Study On The Regulation Of Fructose Metabolism By Liver ChREBP

With the development of social economy and changes in people's lifestyles,the prevalence of metabolic diseases such as obesity,diabetes,fatty liver and hyperlipidemia is rapidly increasing globally.These metabolic diseases often interact and accompany each other,appearing in the form of metabolic syndrome,and further develop into cardiovascular diseases such as atherosclerosis,hypertension,and coronary heart disease,which seriously endanger people's physical and mental health and social development.In the past decade,the incidence of obesity and diabetes in China has increased rapidly,and it is trending towards a younger age.Therefore,metabolic diseases and related cardiovascular diseases have become a major challenge in the field of public health in China,as well as the top priority in the prevention and control of major chronic diseases.A large number of epidemiological data and experimental studies have shown that excessive intake of fructose may be an important factor leading to an increase in the incidence of metabolic diseases.The carbohydrate response element binding protein^[1]is an important transcription factor that regulates glucose and fructose metabolism.Previous studies have found that ChREBP regulates glycolysis and lipid synthesis and acts as an important metabolic coordinating molecule in many cell types.It responds to environmental and hormonal signals and has a wide range of effects on both health and disease states.However,most of the current researches on ChREBP are based on the experimental system of global knockout mouse model,siRNA interference or overexpression of adenovirus,and there are many contradictions among the results of different laboratories.It is difficult to clarify the specific function of ChREBP in different tissues.As a key transcription factor regulating fructose metabolism,the cellular and molecular mechanisms of ChREBP regulating fructose metabolism remain unclear.The liver acts as an important organ of glucose and lipid metabolism,and the role of liver ChREBP in fructose metabolism and fructose tolerance and its related mechanisms are also unclear.To analyze the metabolic regulation role of liver ChREBP,we first established ChREBP liver-specific knockout mouse model using the Cre/loxP recombinase system.The tissue specificity and knockout efficiency of ChREBP knockdown in the liver were verified by qRT-PCR and Western Blot respectively.The results showed that ChREBP was knocked out specificity in the liver and the expression in other tissues was not affected.The liver-specific ChREBP knockout?ChLO?mouse model was successfully established.Using the ChLO mouse model,we systematically investigated the role of liver ChREBP in glucose and lipid metabolism and the pathological changes caused by high sucrose and high fructose diets.The main findings can be divided into the following three aspects.1.ChREBP is involved in maintaining liver glycogen homeostasis.The loss of liver ChREBP does not affect the body weight and blood glucose levels of the mice,nor does it affect the systemic glucose tolerance and insulin sensitivity of the mice under normal chow diet.The liver cells of ChLO mice are normal in morphology,no obvious liver damage,and no significant changes in liver lipid content.However,the liver glycogen content of ChLO mice increased by two times,while the skeletal muscle glycogen content did not change significantly,suggesting that ChREBP plays an important physiological role in regulating liver glycogen homeostasis.2.Liver ChREBP alleviates fatty liver induced by high sucrose.Under high sucrose diet,lipid accumulation in liver of ChLO mice increased significantly.We analyzed all aspects of liver lipid metabolism and found that the ability of ChLO mice liver fatty acid DNL decreased,the ability of ChLO mice liver fatty acid uptake decreased,the ability of ChLO mice liver fatty acid beta oxidation remained unchanged,and the ability of ChLO mice liver lipid secretion decreased.Therefore,we speculated that the increase in liver lipid accumulation in ChLO mice is due to impaired liver lipid secretion.Further analysis of liver lipid secretion-related protein expression revealed that loss of liver ChREBP resulted in decreased mRNA and protein expression of MTTP.3.Liver ChREBP protects mice from fructose-induced glycogenic hepatopathy.The absence of liver ChREBP does not affect the body weight and blood glucose levels under high fructose diet.However,ChLO mice develope severe liver damage,hepatic enlargement and hepatic glycogen accumulation.Meanwhile,there are no obvious inflammatory response,endoplasmic reticulum stress and apoptosis.These results suggest that glycogen accumulation in ChLO mice under high fructose diet leads to glycogenic liver disease.We have shown that liver glycogen accumulation in ChLO mice is due to increased glycogen synthesis rather than reduced glycogen degradation by fasting/refeeding experiments.The results of liver protein expression and metabolite content showed that increased glycogen synthesis in the liver of ChLO mice was associated with increased glycogen synthase activity due to increased liver G-6-P content.In the fed state,the liver ATP content of ChLO mice is significantly reduced,and in the high fructose diet,the decrease in liver ATP content in ChLO mice is further aggravated.In the fasted state,liver ATP levels in ChLO mice were comparable to that of control mice.Fasting/refeeding experiments show that the reduction of liver ATP content in ChLO mice follows liver glycogen accumulation.This suggests that it may be due to the excessive synthesis of glycogen,which consumes a large amount of ATP,resulting in a decrease in liver ATP content in ChLO mice.And there are no changes in the number of liver mitochondria,mitochondrial oxidative phosphorylation protein expression,and mitochondrial respiratory function in ChLO mice.We further analyzed the expression of genes involved in fructose metabolism and found that the expression of liver LPK has the strongest response to fructose feeding in control mice.High fructose stimulation can lead to a significant up-regulation of liver LPK expression.However,other target genes of ChREBP,such as Khk,Aldob,Fbp1 and G6pc,showed poor or no response to fructose at the level of mRNA and protein.And the expression of LPK is dramatically decreased in liver by the deletion of ChREBP both at mRNA and protein levels,while Khk,Fbp1,G6pc,and AldoB are slightly or hardly affected.These results suggest that LPK may be a key checkpoint for controlling fructose metabolism and fructose tolerance in the liver.We performed LPK liver replenishment experiments using the prepared LPK overexpressing adenovirus and expressing only the GFP control adenovirus.Overexpression of LPK in the liver is capable of largely rescuing the phenotypes of hepatomegaly,liver glycogen overload,ATP deficit,and elevated plasma transaminases induced by fructose in ChREBP-mutant mice.In summary,we use the ChLO mouse model to draw the following conclusions:?1?Liver ChREBP plays an important role in the regulation of liver lipid metabolism by regulating MTTP expression.In the case of high sucrose diet,ChREBP can promote MTTP expression and enhance liver lipid secretion,and secrete excess lipids of the liver into the blood in the form of VLDL,thereby preventing liver lipid accumulation induced by high sucrose diet.Our study clarified that liver ChREBP can not only regulate de novo synthesis of liver lipids,but also affect liver lipid metabolism by regulating liver lipid secretion.?2?Liver ChREBP plays an important role in fructose metabolism and fructose tolerance mainly through regulating L-PK.Upon fructose stimulation,ChREBP robustly activates L-PK and glycolytic flux,thereby promoting the entry of fructose metabolites into glycolytic pathway and enhancing ATP production,so as to overcome fructose-induced massive glycogen overload,ATP deficit,and liver injury.Our study clarified the key role of liver ChREBP in fructose metabolism and fructose tolerance.