Effect of oral treatment with a new hypoglycemic agent, AS-6, on the metabolic activities of adipocytes in db/db mice: a comparative study

The mechanism of a new hypoglycemic agent, AS-6, was comparatively studied using the adipocytes from AS-6 treated and untreated genetically obese diabetic mice, db/db. the db/db mice were treated for 1 week with a diet admixture of AS-6 (0.1%). The treatment resulted in the following alterations in metabolic activities; AS-6 treatment increased 125I-insulin binding by 1.4-3.3 fold over the insulin range of 1-1000 microU/ml, the treatment increased the basal activities in 2-deoxyglucose uptake, and in CO2 generation and lipogenesis from U-(14C)-glucose compared with the db/db controls, the treatment partially restored insulin responsiveness in 2-DG uptake and CO2 generation, and 1 mU/ml of insulin greatly stimulated lipogenesis by 5.6 fold above the basal in the control adipocytes while AS-6 treatment changed the lipogenic response less stimulative to the insulin. The results suggest that AS-6 treatment significantly increases insulin binding to the adipocytes associating with an enhancement in glucose metabolism under basal and physiological concentrations of insulin.     

De novo emergence of insulin-stimulated glucose uptake in human aortic endothelial cells incubated with high glucose

Elevated glucose concentrations have profound effects on cell function. We hypothesized that incubation of human aortic endothelial cells (HAEC) with high glucose increases insulin signaling and develops the appearance of insulin-stimulated glucose uptake by the cells. Compared with 5 mM glucose, incubation of HAEC with 30 mM glucose for up to 48 h increased in a time-dependent manner expression of insulin receptor, insulin receptor substrate (IRS)-1, IRS-2, and GLUT1 proteins. High glucose also increased the specific binding of (125)I-labeled insulin in HAEC accompanied by accelerated production of interleukin (IL)-6 and IL-8. Short-term stimulation by 50 microU/ml insulin did not activate [(14)C]glucose uptake by HAEC incubated in 5 mM glucose. However, an addition of insulin to high glucose-exposed endothelial cells led to a significant increase in [(14)C]glucose uptake in a glucose concentration- and time-dependent fashion, reaching a plateau at 48 h of incubation. Furthermore, incubation of HAEC with 30 mM glucose resulted in a new insulin-stimulated extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase phosphorylation and increased lipid peroxidation and production of reactive oxygen species. These studies show for the first time that high glucose increases expression of insulin receptors and downstream elements of the insulin-signaling pathway and transforms "insulin-resistant" aortic endothelial cells into "insulin-sensitive" tissue regarding glucose uptake.     

Effects of insulin on glucose uptake in cultured cells from the central nervous system of rodent.

1. Incubation of C6 glioma cultures with insulin resulted in a time and dose-dependent stimulation of 2-deoxy-D-glucose uptake. The maximal stimulation (160% of the control) was observed with 1 nM insulin and 0.05 nM caused half-maximum effect. 2. Incubation of NG 108-15 (neuroblastoma x glioma hybrid) and N2 neuroblastoma cells with 160 nM insulin did not result in a significant stimulation of this glucose uptake. 3. The basal level and stimulatory effect by insulin on this glucose uptake observed in C6 glioma cells were dependent on the presence of calcium in the medium. 4. Such an increase in glucose uptake in C6 glioma cells was also observed in the presence of diacylglycerol (DG) generating agents, such as carbachol (1 mM) and phospholipase C (0.05 unit/ml) or of DG analogs, such as sn-1,2-dioctanoyl glycerol (250 microM) and phorbol myristate acetate (1 microM). 5. Our results indicated that both calcium ion and DG levels play important roles in the regulation of glucose uptake in the glial cells, but not in neuronal cells from the brain.     

Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells

The inward l-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of l-dopa transport into isolated rat PTCs. Insulin (50-500 microU/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 microU/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 microM). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 microM) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 microM), but not maximal (5 microM), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 microM LY-294002) and protein kinase C (PKC; 1 microM RO-318220). Insulin-induced activation of PKC-zeta was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-zeta, and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.     

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gialpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 approximately 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 microM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [3H]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gialpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gialpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gialpha-2 was found by immunoblot analysis with anti-Gialpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gialpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of Gbetagamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.     

Protein synthesis inhibitors activate glucose transport without increasing plasma membrane glucose transporters in 3T3-L1 adipocytes

In this study, we tested the hypothesis that hexose transport regulation may involve proteins with relatively rapid turnover rates. 3T3-L1 adipocytes, which exhibit 10-fold increases in hexose transport rates within 30 min of the addition of 100 nM insulin, were utilized. Exposure of these cells to 300 microM anisomycin or 500 microM cycloheximide caused a maximal, 7-fold increase in 2-deoxyglucose transport rate after 4-8 h. The effects due to either insulin (0.5 h) or anisomycin (5 h) on the kinetics of zero-trans 3-O-methyl[14C]glucose transport were similar, resulting in 2.5-3-fold increases in apparent Vmax values (control Vmax = 1.6 +/- 0.3 x 10(-7) mmol/s/10(6) cells) coupled with approximately 2-fold decreases in apparent Km values (control Km = 23 +/- 3.3 mM). Insulin elicited the expected increases in plasma membrane levels of HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) transporters (1.6- and 2.8-fold, respectively) as determined by protein immunoblotting. In contrast, neither total cellular contents nor plasma membrane levels of these two transporter isoforms were increased when 3T3-L1 adipocytes were treated with either anisomycin or cycloheximide. 3-[125I]Iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n labeling of glucose transporters in plasma membrane fractions of similarly treated cells was also unaffected by these agents. Thus, a striking discrepancy was observed between the marked increase in cellular hexose transport rates due to these protein synthesis inhibitors and the unaltered amounts of glucose transporter proteins in the plasma membrane fraction. These data indicate that short-term protein synthesis inhibition in 3T3-L1 adipocytes leads to large increases in the intrinsic catalytic activity of one or both of the GLUT1 and GLUT4 transporter isoforms.     

Further evidence for the involvement of a membrane proteolytic step in insulin action.

The hypothesis that insulin action involves a membrane proteolytic step was further explored, by using isolated rat adipocytes and liver plasma membranes. (1) The maximal insulin stimulation of 2-deoxyglucose transport and lipogenesis in fat-cells was selectively inhibited (73-88%) by N alpha-p-tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl; active-site inhibitor of trypsin; 30-125 microM), p-nitrophenyl p'-guanidinobenzoate (active-site inhibitor of serine proteinases; 30-125 microM) and p-tosyl-L-arginine methyl ester (arginine ester substrate analogue of proteinases; 1-2 mM), under conditions where neither the basal rate of each metabolic process nor insulin binding nor cellular ATP content were affected. In contrast, N-acetyl-L-alanyl-L-alanyl-L-alanine methyl ester (alanine ester substrate analogue of proteinases; 1-2 mM) was ineffective. (2) Endoproteinase Arg-C (0.25-40 micrograms/ml) exerted dose-dependent insulin-like effects on both 2-deoxyglucose transport and lipogenesis in fat-cells, whereas endoproteinase Lys-C (5-100 micrograms/ml) was ineffective. The maximal activation by endoproteinase Arg-C of both processes (200 and 177% of control values respectively) was shown to occur under conditions where membrane integrity (assessed by measurement of lactate dehydrogenase leakage and passive glucose diffusion) was preserved. This effect was inhibited by Tos-Lys-CH2Cl (125 microM) and was not additive with the maximal insulin effect. (3) Insulin (1-100 ng/ml) produced a dose-dependent increase in the trichloroacetic acid-soluble 125I radioactivity released after a 30 min incubation at 37 degrees C of 125I-labelled liver plasma membranes, but was ineffective on 125I-labelled bovine serum albumin. Insulin effects on both radio-labelled proteins were reproduced by wheat-germ agglutinin (20 micrograms/ml), an insulin mimicker shown to act through the insulin receptor. These data provide further evidence for the hypothesis that insulin bioeffects involve the activation of a membrane serine proteinase with arginine specificity.     

Effects of insulin and norepinephrine on glucose transport and metabolism in rat brown adipocytes. Potentiation by insulin of norepinephrine-induced glucose oxidation

Glucose is an important fuel for rat brown adipose tissue in vivo and its utilization is highly sensitive to insulin. In this study, the different glucose metabolic pathways and their regulation by insulin and norepinephrine were examined in isolated rat brown adipocytes, using [6-14C]glucose as a tracer. Glucose utilization was stimulated for insulin concentrations in the range of 40-1000 microU/ml. Furthermore, the addition of adenosine deaminase (200 mU/ml) or adenosine (10 microM) did not alter insulin sensitivity of glucose metabolism. The major effect of insulin (1 mU/ml) was a respective 7-fold and 5-fold stimulation of lipogenesis and lactate synthesis, whereas glucose oxidation remained very low. The 5-fold stimulation of total glucose metabolism by 1 mU/ml of insulin was accompanied by an 8-fold increase in glucose transport. In the presence of norepinephrine (8 microM), total glucose metabolism was increased 2-fold. This was linked to a 7-fold increase of glucose oxidation, whereas lipogenesis was greatly inhibited (by 72%). In addition, norepinephrine alone did not modify glucose transport. The addition of insulin to adipocytes incubated with norepinephrine, induced a potentiation of glucose oxidation, while lipogenesis remained very low. In conclusion, in the presence of insulin and norepinephrine glucose is a oxidative substrate for brown adipose tissue. However the quantitative importance of glucose as oxidative fuel remains to be determined.     

Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes

Following the differentiation of 3T3-L1 preadipocytes insulin acutely activates the rate of 2-deoxy-[1-14C]glucose uptake in the mature 3T3-L1 adipocyte by 15- to 20-fold. Phenylarsine oxide, a trivalent arsenical that forms stable ring complexes with vicinal dithiols, prevents insulin-activated hexose uptake in a concentration-dependent manner (Ki = 7 microM) but has no inhibitory effect on basal hexose uptake. 2,3-Dimercaptopropanol at a level nearly stoichiometric to that of phenylarsine oxide prevents or rapidly reverses the inhibition of hexose uptake; 2-mercaptoethanol, even in high stoichiometric excess over the arsenical, does not reverse inhibition of hexose uptake. When phenylarsine oxide is added after adipocytes have been fully activated by insulin, 2-deoxy-[1-14C]glucose uptake rate decays slowly at a rate corresponding to that caused by the withdrawal of insulin (t1/2 = 10 min). Using the same conditions under which phenylarsine oxide blocked activation, the Km for deoxyglucose uptake, the rate at which 125I-insulin became cell-associated, and the 125I-insulin binding isotherm for solubilized insulin receptor were not affected by phenylarsine oxide. These results support the transporter translocation model for insulin-activated hexose transport and implicate vicinal sulfhydryl groups in a post-insulin binding event essential for the translocation of glucose transporters to the plasma membrane.     

Transient effect of platelet-derived growth factor on GLUT4 translocation in 3T3-L1 adipocytes.

We earlier developed a novel method to detect translocation of the glucose transporter (GLUT) directly and simply using c-MYC epitope-tagged GLUT (GLUTMYC). To define the effect of platelet-derived growth factor (PDGF) on glucose transport in 3T3-L1 adipocytes, we investigated the PDGF- and insulin-induced glucose uptake, translocation of glucose transporters, and phosphatidylinositol (PI) 3-kinase activity in 3T3-L1, 3T3-L1GLUT4MYC, and 3T3-L1GLUT1MYC adipocytes. Insulin and PDGF stimulated glucose uptake by 9-10- and 5.5-6.5-fold, respectively, in both 3T3-L1 and 3T3-L1GLUT4MYC adipocytes. Exogenous GLUT4MYC expression led to enhanced PDGF-induced glucose transport. In 3T3-L1GLUT4MYC adipocytes, insulin and PDGF induced an 8- and 5-fold increase in GLUT4MYC translocation, respectively, determined in a cell-surface anti-c-MYC antibody binding assay. This PDGF-induced GLUT4MYC translocation was further demonstrated with fluorescent detection. In contrast, PDGF stimulated a 2-fold increase of GLUT1MYC translocation and 2.5-fold increase of glucose uptake in 3T3-L1GLUT1MYC adipocytes. The PDGF-induced GLUT4MYC translocation, glucose uptake, and PI 3-kinase activity were maximal (100%) at 5-10 min and thereafter rapidly declined to 40, 30, and 12%, respectively, within 60 min, a time when effects of insulin were maximal. Wortmannin (0.1 microM) abolished PDGF-induced GLUT4MYC translocation and glucose uptake in 3T3-L1GLUT4MYC adipocytes. These results suggest that PDGF can transiently trigger the translocation of GLUT4 and stimulate glucose uptake by translocation of both GLUT4 and GLUT1 in a PI 3-kinase-dependent signaling pathway in 3T3-L1 adipocytes.     

Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes.

In chicken thymocytes isolated from 15--40 day-old chickens, after a 2 h incubation at 37 degrees C, insulin stimulated amino isobutyric acid uptake (maximal response: 40--50% of increase at 1 microgram insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 microgram insulin/ml). Binding of 125I-labelled chicken insulin to thymocytes was rapid and higher at 15 degrees C than at 37 degrees C. At steady state, (90 min at 15 degrees C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of 125I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10(8) cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15 degrees C. Degradation of 125I-labelled chicken insulin in the incubation medium was negligible at 15 degrees C but very noticeable at 37 degrees C. Therefore, the low level of insulin binding at 15 degrees C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.     

Leprechaunism: an inherited defect in a high-affinity insulin receptor.

We examined in vivo oral glucose tolerance tests and in vitro insulin binding, cellular response, and insulin-receptor structure of fibroblasts cultured from the skin of a patient with leprechaun syndrome and her parents. In response to oral glucose, the proband exhibited marked hyperinsulinism (maximum plasma insulin = 4,120 microU/ml), the father had mild hyperinsulinism (maximum plasma insulin = 240 microU/ml), and the mother was normal. [125I]insulin binding to monolayers of intact fibroblasts demonstrated complex kinetics that were interpreted using a two-receptor model. Normal high-affinity binding had an apparent KA of 1.6 X 10(10)/molar with 1,100 sites/cell. The proposed low-affinity state receptor had an apparent KA of 6.8 X 10(7)/molar with approximately 30,000 sites/cell. Insulin binding to the proband's cells had no high-affinity binding but had normal low-affinity binding. Cells from the mother had 60%, and cells from the father, 2%, of control insulin binding to the high-affinity receptor, but normal, low-affinity site binding. Two different, insulin-stimulable responses were evaluated under experimental conditions identical with those used for insulin binding. Insulin stimulation of 2-methylaminoisobutyric acid uptake occurred with half-maximal responses between 25 and 50 ng/ml insulin. This response was similar in cells from controls and the patient. By contrast, the uptake and phosphorylation of 2-deoxy-D-glucose was stimulated at half-maximal insulin concentrations between 1 and 10 ng/ml in control cells but was not significantly increased in the proband's cells until 1,000 ng/ml concentrations of insulin were attained. In affinity crosslinking experiments, [125I]insulin was covalently bound to insulin receptors of fibroblast membranes using disuccinimidylsuberate. [125I]insulin specifically bound to 125,000 dalton monomeric subunits and 250,000 dalton dimers. In control cells, the ratio of monomer to dimer was approximately one, but significantly fewer dimers were crosslinked in insulin receptors from the patient's cells. We conclude that in this family two different recessive mutations impair high-affinity insulin-receptor binding and that the proband with leprechaunism is a compound heterozygote for these mutations. The two mutations produced structural changes in the receptor that altered subunit interactions and loss of high-affinity binding and cellular responsivity.     

Phosphorylation of zymogen granule membrane proteins in intact rat pancreatic acinar cells

The comparative effects of insulin and ethanolamine on 14CO2 production and lipid synthesis from [U-14C]-D-glucose in isolated rat adipocytes were studied. Ethanolamine (10 mM) increased 14CO2 production (glucose oxidation) about 5-fold and lipogenesis about 3-fold as compared to the control. Ethanolamine was more efficient than 25 microU/ml insulin regarding both parameters, but it was less efficient than 200 microU/ml insulin in glucose oxidation, and equally potent in lipogenesis. The combination of ethanolamine and insulin was more active than insulin alone. The mechanisms of ethanolamine action include facilitation of glucose transport and increase of pyruvate dehydrogenase activity.     

High-dose oral vitamin C partially replenishes vitamin C levels in patients with Type 2 diabetes and low vitamin C levels but does not improve endothelial dysfunction or insulin resistance

Endothelial dysfunction is a hallmark of Type 2 diabetes related to hyperglycemia and oxidative stress. Nitric oxide-dependent vasodilator actions of insulin may augment glucose disposal. Thus endothelial dysfunction may worsen insulin resistance. Intra-arterial administration of vitamin C improves endothelial dysfunction in diabetes. In the present study, we investigated effects of high-dose oral vitamin C to alter endothelial dysfunction and insulin resistance in Type 2 diabetes. Plasma vitamin C levels in 109 diabetic subjects were lower than healthy (36 +/- 2 microM) levels. Thirty-two diabetic subjects with low plasma vitamin C (<40 microM) were subsequently enrolled in a randomized, double-blind, placebo-controlled study of vitamin C (800 mg/day for 4 wk). Insulin sensitivity (determined by glucose clamp) and forearm blood flow in response to ACh, sodium nitroprusside (SNP), or insulin (determined by plethysmography) were assessed before and after 4 wk of treatment. In the placebo group (n = 17 subjects), plasma vitamin C (22 +/- 3 microM), fasting glucose (159 +/- 12 mg/dl), insulin (19 +/- 7 microU/ml), and SI(Clamp) [2.06 +/- 0.29 x 10(-4) dl x kg(-1) x min(-1)/(microU/ml)] did not change significantly after placebo treatment. In the vitamin C group (n = 15 subjects), basal plasma vitamin C (23 +/- 2 microM) increased to 48 +/- 6 microM (P < 0.01) after treatment, but this was significantly less than that expected for healthy subjects (>80 microM). No significant changes in fasting glucose (156 +/- 11 mg/dl), insulin (14 +/- 2 microU/ml), SI(Clamp) [2.71 +/- 0.46 x 10(-4) dl x kg(-1) x min(-1)/(microU/ml)], or forearm blood flow in response to ACh, SNP, or insulin were observed after vitamin C treatment. We conclude that high-dose oral vitamin C therapy, resulting in incomplete replenishment of vitamin C levels, is ineffective at improving endothelial dysfunction and insulin resistance in Type 2 diabetes.     

Hormonal regulation of amino acid uptake by cultured 3T3-L1 adipocytes

Incubation of the adipocytes for 20 hours with insulin or with Bt2cAMP plus the theophylline stimulated adipocyte uptake of AIB and MeAIB but did not stimulate the uptake of glutamine or cycloleucine. MeAIB uptake by both 3T3-L1 preadipocytes and 3T3-C2 cells was relatively unresponsive to insulin. However, MeAIB uptake by 3T3-C2 cells was stimulated by treatment with Bt2cAMP plus theophylline. Incubation of 3T3 adipocytes for 60 min with insulin yielded maximal stimulation of 2-deoxyglucose uptake but no stimulation of the uptake of AIB, MeAIB or glutamine. Responsiveness of transport to Bt2cAMP does not appear to require adipocyte differentiation. By contrast, adipocyte differentiation may be required for the development of the insulin-responsive transport systems.     

The fate of labelled glucose molecules in the rat adipocyte. Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-3H]glucose or 100 nM [U-14C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-3H]glucose, about two thirds of the cell-associated 3H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-14C]glucose. The only 14C-labelled metabolite escaping to the incubation medium was 14CO2, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-14C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 microM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0-100 microM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED50) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

Reduced glucose transport and increased binding of insulin in adipocytes from diabetic and fasted rats

1. Animals made diabetic by injection of streptozotocin or animals after 3 days of fasting show decreased insulin levels and a decrease in mean cell diameter of adipocytes from epidydymal fat pads in comparison with cells from normal animals. 2. 14CO2 production from D-[U-14C]glucose is impaired in diabetic and fasted animals both in presence or in absence of a concentration of insulin stimulating 14CO2 production maximally. 3. Insulin binding is increased in adipocytes from diabetic and fasted animals due to changes in affinity. 4. Transport studies show that basal and insulin stimulated 2-deoxy[1-14C]-glucose transport is decreased in absolute terms due to a decrease in V and an increase in Km. 5. The relative stimulatory effect of insulin is impaired in adipocytes of diabetic and fasted animals. 6. A shift of the maximal effect of insulin to lower insulin levels is seen in these cells.     

Effects of erythropoietin on glucose metabolism

We purposed to determine the impact of erythropoietin on altering glucose metabolism in the settings of in vitro and in vivo experiments. The acute effect of erythropoietin on lowering blood glucose levels was studied in animal experiments. In [3H]-deoxy-D-glucose isotope studies we measured glucose uptake with insulin and erythropoietin using 3T3-L1 cells cultured under normal or high glucose conditions. Altered activation of Akt and ERK pathways was evaluated in immunoblot analyses. Immunocytochemistry was conducted to determine the glucose transporter 4 translocation to the plasma membrane. Addition of erythropoietin significantly lowered blood glucose levels in vivo in rats. The glucose uptake was markedly increased by erythropoietin treatment (at concentrations 0.15, 0.3, and 0.625 ng/ml) in adipocytes grown in high glucose medium (p<0.05), but it remained unaltered in cells under normal glucose conditions. Significant increase of phosphorylation of ERK and Akt was detected due to erythropoietin (p<0.05). Co-administration of erythropoietin and insulin resulted in higher phosphorylation of Akt and [3H]-deoxy-D-glucose uptake in adipocytes than insulin treatment alone. We found that erythropoietin induced the trafficking of glucose transporter 4 to the plasma membrane. Our data showed that erythropoietin significantly decreased blood glucose levels both in vivo and in vitro, in part, by increasing glucose uptake via the activation of Akt pathway. Preliminary data revealed that adipocytes most likely exhibit a specific receptor for erythropoietin.     

Effects of acute exercise and detraining on insulin action in trained men

Seven endurance-trained subjects [maximal O2 consumption (VO2max) 64 +/- 1 (SE) ml.min-1.kg-1] underwent sequential hyperinsulinemic euglycemic clamps on three occasions: 1) in the "habitual state" 15 h after the last training bout (C), 2) after 60 min of bicycle exercise at 72 +/- 3% of VO2max performed in the habitual state (E), and 3) 5 days after the last ordinary training session (detrained, DT). Sensitivity for insulin-mediated whole-body glucose uptake was not affected by acute exercise [insulin concentrations eliciting 50% of maximal insulin-mediated glucose uptake being 44 +/- 2 (C) vs. 46 +/- 3 (E) microU/ml] but was decreased after detraining (54 +/- 2 microU/ml, P less than 0.05) to levels comparable to those found in untrained subjects [Am. J. Physiol. 254 (Endocrinol. Metab. 17): E248-E259, 1988]. Near-maximal insulin-mediated glucose uptake (responsiveness) was higher than in untrained subjects and not influenced by acute exercise or detraining [13.4 +/- 1.2 (C), 12.2 +/- 0.9 (E), and 12.2 +/- 0.3 (DT) mg.min-1.kg-1]. Calculated by indirect calorimetry, the glucose-to-glycogen conversion was not influenced by E but was reduced during detraining (P less than 0.05) yet remained higher than previously found in untrained subjects (P less than 0.05). However, only on E days did muscle glycogen increase during insulin infusion. Glycogen synthase activity was increased on E and decreased on DT compared with C days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose metabolism in isolated brown adipocytes under beta-adrenergic stimulation. Quantitative contribution of glucose to total thermogenesis.

To quantify the potential of brown adipose tissue as a target organ for glucose oxidation, O2 consumption and glucose metabolism in isolated rat brown adipocytes were measured in the presence and absence of insulin, by using the beta-agonists isoprenaline or Ro 16-8714 to stimulate thermogenesis. Basal metabolic rate (278 mumol of O2/h per g of lipid) was maximally stimulated with isoprenaline (20 nm) and Ro 16-8714 (20 microM) to 1633 and 1024 mumol of O2/h per g respectively, whereas insulin had no effect on O2 consumption. Total glucose uptake, derived from the sum of [U-14C]glucose incorporation into CO2 and total lipids and lactate release, was enhanced with insulin. Isoprenaline and Ro 16-8714 had no effect on insulin-induced glucose uptake, but promoted glucose oxidation while inhibiting insulin-dependent lipogenesis and lactate production. A maximal value for glucose oxidation was obtained under the combined action of Ro 16-8714 and insulin, which corresponded to an equivalent of 165 mumol of O2/h per g of lipid. This makes it clear that glucose is a minor substrate for isolated brown adipocytes, fuelling thermogenesis by a maximum of 16%.