Featured ArticlesVolume 5 | No. 3 | March 2016
|Altered DNA methylation of glycolytic and lipogenic genes in liverKirchner and colleagues show that mRNA expression of targets along the glycolytic pathway are increased in the liver of obese people, concomitant with DNA hypomethylation of the respective genes at an important ATF-motif regulatory site. Constitutive activation of these pathways is associated with protein kinase C epsilon activation and hypomethylation, which may lead to the development of hepatic insulin resistance and hepatic steatosis.|
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Method: We performed genome-wide methylome and transcriptome analysis in liver from severely obese men with or without type 2 diabetes and non-obese men to discover aberrant pathways underlying the development of insulin resistance. Results were validated by pyrosequencing.
Result: We identified hypomethylation of genes involved in hepatic glycolysis and insulin resistance, concomitant with increased mRNA expression and protein levels. Pyrosequencing revealed the CpG-site within ATF-motifs was hypomethylated in four of these genes in liver of severely obese non-diabetic and type 2 diabetic patients, suggesting epigenetic regulation of transcription by altered ATF-DNA binding.
Conclusion: Severely obese non-diabetic and type 2 diabetic patients have distinct alterations in the hepatic methylome and transcriptome, with hypomethylation of several genes controlling glucose metabolism within the ATF-motif regulatory site. Obesity appears to shift the epigenetic program of the liver towards increased glycolysis and lipogenesis, which may exacerbate the development of insulin resistance.[Hide abstract]
|High-fat diet reprograms the epigenome of rat spermatozoaThe results of Barbosa et al. provide insight into mechanisms by which high-fat diet reprograms the epigenome of sperm cells and affects the metabolic phenotype of the offspring throughout several generations. The authors also provide evidence that altered miRNA let-7c expression in sperm is passed down to the adipose tissue of the offspring, leading to a transcriptomic shift in let-7c predicted targets. In conclusion, reprogramming of sperm let-7c expression may constitute a mechanism by which high-fat diet can alter the metabolism of the offspring.|
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Objectives: Chronic and high consumption of fat constitutes an environmental stress that leads to metabolic diseases. We hypothesized that high-fat diet (HFD) transgenerationally remodels the epigenome of spermatozoa and metabolism of the offspring.
Methods: F0-male rats fed either HFD or chow diet for 12 weeks were mated with chow-fed dams to generate F1 and F2 offspring. Motile spermatozoa were isolated from F0 and F1 breeders to determine DNA methylation and small non-coding RNA (sncRNA) expression pattern by deep sequencing.
Results: Newborn offspring of HFD-fed fathers had reduced body weight and pancreatic beta-cell mass. Adult female, but not male, offspring of HFD-fed fathers were glucose intolerant and resistant to HFD-induced weight gain. This phenotype was perpetuated in the F2 progeny, indicating transgenerational epigenetic inheritance. The epigenome of spermatozoa from HFD-fed F0 and their F1 male offspring showed common DNA methylation and small non-coding RNA expression signatures. Altered expression of sperm miRNA let-7c was passed down to metabolic tissues of the offspring, inducing a transcriptomic shift of the let-7c predicted targets.
Conclusion: Our results provide insight into mechanisms by which HFD transgenerationally reprograms the epigenome of sperm cells, thereby affecting metabolic tissues of offspring throughout two generations.[Hide abstract]
|Undernutrition delays a key step in the maturation of hypothalamic feeding circuitsDe Solis and colleagues explore whether effects of moderate postnatal undernutrition (UN) to increase post-weaning growth rates are associated with impairments in the maturation of systems that provide negative feedback to the orexigenic actions of NPY/AgRP/GABA (NAG) neurons. They find that UN delays the development of the homeostatic regulation of feeding, which is tightly correlated with the onset of potassium (KATP) channel expression and the maturation of electrophysiological properties of NAG neurons.|
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Objective: Humans and animals exposed to undernutrition (UN) during development often experience accelerated "catch-up" growth when food supplies are plentiful. Little is known about the mechanisms regulating early growth rates. We previously reported that actions of leptin and presynaptic inputs to orexigenic NPY/AgRP/GABA (NAG) neurons in the arcuate nucleus of the hypothalamus are almost exclusively excitatory during the lactation period, since neuronal and humoral inhibitory systems do not develop until after weaning. Moreover, we identified a critical step that regulates the maturation of electrophysiological responses of NAG neurons at weaning - the onset of genes encoding ATP-dependent potassium (KATP) channel subunits. We explored the possibility that UN promotes subsequent catch-up growth, in part, by delaying the maturation of negative feedback systems to neuronal circuits driving food intake.
Methods: We used the large litter (LL) size model to study the impacts of postnatal UN followed by catch-up growth. We evaluated the maturation of presynaptic and postsynaptic inhibitory systems in NAG neurons using a combination of electrophysiological and molecular criteria, in conjunction with leptin's ability to suppress fasting-induced hyperphagia.
Results: The onset of KATP channel subunit expression and function, the switch in leptin's effect on NAG neurons, the ingrowth of inhibitory inputs to NAG neurons, and the development of homeostatic feedback to feeding circuits were delayed in LL offspring relative to controls. The development of functional KATP channels and the establishment of leptin-mediated suppression of food intake in the peri-weaning period were tightly linked and were not initiated until growth and adiposity of LL offspring caught up to controls.
Conclusion: Our data support the idea that initiation of KATP channel subunit expression in NAG neurons serves as a molecular gatekeeper for the maturation of homeostatic feeding circuits.[Hide abstract]
|Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivityBowman et al. demonstrate in a newly generated line of acyl-CoA synthetase 5 (ACSL5) deficient mice that the in vivo consequences of ACSL ablation are reduced adiposity, circulating triglyceride and glucose levels, increased insulin sensitivity and energy expenditure, and reduced rate of intestinal triglyceride absorption. They find potent increases in hepatic mRNA expression and circulating serum levels of fibroblast growth factor 21 (FGF21), The increased circulating level of FGF21 is associated with the increased mRNA levels of Ucp1, consistent with effects of FGF21 to promote conversion of white to beige adipocytes, leading to increased rates of energy expenditure.|
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Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ACSL isoforms. In vitro studies have suggested a role for ACSL5 in triglyceride synthesis; however, we have limited understanding of the in vivo actions of this ACSL isoform.
Methods: To elucidate the in vivo actions of ACSL5 we generated a line of mice in which ACSL5 expression was ablated in all tissues (ACSL5-/-).
Results: Ablation of ACSL5 reduced ACSL activity by ~80% in jejunal mucosa, ~50% in liver, and ~37% in brown adipose tissue lysates. Body composition studies revealed that ACSL5-/-, as compared to control ACSL5loxP/loxP, mice had significantly reduced fat mass and adipose fat pad weights. Indirect calorimetry studies demonstrated that ACSL5-/- had increased metabolic rates, and in the dark phase, increased respiratory quotient. In ACSL5-/- mice, fasting glucose and serum triglyceride were reduced; and insulin sensitivity was improved during an insulin tolerance test. Both hepatic mRNA (~16-fold) and serum levels of fibroblast growth factor 21 (FGF21) (~13-fold) were increased in ACSL5-/- as compared to ACSL5loxP/loxP. Consistent with increased FGF21 serum levels, uncoupling protein-1 gene (Ucp1) and PPAR-gamma coactivator 1-alpha gene (Pgc1a) transcript levels were increased in gonadal adipose tissue. To further evaluate ACSL5 function in intestine, mice were gavaged with an olive oil bolus; and the rate of triglyceride appearance in serum was found to be delayed in ACSL5-/- mice as compared to control mice.
Conclusion: In summary, ACSL5-/- mice have increased hepatic and serum FGF21 levels, reduced adiposity, improved insulin sensitivity, increased energy expenditure and delayed triglyceride absorption. These studies suggest that ACSL5 is an important regulator of whole-body energy metabolism and ablation of ACSL5 may antagonize the development of obesity and insulin resistance.[Hide abstract]
|Impaired glucose metabolism with muscle-specific glycogen synthase 1 deletionXirouchaki, Mangiafico and colleagues suggest that muscle glycogen synthase 1 (gys1) plays a major role in muscle glycogen synthesis and non-oxidative glucose metabolism as a muscle-specific gys1 deletion in the adult mouse can contribute to peripheral insulin resistance and glucose intolerance. In addition, gys1 is crucial for muscle function during contraction, exercise and endurance capacity as muscle glycogen depletion is a leading factor in impaired muscle performance and fatigue development during exercise.|
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Objective: Muscle glucose storage and muscle glycogen synthase (gys1) defects have been associated with insulin resistance. As there are multiple mechanisms for insulin resistance, the specific role of glucose storage defects is not clear. The aim of this study was to examine the effects of muscle-specific gys1 deletion on glucose metabolism and exercise capacity.
Methods: Tamoxifen inducible and muscle specific gys-1 KO mice were generated using the Cre/loxP system. Mice were subjected to glucose tolerance tests, euglycemic/hyperinsulinemic clamps and exercise tests.
Results: gys1-KO mice showed =85% reduction in muscle gys1 mRNA and protein concentrations, 70% reduction in muscle glycogen levels, postprandial hyperglycaemia and hyperinsulinaemia and impaired glucose tolerance. Under insulin-stimulated conditions, gys1-KO mice displayed reduced glucose turnover and muscle glucose uptake, indicative of peripheral insulin resistance, as well as increased plasma and muscle lactate levels and reductions in muscle hexokinase II levels. gys1-KO mice also exhibited markedly reduced exercise and endurance capacity.
Conclusions: Thus, muscle-specific gys1 deletion in adult mice results in glucose intolerance due to insulin resistance and reduced muscle glucose uptake as well as impaired exercise and endurance capacity.
In brief: This study demonstrates why the body prioritises muscle glycogen storage over liver glycogen storage despite the critical role of the liver in supplying glucose to the brain in the fasting state and shows that glycogen deficiency results in impaired glucose metabolism and reduced exercise capacity.[Hide abstract]
|Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genesAckermann and colleagues provide a novel resource for identifying open chromatin regions in human α-, β-, and acinar cells. This dataset builds upon the growing body of genome-wide epigenetic studies performed in purified human α- and β-cells. They show that integrating ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing) data with other epigenetic information enhances interpretation of computational results, and they anticipate that this ATAC-seq dataset will be useful for integration with future genomic analyses of human islets.|
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Objective: Although glucagon-secreting α-cells and insulin-secreting β-cells have opposing functions in regulating plasma glucose levels, the two cell types share a common developmental origin and exhibit overlapping transcriptomes and epigenomes. Notably, destruction of β-cells can stimulate repopulation via transdifferentiation of α-cells, at least in mice, suggesting plasticity between these cell fates. Furthermore, dysfunction of both α- and β-cells contributes to the pathophysiology of type 1 and type 2 diabetes, and β-cell de-differentiation has been proposed to contribute to type 2 diabetes. Our objective was to delineate the molecular properties that maintain islet cell type specification yet allow for cellular plasticity. We hypothesized that correlating cell type-specific transcriptomes with an atlas of open chromatin will identify novel genes and transcriptional regulatory elements such as enhancers involved in α- and β-cell specification and plasticity.
Methods: We sorted human α- and β-cells and performed the "Assay for Transposase-Accessible Chromatin with high throughput sequencing" (ATAC-seq) and mRNA-seq, followed by integrative analysis to identify cell type-selective gene regulatory regions.
Result: We identified numerous transcripts with either α-cell- or β-cell-selective expression and discovered the cell type-selective open chromatin regions that correlate with these gene activation patterns. We confirmed cell type-selective expression on the protein level for two of the top hits from our screen. The "group specific protein" (GC; or vitamin D binding protein) was restricted to α-cells, while CHODL (chondrolectin) immunoreactivity was only present in β-cells. Furthermore, α-cell- and β-cell-selective ATAC-seq peaks were identified to overlap with known binding sites for islet transcription factors, as well as with single nucleotide polymorphisms (SNPs) previously identified as risk loci for type 2 diabetes.
Conclusions: We have determined the genetic landscape of human α- and β-cells based on chromatin accessibility and transcript levels, which allowed for detection of novel α- and β-cell signature genes not previously known to be expressed in islets. Using fine-mapping of open chromatin, we have identified thousands of potential cis-regulatory elements that operate in an endocrine cell type-specific fashion.[Hide abstract]
|Sex difference in physical activity driven by hypothalamic POMC neuronsMelanocortin peptides encoded by the pro-opiomelanocortin gene (Pomc) are key regulators of energy balance. The findings of Burke and Doslikova et al. support the functional heterogeneity of hypothalamic POMC neurons, revealing that the source synthesized within 5-hydroxytryptamine 2c receptor expressing neurons is sufficient to regulate energy intake and insulin sensitivity in male and female mice. Moreover, these data provide evidence for a specific neurochemical basis for levels of reduced physical activity and reveal that the molecular underpinnings of the impetus to engage in physical activity are differentially modulated in males and females.|
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Objective: Obesity is one of the primary healthcare challenges of the 21st century. Signals relaying information regarding energy needs are integrated within the brain to influence body weight. Central among these integration nodes are the brain pro-opiomelanocortin (POMC) peptides, perturbations of which disrupt energy balance and promote severe obesity. However, POMC neurons are neurochemically diverse and the crucial source of POMC peptides that regulate energy homeostasis and body weight remains to be fully clarified.
Methods: Given that a 5-hydroxytryptamine 2c receptor (5-HT2CR) agonist is a current obesity medication and 5-HT2CR agonist's effects on appetite are primarily mediated via POMC neurons, we hypothesized that a critical source of POMC regulating food intake and body weight is specifically synthesized in cells containing 5-HT2CRs. To exclusively manipulate Pomc synthesis only within 5-HT2CR containing cells, we generated a novel 5-HT2CRCRE mouse line and intercrossed it with Cre recombinase-dependent and hypothalamic specific reactivatable PomcNEO mice to restrict Pomc synthesis to the subset of hypothalamic cells containing 5-HT2CRs. This provided a means to clarify the specific contribution of a defined subgroup of POMC peptides in energy balance and body weight.
Results: Here we transform genetically programed obese and hyperinsulinemic male mice lacking hypothalamic Pomc with increased appetite, reduced physical activity and compromised brown adipose tissue (BAT) into lean, healthy mice via targeted restoration of Pomc function only within 5-HT2CR expressing cells. Remarkably, the same metabolic transformation does not occur in females, who despite corrected feeding behavior and normalized insulin levels remain physically inactive, have lower energy expenditure, compromised BAT and develop obesity.
Conclusions: These data provide support for the functional heterogeneity of hypothalamic POMC neurons, revealing that Pomc expression within 5-HT2CR expressing neurons is sufficient to regulate energy intake and insulin sensitivity in male and female mice. However, an unexpected sex difference in the function of this subset of POMC neurons was identified with regard to energy expenditure. We reveal that a large sex difference in physical activity, energy expenditure and the development of obesity is driven by this subpopulation, which constitutes approximately 40% of all POMC neurons in the hypothalamic arcuate nucleus. This may have broad implications for strategies utilized to combat obesity, which at present largely ignore the sex of the obese individual. [Hide abstract]