Maintenance of glucose homeostasis within a narrow range is critical for cellular metabolism. Control of the synthesis of insulin and somatostatin, two of the hormones involved in glucoregulation, occurs at both the transcriptional and the translational level. The latter insures rapid availability of the hormones in response to ambient glucose levels. Glucose stimulates translation of insulin mRNA at the levels of initiation, elongation, and mobilization from a translation incompetent pool. Although the detailed mechanisms responsible for translational enhancement of somatostatin are unknown, we have recently shown that a sequence present in the 5' non-coding region of its mRNA results in translational attenuation in diverse protein synthesizing systems. This implies that a translational repressor may be present in many cell types which can recognize this sequence. We will determine whether sequence and/or structural information present in the 5' untranslated region of ppSRIF 11 can attenuate translation of heterologous """"""""reporter"""""""" proteins such as chloramphenicol acetyl transferase and globin, and investigate if this region can function in an orientatation independent (""""""""enhancer""""""""-like) manner. The putative binding factor(s) will be isolated and characterized, using a variety of techniques that have been employed to successfully isolate DNA binding proteins, such as """"""""footprinting"""""""" analysis and affinity chromatography. We hypothesize that the binding factor sequesters preprosomatostatin mRNA in a non-translating pool, from which it can be rapidly mobilized by appropriate secretagogues. We have established clonal lines of eukaryotic (GH3) cells expressing preprosomatostatin, using retroviral-mediated gene transfer techniques. These cells will be treated with inhibitors of transcription, and the level of preprosomatostatin synthesized in response to hormonal stimulation will be measured. This will determine whether somatostatin synthesis is increased, even in the absence of transcription. Elucidating the mechanisms involved in somatostatin expression will not only contribute to the understanding of this important glucoregulatory hormone, per se, but will also add to the overall knowledge of translational regulation in eukaryotes.
