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O-linked glycosylation on Ser/Thr with single N-acetylglucosamine (O-GlcNAcylation) is a reversible modification of many cytosolic/nuclear proteins, regulated in part by UDP-GlcNAc levels. Transgenic (T) mice that overexpress GLUT1 in muscle show increased basal muscle glucose transport that is resistant to insulin stimulation. Muscle UDP-GlcNAc levels are increased. To assess whether GLUT4 is a substrate for O-GlcNAcylation, we translated GLUT4 mRNA (mutated at the N-glycosylation site) in rabbit reticulocyte lysates supplemented with [[sup.35]S]methionine. O-GlcNAcylated proteins were galactosylated and separated by lectin affinity chromatography; >20% of the translated GLUT4 appeared to be O-GlcNAcylated. To assess whether GLUT4 or GLUT4-associated proteins were O-GlcNAcylated in muscles, muscle membranes were prepared from T and control (C) mice labeled with UDP-[[sup.3]H]galactose and immunoprecipitated with anti-GLUT4 IgG (or nonimmune serum), and N-glycosyl side chains were removed enzymatically. Upon SDS-PAGE, several bands showed consistently two- to threefold increased labeling in T vs. C. Separating galactosylated products by lectin chromatography similarly revealed approximately threefold more O-GlcNAc-modified proteins in T vs. C muscle membranes. RL-2 immunoblots confirmed these results. In conclusion, chronically increased glucose flux, which raises UDP-GlcNAc in muscle, results in enhanced O-GlcNAcylation of membrane proteins in vivo. These may include GLUT4 and/or GLUT4-associated proteins and may contribute to insulin resistance in this model. glucose transporter 4; glucose transporter 4-associated proteins; O-linked glycosylation on serine/threonine with single N-acetylglucosamine of membrane proteins; transgenic mice overexpressing glucose transporter 1 in muscle; rabbit reticulocyte lysate

GLUT-1 glucose transporter overexpression in muscle of transgenic mice minimizes insulin-induced glucose disposal while GLUT-4 overexpression in fat and muscle enhances whole body glucose release under hyperinsulinemic euglycemic clamp conditions. The degree of glucose transporter expression in fat and muscle can influence fuel metabolism and whole body glucose homeostasis.

Ren, Jian-Ming, Nicole Barucci, Bess A. Marshall, Polly Hansen, Mike M. Mueckler, and Gerald I. Shulman. Transgenic mice overexpressing GLUT-1 protein in muscle exhibit increased muscle glycogenesis after exercise. Am J Physiol Endocrinol Metab 278: E588-E592, 2000.--The purpose of the present study was to determine the rates of muscle glycogenolysis and glycogenesis during and after exercise in GLUT-1 transgenic mice and their age-matched littermates. Male transgenic mice (TG) expressing a high level of human GLUT-1 and their nontransgenic (NT) littermates underwent 3 h of swimming. Glycogen concentration was determined in gastrocnemius and extensor digitorum longus (EDL) muscles before exercise and at 0, 5, and 24 h postexercise, during which food (chow) and 10% glucose solution (as drinking water) were provided. Exercise resulted in ~90% reduction in muscle glycogen in both NT (from 11.2 [+ or -] 1.4 to 2.1 [+ or -] 1.3 [micro]mol/g) and TG (from 99.3 [+ or -] 4.7 to 11.8 [+ or -] 4.3 [micro]mol/g) in gastrocnemius muscle. During recovery from exercise, the glycogen concentration increased to 38.2 [+ or -] 7.3 (5 h postexercise) and 40.5 [+ or -] 2.8 [micro]mol/g (24 h postexercise) in NT mice. In TG mice, however, the increase in muscle glycogen concentration during recovery was greater (to 57.5 [+ or -] 7.4 and 152.1 [+ or -] 15.7 [micro]mol/g at 5 and 24 h postexercise, respectively). Similar results were obtained from EDL muscle. The rate of 2-deoxyglucose uptake measured in isolated EDL muscles was 7- to 10-fold higher in TG mice at rest and at 0 and 5 h postexercise. There was no difference in muscle glycogen synthase activation measured in gastrocnemius muscles between NT and TG mice immediately after exercise. These results demonstrate that the rate of muscle glycogen accumulation postexercise exhibits two phases in TG: 1) an early phase (0-5 h), with rapid glycogen accumulation similar to that of NT mice, and 2) a progressive increase in muscle glycogen concentration, which differs from that of NT mice, during the second phase (5-24 h). Our data suggest that the high level of steady-state muscle glycogen in TG mice is due to the increase in muscle glucose transport activity. swimming; glucose transport; GLUT-1; glycogen

Hsieh, Po-Shiuan, Mary Courtney Moore, Bess Marshall, Michael J. Pagliassotti, Brian Shay, Dennis Szurkus, Doss W. Neal, and Alan D. Cherrington. The head arterial glucose level is not the reference site for generation of the portal signal in conscious dogs. Am. J. Physiol. 277 (Endocrinol. Metab. 40): E678-E684, 1999.--Experiments were performed on twelve 42-h-fasted, conscious dogs to determine whether the head arterial glucose level is used as a reference standard for comparison with the portal glucose level in bringing about the stimulatory effect of portal glucose delivery on net hepatic glucose uptake (NHGU). Each experiment consisted of an 80-min equilibration, a 40-min control, and two 90-min test periods. After the control period, somatostatin was given along with insulin (7.2 pmol [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1]; 3.5-fold increase) and glucagon (0.6 ng [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1]; basal) intraportally. Glucose was infused intraportally (22.2 [micro]mol [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1]) and peripherally as needed to double the hepatic glucose load. In one test period, glucose was infused into both vertebral and carotid arteries ([HEAD.sub.G]; 22.2 [+ or -] 0.8 [micro]mol [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1]); in the other test period, saline was infused into the head arteries ([HEAD.sub.S]). One-half of the dogs received [HEAD.sub.G] first. When all dogs are considered, the blood arterial-portal glucose gradients (-0.52 [+ or -] 0.07 vs. -0.49 [+ or -] 0.03 mM) and the hepatic glucose loads (339 [+ or -] 14 vs. 334 [+ or -] 20 [micro]mol [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1]) were similar in [HEAD.sub.G] and [HEAD.sub.S]. NHGU was 24.1 [+ or -] 3.8 and 25.1 [+ or -] 4.6 [micro]mol [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1], and nonhepatic glucose uptake was 46.1 [+ or -] 4.2 and 48.8 [+ or -] 7.0 [micro]mol [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1] in [HEAD.sub.G] and [HEAD.sub.S], respectively. The head arterial glucose level is not the reference standard used for comparison with the portal glucose level in the generation of the portal signal. liver; brain; liver nerve

Experiments were performed to investigate the relationship between the overexpression of glucose transporter isoforms GLUT-1 or GLUT-4 and diabetes mellitus in transgenic mice. Moreover, the ability of the isoforms to induce obesity, glucose tolerance, fasting hyperglycemia, glucose transport, insulin resistance and glucose-transporter content of muscle were measured and analyzed. The findings demonstrated that the upregulation of glucose transporters in skeletal muscle can be an effective strategy in managing human type 2 diabetes.