Phosphorylation of hexokinase in insulin resistance.

Regulation of hexokinase (HK) is important in controlling glucose fluxes in skeletal muscle. We have observed that fractional hexokinase activity (HKf) is stimulated by insulin but that stimulation is abnormally low in insulin-resistant muscle [l]. Preliminary evidence suggests that phosphorylation of tyrosine residues is required normally to increase hexokinase activity [2]. In the present study, we have investigated whether regulation by phosphorylation is normal in insulin-resistant muscle or whether an altered mechanism could account for the abnormal insulinmediated stimulation of HKf. Soleus muscle strips weighing 20-40 mg were isolated from nine to twelve weeks old lean and obese Zucker rats. Rats had been kept under controlled conditions (12h light/l2h dark, 23 * 1 'C) and fed standard laboratory chow and water ad libitum, except for 12-14h immediately prior to experimentation when food was removed. Animals were stunned by a blow to the head and killed by cervical dislocation. Muscles strips, tied to stainless steel clips via their tendons, were each placed in a 25ml Erlenmeyer flask containing 3ml Dulbecco's Modified Eagle's Medium, 5mM HEPES and 1OpU bovine insulin.ml-I. The flasks were gassed continually with 0 2 : CO2 (95%: 5%). After 30 min pre-incubation at 3 7 T , the muscles were transferred to a second flask containing DMEM and HEPES, as before, and 10 or 10,OOOpU insulin.ml-I. After 30 min incubation, soleus muscles were removed from the clips, and homogenised for 1015 s in ice-cold buffer (250mM sucrose, 5mM HEPES pH 7.4, 5mM MgCl2, 1mM dithiothreitol, 5% dextran 70) using a polytron homogeniser. In some experiments the homogenisation buffer also contained 2pM okadaic acid (OKA) or 20pM sodium orthovanadate (VAN). One volume of homogenate was added to one volume of assay buffer to initiate the reaction. The total hexokinase activity (HKt; activity in the absence of glucose 6phosphate) and the fractional hexokinase activity (activity in the presence of glucose 6-phosphate) were measured in each homogenate. For the measurement of HKf the assay medium contained (final concentrations) 75mM triethanolamine, pH 7.4, 7.5mM MgCQ 1.5mM KCI, 2mM dithiothreitol, 0.8mM EDTA, 2.5mM ATP, 1mM glucose (0.2pCi [U-14C]-glucose.ml-1) lOmM phosphocreatine, 7U creatine kinase ml-I. The buffer was identical for the measurement of HKt with the addition of (final concentrations) 3mM NADP+ and 3U/ml G6PDH. Two volumes of 90% ethanol (v/v) were added after three min to stop the reaction. An aliquot of the final sample was pipetted onto DE81 ion-exchange discs. The discs were washed in an excess of water and the amount of phosphorylated product was quantified using a Beckman liquid scintillation counter. One unit of activity is defined by the conversion of lpmol glucose.min-1 at 30°C. HKt was similar in muscle from lean and obese rats and was not affected by any treatment (results not shown). VAN increased HKf in insulin-sensitive muscle (Fig 1A; p4.04) , equivalent to the effect of incubation with 10.000 pU insulin/ml [l] . The combined effects of 10,OOO pU insulin/ml and VAN were not additive (Fig 1A). OKA had no effect on HKf in homogenates from insulin-sensitive muscle (Fig 1A). Insulin-resistant muscle from obese Zucker rats responded very differently. VAN had no effect on HKf but HKf was increased (p4.02) with the addition of OKA to the homogenisation buffer (Fig 1B). The effect of OKA mimicked the effect of a maximal stimulating concentmtion of insulin (Fig lB)[l]. As with the effects of VAN in insulinsensitive muscle the combined effect of 10,OOOpU insulidml and OKA were not additive in insulin-resistant muscle (Fig 1B). The presence of VAN should inhibit endogenous protein tyrosine phosphatases although it may also act to increase tyrosine kinase activity. Thus, VAN will increase the level of tyrosine phosphorylation. The results support our previous A ".