Insulin-induced Gene Expression Research Articles

FRI020 Adipocyte Subpopulations Regulate Growth Hormone-induced Lipolysis And Insulin Resistance

Abstract Disclosure: S. Siyar: None. J. Huang: None. R. Sharma: None. J.J. Kopchick: None. V. Puri: None. K.Y. Lee: None. Growth Hormone (GH) administration has long been known to increase lipolysis in adipose tissue leading to increased circulating free fatty acid levels and insulin resistance. Previous studies from our lab have found that this effect is mediated, at least in part, through transcriptional repression of the lipid droplet associated protein Fat specific protein (FSP27 also known as CIDEC). Furthermore, recent studies suggest that within a single adipose tissue depot there are different subpopulations of adipocytes. We have identified three distinct subpopulations of adipocytes with unique gene expression profiles and metabolic activities. We have found that one of these subpopulations of adipocytes, termed Type 2 adipocytes, are highly responsive to GH stimulation. In order to assess the physiological role of GH-mediated lipolysis mediated by this subpopulation, we specifically expressed human FSP27 (hFSP27) in Type 2 adipocytes (Type2Ad-hFSP27tg mice). Type2AdhFSP27tg mice had reduced systemic plasma glycerol release after acute GH treatment, and improvement in acute, but not chronic, GH-induced glucose intolerance. These mice exhibited an 45% increase in fat mass and significant increase in adipocyte cell size further indicating a decreased lipolytic response to GH. This decreased lipolytic response was associated with improved hepatic insulin signaling and insulin-induced gene expression in Type2Ad-hFSP27tg mice. Taken together, these results indicate that specifically targeting a subpopulation of adipocytes is sufficient to reduce GH-induced lipolysis and insulin resistance and may provide a platform for improved medical therapies for growth and endocrine disorders. Presentation: Friday, June 16, 2023

Renal tubule ectopic lipid deposition in diabetic kidney disease rat model and in vitro mechanism of leptin intervention.

Diabetic kidney disease (DKD) is a major health burden closely related to lipid metabolism disorders. Leptin has lipid-lowering efficacy, but the specific mechanism of its local effects on kidney is still unclear. This study aims to investigate the role of ectopic lipid deposition (ELD) in DKD and evaluate the lipid-lowering efficacy of leptin in the palmitic acid (PA)-induced renal tubular epithelial cells (NRK-52E). DKD model was established in Sprague-Dawley (SD) rats by giving single intraperitoneal injection of streptozotocin (STZ, 30mg/kg) after high-fat diet for 8weeks. Then, the expression changes of lipid metabolism-related markers were observed. At week 12, the protein expression level of lipid-deposited marker adipose differentiation-related protein (ADRP) was significantly increased. Besides, the lipid synthesis marker sterol regulatory element-binding protein 1c (SREBP 1c) was highly expressed while the expression of insulin-induced gene 1 (Insig-1), a key molecular of inhibiting SREBP 1c, was decreased. Leptin and compound c were incubated with the PA-induced NRK-52E cells to investigate the lipid-lowering effects and whether this effect was mediated by the AMPK/Insig-1/SREBP 1c signaling pathways. mRNA and proteinof ADRP and SREBP 1c were reduced after leptin treatment, while Insig-1 and phosphorylated AMP-activated protein kinase (AMPK) were increased. Conversely, inhibition of AMPK phosphorylation by compound c mostly eliminated lipid-lowering efficacy of leptin in PA-induced cells. Collectively, these results suggested that there was ELD of renal tubular epithelial cells in DKD rats. Leptin upregulated the expression level of Insig-1 by activating AMPK to attenuate ELD in PA-induced NRK-52E cells.

Hypomorphic ASGR1 modulates lipid homeostasis via INSIG1-mediated SREBP signaling suppression.

A population genetic study identified that the asialoglycoprotein receptor 1 (ASGR1) mutation carriers had substantially lower non–HDL-cholesterol (non–HDL-c) levels and reduced risks of cardiovascular diseases. However, the mechanism behind this phenomenon remained unclear. Here, we established Asgr1-knockout mice that represented a plasma lipid profile with significantly lower non–HDL-c and triglyceride (TG) caused by decreased secretion and increased uptake of VLDL/LDL. These 2 phenotypes were linked with the decreased expression of microsomal triglyceride transfer protein and proprotein convertase subtilisin/kexin type 9, 2 key targeted genes of sterol regulatory element–binding proteins (SREBPs). Furthermore, there were fewer nuclear SREBPs (nSREBPs) on account of more SREBPs being trapped in endoplasmic reticulum, which was caused by an increased expression of insulin-induced gene 1 (INSIG1), an anchor of SREBPs. Overexpression and gene knockdown interventions, in different models, were conducted to rescue the ASGR1-deficient phenotypes, and we found that INSIG1 knockdown independently reversed the ASGR1-mutated phenotypes with increased serum total cholesterol, LDL-c, TG, and liver cholesterol content accompanied by restored SREBP signaling. ASGR1 rescue experiments reduced INSIG1 and restored the SREBP network defect as manifested by improved apolipoprotein B secretion and reduced LDL uptake. Our observation demonstrated that increased INSIG1 is a critical factor responsible for ASGR1 deficiency–associated lipid profile changes and nSREBP suppression. This finding of an ASGR1/INSIG1/SREBP axis regulating lipid hemostasis may provide multiple potential targets for lipid-lowering drug development.

Hepatic Levels of DHA-Containing Phospholipids Instruct SREBP1-Mediated Synthesis and Systemic Delivery of Polyunsaturated Fatty Acids.

SummaryPolyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), play fundamental roles in mammalian physiology. Although PUFA imbalance causes various disorders, mechanisms of the regulation of their systemic levels are poorly understood. Here, we report that hepatic DHA-containing phospholipids (DHA-PLs) determine the systemic levels of PUFAs through the SREBP1-mediated transcriptional program. We demonstrated that liver-specific deletion of Agpat3 leads to a decrease of DHA-PLs and a compensatory increase of ARA-PLs not only in the liver but also in other tissues including the brain. Together with recent findings that plasma lysophosphatidylcholine (lysoPC) is the major source of brain DHA, our results indicate that hepatic AGPAT3 contributes to brain DHA accumulation by supplying DHA-PLs as precursors of DHA-lysoPC. Furthermore, dietary fish oil-mediated suppression of hepatic PUFA biosynthetic program was blunted in liver-specific Agpat3 deletion. Our findings highlight the central role of hepatic DHA-PLs as the molecular rheostat for systemic homeostasis of PUFAs.

206-OR: Generation of Induced Pluripotent Stem Cells Derived from a Patient with PIK3R1 Mutation and Analysis of Defects in Insulin Action in Hepatocytes Differentiated from These Cells

Background: A protocol of differentiation form induced pluripotent stem cells (iPSCs) to hepatocytes has been previously reported, and such iPSCs-derived hepatocytes possessed morphological and biochemical characteristics similar to authentic hepatocytes. It is however unknown whether iPSCs-derived hepatocytes normally respond to metabolic regulatory hormones including insulin. Mutations in the gene for p85α subunit of PI3K (PIK3R1), an essential element of insulin action, give rise to severe insulin resistance in human. Such cases are very rare, and only ∼30 families with PIK3R1 mutations have been reported as of today. Methods and Results: We established a protocol of differentiation from iPSCs to hepatocytes that respond to insulin. In hepatocytes differentiated from iPSCs derived from healthy subject with this protocol, phosphorylation of AKT and the expression of the lipogenic genes FASN as well as SREBF1c were induced by insulin. Furthermore, the expression of the gluconeogenic gene G6PC was upregulated by a combination of cAMP and dexamethasone, which was markedly impaired by the addition of insulin. We then generated iPSCs from a severe insulin resistant diabetic patient with the PIK3R1 c.1945C>T mutation, a hot-spot mutation in this gene. Insulin-induced gene expression along with phosphorylation of AKT was impaired in hepatocytes differentiated from the patient-derived iPSCs. We also repaired the mutation of PIK3R1 in the patient-derived iPSCs by the CRISPR/Cas9 genome editing. Insulin responsiveness was recovered in hepatocytes differentiated from the resultant iPSCs in which the mutation was repaired. Conclusion: We established a differentiation protocol of hepatocytes in which insulin’s metabolic actions can be evaluated. The usefulness of this protocol was validated with the use of iPSCs derived from a severe insulin-resistant patient with PIK3R1 mutation. Disclosure T. Hamaguchi: None. Y. Hirota: Other Relationship; Self; Sanofi. T. Takeuchi: None. M. Koyanagi-Aoi: None. T. Aoi: None. W. Ogawa: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharma GmbH & Co.KG, Eli Lilly Japan K.K., Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Other Relationship; Self; Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

Silencing an insulin-induced lncRNA, LncASIR, impairs the transcriptional response to insulin signalling in adipocytes

Long noncoding RNA(lncRNA)s are new regulators governing the metabolism in adipose tissue. In this study, we aimed to understand how lncRNAs respond to insulin signalling and explore whether lncRNAs have a functional role in insulin signalling pathway. We treated primary adipocyte cultures with insulin and collected RNA for RNA-sequencing to profile the non-coding transcriptome changes, through which we identified a top Adipose Specific Insulin Responsive LncRNA (LncASIR). To determine its biological function, we knocked down LncASIR using dcas9-KRAB, followed by RNA-seq to examine the effect on insulin-induced gene expression program. We identified a set of lncRNAs regulated by insulin signalling pathway. LncASIR is transcribed from a super enhancer region and responds robustly to insulin treatment. Silencing LncASIR resulted in an impaired global insulin-responsive gene program. LncASIR is a novel and integral component in the insulin signalling pathway in adipocytes.

MiR-24 is involved in vertebrate LC-PUFA biosynthesis as demonstrated in marine teleost Siganus canaliculatus

Recently, microRNAs (miRNAs) have emerged as crucial regulators of lipid metabolism. However, the miRNA-mediated regulatory mechanism on long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis in vertebrates remains largely unknown. Here, we address a potentially important role of miRNA-24 (miR-24) in the regulation of LC-PUFA biosynthesis in rabbitfish Siganus canaliculatus. miR-24 showed significantly higher abundance in liver of rabbitfish reared in brackish water than in seawater for fish fed vegetable oil diets and in S. canaliculatus hepatocyte line (SCHL) cells incubated with alpha-linolenic acid (ALA) than the control group. Similar expression patterns were also observed on the expression of sterol regulatory element-binding protein-1 (srebp1) and LC-PUFA biosynthesis related genes. While opposite results were observed on the expression of insulin-induced gene 1 (insig1), an endoplasmic reticulum membrane protein blocking Srebp1 proteolytic activation. Luciferase reporter assays revealed rabbitfish insig1 as a target of miR-24. Knockdown of miR-24 in SCHL cells resulted in increased Insig1 protein, and subsequently reduced mature Srebp1 protein and expression of genes required for LC-PUFA biosynthesis, and these effects could be attenuated after additional insig1 knockdown. Opposite results were observed with overexpression of miR-24. Moreover, increasing endogenous insig1 by knockdown of miR-24 inhibited Srebp1 processing and consequently suppressed LC-PUFA biosynthesis in rabbitfish hepatocytes. These results indicate a potentially critical role for miR-24 in regulating LC-PUFA biosynthesis through the Insig1/Srebp1 pathway by targeting insig1. This is the first report of miR-24 involved in LC-PUFA biosynthesis and thus may provide knowledge on the regulatory mechanisms of LC-PUFA biosynthesis in vertebrates.

Enhanced TGF-β Signaling Contributes to the Insulin-Induced Angiogenic Responses of Endothelial Cells

SummaryAngiogenesis, the development of new blood vessels, is a key process in disease. We reported that insulin promotes translocation of transforming growth factor β (TGF-β) receptors to the plasma membrane of epithelial and fibroblast cells, thus enhancing TGF-β responsiveness. Since insulin promotes angiogenesis, we addressed whether increased autocrine TGF-β signaling participates in endothelial cell responses to insulin. We show that insulin enhances TGF-β responsiveness and autocrine TGF-β signaling in primary human endothelial cells, by inducing a rapid increase in cell surface TGF-β receptor levels. Autocrine TGF-β/Smad signaling contributed substantially to insulin-induced gene expression associated with angiogenesis, including TGF-β target genes encoding angiogenic mediators; was essential for endothelial cell migration; and participated in endothelial cell invasion and network formation. Blocking TGF-β signaling impaired insulin-induced microvessel outgrowth from neonatal aortic rings and modified insulin-stimulated blood vessel formation in zebrafish. We conclude that enhanced autocrine TGF-β signaling is integral to endothelial cell and angiogenic responses to insulin.

RNA-binding protein HuD reduces triglyceride production in pancreatic β cells by enhancing the expression of insulin-induced gene 1

Although triglyceride (TG) accumulation in the pancreas leads to β-cell dysfunction and raises the chance to develop metabolic disorders such as type 2 diabetes (T2DM), the molecular mechanisms whereby intracellular TG levels are regulated in pancreatic β cells have not been fully elucidated. Here, we present evidence that the RNA-binding protein HuD regulates TG production in pancreatic β cells. Mouse insulinoma βTC6 cells stably expressing a small hairpin RNA targeting HuD (shHuD) (βTC6-shHuD) contained higher TG levels compared to control cells. Moreover, downregulation of HuD resulted in a decrease in insulin-induced gene 1 (INSIG1) levels but not in the levels of sterol regulatory element-binding protein 1c (SREBP1c), a key transcription factor for lipid production. We identified Insig1 mRNA as a direct target of HuD by using ribonucleoprotein immunoprecipitation (RIP) and biotin pulldown analyses. By associating with the 3′-untranslated region (3ʹUTR) of Insig1 mRNA, HuD promoted INSIG1 translation; accordingly, HuD downregulation reduced while ectopic HuD expression increased INSIG1 levels. We further observed that HuD downregulation facilitated the nuclear localization of SREBP1c, thereby increasing the transcriptional activity of SREBP1c and the expression of target genes involved in lipogenesis; likewise, we observed lower INSIG1 levels in the pancreatic islets of HuD-null mice. Taken together, our results indicate that HuD functions as a novel repressor of lipid synthesis in pancreatic β cells.

Hyperinsulinemia reduces osteoblast activity in vitro via upregulation of TGF-β

Affecting more than 230,000,000 patients, diabetes mellitus is one of the most frequent metabolic disorders in developed countries. Among other complications, diabetic patients have an increased fracture risk and show delayed fracture healing. During the disease progression, these patients' blood glucose and insulin levels vary significantly. Thus, the aim of this study was to analyze the effects of glucose and insulin on primary human osteoblasts. Although, in the presence of insulin and glucose, proliferation of osteoblasts was increased (1.2- to 1.7-fold), their alkaline phosphatase activity and, consequently, production of mineralized matrix were significantly reduced down to 55 % as compared to control cells (p < 0.001). Interestingly, the observed effects were mainly due to stimulation with insulin. Increase in glucose did not alter osteoblasts' function significantly but further enhanced the effects of insulin. Expression of active and total transforming growth factor beta (TGF-β) was increased by glucose and insulin. Stimulation with both glucose and insulin induced gene expression changes (e.g., osteocalcin, Runx2, Satb2, or Stat1) comparable to treatment with recombinant TGF-β(1), further indicating osteoblasts' dysfunction. Inhibition of TGF-β signaling completely abolished the negative effects of glucose and insulin. In summary, glucose and insulin treatment causes osteoblast dysfunction, which is accompanied by an increased TGF-β expression. Blocking TGF-β signaling abrogates the functional loss observed in glucose- and insulin-treated osteoblasts, thus identifying TGF-β as a key regulator. Therefore, increased TGF-β expression during diabetes may be a feasible pathogenic mechanism underlying poor bone formation in uncontrolled diabetes mellitus.

High-starch diets induce precocious adipogenic gene network up-regulation in longissimus lumborum of early-weaned Angus cattle

Adipocyte differentiation is probably controlled by transcriptional and post-transcriptional regulation. Longissimus lumborum from Angus steers (aged 155 d; seven animals per diet) fed high-starch or low-starch diets for 112 d (growing phase) followed by a common high-starch diet for an additional 112 d (finishing phase) was biopsied at 0, 56, 112 and 224 d for transcript profiling via quantitative PCR of twenty genes associated with adipogenesis and energy metabolism. At 56 d steers fed high starch had greater expression of PPARgamma as well as the lipogenic enzymes ATP citrate lyase (ACLY), glucose-6-phosphate dehydrogenase (G6PD), fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4), stearoyl-CoA desaturase (SCD), glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), and diacylglycerol O-acyltransferase homologue 2 (DGAT2), and the adipokine adiponectin (ADIPOQ). Expression of insulin-induced gene 1 (INSIG1) was also greater with high starch at 56 d. Steers fed low starch experienced a marked increase in FASN, FABP4, SCD, DGAT2 and thyroid hormone-responsive (SPOT14 homologue, rat) (THRSP) between 56 and 112 d of feeding. A greater expression of the transcription factors sterol regulatory element-binding transcription factor 1 (SREBF1) and MLX interacting protein-like (MLXIPL) was observed at 224 d in steers fed high starch, suggesting a nutritional imprinting effect. Carryover effects of low starch feeding were discerned by greater expression at 224 d of THRSP, FABP4, SCD and DGAT2. These steers also had greater PPARgamma at 224 d. Despite these responses, low starch led to greater expression at 224 d of nuclear receptor subfamily 2, group F, member 2 (NR2F2), a known repressor of rodent adipocyte differentiation through its negative effects on PPARgamma, ADIPOQ and FABP4. Results suggested that early exposure to high starch induced precocious intramuscular adipocyte proliferation and metabolic imprinting of lipogenic transcription regulators. Low starch might have blunted the PPARgamma-driven adipogenic response through up-regulation of NR2F2 but the endogenous ligand for this nuclear receptor remains unknown.

Enhanced phosphorylation of FOXO and GSK‐3 by organo‐vanadium complexes: potential role in insulino‐mimesis

Several organo‐vanadium complexes (OVCs) have been shown to exert multiple insulino‐mimetic effects in rodent models of diabetes. We have shown that Bis(maltolato) OxoVanadium(IV) (BMOV) activates several components of the insulin signaling pathway such as phosphatidyl inositol‐3 kinase (PI3‐K) and protein kinase B (PKB). Forkhead Box Proteins (FOXO) and Glycogen Synthase Kinase‐3 (GSK‐3), downstream substrates of PKB, are thought to contribute to insulin‐induced gene expression of key gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose‐6‐phosphatase (G6Pase). OVCs are known to modulate the expression of PEPCK and G6Pase, hence in the present studies we investigated BMOV‐induced FOXO and GSK‐3 regulation. Treatment of Chinese hamster ovary cells overexpressing human insulin receptor (CHO‐HIR) enhanced FOXO and GSK‐3 phosphorylation. Wortmannin, a PI3‐K inhibitor, decreased FOXO and GSK‐3 phosphorylation induced by both insulin and OVCs, as did AG1024, an inhibitor of IR/IGF‐1R‐PTK activity. In summary, these data demonstrate that OVCs potently enhance the phosphorylation of FOXO and GSK‐3 via IGF‐1R‐PTK/PI3K‐dependent pathways, suggesting that OVCs may contribute towards the insulino‐mimetic effects of BMOV by transcription regulation of gluconeogenic enzymes.(Supported by grants from Canadian Institutes of Health Research).

Microarray Analysis of Thyroid Stimulating Hormone, Insulin-Like Growth Factor-1, and Insulin-Induced Gene Expression in FRTL-5 Thyroid Cells

To determine which genes are regulated by thyroid stimulating hormone (thyrotropin, TSH), insulin and insulin-like growth factor-1 (IGF-1) in the rat thyroid, we used the microarray technology and observed the changes in gene expression. The expressions of genes for bone morphogenetic protein 6, the glucagon receptor, and cyclin D1 were increased by both TSH and IGF-1; for cytochrome P450, 2c37, the expression was decreased by both. Genes for cholecystokinin, glucuronidase, beta, demethyl-Q 7, and cytochrome c oxidase, subunit VIIIa, were up-regulated; the genes for ribosomal protein L37 and ribosomal protein L4 were down-regulated by TSH and insulin. However, there was no gene observed to be regulated by all three: TSH, IGF-1, and insulin molecules studied. These findings suggest that TSH, IGF-1, and insulin stimulate different signal pathways, which can interact with one another to regulate the proliferation of thyrocytes, and thereby provide additional influence on the process of cellular proliferation.

Blockade of Rapid Versus Prolonged Extracellularly Regulated Kinase 1/2 Activation Has Differential Effects on Insulin-Induced Gene Expression

In the present work, insulin's regulation of expression of activating transcription factor 3 (ATF-3), the putative transcription factor proline-rich induced protein (Pip)92, and insulin-inducible gene-1 (Insig-1) (an ER resident protein involved in regulation of sterol-responsive element-binding protein 1 activation) have been examined in a liver-derived cell line (rat H4IIE hepatoma cells). We report that: 1) insulin-induced transcription of ATF-3, Pip92, and Insig-1 required MEK-ERK activation; 2) insulin-induced transcription of ATF-3 and Pip92 reached maximum levels within 15 min and was blocked by wortmannin but not LY294002; 3) in contrast, the maximum level of insulin-induced transcription of Insig-1 was delayed and was not blocked by either wortmannin or LY294002; 4) insulin activated ERK1/2 in two distinct phases, a rapid peak and a later plateau; 5) the delayed plateau phase of insulin-induced ERK1/2 activation was partially phosphatidylinositol 3-OH-kinase dependent; and 6) however, the rapid, insulin-induced peak of ERK1/2 activation was blocked by wortmannin but not LY294002.

The ubiquitin-proteasome pathway is a new partner for the control of insulin signaling.

Insulin signaling is a transitory effect that has to be tightly controlled in magnitude and duration in order to maintain cell homeostasis. Recent reports have demonstrated that members of the ubiquitin-proteasome pathway represent new partners that have to be taken into account for the regulation of insulin action. The protein amounts of the different signaling molecules involved in insulin action are regulated by their rates of synthesis and degradation. The ubiquitin-proteasome system is involved in the internalization of the insulin receptor, in the control of the amount of insulin receptor substrates 1 and 2, and in insulin degradation. Finally, ubiquitination and sumoylation regulate transcription factors and nuclear receptors that mediate insulin-induced gene expression. It is well known from transgenic models that inappropriate levels of signaling molecules strongly affect insulin action. In humans also, several reports have provided evidence of altered levels of key proteins involved in insulin action in pathologies such as type 2 diabetes. The relationship between these abnormalities and the ubiquitin-proteasome pathway has yet to be clarified, but clarifying the role of ubiquitination in insulin action will certainly lead to a better understanding of insulin resistance.

Insulin and Hypoxia Share Common Target Genes but Not the Hypoxia-inducible Factor-1α

Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.

Insulin Signal Transduction Pathways and Insulin-induced Gene Expression

Insulin regulates metabolic activity, gene transcription, and cell growth by modulating the activity of several intracellular signaling pathways. Insulin activation of one mitogen-activated protein kinase cascade, the MEK/ERK kinase cascade, is well described. However, the effect of insulin on the parallel p38 pathway is less well understood. The present work examines the effect of inhibiting the p38 signaling pathway by use of specific inhibitors, either alone or in combination with insulin, on the activation of ERK1/2 and on the regulation of gene transcription in rat hepatoma cells. Activation of ERK1/2 was induced by insulin and was dependent on the activation of MEK1, the kinase upstream of ERK in this pathway. Treatment of cells with p38 inhibitors also induced ERK1/2 activation/phosphorylation. The addition of p38 inhibitors followed by insulin addition resulted in a greater than additive activation of ERK1/2. The two genes studied, c-Fos and Pip92, are immediate-early genes that are dependent on the ERK1/2 pathway for insulin-regulated induction because the insulin effect was inhibited by pretreatment with a MEK1 inhibitor. The addition of p38 inhibitors induced transcription of both genes in a dose-dependent manner, and insulin stimulation of both genes was enhanced by prior treatment with p38 inhibitors. The ability of the p38 inhibitors to induce ERK1/2 and gene transcription, both alone and in combination with insulin, was abolished by prior inhibition of MEK1. These data suggest possible cross-talk between the p38 and ERK1/2 signaling pathways and a potential role of p38 in insulin signaling.

ADD1/SREBP-1c Mediates Insulin-Induced Gene Expression Linked to the MAP Kinase Pathway

The aim of this study was to define the role of sterol regulatory element binding protein (SREBP)-1c, the human homologue to ADD1 (adipocyte determination- and differentiation-dependent factor 1), in insulin-induced gene expression. Transfection studies using SREBP-1-deficient cells and a LDL receptor promoter fragment containing the ADD1/SREBP-1c binding side showed that the effects of insulin and PDGF were abolished compared to control cells and completely reconstituted by overexpressing ADD1/SREBP-1c. Overexpression of upstream activators of MAP kinases, like MEKK1 or MEK1, demonstrated that ADD1/SREBP-1c-mediated effects of insulin and PDGF might be linked to the MAP kinase cascade. The recombinant N-terminal domain of ADD1/SREBP-1c was phosphorylated predominantly on serine and slightly on threonine residues by MAP kinases ERK1 and ERK2in vitro.This was reversible by alkaline phosphatase. We conclude that ADD1/SREBP-1c mediates gene regulatory effects of insulin as well as PDGF and that this signalling is linked to the MAP kinase cascade.

Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression.

Insulin induces a rapid activation of p21ras in NIH 3T3 and Chinese hamster ovary cells that overexpress the insulin receptor. Previously, we suggested that p21ras may mediate insulin-induced gene expression. To test such a function of p21ras more directly, we studied the effect of different dominant inhibitory mutants of p21ras on the induction of gene expression in response to insulin. We transfected a collagenase promoter-chloramphenicol acetyltransferase (CAT) gene or a fos promoter-luciferase gene into NIH 3T3 cells that overexpressed the insulin receptor. The activities of both promoters were strongly induced after treatment with insulin. This induction could be suppressed by cotransfection of two inhibitory mutant ras genes, H-ras(Asn-17) or H-ras(Leu-61,Ser-186). In particular, insulin-induced activation of the fos promoter was inhibited completely by H-ras(Asn-17). These results show that p21ras functions as an intermediate in the insulin signal transduction route leading to the induction of gene expression.

Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression.

Insulin induces a rapid activation of p21ras in NIH 3T3 and Chinese hamster ovary cells that overexpress the insulin receptor. Previously, we suggested that p21ras may mediate insulin-induced gene expression. To test such a function of p21ras more directly, we studied the effect of different dominant inhibitory mutants of p21ras on the induction of gene expression in response to insulin. We transfected a collagenase promoter-chloramphenicol acetyltransferase (CAT) gene or a fos promoter-luciferase gene into NIH 3T3 cells that overexpressed the insulin receptor. The activities of both promoters were strongly induced after treatment with insulin. This induction could be suppressed by cotransfection of two inhibitory mutant ras genes, H-ras(Asn-17) or H-ras(Leu-61,Ser-186). In particular, insulin-induced activation of the fos promoter was inhibited completely by H-ras(Asn-17). These results show that p21ras functions as an intermediate in the insulin signal transduction route leading to the induction of gene expression.