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1.
J Biol Chem ; 291(6): 2602-15, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26644473

RESUMO

Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3(-/-)) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3(-/-) mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3(-/-) mice. Gpat3(-/-) enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3(-/-) mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism.


Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Ácidos e Sais Biliares/metabolismo , Gorduras na Dieta/farmacologia , Enterócitos/enzimologia , Metabolismo dos Lipídeos/fisiologia , Triglicerídeos/farmacologia , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Animais , Camundongos , Camundongos Knockout , Fosfolipídeos/genética , Fosfolipídeos/metabolismo
2.
iScience ; 24(6): 102554, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34189431

RESUMO

Growth differentiation factor 15 (GDF15) causes anorexia and weight loss in animal models, and higher circulating levels are associated with cachexia and reduced survival in cancer and other chronic diseases such as sepsis. To investigate the role of sepsis-induced GDF15, we examined whether GDF15 neutralization via a validated and highly potent monoclonal antibody, mAB2, modulates lipopolysaccharide (LPS)-induced anorexia, weight loss, and mortality in rodents. LPS injection transiently increased circulating GDF15 in wild-type mice, decreased food intake and body weight, and increased illness behavior and mortality at a high dose. GDF15 neutralization with mAB2 did not prevent or exacerbate any of the effects of LPS. Similarly, in GDF15 knockout mice, the LPS effect on appetite and survival was comparable with that observed in wild-type controls. Therefore, effective inhibition of circulating active GDF15 via an antibody or via gene knockout demonstrated that survival in the LPS acute inflammation model was independent of GDF15.

3.
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
4.
Methods Mol Biol ; 2084: 95-101, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31729655

RESUMO

Differential mobility spectrometry (DMS) is capable of separating molecules based on their size and shape. When coupled to mass spectrometry (MS), DMS reduces chemical background and enhances signal-to-noise (S/N) ratio. Flow injection analysis (FIA) is a technique used to introduce samples into the source of the DMS-MS platform. Here we describe the application of FIA-DMS-MS/MS for the analysis of urinary acylcarnitine species. More than 20 acylcarnitine species can be detected and quantified during a single FIA-DMS-MS/MS acquisition.


Assuntos
Carnitina/análogos & derivados , Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Carnitina/análise , Carnitina/urina , Humanos , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos
5.
J Am Soc Mass Spectrom ; 31(3): 498-507, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32013416

RESUMO

Acylcarnitines have been identified in human and animal metabolomic-profiling studies as urinary markers of radiation exposure, a result which is consistent with their cytoprotective effects and roles in energy metabolism. In the present work, a rapid method for quantitation of the more abundant acylcarnitines in human urine is developed using a valuable set of samples from cancer patients who received total body irradiation (TBI) at Memorial Sloan Kettering Cancer Center. The method uses solid-phase extraction (SPE) processing followed by differential mobility spectrometry (DMS with ethyl acetate modifier) tandem mass spectrometry (ESI-DMS-MS/MS) with deuterated internal standards. The analyzed human urine samples were collected from 38 individual patients at three time points over 24 h during and after the course of radiation treatment, a design allowing each patient to act as their own control and creatinine normalization. Creatinine-normalized concentrations for nine urinary acylcarnitine (acyl-CN) species are reported. Six acyl-CN species were reduced at the 6 h point. Acetylcarnitine (C2:0-CN) and valerylcarnitine (C5:0-CN) showed recovery at 24 h, but none of the other acyl-CN species showed recovery at that point. Levels of three acyl-CN species were not significantly altered by radiation. This rapid quantitative method for clinical samples covers the short- and medium-chain acylcarnitines and has the flexibility to be expanded to cover additional radiation-linked metabolites. The human data presented here indicates the utility of the current approach as a rapid, quantitative technique with potential applications by the medical community, by space research laboratories concerned with radiation exposure, and by disaster response groups.


Assuntos
Carnitina/análogos & derivados , Neoplasias/radioterapia , Neoplasias/urina , Espectrometria de Massas em Tandem/métodos , Animais , Biomarcadores/urina , Carnitina/urina , Humanos , Projetos Piloto , Ratos Sprague-Dawley , Irradiação Corporal Total/efeitos adversos
6.
Nat Metab ; 2(10): 1163-1178, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32929234

RESUMO

Acetyl-CoA carboxylase (ACC) catalyses the first step of de novo lipogenesis (DNL). Pharmacologic inhibition of ACC has been of interest for therapeutic intervention in a wide range of diseases. We demonstrate here that ACC and DNL are essential for platelet production in humans and monkeys, but in not rodents or dogs. During clinical evaluation of a systemically distributed ACC inhibitor, unexpected dose-dependent reductions in platelet count were observed. While platelet count reductions were not observed in rat and dog toxicology studies, subsequent studies in cynomolgus monkeys recapitulated these platelet count reductions with a similar concentration response to that in humans. These studies, along with ex vivo human megakaryocyte maturation studies, demonstrate that platelet lowering is a consequence of DNL inhibition likely to result in impaired megakaryocyte demarcation membrane formation. These observations demonstrate that while DNL is a minor quantitative contributor to global lipid balance in humans, DNL is essential to specific lipid pools of physiological importance.


Assuntos
Plaquetas , Lipogênese/fisiologia , Acetil-CoA Carboxilase/antagonistas & inibidores , Acetil-CoA Carboxilase/metabolismo , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cães , Relação Dose-Resposta a Droga , Método Duplo-Cego , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos , Macaca fascicularis , Megacariócitos/fisiologia , Contagem de Plaquetas , Ratos
7.
JCI Insight ; 52019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393852

RESUMO

Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are liver manifestations of the metabolic syndrome and can progress to hepatocellular carcinoma (HCC). Loss of Growth Hormone (GH) signaling is reported to predispose to NAFLD and NASH through direct actions on the liver. Here, we report that aged mice lacking hepatocyte Jak2 (JAK2L), an obligate transducer of Growth Hormone (GH) signaling, spontaneously develop the full spectrum of phenotypes found in patients with metabolic liver disease, beginning with insulin resistance and lipodystrophy and manifesting as NAFLD, NASH and even HCC, independent of dietary intervention. Remarkably, insulin resistance, metabolic liver disease, and carcinogenesis are prevented in JAK2L mice via concomitant deletion of adipocyte Jak2 (JAK2LA). Further, we demonstrate that GH increases hepatic lipid burden but does so indirectly via signaling through adipocyte JAK2. Collectively, these data establish adipocytes as the mediator of GH-induced metabolic liver disease and carcinogenesis. In addition, we report a new spontaneous model of NAFLD, NASH, and HCC that recapitulates the natural sequelae of human insulin resistance-associated disease progression. The work presented here suggests a attention be paid towards inhibition of adipocyte GH signaling as a therapeutic target of metabolic liver disease.


Assuntos
Adipócitos/metabolismo , Carcinoma Hepatocelular/metabolismo , Fígado Gorduroso/metabolismo , Janus Quinase 2/metabolismo , Neoplasias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Adipócitos/patologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Carcinogênese/metabolismo , Modelos Animais de Doenças , Fígado Gorduroso/patologia , Hormônio do Crescimento , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Resistência à Insulina , Janus Quinase 2/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Síndrome Metabólica/complicações , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais
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.
J Mass Spectrom ; 53(7): 548-559, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29596720

RESUMO

Acetylcarnitine has been identified as one of several urinary biomarkers indicative of radiation exposure in adult rhesus macaque monkeys (non-human primates, NHPs). Previous work has demonstrated an up-regulated dose-response profile in a balanced male/female NHP cohort. As a contribution toward the development of metabolomics-based radiation biodosimetry in human populations and other applications of acetylcarnitine screening, we have developed a quantitative, high-throughput method for the analysis of acetylcarnitine. We employed the Sciex SelexIon DMS-MS/MS QTRAP 5500 platform coupled to flow injection analysis (FIA), thereby allowing for fast analysis times of less than 0.5 minutes per injection with no chromatographic separation. Ethyl acetate is used as a DMS modifier to reduce matrix chemical background. We have measured NHP urinary acetylcarnitine from the male cohorts that were exposed to the following radiation levels: control, 2, 4, 6, 7, and 10 Gy. Biological variability, along with calibration accuracy of the FIA-DMS-MS/MS method, indicates LOQ of 20 µM, with observed biological levels on the order of 600 µM and control levels near 10 µM. There is an apparent onset of intensified response in the transition from 6 to 10 Gy. The results demonstrate that FIA-DMS-MS/MS is a rapid, quantitative technique that can be utilized for the analysis of urinary biomarker levels for radiation biodosimetry.


Assuntos
Acetilcarnitina/urina , Espectrometria de Massas em Tandem/métodos , Animais , Biomarcadores/urina , Relação Dose-Resposta à Radiação , Análise de Injeção de Fluxo , Macaca mulatta , Masculino , Exposição à Radiação
10.
Nat Commun ; 9(1): 5103, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504766

RESUMO

Palatable foods (fat and sweet) induce hyperphagia, and facilitate the development of obesity. Whether and how overnutrition increases appetite through the adipose-to-brain axis is unclear. O-linked beta-D-N-acetylglucosamine (O-GlcNAc) transferase (OGT) couples nutrient cues to O-GlcNAcylation of intracellular proteins at serine/threonine residues. Chronic dysregulation of O-GlcNAc signaling contributes to metabolic diseases. Here we show that adipocyte OGT is essential for high fat diet-induced hyperphagia, but is dispensable for baseline food intake. Adipocyte OGT stimulates hyperphagia by transcriptional activation of de novo lipid desaturation and accumulation of N-arachidonyl ethanolamine (AEA), an endogenous appetite-inducing cannabinoid (CB). Pharmacological manipulation of peripheral CB1 signaling regulates hyperphagia in an adipocyte OGT-dependent manner. These findings define adipocyte OGT as a fat sensor that regulates peripheral lipid signals, and uncover an unexpected adipose-to-brain axis to induce hyperphagia and obesity.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Hiperfagia/metabolismo , Hiperfagia/patologia , Obesidade/metabolismo , Obesidade/patologia , Acetilglucosamina/metabolismo , Tecido Adiposo/patologia , Animais , Western Blotting , Peso Corporal/genética , Peso Corporal/fisiologia , Canabinoides/metabolismo , Linhagem Celular , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real
11.
Diabetes ; 67(2): 208-221, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29203511

RESUMO

Disruption of hepatocyte growth hormone (GH) signaling through disruption of Jak2 (JAK2L) leads to fatty liver. Previously, we demonstrated that development of fatty liver depends on adipocyte GH signaling. We sought to determine the individual roles of hepatocyte and adipocyte Jak2 on whole-body and tissue insulin sensitivity and liver metabolism. On chow, JAK2L mice had hepatic steatosis and severe whole-body and hepatic insulin resistance. However, concomitant deletion of Jak2 in hepatocytes and adipocytes (JAK2LA) completely normalized insulin sensitivity while reducing liver lipid content. On high-fat diet, JAK2L mice had hepatic steatosis and insulin resistance despite protection from diet-induced obesity. JAK2LA mice had higher liver lipid content and no protection from obesity but retained exquisite hepatic insulin sensitivity. AKT activity was selectively attenuated in JAK2L adipose tissue, whereas hepatic insulin signaling remained intact despite profound hepatic insulin resistance. Therefore, JAK2 in adipose tissue is epistatic to liver with regard to insulin sensitivity and responsiveness, despite fatty liver and obesity. However, hepatocyte autonomous JAK2 signaling regulates liver lipid deposition under conditions of excess dietary fat. This work demonstrates how various tissues integrate JAK2 signals to regulate insulin/glucose and lipid metabolism.


Assuntos
Tecido Adiposo/enzimologia , Resistência à Insulina , Janus Quinase 2/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade/metabolismo , Tecido Adiposo/metabolismo , Adiposidade , Animais , Dieta Hiperlipídica/efeitos adversos , Janus Quinase 2/genética , Metabolismo dos Lipídeos , Fígado/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Obesidade/etiologia , Obesidade/fisiopatologia , Especificidade de Órgãos , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Treonina/metabolismo
12.
PLoS One ; 12(1): e0169566, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28081256

RESUMO

Tumor cell proliferation and migration processes are regulated by multiple metabolic pathways including glycolysis and de novo lipogenesis. Since acetyl-CoA carboxylase (ACC) is at the junction of lipids synthesis and oxidative metabolic pathways, we investigated whether use of a dual ACC inhibitor would provide a potential therapy against certain lipogenic cancers. The impact of dual ACC1/ACC2 inhibition was investigated using a dual ACC1/ACC2 inhibitor as well as dual siRNA knock down on the cellular viability and metabolism of two glioblastoma multiform cancer cell lines, U87 and a more aggressive form, U87 EGFRvIII. We first demonstrated that while ACCi inhibited DNL in both cell lines, ACCi preferentially blunted the U87 EGFRvIII cellular proliferation capacity. Metabolically, chronic treatment with ACCi significantly upregulated U87 EGFRvIII cellular respiration and extracellular acidification rate, a marker of glycolytic activity, but impaired mitochondrial health by reducing maximal respiration and decreasing mitochondrial ATP production efficiency. Moreover, ACCi treatment altered the cellular lipids content and increased apoptotic caspase activity in U87 EGFRvIII cells. Collectively these data indicate that ACC inhibition, by reducing DNL and increasing cellular metabolic rate, may have therapeutic utility for the suppression of lipogenic tumor growth and warrants further investigation.


Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Receptores ErbB , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Lipogênese/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Lipogênese/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/genética
13.
Endocrinology ; 157(2): 570-85, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26650570

RESUMO

CD36/FAT (fatty acid translocase) is associated with human and murine nonalcoholic fatty liver disease, but it has been unclear whether it is simply a marker or whether it directly contributes to disease pathogenesis. Mice with hepatocyte-specific deletion of Janus kinase 2 (JAK2L mice) have increased circulating free fatty acids (FAs), dramatically increased hepatic CD36 expression and profound fatty liver. To investigate the role of elevated CD36 in the development of fatty liver, we studied two models of hepatic steatosis, a genetic model (JAK2L mice) and a high-fat diet (HFD)-induced steatosis model. We deleted Cd36 specifically in hepatocytes of JAK2L mice to generate double knockouts and from wild-type mice to generate CD36L single-knockout mice. Hepatic Cd36 disruption in JAK2L livers significantly improved steatosis by lowering triglyceride, diacylglycerol, and cholesterol ester content. The largest differences in liver triglycerides were in species comprised of oleic acid (C18:1). Reduction in liver lipids correlated with an improvement in the inflammatory markers that were elevated in JAK2L mice, namely aspartate aminotransferase and alanine transaminase. Cd36 deletion in mice on HFD (CD36L-HFD) reduced liver lipid content and decreased hepatic 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-FA uptake as compared with CON-HFD. Additionally, CD36L-HFD mice had improved whole-body insulin sensitivity and reduced liver and serum inflammatory markers. Therefore, CD36 directly contributes to development of fatty liver under conditions of elevated free FAs by modulating the rate of FA uptake by hepatocytes. In HFD-fed animals, disruption of hepatic Cd36 protects against associated systemic inflammation and insulin resistance.


Assuntos
Antígenos CD36/genética , Dieta Hiperlipídica , Hepatócitos/metabolismo , Resistência à Insulina/genética , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Antígenos CD36/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Especificidade de Órgãos/genética , Triglicerídeos/metabolismo
14.
J Med Chem ; 59(3): 1165-75, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26734723

RESUMO

Inhibition of the sodium-coupled citrate transporter (NaCT or SLC13A5) has been proposed as a new therapeutic approach for prevention and treatment of metabolic diseases. In a previous report, we discovered dicarboxylate 1a (PF-06649298) which inhibits the transport of citrate in in vitro and in vivo settings via a specific interaction with NaCT. Herein, we report the optimization of this series leading to 4a (PF-06761281), a more potent inhibitor with suitable in vivo pharmacokinetic profile for assessment of in vivo pharmacodynamics. Compound 4a was used to demonstrate dose-dependent inhibition of radioactive [(14)C]citrate uptake in liver and kidney in vivo, resulting in modest reductions in plasma glucose concentrations.


Assuntos
Citratos/metabolismo , Malatos/química , Malatos/farmacologia , Fenilbutiratos/química , Fenilbutiratos/farmacologia , Piridinas/química , Piridinas/farmacologia , Simportadores/antagonistas & inibidores , Animais , Transporte Biológico/efeitos dos fármacos , Glicemia/metabolismo , Citratos/farmacocinética , Relação Dose-Resposta a Droga , Células HEK293 , Hepatócitos/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Malatos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Estrutura Molecular , Fenilbutiratos/administração & dosagem , Piridinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Simportadores/metabolismo
15.
ACS Chem Biol ; 11(9): 2529-40, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27391855

RESUMO

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.


Assuntos
Hidrolases/metabolismo , Lisofosfolipase/antagonistas & inibidores , Serina/metabolismo , Animais , Cristalização , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Lisofosfolipase/química
16.
Sci Rep ; 5: 17391, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26620127

RESUMO

Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.


Assuntos
Ácido Cítrico/metabolismo , Simportadores/antagonistas & inibidores , Células HEK293 , Humanos , Transporte de Íons/efeitos dos fármacos , Simportadores/genética , Simportadores/metabolismo
17.
PLoS One ; 9(5): e97139, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24858947

RESUMO

Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the ß-cells and hepatocytes. Since activation of glucokinase in ß-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase activation may offer glycemic control without inducing hepatic steatosis supporting the evaluation of tissue specific activators in clinical trials.


Assuntos
Diabetes Mellitus Experimental/complicações , Ativadores de Enzimas/farmacologia , Glucoquinase/metabolismo , Hiperglicemia/complicações , Hiperglicemia/tratamento farmacológico , Imidazóis/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Ácidos Nicotínicos/farmacologia , Animais , Ativadores de Enzimas/efeitos adversos , Ativadores de Enzimas/uso terapêutico , Glucose/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Imidazóis/efeitos adversos , Imidazóis/uso terapêutico , Fígado/patologia , Masculino , Ácidos Nicotínicos/efeitos adversos , Ácidos Nicotínicos/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Especificidade de Órgãos , Ratos
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