TFE3 transcriptionally activates hepatic IRS-2, participates in insulin signaling and ameliorates diabetes

Using an expression cloning strategy, we have identified TFE3, a basic helix-loop-helix protein, as a transactivator of metabolic genes that are regulated through an E-box in their promoters. Adenovirus-mediated expression of TFE3 in hepatocytes in culture and in vivo strongly activated expression of IRS-2 and Akt and enhanced phosphorylation of insulin-signaling kinases such as Akt, glycogen synthase kinase 3beta and p70S6 kinase. TFE3 also induced hexokinase II (HK2) and insulin-induced gene 1 (INSIG1). These changes led to metabolic consequences, such as activation of glycogen and protein synthesis, but not lipogenesis, in liver. Collectively, plasma glucose levels were markedly reduced both in normal mice and in different mouse models of diabetes, including streptozotocin-treated, db/db and KK mice. Promoter analyses showed that IRS2, HK2 and INSIG1 are direct targets of TFE3. Activation of insulin signals in both insulin depletion and resistance suggests that TFE3 could be a therapeutic target for diabetes.     

Hepatocyte-specific deletion of BAP31 promotes SREBP1C activation,promotes hepatic lipid accumulation,and worsens IR in mice

Conditional knockout mice with targeted disruption of B-cell associated protein (BAP)31 in adult mouse liver were generated and challenged with a high-fat diet (HFD) for 36 or 96 days and markers of obesity, diabetes, and hepatic steatosis were determined. Mutant mice were indistinguishable from WT littermates, but exhibited increased HFD-induced obesity. BAP31-deletion in hepatocytes increased the expression of SREBP1C and the target genes, including acetyl-CoA carboxylase 1 and stearoyl-CoA desaturase-1, and increased hepatic lipid accumulation and HFD-induced liver steatosis. Immunoprecipitation assay showed that BAP31 interacts with SREBP1C and insulin-induced gene 1 (INSIG1), and BAP31-deletion reduces INSIG1 expression, suggesting that BAP31 may regulate SREBP1C activity by modulating INSIG1 protein levels. Additionally, BAP31-deletion induced glucose and insulin intolerance, decreased Akt and glycogen synthase kinase 3β phosphorylation, and enhanced hepatic glucose production in mice. Expression of endoplasmic reticulum (ER) stress markers was significantly induced in BAP31-mutant mice. HFD-induced inflammation was aggravated in mutant mice, along with increased c-Jun N-terminal kinase and nuclear factor-κB activation. These findings demonstrate that BAP31-deletion induces SREBP activation and promotes hepatic lipid accumulation, reduces insulin signaling, impairs glucose/insulin tolerance, and increases ER stress and hepatic inflammation, explaining the protective roles of BAP31 in the development of liver steatosis and insulin resistance in HFD-induced obesity in animal models.     

Regulation of INSIG2 by microRNA-96

Mature forms of the microRNAs miR-96, -182, and -183 originate from a single genomic locus and have been shown to be elevated approximately 50-fold in the livers of sterol regulatory element-binding protein-1a and -2 (SREBP-1a and -2) transgenic mice. Our study attempted to identify the possible targets of these microRNAs using miRNA target prediction software. This revealed putative sites in insulin-induced genes (INSIGs). The 3′ untranslated region (UTR) of insulin-induced gene 1 (INSIG1) contained sites corresponding to miR-182, and -183, while the 3′ UTR of INSIG2 featured an miR-96 site. Among these putative sites, only miR-96 demonstrated an inhibitory effect that was specific to the 3′ UTR of INSIG2. As INSIG proteins are the main components of SREBP cleavage complexes that act to release active SREBPs, we assessed the effects of miR-96 on INSIG and SREBP levels and activities. We found that miR-96 reduced the levels of INSIG2 in INSIG1 knockout human fibroblasts, resulting in an increase in SREBP-1 and -2 nuclear forms and a subsequent increase in the abundance of the mRNA of their target genes. These results suggest that miR-96, an miRNA induced by SREBP-2 activation, regulates downstream targets of SREBPs and may increase the abundance of active SREBP.     

大鼠再生肝中hsbp1、hsf1、hsf2、hsp70表达水平改变的分析

在克隆了大鼠热休克因子结合蛋白1基因(hsbp1)全长cDNA基础上,进一步分析它在肝再生中作用。用SD纯系大鼠为材料,按Higgens等方法建立大鼠部分肝切除(PH)模型;用原位杂交等方法分析hsbp1在肝再生中表达变化;用基因表达谱芯片分析hsbp1、hsf1、hsf2和hsp70在肝再生中表达变化。原位杂交和基因表达谱芯片分析表明,PH后6h和66-144h,hsbp1表达发生了有意义上调;8-16h,hsf1表达发生了有意义上调;2-16h,hsf2表达发生了有意义上调;0.5-24h,hsp70表达发生了有意义上调。假手术(只打开腹腔和翻动肝叶,但不进行部分肝切除)后0.5-2h,hsbp1表达发生了有意义下调;8-16h,hsf1表达发生了有意义上调;0-144h,hsf2未发生有意义表达变化;0.5-30h,hsp70表达发生了有意义上调。根据实验结果推测,PH后hsbp1表达上调可增加细胞内HSBP1量,促进生长、发育、分化相关基因表达和再生肝的组织结构功能重建;(假)手术后hsbp1表达下调可减少细胞内HSBP1量,有利于HSF1上调hsp70表达,提高机体和肝脏抗损伤能力。     

大鼠再生肝中hsbp1、hsf1、hsf2、shp70表达水平改变的分析

在克隆了大鼠热休克因子结合蛋白1基因（hsbp1）全长cDNA基础上,进一步分析它在肝再生中作用。用SD纯系大鼠为材料。按Higgens等方法建立大鼠部分肝切除（PH）模型;用原位杂交等方法分析凤6pJ在肝再生中表达变化;用基因表达谱芯片分析凤如1、hsf1、如,2和hsp70在肝再生中表达变化。原位杂交和基因表达谱芯片分析表明。PH后6h和66-144h,hsbp1表达发生了有意义上调;8-16h,nsf1表达发生了有意义上调;2-16h,hsf2表达发生了有意义上调;0．5—24h,hsp70表达发生了有意义上调。假手术（只打开腹腔和翻动肝叶。但不进行部分肝切除）后0．5-2h,hsbp1表达发生了有意义下调;8—16h,hsf1表达发生了有意义上调;0—144h,hsf2未发生有意义表达变化;0．5—30h,hsp70表达发生了有意义上调。根据实验结果推测,PH后hsbp1表达上调可增加细胞内HSBP1量。促进生长、发育、分化相关基因表达和再生肝的组织结构功能重建;（假）手术后hsbp1表达下调可减少细胞内HSBP1量,有利于HSF1上调hsp70表达,提高机体和肝脏抗损伤能力。     

A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape

We characterized three-dimensional human hepatoma cell lines, functional liver cell (FLC) cell lines, to establish a highly differentiated hepatoma cell line. We investigated the effect of extracellular matrix and cell morphology on liver-specific gene expression in FLC cells. The hepatocyte nuclear factor-4α (HNF-4α) and other liver-specific gene expressions were enhanced in spherical FLC-4 cells on EHS-gel, but other human hepatoma cells such as HepG2 did not show the enhancement. Importantly, the liver-specific gene expression levels in spherical FLC-4 cells cultured on EHS-gel were comparable to those of human liver and were much higher than those of other human hepatoma cell lines. The major matrix components and growth factors in EHS-gel did not affect cell shape and liver functions. To exclude any effect of the extracellular matrix, we made spherical FLC-4 cells by actin filament disruption. The actin-disrupted spherical cells also showed an enhanced liver-specific gene expression. We concluded that three-dimensional cell shape per se is one of the most important determinants of liver differentiation functions in FLC-4 cells. Cell morphology-dependent induction of liver-specific gene expression was mediated through microtubule organization. In conclusion, differentiation of FLC-4 human hepatoma cell line can be enhanced to a human liver-like level through the three-dimensional cell shape in a microtubule-dependent manner.     

The alpha1-6-fucosyltransferase gene and its biological significance

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-6-fucosyltransferase (alpha1-6FucT) catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycoproteins. This enzyme was purified from a human fibroblast cell line, porcine brain, a human gastric cancer cell line and human blood platelets. cDNA cloning of porcine and human alpha1-6FucT was performed from a porcine brain and gastric cancer cell cDNA libraries, respectively. Their homology is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence. No homology to other fucosyltransferases such as alpha1-2FucT, alpha1-3FucT and alpha1-4FucT was found except for a region consisting of nine amino acids. The alpha1-6FucT gene is located at chromosome 14q24.3, which is also a different location from other fucosyltransferases reported to date. The alpha1-6FucT gene is the oldest gene family in the phylogenic trees among the nine cloned fucosyltransferase genes. alpha1-6FucT is widely expressed in various rat tissues and the expression of alpha1-6FucT in the liver is enhanced during hepatocarcinogenesis of LEC rats which develop hereditary hepatitis and hepatomas. In cases of human liver diseases, alpha1-6FucT is expressed in both hepatoma tissues and their surrounding tissues with chronic liver disease, but not in the case of normal liver. Serum alpha1-6-fucosylated alpha-fetoprotein (AFP) has been employed for an early diagnosis of patients with hepatoma. The mechanisms by which alpha1-6 fucosylation of AFP occurs in the hepatoma is not due to the up-regulation of alpha1-6FucT alone. Interestingly, when the alpha1-6FucT gene is transfected into Hep3B, a human hepatoma cell line, tumor formation in the liver of nude mice after splenic injection is dramatically suppressed. In this review, we focus on alpha1-6FucT and summarize its properties, gene expression and biological significance.     

Expression and functional features of NaCT, a sodium-coupled citrate transporter, in human and rat livers and cell lines

In this article, we report on the expression and function of a Na(+)-coupled transporter for citrate, NaCT, in human and rat liver cell lines and in primary hepatocytes from the rat liver. We also describe the polarized expression of this transporter in human and rat livers. Citrate uptake in human liver cell lines HepG2 and Huh-7 was obligatorily dependent on Na+. The uptake system showed a preference for citrate over other intermediates of the citric acid cycle and exhibited a Michaelis constant of approximately 6 mM for citrate. The transport activity was stimulated by Li+, and the activation was associated with a marked increase in substrate affinity. Citrate uptake in rat liver cell line MH1C1 was also Na+ dependent and showed a preference for citrate. The Michaelis constant for citrate was approximately 10 microM. The transport activity was inhibited by Li+. Primary hepatocytes from the rat liver also showed robust activity for Na+-coupled citrate uptake, with functional features similar to those described in the rat liver cell line. Immunolabeling with a specific anti-NaCT antibody showed exclusive expression of the transporter in the sinusoidal membrane of hepatocytes in human and rat livers. This constitutes the first report on the expression and function of NaCT in liver cells.     

Expression of the Hoxa-13 gene correlates to hepatitis B and C virus associated HCC

To study the Hoxa-13 gene in the liver, we examined its expression by RT-PCR in various liver cell lines, rat livers under different conditions, and human primary hepatocellular carcinomas (HCCs). The gene was found to be expressed in cell lines originating from liver stem-like cells, but not in cell lines originating from hepatocytes and bile duct epithelia. Expression was induced in rat livers after treatment with d-galactosamine, which is known to induce oval cell proliferation, but not after a two-thirds partial hepatectomy (2/3 PH) where induction of oval cell proliferation is thought not to occur. Expression of the gene correlated with human HCC samples associated with Hepatitis B or C virus infection in this small series. These results suggest that the Hoxa-13 gene may provide a potentially useful tool for elucidation of mechanisms involved in lineage-specific differentiation and carcinogenesis of liver stem cells.     

脂肪细胞分化相关基因在大鼠再生肝中表达变化

肝脏由多种细胞构成,肝再生与细胞分化密切相关,细胞分化受基因转录水平调控。为在基因转录水平了解脂肪细胞分化基因在大鼠肝再生中作用,本文用搜集网站资料和查阅相关论文等方法获得上述基因,用Rat Genome2302.0芯片检测它们在大鼠肝再生(liver regeneration,LR)中表达情况,将三次检验结果相同或相似、在肝再生中表达变化2倍以上、真手术组和假手术组相比差异显著的基因视为肝再生相关基因。初步证实上述基因中75个基因与肝再生相关。肝再生启动(PH后0.5-4h)、G0/G1过渡(PH后4-6h)、细胞增殖(PH后6-66h)、细胞分化和组织结构功能重建(PH后72-168h)等四个阶段起始表达的基因数为44、13、30和1;基因的总表达次数为88、58、302和90。表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用。它们共表达上调313次、下调167次,分为43种表达方式。表明肝再生中脂肪细胞发生和分化相关基因活动多样和复杂。根据本文研究结果推测,上述基因不仅调节脂肪细胞分化,而且参与肝再生的生理生化活动。     

ABCC13, an unusual truncated ABC transporter,is highly expressed in fetal human liver

In the present study, we have cloned the cDNA of ABCC13, a novel ABC transporter, from the cDNA library of adult human placenta. The ABCC13 gene spans approximately 70kb on human chromosome 21q11.2 and consists of 14 exons. The open reading frame of the ABCC13 cDNA encodes a peptide consisting of 325 amino acid residues. The amino acid sequence corresponding to putative membrane-spanning domains was remarkably similar to ABCC1, ABCC2, ABCC3, and ABCC6. The ABCC13 gene was expressed in the fetal liver at the highest level among the organs studied. While ABCC13 was expressed in the bone marrow, its expression in peripheral blood leukocytes of adult humans was much lower and no detectable levels were observed in differentiated hematopoietic cells. The expression of ABCC13 in K562 cells decreased during cell differentiation induced by TPA. These results suggest that the expression of human ABCC13 is related with hematopoiesis.     

Structure, hormonal regulation, and identification of the interleukin-6- and dexamethasone-responsive element of the rat haptoglobin gene.

Hepatic expression of the haptoglobin (Hp) gene in mammalian species is stimulated severalfold during an acute-phase reaction. To identify the molecular mechanism responsible for this regulation, the single-copy rat Hp gene has been isolated. The genomic sequences showed a high degree of homology with the primate Hp gene. Activity of the rat Hp gene was increased in cultured liver cells by interleukin-1 (IL-1), IL-6, and glucocorticoids. The genomic Hp gene sequence spanning from -6500 to +6500, when transiently introduced into human hepatoma (HepG2) cells, directed IL-6- and dexamethasone-stimulated expression of rat Hp mRNA and protein. No response to IL-1 was detected, suggesting that the corresponding regulatory element(s) might lie outside of the tested gene sequences. An IL-6- and dexamethasone-responsive element has been localized to the promoter proximal region -146 to -55. Although the nucleotide sequences of this rat Hp gene region showed substantial divergence from that of the human gene, analysis of sequential 5' and 3' deletion constructs indicated an arrangement of functional IL-6 response elements in the rat Hp promoter sequence comparable to that of the human homolog. The magnitude of IL-6 regulation through the rat Hp gene promoter was severalfold lower than that of the human Hp gene. The reduced activity could be ascribed to a single-base difference in an otherwise conserved sequence corresponding to an active element in the human gene. The IL-6 response of the rat Hp element was improved severalfold by substituting that base with the human nucleotide.     

The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization

The Type I (mineralocorticoid) receptor has identical affinities in vitro for cortisol and aldosterone. It has been suggested that the selective role of aldosterone in regulating sodium homeostasis relies on the microsomal enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD). This enzyme converts cortisol to its inactive metabolite, cortisone, preventing cortisol from binding to the Type I receptor. We have isolated human cDNA clones encoding 11-HSD from a human testis cDNA library by hybridization with a previously isolated rat 11-HSD cDNA clone. The cDNA contains an open reading frame of 876 bases, which predicts a protein of 292 amino acids. The sequence is 77% identical at the amino acid level to rat 11-HSD cDNA. The mRNA is widely expressed, but the level of expression is highest in the liver. Hybridization of the human 11-HSD cDNA to a human-hamster hybrid cell panel localized the single corresponding HSD11 gene to chromosome 1. This gene was isolated from a chromosome 1 specific library using the cDNA as a probe. HSD11 consists of 6 exons and is at least 9 kilobases long. The data developed in this study should be applicable to the study of patients with hypertension due to apparent mineralocorticoid excess, a deficiency in 11-HSD activity.     

The house musk shrew (Suncus murinus): a unique animal with extremely low level of expression of mRNAs for CYP3A and flavin-containing monooxygenase

Expression of drug-metabolizing enzymes including cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) in various tissues of Suncus murinus (Suncus) were examined. Northern blot analysis showed that mRNAs hybridizable with cDNAs for rat CYP1A2, human CYP2A6, rat CYP2B1, human CYP2C8, human CYP2D6, rat CYP2E1, human CYP3A4 and rat CYP4A1 were expressed in various tissues from Suncus. The mRNA level of CYP2A in the Suncus lung was very high. Furthermore, it was found that the level of CYP2A mRNA in the Suncus lung was higher compared to the Suncus liver. The expression level of mRNA hybridizable with cDNA for human CYP3A4 was very low. The presence of CYP3A gene in Suncus was proven by the induction of the CYP with dexamethasone. Very low expression levels of mRNAs hybridizable with cDNAs for rat FMO1, rat FMO2, rat FMO3 and rat FMO5 were also seen in Suncus liver. No apparent hybridization band appeared when human FMO4 cDNA was used as a probe. The hepatic expression of mRNAs hybridizable with cDNAs for UDP-glucuronosyltransferase 1*6, aryl sulfotransferase, glutathione S-transferase 1, carboxyesterase and microsomal epoxide hydrolase in the Suncus were observed. These results indicate that the Suncus is a unique animal species in that mRNAs for CYP3A and FMO are expressed at very low levels.