Prolactin improves hepatic steatosis via CD36 pathway

Accepted Manuscript Prolactin improves hepatic steatosis via CD36 pathway Pengzi Zhang, Zhijuan Ge, Hongdong Wang, Wenhuan Feng, Xitai Sun, Xuehui Chu, Can Jiang, Yan Wang, Dalong Zhu, Yan Bi PII: DOI: Reference:

S0168-8278(18)30115-6 https://doi.org/10.1016/j.jhep.2018.01.035 JHEPAT 6858

To appear in:

Journal of Hepatology

Received Date: Revised Date: Accepted Date:

4 October 2017 23 January 2018 25 January 2018

Please cite this article as: Zhang, P., Ge, Z., Wang, H., Feng, W., Sun, X., Chu, X., Jiang, C., Wang, Y., Zhu, D., Bi, Y., Prolactin improves hepatic steatosis via CD36 pathway, Journal of Hepatology (2018), doi: https://doi.org/ 10.1016/j.jhep.2018.01.035

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Prolactin improves hepatic steatosis via CD36 pathway Pengzi Zhang1†, Zhijuan Ge1†, Hongdong Wang 1†, Wenhuan Feng1, Xitai Sun2, Xuehui Chu2, Can Jiang1, Yan Wang 1, Dalong Zhu1*, Yan Bi1* Affiliation:
 
 1

Department of Endocrinology, Drum Tower Hospital Affiliated to Nanjing University

Medical School, Nanjing, China. 2

Department of General Surgery, Drum Tower Hospital Affiliated to Nanjing

University Medical School, Nanjing, China. †

Pengzi Zhang, Zhijuan Ge, Hongdong Wang contributed equally to the manuscript.

*Corresponding authors: Yan Bi, [email protected], and Dalong Zhu, [email protected]. Department of Endocrinology, Drum Tower Hospital affiliated to Nanjing University Medical School, No. 321, Zhongshan Road, Nanjing, 210008, China Tel: +86 25 83106666-61434 Fax: +86 25 68182474

Keywords Prolactin; Prolactin receptor; Non-alcoholic fatty liver disease; CD36; STAT5;

Electronic word count: 6700 Number of figures: 5 Number of tables: 3 Supplementary figures: 5 1

Supplementary tables: 7

Conflict of interest statements All authors declared that there are no potential conflicts of interest with respect to this manuscript.

Financial support statement This work was supported by the National Natural Science Foundation of China Grant Awards (81770819, 81570736, 81570737, 81370947, 81500612, 81400832, 81600637, 81600632, and 81703294), the National Key Research and Development Program of China (2016YFC1304804), Jiangsu Provincial Medical Talent (ZDRCA2016062), the Jiangsu Provincial Key Medical Discipline (ZDXKB2016012), the Key Project of Nanjing Clinical Medical Science, the Key Research and Development Program of Jiangsu Province of China (BE2015604 and BE2016606), the Jiangsu Provincial Medical Talent (ZDRCA2016062), the Nanjing Science and Technology Development Project (201605019). Translational Medicine Core Facilities, Medical School of Nanjing University and Liver Disease Collaborative Research Platform of Medical School of Nanjing University.

Author Contributions P.Z. contributed to the study design, data acquisition, data interpretation and drafting the manuscript. Z.G. and H.W. contributed to data interpretation and drafting 2

the manuscript. W.F., X.S and X.C contributed to the collection of liver biopsy samples. C.J and Y.W contributed to data acquisition. D.Z. and Y.B. contributed to the study design, data interpretation, revision of the manuscript and approval of the final version approval of the final version for publishing.

Clinical trial number: NCT03296605

Abstract Background & Aims: Prolactin (PRL) is a multifunctional polypeptide with effects 3

on metabolism; however, little is known about its effect on hepatic steatosis and lipid metabolism. Methods: The serum PRL levels of 456 non-alcoholic fatty liver disease (NAFLD) patients and 403 non-NAFLD controls diagnosed by ultrasound, and 85 subjects with liver histology obtained during metabolic surgery (44 female and 30 male NAFLD patients and 11 age-matched non-NAFLD female subjects) were evaluated. The gene expressions of prolactin receptor (PRLR) and signaling molecules involved in hepatic lipid metabolism were evaluated in human liver and HepG2 cells. The effects of overexpression of PRLR or FAT (fatty acid translocase)/CD36 or knockdown of PRLR on hepatic lipid metabolism were tested in free fatty acid (FFA)-treated HepG2 cells. 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). 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/l vs. 9.7 [7.1-12.3] µg/l, P=0.031) and women (8.5 [4.2-10.6] µg/l vs. 9.8 [8.2-15.7] µg/l, P=0.027). Furthermore, human hepatic PRLR gene expression was significantly reduced in NAFLD patients and negatively correlated with CD36 gene expression. In FFA-induced HepG2 cells, PRL treatment or PRLR overexpression significantly reduced the expression of CD36 and lipid content, which was abrogated after silencing of PRLR. Furthermore, overexpression of CD36 significantly reduced 4

the PRL-mediated improvement in lipid content. Conclusions: A novel association between central nervous system and liver was reported; PRL/PRLR improved the hepatic lipid accumulation via the CD36 pathway. Lay summary Our clinical study suggested a negative association between prolactin (PRL) /prolactin receptor (PRLR) and the presence of non-alcoholic fatty liver disease (NAFLD). Using cell experiments, we found that PRL ameliorates hepatic steatosis via hepatic prolactin receptor (PRLR) and FAT (fatty acid translocase)/CD36, a key transporter of free fatty acid uptake in liver. Our findings suggested a novel mechanism of PRL and PRLR in improving NAFLD.

Introduction Non-alcoholic fatty liver disease (NAFLD) is a common public health problem 5

affecting up to 25% of the adults around the world [1]. It is associated with a series of metabolic comorbidities. NAFLD is initially caused by an imbalance between lipid demand and supply; however, the pathogenesis of the disease also involves crosstalk between liver and extra-hepatic organs, including adipose tissue and central nervous system (CNS) [2]. By sensing and integration of the peripheral signals (such as leptin), hypothalamic arcuate nucleus (ARC) neurons in CNS can induce hepatic fatty acid oxidation [3]. On the other hand, pituitary released hormone such as growth hormone has been shown to suppress lipid uptake in liver [4, 5]. Prolactin (PRL), a polypeptide produced predominantly by the anterior pituitary gland, is known for its potent stimulation effects in lactation and reproduction by binding to its cell surface receptors (PRLRs) in the mammary gland [6]. Nonetheless, PRLR is also expressed in metabolic tissues such as liver, pancreas, and adipose tissues [7]. Previous studies documented favorable roles of PRL/PRLR in metabolic homeostasis. The PRL levels in the serum in both genders were markedly decreased in patients with type 2 diabetes mellitus (T2DM) [8]. In addition, it was inversely correlated with triglycerides (TG) in females with polycystic ovary syndrome [9] and positively associated with the high-density lipoprotein (HDL) in obese children [10]. The studies investigating the underlying mechanism found that PRL inhibited the mRNA and protein expressions of fatty acid synthase in adipocytes and showed an antilipogenic function [11]. PRL can upregulate the expression of PRLR in human adipose explant through which, PRL can exert its anti-lipolytic action [12]. Moreover, Prlr-/- mice displayed greater fat mass under high fat diet (HFD) [13]. However, the 6

role of PRL/PRLR in hepatic lipid metabolism is yet to be elucidated. Thus, the present study aimed to clarify whether PRL/PRLR is involved in the development of NAFLD. Here, we also explored the relationship between PRL and the severity of hepatic steatosis by liver histology. In addition, the potential mechanisms of PRL/PRLR signaling in regulating hepatic lipid were determined in HepG2 cells. Methods Clinical study. The Ethics Committee of Nanjing Drum Tower Hospital approved the study protocol, and all subjects provided the written informed consent. The study protocol conforms to the guidelines of the Declaration of Helsinki. This study was registered on ClinicalTrials.gov (number NCT03296605). Clinical cohort study. A cohort of 859 adult patients (441 men and 418 women) aged between 18 and 80-year-old, eligible for the study, were collected from September 2015 to January 2017 from the Endocrinology Department of Drum Tower Hospital affiliated to Nanjing University Medical School. The exclusion criteria for the study were as follows: hepatic enzymes levels >5-fold than the upper normal [14], history of alcohol consumption (≥140 g/week for males or 70 g/week for females), other liver diseases including chronic hepatitis B or C infection, biliary obstructive diseases and autoimmune hepatitis, history of using steatogenic medications, known hyperthyroidism or hypothyroidism, systematic corticosteroids, pregnancy, type 1 7

diabetes, malignant tumor, and pituitary diseases. All the enrolled patients underwent abdominal ultrasound under fasting conditions. The ultrasonography was conducted using Philips HD15 Ultrasound Unit (Netherlands) by the same sonographer, blinded to the patients’ information. According to the abdominal ultrasound, clinical NAFLD was diagnosed based on the characteristic echo patterns, including (1) brightness of the liver, (2) diffuse increased echogenicity of the liver parenchyma relative to the kidney, (3) echo attenuation into the deep hepatic portion, (4) blurring of intrahepatic vessels, and (5) poor visualization of diaphragm. NAFLD was diagnosed with at least the presence of the first and second criteria [15]. Bariatric surgery and human liver specimens’ study. A total of 85 eligible obese patients (30 men and 55 women) who underwent metabolic surgery (Roux-en-Y gastric bypass) in our hospital from March to June 2017 were analyzed. Liver tissues samples (approximately 1.5×2.0×2.0 cm3) from patients, who underwent metabolic surgery, were snap-frozen in liquid nitrogen or fixed in 5% formaldehyde and embedded in paraffin for hematoxylin-eosin (H&E) staining. Then, the sections (5-µm thickness) were stained, and liver histology was assessed according to the Nonalcoholic Steatohepatitis Clinical Research Network scoring system, described previously[16], by two liver pathologists, blinded to the patients’ clinical data. Hepatic steatosis was defined according to the proportion of affected hepatocytes: S1 (5–33%, “mild”), S2 (33–66%, “moderate”), and S3 (>66%,“severe”). NAFLD activity score (NAS) including steatosis, hepatocyte ballooning and lobular 8

inflammation, as well as fibrosis stage were also evaluated[16]. For the immunohistochemical assay, the liver sections were deparaffinized and rehydrated, followed by 5% BSA blocking. Then, the sections were probed with primary antibody to PRLR (ab170935, Abcam, USA) at 4 °C in a humidified chamber overnight followed by 2 h incubation with the secondary antibody at 37 °C. The images were captured using a Panoramic 250 slide scanner (3DHistech Ltd., Hungary), and the brown staining of PRLR was considered as positive. Total RNA, extracted from human liver tissue using TRIzol reagent (Invitrogen, USA), was reverse transcribed into cDNA using a Takara RT reagent Kit (Bio, Otsu, Japan). Real-time PCR was performed on a Light Cycler 480 (Roche, Switzerland). β-actin was used as the housekeeping gene for normalization of gene expressions. Primers used are listed in Supplementary Table 1. Clinical measurements. The anthropometric characteristics and fasting venous blood samples were collected for all patients. Individuals without previously diagnosed T2DM were subjected to a 75 g oral glucose tolerance test (OGTT) after an overnight fast of at least 8 h. Impaired glucose regulation (IGR): 5.6 mmol/l ≤ fasting blood glucose (FBG) <7.0 mmol/l and 2 h blood glucose (BG) <7.8 mmol/l; or FBG < 5.6mmol/l and 7.8 mmol/l ≤2 h BG <11.1 mmol/l; T2DM was defined as patients having at least one of the following: history or current use of antidiabetic medication, or FBG ≥7.0 mmol/L or 2 h BG ≥11.1 mmol/L, or HbA1c ≥6.5% [17]. The plasma glucose concentration was measured using a hexokinase method (TBA-200FR, Tokyo, Japan). The level of HbA1c was measured by high-performance liquid 9

chromatography (HLC-73G8, Tosoh, Japan). PRL was estimated in an automated chemiluminescent immunoassay (Siemens Immulite 2000, Siemens Healthcare Diagnostic Products Ltd, UK). Fasting insulin (FINS) was determined by electrochemiluminescent immunoassay (Roche). Serum total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, HDL cholesterol, and TG were detected using an autoanalyzer (Abbott Laboratories, USA). Insulin resistance was measured by the homeostasis model assessment of HOMA-IR index by the following formula: HOMA-IR = FINS (µIU/mL) × FBG (mmol/L) / 22.5 [18]. For diabetic patients, medication information were collected. In female patients who received metabolic surgery, serum free fatty acids (FFAs), IL-6, adiponectin and leptin levels were tested (Supplementary Methods). Cell experiments Cell lines. HepG2 cells were bought from Cell Bank of Shanghai Institute of Cell Biology (Chinese Academy of Sciences), and were characterized by Genetic Testing Biotechnology Corporation (Suzhou, China) via short tandem repeat (STR) markers. Mycoplasm was dected through Hoechst DNA stain method. HepG2 cells were incubated with a mixture of FFA (oleic acid and palmitic acid at the proportion of 1:1, 500 µmol/l, Sigma, USA) or an increasing concentration of PRL (10–200 µg/l) for 24 h, as indicated. Moreover, an adenoviral vector-mediated overexpression of PRLR was transfected in HepG2 cells 24 h before the treatment with FFA or PRL. For the downregulation of PRLR or promotion of CD36 gene expression, short hairpin RNA (shRNA) against PRLR or the plasmid with CD36 10

overexpression was transfected into HepG2 cells using Lipofectamine 2000 (Invitrogen). The shRNA sequence of PRLR was 5’- TAGTGACTTCACCATGAAT -3’, and the scrambled sequence was 5’- TTCTCCGAACGTGTCACGT’ -3’. Also, 500 µmol/l FFA or 100 µg/l PRL was added post-transfection, as indicated. The expression of PRLR, signal transducer and activator of transcription 5 (STAT5), phosphorylated STAT5 (pSTAT5), and CD36 were detected by Western blot and qRT-PCR. Briefly, the cells were lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The lysate was collected by centrifugation, and the total protein extract was denatured. LY294002 and primary antibodies to β-actin, STAT5, pSTAT5, AKT, pAKT (Ser473) were purchased from Cell Signaling Technology (CST; Danvers, MA, USA). Anti-PRLR and anti-CD36 were obtained from Abcam. The band intensities were quantified by Image J (National Institutes of Health, USA) software. qRT-PCR was carried out as described above. Oil Red O (ORO) staining and TG Kits (Applygen, Beijing, China) were utilized for the detection of the TG content in HepG2 cells. Statistical analysis Data with abnormal distribution were presented as median with interquartile range (IQR) and analyzed. Mann-Whitney analysis was applied to test non-normal distribution data between two groups. Kruskal Wallis analysis was used to compare non-normal distribution data between three or more groups. Categorical data were analyzed using Chi-square tests of Fisher’s exact test as indicated. The association between PRL and NAFLD was analyzed using the multivariate logistic regression 11

method with adjustment for age, BMI, and HbA1c, HOMA-IR and the presence of diabetes. 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). All analyses were performed using SPSS software (Version 17.0; SPSS Inc., USA). The significance level was set at P<0.05, and P-values were provided for two-sided tests. Results Serum PRL concentration was negatively associated with NAFLD The clinical and biochemical variables of the patients are summarized in Table 1. Compared to those with non-NAFLD (198 men and 205 women), NAFLD patients (243 men and 213 women) presented higher levels of body mass index (BMI), waist-to-hip ratio (WHR), FBG, HbA1c, alanine aminotransferase (ALT), aspartate aminotransferase (AST), TG, TC, and lower levels of HDL (all P<0.05). The serum PRL levels were significantly decreased in patients with NAFLD in both genders (Men: 7.9 [5.9-10.3] µg/l vs. 9.1 [6.8-13.0] µg/l, P=0.002; Women: 8.7 [6.1-12.4] µg/l vs. 11.6 [8.2-16.1] µg/l, P<0.001) than in those with non-NAFLD. Since an inverse relationship was observed between serum glucose and PRL levels [8], the concentration of PRL in the subgroup of subjects with normal glucose tolerance were compared. We found that NAFLD patients exhibited remarkably lower PRL levels than those without NAFLD in both genders (Supplementary Table 2).

12

Furthermore, the subjects were divided into four groups based on the quartiles of PRL concentrations. The quartile ranges of Q1, Q2, Q3, and Q4 of serum PRL level were ≤6.3, 6.3–8.4, 8.4–11.5, and >11.5 µg/l in men and ≤6.7, 6.7–10.0, 10.0–14.2, and >14.2 µg/l in women, respectively (Supplementary Table 3, 4). A gradual decrease was noted in the incidence 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: 28.1%, P<0.001 in women). Multivariate analysis of age, sex, BMI, HbA1c, HOMA-IR, the presence of diabetes and IGR as well as PRL for NAFLD in total subjects was conducted. The results showed that increasing BMI was a risk factor for NAFLD, and age and PRL were negatively and significantly associated with NAFLD (Supplemetary Table 5). We then further analyzed odds ratios ORs (95% CIs) for NAFLD across the PRL quartiles. The ORs 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 (Fig. 1). Circulating PRL level and hepatic PRLR expression were decreased in patients with biopsy-proven NAFLD Serum PRL levels in all 85 patients (55 women and 30 men) received liver biopsy during metabolic surgery were explored. Firstly, we compared circulating PRL levels in 44 obese women with NAFLD and age-matched 11 obese women with non-NAFLD (Table 2), and the results showed that patients with biopsy-proven NAFLD exhibited statistically lower levels of PRL (9.6 [6.7-14.1] µg/l) than patients with non-NAFLD (15.8 [12.1-18.2] µg/l, P=0.003) (Fig. 2A). Then, the PRL levels in 13

patients with different severities of steatosis (44 women and 30 men) were evaluated based on the histological diagnosis (Table 3). Importantly, the PRL levels in patients with severe hepatic steatosis (affected hepatocytes > 66%) were lower as compared to those with mild-to-moderate hepatic steatosis (affected hepatocytes 5–66%) in both women (8.5 [4.2-10.6] µg/l vs. 9.8 [8.2-15.7] µg/l, P=0.027) and men (8.3 [5.4-9.5] µg/l vs. 9.7 [7.1-12.3] µg/l, P=0.031) (Fig. 2B, C). Hepatic PRLR gene and protein expression in patients, who underwent liver biopsy, were evaluated via qRT-PCR and immunostaining, respectively. As shown in Fig. 2, the NAFLD patients exhibited a reduction in the hepatic PRLR mRNA levels (Fig. 2D) and protein expression (Fig. 2E) as compared to the subjects with non-NAFLD. Hepatic PRLR gene expression was negatively correlated with CD36 in patients with biopsy-proven NAFLD. Next we sought to investigate the role of PRL/PRLR in hepatic lipid metabolism. Hepatosteatosis results from increased de novo lipogenesis and FFA uptake or decreased FFA β-oxidation, and very low-density lipoprotein (VLDL) export. CD36 is a major mediator of hepatic FFA uptake and a critical target downstream of class I cytokine receptors, including PRLR [19]. In the human liver tissue, NALFD patients (44 females) exhibited a significant decrease in the PRLR gene levels (Fig. 2D) and an increase in hepatic CD36 (Fig. 3A) gene expression compared with non NAFLD patients (11 females). Moreover, we

14

found that the mRNA level of PRLR was significantly and negatively correlated with CD36 in patients with NAFLD (Fig. 3B, 44 females and 30 males), whereas no correlations were established between PRLR and genes in fatty acid oxidation and de novo lipogenesis, including acetyl coenzyme A carboxylase (ACC) (Fig. 3C), sterol response

element-binding

protein

1c

(SREBP1c)

(Fig.

3D),

peroxisome

proliferator-activated receptor α (PPARα) (Fig. 3E), and carnitine palmitoyltransferase 1 (CPT1a) (Fig. 3F) . PRL alleviated the lipid accumulation via hepatic PRLR/CD36 pathway. Further, we explored the mechanisms of PRL/PRLR on lipid metabolism in vitro. In HepG2 cells, we demonstrated that the PRLR protein levels increased with PRL treatment from 10–100 µg/l in a dose-dependent manner; however, the level was not further

enhanced

from

100–200

µg/l

(Fig.

4A).

Interestingly,

both

adenovirus-mediated overexpression of PRLR (Supplementary Fig.1) and PRL treatment reduced the lipid accumulation, as revealed by ORO staining (Fig. 4B) and TG quantification assay (Fig. 4C), while the PRLR silencing (Supplementary Fig. 2) via shRNA significantly ameliorated the PRL-reduced lipid levels in cells (Fig. 4C). In FFA-induced HepG2 cells, PRL treatment significantly decreased the protein expression of CD36, and stimulated STAT5 phosphorylation, a critical upstream regulator of CD36 (Fig. 5A). Similarly, PRLR overexpression significantly reduced the mRNA (Fig. 5B) and protein expression of CD36 (Fig. 5C), and enhanced the phosphorylation of STAT5 (Fig. 5C), while the gene expression levels of ACC, SREBP1c, PPARα, and CPT1a were not affected significantly (Fig. 5D). In addition, 15

the effect of PRL on CD36 and phosphorylation of STAT5 was markedly reduced after PRLR silencing (Fig. 5E). Moreover, CD36 overexpression (Supplementary Fig. 3) partially reversed the PRL-induced reduction in hepatic lipid levels, as revealed by TG measurements (Fig. 5F). These data indicated that CD36 was essential for the PRL/PRLR-mediated amelioration of hepatic steatosis. Discussion Besides its well-known lactogenic action, PRL is recently identified as a metabolic hormone as well [20]. In this study, we showed that low PRL level is a risk factor for the development and progression of NAFLD. PRL ameliorates lipid accumulation through PRLR-mediated inhibition of CD36. The current data not only provides the first evidence regarding the lipid-lowering role of PRL in hepatocytes but also emphasizes the crucial regulation of central neuroendocrine system on liver homeostasis. Cumulative evidence has identified PRL as a protective regulator against dysregulations of glucose and lipid metabolism. A high level of PRL was associated with the reduced risk of hyperglycemia in men [21, 22]. A low-dose PRL injection potentiated the glucose-stimulated insulin secretion in diabetic rats [23]. Moreover, chronically elevated serum PRL levels induced by ectopic pituitary graft led to decreased circulating FFA concentrations in mice [24]. Here, we used two clinical cohorts illustrating that PRL was significantly and negatively correlated with NAFLD. In the second cohort study, patients were well-phenotyped for hepatic lesions since each subject underwent liver biopsy, the gold standard for diagnosing NAFLD. 16

Moreover, our data showed that PRL levels were further decreased as the disease progressed from mild and moderate to severe steatosis. PRL is not only secreted by pituitary gland but also by adipose tissue [25]. A previous study illustrated that the amount of PRL released by a pituitary lactotroph was 4-5-fold higher than that of adipocytes. Also, adipocytes-derived PRL was retained locally, acting as an autocrine/paracrine factor [26]. Therefore, in this study, the influence of PRL on hepatic metabolism was largely attributed to pituitary rather than adipose tissue. Previous studies have proposed that PRL regulated adipokine secretion, such as adiponectin and leptin [13, 27]. To test whether these factors play a role regarding PRL levels and NAFLD, To test whether these factors play a role regarding PRL levels and NAFLD, we examined serum concentrations of FFAs, IL-6, adiponectin and leptin in female patients who received liver biopsy. We found that there were no significant difference of these factors between non NAFLD and NAFLD group (Table 2), hence the negative association between PRL and NAFLD may not be influenced by these factors. An earlier report revealed the inhibitory effect of PRL on lipid synthesis in maternal rat liver [28]. Mice with liver-specific knockdown of PRLR manifested impaired glucose tolerance, while the overexpression of hepatic PRLR can reverse the hepatic insulin resistance [29], thereby highlighting a vital role of PRL and PRLR in hepatic metabolism. In our study, human hepatic PRLR was concomitantly downregulated in NAFLD patients when circulating PRL levels were reduced, and PRL (<100 µg/l) intervention enhanced the PRLR expression in HepG2 cells. Since 17

100 µg/l is higher than physiological concentration range, we have also investigated whether PRL at 20 µg/l (near normal range in humans) could exert beneficial effect on hepatic lipid metabolism. The results showed that 20 µg/l PRL also exhibited similar effect with 100 µg/l in reducing CD36 protein expression and TG content in HepG2 cells (Supplementary Fig. 4). However, a high concentration of PRL (>100 µg/l) treatment in vitro cannot further upregulate the expression PRLR, implying that further upregulation of PRLR is not likely to occur in prolactinoma patients. Thus, it may not be able to exert a beneficial effect on lipid metabolism, and that the physiologically elevated PRL is different from the pathological hyperprolactinemia with respect to metabolism. This phenomenon might partially explain the fact that patients with hyperprolactinemia also exhibited metabolic disturbances [30]. Although moderately increased PRL level promoted insulin sensitivity, high-dose PRL administration aggravated the hepatic insulin resistance in diabetic murine models [23, 31]. CD36, also known as FAT, is an FFA transporter that facilitates FFA uptake and has a profound impact on the development of NAFLD. Increased hepatic CD36 expression causes lipid deposition, while the suppression of CD36 is resistant to hepatic lipid accumulation [32]. Herein, we showed that human hepatic PRLR gene expressions were significantly decreased in obese patients with NAFLD and negatively correlated with CD36 gene levels; however, genes involved in hepatic de novo lipogenesis and FFA β oxidation were not identified distinctly. In FFA-treated HepG2 cells, both PRL administration and PRLR overexpression reduced the CD36 18

expression and TG content and knocking down PRLR attenuated these effects. Moreover, CD36 overexpression partially reversed the PRL-induced reduction in hepatic lipid levels. Thus, these results indicated that the lipid-reducing effect of PRL was mediated by PRLR and CD36. Reportedly, PRLR exerts its biological function via cellular kinases including PI3K/AKT, STAT5, and Ras/MAPK [33]. Among the three signaling pathways, the activation of STAT5 can improve hepatic steatosis by inhibiting CD36 in HFD mice [34-36]. Furthermore, we observed that phosphorylated STAT5 was increased after PRL intervention or PRLR overexpression in HepG2 cells. Besides STAT5, PI3K/AKT pathway also participates in the regulation of hepatic lipid metabolism [37]. Therefore we used PI3K inhibitor LY294002 and found that PI3K inhibitor did not affect the levels of CD36 protein after PRL intervention (Supplementary Fig. 5), indicating that the effect of PRL on hepatic lipid metabolism was independent of PI3K/AKT pathway. Taken together, PRL/PRLR may protect the liver against lipid accumulation by inhibiting STAT5/CD36 in hepatocytes. Nevertheless, the present study has several limitations. First, the clinical cohort study was carried out in a group of participants with high prevalence (>80%) of T2DM; however, the negative association between PRL and NAFLD persisted after adjusting for HbA1c levels, HOMA-IR and the presence of diabetes. On the other hand, the subgroup analysis in subjects with normal glucose tolerance showed similar results, suggesting that decreased PRL can lead to the fatty liver that is independent of T2DM. Additionally, in the first clinical cohort study, the medication use of metformin was statistically different in diabetic males between non-NAFLD and 19

NAFLD group, whereas comparable in diabetic females between these groups (Supplementary Table 6). Particularly, a comparison of the anti-diabetic treatment in female patients with liver biopsy showed no significant difference between non-NAFLD and NAFLD group (Supplementary Table 7). Moreover, metformin and insulin were reported with little effect on liver histology although with the amelioration of liver enzymes [38, 39], hence the effect of glucose-lowering medication on the association between PRL and NAFLD might not be clinical significance in this study. Still, further studies determining their relationship in the general population without the history of using anti-diabetic medication, with a large sample size, are essential. Second, the secretion of PRL is pulsatile, and a single measurement of serum may not be adequate to represent the PRL levels during the whole day; nonetheless, the pulsatile secretion occurs primarily during the night and is relatively constant during the day [40]. All subjects underwent the PRL examination at 8:00 a.m., and hence, the variation of PRL secretion at different time points during the day could be avoided. In conclusion, we demonstrated that PRL/PRLR improved the hepatic steatosis via suppression of CD36. The current in vivo human study and in vitro study has provided insight regarding the key modulation of the physiopathological process of NAFLD, and the activation of hepatic PRL/PRLR signaling may serve as a novel potential target for the treatment of NAFLD.

References 20

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[20] Bernard V, Young J, Chanson P, Binart N. New insights in prolactin: pathological implications. Nature reviews Endocrinology 2015;11:265-275. [21] Haring R, Völzke H, Vasan RS, Felix SB, Nauck M, Dörr M, et al. Sex-specific associations of serum prolactin concentrations with cardiac remodeling: Longitudinal results from the Study of Health Pomerania (SHIP). Atherosclerosis 2012;221:570-576. [22] Corona G, Wu FC, Rastrelli G, Lee DM, Forti G, O'Connor DB, et al. Low Prolactin Is Associated with Sexual Dysfunction and Psychological or Metabolic Disturbances in Middle-Aged and Elderly Men: The European Male Aging Study (EMAS). The Journal of Sexual Medicine 2014;11:240-253. [23] Park S, Kim DS, Daily JW, Kim SH. Serum prolactin concentrations determine whether they improve or impair beta-cell function and insulin sensitivity in diabetic rats. Diabetes/metabolism research and reviews 2011;27:564-574. [24] Matsuda M, Mori T, Sassa S, Sakamoto S, Min KP, Kawashima S. Chronic effect of hyperprolactinemia on blood glucose and lipid levels in mice. Life Sciences 1996;58:1171-1177. [25] Harvey S, Arámburo C, Sanders EJ. Extrapituitary production of anterior pituitary hormones: an overview. Endocrine 2011;41:19-30. [26] Hugo ER, Borcherding DC, Gersin KS, Loftus J, Ben-Jonathan N. Prolactin Release by Adipose Explants, Primary Adipocytes, and LS14 Adipocytes. The Journal of Clinical Endocrinology & Metabolism 2008;93:4006-4012. [27] Viengchareun S, Bouzinba-Segard H, Laigneau JP, Zennaro MC, Kelly PA, Bado A, et al. Prolactin potentiates insulin-stimulated leptin expression and release from differentiated brown adipocytes. Journal of molecular endocrinology 2004;33:679-691. [28] Lorenzo M, Roncero C, Benito M. The role of prolactin and progesterone in the regulation of lipogenesis in maternal and foetal rat liver in vivo and in isolated hepatocytes during the last day of gestation. The Biochemical journal 1986;239:135-139. [29] Yu J, Xiao F, Zhang Q, Liu B, Guo Y, Lv Z, et al. PRLR Regulates Hepatic Insulin Sensitivity in Mice via STAT5. Diabetes 2013;62:3103-3113. [30] dos Santos Silva CM, Barbosa FRP, Lima GAB, Warszawski L, Fontes R, Domingues RC, et al. BMI and Metabolic Profile in Patients With Prolactinoma Before and After Treatment With Dopamine Agonists. Obesity 2010;19:800-805. [31] Park S, Kang S, Lee HW, Ko BS. Central prolactin modulates insulin sensitivity and insulin secretion in diabetic rats. Neuroendocrinology 2012;95:332-343. [32] Miquilena-Colina

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Fernandez-Bermejo M, Lozano-Rodriguez T, et al. Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance, hyperinsulinaemia and increased steatosis in non-alcoholic steatohepatitis and chronic hepatitis C. Gut 2011;60:1394-1402. [33] Wilson CG, Tran JL, Erion DM, Vera NB, Febbraio M, Weiss EJ. Hepatocyte-Specific Disruption of CD36 Attenuates Fatty Liver and Improves Insulin Sensitivity in HFD-Fed Mice. Endocrinology 2016;157:570-585. [34] Mueller KM, Themanns M, Friedbichler K, Kornfeld J-W, Esterbauer H, Tuckermann JP, et al. Hepatic growth hormone and glucocorticoid receptor signaling in body growth, steatosis and metabolic liver cancer development. Molecular and Cellular Endocrinology 2012;361:1-11. [35] Baik M, Nam YS, Piao MY, Kang HJ, Park SJ, Lee JH. Liver-specific deletion of the signal transducer and activator of transcription 5 gene aggravates fatty liver in response to a high-fat diet in mice. The Journal of nutritional biochemistry 2016;29:56-63. 22

[36] Hosui A, Tatsumi T, Hikita H, Saito Y, Hiramatsu N, Tsujii M, et al. Signal transducer and activator of transcription 5 plays a crucial role in hepatic lipid metabolism through regulation of CD36 expression. Hepatology research : the official journal of the Japan Society of Hepatology 2016. [37] Titchenell PM, Lazar MA, Birnbaum MJ. Unraveling the Regulation of Hepatic Metabolism by Insulin. Trends in endocrinology and metabolism: TEM 2017;28:497-505. [38] Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology 2012;142:1592-1609. [39] Ratziu V, Goodman Z, Sanyal A. Current efforts and trends in the treatment of NASH. Journal of hepatology 2015;62:S65-75. [40] Katznelson L, Riskind PN, Saxe VC, Klibanski A. Prolactin pulsatile characteristics in postmenopausal women. The Journal of clinical endocrinology and metabolism 1998;83:761-764.

23

Table1. Baseline characteristics of the study subjects. Men

Women

Non-NAFLD

NAFLD

198

243

121 (61.1%)

205 (84.4%)

IGR (n,%)

11 (5.6%)

Age (years)

P

P

Non-NAFLD

NAFLD

205

213

0.000

56 (27.3%)

182 (85.4%)

0.000

15 (6.2%)

0.784

19 (9.3%)

12 (5.6%)

0.156

53 (40, 64)

51 (40, 60)

0.058

51 (40, 61.5)

58 (51, 67)

0.000

BMI (kg/m2)

23.5 (21.8, 25.5)

26.3 (24.7, 28.4)

0.000

22.6 (20.8, 24.9)

25.7 (23.8, 28.6) 0.000

SBP (mmHg)

132 (120, 144)

135 (125, 147)

0.101

128.5 (118, 145)

138 (128, 150.5) 0.000

DBP (mmHg)

79 (71, 88)

84 (77, 91)

0.000

79.5 (70, 88)

81 (71.5, 90.5)

0.186

Waist (cm)

90 (84, 95)

97 (91, 100)

0.000

82 (76, 88)

94 (87, 100)

0.000

0.92 (0.88, 0.95)

0.94 (0.92, 0.98)

0.000

0.87 (0.82, 0.90)

HbA1c (%)

6.8 (5.6, 9.9)

7.6 (6.2, 9.5)

0.033

5.7 (5.2, 7.4)

8.1 (6.6, 9.8)

0.000

FBG (mmol/l)

5.7 (4.9, 8.1)

7.3 (5.6, 9.5)

0.000

5.0 (4.6, 5.8)

7.2 (5.6, 9.9)

0.000

HOMA-IR

1.9 (1.1, 3)

3.2 (1.8, 5.1)

0.000

1.8 (1.1, 2.9)

3.4 (2.2, 5.8)

0.000

ALT (U/l)

19.4 (14.8, 28.7)

30.3 (20, 45.6)

0.000

15.3 (12, 22.5)

23.5 (16.1, 35.8) 0.000

AST (U/l)

17.7 (15, 22)

20.5 (16.9, 27.8)

0.000

17.8 (14.7, 22.5)

20.4 (16.1, 26.3) 0.000

TG (mmol/l)

1.2 (0.9, 1.7)

1.9 (1.3, 2.7)

0.000

1.2 (0.9, 1.8)

1.7 (1.2, 2.4)

0.000

TC (mmol/l)

4.3 (3.6, 4.9)

4.5 (3.9, 5.1)

0.043

4.4 (3.8, 5)

4.6 (3.9, 5.3)

0.028

HDL (mmol/l)

1.1 (0.9, 1.3)

0.9 (0.8, 1.1)

0.000

1.2 (1, 1.5)

1 .0 (0.9, 1.2)

0.000

LDL (mmol/l)

2.5 (1.8, 2.9)

2.5 (1.9, 3)

0.267

2.3 (1.9, 3)

2.6 (2, 3.2)

0.013

PRL (µg/l)

9.1 (6.8, 13.0)

7.9 (5.9, 10.3)

0.002

11.6 (8.2, 16.1)

8.7 (6.1, 12.4)

0.000

N Diabetes (n, %)

WHR

0.93 (0.89, 0.97) 0.000

IGR: Impaired glucose regulation; BMI: body mass index; WHR: waist-to-hip ratio; SBP: systolic blood pressure; DBP: diastolic blood pressure; FBG: fasting blood glucose; ALT: alanine 24

aminotransferase; AST: aspartate aminotransferase; TG: triglyceride; TC: total cholesterol; HOMA-IR: the homeostatic model assessment of insulin resistance; PRL: prolactin. Data are shown as median with interquartile range (IQR) and categorical data as n (%). P values are based on Mann-Whitney U test for continuous data or the x2 test for categorical data.

25

Table 2. Characteristics of non NAFLD patients and age- and gender- matched NAFLD patients in women.

P

Non-NAFLD

NAFLD

11

44

Age (years)

32.0 (28.0, 39.0)

31.0 (27.0, 38.0)

0.924

BMI (kg/m2)

32.8 (30.5, 39.3)

35.9 (33.2, 39.2)

0.214

SBP (mmHg)

123 (114, 145)

132 (123.5, 140.8)

0.256

DBP (mmHg)

79.0 (71.0, 100.0)

84.0 (77.0, 90.0)

0.343

Waist (cm)

107.0 (90.0, 123.5)

110 (104.3, 120.5)

0.361

0.92 (0.84, 0.99)

0.95 (0.90, 1.01)

0.254

HbA1c (%)

5.3 (4.9, 6.3)

5.8 (5.5, 6.3)

0.065

FBG (mmol/l)

5.1 (4.8, 6.0)

5.4 (5.0, 6.8)

0.293

HOMA-IR

4.2 (3.4, 4.8)

5.7 (4.4, 9.6)

0.048

ALT (U/l)

18.7 (13.2, 29.3)

30.9 (21.7, 68.0)

0.014

AST (U/l)

18.7 (14.7, 26.1)

20.8 (15.5, 35.5)

0.307

TG (mmol/l)

1.5 (1.0, 1.9)

1.5 (1.1, 2.0)

0.674

TC (mmol/l)

4.6 (3.6, 5.3)

4.5 (3.7, 5.1)

0.760

HDL (mmol/l)

1.0 (0.9, 1.5)

1.0 (0.9, 1.2)

0.689

LDL (mmol/l)

2.4 (1.8, 3.3)

2.7 (2.1, 3.2)

0.721

PRL (µg/l)

15.8 (12.1, 18.2)

9.6 (6.7, 14.1)

0.003

FFA (mmol/l)

0.11 (0.08, 0.15)

0.11 (0.05, 0.18)

0.669

Adiponectin (µg/ml)

7.7 (4.9, 11.0)

6.9 (5.4, 8.4)

0.687

Leptin (ng/dl)

9.1 (9.0, 9.2)

9.2 (9.0, 9.4)

0.739

N

WHR

26

IL-6 (pg/ml)

1.5 (1.0, 2.4)

1.8 (1.4, 3.0)

0.169

NAS

1.0 (1.0, 3.0)

4.0 (2.0, 5.0)

<0.001

Fibrosis

1.0 (0.0, 1.0)

1.0 (0.0, 1.0)

0.162

BMI: body mass index; WHR: waist-to-hip ratio; SBP: systolic blood pressure; DBP: diastolic blood pressure; FBG: fasting blood glucose; ALT: alanine aminotransferase; AST: aspartate aminotransferase; TG: triglyceride; TC: total cholesterol; HOMA-IR: the homeostatic model assessment of insulin resistance; PRL: prolactin; NAS: NAFLD activity score. Data are shown as median with interquartile range (IQR). P values are based on Mann-Whitney U test for continuous data.

27

Table 3. Characteristics of patients stratified with different severities of NAFLD.

Men

Women

Mild-moderate NAFLD

Severe NAFLD

Mild-moderate NAFLD

Severe NAFLD

13

17

30

14

Age (years)

37.0 (27.5, 44.0)

35 (27.5, 37.5)

0.675

32.5 (27.8, 38.8)

30.0 (27.0, 39.5)

0.472

BMI (kg/m2)

39.2 (35.9, 42.8)

39.5 (35.5, 49.2)

0.902

35.6 (33, 37.8)

38.8 (32.7, 42.6)

0.212

SBP (mmHg)

146 (125, 153.5)

148 (133.5, 150)

0.805

131 (121.3, 138.5)

137.5 (124.5, 158.8)

0.137

DBP (mmHg)

91 (80.5, 96.5)

94 (80.5, 101.5)

0.509

80 (72.8, 86)

90.5 (83.5, 102.3)

0.002

123.0 (112.5, 128)

123.5 (114.5, 145)

0.320

107.0 (104.3, 117.5)

118.0 (103.3, 130.8)

0.157

1.03 (0.99, 1.05)

1.04 (1.01, 1.09)

0.198

0.95 (0.88, 0.99)

0.97 (0.89, 1.03)

0.392

HbA1c (%)

7.3 (6.1, 8.6)

6.6 (5.9, 8.6)

0.550

5.7 (5.5, 6.1)

6.0 (5.5, 7.5)

0.300

FBG (mmol/l)

6.8 (5.4, 9.6)

7.0 (5.5, 10.5)

0.809

5.4 (4.9, 6.1)

6.0 (5, 8.5)

0.166

HOMA-IR

8.8 (5.3, 11.9)

8.0 (6.2, 14.3)

0.660

5.3 (4.3, 6.9)

7.8 (5, 14.7)

0.055

ALT (U/l)

45.5 (28.4, 90.5)

51.7 (40.1, 92.7)

0.187

28.0 (20.6, 57.2)

42.6 (27, 78.1)

0.182

AST (U/l)

24.0 (19.9, 36.5)

29.7 (26.1, 40.4)

0.155

20.0 (15.5, 33.1)

24.3 (15.3, 46.3)

0.262

TG (mmol/l)

2.5 (2.1, 3.6)

2.0 (1.2, 3.5)

0.267

1.3 (1.1, 2.0)

1.7 (1.1, 2.6)

0.222

TC (mmol/l)

4.8 (4.3, 5.6)

4.6 (4.3, 6.4)

0.900

4.5 (3.7, 4.9)

4.4 (3.8, 5.4)

0.632

HDL (mmol/l)

1.0 (0.8, 1.2)

0.9 (0.7, 1)

0.102

1.0 (0.9, 1.3)

1.0 (0.9, 1.1)

0.319

LDL (mmol/l)

2.7 (2.5, 3.2)

2.5 (2, 3.3)

0.660

2.7 (2.3, 3.1)

2.7 (2.1, 3.5)

0.920

PRL (µg/l)

9.8 (8.2, 15.7)

8.5 (4.2, 10.6)

0.027

9.7 (7.1, 12.3)

8.3 (5.4, 9.5)

0.031

NAS

3.0 (2.0, 3.8)

5.0 (4.0, 6.0)

<0.001

3.0 (2.0, 4.0)

5.0 (4.0, 6.0)

<0.001

Fibrosis

1.0 (0.0, 1.5)

1.0 (1.0, 1.0)

0.320

1.0 (0.0, 1.0)

1.0 (0.0, 2.0)

0.202

N

Waist (cm) WHR

P

P

28

BMI: body mass index; WHR: waist-to-hip ratio; SBP: systolic blood pressure; DBP: diastolic blood pressure; FBG: fasting blood glucose; ALT: alanine aminotransferase; AST: aspartate aminotransferase; TG: triglyceride; TC: total cholesterol; HOMA-IR: the homeostatic model assessment of insulin resistance; PRL: prolactin; NAS: NAFLD activity score. Data are shown as median with interquartile range (IQR). P values are based on Mann-Whitney U test for continuous data.

29

Figure legends Fig. 1. Adjusted ORs of NAFLD according to quartiles of serum PRL levels in men and women. The ORs with corresponding 95% CIs were adjusted for age, BMI, HbA1c, HOMA-IR and the presence of diabetes. The quartile ranges of Q1, Q2, Q3, and Q4 of serum PRL level were ≤ 6.3, 6.3–8.4, 8.4–11.5, and >11.5 µg/l in men and ≤ 6.7, 6.7–10.0, 10.0–14.2, and > 14.2 µg/l in women, respectively. Q1 is the reference group. Binary logistic regression study was applied. Fig. 2. Circulating PRL level was evaluated in 85 subjects (55 female and 30 male) diagnosed by liver biopsy. (A) Serum PRL levels in female patients with (n=44) and without (n=11) NAFLD. (B) Serum PRL levels in female patients with mild-moderate NAFLD (n=30) and severe NAFLD (n=14). (C) Serum PRL levels in male patients with mild-moderate NAFLD (n=13) and severe NAFLD (n=17). Data are presented as median with interquartile range. Mann-Whitney analysis was applied for statistical analysis. (D) Hepatic mRNA expression of PRLR in female patients with NAFLD (n=44) and age-, gender-matched non-NALFD patients (n=11). Data are presented as mean ± SEM. Student’s t test was used for statistical analysis.(E) Hepatic protein expression of PRLR in female patients with NAFLD (n=44) and age-, gender-matched non-NALFD patients (n=11). Left panel: H&E staining. Middle panel: Immunohistochemical analysis. Right panel: Immunofluorescence analysis. Scale bar =100 µm. Fig. 3. The mRNA level of CD36 in obese females with and without NAFLD, and correlations between mRNA expressions of PRLR and genes involved in 30

lipid metabolism in female NAFLD patients. (A) Hepatic CD36 mRNA levels in liver tissues of females with (n=44) and without (n=11) NAFLD. Student’s t test was used for statistical analysis. (B) Correlation analysis of mRNA levels between PRLR and CD36 in NAFLD patients (44 females and 30 males). (C-F) Correlation analysis of PRLR mRNA levels and other genes involved in hepatic lipid metabolism, including ACC (C), SREBP1c (D), CPT1a (E), PPARα (F) in NAFLD patients (44 females and 30 males). Hollow points and solid points represent males and females, respectively. Spearman correlation analysis was used (B-F). Fig. 4. PRL alleviated hepatic lipid accumulation via PRLR. (A) Western blot analysis of dose-dependent changes of PRL treatment on PRLR expression in HepG2 cells. The quantification of protein levels is expressed as mean ± SEM (n=3). *P<0.05 compared with first group. # P<0.05 compared with the second group. & P<0.05 compared with the third group. (B) TG content was determined using ORO staining in HepG2 cells after adenovirus mediated PRLR overexpression. Scale bar =100µm. (C) TG content was measured after PRL treatment and PRLR RNAi. One-way ANOVA was applied for statistical analysis. Fig. 5. PRLR and CD36 mediated the effects of PRL on FFA-treated HepG2 cells. (A) Protein levels of pSTAT5, STAT5, and CD36 after PRL intervention. (B) The mRNA levels of CD36 after adenovirus mediated PRLR overexpression. (C) Protein expressions of pSTAT5, STAT5 and CD36 after PRLR overexpression. (D) The mRNA levels of lipid oxidation- and synthesis-related genes after PRLR overexpression in FFA-treated HepG2 cells. (E) Expressions of pSTAT5, STAT5, 31

and CD36 after PRLR silencing. (F) Effect of plasmid mediated CD36 overexpression on TG content. Data are presented as mean ± SEM (n=3), statistical analysis was based on Student’s t test or one-way ANOVA

32

Figure 1

PRL quartiles

OR (95% CI)

β

S.E

p

Men Q2

0.59 (0.29, 1.21)

-0.529 0.369 0.151

Women Q2

0.82 (0.35, 1.88)

-0.203 0.424 0.632

Men Q3

0.54 (0.25, 1.15)

-0.614 0.386 0.112

Women Q3

0.48 (0.21, 1.13)

-0.726 0.432 0.093

Men Q4

0.48 (0.23, 0.99)

-0.739 0.372 0.047

Women Q4

0.36 (0.13, 0.97)

-1.021 0.528 0.044

0

1

2

Figure 2

Female p = 0.003

A 30

C

Female p = 0.027

B 25

Male

p = 0.031

15

10

PRL (ug/l)

PRL (ug/l)

20

15 10

10

5

5 0

0

NonNAFLD

1.5

NAFLD

0

Mild-moderate NAFLD

E

D

PRLR mRNA expression

PRL (ug/l)

20

p = 0.013 Non NAFLD

1.0

0.5

NAFLD 0.0

NonNAFLD NAFLD

Severe NAFLD

H&E staining

Mild-moderate NAFLD

Immunohistochemical staining

Severe NAFLD

Immunofluorescent staining

Figure 3

B

p = 0.033

1.5

1.0

0.0

r = - 0.215 p = 0.066

D

2

4

6

8

r = - 0.041 p = 0.727

4 3

SREBP1c

ACC

0

PRLR

3 2

2 1

1

0 0

2

4

6

8

0

2

E

r = 0.124 p = 0.291

4

F

6

8

r = -0.016 p = 0.889

4 3

CPT1a

3 2

2 1

1 0

4

PRLR

PRLR

PPAR α

r = - 0.247 p = 0.034

2

0

4

0

Male Female

1

0.5

Non- NAFLD NAFLD

C

4 3

CD36

CD3 6 mRNA expression

A

0 0

2

4

PRLR

6

8

0

2

4

PRLR

6

8

Figure 4

PRLR β-actin PRL (ng/ml)

B 0

10

20

3

*# *

200

Con

*#

*

PRLR

2

50 100

FFA+ Ad-GFP

p = 0.012 0.8

p = 0.008

p = 0.043 p = 0.028

0.6 0.4 0.2 0.0

1

0

C Triglycerrides (umol/mg protein)

A

0

10

20 50 100 200

FFA+ Ad-PRLR

FFA PRL Scramble PRLR RNAi -

+

+

+

-

+

-

+

+ -

-

-

+

Figure 5

B 1.5

PRLR

p < 0.001

1.5

p < 0.001

CD36

0.5

β-actin

+

-

PRL FFA

0.0

+ +

0.5

p = 0.012 p = 0.009

1.5 1.0 0.5

0.0

- + + +

-

PRL FFA

p < 0.001

1.0

p = 0.828

2.0

p < 0.001

p < 0.001

PRLR

STAT5

2.5

CD36

1.0

pSTAT5/STAT5

pSTAT5

p = 0.003

CD36 mRNA expression

A

PRL FFA

0.0

- - + - + +

PRL FFA -

+

+ +

Ad-GFP

1.5

Ad-PRLR

p = 0.001 1.0

0.5

0.0

D

C 2.5

CD36 β-actin

1.0 1.5 1.0

0.5

0.5 0.0

+

FFA

Ad-PRLR FFA -

+ +

0.0

Ad-PRLR FFA

+ +

E

-

0.5

0.0

ACC

PRLR

1.0

0.5

0.5 0.0

β-actin -

FFA PRL Scramble PRLR RNAi

p = 0.006

1.5

+ -

+ + + -

+ + + +

-

FFA PRL Scramble PRLR RNAi

2.0

p = 0.001

p < 0.001 p < 0.001 p = 0.01

1.5

CD36

1.0

+ + + -

+ -

p = 0.005

p < 0.001 p = 0.001

+ + +

+ -

+ + + -

+ + +

FFA PRL Scramble PRLR RNAi

0.1 0.0

Scramble

-

+ -

+ +

+

-

+ -

+

+

p = 0.003

0.2

CD36 O.E. -

p < 0.001 p < 0.001

0.3

PRL

1.0 0.5

FFA PRL Scramble PRLR RNAi

CPT1a

0.4

FFA

0.5

0.0

PPARa

p < 0.001

p = 0.008

CD36

0.0

SREBP1c

1.5

STAT5

pSTAT5/STAT5

1.0

p < 0.001 p < 0.001

PRLR pSTAT5

p = 0..055

F

p = 0.001 p < 0.001

2.0

+ +

p = 0..876

p = 0.251 p = 0.090

Triglycerrides (umol/mg protein)

Ad -PRLR

1.5

p = 0.001

Relative mRNA expression

STAT5

p = 0.002

Ad-GFP Ad-PRLR

2.0

CD36

pSTAT5/STAT5

pSTAT5

1.5

-

+ -

+ + + -

+ + +

Graphical abstract

33

Highlights 1. Circulating prolactin levels are negatively association in patients with NAFLD. 2. Prolactin receptor expressions are decreased in livers of obese patients in the presence of NAFLD. 3. Prolactin/prolactin receptor improved the hepatic steatosis via suppression of hepatic CD36.

34