Multiple abnormalities of myocardial insulin signaling in a porcine model of diet-induced obesity.

Heightened cardiovascular risk among patients with systemic insulin resistance is not fully explained by the extent of atherosclerosis. It is unknown whether myocardial insulin resistance accompanies systemic insulin resistance and contributes to increased cardiovascular risk. This study utilized a porcine model of diet-induced obesity to determine if myocardial insulin resistance develops in parallel with systemic insulin resistance and investigated potential mechanisms for such changes. Micropigs (n = 16) were assigned to control (low fat, no added sugars) or intervention (25% wt/wt coconut oil and 20% high-fructose corn syrup) diet for 7 mo. Intervention diet resulted in obesity, hypertension, and dyslipidemia. Systemic insulin resistance was manifest by elevated fasting glucose and insulin, abnormal response to intravenous glucose tolerance testing, and blunted skeletal muscle phosphatidylinositol-3-kinase (PI 3-kinase) activation and protein kinase B (Akt) phosphorylation in response to insulin. In myocardium, insulin-stimulated glucose uptake, PI 3-kinase activation, and Akt phosphorylation were also blunted in the intervention diet group. These findings were explained by increased myocardial content of p85alpha (regulatory subunit of PI 3-kinase), diminished association of PI 3-kinase with insulin receptor substrate (IRS)-1 in response to insulin, and increased serine-307 phosphorylation of IRS-1. Thus, in a porcine model of diet-induced obesity that recapitulates many characteristics of insulin-resistant patients, myocardial insulin resistance develops along with systemic insulin resistance and is associated with multiple abnormalities of insulin signaling.

Pioglitazone acutely stimulates adiponectin secretion from mouse and human adipocytes via activation of the phosphatidylinositol 3'-kinase.

AIMS: Thiazolidinediones increase circulating adiponectin. We have previously demonstrated the involvement of the phosphatidylinositol 3-kinase (PI3K) signaling pathway in insulin-stimulated adiponectin secretion. We therefore investigated the effects of the thiazolidinedione pioglitazone on acute adiponectin secretion, and the involvement of the PI3K signaling pathway in this action. MAIN METHODS: We treated murine 3T3-L1 and human primary adipocytes with 1-10 uM pioglitazone for 2 h, +/-PI3K inhibition by Wortmannin (WT). Secreted adiponectin was measured by Western blot. PI3K activity following 15-minute treatments with 1-10 uM pioglitazone was measured by thin layer chromatography. Pioglitazone's effect on adiponectin synthesis and on secretion of newly synthesized adiponectin was studied in 3T3-L1 adipocytes using a pulse-chase technique. KEY FINDINGS: Pioglitazone was found to increase adiponectin secretion and PI3K activity in a dose-dependent manner from 3T3-L1 and human adipocytes. In 3T3-L1 adipocytes, 10 uM pioglitazone increased adiponectin secretion by 84+/-14% (p<0.0001) at 2 h. Similarly, in human adipocytes there was a 56+/-18% (p<0.02) increase in secretion. WT blocked the pioglitazone effect and decreased adiponectin secretion at 2 h (47% of pioglitazone treated, p<0.006). Pioglitazone increased PI3K activity in a dose-dependent manner in both 3T3-L1 (1.7 vs. 2.7-fold increase over control at 2 uM vs. 10 uM dose, p=0.02) and human adipocytes. SIGNIFICANCE: Our data show that pioglitazone acutely stimulates adiponectin secretion from both 3T3-L1 and human adipocytes. This acute effect of pioglitazone is PI3K-dependent.

Enhanced insulin signaling via Shc in human breast cancer.

Insulin is a mild mitogen and has been shown to potentiate mitogenic influence of other growth factors. Because hyperinsulinemia and/or overexpression of insulin receptors have been linked to development, progression, and outcome of breast cancer, we attempted to evaluate the mechanism of these associations. We have compared the expression of insulin receptors and the magnitude of insulin signaling in breast tumors and adjacent normal mammary tissue samples obtained from 20 patients. We observed that insulin binding more than doubled in the tumors as compared with the normal tissue (P <.01 by paired t test). Insulin signaling to Shc, judged by the magnitude of its phosphorylation, was also significantly enhanced in the tumors. In contrast, the phosphorylation of the insulin-receptor substrate-1 (IRS-1), Akt, and mitogen-activated protein (MAP) kinase were identical in the tumorous and normal mammary tissues. Finally, tumors displayed significantly increased amounts of farnesylated p21 Ras and geranylgeranylated Rho-A (P <.01), consistent with Shc-dependent activation of farnesyl (FTase) and geranylgeranyl transferases (GGTase) in the tumor tissue. We conclude that the mechanism of the mitogenic influence of insulin in breast cancer may include increased expression of insulin receptors, preferential hyperphosphorylation of Shc, and increased amounts of prenylated p21 Ras and Rho-A in tumor tissue as compared with adjacent normal mammary tissue.

Acute effects of different diet compositions on skeletal muscle insulin signalling in obese individuals during caloric restriction.

OBJECTIVE: The cellular effects of restricting fat versus carbohydrate during a low-calorie diet are unclear. The aim of this study was to examine acute effects of energy and macronutrient restriction on skeletal muscle insulin signalling in obesity. MATERIALS/METHODS: Eighteen obese individuals without diabetes underwent euglycemic-hyperinsulinemic clamp and skeletal muscle biopsy after: (a) 5days of eucaloric diet (30% fat, 50% carbohydrate), and (b) 5days of a 30% calorie-restricted diet, either low fat/high carbohydrate (LF/HC: 20% fat, 60% carbohydrate) or high-fat/low carbohydrate (HF/LC: 50% fat, 30% carbohydrate). RESULTS: Weight, body composition, and insulin sensitivity were similar between groups after eucaloric diet. Weight loss was similar between groups after hypocaloric diet, 1.3±1.3kg (p<0.0001 compared with eucaloric). Whole-body insulin sensitivity was unchanged after calorie restriction and similar between groups. However, ex vivo skeletal muscle insulin signalling differed depending on macronutrient composition of calorie-restricted diet. Skeletal muscle of the LF/HC group had increased insulin-stimulated tyrosine phosphorylation of IRS-1, decreased insulin-stimulated Ser307 phosphorylation of IRS-1, and increased IRS-1-associated phosphatidylinositol (PI)3-kinase activity. Conversely, insulin stimulation of tyrosine phosphorylated IRS-1 was absent and serine 307 phosphorylation of IRS-1 was increased on HF/LC, with blunting of IRS-1-associated PI3-kinase activity. CONCLUSION: Acute caloric restriction with an LF/HC diet alters skeletal muscle insulin signalling in a way that improves insulin sensitivity, while acute caloric restriction with an HF/LC diet induces changes compatible with insulin resistance. In both cases, ex vivo changes in skeletal muscle insulin signalling appear prior to changes in whole body insulin sensitivity.

Arterial insulin resistance in Yucatan micropigs with diet-induced obesity and metabolic syndrome.

AIM: Metabolic syndrome affects a large proportion of the population and increases cardiovascular disease risk. Because metabolic syndrome often co-exists clinically with atherosclerosis, it is difficult to distinguish the respective contributions of the components to vascular abnormalities. Accordingly, we utilized a porcine dietary model of metabolic syndrome without atherosclerosis to investigate early abnormalities of vascular function and signaling. METHODS: Thirty-two Yucatan micropigs were fed either a high-fat, high-simple-sugar, high-calorie (HFHS) or standard chow diet (STD) for 6 months. Neither diet contained added cholesterol. Blood pressure and flow-mediated vasodilatation were assessed at baseline and 6 months. Aortas were harvested at 6 months to assess histology, insulin signaling, and endothelial nitric oxide (eNOS) phosphorylation. RESULTS: HFHS pigs developed characteristics of metabolic syndrome including obesity, dyslipidemia, and insulin resistance, but without histologic evidence of atherosclerosis. Although arterial intima-media thickness did not differ between groups, vascular dysfunction in HFHS was manifest by increased blood pressure and impaired flow-mediated vasodilation of the femoral artery. Compared with STD, aortas from HFHS exhibited increased p85α expression and Ser307 IRS-1 phosphorylation, and blunted insulin-stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase activity. In the absence of insulin stimulation, aortic Akt Ser473-phosphorylation was greater in HFHS than in STD. With insulin stimulation, Akt phosphorylation increased in STD, but not HFHS. Insulin-induced Ser1177-phosphorylation of eNOS was decreased in HFHS, compared with STD. CONCLUSIONS: Pigs with metabolic syndrome develop early vascular dysfunction and aortic insulin signaling abnormalities, and could be a useful model for early human vascular abnormalities in this condition.

Rescuing 3T3-L1 adipocytes from insulin resistance induced by stimulation of Akt-mammalian target of rapamycin/p70 S6 kinase (S6K1) pathway and serine phosphorylation of insulin receptor substrate-1: effect of reduced expression of p85alpha subunit of phosphatidylinositol 3-kinase and S6K1 kinase.

Phosphorylation of insulin receptor substrate-1 (IRS-1) on serine residues has been recognized as a mechanism responsible for a diminution of insulin action and insulin resistance. Potential approaches to improve insulin sensitivity may include interference with and/or reduction in expression of certain signaling intermediates that participate in the pathogenesis of insulin resistance. In this study, we transduced fully differentiated 3T3-L1 adipocytes with a constitutively active myristoylated Akt that led to hyperactivation of mammalian target of rapamycin and p70 S6 kinase (S6K1), increased serine phosphorylation of IRS-1, and reduction in insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and glucose transport. We then reduced expression of the PI 3-kinase regulatory subunit, p85alpha, or expression of S6K1 kinase using small interfering RNA transfections, which led to a reduction in p85alpha expression of 70% at 48 h (P < 0.05) and S6K1 of 49% (P < 0.05). Reduction in expression of either p85alpha or S6K1 achieved with small interfering RNA in the presence of myristoylated Akt rescued 3T3-L1 adipocytes from the insulin resistance induced by serine phosphorylation of IRS-1 and completely restored insulin-stimulated activation of PI 3-kinase and glucose uptake. We conclude that reduction in expression of p85alpha or S6K1 could represent therapeutic targets to mitigate insulin resistance.

Early responses of insulin signaling to high-carbohydrate and high-fat overfeeding.

BACKGROUND: Early molecular changes of nutritionally-induced insulin resistance are still enigmatic. It is also unclear if acute overnutrition alone can alter insulin signaling in humans or if the macronutrient composition of the diet can modulate such effects. METHODS: To investigate the molecular correlates of metabolic adaptation to either high-carbohydrate (HC) or high-fat (HF) overfeeding, we conducted overfeeding studies in 21 healthy lean (BMI < 25) individuals (10 women, 11 men), age 20-45, with normal glucose metabolism and no family history of diabetes. Subjects were studied first following a 5-day eucaloric (EC) diet (30% fat, 50% CHO, 20% protein) and then in a counter balanced manner after 5 days of 40% overfeeding of both a HC (20% fat, 60% CHO) diet and a HF (50% fat, 30% CHO) diet. At the end of each diet phase, in vivo insulin sensitivity was assessed using the hyperinsulinemic-euglycemic clamp technique. Ex vivo insulin action was measured from skeletal muscle tissue samples obtained 15 minutes after insulin infusion was initiated. RESULTS: Overall there was no change in whole-body insulin sensitivity as measured by glucose disposal rate (GDR, EC: 12.1 ± 4.7; HC: 10.9 ± 2.7; HF: 10.8 ± 3.4). Assessment of skeletal muscle insulin signaling demonstrated increased tyrosine phosphorylation of IRS-1 (p < 0.001) and increased IRS-1-associated phosphatidylinositol 3 (PI 3)-kinase activity (p < 0.001) following HC overfeeding. In contrast, HF overfeeding increased skeletal muscle serine phosophorylation of IRS-1 (p < 0.001) and increased total expression of p85α (P < 0.001). CONCLUSION: We conclude that acute bouts of overnutrition lead to changes at the cellular level before whole-body insulin sensitivity is altered. On a signaling level, HC overfeeding resulted in changes compatible with increased insulin sensitivity. In contrast, molecular changes in HF overfeeding were compatible with a reduced insulin sensitivity.

Growth hormone regulation of p85alpha expression and phosphoinositide 3-kinase activity in adipose tissue: mechanism for growth hormone-mediated insulin resistance.

Phosphoinositide (PI) 3-kinase is involved in insulin-mediated effects on glucose uptake, lipid deposition, and adiponectin secretion from adipocytes. Genetic disruption of the p85alpha regulatory subunit of PI 3-kinase increases insulin sensitivity, whereas elevated p85alpha levels are associated with insulin resistance through PI 3-kinase-dependent and -independent mechanisms. Adipose tissue plays a critical role in the antagonistic effects of growth hormone (GH) on insulin actions on carbohydrate and lipid metabolism through changes in gene transcription. The objective of this study was to assess the role of the p85alpha subunit of PI 3-kinase and PI 3-kinase signaling in GH-mediated insulin resistance in adipose tissue. To do this, p85alpha mRNA and protein expression and insulin receptor substrate (IRS)-1-associated PI 3-kinase activity were measured in white adipose tissue (WAT) of mice with GH excess, deficiency, and sufficiency. Additional studies using 3T3-F442A cells were conducted to confirm direct effects of GH on free p85alpha protein abundance. We found that p85alpha expression 1) is decreased in WAT from mice with isolated GH deficiency, 2) is increased in WAT from mice with chronic GH excess, 3) is acutely upregulated in WAT from GH-deficient and -sufficient mice after GH administration, and 4) is directly upregulated by GH in 3T3-F442A adipocytes. The insulin-induced increase in PI 3-kinase activity was robust in mice with GH deficiency, but not in mice with GH excess. In conclusion, GH regulates p85alpha expression and PI 3-kinase activity in WAT and provides a potential explanation for 1) the insulin hypersensitivity and associated obesity and hyperadiponectinemia of GH-deficient mice and 2) the insulin resistance and associated reduced fat mass and hypoadiponectinemia of mice with GH excess.

Increased farnesylation of p21-Ras and neonatal macrosomia in women with gestational diabetes.

Six of 22 mothers with gestational diabetes mellitus had infants with macrosomia, cord blood hyperinsulinemia, and increased amounts of a key mitogenic intermediate, farnesylated p21-Ras. The ability of fetal hyperinsulinemia to increase the availability of farnesylated p21-Ras may represent one mechanism of the growth-promoting action of insulin during fetal development.

Increased P85alpha is a potent negative regulator of skeletal muscle insulin signaling and induces in vivo insulin resistance associated with growth hormone excess.

Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85alpha, p85beta, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85alpha is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85alpha-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85alpha, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85alpha in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85alpha or p85beta, respectively (p85alpha (+/-) and p85beta(-/-)). Wild type and p85beta(-/-) mice developed in vivo insulin resistance and demonstrated overexpression of p85alpha and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85alpha(+/-)mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85alpha expression. These findings demonstrated the importance of increased p85alpha in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85alpha as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle.

Insulin regulation of sterol regulatory element-binding protein-1 expression in L-6 muscle cells and 3T3 L1 adipocytes.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate enzymes required for cholesterol and fatty acid synthesis. Expression of SREBP-1 is enhanced by insulin; however, the actual insulin-signaling cascades employed are yet unclear. We determined the roles of the phosphatidylinositol (PI) 3-kinase and mitogen-activated protein (MAP) kinase-dependent pathways in the effect of mediating insulin on SREBP-1 in L-6 skeletal muscle cells and 3T3 L1 adipocytes, using wortmannin or LY294002 to inhibit the PI 3-kinase pathway, and PD98059 to inhibit the MAP kinase-dependent pathway. In myocytes, insulin increased SREBP-1 protein in a dose-dependent manner. 1 and 10 nm insulin significantly increased expression of total cellular SREBP-1 protein at 24 and 48 h, nuclear SREBP-1 protein at 24 h, and SREBP-1a mRNA at 24 h. Although wortmannin and LY294002 had no effect on this aspect of insulin action, PD98059 completely blocked each of these responses. Transfection of a dominant negative mutant of Ras similarly blocked the insulin effect on SREBP-1. In contrast, in adipocytes, the insulin effect on SREBP-1 was mediated via the PI 3-kinase and not the MAP kinase pathway. In conclusion, although insulin increases skeletal muscle SREBP-1 expression in a dose-dependent fashion via the MAP kinase-dependent signaling pathway, insulin action on adipocyte SREBP-1 is mediated via the PI 3-kinase signaling pathway. In the state of insulin resistance, characterized by selective inhibition of the PI 3-kinase pathway, the usual stimulation of lipogenesis by insulin in adipocytes may be inhibited, whereas intramyocellular lipogenesis via the MAP kinase pathway of insulin may continue unabated.