ABSTRACT
INTRODUCTION: Hepatitis B virus (HBV) infection is a global epidemic that can lead to several liver diseases, seriously affecting people's health. This study aimed to investigate the clinical potential of serum ß-klotho (KLB) as a promising biomarker in HBV-related liver diseases. METHODOLOGY: This study enrolled 30 patients with chronic hepatitis B (CHB), 35 with HBV-related cirrhosis, 66 with HBV-related hepatocellular carcinoma (HCC), and 48 healthy individuals. ELISA measured the levels of serum KLB in the four groups. We then compared the differences in serum KLB levels among the groups and analyzed the relationship between serum KLB and routine clinical parameters. RESULTS: The concentrations of serum KLB levels were increased sequentially among the healthy subjects, the HBV-related CHB group, the HBV-related cirrhosis group, and the HBV-related HCC group (p < 0.05). Expression of KLB was positively correlated with alpha-fetoprotein (AFP), total bilirubin, direct bilirubin, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl-transferase, alkaline phosphatase, total bile acid, serum markers for liver fibrosis, ascites, cirrhosis, splenomegaly, and model for end-stage liver disease sodium, while negatively correlated with platelet count, albumin, and prothrombin activity (p < 0.05). In addition, serum KLB has better sensitivity in diagnosing HCC than AFP, and serum KLB combined with AFP has higher sensitivity and specificity than AFP alone in diagnosing HCC. CONCLUSIONS: Serum KLB level is associated with the severity of HBV-related liver diseases and has important diagnostic value for HCC. Therefore, it could be a predictive biomarker for monitoring disease progression.
Subject(s)
Biomarkers , Carcinoma, Hepatocellular , Hepatitis B, Chronic , Klotho Proteins , Humans , Male , Female , Biomarkers/blood , Middle Aged , Adult , Hepatitis B, Chronic/blood , Hepatitis B, Chronic/complications , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/virology , Glucuronidase/blood , Liver Neoplasms/blood , Liver Neoplasms/diagnosis , Liver Neoplasms/virology , Liver Cirrhosis/blood , Liver Cirrhosis/diagnosis , Liver Cirrhosis/virology , Disease Progression , Enzyme-Linked Immunosorbent Assay , AgedABSTRACT
OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5'tRF transfer RNA fragments and microRNA miR-194-5p. METHODS: Combined use of diet induced obese mice with human-derived oleic acid-exposed Hep G2 cells revealed new NAFLD roles of LysTTT-5'tRF and miR-194-5p. RESULTS: Unlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5'tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5'tRF levels while increasing lipid accumulation. Inversely, transfecting fattened cells with a synthetic LysTTT-5'tRF mimic elevated mRNA levels of the metabolic regulator ß-Klotho while decreasing triglyceride amounts by 30% within 24 h. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5'tRF levels. CONCLUSION: Our findings highlight the different yet complementary roles of miR-194-5p and LysTTT-5'tRF and offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis.
Subject(s)
MicroRNAs , Non-alcoholic Fatty Liver Disease , Animals , Humans , Mice , Lysine , MicroRNAs/genetics , MicroRNAs/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Oleic Acid , Perilipin-2ABSTRACT
Metabolic functions of GLP-1 and its analogues have been extensively investigated. In addition to acting as an incretin and reducing body weight, we and others have suggested the existence of GLP-1/fibroblast growth factor 21 (FGF21) axis in which liver mediates certain functions of GLP-1 receptor agonists. In a more recent study, we found with surprise that four-week treatment with liraglutide but not semaglutide stimulated hepatic FGF21 expression in HFD-challenged mice. We wondered whether semaglutide can also improve FGF21 sensitivity or responsiveness and hence triggers the feedback loop in attenuating its stimulation on hepatic FGF21 expression after a long-term treatment. Here, we assessed effect of daily semaglutide treatment in HFD-fed mice for 7 days. HFD challenge attenuated effect of FGF21 treatment on its downstream events in mouse primary hepatocytes, which can be restored by 7-day semaglutide treatment. In mouse liver, 7-day semaglutide treatment stimulated FGF21 as well as genes that encode its receptor (FGFR1) and the obligatory co-receptor (KLB), and a battery of genes that are involved in lipid homeostasis. In epididymal fat tissue, expressions of a battery genes including Klb affected by HFD challenge were reversed by 7-day semaglutide treatment. We suggest that semaglutide treatment improves FGF21 sensitivity which is attenuated by HFD challenge.
Subject(s)
Diet, High-Fat , Fibroblast Growth Factors , Glucagon-Like Peptides , Hepatocytes , Animals , Mice , Fibroblast Growth Factors/metabolism , Hepatocytes/metabolism , Liver/metabolism , Mice, Inbred C57BL , Transcription Factors/metabolism , Glucagon-Like Peptides/pharmacologyABSTRACT
Age-related deterioration in the reproductive capacity of women is directly related to the poor developmental potential of ovarian follicles. Although telomerase plays a key role in female fertility, TERT-targeting therapeutic strategies for age-related female infertility have yet to be investigated. This study elucidated the effect of Telomerase activation on mice ovaries and more specifically on Klb (ß-Klotho) gene expression, which is linked to ageing, female hormonal regulation, and cyclicity. The homology-based 3D model of hTERT was used to predict its binding mode of Cycloastragenol (CAG) using molecular docking and molecular dynamics simulations. Based on docking score, simulation behavior, and interaction with hTERT residues it was observed that CAG could bind with the hTERT model. CAG treatment to primary cultured mouse granulosa cells and activation of telomerase was examined via telomerase activity assay (Mouse TE (telomerase) ELISA Kit) and telomere length by quantitative fluorescence in situ hybridization. CAG mediated telomerase also significantly improved ß-Klotho protein level in the aged granulosa cells. To demonstrate that ß-Klotho is telomerase dependent, the TERT was knocked down via siRNA in granulosa cells and protein level of ß-Klotho was examined. Furthermore, CAG-mediated telomerase activation significantly enhanced the level of Klb and recovered ovarian follicles in the D-galactose (D-gal)-induced ovarian ageing mouse model. Moreover, Doxorubicin-induced ovarian damage, which changes ovarian hormones, and inhibit follicular growth was successfully neutralized by CAG activated telomerase and its recovery of ß-Klotho level. In conclusion, TERT dependent ß-Klotho regulation in ovarian tissues is one of the mechanisms, which can overcome female infertility.
Subject(s)
Infertility, Female , Telomerase , Humans , Female , Mice , Animals , Telomerase/genetics , Telomerase/metabolism , In Situ Hybridization, Fluorescence , Klotho Proteins , Molecular Docking SimulationABSTRACT
Objective@#To examine the associations of fibroblast growth factor 19 (FGF19), its co-receptor KLB gene and its receptor FGFR4 with susceptibility to sarcopenia, so as to provide insights into elucidation of sarcopenia pathogenesis and formulation of precision interventions for sarcopenia.@*Methods@#A case-control study was conducted. Patients with sarcopenia at ages of 60 years and older included in the Zhejiang Provincial Elderly Health Surveillance Cohorts were selected as the sarcopenia group, and normal residents at ages of 60 years and older were served as controls. Subjects' demographics were collected using questionnaire surveys, and the height, body weight, appendicular skeletal muscle mass and grip strength were measured. Genomic DNA was extracted from blood samples for multiplex PCR targeted capture. The associations between the KLB gene single-nucleotide polymorphisms (SNPs) and susceptibility to sarcopenia were evaluated using multivariable logistic regression models. @*Results@#There were 200 cases in the sarcopenia group, including 91 men and 109 women, and 180 cases in the control group, including 70 men and 110 women. All SNPs satisfied the Hardy-Weinberg equilibrium, and the minor allele frequencies were all > 0.05. There were no significant differences in the distribution of SNPs between the sarcopenia and control groups (all P>0.05). Multivariable logistic regression analysis showed that the SNP rs2687968 locus in the KLB gene was significantly associated with the susceptibility to sarcopenia among the elderly men (superdominant model), and individuals carrying the AC allele had a 2.332-fold higher risk of sarcopenia than those carrying the AA/CC allele (95%CI: 1.882-3.313). @*Conclusions@#KLB gene may correlate with the susceptibility to sarcopenia among the elderly men.
ABSTRACT
Aberrant fibroblast growth factor 19 (FGF19) signaling mediated by its receptor, FGF receptor 4 (FGFR4), and coreceptor, klotho ß (KLB), is a driver of hepatocellular carcinoma (HCC). Several potent FGFR4-selective inhibitors have been developed but have exhibited limited efficacy in HCC clinical trials. Here, by using HCC cell line models from the Cancer Cell Line Encyclopedia (CCLE) and the Liver Cancer Model Repository (LIMORE), we show that selective FGFR4 inactivation was not sufficient to inhibit cancer cell proliferation and tumor growth in FGF19-positive HCC. Moreover, genetic inactivation of KLB in these HCC cells resulted in a fitness defect more severe than that resulting from inactivation of FGFR4. By a combination of biochemical and genetic approaches, we found that KLB associated with FGFR3 and FGFR4 to mediate the prosurvival functions of FGF19. KLB mutants defective in interacting with FGFR3 or FGFR4 could not support the growth or survival of HCC cells. Genome-wide CRISPR loss-of-function screening revealed that FGFR3 restricted the activity of FGFR4-selective inhibitors in inducing cell death; the pan-FGFR inhibitor erdafitinib displayed superior potency than FGFR4-selective inhibitors in suppressing the growth and survival of FGF19-positive HCC cells. Among FGF19-positive HCC cases from The Cancer Genome Atlas (TCGA), FGFR3 is prevalently coexpressed with FGFR4 and KLB, suggesting that FGFR redundancy may be a common mechanism underlying the de novo resistance to FGFR4 inhibitors. Our study provides a rationale for clinical testing of pan-FGFR inhibitors as a treatment strategy for FGF19-positive HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Proliferation , Fibroblast Growth Factors/metabolism , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Receptor, Fibroblast Growth Factor, Type 4/genetics , Receptor, Fibroblast Growth Factor, Type 4/metabolismABSTRACT
BACKGROUND: Deregulation of FGF19-FGFR4 signaling is found in several cancers, including hepatocellular carcinoma (HCC), nominating it for therapeutic targeting. FGF401 is a potent, selective FGFR4 inhibitor with antitumor activity in preclinical models. This study was designed to determine the recommended phase 2 dose (RP2D), characterize PK/PD, and evaluate the safety and efficacy of FGF401 alone and combined with the anti-PD-1 antibody, spartalizumab. METHODS: Patients with HCC or other FGFR4/KLB expressing tumors were enrolled. Dose-escalation was guided by a Bayesian model. Phase 2 dose-expansion enrolled patients with HCC from Asian countries (group1), non-Asian countries (group2), and patients with other solid tumors expressing FGFR4 and KLB (group3). FGF401 and spartalizumab combination was evaluated in patients with HCC. RESULTS: Seventy-four patients were treated in the phase I with single-agent FGF401 at 50 to 150 mg. FGF401 displayed favorable PK characteristics and no food effect when dosed with low-fat meals. The RP2D was established as 120 mg qd. Six of 70 patients experienced grade 3 dose-limiting toxicities: increase in transaminases (n = 4) or blood bilirubin (n = 2). In phase 2, 30 patients in group 1, 36 in group 2, and 20 in group 3 received FGF401. In total, 8 patients experienced objective responses (1 CR, 7 PR; 4 each in phase I and phase II, respectively). Frequent adverse events (AEs) were diarrhea (73.8%), increased AST (47.5%), and ALT (43.8%). Increase in levels of C4, total bile acid, and circulating FGF19, confirmed effective FGFR4 inhibition. Twelve patients received FGF401 plus spartalizumab. RP2D was established as FGF401 120 mg qd and spartalizumab 300 mg Q3W; 2 patients reported PR. CONCLUSIONS: At biologically active doses, FGF401 alone or combined with spartalizumab was safe in patients with FGFR4/KLB-positive tumors including HCC. Preliminary clinical efficacy was observed. Further clinical evaluation of FGF401 using a refined biomarker strategy is warranted. TRIAL REGISTRATION: NCT02325739 .
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Antibodies, Monoclonal, Humanized , Bayes Theorem , Biomarkers , Carcinoma, Hepatocellular/drug therapy , Humans , Liver Neoplasms/drug therapy , Piperazines , PyridinesABSTRACT
Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal muscle in IUGR lead to increased risk of premature muscle loss and metabolic disease in later life. Here, we identified ß-Klotho (KLB), a fibroblast growth factor 21 (FGF21) co-receptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturally-occurring disease model, we performed transcriptome-wide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normal-weight (NW) littermates. We found that, alongside large-scale transcriptional changes comprising multiple developmental, tissue injury and metabolic gene pathways, KLB was increased in IUGR muscle. Moreover, FGF21 concentrations were increased in plasma in IUGR fetuses. Using cultures of fetal muscle progenitor cells (MPCs), we showed reduced myogenic capacity of IUGR compared to NW muscle in vitro, as evidenced by differences in fusion indices and myogenic transcript levels, as well as mechanistic target of rapamycin (mTOR) activity. Moreover, transfection of MPCs with KLB small interfering RNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose-dependent effects in porcine and also in human fetal MPCs. In conclusion, our results identify KLB as a novel and potentially critical mediator of impaired muscle development in IUGR, through conserved mechanisms in pigs and humans. Our data shed new light onto the pathogenesis of IUGR, a significant cause of lifelong ill-health in humans and animals. KEY POINTS: Intrauterine growth restriction (IUGR) is associated with large-scale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle. Levels of the fibroblast growth factor 21 (FGF21) co-receptor, ß-Klotho (KLB) are increased in IUGR fetal muscle, and FGF21 concentrations are increased in IUGR fetal plasma. KLB mediates a reduction in muscle development through inhibition of mechanistic target of rapamycin signalling. These effects of KLB on muscle cells are conserved in pig and human, suggesting a vital role of this protein in the regulation of muscle development and function in mammals.
Subject(s)
Fetal Growth Retardation , Muscle Development , Animals , Female , Mammals , Muscle, Skeletal/metabolism , Pregnancy , Signal Transduction , SwineABSTRACT
Diphtheria is an acute infectious upper respiratory tract disease caused by toxigenic strains of Corynebacterium diphtheria and can lead to significant morbidity and mortality in all the age groups. Most of the time diagnosis of diphtheria is clinical. There may be a dirty white patch covering one or both tonsils on examination for which throat swabs are collected for Kleb's-Loeffler's Bacillus (KLB) by direct microscopy and for culture and sensitivity of the organism. To find out the association between clinical diagnosis of diphtheria with smear and culture positivity. 674 cases of Clinical diphtheria were admitted from June 2017 to September 2020 at a tertiary care hospital, Sawai Mansingh Hospital, Jaipur. throat, difficulty in swallowing and swelling in the neck. Out of 674 patients of clinical diphtheria, majority 610 cases (90.5%) were found to have both KLB smear and culture negative. 13 cases (1.9%) were found to have both KLB smear and culture positive. 19 cases (2.8%) were found to have KLB smear positive and culture negative and remaining 32 cases (4.8%) were found to have KLB smear negative and culture positive. Out of 19 patients of KLB smear positive,11 cases (3.5%) were found to have complications. Out of 32 culture positive patients,24 cases (7.7%) were found to have complications. Out of 13 patients of both KLB smear and culture positive,11 cases (3.5%) were found to have complications. Our study concluded that the negative report of KLB smear and culture does not rule out diphtheria and it is evident that percentage of complication is high in patients with either KLB smear or culture or both positive with respect to both being negative. The correlation is found to be significant (p < 0.001).
ABSTRACT
BACKGROUND: We aimed to investigate the ß-klotho (KLB) expression in non-small cell lung cancer (NSCLC) and to determine its value as a novel molecular target for survival prognosis in patients with NSCLC. METHODS: The serum KLB concentrations in 50 patients with NSCLC and the 20 healthy persons were measured by enzyme-linked immunosorbent assay (ELISA) methods. The relationship between serum KLB level, including the level change after therapy, and the progression-free survival (PFS) and overall survival (OS) were analyzed. The KLB expression in A549 cells was measured by real-time polymerase chain reaction (RT-PCR) and western blotting. The function of cells was revealed by in vitro studies. RESULTS: The concentrations of serum KLB in patients with NSCLC were obviously lower than those in healthy subjects. KLB expression was significantly increased in patients after chemotherapy and epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) targeted therapy. In addition, expression of KLB was positively related with PFS and OS. Compared with 16-human bronchial epithelial (HBE) cells, the expression level of KLB was significantly decreased in A549 cells. Overexpression of KLB suppressed the proliferation of A549 cells, along with G1-to-S phase arrest and apoptosis induction. CONCLUSIONS: KLB plays an anti-tumorigenic role in NSCLC. KLB may be a candidate target for the diagnosis and treatment of NSCLC and may serve a potentially significant role in future clinical applications.
ABSTRACT
Hepatoblastoma (HB) is the most frequent pediatric liver malignancy. However, the treatment outcome for patients with advanced-stage HB remains unsatisfactory. Accumulating evidence indicates that ßKlotho (KLB) acts as an oncogene or a tumor-suppressor gene in a context-dependent manner. Despite this, the expression profile and effects of KLB on the growth of HB are still elusive. This study aimed to explore the effect of miR-206/KLB axis on HB growth. The expression of KLB was explored in HB cells (HepG2 and HuH6) and tissues using quantitative polymerase chain reaction (qPCR), Western blot analysis, and immunohistochemistry. Besides, miR-206 expression was determined in HB cells and tissues using qPCR and fluorescence in situ hybridization. The prognostic value of KLB or miR-206 in our patients with HB was investigated using the Kaplan-Meier method. The biological effects of KLB or miR-206 on HB cells were identified in vitro. The proliferative effects of KLB on HuH6 cells were also investigated in vivo. Moreover, the mechanical signaling of KLB in HB was determined through bioinformatics analysis followed by experimental validation. The results showed a significant upregulation of KLB in HB tissues and cells. Elevated level of KLB was found to be significantly correlated with the aggressive phenotype and poor overall survival for children with HB. The in vitro function assay demonstrated that KLB knockdown promoted apoptosis and suppressed the proliferation, migration, and invasion of HB cells. Besides, KLB knockdown inhibited the proliferation of HuH6 cells in vivo, while KLB overexpression had the opposite effect. Furthermore, KLB was proved to be the direct target of miR-206. Low level of miR-206 served as an independent risk factor for poor prognosis in children with HB. The overexpression of miR-206 negatively regulated the aggressive biological behaviors of HB cells, which was partially rescued by KLB overexpression. Mechanically, the miR-206/KLB axis played a vital role in HB growth through augmenting the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling. In conclusion, the data demonstrated that the miR-206/KLB axis might serve as an important biomarker/therapeutic target for HB.
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BACKGROUND: Kallmann syndrome (KS) is a hypogonadotropic hypogonadism accompanied by anosmia or hyposmia. It is associated with the low secretion of gonadotropins which can lead to other abnormal endocrine metabolism disorders such as diabetes. Through genetic and molecular biological methods, more than 10 KS pathogenic genes have been found. AIM: To identify the existing mutation sites of KS with diabetes and reveal the relationship between genotype and phenotype. METHODS: We studied KS pathogenesis through high-throughput exome sequencing on four diabetes' patients with KS for screening the potential pathogenic sites and exploring the genotype-phenotype correlation. Clinical data and peripheral blood samples were collected from the patients. White blood cells were separated and genomic DNA was extracted. High-throughput sequencing of all exons in the candidate pathogenic genes of probands was performed, and the results obtained were analyzed. RESULTS: Sequencing revealed mutations in the KLB p.T313M, ANOS1 p.C172F, and IGSF10 gene (p.Lys1819Arg and p.Arg1035Thr) at different sites, which may have been associated with disease onset. CONCLUSION: The diagnosis of KS is challenging, especially in early puberty, and the clinical manifestations reflect physical delays in development and puberty. Timely diagnosis and treatment can induce puberty, thereby improving sexual, bone, metabolic and mental health.
ABSTRACT
BACKGROUND & AIMS: Obesity is a risk factor for pancreatic cancer. In mice, a high-fat diet (HFD) and expression of oncogenic KRAS lead to development of invasive pancreatic ductal adenocarcinoma (PDAC) by unknown mechanisms. We investigated how oncogenic KRAS regulates the expression of fibroblast growth factor 21, FGF21, a metabolic regulator that prevents obesity, and the effects of recombinant human FGF21 (rhFGF21) on pancreatic tumorigenesis. METHODS: We performed immunohistochemical analyses of FGF21 levels in human pancreatic tissue arrays, comprising 59 PDAC specimens and 45 nontumor tissues. We also studied mice with tamoxifen-inducible expression of oncogenic KRAS in acinar cells (KrasG12D/+ mice) and fElasCreERT mice (controls). KrasG12D/+ mice were placed on an HFD or regular chow diet (control) and given injections of rhFGF21 or vehicle; pancreata were collected and analyzed by histology, immunoblots, quantitative polymerase chain reaction, and immunohistochemistry. We measured markers of inflammation in the pancreas, liver, and adipose tissue. Activity of RAS was measured based on the amount of bound guanosine triphosphate. RESULTS: Pancreatic tissues of mice expressed high levels of FGF21 compared with liver tissues. FGF21 and its receptor proteins were expressed by acinar cells. Acinar cells that expressed KrasG12D/+ had significantly lower expression of Fgf21 messenger RNA compared with acinar cells from control mice, partly due to down-regulation of PPARG expression-a transcription factor that activates Fgf21 transcription. Pancreata from KrasG12D/+ mice on a control diet and given injections of rhFGF21 had reduced pancreatic inflammation, infiltration by immune cells, and acinar-to-ductal metaplasia compared with mice given injections of vehicle. HFD-fed KrasG12D/+ mice given injections of vehicle accumulated abdominal fat, developed extensive inflammation, pancreatic cysts, and high-grade pancreatic intraepithelial neoplasias (PanINs); half the mice developed PDAC with liver metastases. HFD-fed KrasG12D/+ mice given injections of rhFGF21 had reduced accumulation of abdominal fat and pancreatic triglycerides, fewer pancreatic cysts, reduced systemic and pancreatic markers of inflammation, fewer PanINs, and longer survival-only approximately 12% of the mice developed PDACs, and none of the mice had metastases. Pancreata from HFD-fed KrasG12D/+ mice given injections of rhFGF21 had lower levels of active RAS than from mice given vehicle. CONCLUSIONS: Normal acinar cells from mice and humans express high levels of FGF21. In mice, acinar expression of oncogenic KRAS significantly reduces FGF21 expression. When these mice are placed on an HFD, they develop extensive inflammation, pancreatic cysts, PanINs, and PDACs, which are reduced by injection of FGF21. FGF21 also reduces the guanosine triphosphate binding capacity of RAS. FGF21 might be used in the prevention or treatment of pancreatic cancer.
Subject(s)
Acinar Cells/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Cell Transformation, Neoplastic/metabolism , Diet, High-Fat , Fibroblast Growth Factors/metabolism , Pancreatic Intraductal Neoplasms/metabolism , Pancreatic Neoplasms/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Acinar Cells/pathology , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/prevention & control , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Down-Regulation , Fibroblast Growth Factors/genetics , Gene Expression Regulation, Neoplastic , Humans , Klotho Proteins , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Transgenic , Mutation , PPAR gamma/genetics , PPAR gamma/metabolism , Pancreatic Cyst/genetics , Pancreatic Cyst/metabolism , Pancreatic Cyst/pathology , Pancreatic Intraductal Neoplasms/genetics , Pancreatic Intraductal Neoplasms/pathology , Pancreatic Intraductal Neoplasms/prevention & control , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/prevention & control , Pancreatitis/genetics , Pancreatitis/metabolism , Pancreatitis/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Reduced dietary protein intake induces adaptive physiological changes in macronutrient preference, energy expenditure, growth, and glucose homeostasis. We demonstrate that deletion of the FGF21 co-receptor ßKlotho (Klb) from the brain produces mice that are unable to mount a physiological response to protein restriction, an effect that is replicated by whole-body deletion of FGF21. Mice forced to consume a low-protein diet exhibit reduced growth, increased energy expenditure, and a resistance to diet-induced obesity, but the loss of FGF21 signaling in the brain completely abrogates that response. When given access to a higher protein alternative, protein-restricted mice exhibit a shift toward protein-containing foods, and central FGF21 signaling is essential for that response. FGF21 is an endocrine signal linking the liver and brain, which regulates adaptive, homeostatic changes in metabolism and feeding behavior during protein restriction.
Subject(s)
Brain/metabolism , Diet, Protein-Restricted , Feeding Behavior , Fibroblast Growth Factors/metabolism , Liver/metabolism , Adipose Tissue/metabolism , Animals , Diet, High-Fat/adverse effects , Fibroblast Growth Factors/genetics , Food Preferences/physiology , Glucose Intolerance/metabolism , Insulin Resistance , Klotho Proteins , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/metabolismABSTRACT
Endothelial-mesenchymal transition (EndMT) has emerged as an essential bioprocess responsible for the development of organ fibrosis. We have previously reported that fibroblast growth factor receptor 1 (FGFR1) is involved in the anti-EndMT effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). FGFR1 is expressed on the cell membrane and performs its biological function through interaction with co-receptors, including ßklotho (KLB). However, it remains unknown whether KLB is involved in the anti-EndMT effects of AcSDKP. Here, we demonstrated that AcSDKP increased KLB expression in an FGFR1-dependent manner and that KLB deficiency induced AcSDKP-resistant EndMT via the induction of the mitogen-activated protein kinase (MAPK) pathway. In cultured endothelial cells, AcSDKP increased KLB protein level in an FGFR1-dependent manner through induction of the FGFR1-KLB complex. KLB suppression by small interfering RNA transfection did not affect FGFR1 levels and resulted in the induction of EndMT. In contrast to the EndMT observed under FGFR1 deficiency, the EndMT induced by KLB suppression was not accompanied by the induction of Smad3 phosphorylation; instead, KLB-deficient cells exhibited induced activation of the MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) and ERK pathways. Treatment with the specific MEK inhibitor U0126 diminished KLB deficiency-induced EndMT. Consistent with this finding, AcSDKP did not suppress either EndMT or MEK/ERK activation induced by KLB deficiency. Application of either FGF19 or FGF21 synergistically augmented the anti-EndMT effects of AcSDKP. Taken together, these results indicate that endogenous peptide AcSDKP exerts its activity through induction of the FGFR1-KLB complex in vascular endothelial cells.
Subject(s)
Epithelial-Mesenchymal Transition/drug effects , Growth Inhibitors/pharmacology , Membrane Proteins/genetics , Oligopeptides/pharmacology , Animals , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Klotho Proteins , Male , Membrane Proteins/metabolism , MiceABSTRACT
Exercise promotes adipose remodeling and improves obesity-induced metabolic disorders through mechanisms that remain obscure. Here, we identify the FGF21 signaling in adipose tissues as an obligatory molecular transducer of exercise conferring its metabolic benefits in mice. Long-term high fat diet-fed obese mice exhibit compromised effects of exogenous FGF21 on alleviation of hyperglycemia, hyperinsulinemia, and hyperlipidemia, accompanied with markedly reduced expression of FGF receptor-1 (FGFR1) and ß-Klotho (KLB) in adipose tissues. These impairments in obese mice are reversed by treadmill exercise. Mice lacking adipose KLB are refractory to exercise-induced alleviation of insulin resistance, glucose dysregulation, and ectopic lipid accumulation due to diminished adiponectin production, excessive fatty acid release, and enhanced adipose inflammation. Mechanistically, exercise induces the adipose expression of FGFR1 and KLB via peroxisome proliferator-activated receptor-gamma-mediated transcriptional activation. Thus, exercise sensitizes FGF21 actions in adipose tissues, which in turn sends humoral signals to coordinate multi-organ crosstalk for maintaining metabolic homeostasis.
Subject(s)
Adipokines/metabolism , Adipose Tissue/metabolism , Diet, High-Fat/adverse effects , Exercise/physiology , Fibroblast Growth Factors/metabolism , Obesity/complications , Physical Conditioning, Animal/methods , Animals , Humans , Male , MiceABSTRACT
BACKGROUND AND AIMS: Fibroblast growth factor 19 (FGF19) and fibroblast growth factor receptor 4 (FGFR4) signalling play critical roles in hepatocarcinogenesis. This study explored the potential of FGF19- and FGFR4-related biomarkers in predicting early tumour recurrence (ETR) and survival in patients with resectable hepatocellular carcinoma (HCC). METHODS: We examined the mRNA expressions of FGF19, FGFR4, klotho-beta (KLB), cyclin D1 (CCND1) and FGF4 in 151 surgically resected, primary unifocal HCCs through quantitative real-time polymerase chain reaction. Generalized additive models were fitted to detect nonlinear effects of continuous covariates and define thresholds of biomarker expressions. Univariate and multivariate analyses were performed to evaluate prognostic values of these biomarkers for tumour recurrence and patient survival. RESULTS: Overexpression of FGF19, FGFR4, KLB, CCND1 and FGF4 mRNA was detected in 40%, 32%, 26%, 15% and 35% of 151 tumours respectively. ETR was the strongest prognostic factor predicting worse overall survival (hazard ratio [HR], 5.678; 95% confidence interval, 3.7-8.713; P < 0.001). Furthermore, we revealed that mRNA expression levels of KLB (HR, 3.857; P = 0.021) and FGF19 (HR, 3.248; P = 0.017) were significantly associated with the occurrence of ETR. CONCLUSIONS: Frequent overexpression of FGF19/FGFR4-related biomarkers was detected in resectable HCC. Expression levels of KLB and FGF19 may determine patient survival outcomes through their effects on ETR.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Fibroblast Growth Factors/metabolism , Liver Neoplasms/metabolism , Membrane Proteins/metabolism , Neoplasm Recurrence, Local/metabolism , Adult , Aged , Aged, 80 and over , Carcinogenesis , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/surgery , Cell Proliferation/drug effects , Female , Fibroblast Growth Factors/genetics , Humans , Klotho Proteins , Liver Neoplasms/mortality , Liver Neoplasms/surgery , Logistic Models , Male , Membrane Proteins/genetics , Middle Aged , Neoplasm Recurrence, Local/genetics , Prognosis , Receptor, Fibroblast Growth Factor, Type 4/genetics , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Signal Transduction/drug effects , Survival Analysis , Taiwan , Young AdultABSTRACT
Previous studies suggest that specific binding to the complex consisting of fibroblast growth factor receptor-1 (FGFR1) and the coreceptor beta-Klotho (KLB) is the premise for human FGF19 and FGF21 activating the downstream signaling cascades, and regulating the metabolic homeostasis. However, it was found that human FGF21 loses its ability to bind to FGFR1-KLB after iodination with Na125 I and chloramine T, whereas human FGF19 retained its affinity for FGFR1-KLB even after iodination. The molecular mechanisms underlying these differences remained elusive. In this study, we first demonstrated that an intramolecular disulfide bond was formed between cysteine-102 and cysteine-121 in FGF21, implying that the oxidation of the cysteine to cysteic acid, which may interfere with the active conformation of FGF21, did not occur during the iodination procedures, and thus ruled out the possibility of the two conserved cysteine residues mediating the loss of FGF21 binding affinity to FGFR1-KLB upon iodination. Site-directed mutagenesis and molecular modeling were further applied to determine the residue(s) responsible for the loss of FGFR1-KLB affinity. The results showed that mutation of a single tyrosine-207, but not the other five tyrosine residues in FGF21, to a phenylalanine retained the FGFR1-KLB affinity of FGF21 even after iodination, whereas replacing the corresponding phenylalanine residue with tyrosine in FGF19 did not alter its binding affinity to FGFR1-KLB, but decreased the receptor binding ability of the iodinated protein, suggesting that tyrosine-207 is the crucial amino acid responsible for the loss of specifying FGFR1-KLB affinity of the iodinated FGF21.
Subject(s)
Fibroblast Growth Factors/genetics , Membrane Proteins/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Amino Acids/drug effects , Amino Acids/genetics , Cell Line , Chloramines/pharmacology , Fibroblast Growth Factors/drug effects , Halogenation , Homeostasis/genetics , Humans , Klotho Proteins , Oxidation-Reduction/drug effects , Phenylalanine/genetics , Protein Binding/drug effects , Signal Transduction/drug effects , Sodium Iodide/pharmacology , Tosyl Compounds/pharmacology , Tyrosine/drug effectsABSTRACT
OBJECTIVE: To signal, FGF19 and FGF21 require co-receptor ßKlotho (KLB) to act in concert with FGF receptors, and yet there is appreciable variance in the C-terminal sequences of these two novel metabolic hormones where binding is believed to be primary. We seek to determine the functional consequences for these amino acid differences and determine whether such information can be used to design high potency antagonists and agonists. METHODS: We employed a functional in vitro assay to identify C-terminal protein fragments capable of fully blocking KLB-mediated FGF19 and 21 receptor signaling. The key residues in each hormone responsible for support full bioactivity were identified through peptide-based Ala-scanning. Chemical optimization of the peptides was employed to increase their antagonistic potency. An optimized sequence as a substituted part of a full length FGF21 was assessed for enhanced FGFR/KLB-mediated agonism using tissue culture and obese mice. RESULTS: C-terminal FGF19 and FGF21 peptides of relatively short length were observed to potently inhibit the activity of these two hormones, in vitro and in vivo. These FGFs of different sequence also demonstrated a striking conservation of structural determinants to maintain KLB binding. A single C-terminal amino acid in FGF19 was observed to modulate relative activity through FGFR1 and FGFR4. The substitution of native FGF21 C-terminal sequence with a peptide optimized for the highest antagonistic activity resulted in significantly enhanced FGF potency, as measured by in vitro signaling and improvements in metabolic outcomes in diet-induced obese mice. CONCLUSIONS: We report here the ability of short C-terminal peptides to bind KLB and function as antagonists of FGF19 and 21 actions. These proteins maintain high conservation of sequence in those residues central to KLB binding. An FGF21 chimeric protein possessing an optimized C-terminal sequence proved to be a super-agonist in delivery of beneficial metabolic effects in obese mice.
Subject(s)
Fibroblast Growth Factors/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Animals , Fibroblast Growth Factors/antagonists & inhibitors , Fibroblast Growth Factors/physiology , Glucuronidase , HEK293 Cells , Humans , Klotho Proteins , Liver , Male , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Mice, Obese , Peptides , Phosphorylation , Receptor, Fibroblast Growth Factor, Type 1 , Signal TransductionABSTRACT
INTRODUCTION: bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Factor Receptor 1 (FGFR1) half-antibody to an anti-Klothoß (KLB) half-antibody, using the knobs-into-holes strategy. bFKB1 acts as a highly selective agonist for the FGFR1/KLB receptor complex and is intended to ameliorate obesity-associated metabolic defects by mimicking the activity of the hormone FGF21. An important aspect of the biologics product manufacturing process is to establish meaningful product specifications regarding the tolerable levels of impurities that copurify with the drug product. The aim of the current study was to determine acceptable levels of product-related impurities for bFKB1. METHODS: To determine the tolerable levels of these impurities, we dosed obese mice with bFKB1 enriched with various levels of either HMW impurities or anti-FGFR1-related impurities, and measured biomarkers for KLB-independent FGFR1 signaling. RESULTS: Here, we show that product-related impurities of bFKB1, in particular, high molecular weight (HMW) impurities and anti-FGFR1-related impurities, when purposefully enriched, stimulate FGFR1 in a KLB-independent manner. By taking this approach, the tolerable levels of product-related impurities were successfully determined. DISCUSSION: Our study demonstrates a general pharmacology-guided approach to setting a product specification for a bispecific antibody whose homomultimer-related impurities could lead to undesired biological effects.