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Inosine-5'-Monophosphate Dehydrogenase Is Required for Mitogenic Competence of Transformed Pancreatic ß Cells¹

From The Diabetes Laboratories (S.M., S.H., L.H., E.E., V.S., J.S.B., P.O.T.), Pacific Northwest Research Institute, Seattle, Washington 98122; and the Division of Endocrinology (M.R.), the University of Wisconsin, Madison, Wisconsin 53792

Address all correspondence and requests for reprints to: Dr. Stewart Metz, Pacific Northwest Research Institute, 720 Broadway, Seattle, Washington 98122. E-mail: smetz{at}pnri.org

The relation of inosine-5'-monophosphate dehydrogenase (IMPDH; the rate-limiting enzyme in GTP synthesis) to mitogenesis was studied by enzymatic assay, immunoblots, and RT-PCR in several dissimilar transformed pancreatic ß-cell lines, using intact cells. Both of the two isoforms of IMPDH (constitutive type 1 and inducible type 2) were identified using RT-PCR in transformed ß cells or in intact islets. IMPDH 2 messenger RNA (mRNA) and IMPDH protein were both regulated reciprocally by changes in levels of their end-products. Flux through IMPDH was greatest in rapidly growing cells, due mostly to increased uptake of precursor. Glucose (but not 3–0-methylglucose, L-glucose, or fructose) further augmented substrate uptake and also increased IMPDH enzymatic activity after either 4 or 21 h of stimulation. Serum or ketoisocaproate also increased IMPDH activity (but not uptake). Two selective IMPDH inhibitors (mycophenolic acid and mizoribine) reduced IMPDH activity in all cell lines, and, with virtually identical concentration-response curves, inhibited DNA synthesis (assessed as bromodeoxyuridine incorporation) in response to glucose, serum, or ketoisocaproate. Inhibition of DNA synthesis was reversible, completely prevented by repletion of cellular guanine (but not adenine) nucleotides, and could not be attributed to toxic effects. Despite the fact that modulation of IMPDH expression by guanine nucleotides was readily detectable, glucose and/or serum failed to alter IMPDH mRNA or protein, indicating that their effects on IMPDH activity were largely at the enzyme level. Precursors of guanine nucleotides failed, by themselves, to induce mitogenesis. Thus, adequate IMPDH activity (and thereby, availability of GTP) is a critical requirement for ß-cell proliferation. Although it is unlikely that further increases in GTP can, by themselves, initiate DNA synthesis, such increments may be needed to sustain mitogenesis.

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