RESUMO
Insulin resistance is a major public health burden that often results in other comorbidities including type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), and cardiovascular disease. An insulin sensitizer has the potential to become a disease-modifying therapy. It remains an unmet medical need to identify therapeutics that target the insulin signaling pathway to treat insulin resistance. Low-molecular-weight protein tyrosine phosphatase (LMPTP) negatively regulates insulin signaling and has emerged as a potential therapeutic target for insulin sensitization. Genetic studies have demonstrated that LMPTP is positively associated with obesity in humans and promotes insulin resistance in rodents. A recent study showed that pharmacological inhibition or genetic deletion of LMPTP protects mice from high-fat diet-induced insulin resistance and diabetes. Here, we show that loss of LMPTP by genetic deletion has no significant effects on improving glucose tolerance in lean or diet-induced obese mice. Furthermore, our data demonstrate that LMPTP deficiency potentiates cardiac hypertrophy that leads to mild cardiac dysfunction. Our findings suggest that the development of LMPTP inhibitors for the treatment of insulin resistance and type 2 diabetes should be reevaluated, and further studies are needed to characterize the molecular and pathophysiological role of LMPTP.NEW & NOTEWORTHY Inhibition of LMPTP with a small-molecule inhibitor, Cmpd23, improves glucose tolerance in mice as reported earlier. However, genetic deficiency of the LMPTP-encoding gene, Acp1, has limited effects on glucose metabolism but leads to mild cardiac hypertrophy in mice. The findings suggest the potential off-target effects of Cmpd23 and call for reevaluation of LMPTP as a therapeutic target for the treatment of insulin resistance and type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica , Glucose/metabolismo , Insulina/metabolismo , Resistência à Insulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/uso terapêutico , MagrezaRESUMO
The endocrine hormone fibroblast growth factor 21 (FGF21) is a powerful modulator of glucose and lipid metabolism and a promising drug for type 2 diabetes. Here we identify FGF21 as a potent regulator of skeletal homeostasis. Both genetic and pharmacologic FGF21 gain of function lead to a striking decrease in bone mass. In contrast, FGF21 loss of function leads to a reciprocal high-bone-mass phenotype. Mechanistically, FGF21 inhibits osteoblastogenesis and stimulates adipogenesis from bone marrow mesenchymal stem cells by potentiating the activity of peroxisome proliferator-activated receptor γ (PPAR-γ). Consequently, FGF21 deletion prevents the deleterious bone loss side effect of the PPAR-γ agonist rosiglitazone. Therefore, FGF21 is a critical rheostat for bone turnover and a key integrator of bone and energy metabolism. These results reveal that skeletal fragility may be an undesirable consequence of chronic FGF21 administration.
Assuntos
Reabsorção Óssea/patologia , Fatores de Crescimento de Fibroblastos/metabolismo , PPAR gama/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Reabsorção Óssea/metabolismo , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Resistência a Medicamentos/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/administração & dosagem , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Camundongos , Camundongos Knockout , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Rosiglitazona , Tiazolidinedionas/farmacologiaRESUMO
The liver plays a crucial role in mobilizing energy during nutritional deprivation. During the early stages of fasting, hepatic glycogenolysis is a primary energy source. As fasting progresses and glycogen stores are depleted, hepatic gluconeogenesis and ketogenesis become major energy sources. Here, we show that fibroblast growth factor 21 (FGF21), a hormone that is induced in liver by fasting, induces hepatic expression of peroxisome proliferator-activated receptor gamma coactivator protein-1alpha (PGC-1alpha), a key transcriptional regulator of energy homeostasis, and causes corresponding increases in fatty acid oxidation, tricarboxylic acid cycle flux, and gluconeogenesis without increasing glycogenolysis. Mice lacking FGF21 fail to fully induce PGC-1alpha expression in response to a prolonged fast and have impaired gluconeogenesis and ketogenesis. These results reveal an unexpected relationship between FGF21 and PGC-1alpha and demonstrate an important role for FGF21 in coordinately regulating carbohydrate and fatty acid metabolism during the progression from fasting to starvation.
Assuntos
Adaptação Fisiológica/fisiologia , Metabolismo dos Carboidratos , Ácidos Graxos/metabolismo , Fatores de Crescimento de Fibroblastos/fisiologia , Inanição/fisiopatologia , Transativadores/metabolismo , Adaptação Fisiológica/genética , Animais , Glicemia/metabolismo , Western Blotting , Peso Corporal/genética , Peso Corporal/fisiologia , Jejum/sangue , Jejum/fisiologia , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/genética , Insulina/sangue , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inanição/sangue , Transativadores/genética , Fatores de Transcrição , Triglicerídeos/sangueRESUMO
Insulin resistance increases patients' risk of developing type 2 diabetes (T2D), non-alcoholic steatohepatitis (NASH) and a host of other comorbidities including cardiovascular disease and cancer. At the molecular level, insulin exerts its function through the insulin receptor (IR), a transmembrane receptor tyrosine kinase. Data from human genetic studies have shown that Grb14 functions as a negative modulator of IR activity, and the germline Grb14-knockout (KO) mice have improved insulin signaling in liver and skeletal muscle. Here, we show that Grb14 knockdown in liver, white adipose tissues, and heart with an AAV-shRNA (Grb14-shRNA) improves glucose homeostasis in diet-induced obese (DIO) mice. A previous report has shown that germline deletion of Grb14 in mice results in cardiac hypertrophy and impaired systolic function, which could severely limit the therapeutic potential of targeting Grb14. In this report, we demonstrate that there are no significant changes in cardiac function as measured by echocardiography in the Grb14-knockdown mice fed a high-fat diet for a period of four months. While additional studies are needed to further confirm the efficacy and to de-risk potential negative cardiac effects in preclinical models, our data support the therapeutic strategy of inhibiting Grb14 to treat diabetes and related conditions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glucose/metabolismo , Homeostase , Insulina/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Técnicas de Silenciamento de Genes , Insulina/genética , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/metabolismoRESUMO
Zearalenone is a mycoestrogen that is produced in the fungi Fusarium graminearum, Fusarium culmorum, Fusarium equiseti, and Fusarium crookwellense. These fungi commonly exist in agricultural products. Human pregnane X receptor (hPXR) is a ligand-activated transcription factor that regulates the expression of numerous hepatic drug-metabolizing enzymes, including several clinically important cytochrome P450s. In this report, we show that zearalenone is an efficacious ligand for hPXR. We also describe the creation and validation of a novel adenoviral-mediated transduction protocol used to express functional FLAG-tagged-hPXR protein in a transformed cell line (HepG2) and primary cell types (cultured hepatocytes). Treatment of hPXR-transduced HepG2 cells with zearalenone induces expression of CYP3A4, the "prototypical" PXR-target gene in human liver. Treatment of hPXR-transduced cultured hepatocytes isolated from PXR-knockout mice with zearalenone induces the expression of Cyp3a11, the prototypical murine hepatic PXR-target gene. Using mammalian two-hybrid assays, we show that zearalenone displaces the nuclear receptor corepressor protein N-CoR from hPXR, while it recruits coactivator proteins steroid receptor coactivator-1, Glucocorticoid Receptor-Interacting Protein 1 and PPAR-Binding protein (GRIP1) and PBP to hPXR. Concentration-response analysis using a PXR-responsive reporter gene assay reveals that zearalenone activates hPXR with an EC50 value of approximately 1.5 microM. Because activation of hPXR represents the molecular basis of an important class of drug interactions, our findings suggest that studies to investigate the potential of zearalenone to induce the metabolism of other drugs in humans are warranted. In addition, due to the limited availability of primary human hepatocytes, our adenoviral-mediated hPXR expression protocol will likely prove useful in studies of the xenobiotic response.
Assuntos
Estrogênios não Esteroides/toxicidade , Hepatócitos/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Zearalenona/toxicidade , Adenoviridae/genética , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células Cultivadas , Citocromo P-450 CYP3A/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Receptor alfa de Estrogênio/metabolismo , Hepatócitos/metabolismo , Humanos , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Knockout , Modelos Animais , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/deficiência , Receptores de Esteroides/genética , Transdução GenéticaRESUMO
The pregnane X receptor (PXR, NR1I2) is a member of the nuclear receptor superfamily that is activated by a myriad of compounds and natural products in clinical use. Activation of PXR represents the basis for several clinically important drug-drug interactions. Although PXR activation has undesirable effects in patients on combination therapy, it also mediates the hepatoprotective effects exhibited by some herbal remedies. This review focuses on PXR activation by natural products and the potential therapeutic opportunities presented. In particular, the biological effects of St. John's Wort, gugulipid, kava kava, Coleus forskolii, Hypoxis, Sutherlandia, qing hao, wu wei zi, gan cao and other natural products are discussed. The impact of these natural products on drug metabolism and hepatoprotection is highlighted in the context of activation and antagonism of PXR.
Assuntos
Produtos Biológicos/farmacologia , Receptores de Esteroides/metabolismo , Xenobióticos/farmacologia , Animais , Produtos Biológicos/metabolismo , Interações Medicamentosas , Regulação da Expressão Gênica/fisiologia , Humanos , Modelos Biológicos , Receptor de Pregnano X , Ligação Proteica/fisiologia , Receptores de Esteroides/agonistas , Receptores de Esteroides/antagonistas & inibidores , Receptores X de Retinoides/metabolismo , Xenobióticos/metabolismoRESUMO
Pregnane X receptor (PXR, NR1I2) regulates the inducible expression of the 3A sub-family of cytochrome P450 genes (CYP3A). CYP3A enzymes are responsible for the oxidative metabolism of a wide array of endobiotic and xenobiotic compounds. Hepatic CYP3A gene expression is rapidly down-regulated during inflammation and sepsis. There are twelve protein kinase C (PKC) isoforms, classified into three subfamilies according to the structure of the N-terminal regulatory domain and their sensitivity to calcium and diacylglycerol. It is now well accepted that cytokine stimulation of hepatocytes increases intracellular PKC activity during inflammation and sepsis. We show here that protein kinase C alpha (PKC alpha) and phorbol ester-dependent PKC signaling dramatically repressed PXR activity in both, cell-based reporter gene assays and in hepatocytes. Moreover, treatment with the protein phosphatase PP1/PP2A inhibitor okadaic acid (OA) totally abolished PXR activity in reporter gene assays and in cultured hepatocytes. In mammalian two-hybrid assays, treatment with phorbol 12-myristate 13-acetate (PMA) increased the strength of interaction between PXR and the nuclear receptor co-repressor protein (NCoR). Treatment with PMA also abolished the ligand-dependent interaction between PXR and the steroid receptor co-activator 1 protein (SRC1). Our findings suggest that activation of the protein kinase C signaling pathway represses PXR activity through alterations in PXR-protein co-factor complexes, possibly through direct alterations in the phosphorylation status of one or all of these proteins. In addition, our data potentially provide important insights into the molecular mechanism of the repression of hepatic CYP3A gene expression that occurs during the inflammatory response.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Regulação Enzimológica da Expressão Gênica , Fígado/enzimologia , Oxirredutases N-Desmetilantes/genética , Proteína Quinase C/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Animais , Citocromo P-450 CYP3A , Masculino , Camundongos , Receptor de Pregnano X , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Fibroblast growth factor 15 (FGF15) has been proposed as a postprandial hormone that signals from intestine to liver to regulate bile acid and carbohydrate homeostasis. However, detecting FGF15 in blood using conventional techniques has proven difficult. Here, we describe a stable isotope standards and capture by anti-peptide antibodies (SISCAPA) assay that combines immuno-enrichment with selected reaction monitoring (SRM) mass spectrometry to overcome this issue. Using this assay, we show that FGF15 circulates in plasma in an FXR and circadian rhythm-dependent manner at concentrations that activate its receptor. Consistent with the proposed endocrine role for FGF15 in liver, mice lacking hepatocyte expression of the obligate FGF15 co-receptor, ß-Klotho, have increased bile acid synthesis and reduced glycogen storage despite having supraphysiological plasma FGF15 concentrations. Collectively, these data demonstrate that FGF15 functions as a hormone and highlight the utility of SISCAPA-SRM as a sensitive assay for detecting low-abundance proteins in plasma.
Assuntos
Anticorpos/química , Fatores de Crescimento de Fibroblastos/sangue , Espectrometria de Massas/métodos , Peptídeos/sangue , Animais , Camundongos , Camundongos Knockout , CoelhosRESUMO
The mechanism by which pharmacologic administration of the hormone FGF21 increases energy expenditure to cause weight loss in obese animals is unknown. Here we report that FGF21 acts centrally to exert its effects on energy expenditure and body weight in obese mice. Using tissue-specific knockout mice, we show that ßKlotho, the obligate coreceptor for FGF21, is required in the nervous system for these effects. FGF21 stimulates sympathetic nerve activity to brown adipose tissue through a mechanism that depends on the neuropeptide corticotropin-releasing factor. Our findings provide an unexpected mechanistic explanation for the strong pharmacologic effects of FGF21 on energy expenditure and weight loss in obese animals.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Sistema Nervoso Simpático/efeitos dos fármacos , Redução de Peso/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Animais , Hormônio Liberador da Corticotropina/genética , Hormônio Liberador da Corticotropina/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Hipotálamo/metabolismo , Proteínas Klotho , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Sistema Nervoso Simpático/metabolismo , Termogênese/genéticaRESUMO
Hepatocellular carcinoma (HCC), one of the leading causes of cancer-related death, develops from premalignant lesions in chronically damaged livers. Although it is well established that FGF19 acts through the receptor complex FGFR4-ß-Klotho (KLB) to regulate bile acid metabolism, FGF19 is also implicated in the development of HCC. In humans, FGF19 is amplified in HCC and its expression is induced in the liver under cholestatic and cirrhotic conditions. In mice, ectopic overexpression of FGF19 drives HCC development in a process that requires FGFR4. In this study, we describe an engineered FGF19 (M70) that fully retains bile acid regulatory activity but does not promote HCC formation, demonstrating that regulating bile acid metabolism is distinct and separable from tumor-promoting activity. Mechanistically, we show that FGF19 stimulates tumor progression by activating the STAT3 pathway, an activity eliminated by M70. Furthermore, M70 inhibits FGF19-dependent tumor growth in a rodent model. Our results suggest that selectively targeting the FGF19-FGFR4 pathway may offer a tractable approach to improve the treatment of chronic liver disease and cancer.
Assuntos
Ácidos e Sais Biliares/metabolismo , Carcinogênese/metabolismo , Carcinoma Hepatocelular/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Neoplasias Hepáticas Experimentais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Colesterol 7-alfa-Hidroxilase/metabolismo , Dependovirus/genética , Fatores de Crescimento de Fibroblastos/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Dados de Sequência Molecular , Ligação Proteica , Ratos , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Deleção de Sequência , Transdução de SinaisRESUMO
Preventing reproduction during nutritional deprivation is an adaptive process that is conserved and essential for the survival of species. In mammals, the mechanisms that inhibit fertility during starvation are complex and incompletely understood. Here we show that exposure of female mice to fibroblast growth factor 21 (FGF21), a fasting-induced hepatokine, mimics infertility secondary to starvation. Mechanistically, FGF21 acts on the suprachiasmatic nucleus (SCN) in the hypothalamus to suppress the vasopressin-kisspeptin signaling cascade, thereby inhibiting the proestrus surge in luteinizing hormone. Mice lacking the FGF21 co-receptor, ß-Klotho, in the SCN are refractory to the inhibitory effect of FGF21 on female fertility. Thus, FGF21 defines an important liver-neuroendocrine axis that modulates female reproduction in response to nutritional challenge.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Infertilidade Feminina/metabolismo , Proteínas de Membrana/metabolismo , Reprodução , Inanição/metabolismo , Animais , Metabolismo Energético , Feminino , Hipotálamo , Kisspeptinas/antagonistas & inibidores , Kisspeptinas/metabolismo , Proteínas Klotho , Hormônio Luteinizante/biossíntese , Hormônio Luteinizante/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proestro/fisiologia , Transdução de Sinais , Núcleo Supraquiasmático , Vasopressinas/antagonistas & inibidores , Vasopressinas/metabolismoRESUMO
Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes that are lethal to humans. Near freezing body temperatures, heart rates of 3-10 beats per minute, absence of food consumption, and depressed metabolism are characteristic of hibernation torpor bouts that are periodically interrupted by brief interbout arousals (IBAs). The molecular basis of torpor induction is unknown, however starved mice overexpressing the metabolic hormone fibroblast growth factor 21 (FGF21) promote fat utilization, reduce body temperature, and readily enter torpor-all hallmarks of mammalian hibernation. In this study we cloned FGF21 from the naturally hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and found that levels of FGF21 mRNA in liver and FGF21 protein in serum are elevated during hibernation torpor bouts and significantly elevated during IBAs compared to summer active animals. The effects of artificially elevating circulating FGF21 concentrations 50 to 100-fold via adenoviral-mediated overexpression were examined at three different times of the year. This is the first time that a transgenic approach has been used in a natural hibernator to examine mechanistic aspects of hibernation. Surgically implanted transmitters measured various metrics of the hibernation phenotype over a 7-day period including changes in motor activity, heart rate and core body temperature. In April fed-state animals, FGF21 overexpression decreased blood insulin and free fatty acid concentrations, effects similar to those seen in obese mice. However, elevated FGF21 concentrations did not cause torpor in these fed-state animals nor did they cause torpor or affect metabolic parameters in fasted-state animals in March/April, August or October. We conclude that FGF21 is strongly regulated during torpor and IBA but that its overexpression is not sufficient to cause torpor in naturally hibernating ground squirrels.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Hibernação , Sciuridae/metabolismo , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Temperatura Corporal , Meios de Cultivo Condicionados/farmacologia , DNA Complementar/metabolismo , Frequência Cardíaca , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Estações do Ano , Homologia de Sequência de AminoácidosRESUMO
Fibroblast growth factor 21 (FGF21) is a hepatokine that acts as a global starvation signal to modulate fuel partitioning and metabolism and repress growth; however, the site of action of these diverse effects remains unclear. FGF21 signals through a heteromeric cell-surface receptor composed of one of three FGF receptors (FGFR1c, FGFR2c or FGFR3c) in complex with ß-Klotho, a single-pass transmembrane protein that is enriched in metabolic tissues. Here we show that in addition to its known effects on peripheral metabolism, FGF21 increases systemic glucocorticoid levels, suppresses physical activity and alters circadian behavior, which are all features of the adaptive starvation response. These effects are mediated through ß-Klotho expression in the suprachiasmatic nucleus of the hypothalamus and the dorsal vagal complex of the hindbrain. Mice lacking the gene encoding ß-Klotho (Klb) in these regions are refractory to these effects, as well as those on metabolism, insulin and growth. These findings demonstrate a crucial role for the nervous system in mediating the diverse physiologic and pharmacologic actions of FGF21.
Assuntos
Ritmo Circadiano/fisiologia , Metabolismo Energético , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Sistema Nervoso/metabolismo , Animais , Glucocorticoides/metabolismo , Hipotálamo/metabolismo , Proteínas Klotho , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Inanição , Núcleo Supraquiasmático/metabolismoRESUMO
Fibroblast growth factor 21 (FGF21) is a fasting-induced hepatokine that has potent pharmacologic effects in mice, which include improving insulin sensitivity and blunting growth. The single-transmembrane protein ßKlotho functions as a coreceptor for FGF21 in vitro. To determine if ßKlotho is required for FGF21 action in vivo, we generated whole-body and adipose tissue-selective ßKlotho-knockout mice. All of the effects of FGF21 on growth and metabolism were lost in whole-body ßKlotho-knockout mice. Selective elimination of ßKlotho in adipose tissue blocked the acute insulin-sensitizing effects of FGF21. Taken together, these data demonstrate that ßKlotho is essential for FGF21 activity and that ßKlotho in adipose tissue contributes to the beneficial metabolic actions of FGF21.
Assuntos
Tecido Adiposo/metabolismo , Metabolismo Energético/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Crescimento/fisiologia , Resistência à Insulina/fisiologia , Proteínas de Membrana/metabolismo , Animais , Composição Corporal/efeitos dos fármacos , Cromatografia em Gel , Metabolismo Energético/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/fisiologia , Teste de Tolerância a Glucose , Crescimento/genética , Resistência à Insulina/genética , Proteínas Klotho , Proteínas de Membrana/deficiência , Proteínas de Membrana/fisiologia , Camundongos , Camundongos KnockoutRESUMO
It has been recently established that Klotho coreceptors associate with fibroblast growth factor (FGF) receptor tyrosine kinases (FGFRs) to enable signaling by endocrine-acting FGFs. However, the molecular interactions leading to FGF-FGFR-Klotho ternary complex formation remain incompletely understood. Here, we show that in contrast to αKlotho, ßKlotho binds its cognate endocrine FGF ligand (FGF19 or FGF21) and FGFR independently through two distinct binding sites. FGF19 and FGF21 use their respective C-terminal tails to bind to a common binding site on ßKlotho. Importantly, we also show that Klotho coreceptors engage a conserved hydrophobic groove in the immunoglobulin-like domain III (D3) of the "c" splice isoform of FGFR. Intriguingly, this hydrophobic groove is also used by ligands of the paracrine-acting FGF8 subfamily for receptor binding. Based on this binding site overlap, we conclude that while Klotho coreceptors enhance binding affinity of FGFR for endocrine FGFs, they actively suppress binding of FGF8 subfamily ligands to FGFR.
Assuntos
Fator 8 de Crescimento de Fibroblasto/metabolismo , Glucuronidase/metabolismo , Comunicação Parácrina , Transdução de Sinais , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Proteínas Klotho , Ligantes , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
Although members of the fibroblast growth factor (FGF) family and their receptors have well-established roles in embryogenesis, their contributions to adult physiology remain relatively unexplored. Here, we use real-time quantitative PCR to determine the mRNA expression patterns of all 22 FGFs, the seven principal FGF receptors (FGFRs), and the three members of the Klotho family of coreceptors in 39 different mouse tissues. Unsupervised hierarchical cluster analysis of the mRNA expression data reveals that most FGFs and FGFRs fall into two groups the expression of which is enriched in either the central nervous system or reproductive and gastrointestinal tissues. Interestingly, the FGFs that can act as endocrine hormones, including FGF15/19, FGF21, and FGF23, cluster in a third group that does not include any FGFRs, underscoring their roles in signaling between tissues. We further show that the most recently identified Klotho family member, Lactase-like, is highly and selectively expressed in brown adipose tissue and eye and can function as an additional coreceptor for FGF19. This FGF atlas provides an important resource for guiding future studies to elucidate the physiological functions of FGFs in adult animals.
Assuntos
Atlas como Assunto , Fatores de Crescimento de Fibroblastos/fisiologia , Camundongos/fisiologia , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Animais , Análise por Conglomerados , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica , Humanos , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Análise em Microsséries , RNA Mensageiro/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Distribuição TecidualRESUMO
Peroxisome proliferator-activated receptor alpha (PPARalpha) regulates the utilization of fat as an energy source during starvation and is the molecular target for the fibrate dyslipidemia drugs. Here, we identify the endocrine hormone fibroblast growth factor 21 (FGF21) as a mediator of the pleiotropic actions of PPARalpha. FGF21 is induced directly by PPARalpha in liver in response to fasting and PPARalpha agonists. FGF21 in turn stimulates lipolysis in white adipose tissue and ketogenesis in liver. FGF21 also reduces physical activity and promotes torpor, a short-term hibernation-like state of regulated hypothermia that conserves energy. These findings demonstrate an unexpected role for the PPARalpha-FGF21 endocrine signaling pathway in regulating diverse metabolic and behavioral aspects of the adaptive response to starvation.
Assuntos
Jejum/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Hepatócitos/metabolismo , Fígado/metabolismo , PPAR alfa/metabolismo , Adenoviridae/genética , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Feminino , Humanos , Immunoblotting , Lipólise/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plasmídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
Animal studies reveal that fasting and caloric restriction produce increased activity of specific metabolic pathways involved in resistance to weight loss in liver. Evidence suggests that this phenomenon may in part occur through the action of the constitutive androstane receptor (CAR, NR1I3). Currently, the precise molecular mechanisms that activate CAR during fasting are unknown. We show that fasting coordinately induces expression of genes encoding peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), CAR, cytochrome P-450 2b10 (Cyp2b10), UDP-glucuronosyltransferase 1a1 (Ugt1a1), sulfotransferase 2a1 (Sult2a1), and organic anion-transporting polypeptide 2 (Oatp2) in liver in mice. Treatments that elevate intracellular cAMP levels also produce increased expression of these genes in cultured hepatocytes. Our data show that PGC-1alpha interaction with hepatocyte nuclear factor 4alpha (HNF4alpha, NR2A1) directly regulates CAR gene expression through a novel and evolutionarily conserved HNF4-response element (HNF4-RE) located in its proximal promoter. Expression of PGC-1alpha in cells increases CAR expression and ligand-independent CAR activity. Genetic studies reveal that hepatic expression of HNF4alpha is required to produce fasting-inducible CAR expression and activity. Taken together, our data show that fasting produces increased expression of genes encoding key metabolic enzymes and an uptake transporter protein through a network of interactions involving cAMP, PGC-1alpha, HNF4alpha, CAR, and CAR target genes in liver. Given the recent finding that mice lacking CAR exhibit a profound decrease in resistance to weight loss during extended periods of caloric restriction, our findings have important implications in the development of drugs for the treatment of obesity and related diseases.
Assuntos
AMP Cíclico/metabolismo , Privação de Alimentos , Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Agonistas Adrenérgicos/metabolismo , Animais , Restrição Calórica , Células Cultivadas , Receptor Constitutivo de Androstano , AMP Cíclico/química , Epinefrina/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Camundongos , Camundongos Knockout , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptor de Pregnano X , Regiões Promotoras Genéticas , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
Guggulsterone is the active ingredient in gugulipid, an organic extract of the Commiphora mukul plant. Gugulipid has been used for nearly 3000 years in Ayurvedic medicine, mainly as a treatment for arthritis. Herbal practitioners currently use gugulipid therapy in conditions as diverse as rheumatism, coronary artery disease, arthritis, hyperlipidemia, acne, and obesity. The active ingredient in gugulipid is guggulsterone, a plant sterol compound recently identified as a pregnane X receptor (PXR; NR1I2) ligand. We show herein that guggulsterone treatment represses the expression of cytochrome P450 2b10 (Cyp2b10) gene expression by inhibiting constitutive androstane receptor (CAR; NR1I3) activity in hepatocytes lacking functional PXR (PXR-knockout). We also show that PXR-CAR cross-talk determines the net activity of guggulsterone treatment toward Cyp2b10 gene expression. Using mammalian two-hybrid assays, we show that treatment with guggulsterone differentially affects protein cofactor recruitment to these two nuclear receptors. These data identify guggulsterone as an inverse agonist of the nuclear receptor CAR. When viewed together with the data showing that PXR and CAR expression is highly variable in different ethnic populations and that CAR expression is under the control of a circadian rhythm, our data provide important insight into the molecular mechanism of interindividual variability of drug metabolism. These data, together with the recent resolution of the crystal structures of PXR and CAR, will likely aid in the rational design of more specific CAR inverse agonists that are currently viewed as potential antiobesity drugs.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Pregnenodionas/farmacologia , Regiões Promotoras Genéticas/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Esteroide Hidroxilases/genética , Fatores de Transcrição/fisiologia , Animais , Northern Blotting , Células Cultivadas , Receptor Constitutivo de Androstano , Família 2 do Citocromo P450 , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Luciferases/genética , Camundongos , Camundongos Knockout , Plasmídeos/genética , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/efeitos dos fármacos , Receptores de Esteroides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/efeitos dos fármacos , TransfecçãoRESUMO
An extract of the plant Coleus forskohlii has been used for centuries in Ayurvedic medicine to treat various diseases such as hypothyroidism, heart disease, and respiratory disorders. Additionally, complex herbal mixtures containing this extract are gaining popularity in United States for their putative "fat-burning" properties. The active ingredient in C. forskohlii extract is the diterpene compound forskolin. Forskolin is a widely used biochemical tool that activates adenyl cyclase, thereby increasing intracellular concentration of cAMP and thus activating the protein kinase A (PKA) signal transduction pathway. We show herein that both forskolin and its nonadenyl cyclase-activating analog 1,9 dideoxyforskolin induce CYP3A gene expression in primary hepatocytes by functioning as agonists of the pregnane X receptor (PXR). We show that activation of PKA signaling potentiates PXR-mediated induction of CYP3A gene expression in cultured hepatocytes and increases the strength of PXR-coactivator protein-protein interaction in cell-based assays. Kinase assays show that PXR can serve as a substrate for catalytically active PKA in vitro. Our data provide important insights into the molecular mechanism of both the PKA-dependent and -independent effects of forskolin on the expression of drug-metabolizing enzymes in liver. Finally, our data suggest that herbal therapy with C. forskohlii extract should be approached cautiously due to the potential for herb-drug interactions in patients on combination therapy.