Contrasting effects of IRS-1 versus IRS-2 gene disruption on carbohydrate and lipid metabolism in vivo

To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1(-/-)) or IRS-2 (IRS-2(-/-)) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1(-/-), and IRS-2(-/-)) with [3-(3)H]glucose and [(2)H(5)]glycerol and assessed rates of glucose and glycerol turnover under basal (0-90 min) and hyperinsulinemic-euglycemic clamp (90-210 min; 5 mm glucose, and 5 milliunits of insulin.kg(-)(1).min(-)(1)) conditions. Both IRS-1(-)(/-) and IRS-2(-)(/-) mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1(-)(/-) mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2(-)(/-) mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2(-)(/-) mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.     

Identification of an intracellular pool of glucose transporters from basal and insulin-stimulated rat skeletal muscle

The purpose of this study was to simultaneously isolate skeletal muscle plasma and microsomal membranes from the hind limbs of male Sprague-Dawley rats perfused either in the absence or presence of 20 milliunits/ml insulin and to determine the effect of insulin on the number and distribution of glucose transporters in these membrane fractions. Insulin increased hind limb glucose uptake greater than 3-fold (2.4 +/- 0.7 versus 9.2 +/- 1.0 mumol/g x h, p less than 0.001). Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased 2-fold (9.1 +/- 1.0 to 20.4 +/- 3.1 pmol/mg protein, p less than 0.005) in insulin-stimulated muscle while microsomal membrane transporters decreased significantly (14.8 +/- 1.6 to 9.8 +/- 1.4 pmol/mg protein, p less than 0.05). No change in the dissociation constant (Kd approximately 120 nm) was observed. K+-stimulated-p-nitrophenol phosphatase, 5'-nucleotidase, and galactosyltransferase specific activity, enrichment, and recovery in the plasma and microsomal membrane fractions were not altered by insulin treatment. Western blot analysis using the monoclonal antibody mAb 1F8 (specific for the insulin-regulatable glucose transporter) demonstrated increased glucose transporter densities in plasma membranes from insulin-treated hind limb skeletal muscle compared with untreated tissues, while microsomal membranes from the insulin-treated hind limb skeletal muscle had a concomitant decrease in transporter density. We conclude that the increase in plasma membrane glucose transporters explains, at least in part, the increase in glucose uptake associated with insulin stimulation of hind limb skeletal muscle. Our data further suggest that these recruited transporters originate from an intracellular microsomal pool, consistent with the translocation hypothesis.     

Peripheral insulin insensitivity in the hyperglycemic athymic nude mouse: similarity to noninsulin-dependent diabetes mellitus

Previous studies in the homozygous athymic nude mouse (USC colony) have indicated a diabetic state characterized by spontaneous hyperglycemia, abnormal glucose tolerance, and normal or relatively decreased plasma insulin levels. Pancreatic islet cell population assessed by morphometric and immunohistochemical studies demonstrated normal insulin-secreting cells in the hyperglycemic nude mouse. To further elucidate the pathogenesis of the hyperglycemic state in the athymic nude mouse, we have studied the effects of insulin on the transport of glucose in skeletal muscle by measuring basal and insulin-stimulated uptake of a nonmetabolizable glucose analogue, 2-deoxy-D-glucose by using the perfused hindquarter preparation. Although basal 2-deoxy-D-glucose uptake by peripheral skeletal muscle was similar in the hyperglycemic and control mice, the insulin-stimulated uptake of 2-deoxy-D-glucose was significantly decreased in the athymic nude mouse at both 0.1 milliunits/ml and supraphysiologic concentrations of insulin (1 milliunit/ml) when compared with control mice (P less than 0.05 and P less than 0.001, respectively). This form of peripheral insulin insensitivity with normal pancreatic beta cell reserve, in addition to the lean body mass of the diabetic animal, mimics in part the peripheral insulin insensitivity seen in non-insulin-dependent diabetes mellitus.     

Lack of REDD1 reduces whole body glucose and insulin tolerance,and impairs skeletal muscle insulin signaling

A lack of the REDD1 promotes dysregulated growth signaling, though little has been established with respect to the metabolic role of REDD1. Therefore, the goal of this study was to determine the role of REDD1 on glucose and insulin tolerance, as well as insulin stimulated growth signaling pathway activation in skeletal muscle. First, intraperitoneal (IP) injection of glucose or insulin were administered to REDD1 wildtype (WT) versus knockout (KO) mice to examine changes in blood glucose over time. Next, alterations in skeletal muscle insulin (IRS-1, Akt, ERK 1/2) and growth (4E-BP1, S6K1, REDD1) signaling intermediates were determined before and after IP insulin treatment (10 min). REDD1 KO mice were both glucose and insulin intolerant when compared to WT mice, evident by higher circulating blood glucose concentrations and a greater area under the curve following IP injections of glucose or insulin. While the REDD1 KO exhibited significant though blunted insulin-stimulated increases (p < 0.05) in Akt S473 and T308 phosphorylation versus the WT mice, acute insulin treatment has no effect (p < 0.05) on REDD1 KO skeletal muscle 4E-BP1 T37/46, S6K1 T389, IRS-1 Y1222, and ERK 1/2 T202/Y204 phosphorylation versus the WT mice. Collectively, these novel data suggest that REDD1 has a more distinct role in whole body and skeletal muscle metabolism and insulin action than previously thought.     

Time-dependent effects of fatty acids on skeletal muscle metabolism

Increased plasma levels of free fatty acids (FFA) occur in states of insulin resistance such as type 2 diabetes mellitus, obesity, and metabolic syndrome. These high levels of plasma FFA seem to play an important role for the development of insulin resistance but the mechanisms involved are not known. We demonstrated that acute exposure to FFA (1 h) in rat incubated skeletal muscle leads to an increase in the insulin-stimulated glycogen synthesis and glucose oxidation. In conditions of prolonged exposure to FFA, however, the insulin-stimulated glucose uptake and metabolism is impaired in skeletal muscle. In this review, we discuss the differences between the effects of acute and prolonged exposure to FFA on skeletal muscle glucose metabolism and the possible mechanisms involved in the FFA-induced insulin resistance.     

Insulin receptor substrate-2 is not necessary for insulin- and exercise-stimulated glucose transport in skeletal muscle.

Insulin receptor substrate-2-deficient (IRS2(-/-)) mice develop type 2 diabetes. The purpose of this study was to determine whether there is a defect in basal, insulin-, and exercise-stimulated glucose transport in the skeletal muscle of these animals. IRS2(-/-) and wild-type (WT) mice (male, 8-10 weeks) exercised on a treadmill for 1 h or remained sedentary. 2-Deoxyglucose (2DG) uptake was measured in isolated soleus muscles incubated in vitro in the presence or absence of insulin. Resting blood glucose concentration in IRS2(-/-) mice (10.3 mM) was higher than WT animals (4.1 mM), but there was a wide range among the IRS2(-/-) mice (3-25 mM). Therefore, IRS2(-/-) mice were divided into two subgroups based on blood glucose concentrations (IRS2(-/-)L < 7.2 mM, IRS2(-/-)H > 7.2 mM). Only IRS2(-/-)H had lower basal, exercise-, and submaximally insulin-stimulated 2DG uptake, while maximal insulin-stimulated 2DG uptake was similar among the three groups. The ED(50) for insulin to stimulate 2DG uptake above basal in IRS2(-/-)H was higher than WT and IRS2(-/-)L mice, suggesting insulin resistance in the skeletal muscle from the IRS2(-/-) mice with high blood glucose concentrations. Furthermore, resting blood glucose concentrations from all groups were negatively correlated to submaximally insulin-stimulated 2DG uptake (r(2) = 0.33, p < 0.01). Muscle GLUT4 content was significantly lower in IRS2(-/-)H mice compared with WT and IRS2(-/-)L mice. These results demonstrate that the IRS2 protein in muscle is not necessary for insulin- or exercise-stimulated glucose transport, suggesting that the onset of diabetes in the IRS2(-/-) mice is not due to a defect in skeletal muscle glucose transport; hyperglycemia may cause insulin resistance in the muscle of IRS2(-/-) mice.     

Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling

Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding insulin resistance and whether lactate-induced insulin resistance is accompanied by altered insulin signaling and/or insulin-stimulated glucose transport in skeletal muscle. Hyperinsulinemic euglycemic clamps were conducted for 6 h in conscious, overnight-fasted rats with or without lactate infusion (120 micromol x kg(-1) x min(-1)) during the final 3.5 h. Lactate infusion increased plasma lactate levels about fourfold. The elevation of plasma lactate had rapid effects to suppress insulin-stimulated glycolysis, which clearly preceded its effect to decrease insulin-stimulated glucose uptake. Both submaximal and maximal insulin-stimulated glucose transport decreased 25-30% (P < 0.05) in soleus but not in epitrochlearis muscles of lactate-infused rats. Lactate infusion did not alter insulin's ability to phosphorylate the insulin receptor, the insulin receptor substrate (IRS)-1, or IRS-2 but decreased insulin's ability to stimulate IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activities and Akt/protein kinase B activity by 47, 75, and 55%, respectively (P < 0.05 for all). In conclusion, elevation of plasma lactate suppressed glycolysis before its effect on insulin-stimulated glucose uptake, consistent with the hypothesis that suppression of glucose metabolism could precede and cause insulin resistance. In addition, lactate-induced insulin resistance was associated with impaired insulin signaling and decreased insulin-stimulated glucose transport in skeletal muscle.     

Transgenic overexpression of hexokinase II in skeletal muscle does not increase glucose disposal in wild-type or Glut1-overexpressing mice

Glut1 transgenic mice were bred with transgenic mice that overexpress hexokinase II in skeletal muscle in order to determine whether whole-body glucose disposal could be further augmented in mice overexpressing glucose transporters. Overexpression of hexokinase alone in skeletal muscle had no effect on glucose transport or metabolism in isolated muscles, nor did it alter blood glucose levels or the rate of whole-body glucose disposal. Expression of the hexokinase transgene in the context of the Glut1 transgenic background did not alter glucose transport in isolated muscles but did cause additional increases in steady-state glucose 6-phosphate (3.2-fold) and glycogen (7.5-fold) levels compared with muscles that overexpress the Glut1 transporter alone. Surprisingly, however, these increases were not accompanied by a change in basal or insulin-stimulated whole-body glucose disposal in the doubly transgenic mice compared with Glut1 transgenic mice, probably due to an inhibition of de novo glycogen synthesis as a result of the high levels of steady-state glycogen in the muscles of doubly transgenic mice (430 micromol/g versus 10 micromol/g in wild-type mice). We conclude that the hexokinase gene may not be a good target for therapies designed to counteract insulin resistance or hyperglycemia.     

Endothelium-dependent delivery of insulin to muscle interstitium

Insulin contributes to skeletal muscle glucose uptake by increasing blood flow and recruiting perfused capillaries. In this issue of Cell Metabolism, Kubota et al. (2011) show that deletion of IRS-2 in endothelial cells in mice causes impaired transcapillary insulin transport, decreased insulin-stimulated glucose uptake in muscle, and mild glucose intolerance.     

Increased glucose disposal and AMP-dependent kinase signaling in a mouse model of hemochromatosis

Hereditary hemochromatosis is an inherited disorder of increased iron absorption that can result in cirrhosis, diabetes, and other morbidities. We have investigated the mechanisms underlying supranormal glucose tolerance despite decreased insulin secretion in a mouse model of hemochromatosis with deletion of the hemochromatosis gene (Hfe(-/-)). Hfe(-/-) mice on 129Sv or C57BL/6J backgrounds have decreased glucose excursions after challenge compared with controls. In the C57BL/6J/ Hfe(-/-), for example, incremental area under the glucose curve is reduced 52% (p < 0.001) despite decreased serum insulin, and homeostasis model assessment insulin resistance is decreased 50% (p < 0.05). When studied by the euglycemic clamp technique 129Sv/Hfe(-/-) mice exhibit a 20% increase in glucose disposal (p < 0.05) at submaximal insulin but no increase at maximal insulin compared with wild types. [1,2-(13)C]D-glucose clearance from plasma is significantly increased in Hfe(-/-) mice (19%, p < 0.05), and lactate derived from glycolysis is elevated 5.1-fold in Hfe(-/-) mice (p < 0.0001). Basal but not insulin-stimulated glucose uptake is elevated in isolated soleus muscle from Hfe(-/-) mice (p < 0.03). Compared with controls Hfe(-/-) mice exhibit no differences in serum lipid, insulin, glucagon, or thyroid hormone levels; adiponectin levels are elevated 41% (p < 0.05), and the adiponectin message in adipocytes is increased 83% (p = 0.04). Insulin action measured by phosphorylation of Akt is not enhanced in muscle, but phosphorylation of AMP-dependent kinase is increased. We conclude that supranormal glucose tolerance in iron overload is characterized by increased glucose disposal that does not result from increased insulin action. Instead, the Hfe(-/-) mice demonstrate increased adiponectin levels and activation of AMP-dependent kinase.     

Discordant effects of glucosamine on insulin-stimulated glucose metabolism and phosphatidylinositol 3-kinase activity.

The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to GlcN-induced defects in peripheral insulin action. Primed continuous GlcN infusion (750 micromol/kg bolus; 30 micromol/kg.min) in conscious rats limited both maximal stimulation of muscle PI3K by acute insulin (I) (1 unit/kg) bolus (I + GlcN = 1.9-fold versus saline = 3.3-fold above fasting levels; p < 0.01) and chronic activation of PI3K following 3-h euglycemic, hyperinsulinemic (18 milliunits/kg.min) clamp studies (I + GlcN = 1.2-fold versus saline = 2.6-fold stimulation; p < 0.01). To determine the time course of GlcN-induced defects in insulin-stimulated PI3K activity and peripheral insulin action, GlcN was administered for 30, 60, 90, or 120 min during 2-h euglycemic, hyperinsulinemic clamp studies. Activation of muscle PI3K by insulin was attenuated following only 30 min of GlcN infusion (GlcN 30 min = 1.5-fold versus saline = 2.5-fold stimulation; p < 0.05). In contrast, the first impairment in insulin-mediated glucose uptake (Rd) developed following 110 min of GlcN infusion (110 min = 39.9 +/- 1.8 versus 30 min = 42.8 +/- 1.4 mg/kg.min, p < 0.05). However, the ability of insulin to stimulate phosphatidylinositol 3,4, 5-trisphosphate production and to activate glycogen synthase in skeletal muscle was preserved following up to 180 min of GlcN infusion. Thus, increased GlcN availability induced (a) profound and early inhibition of proximal insulin signaling at the level of PI3K and (b) delayed effects on insulin-mediated glucose uptake, yet (c) complete sparing of insulin-mediated glycogen synthase activation. The pattern and time sequence of GlcN-induced defects suggest that the etiology of peripheral insulin resistance may be distinct from the rapid and marked impairment in insulin signaling.     

Phosphoenolpyruvate carboxykinase overexpression selectively attenuates insulin signaling and hepatic insulin sensitivity in transgenic mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.     

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.     

Discordant effects of a chronic physiological increase in plasma FFA on insulin signaling in healthy subjects with or without a family history of type 2 diabetes

Muscle insulin resistance develops when plasma free fatty acids (FFAs) are acutely increased to supraphysiological levels (approximately 1,500-4,000 micromol/l). However, plasma FFA levels >1,000 micromol/l are rarely observed in humans under usual living conditions, and it is unknown whether insulin action may be impaired during a sustained but physiological FFA increase to levels seen in obesity and type 2 diabetes mellitus (T2DM) (approximately 600-800 micromol/l). It is also unclear whether normal glucose-tolerant subjects with a strong family history of T2DM (FH+) would respond to a low-dose lipid infusion as individuals without any family history of T2DM (CON). To examine these questions, we studied 7 FH+ and 10 CON subjects in whom we infused saline (SAL) or low-dose Liposyn (LIP) for 4 days. On day 4, a euglycemic insulin clamp with [3-3H]glucose and indirect calorimetry was performed to assess glucose turnover, combined with vastus lateralis muscle biopsies to examine insulin signaling. LIP increased plasma FFA approximately 1.5-fold, to levels seen in T2DM. Compared with CON, FH+ were markedly insulin resistant and had severely impaired insulin signaling in response to insulin stimulation. LIP in CON reduced insulin-stimulated glucose disposal (Rd) by 25%, insulin-stimulated insulin receptor tyrosine phosphorylation by 17%, phosphatidylinositol 3-kinase activity associated with insulin receptor substrate-1 by 20%, and insulin-stimulated glycogen synthase fractional velocity over baseline (44 vs. 15%; all P < 0.05). In contrast to CON, a physiological elevation in plasma FFA in FH+ led to no further deterioration in Rd or to any additional impairment of insulin signaling. In conclusion, a 4-day physiological increase in plasma FFA to levels seen in obesity and T2DM impairs insulin action/insulin signaling in CON but does not worsen insulin resistance in FH+. Whether this lack of additional deterioration in insulin signaling in FH+ is due to already well-established lipotoxicity, or to other molecular mechanisms related to insulin resistance that are nearly maximally expressed early in life, remains to be determined.     

Knockdown of NYGGF4 increases glucose transport in C2C12 mice skeletal myocytes by activation IRS-1/PI3K/AKT insulin pathway

NYGGF4, an obesity-related gene, is proposed to be involved in the development of insulin resistance. Skeletal muscle is a primary target organ for insulin and NYGGF4 showed a relatively high expression level in skeletal muscle. Therefore, this study aimed to explore the effect of NYGGF4 on insulin sensitivity of skeletal muscle cells. RNA interference (RNAi) was adopted to silence NYGGF4 expression in mice C2C12 skeletal myocytes. A remarkably increased insulin-stimulated glucose uptake and GLUT4 translocation was observed in NYGGF4 silencing C2C12 cells. Importantly, the enhanced glucose uptake induced by NYGGF4 silencing could be abrogated by the PI3K inhibitor LY294002. In addition, the crucial molecules involved in PI3K insulin signaling pathway were detected by western blotting. The results showed that NYGGF4 knockdown dramatically activate the insulin-stimulated phosphorylation of IRS-1 and AKT. Taken together, these data demonstrate that NYGGF4 knockdown increases glucose transport in myocytes by activation of the IRS-1/PI3K/AKT insulin pathway.     

Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle

Inducible nitric oxide synthase (iNOS) is induced by inflammatory cytokines in skeletal muscle and fat. It has been proposed that chronic iNOS induction may cause muscle insulin resistance. Here we show that iNOS expression is increased in muscle and fat of genetic and dietary models of obesity. Moreover, mice in which the gene encoding iNOS was disrupted (Nos2-/- mice) are protected from high-fat-induced insulin resistance. Whereas both wild-type and Nos2-/- mice developed obesity on the high-fat diet, obese Nos2-/- mice exhibited improved glucose tolerance, normal insulin sensitivity in vivo and normal insulin-stimulated glucose uptake in muscles. iNOS induction in obese wild-type mice was associated with impairments in phosphatidylinositol 3-kinase and Akt activation by insulin in muscle. These defects were fully prevented in obese Nos2-/- mice. These findings provide genetic evidence that iNOS is involved in the development of muscle insulin resistance in diet-induced obesity.     

p50alpha/p55alpha phosphoinositide 3-kinase knockout mice exhibit enhanced insulin sensitivity

Class Ia phosphoinositide (PI) 3-kinases are heterodimers composed of a regulatory and a catalytic subunit and are essential for the metabolic actions of insulin. In addition to p85alpha and p85beta, insulin-sensitive tissues such as fat, muscle, and liver express the splice variants of the pik3r1 gene, p50alpha and p55alpha. To define the role of these variants, we have created mice with a deletion of p50alpha and p55alpha by using homologous recombination. These mice are viable, grow normally, and maintain normal blood glucose levels but have lower fasting insulin levels. Results of an insulin tolerance test indicate that p50alpha/p55alpha knockout mice have enhanced insulin sensitivity in vivo, and there is an increase in insulin-stimulated glucose transport in isolated extensor digitorum longus muscle tissues and adipocytes. In muscle, loss of p50alpha/p55alpha results in reduced levels of insulin-stimulated insulin receptor substrate 1 (IRS-1) and phosphotyrosine-associated PI 3-kinase but enhanced levels of IRS-2-associated PI 3-kinase and Akt activation, whereas in adipocytes levels of both insulin-stimulated PI 3-kinase and Akt are unchanged. Despite this, adipocytes of the knockout mice are smaller and have increased glucose uptake with altered glucose metabolic pathways. When treated with gold thioglucose, p50alpha/p55alpha knockout mice become hyperphagic like their wild-type littermates. However, they accumulate less fat and become mildly less hyperglycemic and markedly less hyperinsulinemic. Taken together, these data indicate that p50alpha and p55alpha play an important role in insulin signaling and action, especially in lipid and glucose metabolism.     

Deletion of insulin-degrading enzyme elicits antipodal, age-dependent effects on glucose and insulin tolerance

Background

Insulin-degrading enzyme (IDE) is widely recognized as the principal protease responsible for the clearance and inactivation of insulin, but its role in glycemic control in vivo is poorly understood. We present here the first longitudinal characterization, to our knowledge, of glucose regulation in mice with pancellular deletion of the IDE gene (IDE-KO mice).

Methodology

IDE-KO mice and wild-type (WT) littermates were characterized at 2, 4, and 6 months of age in terms of body weight, basal glucose and insulin levels, and insulin and glucose tolerance. Consistent with a functional role for IDE in insulin clearance, fasting serum insulin levels in IDE-KO mice were found to be ∼3-fold higher than those in wild-type (WT) controls at all ages examined. In agreement with previous observations, 6-mo-old IDE-KO mice exhibited a severe diabetic phenotype characterized by increased body weight and pronounced glucose and insulin intolerance. In marked contrast, 2-mo-old IDE-KO mice exhibited multiple signs of improved glycemic control, including lower fasting glucose levels, lower body mass, and modestly enhanced insulin and glucose tolerance relative to WT controls. Biochemically, the emergence of the diabetic phenotype in IDE-KO mice correlated with age-dependent reductions in insulin receptor (IR) levels in muscle, adipose, and liver tissue. Primary adipocytes harvested from 6-mo-old IDE-KO mice also showed functional impairments in insulin-stimulated glucose uptake.

Conclusions

Our results indicate that the diabetic phenotype in IDE-KO mice is not a primary consequence of IDE deficiency, but is instead an emergent compensatory response to chronic hyperinsulinemia resulting from complete deletion of IDE in all tissues throughout life. Significantly, our findings provide new evidence to support the idea that partial and/or transient inhibition of IDE may constitute a valid approach to the treatment of diabetes.