Metabolic dysregulation in the Atp7b-/- Wilson's disease mouse model.

Inactivating mutations in the copper transporter Atp7b result in Wilson's disease. The Atp7b-/- mouse develops hallmarks of Wilson's disease. The activity of several nuclear receptors decreased in Atp7b-/- mice, and nuclear receptors are critical for maintaining metabolic homeostasis. Therefore, we anticipated that Atp7b-/- mice would exhibit altered progression of diet-induced obesity, fatty liver, and insulin resistance. Following 10 wk on a chow or Western-type diet (40% kcal fat), parameters of glucose and lipid homeostasis were measured. Hepatic metabolites were measured by liquid chromatography-mass spectrometry and correlated with transcriptomic data. Atp7b-/- mice fed a chow diet presented with blunted body-weight gain over time, had lower fat mass, and were more glucose tolerant than wild type (WT) littermate controls. On the Western diet, Atp7b-/- mice exhibited reduced body weight, adiposity, and hepatic steatosis compared with WT controls. Atp7b-/- mice fed either diet were more insulin sensitive than WT controls; however, fasted Atp7b-/- mice exhibited hypoglycemia after administration of insulin due to an impaired glucose counterregulatory response, as evidenced by reduced hepatic glucose production. Coupling gene expression with metabolomic analyses, we observed striking changes in hepatic metabolic profiles in Atp7b-/- mice, including increases in glycolytic intermediates and components of the tricarboxylic acid cycle. In addition, the active phosphorylated form of AMP kinase was significantly increased in Atp7b-/- mice relative to WT controls. Alterations in hepatic metabolic profiles and nuclear receptor signaling were associated with improved glucose tolerance and insulin sensitivity as well as with impaired fasting glucose production in Atp7b-/- mice.

Ghrelin receptor regulates appetite and satiety during aging in mice by regulating meal frequency and portion size but not total food intake.

Aging is often associated with overweight and obesity. There exists a long-standing debate about whether meal pattern also contributes to the development of obesity. The orexigenic hormone ghrelin regulates appetite and satiety by activating its receptor, growth hormone secretagogue receptor (GHS-R). In mice, circulating ghrelin concentrations and brain GHS-R expression were shown to increase with aging. To assess whether GHS-R regulates feeding pattern during aging, we studied meal patterns for the following cohorts of male mice fed a normal unpurified diet: 1) 3-4 mo, young wild-type (WT) mice; 2) 3-4 mo, young Ghsr-null (Ghsr(-/-)) mice; 3) 12-14 mo, middle-aged WT (WT-M) mice; 4) 12-14 mo, middle-aged Ghsr(-/-) (Ghsr(-/-)-M) mice; 5) 24-26 mo, old WT (WT-O) mice; and 6) 24-26 mo, old Ghsr(-/-) (Ghsr(-/-)-O) mice. Although the total daily food intake of Ghsr(-/-) mice was similar to that of WT controls, Ghsr(-/-)-M and Ghsr(-/-)-O mice had 9% (P = 0.07) and 16% (P < 0.05) less body weight compared with WT-M and WT-O mice, respectively, primarily due to reduced fat mass (P < 0.05, WT-M vs. Ghsr(-/-)-M and WT-O vs. Ghsr(-/-)-O). Intriguingly, Ghsr(-/-)-M mice ate larger meals (on average, Ghsr(-/-)-M mice ate 0.117 g/meal and WT-M mice ate 0.080 g/meal; P < 0.01) and took a longer time to eat (Ghsr(-/-)-M, 196.0 s and WT-M, 128.9 s; P < 0.01), but ate less frequently (Ghsr(-/-)-M, 31.0 times/d and WT-M, 42.3 times/d; P < 0.05) than WT-M controls. In addition, we found that expression of hypothalamic orexigenic peptides, neuropeptide Y (NPY) and agouti-related peptide (AgRP), was relatively lower in aged WT mice (P = 0.09 for NPY and P = 0.06 for AgRP), but anorexic peptide pro-opiomelanocortin (POMC) expression remained unchanged between the WT age groups. Interestingly, old Ghsr(-/-) mice had greater hypothalamic NPY expression (102% higher; P < 0.05) and AgRP expression (P = 0.07) but significantly lower POMC expression (P < 0.05) when compared with age-matched WT-O controls. Thus, our results indicate that GHS-R plays an important role in the regulation of meal pattern and that GHS-R ablation may modulate feeding behavior through the regulation of hypothalamic neuropeptides. Our results collectively suggest that ghrelin receptor antagonism may have a beneficial effect on metabolism during aging.

Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress.

Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Our preliminary analyses uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. Motivated to understand how BAT function is regulated epigenetically, we developed a novel method for the first-ever unbiased top-down proteomic quantitation of histone modifications in BAT and validated our results with a multi-omic approach. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28.8°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C) and BAT was analyzed for DNA methylation and histone modifications. Methylation of promoters and intragenic regions in genomic DNA decrease in response to chronic cold exposure. Integration of DNA methylation and RNA expression data suggest a role for epigenetic modification of DNA in gene regulation in response to cold. In response to cold housing, we observe increased bulk acetylation of histones H3.2 and H4, increased histone H3.2 proteoforms with di- and trimethylation of lysine 9 (K9me2 and K9me3), and increased histone H4 proteoforms with acetylation of lysine 16 (K16ac) in BAT. Taken together, our results reveal global epigenetically-regulated transcriptional "on" and "off" signals in murine BAT in response to varying degrees of chronic cold stimuli and establish a novel methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Additionally, we make histone PTM and proteoform quantitation, RNA splicing, RRBS, and transcriptional footprint datasets available as a resource for future research.

Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress.

BACKGROUND: Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Our preliminary analyses of RNA-Seq data uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. RESULTS: Motivated to understand how BAT function is regulated epigenetically, we developed a novel method for the first-ever unbiased top-down proteomic quantitation of histone modifications in BAT and validated our results with a multi-omic approach. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C) and BAT was analyzed for DNA methylation and histone modifications. Methylation of promoters and intragenic regions in genomic DNA decrease in response to chronic cold exposure. Integration of DNA methylation and RNA expression datasets suggest a role for epigenetic modification of DNA in regulation of gene expression in response to cold. In response to cold housing, we observe increased bulk acetylation of histones H3.2 and H4, increased histone H3.2 proteoforms with di- and trimethylation of lysine 9 (K9me2 and K9me3), and increased histone H4 proteoforms with acetylation of lysine 16 (K16ac) in BAT. CONCLUSIONS: Our results reveal global epigenetically-regulated transcriptional "on" and "off" signals in murine BAT in response to varying degrees of chronic cold stimuli and establish a novel methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Additionally, we make histone PTM and proteoform quantitation, RNA splicing, RRBS, and transcriptional footprint datasets available as a resource for future research.

Vitamin B2 enables regulation of fasting glucose availability.

Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. We also found PPARα knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs.

Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP).

Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

Lymphocytes upregulate CD36 in adipose tissue and liver.

CD36 is a multifunctional scavenger receptor and lipid transporter implicated in metabolic and inflammatory pathologies, as well as cancer progression. CD36 is known to be expressed by adipocytes and monocytes/macrophages, but its expression by T cells is not clearly established. We found that CD4 and CD8 T cells in adipose tissue and liver of humans, monkeys, and mice upregulated CD36 expression (ranging from ~5-40% CD36+), whereas little to no CD36 was expressed by T cells in blood, spleen, and lymph nodes. CD36 was expressed predominantly by resting CD38-, HLA.DR-, and PD-1- adipose tissue T cells in monkeys, and increased during high-fat feeding in mice. Adipose tissue and liver promote a distinct phenotype in resident T cells characterized by CD36 upregulation.

Identification and characterization of a supraclavicular brown adipose tissue in mice.

A fundamental challenge to our understanding of brown adipose tissue (BAT) is the lack of an animal model that faithfully represents human BAT. Such a model is essential for direct assessment of the function and therapeutic potential of BAT depots in humans. In human adults, most of the thermoactive BAT depots are located in the supraclavicular region of the neck, while mouse studies focus on depots located in the interscapular region of the torso. We recently discovered BAT depots that are located in a region analogous to that of human supraclavicular BAT (scBAT). Here, we report that the mouse scBAT depot has morphological characteristics of classical BAT, possesses the potential for high thermogenic activity, and expresses a gene signature that is similar to that of human scBAT. Taken together, our studies reveal a mouse BAT depot that represents human BAT and provides a unique tool for developing new translatable approaches for utilizing human scBAT.

Plasma insulin levels predict atherosclerotic lesion burden in obese hyperlipidemic mice.

Despite a clear association between obesity, insulin resistance and atherosclerosis in humans, to date, no animal models have been described in which insulin resistance is associated with atherosclerotic lesion burden. Using two mouse models of obesity-induced hyperlipidemia:leptin deficient (ob/ob) mice on an apolipoprotein E deficient (apoE-/-) or low density lipoprotein receptor deficient (LDLR-/-) background, we sought to determine metabolic parameters most closely associated with atherosclerotic lesion burden. Total plasma cholesterol (TC) levels in ob/ob;apoE-/- mice and ob/ob;LDLR-/- mice were indistinguishable (682+/-48 versus 663+/-16, respectively). Analysis of lipoprotein profiles showed that cholesterol was carried primarily on VLDL in the ob/ob;apoE-/- mice and on LDL in the ob/ob;LDLR-/- mice. Plasma triglycerides (TG) were 55% lower (P<0.001), non-esterified fatty acids (NEFA) were 1.5-fold higher (P<0.01), and insulin levels were 1.7-fold higher (NS) in ob/ob;apoE-/- mice compared to ob/ob;LDLR-/- mice. Other parameters such as body weight, fat pad weight, and glucose levels were not different between the groups. Aortic sinus lesion area of ob/ob;apoE-/- mice was increased 3.2-fold above ob/ob;LDLR-/- mice (102,455+/-8565 microm2/section versus 31,750+/-4478 microm2/section, P<0.001). Lesions in ob/ob;apoE-/- mice were also more complex as evidenced by a 7.7-fold increase in collagen content (P<0.001). Atherosclerotic lesion area was positively correlated with body weight (P<0.005), NEFA (P=0.007), and insulin (P=0.002) levels in the ob/ob;LDLR-/- mice and with insulin (P=0.014) in the ob/ob;apoE-/- mice. In contrast, lesion burden was neither associated with TC and TG, nor with individual lipoprotein pools, in either animal model. These data provide a direct demonstration of the pathophysiologic relevance of hyperinsulinemia, NEFA, and increased body weight to atherosclerotic lesion formation.

Quantitation and cellular localization of 11beta-HSD1 expression in murine thymus.

11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1), an NADPH-dependent reductase, functions in intact cells to convert inactive 11-keto metabolites of glucocorticoids into biologically active glucocorticoids. The enzyme is thus capable of amplifying glucocorticoid action in tissues in which it is expressed. In the experiments presented here, we show that 11beta-HSD1 is expressed in the murine thymus and that expression increases from late fetal development to maximal levels in the adult thymus. Quantitative real time-PCR, immunoblots, and assays of enzymatic activity reveal adult thymic expression of 11beta-HSD1 mRNA and protein at levels approximately 6-7% of those observed in liver. Immunofluorescence experiments show that the enzyme is expressed in the medullary thymocytes and thymocytes present at the corticomedullary junction. These experiments extend our recognition of 11beta-HSD1 expression in cells of the immune system and lend support to the notion that glucocorticoid signaling and amplification of those signals by regeneration of active glucocorticoids from inactive 11-keto metabolites might impact intrathymic T cell development and the establishment of the immune repertoire.

FABP4-Cre Mediated Expression of Constitutively Active ChREBP Protects Against Obesity, Fatty Liver, and Insulin Resistance.

Carbohydrate response element binding protein (ChREBP) regulates cellular glucose and lipid homeostasis. Although ChREBP is highly expressed in many key metabolic tissues, the role of ChREBP in most of those tissues and the consequent effects on whole-body glucose and lipid metabolism are not well understood. Therefore, we generated a transgenic mouse that overexpresses a constitutively active ChREBP isoform under the control of the fatty acid binding protein 4-Cre-driven promoter (FaChOX). Weight gain was blunted in male, but not female, FaChOX mice when placed on either a normal chow diet or an obesogenic Western diet. Respiratory exchange ratios were increased in Western diet-fed FaChOX mice, indicating a shift in whole-body substrate use favoring carbohydrate metabolism. Western diet-fed FaChOX mice showed improved insulin sensitivity and glucose tolerance in comparison with controls. Hepatic triglyceride content was reduced in Western diet-fed FaChOX mice in comparison with controls, suggesting protection from fatty liver. Epididymal adipose tissue exhibited differential expression of genes involved in differentiation, browning, metabolism, lipid homeostasis, and inflammation between Western diet-fed FaChOX mice and controls. Our findings support a role for ChREBP in modulating adipocyte differentiation and adipose tissue metabolism and inflammation as well as consequent risks for obesity and insulin resistance.

The Glucose Sensor ChREBP Links De Novo Lipogenesis to PPARγ Activity and Adipocyte Differentiation.

Reduced de novo lipogenesis in adipose tissue, often observed in obese individuals, is thought to contribute to insulin resistance. Besides trapping excess glucose and providing for triglycerides and energy storage, endogenously synthesized lipids can function as potent signaling molecules. Indeed, several specific lipids and their molecular targets that mediate insulin sensitivity have been recently identified. Here, we report that carbohydrate-response element-binding protein (ChREBP), a transcriptional inducer of glucose use and de novo lipogenesis, controls the activity of the adipogenic master regulator peroxisome proliferator-activated receptor (PPAR)γ. Expression of constitutive-active ChREBP in precursor cells activated endogenous PPARγ and promoted adipocyte differentiation. Intriguingly, ChREBP-constitutive-active ChREBP expression induced PPARγ activity in a fatty acid synthase-dependent manner and by trans-activating the PPARγ ligand-binding domain. Reducing endogenous ChREBP activity by either small interfering RNA-mediated depletion, exposure to low-glucose concentrations, or expressing a dominant-negative ChREBP impaired differentiation. In adipocytes, ChREBP regulated the expression of PPARγ target genes, in particular those involved in thermogenesis, similar to synthetic PPARγ ligands. In summary, our data suggest that ChREBP controls the generation of endogenous fatty acid species that activate PPARγ. Thus, increasing ChREBP activity in adipose tissue by therapeutic interventions may promote insulin sensitivity through PPARγ.

Carbenoxolone treatment attenuates symptoms of metabolic syndrome and atherogenesis in obese, hyperlipidemic mice.

Glucocorticoids, which are well established to regulate body fat mass distribution, adipocyte lipolysis, hepatic gluconeogenesis, and hepatocyte VLDL secretion, are speculated to play a role in the pathology of metabolic syndrome. Recent focus has been on the activity of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which is capable of regenerating, and thus amplifying, glucocorticoids in key metabolic tissues such as liver and adipose tissue. To determine the effects of global 11beta-HSD1 inhibition on metabolic syndrome risk factors, we subcutaneously injected "Western"-type diet-fed hyperlipidemic mice displaying moderate or severe obesity [LDL receptor (LDLR)-deficient (LDLR(-/-)) mice and mice derived from heterozygous agouti (A(y)/a) and homozygous LDLR(-/-) breeding pairs (A(y)/a;LDLR(-/-) mice)] with the nonselective 11beta-HSD inhibitor carbenoxolone for 4 wk. Body composition throughout the study, end-point fasting plasma, and extent of hepatic steatosis and atherosclerosis were assessed. This route of treatment led to detection of high levels of carbenoxolone in liver and fat and resulted in decreased weight gain due to reduced body fat mass in both mouse models. However, only A(y)/a;LDLR(-/-) mice showed an effect of 11beta-HSD1 inhibition on fasting insulin and plasma lipids, coincident with a reduction in VLDL due to mildly increased VLDL clearance and dramatically decreased hepatic triglyceride production. A(y)/a;LDLR(-/-) mice also showed a greater effect of the drug on reducing atherosclerotic lesion formation. These findings indicate that subcutaneous injection of an 11beta-HSD1 inhibitor allows for the targeting of the enzyme in not only liver, but also adipose tissue, and attenuates many metabolic syndrome risk factors, with more pronounced effects in cases of severe obesity and hyperlipidemia.