Insulin synthesis, secretory competence, and glucose utilization are sensitized by transgenic yeast hexokinase.

M.E Voss-Mccowan,B Xu,P.N Epstein

doi:10.1016/s0021-9258(17)40753-8

M.E Voss-Mccowan, B Xu + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(17)40753-8

Copy DOI

Abstract

Glucokinase regulates insulin secretion by controlling the rate of glucose phosphorylation. In this report we utilize islets transgenic for high affinity yeast hexokinase to examine the role of glucose phosphorylation on other beta cell functions. Normal pancreatic islets responded to culture in low glucose by lowering insulin synthetic rates, becoming depleted of insulin and insulin mRNA, losing competence to respond to glucose with increased insulin secretion, and lowering glucokinase levels by one-half. In transgenic islets, increased high affinity hexokinase activity provided significant protection against reductions in all parameters of insulin synthesis and helped preserve the competence of beta cells to secrete insulin. The transgenic hexokinase also increased the rate of glucose utilization. These results demonstrate that glucose phosphorylation and presumably glucokinase mediate these glucose regulated responses. Of the parameters measured, only the change in glucokinase activity did not show an effect of the yeast hexokinase transgene. We also found that yeast hexokinase transgene expression was regulated 10-fold by glucose. This is the first demonstration of glucose inducibility of the insulin promoter in transgenic mice.

Highlights

From the Department of Pharmacology a n d Toxicology, Uniuersity of North Dakota School of Medicine
Normal pancreatic islets re- tion was enhanceadt low glucose concentrations. Those results sponded to culture in low glucose by lowering insulin indicated that transgenic isletswith elevated glucose phosphosynthetic rates, becoming depleted ofinsulin and insu- rylation could provide a suitable model for examining the relin mRNA, losing competenceto respond to glucose with lationship between glucose phosphorylationand other glucoseincreased insulin secretion, and lowering glucokinase levels by one-half
In the studies described in this report, we have demonstrate that glucose phosphorylation and presum-cultured normal islets and transgenicisletscontaininginablyglucokinasemediate these glucoseregulated re- creased glucose phosphorylation activity in low glucose media sponses

Summary

Hexokinase activity was measured in islet extracbtys a modification of

For 30 minat 4 "C and the pellet saved for quantitatioofnDNA. One-pl aliquots of the extract were thceonmbined with 1pl of incubation buffer. (50 mM Hepes, pH 7.6, 120 mM KCl, 8 mM MgCl,, 14 mM 2-mercaptoethanol, 5 mM ATP, 0.1% BSA, and 1 pCi of ~-[2-~H]glucoseM).ost mM Glucose incubations contained 3 mM glucose 6-phosphate to suppress endog- FIG..Dose-response curves for glucose-stimulated insuslei-n enous (murine) high affinity hexokinasaectivity. Incubations for meas- cretion of normal (A) and transgenic ( B )islets cultured at difurement of high affinity yeast hexokinase activity contained 0.5 mM glucose as substrate.Low affinity glucolunase activity was determined from assays performedat substratelevels of 20 mM glucose, followed by subtraction of the high affinity hexokinase activity. Thirty-five islets were incubatefdor 2 h at 37 "C in 20 pl of ferent glucose concentrationFso.ur independent setsof normal and transgenicisletswereisolatedandassayedinparallel.Isletswere cultured for 48 h at 1.5,3.0,5.5, an1d1mM glucose, and each group of islets was assayefdor secretion at 4,8,16,and 32mM glucose.

RESULTS

Hexokinase on Insulin Synthesis and SecretoryCompetence

DISCUSSION

It was somewhat unexpected that theeffect of the transgene

Glucose ducruilntgure

SecreCtormy petence