Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin

Muscle and fat cells develop insulin resistance when cultured under hyperinsulinemic conditions for sustained periods. Recent data indicate that early insulin signaling defects do not fully account for the loss of insulin action. Given that cortical filamentous actin (F-actin) represents an essential aspect of insulin regulated glucose transport, we tested to see whether cortical F-actin structure was compromised during chronic insulin treatment. The acute effect of insulin on GLUT4 translocation and glucose uptake was diminished in 3T3-L1 adipocytes exposed to a physiological level of insulin (5 nm) for 12 h. This insulin-induced loss of insulin responsiveness was apparent under both low (5.5 mm) and high (25 mm) glucose concentrations. Microscopic and biochemical analyses revealed that the hyperinsulinemic state caused a marked loss of cortical F-actin. Since recent data link phosphatidylinositol 4,5-bisphosphate (PIP(2)) to actin cytoskeletal mechanics, we tested to see whether the insulin-resistant condition affected PIP(2) and found a noticeable loss of this lipid from the plasma membrane. Using a PIP(2) delivery system, we replenished plasma membrane PIP(2) in cells following the sustained insulin treatment and observed a restoration in cortical F-actin and insulin responsiveness. These data reveal a novel molecular aspect of insulin-induced insulin resistance involving defects in PIP(2)/actin regulation.     

Loss of cortical actin filaments in insulin-resistant skeletal muscle cells impairs GLUT4 vesicle trafficking and glucose transport

Study has demonstrated an essential role of cortical filamentous actin (F-actin) in insulin-regulated glucose uptake by skeletal muscle. Here, we tested whether perturbations in F-actin contributed to impaired insulin responsiveness provoked by hyperinsulinemia. In L6 myotubes stably expressing GLUT4 that carries an exofacial myc-epitope tag, acute insulin stimulation (20 min, 100 nM) increased GLUT4myc translocation and glucose uptake by 2-fold. In contrast, a hyperinsulinemic state, induced by inclusion of 5 nM insulin in the medium for 12 h decreased the ability of insulin to stimulate these processes. Defects in insulin signaling did not readily account for the observed disruption. In contrast, hyperinsulinemia reduced cortical F-actin. This occurred concomitant with a loss of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP₂), a lipid involved in cytoskeletal regulation. Restoration of plasma membrane PIP₂ in hyperinsulinemic cells restored F-actin and insulin responsiveness. Consistent with these in vitro observations suggesting that the hyperinsulinemic state negatively affects cortical F-actin structure, epitrochlearis skeletal muscle from insulin-resistant hyperinsulinemic Zucker fatty rats displayed a similar loss of F-actin structure compared with that in muscle from lean insulin-sensitive littermates. We propose that a component of insulin-induced insulin resistance in skeletal muscle involves defects in PIP₂/F-actin structure essential for insulin-regulated glucose transport. hyperinsulinemia; phosphatidylinositol 4,5-bisphosphate     

Chromium enhances insulin responsiveness via AMPK

Trivalent chromium (Cr³⁺) is known to improve glucose homeostasis. Cr³⁺ has been shown to improve plasma membrane-based aspects of glucose transporter GLUT4 regulation and increase activity of the cellular energy sensor 5’ AMP-activated protein kinase (AMPK). However, the mechanism(s) by which Cr³⁺ improves insulin responsiveness and whether AMPK mediates this action is not known. In this study we tested if Cr³⁺ protected against physiological hyperinsulinemia-induced plasma membrane cholesterol accumulation, cortical filamentous actin (F-actin) loss and insulin resistance in L6 skeletal muscle myotubes. In addition, we performed mechanistic studies to test our hypothesis that AMPK mediates the effects of Cr³⁺ on GLUT4 and glucose transport regulation. Hyperinsulinemia-induced insulin-resistant L6 myotubes displayed excess membrane cholesterol and diminished cortical F-actin essential for effective glucose transport regulation. These membrane and cytoskeletal abnormalities were associated with defects in insulin-stimulated GLUT4 translocation and glucose transport. Supplementing the culture medium with pharmacologically relevant doses of Cr³⁺ in the picolinate form (CrPic) protected against membrane cholesterol accumulation, F-actin loss, GLUT4 dysregulation and glucose transport dysfunction. Insulin signaling was neither impaired by hyperinsulinemic conditions nor enhanced by CrPic, whereas CrPic increased AMPK signaling. Mechanistically, siRNA-mediated depletion of AMPK abolished the protective effects of CrPic against GLUT4 and glucose transport dysregulation. Together these findings suggest that the micronutrient Cr³⁺, via increasing AMPK activity, positively impacts skeletal muscle cell insulin sensitivity and glucose transport regulation.     

Effects of chromium picolinate on glucose uptake in insulin-resistant 3T3-L1 adipocytes involve activation of p38 MAPK

Chromium picolinate (CrPic) has been discovered as a supplemental or alternative medication for type 2 diabetes, but its mechanism of action is not well understood. The purpose of this study was to explore the possible anti-diabetic mechanisms of CrPic in insulin-resistant 3T3-L1 adipocytes; the insulin resistance was induced by treatment with high glucose and insulin for 24 h. The effects of CrPic on glucose metabolism and the glucose uptake-inducing activity of CrPic were investigated. Meanwhile, the effects of CrPic on glucose transporter 4 (GLUT4) translocation were visualized by immonofluorescence microscopy. In addition, its effects on insulin signaling pathways and mitogen-activated protein kinase (MAPK) signaling cascades were assessed by immunoblotting analysis and real-time PCR. The results showed that CrPic induced glucose metabolism and uptake, as well as GLUT4 translocation to plasma membrane (PM) in both control and insulin-resistant 3T3-L1 adipocytes without any changes in insulin receptor β (IR-β), protein kinase B (AKt), c-Cbl, extracellular signal-regulated kinase (ERK), c-Jun phosphorylation and c-Cbl-associated protein (CAP) mRNA levels. Interestingly, CrPic was able to increase the basal and insulin-stimulated levels of p38 MAPK activation in the control and insulin-resistant cells. Pretreatment with the specific p38 MAPK inhibitor SB203580 partially inhibited the CrPic-induced glucose transport, but CrPic-activated translocation of GLUT4 was not inhibited by SB203580. This study provides an experimental evidence of the effects of CrPic on glucose uptake through the activation of p38 MAPK and it is independent of the effect on GLUT4 translocation. The findings also suggest exciting new insights into the role of p38 MAPK in glucose uptake and GLUT4 translocation.     

Insulin and Chromium Picolinate Induce Translocation of CD36 to the Plasma Membrane Through Different Signaling Pathways in 3T3-L1 Adipocytes,and with a Differential Functionality of the CD36

Chromium picolinate (CrPic) has been indicated to activate glucose transporter 4 (GLUT4) trafficking to the plasma membrane (PM) to enhance glucose uptake in 3T3-L1 adipocytes. In skeletal and heart muscle cells, insulin directs the intracellular trafficking of the fatty acid translocase/CD36 to induce the uptake of cellular long-chain fatty acid (LCFA). The current study describes the effects of CrPic and insulin on the translocation of CD36 from intracellular storage pools to the PM in 3T3-L1 adipocytes in comparison with that of GLUT4. Immunofluorescence microscopy and immunoblotting revealed that both CD36 and GLUT4 were expressed and primarily located intracellularly in 3T3-L1 adipocytes. Upon insulin or CrPic stimulation, PM expression of CD36 increased in a similar manner as that for GLUT4; the CrPic-stimulated PM expression was less strong than that of insulin. The increase in PM localization for these two proteins by insulin paralleled LCFA ([1-¹⁴C]palmitate) or [³H]deoxyglucose uptake in 3T3-L1 adipocytes. The induction of the PM expression of GLUT4, but not CD36, or substrate uptake by insulin and CrPic appears to be additive in adipocytes. Furthermore, wortmannin completely inhibited the insulin-stimulated translocation of GLUT4 or CD36 and prevented the increased uptake of glucose or LCFA in these cells. Taken together, for the first time, these findings suggest that both insulin and CrPic induce CD36 translocation to the PM in 3T3-L1 adipocytes and that their translocation-inducing effects are not additive. The signaling pathway inducing the translocations is different, apparently resulting in a differential activity of CD36.     

Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism

Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction.     

Impairment in insulin sensitivity after early androgenization in the post-pubertal female rat

A link is known to exist between hyperandrogenicity and insulin resistance in mammals. We explored whether androgenization, early in reproductive life, in the female rat has any impact on later peripheral insulin sensitivity and parametrial (PM) fat function. Female, 60 day-old, rats were injected (i.m.) with 100 mul of sterile corn oil either alone (CT) or containing 2 mg of testosterone propionate (TP); rats were then used for experimentation at age 120 days. Daily food intake and body weight were recorded. Different groups of CT and TP rats were subjected to a high glucose load test or 24 h fasting for evaluation of changes in circulating levels of several metabolites and body composition. In vitro experiments were run to study the impact of androgenization on isolated PM adipocyte response to insulin. Finally, the direct effect of testosterone on insulin-induced leptin secretion by normal PM adipocytes was also evaluated. Androgenization induced a significant increase in daily food intake and body weight for the first 20 days after treatment. In vivo experiments indicate that TP rats released more (P<0.05) insulin than CT animals after high glucose load in order to maintain similar circulating glucose levels, a characteristic accompanied by decreased (P<0.05) overall corticoadrenal response in TP rats. Several metabolic responses to fasting were similar in both groups, although impaired adrenal response and changes in body composition were observed only in TP rats. Interestingly, cultured PM adipocytes from TP rats were less (P<0.05) sensitive than CT cells to insulin-induced leptin secretion. Also, we found that 48 h exposure of normal PM adipocytes to high testosterone concentration also impaired adipocyte endocrine function. Our study strongly supports that development of insulin resistance, in the female gender, can be established after an early, even transient, hyperandrogenemia.     

Altered subcellular distribution of IRS-1 and IRS-3 is associated with defective Akt activation and GLUT4 translocation in insulin-resistant old rat adipocytes

Insulin receptor signal transduction depends on the precise intracellular localization of signalling molecules. This study examines the compartmentalization and the insulin-induced translocation and tyrosine phosphorylation of insulin receptor substrates (IRS-1 and IRS-3) in epididymal white adipose tissue from adult and insulin-resistant old rats. We found that insulin induces the translocation of IRS-1 from plasma membrane (PM) and light microsomes (LM) to cytosol, whereas IRS-3 translocates from PM to LM and cytosol upon insulin stimulation. Old rat adipocytes are characterized by higher relative levels of IRS proteins, under basal conditions, in those fractions where they are intended to translocate in response to insulin and exhibit a higher phosphotyrosine content of IRS-1 and -3 in basal conditions and a lower maximal phosphorylation in response to insulin. Furthermore, old rat adipocytes are also characterized by a reduced ability of insulin to stimulate both, Akt/PKB activity and translocation of GLUT4 to the PM. We conclude that the lower stimulation of downstream insulin signalling involved in glucose metabolism in old rat adipocytes may be explained, at least in part, by the altered subcellular distribution of IRS-1 and -3 proteins. In addition, our data suggest that the mechanism of turning on/off insulin receptor-mediated signal is impaired with aging.     

Effect of insulin on caveolin-enriched membrane domains in rat liver

Compartmentalization of signaling molecules may explain, at least in part, how insulin or growth factors achieve specificity. Caveolae/rafts are specialized lipid compartments that have been implicated in insulin signaling. In the present study, we investigated the role of caveolin-enriched membrane domains (CMD) in mediating insulin signaling in rat liver. We report the existence of at least two different populations of CMD in rat liver plasma membranes (PM). One population is soluble in Triton X-100 and seems to be constitutively associated with cytoskeletal elements. The other population of CMD is located in a membrane compartment insoluble in Triton X-100 with light buoyant density and is hence designated CMD/rafts. We found evidence of rapid actin reorganization in rat liver PM in response to insulin, along with the association of CMD/rafts and insulin signaling molecules with a cell fraction enriched in cytoskeletal elements. The presence of CMD in liver parenchyma cells was confirmed by the presence of caveolin-1 in primary rat hepatocyte cultures. Cholesterol depletion, effected by incubating hepatocytes with 2 mm methyl-beta-cyclodextrin, did not permeabilize the cells or interfere with clathrin-dependent internalization. However, at this concentration, methyl-beta-cyclodextrin perturbed CMD of hepatocyte PM and inhibited insulin-induced Akt activation and glycogen synthesis but did not affect insulin-induced insulin receptor kinase tyrosine phosphorylation. These events, together with the presence of a functional insulin receptor in CMD of rat liver PM, suggest that insulin signaling is influenced by the interaction of caveolae with cytoskeletal elements in liver.     

Fatty aldehyde dehydrogenase: potential role in oxidative stress protection and regulation of its gene expression by insulin

Phosphatidylinositol 3-kinase signaling regulates the expression of several genes involved in lipid and glucose homeostasis; deregulation of these genes may contribute to insulin resistance and progression toward type 2 diabetes. By employing RNA arbitrarily primed-PCR to search for novel phosphatidylinositol 3-kinase-regulated genes in response to insulin in isolated rat adipocytes, we identified fatty aldehyde dehydrogenase (FALDH), a key component of the detoxification pathway of aldehydes arising from lipid peroxidation events. Among these latter events are oxidative stresses associated with insulin resistance and diabetes. Upon insulin injection, FALDH mRNA expression increased in rat liver and white adipose tissue and was impaired in two models of insulin-resistant mice, db/db and high fat diet mice. FALDH mRNA levels were 4-fold decreased in streptozotocin-treated rats, suggesting that FALDH deregulation occurs both in hyperinsulinemic insulin-resistant state and hypoinsulinemic type 1 diabetes models. Moreover, insulin treatment increases FALDH activity in hepatocytes, and expression of FALDH was augmented during adipocyte differentiation. Considering the detoxifying role of FALDH, its deregulation in insulin-resistant and type 1 diabetic models may contribute to the lipid-derived oxidative stress. To assess the role of FALDH in the detoxification of oxidized lipid species, we evaluated the production of reactive oxygen species in normal versus FALDH-overexpressing adipocytes. Ectopic expression of FALDH significantly decreased reactive oxygen species production in cells treated by 4-hydroxynonenal, the major lipid peroxidation product, suggesting that FALDH protects against oxidative stress associated with lipid peroxidation. Taken together, our observations illustrate the importance of FALDH in insulin action and its deregulation in states associated with altered insulin signaling.     

Mitochondrial dysfunction and insulin resistance from the outside in: extracellular matrix, the cytoskeleton, and mitochondria

Insulin resistance in skeletal muscle is a prominent feature of obesity and type 2 diabetes. The association between mitochondrial changes and insulin resistance is well known. More recently, there is growing evidence of a relationship between inflammation, extracellular remodeling, and insulin resistance. The intent of this review is to propose a potentially novel mechanism for the development of insulin resistance, focusing on the underappreciated connections among inflammation, extracellular remodeling, cytoskeletal interactions, mitochondrial function, and insulin resistance in human skeletal muscle. Several sources of inflammation, including expansion of adipose tissue resulting in increased lipolysis and alterations in pro- and anti-inflammatory cytokines, contribute to the insulin resistance observed in obesity and type 2 diabetes. In the experimental model of lipid oversupply, an inflammatory response in skeletal muscle leads to altered expression extracellular matrix-related genes as well as nuclear encoded mitochondrial genes. A similar pattern also is observed in "naturally" occurring insulin resistance in muscle of obese nondiabetic individuals and patients with type 2 diabetes mellitus. More recently, alterations in proteins (including α-actinin-2, desmin, proteasomes, and chaperones) involved in muscle structure and function have been observed in insulin-resistant muscle. Some of these cytoskeletal proteins are mechanosignal transducers that allow muscle fibers to sense contractile activity and respond appropriately. The ensuing alterations in expression of genes coding for mitochondrial proteins and cytoskeletal proteins may contribute to the mitochondrial changes observed in insulin-resistant muscle. These changes in turn may lead to a reduction in fat oxidation and an increase in intramyocellular lipid, which contributes to the defects in insulin signaling in insulin resistance.     

Disruption of cortical actin in skeletal muscle demonstrates an essential role of the cytoskeleton in glucose transporter 4 translocation in insulin-sensitive tissues

Cell culture work suggests that signaling to polymerize cortical filamentous actin (F-actin) represents a required pathway for the optimal redistribution of the insulin-responsive glucose transporter, GLUT4, to the plasma membrane. Recent in vitro study further suggests that the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) mediates the effect of insulin on the actin filament network. Here we tested whether similar cytoskeletal mechanics are essential for insulin-regulated glucose transport in isolated rat epitrochlearis skeletal muscle. Microscopic analysis revealed that cortical F-actin is markedly diminished in muscle exposed to latrunculin B. Depolymerization of cortical F-actin with latrunculin B caused a time- and concentration-dependent decline in 2-deoxyglucose transport. The loss of cortical F-actin and glucose transport was paralleled by a decline in insulin-stimulated GLUT4 translocation, as assessed by photolabeling of cell surface GLUT4 with Bio-LC-ATB-BMPA. Although latrunculin B impaired insulin-stimulated GLUT4 translocation and glucose transport, activation of phosphatidylinositol 3-kinase and Akt by insulin was not rendered ineffective. In contrast, the ability of insulin to elicit the cortical F-actin localization of N-WASP was abrogated. These data provide the first evidence that actin cytoskeletal mechanics are an essential feature of the glucose transport process in intact skeletal muscle. Furthermore, these findings support a distal actin-based role for N-WASP in insulin action in vivo.     

Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes

Both insulin and the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. However, regulation of the CIDE family by insulin and the contribution of the CIDE family to insulin actions remain unclear. Here, we investigated whether insulin regulates expression of the CIDE family and which subtypes contribute to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. Insulin decreased CIDEA and increased CIDEC but not CIDEB mRNA expression. Starvation-induced apoptosis in adipocytes was significantly inhibited when insulin decreased the CIDEA mRNA level. Small interfering RNA-mediated depletion of CIDEA inhibited starvation-induced apoptosis similarly to insulin and restored insulin deprivation-reduced adipocyte number, whereas CIDEC depletion did not. Lipid droplet size of adipocytes was increased when insulin increased the CIDEC mRNA level. In contrast, insulin-induced enlargement of lipid droplets was markedly abrogated by depletion of CIDEC but not CIDEA. Furthermore, depletion of CIDEC, but not CIDEA, significantly increased glycerol release from adipocytes. These results suggest that CIDEA and CIDEC are novel genes regulated by insulin in human adipocytes and may play key roles in the effects of insulin, such as anti-apoptosis and lipid droplet formation.     

SH2-containing inositol phosphatase 2 predominantly regulates Akt2, and not Akt1, phosphorylation at the plasma membrane in response to insulin in 3T3-L1 adipocytes

SH2-containing inositol phosphatase 2 (SHIP2) is a physiologically important negative regulator of insulin signaling by hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI 3,4,5-trisphosphate in the target tissues of insulin. Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity without affecting biological systems other than insulin signaling. Therefore, we investigated the molecular mechanisms by which SHIP2 specifically regulates insulin-induced metabolic signaling in 3T3-L1 adipocytes. Insulin-induced phosphorylation of Akt, one of the molecules downstream of PI3-kinase, was inhibited by expression of wild-type SHIP2, whereas it was increased by expression of 5'-phosphatase-defective (DeltaIP) SHIP2 in whole cell lysates. The regulatory effect of SHIP2 was mainly seen in the plasma membrane (PM) and low density microsomes but not in the cytosol. In this regard, following insulin stimulation, a proportion of Akt2, and not Akt1, appeared to redistribute from the cytosol to the PM. Thus, insulin-induced phosphorylation of Akt2 at the PM was predominantly regulated by SHIP2, whereas the phosphorylation of Akt1 was only minimally affected. Interestingly, insulin also elicited a subcellular redistribution of both wild-type and DeltaIP-SHIP2 from the cytosol to the PM. The degree of this redistribution was inhibited in part by pretreatment with PI3-kinase inhibitor. Although the expression of a constitutively active form of PI3-kinase myr-p110 also elicited a subcellular redistribution of SHIP2 to the PM, expression of SHIP2 appeared to affect the myr-p110-induced phosphorylation, and not the translocation, of Akt2. Furthermore, insulin-induced phosphorylation of Akt was effectively regulated by SHIP2 in embryonic fibroblasts derived from knockout mice lacking either insulin receptor substrate-1 or insulin receptor substrate-2. These results indicate that insulin specifically stimulates the redistribution of SHIP2 from the cytosol to the PM independent of 5'-phosphatase activity, thereby regulating the insulin-induced translocation and phosphorylation of Akt2 at the PM.     

Defective Akt activation is associated with glucose- but not glucosamine-induced insulin resistance

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high glucose or glucosamine, provided insulin (0.6 nM) is present during preincubation. Insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity is unaffected (30). Total cellular IRS-1, PI 3-kinase, or Akt concentrations were unchanged. Akt activation in subcellular fractions was assessed by immunoblotting with two phospho-Akt-specific antibodies. Upon acute 100 nM insulin stimulation, plasma membrane (PM)-associated phospho-Akt was highest in cells preincubated in low glucose with no insulin, less in high glucose with no insulin, even less in low glucose+insulin, and lowest in high glucose+insulin. Only high glucose+insulin caused insulin-resistant glucose transport. Acute insulin stimulation increased total PM-Akt about twofold after preincubation without insulin in low or high glucose. Preincubation with 0.6 nM insulin decreased Akt PM translocation by approximately 25% in low and approximately 50% in high glucose. Preincubation with glucosamine did not affect Akt phosphorylation or translocation. Conclusions: chronic exposure to high glucose or insulin downregulates acute insulin-stimulated Akt activation, acting synergistically distal to PI 3-kinase. Maximal insulin activates more Akt than required for maximal glucose transport stimulation. Insulin resistance may ensue when PM-associated phospho-Akt decreases below a threshold. High glucose and glucosamine cause insulin resistance by different mechanisms in 3T3-L1 adipocytes.     

Epinephrine-induced hyperpolarization of islet cells without KATP channels

ID-1101 (4-hydroxyisoleucine), an amino acid extracted from fenugreek seeds, exhibits an interesting glucose-dependent insulin-stimulating activity. The present study was undertaken to investigate a possible extrapancreatic effect of ID-1101 on insulin signaling and action besides its previously described insulinotropic action. Insulin-sensitizing effects of ID-1101 were investigated in rat in vivo by three different approaches: 1) using euglycemic hyperinsulinemic clamps in two different rat models of insulin resistance, i.e., Zucker fa/fa rats and rats fed a sucrose-lipid diet; 2) measuring liver and muscle phosphatidylinositol (PI) 3-kinase activity after an acute injection of ID-1101 in normal and insulin-resistant diabetic rats; and 3) after chronic treatment in two rat models of insulin resistance. Euglycemic hyperinsulinemic clamp experiments revealed that ID-1101 can improve insulin resistance through an increase of peripheral glucose utilization rate in sucrose-lipid-fed rats and by decreasing hepatic glucose production in Zucker fa/fa rats. Moreover, we demonstrated that a single injection of ID-1101 activates the PI 3-kinase activity in liver and muscle from normal rats but also in muscle from diabetic rats. Finally, chronic ID-1101 treatment significantly reduced insulinemia in type 2 diabetic rats and reduced the progression of hyperinsulinemia in insulin-resistant obese Zucker fa/fa rats. These findings clearly demonstrate that ID-1101 can reduce insulin resistance through activation of the early steps of insulin signaling in peripheral tissues and in liver. In summary, ID-1101, besides its insulinotropic effect, directly improves insulin sensitivity, making it a potentially very valuable therapeutic agent for diabetes treatment.     

Higher Protein Kinase C ζ in Fatty Rat Liver and Its Effect on Insulin Actions in Primary Hepatocytes

We previously showed the impairment of insulin-regulated gene expression in the primary hepatocytes from Zucker fatty (ZF) rats, and its association with alterations of hepatic glucose and lipid metabolism. However, the molecular mechanism is unknown. A preliminary experiment shows that the expression level of protein kinase C ζ (PKCζ), a member of atypical PKC family, is higher in the liver and hepatocytes of ZF rats than that of Zucker lean (ZL) rats. Herein, we intend to investigate the roles of atypical protein kinase C in the regulation of hepatic gene expression. The insulin-regulated hepatic gene expression was evaluated in ZL primary hepatocytes treated with atypical PKC recombinant adenoviruses. Recombinant adenovirus-mediated overexpression of PKCζ, or the other atypical PKC member PKCι/λ, alters the basal and impairs the insulin-regulated expressions of glucokinase, sterol regulatory element-binding protein 1c, the cytosolic form of phosphoenolpyruvate carboxykinase, the catalytic subunit of glucose 6-phosphatase, and insulin like growth factor-binding protein 1 in ZL primary hepatocytes. PKCζ or PKCι/λ overexpression also reduces the protein level of insulin receptor substrate 1, and the insulin-induced phosphorylation of AKT at Ser473 and Thr308. Additionally, PKCι/λ overexpression impairs the insulin-induced Prckz expression, indicating the crosstalk between PKCζ and PKCι/λ. We conclude that the PKCζ expression is elevated in hepatocytes of insulin resistant ZF rats. Overexpressions of aPKCs in primary hepatocytes impair insulin signal transduction, and in turn, the down-stream insulin-regulated gene expression. These data suggest that elevation of aPKC expression may contribute to the hepatic insulin resistance at gene expression level.     

Insulin Signaling in Type 2 Diabetes: EXPERIMENTAL AND MODELING ANALYSES REVEAL MECHANISMS OF INSULIN RESISTANCE IN HUMAN ADIPOCYTES*

Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems level mechanistic understanding of insulin resistance, using systems wide and internally consistent data from human adipocytes. Based on quantitative steady-state and dynamic time course data on signaling intermediaries, normally and in diabetes, we developed a dynamic mathematical model of insulin signaling. The model structure and parameters are identical in the normal and diabetic states of the model, except for three parameters that change in diabetes: (i) reduced concentration of insulin receptor, (ii) reduced concentration of insulin-regulated glucose transporter GLUT4, and (iii) changed feedback from mammalian target of rapamycin in complex with raptor (mTORC1). Modeling reveals that at the core of insulin resistance in human adipocytes is attenuation of a positive feedback from mTORC1 to the insulin receptor substrate-1, which explains reduced sensitivity and signal strength throughout the signaling network. Model simulations with inhibition of mTORC1 are comparable with experimental data on inhibition of mTORC1 using rapamycin in human adipocytes. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling, both at the cellular level and, using a multilevel model, at the whole body level. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis.