Jorge E. Rincón Viatela, Karolinska, Stockholm den 12 :e januari, 1999 kl 00:03

REGULATION OF GLUCOSE TRANSPORT AND INSULIN-STIMULATED GLUT4 TRANSLOCATION IN SKELETAL MUSCLE
by Jorge E. Rincón Viatela
Doctoral thesis from the Department of Surgical Sciences Division of Clinical Physiology, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden.

SUMMARY
Skeletal muscle represents about 40% of body mass and accounts for at least 80% of glucose disposal under insulin-stimulated conditions. Furthermore, GLUT4 transport protein is the primary component of the glucose transport system in skeletal muscle. The objective of this thesis has been to study the regulation of the glucose transport process in skeletal muscle under different conditions, such as hyperglycaemia, perturbations of steroid hormones and smoking. Conditions associated with insulin resistance in skeletal muscle. Isolated skeletal muscle obtained from healthy subjects were incubated in vitro. The glucose transport rate was assessed using the glucose analogue 3-O-methylglucose and the bis-mannose photolabelling technique was used to estimate the total molar concentration of GLUT4 in skeletal muscle. Maximal insulin stimulation induced a two-fold increase in both cell-surface GLUT4 content and 3-O-methylglucose transport rate compared with basal conditions. The turnover rate of GLUT4 was unaffected by insulin. In isolated rat skeletal muscle, acute exposure to hyperglycaemia resulted in increased rates of glucose transport. Dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, blocked the hyperglycaemia effect on glucose transport. The protein kinase C inhibitors, H-7 and GF109203X blocked the stimulatory effect of hyperglycaemia on glucose transport. Hyperglycaemia induced a three-fold increase in PKCb2 on total membranes. Hyperglycaemia-induced glucose transport did not lead to changes in the recruitment of GLUT4 or GLUT1 to the plasma membrane, as determined using the sensitive exofacial bis-mannose photolabelling technique. Oopherectomised rats (OVX) with (OVX+T) or without 8 week testosterone treatment were studied to determine cellular mechanisms behind the effects of steroid hormones on whole-body insulin-mediated glucose uptake. The glucose uptake was lower in OVX rats and further reduced in OVX-T rats as compared to controls. Glycogen synthase protein expression in muscle homogenates was decreased by 25% in the OVX rats and by 37% in the OVX+T group when compared to the control group In muscle from habitual smokers, basal 3-O-methylglucose transport was two-fold higher as compared to controls with no significant increase noted in response to insulin. Circulating FFA and triglyceride levels were abnormally elevated in the smokers. Using rat skeletal muscle, no effect of nicotine was observed either on basal or insulin-stimulated glucose transport. However, the nicotine metabolite 5’-hydroxicotine exerted a stimulatory effect on both basal glucose transport and glycogen synthase activity in isolated rat soleus muscle. In conclusion, the bis-mannose photolabelling technique is a sensitive technique, demonstrating that GLUT4 content on the cell surface correlates with glucose transport under insulin-stimulated conditions. The hyperglycaemia-induced increase in glucose transport in skeletal muscle is independent of GLUT4 and GLUT1 translocation to the plasma membrane. Steroid hormones are potent regulators of insulin-stimulated events in rat skeletal muscles, specifically at the level of glycogen synthase. Cigarette smoking appears to have a dual effect on glucose homeostasis, leading to elevated basal rates of glucose uptake and insulin resistance

Key words: skeletal muscle, glucose transport, insulin resistance, hyperglycaemia, steroid hormones, smoking, nicotine.

This thesis is based on the following papers, which are referred to in the text by their Roman numerals I–VI.

I. Lund S., Holman G.D., Zierath J.R., Rincón J, Nolte L.A., Clark A.E., Schmitz O., Pedersen O. and Wallberg-Henriksson H. Effect of insulin on GLUT4 cell surface content and turnover rate in human skeletal muscle as measured by the exofacial bismannose photolabelling technique. Diabetes 46: 1965-1969, 1997.

II. Nolte L.A., Rincón J., Odegaard Whalström E., Craig B.W., Zierath J.R. and Wallberg-Henriksson H. Hyperglycemia activates glucose transport in rat skeletal muscle via a Ca2+-dependent mechanism. Diabetes 44: 1345--1348, 1995.

III. Kawano Y., Rincón J., Ryder J.W., Soler A., Nolte L., Holman G.D., Zierath J.R. and Wallberg-Henriksson H. Hyperglycemia activates glucose transport in rat skeletal muscle by a mechanism involving protein kinase Cß2. Submitted.

IV. Rincón J., Holmäng A., Odegaard E., Lönnroth P., Björntorp P., Zierath J.R. and Wallberg-Henriksson H. Mechanism behind insulin resistance in rat skeletal muscle following oophorectomy and additional testosterone treatment. Diabetes 45: 615-621, 1996.

V. Rincón J., Krook A., Galuska D., Wallberg-Henriksson H. and Zierath J.R. Altered skeletal muscle glucose transport and blood lipid levels in habitual cigarette smoking. Clin. Physiol. In press 1998. Rincón J., Ryder J.W., Kawano Y., Galuska D., Wallberg-Henriksson H. Garrod J., and Zierath J.R. Effect of the nicotine metabolite 5’-hydroxycotinine on glucose transport and glycogen synthase activity in skeletal muscle. In manuscript.