Long-term regulation of hexose transport by insulin in cultured mouse (3T3) adipocytes

Summary Observations in vivo suggest that insulin acts as a long-term regulator of hexose uptake in fat cells. In the present study, we examined the long-term effect of insulin on hexose uptake in vitro. Exposure of fully differentiated mouse 3T3-L₁ adipocytes to insulin induced a time-, concentration-, and protein synthesis-dependent increase in basal 2-deoxyglucose uptake (up to 40%) and a decrease in the acute insulin response. The decrease in insulin effect was due to post-receptor alterations, since insulin binding was not substantially altered. The increase in basal 2-deoxyglucose uptake was due to an increase in the apparent V_max of the transport system rather than to the observed increase (30%) in hexokinase activity, since the concentration of non-phosphorylated 2-deoxyglucose inside the cell was far below the extracellular concentration. The increase in apparent V_max was most likely due to a protein synthesis-dependent increase in de novo synthesis of hexose transporters. Glucose was not essential for the effect. The mechanism responsible for the loss in insulin response remains to be solved. It can be concluded that insulin has the ability to act as a long-term regulator of hexose uptake in fat cells in vitro.     

Insulin binding and hexose transport in rat adipocytes

Summary Insulin binding, initial velocity of [¹⁴C]methylglucose transport, uptake of [¹⁴C]deoxy-glucose and conversion of [U-¹⁴C]glucose to CO₂, glyceride-glycerol and fatty acids were measured at 37 °C in adipocytes from rats of different weights (135–450 g) and therefore with different mean cell volumes (53–389 pl). Insulin binding per cell increased with increasing cell size and binding was 2.3 times higher in the largest cells than in the smallest cells with tracer alone. The difference was largely accounted for by an increase in the apparent affinity. Influx of methylglucose per cell increased with increasing cell size in the absence of insulin and remained constant as a function of cell size in its presence. The effect of insulin ranged from 11 fold in small cells to 3.5 fold in large cells. The rate of conversion of [U-¹⁴C]glucose to CO₂ and lipids was about half of the rate of methylglucose transport under all conditions. In contrast, the uptake of deoxyglucose in insulin-stimulated cells decreased markedly with increasing cell size. Increasing cell size caused a small decrease in sensitivity which could be explained by a smaller amount of insulin bound per unit surface area. The results show that increasing cell size/animal weight causes changes in insulin binding which may explain changes in sensitivity. In addition, the hexose transport system is modified in a way which is not explained by changes in insulin binding. Finally, changes in deoxyglucose uptake with cell size do not parallel changes in methylglucose transport.     

Hexose specificity for downregulation of HepG2/brain-type glucose transporter gene expression in L6 myocytes

Summary Glucose deprivation of L6 myocytes results in the upregulation of glucose transporter activity, protein and mRNA. We have investigated the downregulation of transporter gene expression by glucose and other hexoses in glucose-deprived L6 myocytes. Glucose transport activity was measured as the uptake of ³H-2-deoxyglucose. Transporter protein and mRNA were detected by immunoblot and Northern blot analysis, respectively, with probes to the rat brain glucose transporter. Glucose deprivation of myocytes, in the absence and presence of insulin, increased ³H-2-deoxyglucose uptake, transporter protein and mRNA levels. Refeeding with glucose reversed the glucose deprivation effects on transport activity and mRNA within 12 h, with half-maximal effects at 1–2 mmol/l glucose. Mannose fully substituted for glucose. Refeeding with the non-metabolisable glucose analogues 2-deoxyglucose and 3-0-methylglucose, or with glucosamine or mannitol, downregulated ³H-2-deoxyglucose uptake but had little or no effect on transporter protein and mRNA expression. In contrast, glucose-6-phosphate markedly increased ³H-2-deoxyglucose uptake but partly downregulated transporter mRNA levels, whereas galactose had a small stimulatory effect on both ³H-2-deoxyglucose uptake and transporter mRNA; neither affected transporter protein levels. The transporter mRNA level was not affected by several metabolites (pyruvate, glyceraldehyde, glycerol) and amino acids (alanine, glutamine). These findings indicate that (i) there are independent pathways for hexose regulation of transport activity, protein and mRNA and (ii) down-regulation of transporter mRNA requires metabolism beyond hexose phosphate whereas glucose uptake may be regulated by direct interaction of hexoses with the transporter.     

The ras signaling pathway mimics insulin action on glucose transporter translocation.

Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein. We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with non-transfected cells. Insulin stimulated hexose uptake by approximately 2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras61K-expressing fibroblasts. Overexpression of N-Ras61K also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras61K-expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras61K-expressing compared with parental adipocytes. However, expression of N-Ras61K in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras61K expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway.     

Antagonistic effects of a covalently dimerized insulin derivative on insulin receptors in 3T3-L1 adipocytes.

In the present study we describe the antagonistic effects of the covalently dimerized insulin derivative B29,B29'-suberoyl-insulin on insulin receptors in 3T3-L1 mouse cells. In differentiated 3T3-L1 adipocytes, the derivative fully inhibits binding of 125I-labeled insulin to its receptor with about the same affinity as unlabeled insulin. In contrast, the dimerized derivative only partially (approximately 20%) mimics insulin's effects on glucose transport and DNA synthesis in the absence of insulin. In the presence of insulin, the agent competitively inhibits insulin-stimulated DNA synthesis ([3H]thymidine incorporation into total DNA), glucose transport activity (2-deoxyglucose uptake rate), and insulin receptor tyrosine kinase activity. In rat adipocytes, in contrast, the dimerized derivative stimulates glucose transport (initial 3-O-methylglucose as well as 2-deoxyglucose uptake rates) to the same extent as insulin does, and it fails to inhibit the effect of insulin. The data indicate that the dimerized insulin derivative B29,B29'-suberoyl-insulin is an insulin receptor antagonist (partial agonist) which retains a moderate intrinsic activity. The effects of this agent reveal a striking difference in insulin receptor-mediated stimulation of glucose transport between 3T3-L1 fatty fibroblasts and the mature rat adipocyte.     

Phospholipid-induced inhibition of insulin-stimulated glucose transport in isolated adipocytes: interactions of phospholipids with inhibitors of glucose transport and insulinmimetic agents

Upon interaction with phospholipid vesicles containing phosphatidylserine, isolated rat adipocytes demonstrate an inhibition of insulin-stimulated hexose uptake. In order to elucidate the mechanism of this effect, adipocytes were treated with agents, alone or in combination with vesicles, which affected the insulin-sensitive response at the receptor and post-receptor level. The effect of vesicles at a maximal inhibitory concentration proved to be non-additive with dexamethasone, suggesting that vesicles may act in a manner similar to this agent. In contrast, fat cells treated with vesicles and N-ethylmaleimide (NEM) or trypsin at submaximally effective concentrations demonstrate a partially additive inhibition of insulin-stimulated 2-deoxyglucose uptake. Vesicle treatment of adipocytes before stimulation with agents which mimic insulin, such as Con A and H2O2, demonstrates the same effects as insulin with respect to hexose uptake. These results support the contention that vesicles inhibit insulin action at least partially at the post-receptor level, and may directly interfere with the hexose transport site.     

Partial insulin resistance in the mouse BC3H-1 cell line: absent hexose-independent actions of insulin

We have studied the regulation of glycogen metabolism by insulin in the insulin-sensitive nonfusing muscle cell line BC3H-1. The basal percentage of glycogen synthase I activity was not altered by insulin alone at any concentration, time of exposure, or age of cells tested. The addition of glucose or 2-deoxyglucose to the glucose- and serum-free incubation medium caused a 2-fold increase in glycogen synthase I activity over basal levels, and the effect was enhanced to 3-fold if insulin was added to the medium. Glycogen phosphorylase a activity was not altered by incubation in the presence of insulin, but was lowered by the addition of 2-deoxyglucose. This effect was also enhanced in the presence of insulin. The effect of exogenously added sugar occurred only if a 6-phosphorylatable hexose was used. The effect seen with 2-deoxyglucose was stable to Sephadex G-25 desalting, suggesting that activation of glycogen synthase was the result of a stable (covalent) modification of the enzyme. We were also able to demonstrate the presence of glucose-6-phosphate-activatable glycogen synthase phosphatase activity in the myocytes. The effect of 2-deoxyglucose in the presence or absence of insulin could be completely reversed by including cytochalasin B in the medium, suggesting that both the effect of hexose and the insulin enhancement of its effect were entirely dependent on carrier-mediated hexose uptake. Four insulin-mimetic agents, H2O2 Concanavalin A, Na orthovanadate, and antiinsulin receptor B2 serum, were also tested. Despite different mechanisms of action, each agent qualitatively mimicked insulin in the myocytes. All stimulated hexose transport, glucose incorporation into glycogen, and hexose-dependent activation of glycogen synthase in a manner not additive with insulin, but none increased basal glycogen synthase I activity in the absence of hexose. These results suggest that although insulin is capable of regulating glycogen metabolism both by increasing the uptake of sugar and by altering the activation state of glycogen synthase and phosphorylase, these effects are entirely due to the stimulation of hexose uptake, and hexose-independent actions of insulin are absent in BC3H-1 cells.     

Insulin binding, glucose oxidation, and methylglucose transport in isolated adipocytes from pregnant rats near term

To elucidate the mechanism of insulin resistance during late pregnancy, we studied [125I]iodoinsulin binding, [1-14C]glucose oxidation, and 3-O-[methyl-14C]glucose transport in adipocytes isolated from pregnant rats on day 19 or 20 of gestation. Neither the affinity or number of insulin receptors on pregnant rat adipocytes differed from those on age-matched nonpregnant female rats. Insulin-stimulated glucose oxidation was reduced in the pregnant rat adipocytes. The maximum velocity of insulin-stimulated methylglucose transport was also significantly reduced in the pregnant rat adipocytes. These results suggest that insulin resistance in isolated adipocytes from pregnant rats near term is caused by some postreceptor changes, one of which is a reduction in the number and/or mobility of insulin-stimulated hexose transporters.     

The alpha subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphorylation-independent pathway.

The receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of an alpha and beta subunit, which together form the high-affinity receptor. The alpha subunit by itself binds ligand at low affinity, whereas the isolated beta subunit does not bind GM-CSF. It is generally believed that the high-affinity receptor is responsible for the multiple functions of GM-CSF and that the isolated alpha subunit (GMR alpha) does not transduce a signal. Xenopus laevis oocytes injected with RNA encoding human GMR alpha expressed up to 10(10) low-affinity sites for GM-CSF (Kd = 6 nM). GM-CSF binding to the alpha subunit expressed in Xenopus oocytes caused activation of 2-deoxyglucose transport through endogenous glucose transporters. 2-Deoxyglucose transport was stimulated by similar low concentrations of GM-CSF in HL-60 leukemia cells as well as normal human neutrophils and Xenopus oocytes expressing GMR alpha. Engagement of the isolated alpha subunit in oocytes did not lead to protein phosphorylation or tyrosine phosphorylation of mitogen-activated protein kinase (MAP kinase). Staurosporin and genistein inhibited GM-CSF-induced tyrosine phosphorylation of MAP kinase in human neutrophils and HL-60 cells without affecting GM-CSF-stimulated uptake of 2-deoxyglucose. These results provide direct evidence that the isolated alpha subunit signals for hexose transport and can do so without engagement of the kinase cascade. Our data also indicate that signaling for hexose uptake may occur in a phosphorylation-independent manner in cells expressing the high-affinity GM-CSF receptor.     

Effect of duration of diabetic state on insulin action in isolated rat soleus muscles

We studied the effect of the duration of diabetic state on insulin action in skeletal muscle by measuring insulin binding, 2-deoxyglucose uptake, and intracellular glucose metabolism in isolated soleus muscles from streptozotocin-induced diabetic rats. Insulin binding to soleus muscles from diabetic rats was increased over that from controls. Glucose transport activity was determined by measuring the 2-deoxyglucose uptake at the concentration of 1 mmol/L at 25 degrees C. In the rats with diabetes of one week duration, insulin-stimulated 2-deoxyglucose uptake was not impaired, whereas basal 2-deoxyglucose uptake was decreased. However, the diabetic rats with two weeks duration revealed a 35.6% decrease in the insulin-stimulated 2-deoxyglucose uptake. Furthermore, four week duration of diabetic state led to a 60% decrease both in basal and insulin-stimulated 2-deoxyglucose uptake. Total glucose utilization was estimated as the total amount of glucose incorporated into muscle and lactate released into the medium following incubation at 37 degrees C, with 5 mmol/L glucose. The diabetic rats with one week duration did not demonstrate any changes in total glucose utilization both in basal and insulin-stimulated state. However more than two weeks duration of diabetes led to a 30% to 35% decrease both in basal and insulin-stimulated total glucose utilization, similar to the findings in the 2-deoxyglucose uptake study. We concluded that prolonged insulinopenia led to decreased glucose transport and intracellular glucose metabolism and resulted in insulin resistance in skeletal muscles.     

Dexamethasone inhibition of hydrogen peroxide-stimulated glucose transport

Although an action of corticosteroids to inhibit glucose transport is well known, the mechanism by which this is brought about has been unclear. Some evidence has suggested an action on insulin receptors, but a postbinding or postreceptor effect has also been reported. As hydrogen peroxide acts at a postbinding site to stimulate glucose transport, studies were carried out in 3T3-L1 fibroblasts to determine whether corticosteroids would inhibit hydrogen peroxide-induced glucose transport. In this cell type, both insulin and hydrogen peroxide produced a marked increase in glucose transport after a 30-min incubation. The increase produced by hydrogen peroxide, as well as insulin, was inhibited by previous incubation of the cells with dexamethasone. These findings give further support to the conclusion that dexamethasone has effects on glucose transport at a postbinding site. As changes in membrane lipids influence the movement and/or activity of glucose transporters, and dexamethasone alters membrane lipids, dexamethasone-induced changes in the lipids of the plasma membrane may be important in the mediation of the steroid effect upon glucose transport.     

Glucose starvation is required for insulin stimulation of glucose uptake and metabolism in cultured microvascular endothelial cells

In the present study we determined the uptake and disposition of glucose in serum-deprived rabbit coronary microvessel endothelial (RCME) cells. RCME cells exhibited stereospecific hexose uptake inhibited by cytochalasin B. Pretreatment of the RCME cells with potassium cyanide or 2,4-dinitrophenol inhibited 2-deoxyglucose uptake but not 3-O-methylglucose transport. A major proportion (30-60%) of the 2-deoxyglucose present in the RCME cells was not phosphorylated. These two observations suggested that the rate-limiting step in the uptake of 2-deoxyglucose was not transport but rather the phosphorylation of 2-deoxyglucose to 2-deoxyglucose 6-phosphate. When glucose-deprived cells were incubated 2 hr with [U-14C]glucose the disposition of the label was as follows: glycogen 60%, acid-soluble fraction 30%, and lipid less than 5%. In contrast glucose-fed cells exhibited lower overall glucose incorporation, and a slightly different disposition: glycogen 45%, acid-soluble fraction 50%, and lipid 5%. Glucose-deprived RCME cells also exhibited greater basal levels of 2-deoxyglucose uptake compared to glucose-fed cells. RCME cells incubated in the absence of glucose and serum for 16 hr exhibited dose-dependent insulin stimulation of hexose uptake and subsequent metabolism to macromolecules (i.e., glycogen and the acid-soluble fraction). Significant effects of insulin were observed with concentrations as low as 2 x 10(-10) M, well within the physiological range. In contrast, cells preincubated in serum-free culture medium containing 5.5 mM glucose did not exhibit insulin-enhanced hexose uptake or glucose metabolism (even at doses as high as 10(-7) M). These studies indicate that the effects of insulin on rabbit coronary microvascular endothelial cell glucose uptake and metabolism require both serum and glucose deprivation.     

Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes.

Previous studies indicated that the erythroidtype (GLUT1) glucose transporter isoform contributes to basal but not insulin-stimulated hexose transport in mouse 3T3-L1 adipocytes. In the present studies it was found that basal hexose uptake in 3T3-L1 adipocytes was about 50% lower than that in 3T3-L1 or CHO-K1 fibroblasts. Intrinsic catalytic activities of GLUT1 transporters in CHO-K1 and 3T3-L1 cells were compared by normalizing these hexose transport rates to GLUT1 content on the cell surface, as measured by two independent methods. Cell surface GLUT1 levels in 3T3-L1 fibroblasts and adipocytes were about 10- and 25-fold higher, respectively, than in CHO-K1 fibroblasts, as assessed with an anti-GLUT1 exofacial domain antiserum, delta. The large excess of cell surface GLUT1 transporters in 3T3-L1 adipocytes relative to CHO-K1 fibroblasts was confirmed by GLUT1 protein immunoblot analysis and by photoaffinity labelling (with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n) of glucose transporters in isolated plasma membranes. Thus, GLUT1 intrinsic activity is markedly reduced in 3T3-L1 fibroblasts compared with the CHO-K1 fibroblasts, and further reduction occurs upon differentiation to adipocytes. Intrinsic catalytic activities specifically associated with heterologously expressed human GLUT1 protein in transfected CHO-K1 versus 3T3-L1 cells were determined by subtracting appropriate control cell values for hexose transport and delta-antibody binding from those determined in the transfected cells expressing high levels of human GLUT1. The results confirmed a greater than 90% inhibition of the intrinsic catalytic activity of human GLUT1 transporters on the surface of mouse 3T3-L1 adipocytes relative to CHO-K1 fibroblasts. We conclude that a mechanism that markedly suppresses basal hexose transport catalyzed by GLUT1 is a major contributor to the dramatic insulin sensitivity of glucose uptake in 3T3-L1 adipocytes.     

Dexamethasone regulates the glucose transport system in primary cultured adipocytes: different mechanisms of insulin resistance after acute and chronic exposure

We have studied the ability of dexamethasone to regulate the glucose transport system in primary cultured adipocytes and delineated the mechanisms of insulin resistance after both acute and chronic treatment. Acutely, 20 nM dexamethasone led to a 65% decrease in basal and a 31% decrement in maximally insulin-stimulated glucose transport (ED50 = 3-4 nM; t1/2 = 50 min). These effects were maximal by 90-120 min, and a plateau was maintained over an additional 1-1.5 h. Chronic dexamethasone exposure (24 h) led to a more profound decrease in basal (77%; ED50 = 0.4 nM) and maximally stimulated (55%; ED50 = 1.0 nM) rates of glucose transport and shifted the transport: insulin dose-response curve to the right by increasing the half-maximally effective insulin concentration from 0.2 to 0.4 ng/ml. Dexamethasone did not affect cell surface insulin binding over 24 h. Both the short and long term effects of dexamethasone were partially blocked by the combined presence of insulin during preincubation and were not modulated by glucose. We also assessed effects on the number and cellular distribution of glucose transporter proteins using the cytochalasin-B binding assay. After 2 h, dexamethasone (30 nM) decreased the number of glucose transporters in plasma membranes by 30% in basal cells and by 41% in maximally insulin-stimulated cells, while increasing the number of low density microsomal transporters by 22-23% (P = NS). Transporter number in a total cellular membrane fraction was unaltered by short term dexamethasone. Chronic dexamethasone exposure (24 h) decreased plasma membrane and low density microsomal transporters by 30-50% in both basal and insulin-stimulated cells and depleted transporters by 43% in a total cellular membrane fraction. In conclusion, 1) dexamethasone induces progressive insulin resistance by sequentially regulating multiple aspects of the insulin-responsive glucose transport system. At early times (2 h) dexamethasone impairs insulin's ability to translocate intracellular glucose transporters to the cell surface and with more chronic exposure (24 h), depletes the total number of cellular transporters. 2) Glucose modulates desensitization of the glucose transport system by insulin, but not by dexamethasone, and thus, there are both glucose-dependent and -independent mechanisms of insulin resistance. 3) Insulin can heterologously inhibit dexamethasone's effects on glucose transport at both early and late phases of desensitization. These studies highlight the complex hormonal regulation at the glucose transport system.     

Receptor binding and biologic activity of biosynthetic human insulin and mini-proinsulin produced by recombinant gene technology

Human insulin and its precursor, mini-proinsulin, made with a new biosynthetic method, were tested for their receptor binding, biologic action, and antibody binding ability. The structure of mini-proinsulin is similar to that of proinsulin with a shortened C-peptide, B(1-29)-Ala-Ala-Lys-A(1-21) insulin. The ability of biosynthetic human insulin to bind to receptors, to stimulate 2-deoxyglucose uptake in isolated adipocytes, and to bind to insulin antibody was comparable to that of semisynthetic human insulin. The ability of mini-proinsulin to bind to insulin receptors and to stimulate 2-deoxyglucose uptake in adipocytes was 0.5 and 0.2% that of human insulin, whereas the corresponding abilities of proinsulin were 5 and 3%, respectively. Despite having less receptor binding and biologic activity, mini-proinsulin demonstrated higher affinity for the insulin antibody than did proinsulin. These results suggest that biosynthetic human insulin behaves similarly to semisynthetic human insulin in its receptor binding and biologic activity, and that the shortened C-peptide region reduces receptor binding by fixing or covering the N-terminal region of the A chain, which is important for receptor binding.     

Expression of a dominant-negative Ras mutant does not affect stimulation of glucose uptake and glycogen synthesis by insulin

Summary It has previously been shown that insulin-induced stimulation of glucose uptake and glycogen synthesis requires activation of phosphatidylinositol-3-kinase (PI3kinase). Insulin also induces formation of RasGTP in cells and various studies have yielded inconsistent data with respect to the contribution of signalling pathways activated by RasGTP, to insulin-stimulated glucose uptake and glycogen synthesis. We have examined the requirement of RasGTP-mediated signalling for these insulin responses by expression of a dominant negative mutant of Ras (RasN17) in cells by vaccinia virus mediated gene transfer. This Ras-mutant abrogates the signalling pathways mediated by endogenous RasGTP. Subsequently, the ability of insulin to stimulate 2-deoxyglucose uptake and glycogen was examined. We observed that expression of RasN17 in 3T3L1 adipocytes did not affect the stimulation of hexose uptake by insulin. Similarly, expression of RasN17 in A14 cells, an NIH 3T3-derived cell line with high expression of insulin receptors, did not affect insulin-induced stimulation of glycogen synthesis. In both cell lines, insulin-induced phosphorylation of Mapkinase (Erk1,2) was abrogated after expression of RasN17, demonstrating the functional interference by RasN17 with signalling mediated by endogenous RasGTP. Wortmannin, an inhibitor of PBkinase, abolished dose-dependently the insulin-induced stimulation of hexose uptake and glycogen synthesis without an effect on RasGTP levels in both cell types. We conclude that stimulation of glucose transport and glycogen synthesis by insulin occurs independently of RasGTP-mediated signalling.Abbreviations DMEM Dulbecco's modified Eagle's medium - ECL enhanced chemiluminescence - MAPkinase mitogen-activated protein kinase - PI3kinase phosphatidylinositol-3 kinase - IRS insulin receptor substrate - BSA bovine serum albumin     

Protein kinase C inhibitors block insulin and PMA-stimulated hexose transport in isolated rat adipocytes and BC3H-1 myocytes

Effects of protein kinase C (PKC) inhibitors and "down-regulation" on insulin and PMA-stimulated 2-deoxyglucose transport were determined in isolated rat adipocytes or BC3H-1 myocytes. In both model systems, H-7, sangivamycin, and staurosporine, inhibitors of the catalytic domain of PKC, each effectively blocked insulin and PMA-stimulated hexose uptake at similar concentrations. In the myocytes, staurosporine completely blocked the insulin effect retained post-chronic phorbol myristate acetate (PMA)-induced "down-regulation." These findings indicate (1) that chronic pretreatment with PMA may not lead to a complete loss of PKC activity in the myocyte, and (2) that PKC is involved in insulin-stimulated hexose transport in both isolated rat adipocytes and BC3H-1 myocytes.     

Mechanism of insulin resistance induced by sustained levels of cytosolic free calcium in rat adipocytes

We have recently provided evidence that elevated levels of cytosolic free Ca2+ ([Ca2+]i) decreased insulin-stimulated glucose uptake in isolated rat adipocytes. To investigate the mechanism of Ca2+ action, we examined the effects of elevated levels of [Ca2+]i on insulin binding, autophosphorylation, and tyrosine kinase activity (TKA) of insulin receptors as well as basal and insulin-stimulated cellular distribution of glucose transporters. The latter was assessed by cytochalasin-B binding to plasma membrane and cytosolic fractions. Elevated concentrations of [Ca2+]i were maintained by incubating adipocytes with a depolarizing concentration of K+ (40 mM). Basal nonstimulated glucose uptake was not altered by increased levels of [Ca2+]i. Adipocytes with higher [Ca2+]i (220 +/- 15 nM) showed 30% reduction in insulin-stimulated 2-deoxyglucose uptake compared with control cells ([Ca2+]i, 140 +/- 18 nM). Moreover, adipocytes with higher levels of [Ca2+]i demonstrated an approximately 10% reduction in autophosphorylation and TKA of insulin receptors without a change in insulin binding. Both basal and insulin-stimulated distributions of glucose transporters were unaffected by sustained levels of [Ca2+]i. The effects of elevated [Ca2+]i were not mimicked by protein kinase-C activation. These observations suggest that 1) elevated or sustained levels of [Ca2+]i impair insulin-stimulated glucose uptake; and 2) Ca2+-induced impairment appears to reside at the postbinding steps of insulin action and probably interferes with the TKA of insulin receptors and the intrinsic activity of glucose transporters.     

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.     

Insulin binding and insulin action in cultured fibroblasts: significant differences between a phosphoglucose isomerase-deficient mutant and the parental strain.

The interrelationships of hexose feeding and insulin action were studied in the Chinese hamster fibroblast cell lines 023 and DS-7. The latter, derived from 023 and deficient in phosphoglucose isomerase, has been used to map the metabolic requirements for aldohexose-mediated down-regulation or "curbing" of hexose transport. We have characterized insulin binding and the response to insulin in both cell lines to determine if the insulin-mediated stimulation of transport is similarly dependent on hexose metabolism. DS-7 cells exhibited 5-6 times as many high-affinity insulin binding sites as the parental strain. Apart from this difference, 023 and DS-7 cells showed comparable insulin binding characteristics, which are similar to those observed in other cell types. Insulin at a concentration of 1 microgram/ml (167 nM) was found to stimulate 3-O-methylglucose uptake by approximately equal to 50% in glucose-fed cells of both lines. In neither line did glucose starving significantly alter insulin binding or the insulin-induced stimulation of transport. Feeding with mannose or fructose was found to increase both parameters in 023 cells but had no effect on DS-7 cells. The increase in hexose uptake with the administration of insulin or with glucose starving was shown to be due to an increase in Vmax. Our studies suggest that insulin binding and effect are not regulated by hexose metabolism in the same manner as in the curbing process and insulin induces the recruitment of a transporter pool that is insensitive to hexose curbing.