Role of liver FGF21-KLB signaling in ketogenic diet-induced amelioration of hepatic steatosis

Nutrition & Diabetes ARTICLE www.nature.com/nutd OPEN Role of liver FGF21-KLB signaling in ketogenic diet-induced amelioration of hepatic steatosis Wanrong Guo1,2,3,4,8, Huanyi Cao2,5,8, Yunfeng Shen6, Wuguo Li7, Wei Wang2,6, Lidan Cheng2,6, Mengyin Cai1,2,3 and Fen Xu 1,2,3 ✉ 1234567890();,: © The Author(s) 2024 BACKGROUND: The effectiveness of ketogenic diet (KD) in ameliorating fatty liver has been established, although its mechanism is under investigation. Fibroblast growth factor 21 (FGF21) positively regulates obesity-associated metabolic disorders and is elevated by KD. FGF21 conventionally initiates its intracellular signaling via receptor β-klotho (KLB). However, the mechanistic role of FGF21KLB signaling for KD-ameliorated fatty liver remains unknown. This study aimed to delineate the critical role of FGF21 signaling in the ameliorative effects of KD on hepatic steatosis. METHODS: Eight-week-old C57BL/6 J mice were fed a chow diet (CD), a high-fat diet (HFD), or a KD for 16 weeks. Adeno-associated virus-mediated liver-specific KLB knockdown mice and control mice were fed a KD for 16 weeks. Phenotypic assessments were conducted during and after the intervention. We investigated the mechanism underlying KD-alleviated hepatic steatosis using multi-omics and validated the expression of key genes. RESULTS: KD improved hepatic steatosis by upregulating fatty acid oxidation and downregulating lipogenesis. Transcriptional analysis revealed that KD dramatically activated FGF21 pathway, including KLB and fibroblast growth factor receptor 1 (FGFR1). Impairing liver FGF21 signaling via KLB knockdown diminished the beneficial effects of KD on ameliorating fatty liver, insulin resistance, and regulating lipid metabolism. CONCLUSION: KD demonstrates beneficial effects on diet-induced metabolic disorders, particularly on hepatic steatosis. Liver FGF21-KLB signaling plays a critical role in the KD-induced amelioration of hepatic steatosis. Nutrition and Diabetes (2024)14:18 ; https://doi.org/10.1038/s41387-024-00277-3 INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) stands as one of the most common chronic liver diseases globally, with an estimated prevalence of 32.4% [1]. Lifestyle interventions, including dietary adjustments and physical activity, are the main recommendations for managing NAFLD. Within this context, caloric intake, dietary composition, and meal timing have emerged as focal points in NAFLD treatment [2]. The ketogenic diet (KD), initially developed for epilepsy treatment, is characterized by a composition with over 90% of calories from fat and less than 1% of calories from carbohydrates [3]. Clinical studies have demonstrated that KD induced a remarkable reduction in body weight and the amelioration of fatty liver, accompanied by decreased alanine aminotransferase and aspartate aminotransferase levels [4–6]. Rodent models fed a KD have showcased weight loss, improved insulin sensitivity, lipid metabolism, and glucose metabolism [7–10]. In hepatology studies, KD has been noted to enhance hepatic energy expenditure, promote fatty acid oxidation, and suppress lipogenesis compared with high-fat diets (HFD) or western diets [7, 8]. Mechanistically, KD activates AMP-activated protein kinase and inhibits acetyl-CoA carboxylase (ACC) activity in the liver and muscle [7]. Moreover, our previous study has elucidated that KD reduces malonylation of acetyl-CoA carboxylase 1 (ACC1), critical for activity and stability of ACC1, thereby alleviating fatty liver [11]. Besides the liver, KD activates brown adipose tissue (BAT) function by upregulating heat-related gene expression [12] and mitigates inflammation in white adipose tissues (WATs) [13]. Fibroblast growth factor 21 (FGF21), a crucial metabolic regulator primarily synthesized in the liver, modulates the metabolism of multiple tissues [14]. Elevating serum FGF21 levels through exogenous administration or liver overexpression induces weight loss, enhances energy expenditure, improves glucose metabolism and insulin sensitivity, alleviates hepatic steatosis, promotes lipolysis, induces browning of WAT, and activates BAT function [15–21]. A phase 2a clinical trial has further validated the beneficial effects of FGF21 on steatohepatitis [22]. FGF21 is upregulated by KD [23, 24]. FGF21-deficient mice fed a KD gained weight and developed hepatic steatosis, whereas control mice exhibited weight loss and reduced fatty liver [25]. 1 Department of Endocrinology and Metabolism, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. 2Guangdong Provincial Key Laboratory of Diabetology, Guangzhou, China. 3Guangzhou Municipal Key Laboratory of Mechanistic and Translational Obesity Research, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. 4Medical Intensive Care Unit, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. 5Department of Endocrinology, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou, China. 6Department of Endocrinology and Metabolism, The Second Affiliated Hospital of Nanchang University, Nanchang, China. 7Animal Experiment Center, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. 8These authors contributed equally: Wanrong Guo, Huanyi Cao. ✉email: Received: 21 October 2023 Revised: 1 April 2024 Accepted: 4 April 2024 W. Guo et al. 2 Liver-specific FGF21-knockdown mice fed a KD also displayed fatty liver compared with control mice [23]. FGF21 signals target cells through a receptor complex composed of FGF receptor (FGFR), FGFR1c, and a co-receptor called β-klotho (KLB). Elimination of either FGFR1 or KLB impairs the acute insulin-sensitizing effects of FGF21 [26, 27]. Thus, we hypothesized that KD might ameliorate hepatic steatosis via hepatic FGF21-KLB signaling. In this study, we aimed to delineate the critical role of FGF21 signaling in the ameliorative effects of KD on hepatic steatosis in order to offer new insights into the potential of KD in the treatment of NAFLD. MATERIALS AND METHODS Animal experiments Seven-week-old male C57BL/6 J mice were purchased from GemPharmatech (Nanjing, China). Mice were housed in a standard specific pathogenfree facility with free access to food and water. After a 1-week acclimatization period, eight-week-old mice were used in two experiments. In the first experiment, mice were divided randomly into three groups (n = 5 per group), which were exposed to a chow diet (CD, 11% fat [kcal%]; Guangdong Medical Laboratory Animal Center, Guangzhou, China), an HFD (58% fat [kcal%]; D12331; Research Diets, New Brunswick, NJ, USA), or an HFD and a ketogenic diet (KD, 90.5% fat [kcal%], TD.160153, Envigo, USA) alternating every 2 weeks for a total period of 16 weeks. In the second experiment, adeno-associated virus (AAV) expressing short-hairpin RNAs targeting β-klotho (shKlb) and negative control (shCtrl) were administered to (...truncated)