583rd MEETING, CAMBRIDGE

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The Effects of Chronic Hyperinsulinaemia on Insulin Binding and Glucose Metabolism in Rat Adipocytes JONATHAN WHITTAKER and K. GEORGE M. M. ALBERT1 Department of Clinical Biochemistry, Royal Victoria Infirmary, Newcastle upon Tyne NEI 4LP, U.K.

and DAVID A. YORK and JO SINGH Department of Nutrition, School of Biological Sciences, University of Southampton, Southampton, U.K.

Studies with animal and human obesity (Sol1 et al., 1975 ; Bar et al., 1976), have suggested that insulin may regulate its receptor concentration in target tissues. This has recently been confirmed in vitro in primary cultures of hepatocytes (Blackard et a[., 1978) and in vivo in the rat (Kobayashi & Olefsky, 1978a).We have adapted the hyperinsulinaemic model to study further this regulatory effect and its consequence for glucose metabolism by adipocytes. Male Wistar rats (1 10-14@g)were divided into two groups. One group was injected subcutaneously once dailywith increasing doses of Ultratard insulin (Novo,Copenhagen, Denmark), reaching a dose of 6 units/day by the tenth day. The rats were then maintained on this dose for a further 4 days. The second group was injected with equal volumes of 0.154~-NaCI.Animals were allowed free access to chow and 5 % sucrose in water throughout. On day 15, 24h after the last injection, rats were killed and adipocytes isolated from the epididymal fat pads by the method of Gliemann (1967). For insulin-binding studies, adipocytes were suspended in a Krebs-Ringer Hepes (N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid) buffer, pH7.4 (Vinten et al., 1976), containing bacitracin (1 mg/ml) and bovine serum albumin (1 g/100ml). Cells were incubated for 30min at 37°C with 0.2,ug of 1251-labelledinsulin (specific radioactivity 150Ci/mg) and variousconcentrations of unlabelled insulin. Cells were separated from buffer by centrifugation through silicon oil MS 200. 2-Deoxyglucose-transport studies were performed at 37°C (Kahn et al., 1977); glucose incorporation into total lipid was also measured (Gliemann et al., 1975). The final weight of the rats was similar in both insulin- and saline-treated rats (215+ 5g versus 216+ 5g; n = 20), as was the weight gain per day. Both groups consumed similar quantities of sucrose and chow. Fat-cell volume was increased in the hyperinsulinaemic animals (120 13 versus 86 f lOpl). Blood glucose and plasma insulin values at the time of death were 6.8 + 0.5 mM and 146 f 20munits/l in the hyperinsulinaemic animals and 6.0+ 0.1 mM and 26+ 2munits/l respectively in the saline-treated group. Specific insulin binding was decreased by 50-60 % in the hyperinsulinaemic animals (1.01 f 0.12% versus 2.10f 0.25 % per 2 x lo5 cells; n = 5 ) . Similar decreases were seen

+

Table 1. Plasma insulin, insulin binding and EDso of 2-deoxyglucose transport in controls and hyperinsulinaemic animals 24 and 48 h after last insulin injection For experimental details see the text. Results are given as means f S.E.M.

Control (n = 5 ) Hyperinsulinaemic, after 24h (n = 5) Hyperinsulinaemic, after 48h(n=5) VOl. 7

Plasma insulin (munitsil) 22+ 5 180f40 18f4

Insulin specifically EDs0 for 2-deoxybound glucose transport (ng/ml) (%/2 x lo5cells) 2.1 k 0.25 0.08 0.01 1.01 f0.12 0.24 + 0.02

+

2.18 & 0.4

0.10f0.02

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BIOCHEMICAL SOCIETY TRANSACTIONS

at all concentrations of insulin studied, suggesting that the decrease in binding was due to a decrease in receptor concentration. This was confirmed by Scatchard analysis. There was a proportionate right shift in the insulin dose-response curve for 2-deoxyglucose transport. There was, however, no significant difference in maximal response in either group. In animals studied 48 h after the last insulin injection, both insulin binding and the 2-deoxyglucose transport dose-response curve had returned to normal (Table 1). There was an increase in [U-14C]glucoseincorporation into total lipids in the presence of 40pgof insulin/ml in the hyperinsulinaemic group (35000 f 2000c.p.m./h per 2 x lo5 cells versus 29000f 3000c.p.m./h per 2 x lo5 cells; P

The effects of chronic hyperinsulinaemia on insulin binding and glucose metabolism in rat adipocytes.

583rd MEETING, CAMBRIDGE 1055 The Effects of Chronic Hyperinsulinaemia on Insulin Binding and Glucose Metabolism in Rat Adipocytes JONATHAN WHITTAKE...
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