Abstract
The insulin receptor (IR) and its signaling appear to be essential for insulin secretion from pancreatic beta-cells. However, much less is known about the role of the IR in alpha-cells. To assess the role of the IR in glucagon and insulin secretion, we engineered adeno-viruses for high efficiency small interference RNA (siRNA)-IR expression in isolated mouse pancreatic islets and lentiviruses for siRNA-IR expression in pancreatic alpha- and beta-cell lines (alpha-TC6 and MIN6) with specific, long term stable IR knockdown. Western blot analysis showed that these strategies resulted in 60-80% reduction of IR protein in islets and alpha- and beta-cell lines. Cell growth was reduced by 35-50% in alpha-TC and MIN6 cells stably expressing siRNA-IR, respectively. Importantly, glucagon secretion, in response to glucose (25 to 2.8 mm), was completely abolished in islets expressing siRNA-IR, whereas secretion increased 1.7-fold in islets expressing control siRNA. In contrast, there was no difference in glucose-stimulated insulin secretion when comparing siRNA-IR and siRNA control, with both groups showing a 1.7-fold increase. Islet glucagon and insulin content were also unaffected by IR knockdown. To further explore the role of the IR, siRNA-IR was stably expressed in pancreatic cell lines, which dramatically suppressed glucose-regulated glucagon secretion in alpha-TC6 cells (3.4-fold) but did not affect GSIS in MIN6 cells. Defects in siRNA-IR-expressing alpha-cells were associated with an alteration in the activity of Akt and p70S6K where insulin-induced phosphorylation of protein kinase B/AKt was greatly reduced while p70S6K activation was enhanced, suggesting that the related pathways play important roles in alpha cell function. This study provides direct evidence that appropriate expression of the IR in alpha-cells is required for glucose-dependent glucagon secretion.
Highlights
The pathology of diabetes involves a combination of insulin resistance, defects in pancreatic -cell function and mass, and serious abnormalities in glucagon secretion [1, 2]
This study reveals the importance of the insulin receptor (IR) in ␣-cell function and suggests that the insulinrelated pathway in the ␣-cell could be a potential target for controlling glucagon secretion and glucose counter-regulation
To verify the efficiency of viral transduction, a GFP-expressing adenovirus (ADLOX.HTM, the same vector for generating small interference RNA (siRNA) in this study) was used to infect islets isolated from CD-1 mice, employing the same protocol used for other islet experiments in this study
Summary
The pathology of diabetes involves a combination of insulin resistance, defects in pancreatic -cell function and mass, and serious abnormalities in glucagon secretion [1, 2]. More recent observations suggest that signaling through receptor tyrosine kinases in the -cell controls insulin synthesis and release [4] Both insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR) signaling pathways are known to play important roles in maintaining cell responsiveness to nutrients [5, 6]. Diabetic patients often display elevated levels of circulating glucagon despite hyperglycemia and become unresponsive to the stimulation of glucagon release by low glucose, suggesting that ␣-cell dysfunction occurs during the pathogenesis of diabetes [13,14,15,16] The mechanism of this functional defect is unknown but involves selective failure in cell signaling by glucose, because the glucagon secretory response to other stimuli, such as amino acid administration, remains intact [17]. This study reveals the importance of the IR in ␣-cell function and suggests that the insulinrelated pathway in the ␣-cell could be a potential target for controlling glucagon secretion and glucose counter-regulation
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