The arrangement of the human insulin gene in DNA from 87 individuals was analyzed by the Southern blot hybridization technique with a cloned genomic human insulin probe. Insertions of 1.5 to 3.4 kilobase pairs in the 5'-flanking region of the gene were found in DNA from 38 individuals. These insertions occurred within 1.3 kilobase pairs of the transcription initiation site. In contrast, no insertions were observed in the region 3' to the coding sequence. The prevalence of these insertions in type 2 diabetes was significantly greater than in the other groups (P less than .001). The limitation of this striking length polymorphism to a potential promoter region suggests that these insertions may play a role in insulin gene expression.
These studies of partial pancreatectomy assess pancreatic proinsulin messenger RNA (mRNA) levels as an index of in vivo insulin biosynthesis, and show relationships to glucose homeostasis. Rats were subjected to sham operation, 50% pancreatectomy (Px), or 90% Px, and were examined after 1, 3, or 14 wk. Proinsulin mRNA was measured by dot hybridization to complementary DNA. After 50% Px there was a nearly complete adaptation of proinsulin mRNA. After 90% Px a marked increase of proinsulin mRNA occurred, but it was insufficient and it was not maintained with time. The deficit in insulin production is related to development of hyperglycemia.Sham-operated controls showed no worsening of fasting or fed blood glucose or of intraperitoneal glucose tolerance within the period of observation. Total proinsulin mRNA and pancreatic insulin content rose in proportion to body weight. 50% PN produced no change from controls in body weight or blood glucose. The concentration of proinsulin mRNA in the 50% pancreatic remnant paralleled that of controls after 1 and 3 wk, but then increased after 14 wk, such that total proinsulin mRNA approached control levels. This adaptive response was reflected by changes in serum insulin, but not by pancreatic insulin content, which was only 30% of control after 14 wk. Intraperitoneal glucose tolerance was impaired mildly, and did not worsen with time after pancreatectomy.90% Px led to elevated fed blood glucose and reduced serum insulin after 3 wk, and fasting hyperglycemia was seen after 14 wk. Proinsulin mRNA concentration in the 10% pancreatic remnant showed an adaptive increase after 1 and 3 wk, such that total proinsulin mRNA reached 40% of control. After 14 wk, however, remnant proinsulin mRNA concentration was no longer increased; total proinsulin mRNA and pancreatic insulin content were severely reduced. Intraperitoneal glucose tolerance was impaired more dramatically than with the 50% Px animals, and worsened with time after operation.These observations indicate ability to increase proinsulin mRNA levels as an adaptation to pancreatectomy. Insufficiency of this adaptation is associated with the development of hyperglycemia, and the loss of this adaptation correlates with a worsening of glucose tolerance.
These studies were undertaken to characterize the high molecular weight immunoreactive insulins in plasma and tumor extracts of two patients with insulinoma, and to determine whether they are biosynthetic precursors of proinsulin. Plasma was chromatographed on a Sephadex G-50 column and fractions assayed for IRI. Three peaks were observed, one in the void volume region (6%), a peak in the proinsulin region (25%), and the largest peak comigrating with insulin standard (69%). Insulinoma slices were incubated for 4 h and high molecular weight IRI was observed in chromatographed extracts of the tumor, as well as in extracts of the incubation medium, which comprised up to 3% of the total IRI. Gel filtration chromatography of a pre-operative fasting plasma from the second patient revealed a heterogeneous distribution of IRI, with approximately 53% in the high molecular weight region. At surgery an insulinoma was removed. Slices were incubated for 4 h with 14C-amino acids and extracts chromatogrphed. 14C-labeled protein was observed in the V0, and in the proinsulin and insulin regions. These 14C-labeled proteins were precipitated with anti-insulin serum, and it was determined that whereas 60-90% of 14C-protein in the proinsulin-insulin region was precipitated, virtually none of the 14C-protein in the V0 was immunoprecipitated with anti-insulin serum. The specific activities (CPM precipitated by anti-insulin serum/muU IRI) were 1, 11.4, and 3.1 for V0, proinsulin, and insulin regions, respectively. These results suggest that proinsulin is synthesized first, followed by insulin, and then the high molecular weight IRI. The high molecular weight IRI is not a biosynthetic precursor of proinsulin. Rechromatography of the high-molecular weight IRI in the presence of 8M urea dissociated 90% into smaller immunoreactive components which chromatographed in the proinsulin and insulin region. It was concluded that high molecular weight IRI's are present in plasma, tumor extracts, and incubation medium from insulinoma patients. These high molecular weight IRI's are not biosynthetic precursors, but either aggregates of proinsulin and insulin, or insulin bound to larger proteins. These studies do not rule out the existence in humans of a smaller rapidly-turning over precursor to proinsulin similar to the pre-proinsulin discovered in a rat insulinoma by Chan et al.
Poly(a)-rich mRNA has been isolated from catfish pancreatic islet total nucleic acid. Cell-free translation of the mRNA by wheat germ extracts yielded a protein of 11 000-12 000 molecular weight, estimated by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. This peptide is larger than catfish proinsulin, but contains tryptic peptides of proinsulin. Its synthesis comprises up to 23% of the cell-free product, depending on the conditions of cell-free synthesis. Synthesis is inhibited by 7-methylguanosine 5'-monophosphate suggesting the presence of a 7-methylguanosine cap on the 5' end of catfish proinsulin mRNA. Sucrose gradient centrifugation of the islet poly(A)-rich mRNA yielded 8S and 12S peaks. These fractions were translated with wheat germ extracts and it was determined that over 60% of the islet mRNA-dependent protein from the 8S fraction was preproinsulin. The 8S mRNA fraction was electrophoresed on 3% agarose-6 M urea gels and demonstrated to be several bands, ranging from 100 000-200 000 molecular weight.
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