IRS-1 Gene Expression Research Articles

Abstract P073: Chemerin Receptor Antagonism Improves Vascular Insulin Signaling in Diabetic Mice

Adipose tissue releases many adipokines, including chemerin, which produces its effects through activation of chemokine-like receptor 1 (ChemR23). Chemerin influences major aspects of the metabolic syndrome, including adipogenesis and insulin sensitivity in adipocytes and skeletal muscle cells. Considering that chemerin impairs vascular reactivity and that amongst its many actions, insulin also influences vascular function, we postulate that chemerin decreases insulin-induced vasodilatation, by reducing PI3K/Akt signaling, and impairs glucose uptake by vascular smooth muscle cells (VSMC). Mesenteric arteries, endothelial cells (EC) or VSMC from C57Bl6 mice were incubated with chemerin (0.5 ng/mL, 1 h) or vehicle (veh). Chemerin decreased insulin-induced relaxation (0.1 - 3000 ng/mL, pD2: chemerin: 94.5±0.1 vs. veh: 23.6±0,1), which was prevented by the ChemR23 antagonist CCX 832 (pD2: veh= 16.9±0.1; chem= 174.1±0.1, CCX+chem= 37.4±0.1) and a PI3K activator (YS-49) (pD2: veh= 23.0±0.1; chem= 108.5±0.1, YS-49+chem= 28.5±0.1). Chemerin also decreased PI3K (0.8±0.1 vs. veh 1.1±0.1), Akt (0.8±0.1 vs. veh 1.2±0.1) and AMPK [0.7±0.2 vs. veh 1.0 ±0.1] phosphorylation in VSMC. IRS1 and IRS2 gene expression was decreased in VSMC stimulated with chemerin. In addition, chemerin decreased insulin-stimulated NO production in EC through activation of ChemR23 and PI3K and inhibited insulin-stimulated glucose uptake by VSMC (counts per minute: 5668±729.0 vs. veh 9923±662.7), which was mediated by ChemR23, AMPK and PI3K. Importantly, ChemR23 antagonism in diabetic (db/db) mice partially reversed decreased insulin-induced vasodilatation (mesenteric resistance arteries; pD2: db/m: 6.1±1.0; db/db: 7.3±0.2; db/db+CCX832: 6.9±0.3). In conclusion, chemerin decreases vascular insulin responses by reducing PI3K/Akt and AMPK signaling. Moreover, chemerin/ChemR23 system plays a crucial role in impaired vascular insulin signaling in diabetes, suggesting its involvement in the pathogenesis of vascular insulin resistance. Our study may contribute to a better understanding of the role of chemerin in vascular insulin dysfunction in diabetes- and obesity-associated diseases. Financial Support: FAPESP, Brazil.

Dietary approaches to stop hypertension influence on insulin receptor substrate-1gene expression: A randomized controlled clinical trial.

Background:Insulin receptor substrate (IRS) Type 1 is a main substrate for the insulin receptor, controls insulin signaling in skeletal muscle, adipose tissue, and the vascular, so it is an important candidate gene for insulin resistance (IR). We aimed to compare the effects of the Dietary Approaches to Stop Hypertension (DASH) and Usual Dietary Advices (UDA) on IRS1 gene expression in women at risk for cardiovascular disease.Materials and Methods:A randomized controlled clinical trial was performed in 44 women at risk for cardiovascular disease. Participants were randomly assigned to a UDA diet or the DASH diet. The DASH diet was rich in fruits, vegetables, whole grains, and low-fat dairy products and low in saturated fat, total fat, cholesterol, refined grains, and sweets, with a total of 2400 mg/day sodium. The UDA diet was a regular diet with healthy dietary advice. Gene expression was assessed by the real-time polymerase chain reaction at the first of study and after 12 weeks. Independent sample t-test and paired-samples t-test were used to compare means of all variables within and between two groups respectively.Results:IRS1 gene expression was increased in DASH group compared with UDA diet (P = 0.00). Weight and waist circumference decreased in DASH group significantly compared to the UDA group (P < 0.05) but the results between the two groups showed no significant difference.Conclusion:DASH diet increased IRS1 gene expression and probably has beneficial effects on IR risks.

Enhancement of glucose uptake by Ficus hispida methanolic extract through phosphatidylinositol 3 kinase and inhibiting aldose reductase – An in vitro analysis

IntroductionType 2 diabetes mellitus is a polygenic metabolic syndrome characterized by impaired carbohydrate and lipid metabolism. Diabetes mellitus is commonly associated with secondary disorders such as obesity and genetic predisposition. Prolonged hyperglycemia can also lead to secondary complications such as cataractogenesis, retinopathy and nephropathy through the induction of the polyol pathway. Ficus hispida (FH) is a medium size tree found in India, Sri Lanka and the Andaman Islands in damp localities. Traditionally, different parts of the FH plant have been used in the treatment of various diseases and disorders. MethodologyFH bark was sequentially extracted using different organic solvents ranging from non polar to polar. Glucose uptake assays were performed using 2-deoxy-d-1-[3H] for the FH extracts. Fluorimetric analysis was adopted to assess the aldose reductase inhibitory effect. The gene and protein level expression of the markers were analyzed using RT-PCR and Western blot analysis. ResultsF. hispida methanolic extract (FHME) showed an enhancement in the glucose uptake and upregulated the gene and protein level expression of IRβ, IRS-1, PI3K, GLUT4 and PPARγ. Comparison with wortmannin, a specific PI3K inhibitor confirmed that the active FHME recruits glucose uptake through a PI3K dependent pathway. Subsequently, FHME possesses a significant aldose reductase inhibitory activity. ConclusionThe current study emphasizes the cellular level mechanism of FHME on glucose transport in L6 myotubes and the inhibition of aldose reductase activity.

11β-Hydroxysteroid dehydrogenase type 1 shRNA ameliorates glucocorticoid-induced insulin resistance and lipolysis in mouse abdominal adipose tissue.

Long-term glucocorticoid exposure increases the risk for developing type 2 diabetes. Prereceptor activation of glucocorticoid availability in target tissue by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) coupled with hexose-6-phosphate dehydrogenase (H6PDH) is an important mediator of the metabolic syndrome. We explored whether the tissue-specific modulation of 11β-HSD1 and H6PDH in adipose tissue mediates glucocorticoid-induced insulin resistance and lipolysis and analyzed the effects of 11β-HSD1 inhibition on the key lipid metabolism genes and insulin-signaling cascade. We observed that corticosterone (CORT) treatment increased expression of 11β-HSD1 and H6PDH and induced lipase HSL and ATGL with suppression of p-Thr(172) AMPK in adipose tissue of C57BL/6J mice. In contrast, CORT induced adipose insulin resistance, as reflected by a marked decrease in IR and IRS-1 gene expression with a reduction in p-Thr(308) Akt/PKB. Furthermore, 11β-HSD1 shRNA attenuated CORT-induced 11β-HSD1 and lipase expression and improved insulin sensitivity with a concomitant stimulation of pThr(308) Akt/PKB and p-Thr(172) AMPK within adipose tissue. Addition of CORT to 3T3-L1 adipocytes enhanced 11β-HSD1 and H6PDH and impaired p-Thr(308) Akt/PKB, leading to lipolysis. Knockdown of 11β-HSD1 by shRNA attenuated CORT-induced lipolysis and reversed CORT-mediated inhibition of pThr(172) AMPK, which was accompanied by a parallel improvement of insulin signaling response in these cells. These findings suggest that elevated adipose 11β-HSD1 expression may contribute to glucocorticoid-induced insulin resistance and adipolysis.

BH3-only molecule Bim mediates β-cell death in IRS2 deficiency.

Irs2-deficient mice develop type 2–like diabetes due to a reduction in β-cell mass and a failure of pancreatic islets to undergo compensatory hyperplasia in response to insulin resistance. In order to define the molecular mechanisms, we knocked down Irs2 gene expression in mouse MIN6 insulinoma cells. Insulin receptor substrate 2 (IRS2) suppression induced apoptotic cell death, which was associated with an increase in expression of the BH3-only molecule Bim. Knockdown (KD) of Bim reduced apoptotic β-cell death induced by IRS2 suppression. In Irs2-deficient mice, Bim ablation restored β-cell mass, decreased the number of TUNEL-positive cells, and restored normal glucose tolerance after glucose challenge. FoxO1 mediates Bim upregulation induced by IRS2 suppression, and FoxO1 KD partially inhibits β-cell death induced by IRS2 suppression. These results suggest that Bim plays an important role in mediating the increase in β-cell apoptosis and the reduction in β-cell mass that occurs in IRS2-deficient diabetes.

Growth hormone abolishes beneficial effects of calorie restriction in long-lived Ames dwarf mice

Disruption of the growth hormone (GH) axis promotes longevity and delays aging. In contrast, GH over-expression may lead to accelerated aging and shorter life. Calorie restriction (CR) improves insulin sensitivity and may extend lifespan. Long-lived Ames dwarf (df/df) mice have additional extension of longevity when subjected to 30% CR. The aim of the study was to assess effects of CR or GH replacement therapy separately and as a combined (CR+GH) treatment in GH-deficient df/df and normal mice, on selected metabolic parameters (e.g., insulin, glucose, cholesterol), insulin signaling components (e.g., insulin receptor [IR] β-subunit, phosphorylated form of IR [IR pY1158], protein kinase C ζ/λ [p-PKCζ/λ] and mTOR [p-mTOR]), transcription factor p-CREB, and components of the mitogen-activated protein kinase (MAPK) signaling (p-ERK1/2, p-p38), responsible for cell proliferation, differentiation and survival. CR decreased plasma levels of insulin, glucose, cholesterol and leptin, and increased hepatic IR β-subunit and IR pY1158 levels as well as IR, IRS-1 and GLUT-2 gene expression compared to ad libitum feeding, showing a significant beneficial diet intervention effect. Moreover, hepatic protein levels of p-PKCζ/λ, p-mTOR and p-p38 decreased, and p-CREB increased in CR mice. On the contrary, GH increased levels of glucose, cholesterol and leptin in plasma, and p-mTOR or p-p38 in livers, and decreased plasma adiponectin and hepatic IR β-subunit compared to saline treatment. There were no GH effects on adiponectin in N mice. Moreover, GH replacement therapy did not affect IR, IRS-1 and GLUT-2 gene expression. GH treatment abolishes the beneficial effects of CR; it may suggest an important role of GH–IGF1 axis in mediating the CR action. Suppressed somatotrophic signaling seems to predominate over GH replacement therapy in the context of the examined parameters and signaling pathways.

Subcutaneous adipose tissue zinc-α2-glycoprotein is associated with adipose tissue and whole-body insulin sensitivity.

To examine the regulatory aspects of zinc-α2-glycoprotein (ZAG) association with obesity-related insulin resistance. ZAG mRNA and protein were analyzed in subcutaneous adipose tissue (AT) and circulation of lean, obese, prediabetic, and type 2 diabetic men; both subcutaneous and visceral AT were explored in lean and extremely obese. Clinical and ex vivo findings were corroborated by results of in vitro ZAG silencing experiment. Subcutaneous AT ZAG was reduced in obesity, with a trend to further decrease with prediabetes and type 2 diabetes. ZAG was 3.3-fold higher in subcutaneous than in visceral AT of lean individuals. All differences were lost in extreme obesity. Obesity-associated changes in AT were not paralleled by alterations of circulating ZAG. Subcutaneous AT ZAG correlated with adiposity, adipocyte hypertrophy, whole-body and AT insulin sensitivity, mitochondrial content, expression of GLUT4, PGC1α, and adiponectin. Subcutaneous AT ZAG and adipocyte size were the only predictors of insulin sensitivity, independent on age and BMI. Silencing ZAG resulted in reduced adiponectin, IRS1, GLUT4, and PGC1α gene expression in primary human adipocytes. ZAG in subcutaneous, but not in visceral AT, was markedly reduced in obesity. Clinical, cellular, and molecular evidence indicate that ZAG plays an important role in modulating whole-body and AT insulin sensitivity.

PRAS40 prevents development of diabetic cardiomyopathy and improves hepatic insulin sensitivity in obesity

Diabetes is a multi-organ disease and diabetic cardiomyopathy can result in heart failure, which is a leading cause of morbidity and mortality in diabetic patients. In the liver, insulin resistance contributes to hyperglycaemia and hyperlipidaemia, which further worsens the metabolic profile. Defects in mTOR signalling are believed to contribute to metabolic dysfunctions in diabetic liver and hearts, but evidence is missing that mTOR activation is causal to the development of diabetic cardiomyopathy. This study shows that specific mTORC1 inhibition by PRAS40 prevents the development of diabetic cardiomyopathy. This phenotype was associated with improved metabolic function, blunted hypertrophic growth and preserved cardiac function. In addition PRAS40 treatment improves hepatic insulin sensitivity and reduces systemic hyperglycaemia in obese mice. Thus, unlike rapamycin, mTORC1 inhibition with PRAS40 improves metabolic profile in diabetic mice. These findings may open novel avenues for therapeutic strategies using PRAS40 directed against diabetic-related diseases.

The MRC1/CD68 Ratio Is Positively Associated with Adipose Tissue Lipogenesis and with Muscle Mitochondrial Gene Expression in Humans

BackgroundAlternative macrophages (M2) express the cluster differentiation (CD) 206 (MCR1) at high levels. Decreased M2 in adipose tissue is known to be associated with obesity and inflammation-related metabolic disturbances. Here we aimed to investigate MCR1 relative to CD68 (total macrophages) gene expression in association with adipogenic and mitochondrial genes, which were measured in human visceral [VWAT, n = 147] and subcutaneous adipose tissue [SWAT, n = 76] and in rectus abdominis muscle (n = 23). The effects of surgery-induced weight loss were also longitudinally evaluated (n = 6).Results MCR1 and CD68 gene expression levels were similar in VWAT and SWAT. A higher proportion of CD206 relative to total CD68 was present in subjects with less body fat and lower fasting glucose concentrations. The ratio MCR1/CD68was positively associated with IRS1gene expression and with the expression of lipogenic genes such as ACACA, FASN and THRSP, even after adjusting for BMI. The ratio MCR1/CD68 in SWAT increased significantly after the surgery-induced weight loss (+44.7%; p = 0.005) in parallel to the expression of adipogenic genes. In addition, SWAT MCR1/CD68ratio was significantly associated with muscle mitochondrial gene expression (PPARGC1A, TFAM and MT-CO3). AT CD206 was confirmed by immunohistochemistry to be specific of macrophages, especially abundant in crown-like structures.ConclusionA decreased ratio MCR1/CD68 is linked to adipose tissue and muscle mitochondrial dysfunction at least at the level of expression of adipogenic and mitochondrial genes.

Regulation of IL-4-induced insulin receptor substrate-2 activation in allergic inflammation (P6294)

Abstract Allergic asthma is a chronic airway disease driven by Th2 cytokines, IL-4, -5 and -13. IL-4 receptors activate IRS-2/STAT6 signaling to induce differentiation of M2 macrophages, cells associated with allergic lung inflammation in mice and humans. IL-4, but not IL-13, strongly induced tyrosine phosphorylation (pY) of IRS-2 and M2 gene expression. Understanding regulation of IRS-2 activation is essential for designing novel therapeutics. We hypothesized that both induction of SOCS proteins and serine phosphorylation (pS) of IRS-2 downregulates IRS-2 signaling. To test this, we stimulated both human monocytes (U937 cells) and mouse primary macrophages with IL-4 and observed robust pY-IRS2 that peaked at 60 min and returned to baseline by 150 min. Kinetics of induction of mRNA for SOCS1, SOCS3 and CIS by IL-4 matched the decrease in pY-IRS-2. Using a siRNA knockdown approach, we found that SOCS1 is responsible for downregulating pY-IRS2 but not CIS and SOCS3. SOCS proteins bind to ubiquitin ligase complexes and target proteins for proteasomal degradation. Proteasome inhibitor-treated, IL-4-stimulated U937 cells showed extended pY-IRS2 signaling. In U937 cells, IL-4-induced pS-IRS-2 had an opposite pattern to pY-IRS-2 and when serine phosphatases were inhibited, pY-IRS-2 dramatically decreased. Using serine kinase inhibitors, we found that inhibition of mTORC1 prolonged pY-IRS-2 signaling in BMM and U937 cells, thus uncovering novel, targetable regulators of allergic inflammation.

The Hepatitis C Virus Modulates Insulin Signaling Pathway In Vitro Promoting Insulin Resistance

Insulin is critical for controlling energy functions including glucose and lipid metabolism. Insulin resistance seems to interact with hepatitis C promoting fibrosis progression and impairing sustained virological response to peginterferon and ribavirin. The main aim was to elucidate the direct effect of hepatitis C virus (HCV) infection on insulin signaling both in vitro analyzing gene expression and protein abundance. Huh7.5 cells and JFH-1 viral particles were used for in vitro studies. Experiments were conducted by triplicate in control cells and infected cells. Genes and proteins involved in insulin signaling pathway were modified by HCV infection. Moreover, metformin treatment increased gene expression of PI3K, IRS1, MAP3K, AKT and PTEN more than >1.5 fold. PTP1B, encoding a tyrosin phosphatase, was found highly induced (>3 fold) in infected cells treated with metformin. However, PTP1B protein expression was reduced in metformin treated cells after JFH1 infection. Other proteins related to insulin pathway like Akt, PTEN and phosphorylated MTOR were also found down-regulated. Viral replication was inhibited in vitro by metformin. A strong effect of HCV infection on insulin pathway-related gene and protein expression was found in vitro. These results could lead to the identification of new therapeutic targets in HCV infection and its co-morbidities.

Recombinant Acylation Stimulating Protein Administration to C3−/− Mice Increases Insulin Resistance via Adipocyte Inflammatory Mechanisms

BackgroundComplement 3 (C3), a key component of the innate immune system, is involved in early inflammatory responses. Acylation stimulating protein (ASP; aka C3adesArg), a C3 cleavage product, is produced in adipose tissue and stimulates lipid storage. We hypothesized that, depending on the diet, chronic ASP administration in C3−/− mice would affect lipid metabolism and insulin sensitivity via an adaptive adipose tissue inflammatory response.Methodology/Principal FindingsC3−/− mice on normal low fat diet (ND) or high fat diet (HFD) were chronically administered recombinant ASP (rASP) for 25 days via an osmotic mini-pump. While there was no effect on food intake, there was a decrease in activity, with a relative increase in adipose tissue weight on ND, and a shift in adipocyte size distribution. While rASP administration to C3−/− mice on a ND increased insulin sensitivity, on a HFD, rASP administration had the opposite effect. Specifically, rASP administration in C3−/− HFD mice resulted in decreased gene expression of IRS1, GLUT4, SREBF1 and NFκB in muscle, and decreased C5L2 but increased JNK, CD36, CD11c, CCR2 and NFκB gene expression in adipose tissue as well as increased secretion of proinflammatory cytokines (Rantes, KC, MCP-1, IL-6 and G-CSF). In adipose tissue, although IRS1 and GLUT4 mRNA were unchanged, insulin response was reduced.ConclusionThe effects of chronic rASP administration are tissue and diet specific, rASP administration enhances the HFD induced inflammatory response leading to an insulin-resistant state. These results suggest that, in humans, the increased plasma ASP associated with obesity and cardiovascular disease could be an additional factor directly contributing to development of metabolic syndrome, insulin resistance and diabetes.

Phosphorylated S6K1 (Thr389) is a molecular adipose tissue marker of altered glucose tolerance

Molecular tissue markers of altered glucose metabolism will be useful as potential targets for antidiabetic drugs. S6K1 is a downstream signal of insulin action. We aimed to evaluate pThr389S6K1 and total S6K1 levels in human and rat fat depots as candidate markers of altered glucose metabolism. pThr389S6K1 and total S6K1 levels were measured using enzyme linked immune sorbent assay (ELISA) in 49 adipose tissue samples from subjects with morbid obesity and in 18 peri-renal white adipose tissue samples from rats. The effects of high glucose and rosiglitazone have been explored in human preadipocytes. pThr389S6K1/totalS6K1 in subcutaneous adipose tissue was significantly increased subjects with Type 2 diabetes (0.78±0.26 vs. 0.55±0.14, P=.02) and associated with fasting glucose (r=0.46, P=.04) and glycated hemoglobin (r=0.63, P=.02) in SAT. Similar associations with fasting glucose (r=0.43, P=.03) and IRS1 (r=-0.41, P=.04) gene expression were found in visceral adipose tissue. In addition, rat experiments confirmed the higher pThr389S6K1/totalS6K1 levels in adipose tissue in association with obesity-associated metabolic disturbances. pThr389S6K1/totalS6K1 was validated using western blot in rat adipose tissue. Both ELISA and western blot data significantly correlated (r=0.85, P=.005). In human preadipocytes, high glucose medium led to increased pThr389S6K1/total S6K1 levels in comparison with normal glucose medium, which was significantly decreased under rosiglitazone administration. In conclusion, in human and rat adipose tissue, phosphorylated S6K1 is a marker for increased glucose levels.

Initial Stages of the Insulin Signaling System in the Brain of Rats with Experimental Diabetes Mellitus

We studied reactivity of insulin signal pathway elements, insulin receptor and insulin receptor substrate protein-2 (IRS2 protein), in rat brain in response to insulin insufficiency and insulin resistance during the development of experimental type 1 or type 2 diabetes mellitus. In type 1 diabetes mellitus characterized by acute insulin insufficiency, specific binding of insulin in rat brain increased 2-fold in comparison with the control and IRS2-gene expression in rat hypothalamus and cortex 2-4 fold surpassed the normal values. In type 2 diabetes mellitus (110 and 190 days of development), changes in the test parameters in rat brain were less pronounced. These findings attest to involvement of the brain insulin signal pathway into the response to systemic insulin deficiency in type 1 diabetes mellitus.

Isosteviol Has Beneficial Effects on Palmitate-Induced α-Cell Dysfunction and Gene Expression

BackgroundLong-term exposure to high levels of fatty acids impairs insulin secretion and exaggerates glucagon secretion. The aim of this study was to explore if the antihyperglycemic agent, Isosteviol (ISV), is able to counteract palmitate-induced α-cell dysfunction and to influence α-cell gene expression.Methodology/Principal FindingsLong-term incubation studies with clonal α-TC1–6 cells were performed in the presence of 0.5 mM palmitate with or without ISV. We investigated effects on glucagon secretion, glucagon content, cellular triglyceride (TG) content, cell proliferation, and expression of genes involved in controlling glucagon synthesis, fatty acid metabolism, and insulin signal transduction. Furthermore, we studied effects of ISV on palmitate-induced glucagon secretion from isolated mouse islets. Culturing α-cells for 72-h with 0.5 mM palmitate in the presence of 18 mM glucose resulted in a 56% (p<0.01) increase in glucagon secretion. Concomitantly, the TG content of α-cells increased by 78% (p<0.01) and cell proliferation decreased by 19% (p<0.05). At 18 mM glucose, ISV (10−8 and 10−6 M) reduced palmitate-stimulated glucagon release by 27% (p<0.05) and 27% (p<0.05), respectively. ISV (10−6 M) also counteracted the palmitate-induced hypersecretion of glucagon in mouse islets. ISV (10−6 M) reduced α-TC1–6 cell proliferation rate by 25% (p<0.05), but ISV (10−8 and 10−6 M) had no effect on TG content in the presence of palmitate. Palmitate (0.5 mM) increased Pcsk2 (p<0.001), Irs2 (p<0.001), Fasn (p<0.001), Srebf2 (p<0.001), Acaca (p<0.01), Pax6 (p<0.05) and Gcg mRNA expression (p<0.05). ISV significantly (p<0.05) up-regulated Insr, Irs1, Irs2, Pik3r1 and Akt1 gene expression in the presence of palmitate.Conclusions/SignificanceISV counteracts α-cell hypersecretion and apparently contributes to changes in expression of key genes resulting from long-term exposure to palmitate. ISV apparently acts as a glucagonostatic drug with potential as a new anti-diabetic drug for the treatment of type 2 diabetes.

Decreased IRS2 and TIMP3 Expression in Monocytes From Offspring of Type 2 Diabetic Patients Is Correlated With Insulin Resistance and Increased Intima-Media Thickness

OBJECTIVEIn humans, it is unclear if insulin resistance at the monocyte level is associated with atherosclerosis in vivo. Here we have studied first-degree relatives of patients with type 2 diabetes to investigate whether a reduction in components of the insulin signal transduction pathways, such as the insulin receptor (InsR) or InsR substrate 1 or 2 (IRS1 or IRS2), or a reduction in genetic modifiers of insulin action, such as the TIMP3/ADAM17 (tissue inhibitor of metalloproteinase 3/A disintegrin and metalloprotease domain 17) pathway, is associated with evidence of atherosclerosis.RESEARCH DESIGN AND METHODSInsulin sensitivity was analyzed through euglycemic-hyperinsulinemic clamp, and subclinical atherosclerosis was analyzed through intimal medial thickness. Monocytes were isolated through magnetic cell sorting, and mRNA and proteins were extracted and analyzed by quantitative PCR and pathscan enzyme-linked immunosorbent assays, respectively.RESULTSIn monocyte cells from human subjects with increased risk for diabetes and atherosclerosis, we found that gene expression, protein levels, and tyrosine phosphorylation of IRS2, but not InsR or IRS1, were decreased. TIMP3 was also reduced, along with insulin resistance, resulting in increased ectodomain shedding activity of the metalloprotease ADAM17.CONCLUSIONSSystemic insulin resistance and subclinical atherosclerosis are associated with decreased IRS2 and TIMP3 expression in circulating monocytes.

Specific Glucose-Induced Control of Insulin Receptor Substrate-2 Expression Is Mediated via Ca2+-Dependent Calcineurin/NFAT Signaling in Primary Pancreatic Islet β-Cells

OBJECTIVEInsulin receptor substrate-2 (IRS-2) plays an essential role in pancreatic islet β-cells by promoting growth and survival. IRS-2 turnover is rapid in primary β-cells, but its expression is highly regulated at the transcriptional level, especially by glucose. The aim was to investigate the molecular mechanism on how glucose regulates IRS-2 gene expression in β-cells.RESEARCH DESIGN AND METHODSRat islets were exposed to inhibitors or subjected to adenoviral vector–mediated gene manipulations and then to glucose-induced IRS-2 expression analyzed by real-time PCR and immunoblotting. Transcription factor nuclear factor of activated T cells (NFAT) interaction with IRS-2 promoter was analyzed by chromatin immunoprecipitation assay and glucose-induced NFAT translocation by immunohistochemistry.RESULTSGlucose-induced IRS-2 expression occurred in pancreatic islet β-cells in vivo but not in liver. Modulating rat islet β-cell Ca2+ influx with nifedipine or depolarization demonstrated that glucose-induced IRS-2 gene expression was dependent on a rise in intracellular calcium concentration derived from extracellular sources. Calcineurin inhibitors (FK506, cyclosporin A, and a peptide calcineurin inhibitor [CAIN]) abolished glucose-induced IRS-2 mRNA and protein levels, whereas expression of a constitutively active calcineurin increased them. Specific inhibition of NFAT with the peptide inhibitor VIVIT prevented a glucose-induced IRS-2 transcription. NFATc1 translocation to the nucleus in response to glucose and association of NFATc1 to conserved NFAT binding sites in the IRS-2 promoter were demonstrated.CONCLUSIONSThe mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet β-cell is mediated by the Ca2+/calcineurin/NFAT pathway. This insight into the IRS-2 regulation could provide novel therapeutic means in type 2 diabetes to maintain an adequate functional mass.

DecreasedSTAMP2Expression in Association with Visceral Adipose Tissue Dysfunction

Six-transmembrane protein of prostate 2 (STAMP2) is a counter-regulator of inflammation and insulin resistance according to findings in mice. However, there have been contradictory reports in humans. We aimed to explore STAMP2 in association with inflammatory and metabolic status of human obesity. STAMP2 gene expression was analyzed in adipose tissue samples (171 visceral and 67 sc depots) and during human preadipocyte differentiation. Human adipocytes were treated with macrophage-conditioned medium, TNF-α, and rosiglitazone. In visceral adipose tissue, STAMP2 gene expression was significantly decreased in obese subjects, mainly in obese subjects with type 2 diabetes. STAMP2 gene expression and protein were significantly and inversely associated with obesity phenotype measures (body mass index, waist, hip, and fat mass) and obesity-associated metabolic disturbances (systolic blood pressure and fasting glucose). In addition, STAMP2 gene expression was positively associated with lipogenic (FASN, ACC1, SREBP1, THRSP14, TRα, and TRα1), CAV1, IRS1, GLUT4, and CD206 gene expression. In sc adipose tissue, STAMP2 gene expression was not associated with metabolic parameters. In both fat depots, STAMP2 gene expression in stromovascular cells was significantly higher than in mature adipocytes. STAMP2 gene expression was significantly increased during the differentiation process in parallel to adipogenic genes, being increased in preadipocytes derived from lean subjects. Macrophage-conditioned medium (25%) and TNF-α (100 ng/ml) administration increased whereas rosiglitazone (2 μM) decreased significantly STAMP2 gene expression in human differentiated adipocytes. Decreased STAMP2 expression (mRNA and protein) might reflect visceral adipose dysfunction in subjects with obesity and type 2 diabetes.

Reciprocal interference between insulin and interferon-alpha signaling in hepatic cells: A vicious circle of clinical significance?

Insulin resistance (IR) is common in chronic hepatitis C (CHC) and associates with reduced virological response to pegylated-interferon (PEG-IFN)/ribavirin therapy, but the underlying mechanisms are still unclear. We have previously shown that, in CHC patients, insulin plasma levels are inversely related to antiviral effect induced by PEG-IFN. Therefore, we investigated the in vitro effect of insulin on interferon alpha (IFN-α) intracellular signaling as well as that of IFN-α on insulin signaling. HepG2 cells, preincubated with or without insulin, were stimulated with IFN-α2b and messenger RNA (mRNA) and protein expression of IFN-stimulated genes (ISGs) were measured at different timepoints. The role of intracellular suppressors of cytokine signaling 3 (SOCS3) was evaluated with the small interfering RNA (siRNA) strategy. To assess the effect of IFN-α on insulin signaling, HepG2 were preincubated with or without IFN before addition of insulin and cells were then analyzed for IRS-1 and for Akt/PKB Ser473 phosphorylation. Insulin (100 and 1000 nM) significantly reduced in a dose-dependent fashion IFN-induced gene expression of PKR (P=0.017 and P=0.0017, respectively), MxA (P=0.0103 and P=0.00186), and 2'-5' oligoadenylatesynthetase 1 (OAS-1) (P=0.002 and P=0.006). Insulin also reduced IFN-α-induced PKR protein expression. Although insulin was confirmed to increase SOCS3 expression, siRNA SOCS3 did not restore ISG expression after insulin treatment. IFN-α was found to reduce, in a dose-dependent fashion, IRS-1 gene expression as well as Akt/PKB Ser473 phosphorylation induced by insulin. These results provide evidence of reciprocal interference between insulin and IFN-α signaling in liver cells. These findings may contribute to understand the role of insulin in CHC: IR might be favored by endogenous cytokines including IFN-α, and the resulting hyperinsulinemia then reduces the antiviral response to exogenous IFN in a vicious circle of clinical significance.

IRS2 Is Dowregulated In Primary MDS Cells and During MDS Erythroid Differentiation

AbstractAbstract 1886Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia (AML). The abnormal differentiation of myeloid and erythroid cells is probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling, from upstream activators, to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R) and Erythropoietin receptor (EPOR). It is upregulated and phosphorylated by EPO in normal bone marrow erythroblasts and in UT-7 leukemic cells, as well as in HL60 leukemic cells during granulocytic-differentiation upon DMSO induction. IGF1 signaling is capable of inducing differentiation in several cell types and plays an important role in the regulation of human erythropoiesis. EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. In view of the role of IRS2 in the erythroid and granulocytic differentiation process, we hypothesized that IRS2 might be related to the deficient differentiation of MDS cells and disease progression. The aim of the present study was to characterize the mRNA and protein expression levels of IRS2 in cells of MDS patients and normal donors and to analyze the IRS2 expression levels between low-risk and high-risk of MDS. We also elucidated the expression levels of IRS2 transcripts during erythroid differentiation of CD34+ cells from normal donors and MDS patients. We studied 12 healthy donors and 29 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification; 15 low-risk [RCUD/RCMD/RARS] and 11 high-risk [RAEB-1/RAEB-2] according to WHO classification; 22 low/INT-1 risk and 7 INT-2/high risk according to IPSS; 25 low risk and 4 intermediate/high risk cytogenetic). RT-PCR was performed in total cell from bone marrow samples for gene expression studies. Protein expression was evaluated by Western blot in bone marrow mononuclear cells from MDS patients or in peripheral blood CD34+ from normal donors. Erythroid-differentiation was performed in CD34+ bone marrow cells from 4 normal donors and 4 MDS patients. IRS2 gene expression was significantly decreased in primary MDS cells compared with normal cells (0.74 [4.06-0.15] vs. 4.71 [11.78-0.66], P<0.0001). Western blot analysis corroborated these findings. According to FAB and WHO classifications, real time RT-PCR demonstrated a significantly lower expression of IRS2 in high-risk MDS samples when compared with low-risk: FAB, 0.34 [0.15–1.56] vs. 1.05 [0.19–4.06], P=0.0172; WHO, 0.30 [0.15–1.44] vs. 1.00 [0.19–4.06], P=0.0204. Based on the IPSS classification and cytogenetic risk group, the expression levels of IRS2 were similar between the low and high-risk groups. During erythroid differentiation, we evaluated IRS2 gene expression of CD34+ cells on days 6, 8 and 12 of culture. On day 12 of normal CD34+ erythroid differentiation, there was an 8.25-fold increased in IRS2 expression, compared to day 6. Interesting, MDS CD34+ cells showed a lower increment in IRS2 transcripts at the same time point (3.89-fold increase only). In conclusion, the down regulation of IRS2 in primary MDS cells and the lower increase in its expression during MDS erythroid differentiation, suggest a role of IRS2 to maintain the effective hematopoiesis. Moreover, IRS2 lower expression in high-risk group, suggests that IRS2 plays a role in the MDS pathophysiology and disease progression. Supported by FAPESP and CNPq. Disclosures:No relevant conflicts of interest to declare.