β-羟基丁酸对牦牛脂肪细胞脂肪代谢和脂联素分泌的影响

Effects ofβ-Hydroxybutyric Acid on Fat Metabolism and Adiponectin Secretion in Yak Adipocytes

本试验旨在探究饥饿状态下β-羟基丁酸(BHBA)对牦牛脂肪细胞脂肪代谢以及脂联素(ADPN)分泌的影响,从细胞水平丰富牦牛脂肪代谢调控理论。采取4头1岁左右的体况相近且健康的雄性麦洼牦牛的脂肪组织,采用酶消化法和鸡尾酒法诱导分化获取牦牛原代脂肪细胞进行培养,通过不同浓度BHBA和血清饥饿共处理后,对其成脂情况、脂肪代谢关键酶表达和ADPN分泌情况进行分析。结果显示:成脂情况表明,饥饿状态下,当BHBA浓度>4 mmol/L时,脂滴油红O染色量化值[光密度(OD)值]显著升高(P<0.05)。脂肪分解代谢关键酶检测发现,饥饿状态下,32 mmol/L BHBA能够显著降低脂酰辅酶A氧化酶(ACOX)和激素敏感脂酶(HSL)的基因表达量和浓度(P<0.05)。脂肪分解代谢相关转录因子检测发现,饥饿状态下,4、8、16、32 mmol/L BHBA均能够显著降低叉头框O蛋白1(FoxO1)的基因和蛋白表达量(P<0.05),但并未出现出剂量依赖效应;4、8、16 mmol/L BHBA能够显著促进过氧化物酶增殖物激活受体α(PPARα)蛋白的表达(P<0.05),但32 mmol/L BHBA则显著抑制PPARα基因和蛋白的表达(P<0.05)。脂肪合成代谢关键酶检测发现,饥饿状态下,32 mmol/L BHBA显著提高乙酰辅酶A羧化酶(ACACA)、二酰基甘油酰基转移酶1(DGAT1)的浓度(P<0.05),而BHBA浓度在8、16和32 mmol/L时,FASN浓度均显著提高(P<0.05)。脂肪合成代谢相关转录因子检测发现,饥饿状态下,32 mmol/L BHBA显著促进了CCAAT增强子结合蛋白α(C/EBPα)的基因表达(P<0.05),4、8、16和32 mmol/L BHBA显著提高了过氧化物酶增殖物激活受体γ(PPARγ)和固醇调节元件结合蛋白1c(SREBP1c)浓度(P<0.05),但未出现剂量依赖效应。ADPN以及G蛋白偶联受体109A(GPR109A)检测发现,经高浓度(32 mmol/L)BHBA处理后,ADPN和GPR109A的基因表达量均出现显著下降(P<0.05),但低浓度BHBA则显著促进其表达(P<0.05)。综上所述,在本试验条件下,32 mmol/L BHBA能够抑制牦牛脂肪细胞的脂肪分解代谢,加强脂肪合成代谢,低浓度的BHBA能够促进牦牛脂肪细胞对ADPN的分泌,而高浓度BHBA(>8 mmol/L)则抑制ADPN的分泌。

This experiment aimed to explore the effects ofβ-hydroxybutyric acid(BHBA)on fat metabolism and adiponectin(ADPN)secretion of yak adipocytes under starvation,so as to enrich the regulation theory of yak fat metabolism at the cellular level.The adipose tissue of four male Maiwa yaks aged about 1 year old with similar and healthy body condition were used to obtain yak primary adipocytes by enzyme digestion method and cocktail induced differentiation method.After co-treatment with different concentrations of BHBA and serum starvation,the adipogenesis situation,the expression of key enzymes of fat metabolism and ADPN secretion were detected.The results showed that,under starvation,the quantitative staining value[optical density(OD)value]of oil red O of lipid droplets was significantly increased when the concentration of BHBA was>4 mmol/L(P<0.05).The detection of key enzymes of fat catabolism showed that 32 mmol/L BHBA could significantly decrease the gene expression levels and concentrations of acyl-CoA oxidase(ACOX)and hormone-sensitive triglyceride lipase(HSL)under starvation(P<0.05).The detection of transcription factors related to fat catabolism showed that 4,8,16 and 32 mmol/L BHBA could significantly decrease the gene and protein expression levels of fork head box protein O1(FoxO1)under starvation(P<0.05),but there was no dose-dependent effect;under starvation 4,8 and 16 mmol/L BHBA could significantly promote the protein expression of peroxisome proliferator-activated receptorα(PPARα),but 32 mmol/L could significantly inhibit the gene and protein expression of PPARα(P<0.05).The detection of key enzymes of fat anabolism showed that the concentrations of acetyl coa carboxylase(ACACA)and diacylglycerol acyltransferase 1(DGAT1)were significantly increased when the concentration of BHBA was 32 mmol/L(P<0.05),while the concentration of fatty acid synthase(FASN)was significantly increased when the concentration of BHBA were 8,16 and 32 mmol/L under starvation(P<0.05).The detection of transcription factors related to fat anabolism found that 32 mmol/L BHBA could significantly promote the gene expression of CCAAT/enhancer-binding proteinα(C/EBPα)under starvation(P<0.05),and 4,8,16 and 32 mmol/L BHBA could significantly increase the concentrations of peroxisome proliferator-activated receptorγ(PPARγ)and sterol-regulatory element-binding protein-1c(SREBP1c)(P<0.05),but there was no dose-dependent effect.The detection of ADPN and G-protein coupled receptor 109A(GPR109A)showed that the gene expression levels of ADPN and GPR109A were significantly decreased after treatment with high concentration of BHBA(32 mmol/L)(P<0.05),but they were promoted by low concentration of BHBA(P<0.05).In conclusion,under the condition of this experiment,32 mmol/L BHBA can inhibit the fat catabolism and enhance the fat anabolism of yak adipocytes;low concentration BHBA can promote the secretion of ADPN in yak adipocytes,while high concentration BHBA(>8 mmol/L)can inhibit the secretion of ADPN.