Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[ 14 C]deoxyglucose to assess tissue-specific glucose uptake and [3-3 H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).

The aim of the present study was to determine the specific sites of impairment to muscle glucose uptake (MGU) in the insulin-resistant high-fat-fed, conscious C57BL/6J mouse. Wild type (WT) and hexokinase II overexpressing (HK Tg ) mice were fed either a standard diet or high-fat diet and studied at 4 months of age. A carotid artery and jugular veins had catheters chronically implanted for sampling and infusions, respectively, and mice were allowed to recovery for at least 5 days. Mice were fasted for 5 h and underwent a hyperinsulinemic-euglycemic clamp or saline infusion for 120 min. Separate groups of mice were studied during 30-min sedentary or treadmill exercise periods. A bolus of 2-deoxy[ I nsulin resistance induced by high-fat feeding is characterized by a decrease in insulin-stimulated glucose disposal (1-3). The impaired glucose disposal is likely to be due to deficits in one or more of the steps required for skeletal muscle glucose uptake (MGU). Specifically, these steps are 1) delivery of glucose to the muscle membrane, 2) facilitated transport across the muscle membrane, and 3) intracellular phosphorylation to glucose-6-phosphate (G6P) by a hexokinase (HK) isozyme. It is difficult, in the context of the whole animal, to elucidate which sites of the glucose uptake pathway are functionally altered by high-fat feeding. One approach is to alter protein levels by transgenic manipulation and measure the effect on glucose flux. Physiological stimuli can then be applied to better expose perturbations caused by a transgene. For example, overexpressing HK II in skeletal muscle increases glucose phosphorylation capacity and results in increased MGU in high-flux states created by insulin stimulation or exercise in standard diet-fed FVB/ NJ mice but not under basal glucose flux conditions (4).Transgenic manipulation can also isolate sites of impairment to MGU in insulin-resistant states, and high-flux states (e.g., insulin stimulation and exercise) can be used to amplify the signal resulting from such a deficit. In the present study, exercise and insulin were used in combination with HK II overexpression to determine the role of glucose phosphorylation in the impairment of MGU associated with the insulin resistance of the high-fat-fed C57BL/6J mouse (5,6). Previous investigations in the conscious rat have suggested that high-fat feeding leads to a functional impairment in muscle glucose phosphorylation (7). Therefore, it was hypothesized that impaired MGU resulting from high-fat feeding would be exposed in highflux states and could be corrected by HK II overexpression. The unique surgical catheterization used in this study allows for these hypotheses to be tested in the conscious, unrestrained mouse. Determining the site(s) of impairment of MGU manifested by high-fat feeding will provide insight to the pathogenesis of insulin resistance and lead to the identification of potential therapeutic targets. RESEARCH DESIGN AND METHODSAll procedures performed were approved by the Vanderbilt University Animal Care and Us...

; 10.1152/ ajpendo.00309.2003.-Muscle glucose uptake (MGU) is determined by glucose delivery, transport, and phosphorylation. C57Bl/6J mice overexpressing GLUT4, hexokinase II (HK II), or both were used to determine the barriers to MGU. A carotid artery and jugular vein were catheterized for arterial blood sampling and venous infusions. Experiments were conducted in conscious mice ϳ7 days after surgery. 2-Deoxy-[3 H]glucose was administered during rest or treadmill exercise to calculate glucose concentration-dependent (R g) and -independent (K g) indexes of MGU. Compared with wild-type controls, GLUT4-overexpressing mice had lowered fasting glycemia (165 Ϯ 6 vs. 115 Ϯ 6 mg/dl) and increased R g by 230 and 166% in the gastrocnemius and superficial vastus lateralis (SVL) muscles under sedentary conditions. GLUT4 overexpression was not able to augment exercise-stimulated R g or Kg. Whereas HK II overexpression had no effect on fasting glycemia (170 Ϯ 6 mg/dl) or sedentary R g, it increased exercise-stimulated R g by 82, 60, and 169% in soleus, gastrocnemius, and SVL muscles, respectively. Combined GLUT4 and HK II overexpression lowered fasting glycemia (106 Ϯ 6 mg/dl), increased nonesterified fatty acids, and increased sedentary R g. Combined GLUT4 and HK II overexpression did not enhance exercisestimulated Rg compared with HK II-overexpressing mice because of the reduced glucose concentration. GLUT4 combined with HK II overexpression resulted in a marked increase in exercise-stimulated K g. In conclusion, control of MGU shifts from membrane transport at rest to phosphorylation during exercise. Glucose transport is not normally a significant barrier during exercise. However, when the phosphorylation barrier is lowered by HK II overexpression, glucose transport becomes a key site of control for regulating MGU during exercise.delivery; glucose transporter 4; hexokinase; exercise; 2-deoxyglucose THE CONTROL OF skeletal muscle glucose uptake (MGU) is distributed over the following three serial steps: delivery of glucose from the blood to the sarcolemma, transport across the sarcolemmal membrane, and intracellular phosphorylation by hexokinase (HK; see Ref. 42). Both blood flow and capillary recruitment are important determinants of glucose delivery. Glucose transport is mediated by the facilitated diffusion of glucose through a GLUT family member. Under basal and sedentary conditions, GLUT1 facilitates much of the transport into skeletal muscle (10,30,38); however, after stimulation by contractions (3,8,37), GLUT4 translocates from an intracellular pool to the cell surface, thereby increasing the permeability of the sarcolemma to glucose. Whether HK activity or localization and hence the functional rate of glucose phosphorylation to glucose 6-phosphate (G-6-P) changes during acute stimulation by exercise remains to be clearly determined. While one step may exert dominance in controlling MGU under one condition, the primary control step may shift with physiological stimuli (11)(12)(13)(14)34).During sedentary conditions...

Isotopic techniques were used to test the hypothesis that exercise and nitric oxide synthase (NOS) inhibition have distinct effects on tissue-specific fatty acid and glucose uptakes in a conscious, chronically catheterized mouse model. Uptakes were measured using the radioactive tracers (125)I-labeled beta-methyl-p-iodophenylpentadecanoic acid (BMIPP) and deoxy-[2-(3)H]glucose (DG) during treadmill exercise with and without inhibition of NOS. [(125)I]BMIPP uptake at rest differed substantially among tissues with the highest levels in heart. With exercise, [(125)I]BMIPP uptake increased in both heart and skeletal muscles. In sedentary mice, NOS inhibition induced by nitro-L-arginine methyl ester (L-NAME) feeding increased heart and soleus [(125)I]BMIPP uptake. In contrast, exercise, but not L-NAME feeding, resulted in increased heart and skeletal muscle [2-(3)H]DG uptake. Significant interactions were not observed in the effects of combined exercise and L-NAME feeding on [(125)I]BMIPP and [2-(3)H]DG uptakes. In the conscious mouse, exercise and NOS inhibition produce distinct patterns of tissue-specific fatty acid and glucose uptake; NOS is not required for important components of exercise-associated metabolic signaling, or other mechanisms compensate for the absence of this regulatory mechanism.