AMPK Negatively Regulates Heat Treatment-induced Lactate Secretion In Cultured Boar Sertoli Cells

AMP-activated protein kinase(AMPK)is responsible for cel ular metabolism and energy homeostasis in mammalian tissues,and activation of AMPK enhances catabolism such as fatty acid oxidation and glycolysis.It also plays an important role in energy homeostasis in response to metabolic stress.Similar to tumor cel s,Sertoli cel s shows Warburg-like effects in the metabolic characteristics,which convert glucose to lactate by a high glycolytic fiux even in the presence of sufficient and physiological oxygen levels.Lactate,secreted by Sertoli cel s,can not only be used by developing sperm cel s as important metabolism substrates,but also shows characteristic of anti-apoptosis during spermatogenesis.Secretion of lactate in boar Sertoli cel s is regulated by GLUT3(glucose transporters 3);LDHA(lactate dehydrogenases A)and MCT1(monocarboxylate transporter 1).In our previous studies,heat stress(43 °C,30 min)enhanced lactate secretion in boar immature SCs.Increasing evidence shows that heat stress changes AMPK activity in various tissues and cel s.However,it is unclear whether heat stress affects the lactate secretion by regulating AMPK activity in boar immature Sertoli cel s.Therefore,firstly we examined AMPK activity after heat stress;then explored the role of AMPK in HS-induced the lactate production,the expressions of related genes and LDH activity using AICAR,an AMPK agonist and overexpression of AMPK.In this study,Sertoli cel s were isolated and purified from 3-week-old immature boar testis.Heat stress(43 °C incubator)treated Sertoli cel s for 30 min.Then,SCs were recovered in 32 °C incubator for 0,3,6,12,24 or 48 h.To make sure the optional recovery time,we analyzed the mRNA and proteins of lactate secretion related genes by RT-PCR and western blot,respectively;detected the phosphorylation level of AMPK by western blotting,and measured lactate secretion using Lactate detection kit,according to manufacturer introductions.Then,AICAR and transfected with AMPK overexpressio n vector before heat stress were used to enhance the activity of AMPK;cells were collected to detect mRNA and protein expression of relative enzymes and LDH activity.These results were as followed:(1)Heat stress enhanced SCs lactate secretion with the maximum level(175% of control,P < 0.01)at12 h after heat stress.With time of further duration,the lactate secretion gradually reduced and recovered to basal levels at 48 h(P > 0.05).(2)The phosphorylation level of AMPK had a decreased trend in a time-dependent manner with the val ey at 12 h after heat stress(58% of control,P < 0.01).With the recovery time duration after heat treatment,the p-AMPK activity gradually increased and returned to control group's levels at 48 h(P > 0.05).(3)AICAR(2 mmol/L,2 h),an AMPK agonist significantly increased the phosphorylation level of AMPK(215% of the HS,P < 0.01).Compared with heat treatment alone,pretreatment with AICAR downregulated the lactate secretion by 42.6% and decreased LDH activity by 8.6%.The mRNA and protein expression of heat-induced GLUT3(53.4%,44.7%,respectively),LDHA(54.1%,58.6%,respectively)and MCT1(33.8%,60.3%,respectively)were also reduced significantly(all P < 0.01).(4)Transfection of over AMPK overexpression vector,pcDNA-AMPK resulted in an increase on the phosphorylation level of AMPK(160% of the control,P < 0.01).The mRNA expression of AMPK was also increased by 230% with the pcDNA – AMPK treatment,compared with the control(P < 0.01).However,pcDNA had no significa nt effect on the expression of AMPK(P > 0.05).(5)Compared with heat treatment alone,AMPK overexpression could decrease the heat-induced upregulation of lactate production and LDH activity by22.5% and 10.4% respectively(P < 0.01),lowered the mRNA expression of SLC2A3,LDHA and SLC16A1 by 41.4%,32.1%,31.5% respectively(P < 0.01),reduced the protein levels of GLUT3,LDHA and MCT1 by 29.1%,30.4% and 16.2% respectively(P < 0.01).In conclusion,heat stress reduces the activity of AMPK in Sertoli cel s;reduced AMPK activity enhances lactate secretion in cultured boar Sertoli cel s;upregulates the mRNA and protein expressions of related genes and LDH activity;increases lactate secretion.Therefore,AMPK is a negative regulator of heat treatment-induced lactate secretion in cultured boar Sertoli cel s.Downregulation of the activity of AMPK is an effective way of increasing lactate secretion,inhibiting germ cell apoptosis.