Featured ArticlesVolume 4 | No. 5 | May 2015
|Adipocyte SIRT1 knockout improves metabolic functions in obesityMayoral and colleagues establish that SIRT1 downregulation in adipose tissue plays a previously unknown role in long-term inflammation resolution mediated by PPARγ activation. During chronic high-fat diet Sirt1 adipocyte-specific knockout mice became more insulin sensitive, glucose tolerant, and less inflamed than wildtype controls. The data presented may provide novel approaches to the treatment of type 2 diabetes.|
Abstract | PDF
Objective: Adipose tissue is the primary site for lipid deposition that protects the organisms in cases of nutrient excess during obesogenic diets. The histone deacetylase Sirtuin 1 (SIRT1) inhibits adipocyte differentiation by targeting the transcription factor peroxisome proliferator activated-receptor gamma (PPARγ).
Methods: To assess the specific role of SIRT1 in adipocytes, we generated Sirt1 adipocyte-specific knockout mice (ATKO) driven by aP2 promoter onto C57BL/6 background. Sirt1flx/flx aP2Cre+ (ATKO) and Sirt1flx/flx aP2Cre- (WT) mice were fed high-fat diet for 5 weeks (short-term) or 15 weeks (chronic-term). Metabolic studies were combined with gene expression analysis and phosphorylation/acetylation patterns in adipose tissue.
Results: On standard chow, ATKO mice exhibit low-grade chronic inflammation in adipose tissue, along with glucose intolerance and insulin resistance compared with control fed mice. On short-term HFD, ATKO mice become more glucose intolerant, hyperinsulinemic, insulin resistant and display increased inflammation. During chronic HFD, WT mice developed a metabolic dysfunction, higher than ATKO mice, and thereby, knockout mice are more glucose tolerant, insulin sensitive and less inflamed relative to control mice. SIRT1 attenuates adipogenesis through PPARγ repressive acetylation and, in the ATKO mice adipocyte PPARγ was hyperacetylated. This high acetylation was associated with a decrease in Ser273-PPARγ phosphorylation. Dephosphorylated PPARγ is constitutively active and results in higher expression of genes associated with increased insulin sensitivity.
Conclusion: Together, these data establish that SIRT1 downregulation in adipose tissue plays a previously unknown role in long-term inflammation resolution mediated by PPARγ activation. Therefore, in the context of obesity, the development of new therapeutics that activate PPARγ by targeting SIRT1 may provide novel approaches to the treatment of T2DM.[Hide abstract]
|Nuclear Factor-Y is an adipogenic factor that regulates leptin gene expressionLu and colleagues advance our understanding of leptin gene expression and show that NF-Y controls the expression of leptin and other adipocyte genes. Animals with a knockout of NF-YA develop a moderately severe lipodystrophy with low leptin levels and a metabolic phenotype that is remediable with leptin therapy.|
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Objective: Leptin gene expression is highly correlated with cellular lipid content in adipocytes but the transcriptional mechanisms controlling leptin expression in vivo are poorly understood. In this report, we set out to identify cis- and trans-regulatory elements controlling leptin expression.
Methods: Leptin-BAC luciferase transgenic mice combining with other computational and molecular techniques were used to identify transcription regulatory elements including a CCAAT-binding protein Nuclear Factor Y (NF-Y). The function of NF-Y in adipocyte was studied in vitro with 3T3-L1 cells and in vivo with adipocyte-specific knockout of NF-Y.
Results: Using Leptin-BAC luciferase mice, we showed that DNA sequences between −22 kb and +8.8 kb can confer quantitative expression of a leptin reporter. Computational analysis of sequences and gel shift assays identified a 32 bp sequence (chr6: 28993820–2899385) consisting a CCAAT binding site for Nuclear Factor Y (NF-Y) and this was confirmed by a ChIP assay in vivo. A deletion of this 32 bp sequence in the −22 kb to +8.8 kb leptin-luciferase BAC reporter completely abrogates luciferase reporter activity in vivo. RNAi mediated knockdown of NF-Y interfered with adipogenesis in vitro and adipocyte-specific knockout of NF-Y in mice reduced expression of leptin and other fat specific genes in vivo. Further analyses of the fat specific NF-Y knockout revealed that these animals develop a moderately severe lipodystrophy that is remediable with leptin therapy.
Conclusions: These studies advance our understanding of leptin gene expression and show that NF-Y controls the expression of leptin and other adipocyte genes and identifies a new form of lipodystrophy.[Hide abstract]
|Reduced neutral amino acid availability improves glycaemic controlJiang and colleagues identify the neutral amino acid transporter B0AT1 as a potential target to treat type 2 diabetes. Mice lacking intestinal and renal B0AT1 present with an overload of amino acids in the lumen of the intestine and reduced systemic amino acid availability. The resulting phenotype mediated by GLP-1 and FGF21 is characterised by improved glycaemic control.|
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Objective: Type 2 diabetes arises from insulin resistance of peripheral tissues followed by dysfunction of β-cells in the pancreas due to metabolic stress. Both depletion and supplementation of neutral amino acids have been discussed as strategies to improve insulin sensitivity. Here we characterise mice lacking the intestinal and renal neutral amino acid transporter B0AT1 (Slc6a19) as a model to study the consequences of selective depletion of neutral amino acids.
Methods: Metabolic tests, analysis of metabolite levels and signalling pathways were used to characterise mice lacking the intestinal and renal neutral amino acid transporter B0AT1 (Slc6a19).
Results: Reduced uptake of neutral amino acids in the intestine and loss of neutral amino acids in the urine causes an overload of amino acids in the lumen of the intestine and reduced systemic amino acid availability. As a result, higher levels of glucagon-like peptide 1 (GLP-1) are produced by the intestine after a meal, while the liver releases the starvation hormone fibroblast growth factor 21 (FGF21). The combination of these hormones generates a metabolic phenotype that is characterised by efficient removal of glucose, particularly by the heart, reduced adipose tissue mass, browning of subcutaneous white adipose tissue, enhanced production of ketone bodies and reduced hepatic glucose output.
Conclusions: Reduced neutral amino acid availability improves glycaemic control. The epithelial neutral amino acid transporter B0AT1 could be a suitable target to treat type 2 diabetes.[Hide abstract]
|Munc18c mediates exocytosis of pre-docked and newcomer insulin granulesZhu and colleagues illuminate in human beta-cells that Munc18c acts on distinct pools of secretory granules (SGs) to mediate biphasic glucose-stimulated insulin secretion (GSIS). Depletion of endogenous Munc18c with consequent reduction of Syn-4 levels in islet beta-cells reduces exocytosis of predocked and newcomer SGs in first-phase GSIS and newcomer SGs in second-phase GSIS.|
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Objective: Pancreatic beta-cells express three Munc18 isoforms. Much is known about the roles of Munc18a (pre-docked secretory granules-SGs) and Munc18b (newcomer SGs and SG–SG fusion) in insulin exocytosis. Although shown to influence glucose-stimulated insulin secretion (GSIS) in rodents the precise role of Munc18c in insulin SG exocytosis has not been elucidated. We here examined the role of Munc18c in human pancreatic beta-cells.
Methods: Munc18c-shRNA/RFP lenti-virus (versus control virus) was used to knock down the expression level of Munc18c in human islets or single beta-cells. Insulin secretion and granule exocytosis were measured by performing islets perifusion, single-cell patch clamp capacitance measurements and total internal reflection fluorescence microscopy (TIRFM).
Results: Munc18c is most abundant in the cytosol of human beta-cells. Endogenous function of Munc18c was assessed by knocking down (KD) its islet expression by 70% employing lenti-shRNA virus. Munc18c-KD caused reduction in cognate syntaxin-4 islet expression but not in other exocytotic proteins, resulting in the reduction in GSIS in first- (by 42%) and second phases (by 35%). Single cell analyses of RFP-tagged Munc18c-KD beta-cells by patch clamp capacitance measurements showed inhibition in both readily-releasable pool (by 52%) and mobilization from the reserve pool (by 57%). TIRFM to assess single SG behavior showed that Munc18c-KD inhibition of first phase GSIS was attributed to reduction in exocytosis of pre-docked and newcomer SGs, and second phase inhibition attributed entirely to reduction in newcomer SG fusion (SGs that undergo minimal residence or docking time at the plasma membrane before fusion).
Conclusion: Munc18c is involved in the distinct molecular machineries that affect exocytosis of both predocked and newcomer SG pools that underlie Munc18c's role in first and second phases of GSIS, respectively.[Hide abstract]
|Roux-en-Y gastric bypass is associated with gonadal adipose tissues browningNeinast and colleagues suggest a possible mechanism by which Roux-en-Y gastric bypass (RYGB) improves obesity-related metabolic sequelae. Their results indicate that RYGB may affect beige adipocyte enrichment of adipose tissues by altering sympathetic and natriuretic peptide-related circuits.|
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Objective: Roux-en-Y gastric bypass (RYGB) is an effective method of weight loss and remediation of type-2 diabetes; however, the mechanisms leading to these improvements are unclear. Additionally, adipocytes within white adipose tissue (WAT) depots can manifest characteristics of brown adipocytes. These ‘BRITE/beige’ adipocytes express uncoupling protein 1 (UCP1) and are associated with improvements in glucose homeostasis and protection from obesity. Interestingly, atrial and B-type natriuretic peptides (NPs) promote BRITE/beige adipocyte enrichment of WAT depots, an effect known as “browning.” Here, we investigate the effect of RYGB surgery on NP, NP receptors, and browning in the gonadal adipose tissues of female mice. We propose that such changes may lead to improvements in metabolic homeostasis commonly observed following RYGB.
Methods: Wild type, female, C57/Bl6 mice were fed a 60% fat diet ad libitum for six months. Mice were divided into three groups: Sham operated (SO), Roux-en-Y gastric bypass (RYGB), and Weight matched, sham operated (WM-SO). Mice were sacrificed six weeks following surgery and evaluated for differences in body weight, glucose homeostasis, adipocyte morphology, and adipose tissue gene expression.
Results: RYGB and calorie restriction induced similar weight loss and improved glucose metabolism without decreasing food intake. β3-adrenergic receptor expression increased in gonadal adipose tissue, in addition to Nppb (BNP), and NP receptors, Npr1, and Npr2. The ratio of Npr1:Npr3 and Npr2:Npr3 increased in RYGB, but not WM-SO groups. Ucp1 protein and mRNA, as well as additional markers of BRITE/beige adipose tissue and lipolytic genes increased in RYGB mice to a greater extent than calorie-restricted mice.
Conclusions: Upregulation of Nppb, Npr1, Npr2, and β3-adrenergic receptors in gonadal adipose tissue following RYGB was associated with increased markers of browning. This browning of gonadal adipose tissue may underpin the positive effect of RYGB on metabolic parameters and may in part be mediated through upregulation of natriuretic peptides.[Hide abstract]