Selective Insulin Resistance Research Articles

The exoWAT, endoWAT and ectoWAT Dictate Dysmetabolism, Dyslipidemia and Diabetes

Background & Objective: Three-tier model depicting physiology of metabolism and a timeline of dyslipidemia and diabetes is proposed. Methods & Results: OGTT glucose profiles of 1456 individuals, aged 15 to 80 years, who were not previously diagnosed as having T2D are analysed. A clinical model is conceptualized to explain the integrated physiology of pancreatic islet, hypothalamus, gut and peripheral tissues regulating the carbohydrate, fat, protein and energy metabolism. The beta cells regulate anabolism via insulin, somatostatin (delta) and amylin (gamma). The ‘Delta-Gamma system’ controls catabolism via glucagon and pancreatic polypeptide. Leptin regulates satiety and lipogenesis while ghrelin and fibroblast growth factor-21 regulate hunger and lipolysis by modulating insulin secretion. Dietary fat is deposited in the white adipose tissue (WAT) as exoWAT. Leptin induces ‘selective insulin resistance’ by simultaneously stimulating beta cells and microsomal triglyceride transfer protein (MTTP). Leptin regulates de-novo lipogenesis, endogenous fat deposition as endoWAT and promotes dyslipidemia and euglycemic hyperinsulinemia. WAT storage capacity (cWAT) is genetically determined. When cWAT is exceeded, fat deposition occurs ectopically in non-adipose tissues as ectoWAT and stimulates growth hormone, ghrelin and hyperglycemia. EctoWAT suppresses insulin, stimulates glucagon and ghrelin, leading to lipolysis, hyperglycemia, beta cell suppression-cum-failure. Calorie surplus, superimposed on genetic insulin resistance drives the timeline of dysmetabolism, which marches from healthy (exoWAT) and unhealthy (endoWAT) WAT stage, hepatic followed by peripheral ectoWAT stage, and finally the lipolytic stage. Conclusions: This model can explain the pathogenesis of T2D, its phenotypes and complications, GDM, PCOS as well as the mechanism of action of most antidiabetic drugs.

Emerging Roles of Ceramide in Cardiovascular Diseases.

Ceramide is a core molecule of sphingolipid metabolism that causes selective insulin resistance and dyslipidemia. Research on its involvement in cardiovascular diseases has grown rapidly. In resting cells, ceramide levels are extremely low, while they rapidly accumulate upon encountering external stimuli. Recently, the regulation of ceramide levels under pathological conditions, including myocardial infarction, hypertension, and atherosclerosis, has drawn great attention. Increased ceramide levels are strongly associated with adverse cardiovascular risks and events while inhibiting the synthesis of ceramide or accelerating its degradation improves a variety of cardiovascular diseases. In this article, we summarize the role of ceramide in cardiovascular disease, investigate the possible application of ceramide as a new diagnostic biomarker and a therapeutic target for cardiovascular disorders, and highlight the remaining problems.

Oral Subacute Exposure to Cadmium LOAEL Dose Induces Insulin Resistance and Impairment of the Hormonal and Metabolic Liver-Adipose Axis in Wistar Rats.

Cadmium is a nonessential transition metal considered one of the more hazardous environmental contaminants. The population is chronically exposed to this metal at low concentrations, designated as the LOAEL (lowest observable adverse effect level) dose. We aimed to investigate whether oral subacute exposure to cadmium LOAEL disrupts hormonal and metabolic effects of the liver-adipose axis in Wistar rats. Fifty male Wistar rats were separated into two groups: control (standard normocalorie diet + water free of cadmium) and cadmium (standard normocalorie diet + drinking water with 32.5 ppm CdCl2). After 1 month, zoometry, a serum lipid panel, adipokines, and proinflammatory cytokines were evaluated. Tests of glucose and insulin tolerance (ITT) and insulin resistance were performed. Histological studies on structure, triglyceride distribution, and protein expression of the insulin pathway were performed in the liver and retroperitoneal adipose tissue. In both tissues, the cadmium, triglyceride, glycogen, and proinflammatory cytokine contents were also quantified. The cadmium group developed dyslipidemia, glucose intolerance, hyperinsulinemia, hyperleptinemia, inflammation, and selective insulin resistance in the liver and adipose tissue. In the liver, glycogen synthesis was diminished, while de novo lipogenesis increased, which was associated with low GSK3β-pS9 and strong expression of SREBP-1c. Dysfunctional adipose tissue was observed with hypertrophy and lipolysis, without changes in SREBP-1c expression and low glycogen synthesis. Both tissues accumulated cadmium and developed inflammation. In conclusion, oral subacute cadmium LOAEL dose exposure induces inflammation, insulin signaling modifications, an early insulin resistance stage (insensibility), and impairment of the hormonal and metabolic liver-adipose axis in Wistar rats.

Soluble Klotho-integrin β1/ERK1/2 pathway ameliorates myocardial fibrosis in diabetic cardiomyopathy.

Soluble Klotho (sKL) is closely related to insulin resistance, which is a major factor in the progression of diabetic cardiomyopathy (DCM). The purpose of this study was to investigate the role of sKL in the regulation of DCM and the mechanism involved. A mouse model of type 2 diabetes was induced by high-fat diet and streptozotocin injection. An insulin-resistant cardiac fibroblast model was established by high glucose and high insulin. KL gene overexpression was achieved in vivo and vitro through transfection with an adenovirus-harboring KL-cDNA. Gene overexpression was used to evaluate the role of sKL in the pathophysiologic characteristics of DCM. Insulin-resistant cardiac fibroblasts reduced sKL expression and collagen deposition. Diabetic mice constructed by streptozotocin exhibited severe insulin resistance, inflammation, fibrosis, left ventricular dysfunction, and sKL downregulation. The overexpression of sKL mitigated insulin resistance and metabolic disturbance; inflammation, fibrosis, and upregulated collagen I/III content ratio in diabetic state were significantly reduced. Our findings were accompanied by notable moderation of cardiac function. Further, blunted phosphorylation of Akt was restored with sKL gene overexpression, and activated phosphorylation of extracellular signal-regulated kinase 1/2 in DCM was reduced. Our results suggest that sKL protein overexpression exerts a defensive measure by ameliorating selective insulin resistance in mouse DCM, thus revealing its underlying mechanism for potential human DCM treatment.

Pathophysiological Association between Diabetes Mellitus and Endothelial Dysfunction.

Endothelial dysfunction plays a critical role in atherosclerosis progression, leading to cardiovascular complications. There are significant associations between diabetes mellitus, oxidative stress, and endothelial dysfunction. Oxidative stress is increased by chronic hyperglycemia and acute glucose fluctuations induced by postprandial hyperglycemia in patients with diabetes mellitus. In addition, selective insulin resistance in the phosphoinositide 3-kinase/Akt/endothelial nitric oxide (NO) synthase pathway in endothelial cells is involved in decreased NO production and increased endothelin-1 production from the endothelium, resulting in endothelial dysfunction. In a clinical setting, selecting an appropriate therapeutic intervention that improves or augments endothelial function is important for preventing diabetic vascular complications. Hypoglycemic drugs that reduce glucose fluctuations by decreasing the postprandial rise in blood glucose levels, such as glinides, α-glucosidase inhibitors and dipeptidyl peptidase 4 inhibitors, and hypoglycemic drugs that ameliorate insulin sensitivity, such as thiazolidinediones and metformin, are expected to improve or augment endothelial function in patients with diabetes. Glucagon-like peptide 1 receptor agonists, metformin, and sodium-glucose cotransporter 2 inhibitors may improve endothelial function through multiple mechanisms, some of which are independent of glucose control or insulin signaling. Oral administration of antioxidants is not recommended in patients with diabetes due to the lack of evidence for the efficacy against diabetic complications.

Enhanced Insulin Signaling Through Its Receptor Is Needed for the Development of Hyperuricemia in Humans

Background: There is an association between insulin resistance (IR) and hyperuricemia but the direction of causalityand mechanisms are unclear. In obesity and lipodystrophy, IR is “selective”, with lack of insulin signalingcausing hyperglycemia, but enhanced insulin signaling causing dyslipidemia and possibly hyperuricemia. Rare human conditions exist in which there is extreme, non-selective, IR impairing all insulin signalingpathways (e.g. mutations of the insulin receptor, INSR). We hypothesized that hyperinsulinemia withenhanced signaling through the insulin receptor causes hyperuricemia in selective IR, whereas lack ofinsulin signaling through its receptor despite hyperinsulinemia results in normal uricemia in non-selective IR. Method: Cross-sectional retrospective analysis comparing fasting insulin and serum uric acid in patients withsevere IR that was either selective (due to lipodystrophy) or non-selective (due to INSR mutation orautoantibody to the insulin receptor). Visits were chosen based on availability of serum uric acid andinsulin. If multiple visits were available, the visit with the highest insulin value was chosen. Results: We analyzed data of 68 patients with selective IR and 14 patients with non-selective IR. Fasting seruminsulin median [interquartile range] was 23.4 [14, 53] mcU/mL in selective IR vs 215 [73.7, 604] in non-selective IR, p <0.0001. Serum uric acid was 4.8 [2, 6] mg/dL in selective IR vs 3.5 [2, 4] in non-selectiveIR, p=0.0003. Conclusion: Patients with selective IR due to lipodystrophy had elevated serum uric acid, consistent with thehyperuricemia seen in selective IR caused by obesity. By contrast, patients with non-selective IR hadlower serum uric acid despite more severe IR as evidenced by almost 10-fold higher fasting insulin. These observations are consistent with a model in which enhanced insulin signaling through its receptorleads to hyperuricemia in post-receptor IR. Further studies are needed to understand the mechanism bywhich insulin leads to hyperuricemia.

Human White Adipose Tissue Displays Selective Insulin Resistance in the Obese State

Selective hepatic insulin resistance is a feature of obesity and type 2 diabetes. Whether similar mechanisms operate in white adipose tissue (WAT) of those with obesity and to what extent these are normalized by weight loss are unknown. We determined insulin sensitivity by hyperinsulinemic euglycemic clamp and insulin response in subcutaneous WAT by RNA sequencing in 23 women with obesity before and 2 years after bariatric surgery. To control for effects of surgery, women postsurgery were matched to never-obese women. Multidimensional analyses of 138 samples allowed us to classify the effects of insulin into three distinct expression responses: a common set was present in all three groups and included genes encoding several lipid/cholesterol biosynthesis enzymes; a set of obesity-attenuated genes linked to tissue remodeling and protein translation was selectively regulated in the two nonobese states; and several postobesity-enriched genes encoding proteins involved in, for example, one-carbon metabolism were only responsive to insulin in the women who had lost weight. Altogether, human WAT displays a selective insulin response in the obese state, where most genes are normalized by weight loss. This comprehensive atlas provides insights into the transcriptional effects of insulin in WAT and may identify targets to improve insulin action.

Hepatic Insulin Resistance Is Not Pathway Selective in Humans With Nonalcoholic Fatty Liver Disease.

Both glucose and triglyceride production are increased in type 2 diabetes and nonalcoholic fatty liver disease (NAFLD). For decades, the leading hypothesis to explain these paradoxical observations has been selective hepatic insulin resistance wherein insulin drives de novo lipogenesis (DNL) while failing to suppress glucose production. Here, we aimed to test this hypothesis in humans. We recruited obese subjects who met criteria for bariatric surgery with (n = 16) or without (n = 15) NAFLD and assessed 1) insulin-mediated regulation of hepatic and peripheral glucose metabolism using hyperinsulinemic-euglycemic clamps with [6,6-2H2]glucose, 2) fasting and carbohydrate-driven hepatic DNL using deuterated water (2H2O), and 3) hepatocellular insulin signaling in liver biopsy samples collected during bariatric surgery. Compared with subjects without NAFLD, those with NAFLD demonstrated impaired insulin-mediated suppression of glucose production and attenuated-not increased-glucose-stimulated/high-insulin lipogenesis. Fructose-stimulated/low-insulin lipogenesis was intact. Hepatocellular insulin signaling, assessed for the first time in humans, exhibited a proximal block in insulin-resistant subjects: Signaling was attenuated from the level of the insulin receptor through both glucose and lipogenesis pathways. The carbohydrate-regulated lipogenic transcription factor ChREBP was increased in subjects with NAFLD. Acute increases in lipogenesis in humans with NAFLD are not explained by altered molecular regulation of lipogenesis through a paradoxical increase in lipogenic insulin action; rather, increases in lipogenic substrate availability may be the key.

Ceramides in Metabolism: Key Lipotoxic Players.

The global prevalence of metabolic diseases such as type 2 diabetes mellitus, steatohepatitis, myocardial infarction, and stroke has increased dramatically over the past two decades. These obesity-fueled disorders result, in part, from the aberrant accumulation of harmful lipid metabolites in tissues not suited for lipid storage (e.g., the liver, vasculature, heart, and pancreatic beta-cells). Among the numerous lipid subtypes that accumulate, sphingolipids such as ceramides are particularly impactful, as they elicit the selective insulin resistance, dyslipidemia, and ultimately cell death that underlie nearly all metabolic disorders. This review summarizes recent findings on the regulatory pathways controlling ceramide production, the molecular mechanisms linking the lipids to these discrete pathogenic events, and exciting attempts to develop therapeutics to reduce ceramide levels to combat metabolic disease.

Endothelial dysfunction due to selective insulin resistance in vascular endothelium: insights from mechanistic modeling.

Previously, we have used mathematical modeling to gain mechanistic insights into insulin-stimulated glucose uptake. Phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling required for metabolic actions of insulin also regulates endothelium-dependent production of the vasodilator nitric oxide (NO). Vasodilation increases blood flow that augments direct metabolic actions of insulin in skeletal muscle. This is counterbalanced by mitogen-activated protein kinase (MAPK)-dependent insulin signaling in endothelium that promotes secretion of the vasoconstrictor endothelin-1 (ET-1). In the present study, we extended our model of metabolic insulin signaling into a dynamic model of insulin signaling in vascular endothelium that explicitly represents opposing PI3K/NO and MAPK/ET-1 pathways. Novel NO and ET-1 subsystems were developed using published and new experimental data to generate model structures/parameters. The signal-response relationships of our model with respect to insulin-stimulated NO production, ET-1 secretion, and resultant vascular tone, agree with published experimental data, independent of those used for model development. Simulations of pathological stimuli directly impairing only insulin-stimulated PI3K/Akt activity predict altered dynamics of NO and ET-1 consistent with endothelial dysfunction in insulin-resistant states. Indeed, modeling pathway-selective impairment of PI3K/Akt pathways consistent with insulin resistance caused by glucotoxicity, lipotoxicity, or inflammation predict diminished NO production and increased ET-1 secretion characteristic of diabetes and endothelial dysfunction. We conclude that our mathematical model of insulin signaling in vascular endothelium supports the hypothesis that pathway-selective insulin resistance accounts, in part, for relationships between insulin resistance and endothelial dysfunction. This may be relevant for developing novel approaches for the treatment of diabetes and its cardiovascular complications.

Multi-layered epigenetic regulation of IRS2 expression in the liver of obese individuals with type 2 diabetes

Aims/hypothesisIRS2 is an important molecular switch that mediates insulin signalling in the liver. IRS2 dysregulation is responsible for the phenomenon of selective insulin resistance that is observed in type 2 diabetes. We hypothesise that epigenetic mechanisms are involved in the regulation of IRS2 in the liver of obese and type 2 diabetic individuals.MethodsDNA methylation of seven CpG sites was studied by bisulphite pyrosequencing and mRNA and microRNA (miRNA) expression was assessed by quantitative real-time PCR in liver biopsies of 50 obese non-diabetic and 31 obese type 2 diabetic participants, in a cross-sectional setting. Methylation-sensitive luciferase assays and electrophoretic mobility shift assays were performed. Furthermore, HepG2 cells were treated with insulin and high glucose concentrations to induce miRNA expression and IRS2 downregulation.ResultsWe found a significant downregulation of IRS2 expression in the liver of obese individuals with type 2 diabetes (0.84 ± 0.08-fold change; p = 0.0833; adjusted p value [pa] = 0.0417; n = 31) in comparison with non-diabetic obese participants (n = 50). This downregulation correlated with hepatic IRS2 DNA methylation at CpG5. Additionally, CpG6, which is located in intron 1 of IRS2, was hypomethylated in type 2 diabetes; this site spans the sterol regulatory element binding transcription factor 1 (SREBF1) recognition motif, which likely acts as transcriptional repressor. The adjacent polymorphism rs4547213 (G>A) was significantly associated with DNA methylation at a specificity-protein-1 (SP1) binding site (CpG3). Moreover, DNA methylation of cg25924746, a CpG site located in the shore region of the IRS2 promoter-associated CpG island, was increased in the liver of individuals with type 2 diabetes, as compared with those without diabetes. A second epigenetic mechanism, upregulation of hepatic miRNA hsa-let-7e-5p (let-7e-5p) in obese individuals with type 2 diabetes (n = 29) vs non-diabetic obese individuals (n = 49) (1.2 ± 0.08-fold change; p = 0.0332; pa = 0.0450), is likely to act synergistically with altered IRS2 DNA methylation to decrease IRS2 expression. Mechanistic in vitro experiments demonstrated an acute upregulation of let-7e-5p expression and simultaneous IRS2 downregulation in a liver (HepG2) cell line upon hyperinsulinaemic and hyperglycaemic conditions.Conclusions/interpretationOur study highlights a new multi-layered epigenetic network that could be involved in subtle dysregulation of IRS2 in the liver of individuals with type 2 diabetes. This might lead to fine-tuning of IRS2 expression and is likely to be supplementary to the already known factors regulating IRS2 expression. Thereby, our findings could support the discovery of new diagnostic and therapeutic strategies for type 2 diabetes.Graphical abstract

OR08-04 Differences in Advanced Lipoprotein Profile Between Rabson-Mendenhall Syndrome and Lipodystrophy

Insulin resistance (IR) is associated with metabolic dyslipidemia (high triglycerides [TG] and low HDL) and increased cardiovascular disease (CVD) risk. In obesity-associated IR, dyslipidemia is thought to be caused by increased insulin-mediated stimulation of hepatic lipogenesis, whereas IR in glucoregulatory pathways leads to hyperglycemia. This dichotomy in insulin signaling pathways is termed selective insulin resistance. Rare human conditions exist in which there is extreme, non-selective, IR impairing all insulin signaling pathways (e.g. mutations of the insulin receptor, INSR) or extreme IR affecting only selected intracellular insulin signaling pathways analogous to obesity (e.g. lipodystrophy). Lipodystrophy leads to very high TG, low HDL, and increased CVD, while INSR mutation leads to low TG and high HDL, with unknown CVD risk. We sought to further characterize the lipid phenotype and atherogenicity in these conditions in order to understand effects of different insulin signaling pathways on CVD risk.We studied 7 patients with INSR mutation (42% female; 5 homozygous; 2 heterozygous) and 21 with lipodystrophy (85% female; 5 generalized; 16 partial). Fasting lipoprotein profiles were assessed by NMR using the LP4 deconvolution algorithm. The major lipoprotein particle categories defined by this method are small, medium, and large HDL and LDL particles (HDLP and LDLP) and very small, small, medium, large, and very large TG rich lipoprotein particles (TRLP).Very small TRLP (median 189.6 [68.7, 315.0] vs 4.5 [0.00, 9.4], p=0.0001), small LDLP (mean 1425.0 ± 636.2 vs 612.8 ± 233.9, p=0.003), small HDLP (mean 14.0 ± 4.7 vs 9.0 ± 3.2, p=0.014) were more elevated in patients with lipodystrophy vs INSR mutation. This lipoprotein profile has been associated with increased atherosclerotic coronary artery disease. GlycA, a marker of inflammation was also more elevated in lipodystrophy vs INSR mutation (435.9 ± 107.2 vs 315.7 ± 74.4, p=0.01). Insulin resistance assessed by HOMA-IR was higher in patients with INSR mutation vs lipodystrophy (mean 93.5 ± 94.4 vs 15.6 ± 14.7, p=0. 00085).) Lipoprotein insulin resistance (LPIR), an index of IR based on lipoprotein particles, was lower in patients with INSR mutation (25.0 ± 19.0 vs 84.0 ± 9.0, p < 0.0001) despite their higher HOMA-IR.In conclusion, severe, selective insulin resistance in patients with lipodystrophy was associated with a more atherogenic lipoprotein particle profile and increased inflammation compared to severe, non-selective insulin resistance caused by INSR mutations. Patients with INSR mutations had a striking discrepancy between a glucose/insulin-based index of insulin resistance (HOMA-IR) and a lipid-based marker of insulin resistance (LPIR). These findings point toward a key role of selective insulin resistance in the development of an atherogenic lipid profile, which should lead to increased CVD risk.

Hepatic DNAJB9 Drives Anabolic Biasing to Reduce Steatosis and Obesity

Nutrients stimulate the anabolic synthesis of proteins and lipids, but selective insulin resistance in obesity biases the anabolic program toward lipogenesis. Here, we report the identification of a DNAJB9-driven program that favors protein synthesis and energy production over lipid accumulation. We show there are two pools of DNAJB9 cochaperone. DNAJB9 in the ER lumen promotes the degradation of the lipogenic transcription factor SREBP1c through ERAD, whereas its counterpart on the ER membrane promotes the assembly of mTORC2 in the cytosol and stimulates the synthesis of proteins and ATP. The expression of Dnajb9 is induced by nutrients and downregulated in the obese mouse liver. Restoration of hepatic DNAJB9 expression effectively improves insulin sensitivity, restores protein synthesis, and suppresses food intake, accompanied by reduced hepatic steatosis and adiposity in multiple mouse models of obesity. Therefore, targeting the anabolic balance may provide a unique opportunity to tackle obesity and diabetes.

Insulin and β-adrenergic receptors mediate lipolytic and anti-lipolytic signalling that is not altered by type 2 diabetes in human adipocytes.

Control of fatty acid storage and release in adipose tissue is fundamental in energy homeostasis and the development of obesity and type 2 diabetes. We here take the whole signalling network into account to identify how insulin and β-adrenergic stimulation in concert controls lipolysis in mature subcutaneous adipocytes obtained from non-diabetic and, in parallel, type 2 diabetic women. We report that, and show how, the anti-lipolytic effect of insulin can be fully explained by protein kinase B (PKB/Akt)-dependent activation of the phosphodiesterase PDE3B. Through the same PKB-dependent pathway β-adrenergic receptor signalling, via cAMP and PI3Kα, is anti-lipolytic and inhibits its own stimulation of lipolysis by 50%. Through this pathway both insulin and β-adrenergic signalling control phosphorylation of FOXO1. The dose–response of lipolysis is bell-shaped, such that insulin is anti-lipolytic at low concentrations, but at higher concentrations of insulin lipolysis was increasingly restored due to inhibition of PDE3B. The control of lipolysis was not altered in adipocytes from diabetic individuals. However, the release of fatty acids was increased by 50% in diabetes due to reduced reesterification of lipolytically liberated fatty acids. In conclusion, our results reveal mechanisms of control by insulin and β-adrenergic stimulation — in human adipocytes — that define a network of checks and balances ensuring robust control to secure uninterrupted supply of fatty acids without reaching concentrations that put cellular integrity at risk. Moreover, our results define how selective insulin resistance leave lipolytic control by insulin unaltered in diabetes, while the fatty acid release is substantially increased.

283-LB: Dissociating Insulin Signaling and SREBP1c Action from the Lipogenic Drive Seen in Human and Murine Hepatic Insulin Resistance

The ongoing debate regarding pathway selective hepatic insulin resistance (IR) focuses on potential insulin signaling branchpoints beyond which regulation of glucose metabolism is attenuated and regulation of de novo lipogenesis (DNL) remains intact. These models suggest that impaired insulin receptor kinase activity, proximal to any potential branchpoint, only plays a minor role in clinical hepatic IR. We compared insulin signaling in liver biopsies from human subjects with nonalcoholic fatty liver disease (NAFLD) and hepatic IR vs. weight matched controls, using glucose ingestion to drive insulin secretion and fructose ingestion as a low insulin comparator. Insulin signaling through phosphorylation of insulin receptor β, Akt, and GSK3β was suppressed in NAFLD (all P &amp;lt; 0.05). Insulin regulation of mTORC1 via PRAS40 phosphorylation was absent in NAFLD subjects; no differences were seen between controls and NAFLD subjects in AMPK dependent RAPTOR phosphorylation. Pathway selective IR implies SREBP1c is primarily responsible for DNL in the insulin resistant liver; we probed the transcriptional regulation of DNL by assessing the in vivo occupancy of lipogenic target gene promoters by SREBP1c and ChREBP using ChIP experiments in mice with diet-induced IR. Fasted-refed mice were refed with high fat diet (HFD) and 1% dextrose drinking water. Mice with hepatic IR (9 day HFD fed) or hepatic and peripheral IR (4 week HFD fed) were compared with more insulin sensitive 2 day HFD fed mice. SREBP1c was not bound to the Fasn promoter in insulin resistant livers, while ChREBP was bound to the Fasn promoter equally in all three groups of mice. Conclusions: There is a proximal block in insulin signaling in humans with hepatic IR and NAFLD, dissociating previously measured changes in DNL rates from insulin signaling. SREBP does not mediate DNL in insulin resistant mice while ChREBP action can explain the maintenance of the transcriptional program of lipogenesis. Disclosure D.F. Vatner: None. K.W. ter Horst: None. L. Goedeke: None. D. Zhang: None. V. Samuel: None. M.J. Serlie: Advisory Panel; Self; Fresenius Medical Care. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, Merck &amp; Co., Inc. Advisory Panel; Spouse/Partner; Merck &amp; Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health

MON-159 Increased Gluconeogenesis and Lower Glycogenolysis in Patients with Non-Selective Compared Selective Insulin Resistance

Type 2 diabetes is characterized by increased hepatic glucose production and selective insulin resistance that occurs downstream of the insulin receptor. However, the differential pathways and exact molecules implicated in selective insulin signaling are unclear. To elucidate the hepatic mechanistic pathways affected by selective versus non-selective insulin resistance, we compared basal rates of hepatic glucose production and its components, gluconeogenesis and glycogenolysis, in patients with severe non-selective insulin resistance (insulin receptor mutations (INSR)) to patients with selective insulin resistance (lipodystrophy and type 2 diabetes). In 7 patients with INSR, 14 with lipodystrophy and 9 with type 2 diabetes, we measured rates of hepatic glucose production (via [6,6-2H2] glucose) and fractional gluconeogenesis (via 2H2O) after an overnight fast. Absolute rates of gluconeogenesis were calculated as the product of fractional gluconeogenesis and hepatic glucose production, and glycogenolysis were calculated as hepatic glucose production minus gluconeogenesis. In patients with INSR, lipodystrophy and type 2 diabetes, HbA1c (8.2 ± 2.8, 8.4 ± 1.9, 7.4 ± 0.9%) and fasting glucose (120 ± 70, 159 ± 55, 127 ± 32 mg/dl) were comparable (P>0.05). Fasting serum insulin was higher in INSR vs. lipodystrophy and type 2 diabetes (332 ± 355, 48 ± 50, 50 ± 16 μU/ml; P<0.01). Patients with INSR and lipodystrophy had higher hepatic glucose production vs. type 2 diabetes (24.0 ± 4.0, 24.1 ± 6.2, 15.8 ± 4.3 µmol/kgLEAN/min; P<0.01). Fractional gluconeogenesis was higher in INSR vs. lipodystrophy and type 2 diabetes (77.1 ± 9.4, 55.4 ± 11.5, 59.1 ± 5.9%; P<0.01). Absolute gluconeogenesis was highest in INSR, intermediate in lipodystrophy, and lowest in type 2 diabetes (19.1 ± 4.4, 13.5 ± 4.2, 9.5 ± 1.9 µmol/kgLEAN/min; P<0.01). Glycogenolysis was lowest in INSR, highest in lipodystrophy, and intermediate in type 2 diabetes (4.9 ± 2.2 vs. 10.6 ± 4.8, 6.3 ± 2.6 µmol/kgLEAN/min; P<0.01). Higher hepatic glucose production in lipodystrophy vs. type 2 diabetes is consistent with higher hepatic insulin resistance in patients with lipodystrophy, but comparable fractional rates of gluconeogenesis and glycogenolysis confirm similar mechanisms of selective post-receptor insulin resistance. By contrast, the markedly higher contribution of gluconeogenesis to hepatic glucose production in INSR may reflect complete loss of insulin signaling for glycogen accumulation and/or suppression of gluconeogenesis. Further studies should evaluate whether greater substrate and fuel availability for gluconeogenesis, and/or lower hepatic glycogen stores are important determinants of the components of glucose production in patients with non-selective insulin resistance due to INSR mutation.

Evidence against Pathway-Selective Hepatic Insulin Resistance in Mice

Insulin suppresses hepatic glucose production and increases hepatic de novo lipogenesis (DNL). Paradoxically, hepatic DNL remains elevated in insulin-resistant subjects, leading to the hypothesis that hepatic insulin resistance is pathway-selective. Prior studies of DNL in hepatic insulin resistance are complicated by confounders, such as use of markedly unphysiologic animal models or comparison of animals on different diets and thus different availability of DNL precursors. We measured DNL in InsrT1150A knockin (KI) mice, a strain protected from diacylglycerol-mediated hepatic insulin resistance, and in littermate controls. Diets were matched: 60% high fat diet (HFD) with 1% dextrose in drinking water. After two days of fat-feeding, before the development of significant insulin resistance, KI and WT mice displayed similar rates of DNL (KI 24.3% ± 3.6; WT 22.2% ± 3.5). After 9 days of HFD, when WT but not KI mice have developed hepatic insulin resistance, rates of DNL were reduced in WT mice but preserved in KI mice (KI 16.4% ± 2.4; WT 7.4% ± 1.3; P &amp;lt; 0.01). After 4 weeks of HFD, with the development of skeletal muscle resistance, rates of DNL increased in WT mice to reach those observed in KI mice (KI 19.3% ± 2.6; WT 15.9% ± 2.3). Consistent with the insulin-resistant phenotype, Srebp-1c cleavage was reduced in the setting of hepatic insulin resistance in WT mice; this effect was attenuated in KI mice. The Srebp-regulated lipogenic proteins Fasn and Scd1 were both reduced in 9d HFD fed livers regardless of genotype; at 4 weeks, protein abundance remained suppressed in WT mice but was restored in KI mice. Pklr, a protein associated with DNL but not regulated by Srebp, was unaffected by HFD-induced hepatic insulin resistance. Conclusion: Regulation of DNL is subject to lipid-induced hepatic insulin resistance, challenging the selective hepatic insulin resistance hypothesis. Increased DNL seen in insulin resistant subjects may be due to hyperinsulinemia and diversion of substrate from the insulin resistant periphery. Disclosure D.F. Vatner: None. M.C. Petersen: None. X. Li: None. J.C. Rogers: None. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, Merck &amp; Co., Inc., Novo Nordisk Inc.. Research Support; Self; Gilead Sciences, Inc..

Hepatic CREBZF couples insulin to lipogenesis by inhibiting insig activity and contributes to hepatic steatosis in diet-induced insulin-resistant mice.

These findings uncover an unexpected mechanism that couples changes in extracellular hormonal signals to hepatic lipid homeostasis; disrupting CREBZF function may have the therapeutic potential for treating fatty liver disease and insulin resistance. (Hepatology 2018).

Selective insulin resistance with differential expressions of IRS-1 and IRS-2 in human NAFLD livers

Background/objective:Insulin signals, via the regulation of key enzyme expression, both suppress gluconeogenesis and enhance lipid synthesis in the liver. Animal studies have revealed insulin signaling favoring gluconeogenesis suppression to be selectively impaired in steatotic livers. However, whether, and if so how, such selective insulin resistance occurs in human steatotic livers remains unknown. Our aim was to investigate selective insulin resistance in human livers with non-alcoholic fatty liver disease (NAFLD).Subjects/methods:We examined mRNA expressions of key molecules for insulin signaling, gluconeogenesis and lipogenesis in human liver biopsy samples obtained from 51 non-diabetic subjects: 9 healthy controls and 42 NAFLD patients, and analyzed associations of these molecules with each other and with detailed pathological and clinical biochemistry data.Results:In NAFLD patients, insulin receptor substrate (IRS)-2 expression was decreased, while those of key enzymes for gluconeogenesis were increased. These alterations of IRS-2 and gluconeogenesis enzymes were induced both in simple steatosis (SS) and non-alcoholic steatohepatitis (NASH), while these expression levels did not differ between SS and NASH. Furthermore, alterations in the expressions of IRS-2 and gluconeogenesis enzymes showed strong negative correlations and were concurrently induced in the early histological stage of NAFLD. In contrast, fatty acid synthase (FAS) expression was not decreased in NAFLD, despite IRS-2 downregulation, but correlated strongly with IRS-1 expression. Furthermore, no histological scores were associated with these molecules. Thus, IRS-1 signaling, which is not impaired in NAFLD, appears to modulate FAS expression.Conclusion:These analyses revealed that selective insulin resistance is present in human NAFLD livers and occurs in its early phases. The effect of insulin, during the IRS step, on gene expressions for lipogenesis and gluconeogenesis are apparently distinct and preferential downregulation of IRS-2 may contribute to selective resistance to the suppressive effects of insulin on gluconeogenesis.

Global Analyses of Selective Insulin Resistance in Hepatocytes Caused by Palmitate Lipotoxicity

Obesity is tightly linked to hepatic steatosis and insulin resistance. One feature of this association is the paradox of selective insulin resistance: insulin fails to suppress hepatic gluconeogenesis but activates lipid synthesis in the liver. How lipid accumulation interferes selectively with some branches of hepatic insulin signaling is not well understood. Here we provide a resource, based on unbiased approaches and established in a simple cell culture system, to enable investigations of the phenomenon of selective insulin resistance. We analyzed the phosphoproteome of insulin-treated human hepatoma cells and identified sites in which palmitate selectively impairs insulin signaling. As an example, we show that palmitate interferes with insulin signaling to FoxO1, a key transcription factor regulating gluconeogenesis, and identify altered FoxO1 cellular compartmentalization as a contributing mechanism for selective insulin resistance. This model system, together with our comprehensive characterization of the proteome, phosphoproteome, and lipidome changes in response to palmitate treatment, provides a novel and useful resource for unraveling the mechanisms underlying selective insulin resistance.