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D M Thomas, S D Rogers, K W Ng, and J D Best

ABSTRACT

Corticosteroids have profound effects on bone metabolism, though the underlying mechanisms remain unclear. They are also known to alter glucose metabolism, in part by induction of insulin resistance. To determine whether corticosteroids impair glucose metabolism in bone cells, we have examined the actions of dexamethasone (DEX) on glucose transport and insulin receptor expression using osteoblast-like UMR 106-01 cells. DEX was shown to inhibit basal 2-deoxyglucose uptake by up to 30% in a time- and dose-dependent manner. It inhibited insulin-stimulated glucose transport by 13%. By Northern and Western blot analysis, DEX was shown to stimulate insulin receptor mRNA and protein by up to 5·6-fold, but it had no effect on expression of the glucose transporter GLUT 1 mRNA or protein under basal conditions. However, DEX augmented insulin-stimulated GLUT 1 mRNA and protein levels. By Scatchard analysis of labelled insulin binding, DEX increased insulin receptor number per cell by 54%. Subcellular fractionation and Western blot analysis demonstrated that DEX caused a redistribution of immunoreactive GLUT 1 from plasma membrane to intracellular microsomes, resulting in a 21% decrease in GLUT 1 at the plasma membrane. These data suggest that (i) DEX impairs basal glucose transport by post-translational mechanisms in UMR 106-01 cells, (ii) DEX increases insulin receptor mRNA, protein and insulin binding and (iii) the inhibition of glucose transport by DEX dominates its effects on the insulin receptor. It is possible that DEX inhibition of glucose transport in osteoblasts may contribute to steroid-induced osteoporosis.

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J. E. Meats, M. D. Tuersley, L. Best, A. M. Lynch, and S. Tomlinson

ABSTRACT

We have studied the effect of lactate on a number of intracellular events which may be important in controlling the secretion of insulin by the hamster β-cell line HIT-T15. Using the fluorescent dye Oxonol V, as well as intracellular recording techniques to measure changes in membrane potential, we found that lactate, glucose, K+ and tolbutamide caused depolarization of HIT cells, while valinomycin resulted in hyperpolarization. Consistent with these findings was the observation that 10 mm lactate caused an increase of 69·0±18·4% (s.e.m., n=6) in the level of free cytosolic Ca2+ within HIT cells (assessed by fluorescence of quin 2). This was probably due to influx of Ca2+ through voltage-sensitive calcium channels, since it was dependent upon the concentration of extracellular Ca2+ and inhibited by verapamil. Lactate also caused cytosolic acidification in HIT cells and increased the secretion of insulin. These findings are consistent with the view that the electrogenic efflux of lactate could be a determinant in the activation of HIT cells by lactate and possibly by glucose.

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D M Thomas, S D Rogers, M W Sleeman, G M Pasquini, F R Bringhurst, K W Ng, J D Zajac, and J D Best

ABSTRACT

This study characterizes the actions of insulin and parathyroid hormone (PTH) on the glucose transport system in the rat osteogenic sarcoma cell line UMR 106–01, which expresses a number of features of the osteoblast phenotype. Using [1,2-3H]2-deoxyglucose (2-DOG) as a label, UMR 106–01 cells were shown to possess a glucose transport system which was enhanced by insulin. In contrast, PTH influenced glucose transport in a biphasic manner with a stimulatory effect at 1 h and a more potent inhibitory effect at 16 h on basal and insulin-stimulated 2-DOG transport. To explore the mechanism of PTH action, a direct agonist of cAMP-dependent protein kinase (PKA) was tested. 8-Bromo-cAMP had no acute stimulatory effect but inhibited basal and insulin-stimulated 2-DOG transport at 16 h. This result suggested that the prolonged, but not the acute, effect of PTH was mediated by the generation of cAMP. Further studies with the cell line UMR 4–7, a UMR 106–01 clone stably transfected with an inducible mutant inactive regulatory subunit of PKA, confirmed that the inhibitory but not the stimulatory effect of PTH was mediated by the PKA pathway. Northern blot data indicated that the prolonged inhibitory effects of PTH and 8-bromo-cAMP on glucose transport were likely to be mediated in part by reduction in the levels of GLUT1 (HepG2/brain glucose transporter) mRNA.