Takashi Matsuzaka

Identification of Liver X Receptor-Retinoid X Receptor as an Activator of the Sterol Regulatory Element-Binding Protein 1c Gene Promoter

ABSTRACT In an attempt to identify transcription factors which activate sterol-regulatory element-binding protein 1c (SREBP-1c) transcription, we screened an expression cDNA library from adipose tissue of SREBP-1 knockout mice using a reporter gene containing the 2.6-kb mouse SREBP-1 gene promoter. We cloned and identified the oxysterol receptors liver X receptor (LXRα) and LXRβ as strong activators of the mouse SREBP-1c promoter. In the transfection studies, expression of either LXRα or -β activated the SREBP-1c promoter-luciferase gene in a dose-dependent manner. Deletion and mutation studies, as well as gel mobility shift assays, located an LXR response element complex consisting of two new LXR-binding motifs which showed high similarity to an LXR response element recently found in the ABC1 gene promoter, a reverse cholesterol transporter. Addition of an LXR ligand, 22(R)-hydroxycholesterol, increased the promoter activity. Coexpression of retinoid X receptor (RXR), a heterodimeric partner, and its ligand 9-cis-retinoic acid also synergistically activated the SREBP-1c promoter. In HepG2 cells, SREBP-1c mRNA and precursor protein levels were induced by treatment with 22(R)-hydroxycholesterol and 9-cis-retinoic acid, confirming that endogenous LXR-RXR activation can induce endogenous SREBP-1c expression. The activation of SREBP-1c by LXR is associated with a slight increase in nuclear SREBP-1c, resulting in activation of the gene for fatty acid synthase, one of its downstream genes, as measured by the luciferase assay. These data demonstrate that LXR-RXR can modify the expression of genes for lipogenic enzymes by regulating SREBP-1c expression, providing a novel link between fatty acid and cholesterol metabolism.

Crucial role of a long-chain fatty acid elongase, Elovl6, in obesity-induced insulin resistance

Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 and suppression of hepatic protein kinase C ε activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity.

SREBPs suppress IRS-2-mediated insulin signalling in the liver

Insulin receptor substrate 2 (IRS-2) is the main mediator of insulin signalling in the liver, controlling insulin sensitivity. Sterol regulatory element binding proteins (SREBPs) have been established as transcriptional regulators of lipid synthesis. Here, we show that SREBPs directly repress transcription of IRS-2 and inhibit hepatic insulin signalling. The IRS-2 promoter is activated by forkhead proteins through an insulin response element (IRE). Nuclear SREBPs effectively replace and interfere in the binding of these transactivators, resulting in inhibition of the downstream PI(3)K/Akt pathway, followed by decreased glycogen synthesis. These data suggest a molecular mechanism for the physiological switching from glycogen synthesis to lipogenesis and hepatic insulin resistance that is associated with hepatosteatosis.

The up-regulation of microRNA-335 is associated with lipid metabolism in liver and white adipose tissue of genetically obese mice.

MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. Some miRNAs have been proposed to be associated with obesity. However, miRNAs, which are related to the development of obesity in vivo remains unknown. Here in, we found the up-regulation of miR-335 in obesity using microarray analysis for miRNA. The expressions of miR-335 in liver and white adipose tissue (WAT) were up-regulated in obese mice including ob/ob, db/db, and KKAy mice. Increased miR-335 expressions were associated with an elevated body, liver and WAT weight, and hepatic triglyceride and cholesterol. Furthermore, miR-335 levels were closely correlated with expression levels of adipocyte differentiation markers such as PPARgamma, aP2, and FAS in 3T3-L1 adipocyte. These findings provide the first evidence that the up-regulated expressions of miR-335 in liver and WAT of obese mice might contribute to the pathophysiology of obesity.

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 3β 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.

SREBP-1 Interacts with Hepatocyte Nuclear Factor-4α and Interferes with PGC-1 Recruitment to Suppress Hepatic Gluconeogenic Genes

The hepatocyte nuclear factor-4α (HNF-4α)/PGC-1 pathway plays a crucial role in the transcriptional regulation of hepatic gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and Glc-6-Pase, genes that are activated at fasting and suppressed in a fed state. SREBP-1c dominates the nutritional regulation of lipogenic genes inverse to gluconeogenesis. Here we show the mechanism by which SREBP-1 suppresses expression of gluconeogenic genes. A series of luciferase reporter assays demonstrated that SREBP-1a and -1c effectively inhibited the PEPCK promoter activity that was induced by HNF-4α. The HNF-4α-binding site in the glucocorticoid-response unit was responsible for the SREBP-1 inhibition, although SREBP-1 did not bind to the PEPCK promoter as demonstrated by electrophoretic mobility shift assays. The inhibitory effect was more potent in the isoform of SREBP-1a than SREBP-1c and was eliminated by deletion of the amino-terminal transactivation domain of SREBP-1. Coimmunoprecipitation experiments demonstrated that these two transcription factors directly interact through the transactivation domain of SREBP-1 and the ligand binding/AF2 domains of HNF-4α. Estimation of coactivator recruitment using HNF-4α-Gal4DBD fusion assay showed that SREBP-1 competitively inhibited PGC-1 recruitment, a requirement for HNF-4α activation. Consistent with these results, hepatic PEPCK and Glc-6-Pase mRNA levels are suppressed by overexpression of SREBP-1a and -1c in the transgenic mice. Our data indicate that SREBP-1 has a novel role as negative regulator of gluconeogenic genes through a cross-talk with HNF-4α interference with PGC-1 recruitment.

Protein Kinase A Suppresses Sterol Regulatory Element-binding Protein-1C Expression via Phosphorylation of Liver X Receptor in the Liver

Sterol regulatory element-binding protein (SREBP)-1c is a transcription factor that controls synthesis of fatty acids and triglycerides in the liver and is highly regulated by nutrition and hormones. In the current studies we show that protein kinase A (PKA), a mediator of glucagon/cAMP, a fasting signaling, suppresses SREBP-1c by modulating the activity of liver X receptor α (LXRα), a dominant activator of SREBP-1c expression. Activation of PKA repressed LXR-induced SREBP-1c expression both in rat primary hepatocytes and mouse livers. Promoter analyses revealed that the LXRα-binding site in the SREBP-1c promoter is responsible for PKA inhibitory effect on SREBP-1c transcription. In vitro and in vivo PKA directly phosphorylated LXRα, and the two consensus PKA target sites (195, 196 serines and 290, 291 serines) in its ligand binding/heterodimerization domain were crucial for the inhibition of LXR signaling. PKA phosphorylation of LXRα caused impaired DNA binding activity by preventing LXRα/RXR dimerization and decreased its transcription activity by inhibiting recruitment of coactivator SCR-1 and enhancing recruitment of corepressor NcoR1. These results indicate that LXRα is regulated not only by oxysterol derivatives but also by PKA-mediated phosphorylation, which suggests that nutritional regulation of SREBP-1c and lipogenesis could be regulated at least partially through modulation of LXR.

Palmitate impairs and eicosapentaenoate restores insulin secretion through regulation of SREBP-1c in pancreatic islets.

OBJECTIVE—Chronic exposure to fatty acids causes β-cell failure, often referred to as lipotoxicity. We investigated its mechanisms, focusing on contribution of SREBP-1c, a key transcription factor for lipogenesis. RESEARCH DESIGN AND METHODS—We studied in vitro and in vivo effects of saturated and polyunsaturated acids on insulin secretion, insulin signaling, and expression of genes involved in β-cell functions. Pancreatic islets isolated from C57BL/6 control and SREBP-1–null mice and adenoviral gene delivery or knockdown systems of related genes were used. RESULTS—Incubation of C57BL/6 islets with palmitate caused inhibition of both glucose- and potassium-stimulated insulin secretion, but addition of eicosapentaenoate (EPA) restored both inhibitions. Concomitantly, palmitate activated and EPA abolished both mRNA and nuclear protein of SREBP-1c, accompanied by reciprocal changes of SREBP-1c target genes such as insulin receptor substrate-2 (IRS-2) and granuphilin. These palmitate-EPA effects on insulin secretion were abolished in SREBP-1–null islets. Suppression of IRS-2/Akt pathway could be a part of the downstream mechanism for the SREBP-1c–mediated insulin secretion defect because adenoviral constitutively active Akt compensated it. Uncoupling protein-2 (UCP-2) also plays a crucial role in the palmitate inhibition of insulin secretion, as confirmed by knockdown experiments, but SREBP-1c contribution to UCP-2 regulation was partial. The palmitate-EPA regulation of insulin secretion was similarly observed in islets from C57BL/6 mice pretreated with dietary manipulations. Furthermore, administration of EPA to diabetic KK-Ay mice ameliorated impairment of insulin secretion in their islets. CONCLUSIONS—SREBP-1c plays a dominant role in palmitate-mediated insulin secretion defect, and EPA prevents it through SREBP-1c inhibition, implicating a therapeutic potential for treating diabetes related to lipotoxicity.

SREBP-1-independent regulation of lipogenic gene expression in adipocytes

Sterol regulatory element-binding protein (SREBP)-1c is now well established as a key transcription factor for the regulation of lipogenic enzyme genes such as FAS in hepatocytes. Meanwhile, the mechanisms of lipogenic gene regulation in adipocytes remain unclear. Here, we demonstrate that those in adipocytes are independent of SREBP-1c. In adipocytes, unlike in hepatocytes, the stimulation of SREBP-1c expression by liver X receptor agonist does not accompany lipogenic gene upregulation, although nuclear SREBP-1c protein is concomitantly increased, indicating that the activation process of SREBP-1c by the cleavage system is intact in adipocytes. Supportively, transcriptional activity of the mature form of SREBP-1c for the FAS promoter was negligible when measured by reporter analysis. As an underlying mechanism, accessibility of SREBP-1c to the functional elements was involved, because chromatin immunoprecipitation assays revealed that SREBP-1c does not bind to the functional SRE/E-box site on the FAS promoter in adipocytes. Moreover, genetic disruption of SREBP-1 did not cause any changes in lipogenic gene expression in adipose tissue. In summary, in adipocytes, unlike in hepatocytes, increments in nuclear SREBP-1c are not accompanied by transactivation of lipogenic genes; thus, SREBP-1c is not committed to the regulation of lipogenesis.

Elovl6 promotes nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is associated with obesity and type 2 diabetes, and an increased risk for liver cirrhosis and cancer. ELOVL family member 6, elongation of very long chain fatty acids (Elovl6), is a microsomal enzyme that regulates the elongation of C12‐16 saturated and monounsaturated fatty acids (FAs). We have shown previously that Elovl6 is a major target for sterol regulatory element binding proteins in the liver and that it plays a critical role in the development of obesity‐induced insulin resistance by modifying FA composition. To further investigate the role of Elovl6 in the development of NASH and its underlying mechanism, we used three independent mouse models with loss or gain of function of Elovl6, and human liver samples isolated from patients with NASH. Our results demonstrate that (1) Elovl6 is a critical modulator for atherogenic high‐fat diet–induced inflammation, oxidative stress, and fibrosis in the liver; (2) Elovl6 expression is positively correlated with severity of hepatosteatosis and liver injury in NASH patients; and (3) deletion of Elovl6 reduces palmitate‐induced activation of the NLR family pyrin domain‐containing 3 inflammasome; this could be at least one of the underlying mechanisms by which Elovl6 modulates the progress of NASH. Conclusion: Hepatic long‐chain fatty acid composition is a novel determinant in NASH development, and Elovl6 could be a potential therapeutic target for the prevention and treatment of NASH. (HEPATOLOGY 2012;56:2199–2208)