Effects Of High Glucose On The Secretory Function Of Rat Islets And TC1-6 Cell And Its Mechanism

Partâ… Involvement of the PI3K/Akt pathway in high glucose-induced insulin resistance in rat islets and TC1-6 cellsobjective In order to clarify whether long-term exposure to high glucose affects a cell function and induces hyperglucagonemia via insulin resistance.Method We intraperitoneal injected Sprague Dawley rats with dose of 100mg/kg streptozotocin and administrated the diabetic rats with NPH insulin to keep their glucose level from 5mmol/L-10mmol/L to establishβcell deleted rat model,then we isolated rat islet and cultured for 1d,3d and 5d at 37℃with RPMI 1640 medium containing 5.5mM,11.1mM and 25mM glucose,similarly,TC1-6 cells were exposed to DMEM media containing 5.5mM,11.1mM and 25mM glucose for 1d,3d and 5d.On the following experiment day,the secretion and gene expressions of glucagon were measured and we also detected the glucagon secretion when rat islets and TC1-6 cells cultured for 5 d stimulating with three different concentrations of insulin.Then we used Western Blot method to confirm the effect of high glucose on phosphorylated of Akt in rat islets and TC1-6 cells.Result(1) There was no significant difference in glucagon secretion in response to the three glucose levels in the rat islets or in TC1-6 cells at the end of one day's high glucose exposure,At day 3,compared with the control group(5.5 mM),the glucagon secretion in rat islets exposed to 25 mM glucose was markedly increased by 42%(2252.64±41.71 vs 3205.97±45.53 pg/mg protein;P<0.05),but there was no difference in TC1-6 cells.After 5d exposure,compared with the control group(5.5mmol/l),the glucagon secretion in rat islets was increased by 52%in response to 11 mM glucose(2452.22±67.89 vs 3735.44±54.89 pg/mg protein;P<0.05) and by 117%in response to 25 mM glucose (2452.22±67.89 vs 5320.20±398.51 pg/mg protein;P<0.05).However,with respect to TC1-6 cells,compared with 5.5 mM glucose,glucagon secretion was increased by 74% at day 5 in response to 25 mM glucose(78.62±4.72 vs 136.8±10.94 ng/10⁶ protein;P< 0.05).(2) Acute(1 day) exposure of TC1-6 cells and rat islets to high glucose had no effect on glucagon mRNA levels measured by RT-PCR relative to cells and islets incubated with 5.5 mM glucose(control group).However,at day 3,the glucagon mRNA levels of rat islets incubated with 25 mM glucose increased by 125%compared with the control group(P<0.05),whereas there was little change in glucagon mRNA levels in TC1-6 cells.Meanwhile,at day 5,the glucagon mRNA levels of rat islets and TC1-6 cells exposed to 25 mM glucose increased by 272%and 78%,respectively,relative to the control groups(both:P<0.05).(3) 10^-7mmol/L insulin can significantly inhibit glucagon secretion from rat islets and TC1-6 cells cultured with containing 5.5mM glucose by 60% (2315.02±48.12 vs 915.99±32.54 pg/mg protein;P<0.05) and 61%(55.12±3.86 vs 21.59±1.30ng/10⁶ protein;P < 0.05),accordingly,but just can slightly inhibit glucagon secretion both in rat islets and TC1-6 cells containing 25mM glucose(P>0.05).when insulin concentration added to 10^-5 mmol/L,glucagon secretion of rat islets and TC1-6 cells cultured with 25mM were both suppressed by 66%(3872.34±29.66 vs 1307.65±23.34 pg/mg protein;P<0.05)and 61%(118.61±10.68 vs 46.55 + 3.72 ng/10⁶ protein;P<0.05);(4) when 10^-5mmol/L insulin were administrated for 2h,the phosphorylated ser473-Akt protein levels of rat islets and TC1-6 cells exposure to 5.5mM glucose increased by 200%and 180%accordingly(P<0.05),those of rat islets and TC1-6 cells exposure to 25mM glucose also increased by 137%and 70%in response to 5.5mM after 5d(P<0.05 ).While pretreatment with 10^-5mmol/L wortmannin, phosphorylated ser473-Akt protein expressions of rat islets and TC1-6 cells exposure to 5.5mM glucose and 25mM glucose were both down regulated(P<0.05),but the inhibition in low high group is more significant than high glucose group.Conclusion Chronic glucose toxicity can cause hypersecretion of glucagon,which may be due toαcell insulin resistance induced by impaired activity of the a cell insulin signaling pathway mediated by glucose toxicity. Partâ…¡The role of Syntaxin1A/SNAP-25 proteins in reversing hyperglycemia-induced a cell toxicityObjective To examine the effects of reversing hyperglycemia-induced a cell toxicity and explore the possible mechanisms.Method TC1-6 cell were cultured 10 days in media containing 5.5mmol/L glucose(Low glucose groups,LG),10 days in media containing 25mmol/L glucose(High glucose groups,HG),5 days in 25mmol/L glucose plus 5 days in 5.5mmol/L glucose(HL groups), 5 days in 5.5mmol/L glucose plus 5 days in 25mmol/L glucose(LH groups),The secretion and gene expression of glucagon were measured,we also used Western Blot analysis to confirm the effects of high glucose and reversible glucose induced effects on the expression of Snare proteins(syntaxinl A and SNAP-25).Result(1)compared with TC1-6 cells of HG group,HL group cells Glucagon secretion dropped by 29%(P<0.05);(2)compared with TC1-6 cells of HG group,HL group cells Glucagon mRNA was decreased by 52.6%±2.8%(P<0.05);(3)compared with TC1-6 cells of LG group,the expressions of syntaxin1A and SNAP-25 of HG group cells were increased by 36%and 69%,and HL group cells were decreased by 49%and 56% respectively(P<0.05);Conclusionα-cells abnormal glucagon were ameliorated after removing glucose toxicity, also the expressions of syntaxinlA and SNAP-25 proteins which regulating secretion of glucagon were reduced accordingly.