Length of acute exposure to insulin regulates the rate of deactivation of stimulated glucose transport in isolated rat adipocytes

We have studied the effects of the duration of exposure of rat adipocytes to insulin on the temporal relationships between insulin dissociation from receptors and deactivation of insulin-stimulated glucose transport. Cells were incubated with [125I]insulin plus unlabeled insulin at a total insulin concentration of either 1 or 10 ng/ml at 37 C. After various times of association, the cells were washed, and the rate of dissociation of bound hormone and subsequent decrease in insulin-stimulated glucose transport activity (deactivation) were measured. One nanogram of insulin/ml is a submaximally effective concentration and stimulates glucose transport to approximately 80% of maximal values. Dissociation of insulin from receptors and deactivation of glucose transport activity were measured after 10, 30, and 60 min of exposure of cells to 1 ng/ml insulin. Dissociation rates were unchanged after the various times of exposure to insulin, whereas prolonging the preincubation time from 10 to 60 min slowed the rate of deactivation 5-fold. Dissociation and deactivation were also measured after 5-, 10-, 30-, and 60-min periods of incubation with a maximally stimulating hormone concentration (10 ng/ml). The dissociation rates were similar after the various periods of association, whereas the deactivation rate was 2-fold slower after 30 and 60 min of preincubation compared to that after 5 min. Thus, there is a time-dependent slowing of the rate of deactivation of insulin-stimulated glucose transport that is not accounted for by differences in the amount of insulin bound or the dissociation rate of insulin from its receptors. Therefore, the length of exposure to insulin can influence the rate of decline in glucose transport activity, and this may be important in determining biological effects of pulsatile insulin secretion vs. prolonged insulin exposure on insulin target tissues. Furthermore, the data suggest that with time, insulin causes an increase in some intracellular factor or other cellular perturbation, the level of which modulates the rate of deactivation of the glucose transport system.     

Leukaemia inhibitory factor stimulates glucose transport in isolated cardiomyocytes and induces insulin resistance after chronic exposure

Aims/hypothesis Hypertrophic and failing hearts have increased utilisation of glucose, but also develop insulin resistance and reduced ability to produce ATP. Increased levels of the IL-6-related cytokine leukaemia inhibitory factor (LIF) are found in failing hearts, and we have recently shown that LIF reduces ATP production in isolated cardiomyocytes. In the present study we investigated effects of LIF on glucose metabolism, and how LIF-treated cells respond to insulin stimulation. Methods Cardiomyocytes were isolated from adult Wistar rats by collagen digestion, maintained in culture for 48 h, and then treated with 1 nmol/l LIF. Results Acute LIF treatment increased deoxyglucose uptake compared with controls, but no additive effect was observed in cardiomyocytes treated with LIF and insulin. The phosphatidylinositol 3-kinase inhibitor wortmannin did not affect LIF-induced glucose uptake. LIF had no effect on AMP-activated protein kinase phosphorylation. Cardiomyocytes treated with LIF for 48 h did not respond to insulin by increasing deoxyglucose uptake and showed a reduced insulin-mediated uptake of oleic acid and formation of complex lipids compared with control cells. Chronic LIF treatment increased gene expression of the suppressor of cytokine signalling (Socs) 3 and reduced expression of solute carrier family 2, member 4 (Slc2a4, previously known as glucose transporter 4 [Glut4]). In line with these observations, chronic LIF treatment reduced insulin-mediated phosphorylation of both Akt/protein kinase B (PKB) and glycogen synthase kinase (GSK)-3. Conclusions/interpretation Acute LIF treatment increased glucose uptake in isolated cardiomyocytes by a pathway different from that of insulin. Chronic LIF treatment induced insulin resistance, possibly mediated by altered expression of Socs3 and Slc2a4, and impaired insulin-mediated phosphorylation of GSK-3 and Akt/PKB.     

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.     

Transferrin and iron induce insulin resistance of glucose transport in adipocytes

Normal serum can increase the rate of lipolysis in isolated adipocytes. Recently, we reported that the lipolytic effect of serum could be partly explained by effects of iron and transferrin. To further investigate these effects on fat cell metabolism, we have investigated effects of serum, iron, and transferrin on glucose transport in isolated rat adipocytes. Adipocytes were isolated by collagenase digestion of rat epididymal fat pads, and glucose transport was measured as uptake of [3H]2-deoxyglucose, measured in the presence of 0 to 25 ng/mL insulin. Insulin stimulated glucose transport approximately 8- to 10-fold, with a half-maximally effective concentration (EC50) of approximately 0.15 ng/mL. This was not affected by 45-minute treatment with normal human serum. However, when adipocytes were incubated with serum for 4 hours, cells became markedly insulin resistant. This was manifested as decrease in maximally stimulated glucose transport and a rightward shift in the dose-response curve. Both FeS04 (3 microg/mL) and transferrin (100 microg/mL) had similar, although less pronounced effects on insulin-stimulated glucose transport. Treatment of adipocytes with palmitic acid (120 micromol/L), representing the concentration of fatty acids released into the media after 4 hours of serum treatment, did not alter the effect of insulin on glucose transport. We conclude that transferrin and iron induce insulin resistance of glucose transport in adipocytes through a mechanism independent of fatty acids. These findings may further explain the association between body iron stores and risk of type 2 diabetes mellitus.     

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.     

beta-Adrenergic desensitization by chronic insulin exposure in 3T3-L1 cultured adipocytes

Cultured 3T3-L1 cells provide a model system for studies of the long-term regulation of lipolysis. Insulin acutely inhibits isoproterenol-stimulated lipolysis primarily by decreasing the apparent affinity apparent Km for isoproterenol. In contrast, chronic insulin exposure inhibits lipolysis by a reduction in the maximal effect of isoproterenol Vmax. The decrease in Vmax can be observed with insulin concentrations that are as low as 10(-9) mol/L at the time of addition. The effect is stable to washing, and the cells' responsiveness to isoproterenol returns partially with continued culture. Chronic insulin exposure also markedly reduced dibutyryl-cAMP-stimulated lipolysis indicating an insulin-induced change distal to cAMP concentration in the cascade of reactions controlling lipolysis in these cells. Time course and insulin dose-response experiments indicate an additional proximal alteration. These results indicate that: (1) 3T3-L1 cells are a useful model for studying the long-term regulation of lipolysis. (2) Chronic insulin exposure inhibits lipolysis by a mechanism that differs from the acute effect of insulin. (3) The chronic effects of insulin may be mediated through changes at multiple levels in the lipolytic cascade.     

Implantation of primary cultured adipocytes that secrete insulin modifies blood glucose levels in diabetic mice

Aim/hypothesis In type 1 diabetic patients, basal insulin supplementation plays a central role in tight glycaemic control. Therefore, safe and steady supplementation of basal insulin is strongly desirable, despite the need for multiple injections. The aim of this study was to investigate a procedure for supplementation using genetically engineered, primary-cultured adipocytes in diabetic mice.Methods Furin-cleavable human proinsulin cDNA was transferred into murine primary-cultured adipocytes using a retroviral vector. The cells were implanted subcutaneously into streptozotocin-induced diabetic mice.Results The transfected cells secreted substantial amounts of mature insulin, as well as C-peptide, into conditioned medium. Syngeneic implantation of the cells significantly improved hyperglycaemia and blood HbA₁c concentrations in a manner that was dependent on cell number, without causing hypoglycaemia. The plasma insulin concentration was dependent on the implanted cell number, and the systemic effect of the circulating insulin was confirmed by marked improvement of body weight reduction and liver glycogen content. Additionally, surgical resection of the implants, in which the insulin secretion was immunologically confirmed after transplantation, diminished the glucose-lowering effect, suggesting that in vivo expression could be eliminated if necessary.Conclusions/interpretation These results indicate that the autotransplantation of functionalised adipocytes may lead to a clinical application in the treatment of diabetes.     

Regulation of insulin-stimulated glucose transport by chronic glucose exposure in 3T3-L1 adipocytes.

Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24 h with low (1 mM) or high (25 mM) glucose, and glucose transport was subsequently analyzed. 100 nM insulin was present throughout the experiments. 24 h incubation with 1 mM glucose caused a 2.3+/-0.4 fold increase in glucose transport activity, compared to the values obtained with 25 mM glucose. This difference was not observed when 24 h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6 h incubation with 1 mM glucose, but was increased at 12 h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1 mM glucose compared to 25 mM glucose. The addition of azaserin or deprivation of glutamine at 25 mM glucose did not increase the glucose transport activity to the level obtained with 1 mM glucose. PD98059 did not affect glucose transport activity when incubated with 1 mM or 25 mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1 mM) and high (25 mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.     

Intermittent and rhythmic exposure to melatonin in primary cultured adipocytes enhances the insulin and dexamethasone effects on leptin expression

Considering the cyclic characteristic of production and secretion of pineal melatonin, it is reasonable to assume that this oscillation might be important in determining the variety of its circadian and seasonal effects. To simulate this physiological condition in vitro, isolated adipocytes were exposed to melatonin in a circadian-like pattern by adding the hormone to the incubating medium during 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day). This intermittent procedure was interrupted when three cycles with melatonin were fulfilled (60-hr incubation). Here, we report the effects of melatonin (1 nM) added intermittently or continuously to the incubating medium alone or in combination with insulin (5 nM) and/or dexamethasone (7 nM) on leptin release and expression by rat adipocytes. After acute 12-hr incubation neither melatonin nor insulin alone affected leptin expression, but together they increased it by 105%. Dexamethasone increased leptin mRNA content and release (70%) but this effect was not enhanced by melatonin. Nevertheless, after 60 hr under intermittent melatonin, we observed a synergism between melatonin and dexamethasone. This interaction promoted an increment (75% compared with dexamethasone alone) in leptin release and expression. Our results suggest that circadian-like exposure to melatonin potentiates the dexamethasone action and is important to the effects promoted by insulin on leptin expression. Based on an in vitro approach, this work helps to clarify the physiological relevance and the repercussions of the in vivo circadian pattern of melatonin secretion.     

Insulin-mediated cellular insulin resistance decreases osmotic shock-induced glucose transport in 3T3-L1 adipocytes

Similar to insulin, osmotic shock treatment of 3T3-L1 adipocytes causes translocation of GLUT4 protein to the plasma membrane and an increase in glucose transport activity. In our study, we evaluated the effect of chronic insulin treatment on the osmotic shock signaling pathway leading to GLUT4 translocation and glucose uptake. We found that chronic administration of insulin to the adipocytes induced cellular resistance to osmotic shock-stimulated GLUT4 translocation and glucose transport. We found that chronic insulin treatment attenuated shock-induced Gab-1 tyrosine phosphorylation. Furthermore, chronic insulin exposure led to a marked impairment in the ability of Gab-1 to associate with p85 subunit of PI 3-kinase in response to acute shock and insulin stimulation. Cells that were chronically treated with insulin showed a 70% and a 61% decrease in Gab-1 associated PI 3-kinase activity in shock- vs. insulin-treated cells, respectively. In addition, we found that chronic insulin treatment inhibited both insulin- and osmotic shock-induced membrane ruffling, indicating that two PI 3-kinase dependent effects, GLUT4 translocation and membrane ruffling are decreased in chronically insulin-treated cells. The results described above clearly demonstrate that chronic insulin treatment induces a state of cellular resistance to osmotic shock signal transduction.     

Hexose transport in human adipocytes: Factors influencing the response to insulin and kinetics of methylglucose and glucose transport

Summary Optimal experimental conditions were defined for measuring the initial uptake rate of the non-metabolizable sugar analogue 3-O-methylglucose in non-stimulated and insulin-stimulated human adipocytes. The permeability of the adipocyte plasma membrane for tracer methylglucose (100 mol/l) was 2.9×10^-7 cm x s^-1 at 37 °C and slightly lower at 20 °C. At 37 °C and pH 7.4 insulin (5 nmol/l) increased the permeability about twofold (range 1.5 to fivefold) with half maximal effect at about 100 pmol/l. At pH 7.0 the dose response curve for the insulin effect on the uptake rate of methylglucose was shifted about 2.5-fold to the right. The permeability to L-glucose due to simple diffusion was estimated as 3.0×10^-10 cm x s^-1 suggesting that uptake of methylglucose occurs almost exclusively by facilitated diffusion. The K_m for methylglucose equilibrium exchange in insulin stimulated cells was about 4.8 mmol/l. The initial uptake of tracer methylglucose in insulin-stimulated cells was inhibited by unlabelled methylglucose and by D-glucose with inhibition constants of about 3.8 and 7.7 mmol/l, respectively. Uptake of tracer 2-deoxyglucose (50 umol/l) in insulin-stimulated adipocytes was linear from 10 s to 5 min whereas the rate of uptake in the presence of 3 mmol/l of D-glucose was markedly decreased suggesting that deoxyglucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose.     

Glucose and insulin regulate insulin sensitivity in primary cultured adipocytes without affecting insulin receptor kinase activity

We have previously shown in primary cultured adipocytes that chronic insulin exposure decreases insulin's subsequent ability to maximally restimulate the glucose transport system, and that extracellular glucose potentiates this ligand-induced defect in maximal insulin responsiveness. To examine whether glucose could also modulate insulin sensitivity (i.e. acute insulin effects at submaximal concentrations), adipocytes were cultured for 5 and 24 h in the absence and presence of various glucose and insulin concentrations. Then, after washing cells to remove any insulin and allow for full deactivation of transport, we assessed the dose response of insulin's acute ability to stimulate 2-deoxyglucose transport, bind to cell surface receptors, and activate insulin receptor tyrosine kinase activity. After 5 h, glucose and insulin alone had no chronic regulatory effects; however, in combination, these agents were able to decrease insulin sensitivity. In cells preincubated with 50 ng/ml insulin, the insulin ED50 for acute stimulation of glucose transport was increased by 65% and 116% as medium glucose was raised to 5 and 20 mM, respectively, relative to that at 0 mM glucose. After 24 h, chronic exposure to either glucose (20 mM) or insulin (50 ng/ml) alone increased the ED50 value by 52%, and, together they acted synergistically to increase the ED50 by 183%. While glucose and insulin independently and synergistically impaired insulin sensitivity, both agents were necessary for coregulation of maximal insulin responsiveness (confirming our previous observation). Insulin receptor down-regulation (18%) was observed after 24 h (but not 5 h) in insulin-treated cells; however, the major portion of the decrease in insulin sensitivity was due to uncoupling of occupied insulin receptors from stimulation of the glucose transport system. To further determine the mechanism for postbinding desensitization, we tested for concordant regulation of insulin receptor kinase activity. Insulin's ability to stimulate the receptor tyrosine kinase was assessed by multiple methods, including 1) receptor autophosphorylation and phosphorylation of Glu4-Tyr1 by solubilized insulin receptors activated in vitro, 2) histone-2B phosphorylation by receptors that were stimulated in intact cells and then solubilized under conditions that preserve the in cellulo phosphorylation state, and 3) receptor autophosphorylation and phosphorylation of pp180 in intact cells. Long term treatment (24 h) with glucose (10 mM) and insulin (50 ng/ml) markedly decreased insulin sensitivity (and receptor coupling), but did not affect insulin receptor kinase activity in any of these studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin receptor substrates-1 and -2 are both depleted but via different mechanisms after down-regulation of glucose transport in rat adipocytes

Alterations in muscle and adipose tissue insulin receptor substrate (IRS)-1 and IRS-2 are associated with, and commonly believed to contribute to, development of insulin resistance. In this study, we investigated the mechanisms behind previously observed reductions in IRS levels due to high concentrations of glucose and insulin and their significance in the impairment of glucose uptake capacity in primary rat adipocytes. Semiquantitative RT-PCR analysis showed that insulin (10(4) microU/ml) alone or in combination with glucose (15 mm) markedly suppressed IRS-2 gene expression, whereas IRS-1 mRNA was unaffected by the culture conditions. The negative effect of a high glucose/high insulin setting on IRS-1 protein level was still exerted when protein synthesis was inhibited with cycloheximide. Impairment of glucose uptake capacity after treatment with high glucose and insulin was most pronounced after 3 h, whereas IRS-1 and IRS-2 protein levels were unaffected up to 6 h but were reduced after 16 h. Moreover, impaired glucose uptake capacity could only partially be reversed by subsequent incubation at physiological conditions. These novel results suggest that: 1) in a high glucose/high insulin setting depletion of IRS-1 and IRS-2 protein, respectively, occurs via different mechanisms, and IRS-2 gene expression is suppressed, whereas IRS-1 depletion is due to posttranslational mechanisms; 2) IRS-1 and IRS-2 protein depletion is a secondary event in the development of insulin resistance in this model of hyperglycemia/hyperinsulinemia; and 3) depletion of cellular IRS in adipose tissue may be a consequence rather than a cause of insulin resistance and hyperinsulinemia in type 2 diabetes.     

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.     

Sex differences in insulin action on glucose transport and transporters in human omental adipocytes

We examined the effects of insulin on glucose transport and subcellular glucose transporter distribution in isolated omental adipose cells from men and women. 3-O-Methylglucose transport was measured in intact cells, and the number of glucose transporters in plasma membranes and low density microsomal membranes was determined using the cytochalasin B binding assay. Compared to adipocytes from women, omental adipocytes from men were characterized by 1) 2-fold larger cell volume; 2) 4- to 5- and 2.5-fold higher glucose transport rates when calculated per cell or per cell surface area, respectively, in either basal or insulin-stimulated cells; 3) similar 2-fold insulin stimulating effect per se; and 4) equal concentrations of transporters in both fractions examined, but a 2-fold increase in their total number per cell. Additionally, although not directly measured, the calculated glucose transporter activity in basal plasma membranes prepared from adipocytes from men was 2.7-fold higher than that in women, and insulin further induced a 30% increase in that activity. Thus, a sex-related difference was found between the number of glucose transporters per cell and the resultant glucose transport activity of the intact cells. Together with the increased specific activity of glucose transporters in men compared to women, our findings indicate a sex-related difference in adipocyte glucose transport, mainly due to an increase in the number and modulation of the intrinsic activity of glucose transporters in the plasma membrane.     

Increased insulin binding to adipocytes and monocytes and increased insulin sensitivity of glucose transport and metabolism in adipocytes from non-insulin-dependent diabetics after a low-fat/high-starch/high-fiber diet

Nine non-insulin-dependent diabetics were studied before and after 3 weeks on an isoenergetic high-fiber/high-starch/low-fat diet (alternative diet), and nine non-insulin-dependent diabetics were studied on their usual diet. In the group that ate the alternative diet, the intake of fiber and starch increased 120% and 53%, whereas fat intake decreased 31%. Diabetes control improved as demonstrated by decreased fasting plasma glucose (P < 0.05) and 24-hour urinary glucose excretion (P < 0.05). The in vivo insulin action increased (K_IVITT increased, P < 0.05) with no change in fasting serum insulin levels. In fat cells obtained from patients in the alternative-diet group, insulin receptor binding increased (P < 0.05) after the change of diet. Insulin binding to purified monocytes (more than 95% monocytes) also increased (P < 0.05), whereas no change was found in insulin binding to erythrocytes. When lipogenesis was studied at a tracer glucose concentration at which glucose transport seems to be rate limiting, insulin sensitivity increased (P < 0.02). This is the predicted consequence of increased receptor binding. Moreover, when CO₂ production and lipogenesis were studied at a higher glucose concentration, where steps beyond transport seem to be rate limiting for glucose metabolism, increased insulin sensitivity was also observed. In contrast, no change was found in maximal insulin responsiveness. Fat and blood cells from the patients who continued on their usual diet showed no changes of the mentioned quantities. In the total group of non-insulin-dependent diabetics, fat cell insulin binding as well as rates (both basal and maximal insulin-stimulated rates) of glucose transport, CO₂ production, and lipogenesis were very heterogeneous, but when results from the first and second fat biopsy from the same patient were compared, these values varied only slightly. We conclude that the beneficial effect of a high-fiber/high-starch/low-fat diet on metabolic control in diabetics may in part be mediated through an increased insulin-binding ability of target cells for insulin, which causes an increased insulin sensitivity in these cells.     

Tissue-specific difference in the molecular mechanisms for the development of acute insulin resistance after injury

Acute insulin resistance occurs after injury, hemorrhage, infection, and critical illness. However, little is known about the development of this acute insulin-resistant state. In the current study, we found that insulin resistance develops rapidly in skeletal muscle, with the earliest insulin signaling defects at 60 min. However, defects in insulin signaling were measurable even earlier in liver, by as soon as 15 min after hemorrhage. To begin to understand the mechanisms for the development of acute insulin resistance, serine phosphorylation of insulin receptor substrate (IRS)-1 and c-Jun N-terminal kinase phosphorylation/activation was investigated. These markers (and possible contributors) of insulin resistance were increased in the liver after hemorrhage but not measurable in skeletal muscle. Because glucocorticoids are important counterregulatory hormones responsible for glucose homeostasis, a glucocorticoid synthesis inhibitor, metyrapone, and a glucocorticoid receptor antagonist, RU486, were administered to adult rats prior to hemorrhage. In the liver, the defects of insulin signaling after hemorrhage, including reduced tyrosine phosphorylation of the insulin receptor and IRS-1, association between IRS-1 and phosphatidylinositol 3-kinase and serine phosphorylation of Akt in response to insulin were not altered by pretreatment of rats with metyrapone or RU486. In contrast, hemorrhage-induced defects in insulin signaling were dramatically reversed in skeletal muscle, indicating a prevention of insulin resistance in muscle. These results suggest that distinct mechanisms for hemorrhage-induced acute insulin resistance are present in these two tissues and that glucocorticoids are involved in the rapid development of insulin resistance in skeletal muscle, but not in the liver, after hemorrhage.     

Rapid increase in the insulin sensitivity of rat adipocytes after intravenous glucose administration

To study the mechanism underlying the enhancement of insulin action after carbohydrate challenge, rats were given an iv infusion of 100 mg glucose or saline. Adipocytes were isolated 1, 2, and 3 h after the infusions, and their ability to respond to insulin was examined. Two and 3 h postglucose, the cells showed a 50% increase in insulin sensitivity, as measured by glucose incorporation into total lipid and 2-deoxyglucose uptake. The effect was characterized by a leftward shift in the insulin dose-response curve. There was no change in maximum insulin stimulation. Insulin binding by the fat cells and by liver plasma membranes was not altered by glucose administration. These results demonstrate the presence of a physiological process that acutely enhances the insulin sensitivity of rat adipocytes after iv glucose infusion.     

Molecular mechanisms of insulin resistance in chronic hepatitis C

It is now widely recognized that chronic hepatitis C （CHC） is associated with insulin resistance （IR） and type 2 diabetes, so can be considered a metabolic disease. IR is most strongly associated with hepatitis C virus （HCV） genotype 1, in contrast to hepatic steatosis, which is associated with genotype 3 infection. Apart from the well-described complications of diabetes, IR in CHC predicts faster progression to fibrosis and cirrhosis that may culminate in liver failure and hepatocellular carcinoma. More recently, it has been recognized that IR in CHC predicts a poor response to antiviral therapy. The molecular mechanisms for the association between IR and HC＇V infection are not well defined. This review will elaborate on the clinical associations between CHC and IR and summarize current knowledge regarding the molecular mechanisms that potentially mediate HCV-associated IR.     

Characterization of the insulin resistance of glucose utilization in adipocytes from patients with hyper- and hypothyroidism

Insulin action on glucose utilization was characterized in adipocytes from 10 thyrotoxic patients, 6 hypothyroid patients and 10 age- and sex-matched control subjects. In thyrotoxic patients insulin binding at low insulin concentrations was reduced (P less than 0.05) and accompanied by impaired insulin sensitivity of glucose transport (P less than 0.02), glucose oxidation (P less than 0.05) and lipogenesis (P less than 0.05). Glucose transport and glucose oxidation rates also exhibited depressed maximal insulin responsiveness (P less than 0.05). In hypothyroid patients insulin binding was reduced, too, (P less than 0.05) and associated with impaired sensitivity to insulin of glucose transport (P less than 0.05). Both glucose transport and lipogenesis rates showed decreased maximal insulin responsiveness (P less than 0.05). In conclusion: In man, both hyper- and hypothyroidism are characterized by insulin resistance of adipocyte glucose utilization localized to insulin binding as well as to insulin-stimulated glucose transport and metabolism.