The Effect Of Extracellular Signal-regulated Kinase1/2 Signal Transduction Pathway On Glucose Stimulated Insulin Secretion In The β-TC-6 Cell Line

Part1 Identification of Biological Characteristics and Insulin Secretion in Response to Glucose Stimulation in Theβ-TC-6 PancreaticβCell LineObjective To investigate biological characteristics and insulin secretion in response to glucose stimulation of the B-TC-6 cell line. Methods Bate-TC-6 cells were cultured,morphological characteristics and growth curve were observed to evaluate the proliferative and differentiation ability of the B-TC-6 cells. The level of insulin was measured in supernatant in response to different glucose concentrations with OmM,1.38mM,5.5mM,11.1mM glucose stimulation in the B-TC-6 cells by radioimmunoassay. Results The B-TC-6 cell line has high proliferative and differentiation ability. The insulin level reaches a peak in response to 1.38 mM glucose stimulation compared with 0 mM,5.5 mM or 11.1 mM glucose stimulation. Conclusion In vitro, the B-TC-6 cell line has high proliferative and differentiation ability and maintain their biological characteristics. The most optimum concentration of glucose stimulation is 1.38mM inβ-TC-6 cells. Part2 Activation of Extracellular Signal-regulated Kinasel/2 Signal Transduction Pathway Induced by Glucose Stimulation in Theβ-TC-6 Cell LineObjective To investigate the activation of extracellular signal-regulated kinasel/2 signal transduction pathway in theβ-TC-6 cell line in response to glucose stimulation. Methods 1,Bate-TC-6 cells were seeded in 6-well dishes and incubated for 48 hours in complete culture medium then washed in KRBH buffer supplemented with 0.1% BSA and preincubated for 2 hours in the same buffer. KRBH was then discarded and replaced with fresh buffer containing OmM,1.38 mM,5.5 mM,11.1 mM glucose for 5 minutes. At the end of the incubations, cells were lysed on ice. The supernatants containing whole cell lysates were used for Western blotting.2,Bate-TC-6 cells were seeded in 6-well dishes and incubated for 48 hours in complete culture medium then washed in KRBH buffer supplemented with 0.1% BSA and preincubated for 2 hours in the same buffer. KRBH was then discarded and replaced with fresh buffer containing 1.38 mM glucose for 0 minutes,5 minutes,15 minutes, 30 minutes. At the end of the incubations, cells were lysed on ice. The supernatants containing whole cell lysates were used for Western blotting. Results 1,The ERK1/2 phosphorylation level reaches a peak in response to 1.38 mM glucose stimulation compared with OmM,5.5mM and 11.1mM glucose stimulation in theβ-TC-6 cell line.2,The ERK1/2 phosphorylation level reaches a peak in response to 1.38 mM glucose stimulation for 5min compared with Omin,15min and 30min glucose stimulation in bate-TC-6 cell line. Conclusion Glucose stimulation can activate extraccllular signal-regulated kinase signal transduction pathway in theβ-TC-6 cell line. Part3 The Effect of Extracellular Signal-regulated Kinasel/2 Signal Transduction Pathway on Glucose Stimulated Insulin Secretion in Theβ-TC-6 Cell LineObjective To investigate the effect of extracellular signal-regulated kinasel/2 signal transduction pathway on glucose stimulated insulin secretion in the B-TC-6 cell line.Methods 1,Bate-TC-6 cells were seeded in 6-well dishes and incubated for 47.5hours in complete culture medium.MEK inhibitor PD98059 (2uM or10uM or 50uM) was included into the culture medium 30minutes prior to washed in KRBH buffer supplemented with 0.1% BSA and preincubated for 2 hours in the same buffer. KRBH was then discarded and replaced with fresh buffer containing 1.38 mM glucose for 5 minutes. At the end of the incubations, cells were lysed on ice. The supernatants containing whole cell lysates were used for Western blotting.2,Bate-TC-6 cells were seeded in 24-well dishes and incubated for 47.5 hours in complete culture medium. MEK inhibitor PD98059 (2uM or10uM or 50uM) was included into the culture medium 30minutes prior to washed in KRBH buffer supplemented with 0.1% BSA and preincubated for 30 min in the same buffer. KRBH was then discarded and replaced with fresh buffer containing 1.38 mM glucose for 1 hour. Supernatants were collected and frozen for insulin assays.Results 1,MEK inhibitor PD98059 can inhibit ERK1/2 phosphorylation induced by glucose stimulation in a dose-dependent manner in the B-TC-6 cell line.2,PD98059 can inhibit insulin secretion induced by glucose stimulation in a dose-dependent manner in theβ-TC-6 cell line. Conclusion We propose that extracellular signal-regulated kinase signal transduction pathway activation may have an important effect on glucose stimulated insulin secretion in the B-TC-6 cell line. Part4 Impairment of Insulin Secretion Induced by Glucose in Response to Inflammatory Cytokines in Theβ-TC-6 Cell LineObjective To investigate the impairment of insulin secretion induced by i glucose in response to inflammatory cytokines IL-1βand IFN-y in the B-TC-6 cell line. Methods Bate-TC-6 cells were seeded in 24-well dishes and incubated for 24hours in complete culture medium. IL-1β(0.15ng/ml,1.5ng/ml) and/or IFN-γ(10u/ml,100u/ml) was included into the culture medium 24 hours prior to washed in KRBH buffer supplemented with 0.1% BSA and preincubated for 30 min in the same buffer. KRBH was then discarded and replaced with fresh buffer containing OmM or 1.38 mM glucose for 1 hour. Supernatants were collected and frozen for insulin assays by radioimmunoassay. Results Insulin secretion in response to glucose stimulation was significantly inhibited by IL-1β(0.15ng/ml,1.5ng/ml) and/or IFN-γ(10u/ml,100u/ml). Conclusion Glucose-induced insulin secretion was impaired by inflammatory cytokines IL-1βand/or IFN-γin the B-TC-6 cell line.