TNF-alpha induces hepatic steatosis in mice by enhancing gene expression of sterol regulatory element binding protein-1c (SREBP-1c)

We investigated the effect of tumor necrosis factor-alpha (TNF-alpha), a member of the proinflammatory cytokine family, on steatosis of the mouse liver by analyzing morphological changes and hepatic triglyceride content in response to TNF-alpha. We also examined expression of the sterol regulatory element binding protein-1c gene. Intraperitoneal injection of TNF-alpha acutely and dramatically accelerated the accumulation of fat in the liver, as evidenced by histological analysis and hepatic triglyceride content. This treatment increased liver weight, increased serum levels of free fatty acids, and increased fatty acid synthase and sterol regulatory element binding protein-1c mRNA expression. Furthermore, intraperitoneal injection of lipopolysaccaride (LPS) to induce TNF-alpha expression also accelerated hepatic fat accumulation. Pretreatment with anti-TNF-alpha antibody attenuated the development of LPS-induced fatty change in the liver. Antibody pretreatment not only decreased sterol regulatory element binding protein-1c expression in LPS-treated mice but also attenuated the expression of suppressors of cytokine signaling-3 mRNA. This study suggests that TNF-alpha, acting downstream of LPS, increases intrahepatic fat deposition by affecting hepatic lipogenetic metabolism involving sterol regulatory element binding protein-1c.     

A modest glucokinase overexpression in the liver promotes fed expression levels of glycolytic and lipogenic enzyme genes in the fasted state without altering SREBP-1c expression

Hepatic genes crucial for carbohydrate and lipid homeostasis are regulated by insulin and glucose metabolism. However, the relative contributions of insulin and glucose to the regulation of metabolic gene expression are poorly defined in vivo. To address this issue, adenovirus-mediated hepatic overexpression of glucokinase was used to determine the effects of increased hepatic glucose metabolism on gene expression in fasted or ad libitum fed rats. In the fasted state, a 3 fold glucokinase overexpression was sufficient to mimic feeding-induced increases in pyruvate kinase and acetyl CoA carboxylase mRNA levels, demonstrating a primary role for glucose metabolism in the regulation of these genes in vivo. Conversely, glucokinase overexpression was unable to mimic feeding-induced alterations of fatty acid synthase, glucose-6-phosphate dehydrogenase, carnitine palmitoyl transferase I or PEPCK mRNAs, indicating insulin as the primary regulator of these genes. Interestingly, glucose-6-phosphatase mRNA was increased by glucokinase overexpression in both the fasted and fed states, providing evidence, under these conditions, for the dominance of glucose over insulin signaling for this gene in vivo. Importantly, glucokinase overexpression did not alter sterol regulatory element binding protein 1-c mRNA levels in vivo and glucose signaling did not alter the expression of this gene in primary hepatocytes. We conclude that a modest hepatic overexpression of glucokinase is sufficient to alter expression of metabolic genes without changing the expression of SREBP-1c.     

RBMX is a novel hepatic transcriptional regulator of SREBP-1c gene response to high-fructose diet

In rodents a high-fructose diet induces metabolic derangements similar to those in metabolic syndrome. Previously we suggested that in mouse liver an unidentified nuclear protein binding to the sterol regulatory element (SRE)-binding protein-1c (SREBP-1c) promoter region plays a key role for the response to high-fructose diet. Here, using MALDI-TOF MASS technique, we identified an X-chromosome-linked RNA binding motif protein (RBMX) as a new candidate molecule. In electrophoretic mobility shift assay, anti-RBMX antibody displaced the bands induced by fructose-feeding. Overexpression or suppression of RBMX on rat hepatoma cells regulated the SREBP-1c promoter activity. RBMX may control SREBP-1c expression in mouse liver in response to high-fructose diet.     

Involvement of glomerular SREBP-1c in diabetic nephropathy

The role of glomerular SREBP-1c in diabetic nephropathy was investigated. PEPCK-promoter transgenic mice overexpressing nuclear SREBP-1c exhibited enhancement of proteinuria with mesangial proliferation and matrix accumulation, mimicking diabetic nephropathy, despite the absence of hyperglycemia or hyperlipidemia. Isolated transgenic glomeruli had higher expression of TGFβ-1, fibronectin, and SPARC in the absence of marked lipid accumulation. Gene expression of P47phox, p67phox, and PU.1 were also activated, accompanying increased 8-OHdG in urine and kidney, demonstrating that glomerular SREBP-1c could directly cause oxidative stress through induced NADPH oxidase. Similar changes were observed in STZ-treated diabetic mice with activation of endogenous SREBP-1c. Finally, diabetic proteinuria and oxidative stress were ameliorated in SREBP-1-null mice. Adenoviral overexpression of active and dominant-negative SREBP-1c caused consistent reciprocal changes in expression of both profibrotic and oxidative stress genes in MES13 mesangial cells. These data suggest that activation of glomerular SREBP-1c could contribute to emergence and/or progression of diabetic nephropathy.     

Consumption of soy protein isolate reduces hepatic SREBP-1c and lipogenic gene expression in wild-type mice, but not in FXR-deficient mice

We examined to determine whether hepatic gene expression is affected in mice in which blood lipid levels remain unchanged fed soy protein isolate (SPI) for a short time. We also examined SPI-mediated effects in farnesoid X receptor (FXR)-deficient mice. Compared with casein, SPI affected the expression of various hepatic genes related to lipid metabolism in the wild-type mice. No effects of SPI were observed in the FXR-deficient mice, suggesting the importance of FXR. Hepatic peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) gene expression was reduced by SPI, and this might be associated with a decrease in FXR expression. Decreased FXR led to decreased expression of its target, the bile-salt export pump necessary for bile acid secretion and dietary lipid absorption. The earliest response to SPI was a decrease in hepatic sterol regulatory element-binding protein (SREBP)-1c mRNA, on day 3. SPI activated hepatic adenosine monophosphate-activated protein kinase (AMPK), which can lead to a reduction in SREBP-1c mRNA. These data indicate the importance of SREBP-1c and PGC-1α/FXR in SPI-mediated alterations in hepatic gene expression.     

Hepatic insulin signaling is required for obesity-dependent expression of SREBP-1c mRNA but not for feeding-dependent expression

Dissecting the role of insulin in the complex regulation of triglyceride metabolism is necessary for understanding dyslipidemia and steatosis. Liver insulin receptor knockout (LIRKO) mice show that in the physiological context of feeding, hepatic insulin signaling is not required for the induction of mTORC1, an upstream activator of the lipogenic regulator, SREBP-1c. Feeding induces SREBP-1c mRNA in LIRKO livers, though not to the extent observed in controls. A high fructose diet also partially induces SREBP-1c and lipogenic gene expression in LIRKO livers. Insulin signaling becomes more important in the pathological context of obesity, as knockdown of the insulin receptor in ob/ob mice, a model of Type 2 diabetes, using antisense oligonucleotides, abolishes the induction of SREBP-1c and its targets by obesity and ameliorates steatosis. Thus, insulin-independent signaling pathways can partially compensate for insulin in the induction of SREBP-1c by feeding but the further induction by obesity/Type 2 diabetes is entirely dependent upon insulin.     

mRNA levels of SREBP-1c do not coincide with the changes in adipose lipogenic gene expression

Physiological differences in lipid metabolism exist according to adipose sites. To delineate at which step such gene regulation could occur, mRNA levels of various proteins involved in the overall lipogenic process were determined in subcutaneous (SC) and retroperitoneal (RP) adipose tissues. Fatty acid synthase, malic enzyme, ATP citrate lyase, insulin-sensitive glucose transporter, and glucose-6-phosphate dehydrogenase mRNA levels were coordinately reduced (by up to 50-fold) during fasting in RP and in SC relative to fed rats, and restored or overexpressed (by up to 5- to 6-fold) during refeeding. The response was most often delayed and lower in SC compared to RP. This could contribute to site-specific differences. Interestingly, SREBP-1c mRNA levels were markedly decreased by fasting in SC but remained unchanged in RP. Refeeding tended to restore levels close to fed group values. We conclude that mRNA levels of SREBP-1c do not coincide with the expected changes in adipose lipogenic gene expression of fasted/refed rats.     

Endoplasmic reticulum stress leads to lipid accumulation through upregulation of SREBP-1c in normal hepatic and hepatoma cells

Endoplasmic reticulum stress (ERS) has been found in non-alcoholic fatty liver disease. The study was to further explore the mechanistic relationship between ERS and lipid accumulation. To induce ERS, the hepatoblastoma cell line HepG2 and the normal human L02 cell line were exposed to Tg for 48 h. RT-PCR and Western blot were performed to evaluate glucose-regulated protein (GRP-78) expression as a marker of ERS. ER ultrastructure was assessed by electron microscopy. Triglyceride content was examined by Oil Red O staining and quantitative intracellular triglyceride assay. The hepatic nuclear sterol regulatory element-binding protein (SREBP-1c), liver X receptor (LXRs), fatty acid synthase (FAS), and acetyl-coA carboxylase (ACC1) expressions were examined by real-time PCR and Western blot. 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) was used to inhibit S1P serine protease inhibitor, and SREBP-1c cleavage was evaluated under ERS. SREBP-1c was knockdown and its effect on lipid metabolism was observed. Tg treatment upregulated GRP-78 expression and severely damaged the ER structure in L02 and HepG2 cells. ERS increased triglyceride deposition and enhanced the expression of SREBP-1c, FAS, and ACC1, but have no influence on LXR. AEBSF pretreatment abolished Tg-induced SREBP-1c cleavage. Moreover, SREBP-1c silencing reduced triglycerides and downregulated FAS expression. Pharmacological ERS induced by Tg leads to lipid accumulation through upregulation of SREBP-1c in L02 and HepG2 cells.     

Purification of rat hepatic glucokinase

We have developed a rapid, reliable procedure for the purification of rat hepatic glucokinase. The purification utilizes DEAE-cellulose, two affinity chromatography steps, and high-performance liquid chromatography. Glucokinase with a specific activity of 240 units/mg, a 42 K-fold purification, and a yield of 60% is obtained. The enzyme appears as a homogeneous band, with over 99% purity as assessed by polyacrylamide gel electrophoresis. The purification procedure can be completed in 5 days.