Role Of Prolactin And Its Receptor In Non-Alcoholic Fatty Liver Disease And Hepatic Lipid Metabolism

Aims:Prolactin(PRL)is a polypeptide produced predominantly by the anterior pituitary gland that plays an important role in reproduction and lactation.Recently,PRL was also shown to be closely associated with glucose and lipid metabolism.However,little information is available on its effect in the regulation of hepatic lipid metabolism.Herein,we assessed the effect and mechanism of PRL and its receptor on non-alcoholic fatty liver disease(NAFLD)and hepatic lipid metabolism via human studies as well as animal and cell experiments.Methods:From September 2015 to January 2017,454 NAFLD patients(241 men and 213 women)and 405 non-NAFLD controls(200 men and 205 women)diagnosed by abdominal ultrasound were enrolled.Their serum PRL levels were evaluated by chemiluminescent immunoassay,and other metabolic parameters were also measured.Obese subjects received metabolic surgery from March to June 2017 in our hospital were also recruited for analysis.Their liver samples were obtained during surgery,and NALFD diagnosis were performed based on hematoxylin-eosin staining by two liver pathologists.A total of 55 females(44 NAFLD and 11 age-matched non-NAFLD)as well as 30 male NAFLD patients were evaluated.According to liver histology,NAFLD patients were further classified as mild to moderate NAFLD,and severe NAFLD.Circulating PRL concentrations in these participants were determined.In addition,the mRNA and protein level of prolactin receptor(PRLR)of human liver tissues were examined via qRT-PCR and immunostaining,respectively.Important genes involved in hepatic lipid synthesis,oxidation and uptake were also tested.PRLR expression was detected in livers of ob/ob,db/db and high-fat-diet(HFD)fed mice.Adeno-associated virus that overexpressed PRLR was injected via tail vein in C57BL/6 mice,and Oil Red O staining was used to assess the effect of PRLR on hepatic lipid content.In vitro,free fatty acid induced HepG2 cells were employed to study the mechanism of PRL/PRLR in hepatic lipid metabolism.After PRL intervention,triglyceride(TG)kit and Western blot analysis were used to explore TG content and protein expressions of involved molecules,respectively.PRLRoverexpression or silencing of PRLR was induced by adenoviral vector delivery or small interfering RNA(siRNA)transfection in FFA-treated HepG2 cells.Plasmid of overexpression of fatty acid translocase(FAT/CD36)was transfected into HepG2 cells to promote CD36 gene expression.Data with abnormal distribution were presented as median with interquartile range(IQR)and analyzed.Mann-Whitney and Kruskal Wallis analysis was applied to test non-normal distribution data between two or more groups,respectively.Categorical data were analyzed using Chi-square tests or Fisher’s exact test as indicated.The association between PRL and NAFLD was analyzed using the multivariate logistic regression method with adjustment for age,body mass index(BMI),HbA1c,homeostasis model assessment of insulin resistance(HOMA-IR),the presence of diabetes and imparied glucose tolerance(IGR).Spearman analysis was used to evaluate correlations between PRLR gene expression levels in liver tissue and genes involved in hepatic lipid metabolism.For cell experiments,data were analyzed by unpaired Student’s t test or one-way analysis of variance(ANOVA).Results:Circulating PRL levels were lower in subjects with ultrasound-diagnosed NAFLD(Men:7.9[5.9-10.3]μg/l;Women:8.7[6.1-12.4]μg/l)than those with non-NAFLD(Men:9.1[6.8-13.0]μg/l,P=0.002;Women:11.6[8.2-16.1]μg/l,P<0.001).Subgroups analysis showed that in subjects with normal glucose tolerance,T2DM and IGR,overweight and obese,females with polycystic ovary syndrome,premenopausal,postmenopausal,NAFLD patients still exhibited remarkably lower PRL levels than those without NAFLD in both genders.The subjects were then divided into four groups based on the quartiles of PRL concentrations.A gradual decrease was noted in the prevalence of NAFLD in both genders along with the increased quartile of PRL(Q1:64.9%,Q2:59.5%,Q3:53.2%,Q4:42.7%,P=0.007 in men;Q1:66.7%,Q2:59.4%,Q3:49%,Q4:28.2%,P<0.001 in women).After adjusting for age,sex,BMI,HbAlc,HOMA-IR,the presence of diabetes and IGR as well as PRL,the odds ratios(95%confidential interval)in the highest quartile of PRL level as compared to the lowest quartile was 0.48(0.23,0.99)in men and 0.36(0.13,0.97)in women.Female patients with biopsy-proven NAFLD exhibited statistically lower levels of PRL(9.6[6.7-14.1]μg/1)than patients with non-NAFLD(15.8[12.1-18.2]μg/l,P=0.003).Further,PRL levels in patients with biopsy-proven severe hepatic steatosis were lower as compared to those with mild-to-moderate hepatic steatosis in both men(8.3[5.4-9.5]μg/1 vs.9.7[7.1-12.3]μg/1,P=0.031)and women(8.5[4.2-10.6]μg/1 vs.9.8[8.2-15.7]μg/l,P=0.027).Furthermore,qRT-PCR data showed that human hepatic PRLR gene expression was significantly reduced in NAFLD patients and negatively correlated with CD36 gene expression.Compared with normal mice,Westernblot analysis showed that the expression of PRLR was downregulated in livers of ob/ob,db/db,and HFD fed mice.In addition,Oil Red O staining demonstrated that PRLR overexpression reduced lipid content in liver of HFD mice.In FFA induced HepG2 cells,PRL treatment and PRLR overexpression activated Signal Transducer and Activator of Transcription 5(STAT5),followed by reduction of CD36 expression and the content of triglycerides(TG)content.However,PRLR overexpression did not affect lipid synthesis and oxidation related gene levels.Furthermore,knock down of PRLR or overexpression of CD36 significantly reduced the PRL-mediated improvement in lipid content.Conclusions:For the first time,we reported that lower levels of serum PRL and hepatic PRLR contribute to the development of NAFLD.PRL/PRLR can activate the phosphorylation of STAT5,followed by inhibition of CD36 expression,a key transporter of FFA uptake in liver,and thereby reduce TG levels in hepatocytes.Our in vivo and in vitro results suggested a novel interplay between central nervous system and liver,and hepatic PRL/PRLR pathway may serve as a newly potential target against NAFLD.