Functional inactivation by interleukin-1beta of glyceraldehyde-3-phosphate dehydrogenase in insulin-secreting cells.
OBJECTIVE
It is well established that long-term exposure of isolated beta cells to cytokines [e.g., IL-1beta] results in increased expression of inducible nitric oxide synthase and subsequent release of nitric oxide, which in turn, has been shown to mediate a wide array of effects, including alterations in cellular high-energy metabolism. In this context, several extant studies have demonstrated significant reduction in adenine and guanine nucleotide triphosphate levels in beta cells exposed to IL-1beta. Herein, we examined the functional status of glyceraldehyde-3-phosphate dehydrogenase [GAPDH] in insulin-secreting cells exposed to IL-1beta, since it represents the first enzyme in the glycolytic pathway that is involved in the generation of ATP.


METHODS
GAPDH was assayed spectrophotometrically in the cytosolic fraction derived from control and IL-1beta -treated [300 pM for 24 hrs] insulin-secreting cell lines [HIT-T15 and RINm5F].


RESULTS
IL-treatment resulted in marked attenuation of GAPDH activity in HIT and RIN cells; such a reduction in this activity was not due to inhibition of its expression by IL-1. Instead, we observed that incubation of HIT and RIN lysates with peroxynitrite, a reactive intermediate of nitric oxide with superoxide anion, resulted in significant reduction in the GAPDH activity.


CONCLUSIONS
These results identify a GAPDH as one of the biochemical loci for the effects of IL-derived peroxynitrite in the islet beta cell. The previously reported reduction in high-energy phosphate levels in an IL-treated beta cell may, in part, be due to inhibition of GAPDH activity, and subsequent reduction in the glycolytic efficiency of the beta cell.
Veluthakal
R
R
Department of Pharmaceutical Sciences, Wayne State University and beta Cell Biochemistry Research Laboratory, John D. Dingell VA Medical Center, Detroit, MI 48201, USA.
Khan
I
I
Tannous
M
M
Kowluru
A
A
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D003593:Cytoplasm / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007375:Interleukin-1 / Q000378:metabolism / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007328:Insulin; D007375:Interleukin-1; D030421:Peroxynitrous Acid; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005033:Ethylmaleimide