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Insulin and Glucose Concentration Affect Expression of Stem Cell Factor in Colonic Smooth Muscle Cells

Author: YuYingJuan
Tutor: LinLin
School: Nanjing Medical University
Course: Internal Medicine
Keywords: Insulin Stem cell factor Smooth muscle cell Glucose PI3K MAPK
CLC: R587.2
Type: Master's thesis
Year: 2011
Downloads: 18
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Abstract

Interstitial cells of Cajal (ICC), smooth muscle cells (SMC) and the enteric nervous system (ENS) composed as special functional elements regulate gastrointestinal motility. ICC are specific mesenchymal cells throughout the smooth muscle layer of gastrointestinal tract which show network-like structure. As gastrointestinal pacemaker cells, ICC also feel the pressure of smooth muscle and transmit the neural signal. The maintenance of ICC requires a variety of cytokines of which stem cell factor (SCF) plays an important role. SCF is a multi-functional marrow-derived cell growth factor, it can be synthesized by the gastrointestinal SMC, and play an important regulatory role on a variety of cell growth and development. Diabetes is closely related to the levels of insulin and glucose. Studies on animal and human of DM have shown that both ultrastructure and quantity of ICC is abnormal. Insulin (Ins) can protect / reverse the abnormal changes in ICC. Horvath considered that high glucose had protective effects on the ICC network. This study aims at discussing the impact of insulin and glucose on colonic SMC proliferation and synthesis of SCF and investigating the signaling mechanism of insulin-induced SCF of colonic SMC.Aim:1. To investigate the influence of insulin on the proliferation and the SCF expression of colonic SMC.2. To investigate the signal mechanism of Ins-induced SCF of colonic SMC.3. To investigate the influence of glucose on the ultrastructure, proliferation, SCF expression of colonic SMC.Methods:1. The separation, culture,identification of rat’s colon SMC: sacrificing normal SD rat via broking vertebral, getting colon tissue under aseptic conditions, separating colon SMC with enzymatic hydrolysis method. DMEM which including 10% fetal bovine serum was used to culture the primary generation and passage colon SMC. SMC was identified byα-actin.2. SMCs were cultured: at different Insulin concentrations(0, 2.5, 5, 20, 40 and 80 mg/L) for different durations (0, 8, 16 and 24 hours); MTT was used to test the proliferation of SMC, Western blot and quantitative reverse transcription-polymerase chain reaction were used to examine the expression of SCF at different Insulin levels.3. SMCs were pretreated with specific phosphatidylinositol 3-kinase(PI3K)inhibitor LY-294002 and mitogen-activated protein kinase(MEK)inhibitor PD-98059, and then incubated with insulin(40 mg/L for 16 h). The expression of SCF was examined by Western Blot and RT-PCR.4. SMCs were cultured at different glucose concentration(5.55, 25, 33.3, 55.5 mmol/L)for 20 days. SMC ultrastructure was observed under the electron microscope, the proliferation of SMC was tested by MTT; excluding the influence of glucose seepage pressure on Ins-induced SCF via supposing three matched groups: 5.55 mmol/L glucose plus 19.45 mmol/L mannitol, 5.55 mmol/L glucose plus 27.75 mmol/L mannitol, 5.55 mmol/L glucose plus 49.95 mmol/L mannitol. The expression of SCF protein was testde with Western-Blot.Results:1. Culture and identification of colonic SMC: In primary culture, colonic SMC were attached to the culture vessels by 24h, triangle-shaped or spindle-shaped with centrally located nuclei. SMC were proliferated 3-5d later, and reached confluence after 14 days with a“hill-and-valley”pattern. Under the microscope of 200 times the ecphyma of SMC was visible. Red immunofluorescence was found in cytoplasm of most cultured cells which denoted positive reaction. The nuclei showed blue which was treated Hoechst.2. The effect of different concentrations of Ins on the proliferation and the SCF expression of colonic SMC: At concentration of 5 mg/L, Ins showed remarkable role in SMCs proliferation, the same to the concentrations of 2o, 40, 80 mg/L; 2.5, 5, 20 mg/L of Insulin concentrations presented certain effect on the expression of SCF, optimum concentration of Insulin to upregulate SCF expression was 40 mg/L and the peak expression of SCF occurred 16 hours after incubation with Insulin. 3. The effect of PD-98059 and LY-294002 on expression of the Ins-induced SCF in rat colonic smooth muscle cell: Treatment of colonic SMC with specific inhibitor of both MAPK (PD-98059) and PI3K (LY-294002) significantly suppressed Ins- induced SCF expression.4. The effect of different concentrations of glucose on the ultrastructure, proliferation, SCF expression of colonic SMC: At glucose concentrations of 5.55 and 25 mmol / L, SMC organelles arranged regularly and maintained structural integrity with the latter more abundant mitochondria; When it was at 33.3 mmol / L, mitochondrial degeneration were observed; At 55.5 mmol / L, both the swelling of the endoplasmic reticulum and cytoplasm dissolved occured. The SMCs incubated at glucose concentrations of 25, 33.3 mmol/L proliferated relative rapidly; At glucose concentrations of 55.5mmol/L, the SMCs increased slowly. Compared with 5.55 mmol/L, the expression of SCF was abundant at glucose concentrations of 25 and 33.3 mmol/L, scanty at 55.5 mmol/L.Conclusions:1. A certain concentration of Ins can promote SMC proliferation and induce the expression of SCF, the latter effect may be associated with both PI3K/AKT and MEK/ERKs signal transductions.2. Some degree of hyperglucose is in favour of SMC proliferation and the expression of SCF in SMC. Excessively hyperglucose can damage the SMC ultrastructure.

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