Mice and rats are unusual amongst mammals for having 2 structurally similar but non-allelic insulin genes. Both genes are functional but their expression patterns relative to each other have not been conclusively determined. The insulin 1 gene (ins 1) has been shown in several studies to be selectively glucose responsive in mice and rats. We have used the MIN6 cell line to examine passage-related changes in ins 1 and 2 expression and to test glucose responsiveness of the 2 genes. Quantitative PCR techniques were used to measure mRNA levels for ins 1, ins 2 and beta-actin in MIN6 pseudoislets (cell clusters) at early (P32), intermediate (P40) and late (P64) passage and in primary mouse islets. When expressed per fg beta-actin mRNA, mouse islets displayed similar ratios of ins 1:ins 2 expression (57:43% total insulin mRNA). Passage 32 MIN6 cells also showed similar expression patterns to islets (60 : 40% total insulin mRNA). However by passage 40 ins 1 expression fell to less than 10% of total insulin mRNA and at passage 64 this value was reduced to 2% of total insulin mRNA. Glucose responsiveness of the 2 genes was assessed by incubating pseudoislets at either 0 or 20mM glucose for 24 hours. Significant increases in ins 1 expression were detected in both passage 40 but not P64 MIN6 pseudoislets (P40 at 0mM glucose 80.8 plus/minus 4.4, at 20mM glucose 125.3 plus/minus 7.8 fg ins 1/fg actin, P<0.01) but no effect of glucose was observed on expression of the ins 2 gene. Our results suggest that ins 1 and 2 are differentially regulated in MIN6 cells. Expression patterns of the 2 genes change with passage, with expression of the glucose responsive gene (ins 1) diminishing to low levels at relatively early passages.
03 - 04 Dec 2001
Society for Endocrinology