DIABETES-INDUCED CHANGES IN GUANINE-NUCLEOTIDE-REGULATORY-PROTEIN MESSENGER-RNA DETECTED USING SYNTHETIC OLIGONUCLEOTIDE PROBES

Synthetic oligonucleotide probes were designed to detect the α‐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 β‐subunits, β1 and β2. This probe hybridised with a single 1.8‐kb transcript (β2) in RNA from all tissues studied except for brain, where a less‐abundant 3.4‐kb transcript (β1) 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 α‐Gs, α‐Gi‐2 and α‐Gi‐3; mRNA for α‐Gi‐1 was undetectable. In adipocytes, diabetes increased dramatically the mRNA coding for α‐Gi‐1 and α‐Gi‐3, whilst no significant changes occurred in the fractions coding for α‐Gi‐2 and α‐Gs. No significant changes in the mRNA coding for G‐protein α‐subunits were observed in either brain, heart, skeletal muscle or kidney. Diabetes did not cause any significant changes in the mRNA coding for β2 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. Copyright © 1990, Wiley Blackwell. All rights reserved