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1.
ACS Med Chem Lett ; 14(10): 1427-1433, 2023 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-37849537

RESUMO

Diacylglycerol O-acyltransferase 2 (DGAT2) inhibitors have been shown to lower liver triglyceride content and are being explored clinically as a treatment for non-alcoholic steatohepatitis (NASH). This work details efforts to find an extended-half-life DGAT2 inhibitor. A basic moiety was added to a known inhibitor template, and the basicity and lipophilicity were fine-tuned by the addition of electrophilic fluorines. A weakly basic profile was required to find an appropriate balance of potency, clearance, and permeability. This work culminated in the discovery of PF-07202954 (12), a weakly basic DGAT2 inhibitor that has advanced to clinical studies. This molecule displays a higher volume of distribution and longer half-life in preclinical species, in keeping with its physicochemical profile, and lowers liver triglyceride content in a Western-diet-fed rat model.

2.
Heliyon ; 9(3): e13985, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36915476

RESUMO

Background: NAFLD progression, from steatosis to inflammation and fibrosis, results from an interplay of intra- and extrahepatic mechanisms. Disease drivers likely include signals from white adipose tissue (WAT) and gut. However, the temporal dynamics of disease development remain poorly understood. Methods: High-fat-diet (HFD)-fed Ldlr-/-.Leiden mice were compared to chow-fed controls. At t = 0, 8, 16, 28 and 38w mice were euthanized, and liver, WAT depots and gut were analyzed biochemically, histologically and by lipidomics and transcriptomics together with circulating factors to investigate the sequence of pathogenic events and organ cross-talk during NAFLD development. Results: HFD-induced obesity was associated with an increase in visceral fat, plasma lipids and hyperinsulinemia at t = 8w, along with increased liver steatosis and circulating liver damage biomarkers. In parallel, upstream regulator analysis predicted that lipid catabolism regulators were deactivated and lipid synthesis regulators were activated. Subsequently, hepatocyte hypertrophy, oxidative stress and hepatic inflammation developed. Hepatic collagen accumulated from t = 16 w and became pronounced at t = 28-38 w. Epididymal WAT was maximally hypertrophic from t = 8 w, which coincided with inflammation development. Mesenteric and subcutaneous WAT hypertrophy developed slower and did not appear to reach a maximum, with minimal inflammation. In gut, HFD significantly increased permeability, induced a shift in microbiota composition from t = 8 w and changed circulating gut-derived metabolites. Conclusion: HFD-fed Ldlr-/-.Leiden mice develop obesity, dyslipidemia and insulin resistance, essentially as observed in obese NAFLD patients, underlining their translational value. We demonstrate that marked epididymal-WAT inflammation, and gut permeability and dysbiosis precede the development of NAFLD stressing the importance of a multiple-organ approach in the prevention and treatment of NAFLD.

3.
J Med Chem ; 65(22): 15000-15013, 2022 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-36322383

RESUMO

Discovery efforts leading to the identification of ervogastat (PF-06865571), a systemically acting diacylglycerol acyltransferase (DGAT2) inhibitor that has advanced into clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) with liver fibrosis, are described herein. Ervogastat is a first-in-class DGAT2 inhibitor that addressed potential development risks of the prototype liver-targeted DGAT2 inhibitor PF-06427878. Key design elements that culminated in the discovery of ervogastat are (1) replacement of the metabolically labile motif with a 3,5-disubstituted pyridine system, which addressed potential safety risks arising from a cytochrome P450-mediated O-dearylation of PF-06427878 to a reactive quinone metabolite precursor, and (2) modifications of the amide group to a 3-THF group, guided by metabolite identification studies coupled with property-based drug design.


Assuntos
Diacilglicerol O-Aciltransferase , Hepatopatia Gordurosa não Alcoólica , Humanos , Desenho de Fármacos , Cirrose Hepática , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico
4.
Nat Metab ; 3(12): 1608-1620, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34845393

RESUMO

Carbohydrate can be converted into fat by de novo lipogenesis, a process upregulated in fatty liver disease. Chemically, de novo lipogenesis involves polymerization and reduction of acetyl-CoA, using NADPH as the electron donor. The feedstocks used to generate acetyl-CoA and NADPH in lipogenic tissues remain, however, unclear. Here we show using stable isotope tracing in mice that de novo lipogenesis in adipose is supported by glucose and its catabolism via the pentose phosphate pathway to make NADPH. The liver, in contrast, derives acetyl-CoA for lipogenesis from acetate and lactate, and NADPH from folate-mediated serine catabolism. Such NADPH generation involves the cytosolic serine pathway in liver running in the opposite direction to that observed in most tissues and tumours, with NADPH made by the SHMT1-MTHFD1-ALDH1L1 reaction sequence. SHMT inhibition decreases hepatic lipogenesis. Thus, liver folate metabolism is distinctively wired to support cytosolic NADPH production and lipogenesis. More generally, while the same enzymes are involved in fat synthesis in liver and adipose, different substrates are used, opening the door to tissue-specific pharmacological interventions.


Assuntos
Lipogênese , Fígado/metabolismo , NADP/metabolismo , Serina/metabolismo , Acetilcoenzima A/metabolismo , Tecido Adiposo/metabolismo , Aminoidrolases/metabolismo , Animais , Ácidos Graxos/metabolismo , Feminino , Ácido Fólico/metabolismo , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Glutamina/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Masculino , Redes e Vias Metabólicas , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Camundongos , Complexos Multienzimáticos/metabolismo , Fosforilação Oxidativa , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo
5.
Mol Metab ; 48: 101196, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33667726

RESUMO

OBJECTIVE: Recent studies suggest that excess dietary fructose contributes to metabolic dysfunction by promoting insulin resistance, de novo lipogenesis (DNL), and hepatic steatosis, thereby increasing the risk of obesity, type 2 diabetes (T2D), non-alcoholic steatohepatitis (NASH), and related comorbidities. Whether this metabolic dysfunction is driven by the excess dietary calories contained in fructose or whether fructose catabolism itself is uniquely pathogenic remains controversial. We sought to test whether a small molecule inhibitor of the primary fructose metabolizing enzyme ketohexokinase (KHK) can ameliorate the metabolic effects of fructose. METHODS: The KHK inhibitor PF-06835919 was used to block fructose metabolism in primary hepatocytes and Sprague Dawley rats fed either a high-fructose diet (30% fructose kcal/g) or a diet reflecting the average macronutrient dietary content of an American diet (AD) (7.5% fructose kcal/g). The effects of fructose consumption and KHK inhibition on hepatic steatosis, insulin resistance, and hyperlipidemia were evaluated, along with the activation of DNL and the enzymes that regulate lipid synthesis. A metabolomic analysis was performed to confirm KHK inhibition and understand metabolite changes in response to fructose metabolism in vitro and in vivo. Additionally, the effects of administering a single ascending dose of PF-06835919 on fructose metabolism markers in healthy human study participants were assessed in a randomized placebo-controlled phase 1 study. RESULTS: Inhibition of KHK in rats prevented hyperinsulinemia and hypertriglyceridemia from fructose feeding. Supraphysiologic levels of dietary fructose were not necessary to cause metabolic dysfunction as rats fed the American diet developed hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis, which were all reversed by KHK inhibition. Reversal of the metabolic effects of fructose coincided with reductions in DNL and inactivation of the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP). We report that administering single oral doses of PF-06835919 was safe and well tolerated in healthy study participants and dose-dependently increased plasma fructose indicative of KHK inhibition. CONCLUSIONS: Fructose consumption in rats promoted features of metabolic dysfunction seen in metabolic diseases such as T2D and NASH, including insulin resistance, hypertriglyceridemia, and hepatic steatosis, which were reversed by KHK inhibition.


Assuntos
Inibidores Enzimáticos/administração & dosagem , Frutoquinases/antagonistas & inibidores , Frutose/efeitos adversos , Hipertrigliceridemia/etiologia , Hipertrigliceridemia/prevenção & controle , Síndrome Metabólica/etiologia , Síndrome Metabólica/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Adulto , Animais , Células Cultivadas , Estudos de Coortes , Dieta da Carga de Carboidratos/efeitos adversos , Frutose/administração & dosagem , Frutose/metabolismo , Voluntários Saudáveis , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
6.
J Med Chem ; 63(22): 13546-13560, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-32910646

RESUMO

Increased fructose consumption and its subsequent metabolism have been implicated in metabolic disorders such as nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) and insulin resistance. Ketohexokinase (KHK) converts fructose to fructose-1-phosphate (F1P) in the first step of the metabolic cascade. Herein we report the discovery of a first-in-class KHK inhibitor, PF-06835919 (8), currently in phase 2 clinical trials. The discovery of 8 was built upon our originally reported, fragment-derived lead 1 and the recognition of an alternative, rotated binding mode upon changing the ribose-pocket binding moiety from a pyrrolidinyl to an azetidinyl ring system. This new binding mode enabled efficient exploration of the vector directed at the Arg-108 residue, leading to the identification of highly potent 3-azabicyclo[3.1.0]hexane acetic acid-based KHK inhibitors by combined use of parallel medicinal chemistry and structure-based drug design.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Frutoquinases/antagonistas & inibidores , Frutoquinases/metabolismo , Frutose/efeitos adversos , Doenças Metabólicas/enzimologia , Animais , Cristalografia por Raios X , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Frutose/administração & dosagem , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Resistência à Insulina/fisiologia , Masculino , Doenças Metabólicas/induzido quimicamente , Doenças Metabólicas/tratamento farmacológico , Estrutura Secundária de Proteína , Ratos , Ratos Wistar
7.
Cell Metab ; 32(1): 6-8, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32640246

RESUMO

The abundance of dietary sweeteners and overconsumption of fructose are widely thought to promote metabolic disease. In this issue of Cell Metabolism, Andres-Hernando et al. (2020) identify the liver as the major site of fructose metabolism-mediated metabolic dysfunction and identify a surprising role for intestinal fructose metabolism in driving fructose intake.


Assuntos
Frutoquinases , Açúcares , Frutose , Intestinos , Fígado
8.
Sci Transl Med ; 11(492)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092695

RESUMO

Sebum plays important physiological roles in human skin. Excess sebum production contributes to the pathogenesis of acne vulgaris, and suppression of sebum production reduces acne incidence and severity. We demonstrate that sebum production in humans depends on local flux through the de novo lipogenesis (DNL) pathway within the sebocyte. About 80 to 85% of sebum palmitate (16:0) and sapienate (16:1n10) were derived from DNL, based on stable isotope labeling, much higher than the contribution of DNL to triglyceride palmitate in circulation (~20%), indicating a minor contribution by nonskin sources to sebum lipids. This dependence on local sebocyte DNL was not recapitulated in two widely used animal models of sebum production, Syrian hamsters and Göttingen minipigs. Confirming the importance of DNL for human sebum production, an acetyl-CoA carboxylase inhibitor, ACCi-1, dose-dependently suppressed DNL and blocked synthesis of fatty acids, triglycerides, and wax esters but not free sterols in human sebocytes in vitro. ACCi-1 dose-dependently suppressed facial sebum excretion by ~50% (placebo adjusted) in human individuals dosed orally for 2 weeks. Sebum triglycerides, wax esters, and free fatty acids were suppressed by ~66%, whereas non-DNL-dependent lipid species, cholesterol, and squalene were not reduced, confirming selective modulation of DNL-dependent lipids. Last, individuals with acne vulgaris exhibited increased sebum production rates relative to individuals with normal skin, with >80% of palmitate and sapienate derived from DNL. These findings highlight the importance of local sebocyte DNL for human skin sebaceous gland biology and illuminate a potentially exploitable therapeutic target for the treatment of acne vulgaris.


Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Acne Vulgar/enzimologia , Inibidores Enzimáticos/farmacologia , Lipogênese , Sebo/metabolismo , Acetil-CoA Carboxilase/metabolismo , Adolescente , Adulto , Animais , Células Cultivadas , Cricetinae , Inibidores Enzimáticos/química , Feminino , Humanos , Lipogênese/efeitos dos fármacos , Masculino , Malonil Coenzima A/metabolismo , Pessoa de Meia-Idade , Ratos Wistar , Glândulas Sebáceas/efeitos dos fármacos , Glândulas Sebáceas/metabolismo , Glândulas Sebáceas/patologia , Sebo/efeitos dos fármacos , Suínos , Porco Miniatura , Triglicerídeos/biossíntese , Adulto Jovem
9.
Cell Metab ; 27(2): 351-361.e3, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29414685

RESUMO

Excessive consumption of sweets is a risk factor for metabolic syndrome. A major chemical feature of sweets is fructose. Despite strong ties between fructose and disease, the metabolic fate of fructose in mammals remains incompletely understood. Here we use isotope tracing and mass spectrometry to track the fate of glucose and fructose carbons in vivo, finding that dietary fructose is cleared by the small intestine. Clearance requires the fructose-phosphorylating enzyme ketohexokinase. Low doses of fructose are ∼90% cleared by the intestine, with only trace fructose but extensive fructose-derived glucose, lactate, and glycerate found in the portal blood. High doses of fructose (≥1 g/kg) overwhelm intestinal fructose absorption and clearance, resulting in fructose reaching both the liver and colonic microbiota. Intestinal fructose clearance is augmented both by prior exposure to fructose and by feeding. We propose that the small intestine shields the liver from otherwise toxic fructose exposure.


Assuntos
Ácidos Carboxílicos/metabolismo , Carboidratos da Dieta/metabolismo , Frutose/metabolismo , Glucose/metabolismo , Intestino Delgado/metabolismo , Animais , Comportamento Alimentar , Marcação por Isótopo , Fígado/metabolismo , Metaboloma , Camundongos Endogâmicos C57BL , Microbiota , Modelos Biológicos
10.
J Med Chem ; 60(18): 7835-7849, 2017 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-28853885

RESUMO

Increased fructose consumption and its subsequent metabolism have been implicated in hepatic steatosis, dyslipidemia, obesity, and insulin resistance in humans. Since ketohexokinase (KHK) is the principal enzyme responsible for fructose metabolism, identification of a selective KHK inhibitor may help to further elucidate the effect of KHK inhibition on these metabolic disorders. Until now, studies on KHK inhibition with small molecules have been limited due to the lack of viable in vivo pharmacological tools. Herein we report the discovery of 12, a selective KHK inhibitor with potency and properties suitable for evaluating KHK inhibition in rat models. Key structural features interacting with KHK were discovered through fragment-based screening and subsequent optimization using structure-based drug design, and parallel medicinal chemistry led to the identification of pyridine 12.


Assuntos
Desenho de Fármacos , Frutoquinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Cristalografia por Raios X , Frutoquinases/química , Frutoquinases/metabolismo , Humanos , Masculino , Simulação de Acoplamento Molecular , Piridinas/química , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley
11.
J Med Chem ; 58(18): 7164-72, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26258602

RESUMO

Inhibition of triacylglycerol (TAG) biosynthetic enzymes has been suggested as a promising strategy to treat insulin resistance, diabetes, dyslipidemia, and hepatic steatosis. Monoacylglycerol acyltransferase 3 (MGAT3) is an integral membrane enzyme that catalyzes the acylation of both monoacylglycerol (MAG) and diacylglycerol (DAG) to generate DAG and TAG, respectively. Herein, we report the discovery and characterization of the first selective small molecule inhibitors of MGAT3. Isoindoline-5-sulfonamide (6f, PF-06471553) selectively inhibits MGAT3 with high in vitro potency and cell efficacy. Because the gene encoding MGAT3 (MOGAT3) is found only in higher mammals and humans, but not in rodents, a transgenic mouse model expressing the complete human MOGAT3 was used to characterize the effects of 6f in vivo. In the presence of a combination of diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2) inhibitors, an oral administration of 6f exhibited inhibition of the incorporation of deuterium-labeled glycerol into TAG in this mouse model. The availability of a potent and selective chemical tool and a humanized mouse model described in this report should facilitate further dissection of the physiological function of MGAT3 and its role in lipid homeostasis.


Assuntos
Aciltransferases/antagonistas & inibidores , Isoindóis/química , Sulfonamidas/química , Aciltransferases/genética , Animais , Células Cultivadas , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Cães , Humanos , Isoindóis/farmacocinética , Isoindóis/farmacologia , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Triglicerídeos/biossíntese
12.
Nat Rev Endocrinol ; 7(8): 473-84, 2011 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-21502982

RESUMO

RNA interference (RNAi) is a robust gene silencing mechanism that degrades mRNAs complementary to the antisense strands of double-stranded, short interfering RNAs (siRNAs). As a therapeutic strategy, RNAi has an advantage over small-molecule drugs, as virtually all genes are susceptible to targeting by siRNA molecules. This advantage is, however, counterbalanced by the daunting challenge of achieving safe, effective delivery of oligonucleotides to specific tissues in vivo. Lipid-based carriers of siRNA therapeutics can now target the liver in metabolic diseases and are being assessed in clinical trials for the treatment of hypercholesterolemia. For this indication, a chemically modified oligonucleotide that targets endogenous small RNA modulators of gene expression (microRNAs) is also under investigation in clinical trials. Emerging 'self-delivery' siRNAs that are covalently linked to lipophilic moieties show promise for the future development of therapies. Besides the liver, inflammation of the adipose tissue in patients with obesity and type 2 diabetes mellitus may be an attractive target for siRNA therapeutics. Administration of siRNAs encapsulated within glucan microspheres can silence genes in inflammatory phagocytic cells, as can certain lipid-based carriers of siRNA. New technologies that combine siRNA molecules with antibodies or other targeting molecules also appear encouraging. Although still at an early stage, the emergence of RNAi-based therapeutics has the potential to markedly influence our clinical future.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Síndrome Metabólica/genética , Síndrome Metabólica/terapia , Interferência de RNA , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/terapia , Humanos , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Doenças Metabólicas/terapia , Síndrome Metabólica/metabolismo
13.
Biochem J ; 436(2): 351-62, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21418037

RESUMO

Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi (RNA interference) therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We recently engineered a complex 5 component glucan-based encapsulation system for siRNA (small interfering RNA) delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA that can mediate potent gene silencing in multiple cell types. In order to restrict such gene silencing to phagocytes, a method was developed to entrap siRNA-Endo-Porter complexes in glucan shells of 2-4 µm diameter in the absence of other components. The resulting glucan particles containing fluorescently labelled siRNA were readily internalized by macrophages, but not other cell types, and released the labelled siRNA into the macrophage cytoplasm. Intraperitoneal administration of such glucan particles containing siRNA-Endo-Porter complexes to mice caused gene silencing specifically in macrophages that internalized the particles. These results from the present study indicate that specific targeting to phagocytes is mediated by the glucan, whereas Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyse escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice.


Assuntos
Técnicas de Transferência de Genes , Fagócitos/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , beta-Glucanas/administração & dosagem , Células 3T3-L1 , Animais , Células COS , Chlorocebus aethiops , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Fagócitos/efeitos dos fármacos , Proteoglicanas , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo
14.
Nature ; 458(7242): 1180-4, 2009 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-19407801

RESUMO

Gene silencing by double-stranded RNA, denoted RNA interference, represents a new paradigm for rational drug design. However, the transformative therapeutic potential of short interfering RNA (siRNA) has been stymied by a key obstacle-safe delivery to specified target cells in vivo. Macrophages are particularly attractive targets for RNA interference therapy because they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes. Here we report the engineering of beta1,3-D-glucan-encapsulated siRNA particles (GeRPs) as efficient oral delivery vehicles that potently silence genes in mouse macrophages in vitro and in vivo. Oral gavage of mice with GeRPs containing as little as 20 microg kg(-1) siRNA directed against tumour necrosis factor alpha (Tnf-alpha) depleted its messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum Tnf-alpha levels. Screening with GeRPs for inflammation genes revealed that the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) is a previously unknown mediator of cytokine expression. Importantly, silencing Map4k4 in macrophages in vivo protected mice from lipopolysaccharide-induced lethality by inhibiting Tnf-alpha and interleukin-1beta production. This technology defines a new strategy for oral delivery of siRNA to attenuate inflammatory responses in human disease.


Assuntos
Sistemas de Liberação de Medicamentos , Inativação Gênica , Inflamação/prevenção & controle , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/administração & dosagem , Administração Oral , Animais , Ativação Enzimática/efeitos dos fármacos , Glucanos/metabolismo , Inflamação/genética , Interleucina-1beta/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Especificidade de Órgãos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Especificidade por Substrato , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinase Induzida por NF-kappaB
15.
J Biol Chem ; 284(25): 17082-17091, 2009 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-19321447

RESUMO

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that acts as a primary regulator of adipogenesis and controls adipocyte metabolism and insulin action. Increased expression of tumor necrosis factor (TNFalpha) in adipose tissue of obese subjects potently suppresses the expression of PPARgamma and attenuates adipocyte functions. Here we show that PPARgamma is a substrate of caspase-3 and caspase-6 during TNFalpha receptor signaling in adipocytes, and the consequent PPARgamma cleavage disrupts its nuclear localization. TNFalpha treatment of 3T3-L1 adipocytes decreases full-length PPARgamma while increasing the level of a 45-kDa immunoreactive PPARgamma fragment. Specific inhibitors of caspase-3 and caspase-6 attenuate the cleavage of PPARgamma protein in response to TNFalpha in cultured adipocytes. Incubation of nuclear fractions with recombinant caspase-3 and caspase-6 also generates a 45-kDa PPARgamma cleavage product. Dispersion of nuclear PPARgamma to the cytoplasm in response to TNFalpha treatment occurs in parallel with detection of activated caspase-3. We suggest that activation of the caspase cascade by TNFalpha down-regulates PPARgamma protein and PPARgamma-mediated metabolic processes in adipose cells.


Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Caspases/metabolismo , PPAR gama/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Células 3T3-L1 , Animais , Caspase 3/metabolismo , Caspase 6/metabolismo , Caspase 8/metabolismo , Cinética , Camundongos , Modelos Biológicos , PPAR gama/química , PPAR gama/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo
16.
Cell ; 136(5): 939-51, 2009 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-19249087

RESUMO

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in regulating life span, dauer, metabolism, and stress. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. An RNAi screen for serine/threonine protein phosphatases that counterbalance the effect of the kinases in the IIS pathway identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects IIS pathway-associated phenotypes including life span, dauer, stress resistance, and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56beta regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in regulating insulin signaling through AKT dephosphorylation, thereby having widespread implications in cancer and diabetes research.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Longevidade , Monoéster Fosfórico Hidrolases/análise , Fosforilação , Receptores de Superfície Celular/metabolismo
17.
J Biol Chem ; 282(27): 19302-12, 2007 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-17500068

RESUMO

Tumor necrosis factor alpha (TNFalpha) is a cytokine secreted by macrophages and adipocytes that contributes to the low grade inflammation and insulin resistance observed in obesity. TNFalpha signaling decreases peroxisome proliferator-activated receptor gamma and glucose transporter isoform 4 (GLUT4) expression in adipocytes, impairing insulin action, and this is mediated in part by the yeast Ste20 protein kinase ortholog Map4k4. Here we show that Map4k4 expression is selectively up-regulated by TNFalpha, whereas the expression of the protein kinases JNK1/2, ERK1/2, p38 stress-activated protein kinase, and mitogen-activated protein kinase kinases 4/7 shows little or no response. Furthermore, the cytokines interleukin 1beta (IL-1beta) and IL-6 as well as lipopolysaccharide fail to increase Map4k4 mRNA levels in cultured adipocytes under conditions where TNFalpha elicits a 3-fold effect. Using agonistic and antagonistic antibodies and small interfering RNA (siRNA) against TNFalpha receptor 1 (TNFR1) and TNFalpha receptor 2 (TNFR2), we show that TNFR1, but not TNFR2, mediates the increase in Map4k4 expression. TNFR1, but not TNFR2, also mediates a potent effect of TNFalpha on the phosphorylation of JNK1/2 and p38 stress-activated protein kinase and their downstream transcription factor substrates c-Jun and activating transcription factor 2 (ATF2). siRNA-based depletion of c-Jun and ATF2 attenuated TNFalpha action on Map4k4 mRNA expression. Consistent with this concept, the phosphorylation of ATF2 along with the expression and phosphorylation of c-Jun by TNFalpha signaling was more robust and prolonged compared with that of IL-1beta, which failed to modulate Map4k4. These data reveal that TNFalpha selectively stimulates the expression of a key component of its own signaling pathway, Map4k4, through a TNFR1-dependent mechanism that targets the transcription factors c-Jun and ATF2.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Células 3T3 , Adipócitos/metabolismo , Animais , Transportador de Glucose Tipo 4/metabolismo , Inflamação/metabolismo , Resistência à Insulina , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , PPAR gama/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Receptores Tipo I de Fatores de Necrose Tumoral/agonistas , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Quinase Induzida por NF-kappaB
18.
Metabolism ; 55(1): 35-42, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16324917

RESUMO

Dexamethasone inhibits insulin secretion from isolated islets. In the present experiments, possible underlying biochemical mechanisms responsible for defective secretion were explored. Dexamethasone (1 micromol/L) had no immediate deleterious effect on 15 mmol/L glucose-induced insulin release from perifused rat islets. However, a 3-hour preincubation period with 1 micromol/L dexamethasone resulted in parallel reductions in both the first (64%) and second phases (74%) of 15 mmol/L glucose-induced insulin secretion monitored during a dynamic perifusion. When measured after the perifusion, there were no differences in insulin content or in the capacity of control or dexamethasone-treated islets to use glucose. Dexamethasone (1 micromol/L) preexposure also reduced phorbol ester- and potassium-induced secretion. In additional experiments, islets were labeled for 3 hours with 3H-inositol in the presence or absence of 1 micromol/L dexamethasone. The steroid did not affect total 3H-inositol incorporation during the labeling period. However, the capacity of 15 mmol/L glucose, 30 mmol/L KCl, and 100 micromol/L carbachol to activate phospholipase C (PLC), monitored by the accumulation of labeled inositol phosphates, was significantly reduced in dexamethasone-pretreated islets. Inclusion of the nuclear glucocorticoid receptor antagonist RU486 (mifepristone, 10 micromol/L) abolished the adverse effects of dexamethasone on both glucose-induced inositol phosphate accumulation and insulin secretion. Quantitative Western blot analyses revealed that the islet contents of PLCdelta1, PLCbeta1, beta2, beta3, and protein kinase C alpha were unaffected by dexamethasone pretreatment. These findings demonstrate that dexamethasone pretreatment impairs insulin secretion via a genomic action and that impaired activation of the PLC/protein kinase C signaling system is involved in the evolution of its inhibitory effect on secretion.


Assuntos
Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Insulina/metabolismo , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Animais , Western Blotting , Depressão Química , Glucose/metabolismo , Antagonistas de Hormônios/farmacologia , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Isoenzimas/metabolismo , Cinética , Masculino , Mifepristona/farmacologia , Potássio/farmacologia , Proteína Quinase C/fisiologia , Ratos , Ratos Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/fisiologia
19.
Endocrine ; 23(1): 11-6, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15034191

RESUMO

Glucose-induced insulin secretion is inhibited by 5-hydroxytryptamine (5HT). In the present studies the specificity of 5HT inhibition of release and the potential biochemical mechanisms involved were investigated. Dose-dependent inhibition of 15 mM glucose-induced secretion was induced by a prior 3 h incubation with 5HT. At the highest 5HT concentration (500 microM) employed, both first and second phase responses to 15 mM glucose were reduced 50-60%. In addition, this level (500 microM) of 5HT virtually abolished 10 mM glucose-induced secretion. In contrast, secretion in response to the protein kinase C activator phorbol 12-myristate 13-acetate (500 nM) was immune to 500 microM 5HT pre-treatment. Glucose usage rates were comparable in both control and 500 microM 5HT-pretreated islets. However, the generation of inositol phosphates and the efflux of 3H-inositol from 3H-inositol-prelabeled islets in response to stimulatory glucose were impaired in parallel with insulin secretion. Based on these observations the following conclusions were reached: (1) 5HT impairs glucose-induced insulin release by altering glucose-induced activation of phospholipase C. (2) Biochemical events distal to phospholipase C remain intact despite this proximal biochemical lesion. (3) Amperometric analysis of 5HT release from 5HT-pretreated islets must take into consideration its profound adverse impact on glucose-induced insulin secretion.


Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Serotonina/farmacologia , Fosfolipases Tipo C/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Fosfatos de Inositol/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Taxa Secretória/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia
20.
Biochem Biophys Res Commun ; 315(4): 872-6, 2004 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-14985093

RESUMO

The impact of muscarinic type 3 receptor knockout (M3KO) on the cholinergic regulation of insulin secretion and phospholipase C (PLC) activation was determined. Islets isolated from control, wild-type mice or heterozygotes responded with comparable insulin secretory responses to 15 mM glucose. This response was markedly amplified by the inclusion of 10 microM carbachol. While 15 mM glucose-induced release remained similar to wild-type and heterozygote responses in M3KO mice, the stimulatory impact of carbachol was abolished. Stimulation with 15 mM glucose plus 50 microM carbachol increased fractional efflux rates of myo-[2-3H]inositol from control wild-type and heterozygote islets but not from M3KO islets. Fed plasma insulin levels of M3KO mice were reduced 68% when compared to values obtained from combined wild-type and heterozygote animals. These studies support the conclusion that the M3 receptor in islets is coupled to PLC activation and insulin secretion and that cholinergic stimulation of the islets may play an important role in the regulation of plasma insulin levels.


Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Receptor Muscarínico M3/deficiência , Animais , Carbacol/metabolismo , Carbacol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Glucose/metabolismo , Glucose/farmacologia , Heterozigoto , Inositol/análogos & derivados , Inositol/metabolismo , Insulina/sangue , Secreção de Insulina , Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Muscarínico M3/genética , Fosfolipases Tipo C/metabolismo
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