Studies on naturally occurring and man-made mutations in the insulin gene have provided new insights into insulin biosynthesis, action, and metabolism. Ten families have been identified in which one or more members have single-point mutations in their insulin genes that result in amino acid substitutions within the proinsulin molecule. Six of these cause the secretion of biologically defective insulin molecules due to changes within the A or B chains. Replacing A3-Val with Leu, B24-Phe with Ser, or B25-Phe with Leu results in molecules that have essentially normal immunoreactivity but greatly reduced insulin-receptor-binding potency. Individuals with these mutations have a syndrome of mild diabetes or glucose intolerance, which is inherited in an autosomal-dominant mode and is associated with hyperinsulinemia and altered insulin-C-peptide ratios. Although affected individuals are heterozygous and coexpress both normal and abnormal molecules, the elevated circulating insulin consists mainly of the biologically defective form, which accumulates because it fails to be rapidly metabolized via receptor-mediated endocytosis. Four additional families have mutations that are associated with relatively asymptomatic hyperproinsulinemia. A point mutation affecting proinsulin occurs in 3 of the 4 families, leading to replacement of Arg-65 by His, which prevents recognition of the C-peptide-A-chain dibasic cleavage site by the appropriate (β-cell processing protease and results in the circulation of a type II proinsulin intermediate form (des 64, 65 HPI). Members of a fourth family with hyperproinsulinemia have a substitution of B10-His with Asp, resulting in a proinsulin that exhibitsmarkedly altered subcellular sorting behavior. A significant proportion of the newly synthesized Asp-10 proinsulin is secreted in an unprocessed form via an unregulated or constitutive secretory pathway. This syndrome has been modeled in transgenic mice by introduction of this abnormal gene into the germ line, resulting in its expression at high levels along with the normal mouse insulin genes in the β-cells. These animals have not only reproduced the hyperproinsulinemia syndrome, thus allowing us to examine its mechanism in considerable detail, but have also provided opportunities to examine other aspects of insulin-gene expression. Various molecular expression techniques are now available that allow normal or mutated insulin genes to be expressed via transfection of DNA in cultured cells, injections of in vitro-generated mRNA into Xenopus oocytes, or translation of mRNA in reticulocyte cell-free systems so that their altered properties can be assessed. Application of these and other molecular biological techniques to the expression of naturally occurring mutant proinsulins and others made in the laboratory has provided new forms of insulin for therapy of diabetes and a deeper understanding of the mechanisms of biosynthesis, intracellular sorting, processing, and secretion of insulin under normal and abnormal conditions.

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