Expression of an islet regenerating (reg) gene in isolated rat islets: effects of nutrient and non-nutrient growth factors

Abstract

The expression of a novel regenerating (reg) gene has been reported previously in the regenerating islets of a surgical model of diabetes in rats. We exposed collagenase-isolated rat islets for three days to nutrient and non-nutrient growth factors in minimally supplemented RPMI medium (2.7 mmol/l glucose, 2% fetal calf serum), and investigated the relationship between reg gene expression and islet cell replication. RNA was prepared from half of the islets by homogenisation in guanidinium isothiocyanate followed by phenol/chloroform extraction. Northern/dot blot analyses were used to semi-quantify reg mRNA. Islet cell replication was estimated by culturing the remaining islets in radiolabelled thymidine to determine de novo DNA synthesis. Thymidine uptake was stimulated by the following factors: 11 mmol/l glucose (50% increase); 10% amino acids (126% increase); 10% fetal calf serum (39% increase); 100 ng/ml insulin (45% increase); 250 ng/ml growth hormone (65% increase); 1.5 nmol/l aldosterone (29% increase); 2U/ml platelet derived growth factor (116% increase). The results are expressed as a percentage of the thymidine incorporated into control islets cultured in minimal RPMI (1118±100 (SD) cpm/μg protein, n=15). Increased islet cell replication was paralleled in each case by a clear rise in reg mRNA expression compared to controls. Furthermore, the rank order for reg gene expression was the same as that for thymidine uptake (r=0.90). The present findings suggest a clear association between reg gene expression and islet cell replication in vitro, and are the first to demonstrate reg gene expression in response to individual growth factors. © 1992 Springer-Verlag.