Diabetes-induced changes in guanine-nucleotide-regulatory-protein mRNA detected using synthetic oligonucleotide probes

Abstract

Synthetic oligonucleotide probes were designed to detect the alpha-subunits of the guanine-nucleotide-regulatory proteins (G-proteins) Gi-1, Gi-2, Gi-3 and Gs (Gi is inhibitory and Gs is stimulatory). Each probe detected a single major mRNA species in Northern blots of RNA extracted from a variety of tissues. A probe was designed to identify the two forms of G-protein beta-subunits, beta 1 and beta 2. This probe hybridised with a single 1.8-kb transcript (beta 2) in RNA from all tissues studied except for brain, where a less-abundant 3.4-kb transcript (beta 1) was also detected. These probes were used to assess whether the induction of diabetes, using streptozotocin, altered the levels of mRNA coding for specific G-protein components. In hepatocytes, diabetes caused a significant reduction in the number of transcripts coding for alpha-Gs, alpha-Gi-2 and alpha-Gi-3; mRNA for alpha-Gi-1 was undectable. In adipocytes, diabetes increased dramatically the mRNA coding for alpha-Gi-1 and alpha-Gi-3, whilst no significant changes occurred in the fractions coding for alpha-Gi-2 and alpha-Gs. No significant changes in the mRNA coding for G-protein alpha-subunits were observed in either brain, heart, skeletal muscle or kidney. Diabetes did not cause any significant changes in the mRNA coding for beta 2 in any tissue or cell population studied. Such results on the relative levels of mRNA encoding G-protein components was obtained by comparing equal amounts of total RNA from tissues of control and diabetic animals. G-protein mRNA levels were expressed relative to ribosomal 28S RNA levels and, in some instances, relative to transcripts for a structural protein called CHO-B. The total cellular levels of both RNA and DNA were assessed in the various tissues and cells studied. Major falls in RNA levels/cell appeared to occur in hepatocytes and to a lesser extent in adipocytes and skeletal muscle. Thus major reductions in G-protein transcripts occurred in hepatocytes. The detected changes in G-protein mRNA are discussed in relation to the available evidence on G-protein expression. We suggest that diabetes causes tissue-specific changes in the levels of mRNA for particular G-protein species; this may have consequences for the functioning of cellular signal-transduction mechanisms in the affected tissues.