Regulation Mechanism Of Mitochondrial Calcium Uniporter On Fat Metabolism

Lipid metabolism is closely related to animal production and human health,and the liver,as an important organ of lipid metabolism,plays a key role in regulating lipid metabolism.In animal production,excessive accumulation of fat in the liver can lead to fatty liver syndrome,which can seriously damage animal health and reduce breeding efficiency.In human health,excessive accumulation of fat can lead to fatty liver,diabetes and other metabolic systemic diseases.Meanwhile,mitochondria,as the center of cellular energy metabolism,are important for fat metabolism.Recent studies have shown that mitochondrial calcium homeostasis can affect body lipid metabolism,but its mechanism is still unclear,so we investigated the relationship between mitochondrial calcium homeostasis and liver lipid metabolism.In this project,we used human hepatoma cells（HepG2）,alpha mouse liver cells（AML12）,and chicken hepatocellular carcinoma cells（LMH）as cell models,C57BL/6 mice and yellow-feathered broiler as animal models to establish high fat induction models in vivo and in vitro,and used MCU overexpression vector,RNAi technology,ER-Mito Linker vector,MICU1 mutation vector,transmission electron microscopy,MCU inhibitors and other techniques to investigate the key of mitochondrial calcium homeostasis,Mitochondrial Calcium Uniporter（MCU）,to explore the regulatory mechanism of mitochondrial calcium homeostasis on lipid metabolism,and to provide theoretical basis and feasible solutions for the treatment of fatty liver.The following results were obtained:1.MCU affects mitochondrial calcium homeostasis and regulates hepatocyte lipid accumulationUsing HepG2 cells,an oleic acid/palmitic acid（OA/PA）-induced high-fat model was established in vitro,and it was found that high-fat can induce mitochondrial calcium overload in hepatocytes.MCU was overexpressed or inhibited using MCU vector and si RNA while high-fat induction,and it was found that overexpression of MCU elevated mitochondrial calcium and increased lipid deposition;inhibition of MCU decreased mitochondrial calcium levels and reduced lipid accumulation.MCU also affected fatty acid synthase and lipase activities and promoted reactive oxygen species production,suggesting that MCU may regulate hepatocyte lipid metabolism by regulating oxidative stress and lipid metabolizing enzyme activity.The above phenomenon was also found in AML12cells as a model,further demonstrating the regulatory role of MCU on hepatocyte lipid metabolism.2.Inhibition of hepatic MCU alleviates hepatic steatosisTo verify the role of MCU in vivo,mice were used to inhibit hepatic MCU using adeno-associated virus and induced by high-fat diet.The results showed that inhibition of MCU enhanced glucose tolerance,reduced adiposity,and alleviated epididymal adipocyte hypertrophy and hepatic steatosis in mice.Hepatocyte-specific heterozygotes MCU knockout mice(MCU^+/-)were generated by crossing MCU^fl/fl with hepatocyte-specific Cre recombinase transgenic mice.Knockdown of MCU in mouse liver followed by high-fat diet experiment revealed that MCU^+/-significantly reduced adiposity;adipocyte hypertrophy was significantly inhibited;glucose tolerance was enhanced;and hepatic steatosis was effectively alleviated.3.Study on the mechanism of mitochondrial calcium overload in hepatocytes induced by high fatHepG2 cells were used as a model to investigate the mechanism of mitochondrial calcium overload in hepatocytes under high-fat condition.The results showed that high fat causes a decrease in hepatocyte mitochondrial content and promotes the formation of hepatic mitochondrial-endoplasmic reticulum associated-membrane（MAM）.Mitochondrial calcium overload in hepatocytes,along with increased triglyceride accumulation,following the use of a mitochondrial-endoplasmic reticulum Linker vector（ER-Mito-Linker）to mimic MAM;inhibition of key genes PACS2 and IP3R2 in MAM formation by RNAi technology alleviates high-fat-induced mitochondrial calcium overload and reduces triglyceride deposition in hepatocytes;demonstrating that high-fat diet-induced MAM formation is responsible for mitochondrial calcium overload and fat deposition.To explore the opening and closing mechanism of MCU channels under high fat condition,the MCU channel complexes EMRE,MICU1,and MICU2were overexpressed or inhibited,it was found that only MICU1 had an effect on hepatocyte lipid deposition;more interestingly,overexpression of MICU1reduced mitochondrial calcium levels in a no-high-fat condition;while in a high-fat condition,overexpression of MICU1 did not affect mitochondrial calcium levels;demonstrating that a high-fat affects the regulation of mitochondrial calcium levels by MICU1.MICU1 is responsible for controlling MCU channel switching,and when MICU1 is in a high concentration Ca²⁺environment,the EF hand region of MICU1 binds to Ca²⁺,causing the effect of MICU1 in closing MCU channels to disappear.To test this theory,the EF hand region of MICU1bound to Ca²⁺was mutated and transfected into cells,and the results showed that MICU1 failed to reduce mitochondrial calcium levels in a high-fat condition,while the MICU1 mutant acted to prevent mitochondrial calcium overload,and the MICU1 mutant also alleviated high-fat-induced triglyceride deposition in hepatocytes.In summary,high-fat lead to increased MAM formation,which releases calcium from the endoplasmic reticulum via IP3R,leaving MICU1 in a high Ca²⁺environment,and the high Ca²⁺environment dysfunctionalizes MICU1,leading to mitochondrial calcium overload and increased fat accumulation.4.MCU inhibitor alleviates liver steatosis in miceMolecular simulations using Discovery Studio to dock MCU and its inhibitor mitoxantrone showed that mitoxantrone exerted its inhibitory activity by interacting with two key residues,Glu264 and Asp261,thereby preventing Ca²⁺from passing through MCU.To find an effective way to inhibit fatty liver,MCU inhibitors,Ru360 and mitoxantrone,were used in hepatocytes to inhibit MCU.The cellular results showed that MCU inhibitors significantly reduced the calcium levels in hepatocyte mitochondria,alleviated the high-fat-induced reduction in oxygen consumption rate in hepatocytes,and decreased lipid deposition.Mice were administered intraperitoneally for eight weeks using the MCU-specific inhibitors Ru360 and mitoxantrone when establishing a high-fat model.The results showed that both significantly alleviated the high-fat diet-induced increase in adiposity,adipocyte hypertrophy and enhanced glucose tolerance,alleviated hepatic steatosis,and enhanced fatty acidβ-oxidation key enzyme-esteryl coenzyme A dehydrogenase activity,suggesting that MCU inhibitors may reduce lipid accumulation by promoting hepatic fatty acid oxidation.5.MCU inhibitors alleviate liver steatosis in chickensTo investigate how to alleviate fatty liver in animal production,we used MCU inhibitors to treat chicken hepatocytes and broiler chicks.Using LMH chicken hepatocytes as a model,we found that MCU inhibitors could alleviate high-fat-induced mitochondrial calcium overload and reduced lipid accumulation.Injecting MCU inhibitors into yellow-feathered broiler when establishing a high-fat diet model.It was found that high-fat diet induced hepatic hypertrophy in chickens,and MCU inhibitor alleviated hepatic hypertrophy;high-fat diet also induced fat deposition in chickens’liver,while MCU inhibitor alleviated hepatic fat deposition.In conclusion,MCU can regulate hepatic lipid metabolism,and inhibition or knockdown of hepatic MCU can alleviate high-fat-induced hepatic steatosis in mice.High-fat diet induces hepatic MAM formation,and the high Ca²⁺environment generated by MAM dysregulates MICU1 action,leading to mitochondrial calcium overload,resulting in elevated ROS,reduced lipase activity,and decreased fatty acidβoxidation,while MCU inhibitors can alleviate high-fat-induced hepatocyte,mouse,and chicken liver steatosis.This provides new ideas for studying the formation mechanism of fatty liver in animals and humans,discovering new approaches for the treatment of fatty liver and laying the foundation for subsequent drug development.