不同发育阶段雄性小鼠睾丸组织mEppin基因mRNA的表达差异研究

目的实时荧光定量PCR检测雄性小鼠不同发育阶段睾丸组织中m Eppin基因的mRNA表达差异,为探究m Eppin基因表达与雄性生殖相关性提供生物学数据。方法分别选取胚胎期、50日龄、90日龄雄性ICR小鼠各6只,共18只,取各组小鼠睾丸组织提取总RNA,实时荧光定量PCR方法检测m Eppin基因mRNA表达水平。结果不同发育阶段小鼠睾丸m Eppin基因表达水平差异明显。胚胎组小鼠中没有检测到m Eppin基因mRNA表达,50日龄小鼠和90日龄小鼠中均检测到m Eppin基因mRNA有较高表达水平。其中,90日龄小鼠睾丸中m Eppin基因mRNA的相对表达水平是50日龄小鼠的2.65倍。结论本研究结果表明m Eppin基因mRNA的表达在小鼠睾丸组织中具阶段特异性。     

Wnt-4 Expression Induces a Pregnancy-like Growth Pattern in Reconstituted Mammary Glands in Virgin Mice

Expression of Wnt-4, a member of the Wnt gene family, is induced during early pregnancy in the mouse mammary gland. To investigate the function of Wnt-4, we used a recombinant retrovirus to constitutively express the gene in transplanted mammary epithelium grown in virgin animals. In fully grown glands, Wnt-4 expression resulted in ducts that were more highly branched than normal and caused some premature alveolar development. These changes resembled those seen during pregnancy, suggesting that endogenous Wnt-4 expression may regulate epithelial branching in early pregnancy. The modified growth pattern induced by Wnt-4 expression was similar to that induced by Wnt-1, one of the members of the Wnt gene family activated by mouse mammary tumour virus. As Wnt-1 is not normally expressed in the mammary gland, it may exert its effect on the mammary gland by activating a developmental pathway normally regulated by Wnt-4.     

Chronic Endocannabinoid System Stimulation Induces Muscle Macrophage and Lipid Accumulation in Type 2 Diabetic Mice Independently of Metabolic Endotoxaemia

Aims

Obesity and type 2 diabetes are characterised by low-grade inflammation, metabolic endotoxaemia (i.e., increased plasma lipopolysaccharides [LPS] levels) and altered endocannabinoid (eCB)-system tone. The aim of this study was to decipher the specific role of eCB-system stimulation or metabolic endotoxaemia in the onset of glucose intolerance, metabolic inflammation and altered lipid metabolism.

Methods

Mice were treated with either a cannabinoid (CB) receptor agonist (HU210) or low-dose LPS using subcutaneous mini-pumps for 6 weeks. After 3 weeks of the treatment under control (CT) diet, one-half of each group of mice were challenged with a high fat (HF) diet for the following 3-week period.

Results

Under basal conditions (control diet), chronic CB receptor agonist treatment (i.e., 6 weeks) induced glucose intolerance, stimulated metabolic endotoxaemia, and increased macrophage infiltration (CD11c and F4/80 expression) in the muscles; this phenomenon was associated with an altered lipid metabolism (increased PGC-1α expression and decreased CPT-1b expression) in this tissue. Chronic LPS treatment tended to increase the body weight and fat mass, with minor effects on the other metabolic parameters. Challenging mice with an HF diet following pre-treatment with the CB agonist exacerbated the HF diet-induced glucose intolerance, the muscle macrophage infiltration and the muscle''s lipid content without affecting the body weight or the fat mass.

Conclusion

Chronic CB receptor stimulation under basal conditions induces glucose intolerance, stimulates metabolic inflammation and alters lipid metabolism in the muscles. These effects worsen following the concomitant ingestion of an HF diet. Here, we highlight the central roles played by the eCB system and LPS in the pathophysiology of several hallmarks of obesity and type 2 diabetes.     

Nitrogen-Deprivation Elevates Lipid Levels in Symbiodinium spp. by Lipid Droplet Accumulation: Morphological and Compositional Analyses

Stable cnidarian-dinoflagellate (genus Symbiodinium) endosymbioses depend on the regulation of nutrient transport between Symbiodinium populations and their hosts. It has been previously shown that the host cytosol is a nitrogen-deficient environment for the intracellular Symbiodinium and may act to limit growth rates of symbionts during the symbiotic association. This study aimed to investigate the cell proliferation, as well as ultrastructural and lipid compositional changes, in free-living Symbiodinium spp. (clade B) upon nitrogen (N)-deprivation. The cell proliferation of the N-deprived cells decreased significantly. Furthermore, staining with a fluorescent probe, boron dipyrromethane 493/503 (BODIPY 493/503), indicated that lipid contents progressively accumulated in the N-deprived cells. Lipid analyses further showed that both triacylglycerol (TAG) and cholesterol ester (CE) were drastically enriched, with polyunsaturated fatty acids (PUFA; i.e., docosahexaenoic acid, heneicosapentaenoic acid, and oleic acid) became more abundant. Ultrastructural examinations showed that the increase in concentration of these lipid species was due to the accumulation of lipid droplets (LDs), a cellular feature that have previously shown to be pivotal in the maintenance of intact endosymbioses. Integrity of these stable LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Proteomic analyses of these LDs identified proteins putatively involved in lipid metabolism, signaling, stress response and energy metabolism. These results suggest that LDs production may be an adaptive response that enables Symbiodinium to maintain sufficient cellular energy stores for survival under the N-deprived conditions in the host cytoplasm.     

Cholic Acid Induces a Cftr Dependent Biliary Secretion and Liver Growth Response in Mice

The cause of Cystic fibrosis liver disease (CFLD), is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobic bile salts, are related to the development CFLD. To test our hypothesis we studied, in Cftr^-/- and control mice, bile production, bile composition and liver pathology, in normal feeding condition and during cholate exposure, either acute (intravenous) or chronic (three weeks via the diet). In Cftr^-/- and control mice the basal bile production was comparable. Intravenous taurocholate increased bile production to the same extent in Cftr^-/- and control mice. However, chronic cholate exposure increased the bile flow significantly less in Cftr^-/- mice than in controls, together with significantly higher biliary bile salt concentration in Cftr^-/- mice. Prolonged cholate exposure, however, did not induce CFLD like pathology in Cftr^-/- mice. Chronic cholate exposure did induce a significant increase in liver mass in controls that was absent in Cftr^-/- mice. Chronic cholate administration induces a cystic fibrosis-specific hepatobiliary phenotype, including changes in bile composition. These changes could not be associated with CFLD like pathological changes in CF mouse livers. However, chronic cholate administration induces liver growth in controls that is absent in Cftr^-/- mice. Our findings point to an impaired adaptive homeotrophic liver response to prolonged hydrophobic bile salt exposure in CF conditions.     

HILPDA Uncouples Lipid Droplet Accumulation in Adipose Tissue Macrophages from Inflammation and Metabolic Dysregulation

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A Distinct Expression Pattern of Cyclin K in Mammalian Testes Suggests a Functional Role in Spermatogenesis

Germ cell and embryonic stem cells are inextricably linked in many aspects. Remarkably both can generate all somatic cell types in organisms. Yet the molecular regulation accounting for these similarities is not fully understood. Cyclin K was previously thought to associate with CDK9 to regulate gene expression. However, we and others have recently shown that its cognate interacting partners are CDK12 and CDK13 in mammalian cells. We further demonstrated that cyclin K is essential for embryonic stem cell maintenance. In this study, we examined the expression of cyclin K in various murine and human tissues. We found that cyclin K is highly expressed in mammalian testes in a developmentally regulated manner. During neonatal spermatogenesis, cyclin K is highly expressed in gonocytes and spermatogonial stem cells. In adult testes, cyclin K can be detected in spermatogonial stem cells but is absent in differentiating spermatogonia, spermatids and spermatozoa. Interestingly, the strongest expression of cyclin K is detected in primary spermatocytes. In addition, we found that cyclin K is highly expressed in human testicular cancers. Knockdown of cyclin K in a testicular cancer cell line markedly reduces cell proliferation. Collectively, we suggest that cyclin K may be a novel molecular link between germ cell development, cancer development and embryonic stem cell maintenance.     

Effects of Puerarin on Lipid Accumulation and Metabolism in High-Fat Diet-Fed Mice

In order to investigate the mechanisms by which puerarin from kudzu root extract regulates lipid metabolism, fifty mice were randomly assigned to five groups: normal diet, high-fat diet (HFD), and HFD containing 0.2%, 0.4% or 0.8% puerarin for 12 weeks. Body weight, intraperitioneal adipose tissue (IPAT) weight, serum biochemical parameters, and hepatic and feces lipids were measured. Activity and mRNA and protein expressions of hepatic lipid metabolism-related enzymes were analyzed. Compared with HFD, 0.4% and 0.8% puerarin significantly decreased body and IPAT weight. There was a significant decrease in the serum and hepatic concentrations of total cholesterol, triglycerides and leptin in mice fed the 0.4% and 0.8% puerarin diets compared with HFD. Fatty acid synthase activity was suppressed in mice fed the 0.4% and 0.8% puerarin diets, while the activities of AMP-activated protein kinase (AMPK), carnitine acyltransferase (CAT) and hormone-sensitive lipase (HSL) were increased. mRNA expression of peroxisome proliferator-activated receptor γ 2 (PPARγ 2) was down-regulated in liver of mice fed the 0.8% diet compared with HFD, while mRNA expression of CAT and HSL was considerably up-regulated by 0.4% and 0.8% puerarin diets. The protein expression of PPARγ2 in liver was decreased and those of p-AMPK, HSL and p-HSL were increased in mice fed 0.4% and 0.8% puerarin diets. These results suggest that > 0.4% puerarin influenced the activity, mRNA and protein levels of hepatic lipid metabolism-related enzymes, decreasing serum and liver lipids, body weight gain and fat accumulation. Puerarin might be beneficial to prevent lifestyle-related diseases.     

Enhanced Gastrointestinal Expression of Cytosolic Malic Enzyme (ME1) Induces Intestinal and Liver Lipogenic Gene Expression and Intestinal Cell Proliferation in Mice

The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue’s metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.     

Overexpression of miR-155 in the Liver of Transgenic Mice Alters the Expression Profiling of Hepatic Genes Associated with Lipid Metabolism

Hepatic expression profiling has revealed miRNA changes in liver diseases, while hepatic miR-155 expression was increased in murine non-alcoholic fatty liver disease, suggesting that miR-155 might regulate the biological process of lipid metabolism. To illustrate the effects of miR-155 gain of function in transgenic mouse liver on lipid metabolism, transgenic mice (i.e., Rm155LG mice) for the conditional overexpression of mouse miR-155 transgene mediated by Cre/lox P system were firstly generated around the world in this study. Rm155LG mice were further crossed to Alb-Cre mice to realize the liver-specific overexpression of miR-155 transgene in Rm155LG/Alb-Cre double transgenic mice which showed the unaltered body weight, liver weight, epididymal fat pad weight and gross morphology and appearance of liver. Furthermore, liver-specific overexpression of miR-155 transgene resulted in significantly reduced levels of serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL), as well as remarkably decreased contents of hepatic lipid, TG, HDL and free fatty acid in Rm155LG/Alb-Cre transgenic mice. More importantly, microarray data revealed a general downward trend in the expression profile of hepatic genes with functions typically associated with fatty acid, cholesterol and triglyceride metabolism, which is likely at least partially responsible for serum cholesterol and triglyceride lowering observed in Rm155LG/Alb-Cre mice. In this study, we demonstrated that hepatic overexpression of miR-155 alleviated nonalcoholic fatty liver induced by a high-fat diet. Additionally, carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) was identified as a direct miR-155 target gene that is potentially responsible for the partial liver phenotypes observed in Rm155LG/Alb-Cre mice. Taken together, these data from miR-155 gain of function study suggest, for what we believe is the first time, the altered lipid metabolism and provide new insights into the metabolic state of the liver in Rm155LG/Alb-Cre mice.     

Expression Pattern of the LacZ Reporter in Secretogranin III Gene-trapped Mice

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping (SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system:     

Testosterone-Dependent Interaction between Androgen Receptor and Aryl Hydrocarbon Receptor Induces Liver Receptor Homolog 1 Expression in Rat Granulosa Cells

Androgens play a major role in the regulation of normal ovarian function; however, they are also involved in the development of ovarian pathologies. These contrasting effects may involve a differential response of granulosa cells to the androgens testosterone (T) and dihydrotestosterone (DHT). To determine the molecular pathways that mediate the distinct effects of T and DHT, we studied the expression of the liver receptor homolog 1 (LRH-1) gene, which is differentially regulated by these steroids. We found that although both T and DHT stimulate androgen receptor (AR) binding to the LRH-1 promoter, DHT prevents T-mediated stimulation of LRH-1 expression. T stimulated the expression of aryl hydrocarbon receptor (AHR) and its interaction with the AR. T also promoted the recruitment of the AR/AHR complex to the LRH-1 promoter. These effects were not mimicked by DHT. We also observed that the activation of extracellular regulated kinases by T is required for AR and AHR interaction. In summary, T, but not DHT, stimulates AHR expression and the interaction between AHR and AR, leading to the stimulation of LRH-1 expression. These findings could explain the distinct response of granulosa cells to T and DHT and provide a molecular mechanism by which DHT negatively affects ovarian function.     

MicroRNA-185改善非酒精性脂肪肝小鼠胰岛素敏感性并调节脂代谢基因的表达

目的:探讨micro RNA-185(miR-185)对高脂饮食的小鼠模型的HepG2肝细胞脂质代谢和胰岛素信号通路的调节作用。方法:应用定量反转录聚合酶链反应评估过表达或抑制miR-185表达脂质合成相关基因的mRNA水平。此外,应用Western Blot方法测定转染HepG2细胞pre-mir-185后的关键信号通路组分(IRS-1,IRS-2,PI3K、AKT2)和磷酸化PI3K和AKT2的表达情况。结果:诱导的人类HepG2细胞的软脂酸对mir-185水平的下降具有时间和剂量依赖性。经过mir-185转染的HepG2细胞显著降低脂肪酸合成酶,3-hydroxy-3-methylglutaryl-coa还原酶,固醇调节元件结合蛋白和固醇调节元件结合蛋白-1c的mRNA水平,而使用anti-mir-185寡核苷酸抑制mir-185在HepG2细胞中产生相反的作用。在高脂饮食的小鼠模型,与对照组动物相比,mir-185处理后脂质积累明显改善。mir-185诱导后通过上调胰岛素受体底物2增强胰岛素信号通路。结论:miR-185在体内和体外调节肝细胞脂肪酸代谢和胆固醇平衡,以及在改善胰岛素敏感性中起重要作用,miR-185可能成为非酒精性脂肪肝和胰岛素抵抗的新靶点和治疗非酒精性脂肪肝药物作用新靶标。