Maximal Insulin-stimulated Glucose Uptake Research Articles

Gene deletion of γ-actin impairs insulin-stimulated skeletal muscle glucose uptake in growing mice but not in mature adult mice.

The cortical cytoskeleton, consisting of the cytoplasmic actin isoforms β and/or γ‐actin, has been implicated in insulin‐stimulated GLUT4 translocation and glucose uptake in muscle and adipose cell culture. Furthermore, transgenic inhibition of multiple actin‐regulating proteins in muscle inhibits insulin‐stimulated muscle glucose uptake. The current study tested if γ‐actin was required for insulin‐stimulated glucose uptake in mouse skeletal muscle. Based on our previously reported age‐dependent phenotype in muscle‐specific β‐actin gene deletion (−/−) mice, we included cohorts of growing 8–14 weeks old and mature 18–32 weeks old muscle‐specific γ‐actin−/− mice or wild‐type littermates. In growing mice, insulin significantly increased the glucose uptake in slow‐twitch oxidative soleus and fast‐twitch glycolytic EDL muscles from wild‐type mice, but not γ‐actin−/−. In relative values, the maximal insulin‐stimulated glucose uptake was reduced by ~50% in soleus and by ~70% in EDL muscles from growing γ‐actin−/− mice compared to growing wild‐type mice. In contrast, the insulin‐stimulated glucose uptake responses in mature adult γ‐actin−/− soleus and EDL muscles were indistinguishable from the responses in wild‐type muscles. Mature adult insulin‐stimulated phosphorylations on Akt, p70S6K, and ULK1 were not significantly affected by genotype. Hence, insulin‐stimulated muscle glucose uptake shows an age‐dependent impairment in young growing but not in fully grown γ‐actin−/− mice, bearing phenotypic resemblance to β‐actin−/− mice. Overall, γ‐actin does not appear required for insulin‐stimulated muscle glucose uptake in adulthood. Furthermore, our data emphasize the need to consider the rapid growth of young mice as a potential confounder in transgenic mouse phenotyping studies.

Insulin Resistance Is Not Sustained Following Denervation in Glycolytic Skeletal Muscle.

Denervation rapidly induces insulin resistance (i.e., impairments in insulin-stimulated glucose uptake and signaling proteins) in skeletal muscle. Surprisingly, whether this metabolic derangement is long-lasting is presently not clear. The main goal of this study was to determine if insulin resistance is sustained in both oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles following long-term (28 days) denervation. Mouse hindlimb muscles were denervated via unilateral sciatic nerve resection. Both soleus and EDL muscles atrophied ~40%. Strikingly, while denervation impaired submaximal insulin-stimulated [3H]-2-deoxyglucose uptake ~30% in the soleus, it enhanced submaximal (~120%) and maximal (~160%) insulin-stimulated glucose uptake in the EDL. To assess possible mechanism(s), immunoblots were performed. Denervation did not consistently alter insulin signaling (e.g., p-Akt (Thr308):Akt; p-TBC1D1 [phospho-Akt substrate (PAS)]:TBC1D1; or p-TBC1D4 (PAS):TBC1D4) in either muscle. However, denervation decreased glucose transporter 4 (GLUT4) levels ~65% in the soleus but increased them ~90% in the EDL. To assess the contribution of GLUT4 to the enhanced EDL muscle glucose uptake, muscle-specific GLUT4 knockout mice were examined. Loss of GLUT4 prevented the denervation-induced increase in insulin-stimulated glucose uptake. In conclusion, the denervation results sustained insulin resistance in the soleus but enhanced insulin sensitivity in the EDL due to increased GLUT4 protein levels.

Adipose Tissue Transferrin and Insulin Resistance.

Excessive body iron stores are a risk factor for decreased insulin sensitivity (SI) and diabetes. We hypothesized that transcriptional dysregulation of genes involved in iron metabolism in adipocytes causes insulin resistance. To define the genetic regulation of iron metabolism and its role in SI, we used gene expression, genotype, and SI data from an African American cohort (N = 256). Replication studies were performed in independent European ancestry cohorts. In vitro studies in human adipocytes were performed to define the role of a selected gene in causing insulin resistance. Among 62 transcripts representing iron homeostasis genes, expression of 30 in adipose tissue were correlated with SI. Transferrin (TF) and ferritin heavy polypeptide were most positively and negatively associated with SI, respectively. These observations were replicated in two independent European ancestry adipose data sets. The strongest cis-regulatory variant for TF expression (rs6785596; P = 7.84 × 10-18) was identified in adipose but not muscle or liver tissue. Variants significantly affected the normal relationship of serum ferritin to insulin resistance. Knockdown of TF in differentiated Simpson-Golabi-Behmel syndrome adipocytes by short hairpin RNA decreased intracellular iron, reduced maximal insulin-stimulated glucose uptake, and reduced Akt phosphorylation. Knockdown of TF caused differential expression of 465 genes, including genes involved in glucose transport, mitochondrial function, Wnt-pathway/ SI, chemokine activity, and obesity. Iron chelation recapitulated key changes in the expression profile induced by TF knockdown. Genetic regulation of TF expression in adipose tissue plays a novel role in regulating SI.

Adipose Tissue Transferrin and Insulin Resistance

Iron is an important micronutrient involved in several metabolic processes, and excessive iron is a risk factor for insulin resistance (IR) and diabetes. To define the genetic regulation of iron metabolism and its role in IR, we used gene expression, genotype, and insulin sensitivity data from an African American cohort (AAGMEx, N=256). Among the genes in a manually curated list of 62 transcripts representing iron homeostasis genes, expression of 32 in adipose tissue showed significant correlation with SI (p<0.01). The expression levels of transferrin (TF, β= 0.29, p=7.84 x 10-6) and ferritin heavy chain 1 (FTH1, β= -1.2, p=3.75 x 10-13) in adipose tissue were the most positively and negatively associated with SI, respectively. Observations were replicated in independent adipose tissue data sets from individuals of European ancestry (ARCA, N=99; METSIM, N=720). Among these SI-associated transcripts, the strongest cis-regulatory genetic variant (cis-eSNP) was for TF (rs6785596, β= 0.962, p=7.84 x 10-18) in adipose, but not in muscle or liver. To identify the role of TF in adipocytes, we downregulated its expression in a human adipocyte cell model (SGBS). Gene specific shRNA knockdown of TF caused differential expression (log2FC +/-0.4, probability>70%) of 465 genes, involved in mitochondrial function (CPT1B, UCP2), glucose transport (GLUT4), Wnt-pathway/insulin sensitivity (SFRP4, SFRP1), chemokine activity/cell-cell interaction (CXCL1, CXCL12, ICAM1), and genes with possible roles in obesity (CES1, RARRES2). Knockdown of TF mRNA in differentiated SGBS cells impaired mitochondrial respiration (49±10% reduction of maximal oxygen consumption rate) and caused a 34±4% reduction in maximal insulin-stimulated glucose uptake (p<0.05). In summary, genetic regulation of transferrin expression in adipose tissue plays a novel role in regulating insulin sensitivity. Disclosure D. McClain: None. N.K. Sharma: None. F. Lorenzo: None. S. Jain: None. C.D. Langefeld: None. M.E. Comeau: None. L. Salaye: None. S.K. Das: None.

Exercise training is an effective alternative to estrogen supplementation for improving glucose homeostasis in ovariectomized rats

The irreversible loss of estrogen (specifically 17-β-estradiol; E2) compromises whole-body glucose tolerance in women. Hormone replacement therapy (HRT) is frequently prescribed to treat estrogen deficiency, but has several deleterious side effects. Exercise has been proposed as an HRT substitute, however, their relative abilities to treat glucose intolerance are unknown. Thirty ovariectomized (OVX) and 20 SHAM (control) rats underwent glucose tolerance tests (GTT) 10 weeks post surgery. Area under the curve (AUC) for OVX rats was 60% greater than SHAM controls (P = 0.0005). Rats were then randomly assigned to the following treatment groups: SHAM sedentary (sed) or exercise (ex; 60 min, 5×/weeks), OVX sed, ex, or E2 (28 μg/kg bw/day) for 4 weeks. OVX ex rats experienced a ∼45% improvement in AUC relative to OVX sed rats, whereas OVX E2 underwent a partial reduction (17%; P = 0.08). Maximal insulin-stimulated glucose uptake in soleus and EDL was not impaired in OVX rats, or augmented with exercise or E2. Akt phosphorylation did not differ in soleus, EDL, or liver of any group. However, OVX ex and OVX E2 experienced greater increases in p-Akt Ser473 in VAT and SQ tissues compared with SHAM and OVX sed groups. Mitochondrial markers CS, COXIV, and core1 were increased in soleus posttraining in OVX ex rats. The content of COXIV was reduced by 52% and 61% in SQ of OVX sed and E2 rats, compared to SHAM controls, but fully restored in OVX ex rats. In summary, exercise restores glucose tolerance in OVX rats more effectively than E2. This is not reflected by alterations in muscle maximal insulin response, but increased insulin signaling in adipose depots may underlie whole-body improvements.

Feeding butter with elevated content of trans-10, cis-12 conjugated linoleic acid to lean rats does not impair glucose tolerance or muscle insulin response.

BackgroundNumerous studies have investigated the effects of isolated CLA supplementation on glucose homeostasis in humans and rodents. However, both the amount and relative abundance of CLA isomers in supplemental form are not representative of what is consumed from natural sources. No study to date has examined the effects of altered CLA isomer content within a natural food source. Our goal was to increase the content of the insulin desensitizing CLAt10,c12 isomer relative to the CLAc9,t11 isomer in cow’s milk by inducing subacute rumenal acidosis (SARA), and subsequently investigate the effects of this milk fat on parameters related to glucose and insulin tolerance in rats.MethodsWe fed female rats (~2.5 to 3 months of age) CLA t10,c12 –enriched (SARA) butter or non-SARA butter based diets for 4 weeks in either low (10% of kcal from fat; 0.18% total CLA by weight) or high (60% of kcal from fat; 0.55% total CLA by weight) amounts. In an effort to extend these findings, we then fed rats high (60% kcal) amounts of SARA or non-SARA butter for a longer duration (8 weeks) and assessed changes in whole body glucose, insulin and pyruvate tolerance in comparison to low fat and 60% lard conditions.ResultsThere was a main effect for increased fasting blood glucose and insulin in SARA vs. non-SARA butter groups after 4 weeks of feeding (p < 0.05). However, blood glucose and insulin concentration, and maximal insulin-stimulated glucose uptake in skeletal muscle were similar in all groups. Following 8 weeks of feeding, insulin tolerance was impaired by the SARA butter, but not glucose or pyruvate tolerance. The non-SARA butter did not impair tolerance to glucose, insulin or pyruvate.ConclusionsThis study suggests that increasing the consumption of a naturally enriched CLAt10,c12 source, at least in rats, has minimal impact on whole body glucose tolerance or muscle specific insulin response.

Myo1c Regulates Glucose Uptake in Mouse Skeletal Muscle

Contraction and insulin promote glucose uptake in skeletal muscle through GLUT4 translocation to cell surface membranes. Although the signaling mechanisms leading to GLUT4 translocation have been extensively studied in muscle, the cellular transport machinery is poorly understood. Myo1c is an actin-based motor protein implicated in GLUT4 translocation in adipocytes; however, the expression profile and role of Myo1c in skeletal muscle have not been investigated. Myo1c protein abundance was higher in more oxidative skeletal muscles and heart. Voluntary wheel exercise (4 weeks, 8.2 ± 0.8 km/day), which increased the oxidative profile of the triceps muscle, significantly increased Myo1c protein levels by ∼2-fold versus sedentary controls. In contrast, high fat feeding (9 weeks, 60% fat) significantly reduced Myo1c by 17% in tibialis anterior muscle. To study Myo1c regulation of glucose uptake, we expressed wild-type Myo1c or Myo1c mutated at the ATPase catalytic site (K111A-Myo1c) in mouse tibialis anterior muscles in vivo and assessed glucose uptake in vivo in the basal state, in response to 15 min of in situ contraction, and 15 min following maximal insulin injection (16.6 units/kg of body weight). Expression of wild-type Myo1c or K111A-Myo1c had no effect on basal glucose uptake. However, expression of wild-type Myo1c significantly increased contraction- and insulin-stimulated glucose uptake, whereas expression of K111A-Myo1c decreased both contraction-stimulated and insulin-stimulated glucose uptake. Neither wild-type nor K111A-Myo1c expression altered GLUT4 expression, and neither affected contraction- or insulin-stimulated signaling proteins. Myo1c is a novel mediator of both insulin-stimulated and contraction-stimulated glucose uptake in skeletal muscle.

Activation of the A1adenosine receptor increases insulin-stimulated glucose transport in isolated rat soleus muscle

The A1 adenosine receptor (A1AR) has been suggested to participate in insulin- and contraction-stimulated glucose transport in skeletal muscle, but the qualitative and quantitative nature of the effect are controversial. We sought to determine if A1AR is expressed in rat soleus muscle and then characterize its role in glucose transport in this muscle. A1AR mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. To examine the role of adenosine in 3-O-methylglucose transport, isolated muscles were exposed to adenosine deaminase and alpha,beta-methylene adenosine diphosphate to remove endogenous adenosine and were left unstimulated (basal) or stimulated with insulin. To assess the functional participation of A1AR in 3-O-methylglucose transport, muscles were incubated with A1-selective agonist and (or) antagonist in the absence of endogenous adenosine and with or without insulin. A1AR mRNA was expressed in soleus muscle and A1AR was present at the plasma membrane. Removal of endogenous adenosine reduced glucose transport in response to 100 microU/mL insulin (approximately 50%). The A1-selective agonist, N6-cyclopentyladenosine, increased submaximal (100 microU/mL) insulin-stimulated glucose transport in a dose-dependent manner (0.001-1.0 micromol/L). This stimulatory effect was inhibited by the A1-selective receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine in a concentration-dependent manner (0.001-1.0 micromol/L). However, neither activation nor inhibition of A1AR altered basal or maximal (10 mU/mL) insulin-stimulated glucose transport. Our results suggest that adenosine contributes approximately 50% to insulin-stimulated muscle glucose transport by activating the A1AR. This effect is limited to increasing insulin sensitivity, but not to either basal or maximal insulin-stimulated glucose uptake in rat soleus muscle.

Chronic testosterone treatment induces selective insulin resistance in subcutaneous adipocytes of women

Adipose tissue plays a central role in determining whole body insulin sensitivity. Several aspects of adipose cell function are regulated by androgens. Given that high androgen levels and insulin resistance are linked in women, we proposed that androgens may influence insulin-mediated glucose metabolism in adipose cells. Preadipocytes harvested from s.c. adipose tissue of healthy women aged 37 +/- 5 years were differentiated in vitro, then treated with testosterone (T) and/or androgen receptor (AR) antagonists (cyproterone acetate, flutamide) for 48 h. Maximal insulin-stimulated glucose uptake (insulin 10 nM) and increment following insulin stimulation were significantly impaired in cells treated with T 10 and 100 nmol/l. This defect was abolished by cyproterone acetate and partially reversed by flutamide. The effect of T could not be accounted for by altered differentiation status of the adipocytes. In the glucose metabolic pathway of insulin signaling, treatment of cells with T 10 nmol/l did not alter insulin-stimulated phosphorylation of insulin receptor substrate-1 or Akt, but insulin-stimulated phosphorylation of protein kinase C (PKC) zeta was impaired. Insulin signaling via the mitogenic/gene regulatory pathway, as assessed by extracellular signal-regulated kinase phosphorylation, was unchanged. We conclude that (1) T, or an androgenic metabolite of T, induces insulin resistance in adipocytes of women, selective for metabolic signaling pathways; (2) this defect is via AR; and (3) the defect in signaling is independent of phosphatidylinositol 3-kinase activation and involves impaired phosphorylation of PKCzeta. These findings are relevant to understanding the pathogenesis of insulin resistance in hyperandrogenic women.

Adipocytes with increased hexosamine flux exhibit insulin resistance, increased glucose uptake, and increased synthesis and storage of lipid

The hexosamine signaling pathway has been shown to serve a nutrient-sensing function. We have previously shown that overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine-fructose-6-phosphate amidotransferase) in adipose tissue of transgenic mice results in skeletal muscle insulin resistance and altered regulation of leptin and adiponectin. To dissect the pathways by which the hexosamine pathway affects fuel storage and energy homeostasis, we have examined the characteristics of adipocytes from these animals. After 3 mo of age, epididymal fat pads from adult transgenic animals are 42% heavier (P = 0.003) and individual adipocytes are 23% larger in diameter (P < 0.05) than those from littermate wild-type controls. Isolated adipocytes from transgenic mice are insulin resistant, with a 2.5-fold increase in the ED50 for stimulation of 2-deoxy-D-glucose uptake. However, maximal insulin-stimulated glucose uptake is increased in transgenic adipocytes by 39% (P < 0.05). This upregulation of glucose uptake was associated with a 41% increase in the expression of GLUT4 mRNA and a 28% increase in GLUT4 protein in transgenics compared with controls (P < 0.05). GLUT1 mRNA and protein did not significantly differ between fasted control and transgenics. Total lipid synthesis was also increased in epididymal adipocytes from transgenic animals by 206% compared with controls (P < 0.05). Fatty acid oxidation was increased 1.6-fold in the transgenic adipocytes (P < 0.05). We conclude that the hexosamine signaling pathway upregulates fat storage in adipocytes in states of carbohydrate excess, in part by increasing GLUT4 and glucose uptake and by augmenting fatty acid synthesis.

Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice.

Decreased GLUT4 expression, impaired insulin receptor (IR), IRS-1, and pp60/IRS-3 tyrosine phosphorylation are characteristics of adipocytes from insulin-resistant animal models and obese NIDDM humans. However, the sequence of events leading to the development of insulin signaling defects and the significance of decreased GLUT4 expression in causing adipocyte insulin resistance are unknown. The present study used male heterozygous GLUT4 knockout mice (GLUT4(+/-)) as a novel model of diabetes to study the development of insulin signaling defects in adipocytes with the progression of whole body insulin resistance and diabetes. Male GLUT4(+/-) mice with normal fed glycemia and insulinemia (N/N), normal fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with hyperinsulinemia (H/H) exist at all ages. The expression of GLUT4 protein and the maximal insulin-stimulated glucose transport was 50% decreased in adipocytes from all three groups. Insulin signaling was normal in N/N adipose cells. From 35 to 70% reductions in insulin-stimulated tyrosine phosphorylation of IR, IRS-1, and pp60/IRS-3 were noted with no changes in the cellular content of IR, IRS-1, and p85 in N/H adipocytes. Insulin-stimulated protein tyrosine phosphorylation was further decreased to 12-23% in H/H adipose cells accompanied by 42% decreased IR and 80% increased p85 expression. Insulin-stimulated, IRS-1-associated PI3 kinase activity was decreased by 20% in N/H and 68% reduced in H/H GLUT4(+/-) adipocytes. However, total insulin-stimulated PI3 kinase activity was normal in H/H GLUT4(+/-) adipocytes. Taken together, these results strongly suggest that hyperinsulinemia triggers a reduction of IR tyrosine kinase activity that is further exacerbated by the appearance of hyperglycemia. However, the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake. Furthermore, the data indicate that the cellular content of GLUT4 is the rate-limiting factor in mediating maximal insulin-stimulated glucose uptake in GLUT4(+/-) adipocytes.

Chronic leptin administration increases insulin-stimulated skeletal muscle glucose uptake and transport

Leptin, the product of the ob gene, has been shown to reduce fat mass, food intake, hyperglycemia, and hyperinsulinemia and to increase whole-body glucose disposal. However, it is unknown if leptin improves insulin action in skeletal muscle. Therefore, the purpose of this investigation was to determine if chronic leptin administration increases insulin-stimulated skeletal muscle glucose uptake and transport. Sixty-nine female Sprague-Dawley rats (240 to 250 g) were randomly assigned to one of three groups: (1) control, (2) pair-fed, and (3) leptin. All animals were subcutaneously implanted with miniosmotic pumps that delivered 0.5 mg leptin/kg/d to the leptin animals and vehicle to the control and pair-fed animals for 14 days. Following this 14-day period, all animals were subjected to hindlimb perfusion to determine the rates of skeletal muscle glucose uptake and 3- O-methyl- d-glucose (3-MG) transport under basal, submaximal (500 μU/mL), and maximal (10,000 μU/mL) insulin concentrations. Chronic leptin treatment significantly increased ( P < .05) the rate of glucose uptake across the hindlimb by 27%, 32%, and 47% under basal, submaximal, and maximal insulin, respectively, compared with the control and pair-fed condition. However, when the submaximal rate of glucose uptake was expressed as a percentage of maximal insulin-stimulated glucose uptake, no differences existed among the groups, indicating that leptin treatment does not increase insulin sensitivity. Rates of 3-MG transport in the soleus, plantaris, and white and red portions of the gastrocnemius (WG and RG) were significantly increased ( P < .05) in leptin animals under all perfusion conditions. 3-MG transport was not different between control and pair-fed animals. Collectively, these findings suggest that improvements in insulin-stimulated skeletal muscle glucose uptake and transport following chronic leptin treatment result from increased insulin responsiveness.

Limitations to exercise- and maximal insulin-stimulated muscle glucose uptake.

The hypothesis of this investigation was that insulin and muscle contraction, by increasing the rate of skeletal muscle glucose transport, would bias control so that glucose delivery to the sarcolemma (and t tubule) and phosphorylation of glucose intracellularly would exert more influence over glucose uptake. Because of the substantial increases in blood flow (and hence glucose delivery) that accompany exercise, we predicted that glucose phosphorylation would become more rate determining during exercise. The transsarcolemmal glucose gradient (TSGG; the glucose concentration difference across the membrane) is inversely related to the degree to which glucose transport determines the rate of glucose uptake. The TSGG was determined by using isotopic methods in conscious rats during euglycemic hyperinsulinemia [Ins; 20 mU/(kg. min); n = 7], during treadmill exercise (Ex, n = 6), and in sedentary, saline-infused rats (Bas, n = 13). Rats received primed, constant intravenous infusions of trace 3-O-[3H]methyl-D-glucose and [U-14C]mannitol. Then 2-deoxy-[3H]glucose was infused for the calculation of a glucose metabolic index (Rg). At the end of experiments, rats were anesthetized, and soleus muscles were excised. Total soleus glucose concentration and the steady-state ratio of intracellular to extracellular 3-O-[3H]methyl-D-glucose (which distributes on the basis of the TSGG) were used to calculate ranges of possible glucose concentrations ([G]) at the inner and outer sarcolemmal surfaces ([G]im and [G]om, respectively). Soleus Rg was increased in Ins and further increased in Ex. In Ins, total soleus glucose, [G]om, and the TSGG were decreased compared with Bas, while [G]im remained near 0. In Ex, total soleus glucose and [G]im were increased compared with Bas, and there was not a decrease in [G]om as was observed in Ins. In addition, accumulation of intracellular free 2-deoxy-[3H]glucose occurred in soleus in both Ex and Ins. Taken together, these data indicate that, in Ex, glucose phosphorylation becomes an important limitation to soleus glucose uptake. In Ins, both glucose delivery and glucose phosphorylation influence the rate of soleus glucose uptake more than under basal conditions.

The Development of Insulin Resistance with High Fat Feeding in Rats Does Not Involve Either Decreased Insulin Receptor Tyrosine Kinase Activity or Membrane Glycoprotein PC-1

Recent studies have suggested that the insulin receptor tyrosine kinase inhibitor, membrane glycoprotein PC-1, may play a role in certain insulin resistant states. In the present study, we examined whether either insulin receptor function or PC-1 activity was altered during the development of insulin resistance that occurs with high fat feeding in normal rats. Over the course of 14 days of high fat feeding, both maximal and submaximal (physiological) insulin-stimulated skeletal muscle glucose uptake decreased gradually; after 14 days of high fat feeding, submaximal and maximal insulin-stimulated glucose uptake decreased by ∼40 and ∼50%, respectively. In contrast, in the same muscles (tibialis anterior) of these animals, neither insulin receptor content nor insulin-stimulated insulin receptor autophosphorylation was altered after 14 days of high fat feeding. PC-1 has both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) enzyme activities. These enzyme activities showed no changes during the course of 14 days of high fat feeding. Individual data revealed that there was no significant correlation between insulin-stimulated glucose uptake and alkaline phosphodiesterase or nucleotide pyrophosphatase activity (P> 0.05). Together, these data indicate that neither defects in insulin receptor function nor elevated PC-1 activities are involved in the development of insulin resistance in rats with high fat feeding, and the insulin resistance induced with high fat feeding is likely due to postreceptor defects in skeletal muscle.

Eccentric exercise decreases maximal insulin action in humans: muscle and systemic effects.

1. Unaccustomed eccentric exercise decreases whole-body insulin action in humans. To study the effects of one-legged eccentric exercise on insulin action in muscle and systemically, the euglycaemic clamp technique combined with arterial and bilateral femoral venous catheterization was used. Seven subjects participated in two euglycaemic clamps, performed in random order. One clamp was preceded 2 days earlier by one-legged eccentric exercise (post-eccentric exercise clamp (PEC)) and one was without the prior exercise (control clamp (CC)). 2. During PEC the maximal insulin-stimulated glucose uptake over the eccentric thigh was marginally lower when compared with the control thigh, (11.9%, 64.6 +/- 10.3 vs. 73.3 +/- 10.2 mumol kg-1 min-1, P = 0.08), whereas no inter-thigh difference was observed at a submaximal insulin concentration. The glycogen concentration was lower in the eccentric thigh for all three clamp steps used (P < 0.05). The glucose transporter GLUT4 protein content was on average 39% lower (P < 0.05) in the eccentric thigh in the basal state, whereas the maximal activity of glycogen synthase was identical in the two thighs for all clamp steps. 3. The glucose infusion rate (GIR) necessary to maintain euglycaemia during maximal insulin stimulation was lower during PEC compared with CC (15.7%, 81.3 +/- 3.2 vs. 96.4 +/- 8.8 mumol kg-1 min-1, P < 0.05). 4. Our data show that 2 days after unaccustomed eccentric exercise, muscle and whole-body insulin action is impaired at maximal but not submaximal concentrations. The local effect cannot account for the whole-body effect, suggesting the release of a factor which decreases insulin responsiveness systemically.

Overexpression of Hexokinase II in Transgenic Mice: EVIDENCE THAT INCREASED PHOSPHORYLATION AUGMENTS MUSCLE GLUCOSE UPTAKE

Hexokinase II (HKII) is the predominant isozyme expressed in peripheral insulin-responsive tissues. To explore the role of HKII in muscle glucose metabolism, two lines of transgenic mice were generated where overexpression was restricted to striated muscle; HKII protein levels and activity were increased by 3-8-fold. Oral glucose tolerance, intravenous insulin tolerance, and insulin and lactate levels were unaffected in transgenic mice. There was a trend toward increased levels of muscle glycogen; however, glucose-6-phosphate levels were increased by 43% in transgenic skeletal muscle following in vivo glucose and insulin administration. Using 2-[3H]deoxyglucose as a tracer, in vitro basal and insulin-stimulated glucose uptake were determined in extensor digitorum longus, soleus, and epitrochlearis muscles. Maximal insulin-stimulated glucose uptake was increased by 17% (extensor digitorum longus), 34% (soleus), and 90% (epitrochlearis) in transgenic muscles; basal and submaximal glucose uptake was also modestly increased in soleus and epitrochlearis. These data suggest that increased muscle HKII (corresponding to the upper end of the physiologic range) may not be sufficient to augment net in vivo glucose homeostasis. However, glucose phosphorylation can represent a rate-limiting step for skeletal muscle glucose utilization since muscle glucose-6-phosphate levels are increased during in vivo hyperinsulinemia and hyperglycemia; furthermore, basal and insulin-mediated muscle glucose uptake can be increased by a selective increase in HKII expression.

Glucose transporters and in vivo glucose uptake in skeletal and cardiac muscle: fasting, insulin stimulation and immunoisolation studies of GLUT1 and GLUT4

Our aim was to study glucose transporters GLUT1 and GLUT4 in relation to in vivo glucose uptake in rat cardiac and skeletal muscle. The levels of both transporters were of a similar order of magnitude in whole muscle tissue (GLUT1/GLUT4 ratio varied from 0.1 to 0.6), suggesting that both may have an important physiological role in regulating muscle glucose metabolism. GLUT4 correlated very strongly (r2 = 0.97) with maximal insulin-stimulated glucose uptake (Rg' max., estimated using the glucose clamp plus 2-deoxy[3H]glucose bolus technique) in six skeletal muscles and heart. A distinct difference in regulation of the two transporters was evident in heart: in 5 h-fasted rats, basal glucose uptake and GLUT1 levels in heart were very high and both were reduced, by 90 and 60% respectively, by 48 h fasting. However, in heart (and in red skeletal muscle), neither GLUT4 levels nor Rg' max. were reduced by 48 h fasting. GLUT1 was shown to be specifically expressed in cardiac myocytes, because intracellular vesicles enriched in GLUT4 contained significant levels of GLUT1. In conclusion, the high association of muscle GLUT4 content with insulin responsiveness in different muscles, and the preservation of both with fasting, supports a predominant role of GLUT4 in insulin-mediated glucose uptake. GLUT1 may play an important role in mediating cardiac muscle glucose uptake in the basal metabolic state. Marked changes in GLUT1 expression with alterations in the metabolic state, such as prolonged fasting, may play an important role in cardiac glucose metabolism.

Fasting Does Not Impaird Insulin-Stimulated Glucose Uptake But Alters Intracellular Glucose Metabolism in Conscious Rats

Effects of 24-h and 48-h fasting on maximal insulin-stimulated whole-body and muscle glucose uptake, glycogen synthesis, and glycolysis were studied in conscious rats by combining the glucose clamp technique with tracer methods. Fasting decreased body weight and basal plasma glucose, plasma insulin, hepatic glucose output, and glucose clearance (P < 0.05 for all). However, maximal insulin-stimulated whole-body glucose uptake, normalized to body weight, was almost identical in fed, 24-h fasted, and 48-h fasted rats (191 +/- 8, 185 +/- 14, and 182 +/- 5 mumol.kg-1.min-1, respectively; P > 0.7). Similarly, rates of insulin-stimulated glucose uptake by four different skeletal muscles, estimated by the 2-deoxyglucose injection technique, were not different among the three groups. In contrast to glucose uptake, insulin-stimulated whole-body glycolysis was decreased significantly after fasting (36% after 48 h fasting; P < 0.05), whereas insulin-stimulated whole-body glycogen synthesis was increased (44% after 48 h fasting; P < 0.05). In fed rats, glycolysis was the major pathway for glucose metabolism during hyperinsulinemia, accounting for 60 +/- 5% of glucose uptake. This fraction was decreased significantly by fasting (P < 0.01), so that after a 48-h fast, glycolysis accounted for only 40 +/- 3% of insulin-stimulated glucose uptake and glycogen synthesis became predominant pathway, accounting for 60 +/- 3% of whole-body glucose utilization. Whole-body patterns of glucose metabolism during hyperinsulinemia were paralleled by glucose metabolism in individual muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in insulin action as a function of original anatomical site of newly differentiated adipocytes obtained in primary culture.

Stromal vascular cells were isolated from adipose tissue obtained from three different anatomical locations: epididymal (EPI), retroperitoneal (RP), and dorsal subcutaneous (SC), and allowed to differentiate in primary tissue culture. Cell number, protein concentration, glycerophosphate dehydrogenase, and lipoprotein lipase activity were similar in cells obtained from the EPI, RP, and SC regions, as were total insulin binding and the affinity of insulin for its receptor. However, both maximal insulin receptor tyrosine kinase activity and insulin-stimulated phosphorylation of the insulin receptor were significantly lower (P less than 0.05) in cells cultured from the SC region. In addition, newly differentiated adipocytes from the SC region were less sensitive to the ability of insulin to stimulate glucose uptake, and maximal insulin-stimulated glucose uptake by these cells was also significantly lower (P less than 0.05) when compared to cells obtained from the two other regions. Since these studies were performed on adipocyte precursor cells, allowed to differentiate to a similar degree in primary culture, the observed differences in insulin receptor phosphorylating activity, as well as the ability of insulin to stimulate glucose uptake appear to be intrinsic to adipose tissue from the three sites.

Relationship Between Blood Pressure, Plasma Insulin ana Triglyceride Concentration, and Insulin Action in Spontaneous Hypertensive and Wistar-Kyoto Rats

Previous results have shown that spontaneously hypertensive rats (SHR) are insulin resistant, hyperinsulinemic, and hypertriglyceridemic as compared to Wistar-Kyoto rats (WKY). Since SHR rats also have higher blood pressures than WKY rats, the current study was initiated in an attempt to evaluate the relationship between the magnitude of blood pressure and the differences in insulin and lipid metabolism seen in SHR and WKY rats. The experimental variables to be compared were determined in three groups of SHR and WKY rats, varying in age: Group I rats were 6 to 7 weeks of age; Group II rats were 8 to 9 weeks old; whereas Group III rats were 12 to 13 weeks of age. Maximal insulin-stimulated glucose transport by adipocytes isolated from SHR rats was significantly lower (P less than .005) than isolated adipocytes from WKY rats in all three groups, whereas plasma insulin and triglyceride concentrations were significantly higher (P less than .005). Blood pressure was also higher in SHR than in WKY rats within each group. Since absolute values for maximal insulin-stimulated glucose uptake did not change statistically as the rats grew older, the magnitude of the decrease in SHR rats was similar in each group. The increase in plasma insulin and triglyceride concentration observed in SHR rats was also the same in each group, but the absolute values increased with age in both SHR and WKY rats. However, blood pressure rose significantly with age in SHR, but not WKY rats, leading to a progressive increase in the degree of hypertension in SHR rats. These results support the view that hypertension, per se, does not lead to insulin resistance, hyperinsulinemia, and hypertriglyceridemia.(ABSTRACT TRUNCATED AT 250 WORDS)