Sterol Regulatory Element-binding Factor Research Articles

Altered pre-lamin A processing is a common mechanism leading to lipodystrophy

Lipodystrophies are a heterogeneous group of human disorders characterized by the anomalous distribution of body fat associated with insulin resistance and altered lipid metabolism. The pathogenetic mechanism of inherited lipodystrophies is not yet clear; at the molecular level they have been linked to mutations of lamin A/C, peroxisome proliferator-activated receptor (PPARgamma) and other seemingly unrelated proteins. In this study, we examined lamin A/C processing in three laminopathies characterized by lipodystrophic phenotypes: Dunnigan type familial partial lipodystrophy, mandibuloacral dysplasia and atypical Werner's syndrome. We found that the lamin A precursor was specifically accumulated in lipodystrophy cells. Pre-lamin A was located at the nuclear envelope and co-localized with the adipocyte transcription factor sterol regulatory element binding protein 1 (SREBP1). Using co-immunoprecipitation experiments, we obtained the first demonstration of an in vivo interaction between SREBP1 and pre-lamin A. Binding of SREBP1 to the lamin A precursor was detected in patient fibroblasts as well as in control fibroblasts forced to accumulate pre-lamin A by farnesylation inhibitors. In contrast, SREBP1 did not interact in vivo with mature lamin A or C in cultured fibroblasts. To gain insights into the effect of pre-lamin A accumulation in adipose tissue, we inhibited lamin A precursor processing in 3T3-L1 pre-adipocytes. Our results show that pre-lamin A sequesters SREBP1 at the nuclear rim, thus decreasing the pool of active SREBP1 that normally activates PPARgamma and causing impairment of pre-adipocyte differentiation. This defect can be rescued by treatment with troglitazone, a known PPARgamma ligand activating the adipogenic program.

Lack of evidence for a role of TRB3/NIPK as an inhibitor of PKB-mediated insulin signalling in primary hepatocytes.

The protein TRB3 (tribbles 3), also called NIPK (neuronal cell death-inducible protein kinase), was recently identified as a protein-protein interaction partner and an inhibitor of PKB (protein kinase B). To explore the hypothesis that TRB3/NIPK might act as a negative regulator of insulin signalling in the liver, this protein was overexpressed by adenoviral transduction of primary cultures of rat hepatocytes, and various aspects of insulin action were investigated. The insulin-induced phosphorylation of Ser-473 and Thr-308 of PKB was found to be undiminished in transduced hepatocytes with a molar excess of TRB3/NIPK over PKB of more than 25-fold. Consistent with unimpaired insulin activation of PKB, the stimulation of Ser-21 and Ser-9 phosphorylation of glycogen synthase kinase 3-alpha and -beta, and the apparent phosphorylation level of 4E-BP1 (eukaryotic initiation factor 4-binding protein 1), were similar in transduced and control hepatocytes. The induction by insulin of the mRNAs encoding glucokinase and SREBF1 (sterol-regulatory-element-binding factor 1) were also normal in TRB3/NIPK hepatocytes. In contrast, the insulin-dependent induction of these two genes, as well as the activation of PKB, were shown to be suppressed in hepatocytes treated with the lipid ether compound PIA6 (phosphatidylinositol ether lipid analogue 6), a recently discovered specific inhibitor of PKB. Since TRB3/NIPK was reported to be increased in the liver of fasting mice, the effects of glucagon, glucocorticoids and insulin on the level of endogenous TRB3/NIPK mRNA in primary hepatocytes were investigated. No significant change in mRNA level occurred under any of the hormonal treatments. The present study does not support the hypothesis that the physiological role of TRB3/NIPK might be to put a brake on insulin signalling in hepatocytes.

Carbohydrate responsive element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c): two key regulators of glucose metabolism and lipid synthesis in liver

In mammals, the regulation of hepatic metabolism plays a key role in whole body energy balance, since the liver is the major site of carbohydrate metabolism (glycolysis and glycogen synthesis) and triglyceride synthesis (lipogenesis). Lipogenesis is regulated through the acute control of key enzyme activities by means of allosteric and covalent modifications. Moreover, the synthesis of most glycolytic and lipogenic enzymes is regulated in response to dietary status, in which glucose, in particular, is a crucial energy nutrient. This latter response occurs in large part through transcriptional regulation of genes encoding glycolytic and lipogenic enzymes. In the past few years, recent advances have been made in understanding the transcriptional regulation of hepatic glycolytic and lipogenic genes by insulin and glucose. Although insulin is a major regulator of hepatic lipogenesis, there is increasing evidence that glucose also contributes to the coordinated regulation of carbohydrate and lipid metabolism in liver. Here, we review the respective roles of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) in mediating the effect of insulin on hepatic gene expression, and the role of carbohydrate responsive element binding protein (ChREBP) in regulating gene transcription by glucose.

Insulin-activated Erk-mitogen-activated Protein Kinases Phosphorylate Sterol Regulatory Element-binding Protein-2 at Serine Residues 432 and 455 in Vivo

The transcription factor sterol regulatory element binding protein (SREBP)-2 plays a pivotal role in lipid metabolism. Previously, we have shown that the mature form of SREBP-2 is a substrate of Erk-mitogen-activated protein kinases (MAPK). The aim of the present study was to identify Erk-specific phosphorylation sites. Using a protein chemistry approach, we could identify Ser-432 and Ser-455 as major phosphorylation sites. Further characterization by electrophoretic mobility shift assay and promoter reporter gene analyses revealed that phosphorylation does not influence protein/DNA interaction, but enhances trans-activity. In intact cells, SREBP-2 is phosphorylated by insulin, which seems to be related to their bio-responses on low density lipoprotein receptor activity. These results suggest that activation of Erk-MAPK pathways by hormones such as insulin might be related to a novel regulatory principle of SREBP-2.

GH and insulin affect fatty acid synthase activity in isolated porcine adipocytes in culture without any modifications of sterol regulatory element binding protein-1 expression

The ability of GH to decrease fatness and insulin-regulated events such as lipogenic enzyme activities is well known in pigs. Nevertheless, the precise mechanism underlying these actions has not been elucidated yet. Expression of the transcription factor sterol regulatory element binding protein (SREBP)-1 has been reported as a key mediator of insulin action in rat hepatocytes and adipose cell lines. The present study aimed to determine whether the regulation of lipogenesis by GH and/or insulin in porcine adipocytes also involved SREBP-1. Isolated adipocytes, obtained from perirenal or s.c. adipose tissue samples of female pigs (51+/-0.4 kg; n=17), were cultured in serum-free medium in the absence or presence of these hormones for up to 4 days. Glucose incorporation and fatty acid synthase activity were increased by insulin in a dose-dependent manner in adipocytes of both sites. The increase was maximal at 1.7 and 17 nM in s.c. and perirenal adipocytes respectively, suggesting inter-depot differences in the regulation of lipogenesis by insulin. These insulin-stimulated events were decreased by GH (1 nM). No change in SREBP-1 mRNA levels was observed in response to GH and/or insulin. Taken together, these data indicate that the regulation of lipogenesis by insulin and GH appears to not involve changes in SREBP-1 mRNA levels in porcine adipocytes.

Involvement of the SREBP pathway in the mode of action of androgens in sebaceous glands in vivo.

Androgens have profound effects on the physiology of the sebaceous gland. Using the hamster ear sebaceous gland model, we performed a detailed kinetic study to clarify the mechanism of androgen action on sebaceous gland function. We demonstrated that the growth of sebaceous glands observed after androgen treatment was due to both an increase in sebocyte proliferation and a parallel induction of sebocyte terminal differentiation, as evidenced by the induction of the synthesis of specific sebaceous lipids such as cholesterol esters, triglycerides, and squalene. Accordingly, the effect of androgen treatment on the mRNA expression of several key enzymes involved in the synthesis of sebaceous lipids has been studied using semi-quantitative RT-PCR. Up-regulation by androgens of mRNA expression of HMG coenzyme A synthase and reductase, acetyl coenzyme A carboxylase (ACC), glycerol 3-phosphate acyl transferase (GPAT), and FAR-17c (stearoyl coenzyme A desaturase homologous), was demonstrated. Because sterol-response element(s) (SREs) are known to be present in the promoters of these genes, we analyzed the expression by RT-PCR and the activation of the transcription factor sterol regulatory element binding protein (SREBP) using immunoblotting experiments. Our results showed that SREBP-1 was up-regulated and rapidly activated after androgen treatment. Altogether, these results demonstrate for the first time that in sebaceous glands, in vivo, androgen regulates the synthesis of sebum lipids through the SREBP pathway.

New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose: a role for the transcription factor sterol regulatory element binding protein-1c.

The regulation of hepatic glucose metabolism has a key role in whole-body energy metabolism, since the liver is able to store (glycogen synthesis, lipogenesis) and to produce (glycogenolysis, gluconeogenesis) glucose. These pathways are regulated at several levels, including a transcriptional level, since many of the metabolism-related genes are expressed according to the quantity and quality of nutrients. Recent advances have been made in the understanding of the regulation of hepatic glycolytic, lipogenic and gluconeogenic gene expression by pancreatic hormones, insulin and glucagon and glucose. Here we review the role of the transcription factors forkhead and sterol regulatory element binding protein-1c in the inductive and repressive effects of insulin on hepatic gene expression, and the pathway that leads from glucose to gene regulation with the recently discovered carbohydrate response element binding protein. We discuss how these transcription factors are integrated in a regulatory network that allows a fine tuning of hepatic glucose storage or production, and their potential importance in metabolic diseases.

Critical Role for Cataplerosis via Citrate in Glucose-Regulated Insulin Release

The molecular mechanisms mediating acute regulation of insulin release by glucose are partially known. The process involves at least two pathways that can be discriminated on basis of their (in)dependence of closure of ATP-sensitive potassium (K+(ATP)) channels. The mechanism of the K+(ATP) channel-independent pathway was proposed to involve cataplerosis, the export of mitochondrial intermediates into the cytosol and in the induction of fatty acid-derived signaling molecules. In the present article, we have explored in fluorescence-activated cell sorter (FACS)-purified rat beta-cells the molecular steps involved in chronic glucose regulation of the insulin secretory response. When compared with culture in 10 mmol/l glucose, 24 h culture in 3 mmol/l glucose shifts the phenotype of the cells into a state with low further secretory responsiveness to glucose, lower rates of glucose oxidation, and lower rates of cataplerosis. Microarray mRNA analysis indicates that this shift can be attributed to differences in expression of genes involved in the K+(ATP) channel-dependent pathway, in cataplerosis and in fatty acid/cholesterol biosynthesis. This response was paralleled by glucose upregulation of the transcription factor sterol regulatory element binding protein 1c (SREBP1c) (ADD1) and downregulation of peroxisome proliferator-activated receptor (PPAR)-alpha and PPAR-beta (PPARdelta). The functional importance of cataplerosis via citrate for glucose-induced insulin release was further supported by the observation that two ATP-citrate lyase inhibitors, radicicol and (-)-hydroxycitrate, block part of glucose-stimulated release in beta-cells. In conclusion, chronic glucose regulation of the glucose-responsive secretory phenotype is associated with coordinated changes in gene expression involved in the K+(ATP) channel-dependent pathway, in cataplerosis via citrate and in acyl CoA/cholesterol biosynthesis.

Proteolysis and sterol regulation.

The mammalian cell continuously adjusts its sterol content by regulating levels of key sterol synthetic enzymes and levels of LDL receptors that mediate uptake of cholesterol-laden particles. Control is brought about by sterol-regulated transcription of relevant genes and by regulated degradation of the committed step enzyme HMG-CoA reductase (HMGR). Current work has revealed that proteolysis is at the heart of each of these mechanistically distinct axes. Transcriptional control is effected by regulated cleavage of the membrane-bound transcription factor sterol regulatory element binding protein (SREBP), and HMGR degradation is brought about by ubiquitin-mediated degradation. In each case, ongoing cell biological processes are being harnessed to bring about regulation. The secretory pathway plays a central role in allowing sterol-mediated control of transcription. The constitutively active endoplasmic reticulum (ER) quality control apparatus is employed to bring about regulated destruction of HMGR. This review describes the methods and results of various studies to understand the mechanisms and molecules involved in these distinct but interrelated aspects of sterol regulation and the intriguing similarities that appear to exist at the levels of protein sequence and cell biology.

Copper Deficiency Induces Hepatic Fatty Acid Synthase Gene Transcription in Rats by Increasing the Nuclear Content of Mature Sterol Regulatory Element Binding Protein 1

Dietary copper (Cu) deficiency results in an accelerated rate of hepatic fatty acid synthase gene transcription and an enhanced rate of hepatic lipid synthesis. Because the nuclear transcription factor sterol regulatory element binding protein-1 (SREBP-1) is a strong enhancer of fatty acid synthase promoter activity, it was hypothesized that Cu deficiency induces fatty acid synthase gene transcription by increasing the nuclear localization of mature SREBP-1. Male weanling rats were pair-fed a Cu-adequate (6.0 mg/kg) or Cu-deficient (0.6 mg/kg) diet (AIN-93) for 28 d. DNase I hypersensitivity site mapping of the hepatic fatty acid synthase gene revealed the presence of four major hypersensitivity sites located at -8700 to -8600, -7300 to -6900, -600 to -400 and -100 to +50. Although Cu deficiency did not change the hypersensitivity site pattern or intensity, in vitro footprinting of the region between -100 and +50 indicated that Cu deficiency enhanced DNA protein interactions within this region. The sequence between -68 and -58 contains the DNA recognition sequence for SREBP-1 and upstream stimulatory element-1 (USF-1). Western blot analysis revealed that the dietary Cu deficiency increased the hepatic nuclear content of mature SREBP-1 by 150% (P: < 0.05), and it concomitantly decreased the membrane content of precursor SREBP-1 by 45% (P: < 0.05). Changes in the hepatic distribution of SREBP-1 associated with Cu deficiency were not accompanied by changes in SREBP-1 mRNA. The nuclear content of USF-1 was unaffected by dietary Cu status. The hepatic increase in mature SREBP-1 of Cu-deficient rats was accompanied by a 400% increase and an 80% decrease in the abundance of fatty acid synthase and cholesterol 7-alpha hydroxylase mRNA, respectively. hepatic These data indicate that a Cu deficiency stimulates hepatic lipogenic gene expression by increasing the hepatic translocation of mature SREBP-1.

Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes.

Hepatic glucokinase plays a key role in glucose metabolism as underlined by the anomalies associated with glucokinase mutations and the consequences of tissue-specific knock-out. In the liver, glucokinase transcription is absolutely dependent on the presence of insulin. The cis-elements and trans-acting factors that mediate the insulin effect are presently unknown; this is also the case for most insulin-responsive genes. We have shown previously that the hepatic expression of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) is activated by insulin. We show here in primary cultures of hepatocytes that the adenovirus-mediated transduction of a dominant negative form of SREBP-1c inhibits the insulin effect on endogenous glucokinase expression. Conversely, in the absence of insulin, the adenovirus-mediated transduction of a dominant positive form of SREBP-1c overcomes the insulin dependency of glucokinase expression. Hepatic fatty acid synthase and Spot-14 are insulin/glucose-dependent genes. For this latter class of genes, the dominant positive form of SREBP-1c obviates the necessity for the presence of insulin, whereas glucose potentiates the effect of SREBP-1c on their expression. In addition, the insulin dependency of lipid accumulation in cultured hepatocytes is overcome by the dominant positive form of SREBP-1c. We propose that SREBP-1c is a major mediator of insulin action on hepatic gene expression and a key regulator of hepatic glucose/lipid metabolism.

Sterol dependent LDL-receptor gene transcription in lymphocytes from normal and CML patients.

Sterol regulatory element (SRE) has been recognized to regulate various key genes coding for especially low density lipoprotein (LDL)-receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase known to play a crucial role in the cholesterol feedback mechanism. The deranged cholesterol feedback mechanism has been widely recognised in initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Consequently, the present study was addressed to understand this phenomenon and revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when the CML patients achieved complete haematological remission (CHR) through alpha-interferon therapy. Further, an inverse relationship was also observed between sterol modulated LDL-receptor gene transcription and the binding affinity of this 47 kDa factor to the SRE sequence. Based upon these results we propose that alpha-interferon through its receptor initiates phosphatidic acid dependent signalling which in turn regulates the affinity of 47 kDa sterol regulatory element binding factor as well as LDL-receptor gene transcription in lymphocytes from CML patients.