Insulin Receptor Substrates-1 and -2 Are Both Depleted but via Different Mechanisms after Down-Regulation of Glucose Transport in Rat Adipocytes

Abstract Alterations in muscle and adipose tissue insulin receptor substrate (IRS)-1 and IRS-2 are associated with, and commonly believed to contribute to, development of insulin resistance. In this study, we investigated the mechanisms behind previously observed reductions in IRS levels due to high concentrations of glucose and insulin and their significance in the impairment of glucose uptake capacity in primary rat adipocytes. Semiquantitative RT-PCR analysis showed that insulin (104 μU/ml) alone or in combination with glucose (15 mm) markedly suppressed IRS-2 gene expression, whereas IRS-1 mRNA was unaffected by the culture conditions. The negative effect of a high glucose/high insulin setting on IRS-1 protein level was still exerted when protein synthesis was inhibited with cycloheximide. Impairment of glucose uptake capacity after treatment with high glucose and insulin was most pronounced after 3 h, whereas IRS-1 and IRS-2 protein levels were unaffected up to 6 h but were reduced after 16 h. Moreover, impaired glucose uptake capacity could only partially be reversed by subsequent incubation at physiological conditions. These novel results suggest that: 1) in a high glucose/high insulin setting depletion of IRS-1 and IRS-2 protein, respectively, occurs via different mechanisms, and IRS-2 gene expression is suppressed, whereas IRS-1 depletion is due to posttranslational mechanisms; 2) IRS-1 and IRS-2 protein depletion is a secondary event in the development of insulin resistance in this model of hyperglycemia/hyperinsulinemia; and 3) depletion of cellular IRS in adipose tissue may be a consequence rather than a cause of insulin resistance and hyperinsulinemia in type 2 diabetes.

Download Full-text

High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes

Acta Endocrinologica ◽

10.1530/eje.0.1480157 ◽

2003 ◽

Vol 148 (1) ◽

pp. 157-167 ◽

Cited By ~ 43

Author(s):

J Buren ◽

HX Liu ◽

J Lauritz ◽

JW Eriksson

Keyword(s):

Glucose Uptake ◽

High Glucose ◽

Insulin Treatment ◽

Insulin Signalling ◽

Binding Capacity ◽

Insulin Binding ◽

Rat Adipocytes ◽

Glucose Levels ◽

High Insulin ◽

The Right

OBJECTIVE: The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins. DESIGN AND METHODS: Rat adipocytes were cultured for 24 h in different glucose concentrations with 10(4) microU/ml of insulin or without insulin. After washing, (125)I-insulin binding, basal and acutely insulin-stimulated d-[(14)C]glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed. RESULTS: High glucose (15 and 25 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by approximately 40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by approximately 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by approximately 20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination, shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by approximately 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration. CONCLUSIONS: Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose, it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.

Download Full-text

Rosemary Extract Activates AMPK, Inhibits mTOR and Attenuates the High Glucose and High Insulin-Induced Muscle Cell Insulin Resistance

Applied Physiology Nutrition and Metabolism ◽

10.1139/apnm-2020-0592 ◽

2021 ◽

Author(s):

Hesham Shamshoum ◽

Filip Vlavcheski ◽

Rebecca E.K. MacPherson ◽

Evangelia Tsiani

Keyword(s):

Insulin Resistance ◽

Plasma Membrane ◽

Blood Glucose ◽

Glucose Uptake ◽

Muscle Cell ◽

High Glucose ◽

Serine Phosphorylation ◽

Rosemary Extract ◽

Insulin Levels ◽

High Insulin

Impaired action of insulin in skeletal muscle, termed insulin resistance, leads to increased blood glucose levels resulting in compensatory increase in insulin levels. The elevated blood glucose and insulin levels exacerbate insulin resistance and contribute to the pathogenesis of type 2 diabetes mellitus (T2DM). In previous studies we found attenuation of free fatty acid-induced muscle cell insulin resistance by rosemary extract (RE). In the present study we investigated the effects of RE on high glucose (HG) and high insulin (HI)-induced muscle cell insulin resistance. Exposure of L6 myotubes to 25 mM glucose and 100 nM insulin for 24 h, to mimic hyperglycemia and hyperinsulinemia, abolished the acute insulin-stimulated glucose uptake, increased the serine phosphorylation of IRS-1 and the phosphorylation/ activation of mTOR and p70S6K. Treatment with RE significantly improved the insulin-stimulated glucose uptake and increased the acute insulin-stimulated tyrosine phosphorylation while reduced the HG+HI-induced serine phosphorylation of IRS-1 and phosphorylation of mTOR and p70S6K. Additionally, treatment with RE significantly increased the phosphorylation of AMPK, its downstream effector ACC and the plasma membrane GLUT4 levels. Our data indicate a potential of RE to counteract muscle cell insulin resistance and more studies are required to investigate its effectiveness in vivo. Novelty: • Rosemary extract (RE) phosphorylated muscle cell AMPK and ACC under both normal and high glucose (HG)/high insulin (HI) conditions. • The HG/HI-induced serine phosphorylation of IRS-1 and activation of mTOR and p70S6K were attenuated by RE. • RE increased the insulin-stimulated glucose uptake by enhancing GLUT4 glucose transporter translocation to plasma membrane.

Download Full-text

Insulin resistance induced by high glucose and high insulin precedes insulin receptor substrate 1 protein depletion in human adipocytes

Metabolism ◽

10.1016/j.metabol.2006.09.012 ◽

2007 ◽

Vol 56 (2) ◽

pp. 190-198 ◽

Cited By ~ 30

Author(s):

Frida Renström ◽

Jonas Burén ◽

Maria Svensson ◽

Jan W. Eriksson

Keyword(s):

Insulin Resistance ◽

Insulin Receptor ◽

High Glucose ◽

Insulin Receptor Substrate ◽

Human Adipocytes ◽

Insulin Receptor Substrate 1 ◽

Protein Depletion ◽

High Insulin

Download Full-text

Differential gene expression of insulin receptor isoforms A and B and insulin receptor substrates 1, 2 and 3 in rat tissues: modulation by aging and differentiation in rat adipose tissue

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01635 ◽

2005 ◽

Vol 34 (1) ◽

pp. 153-161 ◽

Cited By ~ 39

Author(s):

R Serrano ◽

M Villar ◽

C Martínez ◽

J M Carrascosa ◽

N Gallardo ◽

...

Keyword(s):

Gene Expression ◽

Insulin Resistance ◽

Adipose Tissue ◽

Insulin Receptor ◽

Differential Gene Expression ◽

Target Tissue ◽

Insulin Receptor Isoforms ◽

Alternatively Spliced ◽

Differential Gene ◽

Exon 11

The insulin receptor (IR) occurs as two alternatively spliced isoforms, IR-A (exon 11−) and IR-B (exon 11+), which exhibit functional differences and are expressed in a tissue-specific manner. The IR substrate (IRS) proteins 1, 2 and 3 also differ in function and tissue distribution. Here we show the differential gene expression of IRs and IRSs in several rat target tissues of insulin action. IR-B is significantly higher than IR-A in epididymal white adipose tissue and adipogenesis induces a shift in the alternatively spliced species of IR from the A to the B isoform. Moreover, since aging in the rat is associated with the development of insulin resistance we looked for alterations of expression of these proteins in adipocytes from old rats. Our results reveal that there is a specific decrease in the expression of the IR-B isoform, as well as both mRNA and protein levels of IR, IRS-1 and IRS-3 being significantly decreased, in epididymal adipose tissue from old compared with adult rats. It is concluded that the down-regulation of early components of the insulin transduction pathway in a primary insulin target tissue could be related to the insulin resistance of aging.

Download Full-text

Preweaning Growth Hormone Treatment Ameliorates Adipose Tissue Insulin Resistance and Inflammation in Adult Male Offspring Following Maternal Undernutrition

Endocrinology ◽

10.1210/en.2013-1146 ◽

2013 ◽

Vol 154 (8) ◽

pp. 2676-2686 ◽

Cited By ~ 25

Author(s):

C. M. Reynolds ◽

M. Li ◽

C. Gray ◽

M. H. Vickers

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Adipose Tissue ◽

Insulin Receptor ◽

Glucose Uptake ◽

Saline Control ◽

Low Grade ◽

Adult Offspring ◽

Metabolic Dysfunction ◽

Gh Treatment

Abstract It is well established that early-life nutritional alterations lead to increased risk of obesity and metabolic disorders in adult life. Although it is clear that obesity gives rise to chronic low-grade inflammation, there is little evidence regarding the role of inflammation in the adipose tissue of undernourished (UN) offspring. GH reduces fat mass and has antiinflammatory properties. The present study examined the effect of maternal UN on adipose inflammation in adult offspring and whether GH treatment during a critical period of developmental plasticity could ameliorate metabolic dysfunction associated with a poor start to life. Sprague Dawley rats were assigned to chow (C) or UN (50% ad libitum; UN) diet throughout gestation. Male C and UN pups received saline (control saline [CS]/UN) or GH (2.5 μg/g/d; control growth hormone [CGH]/undernourished growth hormone [UNGH]) from days 3–21. Postweaning males were further randomized and fed either chow or high-fat diet until day 160. An ex vivo glucose uptake assay demonstrated adipose tissue from UN offspring displayed attenuated insulin-stimulated glucose uptake compared with CS, CGH, and UNGH. This was associated with increased insulin receptor, glucose transporter 4, and insulin receptor substrate 1 gene expression. Furthermore, UN demonstrated enhanced TNFα and IL-1β secretion from adipose explants and stromal vascular fraction cultures accompanied by increased adipose tissue gene expression of several key proinflammatory genes and markers of macrophage infiltration. Overall, UN offspring displayed a more potent immunophenotype, which correlated with decreased insulin sensitivity. Preweaning GH treatment negates these detrimental effects, indicating the potential for reversing metabolic dysfunction in UN adult offspring.

Download Full-text

High-fructose feeding suppresses cold-stimulated brown adipose tissue glucose uptake in young men independently of changes in thermogenesis and the gut microbiome

10.1101/2022.01.11.475847 ◽

2022 ◽

Author(s):

Gabriel Richard ◽

Denis P. Blondin ◽

Saad A. Syed ◽

Laura Rossi ◽

Michelle E. Fontes ◽

...

Keyword(s):

Insulin Resistance ◽

Adipose Tissue ◽

Glucose Uptake ◽

High Glucose ◽

Gut Microbiome ◽

Metabolic Diseases ◽

Short Chain Fatty Acids ◽

Alcoholic Fatty Liver ◽

High Fructose ◽

Brown Adipose

Diets rich in added sugars, especially high in fructose, are associated with metabolic diseases such as insulin resistance, and non-alcoholic fatty liver disease. Studies have shown a link between these pathologies and changes in the microbiome and its metabolites. Given the reported associations in animal models between the microbiome and brown or beige adipose tissue (BAT) function, and the alterations in the microbiome induced by high glucose or high fructose diets, we investigated the potential causal link between high glucose or fructose diets and BAT dysfunction in humans. We show that BAT glucose uptake, but not thermogenesis, is impaired by a high fructose but not high glucose diet, in the absence of changes in body mass, the gastrointestinal microbiome, and faecal short-chain fatty acids. We conclude that BAT metabolic dysfunction occurs independently from changes in gut microbiome composition, and earlier than other pathophysiological abnormalities associated with insulin resistance and dyslipidemia during fructose overconsumption in humans.

Download Full-text

Subcutaneous Adipose Tissue Metabolic Function and Insulin Sensitivity in People with Obesity

10.2337/figshare.14939088.v1 ◽

2021 ◽

Author(s):

Han-Chow E. Koh ◽

Stephan van Vliet ◽

Terri A. Pietka ◽

Gretchen A. Meyer ◽

Babak Razani ◽

...

Keyword(s):

Insulin Resistance ◽

Adipose Tissue ◽

Fatty Acid ◽

Glucose Uptake ◽

Subcutaneous Adipose Tissue ◽

Whole Body ◽

Plasma Fatty Acid ◽

Insulin Resistant ◽

High Insulin ◽

Subcutaneous Adipose

We used stable isotope-labeled glucose and palmitate tracer infusions, a hyperinsulinemic-euglycemic clamp, positron-emission tomography of muscles and adipose tissue after [<sup>18</sup>F]fluorodeoxyglucose and [<sup>15</sup>O]water injections, and subcutaneous adipose tissue (SAT) biopsy to test the hypotheses that: i) increased glucose uptake in SAT is responsible for high insulin-stimulated whole-body glucose uptake in people with obesity who are insulin-sensitive, and ii) putative SAT factors thought to cause insulin resistance are present in people with obesity who are insulin-resistant but not in those who are insulin-sensitive. We found high insulin-stimulated whole-body glucose uptake in insulin-sensitive participants with obesity was not due to channeling of glucose into SAT, but was due to high insulin-stimulated muscle glucose uptake. Furthermore, insulin-stimulated muscle glucose uptake was not different between insulin-sensitive obese and lean participants even though adipocytes were larger, SAT perfusion and oxygenation were lower, and markers of SAT inflammation, fatty acid appearance in plasma in relation to fat-free mass, and plasma fatty acid concentration were higher in the insulin-sensitive obese than lean participants. In addition, we observed only marginal or no differences in adipocyte size, SAT perfusion and oxygenation, and markers of SAT inflammation between insulin-resistant and insulin-sensitive obese participants. Plasma fatty acid concentration was also not different between insulin-sensitive and insulin-resistant obese participants even though SAT was resistant to the inhibitory effect of insulin on lipolysis in the insulin-resistant obese group. These data suggest several putative SAT factors that are commonly implicated in causing insulin resistance are normal consequences of SAT expansion unrelated to insulin resistance.

Download Full-text

Influence of TNF-α and IL-6 infusions on insulin sensitivity and expression of IL-18 in humans

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00471.2005 ◽

2006 ◽

Vol 291 (1) ◽

pp. E108-E114 ◽

Cited By ~ 76

Author(s):

Rikke Krogh-Madsen ◽

Peter Plomgaard ◽

Kirsten Møller ◽

Bettina Mittendorfer ◽

Bente K. Pedersen

Keyword(s):

Gene Expression ◽

Insulin Resistance ◽

Adipose Tissue ◽

Insulin Sensitivity ◽

Glucose Uptake ◽

Muscle Tissue ◽

Plasma Concentrations ◽

Whole Body ◽

Euglycemic Hyperinsulinemic Clamp ◽

Tnf Α

Inflammation is associated with insulin resistance, and both tumor necrosis factor (TNF)-α and interleukin (IL)-6 may affect glucose uptake. TNF induces insulin resistance, whereas the role of IL-6 is controversial. High plasma levels of IL-18 are associated with insulin resistance in epidemiological studies. We investigated the effects of TNF and IL-6 on IL-18 gene expression in skeletal muscle and adipose tissue. Nine human volunteers underwent three consecutive interventions, receiving an infusion of recombinant human (rh)IL-6, rhTNF, and saline. Insulin sensitivity was assessed by measurement of whole body glucose uptake with the stable isotope tracer method during a euglycemic hyperinsulinemic clamp (20 mU·min−1·kg−1), which was initiated 1 h after the IL-6-TNF-saline infusion. Cytokine responses were measured in plasma, muscle, and fat biopsies. Plasma concentrations of TNF and IL-6 increased 10- and 38-fold, respectively, during the cytokine infusions. Whole body insulin-mediated glucose uptake was significantly reduced during TNF infusion but remained unchanged during IL-6 infusion. TNF induced IL-18 gene expression in muscle tissue, but not in adipose tissue, whereas IL-6 infusion had no effect on IL-18 gene expression in either tissue. We conclude that TNF-induced insulin resistance of whole body glucose uptake is associated with increased IL-18 gene expression in muscle tissue, indicating that TNF and IL-18 interact, and both may have important regulatory roles in the pathogenesis of insulin resistance.

Download Full-text

Subcutaneous Adipose Tissue Metabolic Function and Insulin Sensitivity in People with Obesity

10.2337/figshare.14939088 ◽

2021 ◽

Author(s):

Han-Chow E. Koh ◽

Stephan van Vliet ◽

Terri A. Pietka ◽

Gretchen A. Meyer ◽

Babak Razani ◽

...

Keyword(s):

Insulin Resistance ◽

Adipose Tissue ◽

Fatty Acid ◽

Glucose Uptake ◽

Subcutaneous Adipose Tissue ◽

Whole Body ◽

Plasma Fatty Acid ◽

Insulin Resistant ◽

High Insulin ◽

Subcutaneous Adipose

Download Full-text

LMO3 reprograms visceral adipocyte metabolism during obesity

Journal of Molecular Medicine ◽

10.1007/s00109-021-02089-9 ◽

2021 ◽

Author(s):

Gabriel Wagner ◽

Anna Fenzl ◽

Josefine Lindroos-Christensen ◽

Elisa Einwallner ◽

Julia Husa ◽

...

Keyword(s):

Gene Expression ◽

Adipose Tissue ◽

Insulin Sensitivity ◽

Glucose Uptake ◽

Visceral Adipose Tissue ◽

Tissue Expansion ◽

Oxidative Capacity ◽

Glut4 Translocation ◽

Mitochondrial Oxidative Capacity ◽

Visceral Adipose

Abstract Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. We recently identified LIM domain only 3 (LMO3) in human mature visceral adipocytes; however, its function in these cells is currently unknown. The aim of this study was to determine the potential involvement of LMO3-dependent pathways in the modulation of key functions of mature adipocytes during obesity. Based on a recently engineered hybrid rAAV serotype Rec2 shown to efficiently transduce both brown adipose tissue (BAT) and white adipose tissue (WAT), we delivered YFP or Lmo3 to epididymal WAT (eWAT) of C57Bl6/J mice on a high-fat diet (HFD). The effects of eWAT transduction on metabolic parameters were evaluated 10 weeks later. To further define the role of LMO3 in insulin-stimulated glucose uptake, insulin signaling, adipocyte bioenergetics, as well as endocrine function, experiments were conducted in 3T3-L1 adipocytes and newly differentiated human primary mature adipocytes, engineered for transient gain or loss of LMO3 expression, respectively. AAV transduction of eWAT results in strong and stable Lmo3 expression specifically in the adipocyte fraction over a course of 10 weeks with HFD feeding. LMO3 expression in eWAT significantly improved insulin sensitivity and healthy visceral adipose tissue expansion in diet-induced obesity, paralleled by increased serum adiponectin. In vitro, LMO3 expression in 3T3-L1 adipocytes increased PPARγ transcriptional activity, insulin-stimulated GLUT4 translocation and glucose uptake, as well as mitochondrial oxidative capacity in addition to fatty acid oxidation. Mechanistically, LMO3 induced the PPARγ coregulator Ncoa1, which was required for LMO3 to enhance glucose uptake and mitochondrial oxidative gene expression. In human mature adipocytes, LMO3 overexpression promoted, while silencing of LMO3 suppressed mitochondrial oxidative capacity. LMO3 expression in visceral adipose tissue regulates multiple genes that preserve adipose tissue functionality during obesity, such as glucose metabolism, insulin sensitivity, mitochondrial function, and adiponectin secretion. Together with increased PPARγ activity and Ncoa1 expression, these gene expression changes promote insulin-induced GLUT4 translocation, glucose uptake in addition to increased mitochondrial oxidative capacity, limiting HFD-induced adipose dysfunction. These data add LMO3 as a novel regulator improving visceral adipose tissue function during obesity. Key messages LMO3 increases beneficial visceral adipose tissue expansion and insulin sensitivity in vivo. LMO3 increases glucose uptake and oxidative mitochondrial activity in adipocytes. LMO3 increases nuclear coactivator 1 (Ncoa1). LMO3-enhanced glucose uptake and mitochondrial gene expression requires Ncoa1.

Download Full-text