Skeletal muscle expression of adipose-specific phospholipase in peripheral artery disease.

Flow-limiting atherosclerotic lesions of arteries supplying the limbs are a cause of symptoms in patients with peripheral artery disease (PAD). Musculoskeletal metabolic factors also contribute to the pathophysiology of claudication, which is manifest as leg discomfort that impairs walking capacity. Accordingly, we conducted a case-control study to determine whether skeletal muscle metabolic gene expression is altered in PAD. Calf skeletal muscle gene expression of patients with PAD and healthy subjects was analyzed using microarrays. The top-ranking gene differentially expressed between PAD and controls (FDR < 0.001) was PLA2G16, which encodes adipose-specific phospholipase A2 (AdPLA) and is implicated in the maintenance of insulin sensitivity and regulation of lipid metabolism. Differential expression was confirmed by qRT-PCR; PLA2G16 was downregulated by 68% in patients with PAD (p < 0.001). Expression of Pla2g16 was then measured in control (db/+) and diabetic (db/db) mice that underwent unilateral femoral artery ligation. There was significantly reduced expression of Pla2g16 in the ischemic leg of both control and diabetic mice (by 51%), with significantly greater magnitude of reduction in the diabetic mice (by 79%). We conclude that AdPLA is downregulated in humans with PAD and in mice with hindlimb ischemia. Reduced AdPLA may contribute to impaired walking capacity in patients with PAD via its effects on skeletal muscle metabolism. Further studies are needed to fully characterize the role of AdPLA in PAD and to investigate its potential as a therapeutic target for alleviating symptoms of claudication.

Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (l50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

Metabolic control of primed human pluripotent stem cell fate and function by the miR-200c-SIRT2 axis.

A hallmark of cancer cells is the metabolic switch from oxidative phosphorylation (OXPHOS) to glycolysis, a phenomenon referred to as the 'Warburg effect', which is also observed in primed human pluripotent stem cells (hPSCs). Here, we report that downregulation of SIRT2 and upregulation of SIRT1 is a molecular signature of primed hPSCs and that SIRT2 critically regulates metabolic reprogramming during induced pluripotency by targeting glycolytic enzymes including aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase. Remarkably, knockdown of SIRT2 in human fibroblasts resulted in significantly decreased OXPHOS and increased glycolysis. In addition, we found that miR-200c-5p specifically targets SIRT2, downregulating its expression. Furthermore, SIRT2 overexpression in hPSCs significantly affected energy metabolism, altering stem cell functions such as pluripotent differentiation properties. Taken together, our results identify the miR-200c-SIRT2 axis as a key regulator of metabolic reprogramming (Warburg-like effect), via regulation of glycolytic enzymes, during human induced pluripotency and pluripotent stem cell function.

What Have Metabolomics Approaches Taught Us About Type 2 Diabetes?

Type 2 diabetes (T2D) is increasing worldwide, making identification of biomarkers for detection, staging, and effective prevention strategies an especially critical scientific and medical goal. Fortunately, advances in metabolomics techniques, together with improvements in bioinformatics and mathematical modeling approaches, have provided the scientific community with new tools to describe the T2D metabolome. The metabolomics signatures associated with T2D and obesity include increased levels of lactate, glycolytic intermediates, branched-chain and aromatic amino acids, and long-chain fatty acids. Conversely, tricarboxylic acid cycle intermediates, betaine, and other metabolites decrease. Future studies will be required to fully integrate these and other findings into our understanding of diabetes pathophysiology and to identify biomarkers of disease risk, stage, and responsiveness to specific treatments.

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.

Severe insulin resistance alters metabolism in mesenchymal progenitor cells.

Donohue syndrome (DS) is characterized by severe insulin resistance due to mutations in the insulin receptor (INSR) gene. To identify molecular defects contributing to metabolic dysregulation in DS in the undifferentiated state, we generated mesenchymal progenitor cells (MPCs) from induced pluripotent stem cells derived from a 4-week-old female with DS and a healthy newborn male (control). INSR mRNA and protein were significantly reduced in DS MPC (for ß-subunit, 64% and 89% reduction, respectively, P < .05), but IGF1R mRNA and protein did not differ vs control. Insulin-stimulated phosphorylation of INSR or the downstream substrates insulin receptor substrate 1 and protein kinase B did not differ, but ERK phosphorylation tended to be reduced in DS (32% decrease, P = .07). By contrast, IGF-1 and insulin-stimulated insulin-like growth factor 1 (IGF-1) receptor phosphorylation were increased in DS (IGF-1, 8.5- vs 4.5-fold increase; INS, 11- vs 6-fold; P < .05). DS MPC tended to have higher oxygen consumption in both the basal state (87% higher, P =.09) and in response to the uncoupler carbonyl cyanide-p-triflouromethoxyphenylhydrazone (2-fold increase, P =.06). Although mitochondrial DNA or mass did not differ, oxidative phosphorylation protein complexes III and V were increased in DS (by 37% and 6%, respectively; P < .05). Extracellular acidification also tended to increase in DS (91% increase, P = .07), with parallel significant increases in lactate secretion (34% higher at 4 h, P < .05). In summary, DS MPC maintain signaling downstream of the INSR, suggesting that IGF-1R signaling may partly compensate for INSR mutations. However, alterations in receptor expression and pathway-specific defects in insulin signaling, even in undifferentiated cells, can alter cellular oxidative metabolism, potentially via transcriptional mechanisms.

Metabolic modeling of muscle metabolism identifies key reactions linked to insulin resistance phenotypes.

OBJECTIVE: Dysregulated muscle metabolism is a cardinal feature of human insulin resistance (IR) and associated diseases, including type 2 diabetes (T2D). However, specific reactions contributing to abnormal energetics and metabolic inflexibility in IR are unknown. METHODS: We utilize flux balance computational modeling to develop the first systems-level analysis of IR metabolism in fasted and fed states, and varying nutrient conditions. We systematically perturb the metabolic network to identify reactions that reproduce key features of IR-linked metabolism. RESULTS: While reduced glucose uptake is a major hallmark of IR, model-based reductions in either extracellular glucose availability or uptake do not alter metabolic flexibility, and thus are not sufficient to fully recapitulate IR-linked metabolism. Moreover, experimentally-reduced flux through single reactions does not reproduce key features of IR-linked metabolism. However, dual knockdowns of pyruvate dehydrogenase (PDH), in combination with reduced lipid uptake or lipid/amino acid oxidation (ETFDH), does reduce ATP synthesis, TCA cycle flux, and metabolic flexibility. Experimental validation demonstrates robust impact of dual knockdowns in PDH/ETFDH on cellular energetics and TCA cycle flux in cultured myocytes. Parallel analysis of transcriptomic and metabolomics data in humans with IR and T2D demonstrates downregulation of PDH subunits and upregulation of its inhibitory kinase PDK4, both of which would be predicted to decrease PDH flux, concordant with the model. CONCLUSIONS: Our results indicate that complex interactions between multiple biochemical reactions contribute to metabolic perturbations observed in human IR, and that the PDH complex plays a key role in these metabolic phenotypes.

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.

Adenylate kinase 2 links mitochondrial energy metabolism to the induction of the unfolded protein response.

The unfolded protein response (UPR) is a homeostatic signaling mechanism that balances the protein folding capacity of the endoplasmic reticulum (ER) with the secretory protein load of the cell. ER protein folding capacity is dependent on the abundance of chaperones, which is increased in response to UPR signaling, and on a sufficient ATP supply for their activity. An essential branch of the UPR entails the splicing of XBP1 mRNA to form the XBP1 transcription factor. XBP1 has been shown to be required during adipocyte differentiation, enabling mature adipocytes to secrete adiponectin, and during differentiation of B cells into antibody-secreting plasma cells. Here we find that adenylate kinase 2 (AK2), a mitochondrial enzyme that regulates adenine nucleotide interconversion within the intermembrane space, is markedly induced during adipocyte and B cell differentiation. Depletion of AK2 by RNAi impairs adiponectin secretion in 3T3-L1 adipocytes, IgM secretion in BCL1 cells, and the induction of the UPR during differentiation of both cell types. These results reveal a new mechanism by which mitochondria support ER function and suggest that specific mitochondrial defects may give rise to impaired UPR signaling. The requirement for AK2 for UPR induction may explain the pathogenesis of the profound hematopoietic defects of reticular dysgenesis, a disease associated with mutations of the AK2 gene in humans.

Paradoxical effect of mitochondrial respiratory chain impairment on insulin signaling and glucose transport in adipose cells.

Adipocyte function is crucial for the control of whole body energy homeostasis. Pathway analysis of differentiating 3T3-L1 adipocytes reveals that major metabolic pathways induced during differentiation involve mitochondrial function. However, it is not clear why differentiated white adipocytes require enhanced respiratory chain activity relative to pre-adipocytes. To address this question, we used small interference RNA to interfere with the induction of the transcription factor Tfam, which is highly induced between days 2 and 4 of differentiation and is crucial for replication of mitochondrial DNA. Interference with Tfam resulted in cells with decreased respiratory chain capacity, reflected by decreased basal oxygen consumption, and decreased mitochondrial ATP synthesis, but no difference in many other adipocyte functions or expression levels of adipose-specific genes. However, insulin-stimulated GLUT4 translocation to the cell surface and subsequent glucose transport are impaired in Tfam knockdown cells. Paradoxically, insulin-stimulated Akt phosphorylation is significantly enhanced in these cells. These studies reveal independent links between mitochondrial function, insulin signaling, and glucose transport, in which impaired respiratory chain activity enhances insulin signaling to Akt phosphorylation, but impairs GLUT4 translocation. These results indicate that mitochondrial respiratory chain dysfunction in adipocytes can cause impaired insulin responsiveness of GLUT4 translocation by a mechanism downstream of the Akt protein kinase.

Enhanced angiogenesis in obesity and in response to PPARgamma activators through adipocyte VEGF and ANGPTL4 production.

PPARgamma activators such as rosiglitazone (RSG) stimulate adipocyte differentiation and increase subcutaneous adipose tissue mass. However, in addition to preadipocyte differentiation, adipose tissue expansion requires neovascularization to support increased adipocyte numbers. Paradoxically, endothelial cell growth and differentiation is potently inhibited by RSG in vitro, raising the question of how this drug can induce an increase in adipose tissue mass while inhibiting angiogenesis. We find that adipose tissue from mice treated with RSG have increased capillary density. To determine whether adipose tissue angiogenesis was stimulated by RSG, we developed a novel assay to study angiogenic sprout formation ex vivo. Angiogenic sprout formation from equally sized adipose tissue fragments, but not from aorta rings, was greatly increased by obesity and by TZD treatment in vivo. To define the mechanism involved in RSG-stimulated angiogenesis in adipose tissue, the expression of proangiogenic factors by adipocytes was examined. Expression of VEGFA and VEGFB, as well as of the angiopoietin-like factor-4 (ANGPTL4), was stimulated by in vivo treatment with RSG. To define the potential role of these factors, we analyzed their effects on endothelial cell growth and differentiation in vitro. We found that ANGPTL4 stimulates endothelial cell growth and tubule formation, albeit more weakly than VEGF. However, ANGPTL4 mitigates the growth inhibitory actions of RSG on endothelial cells in the presence or absence of VEGF. Thus, the interplay between VEGF and ANGPTL4 could lead to a net expansion of the adipose tissue capillary network, required for adipose tissue growth, in response to PPARgamma activators.

Cidea is associated with lipid droplets and insulin sensitivity in humans.

Storage of energy as triglyceride in large adipose-specific lipid droplets is a fundamental need in all mammals. Efficient sequestration of fat in adipocytes also prevents fatty acid overload in skeletal muscle and liver, which can impair insulin signaling. Here we report that the Cide domain-containing protein Cidea, previously thought to be a mitochondrial protein, colocalizes around lipid droplets with perilipin, a regulator of lipolysis. Cidea-GFP greatly enhances lipid droplet size when ectopically expressed in preadipocytes or COS cells. These results explain previous findings showing that depletion of Cidea with RNAi markedly elevates lipolysis in human adipocytes. Like perilipin, Cidea and the related lipid droplet protein Cidec/FSP27 are controlled by peroxisome proliferator-activated receptor gamma (PPARgamma). Treatment of lean or obese mice with the PPARgamma agonist rosiglitazone markedly up-regulates Cidea expression in white adipose tissue (WAT), increasing lipid deposition. Strikingly, in both omental and s.c. WAT from BMI-matched obese humans, expression of Cidea, Cidec/FSP27, and perilipin correlates positively with insulin sensitivity (HOMA-IR index). Thus, Cidea and other lipid droplet proteins define a novel, highly regulated pathway of triglyceride deposition in human WAT. The data support a model whereby failure of this pathway results in ectopic lipid accumulation, insulin resistance, and its associated comorbidities in humans.

Keystone meeting summary: 'Adipogenesis, obesity, and inflammation' and 'Diabetes mellitus and the control of cellular energy metabolism, ' January 21-26, 2006, Vancouver, Canada.

The dysregulation of specific cellular functions in adipocytes, muscle cells, beta cells, and the liver leads to changes in systemic metabolic processes and ultimately to the pathophysiological manifestations that cause type 2 diabetes. The underlying cellular mechanisms are complex. The two meetings summarized here aimed to highlight the recent advances in our understanding of the molecular basis of feeding and nutrient storage and on the molecular consequences of obesity in terms of promoting risk for type 2 diabetes and cardiovascular disease.

Regulation of the SHP-2 tyrosine phosphatase by a novel cholesterol- and cell confluence-dependent mechanism.

Endothelial cells approaching confluence exhibit marked decreases in tyrosine phosphorylation of receptor tyrosine kinases and adherens junctions proteins, required for cell cycle arrest and adherens junctions stability. Recently, we demonstrated a close correlation in endothelial cells between membrane cholesterol and tyrosine phosphorylation of adherens junctions proteins. Here, we probe the mechanistic basis for this correlation. We find that as endothelial cells reach confluence, the tyrosine phosphatase SHP-2 is recruited to a low-density membrane fraction in a cholesterol-dependent manner. Binding of SHP-2 to this fraction was not abolished by phenyl phosphate, strongly suggesting that this binding was mediated by other regions of SHP-2 beside its SH2 domains. Annexin II, previously implicated in cholesterol trafficking, was associated in a complex with SHP-2, and both proteins localized to adhesion bands in confluent endothelial monolayers. These studies reveal a novel, cholesterol-dependent mechanism for the recruitment of signaling proteins to specific plasma membrane domains via their interactions with annexin II.

Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone.

White adipose tissue is an important endocrine organ involved in the control of whole-body metabolism, insulin sensitivity, and food intake. To better understand these functions, 3T3-L1 cell differentiation was studied by using combined proteomic and genomic strategies. The proteomics approach developed here exploits velocity gradient centrifugation as an alternative to isoelectric focusing for protein separation in the first dimension. A 20- to 30-fold increase in the concentration of numerous mitochondrial proteins was observed during adipogenesis, as determined by mass spectrometry and database correlation analysis. Light and electron microscopy confirmed a large increase in the number of mitochondrion profiles with differentiation. Furthermore, mRNA profiles obtained by using Affymetrix GeneChips revealed statistically significant increases in the expression of many nucleus-encoded mitochondrial genes during adipogenesis. Qualitative changes in mitochondrial composition also occur during adipose differentiation, as exemplified by increases in expression of proteins involved in fatty acid metabolism and of mitochondrial chaperones. Furthermore, the insulin sensitizer rosiglitazone caused striking changes in mitochondrial shape and expression of selective mitochondrial proteins. Thus, although mitochondrial biogenesis has classically been associated with brown adipocyte differentiation and thermogenesis, our results reveal that mitochondrial biogenesis and remodeling are inherent to adipose differentiation per se and are influenced by the actions of insulin sensitizers.