The Impacts Of Testosterone On Glucolipid Metabolism And Vessel Injury In Puberty Male Rats And Their Related Molecular Mechanisms

Objective The objective of this study is to investigate the effect of testosterone on glucolipid metabolism and thoracic vascular injuries in male SD rats during adolescence. We use LPS to induce the model of endothelial cell damage. To investigate the possible roles of testosterone on vascular endothelial cells and its underlying mechanism, primary human umbilical vein endothelial cells is used.Method The subjects were 40 male SD rats at the age of 21 days. After adaptive breeding one day, all sublects were divided into normal control group(n=10), high fat diet group(n=10), high fat diet + low dose testosterone replacement group(n=10) and high fat diet + high dose testosterone group(n=10). Normal control group was fed normal diet,the others groups were fed high fat diet. Three weeks later, the rats of high fat diet group, whose weight exceed more than 20% of normal control group was regarded as successful obese rats model. High fat diet + low dose testosterone replacement group was injected 12.5ug/kg testosterone subcutaneously for 4 weeks. High fat diet + high dose testosterone replacement group was injected 25ug/kg testosterone subcutaneously for 4 weeks. At the age of 10 weeks, all the rats were sacrificed.Angular blood was taken to determined total cholesterol, high density lipoprotein,glucose, insulin, estrogen and testosterone level. Hematoxylin and eosin(HE)staining was performed to evaluate morphology change of thoracic aortic tissue.Immunohistochemistry staining was used to detect biomarkers of PI3 K signal pathway including PI3 K, AKt, IRS-1, GLUT-4, biomarkers of the nuclear factor-κB(NF-κB) inflammation pathway including NF-κB and TNF-α. The expression of PI3 K,AKt, IRS-1, GLUT-4, NF-κB and TNF-α in the aortas were determined using quantitative polymerase chain reaction and western blot, respectively. In the aortas,apoptotic cell death were detected by terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling(TUNEL) assay. Statistical methods were used to calculate whether the indexes between the four groups was statistically significant. Primary HUVECS were isolated by type II collagen enzyme and cultured. We detected rabbit anti human VIII factor associated antigen with immunohistochemical method. The studies are done when the cells are over 80% confluence within 6 passages. Used the concentrations of 0 ug/ml、1ug/ml、10ug/ml、100ug/ml LPS at 6 h、12 h and 24 h to stimulate the HUVECs. The cell proliferation activity of HUVECs was detected by MTT method. Then choosed the best concentration of LPS according to the results of MTT to built the model of endothelial cell damage. Given six concentrations of testosterone and divided into normal control group, LPS group, LPS+10-6mol/l T group,LPS+10-7mol/l T group,LPS+10-8mol/l T group,LPS+10-9mol/l T group,LPS+10-10mol/l T group and LPS+10-11mol/l T group. Measure the cell vitality and the ability to migrate by MTT, Transwell and cell wound scratch assay. Then we choosed the best testosterone concentration. At last, we choosed 10ug/ml LPS and10-9mol/l testosterone for next study. HUVECs were randomly divided into three groups: normal control group, LPS group and LPS + 10-9mol/l testosterone. Cell apoptosis were detected by TUNEL staining. The expression of PI3 K 、 NF-κB 、TNF-α、AKT、 IRS-1 and GLUT-4 were detected by immunofluorescence, western blot and PCR.Result1.The body weight of high fat diet group were higher than those in normal control group. The model of obese adolescent rats was established successfully, the outcome was statistically significant(P<0.05). We found that the level of glucose, total cholesterol, non-high density lipoprotein and insulin in high fat diet group were all higher than those in normal control group. The level of high density lipoprotein in high fat diet group was lower than normal control group(P<0.05). With low dosetestosterone treatment, the level of glucose, total cholesterol, non-high density lipoprotein and insulin was nearly rised to normal group. The level of glucose, total cholesterol, non-high density lipoprotein and insulin in FHT group were all higher than those in normal control group. The level of high density lipoprotein in high fat diet group was lower than normal control group(P<0.05).2.HE staining showed that the vascular endothelial and outer membrane boundary were normal and the structure of the vessel wall was complete in normal control group and FLT group. But some disorders were found in FC group and FHT group.3.Immunohistochemical staining, western blot and quantitative real-time polymerase chain reaction show that the expression of IRS-1, AKT and Gl UT-4 in FC group were lower than normal control group(P<0.05).The level of IRS-1, AKT and GLUT-4 in FLT group were dramatically increased, when compared with FC group. What’s more,there was a significant decrease of IRS-1, AKT and GLUT-4 in FHT group, when compared with normal control group. We also found that a significant higher NF-κB,TNF-α and PI3 K level were observed in FC group relative to normal control group.4.Correlation analysis showed that the level of FPG、FINS、HOMA-IR、TC and non-HDL was decreased with the rising of testosterone,but the level of HDL was increased with the rising of testosterone in NC group, FC group and FLT group. The level of FPG、FINS、HOMA-IR、TC and non-HDL was increased with the rising of testosterone, but the level of HDL was decreased with the rising of testosterone in FHT group,5. Immunocytochemical staining of successfully isolated primary HUVECs were shown rabbit anti human VIII factor related antigen positive.6. MTT results showed that cell vitality was suppressed after stimulated by 10ug/ml LPS for 6 h compared with normal control group, the outcome was statistically significant( P <0.05). We choose 10ug/ml LPS stimulating 6 h to make endothelial cell damage model.7. The results of MTT, Transwell and cell wound scratch assay shown that physiologic concentration testosterone can improved the decrease of cell vitality andmigration, which induced by LPS. When the concentration of testosterone <10-9mol/l,the protective effect of testosterone on cell vitality and migration is increased with the rising of testosterone concentration. When the concentration of testosterone >10-9mol/l, the protective effect of testosterone on cell vitality and migration is decreased with the rising of testosterone concentration.8. The result of TUNEL staining shown that the apoptotic indexes of LPS group and LPS+10-9mol/l T group were significantly higher than normal control group(P<0.05).The apoptotic indexes of LPS+10-9mol/l T group was significantly lower than LPS group(P<0.05).9.The immunofluorescence staining results shown that the expression of GLUT-4 in LPS group and LPS+10-9mol/l T group were significantly lower than normal control group(P<0.05). Compared with LPS group, the expression of GLUT-4 were significantly higher in LPS+10-9mol/l T group(P<0.05). The expression of NF-κB in LPS group and LPS+10-9mol/l T group were significantly higher than normal control group(P<0.05). Compared with LPS group, the expression of NF-κB were significantly lower in LPS+10-9mol/l T group(P<0.05).10.The results of western blot and PCR shown that the expression of IRS-1, AKT and GLUT-4 in LPS group were lower than in normal control group(P<0.05). The expression of IRS-1, AKT and GLUT-4 in LPS+10-9mol/l T group were higher than in LPS group(P<0.05). The expression of PI3K、NF-κB and TNF-α in LPS group were higher than in normal control group(P<0.05). The expression of PI3K、NF-κB and TNF-α in LPS+10-9mol/l T group were lower than in LPS group(P<0.05).Conclusion1.High fat diet induced marked disorders of glucolipid metabolism and vascular injuries in puberty male rats.2.Low dose testosterone replacement treatment ameliorates the damages caused by high fat diet. High dose testosterone replacement treatment aggravate the damagescaused by high fat diet.3.The effect of testosterone on glucolipid metabolism and the morphology of vascular wall was induced via PI3K/AKT signal pathway.4. Physiologic concentration testosterone can effectively improve LPS induced endothelial cell apoptosis. Physiologic concentration testosterone can also enhance the damage of cell vitality and migration caused by LPS.5. When the concentration of testosterone <10-9mol/l, the protective effect of testosterone on HUVECs is increased with the rising of testosterone concentration.When the concentration of testosterone >10-9mol/l, the protective effect of testosterone on HUVECs is decreased with the rising of testosterone concentration.6. The protective effect of testosterone on HUVECs was associated with promoting the expression of IRS-1, AKT and GLUT-4 and inhibiting the expression of PI3K、NF-κB and TNF-α. PI3 K /AKT may be a pathway that connecting testosterone and endothelial function.