limits identification of impaired beta-cell function beyond response to prevailing insulin sensitivity. We implemented hyperglycemic clamps (HG) to classify prediabetes … Introduction and Objective: Prediabetes, often classified by static glucose measures, limits identification of impaired beta-cell function beyond response to prevailing insulin sensitivity. We implemented hyperglycemic clamps (HG) to classify prediabetes phenotypes based on stimulated insulin response. Methods: We screened healthy individuals (n=2500, 18-45 yrs, BMI 18.5-25 kg/m2) and recruited (37 normal glycemia, NG; 63 prediabetes, preD) participants based on ADA HbA1c cutoff (5.7). Whole blood was collected at 17 time points during HG clamp. Glucose, insulin, C-peptide, HOMAIR and HOMA B% were measured. Statistical analysis included Wilcoxon test to compare groups and K-means clustering to identify subtypes of prediabetes. Differences between preD clusters at baseline, 1st (10 min) and 2nd phase (120 min) insulin secretion were assessed. Results: Compared to NG, preD had higher glucose (p=0.028) at 120 min, lower C-peptide [baseline (p=0.048), 1st phase (p=0.024), 2nd phase (p=0.006)] and lower HOMA-B at baseline and 2nd phase. K-means clustering using C-peptide and HOMA-B identified two clusters: high insulin cluster (preD1), low insulin cluster (preD2) (Table). Conclusion: HG clamps in prediabetes individuals suggest heterogeneity and majority have lower 2nd phase glucose-stimulated insulin response compared to NG individuals, indicating beta-cell dysfunction as a major contributor to prediabetes in Indians. Disclosure V. Natarajan: None. B. Attunuru: None. D. Shankar: None. P.R. Somvanshi: None. V. Nayanatara: None. S. Pinninti: None. S. Kulkarni: None. K.R. Choudari: None. L. Staimez: None. A.C. Powers: None. K. Narayan: None. A. Kurpad: None. M. Sasikala: None. Funding DBT Wellcome Trust India Alliance (IA/CRC/23/1/600505)

2155-LB: Hepatic BHMT Integrates GIP/GLP-1 Signaling to Ameliorate Metabolic Dysfunction through Coordinated Regulation of Lipid Metabolism and Fibrogenesis

2025-06-13

Han Yan | Diabetes

Introduction and Objective: Metabolic dysfunction-associated steatohepatitis (MASH), a critical stage in NAFLD progression, lacks effective treatments. While GIP/GLP-1R dual agonists (GIP/GLP-1RA) show therapeutic potential, their mechanisms remain unclear. Our preclinical … Introduction and Objective: Metabolic dysfunction-associated steatohepatitis (MASH), a critical stage in NAFLD progression, lacks effective treatments. While GIP/GLP-1R dual agonists (GIP/GLP-1RA) show therapeutic potential, their mechanisms remain unclear. Our preclinical study identified serum BHMT downregulation by GIP/GLP-1RA. This study investigates the BHMT-incretin relationship, its roles in MASH pathogenesis, and its potential as a therapeutic target for metabolic syndrome. Methods: Serum proteomic analysis was performed in obese diabetic mice following GIP/GLP-1RA treatment. Hepatic fibrosis models were established using a high-fat/high-fructose diet or CCl4 injection in mice with hepatocyte-specific BHMT modulation. BHMT expression was assessed in human and murine MAFLD/MASH livers. Adenovirus, AAV vectors, and recombinant BHMT were used to study cytosolic and secretory BHMT functions in hepatocytes and stellate cells. Systemic and hepatic metabolic phenotypes were evaluated following BHMT manipulation. Proteomics, co-IP, and click chemistry were employed to identify BHMT-interacting proteins and elucidate underlying mechanisms. Results: BHMT is predominantly expressed in hepatocytes. Hepatic BHMT expression progressively decreases during disease progression, while serum BHMT levels slightly increase. Mechanistically, cytoplasmic BHMT interacts with 3-KAT to enhance lipid oxidation, while secreted BHMT is internalized by stellate cells, binding to cleaved NOTCH4 to inhibit the NOTCH4-Hes1 pathway and attenuate fibrosis. Both intrahepatic overexpression and exogenous BHMT supplementation improved metabolic disorders. Conclusion: BHMT functions as a nutrient sensor, in response to GIP/GLP-1-mediated metabolic alterations. Hepatocyte-derived cytosolic and secretory BHMT plays a critical role in liver disease pathogenesis, representing a promising therapeutic target for metabolic syndrome management. Disclosure H. Yan: None. Funding National Natural Science Foundation of China (82300957)

2156-LB: Superior Hepatoprotective Effects of OPK-88006, a Novel GLP-1/Glucagon Receptor Dual Agonist, to Semaglutide and Survodutide in the GAN Diet-Induced Obese and Biopsy-Confirmed Mouse Model of MASH

2025-06-13

Ji‐Chang Hsiao , Laura Moschcovich , MIRI GERZON-ZAKAR +5 more | Diabetes

Introduction and Objective: The present study aimed to investigate the hepatoprotective effects of OPK-88006, a novel GLP-1/glucagon receptor agonist, in the GAN diet-induced obese and biopsy-confirmed mouse model of MASH … Introduction and Objective: The present study aimed to investigate the hepatoprotective effects of OPK-88006, a novel GLP-1/glucagon receptor agonist, in the GAN diet-induced obese and biopsy-confirmed mouse model of MASH with liver fibrosis. The late-stage clinical candidates semaglutide and survodutide for the treatment of patients with MASH and liver fibrosis, were included for comparison. Methods: Male GAN DIO-MASH mice with NAFLD Activity Score (NAS≥5) and moderate/advanced fibrosis (stage F2-F3) were administered once daily subcutaneous vehicle, semaglutide (30 nmol/kg), survodutide (15) nmol/kg) and OPK-88006 (20 nmol/kg) for 12 weeks. Histopathological scoring was performed. Other terminal endpoints included quantitative liver histology, blood and liver biochemistry in addition to RNAsequencing-Bioinformatic analysis. Results: GAN DIO-MASH mice demonstrated vehicle-corrected weight loss of 12%, 24% and 26% for OPK-88006, semaglutide and survodutide treatment, respectively. All treatments improved hepatomegaly, plasma transaminases and plasma lipids levels, albeit only OPK-88006 induced 2-point statistically significant improvement in NAS. In agreement, OPK-88006 reduced quantitative liver histology on steatosis and inflammation (galectin-3). Treatments reduced markers for fibrosis (TIMP-1 PIIINP) and hepatic stellate activation (α-SMA), indicating effect on fibrogenesis. Finally, all treatments induced significant gene regulation and suppressed genes involved in extracellular matrix and inflammation pathways. Conclusion: OPK-88006 treatment improved metabolic, biochemical and histopathological parameters of MASH, including hepatic transcriptomic profile, in the GAN DIO-MASH mouse model. Notably, OPK-88006 treatment improved NAFLD Activity Score superior to late-stage clinical candidates semaglutide and survodutide, introducing OPK-88006 as a promising treatment for MASH. Disclosure J. Hsiao: None. L. Moschcovich: None. M. Gerzon-Zakar: None. A. Rivkin: None. S.E. Pors: None. M.R. Madsen: None. T. Cruz: Employee; OPKO Health. M. Feigh: None.

1605-P: Insulin Resistance Promotes Steatosis and Increases the Risk of Clinically Significant Fibrosis in People with Type 2 Diabetes

2025-06-13

NATHALY CUERVO-PARDO , SRILAXMI KALAVALAPALLI , Eddison Godinez Leiva +7 more | Diabetes

Introduction and Objective: Type 2 diabetes (T2D) is often associated with metabolic dysfunction-associated steatotic liver disease (MASLD) which may lead to clinically significant fibrosis (defined as moderate-to-severe fibrosis stage ≥F2 … Introduction and Objective: Type 2 diabetes (T2D) is often associated with metabolic dysfunction-associated steatotic liver disease (MASLD) which may lead to clinically significant fibrosis (defined as moderate-to-severe fibrosis stage ≥F2 or “at-risk” MASLD≥F2) and eventually cirrhosis. The role of insulin resistance (IR) in the development of MASLD≥F2 remains unclear. We examined the relationship of IR to the severity of MASLD in people with T2D. Methods: We studied 190 participants with T2DM (age: 59±10; BMI: 32±7 kg/m2, A1c: 6.6±1.0%) who were divided by into IR (HOMA-IR of ≥2.5) or insulin sensitive (IS). We used transient elastography (VCTE; Fibroscan®) to establish the presence of steatosis/MASLD (CAP ≥274 dB/m). Fibrosis (any stage) was defined as LSM ≥7.0 kPa (≥F1) and clinically significant fibrosis (MASLD≥F2) as LSM ≥8.2 kPa. Results: Having T2DM and IR (n=135), compared to those IS (n=55), was often associated with obesity (69% vs. 32%), higher triglycerides (TG), lower HDL-C, and adipose tissue dysfunction (higher adipo-IR and lower adiponectin). Also, the prevalence of steatosis (IR: 75% vs. IS: 31%) and liver fibrosis ≥F1 (IR: 13% vs. IS: 4%) or ≥F2 (IR: 7% vs. IS: 2%) was 2- to 3-fold higher (all p&lt;0.01). Of interest, 25% of IR did not have steatosis (or fibrosis). In IS individuals with T2D, only 32% had steatosis and none developed MASLD≥F2. In people with steatosis, being IR vs. IS was associated with worse CV risk factors: A1c: 6.8±1.1 vs. 6.3±0.7%; sBP: 133±13 vs. 125±14 mmHg; dBP: 81±8 vs. 76±9 mmHg, TG: 148±78 vs. 101±40 mg/dL; HDL-C: 45±11 vs. 48±12; apoB: 94±23 vs. 83±19 mg/dL (all p&lt;0.05). If IS, having steatosis played a minimal role on CV risk factors. Conclusion: In people with T2D who are insulin-sensitive, steatosis and clinically significant fibrosis are rare. In contrast, IR is a strong driver of both steatosis and MASLD≥F2. Measurement of IR (HOMA-IR) may assist in the risk stratification and prevention of cirrhosis in T2D. Disclosure N. Cuervo-Pardo: None. S. Kalavalapalli: None. E. Godinez Leiva: None. A. Ortiz Rocha: None. A. Sharma: None. D. Barb: Other Relationship; Inventiva Pharma, Boehringer-Ingelheim. T. Nguyen: None. E. Valdez Saenz: None. R. Lomonaco: None. M.A. Connelly: Employee; LabCorp. Stock/Shareholder; LabCorp. K. Cusi: Research Support; Boehringer-Ingelheim, Echosens, Inventiva, Perspectum Ltd, LabCorp. Consultant; Arrowhead Pharmaceuticals, Inc, AstraZeneca, TERNS Pharmaceuticals, 89bio, Inc, Boehringer-Ingelheim, Eli Lilly and Company, Novo Nordisk A/S, Sagimet Biosciences. Funding NIH/NIDDK (R01DK120331) PI: Kenneth Cusi.

893-P: Long-Term Administration of Dorzagliatin Combined with Sitagliptin for the Management of Glucose Homeostasis in High-Fat Diet–Induced Obesity Mice

2025-06-13

DONGNA HAN , Meng Shi , RUYUE LI +3 more | Diabetes

Introduction and Objective: A short-term study in obese T2D patients showed that the combination of dorzagliatin, a glucokinase activator (GKA) and sitagliptin improved glucose controls with increased GLP-1 secretion. This … Introduction and Objective: A short-term study in obese T2D patients showed that the combination of dorzagliatin, a glucokinase activator (GKA) and sitagliptin improved glucose controls with increased GLP-1 secretion. This study aims to assess the impact of long-term administration of dorzagliatin and the combination of dorzagliatin/sitagliptin on glucose homeostasis in high-fat diet-induced obesity/diabetes (DIO) mice. Methods: DIO mice were developed following a 6-month high-fat diet. Prior to treatment, mice were evaluated. Mice were then administered dorzagliatin at a dosage of 30 mg/Kg/day, or a combination of dorzagliatin (same dose) and sitagliptin (20 mg/Kg/day) for 30 days. Mice on a standard diet serve as controls. Results: Compared to controls, DIO mice have elevated random and fasting glucose levels, along with higher insulin and glucagon, decreased GLP-1/glucose ratio. On day 30, the glucose levels were all reduced compared to pre-treatment, and the combination therapy was more effective than dorzagliatin alone. Dorzagliatin alone elevated insulin and GLP-1 secretion, the combination resulted in a further increase. The blood chemistry analysis indicated that DIO mice exhibit increased levels of ALT, AST, lipid profiles, and glycated serum protein (GSP). Drug therapy has significantly improved GSP and AST. The combined therapy also improved the LDL levels. Conclusion: DIO mice have impaired GLP-1 secretion, elevated glucose, insulin and glucagon levels. Following 30 days of treatment, glucose levels were improved with increased secretion of insulin, the combination of GKA and DPP4i was superior to dorzagliatin alone with the improved GLP-1 secretion. Those data indicated that, in addition to increased insulin secretion by GKA, the enhanced GLP-1 secretion also played a significant role in improving glucose homeostasis, and the addition of DPP4i amplified these effects. Disclosure D. Han: None. S. Meng: None. R. Li: None. L. Feng: None. L. Chen: None. C. Li: None.

2169-LB: Comparative Metabolic Effects of Semaglutide, Tirzepatide, and Retatrutide in a Monogenic (db/db) Mouse Model of Obesity

2025-06-13

KATHRYN A. ESCHETE , SARAH OLKKOLA , JASMINE SWAGEL +7 more | Diabetes

Introduction and Objective: Mouse models of obesity exhibit heterogeneity in metabolic flexibility, with diet-induced obesity (DIO) mice showing greater adaptability in energy balance regulation, while monogenic obesity models have impaired … Introduction and Objective: Mouse models of obesity exhibit heterogeneity in metabolic flexibility, with diet-induced obesity (DIO) mice showing greater adaptability in energy balance regulation, while monogenic obesity models have impaired compensatory mechanisms due to genetic mutations. In this study, we assessed the metabolic impacts of mono, dual, and triple GLP-1 agonists on a monogenic (db/db) mouse model of obesity and type-2 diabetes. Methods: Adult male B6.BKS(D)-Leprdb/J mice (6 weeks old) were randomized into four treatment groups (n=20/group): Vehicle (PBS), Semaglutide (10 nmol/kg), Tirzepatide (10 nmol/kg), and Retatrutide (10 nmol/kg). All treatments were administered subcutaneously once daily for four weeks. Metabolic assessments included measurements of body weight, food intake, fat, and lean mass, along with glucose tolerance testing, microCT imaging, and proteomic analysis. Results: At randomization, Leprdb/db mice had an average body weight of 37.2 ± 1.2 g. Over the treatment period, vehicle-treated mice exhibited a 23% increase in body weight, whereas Semaglutide, Tirzepatide, and Retatrutide treatment mitigated weight gain, resulting in increases of 20%, 14.2%, and 14.3%, respectively. The tirzepatide treatment group exhibited the greatest reduction in food intake. Blood glucose levels were significantly reduced in the Tirzepatide-treated group (Vehicle: 297.6 mg/dL vs. Tirzepatide: 124.3 mg/dL, p&lt;0.05). During the glucose tolerance test (GTT), both Semaglutide (p&lt;0.05) and Tirzepatide (p&lt;0.001) exhibited enhanced glucose clearance compared to the vehicle-treated group. Fat mass was reduced by 14.3% with Semaglutide, 17.2% with Tirzepatide, and 17.5% with Retatrutide from baseline. Conversely, lean mass increased by 3.2% in the vehicle group, 6.9% with Semaglutide, 1.75% with Tirzepatide, and 0.5% with Retatrutide. Conclusion: Overall, Tirzepatide demonstrated the most pronounced metabolic benefits in the monogenic model of obesity coupled with type-2 diabetes. Disclosure K.A. Eschete: None. S. Olkkola: None. J. Swagel: None. A. Dunn: None. N. Bethur: None. H. Kaur: None. T. Stodola: None. B. Hoffmann: None. K. O'Connell: None. V. Yin: None. A. .: None.

Abstract PS15-03: Retrospective study of GLP-1 Receptor Agonists in Breast Cancer Survivors: Weight Loss and Patient Outcomes

2025-06-13

Jasmine Sukumar , Akshara Singareeka Raghavendra , Sarah Pasyar +5 more | Clinical Cancer Research

Abstract Background: Obesity in breast cancer (BC) survivors is associated with higher BC recurrence risk and mortality, and weight loss in an important tenet of health promotion. Glucagon-like peptide-1 receptor … Abstract Background: Obesity in breast cancer (BC) survivors is associated with higher BC recurrence risk and mortality, and weight loss in an important tenet of health promotion. Glucagon-like peptide-1 receptor agonists (GLP1-RA) are incretin mimetics with favorable metabolic effects and approved for type 2 diabetes (DM2) or weight loss. While drug utilization is increasing, the implications in cancer survivors are not well elucidated. This study evaluated treatment patterns of GLP1-RA, weight loss trends, and patient outcomes in a cohort of BC survivors. Methods: We retrospectively analyzed patients with non-metastatic (DCIS or invasive stage I-III) BC treated at MD Anderson Cancer Center (MDACC) who received at least 3 months of GLP1-RA from 2005 - 2024. Data was obtained from the MDACC BC and pharmacy databases. Linear regression models estimated the association between weight change and clinical factors. After excluding patients with DCIS, propensity score matching (1:2 ratio) was used to match patients who received GLP1-RA with those who did not, based on baseline body mass index (BMI), DM2, age, clinical stage, and BC receptor status. Kaplan-Meier estimates and log-rank tests estimated and compared overall survival (OS) and disease-free survival (DFS) between patients who received GLP1-RA and those who did not. Results: In total, 1,022 patients met inclusion criteria. Median age was 54 years (23-86) years, and 56.9% were postmenopausal; 79.0% had DM2. Most (91.4%) had stage I-III invasive BC and 8.6% had DCIS; 80.2% had hormone receptor positive BC and 65.9% received adjuvant endocrine therapy with an aromatase inhibitor (AI) or tamoxifen. GLP1-RA was started following definitive BC therapy (chemotherapy, surgery, radiation) in 87.6%; the median time from BC diagnosis to GLP1-RA initiation in these patients was 4.7 (0.0-34.0) years. The median duration of GLP1-RA use was 1.2 (0.3-8.1) years. Median BMI at BC diagnosis was 33.5 (20.1-56) kg/m2. Baseline median weight and median BMI at GLP1-RA initiation (within 90 days prior) was 86.8 (47.2-175.0) kg and 33.6 (18.9-61.8) kg/m2, respectively. In patients (n=442, 43.2%) who received the drugs approved for weight loss (semaglutide or tirzepatide), median weight loss at 3 (+/- 45 days), 6 (+-/45 days), and 12 (+/- 60 days) months was -1.9% (-13.2%-14.9%), -3.1% (-20.2%-19.0%), and -2.6% (-27.8%-11.5%), respectively. On multivariate regression analysis, no significant association was observed between the 6 month change in weight and clinical factors (DM2, metformin use, endocrine therapy use, duration of GLP1-RA, clinical stage); however, the 12 month change in weight was significantly associated with clinical stage (invasive BC was associated with more weight loss vs DCIS, p&lt;0.01) and endocrine therapy (tamoxifen or AI use was associated with weight gain, p=0.019). Patients who received endocrine therapy experienced, on average, a 2.83 kg weight gain at 12 months compared to those who did not, after adjusting for other variables in the model. In those with invasive BC (DCIS excluded); there was no significant difference in DFS but there was a significantly improved OS in patients who received GLP1-RA compared with controls (median survival not reached (0-30.9) vs 27.0 years (0-57.3), p&lt;0.0001). Conclusion: This is the largest study describing real world patterns of GLP1-RA use in BC survivors. These medications were associated with modest weight loss; however, endocrine therapy may decrease this impact. An improved all-cause survival was observed, but there was no difference in DFS. Clinical trials are needed to investigate the role of these agents for weight loss as an adjunct to lifestyle interventions in cancer survivors. Further exploration of potential anti-cancer biological effects for BC risk reduction and cancer control may also be warranted. Citation Format: Jasmine Sukumar, Akshara Raghavendra, Sarah Pasyar, Roland Bassett, Debu Tripathy, Carlos H Barcenas, Karen Basen-Engquist, Banu Arun. Retrospective study of GLP-1 Receptor Agonists in Breast Cancer Survivors: Weight Loss and Patient Outcomes [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS15-03.