Studies from our laboratory using acute pharmacologic blockade of nitric oxide synthase (NOS) activity have suggested that nitric oxide (NO) has an important role in regulating carbohydrate metabolism. We now report on insulin sensitivity in mice with targeted disruptions in endothelial NOS (eNOS) and neuronal NOS (nNOS) genes compared with their wild-type (WT) counterparts. Mice underwent hyperinsulinemic-euglycemic clamp studies after a 24-h fast, during an insulin infusion of 20 mU · k g -1 · min -1 . Glucose levels were measured at baseline and every 10 min during the clamp. Insulin levels were measured at baseline and at the end of the clamp study. Glucose infusion rates (GIRs) during the last 30 min of the clamp study were in a steady state. Tritiated glucose infusion was used to measure rates of endogenous glucose output (EGO) both at baseline and during steady-state euglycemia. Glucose disposal rates (GDRs) were computed from the GIR and EGO. Fasting and steady-state glucose and insulin levels were comparable in the 3 groups of mice. No differences in fasting EGO were noted between the groups. GIR was significantly reduced (37%, P = 0.001) in the eNOS knockout (KO) mice compared with the WT mice, with values for the nNOS mice being intermediate. EGO was completely suppressed in the nNOS and WT mice during insulin infusion, but not in the eNOS mice. Even so, the eNOS mice displayed significantly reduced whole-body GDRs compared with those of the WT mice ( 8 2 . 6 7 ± 10.77 vs. 103.67 ± 3.47 m g · k g -1 · min -1 , P = 0.03). eNOS KO mice are insulin resistant at the level of the liver and peripheral tissues, whereas the nNOS KO mice are insulin resistant only in the latter. These data indicate that NO plays a role in modulating insulin sensitivity and carbohydrate metabolism and that the eNOS isoform may play a dominant role relative to nNOS. Diabetes 49:XXX-XXX, 2000 N itric oxide (NO) has emerged as an important molecule with diverse biological functions. In the blood vessels, NO mediates endotheliumdependent vasodilation (1-3) in response to diverse stimuli such as shear stress (4-6), insulin (7), acetylcholine (8,9), and bradykinin (3,10). In the central nervous system (CNS) and peripheral nervous tissue, NO is an unusual neurotransmitter (11-13). NO is generated when the amino acid L-arginine is converted to citrulline by the enzyme NO synthase (NOS) (14,15). Three separate genes encode the known isoforms of NOS (16): endothelial NOS (eNOS or NOS III) and neuronal NOS (nNOS or NOS II) catalyze the constitutive production of NO in a calcium-dependent manner predominantly in the blood vessels and neural tissues, respect i v e l y. The third isoform, inducible NOS (iNOS or NOS I) is located in macrophages and catalyzes NO formation in i n flammatory cells.Intravenous administration of N G -m o n o m e t h y l -L-a r g i n i n e (L-NMMA), a competitive inhibitor of all NOS isoforms, acutely induces hypertension and insulin resistance in rats ( 1 7 ) . M o r e r e c e n t l y, we reported that acute pharm...

Glucosamine (Glmn), a product of glucose metabolism via the hexosamine pathway, causes insulin resistance in isolated adipocytes by impairing insulin-induced GLUT 4 glucose transporter translocation to the plasma membrane. We hypothesized that Glmn causes insulin resistance in vivo by a similar mechanism in skeletal muscle. We performed euglycemic hyperinsulinemic clamps (12 mU/kg/min + 3H-3-glucose) in awake male Sprague-Dawley rats with and without Glmn infusion at rates ranging from 0.1 to 6.5 mg/ kg/min. After 4 h of euglycemic clamping, hindquarter muscles were quick-frozen and homogenized, and membranes were subfractionated by differential centrifugation and separated on a discontinuous sucrose gradient (25, 30, and 35 % sucrose). Membrane proteins were solubilized and immunoblotted for GLUT 4. With Glmn, glucose uptake (GU) was maximally reduced by 33± 1 %, P < 0.001. The apparent Glmn dose to reduce maximal GU by 50% was 0.1 mg/kg/ min or 1/70th the rate of GU on a molar basis. Control galactosamine and mannosamine infusions had no effect on GU. Relative to baseline, insulin caused a 2.6-fold increase in GLUT 4 in the 25% membrane fraction (f), P < 0.01, and a 40% reduction in the 35%f, P < 0.05, but had no effect on GLUT 4 in the 30%f, P = NS. Addition of Glmn to insulin caused a 41% reduction of GLUT 4 in the 25%f, P < 0.05, a 29% fall in the 30%f, and prevented the reduction of GLUT 4 in the 35%f. The 30%f membranes were subjected to a second separation with a 27 and 30% sucrose gradient. Insulin mobilized GLUT 4 away from the 30%f, P < 0.05, but not the 27%f. In contrast, Glmn reduced GLUT 4 in the 27%f, P < 0.05, but not the 30%f. Thus, Glmn appears to alter translocation of an insulin-insensitive GLUT 4 pool. Coinfusion of Glmn did not alter enrichment of the sarcolemmal markers 5'-nucleotidase, Na+/ K+ATPase, and phospholemman in either 25, 30, or 35%f. Thus, Glmn completely blocked movement of GLUT 4 induced by insulin. Glmn is a potent inducer of insulin resis-

Glucosamine, a metabolite of glucose via the hexosamine biosynthetic pathway, potently induces insulin resistance in skeletal muscle by impairing insulin-induced GLUT4 translocation to the plasma membrane. Activation of phosphoinositide (PI) 3-kinase is necessary for insulin-stimulated GLUT4 translocation, and the serine/threonine kinase Akt/protein kinase B (PKB) is a downstream mediator of some actions of PI 3-kinase. To determine whether glucosamine-induced insulin resistance could be due to impaired signaling, we measured insulin receptor substrate (IRS)-1 and insulin receptor tyrosine phosphorylation; PI 3-kinase activity associated with IRS-1, IRS-2, and phosphotyrosine; and Akt activity and phosphorylation in skeletal muscle of rats infused for 2 h with glucosamine (6.0 mg x kg(-1) x min(-1)) or saline. Euglycemic-hyperinsulinemic clamp studies (12 mU x kg(-1) x min(-1) insulin) in awake rats showed that glucosamine infusion resulted in rapid induction of insulin resistance, with a 33% decrease in glucose infusion rate (P < 0.01). Tissues were harvested after saline alone (basal), 1 min after an insulin bolus (10 U/kg), or after 2 h of insulin clamp in saline- and glucosamine-infused rats. After 1 min of insulin stimulation, phosphorylation of IRS-1 and insulin receptor increased 6- to 8-fold in saline-infused rats and 7- to 10-fold in glucosamine-infused rats. In saline-infused rats, 1 min of insulin stimulation increased PI 3-kinase activity associated with IRS-1, IRS-2, or phosphotyrosine 7.6-, 6.4-, and 10-fold, respectively. In glucosamine-infused rats treated for 1 min with insulin, PI 3-kinase activity associated with IRS-1 was reduced 28% (P < 0.01) and that associated with phosphotyrosine was reduced 43% (P < 0.01). Insulin for 1 min stimulated Akt/PKB activity approximately 5-fold in both saline- and glucosamine-infused rats; insulin-induced hyperphosphorylation of Akt/PKB was not different between groups. Glucosamine infusion alone had no effect on tyrosine phosphorylation of the insulin receptor or IRS-1 or on stimulation of PI 3-kinase or Akt/PKB activity. However, 2 h of insulin clamp reduced PI 3-kinase activity associated with IRS-1, IRS-2, or phosphotyrosine to <30% of that seen with 1 min of insulin. No effect of glucosamine was seen on these signaling events when compared with 2 h of insulin clamp without glucosamine. Our data show that 1) glucosamine infusion in rats is associated with an impairment in the early activation of PI 3-kinase by insulin in skeletal muscle, 2) this insulin-resistant state does not involve alterations in the activation of Akt/PKB, and 3) prolonged insulin infusion under clamp conditions results in a blunting of the PI 3-kinase response to insulin.

To explore the relationship between insulin resistance and hypertension, we examined whether acute induction of hypertension can engender insulin resistance. For this purpose we measured rates of insulin-mediated glucose uptake in awake unstressed rats with the euglycemic hyperinsulinemic (12 microns.kg-1.min-1) clamp technique during infusions of saline alone or after induction of hypertension by bolus administration of NG-monomethyl-L-arginine (L-NMMA, 30 and 15 mg/kg), a competitive inhibitor of nitric oxide synthase. Arterial pressure was approximately 20% greater with L-NMMA bolus than with saline alone. Isotopically determined steady-state rates of glucose uptake were 36 +/- 1 mg.kg-1.min-1 during saline alone and 26 +/- 2 and 19 +/- 1 mg.kg-1.min-1 with low- and high-dose L-NMMA (P < 0.001 vs. saline), respectively. To rule out that insulin resistance induced by L-NMMA was adrenergically mediated, clamp studies were repeated with alpha- and beta-blockade. Rates of glucose uptake remained approximately 20% below those observed with saline alone (P < 0.001). A significant inverse correlation was observed between the height of the blood pressure and the rate of glucose uptake (r = 0.32, P = 0.04). In conclusion, acute induction of hypertension with L-NMMA can cause marked insulin resistance. We postulate that reduced skeletal muscle perfusion and/or sympathetic nervous system activation may contribute to insulin resistance induced by L-NMMA.

Systemic inhibition of nitric oxide synthase (NOS) with N Gmonomethyl-L -arginine ( L -NMMA) causes acute insulin resistance (IR), but the mechanism is unknown. We tested whether L -NMMA-induced IR occurs via NOS blockade in the central nervous system (CNS). Six groups of SpragueDawley rats were studied after chronic implantation of an intracerebroventricular (ICV) catheter into the lateral ventricle and catheters into the carotid artery and jugular vein. Animals were studied after overnight food deprivation, awake, unrestrained, and unstressed; all ICV infusion of L -NMMA or D -NMMA (control) were performed with artificial cerebrospinal fluid. ICV administration of L -NMMA resulted in a 30% rise in the basal glucose level after 2 h, while ICV D -NMMA had no effect on glucose levels. Insulin, epinephrine, and norepinephrine levels were unchanged from baseline in both groups. Tracer ( 3 H-3-glucose)-determined glucose disposal rates during 2 h euglycemic hyperinsulinemic (300 U/ml) clamps performed after ICV administration of L -NMMA were reduced by 22% compared with D -NMMA. Insulin secretory responses to a hyperglycemic clamp and to a superimposed arginine bolus were reduced by 28% in L -NMMA-infused rats compared with D -NMMA. In conclusion, ICV administration of L -NMMA causes hyperglycemia via the induction of defects in insulin secretion and insulin action, thus recapitulating abnormalities observed in type 2 diabetes. The data suggest the novel concept that central NOS-dependent pathways may control peripheral insulin action and secretion. This control is not likely to be mediated via adrenergic mechanisms and could occur via nonadrenergic, noncholinergic nitrergic neural and/or endocrine pathways. These data support previously published data suggesting that CNS mechanisms may be involved in the pathogenesis of some forms of insulin resistance and type 2 diabetes independent of adiposity. ( J. Clin. Invest.

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