Fibroblast Growth Factor 21 Knockout Mice Research Articles

Fibroblast growth factor 21 alleviates acetaminophen induced acute liver injury by activating Sirt1 mediated autophagy

Background and aimsAcetaminophen (APAP) is the main cause of acute liver injury (ALI) in the Western. Our previous study has shown that fenofibrate activated hepatic expression of fibroblast growth factor 21 (FGF21) can protect the liver form APAP injuries by promoting autophagy. However, the underlying mechanism involved in FGF21-mediated autophagy remains unsolved. MethodsThe ALI mice model was established by intraperitoneal injection of APAP. To investigate the influence of FGF21 on autophagy and Sirt1 expression in APAP-induced ALI, FGF21 knockout (FGF21KO) mice and exogenously supplemented mouse recombinant FGF21 protein were used. In addition, primary isolated hepatocytes and the Sirt1 inhibitor EX527 were used to observe whether FGF21 activated autophagy in APAP injury is regulated by Sirt1 at the cellular level. ResultsFGF21, Sirt1, and autophagy levels increased in mice with acute liver injury (ALI) and in primary cultured hepatocytes. Deletion of the FGF21 gene exacerbated APAP-induced liver necrosis and oxidative stress, and decreased mitochondrial potential. It also reduced the mRNA and protein levels of autophagy-related proteins such as Sirt1, LC3-II, and p62, as well as the number of autophagosomes. Replenishment of FGF21 reversed these processes. In addition, EX527 partially counteracted the protective effect of FGF21 by worsening oxidative damage, mitochondrial damage, and reducing autophagy in primary liver cells treated with APAP. ConclusionFGF21 increases autophagy by upregulating Sirt1 to alleviate APAP-induced injuries.

FGF21-dependent alleviation of cholestasis-induced liver fibrosis by sodium butyrate.

The beneficial effects of fibroblast growth factor 21 (FGF21) and sodium butyrate (NaB) on protection against cholestasis-induced liver fibrosis are not well known. This study aimed to explore the effects of FGF21 and NaB on bile duct ligation (BDL)-induced liver fibrosis. Wild-type (WT) and FGF21 knockout (KO) mice received BDL surgery for 14days. Liver fibrosis was assessed by Masson's staining for fibrosis marker expressions at the mRNA or protein levels. Adenovirus-mediated FGF21 overexpression in the WT mice was assessed against BDL damage. BDL surgeries were performed in WT and FGF21 KO mice that were administered either phosphate-buffered saline or NaB. The effects of NaB on the energy metabolism and gut microbiota were assessed using stable metabolism detection and 16S rRNA gene sequencing. BDL-induced liver fibrosis in the WT mice was accompanied by high induction of FGF21. Compared to the WT mice, the FGF21 KO mice showed more severe liver fibrosis induced by BDL. FGF21 overexpression protected against BDL-induced liver fibrosis, as proved by the decreasing α-SMA at both the mRNA and protein levels. NaB administration enhanced the glucose and energy metabolisms as well as remodeled the gut microbiota. NaB alleviated BDL-induced liver fibrosis in the WT mice but aggravated the same in FGF21 KO mice. FGF21 plays a key role in alleviating cholestasis-induced liver damage and fibrosis. NaB has beneficial effects on cholestasis in an FGF21-dependent manner. NaB administration can thus be a novel nutritional therapy for treating cholestasis via boosting FGF21 signaling and regulating the gut microbiota.

FGF21 ameliorates septic liver injury by restraining proinflammatory macrophages activation through the autophagy/HIF-1α axis

IntroductionSepsis, a systemic immune syndrome caused by severe trauma or infection, poses a substantial threat to the health of patients worldwide. The progression of sepsis is heavily influenced by septic liver injury, which is triggered by infection and cytokine storms, and has a significant impact on the tolerance and prognosis of septic patients. The objective of our study is to elucidate the biological role and molecular mechanism of fibroblast growth factor 21 (FGF21) in the process of sepsis. ObjectivesThis study was undertaken in an attempt to elucidate the function and molecular mechanism of FGF21 in therapy of sepsis. MethodsSerum concentrations of FGF21 were measured in sepsis patients and septic mice. Liver injury was compared between mice FGF21 knockout (KO) mice and wildtype (WT) mice. To assess the therapeutic potential, recombinant human FGF21 was administered to septic mice. Furthermore, the molecular mechanism of FGF21 was investigated in mice with myeloid-cell specific HIF-1α overexpression mice (LyzM-CreDIO-HIF-1α) and myeloid-cell specific Atg7 knockout mice (Atg7△mye). ResultsSerum level of FGF21 was significantly increased in sepsis patients and septic mice. Through the use of recombinant human FGF21 (rhFGF21) and FGF21 KO mice, we found that FGF21 mitigated septic liver injury by inhibiting the initiation and propagation of inflammation. Treatment with rhFGF21 effectively suppressed the activation of proinflammatory macrophages by promoting macroautophagy/autophagy degradation of hypoxia-inducible factor-1α (HIF-1α). Importantly, the therapeutic effect of rhFGF21 against septic liver injury was nullified in LyzM-CreDIO-HIF-1α mice and Atg7△mye mice. ConclusionsOur findings demonstrate that FGF21 considerably suppresses inflammation upon septic liver injury through the autophagy/ HIF-1α axis.

Fibroblast growth factor 21 mitigates lupus nephritis progression via the FGF21/Irgm 1/NLRP3 inflammasome pathway

As an endocrine cytokine, fibroblast growth factor 21 (FGF21) exhibits anti-inflammatory properties. With the development of lupus nephritis (LN), which is tightly related to pathogenic factors, including inflammation and immune cell dysregulation, we explored the impact of Fibroblast Growth Factor 21 (FGF21) as well as its underlying mechanism. We induced an in vivo LN model using pristane in both wild-type C57BL/6 and FGF21 knockout (FGF21–/–) mice. LN serum obtained from 32-week-old wild-type LN mice was used to stimulate RAW264.7 and human renal tubular epithelial (HK-2) cells to mimic an in vitro LN model. Moreover, our findings revealed that FGF21–/– mice showed more severe kidney injury compared to wild-type mice, as evidenced by increased levels of renal function markers, inflammatory factors, and fibrosis markers. Notably, exogenous administration of FGF21 to wild-type LN mice markedly mitigated these adverse effects. Additionally, we used tandem mass tag (TMT)-based quantitative proteomics to detect differentially expressed proteins following FGF21 treatment. Results indicated that 121 differentially expressed proteins influenced by FGF21 were involved in biological processes such as immune response and complement activation. Significantly upregulated protein Irgm 1, coupled with modulated inflammatory response, appeared to contribute to the beneficial effects of FGF21. Furthermore, Western blot analysis demonstrated that FGF21 upregulated Irgm 1 while inhibiting nucleotide-binding oligomerization domain-like receptors family pyrin domain including 3 (NLRP3) inflammasome expression. Silencing Irgm 1, in turn, reversed FGF21′s inhibitory effect on NLRP3 inflammasome. In summary, FGF21 can potentially alleviate pristane-induced lupus nephritis in mice, possibly through the FGF21/Irgm 1/NLRP3 inflammasome pathway.

347-OR: Methionine Restriction Differentially Protects against High-Fat Diet-Induced Neuroinflammation and Central Insulin Resistance in Male and Female Mice

High-fat diets (HFD) promote central inflammation and insulin resistance (IR). Methionine restriction (MR) improves peripheral insulin sensitivity, lipid metabolism, and cognitive function, at least in part by increasing fibroblast growth factor 21 (FGF21). However, the ability of MR to protect against neuroinflammation and central IR during adolescence has not been examined. Moreover, whether MR depends on FGF21 to protect against HFD-induced changes in central insulin signaling or neuroinflammation is unknown. We sought to determine whether MR protects against HFD-induced neuroinflammation and central IR during adolescence in males and females and whether MR’s effects are dependent on FGF21. Male and female wild-type (WT) and FGF21 knockout (KO) mice, aged 5 to 6 wk, were fed normal (18.9% fat-derived kcal) or high-fat (60% fat-derived kcal) versions of control (0.86% methionine) or MR (0.17% methionine) diets for 5 wk. To investigate insulin action, ITTs were performed after 4 wk on the diet, and saline or insulin (0.6U/kg body weight) was administered 15 min prior to sacrifice. MR significantly decreased weight gain of HFD-fed mice regardless of sex or genotype (p < 0.05). MR increased serum FGF21 of all males and HFD-fed females (p < 0.05), and strongly tended to increase FGF21 in female mice on the normal-fat diet. HFD feeding tended to promote inflammatory gene expression in the hippocampus of WT males, while MR trended to mitigate inflammation independent of FGF21. MR also significantly reduced fasting blood glucose and improved peripheral insulin response in an FGF21-dependent fashion (p < 0.05). Though, neither HFD nor MR had a notable impact on markers of IR in the hypothalamus or hippocampus. Our findings indicate that short-term MR improves metabolic parameters of adolescent mice fed normal or high-fat diets. In addition, MR acts in an FGF21-independent and sex-dependent manner to improve neuroinflammation. Disclosure H.L.Lail: None. R.Lawrence: None. D.Wanders: None.

FGF21-FGFR1 controls mitochondrial homeostasis in cardiomyocytes by modulating the degradation of OPA1

Fibroblast growth factor 21 (FGF21) is a pleiotropic hormone secreted primarily by the liver and is considered a major regulator of energy homeostasis. Recent research has revealed that FGF21 could play an important role in cardiac pathological remodeling effects and prevention of cardiomyopathy; however, the underlying mechanism remains largely unknown. This study aimed to determine the mechanism underlying the cardioprotective effects of FGF21. We engineered FGF21 knock out mice and subsequently elucidated the effects of FGF21 and its downstream mediators using western blotting, qRT-PCR, and mitochondrial morphological and functional analyses. FGF21 knockout mice showed cardiac dysfunction, accompanied by a decline in global longitudinal strain (GLS) and ejection fraction (EF), independent of metabolic disorders. Mitochondrial quality, quantity, and function were abnormal, accompanied by decreased levels of optic atrophy-1 (OPA1) in FGF21 KO mice. In contrast to FGF21 knockout, cardiac-specific overexpression of FGF21 alleviated the cardiac dysfunction caused by FGF21 deficiency. In an in vitro study, FGF21 siRNA deteriorated mitochondrial dynamics and impaired function induced by cobalt chloride (CoCl2). Both recombinant FGF21 and adenovirus-mediated FGF21 overexpression could alleviate CoCl2-induced mitochondrial impairment by restoring mitochondrial dynamics. FGF21 was essential for maintaining mitochondrial dynamics and function of the cardiomyocytes. As a regulator of cardiomyocyte mitochondrial homeostasis under oxidative stress, FGF21 could be an important new target for therapeutic options for patients with heart failure.

Targeting hepatic serine-arginine protein kinase 2 ameliorates alcohol-associated liver disease by alternative splicing control of lipogenesis.

Lipid accumulation induced by alcohol consumption is not only an early pathophysiological response but also a prerequisite for the progression of alcohol-associated liver disease (ALD). Alternative splicing regulates gene expression and protein diversity; dysregulation of this process is implicated in human liver diseases. However, how the alternative splicing regulation of lipid metabolism contributes to the pathogenesis of ALD remains undefined. Serine-arginine-rich protein kinase 2 (SRPK2), a key kinase controlling alternative splicing, is activated in hepatocytes in response to alcohol, in mice with chronic-plus-binge alcohol feeding, and in patients with ALD. Such induction activates sterol regulatory element-binding protein 1 and promotes lipogenesis in ALD. Overexpression of FGF21 in transgenic mice abolishes alcohol-mediated induction of SRPK2 and its associated steatosis, lipotoxicity, and inflammation; these alcohol-induced pathologies are exacerbated in FGF21 knockout mice. Mechanistically, SRPK2 is required for alcohol-mediated impairment of serine-arginine splicing factor 10, which generates exon 7 inclusion in lipin 1 and triggers concurrent induction of lipogenic regulators-lipin 1β and sterol regulatory element-binding protein 1. FGF21 suppresses alcohol-induced SRPK2 accumulation through mammalian target of rapamycin complex 1 inhibition-dependent degradation of SRPK2. Silencing SRPK2 rescues alcohol-induced splicing dysregulation and liver injury in FGF21 knockout mice. These studies reveal that (1) the regulation of alternative splicing by SRPK2 is implicated in lipogenesis in humans with ALD; (2) FGF21 is a key hepatokine that ameliorates ALD pathologies largely by inhibiting SRPK2; and (3) targeting SRPK2 signaling by FGF21 may offer potential therapeutic approaches to combat ALD.

FGF21 Depletion Attenuates Colitis through Intestinal Epithelial IL-22-STAT3 Activation in Mice.

Fibroblast growth factor 21 (FGF21) is a glucose and lipid metabolic regulator. Recent research revealed that FGF21 was also induced by inflammatory stimuli. Its role in inflammatory bowel disease (IBD) has not been investigated. In this study, an experimental IBD model was established in FGF21 knockout (KO) and wild-type (WT) mice by adding 2.5% (wt/vol) dextran sodium sulfate (DSS) to their drinking water for 7 days. The severity of the colitis and the inflammation of the mouse colon tissues were analyzed. In WT mice, acute DSS treatment induced an elevation in plasma FGF21 and a significant loss of body weight in a time-dependent manner. Surprisingly, the loss of body weight and the severity of the colitis induced by DSS treatment in WT mice were significantly attenuated in FGF21 KO mice. Colon and circulating pro-inflammatory factors were significantly lower in the FGF21 KO mice compared to the WT mice. As shown by BrdU staining, the FGF21 KO mice demonstrated increased colonic epithelial cell proliferation. DSS treatment reduced intestinal Paneth cell and goblet cell numbers in the WT mice, and this effect was attenuated in the FGF21 KO mice. Mechanistically, FGF21 deficiency significantly increased the signal transducer and activator of transcription (STAT)-3 activation in intestinal epithelial cells and increased the expression of IL-22. Further study showed that the expression of suppressor of cytokine signaling-2/3 (SOCS 2/3), a known feedback inhibitor of STAT3, was significantly inhibited in the DSS-treated FGF2 KO mice compared to the WT mice. We conclude that FGF21 deficiency attenuated the severity of DSS-induced acute colitis, which is likely mediated by enhancing the activation of the IL-22-STAT3 signaling pathway in intestinal epithelial cells.

Abstract 638: EPB-53 prevents NASH-HCC transition via regulation of SPHK1-S1P-HIPPO signaling and immune modulation in a murine model

Abstract Background: Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of disorders, ranging from non-progressive bland steatosis to non-alcoholic hepatitis (NASH), cirrhosis, and malignant transformation into hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) exerts a key role in lipid homeostasis and play a promising therapeutic candidate for the treatment of obesity, diabetes, and NASH. Small molecules like EPB-53 that are activators of heme-regulated inhibitor (HRI) of eukaryotic initiation factor 2α (eIF2α) kinase have been synthesized to induce FGF21. Previous study has shown that oral administration of EPB-53 alleviates NASH through increased FGF21 to prevent hepatic steatosis. However, it is unknown whether EPB-53 can prevent NASH-HCC transition. Aim: To investigate the effect of EPB-53 on NASH-HCC transition and the therapeutic mechanism, focusing on the lipid metabolic reprogramming and tumor immune microenvironment (TIM). Methods: NASH-HCC transition model was established in two-week-old male FGF21 knockout (KO) mice and wild type controls (C57BL/6J mice) by diethylnitrosamine (DEN) administration at 40 mg/kg body weight (i.p.). Four weeks after DEN administration, the mice were fed with western-style high fat diet (WSHFD, 60% kcal% fat and 19% kcal% fructose) for 3 months, and gavaged daily with either vehicle, 2% w/v, (2-hydroxypropyl)-β-cyclodextrin or EPB-53 (300 mg/kg/day) in vehicle for 4 weeks before sacrifice. The tumor incidence, lipid metabolism, and carcinogenesis signaling pathways were determined. Mass cytometry (CyTOF) analysis was performed to detect the cell subpopulations in the NASH microenvironment and NASH-HCC TIM. Results: Severe NASH with tumor cell detection, aberrant lipid components and HIPPO-YAP signaling were found in liver tissues of FGF21-KO mice compared to the WT controls. The upregulated sphingosine Kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) in liver subsequently induced YAP dephosphorylation and nuclear localization. EPB-53 treatment prevents NASH-HCC transition evident as decreased HCC incidence in WT mice but not in FGFKO mice. Continuously increased FGF21 production in WT mice contributes to down-regulation of SPHK1-S1P-HIPPO-YAP signaling. Interestingly, EPB-53 treatment can alleviate the compromised function of macrophages/Kupffer cells which render a TIM in the NASH-HCC mice. Conclusion: 1) Lack of FGF21 played a key role in lipid metabolic disorder and SPHK1-S1P-HIPPO signaling to promote NASH-HCC transition; 2) Lack of FGF21 rendered a TIM in NASH liver contributing to the NASH-HCC transition; 3) EPB-53 treatment can not only alleviate NASH, but also prevent NASH-HCC transition via lipid metabolic reprogramming, down-regulation of the SPHK1-S1P-HIPPO axis, and modulation of TIM. Citation Format: Min Tan, Eugenia Pujol, Santiago Vazquez, Jingwen Zhang, Jun Yan, Robert C. Martin, Manuel Vazquez-Carrera, Yan Li. EPB-53 prevents NASH-HCC transition via regulation of SPHK1-S1P-HIPPO signaling and immune modulation in a murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 638.

Social stress alters sleep in FGF21-deficient mice

Although several previous studies have suggested a relationship between sleep and the stress response, the mechanism underlying this relationship remains largely unknown. Here, we show that fibroblast growth factor 21 (FGF21), a lipid metabolism-related hormone, may play a role in this relationship. In this study, we examined differences in the stress response between FGF21 knockout (KO) mice and wild-type (WT) mice after social defeat stress (SDS). When the amount of non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep and wakefulness were averaged over the dark period after SDS, only KO mice showed significant differences in NREM sleep and wakefulness. In the social interaction test, KO mice seemed to be more prone to social avoidance. Our real-time (RT) -PCR results revealed that the mRNA expression of the stress- and sleep-related gene gamma-aminobutyric acid A receptor subunit alpha 2 was significantly lower in WT mice than in KO mice. Moreover, KO mice showed lower plasma levels of ketone bodies, which also affect sleep/wake regulation, than WT mice. These results suggested that FGF21 might influence sleep/wake regulation by inducing production of an anti-stress agent and/or ketone bodies, which may result in resilience to social stress.

Central FGF21 production regulates memory but not peripheral metabolism.

SUMMARYFibroblast growth factor 21 (FGF21) is a liver-derived endocrine hormone that functions to regulate energy homeostasis and macronutrient intake. Recently, FGF21 was reported to be produced and secreted from hypothalamic tanycytes, to regulate peripheral lipid metabolism; however, rigorous investigation of FGF21 expression in the brain has yet to be accomplished. Using a mouse model that drives CRE recombinase in FGF21-expressing cells, we demonstrate that FGF21 is not expressed in the hypothalamus, but instead is produced from the retrosplenial cortex (RSC), an essential brain region for spatial learning and memory. Furthermore, we find that central FGF21 produced in the RSC enhances spatial memory but does not regulate energy homeostasis or sugar intake. Finally, our data demonstrate that administration of FGF21 prolongs the duration of long-term potentiation in the hippocampus and enhances activation of hippocampal neurons. Thus, endogenous and pharmacological FGF21 appear to function in the hippocampus to enhance spatial memory.

FGF21 alleviates pulmonary hypertension by inhibiting mTORC1/EIF4EBP1 pathway via H19.

Long non‐coding RNAs (lncRNAs) play a significant role in pulmonary hypertension (PH). Our preliminary data showed that hypoxia‐induced PH is attenuated by fibroblast growth factor 21 (FGF21) administration. Therefore, we further investigated the regulatory role of long non‐coding RNAs in PH treated with FGF21. RNA sequencing analysis and real‐time PCR identified a significantly up‐regulation of the H19 after FGF21 administration. Moreover, gain‐ and loss‐of‐function assays demonstrated that FGF21 suppressed hypoxia‐induced proliferation of pulmonary artery smooth muscle cells partially through upregulation of H19. In addition, FGF21 deficiency markedly exacerbated hypoxia‐induced increases of pulmonary artery pressure and pulmonary vascular remodelling. In addition, AAV‐mediated H19 overexpression reversed the malignant phenotype of FGF21 knockout mice under hypoxia expose. Further investigation uncovered that H19 also acted as an orchestra conductor that inhibited the function of mechanistic target of rapamycin complex 1 (mTORC1) by disrupting the interaction of mTORC1 with eukaryotic translation initiation factor 4E–binding protein 1 (EIF4EBP1). Our work highlights the important role of H19 in PH treated with FGF21 and suggests a mechanism involving mTORC1/EIF4EBP1 inhibition, which may provide a fundamental for clinical application of FGF21 in PH.

Long-term adjustment of hepatic lipid metabolism after chronic stress and the role of FGF21

Chronic stress leads to post-traumatic stress disorder (PTSD) and metabolic disorders including fatty liver. We hypothesized that stress-induced molecular mechanisms alter energy metabolism, thereby promoting hepatic lipid accumulation even after a stress-free recovery period. In this context, we investigated fibroblast growth factor-21 (FGF21) as protective for energy and glucose homeostasis. FGF21 knockout mice (B6.129S6(SJL)-Fgf21tm1.2Djm; FGF21KO) and control mice (C57BL6; WT) were subjected to chronic variable stress. Mice were examined directly after acute intervention (Cvs) and long-term after 3 months of recovery (3mCvs). In WT, Cvs reduced insulin sensitivity and hepatic lipid accumulation, whilst fatty acid uptake increased. FGF21KO mice responded to Cvs with improved glucose tolerance, insulin resistance but liver triglycerides and plasma lipids were unaltered. Hepatic gene expression was specifically altered by genotype and stress e.g. by PPARa and SREBP-1 regulated genes. The stress-induced alteration of hepatic metabolism persisted after stress recovery. In hepatocytes at 3mCvs, differential gene regulation and secreted proteins indicated a genotype specific progression of liver dysfunction. Overall, at 3mCvs FGF21 was involved in maintaining mitochondrial activity, attenuating de novo lipogenesis, increased fatty acid uptake and histone acetyltransferase activity. Glucocorticoid release and binding to the FGF21 promoter may contribute to prolonged FGF21 release and protection against hepatic lipid accumulation. In conclusion, we showed that stress favors fatty liver disease and FGF21 protected against hepatic lipid accumulation after previous chronic stress loading by i) restored physiological function, ii) modulated gene expression via DNA-modifying enzymes, and iii) maintained energy metabolism.

Liver-Specific Nonviral Gene Delivery of Fibroblast Growth Factor 21 Protein Expression in Mice Regulates Body Mass and White/Brown Fat Respiration.

Viral-mediated in vivo gene delivery methods currently dominate among therapeutic strategies within the clinical and experimental settings, albeit with well documented limitations arising from immunologic constraints. In this study, we demonstrate the utility of nonviral hepatotropic in vivo gene delivery of unpackaged expression constructs, including one encoding fibroblast growth factor 21 (FGF21). FGF21 is an important hepatokine whose expression positively correlates with therapeutic outcomes across various animal models of obesity. Our data demonstrate that FGF21 expression can be restored into the livers of immunocompetent FGF21 knockout mice for at least 2 weeks after a single injection with an FGF21 expression plasmid. In wild-type C57BL6/J mice, in vivo transfection with an FGF21-expressing plasmid induced weight loss, decreased adiposity, and activated thermogenesis in white fat within 2 weeks. Furthermore, in vivo FGF21 gene delivery protected C57BL6/J mice against diet-induced obesity by decreasing adiposity and increasing uncoupling protein 1–dependent thermogenesis in brown fat and by boosting respiratory capacity in subcutaneous and perigonadal white fat. Together, the data illustrate a facile and effective methodology for delivering prolonged protein expression specifically to the liver. We contend that this method will find utility in basic science research as a practical means to enhance in vivo studies characterizing liver protein function. We further believe our data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease.Significance StatementThis study presents a valuable method for nonviral gene delivery in mice that improves upon existing techniques. The data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease and will likely enhance in vivo studies characterizing liver protein function.

Time-restricted feeding improves the reproductive function of female mice via liver fibroblast growth factor 21.

BackgroundThere has been a significant increase, to epidemic levels, of obese and overweight women of reproductive age, causing impairments to reproductive health. Time‐restricted feeding (TRF) including isocaloric intake has shown to be preventive of obesity‐related disorders. However, its therapeutic ability to improve the reproductive function of female remains largely unknown.MethodsHere, we investigated the ability of TRF to improve the reproductive function in wild‐type and liver‐specific FGF21 knockout female mice. To study fertility, a continuous and a short‐term fertility test, gonadotropin releasing‐hormone (GnRH), and Kisspeptin test were performed. Immortalized GnRH neuron was used to examine the direct role of liver fibroblast growth factor 21 (FGF21) on GnRH secretion.ResultsWe found that TRF rescues female mice from bodyweight gain and glucose intolerance, as well as ovarian follicle loss and dysfunction of estrus cyclicity induced by high‐fat diet. Furthermore, the beneficial effects of the TRF regimen on the reproductive performance were also observed in mice fed both chow and high‐fat diet. However, those beneficial effects of TRF on metabolism and reproduction were absent in liver‐specific FGF21 knockout mice. In vitro, FGF21 directly acted on GnRH neurons to modulate GnRH secretion via extracellular regulated protein kinases (ERK1/2) pathway.ConclusionsOverall, time‐restricted feeding improves the reproductive function of female mice and liver FGF21 signaling plays a key role in GnRH neuron activity in female mice.

Abstract 2550: Lack of FGF21 accelerates NASH-HCC transition via up-regulation of hepatic SPHK1-S1P-HIPPO signaling in murine models

Abstract Background: Non-alcoholic fatty liver disease(NAFLD) encompasses a broad spectrum of disorders, ranging from non-progressive bland steatosis to non-alcoholic hepatitis (NASH), cirrhosis, and malignant transformation into hepatocellular carcinoma (HCC). Accumulating evidence indicates that lipid metabolic reprogramming mediates the transition from NASH to HCC, however the NASH related carcinogenesis and role of FGF21 played during NASH-HCC transition are largely unknown. while fibroblast growth factor 21 (FGF21) exerts a key role in lipid homeostasis and play a promising therapeutic candidate for the treatment of obesity and diabetes. Aim: To investigate the effect of FGF21 on lipid metabolic reprogramming during the NASH-HCC carcinogenetic transformation and to reveal the potential NASH-HCC mechanism. Methods: Three NASH-HCC transition models were established in FGF21 knockout (KO) mice, with treatment of DEN at aged 2 weeks at 40 mg/kg body weight (i.p.) and feeding, 1) high fat diet (HFD, 60% kcal% fat); 2) western-style high fat diet (WSHFD, 60% kcal% fat and 19% kcal% fructose); and 3) methionine/choline-deficient diet (MCD). The cellular and molecular events, in turn, de novo lipogenesis, sphingolipid metabolism, white adipose tissue (WAT) lipolysis, insulin resistance (IR), release of free fatty acids (FFAs), metabolites of gut flora, carcinogenesis signaling pathways and NASH-HCC initiation, were determined both in vivo and in vitro. The in vitro studies were performed using shRNA-FGF21 knock down hepatocyte and HCC cell lines. Results: Aberrant lipid components, gut microbiome, hepatic Hippo-YAP and NF-kB signaling were found consistently in the NASH-HCC tissues from FGF21-KO mice with 3 diets treatments, compared to the wild type controls. Increased de novo lipogenesis, FFAs overflow to the liver, and inflammation agents from the metabolites of gut flora upregulated the hepatic sphingosine Kinase 1 (SPHK1) expression and its product sphingosine-1-phosphate (S1P), subsequently induced YAP dephosphorylation and nuclear localization. rhFGF21 or SPHK1 inhibitor (SKI-II) treatment significantly decreased the tumor volumes via alleviation of FFAs and S1P productions. Use of curcumin to modulate gut microbiota resulted in suppression of NASH-HCC growth in mice. Conclusion: 1) lack of FGF21 played a key role in hepatic lipid metabolism disorder in liver; 2) lack of FGF21 accelerated the SPHK1-S1P-HIPPO signaling promoted NASH-HCC transition; 3) administration of rhFGF21 not only attenuates NASH, but also prevents HCC carcinogenetic transformation via down-regulation of the SPHK1-S1P-HIPPO axis. This work was supported by an Institutional Development Award (IDeA) from the NIGMS of the National Institutes of Health under grant number P20GM113226. Key Words: Fibroblast growth factor 21; Steatohepatities; Hepatocellular carcinoma; Free fatty acid; Sphingolipid Citation Format: Xiaoju Shi, Robert C. Martin, Youxi Yu, Yujia Chen, Xingtong Wang, Guangyi Wang, Guoyue Lv, Ping Zhang, Yan Li. Lack of FGF21 accelerates NASH-HCC transition via up-regulation of hepatic SPHK1-S1P-HIPPO signaling in murine models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2550.

Abstract 299: Cardiac Fibroblast-derived FGF21 is a Novel Therapeutic Target for Cardiac Pathological Remodeling

Background: Fibroblast Growth factor 21(FGF21) is an endocrine factor, secreted mainly by the liver, that exerts metabolic actions. It has been shown that FGF21 has an anti-hypertrophic action for cardiac hypertrophy. We previously reported that murine cardiac FGF21 expression was upregulated by pressure-overload. The objective of this study was to clarify the role of cardiac fibroblast-derived FGF21 in cardiac pathological remodeling. Methods and Results: We generated fibroblast-specific/tamoxifen-inducible FGF21 knockout mice crossing by FGF21flox mice and Col1a2-CreERT mice (FB-FGF21KO). FGF21flox control mice and KO mice were induced cardiac hypertrophy by transverse aortic constriction (TAC). After 3-weeks TAC surgery, we evaluated the cardiac function by echocardiography or conductance catheter. TAC-mediated cardiac hypertrophy or impairment of systolic function were exacerbated in FB-FGF21KO mice. Conductance catheter showed that Tau, diastolic relaxation marker, was exacerbated in FB-FGF21KO mice with TAC. FB-FGF21KO heart with TAC showed significant upregulation in profibrotic genes and down-regulation in Ca2+ ATPase in comparison with control TAC heart. Cardiac Sirt1, which plays a central role in energy metabolism or oxidative stress, increased by TAC was significantly attenuated in FB-FGF21KO heart. Conclusion: Deletion of FGF21 in cardiac fibroblasts exacerbates cardiac dysfunction in response to pressure overload suggesting that cardiac fibroblasts regulates pathological remodeling via FGF21 regulation.

Fibroblast Growth Factor 21 Stimulates Pancreatic Islet Autophagy via Inhibition of AMPK-mTOR Signaling.

Background: Islet autophagy plays a role in glucose/lipid metabolism in type 2 diabetes mellitus. Meanwhile, fibroblast growth factor 21 (FGF21) has been found to regulate insulin sensitivity and glucose homeostasis. Whether FGF21 induces islet autophagy, remains to be elucidated. This study aimed to explore the physiological roles and signaling pathways involved in FGF21-stimulated islet autophagy under glucolipotoxic conditions. Methods: C57/BL6J mice were fed a standard diet or high-fat diet (HFD) for 12 weeks, and islets were isolated from normal and FGF21 knockout (KO) mice. Isolated islets and INS-1E cells were exposed to normal and high-concentration glucose and palmitic acid with/without FGF21 or AMPK inhibitor compound C. Real-time PCR, Western blot and immunohistochemistry/transmission electron microscopy were performed for the expression of targeted genes/proteins. Results: HFD-treated mice showed increases in fasting plasma glucose, body weight and impaired glucose tolerance; islet protein expression of FGF21 was induced after HFD treatment. Protein expression levels of FGF21 and LC3-II (autophagy marker) were induced in mouse islets treated with high concentrations of palmitic acid and glucose, while phosphorylation of AMPK was reduced, compared with controls. In addition, induction of LC3-II protein expression was reduced in islets isolated from FGF21 KO mice. Furthermore, exogenous administration of FGF21 diminished phosphorylation of AMPK and stimulated protein expression of LC3-II. Consistently, compound C significantly induced increased expression of LC3-II protein. Conclusions: Our data indicate that glucolipotoxicity-induced FGF21 activation mediates islet autophagy via AMPK inhibition, and further consolidate the evidence for the FGF21/analog being a pharmacotherapeutic target for obesity and its related T2DM.

Fibroblast growth factor 21 controls mitophagy and muscle mass

BackgroundSkeletal muscle is a plastic tissue that adapts to changes in exercise, nutrition, and stress by secreting myokines and myometabolites. These muscle‐secreted factors have autocrine, paracrine, and endocrine effects, contributing to whole body homeostasis. Muscle dysfunction in aging sarcopenia, cancer cachexia, and diabetes is tightly correlated with the disruption of the physiological homeostasis at the whole body level. The expression levels of the myokine fibroblast growth factor 21 (FGF21) are very low in normal healthy muscles. However, fasting, ER stress, mitochondrial myopathies, and metabolic disorders induce its release from muscles. Although our understanding of the systemic effects of muscle‐derived FGF21 is exponentially increasing, the direct contribution of FGF21 to muscle function has not been investigated yet.MethodsMuscle‐specific FGF21 knockout mice were generated to investigate the consequences of FGF21 deletion concerning skeletal muscle mass and force. To identify the mechanisms underlying FGF21‐dependent adaptations in skeletal muscle during starvation, the study was performed on muscles collected from both fed and fasted adult mice. In vivo overexpression of FGF21 was performed in skeletal muscle to assess whether FGF21 is sufficient per se to induce muscle atrophy.ResultsWe show that FGF21 does not contribute to muscle homeostasis in basal conditions in terms of fibre type distribution, fibre size, and muscle force. In contrast, FGF21 is required for fasting‐induced muscle atrophy and weakness. The mass of isolated muscles from control‐fasted mice was reduced by 15–25% (P < 0.05) compared with fed control mice. FGF21‐null muscles, however, were significantly protected from muscle loss and weakness during fasting. Such important protection is due to the maintenance of protein synthesis rate in knockout muscles during fasting compared with a 70% reduction in control‐fasted muscles (P < 0.01), together with a significant reduction of the mitophagy flux via the regulation of the mitochondrial protein Bnip3. The contribution of FGF21 to the atrophy programme was supported by in vivo FGF21 overexpression in muscles, which was sufficient to induce autophagy and muscle loss by 15% (P < 0.05). Bnip3 inhibition protected against FGF21‐dependent muscle wasting in adult animals (P < 0.05).ConclusionsFGF21 is a novel player in the regulation of muscle mass that requires the mitophagy protein Bnip3.

Glucagon-Like Peptide 1 Increases β-Cell Regeneration by Promoting α- to β-Cell Transdifferentiation.

Glucagon-like peptide 1 (GLP-1) can increase pancreatic β-cells, and α-cells could be a source for new β-cell generation. We investigated whether GLP-1 increases β-cells through α-cell transdifferentiation. New β-cells originating from non-β-cells were significantly increased in recombinant adenovirus expressing GLP-1 (rAd-GLP-1)-treated RIP-CreER;R26-YFP mice. Proliferating α-cells were increased in islets of rAd-GLP-1-treated mice and αTC1 clone 9 (αTC1-9) cells treated with exendin-4, a GLP-1 receptor agonist. Insulin+glucagon+ cells were significantly increased by rAd-GLP-1 or exendin-4 treatment in vivo and in vitro. Lineage tracing to label the glucagon-producing α-cells showed a higher proportion of regenerated β-cells from α-cells in rAd-GLP-1-treated Glucagon-rtTA;Tet-O-Cre;R26-YFP mice than rAd producing β-galactosidase-treated mice. In addition, exendin-4 increased the expression and secretion of fibroblast growth factor 21 (FGF21) in αTC1-9 cells and β-cell-ablated islets. FGF21 treatment of β-cell-ablated islets increased the expression of pancreatic and duodenal homeobox-1 and neurogenin-3 and significantly increased insulin+glucagon+ cells. Generation of insulin+glucagon+ cells by exendin-4 was significantly reduced in islets transfected with FGF21 small interfering RNA or islets of FGF21 knockout mice. Generation of insulin+ cells by rAd-GLP-1 treatment was significantly reduced in FGF21 knockout mice compared with wild-type mice. We suggest that GLP-1 has an important role in α-cell transdifferentiation to generate new β-cells, which might be mediated, in part, by FGF21 induction.