A Cell-Autonomous Signature of Dysregulated Protein Phosphorylation Underlies Muscle Insulin Resistance in Type 2 Diabetes.

Skeletal muscle insulin resistance is the earliest defect in type 2 diabetes (T2D), preceding and predicting disease development. To what extent this reflects a primary defect or is secondary to tissue cross talk due to changes in hormones or circulating metabolites is unknown. To address this question, we have developed an in vitro disease-in-a-dish model using iPS cells from T2D patients differentiated into myoblasts (iMyos). We find that T2D iMyos in culture exhibit multiple defects mirroring human disease, including an altered insulin signaling, decreased insulin-stimulated glucose uptake, and reduced mitochondrial oxidation. More strikingly, global phosphoproteomic analysis reveals a multidimensional network of signaling defects in T2D iMyos going beyond the canonical insulin-signaling cascade, including proteins involved in regulation of Rho GTPases, mRNA splicing and/or processing, vesicular trafficking, gene transcription, and chromatin remodeling. These cell-autonomous defects and the dysregulated network of protein phosphorylation reveal a new dimension in the cellular mechanisms underlying the fundamental defects in T2D.

Hematopoietic-Derived Galectin-3 Causes Cellular and Systemic Insulin Resistance.

In obesity, macrophages and other immune cells accumulate in insulin target tissues, promoting a chronic inflammatory state and insulin resistance. Galectin-3 (Gal3), a lectin mainly secreted by macrophages, is elevated in both obese subjects and mice. Administration of Gal3 to mice causes insulin resistance and glucose intolerance, whereas inhibition of Gal3, through either genetic or pharmacologic loss of function, improved insulin sensitivity in obese mice. In vitro treatment with Gal3 directly enhanced macrophage chemotaxis, reduced insulin-stimulated glucose uptake in myocytes and 3T3-L1 adipocytes and impaired insulin-mediated suppression of glucose output in primary mouse hepatocytes. Importantly, we found that Gal3 can bind directly to the insulin receptor (IR) and inhibit downstream IR signaling. These observations elucidate a novel role for Gal3 in hepatocyte, adipocyte, and myocyte insulin resistance, suggesting that Gal3 can link inflammation to decreased insulin sensitivity. Inhibition of Gal3 could be a new approach to treat insulin resistance.

Insulin Resistance in Human iPS Cells Reduces Mitochondrial Size and Function.

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.

Myotubes derived from human-induced pluripotent stem cells mirror in vivo insulin resistance.

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.

Age related skeletal muscle atrophy and upregulation of autophagy in dogs.

Sarcopenia, the age related loss of muscle mass and strength, is a multifactorial condition that occurs in a variety of species and represents a major healthcare concern for older adults in human medicine. In veterinary medicine, skeletal muscle atrophy is often observed in dogs as they reach old age, but the process is not well understood. Autophagy is a mechanism for degradation and recycling of cellular constituents and is potentially involved in sarcopenia. The aim of the present study was to evaluate the expression of three markers of autophagy, Beclin 1, LC3 and p62, in muscle wasting of geriatric dogs, to establish whether the levels of autophagy change with increasing age. Muscle biopsies from 25 geriatric dogs were examined and compared with those from five healthy young dogs. Samples from older dogs, assessed by routine histology, histoenzymatic staining and immunohistochemistry, showed evidence of muscle atrophy, sarcoplasmic vacuolisation and mitochondrial alterations. Furthermore, in 80% of the muscle samples from the older dogs, marked intracytoplasmic staining for Beclin 1 and LC3 was observed. Significantly greater expression of LC3 II and Beclin 1, but lower expression of p62, was found by Western blotting, comparing muscle samples from old vs. young dogs. The results of the study suggest that enhanced autophagy might be one of the factors underlying muscle atrophy in dogs as they age.

Genetic insulin resistance is a potent regulator of gene expression and proliferation in human iPS cells.

Insulin resistance is central to diabetes and metabolic syndrome. To define the consequences of genetic insulin resistance distinct from those secondary to cellular differentiation or in vivo regulation, we generated induced pluripotent stem cells (iPSCs) from individuals with insulin receptor mutations and age-appropriate control subjects and studied insulin signaling and gene expression compared with the fibroblasts from which they were derived. iPSCs from patients with genetic insulin resistance exhibited altered insulin signaling, paralleling that seen in the original fibroblasts. Insulin-stimulated expression of immediate early genes and proliferation were also potently reduced in insulin resistant iPSCs. Global gene expression analysis revealed marked differences in both insulin-resistant iPSCs and corresponding fibroblasts compared with control iPSCs and fibroblasts. Patterns of gene expression in patients with genetic insulin resistance were particularly distinct in the two cell types, indicating dependence on not only receptor activity but also the cellular context of the mutant insulin receptor. Thus, iPSCs provide a novel approach to define effects of genetically determined insulin resistance. This study demonstrates that effects of insulin resistance on gene expression are modified by cellular context and differentiation state. Moreover, altered insulin receptor signaling and insulin resistance can modify proliferation and function of pluripotent stem cell populations.

A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species.

Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3ß inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3ß inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.

Prep1 controls insulin glucoregulatory function in liver by transcriptional targeting of SHP1 tyrosine phosphatase.

OBJECTIVE: We investigated the function of the Prep1 gene in insulin-dependent glucose homeostasis in liver. RESEARCH DESIGN AND METHODS: Prep1 action on insulin glucoregulatory function has been analyzed in liver of Prep1-hypomorphic mice (Prep1(i/i)), which express 2-3% of Prep1 mRNA. RESULTS: Based on euglycemic hyperinsulinemic clamp studies and measurement of glycogen content, livers from Prep1(i/i) mice feature increased sensitivity to insulin. Tyrosine phosphorylation of both insulin receptor (IR) and insulin receptor substrate (IRS)1/2 was significantly enhanced in Prep1(i/i) livers accompanied by a specific downregulation of the SYP and SHP1 tyrosine phosphatases. Prep1 overexpression in HepG2 liver cells upregulated SYP and SHP1 and inhibited insulin-induced IR and IRS1/2 phosphorylation and was accompanied by reduced glycogen content. Consistently, overexpression of the Prep1 partner Pbx1, but not of p160MBP, mimicked Prep1 effects on tyrosine phosphorylations, glycogen content, and on SYP and SHP1 expression. In Prep1 overexpressing cells, antisense silencing of SHP1, but not that of SYP, rescued insulin-dependent IR phosphorylation and glycogen accumulation. Both Prep1 and Pbx1 bind SHP1 promoter at a site located between nucleotides -2,113 and -1,778. This fragment features enhancer activity and induces luciferase function by 7-, 6-, and 30-fold, respectively, in response to Prep1, Pbx1, or both. CONCLUSIONS: SHP1, a known silencer of insulin signal, is a transcriptional target of Prep1. In liver, transcriptional activation of SHP1 gene by Prep1 attenuates insulin signal transduction and reduces glucose storage.

Prep1 deficiency induces protection from diabetes and increased insulin sensitivity through a p160-mediated mechanism.

We have examined glucose homeostasis in mice hypomorphic for the homeotic transcription factor gene Prep1. Prep1-hypomorphic (Prep1(i/i)) mice exhibit an absolute reduction in circulating insulin levels but normal glucose tolerance. In addition, these mice exhibit protection from streptozotocin-induced diabetes and enhanced insulin sensitivity with improved glucose uptake and insulin-dependent glucose disposal by skeletal muscle. This muscle phenotype does not depend on reduced expression of the known Prep1 transcription partner, Pbx1. Instead, in Prep1(i/i) muscle, we find normal Pbx1 but reduced levels of the recently identified novel Prep1 interactor p160. Consistent with this reduction, we find a muscle-selective increase in mRNA and protein levels of PGC-1alpha, accompanied by enhanced expression of the GLUT4 transporter, responsible for insulin-stimulated glucose uptake in muscle. Indeed, using L6 skeletal muscle cells, we induced the opposite effects by overexpressing Prep1 or p160, but not Pbx1. In vivo skeletal muscle delivery of p160 cDNA in Prep1(i/i) mice also reverses the molecular phenotype. Finally, we show that Prep1 controls the stability of the p160 protein. We conclude that Prep1 controls insulin sensitivity through the p160-GLUT4 pathway.

Phorbol esters induce intracellular accumulation of the anti-apoptotic protein PED/PEA-15 by preventing ubiquitinylation and proteasomal degradation.

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA)-15 is an anti-apoptotic protein whose expression is increased in several cancer cells and following experimental skin carcinogenesis. Exposure of untransfected C5N keratinocytes and transfected HEK293 cells to phorbol esters (12-O-tetradecanoylphorbol-13-acetate (TPA)) increased PED/PEA-15 cellular content and enhanced its phosphorylation at serine 116 in a time-dependent fashion. Ser-116 --> Gly (PED(S116G)) but not Ser-104 --> Gly (PED(S104G)) substitution almost completely abolished TPA regulation of PED/PEA-15 expression. TPA effect was also prevented by antisense inhibition of protein kinase C (PKC)-zeta and by the expression of a dominant-negative PKC-zeta mutant cDNA in HEK293 cells. Similar to long term TPA treatment, overexpression of wild-type PKC-zeta increased cellular content and phosphorylation of WT-PED/PEA-15 and PED(S104G) but not of PED(S116G). These events were accompanied by the activation of Ca2+-calmodulin kinase (CaMK) II and prevented by the CaMK blocker, KN-93. At variance, the proteasome inhibitor lactacystin mimicked TPA action on PED/PEA-15 intracellular accumulation and reverted the effects of PKC-zeta and CaMK inhibition. Moreover, we show that PED/PEA-15 bound ubiquitin in intact cells. PED/PEA-15 ubiquitinylation was reduced by TPA and PKC-zeta overexpression and increased by KN-93 and PKC-zeta block. Furthermore, in HEK293 cells expressing PED(S116G), TPA failed to prevent ubiquitin-dependent degradation of the protein. Accordingly, in the same cells, TPA-mediated protection from apoptosis was blunted. Taken together, our results indicate that TPA increases PED/PEA-15 expression at the post-translational level by inducing phosphorylation at serine 116 and preventing ubiquitinylation and proteosomal degradation.