Mary-Elizabeth Patti

Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1

Type 2 diabetes mellitus (DM) is characterized by insulin resistance and pancreatic β cell dysfunction. In high-risk subjects, the earliest detectable abnormality is insulin resistance in skeletal muscle. Impaired insulin-mediated signaling, gene expression, glycogen synthesis, and accumulation of intramyocellular triglycerides have all been linked with insulin resistance, but no specific defect responsible for insulin resistance and DM has been identified in humans. To identify genes potentially important in the pathogenesis of DM, we analyzed gene expression in skeletal muscle from healthy metabolically characterized nondiabetic (family history negative and positive for DM) and diabetic Mexican–American subjects. We demonstrate that insulin resistance and DM associate with reduced expression of multiple nuclear respiratory factor-1 (NRF-1)-dependent genes encoding key enzymes in oxidative metabolism and mitochondrial function. Although NRF-1 expression is decreased only in diabetic subjects, expression of both PPARγ coactivator 1-α and-β (PGC1-α/PPARGC1 and PGC1-β/PERC), coactivators of NRF-1 and PPARγ-dependent transcription, is decreased in both diabetic subjects and family history-positive nondiabetic subjects. Decreased PGC1 expression may be responsible for decreased expression of NRF-dependent genes, leading to the metabolic disturbances characteristic of insulin resistance and DM.

Insulin resistance differentially affects the PI 3-kinase– and MAP kinase–mediated signaling in human muscle

The broad nature of insulin resistant glucose metabolism in skeletal muscle of patients with type 2 diabetes suggests a defect in the proximal part of the insulin signaling network. We sought to identify the pathways compromised in insulin resistance and to test the effect of moderate exercise on whole-body and cellular insulin action. We conducted euglycemic clamps and muscle biopsies on type 2 diabetic patients, obese nondiabetics and lean controls, with and without a single bout of exercise. Insulin stimulation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway, as measured by phosphorylation of the insulin receptor and IRS-1 and by IRS protein association with p85 and with PI 3-kinase, was dramatically reduced in obese nondiabetics and virtually absent in type 2 diabetic patients. Insulin stimulation of the MAP kinase pathway was normal in obese and diabetic subjects. Insulin stimulation of glucose-disposal correlated with association of p85 with IRS-1. Exercise 24 hours before the euglycemic clamp increased phosphorylation of insulin receptor and IRS-1 in obese and diabetic subjects but did not increase glucose uptake or PI 3-kinase association with IRS-1 upon insulin stimulation. Thus, insulin resistance differentially affects the PI 3-kinase and MAP kinase signaling pathways, and insulin-stimulated IRS-1-association with PI 3-kinase defines a key step in insulin resistance.

Bidirectional modulation of insulin action by amino acids.

Amino acids have been shown to stimulate protein synthesis, inhibit proteolysis, and decrease whole-body and forearm glucose disposal. Using cultured hepatoma and myotube cells, we demonstrate that amino acids act as novel signaling elements in insulin target tissues. Exposure of cells to high physiologic concentrations of amino acids activates intermediates important in the initiation of protein synthesis, including p70 S6 kinase and PHAS-I, in synergy with insulin. This stimulatory effect is largely due to branched chain amino acids, particularly leucine, and can be reproduced by its transamination product, ketoisocaproic acid. Concurrently, amino acids inhibit early steps in insulin action critical for glucose transport and inhibition of gluconeogenesis, including decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2, decreased binding of grb 2 and the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2, and a marked inhibition of insulin-stimulated phosphatidylinositol 3-kinase. Taken together, these data support the hypothesis that amino acids act as specific positive signals for maintenance of protein stores, while inhibiting other actions of insulin at multiple levels. This bidirectional modulation of insulin action indicates crosstalk between hormonal and nutritional signals and demonstrates a novel mechanism by which nutritional factors contribute to insulin resistance.

Serum Bile Acids Are Higher in Humans With Prior Gastric Bypass: Potential Contribution to Improved Glucose and Lipid Metabolism

The multifactorial mechanisms promoting weight loss and improved metabolism following Roux‐en‐Y gastric bypass (GB) surgery remain incompletely understood. Recent rodent studies suggest that bile acids can mediate energy homeostasis by activating the G‐protein coupled receptor TGR5 and the type 2 thyroid hormone deiodinase. Altered gastrointestinal anatomy following GB could affect enterohepatic recirculation of bile acids. We assessed whether circulating bile acid concentrations differ in patients who previously underwent GB, which might then contribute to improved metabolic homeostasis. We performed cross‐sectional analysis of fasting serum bile acid composition and both fasting and post‐meal metabolic variables, in three subject groups: (i) post‐GB surgery (n = 9), (ii) without GB matched to preoperative BMI of the index cohort (n = 5), and (iii) without GB matched to current BMI of the index cohort (n = 10). Total serum bile acid concentrations were higher in GB (8.90 ± 4.84 µmol/l) than in both overweight (3.59 ± 1.95, P = 0.005, Ov) and severely obese (3.86 ± 1.51, P = 0.045, MOb). Bile acid subfractions taurochenodeoxycholic, taurodeoxycholic, glycocholic, glycochenodeoxycholic, and glycodeoxycholic acids were all significantly higher in GB compared to Ov (P < 0.05). Total bile acids were inversely correlated with 2‐h post‐meal glucose (r = −0.59, P < 0.003) and fasting triglycerides (r = −0.40, P = 0.05), and positively correlated with adiponectin (r = −0.48, P < 0.02) and peak glucagon‐like peptide‐1 (GLP‐1) (r = 0.58, P < 0.003). Total bile acids strongly correlated inversely with thyrotropic hormone (TSH) (r = −0.57, P = 0.004). Together, our data suggest that altered bile acid levels and composition may contribute to improved glucose and lipid metabolism in patients who have had GB.

Severe hypoglycaemia post-gastric bypass requiring partial pancreatectomy: evidence for inappropriate insulin secretion and pancreatic islet hyperplasia.

Aims/hypothesisPostprandial hypoglycaemia following gastric bypass for obesity is considered a late manifestation of the dumping syndrome and can usually be managed with dietary modification. We investigated three patients with severe postprandial hypoglycaemia and hyperinsulinaemia unresponsive to diet, octreotide and diazoxide with the aim of elucidating the pathological mechanisms involved.MethodsGlucose, insulin, and C-peptide were measured in the fasting and postprandial state, and insulin secretion was assessed following selective intra-arterial calcium injection. Pancreas histopathology was assessed in all three patients.ResultsAll three patients had evidence of severe postprandial hyperinsulinaemia and hypoglycaemia. In one patient, reversal of gastric bypass was ineffective in reversing hypoglycaemia. All three patients ultimately required partial pancreatectomy for control of neuroglycopenia; pancreas pathology of all patients revealed diffuse islet hyperplasia and expansion of beta cell mass.Conclusions/interpretationThese findings suggest that gastric bypass-induced weight loss may unmask an underlying beta cell defect or contribute to pathological islet hyperplasia, perhaps via glucagon-like peptide 1-mediated pathways.

The Role of Mitochondria in the Pathogenesis of Type 2 Diabetes

The pathophysiology of type 2 diabetes mellitus (DM) is varied and complex. However, the association of DM with obesity and inactivity indicates an important, and potentially pathogenic, link between fuel and energy homeostasis and the emergence of metabolic disease. Given the central role for mitochondria in fuel utilization and energy production, disordered mitochondrial function at the cellular level can impact whole-body metabolic homeostasis. Thus, the hypothesis that defective or insufficient mitochondrial function might play a potentially pathogenic role in mediating risk of type 2 DM has emerged in recent years. Here, we summarize current literature on risk factors for diabetes pathogenesis, on the specific role(s) of mitochondria in tissues involved in its pathophysiology, and on evidence pointing to alterations in mitochondrial function in these tissues that could contribute to the development of DM. We also review literature on metabolic phenotypes of existing animal models of impaired mitochondrial function. We conclude that, whereas the association between impaired mitochondrial function and DM is strong, a causal pathogenic relationship remains uncertain. However, we hypothesize that genetically determined and/or inactivity-mediated alterations in mitochondrial oxidative activity may directly impact adaptive responses to overnutrition, causing an imbalance between oxidative activity and nutrient load. This imbalance may lead in turn to chronic accumulation of lipid oxidative metabolites that can mediate insulin resistance and secretory dysfunction. More refined experimental strategies that accurately mimic potential reductions in mitochondrial functional capacity in humans at risk for diabetes will be required to determine the potential pathogenic role in human insulin resistance and type 2 DM.

The Emerging Genetic Architecture of Type 2 Diabetes

Type 2 diabetes is a genetically heterogeneous disease, with several relatively rare monogenic forms and a number of more common forms resulting from a complex interaction of genetic and environmental factors. Previous studies using a candidate gene approach, family linkage studies, and gene expression profiling uncovered a number of type 2 genes, but the genetic basis of common type 2 diabetes remained unknown. Recently, a new window has opened on defining potential type 2 diabetes genes through genome-wide SNP association studies of very large populations of individuals with diabetes. This review explores the pathway leading to discovery of these genetic effects, the impact of these genetic loci on diabetes risk, the potential mechanisms of action of the genes to alter glucose homeostasis, and the limitations of these studies in defining the role of genetics in this important disease.

In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism

Introduction The rapid global rise in metabolic disease suggests that nongenetic environmental factors contribute to disease risk. Early life represents a window of phenotypic plasticity important for adult metabolic health and that of future generations. Epigenetic inheritance has been implicated in the paternal transmission of environmentally induced phenotypes, but the mechanisms responsible remain unknown. In utero undernourishment alters the adult germ cell methylome. Undernourishment during PGC reprogramming results in hypomethylation of discrete loci in adult sperm. These regions are enriched in nucleosomes and are low-methylated regions. Although partially resistant to blastocyst reprogramming, differential methylation does not persist in the next generation. However, dysregulated expression of genes neighboring DMRs is observed in F2 offspring. Rationale We investigated the role of DNA methylation in epigenetic inheritance in an established murine model of intergenerational developmental programming. F1 offspring of undernourished dams (UN) have low birth weight and multiple metabolic defects. Metabolic phenotypic inheritance to the F2 generation is observed through the paternal line, even though the F1 mice did not experience postnatal environmental perturbation. The timing of nutritional restriction coincides with methylation reacquisition in F1 male primordial germ cells (PGCs). Therefore, we assessed F1 sperm whole-genome methylation using immunoprecipitation of methylated DNA, combined with high-throughput sequencing, followed by independent validation. We characterized the regions susceptible to methylation change and investigated the legacy of such methylation change in the phenotypic development of the next generation. Results In UN mice, 111 regions are hypomethylated relative to control sperm, and these changes are validated by bisulfite pyrosequencing. Methylation differences span multiple CpGs, with robust absolute changes of 10 to 30% (relative reduction ~50%). The absolute methylation level is consistent with differentially methylated regions (DMRs) being “low-methylated regions,” known to be enriched in regulatory elements. Indeed, luciferase assays suggest a role for these DMRs in transcriptional regulation. Hypomethylated DMRs are significantly depleted from coding and repetitive regions and enriched in intergenic regions and CpG islands. They are also enriched in nucleosome-retaining regions, which suggests that, at some loci, paternal germline hypomethylation induced by in utero undernutrition is transmitted in a chromatin context. DMRs are late to regain methylation in normal male PGCs. This may render them particularly susceptible to environmental perturbations that delay or impair remethylation in late gestation. Except for imprinted loci, gene-associated male germline methylation has generally been thought to be largely erased in the zygote,although recent studies suggest that resistance to reprogramming is more widespread. Indeed, 43% of hypomethylated DMRs persist and thus have the potential to affect development of the next generation. We show that differential methylation is lost in late-gestation F2 tissues, but considerable tissue-specific differences in expression of metabolic genes neighboring DMRs are present. Thus, it is unlikely that these expression changes are directly mediated by altered methylation; rather, the cumulative effects of dysregulated epigenetic patterns earlier in development may yield sustained alterations in chromatin architecture, transcriptional regulatory networks, differentiation, or tissue structure. Conclusion Prenatal undernutrition can compromise male germline epigenetic reprogramming and thus permanently alter DNA methylation in the sperm of adult offspring at regions resistant to zygotic reprogramming. However, persistence of altered DNA methylation into late-gestation somatic tissues of the subsequent generation is not observed. Nonetheless, alterations in gamete methylation may serve as a legacy of earlier developmental exposures and may contribute to the intergenerational transmission of environmentally induced disease. The nutritional sins of the mother… Prenatal exposures of a mother can affect the health of her offspring, but how? Radford et al. found that the male progeny of undernourished pregnant mice had altered DNA chemistry in their sperm. In addition, the offspring displayed compromised metabolic health. The specific affected genes not only lost DNA methylation but also lacked the normal sperm DNA packaging factors (protamines) and instead were enriched in nucleosomes. Thus, when subjected to a suboptimal prenatal environment, offspring feel the effects of the maternal assault. Science, this issue p. 10.1126/science.1255903 Prenatal assaults change DNA methylation and chromatin structure in sperm and affect offspring. [Also see Perspective by Susiarjo and Bartolomei] Adverse prenatal environments can promote metabolic disease in offspring and subsequent generations. Animal models and epidemiological data implicate epigenetic inheritance, but the mechanisms remain unknown. In an intergenerational developmental programming model affecting F2 mouse metabolism, we demonstrate that the in utero nutritional environment of F1 embryos alters the germline DNA methylome of F1 adult males in a locus-specific manner. Differentially methylated regions are hypomethylated and enriched in nucleosome-retaining regions. A substantial fraction is resistant to early embryo methylation reprogramming, which may have an impact on F2 development. Differential methylation is not maintained in F2 tissues, yet locus-specific expression is perturbed. Thus, in utero nutritional exposures during critical windows of germ cell development can impact the male germline methylome, associated with metabolic disease in offspring.

Intergenerational Transmission of Glucose Intolerance and Obesity by In Utero Undernutrition in Mice

OBJECTIVE—Low birth weight (LBW) is associated with increased risk of obesity, diabetes, and cardiovascular disease during adult life. Moreover, this programmed disease risk can progress to subsequent generations. We previously described a mouse model of LBW, produced by maternal caloric undernutrition (UN) during late gestation. LBW offspring (F1-UN generation) develop progressive obesity and impaired glucose tolerance (IGT) with aging. We aimed to determine whether such metabolic phenotypes can be transmitted to subsequent generations in an experimental model, even in the absence of altered nutrition during the second pregnancy. RESEARCH DESIGN AND METHODS—We intercrossed female and male F1 adult control (C) and UN mice and characterized metabolic phenotypes in F2 offspring. RESULTS—We demonstrate that 1) reduced birth weight progresses to F2 offspring through the paternal line (C♀-C♂ = 1.64 g; C♀-UN♂ = 1.57 g, P < 0.05; UN♀-C♂ = 1.64 g; UN♀-UN♂ = 1.60 g, P < 0.05), 2) obesity progresses through the maternal line (percent body fat: C♀-C♂ = 22.4%; C♀-UN♂ = 22.9%; UN♀-C♂ = 25.9%, P < 0.05; UN♀-UN♂ = 27.5%, P < 0.05), and 3) IGT progresses through both parental lineages (glucose tolerance test area under curve C♀-C♂ = 100; C♀-UN♂ = 122, P < 0.05; UN♀-C♂ = 131, P < 0.05; UN♀-UN♂ = 151, P < 0.05). Mechanistically, IGT in both F1 and F2 generations is linked to impaired β-cell function, explained, in part, by dysregulation of Sur1 expression. CONCLUSIONS—Maternal undernutrition during pregnancy (F0) programs reduced birth weight, IGT, and obesity in both first- and second-generation offspring. Sex-specific transmission of phenotypes implicates complex mechanisms including alterations in the maternal metabolic environment (transmaternal inheritance of obesity), gene expression mediated by developmental and epigenetic pathways (transpaternal inheritance of LBW), or both (IGT).

Peroxisome Proliferator Activator Receptor γ Coactivator-1 Expression Is Reduced in Obesity POTENTIAL PATHOGENIC ROLE OF SATURATED FATTY ACIDS AND p38 MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION

Peroxisome proliferator activator receptor-γ coactivator 1 (PGC-1) is a major candidate gene for diabetes-related metabolic phenotypes, contributing to decreased expression of nuclear-encoded mitochondrial genes in muscle and adipose tissue. We have demonstrated that muscle expression of PGC-1α and -β is reduced in both genetic (Lepob/Lepob) and acquired obesity (high fat diet). In C57BL6 mice, muscle PGC-1α expression decreased by 43% (p < 0.02) after 1 week of a high fat diet and persisted more than 11 weeks. In contrast, PGC-1α reductions were not sustained in obesity-resistant A/J mice. To identify mediators of obesity-linked reductions in PGC-1, we tested the effects of cellular nutrients in C2C12 myotubes. Although overnight exposure to high insulin, glucose, glucosamine, or amino acids had no effect, saturated fatty acids potently reduced PGC-1α and -β mRNA expression. Palmitate decreased PGC-1α and -β expression by 38% (p = 0.01) and 53% (p = 0.006); stearate similarly decreased expression of PGC-1α and -β by 22% (p = 0.02) and 39% (p = 0.02). These effects were mediated at a transcriptional level, as indicated by an 11-fold reduction of PGC-1α promoter activity by palmitate and reversal of effects by histone deacetylase inhibition. Palmitate also (a) reduced expression of tricarboxylic acid cycle and oxidative phosphorylation mitochondrial genes and (b) reduced oxygen consumption. These effects were reversed by overexpression of PGC-1α or -β, indicating PGC-1 dependence. Palmitate effects also required p38 MAPK, as demonstrated by 1) palmitate-induced increase in p38 MAPK phosphorylation, 2) reversal of palmitate effects on PGC-1 and mitochondrial gene expression by p38 MAPK inhibitors, and 3) reversal of palmitate effects by small interfering RNA-mediated decreases in p38α MAPK. These data indicate that obesity and saturated fatty acids decrease PGC-1 and mitochondrial gene expression and function via p38 MAPK-dependent transcriptional pathways.