Differential induction of genes in liver and brown adipose tissue regulated by peroxisome proliferator-activated receptor-alpha during fasting and cold exposure in acyl-CoA dehydrogenase-deficient mice

Mice deficient for either long-chain acyl-CoA dehydrogenase (LCAD-/-) or very-long-chain acyl-CoA dehydrogenase (VLCAD-/-) develop hepatic steatosis upon fasting, due to disrupted mitochondrial fatty acid oxidation. Moreover, neither mouse model can maintain core body temperature when exposed to cold. We investigated the effects of fasting and cold exposure on gene expression in these mice. Non-fasted LCAD-/- mice showed gene expression changes indicative of fatty liver, including elevated mRNA levels for peroxisome proliferator-activated receptor-gamma (PPARgamma) and genes involved in lipogenesis. In LCAD-/- and VLCAD-/- mice challenged with fasting and cold exposure, expression of fatty acid oxidation genes was elevated in liver, consistent with increased PPARalpha activity. This effect was not seen in brown adipose tissue, suggesting that expression of these genes may be regulated differently than in liver. The effect of acute cold exposure on expression of fatty acid oxidation genes was measured in peroxisome proliferator-activated receptor (PPAR)-alpha-deficient mice (PPARalpha-/-) and controls. In PPARalpha-/- mice, basal expression of the acyl-CoA dehydrogenases was reduced in liver but was not altered in brown adipose tissue. While cold altered the expression of PPARgamma, sterol-regulatory element binding protein-1 (SREBP-1), ATP citrate lyase, and the uncoupling proteins in brown adipose tissue from both PPARalpha-/- and control mice, fatty acid oxidation genes were unaffected. Thus, while fatty acid oxidation appears critical for non-shivering thermogenesis, expression of the acyl-CoA dehydrogenases is not influenced by cold exposure. Moreover, mitochondrial fatty acid oxidation genes are not regulated by PPARalpha in brown adipose tissue as they are in liver.     

Evaluation of the therapeutic potential of PPARalpha agonists for X-linked adrenoleukodystrophy

Adrenoleukodystrophy protein (ABCD1), a peroxisomal membrane protein, is mutated in patients affected by X-linked adrenoleukodystrophy (X-ALD). Adrenoleukodystrophy-related protein (ABCD2) is the closest relative of ABCD1. Pharmacological induction of ABCD2 gene expression has been proposed as a novel therapy strategy for X-ALD. Fibrates induce peroxisome proliferation and Abcd2 expression in rodent liver. Here we evaluate the possibility of using peroxisome proliferator-activated receptor alpha (PPARalpha) agonists for pharmacological induction of ABCD2 expression. In the liver of PPARalpha-deficient mice, both the constitutive and the fenofibrate-inducible Abcd2 gene expression was found to be PPARalpha-dependent. In the brain, PPARalpha-deficiency has no effect on Abcd2 expression. In mice orally treated with the novel, highly selective, and potent PPARalpha agonists GW 7647, GW 6867, and tetradecylthioacetic acid, Abcd2 expression was induced in liver and adrenal glands, but not in brain and testis. None of four putative PPREs identified in the 5(')-flanking DNA and in intron 1 of the Abcd2 gene conferred fibrate response in luciferase reporter assays. Thus, although fibrate-mediated Abcd2 induction is PPARalpha-dependent, it appears to be an indirect mechanism. Within the mouse Abcd2 promoter, a putative sterol regulatory element (SRE) similar in sequence and position to the characterized SRE sequence of the human ABCD2 promoter, was identified. A PPARalpha dependent induction of the sterol regulatory-binding protein 2 (SREBP2) and a down-regulation of SREBP1c mRNA levels could be demonstrated after fenofibrate treatment of mice. Our results suggest that the PPARalpha agonist-mediated induction of Abcd2 expression seems to be indirect and possibly mediated by SREBP2.     

db/db小鼠肝脏中实时定量逆转录PCR内参照基因及标准化方法的评估与整合（英文）

目的:实时定量逆转录PCR(RT-q PCR)结果的标准化对于保证最终结果的准确性尤其重要。常用的标准化方法包括用加入的核酸量校正(ΔCt法)、用单个内参照基因校正(ΔΔCt法)和用统计学软件计算多个内参照基因的几何平均值进行校正。我们在db/db小鼠肝脏中对各种校正方法进行评估。方法:用ge Norm和NormFinder两种软件评估ACTB、e IF5、GAPDH、HMBS、HPRT1、Polr2A和RPLP0共7个内参照基因,以肝脏脂质合成相关基因Thrsp、SCD、SREBP1c和FAS作为目的基因。结果:应用ΔCt法,db/db小鼠肝脏中所有目的基因及GAPDH的表达显著升高(P0.05)。ge Norm计算认为ACTB和HMBS最稳定。Norm Finder计算认为ACTB最稳定,而GAPDH和RPLP0为最佳组合。以单个基因ACTB或RPLP0,以及ACTB与HMBS,或GAPDH与RPLP0的几何平均值进行校正,db/db小鼠肝脏中除SREBP1c以外,Thrsp、SCD1和FAS的表达均升高(P0.05)。结论:用ΔCt法校正RTq PCR的结果稳定并具有生物学意义。统计学软件ge Norm或Norm Finder应与ΔCt法整合使用。     

GLP-1受体激动剂对肥胖小鼠脂肪组织的脂解作用及机制探讨

目的:探讨胰高血糖素样肽1(GLP-1)受体激动剂艾塞那肽(exendin-4)对肥胖小鼠脂肪组织的作用及机制。方法:8周龄C57BL/6J小鼠高脂喂养12周后随机分为艾塞那肽组和生理盐水对照组,另设正常饮食组。取附睾旁脂肪检测sirtuin 1(SIRT1)、脂肪甘油三酯脂酶(ATGL)、肿瘤坏死因子α(TNF-α)及脂联素mRNA的表达。Exendin-4或联合SIRT1激动剂/抑制剂处理3T3-L1脂肪细胞24 h;小鼠胚胎成纤维细胞(MEF)诱导成脂肪细胞后exendin-4干预24 h;检测SIRT1、ATGL和激素敏感性脂酶(HSL)的蛋白表达水平。结果:与生理盐水对照组相比,艾塞那肽组小鼠附睾旁脂肪量、空腹血糖及血甘油三酯水平降低(均P0.05),体重减轻,血TNF-α水平降低。艾塞那肽干预后,肥胖小鼠脂肪组织SIRT1、ATGL和脂联素mRNA表达明显上调,TNF-αmRNA表达明显下调(P0.05)。Exendin-4剂量依赖性促进3T3-L1脂肪细胞SIRT1、ATGL和HSL脂解相关蛋白的表达。联合SIRT1激动剂后,脂滴数量减少,上述脂解相关蛋白的表达上调。联合SIRT1抑制剂后上述作用减弱。敲除SIRT1后MEF脂肪细胞内脂滴增大,数量增多,exendin-4促进脂解的作用消失。结论:艾塞那肽通过激活SIRT1促进肥胖小鼠脂肪组织脂解作用。     

Reduced plasma free fatty acid availability during exercise: effect on gene expression

Endurance exercise transiently increases the mRNA of key regulatory proteins involved in skeletal muscle metabolism. During prolonged exercise and subsequent recovery, circulating plasma fatty acid (FA) concentrations are elevated. The present study therefore aimed to determine the sensitivity of key metabolic genes to FA exposure, assessed in vitro using L6 myocytes and secondly, to measure the expression of these same set of genes in vivo, following a single exercise bout when the post-exercise rise in plasma FA is abolished by acipimox. Initial studies using L6 myotubes demonstrated dose responsive sensitivity for both PDK4 and PGC-1α mRNA to acute FA exposure in vitro. Nine active males performed two trials consisting of 2 h exercise, followed by 2 h of recovery. In one trial, plasma FA availability was reduced by the administration of acipimox (LFA), a pharmacological inhibitor of adipose tissue lipolysis, and in the second trial a placebo was provided (CON). During the exercise bout and during recovery, the rise in plasma FA and glycerol was abolished by acipimox treatment. Following exercise the mRNA abundance of PDK4 and PGC-1α were elevated and unaffected by either acipimox or placebo. Further analysis of skeletal muscle gene expression demonstrated that the CPT I gene was suppressed in both trials, whilst UCP-3 gene was only modestly regulated by exercise alone. Acipimox ingestion did not alter the response for both CPT I and UCP-3. Thus, this study demonstrates that the normal increase in circulating concentrations of FA during the later stages of exercise and subsequent recovery is not required to induce skeletal muscle mRNA expression of several proteins involved in regulating substrate metabolism.     

Influence of DMBA-induced mammary cancer on the liver CPT I, mit HMG-CoA synthase and PPARalpha mRNA expression in rats fed a low or high corn oil diet

Hepatic mitochondrial outer membrane carnitine palmitoyltransferase I (CPT I) and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) enzymes play a key role in regulation of fatty acid oxidation and in ketogenic pathways, respectively. Their expression are regulated by fatty acids mainly by the peroxisome proliferator-activated receptor alpha (PPARalpha). To investigate possible mechanisms through which cancer alters the lipid metabolism, we analyzed by Northern blot, the mRNA relative abundance of these proteins in liver from healthy and DMBA-induced mammary tumor-bearing rats fed a low or high corn oil diet. Serum levels of lipids, body weight and mass were also determined. Whereas mRNA steady-state levels of CPT I and mit HMG-CoA synthase were unaffected by the presence of the extra-hepatic tumor, the cancer state seemed to modify the regulation of the expression of these genes by high fat diet. We hypothesize that putative changes in PPARalpha mRNA levels could have contributed to such alterations. These results, together with changes in serum lipid profiles, body weight and mass, indicate fat mobilization and non-enhanced oxidation rates despite a high-fat feeding. This effect of the cancer state could be related to tumor aggressiveness and suggest a preferential redirection of long-chain fatty acids into energetic and specific pathways of the cancer cells.     

Characterization of GLUT4 and calpain expression in healthy human skeletal muscle during fasting and refeeding

Aims: Calpain‐10 and calpain‐3 and the diabetes ankyrin repeat protein (DARP) have all been linked to insulin resistance and type 2 diabetes. We set out to measure the expression of these genes in human skeletal muscle and relate them to functional measurements of insulin action during fasting (which induces insulin resistance) and refeeding (which reverses it). Methods: Ten healthy male volunteers underwent 48 h of starvation followed by 24 h of high carbohydrate refeeding. On three occasions, before and after starvation and after refeeding, subjects underwent a 16 min insulin tolerance test to quantify insulin sensitivity. Muscle biopsies were obtained before and after fasting and after refeeding for the analysis of calpain‐10 and calpain‐3, GLUT4 and DARP expression by Western blotting and real‐time PCR. Results: Fasting led to a marked reduction in whole body insulin sensitivity by approx. 45% (P < 0.01) and skeletal muscle GLUT4 gene expression by approx. 40% (P < 0.05). However, fasting had no effect on calpain‐10 and calpain‐3 mRNA or protein levels, or DARP mRNA expression. Refeeding only partly restored insulin sensitivity and GLUT4 gene expression to their pre‐fast values, but did not effect the expression of calpain‐10, calpain‐3 or DARP. Conclusions: These findings demonstrate that in healthy non‐diabetic humans induction of insulin resistance by fasting and its reversal by refeeding with a high CHO diet is mirrored by changes in skeletal muscle GLUT4 but not calpain‐10 and calpain‐3 expression.     

Adiponectin receptor 2 is regulated by nutritional status, leptin and pregnancy in a tissue-specific manner

The aim of the present work was to study the regulation of circulating adiponectin levels and the expression of adiponectin receptor 2 (Adipo-R2) in several rat tissues in relation to fasting, leptin challenge, pregnancy, and chronic undernutrition.Using real-time PCR, we found Adipo-R2 mRNA expression in the liver, stomach, white and brown adipose tissues (WAT and BAT) of adult rats. Immunohistochemical studies confirmed protein expression in the same tissues. Adipo-R2 mRNA levels were decreased in liver after fasting, with no changes in the other tissues. Leptin decreased Adipo-R2 expression in liver and stomach, but increased its expression in WAT and BAT. Chronic caloric restriction in normal rats increased Adipo-R2 gene expression in stomach, while it decreased hepatic Adipo-R2 levels in pregnant rats. Using radioimmunoassay, we found that plasma adiponectin levels were diminished by fasting and leptin. Conversely, circulating adiponectin was increased in food-restricted rats, whereas its levels decreased in food-restricted pregnant rats by the end of gestation.In conclusion our findings provide the first evidence that (a) Adipo-R2 mRNA is regulated in a tissue-specific manner by fasting, but leptin is not responsible for those changes; (b) chronic caloric restriction in normal and pregnant rats also regulate Adipo-R2 mRNA in a tissue-specific manner; and (c) Adipo-R2 mRNA does not show a clear correlation with plasma adiponectin levels.     

Role of macrophages in depot-dependent browning of white adipose tissue

Sympathetic stimulation induces beige adipocytes in white adipose tissue (WAT), known as browning of WAT. In this study, exposure of mice to cold ambient temperature (10 °C) for 24 h induced the mRNA expression of uncoupling protein 1 (UCP1), a marker for beige adipocytes, in inguinal WAT, but not in perigonadal WAT. Thus, we examined the role of macrophages in depot-dependent WAT browning in mice. Flowcytometric analysis showed that total number of macrophages was higher in perigonadal WAT than in inguinal WAT. Cold exposure failed to change the expression of macrophage marker genes in inguinal WAT; however, it increased the mRNA expression of CD11c and tumor necrosis factor-α in perigonadal WAT, indicating that proinflammatory M1 macrophage is activated. The removal of macrophages using clodronate significantly enhanced cold-induced UCP1 mRNA expression in perigonadal WAT. These results suggest that M1 macrophages are involved in the phenotype of perigonadal WAT that hardly undergo browning.     

Re-evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver dysfunction, and its prevalence has markedly increased. We previously evaluated the expression of fatty acid metabolism-related genes in NAFLD and reported changes in expression that could contribute to increased fatty acid synthesis. In the present study, we evaluated the expression of additional fatty acid metabolism-related genes in larger groups of NAFLD (n=26) and normal liver (n=10) samples. The target genes for real-time PCR analysis were as follows: acetyl-CoA carboxylase (ACC) 1, ACC2, fatty acid synthase (FAS), sterol regulatory element-binding protein 1c (SREBP-1c), and adipose differentiation-related protein (ADRP) for evaluation of de novo synthesis and uptake of fatty acids; carnitine palmitoyltransferase 1a; (CPT1a), long-chain acyl-CoA dehydrogenase (LCAD), long-chain L-3-hydroxyacylcoenzyme A dehydrogenase alpha (HADHalpha), uncoupling protein 2 (UCP2), straight-chain acyl-CoA oxidase (ACOX), branched-chain acyl-CoA oxidase (BOX), cytochrome P450 2E1 (CYP2E1), CYP4A11, and peroxisome proliferator-activated receptor (PPAR)alpha for oxidation in the mitochondria, peroxisomes and microsomes; superoxide dismutase (SOD), catalase, and glutathione synthetase (GSS) for antioxidant pathways; and diacylglycerol O-acyltransferase 1 (DGAT1), PPARgamma, and hormone-sensitive lipase (HSL) for triglyceride synthesis and catalysis. In NAFLD, although fatty acids accumulated in hepatocytes, their de novo synthesis and uptake were up-regulated in association with increased expression of ACC1, FAS, SREBP-1c, and ADRP. Fatty acid oxidation-related genes, LCAD, HADHalpha, UCP2, ACOX, BOX, CYP2E1, and CYP4A11, were all overexpressed, indicating that oxidation was enhanced in NAFLD, whereas the expression of CTP1a and PPARalpha was decreased. Furthermore, SOD and catalase were also overexpressed, indicating that antioxidant pathways are activated to neutralize reactive oxygen species (ROS), which are overproduced during oxidative processes. The expression of DGAT1 was up-regulated without increased PPARgamma expression, whereas the expression of HSL was decreased. Our data indicated the following regarding NAFLD: i) increased de novo synthesis and uptake of fatty acids lead to further fatty acid accumulation in hepatocytes; ii) mitochondrial fatty acid oxidation is decreased or fully activated; iii) in order to complement the function of mitochondria (beta-oxidation), peroxisomal (beta-oxidation) and microsomal (omega-oxidation) oxidation is up-regulated to decrease fatty acid accumulation; iv) antioxidant pathways including SOD and catalase are enhanced to neutralize ROS overproduced during mitochondrial, peroxisomal, and microsomal oxidation; and v) lipid droplet formation is enhanced due to increased DGAT expression and decreased HSL expression. Further studies will be needed to clarify how fatty acid synthesis is increased by SREBP-1c, which is under the control of insulin and AMP-activated protein kinase.     

Differential modulation of PPARα and γ target gene expression in the liver and kidney of rats treated with aspirin

Aspirin modified peroxisomal enzymatic activities both in the liver and renal cortex of rats, producing typical effects of peroxisomal proliferators (PPs). Although similar increments in beta-oxidation system and catalase activities were observed in both organs, induction of mRNA-Cyp4a10 and mRNA-FAT/CD36, target genes for peroxisome proliferator-activated receptors alpha (PPARalpha) and gamma (PPARgamma), respectively, was only present in the liver. There was no effect on liver mRNA-PPARalpha, while mRNA-PPARgamma was down-regulated, probably as a result of enzymatic inhibition of cyclooxygenases (COXs) by aspirin which has been shown to decrease the levels of PGJ2 and its metabolites, known as strong endogenous ligands for PPARgamma. Typical PP alterations in cell replication and apoptosis were not found during aspirin treatment or after withdrawal, suggesting that peroxisome proliferation occurs without inducing cell cycle alterations. Probably, the synergic action of both PPARalpha and PPARgamma receptors might reduce the impact on cell proliferation and apoptosis.     

Repeated fasting/refeeding elevates plasma leptin without increasing fat mass in rats

It is known that repeated fasting and refeeding increase capacity of fat storage in adipose tissue as an adaptive response to fasting. However, the amount of weight gain in fasted/refed animals falls behind the control level in most rodent studies. Leptin, an adipocyte-derived hormone that impacts on energy homeostasis, may be up-regulated by repeated cycles of fasting and refeeding. In this study, we investigated the adaptive response of leptin to repeated cycles of 1-day fasting and 1-day refeeding for 42 days in rats. The repeated fasting and refeeding (RFR) rats gained less body weight than the controls. Daily food intake of the RFR rats was decreased after Day 16 and remained suppressed. Circulating leptin levels of the RFR rats were significantly elevated at Day 35 compared with the controls and at Day 44 compared with the controls and pair-fed (PF) rats. Leptin mRNA levels of these rats were also significantly increased in retroperitoneal white adipose tissue (RT-WAT) compared with the controls and PF rats. Moreover, hypothalamic proopiomelanocortic (POMC) gene expression was augmented in the RFR rats compared with the controls and PF rats. However, there was no statistical difference in percent visceral fat mass among the experimental groups. Lipoprotein lipase (LPL) mRNA levels of RFR rats were significantly increased in RT-WAT compared with the controls and PF rats. These data indicated that leptin was up-regulated in response to chronic repeated fasting and refeeding cycles without a concomitant increase in adiposity, and the augmented leptin levels were associated with an increase in POMC gene expression, reduced food intake, and diminished body weight gain.     

脂代谢相关基因在低密度脂蛋白受体基因缺失幼龄小鼠肝脏中的表达分析

目的 探讨脂代谢相关基因在低密度脂蛋白受体(LDLR)基因缺失(LDLR-/-)小鼠肝脏中的表达特征及其与血脂紊乱和动脉粥样硬化早期病变的关系.方法 应用RT-PCR技术分析14、30、60和90天龄LDLR-/-与野生型(WT)小鼠靶基因表达差异,并进行血生化及主动脉形态学检测.结果 与同龄WT小鼠相比,LDLR-/-小鼠肝脏中载脂蛋白AⅣ、脂肪酸转运酶和肉碱棕榈酰转移酶Ⅰ的mRNA水平在14天龄时即显著下调(P＜0.05);30天龄时载脂蛋白A Ⅰ显著上调,载脂蛋白F则显著下调(P＜0.05);60天龄时肝X受体α显著升高(P＜0.05),酰基辅酶A氧化酶1在90天龄时显著下调(P＜0.05);载脂蛋白A Ⅴ、载脂蛋白E、过氧化物增殖物激活受体α和血管生成素样蛋白3差异无统计学意义(P＞0.05).血清总胆固醇、甘油三酯和低密度脂蛋白C含量从14天龄起均显著高于同龄WT小鼠(均P＜0.05),并随年龄增长持续升高.结论 上述脂代谢相关基因在幼龄小鼠即发生表达水平的改变,与血脂紊乱及主动脉病变发生过程呈正相关,说明其可能共同参与幼龄小鼠的脂质代谢紊乱,进而影响动脉内皮细胞功能改变乃至动脉粥样硬化早期病变的发生.