Reduced Expression of the Insulin Receptor in Mouse Insulinoma (MIN6) Cells Reveals Multiple Roles of Insulin Signaling in Gene Expression, Proliferation, Insulin Content, and Secretion

Abstract

The role of insulin signaling in pancreatic ␤ cells has become increasingly apparent. Stably transformed insulinoma cell lines (MIN6) were created with small interfering RNA resulting in the reduction of insulin receptor (IR) expression up to 80% (insulin receptor knockdown, IRKD⌬80). Functionally perturbed IR signaling was confirmed with the absence of insulin-stimulated insulin receptor substrate 1 tyrosine phosphorylation. Additionally, Akt phosphorylation was reduced and responded poorly to glucose stimulation. Gene expression profiling revealed that reduced IR expression was associated with alterations in expression of >1,500 genes with diverse functions. IRKD cells exhibited low rate of proliferation due to delay in transition from G 0 /G 1 to S phase, whereas susceptibility to apoptosis did not differ from that of control cells. Insulin content was reduced in proportion to the reduction of IR. IRKD cells maintained glucose responsiveness as measured by NAD(P)H generation, whereas Ca 2؉ responses and insulin secretion were enhanced. IRKD⌬80 and control cells were treated with glucose (25 mM) or insulin (100 nM) for 45 min, and gene expression profiles were assessed. Transcriptional activation of several hundred early response genes common to both glucose and insulin stimulation was observed in control cells. In IRKD⌬80 cells, insulin failed to activate any genes as anticipated. Importantly, glucose stimulation of gene expression in IRKD⌬80 cells showed that most genes previously activated by glucose were no longer activated, suggesting a major autocrine/paracrine effect of insulin on glucoseregulated gene expression. On the other hand, there were a number of glucose-regulated genes in the IRKD⌬80 cells that were not previously observed in control cells, suggesting a feedback regulation of insulin signaling on glucose-regulated gene expression. These results demonstrate important roles of the insulin receptor in islet ␤ cell gene expression and function and may serve to elucidate molecular defects in animal models with diminished ␤ cell insulin signaling.Pancreatic ␤ cells dynamically adapt to their environment (1, 2). Changes in plasma glucose concentration along with various hormones and growth factors have been shown to be major determinants of insulin secretion, biosynthesis, and islet mass (3, 4). The islet ␤ cell responds to these changes in its environment by altering its transcriptional responses, and these changes in gene expression ultimately result in altered mass and function. However, the signaling pathways activated by these environmental changes and the ensuing transcriptional events that mediate these biological responses are only beginning to be elucidated. Specifically, the relationships between the signaling pathways activated by glucose and those activated by growth factors such as insulin remain unclear.The roles of insulin as a growth factor in the modulation of ␤ cell mass and function have been implied by the results of recent experiments. Glucose stimulation of ␤ cells in culture ha...

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