Heat Stress Regulates Lactate Synthesis And Secretion By Insulin-mediated PI3K/Akt Signal In Immature Boar Sertoli Cells

Severe heat stress has a significantly negative effect on male reproductive functions.Lactate produced by Sertoli cells is not only the most important metabolic substrate of developing germ cells,but also can enhance the anti-apoptotic abilities of germ cells.Therefore,lactate plays an important role in protecting male animals from the adverse effects of heat stress on reproductive functions.Heat stress also increases the basal insulin and insulin sensitivity.PI3K/Akt plays an important role in insulin-mediated glucose metabolism.Furthormore,mild heat stress increases the synthesis and secretion of lactate in Sertoli cells,but it is still unclear whether mild heat stress stimulate Sertoli cells lactate synthesis and secretion through insulin-mediated PI3K/Akt signaling pathway.In this experiment,Sertoli cells isolated from 21-day-old immature boar testis were used in vitro to induce heat stress.Firstly,Sertoli cells were treated with different concentrations of insulin(5 ~ 200 nM)for 24 hours.Cell viability was measured with CCK-8 kit,and the level of lactate secretion was examined by lactate test kits to make sure the concentration to be used in subsequent experiments.Then,cells were treated with the selected concentration of insulin(100 nM)for 0~36 h.The level of lactate secretion at each time point was measured,and determined the treat time of insulin.Secondly,at 2 hours post heat treatment(43℃,30 min),cells were treated by 100 nM insulin for 24 h.The mRNA expression of INSR,IGF1 R,IRS1,and IRS2 was detected by quantitative real-time PCR.Western blot was used to detect the phosphorylation level of PI3 K and Akt in the cells.Finally,Sertoli cells were pretreated for 2 hours by PI3 K inhibitors.Then,cells were treated by heat and insulin successively.The expressions of GLUT3,MCT1,LDHA and the level of lactate secretion were measured to make sure the effct of PI3 K on lactate secretion.The experimental results are as follows:1.Lactate secreted by Sertoli cells reached its peak after treatment with 100 nM insulin for 24 h,which was increased by 133.3%,compared with the control group(P<0.01).When the insulin concentration was 200 nM,lactate secretion began to decrease(P<0.01).When cells were treated with insulin(100 nM)for 6 h,there was no significant change in lactate secretion(P>0.05).As the treatment time prolonged,lactate secreted by Sertoli cells increased gradually,and the peak was at 24 hours,showing a time-dose dependence2.Both heat stress and insulin increased lactate secretion in Sertoli cells.The joint effect was significantly higher than one of them alone,which was 21.19%(P<0.01)and 4.21%(P<0.05)higher than heat treatment group or the insulin group,respectively.3.Heat stress increased the expression of IGF1 R and IRS2 mRNA,but it had no significant effect on the expression of INSR and IRS1 mRNA(P>0.05).Heat stress combined with insulin significantly increased the expression of INSR,IGF1 R,IRS1 and IRS2 mRNA,but the effect on IGF1 R was higher than that of INSR,and the effect on IRS2 mRNA expression was higher than that of IRS1(P<0.01).4.Compared with the control,heat stess significantly increased the phosphorylation levels of PI3 K and Akt.Addition of insulin under heat treatment significantly promoted the phosphorylation of PI3 K and Akt,and the phosphorylation levels of PI3 K and Akt were increased by 41.54%(P<0.01)and 10.99%(P<0.05)respectively.5.Under heat stress cooperating with insulin,PI3 K inhibitors significantly reduced the expression of the lactate secretion-related gene(SLC2A3,LDHA,MCT1)and protein(GLUT3,MCT1,LDHA)(P<0.05).The activity of LDH,and the lactate secreted by Sertoli cells was reduced(P<0.01).In summary,heat stress mainly up-regulates GLUT3,MCT1,and LDHA expression to promote lactate synthesis and secretion via up-regulating the expression of IGF1 R and IRS2 and activating PI3K/Akt signaling pathway.In this process,heat stress and insulin have a synergistic effect.This conclusion can provide a theoretical basis for clinically relieving and treating the adverse effects of heat stress on male reproduction.