Agouti modulation of both adipocyte and pancreatic islet function via a Ca²⁺-dependent mechanism

Dominant mutations at the promoter of the mouse agouti gene results in a yellow coat color as well as obesity, hyperinsulinemia, insulin resistance and gender-dependent hyperglycemia. Both central and peripheral actions of agouti contribute to agouti-induced obesity syndromeThe human analogue of agouti is expressed in adipose tissue and exerts potent peripheral effects. Recombinant agouti protein has been shown to stimulate adipocyte intracellular calcium ([Ca2+]i) and up-regulate fatty acid synthase (FAS), a key enzyme in de novo lipogenesis in human adipocytes via a Ca2+-dependent mechanism, thereby promoting lipid storage in adipose tissue. However, transgenic mice overexpressing agouti only in adipose tissue under the control of aP2 promoter do not become obese unless hyperinsulinemia is concomitantly present, suggesting an interaction between hyperinsulinemia and adipose tissue agouti expression. In addition, adipocyte lipolysis is also impaired in agouti mutants compared to wild type animals. We therefore further investigated the effect of recombinant agouti protein on both human adipocyte and pancreatic islet function. We demonstrated that the human homologue of agouti is expressed in the pancreas. Agouti protein also induced a significant Ca2+ influx into human pancreatic islets, and serves as a potent secretagogue for both insulin and amylin release in human pancreatic islets, which may contribute to hyperinsulinemia and hyperamylinemia. In human adipocyte, recombinant agouti protein potently inhibited both ACTH- and forskolin-induced lipolysis via a Ca2+-dependent mechanism, indicating a post receptor event. Further investigation of the mechanism whereby increasing in [Ca2+]i results in inhibition of lipolysis demonstrated that the anti-lipolytic effect of increasing [Ca2+]i is exerted by the activation of adipocyte phosphodiesterase (PDE 3B), leading to a decrease in cAMP, a net decrease in hormone sensitive lipase phosphorylation and inhibition of lipolysis. In addition, we demonstrated that human adipocyte agouti is up-regulated during adipocyte differentiation. Using human adipose tissue obtained from plastic surgery with body mass index (BMI) ranging from 21 to 31, we found a positive correlation between adipose tissue agouti content and FAS activity, as well as between adipose tissue agouti mRNA level and FAS mRNA levelThese data suggest that agouti may modulate both adipocyte and pancreatic islet function via a Ca2+- dependent mechanismIn the pancreas, agouti-induced hyperinsulinemia and hyperamylinemia may serve as a compensatory system in regulating blood glucose by coordinately regulating glucose input and clearance, respectively. However, agouti- induced hyperinsulinemia may contribute to insulin resistance and obesity. In addition, agouti stimulation of amylin release may result in islet amyloid deposits, impaired B-cell function and diabetes. In the adipose tissue, agouti potently inhibits agonists-stimulated lipolysis by activating adipocyte PDE 3B, thereby resulting in a coordinated regulation of adipocyte lipid metabolism, which may contribute to adipose tissue lipid storageIn addition, we have shown a correlation between human adipose tissue agouti expression and FAS activity and expression. As humans normally express agouti in adipose tissue and pancreas, it is possible that agouti and FAS may be regulated in a similar manner. Alternatively, agouti may be one of adipocyte synthesized factors that may in part contribute to the modulation of adipose tissue lipid metabolism in vivo. Finally, agouti-induced hyperinsulinemia may synergistically act with adipocyte agouti in modulating adipocyte lipid metabolism, thereby contributing to obesity

Omega-3 Polyunsaturated Fatty Acids Antagonize Macrophage Inflammation via Activation of AMPK/SIRT1 Pathway

Macrophages play a key role in obesity-induced inflammation. Omega-3 polyunsaturated fatty acids (v-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exert anti-inflammatory functions in both humans and animal models, but the exact cellular signals mediating the beneficial effects are not completely understood. We previously found that two nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 interact to regulate macrophage inflammation. Here we aim to determine whether v-3 PUFAs antagonize macrophage inflammation via activation of AMPK/SIRT1 pathway. Treatment of v-3 PUFAs suppresses lipopolysaccharide (LPS)-induced cytokine expression in macrophages. Luciferase reporter assays, electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) assays show that treatment of macrophages with v-3 PUFAs significantly inhibits LPS-induced NF-kB signaling. Interestingly, DHA also increases expression, phosphorylation and activity of the major isoform a1AMPK, which further leads to SIRT1 overexpression. More importantly, DHA mimics the effect of SIRT1 on deacetylation of the NF-kB subunit p65, and the ability of DHA to deacetylate p65 and inhibit its signaling and downstream cytokine expression require SIRT1. In conclusion, v-3 PUFAs negatively regulate macrophage inflammation by deacetylating NF-kB, which acts through activation of AMPK/SIRT1 pathway. Our study defines AMPK/SIRT1 as a novel cellular mediator for the anti-inflammatory effects of v-3 PUFAs

A comparative study of the proventricular structure in twenty Chinese Tettigoniidae (Orthoptera) species

This study focuses on the proventriculus and the alimentary canal of twenty Tettigoniidae species among three subfamilies, Tettigoniinae, Phaneropterinae and Conocephalinae. Each part of the alimentary canal and the inner structure of proventriculus were examined under optic microscope and scanning electron microscopy. As a result, the length of each part of the alimentary canal and the inner structure of proventriculus were highly associated with feeding habits. Carnivorous species always had a short foregut and long cilia on the base of the sclerotized appendix in proventriculus, whereas herbivorous species always had a longer foregut and a highly sclerotized proventriculus. These results increase understanding of the alimentary canal in Tettigoniidae and will be useful in future studies of their feeding habits

Histone Deacetylase 1 (HDAC1) Negatively Regulates Thermogenic Program in Brown Adipocytes via Coordinated Regulation of H3K27 Deacetylation and Methylation

Inhibiting class I histone deacetylases (HDACs) increases energy expenditure, reduces adiposity and improves insulin sensitivity in obese mice. However, the precise mechanism is poorly understood. Here, we demonstrate that HDAC1 is a negative regulator of brown adipocyte thermogenic program. HDAC1 level is lower in mouse brown fat (BAT) than white fat, is suppressed in mouse BAT during cold exposure or β3-adrenergic stimulation, and is down-regulated during brown adipocyte differentiation. Remarkably, overexpressing HDAC1 profoundly blocks, whereas deleting HDAC1 significantly enhances β-adrenergic activation-induced BAT-specific gene expression in brown adipocytes. β-adrenergic activation in brown adipocytes results in a dissociation of HDAC1 from promoters of BAT-specific genes, including uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α), leading to increased acetylation of histone H3 lysine 27 (H3K27), an epigenetic mark of gene activation. This is followed by dissociation of the polycomb repressive complexes, including the H3K27 methyltransferase enhancer of zeste homologue (EZH2), suppressor of zeste 12 (SUZ12), and ring finger protein 2 (RNF2) from, and concomitant recruitment of H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) to UCP1 and PGC1α promoters, leading to decreased H3K27 trimethylation, a histone transcriptional repression mark. Thus, HDAC1 negatively regulates brown adipocyte thermogenic program, and inhibiting HDAC1 promotes BAT-specific gene expression through a coordinated control of increased acetylation and decreased methylation of H3K27, thereby switching transcriptional repressive state to active state at the promoters of UCP1 and PGC1α. Targeting HDAC1 may be beneficial in prevention and treatment of obesity by enhancing BAT thermogenesis

Histone Deacetylase 1 (HDAC1) Negatively Regulates Thermogenic Program in Brown Adipocytes via Coordinated Regulation of H3K27 Deacetylation and Methylation

Inhibiting class I histone deacetylases (HDACs) increases energy expenditure, reduces adiposity and improves insulin sensitivity in obese mice. However, the precise mechanism is poorly understood. Here, we demonstrate that HDAC1 is a negative regulator of brown adipocyte thermogenic program. HDAC1 level is lower in mouse brown fat (BAT) than white fat, is suppressed in mouse BAT during cold exposure or β3-adrenergic stimulation, and is down-regulated during brown adipocyte differentiation. Remarkably, overexpressing HDAC1 profoundly blocks, whereas deleting HDAC1 significantly enhances β-adrenergic activation-induced BAT-specific gene expression in brown adipocytes. β-adrenergic activation in brown adipocytes results in a dissociation of HDAC1 from promoters of BAT-specific genes, including uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α), leading to increased acetylation of histone H3 lysine 27 (H3K27), an epigenetic mark of gene activation. This is followed by dissociation of the polycomb repressive complexes, including the H3K27 methyltransferase enhancer of zeste homologue (EZH2), suppressor of zeste 12 (SUZ12), and ring finger protein 2 (RNF2) from, and concomitant recruitment of H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) to UCP1 and PGC1α promoters, leading to decreased H3K27 trimethylation, a histone transcriptional repression mark. Thus, HDAC1 negatively regulates brown adipocyte thermogenic program, and inhibiting HDAC1 promotes BAT-specific gene expression through a coordinated control of increased acetylation and decreased methylation of H3K27, thereby switching transcriptional repressive state to active state at the promoters of UCP1 and PGC1α. Targeting HDAC1 may be beneficial in prevention and treatment of obesity by enhancing BAT thermogenesis

Intestinal Cgi-58 Deficiency Reduces Postprandial Lipid Absorption

Comparative Gene Identification-58 (CGI-58), a lipid droplet (LD)-associated protein, promotes intracellular triglyceride (TG) hydrolysis in vitro. Mutations in human CGI-58 cause TG accumulation in numerous tissues including intestine. Enterocytes are thought not to store TG-rich LDs, but a fatty meal does induce temporary cytosolic accumulation of LDs. Accumulated LDs are eventually cleared out, implying existence of TG hydrolytic machinery in enterocytes. However, identities of proteins responsible for LD-TG hydrolysis remain unknown. Here we report that intestine-specific inactivation of CGI-58 in mice significantly reduces postprandial plasma TG concentrations and intestinal TG hydrolase activity, which is associated with a 4-fold increase in intestinal TG content and large cytosolic LD accumulation in absorptive enterocytes during the fasting state. Intestine-specific CGI-58 knockout mice also display mild yet significant decreases in intestinal fatty acid absorption and oxidation. Surprisingly, inactivation of CGI-58 in intestine significantly raises plasma and intestinal cholesterol, and reduces hepatic cholesterol, without altering intestinal cholesterol absorption and fecal neutral sterol excretion. In conclusion, intestinal CGI-58 is required for efficient postprandial lipoprotein-TG secretion and for maintaining hepatic and plasma lipid homeostasis. Our animal model will serve as a valuable tool to further define how intestinal fat metabolism influences the pathogenesis of metabolic disorders, such as obesity and type 2 diabetes

Interaction Between Leucine and Phosphodiesterase 5 Inhibition in Modulating Insulin Sensitivity and Lipid Metabolism

Purpose: Leucine activates SIRT1/AMP-activated protein kinase (AMPK) signaling and markedly potentiates the effects of other sirtuin and AMPK activators on insulin signaling and lipid metabolism. Phosphodiesterase 5 inhibition increases nitric oxide–cGMP signaling, which in turn exhibits a positive feedback loop with both SIRT1 and AMPK, thus amplifying peroxisome proliferator-activated receptor γ co-activator α (PGC1α)-mediated effects. Methods: We evaluated potential synergy between leucine and PDE5i on insulin sensitivity and lipid metabolism in vitro and in diet-induced obese (DIO) mice. Results: Leucine (0.5 mM) exhibited significant synergy with subtherapeutic doses (0.1–10 nM) of PDE5-inhibitors (sildenafil and icariin) on fat oxidation, nitric oxide production, and mitochondrial biogenesis in hepatocytes, adipocytes, and myotubes. Effects on insulin sensitivity, glycemic control, and lipid metabolism were then assessed in DIO-mice. DIO-mice exhibited fasting and postprandial hyperglycemia, insulin resistance, and hepatic steatosis, which were not affected by the addition of leucine (24 g/kg diet). However, the combination of leucine and a subtherapeutic dose of icariin (25 mg/kg diet) for 6 weeks reduced fasting glucose (38%, P,0.002), insulin (37%, P,0.05), area under the glucose tolerance curve (20%, P,0.01), and fully restored glucose response to exogenous insulin challenge. The combination also inhibited hepatic lipogenesis, stimulated hepatic and muscle fatty acid oxidation, suppressed hepatic inflammation, and reversed high-fat diet-induced steatosis. Conclusion: These robust improvements in insulin sensitivity, glycemic control, and lipid metabolism indicate therapeutic potential for leucine–PDE5 inhibitor combinations

N‑Linked Glycosylation Prevents Deamidation of Glycopeptide and Glycoprotein

Deamidation has been recognized as a common spontaneous pathway of protein degradation and a prevalent concern in the pharmaceutical industry; deamidation caused the reduction of protein/peptide drug efficacy and shelf life in several cases. More importantly, deamidation of physiological proteins is related to several human diseases and considered a timer for the diseases. N-linked glycosylation has a variety of significant biological functions, and it interestingly occurs right on the deamidation site-asparagine. It has been perceived that N-glycosylation could prevent deamidation, but experimental support is still lacking for clearly understanding the role of N-glycosylation on deamidation. Our results presented that deamidation is prevented by naturally occurring N-linked glycosylation. Glycopeptides and corresponding nonglycosylated peptides were used to compare their deamidation rates. All the nonglycosylated peptides have different half-lives ranging from one to 20 days, for the corresponding glycosylated peptides; all the results showed that the deamidation reaction was significantly reduced by the introduction of N-linked glycosylation. A glycoprotein, RNase B, also showed a significantly elongated deamidation half-life compared to nonglycosylated protein RNase A. At last, N-linked glycosylation on INGAP-P, a therapeutic peptide, increased the deamidation half-life of INGAP-P as well as its therapeutic potency

Loss of Abhd5 Promotes Colorectal Tumor Development and Progression by Inducing Aerobic Glycolysis and Epithelial-Mesenchymal Transition

How cancer cells shift metabolism to aerobic glycolysis is largely unknown. Here, we show that deficiency of a/b-hydrolase domain-containing 5 (Abhd5), an intracellular lipolytic activator that is also known as comparative gene identification 58 (CGI-58), promotes this metabolic shift and enhances malignancies of colorectal carcinomas (CRCs). Silencing of Abhd5 in normal fibroblasts induces malignant transformation. Intestine-specific knockout of Abhd5 in ApcMin/+ mice robustly increases tumorigenesis and malignant transformation of adenomatous polyps. In colon cancer cells, Abhd5 deficiency induces epithelial-mesenchymal transition by suppressing the AMPKa-p53 pathway, which is attributable to increased aerobic glycolysis. In human CRCs, Abhd5 expression falls substantially and correlates negatively with malignant features. Our findings link Abhd5 to CRC pathogenesis and suggest that cancer cells develop aerobic glycolysis by suppressin

Macrophage CGI-58 Deficiency Activates ROS-Inflammasome Pathway to Promote Insulin Resistance in Mice

SummaryOvernutrition activates a proinflammatory program in macrophages to induce insulin resistance (IR), but its molecular mechanisms remain incompletely understood. Here, we show that saturated fatty acid and lipopolysaccharide, two factors implicated in high-fat diet (HFD)-induced IR, suppress macrophage CGI-58 expression. Macrophage-specific CGI-58 knockout (MaKO) in mice aggravates HFD-induced glucose intolerance and IR, which is associated with augmented systemic/tissue inflammation and proinflammatory activation of adipose tissue macrophages. CGI-58-deficient macrophages exhibit mitochondrial dysfunction due to defective peroxisome proliferator-activated receptor (PPAR)γ signaling. Consequently, they overproduce reactive oxygen species (ROS) to potentiate secretion of proinflammatory cytokines by activating NLRP3 inflammasome. Anti-ROS treatment or NLRP3 silencing prevents CGI-58-deficient macrophages from oversecreting proinflammatory cytokines and from inducing proinflammatory signaling and IR in the cocultured fat slices. Anti-ROS treatment also prevents exacerbation of inflammation and IR in HFD-fed MaKO mice. Our data thus establish CGI-58 as a suppressor of overnutrition-induced NLRP3 inflammasome activation in macrophages