Liver-specific Knockout Of Ghr Induced NAFLD And The Underlying Mechanisms

Objective:Non-alcoholic fatty liver disease（NAFLD）is characterized by steatosis,lobular inflammation,ballooning and fibrosis.It is a liver injury induced by liver lesions except alcohol and other known factors.NAFLD is defined as steatosis of more than 5%hepatocytes in the absence of excessive significant alcohol consumption,other liver disease or the consumption of steatogenic drugs.In recent years,the prevalence of NAFLD has been increasing year by year and has seriously threatened human health.According to the Guidelines for the prevention and control of Chinese fatty liver disease（Edition 2015）,the prevalence of fatty liver disease in Chinese adults is about 15%（6.3%-27%）by the time of data publication.Non-alcoholic fatty liver disease is closely related to metabolic syndromes and has become the world’s most common chronic liver disease.Growth hormone（GH）is a peptide hormone secreted by the pituitary gland and consists of 191 amino acids.It binds to growth hormone receptor（GHR）.GH is an important endocrine factor regulating the growth and development of the body.GH also stimulates the liver to produce insulin-like growth factor 1（IGF-1）through GHR.Low serum levels of GH and IGF-1 are correlated with histologic severity of NAFLD.GH and IGF-1 replacement can alleviate the fatty liver condition in obese rodents and in GH-deficient patients.These observations suggest that GH plays a key role in regulating hepatic lipid processing.In this study,hepatic deletion of growth hormone receptor in mice（Li GHRKO）was used to analyse the relationship between GH/IGF-1 axis and NAFLD and to identify potential therapeutic targets of NAFLD.Methods:1.The Cre/Loxp system was used to generate a mouse model with hepatic ablation of GHR（LiGHRKO）,by breeding GHR^fl/fll/fl mice to albumin-cre mice in the C57BL/6J genetic background.The genotype of LiGHRKO was verified by PCR amplification and agarose gel electrophoresis experiment.Floxed GHR littermates were used as control.2.The DNA and RNA levels of GHR in the liver and other organs were detected by PCR and qRT-PCR to determine the efficiency and specificity of the liver-specific knockout of GHR.3.8-week-old LiGHRKO and control male mice（n=8-10）were weighed and sacrificed.The organs were weighed and the livers were photographed.4.Blood samples of inner canthus were collected in 1.5 ml Eppendorf tubes,then centrifuged at 4000 rpm for 10 min to separate the serum.The levels of serum ALT,AST,HDL,LDL and VLDL were measured using ELISA kits.5.RNA was extracted from the liver tissues of the 8-week-old LiGHRKO and control male mice.Purified RNA was converted to c DNA,and the expression levels of glucose and lipid metabolism related genes were examined using qRT-PCR assays.6.Proteins were extracted from the liver tissues of the 8-week-old LiGHRKO and control male mice.Key molecules along the PI3K/AKT/mTOR/p70S6k1 pathway were examined by Western blot and Immuohistochemical staining.7.The mitochondrial functions in livers of Li GHRKO and control male mice were detected by measurements of reactive oxygen species（ROS）,membrane potential（ΔΨm）and the expression levels of mitochondrial related genes.The ultrastructure of hepatic mitochondria in LiGHRKO and control mice was observed by transmission electron microscopy.Results:1.There were no significant changes in body weight and weights and volumes of other organs other than liver in 8-week-old LiGHRKO male mice.Serum lipid,lipoprotein and liver function profile revealed a non-alcoholic fatty liver phenotype in Li GHRKO male mice.2.Decreased glucose tolerance and symptoms of insulin resistance were shown in LiGHRKO male mice,indicating an abnormal glucose metabolism.3.The expressions of lipogenesis markers in the liver tissues of LiGHRKO mice were up-regulated,while the expressions of lipid oxidation markers were decreased.Excessive accumulation of fat in the liver of LiGHRKO mice was also noted,showing increased hepatic lipid uptake and increased de novo lipogenesis.4.In LiGHRKO male mice,the phosphorylation of PI3K ^Tyr607,AKT ^Ser273,mTOR ^Ser2448and p70S6K1 ^Ser371were decreased,suggesting the inhibition of the cascade of PI3K/AKT/mTOR/p70S6K1 by liver-specific GHR disruption.5.Increased ROS and decreasedΔΨm levels,decreased mitochondrial related gene expression and injured mitochondrial structure were found in Li GHRKO male mice,implying the contribution of mitochondria dysfunctions to the NAFLD development in these mice.Conclusion:Our data show that liver GHR ablation leads to typical symptoms of NAFLD in mice,accompanied by imbalances in glucose and lidid metabolism.A mechanism responsible for it is proposed as follows,deregulation of PI3K/AKT/mTOR/p70S6K1 pathway caused by GH/IGF-1 axis impairment in liver promotes gluconeogenesis and results in mitochondrial dysfunction,which initiates steatosis and triggers the pathogenesis of NAFLD.