Hyodeoxycholic acid ameliorates nonalcoholic fatty liver disease by inhibiting RAN-mediated PPARα nucleus-cytoplasm shuttling

Nonalcoholic fatty liver disease (NAFLD) is usually characterized with disrupted bile acid (BA) homeostasis. However, the exact role of certain BA in NAFLD is poorly understood. Here we show levels of serum hyodeoxycholic acid (HDCA) decrease in both NAFLD patients and mice, as well as in liver and intestinal contents of NAFLD mice compared to their healthy counterparts. Serum HDCA is also inversely correlated with NAFLD severity. Dietary HDCA supplementation ameliorates diet-induced NAFLD in male wild type mice by activating fatty acid oxidation in hepatic peroxisome proliferator-activated receptor α (PPARα)-dependent way because the anti-NAFLD effect of HDCA is abolished in hepatocyte-specific Pparα knockout mice. Mechanistically, HDCA facilitates nuclear localization of PPARα by directly interacting with RAN protein. This interaction disrupts the formation of RAN/CRM1/PPARα nucleus-cytoplasm shuttling heterotrimer. Our results demonstrate the therapeutic potential of HDCA for NAFLD and provide new insights of BAs on regulating fatty acid metabolism.

Written informed consent was obtained from all participants before recruiment. Samples of NAFLD subjects and clinical data were from a multicenter, randomized, double-blind clinical trial conducted at four centers in Shanghai, China, in patients with imaging confirmed NAFLD with abnormal liver function. The aim of this trial was to evaluate the efficacy of a traditional Chinese medicine prescription in patients with NAFLD. The samples/data of NAFLD patients were before the intervention of the traditional Chinese medicine. This trial was approved by the Institutional Review Board of Shuguang Hospital Affiliated to Shanghai University of Chinese Medicine (Approval No. 2017-548-31) and was conducted in accordance with the Principles of Good Clinical Practice and the Declaration of Helsinki. Subjects were recruited mainly from outpatient clinics. Recruitment advertisements reviewed by the Ethics Committee are placed in places where potential subjects congregate to facilitate recruitment. All clinical examinations in this clinical trial were free of charge with no participant compensation, and written informed consent is obtained from each patient prior to screening and enrollment. Healthy subjects were recruited from the Phase I clinical program of GCP Center or the Physical Examination Center of Shanghai Shuguang Hospital. This study was conducted in accordance with the Declaration of Helsinki and was approved by Institutional Review Board of Shuguang Hospital Affiliated to Shanghai University of Chinese Medicine (No. 2019-662-17-01).
The diagnostic criteria for NAFLD were referred to the Guidelines for the Diagnosis and Treatment of Non-alcoholic Fatty Liver Disease (2010) issued by Fatty liver and Alcoholic Liver Disease Group, Hepatology Society of Chinese Medical Association.The inclusion criteria for NAFLD participants: (1) Meet the above diagnostic criteria for NAFLD; (2) 18-65 years old; (3) CAP score > 300 (ECHOSENS, FibroScan 502); (4) Serum alanine aminotransferase (ALT) >80. Exclusion criteria for NAFLD participants: (1) Excessive alcohol consumption (140 g/week in men or 70 g/week in women); (2) Combined with alcoholic liver disease, viral hepatitis, Wilson's disease, autoimmune liver disease, or other chronic liver disease; (3) Combined with hypothyroidism, inflammatory bowel disease, Cushing's syndrome, lack of beta lipoprotein hematic disease, encephalopathy, type of lipid deposition disease, fatty liver tumor and some associated with insulin resistance syndrome (lipid atrophic diabetes, Mauriac syndrome); (4) Medicine with drugs that can cause fatty liver, such as tamoxifen, ethamiodarone, valproate, glucocorticoids, methotrexate, etc. or total parenteral nutrition; (5) Combined with other serious diseases include renal insufficiency, heart disease, lung disease, malignant tumors of the liver and other systems, mental illness, and other conditions affecting the metabolic state of the whole body, such as pregnancy, breastfeeding, etc; (6) Antibiotics and proton pump inhibitors were used within a month; (7) Medicine with drugs for lowering triglycerides or cholesterol within three months, such as kinds of statin or fibrate;(8) Gastrointestinal surgery was performed in the last year or weight-loss medications taken with more than 10 percent of body weight lost. Healthy controls were defined as individuals with normal routine laboratory tests and no diagnosis of metabolic diseases such as hypertension, diabetes, hyperlipidemia, hyperuricemia, and other serious conditions. Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. All clinical samples that met the inclusion and exclusion criteria were analyzed for this study. We used as many as we could obtain. For animal experiments, we generally set 6 mice per group, which is a very common number of animals used, consistent with previous published papers (PMID: 37443159, PMID: 37142604). In addition, the number of liver knockout animals in each group is 4-5 per group, because the time for revising the article is limited, we only have those number of mice at that time.
No data were excluded from analysis.
Indicated in figure legends.
The mice were randomly divided and allocated into experimental groups.Cells were grown under the same conditions and randomly allocated into different treatment groups without any bias.
The investigators were blinded to the group allocation during data collection.