Branched Chain Amino Acids Exacerbated Hepatic Glucose And Lipid Metabolic Disorders And Promoted Lipid Ectopic Accumulation In Diet-Induced Obese Mice

Backgrounds:Obesity-related metabolic diseases-including diabetes and non-alcoholic fatty liver disease(NAFLD)has become the major public health burden worldwide.Obesity is associated with increased lipid storage in ectopic tissues,such as the liver,skeletal muscle and kidney.Ectopic lipid accumulation in the liver is known as NAFLD and is closely associated with insulin resistance [1].NAFLD can be divided into simple steatosis and non-alcoholic steatohepatitis(NASH).The clinical evidences sugguest that with the development of NASH,hepatic lipid deposition decreased,hepatic lipogenesis weakened but insulin resistance deteriorated [2].Similar phenomena can also occurred in the liver with the development of hepatic insulin resistance: "selective insulin resistance"-insulin fails to suppress gluconeogenesis but continues to activate lipogenesis,and severer "pure insulin resistance"-insulin fails to suppress gluconeogenesis but fails to activate lipogenesis [3].The obvious difference is the suppressed hepatic lipogenesis in "pure insulin resistance".The effect of severer hepatic insulin resistance upon systemic lipid ectopic accumulation and targeting organ injury has not been reported.The liver is the critical site perfoming glucose,lipid and amino acid metabolisms,hepatic glucose and lipid metabolism is crucial to sustain systemic glucose and lipid homeostasis.Evidences indicate that branched chian amino acids(branched chain amino acids,BCAA)is closely correlated with the development of NAFLD and insulin resistance [4,5].Whether BCAA disrupt hepatic glucose and lipid metabolism,deteriorate hepatic insulin resistance and further contribut to the systemic glucose and lipid homeostasis has been unidentified.Investigating the influence of BCAA on hepatic glucose and lipid metabolism will provide new target for preventing and treating NAFLD and diabetes.Aims:1 To explore the effect of BCAA on blood glucose and lipid levels,lipid ectopic accumulation and targeting organ injury in HF-diet-induced obese mice;2 To study the effect BCAA supplementation on hepatic glucose and lipid metabolism and the correlated mechanisms in HF-diet-induced obese mice;3 To intervene the correlated mechinsm of BCAA-induced hepatic metabolic disorders and observe the whole-body therapeutic effects.Methods:(1)Mice were divided into four groups: normal diet feeding group(ND group),60% high fat diet feeding group(HF group),60% high fat diet plus 4% BCAA drinking water feeding group(HF/BCAA group),HF/BCAA diet-paired group(HF/Paired group),and then observed the effect of BCAA supplementation upon blood glucose and lipid levels,on lipid ectopic accumulation in the liver skeletal muscle and kidney and on targeting organ injury in HF-fat diet induced obese mice.Because BCAA inhibited C57BL/6 mice food intake[6],we included another HF/Paired group in which the food intake is matched to that of HF/BCAA group mice,so as to To dissect the interactions between caloric intake and diet composition as it relates to insulin sensitivity;(2)Liver is the crucial organ in responseto insulin to regulate glucose,lipid and amino acid metabolism.In the liver,we analyze the the effects of BCAA on hepatic gluconeogenesis and lipogenesis,lipid β oxidation,VLDL production and output and hepatic Akt signaling with Western blotting and qPCR,to determined the effects of BCAA upon hepatic glucose and lipid metabolism;(3)Akt signaling is crucial in insulin’s regulation upon hepatic glucose and lipid metabolism [7,8].We investigated the mechanism of BCAA/BCKA inactivating hepatic Akt2 kinase activity and inducing hepatocytes Akt2 protein degradation and further found out the specific E3 ligase that mediates Akt2 protein degradation induced by BCAA/BCKA through Western blotting,immunocoprecipitation and immunofluorescence.The results showed that BCAA/BCKA inhibited Akt kinase phosphorylation through up-regulating mTORC1 pathway and down-regulating mTORC2 pathway;BCAA/BCKA promoted the binding of E3 ubiquitin ligase Mul1 and AKT2 by down-regulating Ritor,the main component of mTORC2,and induced hepatocytes Akt2 protein degradation by ubiquitin-dependent proteasomal degradation pathway.(4)Through tail vein injection of AAV9-myr-Akt2 to restore hepatic Akt2 signaling in HF/BCAA mice,we aimed to clarify whether suppressed Akt2 signaling mediated BCAA-induced hepatic glucose and lipid metabolic disorders and further to explore the molecular mechanism downstream of Akt2 that mediated inhibitied hepatic lipogenesis and enhanced hepatic gluconeogenesis in diet induced obese mice,respectively;(5)In vivo,we systematically observed the influence of hepatic Akt2-restoration upon glucose tolerance,insulin tolerance,pyruvate tolerance,hepatic VLDL-TG production and output,plasma glucose and lipids levels,systemic lipid ectopic accumulation,and the targeting liver and kidney injury.And further clarify the mechanism of BCAA disrupting systemic glucose and lipid metabolic homeostasis.Results:(1)HF-diet feeding for 16 weeks successfully induced C57BL/6 mice insulin resistance,as demonstrated by elevated blood glucose level and impaired glucose tolerance.BCAA supplementation significantly increased the plasma glucose level,plasma TG level,plasma LDL level and plasma free fatty acids(FFA)level in diet-induced obese mice(P < 0.05).BCAA supplementation reduced hepatic lipid accumulation,increased the lipid accumulation in the kidney and skeletal muscle,significantly enhanced the urinary protein and the renal tubular cast(P < 0.05).These results suggest that BCAA supplementation disturbs hepatic and systemic glucose and lipid metabolic homeostasis,exacerbates lipid ectopic accumulation in skeletal muscle and kidney,and damaging targeting organs such as liver and kidney;(2)The liver is "chemical factory" performing glucose,lipid and amino acids metabolism in our body,HF/BCAA mice liver displayed enhanced glycogen deposition and decreased lipid accumulation,we investigated the role of BCAA upon hepatic glucose and lipid metabolism.BCAA supplementation did not significantly increase hepatic lipid β oxidation,but partially up-regulated hepatic VLDL-TG output in diet-induced obese mice.BCAA supplementation evidently promoted hepatic glyconeogenesis and inhibited lipidgenesis in obese mice(P < 0.05),significantly inhibited the activation of hepatic Akt signaling and down-regulating hepatic Akt2 protein by posttranscriptional manner;Akt2 is the key effector of insulin regulating glucose and lipid metabolism[9,10].We found that BCAA/BCKA significantly inhibited the Akt2 phosphorylatory level,evidently decreased hepatic Akt2 protein level,but did not affect the hepatocytes Akt2 m RNA level.These results suggest that BCAA suppressed hepatic lipogenesis and promote hepatic glyconeogenesis in HF diet-induced obese mice,and BCAA/BCKA significantly inhibited hepatic Akt2 signal;(3)The above suggested BCAA/BCKA significantly down regulated the protein level and activity of Akt2,but did not affect the Akt2 mRNA level in hepatocytes.Herein,we explored the mechanism concerning BCAA-induced Akt2 inactivation and investigated whether post-translational modification manner mediated hepatocyte Akt2 degradation.BCAA/BCKA downregulated hepatocytes Rictor protein level(the main component of mTORC2),and induced Akt2 protein degradation through ubiquitin-proteasome-dependent pathway.BCAA/BCKA inhibited Akt activation by upregulating mTORC1 and down-regulating mTORC2 pathways.BCAA/BCKA promoted the binding of mul1 and Akt2;Ad-Rictor overexpression inhibited the binding of mul1 and Akt2.Besides,Mul1 si RNA treatment knocked down hepatocyte Mul1 levels,partially reversed BCAA/BCKA-induced Akt2 degradation in primary hepatocytes;(4)Previously,we elucidated the mechanism of BCAA induced hepatic Akt2 inhibition.Thereafter,we further explored and clarified whether and how Akt2 signaling and its downstream pathway mediated BCAA induced hepatic glucose and lipid metabolic disorders in obese mice.We found that AAV9-myr-Akt2 overexpression through tail vein injection restored hepatic Akt2 signaling,partially reversed BCAA-induced increased hepatic gluconeogenesis and inhibitied hepatic lipogenesis;Morever,Akt2/insig2a/srebp1 c pathway and Akt2/Foxo1 pathway mediated BCAA-induced decreased hepatic lipogenesis and increased hepatic gluconeogenesis,respectively;(5)Intervening the mechanism of BCAA-induced hepatic glucose and lipid metabolic disorders,we systematically observed the effects of BCAA on blood glucose and lipid levels,insulin resistance and lipid ectopic accumulation.We found Akt2 upregulation partially restored the impaired glucose tolerance,insulin tolerance and pyruvate tolerance caused by BCAA supplementation,and partially improved hepatic VLDL output and reduced the blood glucose and lipid levels in HF/BCAA mice(P < 0.05);Akt2 restoration partially reversed BCAA induced muscular and renal lipid ectopic accumulation and improved the urinary microalbumin and tubular cast caused by BCAA supplementation(P < 0.05).Conclusions:BCAA induced severe hepatic insulin resistance,resulted in systemic glucose and lipid metabolic disorders and damaging targeting oragns such as liver and kidney.Mechanistically,BCAA/BCKA inactivated hepatic Akt2 kinase through mTOR pathway,and promoted the binding of E3 ubiquitin ligase Mul1 and Akt2,leading to ubiquitin-dependent degradation of hepatic Akt2 protein.In addition,Akt2/insig2a/srebp1 c pathway and Akt2/Foxo1 pathway mediated BCAA-induced decreased hepatic lipogenesis and enhanced hepatic gluconeogenesis,respectively.BCAA promoted hepatic VLDL-TG production and output,increased blood glucose and blood lipid levels,contributed to lipid ectopic deposition and targeting organ injury.Targeting BCAA metabolism can be a potential strategy for severe insulin resistance related diseases.