Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding

To elucidate cellular mechanisms of insulin resistance induced by excess dietary fat, we studied conscious chronically high-fat-fed (HFF) and control chow diet-fed rats during euglycemic-hyperinsulinemic (560 pmol/l plasma insulin) clamps. Compared with chow diet feeding, fat feeding significantly impaired insulin action (reduced whole body glucose disposal rate, reduced skeletal muscle glucose metabolism, and decreased insulin suppressibility of hepatic glucose production [HGP]). In HFF rats, hyperinsulinemia significantly suppressed circulating free fatty acids but not the intracellular availability of fatty acid in skeletal muscle (long chain fatty acyl-CoA esters remained at 230% above control levels). In HFF animals, acute blockade of beta-oxidation using etomoxir increased insulin-stimulated muscle glucose uptake, via a selective increase in the component directed to glycolysis, but did not reverse the defect in net glycogen synthesis or glycogen synthase. In clamp HFF animals, etomoxir did not significantly alter the reduced ability of insulin to suppress HGP, but induced substantial depletion of hepatic glycogen content. This implied that gluconeogenesis was reduced by inhibition of hepatic fatty acid oxidation and that an alternative mechanism was involved in the elevated HGP in HFF rats. Evidence was then obtained suggesting that this involves a reduction in hepatic glucokinase (GK) activity and an inability of insulin to acutely lower glucose-6-phosphatase (G-6-Pase) activity. Overall, a 76% increase in the activity ratio G-6-Pase/GK was observed, which would favor net hepatic glucose release and elevated HGP in HFF rats. Thus in the insulin-resistant HFF rat 1) acute hyperinsulinemia fails to quench elevated muscle and liver lipid availability, 2) elevated lipid oxidation opposes insulin stimulation of muscle glucose oxidation (perhaps via the glucose-fatty acid cycle) and suppression of hepatic gluconeogenesis, and 3) mechanisms of impaired insulin-stimulated glucose storage and HGP suppressibility are not dependent on concomitant lipid oxidation; in the case of HGP we provide evidence for pivotal involvement of G-6-Pase and GK in the regulation of HGP by insulin, independent of the glucose source.     

The impact of metformin therapy on hepatic glucose production and skeletal muscle glycogen synthase activity in overweight type II diabetic patients

The effect of metformin therapy on glucose metabolism was examined in eight overweight newly presenting untreated type II diabetic patients (five males, three females). Patients were treated for 12 weeks with either metformin (850 mg x 3) or matching placebo using a double-blind crossover study design; patients were studied at presentation and at the end of each treatment period. Insulin action was assessed by measuring activation of skeletal muscle glycogen synthase (GS) before and during a 4-hour hyperinsulinemic euglycemic clamp (100 mU.kg-1 x h-1). Metformin therapy was associated with a significant decrease in fasting blood glucose (6.8 +/- 0.6 v 8.3 +/- 0.9 mmol.L-1, P < .01) and glycosylated hemoglobin ([HbA1] 7.7% +/- 0.4% v 8.5% +/- 0.5%, P < .01) levels. Fasting hepatic glucose production (HGP) was also significantly decreased following metformin therapy (1.98 +/- 0.13 v 2.41 +/- 0.20 mg.kg-1 x min-1, P < .02), whereas fasting insulin and C-peptide concentrations remained unaltered. The decrease in basal HGP correlated closely with the decrease in fasting blood glucose concentration (r = .92, P < .001). Insulin-stimulated glucose uptake was assessed using the hyperinsulinemic euglycemic clamp technique and was increased post-metformin (3.8 +/- 0.6 v 3.1 +/- 0.7 mg.kg-1 x min-1, P < .05). This was primarily the result of increased nonoxidative glucose metabolism (1.1 +/- 0.6 v 0.4 +/- 0.6 mg.kg-1 x min-1, P < .05); oxidative glucose metabolism did not change. Metformin had no measurable effect on insulin activation of skeletal muscle GS, the rate-limiting enzyme controlling muscle glucose storage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some implications for quantitative risk assessment if hormesis exists

Insulin resistance is associated with hyperleptinemia, whilst exposure of hepatoma cells and isolated adipocytes to high concentrations of leptin has been demonstrated to result in attenuated insulin response and a reduced suppression of gluconeogenesis. To determine the acute metabolic effects of hyperleptinemia, we measured whole body glucose uptake (WBU) and hepatic glucose production rate (HGP) in rats using the euglycemic hyperinsulinemic clamping technique. Anesthetised male rats received recombinant murine leptin (1 microg/min) or vehicle into the jugular vein for 90 min. After 30 min of leptin infusion, insulin was infused to a level of 70 microU/ml and a variable-rate glucose infusion was adjusted to maintain blood glucose levels to 4-4.5 mmol/l. Glucose infusion rates during clamping were not different between leptin-infused and control rats, and there were no significant effects on the HPR or WBU measured using [6-(3)H]glucose under basal or clamped conditions. In summary, our data demonstrate that acute hyperleptinemia in normal weight Wistar rats does not appear to reduce insulin sensitivity, in vivo, or to affect HPR under clamp conditions.     

Insulin resistance in cancer patients is associated with enhanced tumor necrosis factor-alpha expression in skeletal muscle

The roles of tumor necrosis factor (TNF)-alpha and the facilitative glucose transporter (GLUT) 4 on the induction of insulin resistance in peripheral tissues of cancer patients was examined by quantitative competitive PCR on biopsies of abdominal rectal muscle from patients with gastrointestinal cancer. The degree of insulin resistance in these patients was measured by the euglycemic hyperinsulinemic glucose clamp using a high physiologic insulin concentration (100 microU/ml). Quantitative competitive PCR was carried out using DNA competitors constructed by deleting 20-30 bp between the two primer annealing sites. Decreased glucose uptake (M value) in peripheral tissues was accompanied by a significantly increased TNF-alpha mRNA in skeletal muscle (r=0.867, p=0.0025). GLUT4 mRNA, however, was positively correlated with M values (r=0.739, p=0.015). The amounts of mRNAs for TNF-alpha and GLUT4 in skeletal muscle were not correlated. Serum TNF-alpha concentrations remained below the limit of detection. These findings suggest that the insulin resistance in peripheral tissues of cancer patients is in part due to the induction TNF-alpha mRNA and the down regulation of GLUT4 mRNA in peripheral tissues.     

Insulin action on whole body glucose utilization and on muscle glucose transporter translocation in mice

Murine models of insulin resistance and diabetes are versatile and have been used to investigate genetic and metabolic disorders. However, the principal assays to assess insulin action, i.e., the euglycemic-hyperinsulinemic clamp and subcellular distribution of glucose transporters, have not been implemented in this species. Here we describe procedures which allow these methods to be adapted to mice. When normal C57bl/6j mice were infused with graded doses of insulin (1, 3, 10 or 30 mU/kg/min) during a euglycemic-hyerinsulinemic clamp, the glucose infusion rate necessary to maintain euglycemia increased in a dose-dependent manner (7.4 +/- 1.7, 13.1 +/- 3.6, 24.1 +/- 2.3 or 34.8 +/- 7.5 mg/kg/min), respectively. Hindlimb muscles were isolated and samples of 2-3 g were subjected to subcellular fractionation finalizing on 25%, 30% and 35% sucrose gradients. Fraction F25 (plasma membranes) was enriched in alpha 2 Na+/K(+)-ATPase and GLUT1 glucose transporters, whereas fraction F35 (intracellular membranes) was enriched in Ca(2+)-ATPase and GLUT4 glucose transporters. Following insulin treatment, GLUT4 increased in F25 and decreased in F35. Insulin treatment had no effect on GLUT1 in F25. However, unlike in rat skeletal muscle, GLUT1 was detectable in F35 and its content decreased in this fraction following insulin treatment. The results demonstrate that whole-body glucose utilization can be assessed in mice using euglycemic-hyperinsulinemic clamps and demonstrate how subcellular fractionation procedures can be applied to murine muscle. Murine muscle GLUT4 translocates from an intracellular storage site to the plasma membrane in response to insulin.     

Characterization of cellular defects of insulin action in type 2 (non-insulin-dependent) diabetes mellitus

Seven non-insulin-dependent diabetes mellitus (NIDDM) patients participated in three clamp studies performed with [3-3H]- and [U-14C]glucose and indirect calorimetry: study I, euglycemic (5.2 +/- 0.1 mM) insulin (269 +/- 39 pM) clamp; study II, hyperglycemic (14.9 +/- 1.2 mM) insulin (259 +/- 19 pM) clamp; study III, euglycemic (5.5 +/- 0.3 mM) hyperinsulinemic (1650 +/- 529 pM) clamp. Seven control subjects received a euglycemic (5.1 +/- 0.2 mM) insulin (258 +/- 24 pM) clamp. Glycolysis and glucose oxidation were quantitated from the rate of appearance of 3H2O and 14CO2; glycogen synthesis was calculated as the difference between body glucose disposal and glycolysis. In study I, glucose uptake was decreased by 54% in NIDDM vs. controls. Glycolysis, glycogen synthesis, and glucose oxidation were reduced in NIDDM patients (P < 0.05-0.001). Nonoxidative glycolysis and lipid oxidation were higher. In studies II and III, glucose uptake in NIDDM was equal to controls (40.7 +/- 2.1 and 40.7 +/- 1.7 mumol/min.kg fat-free mass, respectively). In study II, glycolysis, but not glucose oxidation, was normal (P < 0.01 vs. controls). Nonoxidative glycolysis remained higher (P < 0.05). Glycogen deposition increased (P < 0.05 vs. study I), and lipid oxidation remained higher (P < 0.01). In study III, hyperinsulinemia normalized glycogen formation, glycolysis, and lipid oxidation but did not normalize the elevated nonoxidative glycolysis or the decreased glucose oxidation. Lipid oxidation and glycolysis (r = -0.65; P < 0.01), and glucose oxidation (r = -0.75; P < 0.01) were inversely correlated. In conclusion, in NIDDM: (a) insulin resistance involves glycolysis, glycogen synthesis, and glucose oxidation; (b) hyperglycemia and hyperinsulinemia can normalize total body glucose uptake; (c) marked hyperinsulinemia normalizes glycogen synthesis and total flux through glycolysis, but does not restore a normal distribution between oxidation and nonoxidative glycolysis; (d) hyperglycemia cannot overcome the defects in glucose oxidation and nonoxidative glycolysis; (e) lipid oxidation is elevated and is suppressed only with hyperinsulinemia.     

Dissociation of GLUT4 translocation and insulin-stimulated glucose transport in transgenic mice overexpressing GLUT1 in skeletal muscle

Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism, but impaired stimulation of glucose transport by insulin, contractions, or hypoxia (Gulve, E. A., Ren, J.-M., Marshall, B. A., Gao, J., Hansen, P. A., Holloszy, J. O. , and Mueckler, M. (1994) J. Biol. Chem. 269, 18366-18370). This study examined the relationship between glucose transport and cell-surface glucose transporter content in isolated skeletal muscle from wild-type and GLUT1-overexpressing mice using 2-deoxyglucose, 3-O-methylglucose, and the 2-N-[4-(1-azi-2,2, 2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4-yloxy)-2-propyl amine exofacial photolabeling technique. Insulin (2 milliunits/ml) stimulated a 3-fold increase in 2-deoxyglucose uptake in extensor digitorum longus muscles of control mice (0.47 +/- 0.07 micromol/ml/20 min in basal muscle versus 1.44 micromol/ml/20 min in insulin-stimulated muscle; mean +/- S.E.). Insulin failed to increase 2-deoxyglucose uptake above basal rates in muscles overexpressing GLUT1 (4.00 +/- 0.40 micromol/ml/20 min in basal muscle versus 3.96 +/- 0.37 micromol/ml/20 min in insulin-stimulated muscle). A similar lack of insulin stimulation in muscles overexpressing GLUT1 was observed using 3-O-methylglucose. However, the magnitude of the insulin-stimulated increase in cell-surface GLUT4 photolabeling was nearly identical (approximately 3-fold) in wild-type and GLUT1-overexpressing muscles. This apparently normal insulin-stimulated translocation of GLUT4 in GLUT1-overexpressing muscle was confirmed by immunoelectron microscopy. Our findings suggest that GLUT4 activity at the plasma membrane can be dissociated from the plasma membrane content of GLUT4 molecules and thus suggest that the intrinsic activity of GLUT4 is subject to regulation.     

Insulin increases liver protein phosphatase-1 and protein phosphatase-2C activities in lean, young adult rhesus monkeys

Liver glycogen synthase activity is increased, and glycogen phosphorylase activity and glucose 6-phosphate content reduced by in vivo insulin during a euglycemic hyperinsulinemic clamp in lean young adult rhesus monkeys. To examine the mechanism of dephosphorylation of liver glycogen synthase and glycogen phosphorylase, the enzyme activities of protein phosphatase-1, protein phosphatase-2C, cAMP-dependent protein kinase, glycogen synthase kinase-3, protein kinase C and protein tyrosine kinase were determined before and after three hours of in vivo insulin in these same monkeys. The bioactivity of an inositol phosphoglycan insulin mediator (pH 2.0) and cAMP concentrations were also measured in the liver before and after insulin administration. Insulin caused significant increases in protein phosphatase-1 (p = 0.005) and in protein phosphatase-2C activities (p = 0.001). Insulin-stimulated minus basal bioactivity of the pH 2.0 insulin mediator was strongly inversely related to the insulin-stimulated minus basal glucose 6-phosphate content (r = -0.93, p < 0.0001). These findings suggest that protein phosphatase-1 and protein phosphatase-2C may be involved in the mechanism of in vivo insulin activation of liver glycogen synthase and inactivation of liver glycogen phosphorylase.     

Removal of adenosine decreases the responsiveness of muscle glucose transport to insulin and contractions

Adenosine in the extracellular space modulates stimulated glucose transport in striated muscle. In the heart and in adipocytes, adenosine potentiates insulin-stimulated glucose transport. There is controversy regarding the effect of adenosine in skeletal muscle, with reports of both an inhibitory effect and no effect, on insulin-stimulated glucose transport. We found that, in rat epitrochlearis and soleus muscles, removing adenosine with adenosine deaminase or blocking its action with the adenosine receptor blocker CPDPX markedly reduces the responsiveness of glucose transport to stimulation by 1) insulin alone, 2) contractions alone, and 3) insulin and contractions in combination. Measurement of the increase in GLUT4 at the cell surface in response to a maximally effective insulin stimulus in the epitrochlearis muscle, using the exofacial label ATB-[3H]BMPA, showed that adenosine deaminase treatment markedly reduces cell-surface GLUT4 labeling. The reduction in cell-surface GLUT4 labeling was similar in magnitude to the decrease in maximally insulin-stimulated glucose transport activity in adenosine deaminase-treated muscles. These results show that adenosine potentiates insulin- and contraction-stimulated glucose transport in skeletal muscle by enhancing the increase in GLUT4 at the cell surface and raise the possibility that decreased adenosine production or action could play a causative role in insulin resistance.     

Postabsorptive respiratory quotient and insulin-stimulated glucose storage rate in nondiabetic pima indians are related To glycogen synthase fractional activity in cultured myoblasts

A decreased ratio of fat to carbohydrate oxidation rate (an elevated respiratory quotient) predicts the development of obesity. Skeletal muscle accounts for a major fraction of total body lipid oxidation and is the principle site for reduced glucose storage in insulin-resistant subjects. The potentially important role that muscle has in promoting obesity or insulin resistance may be based on metabolic control intrinsic to skeletal muscle. Cultured skeletal muscle provides a system to examine the importance of inherent metabolic traits in muscle biopsies from obese and insulin-resistant subjects. Glycogen synthase fractional activity (GSFA) was measured in cultured myoblasts from 21 Pima Indians characterized in vivo using indirect calorimetry and a euglycemic hyperinsulinemic clamp. Basal GSFA in cultured muscle cells is inversely correlated with postabsorptive respiratory quotient of the muscle donors (r = -0.66, P = 0.001) and with in vivo high dose insulin-stimulated glucose storage rates (r = 0.47, P = 0.04). These results indicate that the postabsorptive respiratory quotients and insulin-mediated glucose storage rates in vivo share a common regulatory mechanism with GSFA in cultured myoblasts. Abnormal regulation of glycogen synthase phosphorylation state may be an intrinsic defect in skeletal muscle associated with obesity and insulin resistance.     

Leptin increases glucose transport and utilization in skeletal muscle in vitro

1. The present study examines the effect of leptin on glucose transport and metabolism in incubated soleus muscle from male lean albino rats. 2. Insulin (100 microU/ml) increased glucose uptake by twofold while the leptin group (100 nmol/l) reached 75% of the insulin response after 1 hr of incubation. However, leptin did not potentiate the insulin effect on glucose uptake in soleus muscle. 3. Leptin elicited a significant increase (27.7%) in total lactate production, accompanied by a three-fold increment in glycogen synthesis from [U-14C]D-glucose. 4. Insulin raised glycogen synthesis by sixfold. The leptin plus insulin group increased glycogen synthesis by eightfold, which is equivalent to the sum of the separated leptin and insulin groups. 5. Leptin per se exerts an insulin-like effect stimulating glucose uptake, glycogen synthesis, and lactate formation and also seems to potentiate the effect of insulin on glucose incorporation into glycogen in incubated soleus muscle.     

Overexpression of glycogen phosphorylase increases GLUT4 expression and glucose transport in cultured skeletal human muscle

Skeletal muscle glucose utilization, a major factor in the control of whole-body glucose tolerance, is modulated in accordance with the muscle metabolic demand. For instance, it is increased in chronic contraction or exercise training in association with elevated expression of GLUT4 and hexokinase II (HK-II). In this work, the contribution of increased metabolic flux to the regulation of the glucose transport capacity was analyzed in cultured human skeletal muscle engineered to overexpress glycogen phosphorylase (GP). Myocytes treated with an adenovirus-bearing muscle GP cDNA (AdCMV-MGP) expressed 10 times higher GP activity and exhibited a twofold increase in the Vmax for 2-deoxy-D-[3H]glucose (2-DG) uptake, with no effect on the apparent Km. The stimulatory effect of insulin on 2-DG uptake was also markedly enhanced in AdCMV-MGP-treated cells, which showed maximal insulin stimulation 2.8 times higher than control cells. No changes in HKII total activity or the intracellular compartmentalization were found. GLUT4, protein, and mRNA were raised in AdCMV-MGP-treated cells, suggesting pretranslational activation. GLUT4 was immunodetected intracellularly with a perinuclear predominance. Culture in glucose-free or high-glucose medium did not alter GLUT4 protein content in either control cells or AdCMV-MGP-treated cells. Control and GP-overexpressing cells showed similar autoinhibition of glucose transport, although they appeared to differ in the mechanism(s) involved in this effect. Whereas GLUT1 protein increased in control cells when they were switched from a high-glucose to a glucose-free medium, GLUT1 remained unaltered in GP-expressing cells upon glucose deprivation. Therefore, the increased intracellular metabolic (glycogenolytic-glycolytic) flux that occurs in muscle cells overexpressing GP causes an increase in GLUT4 expression and enhances basal and insulin-stimulated glucose transport, without significant changes in the autoinhibition of glucose transport. This mechanism of regulation may be operative in the postexercise situation in which GLUT4 expression is upregulated in coordination with increased glycolytic flux and energy demand.     

Mechanisms of insulin resistance during acute endotoxemia

We characterized the mechanisms underlying acute endotoxin-induced alterations in glucose metabolism and determined the extent to which catecholamines mediate these changes. Acute endotoxemia was induced in chronically catheterized awake rats by a bolus injection of lipopolysaccharide (LPS; 1 mg/kg; LD10). Basal glucose turnover (Rt; infusion of [5-3H]glucose), in vivo insulin action on overall glucose utilization (euglycemic clamp), glycolysis, and glycogen synthesis were determined in four groups of rats. These groups received 1) LPS (LPS rats; n = 6), 2) saline (control rats; n = 6), 3) LPS and alpha beta-blockade (alpha beta-blockade and LPS rats; n = 9), or 4) saline and alpha beta-blockade (alpha beta-blockade control rats; n = 9). In the basal state, LPS induced hypotension and transient hyperglycemia. These changes were associated with glycogen depletion in both skeletal muscle and liver, and increased Rt. During hyperinsulinemia, whole body glucose disposal was 37% decreased (105 vs. 166 mumol/kg.min; P < 0.01). This whole body insulin resistance was characterized by decreased glycogen synthesis and glycogen synthase activity, but not by altered whole body glycolysis. alpha beta-Blockade abolished transient hyperglycemia, increased Rt, and accelerated basal liver glycogen depletion (45 vs. 105 mmol/kg dry, LPS and alpha beta-blockade rats vs. LPS rats; P < 0.05), but inhibited muscle glycogenolysis. alpha beta-Blockade did not reverse the insulin resistance induced by endotoxin. These data suggest that catecholamines counteract the LPS-induced increase in basal glucose turnover and stimulate muscle glycogenolysis during acute endotoxemia. These effects might explain the better preservation of hepatic glycogen in the absence than in the presence of alpha beta-blockade and serve as a defense mechanism against hypoglycemia. Catecholamines do not seem to be the immediate causes of insulin resistance during acute endotoxemia.     

Transgenic mice expressing the human GLUT4/muscle-fat facilitative glucose transporter protein exhibit efficient glycemic control

To examine the physiological role of the GLUT4/muscle-fat specific facilitative glucose transporter in regulating glucose homeostasis, we have generated transgenic mice expressing high levels of this protein in an appropriate tissue-specific manner. Examination of two independent founder lines demonstrated that high-level expression of GLUT4 protein resulted in a marked reduction of fasting glucose levels (approximately 70 mg/dl) compared to wild-type mice (approximately 130 mg/dl). Surprisingly, 30 min following an oral glucose challenge the GLUT4 transgenic mice had only a slight elevation in plasma glucose levels (approximately 90 mg/dl), whereas wild-type mice displayed a typical 2- to 3-fold increase (approximately 250-300 mg/dl). In parallel to the changes in plasma glucose, insulin levels were approximately 2-fold lower in the transgenic mice compared to the wild-type mice. Furthermore, isolated adipocytes from the GLUT4 transgenic mice had increased basal glucose uptake and subcellular fractionation indicated elevated levels of cell surface-associated GLUT4 protein. Consistent with these results, in situ immunocytochemical localization of GLUT4 protein in adipocytes and cardiac myocytes indicated a marked increase in plasma membrane-associated GLUT4 protein in the basal state. Taken together these data demonstrate that increased expression of the human GLUT4 gene in vivo results in a constitutively high level of cell surface GLUT4 protein expression and more efficient metabolic control over fluctuations in plasma glucose concentrations.     

Acute effects of intraperitoneal versus subcutaneous insulin delivery on glucose homeostasis in patients with NIDDM. Veterans Affairs Implantable Insulin Pump Study Group

OBJECTIVE: The objective of this study is to compare the effect of intraperitoneal versus subcutaneous insulin injection on hepatic glucose production (HGP) and systemic glucose utilization (Rd) in patients with NIDDM. RESEARCH DESIGN AND METHODS: Eight male volunteers with NIDDM, each of whom had a programmable-rate, implantable insulin pump, were given an injection of insulin (0.15 units/kg body wt) by intraperitoneal or subcutaneous injection on separate days in randomized order. Plasma glucose was kept constant for 5 h using the glucose clamp technique, and HGP and Rd were measured using isotope dilution. RESULTS: Intraperitoneal insulin injection resulted in higher and earlier peak systemic insulin concentrations (1,469 +/- 245 vs. 454 +/- 48 pmol/l, P < 0.01). Glucose Rd doubled within 1 h after intraperitoneal injection and was greater than that attained with subcutaneous injection (3.91 +/- 0.27 vs. 2.60 +/- 0.19 mg.kg-1.min-1, P < 0.01). Intraperitoneal and subcutaneous injections suppressed HGP and plasma free fatty acid to a similar extent during the first 3 h, effects tht persisted through 5 h after subcutaneous insulin injection but waned 3-4 h after intraperitoneal injection. CONCLUSIONS: In patients with NIDDM, intraperitoneal insulin injection achieves more rapid and greater peak values for stimulation of glucose Rd than subcutaneous insulin injection. With regard to HGP and lipolysis, intraperitoneal and subcutaneous injections achieve similar initial suppression but this is maintained for a more limited duration with intraperitoneal as compared with subcutaneous injection. These differences in insulin action seem directly related to the rapidity of insulin absorption with intraperitoneal injection.     

Neonatal de-afferentation of capsaicin-sensitive sensory nerves increases in vivo insulin sensitivity in conscious adult rats

Sensory neuropeptides, released from the peripheral nervous system, might modulate glucose homeostasis by antagonizing insulin action. The effects of de-afferentation of functional small diameter unmyelinated C-fibres (sensory nerves) on in vivo insulin-mediated intracellular glucose metabolism were investigated by using euglycaemic insulin (6 and 18 mU/kg x min) clamps with [3-(3)H]-glucose infusion in 24 adult rats, treated neonatally with either capsaicin (CAP) (50 mg/kg) or vehicle (CON). Following the clamp, skeletal muscle groups, liver and adipose tissue were freeze-clamped. At plasma insulin levels of approximately 90 mU/l, CAP-rats showed a 21% increase in whole body glucose uptake compared with CON (24.4 +/- 1.6 vs 20.1 +/- 0.8 mg/kg min, p < 0.02), which was paralleled by a 20% increase in whole body glycolysis (12.6 +/- 0.8 vs 10.5 +/- 0.5 mg/ kg.min p < 0.05) (concentration of 3H2O in plasma). Whole body skeletal muscle glycogenesis was increased by 80% in CAP-rats (5.7 +/- 0.7 vs 3.1 +/- 0.7 mg/kg x min, p < 0.05) with increased muscle glycogen synthase activity. Whole body (muscle, liver and adipose tissue combined) de novo lipogenesis also was increased in CAP-rats compared with CON (0.69 +/- 0.10 vs 0.44 +/- 0.06 mg/kg x min, p < 0.05) (incorporation of [3-(3)H]-glucose counts into glycogen or fat). Hepatic glucose production was lower in CAP-rats compared with CON (0.6 +/- 0.6 vs 2.1 +/- 0.7 mg/kg x min, p < 0.05). Plasma glucagon, corticosterone, epinephrine and norepinephrine levels were reduced in CAP-rats: 43 +/- 2 compared with 70 +/- 6 pg/ml, 855 +/- 55 compared with 1131 +/- 138 nmol/l, 513 +/- 136 compared with 1048 +/- 164 pmol/l and 928 +/- 142 compared with 1472 +/- 331 pmol/l, respectively, p < 0.05. At plasma insulin levels of approximately 400 mU/l, CAP-rats showed no differences in peripheral and hepatic insulin action compared with CON. We conclude that the removal of endogenous sensory neuropeptides, by de-afferentation of capsaicin-sensitive sensory nerves, increases in vivo insulin sensitivity, but not responsiveness: 1) primarily through an increased sensitivity of skeletal muscle glycogen synthesis to insulin; 2) through a reduction in the levels of counter-regulatory hormones, thereby creating a milieu which favours overall in vivo insulin sensitivity with respect to glucose uptake, glucose production, glycolysis, glycogenesis and lipogenesis.     

Tinnitus after head injury: evidence from otoacoustic emissions

The ability of portal vein insulin to control hepatic glucose production (HGP) is debated. The aim of the present study was to determine, therefore, if the liver can respond to a selective decrease in portal vein insulin. Isotopic ([3H]glucose) and arteriovenous difference methods were used to measure HGP in conscious overnight fasted dogs. A pancreatic clamp (somatostatin plus basal portal insulin and glucagon) was used to control the endocrine pancreas. A 40-min control period was followed by a 180-min test period. During the latter, the portal vein insulin level was selectively decreased while the arterial insulin level was not changed. This was accomplished by stopping the portal insulin infusion and giving insulin peripherally at half the basal portal rate (PID, n=5). In a control group (n=5), the portal insulin infusion was not changed and glucose was infused to match the hyperglycemia that occurred in the PID group. A selective decrease of 120 pmol/l in portal vein insulin was achieved (basal, 150+/-36 to last 30 min, 30+/-12 pmol/l) in the absence of a change in the arterial insulin level (basal, 30+/-3 to last 30 min, 36+/-4 pmol/l). Neither arterial nor portal insulin levels changed in the control group (30+/-6 and 126+/-30 pmol/l, respectively). In response to the selective decrease in portal vein insulin, net hepatic glucose output (NHGO) increased significantly, from 8+/-1 (basal) to 30+/-6 and 14+/-2 micromol x kg(-1) x min(-1) by 15 min and the last 30 min (P < 0.05) of the experimental period, respectively. Arterial plasma glucose increased from 5.9+/-0.2 (basal) to 10.5+/-0.4 micromol/l (last 30 min). Three-carbon gluconeogenic precursor uptake fell from 11.2+/-2.9 (basal) to 5.9+/-0.7 micromol x kg(-1) x min(-1) (last 30 min), and thus a change in gluconeogenesis could not account for any of the increase in NHGO. With matched hyperglycemia (basal, 5.5+/-0.3 to last 30 min, 10.5+/-0.8 micromol/l) but no change in insulin, NHGO decreased from 12+/-1 (basal) to 0 (-1+/-6 micromol x kg(-1) x min(-1), last 30 min, P < 0.05) and hepatic gluconeogenic precursor uptake did not change (basal, 8.0+/-1.7 to last 30 min, 8.9+/-2.2 micromol x kg[-1] x min[-1]). Thus, the liver responds rapidly to a selective decrease in portal vein insulin by markedly increasing HGP as a result of increased glycogenolysis. These studies indicate that after an overnight fast, basal HGP (glycogenolysis) is highly sensitive to the hepatic sinusoidal insulin level.     

The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import

Transgenic mice that overexpressed IGFBP-1 are hyperinsulinemic in the first week of life and gradually develop fasting hyperglycemia. In adult transgenic mice, the hypoglycemic response to IGF-I but not insulin or des (1-3) IGF-I was attenuated (P < 0.05) compared with wild-type mice. Furthermore, in isolated adipocytes from transgenic mice, the stimulatory effect of IGF-I but not insulin on 2-deoxy-[3H]-glucose uptake was reduced (P < 0.02). In contrast, in isolated soleus muscle, the effects of both IGF-I and insulin on 2-deoxy-3H-glucose uptake and on [3H]-glucose incorporation into glycogen were significantly reduced compared to wild-type mice. The decline in specific activity of the 2-deoxy-3H-glucose, a measure of glucose appearance in the circulation, was more marked in transgenic animals (P < 0.05). In addition, tissue uptake of glucose was significantly higher in diaphragm, heart, intestine, liver, soleus muscle, and adipose tissue from fasting transgenic mice. Plasma concentrations of alanine, lysine, and methionine were also elevated in transgenic mice. These data suggest that overexpression of IGFBP-1 attenuates the hypoglycemic effect of endogenous IGF-I, which is initially compensated for by enhanced pancreatic insulin production. However, in adult mice pancreatic insulin content is reduced, insulin resistance is demonstrable in skeletal muscle and fasting hyperglycemia develops.     

Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism

The relationship between glucokinase (GK) gene copy number and glucose homeostasis was studied in transgenic mice with additional copies of the entire GK gene locus (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22564-22569). The plasma glucose concentration was reduced by 25 +/- 3% and 37 +/- 4% in mice with one or two extra copies of the gene locus, respectively. The basis for the hypoglycemic phenotype was determined using metabolic tracer techniques in chronically cannulated, conscious mice with one extra GK gene copy. Under basal conditions (6-h fasted) transgenic mice had a lower blood glucose concentration (-12 +/- 1%) and a slightly higher glucose turnover rate (+8 +/- 3%), resulting in a significantly higher glucose clearance rate (+21 +/- 2%). Plasma insulin levels were not different, suggesting that increased glucose clearance was due to augmented hepatic, not islet, GK gene expression. Under hyperglycemic clamp conditions the transgenic mice had glucose turnover and clearance rates similar to the controls, but showed a lower plasma insulin response (-48 +/- 5%). Net hepatic glycogen synthesis was markedly elevated (+360%), whereas skeletal muscle glycogen synthesis was decreased (-40%). These results indicate that increased GK gene dosage leads to increased hepatic glucose metabolism and, consequently, a lower plasma glucose concentration. Increased insulin secretion was not observed, even though the transgene is expressed in islets, because hypoglycemia causes a down-regulation in islet GK content (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, in press).     

Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance

Insulin resistance is instrumental in the pathogenesis of type 2 diabetes mellitus and the Insulin Resistance Syndrome. While insulin resistance involves decreased glucose transport activity in skeletal muscle, its molecular basis is unknown. Since muscle GLUT4 glucose transporter levels are normal in type 2 diabetes, we have tested the hypothesis that insulin resistance is due to impaired translocation of intracellular GLUT4 to sarcolemma. Both insulin-sensitive and insulin-resistant nondiabetic subgroups were studied, in addition to type 2 diabetic patients. Biopsies were obtained from basal and insulin-stimulated muscle, and membranes were subfractionated on discontinuous sucrose density gradients to equilibrium or under nonequilibrium conditions after a shortened centrifugation time. In equilibrium fractions from basal muscle, GLUT4 was decreased by 25-29% in both 25 and 28% sucrose density fractions and increased twofold in both the 32% sucrose fraction and bottom pellet in diabetics compared with insulin-sensitive controls, without any differences in membrane markers (phospholemman, phosphalamban, dihydropyridine-binding complex alpha-1 subunit). Thus, insulin resistance was associated with redistribution of GLUT4 to denser membrane vesicles. No effects of insulin stimulation on GLUT4 localization were observed. In non-equilibrium fractions, insulin led to small GLUT4 decrements in the 25 and 28% sucrose fractions and increased GLUT4 in the 32% sucrose fraction by 2.8-fold over basal in insulin-sensitive but only by 1.5-fold in both insulin-resistant and diabetic subgroups. The GLUT4 increments in the 32% sucrose fraction were correlated with maximal in vivo glucose disposal rates (r = +0.51, P = 0.026), and, therefore, represented GLUT4 recruitment to sarcolemma or a quantitative marker for this process. Similar to GLUT4, the insulin-regulated aminopeptidase (vp165) was redistributed to a dense membrane compartment and did not translocate in response to insulin in insulin-resistant subgroups. In conclusion, insulin alters the subcellular localization of GLUT4 vesicles in human muscle, and this effect is impaired equally in insulin-resistant subjects with and without diabetes. This translocation defect is associated with abnormal accumulation of GLUT4 in a dense membrane compartment demonstrable in basal muscle. We have previously observed a similar pattern of defects causing insulin resistance in human adipocytes. Based on these data, we propose that human insulin resistance involves a defect in GLUT4 traffic and targeting leading to accumulation in a dense membrane compartment from which insulin is unable to recruit GLUT4 to the cell surface.