Hepatic fibroblast growth factor 21 is required for curcumin or resveratrol in exerting their metabolic beneficial effect in male mice

Hepatic fibroblast growth factor 21 is required for curcumin or resveratrol in exerting their metabolic beneficial effect in male mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hepatic fibroblast growth factor 21 is required for curcumin or resveratrol in exerting their metabolic beneficial effect in male mice Tianru Jin, Jia Nuo Feng, Weijuan Shao, Lin Yang, Juan Pang, Wenhua Ling, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4432933/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Feb, 2025 Read the published version in Nutrition & Diabetes → Version 1 posted 10 You are reading this latest preprint version Abstract Conclusion and significance: We conclude that hepatic FGF21 is required for curcumin or resveratrol in exerting their major metabolic beneficial effect. The recognition that FGF21 as the common target of dietary interventions brings us a novel angle in understanding metabolic disease treatment and prevention. It remains to be explored how various dietary interventions regulate FGF21 expression and function, via certain common or unique gut-liver or gut-brain-liver axis. Background: Our mechanistic understanding on metabolic beneficial effects of dietary polyphenols has been hampered for decades due to the lack of functional receptors for those compounds and their extremely low plasma concentrations. Recent studies by our team and others have suggested that those dietary polyphenols may target gut microbiome and gut-liver axis and that hepatic fibroblast factor 21 (FGF21) serves as a common target for various dietary interventions. Methods: Utilizing liver-specific FGF21 null mice ( lFgf21 -/- ), we are asking a straightforward question: Is hepatic FGF21 required for curcumin or resveratrol, two typical dietary polyphenols, in exerting their metabolic beneficial effect in obesogenic diet-induced obese mouse models. Results: On low-fat diet feeding, no appreciable defect on glucose disposal was observed in male or female lFgf21 -/- mice, while fat tolerance was impaired in male but not in female lFgf21 -/- mice, associated with elevated serum triglyceride (TG) level, reduced hepatic expression of the Ehhadh and Ppargc1a , which encodes the two downstream effectors of FGF21. On high-fat-high-fructose (HFHF) diet challenge, Fgf21 fl/fl but not lFgf21 -/- mice exhibited response to curcumin intervention on reducing serum TG, and on improving fat tolerance. Resveratrol intervention also affected FGF21 expression or its downstream effectors. Metabolic beneficial effects of resveratrol intervention observed in HFHF diet-challenged Fgf21 fl/fl mice were either absent or attenuated in lFgf21 -/- mice. Dietary polyphenol intervention FGF21 FGFR1 KLB Resveratrol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Except for prescribed drugs and physical exercise, various dietary interventions were also shown to improve metabolic homeostasis (1, 2). One type of dietary intervention is the change of dietary behaviors, such as nutritional restriction and intermittent fasting (3), while another type is the addition of chemical compounds from edible plants into the diet (4). One category of those compounds is dietary polyphenols, with the curry compound curcumin and resveratrol, mostly found in red grapes, as two typical examples (1, 5). Interestingly, observations made by our team and others have shown that both dietary polyphenol intervention and amino acid restriction target the hepatic hormone fibroblast growth factor 21 (FGF21) (3, 6-8). Members of the FGF family interact with FGF receptors (FGFRs) including FGFR1, leading to complicated downstream signaling events. Due to the lack of a heparin binding domain, FGF21, FGF19 (FGF15 in rodents) and FGF23 can be released freely into the bloodstream, serving as endocrine hormones (9). In addition to FGFRs, the obligatory co-receptor β-klotho (KLB) is also required for FGF21 to exert its metabolic function (9). It is generally accepted that plasma FGF21 is liver driven, although FGF21/Fgf21 mRNA expression can be detected in adipose tissues, pancreas, and elsewhere (9-11). Various FGF21 analogues have been tested in clinical trials for treating metabolic disorders including diabetes and fatty liver disorders (9). The most promising effects of those “pre-drugs” are the attenuation of hyperlipidemia and the improvement of insulin sensitivity. We and others have also reported that hepatic FGF21 expression can be regulated by glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1RAs), including both exenatide and liraglutide (12-15). Utilizing the liver specific Fgf21 knockout mouse model, we have demonstrated recently that hepatic FGF21 is required for liraglutide in improving energy homeostasis in male mice with obesogenic diet challenge, making hepatic FGF21 as a “central molecule” for both GLP-1R-based drugs and diet interventions (12). Liraglutide treatment or curcumin intervention is also shown to attenuate high-fat-diet (HFD) induced repression on Fgfr1 , which encodes the FGF21 receptor FGFR1, and Klb , which encodes the obligatory co-receptor KLB (6, 12). These regulatory events are commonly interpreted as the improvement of FGF21 sensitivity (6, 12). Utilizing the similar hepatic FGF21 deficient mice, here we have asked a straightforward question: Is FGF21 also required for curcumin or resveratrol, two typical dietary polyphenols, in exerting their metabolic beneficial effects? Materials And Methods The source of dietary polyphenols The curry compound curcumin was purchased from Organika Health Products (Richmond, BC, Canada; a 95% standardize curcumin extract). Resveratrol was purchased from Combi-Blocks (Catalog #: OR-1053, San Diego, CA, USA). Methods for curcumin and resveratrol intervention have been described in our previous studies (5, 6, 16-19). Contents of experimental diets utilized in this study are shown in Supporting Table 1. Animals and Animal Experimental Design C57BL/6J mice, liver specific Fgf21 knockout ( lFgf21 -/- ) mice and wild type littermates ( Fgf21 fl/fl ) were utilized in this study. lFgf21 -/- mice were generated by mating Fgf21 loxP (Strain #:022361, Jackson lab) with Alb-Cre mice (Strain #:003574, Jackson lab) as we have reported previously (12) (Figure S1A). Mice were maintained at ambient room temperature and relative humidity of 50%, with free access to food and water under a 12 h light:12 h darkness cycle (n=4-5 per cage). The animal experiments and protocol were approved by the University Health Network Animal Care Committee and were performed in accordance with the guidelines of the Canadian Council of Animal Care. For curcumin intervention, curcumin was added to the high fat high fructose diet (60% HFD with 20% fructose) in a dosage of 4g/kg, as we have reported (6). For resveratrol intervention, 0.5% of resveratrol was added to the indicated diet (5). Mice were randomly assigned to either receive or not receive dietary intervention. Based on our animal protocol, mouse with serious body weight loss or shown “sickness” symptoms will be excluded from the study. For the current study, no mice or data points were excluded. The generation of Fgf21 fl/fl , lFgf21 -/- mice were verified by genotyping. The male and female Fgf21 fl/fl and lFgf21 -/- littermates were fed with chow (low fat diet, LFD) diet, metabolic tolerance tests were conducted at the week of 8 th , 10 th , 12 nd and 15 th for glucose tolerance (GTTs), pyruvate tolerance tests (PTTs), insulin tolerance test (ITT) and fat tolerance test (FTT), respectively. Prior to fat tolerance test (FTT), the blood triglyceride levels at random or fast state were assessed at the week of 14 th . Metabolic tolerance tests and triglyceride (TG) measurement Methods for GTT, PTT and ITT have been previously presented (20). For intraperitoneal GTTs and PTTs, both male and female mice were fasted for 16 hrs prior to the intraperitoneal injection of glucose (2g/kg body weight) or pyruvate (2g/kg body weight). For ITTs, male and female mice were fasted for 4 hours prior to the injection of insulin (0.5 U/kg body weight). Blood glucose levels were determined at 0, 15, 30, 60, 90, and 120 min. We adopted the method by Gniuli et al for conducting FTT (21). Briefly, mice were fasted overnight prior to oral gavage of 1% olive oil of body weight. Blood was collected from tail vein at 0, 1, 2 and 4 hours for TG measurement. To determine TG produced by the liver (lipid tolerance test), mice fasted overnight were injected intraperitoneally with poloxamer 407 (Sigma) to block lipolysis, and blood was collected from tail vein at indicated hours to measure TG levels (5). qRT-PCR and Western blotting Methods for qRT-PCR and Western blotting have been described previously (5, 20), with oligo nucleotide primers and antibodies listed in Supporting Table 2 and Table 3. RNAseq sample preparation and data analysis Mice were subjected to a 16-week normal chow diet (LFD), HFHF diet and HFHF diet with resveratrol (HFR). By the end of the experiment, mice liver tissues were collected for RNA isolation. Total RNA was harvested using RNeasy Mini kit (QIAGEN) and further quantified and analyzed using Nanodrop spectrophotometer and Bioanalyzer. One microgram of total RNA was utilized and sent to Center of Applied Genomics (Sickkids Hospital, Canada) for sequencing library construction, as we described previously (12). Data processing and analyzing were conducted through a standardized pipeline called RNA-seq IMmune Analysis Pipeline, per literature description (22). Briefly, unprocessed FASTQ files containing raw data were downloaded and transferred. The RNA-seq reads were aligned against the mm10 reference genome assembly (Genome Reference Consortium Mouse Build 38) obtained from the NCI Genome Data Commons (GDC) using STAR (version 2.4.2a). Quality control was performed on aligned BAM files using RSeQC and then expression levels were quantified using SALMON (V.0.14.0). After converting Ensemble IDs to mouse gene symbols (GRCm38.p6), the reads per gene were normalized and differentially expressed genes were analyzed with DESeq2 (V1.22.2). Raw data and processed data have been submitted to the Gene Expression Omnibus (GEO) database (GSE241713). Statistical analyses Results are expressed as mean ± SEM. Student’s t test or one-way ANOVA followed by Sidak post-hoc correction were applied for calculating the statistical significance. In all cases, P < 0.05 is considered statistically significant. Statistical analyses were performed with GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Results Male but not female lFgf21 -/- mice on regular chow diet (defined as low fat diet, LFD) feeding show impaired fat tolerance We have generated lFgf21 -/- mice and the littermate controls ( Fgf21 fl/fl ) for current study by mating Alb-Cre with Fgf21 fl/fl , as illustrated in Fig. S1A. lFgf21 -/- mouse liver showed barely detectable FGF21 (Fig. S1B-D), while FGF21 expression in both brown adipose tissue and hypothalamus were virtually unaffected (Fig. S1E-F). Six-week-old male and female lFgf21 -/- mice and the control Fgf21 fl/fl mice were fed with regular chow diet (low fat diet, LFD) for 12 weeks, while three metabolic tolerance tests (glucose tolerance test, GTT; insulin tolerance test, ITT; and pyruvate tolerance test, PTT) were conducted at indicated time for all mice, as indicated in Fig. 1A. Apparently, on LFD feeding, male or female lFgf21 -/- mice exhibited comparable glucose, pyruvate, and insulin tolerance when compared with sex-matched control littermates Fgf21 fl/fl mice (Fig. 1B-G). There were no appreciable differences on body weight with hepatic FGF21 knockout in both male and female mice (Fig. 1H-I). The above mice were then fed with LFD for an additional 3-week period (Fig. 1A), followed by collecting random blood samples and conducting fat tolerance tests (FTT). Mice were then fasted overnight before they were sacrificed for tissue sample collections. As shown, male lFgf21 -/- mice exhibited elevated random and fasting plasma TG levels, as well as impaired fat tolerance when compared with correspondent littermate controls (Fig. 2A-B). Such defects were not observed in female lFgf21 -/- mice (Fig. 2C-D). We then assessed the expression of a battery of hepatic genes that are related to the FGF21 cellular signaling. Expression levels of genes that encode FGFR1 ( Fgfr1 ) and KLB ( Klb ), as well as the two FGF21 downstream mediators ( Ehhadh, which encodes enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase; and Ppargc1a, which encodes PPARG coactivator 1 alpha) were reduced in male lFgf21 -/- mice (Fig. 2E). In female lFgf21 -/- mice, only Ppargc1a level was reduced (Fig. 2F). We have also assessed expression of liver Fgf1 and gut (ileum) Fgf15 , asking whether hepatic FGF21 deficiency results in elevated Fgf1 or Fgf15 expression for compensation. No such compensatory elevation was observed in lFgf21 -/- mice (Fig. 2G-2H). Together, we conclude that male but not female lFgf21 -/- mice showed impaired lipid homeostasis in the absence of obesogenic dietary challenge, associated with reduced hepatic expression of Fgfr1 , Klb , and Ehhadh . Our following investigations were then performed on male mice only. Dietary curcumin intervention improves lipid homeostasis in the control Fgf21 fl/fl mice but not in lFgf21 -/- mice with the obesogenic dietary challenge We have reported previously that in HFD-challenged male mice, curcumin or anthocyanin intervention regulated hepatic FGF21 production and improved FGF21 sensitivity in hepatocytes (6, 23). Curcumin intervention stimulated hepatic FGF21 expression in mice on LFD feeding and attenuated HFD-induced hepatic FGF21 over-expression and FGF21 resistance (6). Here we aimed to determine whether hepatic FGF21 is required for curcumin in exerting its major metabolic beneficial effect, especially lipid homeostatic effect in mice on obesogenic dietary challenge. As shown, male lFgf21 -/- mice or the control littermate Fgf21 fl/fl mice were fed with high fat high fructose (HFHF) diet without or with curcumin intervention for 15 weeks (Fig. 3A). In the control littermates, curcumin intervention moderately attenuated HFHF-diet induced body weight gain (Fig. 3B-C), reduced fasting serum and hepatic TG contents (Fig. 3D-E), and improved lipid tolerance (Fig. 3F). Curcumin dietary intervention also reduced hepatic expression of ChREBP, as well as expression of genes that encode ChREBP ( Mlxipl ) and fatty acid synthase ( Fasn ) (Fig. S2A-B). In lFgf21 -/- mice, none of the above regulatory effects of dietary curcumin intervention were observable (Fig. 3G-K and Fig. S2C-D). We hence conclude that liver FGF21 is required for curcumin intervention in exerting its metabolic beneficial effect, especially the improvement of lipid homeostasis. In obesogenic diet challenged male mice, dietary resveratrol intervention also regulates hepatic FGF21 In 2014, Li and colleagues have reported that resveratrol treatment increased the transcriptional activity of the FGF21 gene promoter (7). We hence ask whether in vivo resveratrol intervention in wild type mice with an obesogenic dietary challenge affects hepatic FGF21 expression, FGF21 sensitivity, or FGF21 mediated cellular signaling events. Here we conducted such assessments in two different sets of mice. In the first set, wild type C57BL/6J mice were fed with LFD, HFD, or HFD with resveratrol (HFD+Res) intervention for 8 weeks (Fig. 4A). In such experimental settings, hepatic Fgf21 expression was reduced by HFD feeding, and the reduction was attenuated by resveratrol intervention (Fig. 4B). Hepatic FGF21 protein expression was not significantly affected by HFD while concomitant resveratrol intervention exhibited a stimulatory effect on hepatic FGF21 expression (Fig. 4C). Importantly, resveratrol intervention increased expression of Fgfr1 , which was reduced by HFD challenge (Fig. 4D). Klb level was not significantly affected by 8-week HFD challenge, while resveratrol intervention exhibited a stimulatory effect on hepatic Klb expression (Fig. 4E). Among the four downstream effectors of FGF21 we have assessed, expression of Ehhadh was inhibited by HFD and the inhibition was effectively reversed by concomitant resveratrol intervention. Acox1 (encodes Acyl-CoA Oxidase 1) expression was not affected by HFD challenge while resveratrol intervention elevated its expression level. HFD challenge significantly reduced expression level of Ppargc1a , while resveratrol intervention generated no appreciable reversing effect in the current experimental settings. Finally, hepatic Pdk4 (which encodes pyruvate dehydrogenase lipoamide kinase isozyme 4) level was not affected by HFD challenge or resveratrol intervention in our current experimental settings (Fig. 4F). The above observations suggest that like curcumin intervention previously reported by our team and by others (6, 24, 25), resveratrol intervention can also target hepatic FGF21 or its downstream signaling events. Fructose consumption is known to stimulate hepatic FGF21 expression, associated with the development of insulin resistance (26). In the second set of mice, we challenged Fgf21 fl/fl mice with HFHF-diet without and with resveratrol intervention for an extended period, as indicated in Fig. 5A. As shown, 16-week HFHF-diet challenge significantly increased hepatic Fgf21 levels, while dietary resveratrol intervention attenuated the elevation effectively (Fig. 5B). At FGF21 protein level, elevation was observed in mice with HFHF challenge, without or with 16-week resveratrol intervention (Fig. 5C). We then collected liver tissues from those mice for RNAseq analysis. As we have anticipated (Fig. 5D, Fig. S3, and Table S4), HFHF-diet challenge increased hepatic expression of Fgf21 but reduced the expression of Klb , while those effects were reciprocally reversed by 16-week dietary resveratrol intervention. The attenuation effect of HFHF-diet and reversible effect of resveratrol intervention was also observed on certain downstream effectors of FGF21 signaling, which were then further verified by our qRT-PCR experiment (Fig. 5E-G). As shown in Fig. S3, HFHF diet feeding most significantly repressed expression of genes including Eif4ebp3 (which encodes eukaryotic translation initiation factor 4E binding protein 3) and Zbtb16 (which encodes zinc finger and BTB domain-containing protein 16), and those genes were also significantly restored by resveratrol intervention. Exact metabolic functions of these two genes remain to be further explored. Additional information on the effect of HFHF diet challenge and resveratrol intervention on hepatic gene expression are presented in Fig. S4 and S5. Dietary resveratrol intervention improves glucose tolerance and reduces serum and hepatic TG levels in HFHF challenged Fgf21 fl/fl mice but not in HFHF challenged lFgf21 -/- mice We then directly compared the effect of dietary resveratrol intervention in Fgf21 fl/fl mice and lFgf21 -/- mice with HFHF-diet challenge (Fig. 6A). As shown, glucose tolerance was improved by dietary resveratrol intervention in the control littermate Fgf21 fl/fl mice but not in lFgf21 -/- mice (Fig. 6B). Dietary resveratrol intervention also attenuated HFHF-diet induced fasting hyperglycemia and hyperinsulinemia in Fgf21 fl/fl mice but not in lFgf21 -/- mice (Fig. 6C-D). Interestingly, in both Fgf21 fl/fl and lFgf21 -/- mice, 16-week dietary resveratrol intervention attenuated HFHF-diet induced body weight gain (Fig. 6E-F) and fat accumulation (Fig. S6), although the degree of the attenuation in Fgf21 fl/fl mice appeared much stronger than that in lFgf21 -/- mice (Figure 6E-F and Fig. S6). Nevertheless, in Fgf21 fl/fl mice but not in lFgf21 -/- mice, dietary resveratrol intervention attenuated HFHF diet induced elevation on serum as well as hepatic TG levels (Figure 6G-H). Thus, although hepatic FGF21 is required for resveratrol intervention in exerting its metabolic beneficial effect on improving energy homeostasis, the body weight lowering effect of dietary resveratrol intervention does not completely rely on hepatic FGF21. Whether this involves FGF21 expressed in adipose tissue, or the brain deserves further investigations. Discussion Although native FGF21 and a few FGF21 analogues were shown to bring metabolic and other beneficial effect in various diseases models (9, 27, 28), obese human subjects and animal models, either due to a defined genetic defect or generated by obesogenic diet challenge, show elevated hepatic and plasma FGF21 levels, suggesting that obesity represents an FGF21 resistance state (9, 29, 30). We and others have reported that dietary intervention with curcumin or anthocyanin, or other dietary polyphenols, regulate plasma and hepatic FGF21 levels, as well as FGF21 sensitivity (6, 18, 23, 24). Other edible plant compounds, such betaine, were also shown to regulate hepatic FGF21 (31). Importantly, in mice on LFD feeding, dietary curcumin or anthocyanin intervention stimulated hepatic FGF21 level; while in mice on HFD, the intervention attenuates HFD-induced FGF21 over-expression, associated with the reversion on HFD-induced repression on Fgfr1 or Klb expression (6, 23, 25). These regulatory events are commonly interpreted as the improvement of FGF21 sensitivity (6, 32). We show here that on LFD feeding, male but not female lFgf21 -/- mice exhibited impaired fat tolerance, associated with reduced expression of genes that encode FGF21 receptor and the co-receptor ( Fgfr1 and Klb ), as well as Ehhadh and Ppargc1a . Previously, we and others have also shown that in female mice, the female hormone estradiol (E2) increased FGF21 production (29, 33). How can female hormones including E2 compensate the lack of hepatic FGF21 on lipid homeostasis in the absence of obesogenic dietary challenge remains to be further investigated. In the absence of an obesogenic dietary challenge, extra-hepatic FGF21, including those generated in adipose tissues, brain, and elsewhere, might be sufficient for female mice in maintaining metabolic homeostasis. Nevertheless, we presented here our comprehensive observations in HFD challenged mice on hepatic FGF21 regulation with resveratrol intervention and then further expanded our investigation with HFHF diet induced obese and insulin resistance mouse model, show that resveratrol intervention reversed the repression of HFHF-diet on expression of Klb , as well as genes that encode FGF21 effectors. Importantly, we demonstrated here that hepatic FGF21 is required for either curcumin or resveratrol intervention in exerting their metabolic beneficial effect in the obesogenic diet challenged mouse models, although the body weight lowering effect of resveratrol or curcumin intervention may only partially rely on hepatic FGF21. In a previous investigation, we noticed that hepatic FGF21 is not absolutely required for the GLP-1RA liraglutide in lowering the body weight in mice with HFD challenge (12). How can hepatic FGF21 mediate metabolic beneficial effect of both nutrient restriction (3, 34) and dietary polyphenol interventions (6, 23, 25)? Why hepatic FGF21 is required for both the GLP-1R agonists (including liraglutide, semaglutide and exenatide) and various dietary interventions (8)? The diabetes drug metformin, which is also a chemical isolated from plant, was shown to induce GLP-1 secretion, and such function contributes to the actions of metformin in the treatment of type 2 diabetes (35). As metformin has been shown to exert its function via “reshaping” gut microbiome, it remains to be determined whether gut microbiome is involved in regulating gut GLP-1 production or secretion (36). A recent study by Martin and colleagues showed that in the absence of gut microbiome, FGF21 adaptive pathway is desensitized in response to dietary protein restriction (37). We are aware of the fundamental role of gut microbiome in health and diseases for decades, and a few recent studies have demonstrated that beneficial effects of resveratrol intervention are strongly associated with alterations in gut microbiome (38-40). Targeting gut microbiome or intestinal signaling cascades, including the gut hormone GLP-1, the intestinal Takeda G protein-coupled receptor 5 (TGR5) or Farnesoid X receptor (FXR) medicated bile acid signaling cascades, are also shared with the diabetes drug metformin, other phytomedicine including red ginseng extracts, and theabrownin isolated from Pu-erh tea, and blueberry and cranberry anthocyanin extracts (36, 41-44). Prior to reporting our current investigation, we have reported very recently that resveratrol intervention target gut microbiome, leading to the attenuation of gut bile acid/FXR signaling and chylomicron secretion, and improved lipid homeostasis (5). It is plausible to suggest that gut microbiome mediates functions of the two categories of dietary interventions (Fig. 6I), with the participation of gut metabolites and gut produced hormones, including GLP-1 and gastric inhibitory polypeptide (GIP), as well as gut/brain axis, gut/liver axis, or other axis that links gut and other peripheral organs, as we have reviewed recently (8). Hepatic FGF21 is required for curcumin or resveratrol in exerting their major metabolic beneficial effect. The existence of common targets, such as FGF21, for GLP-1RAs and various types of dietary interventions, makes us to recognize the link between these two categories of “medicines”, between these two lines of biomedical research, brings us a novel angle in understanding and further investigation of metabolic disease treatment and prevention, with prescribed drugs and various phytomedicine. Abbreviations Acox1, Acyl-CoA Oxidase 1; Alb-Cre, albumin-Cre; Ehhadh, enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase; ChREBP, Carbohydrate response element binding protein; Fasn, fatty acid synthase; FGF15, fibroblast growth factor 15; FGF19, fibroblast growth factor 19; FGF21, fibroblast growth factor 21; FGF23, fibroblast growth factor 23; FGFR1, FGF receptor 1; FGFRs, FGF receptors; FTT, fat tolerance test; FXR, Farnesoid X receptor; GEO, Gene Expression Omnibus; GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1 receptor; GLP-1RAs, GLP-1 receptor agonists; GTT, glucose tolerance test; HFD, high fat diet; HFHF, high fat high fructose diet; ITT, insulin tolerance test; KLB, β-klotho; LFD, low fat diet; Pdk4, pyruvate dehydrogenase lipoamide kinase isozyme 4; Ppargc1α, PPARG coactivator 1 alpha; PTT, pyruvate tolerance test; SEM, the standard error of the mean; TG, triglyceride; TGR5, Takeda G protein-coupled receptor 5. Declarations Funding This study was supported by the Canadian Institute of Health Research (PJT159735 to T.J.). JNF is the recipient of the Banting and Best Diabetes Centre- Novo Nordisk Studentship and Ontario Graduate Scholarship. The guarantor of the manuscript is Tianru Jin. CRediT authorship contribution statement Jia Nuo Feng: Conceptualization, Methodology, Investigation, Visualization, Formal analysis, Writing – original draft, Writing – review & editing. Weijuan Shao: Conceptualization, Methodology, Investigation, Visualization, Writing – original draft, Writing – review & editing. Lin Yang: Data curation, Formal Analysis, Methodologies, Software. Juan Pang: Investigation, Formal analysis, Visualization. Wenhua Ling: Writing – review & editing. Dinghui Liu: Investigation, Formal analysis, Visualization. Michael B Wheeler: Writing – review & editing. Housheng Hansen He: Formal Analysis, Methodologies, Software, Writing – review & editing. 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Additional Declarations There is NO conflict of interest to disclose Supplementary Files Supplementaryfigures0524.pdf supportingtablesV1.docx Cite Share Download PDF Status: Published Journal Publication published 10 Feb, 2025 Read the published version in Nutrition & Diabetes → Version 1 posted Editorial decision: revise 18 Sep, 2024 Review # 3 received at journal 12 Sep, 2024 Reviewer # 3 agreed at journal 18 Aug, 2024 Review # 2 received at journal 10 Jun, 2024 Reviewer # 2 agreed at journal 28 May, 2024 Reviewer # 1 agreed at journal 28 May, 2024 Reviewers invited by journal 28 May, 2024 Submission checks completed at journal 17 May, 2024 First submitted to journal 16 May, 2024 Editor assigned by journal 16 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4432933","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":307850834,"identity":"7e200322-def7-435c-8f79-368c8f35219e","order_by":0,"name":"Tianru Jin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYDACZijFxsB8gDgdPAgtbAkMYE1shLQgMQ2I02LPzvzscWUbAzufdM836Q8V9+TM5ZsPMPyowecwNnPDs21Ah8mc3SZx4EyxsWUbWwJjzzG8fjGTbARpkcjdJnGwLSFxwzEeA2Z8ruNhZv8G1ZLzDEnLP3xaeGC25LAhtDC24dFymKdMsuGcBFBLmrHFmTMJxgbH0hIO9vbh1sLef3ybZEOZTbL8jOSHNyoqEuQMDh8++ODHN9xaoEAiGYV7gKAGILAjRtEoGAWjYBSMUAAAEJ9DRisZxVcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0307-7391","institution":"University of Toronto","correspondingAuthor":true,"prefix":"","firstName":"Tianru","middleName":"","lastName":"Jin","suffix":""},{"id":307850835,"identity":"63dcc7ef-ea4e-4689-8b0e-4dd882ad4606","order_by":1,"name":"Jia Nuo Feng","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"Nuo","lastName":"Feng","suffix":""},{"id":307850836,"identity":"41132fd7-e9e5-423d-a540-53a841873c55","order_by":2,"name":"Weijuan Shao","email":"","orcid":"","institution":"University Health Network","correspondingAuthor":false,"prefix":"","firstName":"Weijuan","middleName":"","lastName":"Shao","suffix":""},{"id":307850837,"identity":"bf9c6030-f511-47ea-842a-3ee49ac9d558","order_by":3,"name":"Lin Yang","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Yang","suffix":""},{"id":307850838,"identity":"87f2b753-5702-4e90-b55e-5302df812941","order_by":4,"name":"Juan Pang","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Pang","suffix":""},{"id":307850839,"identity":"54824f60-7749-4fea-a37a-18a4ec3035d1","order_by":5,"name":"Wenhua Ling","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wenhua","middleName":"","lastName":"Ling","suffix":""},{"id":307850840,"identity":"5b5368bc-007b-44fb-b2b6-c07a6d3cc3c1","order_by":6,"name":"Dinghui Liu","email":"","orcid":"","institution":"The Third Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Dinghui","middleName":"","lastName":"Liu","suffix":""},{"id":307850841,"identity":"d5b45cae-9c2a-4a5a-a2a0-2efa0662111f","order_by":7,"name":"Michael Wheeler","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Wheeler","suffix":""},{"id":307850842,"identity":"a77caac6-6248-4fdf-93f9-0ce52e89b8d5","order_by":8,"name":"Housheng He","email":"","orcid":"https://orcid.org/0000-0003-2898-3363","institution":"Princess Margaret Cancer Centre","correspondingAuthor":false,"prefix":"","firstName":"Housheng","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2024-05-16 19:26:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4432933/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4432933/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41387-025-00363-0","type":"published","date":"2025-02-10T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58100521,"identity":"90195006-2213-433c-ba87-14fff66e582d","added_by":"auto","created_at":"2024-06-11 06:30:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":210249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNo appreciable defect on glucose disposal in male or female \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elFgf21\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice when fed with regular chow LFD. (A) \u003c/strong\u003eIllustration of the animal experimental timeline. \u003cstrong\u003e(B-D) \u003c/strong\u003eGlucose level during tolerance test in adult male mice, Glucose tolerance test (GTT) at the age of 8 weeks (n=6 for both \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e)\u003cstrong\u003e (B). \u0026nbsp;\u003c/strong\u003ePyruvate tolerance test (PTT) at the age of 10 weeks (n=4)\u003cstrong\u003e (C). \u003c/strong\u003eInsulin tolerance test, ITT at the age of 12 weeks (n=4)\u003cstrong\u003e (D). (E-G) \u003c/strong\u003eGlucose level during GTT in adult female mice, GTT at the age of 8 weeks (n=6 for \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e, n=5 for \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e)\u003cstrong\u003e (E). \u003c/strong\u003ePTT at the age of 10 weeks (n=3)\u003cstrong\u003e (F). \u003c/strong\u003eITT at the age of 12 weeks (n=3) \u003cstrong\u003e(G). (H-I) \u003c/strong\u003eBody weight in both male \u003cstrong\u003e(H)\u003c/strong\u003e and female mice \u003cstrong\u003e(I)\u003c/strong\u003e.\u0026nbsp; Data are shown as the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4432933/v1/9c933601071bf0c2f379ec55.png"},{"id":58098892,"identity":"5ec6b197-e91c-4f50-8e31-ab4f95998cdf","added_by":"auto","created_at":"2024-06-11 06:22:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMale but not female \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elFgf21\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice show impaired fat tolerance when fed on regular chow diet.\u003c/strong\u003e\u0026nbsp; \u003cstrong\u003e(A)\u003c/strong\u003e Random and fasting serum TG levels in indicated adult (8 weeks) male mice (n=6 for both groups). \u003cstrong\u003e(B)\u003c/strong\u003e Postprandial TG levels during fat tolerance test (FTT) in male mice at the age of 15 weeks (oral gavage 1% olive oil). \u003cstrong\u003e(C)\u003c/strong\u003e Random and fasting serum TG levels in adult female mice (n=7 for both groups, 8 weeks). \u003cstrong\u003e(D)\u003c/strong\u003e Postprandial TG levels during FTT in female mice at the age of 15 weeks. \u003cstrong\u003e(E-F)\u003c/strong\u003e comparison of expression levels of hepatic genes that encode FGFR1 (\u003cem\u003eFgfr1\u003c/em\u003e) and KLB (\u003cem\u003eKlb\u003c/em\u003e), as well as two FGF21 downstream effectors, peroxisomal L-bifunctional enzyme (\u003cem\u003eEhhadh\u003c/em\u003e) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (\u003cem\u003ePpargc1a\u003c/em\u003e) in male \u003cstrong\u003e(E)\u003c/strong\u003e and female mice \u003cstrong\u003e(F)\u003c/strong\u003e. \u003cstrong\u003e(G-H)\u003c/strong\u003e \u003cem\u003eFgf1\u003c/em\u003e \u003cstrong\u003e(G)\u003c/strong\u003e and \u003cem\u003eFgf15\u003c/em\u003e \u003cstrong\u003e(H)\u003c/strong\u003e gene expression levels in the liver of \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u0026nbsp;\u003c/sup\u003e(n= 4-7) and \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 3-6). mice. AUC, area under the curve. *P \u0026lt; 0.05 and **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4432933/v1/1be6f028fef1e4d1751eb657.png"},{"id":58098886,"identity":"c7606004-4ae2-47b2-a1db-5d4d94311338","added_by":"auto","created_at":"2024-06-11 06:22:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":292649,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDietary curcumin intervention exhibits metabolic beneficial effects on obesogenic diet challenged male \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFgf21\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003efl/fl\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice but not male \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elFgf21\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Illustration of the animal experimental timeline. \u003cstrong\u003e(B)\u003c/strong\u003e Body weight of \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice during the experimental period. \u003cstrong\u003e(C)\u003c/strong\u003e Body weight gain of \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eat the end of the 15th week after overnight fasting. \u003cstrong\u003e(D-E)\u003c/strong\u003e Serum \u003cstrong\u003e(D)\u003c/strong\u003e and hepatic \u003cstrong\u003e(E)\u003c/strong\u003e TG content levels in \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003e(F)\u003c/strong\u003e FTT and AUC. Overnight fasted \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice were (intraperitoneal, \u003cem\u003ei.p\u003c/em\u003e) injected with 1 g/kg poloxamer 407 to block lipolysis. Blood samples were then collected from tail vein at indicated time for TG level measurement. \u003cstrong\u003e(G)\u003c/strong\u003e Body weight of \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice during the experimental period. \u003cstrong\u003e(H)\u003c/strong\u003e Body weight gain of \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice at the end of the 15\u003csup\u003eth\u003c/sup\u003e week after overnight fasting. \u003cstrong\u003e(I)\u003c/strong\u003e Serum TG level of \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003e(J)\u003c/strong\u003e Hepatic TG content in the \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice.\u0026nbsp; \u003cstrong\u003e(K)\u003c/strong\u003e FTT and AUC for overnight fasted \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice. Data are shown as the mean ± SEM (n=4-5 each group). *P \u0026lt; 0.05 and **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4432933/v1/9877086044861eca05c03272.png"},{"id":58100520,"identity":"3ebdacc9-3e21-43c1-9858-6083142c5444","added_by":"auto","created_at":"2024-06-11 06:30:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":253727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDietary resveratrol intervention targets hepatic FGF21 and its signaling in HFD challenged male wild type C57BL/6J mice. (A)\u003c/strong\u003e Illustration of the animal experimental timeline in male C57BL/6J mice. \u003cstrong\u003e(B)\u003c/strong\u003e Hepatic \u003cem\u003eFgf21\u003c/em\u003elevel in indicated group of mice. \u003cstrong\u003e(C)\u003c/strong\u003e Hepatic FGF21 protein level. The right panel shows the densitometric analysis data. \u003cstrong\u003e(D-E)\u003c/strong\u003e Liver expression levels of \u003cem\u003eFgfr1\u003c/em\u003e and \u003cem\u003eKlb \u003c/em\u003ein indicated group of mice. \u003cstrong\u003e(F)\u003c/strong\u003e Liver expression levels of \u003cem\u003eEhhadh\u003c/em\u003e, \u003cem\u003eAcox1\u003c/em\u003e, \u003cem\u003ePpargc1a\u003c/em\u003e and \u003cem\u003ePdk4\u003c/em\u003e in indicated group of mice. Data are shown as the mean ± SEM (n=5 each group). *P \u0026lt; 0.05, **P \u0026lt; 0.01 and ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4432933/v1/b5c9b59ba83098c9123ea59c.png"},{"id":58098889,"identity":"da3dc997-b7a6-40a9-981b-b2bf3ce7ada2","added_by":"auto","created_at":"2024-06-11 06:22:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":425605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDietary resveratrol intervention targets hepatic FGF21 and its signaling in HFHF diet challenged \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFgf21\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003efl/fl \u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003emice.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Illustration of the animal experimental timeline in \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice [n=4 for LFD group, and n=6 for HFHF and HFR (HFHF diet plus resveratrol intervention) groups]. \u003cstrong\u003e(B)\u003c/strong\u003e Hepatic \u003cem\u003eFgf21\u003c/em\u003e level in indicated group of mice. \u003cstrong\u003e(C) \u003c/strong\u003eHepatic FGF21 protein levels. The bottom panel shows the densitometric analysis data. \u003cstrong\u003e(D)\u003c/strong\u003e Heat map show comparison of selected FGF21 related genes among mice fed with LFD, HFHF and HFR diet. \u003cstrong\u003e(E-G)\u003c/strong\u003e qRT-PCR assessment on hepatic expression of indicated genes in LFD (n=4), HFHF (n=6) and HFR (n=6) mice. Data are shown as the mean ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01 and ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4432933/v1/693a369e07227927e902c49a.png"},{"id":58100522,"identity":"0664eb86-9caa-48d8-8777-fb8c509a2b80","added_by":"auto","created_at":"2024-06-11 06:30:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":214589,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResveratrol intervention exhibits metabolic beneficial effects on obesogenic diet challenged \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFgf21\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003efl/fl\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice but not \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elFgf21\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emice.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Illustration of the animal experimental timeline. \u003cstrong\u003e(B)\u003c/strong\u003e Blood glucose level and AUC during glucose tolerance test of \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003e(C)\u003c/strong\u003e Fast glucose level at the end of study.\u0026nbsp; \u003cstrong\u003e(D)\u003c/strong\u003e Serum Insulin level. \u003cstrong\u003e(E)\u003c/strong\u003e Body weight during the experimental period. \u003cstrong\u003e(F)\u003c/strong\u003e Body weight gain at the end of the 16th week after overnight fasting. \u003cstrong\u003e(G)\u003c/strong\u003e Serum TG level of indicated group of mice. \u003cstrong\u003e(H)\u003c/strong\u003e Hepatic TG content of indicated group of mice. \u003cstrong\u003e(I)\u003c/strong\u003e Diagram shows our view that it is gut microbiome that mediates functions of dietary interventions, involving the hepatic hormone FGF21. Dietary intervention including dietary polyphenol supplementation or dietary behavior changes alter gut microbiome composition and diversity. This in term triggers organ-organ crosstalk involving the gut-brain, gut-liver or other axis, with altered entero-endocrine hormone (GLP-1, GIP, and others) production and function, and altered levels of gut metabolites (bile acids and others), leading to the alteration on hepatic FGF21 production and sensitivity. Hence, without hepatic FGF21, dietary intervention cannot exert its metabolic beneficial effect. Data are shown as the mean ± SEM (n=5 for \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e, and n = 6 for \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e). *P \u0026lt; 0.05, **P \u0026lt; 0.01 and ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4432933/v1/ba47935140921d461eecd931.png"},{"id":75984561,"identity":"bce1b2ac-bb65-4848-bb7f-0884094f4b4b","added_by":"auto","created_at":"2025-02-11 08:09:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2019352,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4432933/v1/24ea01c2-2849-407a-8f9a-18946835f07a.pdf"},{"id":58098891,"identity":"52520ac4-8bd3-49dc-8103-2d5dc9619290","added_by":"auto","created_at":"2024-06-11 06:22:43","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":925264,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures0524.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4432933/v1/2e456c8ed7972807406618c6.pdf"},{"id":58098885,"identity":"747e9459-bab2-4bba-bf5d-2da7d7443499","added_by":"auto","created_at":"2024-06-11 06:22:43","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33379,"visible":true,"origin":"","legend":"","description":"","filename":"supportingtablesV1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4432933/v1/f701956069dd593e1e159a80.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Hepatic fibroblast growth factor 21 is required for curcumin or resveratrol in exerting their metabolic beneficial effect in male mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExcept for prescribed drugs and physical exercise, various dietary interventions were also shown to improve metabolic homeostasis\u0026nbsp;(1, 2). One type of dietary intervention is the change of dietary behaviors, such as nutritional restriction and intermittent fasting\u0026nbsp;(3), while another type is the addition of chemical compounds from edible plants into the diet\u0026nbsp;(4). One category of those compounds is dietary polyphenols, with the curry compound curcumin and resveratrol, mostly found in red grapes, as two typical examples\u0026nbsp;(1, 5). Interestingly, observations made by our team and others have shown that both dietary polyphenol intervention and amino acid restriction target the hepatic hormone fibroblast growth factor 21 (FGF21)\u0026nbsp;(3, 6-8). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMembers of the FGF family interact with FGF receptors (FGFRs) including FGFR1, leading to complicated downstream signaling events. Due to the lack of a heparin binding domain, FGF21, FGF19 (FGF15 in rodents) and FGF23 can be released freely into the bloodstream, serving as endocrine hormones (9). In addition to FGFRs, the obligatory co-receptor \u0026beta;-klotho (KLB) is also required for FGF21 to exert its metabolic function (9). It is generally accepted that plasma FGF21 is liver driven, although \u003cem\u003eFGF21/Fgf21\u003c/em\u003e mRNA expression can be detected in adipose tissues, pancreas, and elsewhere (9-11). Various FGF21 analogues have been tested in clinical trials for treating metabolic disorders including diabetes and fatty liver disorders (9). The most promising effects of those \u0026ldquo;pre-drugs\u0026rdquo; are the attenuation of hyperlipidemia and the improvement of insulin sensitivity. We and others have also reported that hepatic FGF21 expression can be regulated by glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1RAs), including both exenatide and liraglutide (12-15). Utilizing the liver specific \u003cem\u003eFgf21\u003c/em\u003e knockout mouse model, we have demonstrated recently that hepatic FGF21 is required for liraglutide in improving energy homeostasis in male mice with obesogenic diet challenge, making hepatic FGF21 as a \u0026ldquo;central molecule\u0026rdquo; for both GLP-1R-based drugs and diet interventions (12). Liraglutide treatment or curcumin intervention is also shown to attenuate high-fat-diet (HFD) induced repression on \u003cem\u003eFgfr1\u003c/em\u003e, which encodes the FGF21 receptor FGFR1, and \u003cem\u003eKlb\u003c/em\u003e, which encodes the obligatory co-receptor KLB (6, 12). These regulatory events are commonly interpreted as the improvement of FGF21 sensitivity (6, 12).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUtilizing the similar hepatic FGF21 deficient mice, here we have asked a straightforward question: Is FGF21 also required for curcumin or resveratrol, two typical dietary polyphenols, in exerting their metabolic beneficial effects? \u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eThe source of dietary polyphenols\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe curry compound curcumin was purchased from Organika Health Products (Richmond, BC, Canada; a 95% standardize curcumin extract). Resveratrol was purchased from Combi-Blocks (Catalog #: \u0026nbsp; OR-1053, San Diego, CA, USA). Methods for curcumin and resveratrol intervention have been described in our previous studies\u0026nbsp;(5, 6, 16-19). Contents of experimental diets utilized in this study are shown in Supporting Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals and Animal Experimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6J mice, liver specific \u003cem\u003eFgf21\u003c/em\u003e knockout (\u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e) mice and wild type littermates (\u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e)\u0026nbsp;were utilized in this study. \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice were generated by mating \u003cem\u003eFgf21\u003csup\u003eloxP\u003c/sup\u003e\u003c/em\u003e (Strain #:022361, Jackson lab) with \u003cem\u003eAlb-Cre\u003c/em\u003e mice (Strain #:003574, Jackson lab) as we have reported previously\u0026nbsp;(12)\u0026nbsp;(Figure S1A). Mice were maintained at ambient room temperature and relative humidity of 50%, with free access to food and water under a 12 h light:12 h darkness cycle (n=4-5 per cage). The animal experiments and protocol were approved by the University Health Network Animal Care Committee and were performed in accordance with the guidelines of the Canadian Council of Animal Care. For curcumin intervention, curcumin was added to the high fat high fructose diet (60% HFD with 20% fructose) in a dosage of 4g/kg, as we have reported\u0026nbsp;(6). For resveratrol intervention, 0.5% of resveratrol was added to the indicated diet\u0026nbsp;(5). Mice were randomly assigned to either receive or not receive dietary intervention. Based on our animal protocol, mouse with serious body weight loss or shown \u0026ldquo;sickness\u0026rdquo; symptoms will be excluded from the study. For the current study, no mice or data points were excluded.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe generation of \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e, lFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice were verified by genotyping. The male and female \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e littermates were fed with chow (low fat diet, LFD) diet, metabolic tolerance tests were conducted at the week of 8\u003csup\u003eth\u003c/sup\u003e, 10\u003csup\u003eth\u003c/sup\u003e, 12\u003csup\u003end\u003c/sup\u003e and 15\u003csup\u003eth\u003c/sup\u003e for glucose tolerance (GTTs), pyruvate tolerance tests (PTTs), insulin tolerance test (ITT) and fat tolerance test (FTT), respectively. Prior to fat tolerance test (FTT), the blood triglyceride levels at random or fast state were assessed at the week of 14\u003csup\u003eth\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolic tolerance tests and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etriglyceride (TG) measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMethods for GTT, PTT and ITT have been previously presented\u0026nbsp;(20). For intraperitoneal GTTs and PTTs, both male and female mice were fasted for 16 hrs prior to the intraperitoneal injection of glucose (2g/kg body weight) or pyruvate (2g/kg body weight). For ITTs, male and female mice were fasted for 4 hours prior to the injection of insulin (0.5 U/kg body weight). Blood glucose levels were determined at 0, 15, 30, 60, 90, and 120 min. We adopted the method by Gniuli \u003cem\u003eet al\u0026nbsp;\u003c/em\u003efor conducting FTT\u0026nbsp;(21). Briefly, mice were fasted overnight prior to oral gavage of 1% olive oil of body weight. Blood was collected from tail vein at 0, 1, 2 and 4 hours for TG measurement. To determine TG produced by the liver (lipid tolerance test), mice fasted overnight were injected intraperitoneally with poloxamer 407 (Sigma) to block lipolysis, and blood was collected from tail vein at indicated hours to measure TG levels\u0026nbsp;(5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR and Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMethods for qRT-PCR and Western blotting have been described previously\u0026nbsp;(5, 20), with oligo nucleotide primers and antibodies listed in Supporting Table 2 and Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNAseq sample preparation and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were subjected to a 16-week normal chow diet (LFD), HFHF diet and HFHF diet with resveratrol (HFR). By the end of the experiment, mice liver tissues were collected for RNA isolation. Total RNA was harvested using RNeasy Mini kit (QIAGEN) and further quantified and analyzed using Nanodrop spectrophotometer and Bioanalyzer. One microgram of total RNA was utilized and sent to Center of Applied Genomics (Sickkids Hospital, Canada) for sequencing library construction, as we described previously\u0026nbsp;(12). Data processing and analyzing were conducted through a standardized pipeline called RNA-seq IMmune Analysis Pipeline, per literature description\u0026nbsp;(22). Briefly, unprocessed FASTQ files containing raw data were downloaded and transferred. The RNA-seq reads were aligned against the mm10 reference genome assembly (Genome Reference Consortium Mouse Build 38) obtained from the NCI Genome Data Commons (GDC) using STAR (version 2.4.2a). \u0026nbsp; Quality control was performed on aligned BAM files using RSeQC and then expression levels were quantified using SALMON (V.0.14.0). After converting Ensemble IDs to mouse gene symbols (GRCm38.p6), the reads per gene were normalized and differentially expressed genes were analyzed with DESeq2 (V1.22.2).\u0026nbsp;Raw data and processed data have been submitted to the Gene Expression Omnibus (GEO) database (GSE241713).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults are expressed as mean \u0026plusmn; SEM. Student\u0026rsquo;s t test or one-way ANOVA followed by Sidak post-hoc correction were applied for calculating the statistical significance. In all cases, P \u0026lt; 0.05 is considered statistically significant. Statistical analyses were performed with GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMale but not female \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003emice on regular chow diet (defined as low fat diet, LFD) feeding show impaired fat tolerance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have generated \u003cem\u003elFgf21\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice and the littermate controls (\u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e) for current study by mating \u003cem\u003eAlb-Cre\u0026nbsp;\u003c/em\u003ewith\u003cem\u003e\u0026nbsp;Fgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e, as illustrated in Fig. S1A. \u003cem\u003elFgf21\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emouse liver showed barely detectable FGF21 (Fig. S1B-D), while FGF21 expression in both brown adipose tissue and hypothalamus were virtually unaffected (Fig. S1E-F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSix-week-old male and female\u003cem\u003e\u0026nbsp;lFgf21\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice and the control \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice were fed with regular chow diet (low fat diet, LFD) for 12 weeks, while three metabolic tolerance tests (glucose tolerance test, GTT; insulin tolerance test, ITT; and pyruvate tolerance test, PTT) were conducted at indicated time for all mice, as indicated in Fig. 1A. Apparently, on LFD feeding, male or female \u003cem\u003elFgf21\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice exhibited comparable glucose, pyruvate, and insulin tolerance when compared with sex-matched control littermates \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Fig. 1B-G). There were no appreciable differences on body weight with hepatic FGF21 knockout in both male and female mice (Fig. 1H-I).\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe above mice were then fed with LFD for an additional 3-week period (Fig. 1A), followed by collecting random blood samples and conducting fat tolerance tests (FTT). Mice were then fasted overnight before they were sacrificed for tissue sample collections. As shown, male \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice exhibited elevated random and fasting plasma\u0026nbsp;TG levels, as well as\u0026nbsp;impaired fat tolerance when compared with correspondent littermate controls (Fig. 2A-B). Such defects were not observed in female\u003cem\u003e\u0026nbsp;lFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice (Fig. 2C-D). We then assessed the expression of a battery of hepatic genes that are related to the FGF21 cellular signaling. Expression levels of genes that encode FGFR1 (\u003cem\u003eFgfr1\u003c/em\u003e) and KLB (\u003cem\u003eKlb\u003c/em\u003e), as well as the two FGF21 downstream mediators (\u003cem\u003eEhhadh,\u0026nbsp;\u003c/em\u003ewhich encodes enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase;\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePpargc1a,\u0026nbsp;\u003c/em\u003ewhich encodes PPARG coactivator 1 alpha) were reduced in male \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice (Fig. 2E). In female \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, only \u003cem\u003ePpargc1a\u0026nbsp;\u003c/em\u003elevel was reduced (Fig. 2F). We have also assessed expression of liver \u003cem\u003eFgf1\u0026nbsp;\u003c/em\u003eand gut (ileum) \u003cem\u003eFgf15\u003c/em\u003e, asking whether hepatic FGF21 deficiency results in elevated \u003cem\u003eFgf1 or Fgf15\u0026nbsp;\u003c/em\u003eexpression for compensation. No such compensatory elevation was observed in \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice (Fig. 2G-2H). Together, we conclude that male but not female \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice showed impaired lipid homeostasis in the absence of obesogenic dietary challenge, associated with reduced hepatic expression of \u003cem\u003eFgfr1\u003c/em\u003e,\u0026nbsp;\u003cem\u003eKlb\u003c/em\u003e, and \u003cem\u003eEhhadh\u003c/em\u003e. Our following investigations were then performed on male mice only.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDietary curcumin intervention improves lipid homeostasis in the control \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice but not in \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice with the obesogenic dietary challenge\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have reported previously that in HFD-challenged male mice, curcumin or anthocyanin intervention regulated hepatic FGF21 production and improved FGF21 sensitivity in hepatocytes\u0026nbsp;(6, 23). Curcumin intervention stimulated hepatic FGF21 expression in mice on LFD feeding and attenuated HFD-induced hepatic FGF21 over-expression and FGF21 resistance\u0026nbsp;(6). Here we aimed to determine whether hepatic FGF21 is required for curcumin in exerting its major metabolic beneficial effect, especially lipid homeostatic effect in mice on obesogenic dietary challenge. As shown, male \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice or the control littermate \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice were fed with high fat high fructose (HFHF) diet without or with curcumin intervention for 15 weeks (Fig. 3A). In the control littermates, curcumin intervention moderately attenuated HFHF-diet induced body weight gain (Fig. 3B-C), reduced fasting serum and hepatic\u0026nbsp;TG\u0026nbsp;contents\u0026nbsp;(Fig. 3D-E), and improved lipid tolerance (Fig. 3F). Curcumin dietary intervention also reduced hepatic expression of ChREBP, as well as expression of genes that encode ChREBP (\u003cem\u003eMlxipl\u003c/em\u003e) and fatty acid synthase (\u003cem\u003eFasn\u003c/em\u003e) (Fig. S2A-B). In\u0026nbsp;\u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, none of the above regulatory effects of dietary curcumin intervention were observable (Fig. 3G-K and Fig. S2C-D). We hence conclude that\u0026nbsp;liver FGF21 is required for curcumin intervention in exerting its metabolic beneficial effect, especially the improvement of lipid homeostasis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn obesogenic diet challenged male mice, dietary resveratrol intervention also regulates hepatic FGF21\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;2014,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eLi and colleagues have reported that resveratrol treatment increased the transcriptional activity of the \u003cem\u003eFGF21\u003c/em\u003e gene promoter\u0026nbsp;(7). We hence ask whether \u003cem\u003ein vivo\u003c/em\u003e resveratrol intervention in wild type mice with an obesogenic dietary challenge affects hepatic FGF21 expression, FGF21 sensitivity, or FGF21 mediated cellular signaling events. Here we conducted such assessments in two different sets of mice. In the first set, wild type C57BL/6J mice were fed with LFD, HFD, or HFD with resveratrol (HFD+Res) intervention for 8 weeks (Fig. 4A). In such experimental settings, hepatic \u003cem\u003eFgf21\u003c/em\u003e expression was reduced by HFD feeding, and the reduction was attenuated by resveratrol intervention (Fig. 4B). Hepatic FGF21 protein expression was not significantly affected by HFD while concomitant resveratrol intervention exhibited a stimulatory effect on hepatic FGF21 expression (Fig. 4C). Importantly, resveratrol intervention increased expression of \u003cem\u003eFgfr1\u003c/em\u003e, which was reduced by HFD challenge (Fig. 4D). \u003cem\u003eKlb\u003c/em\u003e level was not significantly affected by 8-week HFD challenge, while resveratrol intervention exhibited a stimulatory effect on hepatic\u003cem\u003e\u0026nbsp;Klb\u003c/em\u003e expression (Fig. 4E). Among the four downstream effectors of FGF21 we have assessed, expression of \u003cem\u003eEhhadh\u0026nbsp;\u003c/em\u003ewas inhibited by HFD and the inhibition was effectively reversed by concomitant resveratrol intervention. \u003cem\u003eAcox1\u003c/em\u003e (encodes Acyl-CoA Oxidase 1) expression was not affected by HFD challenge while resveratrol intervention elevated its expression level. HFD challenge significantly reduced expression level of \u003cem\u003ePpargc1a\u003c/em\u003e, while resveratrol intervention generated no appreciable reversing effect in the current experimental settings. Finally, hepatic \u003cem\u003ePdk4\u003c/em\u003e (which encodes pyruvate dehydrogenase lipoamide kinase isozyme 4) level was not affected by HFD challenge or resveratrol intervention in our current experimental settings (Fig. 4F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe above observations suggest that like curcumin intervention previously reported by our team and by others\u0026nbsp;(6, 24, 25), resveratrol intervention can also target hepatic FGF21 or its downstream signaling events. Fructose consumption is known to stimulate hepatic FGF21 expression, associated with the development of insulin resistance\u0026nbsp;(26). In the second set of mice, we challenged \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice with HFHF-diet without and with resveratrol intervention for an extended period, as indicated in Fig. 5A. As shown, 16-week HFHF-diet challenge significantly increased hepatic \u003cem\u003eFgf21\u003c/em\u003e levels, while dietary resveratrol intervention attenuated the elevation effectively (Fig. 5B). At FGF21 protein level, elevation was observed in mice with HFHF challenge, without or with 16-week resveratrol intervention (Fig. 5C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe then collected liver tissues from those mice for RNAseq analysis. As we have anticipated (Fig. 5D, Fig. S3, and Table S4), HFHF-diet challenge increased hepatic expression of \u003cem\u003eFgf21\u003c/em\u003e but reduced the expression of \u003cem\u003eKlb\u003c/em\u003e, while those effects were reciprocally reversed by 16-week dietary resveratrol intervention.\u0026nbsp;The attenuation effect of HFHF-diet and reversible effect of resveratrol intervention was also observed on certain downstream effectors of FGF21 signaling, which were then further verified by our qRT-PCR experiment (Fig. 5E-G). As shown in Fig. S3, HFHF diet feeding most significantly repressed expression of genes including \u003cem\u003eEif4ebp3\u003c/em\u003e (which encodes eukaryotic translation initiation factor 4E binding protein 3) and \u003cem\u003eZbtb16\u0026nbsp;\u003c/em\u003e(which encodes zinc finger and BTB domain-containing protein 16), and those genes were also significantly restored by resveratrol intervention. Exact metabolic functions of these two genes remain to be further explored. Additional information on the effect of HFHF diet challenge and resveratrol intervention on hepatic gene expression are presented in Fig. S4 and S5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDietary resveratrol intervention improves glucose tolerance and reduces serum and hepatic TG levels in HFHF challenged \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice but not in HFHF challenged \u003cem\u003elFgf21\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then directly compared the effect of dietary resveratrol intervention in \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice and \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice with HFHF-diet challenge (Fig. 6A). As shown, glucose tolerance was improved by dietary resveratrol intervention in the control littermate \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice but not in \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice (Fig. 6B). Dietary resveratrol intervention also attenuated HFHF-diet induced fasting hyperglycemia and hyperinsulinemia in \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice but not in \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice (Fig. 6C-D). Interestingly, in both \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, 16-week dietary resveratrol intervention attenuated HFHF-diet induced body weight gain (Fig. 6E-F) and fat accumulation (Fig. S6), although the degree of the attenuation in \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice appeared much stronger than that in \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice (Figure 6E-F and Fig. S6). Nevertheless, in \u003cem\u003eFgf21\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice but not in \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, dietary resveratrol intervention attenuated HFHF diet induced elevation on serum as well as hepatic TG levels (Figure 6G-H). Thus, although hepatic FGF21 is required for resveratrol intervention in exerting its metabolic beneficial effect on improving energy homeostasis, the body weight lowering effect of dietary resveratrol intervention does not completely rely on hepatic FGF21. Whether this involves FGF21 expressed in adipose tissue, or the brain deserves further investigations. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough native FGF21 and a few FGF21 analogues were shown to bring metabolic and other beneficial effect in various diseases models\u0026nbsp;(9, 27, 28), obese human subjects and animal models, either due to a defined genetic defect or generated by obesogenic diet challenge, show elevated hepatic and plasma FGF21 levels, suggesting that obesity represents an FGF21 resistance state\u0026nbsp;(9, 29, 30). We and others have reported that dietary intervention with curcumin or anthocyanin, or other dietary polyphenols, regulate plasma and hepatic FGF21 levels, as well as FGF21 sensitivity\u0026nbsp;(6, 18, 23, 24). Other edible plant compounds, such betaine, were also shown to regulate hepatic FGF21\u0026nbsp;(31). Importantly, in mice on LFD feeding, dietary curcumin or anthocyanin intervention stimulated hepatic FGF21 level; while in mice on HFD, the intervention attenuates HFD-induced FGF21 over-expression, associated with the reversion on HFD-induced repression on \u003cem\u003eFgfr1\u003c/em\u003e or \u003cem\u003eKlb\u003c/em\u003e expression\u0026nbsp;(6, 23, 25). These regulatory events are commonly interpreted as the improvement of FGF21 sensitivity\u0026nbsp;(6, 32). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe show here that on LFD feeding, male but not female \u003cem\u003elFgf21\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice exhibited impaired fat tolerance, associated with reduced expression of genes that encode FGF21 receptor and the co-receptor (\u003cem\u003eFgfr1\u003c/em\u003e and \u003cem\u003eKlb\u003c/em\u003e), as well as \u003cem\u003eEhhadh\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e. Previously, we and others have also shown that in female mice,\u0026nbsp;the female hormone estradiol\u0026nbsp;(E2) increased FGF21 production\u0026nbsp;(29, 33). How can female hormones including E2 compensate the lack of hepatic FGF21 on lipid homeostasis in the absence of obesogenic dietary challenge remains to be further investigated. In the absence of an obesogenic dietary challenge, extra-hepatic FGF21, including those generated in adipose tissues, brain, and elsewhere, might be sufficient for female mice in maintaining metabolic homeostasis. Nevertheless, we presented here our comprehensive observations in HFD challenged mice on hepatic FGF21 regulation with resveratrol intervention and then further expanded our investigation with HFHF diet induced obese and insulin resistance mouse model, show that resveratrol intervention reversed the repression of HFHF-diet on expression of \u003cem\u003eKlb\u003c/em\u003e,\u0026nbsp;as well as genes that encode FGF21 effectors. Importantly, we demonstrated here that hepatic FGF21 is required for either curcumin or resveratrol intervention in exerting their metabolic beneficial effect in the obesogenic diet challenged mouse models, although the body weight lowering effect of resveratrol or curcumin intervention may only partially rely on hepatic FGF21. In a previous investigation, we noticed that hepatic FGF21 is not absolutely required for the GLP-1RA liraglutide in lowering the body weight in mice with HFD challenge\u0026nbsp;(12).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHow can hepatic FGF21 mediate metabolic beneficial effect of both nutrient restriction\u0026nbsp;(3, 34)\u0026nbsp;and dietary polyphenol interventions\u0026nbsp;(6, 23, 25)? Why hepatic FGF21 is required for both the GLP-1R agonists (including liraglutide, semaglutide and exenatide) and various dietary interventions\u0026nbsp;(8)? The diabetes drug metformin, which is also a chemical isolated from plant, was shown to induce GLP-1 secretion, and such function contributes to the actions of metformin in the treatment of type 2 diabetes\u0026nbsp;(35). As metformin has been shown to exert its function via \u0026ldquo;reshaping\u0026rdquo; gut microbiome, it remains to be determined whether gut microbiome is involved in regulating gut GLP-1 production or secretion\u0026nbsp;(36). A recent study by Martin and colleagues showed that in the absence of gut microbiome, FGF21 adaptive pathway is desensitized in response to dietary protein restriction\u0026nbsp;(37). We are aware of the fundamental role of gut microbiome in health and diseases for decades, and a few\u0026nbsp;recent studies have demonstrated that beneficial effects of resveratrol intervention are strongly associated with alterations in gut microbiome\u0026nbsp;(38-40). Targeting gut microbiome or intestinal signaling cascades, including the gut hormone GLP-1, the intestinal Takeda G protein-coupled receptor 5 (TGR5) or Farnesoid X receptor (FXR) medicated bile acid signaling cascades, are also shared with the diabetes drug metformin, other phytomedicine including red ginseng extracts, and theabrownin isolated from Pu-erh tea, \u0026nbsp;and blueberry and cranberry anthocyanin extracts\u0026nbsp;(36, 41-44). Prior to reporting our current investigation,\u0026nbsp;we have reported very recently that resveratrol intervention target gut microbiome, leading to the attenuation of gut bile acid/FXR signaling and chylomicron secretion, and improved lipid homeostasis\u0026nbsp;(5). It is plausible to suggest that gut microbiome mediates functions of the two categories of dietary interventions (Fig. 6I), with the participation of gut metabolites and gut produced hormones, including GLP-1 and\u0026nbsp;gastric inhibitory polypeptide (GIP), as well as gut/brain axis, gut/liver axis, or other axis that links gut and other peripheral organs, as we have reviewed recently\u0026nbsp;(8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHepatic FGF21 is required for curcumin or resveratrol in exerting their major metabolic beneficial effect. The existence of common targets, such as FGF21, for GLP-1RAs and various types of dietary interventions, makes us to recognize the link between these two categories of \u0026ldquo;medicines\u0026rdquo;, between these two lines of biomedical research, brings us a novel angle in understanding and further investigation of metabolic disease treatment and prevention, with prescribed drugs and various phytomedicine. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eAcox1, Acyl-CoA Oxidase 1; Alb-Cre, albumin-Cre; Ehhadh, enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase; ChREBP, Carbohydrate response element binding protein; Fasn, fatty acid synthase; FGF15, fibroblast growth factor 15; FGF19, fibroblast growth factor 19; FGF21, fibroblast growth factor 21; FGF23, fibroblast growth factor 23; FGFR1, FGF receptor 1; FGFRs, FGF receptors; FTT, fat tolerance test; FXR, Farnesoid X receptor; GEO, Gene Expression Omnibus; GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1 receptor; GLP-1RAs, GLP-1 receptor agonists; GTT, glucose tolerance test; HFD, high fat diet; HFHF, high fat high fructose diet; ITT, insulin tolerance test; KLB, \u0026beta;-klotho; LFD, low fat diet; Pdk4, pyruvate dehydrogenase lipoamide kinase isozyme 4; Ppargc1\u0026alpha;, PPARG coactivator 1 alpha; PTT, pyruvate tolerance test; SEM, the standard error of the mean; TG, triglyceride; TGR5, Takeda G protein-coupled receptor 5.\u003c/em\u003e\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Canadian Institute of Health Research (PJT159735 to T.J.). JNF is the recipient of the Banting and Best Diabetes Centre- Novo Nordisk Studentship and Ontario Graduate Scholarship. The guarantor of the manuscript is Tianru Jin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJia Nuo Feng: Conceptualization, Methodology, Investigation, Visualization, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Weijuan Shao: Conceptualization, Methodology, Investigation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Lin Yang: Data curation, Formal Analysis, Methodologies, Software. Juan Pang: Investigation, Formal analysis, Visualization. Wenhua Ling: Writing \u0026ndash; review \u0026amp; editing. Dinghui Liu: Investigation, Formal analysis, Visualization. Michael B Wheeler: Writing \u0026ndash; review \u0026amp; editing. Housheng Hansen He: Formal Analysis, Methodologies, Software, Writing \u0026ndash; review \u0026amp; editing. Tianru Jin: Conceptualization, Funding acquisition, Resources, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Banting \u0026amp; Best Diabetes Centre and Ontario Graduate Scholarship for doctoral funding toward Jia Nuo Feng.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJin T, Song Z, Weng J, Fantus IG. Curcumin and other dietary polyphenols: potential mechanisms of metabolic actions and therapy for diabetes and obesity. Am J Physiol Endocrinol Metab. 2018;314(3):E201-e5.\u003c/li\u003e\n\u003cli\u003eJin T. 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Eur J Nutr. 2021;60(5):2735-46.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nutrition-and-diabetes","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nutd","sideBox":"Learn more about [Nutrition \u0026 Diabetes](http://www.nature.com/nutd/)","snPcode":"41387","submissionUrl":"https://mts-nutd.nature.com/cgi-bin/main.plex","title":"Nutrition \u0026 Diabetes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dietary polyphenol intervention, FGF21, FGFR1, KLB, Resveratrol","lastPublishedDoi":"10.21203/rs.3.rs-4432933/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4432933/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eConclusion and significance: \u003c/strong\u003eWe conclude that hepatic FGF21 is required for curcumin or resveratrol in exerting their major metabolic beneficial effect. The recognition that FGF21 as the common target of dietary interventions brings us a novel angle in understanding metabolic disease treatment and prevention. It remains to be explored how various dietary interventions regulate FGF21 expression and function, via certain common or unique gut-liver or gut-brain-liver axis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Our mechanistic understanding on metabolic beneficial effects of dietary polyphenols has been hampered for decades due to the lack of functional receptors for those compounds and their extremely low plasma concentrations. Recent studies by our team and others have suggested that those dietary polyphenols may target gut microbiome and gut-liver axis and that hepatic fibroblast factor 21 (FGF21) serves as a common target for various dietary interventions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Utilizing liver-specific FGF21 null mice (\u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e), we are asking a straightforward question: Is hepatic FGF21 required for curcumin or resveratrol, two typical dietary polyphenols, in exerting their metabolic beneficial effect in obesogenic diet-induced obese mouse models. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eOn low-fat diet feeding, no appreciable defect on glucose disposal was observed in male or female \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice, while fat tolerance was impaired in male but not in female \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice, associated with elevated serum triglyceride (TG) level, reduced hepatic expression of the \u003cem\u003eEhhadh\u003c/em\u003e and \u003cem\u003ePpargc1a\u003c/em\u003e, which encodes the two downstream effectors of FGF21. On high-fat-high-fructose (HFHF) diet challenge, \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e but not \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice exhibited response to curcumin intervention on reducing serum TG, and on improving fat tolerance. Resveratrol intervention also affected FGF21 expression or its downstream effectors. Metabolic beneficial effects of resveratrol intervention observed in HFHF diet-challenged \u003cem\u003eFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice were either absent or attenuated in \u003cem\u003elFgf21\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e","manuscriptTitle":"Hepatic fibroblast growth factor 21 is required for curcumin or resveratrol in exerting their metabolic beneficial effect in male mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 06:22:38","doi":"10.21203/rs.3.rs-4432933/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-09-18T10:48:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-12T04:28:18+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-18T09:09:34+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-06-10T13:52:10+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-05-28T17:16:27+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-05-28T17:07:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-05-28T17:03:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-17T10:06:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Nutrition \u0026 Diabetes","date":"2024-05-16T19:24:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-16T19:24:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nutrition-and-diabetes","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nutd","sideBox":"Learn more about [Nutrition \u0026 Diabetes](http://www.nature.com/nutd/)","snPcode":"41387","submissionUrl":"https://mts-nutd.nature.com/cgi-bin/main.plex","title":"Nutrition \u0026 Diabetes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fac1c74e-1729-4a31-8607-ef2334984432","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-11T08:09:12+00:00","versionOfRecord":{"articleIdentity":"rs-4432933","link":"https://doi.org/10.1038/s41387-025-00363-0","journal":{"identity":"nutrition-and-diabetes","isVorOnly":false,"title":"Nutrition \u0026 Diabetes"},"publishedOn":"2025-02-10 05:00:00","publishedOnDateReadable":"February 10th, 2025"},"versionCreatedAt":"2024-06-11 06:22:38","video":"","vorDoi":"10.1038/s41387-025-00363-0","vorDoiUrl":"https://doi.org/10.1038/s41387-025-00363-0","workflowStages":[]},"version":"v1","identity":"rs-4432933","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4432933","identity":"rs-4432933","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}