Effects Of High Glucose And Hypoxia On Expression Of HIF-1α And Biological Function Of MIN6and HUVEC

American Association of Clinical Endocrinologists (AACE) published diabetescomprehensive treatment guidelines in2011, which first proposed that the clinical doctorsshould pay more attention to the diagnosis and treatment of sleep apnea of patients withdiabetes. Obstructive sleep apnea hypopnea syndrome (OSAHS) is a systemic diseasewhich has a high incidence in obese patients with type2diabetes. Hypoxemia caused byOSAHS can lead to multiple organ disorder, of which the most important is cardiovasculardisorder.Therefore, in order to investigate the mechanism of hypoxemia and hyperglycemia inthe development of diabetes and cardiovascular diseases, islet beta cell lines MIN6andhuman umbilical vein endothelial cells (HUVEC) were used in our study. The proliferation,autophagy, insulin secretion of MIN6and proliferation of HUVEC were observed underboth condition of hypoxia and high glucose.Part I Effects of high glucose and hypoxia on proliferation,autophagy and biological function of MIN6Objective To investigate changes of proliferation, autophagy and insulin secretionof MIN6treated by high glucose and hypoxia.Methods (1) MIN6pre-treated with euglycemia and hyperglycemia (5.5,33.3mmol/L) were further divided into6groups with different duration of anoxia:0d,1d,2d,3d,4d, reoxygenation1d after anoxia1d, respectively. The proliferation of MIN6weredetected by Cell Counting Kit-8(CCK-8) assay.(2) MIN6pre-treated with euglycemia andhyperglycemia (5.5,33.3mmol/L) were further divided into2groups: normoxia group andhypoxia group. Expression of HIF-1α and Beclin-1were detected by real-time PCR.(3)MIN6pre-treated with euglycemia and hyperglycemia (5.5,33.3mmol/L) were further dividedinto5groups with different duration of anoxia:0d,1d,2d,3d,4d. The concentration ofinsulin in supernatant in each group were detected by ELISA. Results (1) Compared hyperglycemia group with euglycemia group, there is nodifference of proliferation in normoxia (P>0.05). But under hypoxic condition, theproliferation of MIN6in both euglycemia group and hyperglycemia group were inhibitedin a time-dependent manner (euglycemia group r=-0.966, P<0.01; hyperglycemia group r=-0.912, P<0.05), and the inhibition of proliferation was even more in hyperglycemia groupthan that in euglycemia group (P<0.01). There is significant inhibition of proliferation inreoxygenation after hypoxia compared with normoxia in hyperglycemia group (P<0.01),while not in euglycemia group (P>0.05).(2) The expression of HIF-1α and Beclin-1were upregulated under hypoxia and related (r=0.965, P<0.05).(3) Under hypoxic condition, with thesame duration of anoxia time, the insulin level of hyperglycemia group was lower than thatof euglycemia group (P<0.01). Insulin levels decreased gradually under hypoxia in atime-dependent manner both in euglycemia group and hyperglycemia group (euglycemiagroup r=-0.997, P<0.01; hyperglycemia group r=-0.999, P<0.01).Conclusion Simple high glucose does not inhibit the proliferation of islet beta cellin normoxia. Hypoxia could inhibit the proliferation of islet beta cell. And under thecombination of high glucose with hypoxia, the inhibition is even more. It suggests thathypoxia and high glucose have synergistic effect on inhibition of the proliferation of isletbeta cell. The inhibition of proliferation can be reversed by reoxygenation in euglycemiagroup, while not in hyperglycemia group. Hypoxia induces autophagy by HIF-1α. Inhibition ofproliferation and autophagy lead to decrease and disorder of islet beta cells, which resultsin decreasing of insulin production. Part II Effects of high glucose and hypoxia onproliferation of HUVECObjective Observe the effects of high glucose and hypoxia on proliferation ofHUVEC, to investigate the role they play in occurrence and development of diabeticvascular lesions.Methods HUVEC pre-treated with euglycemia and hyperglycemia (5.5,33.3mmol/L) were further divided into5groups with different duration of anoxia:0h,12h,24h,48h, reoxygenation24h after anoxia24h, respectively. After the culture ended, theproliferation of HUVEC was detected by cell counting kit-8assay. Results Hypoxia obviously inhibited the proliferation of HUVEC in botheuglycemia group and hyperglycemia group in a time-dependent manner (euglycemiagroup r=-1.000, P<0.01; hyperglycemia group r=-0.997, P<0.01). Under hypoxiccondition, the inhibition of proliferation was even more in hyperglycemia group than thatin euglycemia group (P<0.01). There is no obviously difference of proliferation inreoxygenation after hypoxia compared with hypoxia both in euglycemia group andhyperglycemia group (P>0.05).Conclusion High glucose and hypoxia can obviously inhibit the proliferation ofHUVEC and they have synergistic effect on inhibition of cells. Reoxygenation cannotreverse it. It suggests that high glucose and hypoxia could decrease the number of vascularendothelial cells, which may be one of the mechanisms for leading to vascular endothelialinjury and inducing diabetic vascular complications.