PKC-zeta mediates insulin effects on glucose transport in cultured preadipocyte-derived human adipocytes

Insulin-stimulated glucose transport is impaired in the early phases of type 2 diabetes mellitus. Studies in rodent cells suggest that atypical PKC (aPKC) isoforms (zeta, lamda, and iota) and PKB, and their upstream activators, PI3K and 3-phosphoinositide-dependent protein kinase-1 (PDK-1), play important roles in insulin-stimulated glucose transport. However, there is no information on requirements for aPKCs, PKB, or PDK-1 during insulin action in human cell types. Presently, by using preadipocyte-derived adipocytes, we were able to employ adenoviral gene transfer methods to critically examine these requirements in a human cell type. These adipocytes were found to contain PKC-zeta, rather than PKC-lamda/iota, as their major aPKC. Expression of kinase-inactive forms of PDK-1, PKC-zeta, and PKC-lamda (which functions interchangeably with PKC-zeta) as well as chemical inhibitors of PI 3-kinase and PKC-zeta/lamda, wortmannin and the cell-permeable myristoylated PKC-zeta pseudosubstrate, respectively, effectively inhibited insulin-stimulated glucose transport. In contrast, expression of a kinase-inactive, activation-resistant, triple alanine mutant form of PKB-alpha had little or no effect, and expression of wild-type and constitutively active PKC-zeta or PKC-lamda increased glucose transport. Our findings provide convincing evidence that aPKCs and upstream activators, PI 3-kinase and PDK-1, play important roles in insulin-stimulated glucose transport in preadipocyte-derived human adipocytes.     

Rosiglitazone, insulin treatment, and fasting correct defective activation of protein kinase C-zeta/lambda by insulin in vastus lateralis muscles and adipocytes of diabetic rats

Atypical protein kinases C (PKCs), zeta and lambda, and protein kinase B (PKB) are thought to function downstream of phosphatidylinositol 3-kinase (PI 3-kinase) and regulate glucose transport during insulin action in skeletal muscle and adipocytes. Insulin-stimulated glucose transport is defective in type II diabetes mellitus, and this defect is ameliorated by thiazolidinediones and lowering of blood glucose by chronic insulin therapy or short-term fasting. Presently, we evaluated the effects of these insulin-sensitizing modalities on the activation of insulin receptor substrate-1 (IRS-1)-dependent PI 3-kinase, PKC-zeta/lambda, and PKB in vastus lateralis skeletal muscles and adipocytes of nondiabetic and Goto-Kakizaki (GK) diabetic rats. Insulin provoked rapid increases in the activity of PI 3-kinase, PKC-zeta/lambda, and PKB in muscles and adipocytes of nondiabetic rats, but increases in IRS-1-dependent PI 3-kinase and PKC-zeta/lambda, but not PKB, activity were substantially diminished in GK muscles and adipocytes. Rosiglitazone treatment for 10-14 days, 10-day insulin treatment, and 60-h fasting reversed defects in PKC-zeta/lambda activation in GK muscles and adipocytes and increased glucose transport in GK adipocytes, without necessarily increasing IRS-1-dependent PI 3-kinase or PKB activation. Our findings suggest that insulin-sensitizing modalities, viz. thiazolidinediones, chronic insulin treatment, and short-term fasting, similarly improve defects in insulin-stimulated glucose transport at least partly by correcting defects in insulin-induced activation of PKC-zeta/lambda.     

Insulin signal transduction and glucose transport in human adipocytes: effects of obesity and low calorie diet

AIM/HYPOTHESIS: We examined insulin signal transduction at the level of insulin receptor substrates (IRS) 1 and 2, phosphatidylinositol (PI) 3-kinase and glucose transport in isolated subcutaneous adipocytes from obese and lean women. METHODS: Glucose transport and insulin signalling were investigated in isolated adipocytes from six obese women (BMI 36-43 kg/m(2)) (before and after 11 days of very low calorie diet) and from six lean women (BMI 22-26 kg/m(2)). RESULTS: Insulin sensitivity of glucose transport was reduced in adipocytes from obese women (p<0.05), with further reductions in basal and maximal insulin-stimulated glucose transport after a very low calorie diet (p<0.05). In obese women, IRS-1 associated PI 3-kinase activity was markedly impaired (p<0.05), whereas, IRS-2 associated PI 3-kinase activity was normal. IRS-1 associated PI 3-kinase activity remained blunted after a very low calorie diet, whereas IRS-2 associated PI 3-kinase activity was increased. GLUT4 protein was reduced by 37% in obese versus lean subjects (p<0.05), and decreased further after a very low calorie diet (from 19+/-4 to 14+/-4 arbitrary units; p<0.05). CONCLUSION/INTERPRETATION: IRS-1 signalling to PI 3-kinase is a site of insulin resistance in adipocytes from obese women, whereas insulin action on IRS-2 is normal. Thus, IRS-1 and IRS-2 undergo differential regulation in adipocytes from obese insulin resistant subjects. Finally, a very low calorie diet is associated with a further impairment in glucose transport in adipose tissue. The defect in glucose transport after a very low calorie diet occurs independent of further defects in insulin signalling at the level of the PI 3-kinase.     

Amino acids require glucose to enhance, through phosphoinositide-dependent protein kinase 1, the insulin-activated protein kinase B cascade in insulin-resistant rat adipocytes

Aims/hypothesis Amino acids are well known to activate the mammalian target of the rapamycin (mTOR) pathway in synergy with insulin to regulate cell functions. Despite recent important advances, the mTOR signalling pathway is poorly understood. Our previous results revealed a new pathway in which amino acids permit insulin-induced activation of the protein kinase B (PKB)/mTOR pathway in freshly isolated adipocytes when phosphatidylinositol 3-kinase (PI3K) is inhibited. The aim of this study was to further investigate this pathway at the molecular level.Methods We studied the effect of amino acids on PKB phosphorylation in different cellular models or in freshly isolated adipocytes incubated in different buffers, after a time course of insulin and amino acids and in the presence of pharmacological inhibitors. To investigate the potential role of amino acids in insulin action, the effect on glucose transport in obese rat adipocytes following a high-fat diet was assessed.Results Insulin-induced PKB phosphorylation is restored by amino acids in the presence of wortmannin in adipose tissue explants and freshly isolated adipocytes, but not in cultured adipocytes or hepatocytes. Moreover, amino acids require the presence of glucose to phosphorylate PKB and to partially rescue glucose transport in a PI3K-independent manner. The results also suggest that the amino acids act through the phosphoinositide-dependent protein kinase 1. In addition, amino acids were seen to improve insulin-stimulated glucose transport in adipocytes from high-fat-fed rats.Conclusions/interpretation This study suggests that amino acids could enhance adipocyte insulin signalling in pathophysiological situations such as insulin resistance associated with obesity.     

Defective activation of atypical protein kinase C zeta and lambda by insulin and phosphatidylinositol-3,4,5-(PO4)(3) in skeletal muscle of rats following high-fat feeding and streptozotocin-induced diabetes

Insulin-stimulated glucose transport in skeletal muscle is thought to be effected at least partly through atypical protein kinase C isoforms (aPKCs) operating downstream of phosphatidylinositol (PI) 3-kinase and 3-phosphoinositide-dependent protein kinase-1 (PDK-1). However, relatively little is known about the activation of aPKCs in physiological conditions or insulin-resistant states. Presently, we studied aPKC activation in vastus lateralis muscles of normal chow-fed and high-fat-fed rats and after streptozotocin (STZ)-induced diabetes. In normal chow-fed rats, dose-dependent increases in aPKC activity approached maximal levels after 15-30 min of stimulation by relatively high and lower, presumably more physiological, insulin concentrations, achieved by im insulin or ip glucose administration. Insulin-induced activation of aPKCs was impaired in both high-fat-fed and STZ-diabetic rats, but, surprisingly, IRS-1-dependent and IRS-2-dependent PI 3-kinase activation was not appreciably compromised. Most interestingly, direct in vitro activation of aPKCs by PI-3,4,5-(PO(4))(3), the lipid product of PI 3-kinase, was impaired in both high-fat-fed and STZ-diabetic rats. Defects in activation of aPKCs by insulin and PI-3,4,5-(PO(4))(3) could not be explained by diminished PDK-1-dependent phosphorylation of threonine-410 in the PKC-zeta activation loop, as this phosphorylation was increased even in the absence of insulin treatment in high-fat-fed rats. Conclusions: 1) muscle aPKCs are activated at relatively low, presumably physiological, as well as higher supraphysiological, insulin concentrations; 2) aPKC activation is defective in muscles of high-fat-fed and STZ-diabetic rats; and 3) defective aPKC activation in these states is at least partly due to impaired responsiveness to PI-3,4,5-(PO(4))(3), apparently at activation steps distal to PDK-1-dependent loop phosphorylation.     

Combined thiazolidinedione–metformin treatment synergistically improves insulin signalling to insulin receptor substrate-1-dependent phosphatidylinositol 3-kinase, atypical protein kinase C and protein kinase B/Akt in human diabetic muscle

Aims/hypotheses  Insulin-stimulated glucose transport in muscle is impaired in type 2 diabetes, presumably reflecting reduced activation of atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). As previously shown, reductions in aPKC activation are seen at sub-maximal and maximal insulin stimulation, reductions in PKB activation are best seen at sub-maximal insulin stimulation and aPKC reductions at maximal insulin are partly improved by thiazolidinedione or metformin treatment. However, effects of combined thiazolidinedione–metformin treatment on aPKC or PKB activation by sub-maximal and maximal insulin are unknown. Methods  Type 2 diabetic patients were examined before and 5 to 6 weeks after combined thiazolidinedione–metformin therapy for activation of muscle aPKC and PKBβ and their upstream activators, the insulin receptor (IR) and IRS-1-associated phosphatidylinositol 3-kinase (PI3K), during euglycaemic–hyperinsulinaemic clamp studies conducted with sub-maximal (400–500 pmol/l) and maximal (1400 pmol/l) insulin concentrations. Results  Following combined thiazolidinedione–metformin therapy, increases in glucose disposal and increases in sub-maximal and maximal insulin-induced activities of all four muscle signalling factors, IR, IRS-1-dependent PI3K (IRS-1/PI3K), aPKC and PKBβ, were observed. Increases in PKBβ enzyme activity were accompanied by increases in phosphorylation of PKB and its substrate, AS160, which is needed for glucose transport. Despite improved aPKC activity, muscle aPKC levels, which are diminished in type 2 diabetes, were not altered. Conclusions/interpretation  Combined thiazolidinedione–metformin treatment markedly improves sub-maximal and maximal insulin signalling to IR, IRS-1/PI3K, aPKC and PKBβ in type 2 diabetic muscle. These improvements exceed those previously reported after treatment with either agent alone.     

Dexamethasone impairs insulin signalling and glucose transport by depletion of insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase B in primary cultured rat adipocytes

OBJECTIVE: Glucocorticoid excess leads to insulin resistance. This study explores the effects of glucocorticoids on the glucose transport system and insulin signalling in rat adipocytes. The interaction between glucocorticoids and high levels of insulin and glucose is also addressed. DESIGN AND METHODS: Isolated rat adipocytes were cultured for 24 h at different glucose concentrations (5 and 15 mmol/l) with or without the glucocorticoid analogue dexamethasone (0.3 micromol/l) and insulin (10(4) microU/ml). After the culture period, the cells were washed and then basal and insulin-stimulated glucose uptake, insulin binding and lipolysis as well as cellular content of insulin signalling proteins (insulin receptor substrate-1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB)) and glucose transporter isoform GLUT4 were measured. RESULTS: Dexamethasone in the medium markedly decreased both basal and insulin-stimulated glucose uptake at both 5 and 15 mmol/l glucose (by approximately 40-50%, P<0.001 and P<0.05 respectively). Combined long-term treatment with insulin and dexamethasone exerted additive effects in decreasing basal, and to a lesser extent insulin-stimulated, glucose uptake capacity (P<0.05) compared with dexamethasone alone, but this was seen only at high glucose (15 mmol/l). Insulin binding was decreased (by approximately 40%, P<0.05) in dexamethasone-treated cells independently of surrounding glucose concentration. Following dexamethasone treatment a approximately 75% decrease (P<0.001) in IRS-1 expression and an increase in IRS-2 (by approximately 150%, P<0.001) was shown. Dexamethasone also induced a subtle decrease in PI3-K (by approximately 20%, P<0.01) and a substantial decrease in PKB content (by approximately 45%, P<0.001). Insulin-stimulated PKB phosphorylation was decreased (by approximately 40%, P<0.01) in dexamethasone-treated cells. Dexamethasone did not alter the amount of total cellular membrane-associated GLUT4 protein. The effects of dexamethasone per se on glucose transport and insulin signalling proteins were mainly unaffected by the surrounding glucose and insulin levels. Dexamethasone increased the basal lipolytic rate (approximately 4-fold, P<0.05), but did not alter the antilipolytic effect of insulin. CONCLUSIONS: These results suggest that glucocorticoids, independently of the surrounding glucose and insulin concentration, impair glucose transport capacity in fat cells. This is not due to alterations in GLUT4 abundance. Instead dexamethasone-induced insulin resistance may be mediated via reduced cellular content of IRS-1 and PKB accompanied by a parallel reduction in insulin-stimulated activation of PKB.     

Contrasting insulin dose-dependent defects in activation of atypical protein kinase C and protein kinase B/Akt in muscles of obese diabetic humans

Aims/hypothesis Insulin-stimulated glucose transport in muscle is impaired in obesity and type 2 diabetes, but alterations in levels of relevant signalling factors, i.e. atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt), are still uncertain. Clamp studies using maximal insulin concentrations have revealed defects in activation of aPKC, but not PKB, in both obese non-diabetic and obese diabetic subjects. In contrast, clamp studies using submaximal insulin concentrations revealed defects in PKB activation/phosphorylation in obese non-diabetic and diabetic subjects, but changes in aPKC were not reported. The aim of this study was to test the hypothesis that dose-related effects of insulin may account for the reported differences in insulin signalling to PKB in diabetic muscle.Subjects and methods We compared enzymatic activation of aPKC and PKB, and PKB phosphorylation (threonine-308 and serine-473) during hyperinsulinaemic–euglycaemic clamp studies using both submaximal (400–500 pmol/l) and maximal (1400 pmol/l) insulin levels in non-diabetic control and obese diabetic subjects.Results In lean control subjects, the submaximal insulin concentration increased aPKC activity and glucose disposal to approximately 50% of the maximal level and PKBβ activity to 25% of the maximal level, but PKBα activity was not increased. In these subjects, phosphorylation of PKBα and PKBβ was increased to near-maximal levels at submaximal insulin concentrations. In obese diabetic subjects, whereas aPKC activation was defective at submaximal and maximal insulin concentrations, PKBβ activation and the phosphorylation of PKBβ and PKBα were defective at submaximal, but not maximal, insulin concentrations.Conclusions/interpretations Defective PKBβ activation/phosphorylation, seen on submaximal insulin stimulation in diabetic muscle, may largely reflect impaired activation of insulin signalling factors present in concentrations greater than those needed for full PKB activation/phosphorylation. Defective aPKC activation, seen at all insulin levels, appears to reflect, at least partly, an impaired action of distal factors needed for aPKC activation, or poor aPKC responsiveness.     

Defective Activation of Protein Kinase C-z in Muscle by Insulin and Phosphatidylinositol-3,4,5,-(PO(4))(3) in Obesity and Polycystic Ovary Syndrome

Insulin resistance occurs frequently in metabolic syndrome components, obesity, and the polycystic ovary syndrome, and is partly due to impaired glucose transport into skeletal muscle, but underlying mechanisms are uncertain. Atypical protein kinase C and protein kinase B, operating downstream of phosphatidylinositol 3-kinase, mediate insulin effects on glucose transport, but their importance in these syndromes is poorly understood. Presently, we examined these signaling factors in muscle biopsies obtained during euglycemic/hyperinsulinemic clamp studies. In lean subjects, insulin provoked approximately twofold increases in muscle atypical protein kinase C activity. In obese subjects and obese subjects who had evidence of the polycystic ovary syndrome, insulin-stimulated glucose disposal and atypical protein kinase C activation were diminished, whereas activation of insulin receptor substrate-1-dependent phosphatidylinositol 3-kinase and protein kinase B trended lower, but not significantly. Interestingly, direct activation of atypical protein kinase C by phosphatidylinositol-3,4,5-(PO(4))(3), the lipid product of phosphatidylinositol 3-kinase, was readily apparent in immunoprecipitates prepared from muscles of lean subjects, but to a lesser degree or poorly if at all in subjects who were obese or had the obesity/polycystic ovary syndrome. Our findings suggest that activation of muscle atypical protein kinase C by insulin and phosphatidylinositol-3,4,5-(PO(4))(3) is defective and may contribute to skeletal muscle insulin resistance in women who are obese, or have obesity associated with the polycystic ovary syndrome.     

Insulin action kinetics in adipocytes from obese and noninsulin-dependent diabetes mellitus subjects: identification of multiple cellular defects in glucose transport

Recent results from in vivo studies have shown that the kinetics of insulin action are impaired in lean and obese noninsulin-dependent diabetes mellitus (NIDDM) subjects as well as in obese nondiabetic subjects. We have measured the onset and loss of insulin action on glucose transport in adipocytes obtained from obese nondiabetic and obese NIDDM subjects to determine the contributions of obesity and diabetes to these cellular defects in insulin action. Basal and maximally insulin-stimulated rates of 3-O-methylglucose transport in adipocytes from obese and obese NIDDM subjects were reduced to 50% of the values in cells from normal subjects (P less than 0.05). The activation of glucose transport by insulin (4.3 nmol/L) was slower in cells from obese NIDDM patients. Half of the maximal insulin effect (A50) was reached by 23.0 +/- 5.0 min compared to 9.4 +/- 1.1 min in normal cells (P less than 0.05). Conversely, the deactivation of insulin-stimulated glucose transport upon removal of insulin was more rapid in adipocytes from the obese and obese NIDDM subjects. Half of the maximal insulin effect (D50) was lost by 12.4 +/- 1.7 min in obese NIDDM cells and by 8.9 +/- 1.9 min in obese subjects compared to 25.3 +/- 1.9 min in adipocytes from normal subjects (P less than 0.01). In conclusion, 1) basal and insulin-stimulated rates of glucose transport are similarly reduced in adipocytes from obese and obese NIDDM subjects; and 2) adipocytes from obese and obese NIDDM subjects display defects in the kinetics of insulin action, slower activation and accelerated deactivation, that mirror the defects measured in vivo. Both impairments in the kinetics of insulin action may contribute to the insulin resistance in these subject groups.     

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.     

Insulin in Combination with Vanadate Stimulates Glucose Transport in Isolated Cardiomyocytes from Obese Zucker Rats

Insulin stimulates glucose uptake in muscle cells via activation of protein kinase B (PKB). The protein tyrosine phosphatase (PTP) inhibitor vanadate, is a known insulin mimetic agent but the mechanism whereby vanadate exerts its effect is not clearly understood. Vanadate also has beneficial effects in the diabetic myocardium. The aim of this study was to correlate insulin stimulation of glucose uptake and PKB activation with that induced by vanadate in adult ventricular myocytes from lean and obese Zucker fa/fa rats. In lean Zucker rats, 100 nM insulin and 5 mM vanadate stimulated myocardial 2-deoxy-D-[³H]glucose (2-DG) uptake from 27.17 ± 1.72 to 96.52 ± 10.87 and 43.86 ± 4.02 pmole/mg protein p/30 min respectively while a combination of insulin and vanadate could not improve the maximal response of insulin. In obese Zucker hearts, basal as well as insulin and vanadate stimulated glucose uptake were severely impaired (15.49 ± 1.44 vs 25.51 ± 3.11 and 20.11 ± 1.68 pmole/mg protein/30 min respectively). A combination of insulin and vanadate, resulted in a response significantly improved from the maximal response of insulin. This stimulation of 2-DG uptake was, in all instances, blocked by the PI 3-kinase inhibitors wortmannin and LY 294002.Insulin could not activate PKB, as measured by the Ser⁴⁷³ phosphorylated form of the enzyme, in the obese Zucker rats to the same extent as in lean controls. Similar to glucose uptake, activation of PKB by vanadate plus insulin was significantly more than that accomplished by insulin alone in obese rats. Both insulin and vanadate activation of PKB was prevented by wortmannin and LY 294002. Thus, the present study demonstrates that: (i) in cardiomyocytes from lean and obese Zucker rats, both insulin and vanadate stimulate glucose uptake and PKB activation through a PI-3-kinase sensitive pathway. (ii) In obese Zucker rats, neither insulin nor vanadate could induce glucose uptake or activation of PKB to the same extent as in lean controls. (iii) A combination of insulin with vanadate may be beneficial to increase glucose uptake in diabetic hearts, as this gives a better response than insulin alone.     

Role of atypical protein kinase C in activation of sterol regulatory element binding protein-1c and nuclear factor kappa B (NFκB) in liver of rodents used as a model of diabetes, and relationships to hyperlipidaemia and insulin resistance

Aims/hypothesis  Previous findings in rodents used as a model of diabetes suggest that insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but, unexpectedly, conserved in liver, despite impaired hepatic protein kinase B (PKB/Akt) activation. Moreover, aPKC at least partly regulates two major transactivators: (1) hepatic sterol receptor binding protein-1c (SREBP-1c), which controls lipid synthesis; and (2) nuclear factor kappa B (NFκB), which promotes inflammation and systemic insulin resistance. Methods  In Goto–Kakizaki rats used as a model of type 2 diabetes, we examined: (1) whether differences in hepatic aPKC and PKB activation reflect differences in activation of IRS-1- and IRS-2-dependent phosphatidylinositol 3-kinase (PI3K); (2) whether hepatic SREBP-1c and NFκB are excessively activated by aPKC; and (3) metabolic consequences of excessive activation of hepatic aPKC, SREBP-1c and NFκB. Results  In liver, as well as in muscle, IRS-2/PI3K activation by insulin was intact, whereas IRS-1/PI3K activation by insulin was impaired. Moreover, hepatic IRS-2 is known to control hepatic aPKC during insulin activation. Against this background, selective inhibition of hepatic aPKC by adenoviral-mediated expression of mRNA encoding kinase-inactive aPKC or short hairpin RNA targeting Irs2 mRNA and partially depleting hepatic IRS-2 diminished hepatic SREBP-1c production and NFκB activities, concomitantly improving serum lipids and insulin signalling in muscle and liver. Similar improvements in SREBP-1c, NFκB and insulin signalling were seen in ob/ob mice following inhibition of hepatic aPKC. Conclusions/interpretation  In diabetic rodent liver, diminished PKB activation may largely reflect impaired IRS-1/PI3K activation, while conserved aPKC activation reflects retained IRS-2/PI3K activity. Hepatic aPKC may also contribute importantly to excessive SREPB-1c and NFκB activities. Excessive hepatic aPKC-dependent activation of SREBP-1c and NFκB may contribute importantly to hyperlipidaemia and systemic insulin resistance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Defective non-insulin-mediated and insulin-mediated glucose transport and metabolism in adipocytes from obese and lean patients with untreated type 2 diabetes mellitus

Insulin binding, glucose transport, and glucose metabolism were investigated in isolated adipocytes from 11 lean and 13 obese patients with non-insulin-dependent diabetes mellitus. Insulin binding at 15 degrees C was reduced by 35% (p less than 0.01) in both lean and obese diabetic patients, whereas insulin binding (or uptake) at 37 degrees C was similar in diabetic patients and healthy controls. In lean diabetic patients both non-insulin-mediated (basal) and maximally insulin-stimulated glucose transport and metabolism were significantly reduced (all p less than 0.01). The percentage responses to insulin were also markedly reduced (p less than 0.05, p less than 0.02). In obese diabetic patients basal glucose transport was reduced (p less than 0.01) but basal glucose metabolism was not. Insulin-stimulated glucose transport and metabolism were significantly reduced (p less than 0.01, p less than 0.05). The percentage responses were reduced compared to healthy controls (p less than 0.05, p less than 0.05) but higher than in lean diabetic patients (p less than 0.05). We conclude that adipocytes isolated from both lean and obese patients with non-insulin-dependent diabetes mellitus are characterized by severely depressed non-insulin-mediated and insulin-mediated glucose transport and depressed insulin-mediated glucose metabolism. The major defect seems to be a reduced maximal effect of insulin on glucose metabolism, suggesting post-binding and post-transport abnormalities.     

Activation of the mammalian target of rapamycin pathway acutely inhibits insulin signaling to Akt and glucose transport in 3T3-L1 and human adipocytes

The mammalian target of rapamycin (mTOR) pathway has recently emerged as a chronic modulator of insulin-mediated glucose metabolism. In this study, we evaluated the involvement of this pathway in the acute regulation of insulin action in both 3T3-L1 and human adipocytes. Insulin rapidly (t(1/2) = 5 min) stimulated the mTOR pathway, as reflected by a 10-fold stimulation of 70-kDa ribosomal S6 kinase 1 (S6K1) activity in 3T3-L1 adipocytes. Inhibition of mTOR/S6K1 by rapamycin increased insulin-stimulated glucose transport by as much as 45% in 3T3-L1 adipocytes. Activation of mTOR/S6K1 by insulin was associated with a rapamycin-sensitive increase in Ser636/639 phosphorylation of insulin receptor substrate (IRS)-1 but, surprisingly, did not result in impaired IRS-1-associated phosphatidylinositol (PI) 3-kinase activity. However, insulin-induced activation of Akt was increased by rapamycin. Insulin also activated S6K1 and increased phosphorylation of IRS-1 on Ser636/639 in human adipocytes. As in murine cells, rapamycin treatment of human adipocytes inhibited S6K1, blunted Ser636/639 phosphorylation of IRS-1, leading to increased Akt activation and glucose uptake by insulin. Further studies in 3T3-L1 adipocytes revealed that rapamycin prevented the relocalization of IRS-1 from the low-density membranes to the cytosol in response to insulin. Furthermore, inhibition of mTOR markedly potentiated the ability of insulin to increase PI 3,4,5-triphosphate levels concomitantly with an increased phosphorylation of Akt at the plasma membrane, low-density membranes, and cytosol. However, neither GLUT4 nor GLUT1 translocation induced by insulin were increased by rapamycin treatment. Taken together, these results indicate that the mTOR pathway is an important modulator of the signals involved in the acute regulation of insulin-stimulated glucose transport in 3T3-L1 and human adipocytes.     

High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes

OBJECTIVE: The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins. DESIGN AND METHODS: Rat adipocytes were cultured for 24 h in different glucose concentrations with 10(4) microU/ml of insulin or without insulin. After washing, (125)I-insulin binding, basal and acutely insulin-stimulated d-[(14)C]glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed. RESULTS: High glucose (15 and 25 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by approximately 40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by approximately 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by approximately 20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination, shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by approximately 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration. CONCLUSIONS: Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose, it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.     

Leptin secretion and negative autocrine crosstalk with insulin in brown adipocytes

Leptin is an important adipocytokine whose main regulative effects on energy metabolism are exerted via activation of signalling pathways in the central nervous system. Another important regulator of energy homeostasis is insulin. The role of direct autocrine leptin effects on adipose tissue and crosstalk with insulin, in particular in the thermogenically active brown adipose tissue, remains unclear. In the present study, we have investigated leptin secretion and interaction with insulin in highly insulin-responsive immortalised mouse brown adipocytes. Leptin was secreted in a differentiation-dependent manner, and acute leptin treatment of mature adipocytes dose- and time-dependently stimulated phosphorylation of STAT3 and MAP kinase. Interestingly, acute pretreatment of fully differentiated brown adipocytes with leptin (100 nM) significantly diminished insulin-induced glucose uptake by approximately 25%. This inhibitory effect was time-dependent and maximal after 60 min of leptin prestimulation. Furthermore, it correlated with a 35% reduction in insulin-stimulated insulin receptor kinase activity after acute leptin pretreatment. Insulin-induced insulin receptor substrate-1 tyrosine phosphorylation and binding to the regulatory subunit p85 of phosphatidylinositol 3-kinase (PI 3-kinase) were diminished by approximately 60% and 40%, respectively. Taken together, this study has demonstrated strong differentiation-dependent leptin secretion in brown adipocytes and PI 3-kinase-mediated negative autocrine effects of this hormone on insulin action. Direct peripheral leptin-insulin crosstalk may play an important role in the regulation of energy homeostasis.     

Positive and negative regulation of glucose uptake by hyperosmotic stress

This review will provide insight on the current understanding of the intracellular signaling mechanisms by which hyperosmolarity mimics insulin responses such as Glut 4 translocation and glucose transport but also antagonizes insulin effects. Glucose uptake induced by insulin is largely dependent on the PI 3-kinase/PKB pathway. In both adipocyte and muscle cells, hyperosmolarity promotes glucose uptake by multiple mechanisms which do not require PI 3-kinase/PKB pathway but are dependent on the cell type. In muscle, osmotic stress induces glucose uptake by stimulation of AMP-Kinase and/or inhibition of Glut 4 endocytosis. In adipocytes, activation of Gab1-dependent signaling pathway plays an important role in osmotic stress-mediated glucose uptake. Apart of its insulin-like effects, hyperosmolarity can lead to cellular insulin resistance mediated by both prevention of PKB activation and inhibition of the Insulin Receptor Substrate-1 (IRS1) function. Serine phosphorylation and degradation of IRS1 negatively regulate its functions. Understanding how osmotic stress induces glucose transport or mediates insulin resistance may provide novel targets for strategies to enhance glucose transport or to prevent insulin resistance.     

Agent and cell-type specificity in the induction of insulin resistance by HIV protease inhibitors

OBJECTIVE: To test agent and cell-type specificity in insulin resistance induced by prolonged exposure to HIV protease inhibitors (HPI), and to assess its relation to the direct, short-term inhibition of insulin-stimulated glucose uptake. METHODS: Following prolonged (18 h) and short (5-10 min) exposure to HPI, insulin-stimulated glucose transport, protein kinase B (PKB) phosphorylation, and GLUT4 translocation were evaluated in 3T3-L1 adipocytes, fibroblasts, L6 myotubes, and L6 cells overexpressing a myc tag on the first exofacial loop of GLUT4 or GLUT1. RESULTS: Prolonged exposure of 3T3-L1 adipocytes to nelfinavir, but not to indinavir or saquinavir, resulted in increased basal lipolysis but decreased insulin-stimulated glucose transport and PKB phosphorylation. In addition, impaired insulin-stimulated glucose uptake and PKB phosphorylation were also observed in the skeletal muscle cell line L6, and in 3T3-L1 fibroblasts. Interestingly, this coincided with increased basal glucose uptake as well as with elevated total-membrane glucose transporter GLUT1 protein content. In contrast to these unique effects of nelfinavir, the mere presence of any of the agents in the 5 min transport assay inhibited insulin-stimulated glucose-uptake activity. This appeared to be caused by direct and specific interaction of the drugs with GLUT4 fully assembled at the plasma membrane, since insulin-stimulated cell-surface exposure of an exofacial myc epitope on GLUT4 was normal. CONCLUSIONS: Independent mechanisms for HPI-induced insulin resistance exist: prolonged exposure to nelfinavir interferes with insulin signaling and alters cellular metabolism of adipocytes and muscle cells, whereas a direct inhibitory effect on insulin-stimulated glucose uptake may occurs through specific interaction of HPI with GLUT4.     

In vitro insensitivity of glucose transport and antilipolysis to insulin due to receptor and postreceptor abnormalities in obese Pima Indians with normal glucose tolerance

The in vitro sensitivities of glucose transport and antilipolysis to insulin and insulin binding were measured in adipocytes isolated from three groups of normal glycemic male Pima Indians--10 lean (11% to 22% body fat), 11 moderately obese (26% to 34% body fat), and 7 severely obese (37% to 40% body fat) subjects. Both a half-maximum concentration of insulin for the stimulation of glucose transport (ED50 [transport]) and a half-maximum concentration of insulin for the suppression of 25 nmol/L isoproterenol-stimulated lipolysis (ED50 [antilipolysis]) were significantly (P less than 0.05) greater in the moderately obese subjects than in the lean subjects as well as greater in the severely obese group than in the moderately obese group. Mono 125I-(Tyr A14)-insulin binding per cell in the presence of 25, 100, and 200 pm insulin was similar among lean, mildly obese, and severely obese subjects. 125I-insulin binding per cell surface area of adipocytes isolated from either moderately or severely obese Indians was significantly lower (P less than 0.005) than that of lean Indians. However, there was a similar insulin binding per cell surface area between mildly and severely obese subjects. These results indicate that diminished insulin binding per cell surface area may explain decreased sensitivity of transport and antilipolysis to insulin in moderately obese subjects relative to lean subjects. In contrast, these diminished sensitivities in the severely obese subjects relative to moderate obese subjects are not explained by a change in insulin binding and, therefore, are presumably induced by an abnormality of a postbinding step of insulin action.