Proteomics analysis reveals diabetic kidney as a ketogenic organ in type 2 diabetes

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. To date, the molecular mechanisms of DN remain largely unclear. The present study aimed to identify and characterize novel proteins involved in the development of DN by a proteomic approach. Proteomic analysis revealed that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2), the key enzyme in ketogenesis, was increased fourfold in the kidneys of type 2 diabetic db/db mice. Consistently, the activity of HMGCS2 in kidneys and 24-h urinary excretion of the ketone body β-hydroxybutyrate (β-HB) were significantly increased in db/db mice. Immunohistochemistry, immunofluorescence, and real-time PCR studies further demonstrated that HMGCS2 was highly expressed in renal glomeruli of db/db mice, with weak expression in the kidneys of control mice. Because filtered ketone bodies are mainly reabsorbed in the proximal tubules, we used RPTC cells, a rat proximal tubule cell line, to examine the effect of the increased level of ketone bodies. Treating cultured RPTC cells with 1 mM β-HB significantly induced transforming growth factor-β1 expression, with a marked increase in collagen I expression. β-HB treatment also resulted in a marked increase in vimentin protein expression and a significant reduction in E-cadherin protein levels, suggesting an enhanced epithelial-to-mesenchymal transition in RPTCs. Collectively, these findings demonstrate that diabetic kidneys exhibit excess ketogenic activity resulting from increased HMGCS2 expression. Enhanced ketone body production in the diabetic kidney may represent a novel mechanism involved in the pathogenesis of DN.     

外源性脂肪酸对罗非鱼脂肪细胞增殖与分化的影响

为了探究脂肪酸对罗非鱼(Oreochromis niloticus)脂肪细胞增殖和分化的影响, 在体外培养罗非鱼前脂肪细胞, 并在其增殖和分化过程中分别添加100 μmol/L的棕榈酸(Palmitic Acid, PA)、油酸(Oleic Acid, OA), 亚油酸(Linoleic Acid, LA)和α-亚麻酸(α-Linolenic Acid, LNA)进行处理。使用SRB (Sulforhodamine B)染色法和油红O染色法检测外源性脂肪酸对脂肪细胞增殖和分化的影响, Real-time qPCR检测增殖分化过程中基因表达情况。结果显示, 在培养8d时, 外源添加的不饱和脂肪酸可以促进罗非鱼前脂肪细胞增殖, 并且增殖过程中增殖相关基因(c-fos和c-myc)、脂解相关基因(ATGL)和脂合成相关基因(PPARγ和CD36)的表达与对照组相比均显著提高(P<0.05)。此外, 外源脂肪酸的加入可以抑制脂肪细胞的分化。棕榈酸的加入使得脂肪细胞中产生的脂滴面积较少, 数量较多; 分化过程中细胞的β氧化相关基因(CPT-1a)与对照组相比显著上调, 而脂解相关基因(ATGL)则显著下调。外源性不饱和脂肪酸可以促进罗非鱼前脂肪增殖, 而饱和脂肪酸主要抑制细胞分化。在增殖过程中, 过量的脂肪酸先通过脂合成储存在胞内, 再借助脂解等途径进行代谢, 从而帮助细胞适应环境中高浓度的脂肪酸。而在分化过程中, 添加外源脂肪酸, 可能通过抑制脂肪细胞内的脂合成和脂解的发生, 同时促进β氧化等方式来抑制脂肪细胞分化。     

Streptozotocin diabetes increases mRNA expression of ketogenic enzymes in the rat heart

BackgroundDiabetic cardiomyopathy develops in insulin-dependent diabetic patients who have no hypertension, cardiac hypertrophy or vascular disease. Diabetes increases cardiac fatty acid oxidation, but cardiac hypertrophy limits fatty acid oxidation. Here we examined effects of diabetes on gene expression in rat hearts.MethodsWe used oligonucleotide microarrays to examine effects of insulindependent diabetes in the rat heart. RTQ PCR confirmed results of microarrays. Specific antibodies were used to examine changes in the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2).ResultsA surprising result of diabetes was increased mRNA encoding all enzymes of the ketone body synthesis pathway. Increased mRNA expression for these enzymes was confirmed by RTQ PCR. The mRNA encoding HMGCS2, the rate-controlling enzyme, was 27 times greater in diabetic hearts. Total HMGCS2 protein increased 8-fold in diabetic hearts, but no difference was found in HMGCS2 protein in control vs. diabetic liver.ConclusionsInsulin-dependent diabetes induced the enzymes of ketone body synthesis in the heart, including HMGCS2, as well as increasing enzymes of fatty acid oxidation.General significanceThe mammalian heart does not export ketone bodies to other tissues, but rather is a major consumer of ketone bodies. Induction of HMGCS2, which is normally expressed only in the fetal and newborn heart, may indicate an adaptation by the heart to combat “metabolic inflexibility” by shifting the flux of excess intramitochondrial acetyl-CoA derived from elevated fatty acid oxidation into ketone bodies, liberating free CoA to balance the acetyl-CoA/CoA ratio in favor of increased glucose oxidation through the pyruvate dehydrogenase complex.     

Soymorphin-5, a soy-derived μ-opioid peptide, decreases glucose and triglyceride levels through activating adiponectin and PPARα systems in diabetic KKAy mice

Soymorphin-5 (YPFVV) derived from soybean β-conglycinin β-subunit is a μ-opioid agonist peptide having anxiolytic-like activity. Here, we show that soymorphin-5 improves glucose and lipid metabolism after long-term oral administration to KKAy mice, a type 2 diabetes model animal. Soymorphin-5 inhibited hyperglycemia without an increase in plasma insulin levels in KKAy mice. Soymorphin-5 also decreased plasma and liver triglyceride (TG) levels and liver weight, suggesting that soymorphin-5 improved lipid metabolism. Soymorphin-5 increased plasma adiponectin concentration and liver mRNA expression of AdipoR2, a subtype of adiponectin receptor that is involved in stimulating the peroxisome proliferator-activated receptor (PPAR)α pathway and fatty acid β-oxidation. The expressions of the mRNA of PPARα and its target genes acyl-CoA oxidase, carnitine palmitoyltransferase 1 A, and uncoupling protein-2, in the liver were also increased after oral administration of soymorphin-5. Furthermore, des-Tyr-soymorphin-5 (PFVV) without μ-opioid and anxiolytic-like activities did not decrease blood glucose levels in KKAy mice. These results suggest that μ-opioid peptide soymorphin-5 improves glucose and lipid metabolism via activation of the adiponectin and PPARα system and subsequent increases of β-oxidation and energy expenditure in KKAy mice.     

miR‐149 controls non‐alcoholic fatty liver by targeting FGF‐21

Non‐alcoholic fatty liver disease (NAFLD), a lipid metabolism disorder characterized by the accumulation of intrahepatic fat, has emerged as a global public health problem. However, its underlying molecular mechanism remains unclear. We previously have found that miR‐149 was elevated in NAFLD induced by high‐fat diet mice model, whereas decreased by a 16‐week running programme. Here, we reported that miR‐149 was increased in HepG2 cells treated with long‐chain fatty acid (FFA). In addition, miR‐149 was able to promote lipogenesis in HepG2 cells in the absence of FFA treatment. Moreover, inhibition of miR‐149 was capable of inhibiting lipogenesis in HepG2 cells in the presence of FFA treatment. Meanwhile, fibroblast growth factor‐21 (FGF‐21) was identified as a target gene of miR‐149, which was demonstrated by the fact that miR‐149 could negatively regulate the protein expression level of FGF‐21, and FGF‐21 was also responsible for the effect of miR‐149 inhibitor in decreasing lipogenesis in HepG2 cells in the presence of FFA treatment. These data implicate that miR‐149 might be a novel therapeutic target for NAFLD.     

A pan-PPAR ligand induces hepatic fatty acid oxidation in PPARα−/− mice possibly through PGC-1 mediated PPARδ coactivation

Tetradecylthioacetic acid (TTA) is a hypolipidemic modified fatty acid and a peroxisome proliferator-activated receptor (PPAR) ligand. The mechanisms of TTA-mediated effects seem to involve the PPARs, but the effects have not been assigned to any specific PPAR subtype. PPARα−/− mice were employed to study the role of PPARα after TTA treatment. We also performed in vitro transfection assays to obtain mechanistic knowledge of how TTA affected PPAR activation in the presence of PPARγ coactivator (PGC)-1 and steroid receptor coactivators (SRC)-1 and SRC-2, which are associated with energy balance and mitochondrial biogenesis. We show that TTA increases hepatic fatty acid β-oxidation in PPARα−/− mice. TTA acts as a pan-PPAR ligand in vitro, and PGC-1, SRC-1 and SRC-2 have cell type and PPAR-specific effects together with TTA. In the absence of exogenous ligands, SRC-1 did not induce PPAR activity, while PGC-1 was the most potent PPAR coactivator. When the coactivators were overexpressed, pronounced effects of TTA were observed especially for PPARδ and PPARγ. We conclude that PPARα is involved in, but not required for, the hypolipidemic mechanisms of TTA. It appears that the activity of PPARδ, with substantial contribution of nuclear receptor coactivators, PGC-1 in special, is conducive to TTA's mechanism of action.     

Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids

L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid β-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor α (PPARα) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression covaries with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadh's expression by PPARα, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of ω-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting.     

Protective effect of ganoderic acid against the streptozotocin induced diabetes,inflammation, hyperlipidemia and microbiota imbalance in diabetic rats

Diabetes mellitus (DM) is a metabolic disorder with numerous symptoms categorized via serves hyperglycemia effect along with altered fat, protein and carbohydrate metabolism mainly resultant from defects in insulin action/secretion or both. The aim of the current experimental study was to comfort the neuroprotective effect of ganoderic acid against the streptozotocin (STZ)-induced type I diabetes mellitus in mice and explore the underlying mechanism. Differentiation of 3T3-L1 preadipocytes effect; hepatic and glucose consumption effect of ganoderic acid was estimated on HepG2 cell lines and peroxisome proliferator-activated receptor (PPAR). FFA content was estimated in adipose and hepatic tissues. Ganoderic acid induced the 3T3-L1 preadipocytes differentiation. The mRNA expression of PPAR was increased in the high glucose-treated group in HepG2 and ganoderic acid treatment down-regulated the mRNA expression of PPAR. Ganoderic acid exhibited the inhibitory effect of α-glucosidase and α-amylase. Ganoderic acid demonstrated the reduced blood glucose and increase insulin level and also reduced the free fatty in hepatic and adipose tissue. Histopathological study showed the enhancement of β-cells in ganoderic acid-treated mice. Finally, their prebiotic effects on gut microbiota were illustrated via enhancing the population of diabetes resistant bacteria and also reducing the quantity of diabetes sensitive bacteria. Ganoderic acid attenuated STZ induced T1DM in mice via inflammatory pathways.