Insulin-independent induction of sterol regulatory element-binding protein-1c expression in the livers of streptozotocin-treated mice

Insulin and glucose together have been previously shown to regulate hepatic sterol regulatory element-binding protein (SREBP)-1c expression. We sought to explore the nutritional regulation of lipogenesis through SREBP-1c induction in a setting where effects of sugars versus insulin could be distinguished. To do so, mice were insulin depleted by streptozotocin (STZ) administration and subjected to a fasting-refeeding protocol with glucose, fructose, or sucrose. Unexpectedly, the insulin-depleted mice exhibited a marked induction of SREBP-1c on all sugars, and this increase in SREBP-1c was even more dramatic than in the non-STZ-administered controls. The time course of changes in SREBP-1 induction varied depending on the type of sugars in both control and STZ-administered mice. Glucose refeeding gave a peak of SREBP-1c induction, whereas fructose refeeding caused slow and gradual increments, and sucrose refeeding fell between these two responses. Expression of various lipogenic enzymes were also gradually increased over time, irrespective of the types of sugars, with greater intensities in STZ-administered than in nontreated mice. In contrast, induction of hepatic glucokinase and suppression of phoshoenolpyruvate carboxykinase were insulin dependent in an early refed state. These data clearly demonstrate that nutritional regulation of SREBP-1c and lipogenic genes may be completely independent of insulin as long as sufficient carbohydrates are available.     

DNA甲基化在肝脏再生中的研究现状与展望

目的总结目前DNA甲基化与肝脏再生关系的研究现状。方法检索国内外相关文献,对肝细胞甲基化水平、基因表达调控、甲基化相关蛋白与肝脏再生关系的研究进行综述。结果 DNA甲基化作为生物体内一种重要的表观遗传调控方式,近年来在肝脏再生中的作用越来越被重视。现有的研究已经发现,在肝脏再生过程中存在基因组低甲基化、相关增殖基因甲基化改变以及DNA甲基化转移酶、含植物同源结构域和环指结构域泛素样蛋白1调控肝脏再生等表观遗传现象。结论 DNA甲基化与肝脏再生之间存在着诸多的联系,从DNA甲基化水平调节肝脏再生有望在不久的将来成为现实。     

DNA损伤修复基因XRCC1启动子甲基化对肝癌易感性及其预后的影响

目的 探讨原发性肝癌组织和正常肝脏组织的DNA损伤修复基因XRCC1启动子甲基化状态,分析其与肝癌易感性的关系及对患者预后的影响.方法 甲基化特异性PCR检测手术切除的肝癌组织78例及正常肝组织78例的XRCC1基因甲基化情况,并随访3年以上.结果 肝癌组的XRCC1启动子甲基化率远高于对照组（P＜0.05）,肝癌组发生XRCC1启动子甲基化的危险是对照组的13倍（4.089 vs 41.332）;XRCC1启动子甲基化的个体发生肝癌的危险是非甲基化的10.36倍（3.423 vs 31.354）（P＜0.05）;XRCC1启动子甲基化可致肝癌患者无进展期生存率和总体生存率降低（P＜ 0.05）.结论 DNA损伤修复基因XRCC1启动子甲基化在肝癌发生和发展的过程中起着重要作用,并对肝癌患者较差的预后具有提示作用.     

Gene expression profiles of nondiabetic and diabetic obese mice suggest a role of hepatic lipogenic capacity in diabetes susceptibility

Obesity is a strong risk factor for the development of type 2 diabetes. We have previously reported that in adipose tissue of obese (ob/ob) mice, the expression of adipogenic genes is decreased. When made genetically obese, the BTBR mouse strain is diabetes susceptible and the C57BL/6J (B6) strain is diabetes resistant. We used DNA microarrays and RT-PCR to compare the gene expression in BTBR-ob/ob versus B6-ob/ob mice in adipose tissue, liver, skeletal muscle, and pancreatic islets. Our results show: 1) there is an increased expression of genes involved in inflammation in adipose tissue of diabetic mice; 2) lipogenic gene expression was lower in adipose tissue of diabetes-susceptible mice, and it continued to decrease with the development of diabetes, compared with diabetes-resistant obese mice; 3) hepatic expression of lipogenic enzymes was increased and the hepatic triglyceride content was greatly elevated in diabetes-resistant obese mice; 4) hepatic expression of gluconeogenic genes was suppressed at the prediabetic stage but not at the onset of diabetes; and 5) genes normally not expressed in skeletal muscle and pancreatic islets were expressed in these tissues in the diabetic mice. We propose that increased hepatic lipogenic capacity protects the B6-ob/ob mice from the development of type 2 diabetes.     

Aberrant methylation of the TDMR of the GTF2AIL promoter does not affect fertilisation rates via TESE in patients with hypospermatogenesis

Increasing evidence shows a relationship between epigenetic regulation and male infertility. The GTF2A1L gene promoter contains the DNA methylation site of a tissue-specific differentially methylated region （TDMR）. Eighty-six patients with non-obstructive azoospermia were assessed for the DNA methylation state of CpG islands in the GTF2AIL promoter using testicular genomic DNA. Based on histological criteria, 26 of the 86 patients had normal spermatogenesis （controls）, 17 had hypospermatogenesis and 26 had a Sertoli cell-only phenotype or tubular sclerosis. GTF2AIL TDMR methylation was significantly lower in testes DNA from control samples than from hypospermatogenic samples （P=0.029）. Patients with hypospermatogenesis were divided into two subgroups： high DNA methylation （HM, n=5） and low DNA methylation （LM, n= 12）. The GTF2AIL TDMR methylation rate differed significantly between the HM and LM groups （P=0.0019）, and GTF2A 1L expression was significantly higher among the LM than in the HM patients （P=0.023）. High TDMR methylation was correlated with low GTF2AIL gene expression levels. Both groups demonstrated relatively good outcomes with respect to sperm retrieval, fertilisation, pregnancy and childbirth rates. We observed that aberrant GTF2AIL gene expression was not correlated with fertilisation rates. The testicular sperm extraction （TESE） technique may be used to overcome male infertility due to aberrant TDMR methvlation.     

Regulation of renal fatty acid and cholesterol metabolism, inflammation, and fibrosis in Akita and OVE26 mice with type 1 diabetes

In Akita and OVE26 mice, two genetic models of type 1 diabetes, diabetic nephropathy is characterized by mesangial expansion and loss of podocytes, resulting in glomerulosclerosis and proteinuria, and is associated with increased expression of profibrotic growth factors, proinflammatory cytokines, and increased oxidative stress. We have also found significant increases in renal triglyceride and cholesterol content. The increase in renal triglyceride content is associated with 1) increased expression of sterol regulatory element-binding protein (SREBP)-1c and carbohydrate response element-binding protein (ChREBP), which collectively results in increased fatty acid synthesis, 2) decreased expression of peroxisome proliferator-activated receptor (PPAR)-alpha and -delta, which results in decreased fatty acid oxidation, and 3) decreased expression of farnesoid X receptor (FXR) and small heterodimer partner (SHP). The increase in cholesterol content is associated with 1) increased expression of SREBP-2 and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, which results in increased cholesterol synthesis, and 2) decreased expression of liver X receptor (LXR)-alpha, LXR-beta, and ATP-binding cassette transporter-1, which results in decreased cholesterol efflux. Our results indicate that in type 1 diabetes, there is altered renal lipid metabolism favoring net accumulation of triglycerides and cholesterol, which are driven by increases in SREBP-1, ChREBP, and SREBP-2 and decreases in FXR, LXR-alpha, and LXR-beta, which may also play a role in the increased expression of profibrotic growth hormones, proinflammatory cytokines, and oxidative stress.     

Isomer-dependent metabolic effects of conjugated linoleic acid: insights from molecular markers sterol regulatory element-binding protein-1c and LXRalpha

Conjugated linoleic acid (CLA) is a heterogeneous group of positional and geometric isomers of linoleic acid. This study demonstrates the divergent effects of the cis-9 trans-11 (c9,t11-CLA) and trans-10 cis-12 (t10,c12-CLA) isomers of CLA on lipid metabolism and nutrient regulation of gene expression in ob/ob mice. The c9, t11-CLA diet decreased serum triacylglycerol (P = 0.01) and nonesterified fatty acid (NEFA) (P = 0.05) concentrations, and this was associated with reduced hepatic sterol regulatory element-binding protein-1c (SREBP-1c; P = 0.0045) mRNA expression, coupled with reduced levels of both the membrane-bound precursor and the nuclear forms of the SREBP-1 protein. C9,t11-CLA significantly reduced hepatic LXRalpha (P = 0.019) mRNA expression, a novel regulator of SREBP-1c. In contrast, c9,t11-CLA increased adipose tissue SREBP-1c mRNA expression (P = 0.0162) proportionally to the degree of reduction of tumor necrosis factor alpha (TNF-alpha) mRNA (P = 0.012). Recombinant TNF-alpha almost completely abolished adipose tissue SREBP-1c mRNA expression in vivo. The t10,c12-CLA diet promoted insulin resistance and increased serum glucose (P = 0.025) and insulin (P = 0.01) concentrations. T10, c12-CLA induced profound weight loss (P = 0.0001) and increased brown and white adipose tissue UCP-2 (P = 0.001) and skeletal muscle UCP-3 (P = 0.008) mRNA expression. This study highlights the contrasting molecular and metabolic effect of two isomers of the same fatty acids. The ameliorative effect of c9,t11-CLA on lipid metabolism may be ascribed to reduced synthesis and cleavage of hepatic SREBP-1, which in turn may be regulated by hepatic LXRalpha expression.     

Regulation of miR‐200c and miR‐141 by Methylation in Prostate Cancer

BACKGROUND

In prostate cancer (PCa), abnormal expression of several microRNAs (miRNAs) has been previously reported. Increasing evidence shows that aberrant epigenetic regulation of miRNAs is a contributing factor to their altered expression in cancer. In this study, we investigate whether expression of miR‐200c and miR‐141 in PCa is related to the DNA methylation status of their promoter.

METHODS

PCR analysis of miR‐200c and miR‐141, and CpG methylation analysis of their common promoter, was performed in PCa cell‐lines and in archived prostate biopsy specimens. The biological significance of miR‐200c and miR‐141 expression in prostate cancer cells was assessed by a series of in vitro bioassays and the effect on proposed targets DNMT3A and TET1/TET3 was investigated. The effect on promoter methylation status in cells treated with demethylating agents was also examined.

RESULTS

miR‐200c and miR‐141 are both highly elevated in LNCaP, 22RV1, and DU145 cells, but significantly reduced in PC3 cells. This correlates inversely with the methylation status of the miR‐200c/miR‐141 promoter, which is unmethylated in LNCaP, 22RV1, and DU145 cells, but hypermethylated in PC3. In PC3 cells, miR‐200c and miR‐141 expression is subsequently elevated by treatment with the demethylating drug decitabine (5‐aza‐2′deoxycytidine) and by knockdown of DNA methyltransferase 1 (DNMT1), suggesting their expression is regulated by methylation. Expression of miR‐200c and miR‐141 in prostate biopsy tissue was inversely correlated with methylation in promoter CpG sites closest to the miR‐200c/miR‐141 loci. In vitro, over‐expression of miR‐200c in PC3 cells inhibited growth and clonogenic potential, as well as inducing apoptosis. Expression of the genes DNMT3A and TET1/TET3 were down‐regulated by miR‐200c and miR‐141 respectively. Finally, treatment with the soy isoflavone genistein caused demethylation of the promoter CpG sites closest to the miR‐200c/miR‐141 loci resulting in increased miR‐200c expression.

CONCLUSIONS

Our findings provide evidence that miR‐200c and miR‐141 are under epigenetic regulation in PCa cells. We propose that profiling their expression and methylation status may have potential as a novel biomarker or focus of therapeutic intervention in the diagnosis and prognosis of PCa. Prostate 76:1146–1159, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.     

肝硬化组织中p16INK4a和Rb的基因甲基化状态和蛋白表达

目的：探讨肝硬化组织中p16INK4a和视网膜母细胞瘤易感基因（Rb基因）启动子区域的甲基化状态与其蛋白的表达的关系。方法：分别采用PCR和免疫组织化学方法测定65例肝硬化患者和20例正常人肝组织中p16INK4a和Rb启动子区域甲基化状况和蛋白的表达。结果：20例正常肝组织（对照组肝组织）中均未检测到p16INK4a和Rb甲基化异常,肝硬化组肝组织中p16INK4a和Rb基因甲基化率分别为40.0%（26/65）和36.9%（24/65）;p16INK4a和Rb蛋白表达的积分光密度（IOD）在正常肝组织为225.7±27.4和254.7±34.8,在肝硬化组肝组织中为32.4±7.5和45.2±6.4,差异均有统计学意义（均P<0.05）。结论：肝硬化组织中存在p16INK4a和Rb异常甲基化和p16INK4a和Rb蛋白的表达降低,由此导致的细胞周期失控可能参与了肝硬化的发生发展过程。     

Hepatic Akt activation induces marked hypoglycemia, hepatomegaly, and hypertriglyceridemia with sterol regulatory element binding protein involvement

Akt is critical in insulin-induced metabolism of glucose and lipids. To investigate functions induced by hepatic Akt activation, a constitutively active Akt, NH(2)-terminally myristoylation signal-attached Akt (myr-Akt), was overexpressed in the liver by injecting its adenovirus into mice. Hepatic myr-Akt overexpression resulted in a markedly hypoglycemic, hypoinsulinemic, and hypertriglyceridemic phenotype with fatty liver and hepatomegaly. To elucidate the sterol regulatory element binding protein (SREBP)-1c contribution to these phenotypic features, myr-Akt adenovirus was injected into SREBP-1 knockout mice. myr-Akt overexpression induced hypoglycemia and hepatomegaly with triglyceride accumulation in SREBP-1 knockout mice to a degree similar to that in normal mice, whereas myr-Akt-induced hypertriglyceridemia in knockout mice was milder than that in normal mice. The myr-Akt-induced changes in glucokinase, phosphofructokinase, glucose-6-phosphatase, and PEPCK expressions were not affected by knocking out SREBP-1, whereas stearoyl-CoA desaturase 1 induction was completely inhibited in knockout mice. Constitutively active SREBP-1-overexpressing mice had fatty livers without hepatomegaly, hypoglycemia, or hypertriglyceridemia. Hepatic acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and glucose-6-phosphate dehydrogenase expressions were significantly increased by overexpressing SREBP-1, whereas glucokinase, phospho-fructokinase, glucose-6-phosphatase, and PEPCK expressions were not or only slightly affected. Thus, SREBP-1 is not absolutely necessary for the hepatic Akt-mediated hypoglycemic effect. In contrast, myr-Akt-induced hypertriglyceridemia and hepatic triglyceride accumulation are mediated by both Akt-induced SREBP-1 expression and a mechanism involving fatty acid synthesis independent of SREBP-1.