Study The Role Of ERK1 / 2 Signaling Pathway In The Inflammatory Cytokine IL-1β Injury In Mice Insulinoma β-TC-6 Cells

Objective To investigate the impairment of glucose stimulated insulin secretion (GSIS) in response to inflammatory cytokine IL-1β in β-TC-6cells.Methods1. β-TC-6cells were seeded in24-well dishes for48hours in complete culture medium then washed in KRBH buffer without glucose and preincubated for30minutes in the same buffer. KRBH was then discarded and replaced with fresh buffer containing OmM,1.38mM,5.5mM glucose for1hour. Supematants were collected for insulin assays by radioimmunoassay.2. β-TC-6cells were seeded in24-well dishes and incubated for48hours in complete culture medium. IL-1β (0.15ng/ml,1.5ng/ml and15ng/ml) were included into the culture medium separately24hours prior to washed in KRBH buffer without glucose and preincubated for30min in the same buffer. KRBH was then discarded and replaced with fresh buffer containing1.38mM glucose for1hour. Supematants were collected for insulin assays by radioimmunoassay.Results1. The insulin level reaches a peak in response to1.38mM glucose stimulation compared with0mM [(70.31±1.56)vs(45.23±4.07)uIU/ml, t=-25.085,p<0.05] and5.5mM [(70.31±1.56) vs(58.32±1.93)uIU/ml, t=-11.995,p<0.05] glucose stimulation.2. The insulin level in response to glucose stimulation was significantly lower in0.15ng/ml (83.76±1.16uIU/ml),1.5ng/ml (59.46±3.20uIU/ml) and15ng/ml (56.98±1.19ulU/ml) IL-1β groups compared with pure glucose-stimulated group (108.13±3.71uIU/ml)(F=714.573, P<0.05).Conclusions The most optimum glucose concentration of GSIS is1.38mM in β-TC-6cells. Glucose-induced insulin secretion is impaired by inflammatory cytokine IL-1β in a dose-dependent manner. Objective To investigate the effect of extracellular signal-regulated kinase1/2(ERK1/2) signal transduction pathway on impairment of GSIS in response to IL-1β in β-TC-6cells.Methods1. P-TC-6cells were seeded in6-well dishes for48hours in complete culture medium then washed in KRBH buffer without glucose and preincubated for30minutes in the same buffer. KRBH was then discarded and replaced with fresh buffer containing OmM,1.38mM,5.5mM glucose for5minutes. At the end of the incubation, cells were lysed on ice. The supematants containing whole cell lysates were used for phosphorylated ERK1/2detection by Western blotting.2. β-TC-6cells were seeded in6-well dishes and incubated for48hours in complete culture medium. IL-1β (0.15ng/ml,1.5ng/ml and15ng/ml) were, included into the culture medium separately24hours prior to washed in KRBH buffer without glucose and preincubated for30minutes in the same buffer. KRBH was then discarded and replaced with fresh buffer containing1.38mM glucose for5minutes. At the end of the incubation, cells were lysed on ice. The supematants containing whole cell lysates were used for phosphorylated ERK1/2detection by Western blotting.Results1. The level of ERK1/2phosphorylation reaches a peak in response to1.38mM glucose stimulation compared with OmM and5.5mM glucose stimulation in P-TC-6cells.2. IL-1β can inhibit ERK1/2phosphorylation induced by glucose stimulation in a dose-dependent manner in β-TC-6cells.Conclusions Glucose stimulation can activate ERK1/2signal transduction pathway in β-TC-6cells. IL-1β can inhibit glucose-stimulated ERK1/2activation and impaire GSIS in p-TC-6cells. Objective To investigate the protective effect of GLP-1receptor agonist exendin-4on impairment of GSIS in response to IL-1β in β-TC-6cells.Methods1. β-TC-6cells were seeded in24-well dishes and incubated for24hours in complete culture medium. IL-1β (0.15ng/ml,1.5ng/ml and15ng/ml) and exendin-4(0.1nmol/ml and lnmol/ml) were included into the culture medium separately24hours prior to washed in KRBH buffer without glucose and preincubated for30min in the same buffer. KRBH was then discarded and replaced with fresh buffer containing1.38mM glucose for1hour. Supematants were collected for insulin assays by radioimmunoassay.2. β-TC-6cells were seeded in24-well dishes and incubated for24hours in complete culture medium.0.15ng/ml IL-1β was included into the culture medium and lnmol/ml exendin-4was added2hours prior to, simultaneously,18hours and22hours posterior to IL-1β separately24hours prior to washed in KRBH buffer without glucose and preincubated for30min in the same buffer. KRBH was then discarded and replaced with fresh buffer containing1.38mM glucose for1hour. Supematants were collected for insulin assays by radioimmunoassay.3. β-TC-6cells were seeded in6-well dishes and incubated for24hours in complete culture medium. IL-1β, exendin-4were added and cells were processed in the same way with method2. KRBH was discarded and replaced with fresh buffer containing1.38mM glucose for5minutes. At the end of the incubations, cells were lysed on ice. The supematants containing whole cell lysates were used for phosphorylated ERK1/2detection by Western blotting.Results1. The insulin level was56.97±1.20uIU/ml in glucose free group,94.22±4.09uIU/ml in1.38mM glucose stimulation group,70.16±2.56ulU/ml in0.15ng/ml IL-1β group,68.32±0.94uIU/ml in1.5ng/ml IL-1β group,55.09±1.46uIU/ml in15ng/ml IL-1β group,70.65±1.20uIU/ml in0.15ng/ml IL-1β with O.lnmol/ml exendin-4group,64.25±1.44ulU/ml in1.5ng/ml IL-1β with0.1nmol/ml exendin-4group,61.99±0.66ulU/ml in15ng/ml IL-1β with0.1nmol/ml exendin-4group,77.23±0.69ulU/ml in0.15ng/ml IL-1β with1nmol/ml exendin-4group,60.34±3.66uIU/ml in1.5ng/ml IL-1β with lnmol/ml exendin-4group,59.48±1.94ulU/ml in15ng/ml IL-1β with1nmol/ml exendin-4group. There was significant difference among groups (F=55.686, P<0.05).2. The insulin level was43.61±1.68ulU/ml in glucose free group,83.95±0.17ulU/ml in1.38mM glucose stimulation group,49.04±0.21ulU/ml in0.15ng/ml IL-1β group,74.45±5.98ulU/ml in exendin-4added2hours prior to IL-1β group,60.71±4.35ulU/ml in exendin-4added simultaneously with IL-1β group,60.21±2.39ulU/ml in exendin-4added18hours posterior to IL-1β group,56.70±0.78ulU/ml in exendin-4added22hours posterior to IL-1β group. There was significant difference among groups (F=42.806, P<0.05).3. IL-1β can inhibit ERK1/2phosphorylation induced by glucose stimulation and exendin-4can restore glucose stimulated ERK1/2phosphorylation inhibited by IL-1β in p-TC-6cells.Conclusions Exendin-4has a protective effect on impairment of GSIS in response to IL-1β inp-TC-6cells. The mechanism may be related to the restoration of glucose stimulated ERK1/2phosphorylation inhibited by IL-1β.