Induced pluripotent stem cells (iPS cells) represent a unique tool for the study of the pathophysiology of human disease, because these cells can be differentiated into multiple cell types in vitro and used to generate patient- and tissue-specific disease models. Given the critical role for skeletal muscle insulin resistance in whole-body glucose metabolism and type 2 diabetes, we have created a novel cellular model of human muscle insulin resistance by differentiating iPS cells from individuals with mutations in the insulin receptor (IR-Mut) into functional myotubes and characterizing their response to insulin in comparison with controls. Morphologically, IR-Mut cells differentiated normally, but had delayed expression of some muscle differentiation-related genes. Most importantly, whereas control iPS-derived myotubes exhibited in vitro responses similar to primary differentiated human myoblasts, IR-Mut myotubes demonstrated severe impairment in insulin signaling and insulin-stimulated 2-deoxyglucose uptake and glycogen synthesis. Transcriptional regulation was also perturbed in IR-Mut myotubes with reduced insulin-stimulated expression of metabolic and early growth response genes. Thus, iPS-derived myotubes from individuals with genetically determined insulin resistance demonstrate many of the defects observed in vivo in insulin-resistant skeletal muscle and provide a new model to analyze the molecular impact of muscle insulin resistance.

Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4(R24C)). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT.

CONTEXT: HIV patients are at an increased risk for cardiometabolic disease secondary to depot-specific alterations in adipose function, but mechanisms remain poorly understood. OBJECTIVE: The endoribonuclease Dicer has been linked to the modulation of brown and white adipocyte differentiation. We previously demonstrated that Dicer knockout mice undergo transformation of brown adipose tissue to white adipose tissue and develop a lipodystrophic phenotype. We hypothesized reduced Dicer and brown adipose tissue gene expression from nonlipomatous sc fat among HIV patients with a lipodystrophic phenotype. DESIGN: Eighteen HIV (nine with and without lipodystrophic changes in fat distribution, characterized by excess dorsocervical adipose tissue [DCAT]) and nine non-HIV subjects underwent punch biopsy of abdominal sc fat to determine expression of Dicer and other adipose-related genes. RESULTS: HIV subjects with long-duration antiretroviral use demonstrated excess DCAT vs non-HIV subjects (9.8 ± 1.0 vs 6.6 ± 0.8 cm(2), P = .02) with similar body mass index. Dicer expression was decreased in abdominal sc fat of HIV vs non-HIV (4.88 [1.91, 11.93] vs 17.69 [10.72, 47.91], P = .01), as were PPARα, ZIC1, PRDM16, DIO2, and HSP60 (all P ≤ .03). Moreover, the expression of Dicer (2.49 [0.02, 4.88] vs 11.20 [4.83, 21.45], P = .006), brown fat (PPARα [P = .002], ZIC1 [P = .004], LHX8 [P = .03], PRDM16 [P = .0008], PAT2 [P = .008], P2RX5 [P = .02]), beige fat (TMEM26 [P = .004], CD137 [P = .008]), and other genes (DIO2 [P = .002], leptin [P = .003], HSP60 [P = .0004]) was further decreased in abdominal sc fat comparing HIV subjects with vs without excess DCAT. Down-regulation of Dicer in the abdominal sc fat correlated with the down-regulation of all brown and beige fat genes (all P ≤ .01). CONCLUSION: Our results demonstrate dysfunctional sc adipose tissue marked by reduced Dicer in relationship to the down-regulation of brown and beige fat-related genes in lipodystrophic HIV patients and may provide a novel mechanism for metabolic dysregulation. A strategy to increase browning of white adipose tissue may improve cardiometabolic health in HIV.

Insulin resistance, a critical component of type 2 diabetes (T2D), precedes and predicts T2D onset. T2D is also associated with mitochondrial dysfunction. To define the cause-effect relationship between insulin resistance and mitochondrial dysfunction, we compared mitochondrial metabolism in induced pluripotent stem cells (iPSC) from 5 healthy individuals and 4 patients with genetic insulin resistance due to insulin receptor mutations. Insulin-resistant iPSC had increased mitochondrial number and decreased mitochondrial size. Mitochondrial oxidative function was impaired, with decreased citrate synthase activity and spare respiratory capacity. Simultaneously, expression of multiple glycolytic enzymes was decreased, while lactate production increased 80%. These perturbations were accompanied by an increase in ADP/ATP ratio and 3-fold increase in AMPK activity, indicating energetic stress. Insulin-resistant iPSC also showed reduced catalase activity and increased susceptibility to oxidative stress. Thus, insulin resistance can lead to mitochondrial dysfunction with reduced mitochondrial size, oxidative activity, and energy production.

Nonalcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome. Overconsumption of high-fat diet (HFD) and increased intake of sugar-sweetened beverages are major risk factors for development of NAFLD. Today the most commonly consumed sugar is high fructose corn syrup. Hepatic lipids may be derived from dietary intake, esterification of plasma free fatty acids (FFA) or hepatic de novo lipogenesis (DNL). A central abnormality in NAFLD is enhanced DNL. Hepatic DNL is increased in individuals with NAFLD, while the contribution of dietary fat and plasma FFA to hepatic lipids is not significantly altered. The importance of DNL in NAFLD is further established in mouse studies with knockout of genes involved in this process. Dietary fructose increases levels of enzymes involved in DNL even more strongly than HFD. Several properties of fructose metabolism make it particularly lipogenic. Fructose is absorbed via portal vein and delivered to the liver in much higher concentrations as compared to other tissues. Fructose increases protein levels of all DNL enzymes during its conversion into triglycerides. Additionally, fructose supports lipogenesis in the setting of insulin resistance as fructose does not require insulin for its metabolism, and it directly stimulates SREBP1c, a major transcriptional regulator of DNL. Fructose also leads to ATP depletion and suppression of mitochondrial fatty acid oxidation, resulting in increased production of reactive oxygen species. Furthermore, fructose promotes ER stress and uric acid formation, additional insulin independent pathways leading to DNL. In summary, fructose metabolism supports DNL more strongly than HFD and hepatic DNL is a central abnormality in NAFLD. Disrupting fructose metabolism in the liver may provide a new therapeutic option for the treatment of NAFLD.

Brown fat is a thermogenic tissue that generates heat to maintain body temperature in cold environments and dissipate excess energy in response to overfeeding. We have addressed the role of the IGFIR in the brown fat development and function. Mice lacking IGFIR exhibited normal brown adipose tissue/body weight in knockout (KO) vs control mice. However, lack of IGFIR decreased uncoupling protein 1 expression in interscapular brown fat and beige cells in inguinal fat. More importantly, the lack of IGFIR resulted in an impaired cold acclimation. No differences in the total fat volume were found in the KO vs control mice. Epididymal fat showed larger adipocytes but with a lower number of adipocytes in KO vs control mice at age 12 months. In addition, KO mice showed a sustained moderate hyperinsulinemia and hypertriglyceridemia upon time and hepatic insulin insensitivity associated with lipid accumulation, with the outcome of a global insulin resistance. In addition, we found that the expression of uncoupling protein 3 in the skeletal muscle was decreased and its expression was increased in the heart in parallel with the expression of beta-2 adrenergic receptors. Upon nonobesogenic high-fat diet, we found a severe insulin resistance in the liver and in the skeletal muscle, but unchanged insulin sensitivity in the heart. In conclusion, our data suggest that IGFIR it is not an essential growth factor in the brown fat development in the presence of the IR and very high plasma levels of IGF-I, but it is indispensable for full brown fat functionality.