The Role Of AKAP1 In Inhibiting Mitochondrial Thermogenesis In Brown Adipocytes

Objective: A-kinase anchoring protein 1(AKAP1)anchors cAMP-dependent protein kinase A(PKA)to the mitochondrial outer membrane and plays an important role in the regulation of mitochondrial function and cell survival.Our previous study has suggested that AKAP1 regulated metabolism in brown adipose tissue(BAT)and cold-induced BAT thermogenesis in obese mice.However,the underlying mechanisms remain unknown.This study was designed to investigate the specific role of AKAP1 and its underlying mechanisms in regulating mitochondrial thermogenesis in brown adipocytes.Methods:(1)To investigate the role of AKAP1 in promoting mitochondrial heat generation in brown adipocytes: The oxygen consumption rate of mitochondria in brown adipose tissue of AKAP1 knockout mice and wild mice under high-fat diet was measured by Seahorse cell energy metabolism detector.The expression of UCP1 in brown adipose tissue of AKAP1 knockout mice and wild mice was detected by WB experiment.The morphological changes of lipid droplets in primary brown adipocytes induced by palmitic acid were detected by oil red staining and BODIPY staining experiments with palmitic acid-induced hypertrophy models of primary brown fat cells.The oxygen consumption rate of primary brown adipocytes of AKAP1 knockout mice and wild mice induced by high concentration of palmitic acid was measured using Seahorse cell energy metabolism detector.(2)To explore the mechanism of AKAP1 promoting mitochondrial heat generation in brown adipocytes: Molecules interacting with AKAP1 were identified using IP-MS,and verified using CO-IP and WB.The effect of interfering with ACSL1 on the oxygen consumption rate of primary brown adipocytes from AKAP1 knockout mice and wildmice treated with high concentration palmitic acid was detected by Seahorse cell energy metabolism detector.(3)Preliminary study on the mechanism of obesity feedback-induced inhibition of AKAP1 expression: Western blot and real-time quantitative PCR were used to detect the expression of AKAP1 in different tissues of obese mice.Verify whether high concentrations of palmitic acid affect the expression of AKAP1.Bioinformatics was used to predict the transcription factors that affect the expression of AKAP1.Western blot and real-time quantitative PCR were used to detect the expression of AKAP1 in wild mice primary brown adipocytes after interference with CEBP-β.GEO database was used to analyze the correlation between AKAP1 and CEBP-β.Results:(1)We found that compared with wild-type mice,mitochondrial oxygen consumption in brown adipose tissue of high-fat fed AKAP1 knockout mice was significantly increased,and UCP1 expression was increased,indicating that mitochondrial heat production in AKAP1 knockout mice was increased under high-fat feeding conditions.However,under normal diet conditions,UCP1 expression was not changed in AKAP1 knockout mice compared to wild type mice,suggesting that AKAP1 had no direct effects on UCP1 expression.Palmitic acid was used to provide a high-fat environment for primary brown adipocytes,and a mouse model of primary brown adipocyte hypertrophy was constructed.The content of lipid droplets in primary brown adipocytes was significantly reduced in primary brown adipocytes from AKAP1 knockout mice.Further,we found that high concentrations of palmitic acid treatment significantly enhanced basal oxygen consumption rate,uncoupled respiration,and maximum oxygen consumption rate in primary brown adipocytes from AKAP1 knockout mice,suggesting that AKAP1 inhibited mitochondrial thermogenesis by reducing mitochondrial respiratory function..(2)CO-IP experiments revealed that ACSL1 interacts with AKAP1,and both are localized on the outer membrane of mitochondria.Although AKAP1 knockout did not affect the mitochondrial localization of ACSL1,AKAP1 knockout significantly increased mitochondrial ACSL activity and reduced mitochondrial PKA activity.Down-regulating ACSL1 expression can significantly inhibit mitochondrial oxygen consumption in AKAP1 knockout brown adipocytes.(3)The expression level of AKAP1 in adipose tissue of obese mice was significantly lower than that in normal mice.Moreover,in primary brown adipocytes,high concentrations of palmitic acid treatment markedly inhibited the expression level of AKAP1 in a concentration-dependent manner..Bioinformatics analysis predicts that the transcription regulator of AKAP1 could be CEBP-β.Down-regulating the expression of CEBP-β in brown adipocytes significantly inhibited the expression of AKAP1.In addition,we found a positive correlation of AKAP1 and CEBP-β mRNA levels in human adipose tissue using a GEO database.Conclusions: In this study,we found that AKAP1 inhibited mitochondrial respiration of brown adipocytes by inhibiting ACSL1 activity.Furthermore,the expression of AKAP1 in adipose tissue of mice was reduced under high-fat diet,which might be related to CEBP-β.