Glucagon Biosynthesis in Human Pancreatic Islets: Preliminary Evidence for a Biosynthetic Intermediate

Abstract

Viable pancreatic islets were isolated from the pancreas of humans using modifications of the collagenase digestion and Ficoll gradient techniques. Gel filtration of tissue extracts following islet incubation in the presence of 3H-tryptophan indicated that radioactivity becomes incorporated into at least two islet pro teins. The larger of the two (LGI) has the approximate molecular size of proinsulin and the sm aller coelutes with glucagon as determined by column standardization. Radioimmunoassay of the gel filtration eluate for glucagon revealed that both molecules have glucagon immunoreactivity. Gel filtration in the presence of 8M urea did not alter the elution pattern of the LGI molecule. Polyacrylamide gel electrophoresis was performed on these glucagon immunoreactive molecules. The 3 H radioactivity and the glucagon immunoreactivity of the sm aller molecule were found to co-migrate electrophoretically with crystalline poreine glucagon and monodesamidoglucagon. With electrophoresis at high pH the electrophoretie mobility of the LGI molecule proved to be lower than that of glucagon. Partial tryptic degradation of the human LGI molecule yields 3H_ tryptophan labeled products having charge and immunologie characteristics indistinguishable from porcine glucagon and monodesamidoglucagon. Although further investigation is indicated, these results suggest that the human LGI molecule contains covalently bound glucagon and may thus serve as a precursor or an intermediate in human glucagon biosynthesis.

Heterogeneity of plasma glucagon. Circulating components in normal subjects and patients with chronic renal failure.

“…The 9,000 mol wt IRG in CRF plasma resembles material which is present in the pancreas of man and animals and which is now believed to be proglucagon (9)(10)(11)(12)(13)(14). Our studies lend support to the possibility that this circulating material might be proglucagon because exposure to trypsin converted it to small molecular weight components, some of which were similar in size to the native hormone.…”

“…In a preliminary communication (7) we have shown that circulating IRG in this condition is heterogenous, and that a significant amount of glucagon immunoreactivity is present in a peak with a molecular weight of approximately 9,000, consistent with descriptions of proglucagon (8)(9)(10)(11)(12)(13)(14). Studies by other investigators have indicated that IRG may exist in several forms in plasma of normal and diabetic subjects (15,16) and of experimental animals (17,18).…”

Secretion by glucagonomas of a possible glucagon precursor.

“…The suggestion that this void volume material is secreted (Table III) cursor has not been conclusively proven, there is persuasive evidence that a larger molecule (or molecules) is sequentially cleaved to glucagon (14). Studies in islet tissue from a number of species including humans (14)(15)(16)(17) indicate that [3H]tryptophane is incorporated into a peptide of about 9,000 daltons. In addition, material of this weight with glucagon immunoreactivity has been found in pancreatic extracts from several species (10).…”

Dominant inheritance of large molecular weight immunoreactive glucagon.

“…In addition to 3,500-dalton IRG, humani plasnma contains variable amounts of 160,000-and 9,000-dalton IRG, with elevated levels of the latter being common in patients with chroniic renal failure (8,22) and in those with glucagononmas (23)(24)(25)(26). Studies with human islets have demonstrated the inicorporation of [3H]tryptophan into both 9,000-and 3,500-dalton IRG (10). In the pedigree reported here, all affected familv members had excessive levels of L-IRG with molecular weights of 9,000 and 10,000-20,000 daltons.…”

“…The L-IRG in affected members consists of species with mol wt 9,000 and approximately 10,000-20,000 daltons, which are similar to the molecular weight of the putative precursors of 3,500-dalton glucagon which have been observed in human and animal in vitro pancreas studies (9,10). In addition, plasma levels of these L-IRG species IAbbreviations used in this paper: IRG, immunoreactive glucagon; L-IRG, large molecular weight immunoreactive glucagon; S-IRG, small molecular weight immunoreactive glucagon.…”