| IntroductionInsulin is secreted inβcells of islets and transported to the target tissues and target organs (such as liver, muscle and fatty tissues) through blood circulation. Insulin modulates its physiological effect by combining with insulin receptor. The main effect of insulin is to promote glucose transmembrane transport, glucose ingestion and glycogen synthesis, and to offer energy for protein or fat synthesis. In addition, insulin plays an essential role in promoting growth.In 1995, Harbeck MC et al.[1] proved the mRNA expression of insulin receptor and insulin receptor substrate in theβcells of islets through reverse transcription PCR (RT-PCR). Then, other signal transduction elements of insulin in theβcells were discovered. So far, there are two main signal transduction pathways. One is phosphatidylinositide-3 kinase (PI3K) signal pathway which mediates metabolic regulation, the other is RAS signal pathway which mediates genetic transcription and cell growth.In 1983, Streb H et al.[2] first reported Inositol-1,4,5- triphosphate (IP3) was recognized as a second messenger. After this, people widely investigated inositol signals and found that hormone, neurotransmitter, cell factor, growth factor and so on combined with receptor of cell surface, activated phosphoinositide specific-phospholipase C (PI-PLC). Activated phospholipase C ( PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2), which produce IP3 and diacylglycerol (DAG). Then IP3 and DAG regulated generation, differentiation, secretion and contraction and vital movement of cell.PLC is a key enzyme in the phosphatidyl inositol signal pathway and generally distributed in the body. As yet, twelve kinds of PLC isozymes have been found in mammalia animal. According to the construction features and regulating activation mechanism, PLC isozymes are divided into PLCβ(1-4), PLCγ(1-2), PLCδ(1-4), PLCεand PLCζ, which have some kinds of differently regulating mechanism. Previous researches have proved [3]that PLCγ1 is activated by receptor tyrosine kinase (RTK). Activated PLCγ1 hydrolyzes PIP2, which produces IP3 and DAG. Level of IP3 increasing inβcells of islets causes Ca2+ release and regulates insulin secretion. Gasa R and Kim SS et al[4,5] reported that PLCγ1 was expressed in islets of rats and mice and islet cell tumor cell line, but whether it participated feedback regulation of insulin secretion is unknown. Our early study[6] hinted hyperinsulinemia could cause damage of first phase insulin secretion. But mechanism was unknown with regard to damage of first phase insulin secretion. Insulin receptor is PTK receptor, whether insulin produces influence of PLCγ1 after insulin incubatesβcells and then affects first phase insulin secretion. Now it not yet has been reported. Insulin usually causes effect when it was combined with IR. But IR is expressed in theβcells. Thus, the study investigates responsivity of PLCγ1 by insulin effect and approach function of insulin signal transduction inβcells of islets in different time. ObjectiveTo observe the effect of insulin on expression of PLCγ1 and phosphorylated PLCγ1 in theβcells of rat islets andβTC-3 cells. And to investigate the insulin signal transduction inβcells of islets.Methods1. Rat islets were isolated and purified. Dithizone solution identified islets.The morphological features were shown with hematoxylin-eosin (HE) dyeing. The expression of PLCγ1 in different times (0, 30, 60, 120 and 240 min)was semiquantitatively analyzed by method of immunocytochemistry combined with laser scanning confocal microscopy and image analyzed technology.2.βTC-3 cells were cultivated. In growing period,βTC-3 cells were divided into three groups, including of insulin group (final concentration 100mIU/L), insulin and Genistein group (final concentration 0.1 mmol/L), insulin and PTX (final concentration 0.1mg/L). Every group was divided into five subsets (0, 30, 60, 120 and 240 min). Every subset cells were counted for 5×106. Protein was extracted and quantitated. PLCγ1 and phosphorylated PLCγ1 were detected by Western blot experiments.Results1. PLCγ1 and phosphorylated PLCγ1 was expressed in theβcells and exocrine pancreas. The expression of PLCγ1 in theβcells for 30 min with 100mIU/L insulin incubation was obviously decreased compared with that at 0 min (P<0.05). And there was no obvious change at 240 min compared with insulin incubation. The expression of phosphorylated PLCγ1 for 30 min with 100μU/mL insulin incubation was obviously increased compared with that at 0 min (P<0.05). Then along with time lasting the expression of phosphorylated PLCγ1 gradually decreased. The result explained Phosphorylation of PLCγ1 was taken place in theβcells of islets after insulin affected it.2. In five subsets ofβTC-3 cells by 100mIU/L insulin incubation, the expression of PLCγ1 for 30 min was obviously decreased; the expression of phosphorylated PLCγ1 for 30 min was obviously increased. After 0.1μmol/mL Genistein (RTK repression) was added into 100mIU/L insulin, the expression of PLCγ1 for 30 min was not decreased; the expression of phosphorylated PLCγ1 for 30 min was not increased. After 0.1mg/L PTX was added into100mIU/L insulin, the expression of PLCγ1 for 30 min was still obviously decreased; the expression of phosphorylated PLCγ1 for 30 min was still obviously increased. The change similar to the change of PLCγ1 by insulin incubation. These results show that insulin function which leads to the expression of PLCγ1 and changes of phosphorylation was modulated by RTK activation, and had nothing to do with G protein coupled receptor (GPCR).Conclusions1. The phosphorylation of PLCγ1 in theβcells for 30 min with insulin incubation was caused.2. The expression of PLCγ1 in theβTC-3 cells for 30 min with insulin incubation was obviously decreased, phosphorylation level was obviously increased. The effect with insulin is mediated by RTK.
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