Hallmark Of Insulin Resistance Research Articles

FADD regulates adipose inflammation, adipogenesis, and adipocyte survival

Adipose tissue, aside from adipocytes, comprises various abundant immune cells. The accumulation of low-grade chronic inflammation in adipose tissue serves as a primary cause and hallmark of insulin resistance. In this study, we investigate the physiological roles of FADD in adipose tissue inflammation, adipogenesis, and adipocyte survival. High levels of Fadd mRNA were observed in mitochondrial-rich organs, particularly brown adipose tissue. To explore its metabolic functions, we generated global Fadd knockout mice, resulting in embryonic lethality, while heterozygous knockout (Fadd+/−) mice did not show any significant changes in body weight or composition. However, Fadd+/− mice exhibited reduced respiratory exchange ratio (RER) and serum cholesterol levels, along with heightened global and adipose inflammatory responses. Furthermore, AT masses and expression levels of adipogenic and lipogenic genes were decreased in Fadd+/− mice. In cellular studies, Fadd inhibition disrupted adipogenic differentiation and suppressed the expression of adipogenic and lipogenic genes in cultured adipocytes. Additionally, Fadd overexpression caused adipocyte death in vitro with decreased RIPK1 and RIPK3 expression, while Fadd inhibition downregulated RIPK3 in iWAT in vivo. These findings collectively underscore the indispensable role of FADD in adipose inflammation, adipogenesis, and adipocyte survival.

THU312 The Type 1 Diabetes Susceptible LEW.1WR1 Rat Develops Insulin Resistance And Severe Non-alcoholic Fatty Liver Disease

Abstract Disclosure: S.T. Love-Rutledge: None. M. Wimalarathne: None. L.D. Mercado: None. A. Smiley: None. C. Apperson: None. Q.C. Vidal: None. A subpopulation of type 1 diabetes patients develop the pathophysiological parameters to be termed double diabetes. This patient population often develops obesity, non-alcoholic fatty liver disease, and other hallmarks of insulin resistance. The LEW.1WR1 rat is a type 1 diabetes susceptible rodent with hyperinsulinemia that has shown signs of fatty liver infiltration1,2. We hypothesized that LEW.1WR1 rats would develop insulin resistance and severe nonalcoholic fatty liver disease due to its increased circulating insulin and predisposition to autoimmune disease. LEW.1WR1 rats and Wistar Furth rats were used for the 18-week long experiment. Peripheral insulin sensitivity was evaluated using insulin tolerance tests at weeks 11 and 17 showing worsening insulin sensitivity as evidenced by the gradual slope compared to the steep slope of the Wistar Furth rats. HOMA-IR values support LEW.1WR1 rats develop worsening insulin sensitivity and are insulin resistant at week 23 of the experiment (2.613± 0.796, p < 0.0001). Terminal liver mass confirmed increased liver mass normalized to body mass and liver triglyceride levels suggested increased ectopic lid accumulation in the liver (Liver Mass 2.65% ±0.32 p = 0.0005 and triglyceride levels 192.8 ± 21.85 mg/dL, p = 0.034). The H&E-stained digital liver slides were scored for liver ballooning, steatosis, and markers of inflammation. Fibrosis and terminal circulating cytokines were also measured to assess severity of condition. While both groups showed increased steatosis and injury ballooning, trichrome blue staining and inflammatory cytokines verify the severity of disease in the LEW.1WR1 rats. LEW rats have increased fibrosis area and high serum TNF-α, IFN-, MCP-1 levels. As hypothesized, we observed that LEW.1WR1 rats show characteristics of insulin resistance and malignant nonalcoholic fatty liver disease development.

Impact of physical activity on brain oxidative metabolism and intrinsic capacities in young swiss mice fed a high fat diet

Type 2 diabetes and obesity characterized by hallmarks of insulin resistance along with an imbalance in brain oxidative metabolism would impair intrinsic capacities (ICs), a new concept for assessing mental and physical functioning. Here, we explored the impact of physical activity on antioxidant responses and oxidative metabolism in discrete brain areas of HFD or standard diet (STD) fed mice but also its consequences on specific domains of ICs. 6-week-old Swiss male mice were exposed to a STD or a HFD for 16 weeks and half of the mice in each group had access to an activity wheel and the other half did not. As expected HFD mice displayed peripheral insulin resistance but also a persistent inhibition of aconitase activity in cortices revealing an increase in mitochondrial reactive oxygen species (ROS) production. Animals with access to the running wheel displayed an improvement of insulin sensitivity regardless of the diet factor whereas ROS production remained impaired. Moreover, although the access of the running wheel did not influence mitochondrial biomass, in the oxidative metabolism area, it produced a slight decrease in brain SOD1 and catalase expression notably in HFD fed mice. At the behavioural level, physical exercise produced anxiolytic/antidepressant-like responses and improved motor coordination in both STD and HFD fed mice. However, this non-pharmacological intervention failed to enhance cognitive performance. These findings paint a contrasting landscape about physical exercise as a non-pharmacological intervention for positively orienting the aging trajectory.

Plasma concentrations of apolipoproteins C3 and C2 do not explain differences in insulin-stimulated endothelial-bound lipoprotein lipase activity in insulin resistant humans

Elevated circulating triglycerides (i.e., dyslipidemia) is a hallmark of insulin resistance and is generally caused by decrease in lipoprotein lipase (LPL) activity. LPL hydrolyzes triglycerides (TG) into free fatty acids in plasma for use and/or storage in tissues (i.e., adipose, skeletal muscle). Plasma apolipoproteins (Apos) C3 and C2 interact with LPL to modulate its function by activating or inhibiting LPL. Previous research shows, in the metabolic environment of insulin resistance, the concentration of ApoC3 is increased in plasma while that of ApoC2 is decreased. Thus, Apos are considered key factors in regulating plasma lipid metabolism via modulation of LPL function. The purpose of this study was to investigate the concentrations of ApoC3 and ApoC2 in plasma and along endothelial-bound LPL activity in insulin resistant and healthy/non-insulin resistant individuals. We evaluated insulin resistance (IR)/insulin sensitivity (IS) using an oral glucose tolerance test (i.e., Matsuda index calculation) in subjects between ages 19 and 45 years old. Subjects were placed in the following two groups: 1) IR, if Matsuda index <4.0 (N=6; 4 males, 2 females; BMI: 23-45 kg/m2), or 2) IS, if Matsuda index >7.0 (N=11, 9 males, 2 females; BMI: 18-26 kg/m2). Subjects received an intravenous insulin infusion (0.5 mU/kg/min) for 30 minutes to resemble increased postprandial plasma insulin response. To release whole-body endothelial-bound LPL, subjects received an injection of intravenous heparin (75 UI/kg). Plasma samples were collected 10 minutes after the heparin infusion and analyzed for LPL concentration and activity, and ApoC3 and ApoC2 concentrations using ELISA. Differences between IS and IR subjects were determined using a t-test, and the P value was set at P < 0.05. Plasma LPL concentrations were not different between groups (IR = 457 ± 17 ng/ml, IS = 453 ± 27 ng/ml, P > 0.05), but plasma LPL activity was higher in the IR subjects (IR = 665 ± 113 nmol/min/ml, IS = 365 ± 59 nmol/min/ml, P = 0.02). IR subjects had higher concentrations of plasma ApoC3 (IR = 3.6 ± 0.5 mg/dl, IS = 2.7 ± 0.2 mg/dl, P=0.03) but ApoC2 was not different between groups (IR = 0.15 ± 0.03 mg/dl, IS = 0.11 ± 0.01 mg/dl, P = 0.11). The ratio of circulating LPL to circulating ApoC3 (IR = 145 ± 22, IS = 176 ± 15) or circulating ApocC2 (IR = 4104 ± 844, IS = 4391 ± 483) were not different between groups (P > 0.05). We conclude that interactions of LPL with APOC3 and ApoC2 may not be main determinants regulating differential whole-body plasma LPL activity under plasma insulin-stimulated conditions in insulin resistant humans. American Diabetes Association Grant #7-12-CT-40 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dietary patterns and fertility status in men: Mediterranean diet does make a difference in ameliorating the rise in male infertility problems due to changing lifestyle

Dietary patterns and fertility status in men: Mediterranean diet does make a difference in ameliorating the rise in male infertility problems due to changing lifestyle

Several Metabolite Families Display Inflexibility during Glucose Challenge in Patients with Type 2 Diabetes: An Untargeted Metabolomics Study.

Metabolic inflexibility is a hallmark of insulin resistance and can be extensively explored with high-throughput metabolomics techniques. However, the dynamic regulation of the metabolome during an oral glucose tolerance test (OGTT) in subjects with type 2 diabetes (T2D) is largely unknown. We aimed to identify alterations in metabolite responses to OGTT in subjects with T2D using untargeted metabolomics of both plasma and subcutaneous adipose tissue (SAT) samples. Twenty subjects with T2D and twenty healthy controls matched for sex, age, and body mass index (BMI) were profiled with untargeted metabolomics both in plasma (755 metabolites) and in the SAT (588) during an OGTT. We assessed metabolite concentration changes 90 min after the glucose load, and those responses were compared between patients with T2D and controls. Post-hoc analyses were performed to explore the associations between glucose-induced metabolite responses and markers of obesity and glucose metabolism, sex, and age. During the OGTT, T2D subjects had an impaired reduction in plasma levels of several metabolite families, including acylcarnitines, amino acids, acyl ethanolamines, and fatty acid derivates (p < 0.05), compared to controls. Additionally, patients with T2D had a greater increase in plasma glucose and fructose levels during the OGTT compared to controls (p < 0.05). The plasma concentration change of most metabolites after the glucose load was mainly associated with indices of hyperglycemia rather than insulin resistance, insulin secretion, or BMI. In multiple linear regression analyses, hyperglycemia indices (glucose area under the curve (AUC) during OGTT and glycosylated hemoglobin (HbA1c)) were the strongest predictors of plasma metabolite changes during the OGTT. No differences were found in the adipose tissue metabolome in response to the glucose challenge between T2D and controls. Using a metabolomics approach, we show that T2D patients display attenuated responses in several circulating metabolite families during an OGTT. Besides the well-known increase in monosaccharides, the glucose-induced lowering of amino acids, acylcarnitines, and fatty acid derivatives was attenuated in T2D subjects compared to controls. These data support the hypothesis of inflexibility in several metabolic pathways, which may contribute to dysregulated substrate partitioning and turnover in T2D. These findings are not directly associated with changes in adipose tissue metabolism; therefore, other tissues, such as muscle and liver, are probably of greater importance.

Changes in the Expression of Insulin Pathway, Neutrophil Elastase and Alpha 1 Antitrypsin Genes from Leukocytes of Young Individuals with Insulin Resistance.

BackgroundChronic hyperinsulinemia is a hallmark of insulin resistance that affects a diversity of cells, including leukocytes modifying the expression of some genes involved in insulin signaling.PurposeThe aim of this study was to evaluate how hyperinsulinemia affects the expression of genes involved in the proximal insulin signaling pathway in leukocytes from 45 young individuals grouped: normal weight with not insulin resistance (NIR), with insulin resistance (IR) and with obesity (OB-IR).MethodsqPCR was performed to analyze the expression of insulin receptor (INSR), insulin receptor substrate 1 and 2 (IRS-1 and IRS-2), neutrophil elastase (NE), alpha 1 antitrypsin (A1AT), glucose transporters 1, 3 and 4 (GLUT-1, GLUT-3 and GLUT-4) by the 2−ΔCt method, and the correlation between the genes was determined by Spearman’s test.ResultsThe mRNA expression analysis of all genes between NIR and IR individuals revealed no differences. However, when comparing NIR and IR individuals with OB-IR, an increase in NE and A1AT expression and a clear trend towards a decrease in IRS-2 expression was observed, whereas the comparison of IR and OB-IR showed a decrease in GLUT-3 expression. Overall, the correlation analysis showed that in the IR group there was a positive correlation only between NE with IRS-1 (r = 0.72, p = 0.003), while in the OB-IR group, there was a positive correlation between the NE and A1AT with INSR (r = 0.62, p = 0.01 and r = 0.74, p = 0.002, respectively) and with IRS-2 (r = 0.74, p = 0.002 and r = 0.76, p = 0.001, respectively).ConclusionThese results suggest that hyperinsulinemia and obesity are associated with changes in the expression of genes in leukocytes involved in the insulin pathway that are related to NE and A1AT.

Endothelial Cell CD36 Reduces Atherosclerosis and Controls Systemic Metabolism.

High-fat Western diets contribute to tissue dysregulation of fatty acid and glucose intake, resulting in obesity and insulin resistance and their sequelae, including atherosclerosis. New therapies are desperately needed to interrupt this epidemic. The significant idea driving this research is that the understudied regulation of fatty acid entry into tissues at the endothelial cell (EC) interface can provide novel therapeutic targets that will greatly modify health outcomes and advance health-related knowledge. Dysfunctional endothelium, defined as activated, pro-inflammatory, and pro-thrombotic, is critical in atherosclerosis initiation, in modulating thrombotic events that could result in myocardial infarction and stroke, and is a hallmark of insulin resistance. Dyslipidemia from high-fat diets overwhelmingly contributes to the development of dysfunctional endothelium. CD36 acts as a receptor for pathological ligands generated by high-fat diets and in fatty acid uptake, and therefore, it may additionally contribute to EC dysfunction. We created EC CD36 knockout (CD36°) mice using cre-lox technology and a cre-promoter that does not eliminate CD36 in hematopoietic cells (Tie2e cre). These mice were studied on different diets, and crossed to the low density lipoprotein receptor (LDLR) knockout for atherosclerosis assessment. Our data show that EC CD36° and EC CD36°/LDLR° mice have metabolic changes suggestive of an uncompensated role for EC CD36 in fatty acid uptake. The mice lacking expression of EC CD36 had increased glucose clearance compared with controls when fed with multiple diets. EC CD36° male mice showed increased carbohydrate utilization and decreased energy expenditure by indirect calorimetry. Female EC CD36°/LDLR° mice have reduced atherosclerosis. Taken together, these data support a significant role for EC CD36 in systemic metabolism and reveal sex-specific impact on atherosclerosis and energy substrate use.

Momordica charantia nanoparticles promote mitochondria biogenesis in the pancreas of diabetic-induced rats: gene expression study

BackgroundMitochondria dysfunction is one of the clinical features of diabetes mellitus (DM), which is a hallmark of insulin resistance (IR). This study investigates the therapeutic effect of Momordica charantia nanoparticles on mitochondria biogenesis in diabetic-induced rats. Forty-two adult wistar rats (average weight of 189 ± 10.32) were grouped as follows: STZ (65 mg/kg), control group, STZ + silver nitrate (10 mg/kg), STZ + M. charantia silver nanoparticles (50 mg/kg), STZ + metformin (100 mg/kg), and STZ + M. charantia aqueous extract (100 mg/kg). DM was induced intraperitoneal using freshly prepared solution of STZ (65 mg/kg), and rats with fasting blood sugar (FBS) above 250 mg/dl after 72 h of induction were considered diabetic. Treatment started after the third day of induction and lasted for 11 days. Effect of M. charantia nanoparticles on glucose level and pancreatic expression of genes involved in mitochondria biogenesis (PGC-1α, AMPK, GSK-3β, PPARϒ), inflammation (IL-1B, TNFα) and glucose sensitivity (PI3K, AKT, PTEN Insulin and Glut2) were quantified using reverse-transcriptase polymerase chain reaction (RT-PCR).ResultsThe results showed that M. charantia nanoparticles promote mitochondria biogenesis, glucose sensitivity and reverse inflammation in the pancreas of diabetes rat model through upregulation of PGC-1α, AMPK, PPARϒ, AKT, Insulin and Glut2 mRNA expression and downregulation of GSK-3β, PI3K, IL-1B and TNFα mRNA expression in the pancreas of diabetic rats.ConclusionThis study thus concludes that M. charantia nanoparticles may provide effective therapeutics against mitochondria dysfunction in the pancreas of diabetic model.

Insulin Resistance and Cancer: In Search for a Causal Link.

Insulin resistance (IR) is a condition which refers to individuals whose cells and tissues become insensitive to the peptide hormone, insulin. Over the recent years, a wealth of data has made it clear that a synergistic relationship exists between IR, type 2 diabetes mellitus, and cancer. Although the underlying mechanism(s) for this association remain unclear, it is well established that hyperinsulinemia, a hallmark of IR, may play a role in tumorigenesis. On the other hand, IR is strongly associated with visceral adiposity dysfunction and systemic inflammation, two conditions which favor the establishment of a pro-tumorigenic environment. Similarly, epigenetic modifications, such as DNA methylation, histone modifications, and non-coding RNA, in IR states, have been often associated with tumorigenesis in numerous types of human cancer. In addition to these observations, it is also broadly accepted that gut microbiota may play an intriguing role in the development of IR-related diseases, including type 2 diabetes and cancer, whereas potential chemopreventive properties have been attributed to some of the most commonly used antidiabetic medications. Herein we provide a concise overview of the most recent literature in this field and discuss how different but interrelated molecular pathways may impact on tumor development.

Organ Crosstalk and the Modulation of Insulin Signaling.

A highly complex network of organ communication plays a key role in regulating metabolic homeostasis, specifically due to the modulation of the insulin signaling machinery. As a paradigm, the role of adipose tissue in organ crosstalk has been extensively investigated, but tissues such as muscles and the liver are equally important players in this scenario. Perturbation of organ crosstalk is a hallmark of insulin resistance, emphasizing the importance of crosstalk molecules in the modulation of insulin signaling, potentially leading to defects in insulin action. Classically secreted proteins are major crosstalk molecules and are able to affect insulin signaling in both directions. In this review, we aim to focus on some crosstalk mediators with an impact on the early steps of insulin signaling. In addition, we also summarize the current knowledge on the role of extracellular vesicles in relation to insulin signaling, a more recently discovered additional component of organ crosstalk. Finally, an attempt will be made to identify inter-connections between these two pathways of organ crosstalk and the potential impact on the insulin signaling network.

The IgG Antibody Paradox in Insulin Resistance: Pathogenic and Therapeutic

Chronic low-grade inflammation and mitochondrial dysfunction are hallmarks of insulin resistance. However, the mechanisms by which the immune system can propagate systemic insulin resistance remains poorly understood. IgG antibodies are a critical component of immunity and display paradoxical properties. IgG can propagate inflammation by crosslinking Fc receptors activating innate immune cells, and conversely, when given intravenously at high doses (1–2 g/kg intravenous immunoglobulin), actively suppress inflammation. Here, we demonstrate that IgG can exert similar paradoxical properties on glucose metabolism. IgG can elicit insulin resistance, and conversely, when given at high doses, promote insulin sensitivity in a diabetic mouse model. IgG, through its Fc-mediated interactions, suppresses insulin-induced mitochondrial function as well as insulin signaling. Modulation of insulin-dependent mitochondrial respiration by serum or purified IgG highly correlates (R2 = 0.70) with the quantitative measurement of insulin sensitivity accessed by the modified insulin suppression test. Our studies indicate that IgG antibody glycosylation is critically important to these conflicting actions. In mice and humans, the progression of insulin resistance is associated with reduced IgG Fc region sialylation, and administration of asialylated IgG is sufficient to cause insulin resistance in IgG null mice. On the other hand, a single administration of high-dose IgG significantly improved insulin and glucose tolerance as well as plasma glucose levels lasting over 72 days post-administration. These results demonstrate new insights into the systemic nature of insulin resistance, a novel mechanism of the disease, and an innovative therapeutic strategy for treating type 2 diabetes.

A Prospective Study of Ultrasonographic Assessment of Non-Alcoholic Fatty Liver Disease (Steatosis) Among Type 2 Diabetes Mellitus With Liver Function Test Correlation

Non-alcoholic fatty liver disease (NAFLD) emerging condition of liver disease. Prevalence of this disease is estimated to be around 9-32% among Indian population with increased incidence rate among obese and diabetes. Insulin resistance is strongly associated with NAFLD. The spectrum varies from simple steatosis (NAFL) to non-alcoholic steatohepatitis (NASH), finally to cirrhosis. It has been regarded as a manifestation of the metabolic syndrome. Ultrasonography (USG) is the simplest and cost-effective imaging technique for the identification of NAFLD. The Aminotransferase levels were also significantly elevated among T2DM. The results from the study reinforced the well established clinical association of NAFLD with higher Aminotransferase levels among T2DM. NAFLD may be considered as the hepatic hallmark of insulin resistance, and its correlation with liver enzymes, an excellent marker to predict disease outcome. This study is conducted to establish the prevalence of(NAFLD) among type 2 Diabetes mellitus (T2DM) along with Liver function test (Aminotransferase levels) correlation. The results from the study reinforced the well established clinical association of NAFLD with other co-morbidities like dyslipidemia, obesity, metabolic syndrome as the prevalence of NAFLD in these co-morbidities condition had a higher level rise in aminotransferases.The prevalence of NAFLD among T2DM in our study is higher when compared with other studies. NAFLD may be considered as a hepatic hallmark of insulin resistance and correlating it with alteration in liver enzymes will be an excellent marker to predict disease outcome.

296-OR: Identification and Characterization of New Players in Insulin-Controlled GLUT4 Trafficking

The anabolic hormone insulin promotes glucose uptake by triggering the translocation of the glucose transporter GLUT4 from intracellular storage vesicles to the cell surface through exocytosis. Upon the termination of insulin signaling, GLUT4 gradually returns to intracellular storage compartments through endocytosis. Defects in GLUT4 translocation are a hallmark of insulin resistance (IR) and type 2 diabetes (T2D). Our team has performed genome-scale genetic screens to systematically dissect GLUT4 exocytosis and endocytosis. The screens recovered known GLUT4 trafficking regulators but most of the hits were not previously linked to the GLUT4 pathway. A regulator of GLUT4 endocytosis identified in the screens is AAGAB, a soluble factor of 34 kDa. AAGAB controls the stepwise assembly of AP2 adaptor, a central cargo adaptor in clathrin-mediated endocytosis. Unexpectedly, we found that AAGAB also binds to Munc18c and directly inhibits SNARE-Munc18-mediated GLUT4 vesicle fusion. Thus, AAGAB plays a dual role in GLUT4 trafficking. I will also discuss OSBPL8 and OSBPL10, two negative regulators of GLUT4 translocation identified in our screens. OSBPL8 and OSBPL10 are putative lipid transfer proteins operating between intracellular organelles. We propose that OSBPL8 and OSBPL10 regulate lipid dynamics required for insulin signaling, thereby influencing the activities of GLUT4 exocytic regulators. These genetic studies will likely facilitate the identification of new therapeutic targets for IR and T2D. Disclosure J. Shen: None. Funding American Diabetes Association (1-19-IBS-305); National Institutes of Health (DK095367, GM126960)

191-OR: New Regulators of Insulin-Stimulated GLUT4 Exocytosis

Insulin promotes glucose uptake by triggering the translocation of the glucose transporter GLUT4 from intracellular storage vesicles to the cell surface through exocytosis. Defects in GLUT4 exocytosis are a hallmark of insulin resistance (IR) and type 2 diabetes (T2D). To develop effective and safe treatments for IR and T2D, it is crucial to gain a comprehensive understanding of insulin-stimulated GLUT4 exocytosis at the molecular level. Recently, our group developed new genetic screen platforms to systematically dissect insulin-stimulated GLUT4 exocytosis, taking advantage of the revolutionary CRISPR-Cas9 genome editing system. Our genome-wide CRISPR screens recovered known GLUT4 exocytic regulators but most of the hits were not previously linked to the GLUT4 exocytic pathway. Here, I will focus on AAGAB, a negative regulator of GLUT4 translocation identified in the screens. We discovered that AAGAB plays a dual role in GLUT4 trafficking: 1) it promotes the endocytosis of GLUT4, and 2) it negatively regulates the SNARE-Munc18-mediated GLUT4 vesicle fusion. In addition, I will discuss OSBPL8 and OSBPL10, another two negative regulators identified in our screens. OSBPL8 and OSBPL10 are putative lipid transfer proteins operating between intracellular organelles. We propose that OSBPL8 and OSBPL10 regulate lipid dynamics required for insulin signaling, thereby influencing the activities of GLUT4 exocytic regulators. These genetic studies will facilitate the identification of new therapeutic targets for IR and T2D. Disclosure J. Shen: None. Funding National Institutes of Health

Cis-9, Trans-11 Conjugated Linoleic Acid Reduces Phosphoenolpyruvate Carboxykinase Expression and Hepatic Glucose Production in HepG2 Cells.

Dysregulated hepatic gluconeogenesis is a hallmark of insulin resistance and type 2 diabetes mellitus (T2DM). Although existing drugs have been proven to improve gluconeogenesis, achieving this objective with functional food is of interest, especially using conjugated linoleic acid (CLA) found in dairy products. Both cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) isomers of CLA were tested in human (HepG2) and rat (H4IIE) hepatocytes for their potential effects on gluconeogenesis. The hepatocytes exposed for 24 h with 20 μM of c9,t11-CLA had attenuated the gluconeogenesis in both HepG2 and H4IIE by 62.5% and 80.1%, respectively. In contrast, t10,c12-CLA had no effect. Of note, in HepG2 cells, the exposure of c9,t11-CLA decreased the transcription of gluconeogenic enzymes, cytosolic phosphoenolpyruvate carboxykinase (PCK1) by 87.7%, and glucose-6-phosphatase catalytic subunit (G6PC) by 38.0%, while t10,c12-CLA increased the expression of G6PC, suggesting the isomer-specific effects of CLA on hepatic glucose production. In HepG2, the peroxisome proliferator-activated receptor (PPAR) agonist, rosiglitazone, reduced the glucose production by 72.9%. However, co-administration of c9,t11-CLA and rosiglitazone neither exacerbated nor attenuated the efficacy of rosiglitazone to inhibit glucose production; meanwhile, t10,c12-CLA abrogated the efficacy of rosiglitazone. Paradoxically, PPARγ antagonist GW 9662 also led to 70.2% reduction of glucose production and near undetectable PCK1 expression by abrogating CLA actions. Together, while the precise mechanisms by which CLA isomers modulate hepatic gluconeogenesis directly or via PPAR warrant further investigation, our findings establish that c9,t11-CLA suppresses gluconeogenesis by decreasing PEPCK on hepatocytes.

Hepatic Insulin Clearance in Regulation of Systemic Insulin Concentrations-Role of Carbohydrate and Energy Availability.

Hyperinsulinemia is the hallmark of insulin resistance in obesity, and the relative importance of insulin clearance, insulin resistance, and insulin hypersecretion has been widely debated. On the basis of recent experimental evidence, we summarize existing evidence to suggest hepatic insulin clearance as a major and immediate regulator of systemic insulin concentrations responding within days to altered dietary energy and, in particular, carbohydrate intake. Hepatic insulin clearance seems to be closely associated with opposite alterations in hepatic lipid content and glucose production, providing a potential mechanistic link to hepatic insulin sensitivity. The molecular regulation of insulin clearance in the liver is likely to involve changes in insulin binding and receptor internalization in response to the dietary alterations, the molecular mechanisms of which await further research.

Nox2 contributes to hyperinsulinemia-induced redox imbalance and impaired vascular function

Insulin resistance promotes vascular endothelial dysfunction and subsequent development of cardiovascular disease. Previously we found that skeletal muscle arteriolar flow-induced dilation (FID) was reduced following a hyperinsulinemic clamp in healthy adults. Therefore, we hypothesized that hyperinsulinemia, a hallmark of insulin resistance, contributes to microvascular endothelial cell dysfunction via inducing oxidative stress that is mediated by NADPH oxidase (Nox) system. We examined the effect of insulin, at levels that are comparable with human hyperinsulinemia on 1) FID of isolated arterioles from human skeletal muscle tissue in the presence and absence of Nox inhibitors and 2) human adipose microvascular endothelial cell (HAMECs) expression of nitric oxide (NO), endothelial NO synthase (eNOS), and Nox-mediated oxidative stress. In six lean healthy participants (mean age 25.5±1.6 y, BMI 21.8±0.9), reactive oxygen species (ROS) were increased while NO and arteriolar FID were reduced following 60min of ex vivo insulin incubation. These changes were reversed after co-incubation with the Nox isoform 2 (Nox2) inhibitor, VAS2870. In HAMECs, insulin-induced time-dependent increases in Nox2 expression and P47phox phosphorylation were echoed by elevations of superoxide production. In contrast, phosphorylation of eNOS and expression of superoxide dismutase (SOD2 and SOD3) isoforms showed a biphasic response with an increased expression at earlier time points followed by a steep reduction phase. Insulin induced eNOS uncoupling that was synchronized with a drop of NO and a surge of ROS production. These effects were reversed by Tempol (SOD mimetic), Tetrahydrobiopterin (BH4; eNOS cofactor), and VAS2870. Finally, insulin induced nitrotyrosine formation which was reversed by inhibiting NO or superoxide generation. In conclusions, hyperinsulinemia may reduce FID via inducing Nox2-mediated superoxide production in microvascular endothelial cells which reduce the availability of NO and enhances peroxynitrite formation. Therefore, the Nox2 pathway should be considered as a target for the prevention of oxidative stress-associated endothelial dysfunction during hyperinsulinemia.

ADP-ribosylation factor-like GTPase 15 enhances insulin-induced AKT phosphorylation in the IR/IRS1/AKT pathway by interacting with ASAP2 and regulating PDPK1 activity

Decreased phosphorylation in the insulin signalling pathway is a hallmark of insulin resistance. The causes of this phenomenon are complicated and multifactorial. Recently, genomic analyses have identified ARL15 as a new candidate gene related to diabetes. However, the ARL15 protein function remains unclear. Here, we show that ARL15 is upregulated by insulin stimulation. This effect was impaired in insulin-resistant pathophysiology in TNF-α-treated C2C12 myotubes and in the skeletal muscles of leptin knockout mice. In addition, ARL15 localized to the cytoplasm in the resting state and accumulated in the Golgi apparatus around the nucleus upon insulin stimulation. ARL15 overexpression can enhance the phosphorylation of the key insulin signalling pathway molecules IR, IRS1 and AKT in C2C12 myotubes. Moreover, ARL15 knockdown can also specifically inhibit the phosphorylation of PDPK1 Ser241, thereby reducing PDPK1 activity and its downstream phosphorylation of AKT Thr308. Co-immunoprecipitation assays identified ASAP2 as an ARL15-interacting protein. In conclusion, we have identified that ARL15 acts as an insulin-sensitizing effector molecule to upregulate the phosphorylation of members of the canonical IR/IRS1/PDPK1/AKT insulin pathway by interacting with its GAP ASAP2 and activating PDPK1. This research may provide new insights into GTPase-mediated insulin signalling regulation and facilitate the development of new pharmacotherapeutic targets for insulin sensitization.

Integrin‐Linked Kinase Enhances Skeletal Muscle Fatty Acid Metabolism In Vivo

Accumulation of extracellular matrix (ECM) components and activation of specific integrin receptors contribute to skeletal muscle (SkM) insulin resistance in the high fat (HF)‐fed mouse. Integrin‐linked kinase (ILK) is an adaptor protein that serves as a hub for the integrin signaling but its role in metabolism is poorly defined. A shift from glucose to fatty acid (FA) oxidation is a hallmark of insulin resistance and diabetes, but the mechanisms responsible for this shift in nutrient utilization are unclear. Integrins have been implicated in the transcriptional regulation of mitochondrial biogenesis and are known to interact with FA transporters at the plasma membrane. The objective of this study was to test the novel hypothesis that integrins, through ILK signaling, enhance SkM FA metabolism under insulin resistant conditions.SkM‐specific ILK knockout mice (mILK‐KO) were generated using mice expressing Cre recombinase on the human SkM actin (HSA) promoter. Body weight was measured weekly and body composition was measured immediately prior to and following energy balance studies. mILK‐KO and their wild type (WT) littermates were fed a chow or HF diet (60% calories from fat) for 27 weeks then placed in metabolic cages for five days (n=6–9/group). Tissues were collected after a 5‐h fast. Pyruvate and FA‐supported mitochondrial respiratory capacity was measured in permeabilized SkM myofibers (PmFBs) from the soleus and EDL. Muscle free fatty acids (FFAs) and triglycerides (TGs) were measured in gastrocnemius with gas chromatography. Comparisons were made using Student's t‐test or 2‐way ANOVA as appropriate.HF‐fed mILK‐KO mice had a decreased reliance on FA oxidation compared to their WT littermates, as suggested by the increase in RQ. This RQ difference was independent of changes in whole body energy expenditure, body composition, food consumption or spontaneous activity. RQ was unchanged in mILK‐KO mice fed a chow diet. FA‐supported PmFB respiration was decreased in EDL, but not soleus, of HF‐fed mILK‐KO mice. Respiration in chow‐fed mice was not affected by muscle ILK deletion. Total muscle FFAs and TGs were increased by HF feeding but lower in mILK‐KO mice fed a HF diet. Muscle lipids were unaffected by ILK deletion in mice on a chow diet.These data are the first to demonstrate a mechanistic role for integrin signaling in the regulation of SkM FA metabolism in vivo and suggest that integrin signaling, through ILK, responds to nutrient overload by enhancing the uptake, storage and oxidation of FAs in SkM. These findings provide strong support for a model where extracellular cues (e.g. ECM expansion) cause a coordinated shift in cellular energetics, a paradigm that may permit for therapeutic targeting of oxidative metabolism through integrin receptors.Support or Funding InformationFunding for this project was provided by the following NIH grants: R01 DK054902, U24 DK059637, P30 DK020593, and T32 DK007061.