RESUMO
Strategies to increase energy expenditure are an attractive approach to reduce excess fat storage and body weight to improve metabolic health. In mammals, uncoupling protein-1 (UCP1) in brown and beige adipocytes uncouples fatty acid oxidation from ATP generation in mitochondria and promotes energy dissipation as heat. We set out to identify small molecules that enhance UCP1 levels and activity using a high-throughput screen of nearly 12,000 compounds in mouse brown adipocytes. We identified a family of compounds that increase Ucp1 expression and mitochondrial activity (including un-coupled respiration) in mouse brown adipocytes and human brown and white adipocytes. The mechanism of action may be through compound binding to A kinase anchoring protein (AKAP) 1, modulating its localization to mitochondria and its interaction with protein kinase A (PKA), a known node in the ß-adrenergic signaling pathway. In mice, the hit compound increased body temperature, UCP1 protein levels, and thermogenic gene expression. Some of the compound effects on mitochondrial function were UCP1- or AKAP1-independent, suggesting compound effects on multiple nodes of energy regulation. Overall, our results highlight a role for AKAP1 in thermogenesis, uncoupled respiration, and regulation energy balance.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Termogênese/efeitos dos fármacos , Proteína Desacopladora 1/biossíntese , Adipócitos Marrons/enzimologia , Adipócitos Marrons/metabolismo , Adipócitos Brancos/enzimologia , Adipócitos Brancos/metabolismo , Animais , Células Cultivadas , Metabolismo Energético , Ativação Enzimática , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
We previously reported the involvement of protein arginine methyltransferase 1 (PRMT1) in adipocyte thermogenesis. Here, we investigate the effects of PRMT1 inhibitors on thermogenesis. Unexpectedly, we find that the PRMT1 inhibitor TC-E 5003 (TC-E) induces the thermogenic properties of primary murine and human subcutaneous adipocytes. TC-E treatment upregulates the expression of Ucp1 and Fgf21 significantly and activates protein kinase A signaling and lipolysis in primary subcutaneous adipocytes from both mouse and humans. We further find that the thermogenic effects of TC-E are independent of PRMT1 and beta-adrenergic receptors. Our data indicate that TC-E exerts strong effects on murine and human subcutaneous adipocytes by activating beige adipocytes via PKA signaling.
Assuntos
Benzenoacetamidas/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Inibidores Enzimáticos/farmacologia , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Termogênese/efeitos dos fármacos , Adipócitos Bege/citologia , Adipócitos Bege/efeitos dos fármacos , Adipócitos Bege/enzimologia , Adipócitos Marrons/citologia , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/enzimologia , Adipócitos Brancos/citologia , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/enzimologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Lipólise/efeitos dos fármacos , Lipólise/genética , Camundongos , Cultura Primária de Células , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Termogênese/genética , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Delphinidin-3-O-ß-glucoside (D3G) is a health-promoting anthocyanin whose anti-obesity activity has not yet been thoroughly investigated. We examined the effects of D3G on adipogenesis and lipogenesis in 3T3-L1 adipocytes and primary white adipocytes using real-time RT-PCR and immunoblot analysis. D3G significantly inhibited the accumulation of lipids in a dose-dependent manner without displaying cytotoxicity. In the 3T3-L1 adipocytes, D3G downregulated the expression of key adipogenic and lipogenic markers, which are known as peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBP1), CCAAT/enhancer-binding protein alpha (C/EBPα), and fatty acid synthase (FAS). Moreover, the relative protein expression of silent mating type information regulation 2 homolog 1 (SIRT1) and carnitine palmitoyltransferase-1 (CPT-1) were increased, alongside reduced lipid levels and the presence of several small lipid droplets. Furthermore, D3G increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), which suggests that D3G may play a role in AMPK and ACC activation in adipocytes. Our data indicate that D3G attenuates adipogenesis and promotes lipid metabolism by activating AMPK-mediated signaling, and, hence, could have a therapeutic role in the management and treatment of obesity.
Assuntos
Adipócitos/citologia , Adipogenia/efeitos dos fármacos , Antocianinas/farmacologia , Glucosídeos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos Brancos/citologia , Adipócitos Brancos/enzimologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , PPAR gama/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
Globally, approximately 10%-25% of women smoke during pregnancy. Since nicotine is highly addictive, women may use nicotine-containing products like nicotine replacement therapies for smoking cessation, but the long-term consequences of early life exposure to nicotine remain poorly defined. Our laboratory has previously demonstrated that maternal nicotine exposed (MNE) rat offspring exhibit hypertriglyceridemia due to increased hepatic de novo lipogenesis. Hypertriglyceridemia may also be attributed to impaired white adipose tissue (WAT) lipid storage; however, the effects of MNE on WAT are not completely understood. We hypothesize that nicotine-induced alterations in adipose function (eg, lipid storage) underlie dyslipidemia in MNE adults. Female 6-month-old rats exposed to nicotine during gestation and lactation exhibited significantly decreased visceral adipocyte cell area by 40%, attributed, in part, to a 3-fold increase in adipose triglyceride lipase (ATGL) protein expression compared with vehicle. Given ATGL has antioxidant properties and in utero nicotine exposure promotes oxidative stress in various tissues, we next investigated if there was evidence of increased oxidative stress in MNE WAT. At both 3 weeks and 6 months, MNE offspring expressed 37%-48% higher protein levels of superoxide dismutase-1 and -2 in WAT. Since oxidative stress can induce inflammation, we examined the inflammatory profile of WAT and found increased expression of cytokines (interleukin-1ß, tumor necrosis factor α, and interleukin-6) by 44%-61% at 6 months. Collectively, this suggests that the expression of WAT ATGL may be induced to counter MNE-induced oxidative stress and inflammation. However, higher levels of ATGL would further promote lipolysis in WAT, culminating in impaired lipid storage and long-term dyslipidemia.
Assuntos
Tecido Adiposo Branco/efeitos dos fármacos , Antioxidantes/metabolismo , Lipase/genética , Exposição Materna/efeitos adversos , Nicotina/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/enzimologia , Tecido Adiposo Branco/embriologia , Tecido Adiposo Branco/enzimologia , Tecido Adiposo Branco/crescimento & desenvolvimento , Animais , Proteínas de Escherichia coli/efeitos dos fármacos , Feminino , Lipogênese/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/enzimologia , Efeitos Tardios da Exposição Pré-Natal/genética , Ratos WistarRESUMO
Mechanistic target of rapamycin complex 1 (mTORC1) loss of function reduces adiposity whereas partial mTORC1 inhibition enhances fat deposition. Herein we evaluated how constitutive mTORC1 activation in adipocytes modulates adiposity in vivo. Mice with constitutive mTORC1 activation in adipocytes induced by tuberous sclerosis complex (Tsc)1 deletion and littermate controls were evaluated for body mass, energy expenditure, glucose and fatty acid metabolism, mitochondrial function, mRNA and protein contents. Adipocyte-specific Tsc1 deletion reduced visceral, but not subcutaneous, fat mass, as well as adipocyte number and diameter, phenotypes that were associated with increased lipolysis, UCP-1 content (browning) and mRNA levels of pro-browning transcriptional factors C/EBPß and ERRα. Adipocyte Tsc1 deletion enhanced mitochondrial oxidative activity, fatty acid oxidation and the expression of PGC-1α and PPARα in both visceral and subcutaneous fat. In brown adipocytes, however, Tsc1 deletion did not affect UCP-1 content and basal respiration. Adipocyte Tsc1 deletion also reduced visceral adiposity and enhanced glucose tolerance, liver and muscle insulin signaling and adiponectin secretion in mice fed with purified low- or high-fat diet. In conclusion, adipocyte-specific Tsc1 deletion enhances mitochondrial activity, induces browning and reduces visceral adiposity in mice.
Assuntos
Adipócitos Marrons/enzimologia , Adipócitos Brancos/enzimologia , Tecido Adiposo Marrom/enzimologia , Adiposidade , Gordura Intra-Abdominal/enzimologia , Mitocôndrias/enzimologia , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adipócitos Marrons/ultraestrutura , Adipócitos Brancos/ultraestrutura , Adiponectina/deficiência , Adiponectina/genética , Tecido Adiposo Marrom/ultraestrutura , Adiposidade/genética , Animais , Respiração Celular , Dieta com Restrição de Gorduras , Dieta Hiperlipídica , Metabolismo Energético , Ativação Enzimática , Regulação da Expressão Gênica , Genótipo , Glucose/metabolismo , Insulina/metabolismo , Gordura Intra-Abdominal/ultraestrutura , Lipólise , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura , Oxirredução , Fenótipo , Transdução de Sinais , Fatores de Tempo , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt(-/-) mice are protected against high-fat diet (HFD)-induced obesity and insulin resistance; however, these mice develop nonalcoholic fatty liver disease (NAFLD). We hypothesized that peroxisomal proliferator-activated receptor-γ (PPARγ) activation by pioglitazone might stimulate adipocyte proliferation, thereby directing lipids from the liver toward white adipose tissue. Pioglitazone might also act directly on PPARγ in the liver to improve NAFLD. Pemt(+/+) and Pemt(-/-) mice were fed a HFD with or without pioglitazone (20 mg·kg(-1)·day(-1)) for 10 wk. Pemt(-/-) mice were protected from HFD-induced obesity but developed NAFLD. Treatment with pioglitazone caused an increase in body weight gain in Pemt(-/-) mice that was mainly due to increased adiposity. Moreover, pioglitazone improved NAFLD in Pemt(-/-) mice, as indicated by a 35% reduction in liver weight and a 57% decrease in plasma alanine transaminase levels. Livers from HFD-fed Pemt(-/-) mice were steatotic, inflamed, and fibrotic. Hepatic steatosis was still evident in pioglitazone-treated Pemt(-/-) mice; however, treatment with pioglitazone reduced hepatic fibrosis, as evidenced by reduced Sirius red staining and lowered mRNA levels of collagen type Iα1 (Col1a1), tissue inhibitor of metalloproteinases 1 (Timp1), α-smooth muscle actin (Acta2), and transforming growth factor-ß (Tgf-ß). Similarly, oxidative stress and inflammation were reduced in livers from Pemt(-/-) mice upon treatment with pioglitazone. Together, these data show that activation of PPARγ in HFD-fed Pemt(-/-) mice improved liver function, while these mice were still protected against diet-induced obesity and insulin resistance.
Assuntos
Anti-Infecciosos/farmacologia , Hepatite/prevenção & controle , Cirrose Hepática Experimental/prevenção & controle , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , PPAR gama/agonistas , Fosfatidiletanolamina N-Metiltransferase/deficiência , Tiazolidinedionas/farmacologia , Actinas/genética , Actinas/metabolismo , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/enzimologia , Adipócitos Brancos/patologia , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/enzimologia , Tecido Adiposo Branco/patologia , Adiposidade/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Dieta Hiperlipídica , Predisposição Genética para Doença , Hepatite/enzimologia , Hepatite/genética , Hepatite/patologia , Resistência à Insulina , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática Experimental/enzimologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/enzimologia , Obesidade/genética , Obesidade/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/metabolismo , Fenótipo , Fosfatidiletanolamina N-Metiltransferase/genética , Pioglitazona , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBß and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBß can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBß assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase.
Assuntos
Adipócitos Brancos/enzimologia , Adipogenia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , AnimaisRESUMO
Protein kinase B (Akt) kinases are critical signal transducers mediating insulin action. Genetic studies revealed that Akt1 and Akt2 signalling differentially contribute to sustain lipid and glucose homoeostasis; however Akt isoform-specific effectors remain elusive due to the lack of a suitable model system to mechanistically interrogate Akt isoform-specific signalling. To overcome those technical limitations we developed a novel model system that provides acute and specific control of signalling by Akt isoforms. We generated mutants of Akt1 and Akt2 resistant to the allosteric Akt inhibitor MK-2206. We then developed adipocyte cell lines, in which endogenous Akt1 or Akt2 has been replaced by their corresponding drug-resistant Akt mutant. Treatment of those cells with MK-2206 allowed for acute and specific control of either Akt1 or Akt2 function. Our data showed that Akt1(W80A) and Akt2(W80A) mutants are resistant to MK-2206, dynamically regulated by insulin and able to signal to Akt downstream effectors. Analyses of insulin action in this cellular system showed that Akt1 and Akt2 are both able to mediate insulin regulation of the transcription factor forkhead box O1 (FoxO1) and the glucose transporter 4 (GLUT4), revealing a redundant role for these Akt kinases in the control of glucose transport into fat cells. In contrast, Akt1 signalling is uniquely required for adipogenesis, by controlling the mitotic clonal expansion (MCE) of pre-adipocytes that precedes white adipose cell differentiation. Our data provide new insights into the role of Akt kinases in glucose transport and adipogenesis and support our model system as a valuable tool for the biochemical characterization of signalling by specific Akt isoforms.
Assuntos
Adipócitos Brancos/enzimologia , Adipogenia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células 3T3-L1 , Adipócitos Brancos/citologia , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/metabolismo , Adipogenia/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Resistência a Medicamentos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Resistência à Insulina , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Mice lacking phosphatidylethanolamine N-methyltransferase (PEMT, Pemt(-/-) mice) are resistant to high-fat diet (HFD)-induced obesity (DIO) but develop non-alcoholic steatohepatitis. PEMT expression is strongly induced during differentiation of 3T3-L1 adipocytes. Hence, we hypothesized that white adipose tissue (WAT) might be a key player in the protection against DIO in Pemt(-/-) mice. We fed Pemt(-/-) and Pemt(+/+) mice the HFD for 2 weeks, after which we examined adipocyte differentiation, adipogenesis and lipolysis in WAT. Pemt(-/-) mice gained less body weight, had reduced WAT mass and had smaller adipocytes than Pemt(+/+) mice. The protein levels of adipose differentiation markers FABP4, PPARγ and C/EBPß were not altered by genotype, but acetyl-CoA carboxylase expression and activation was reduced in the Pemt(-/-) mice. The in vivo conversion of [¹4C]acetate to [¹4C]TG in WAT was also lower in Pemt(-/-) mice. The release of glycerol from WAT explants was comparable between Pemt(+/+) and Pemt(-/-) mice under basal condition and in the presence of isoproterenol, indicating unaffected lipolytic capacity. Furthermore, the amounts of leptin, cytokines and chemokines in WAT were not altered by genotype in mice fed the HFD for 2 weeks. However, after 10 weeks of HFD, WAT from Pemt(-/-) mice had dramatically lower leptin, inflammatory cytokines (IL-1 and TNF-α) and chemokines (MCP-1 and RANTES), and significantly higher anti-inflammatory cytokine IL-10 than Pemt(+/+) mice. Together, our data show that PEMT deficiency did not affect the capability for differentiation and lipolysis in WAT. Decreased lipogenesis in WAT may contribute to the resistance to DIO in Pemt(-/-) mice.
Assuntos
Tecido Adiposo Branco/enzimologia , Dieta Hiperlipídica , Lipogênese , Obesidade/prevenção & controle , Fosfatidiletanolamina N-Metiltransferase/deficiência , Adipócitos Brancos/enzimologia , Adipogenia , Tecido Adiposo Branco/fisiopatologia , Adiposidade , Animais , Biomarcadores/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Genótipo , Lipídeos/sangue , Lipólise , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/sangue , Obesidade/enzimologia , Obesidade/genética , Obesidade/fisiopatologia , Fenótipo , Fosfatidiletanolamina N-Metiltransferase/genética , Fatores de Proteção , Fatores de Tempo , Aumento de PesoRESUMO
Arachidonic acid (AA) is a major PUFA that has been implicated in the regulation of adipogenesis. We examined the effect of a short exposure to AA at different stages of 3T3-L1 adipocyte differentiation. AA caused the upregulation of fatty acid binding protein 4 (FABP4/aP2) following 24 h of differentiation. This was mediated by the prostaglandin F(2α) (PGF(2α)), as inhibition of cyclooxygenases or PGF(2α) receptor signaling counteracted the AA-mediated aP2 induction. In addition, calcium, protein kinase C, and ERK are all key elements of the pathway through which AA induces the expression of aP2. We also show that treatment with AA during the first 24 h of differentiation upregulates the expression of the transcription factor Fos-related antigen 1 (Fra-1) via the same pathway. Finally, treatment with AA for 24 h at the beginning of the adipocyte differentiation is sufficient to inhibit the late stages of adipogenesis through a Fra-1-dependent pathway, as Fra-1 knockdown rescued adipogenesis. Our data show that AA is able to program the differentiation potential of preadipocytes by regulating gene expression at the early stages of adipogenesis.
Assuntos
Adipócitos Brancos/metabolismo , Adipogenia , Ácido Araquidônico/metabolismo , Proteínas de Ligação a Ácido Graxo/agonistas , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Proto-Oncogênicas c-fos/agonistas , Receptores de Prostaglandina/agonistas , Células 3T3-L1 , Adipócitos Brancos/citologia , Adipócitos Brancos/enzimologia , Animais , Sinalização do Cálcio , Dinoprosta/metabolismo , Regulação para Baixo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Cinética , Sistema de Sinalização das MAP Quinases , Camundongos , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Cardiotrophin-1 (CT-1) is a cytokine with antiobesity properties and with a role in lipid metabolism regulation and adipose tissue function. The aim of this study was to analyze the molecular mechanisms involved in the lipolytic actions of CT-1 in adipocytes. Recombinant CT-1 (rCT-1) effects on the main proteins and signaling pathways involved in the regulation of lipolysis were evaluated in 3T3-L1 adipocytes and in mice. rCT-1 treatment stimulated basal glycerol release in a concentration- and time-dependent manner in 3T3-L1 adipocytes. rCT-1 (20 ng/ml for 24 h) raised cAMP levels, and in parallel increased protein kinase (PK)A-mediated phosphorylation of perilipin and hormone sensitive lipase (HSL) at Ser660. siRNA knock-down of HSL or PKA, as well as pretreatment with the PKA inhibitor H89, blunted the CT-1-induced lipolysis, suggesting that the lipolytic action of CT-1 in adipocytes is mainly mediated by activation of HSL through the PKA pathway. In ob/ob mice, acute rCT-1 treatment also promoted PKA-mediated phosphorylation of perilipin and HSL at Ser660 and Ser563, and increased adipose triglyceride lipase (desnutrin) content in adipose tissue. These results showed that the ability of CT-1 to regulate the activity of the main lipases underlies the lipolytic action of this cytokine in vitro and in vivo, and could contribute to CT-1 antiobesity effects.
Assuntos
Adipócitos Brancos/metabolismo , Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Lipase/metabolismo , Lipólise , Fosfoproteínas/metabolismo , Esterol Esterase/metabolismo , Regulação para Cima , Células 3T3-L1 , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/enzimologia , Animais , Proteínas de Transporte/biossíntese , Proteínas de Ciclo Celular/agonistas , Proteínas de Ciclo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocinas/genética , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Inativação Gênica , Lipase/antagonistas & inibidores , Lipase/química , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Camundongos Mutantes , Perilipina-1 , Fosfoproteínas/biossíntese , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esterol Esterase/antagonistas & inibidores , Esterol Esterase/química , Esterol Esterase/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Responses of adipose cells to adrenoceptor regulation, including that of ß-adrenoceptor (AR), and the signalling machinery involved in these responses are not sufficiently understood; information that is helpful for elucidating the adrenoceptor (adrenergic and ß-AR)-responsive machinery is insufficient. We examined phospho-Thr-172 AMPK production in mouse-derived 3T3-L1 adipocytes treated with epinephrine or CL316243 (a ß3-AR agonist) for 15 min. We also examined MAPK activation or G protein-associated PI3K activation or -associated PI3K p85 complex formation in rat epididymal (white) adipocytes treated with CL316243 for 15 min or aluminum fluoride (a G-protein signalling activator) for 20 min. Furthermore, we examined the effect of PTX (a trimeric G-protein inactivator) on p85 complex formation induced by aluminum fluoride treatment. Western blot analysis revealed that epinephrine or CL316243 treatment increased the phospho- Thr-172 AMPK (an active form of AMPK) level in 3T3-L1 adipocytes. Activated kinase analysis with a specific substrate showed that CL316243 or aluminum fluoride treatment activated MAPK in rat adipocytes. Immunoprecipitation experiments with a G-protein ß subunit (Gß) antibody showed that treatment of rat adipocytes with CL316243 activated PI3K and increased the PI3K p85 level in the Gß antibody immunoprecipitates. Such an increase in the p85 level was similarly elicited by aluminum fluoride treatment in a PTX-sensitive manner. Our results provide possible clues for clarifying the signalling machinery involved in adrenoceptor responses, including those of ß3-AR, in mouse-derived adipocytes and rat white adipocytes. Our findings advance the understanding of responses to adrenoceptor regulation in adipose cells and of the cellular signalling machinery present in the cells.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos Brancos/enzimologia , Compostos de Alumínio/farmacologia , Dioxóis/farmacologia , Epinefrina/farmacologia , Fluoretos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células 3T3-L1 , Animais , Imunoprecipitação , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Toxina Pertussis/farmacologia , Fosfotreonina/metabolismo , RatosRESUMO
PURPOSE: The principal function of the adipose tissue is the storage of energy in the form of triglyceride through the process of adipogenesis, as well as the provision of the stored energy through lipolysis. In the present study, we investigated the effect of hydroxytyrosol on lipolysis in 3T3-L1 adipocytes. METHODS: 3T3-L1 adipocytes, used as in vitro model in this study, were treated with several concentration of hydroxytyrosol. Glycerol release was measured to identify the lipolytic rate activation. All factors activation and expression were carried out via Western blotting and qRT-PCR. RESULTS: Our results showed that hydroxytyrosol, over a range of concentrations, attenuated triglyceride accumulation and stimulated glycerol release in fully differentiated adipocytes in a dose- and time-dependent manner. Moreover, hydroxytyrosol had no effect on adipocyte viability. To understand the mechanism underlying hydroxytyrosol-stimulated lipolysis, we used inhibitors of PKA, PKC, PKG, ERK1/2, and nitric oxide production. Pretreatment with a PKA inhibitor (Rp-cAMPs) and an ERK1/2 inhibitor (U0126) significantly attenuated hydroxytyrosol-stimulated lipolysis. In contrast, a PKC inhibitor (Calphostin C), 2 PKG inhibitors (KT 5823 and Rp-cGMPs), and a nitric oxide inhibitor (S-ethyl ITU) had no effect on hydroxytyrosol-stimulated lipolysis. Over the same range of concentrations, hydroxytyrosol downregulated the expression of adipose triglyceride lipase, hormone sensitive lipase (HSL), and adipogenesis-related transcription factors PPARγ and C/EBPα. In addition, hydroxytyrosol increased the phosphorylation rate of HSL at Ser563 and Ser660, as well as perilipin and ERK phosphorylation. CONCLUSION: Hydroxytyrosol induced lipolysis in 3T3-L1 adipocytes via the activation of PKA and ERK1/2 pathway.
Assuntos
Adipócitos Brancos/efeitos dos fármacos , Antioxidantes/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Lipólise/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Álcool Feniletílico/análogos & derivados , Triglicerídeos/metabolismo , Células 3T3-L1 , Adipócitos Brancos/enzimologia , Adipócitos Brancos/metabolismo , Animais , Antioxidantes/efeitos adversos , Antioxidantes/química , Sobrevivência Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/química , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicerol/metabolismo , Cinética , Lipase/antagonistas & inibidores , Lipase/genética , Lipase/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Álcool Feniletílico/efeitos adversos , Álcool Feniletílico/antagonistas & inibidores , Álcool Feniletílico/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Esterol Esterase/genética , Esterol Esterase/metabolismoRESUMO
This study aimed to examine the anti-proliferative effects of α-, γ- and δ-tocotrienols (αT3, γT3 and δT3), and α-tocopherol on 3T3-L1 adipocytes. Results showed that compared with other vitamin E analogues, γT3 demonstrated the most potent anti-proliferative effect on 3T3-L1 cells. It significantly caused a reduction in mitochondrial membrane potential (Δψm) and an increase in ROS formation, as well as inducing cell apoptosis and cell cycle arrest at S phase. Further studies showed that it down-regulated Bcl-2 and PPAR-γ expression, suppressed Akt and ERK activation and phosphorylation, and caused cytochrome c release from mitochondria to cytosol, whereas it up-regulated CD95 (APO-1/CD95) and Bax expression, and caused caspase-3 and JNK activation, PARP cleavage and AMPK phosphorylation. Pretreatments with caspase-3 (z-DEVD-fmk) and AMPK (CC) inhibitors significantly suppressed the γT3-induced ROS production and cell death. Caspase-3 inhibitor also efficiently blocked CD95 (APO-1/CD95) and Bax expression, caspase-3 activation and PARP cleavage, whereas antioxidant N-acetyl-l-cysteine, AMPK inhibitor and AMPK siRNA effectively blocked the AMPK phosphorylation. Taken together, these results conclude that the potent anti-proliferative and anti-adipogenic effects of γT3 on 3T3-L1 adipocytes could be through the Bax-mediated mitochondrial and AMPK signaling pathways.
Assuntos
Adipócitos Brancos/metabolismo , Adipogenia , Apoptose , Cromanos/metabolismo , Suplementos Nutricionais , Mitocôndrias/metabolismo , Transdução de Sinais , Vitamina E/análogos & derivados , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos Brancos/citologia , Adipócitos Brancos/enzimologia , Animais , Fármacos Antiobesidade/metabolismo , Proliferação de Células , Sobrevivência Celular , Cromanos/antagonistas & inibidores , Regulação para Baixo , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/enzimologia , Fosforilação , Processamento de Proteína Pós-Traducional , Interferência de RNA , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Fase S , Vitamina E/antagonistas & inibidores , Vitamina E/metabolismo , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: The purpose of this study is to investigate whether local inflammatory events, such as periodontal disease, are able to increase tumor necrosis factor-alpha (TNF-α) plasmatic concentration and decrease insulin sensitivity and insulin signaling in non-diabetic rats. METHODS: Forty-eight male Wistar rats (2 months old) were divided into two groups, with either ligature-induced periodontal disease (LPD) or control conditions (CN). Experiments were performed in both groups 28 days after ligature placement. Plasmatic concentration of glycemia and TNF-α (n = 10) were analyzed by the glucose oxidase and enzyme-linked immunosorbent assay method, respectively. Insulin sensitivity (n = 7) was measured using the insulin tolerance test. Insulin signal transduction (n = 7) was measured by pp185 tyrosine phosphorylation status in insulin-sensitive tissues using the Western blotting method. RESULTS: The LPD group showed decreased insulin sensitivity (P <0.05), although no glycemic alterations were noted (P >0.05). TNF-α plasmatic concentration was higher in LPD rats compared to CN rats. In addition, a decrease in the pp185 tyrosine phosphorylation status was observed after insulin stimulus in both white adipose and skeletal muscle tissues of the LPD group compared with the CN group. CONCLUSIONS: LPD is able to cause alterations to both insulin signaling and insulin sensitivity, probably because of the elevation of TNF-α plasmatic concentration. Thus, the present results emphasize the importance of the prevention of local inflammatory diseases, such as periodontitis, to prevent diabetes mellitus.
Assuntos
Resistência à Insulina/fisiologia , Insulina/sangue , Periodontite/sangue , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/sangue , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/enzimologia , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/patologia , Processo Alveolar/diagnóstico por imagem , Processo Alveolar/patologia , Animais , Glicemia/análise , Retração Gengival/patologia , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina/farmacologia , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Periodontite/patologia , Fosforilação , Radiografia , Ratos , Ratos Wistar , Receptor de Insulina/efeitos dos fármacos , Colo do Dente/diagnóstico por imagem , Colo do Dente/patologiaRESUMO
We have previously shown that medium-chain triglyceride (MCT) resulted in significantly less body fat mass than long-chain triglyceride (LCT) did in hypertriglyceridimic subjects. The possible mechanism for this was investigated by measuring and analyzing changes in the body fat, blood lipid profile, enzymatic level and activity of hormone-sensitive lipase (HSL) and its mRNA expression, and levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in white adipose tissue (WAT) of C57BL/6J mice fed for 16 weeks on an MCT or LCT diet. MCT induced lower body weight and body fat, and an improved blood lipid profile than LCT did. The enzymatic level and activity of HSL and its mRNA expression, and the levels of cAMP and PKA were significantly higher in WAT of mice fed with the MCT diet. No significant differences in the levels of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in WAT were apparent between the effects of MCT and LCT. It is concluded that lipolysis by the increased level and activity of HSL, which was induced by the activation of cAMP-dependent PKA in WAT, was partially responsible for the lower fat accumulation in C57BL/6J mice fed with MCT.
Assuntos
Adipócitos Brancos/efeitos dos fármacos , Ácidos Graxos/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Esterol Esterase/genética , Esterol Esterase/metabolismo , Triglicerídeos/química , Triglicerídeos/farmacologia , Adipócitos Brancos/enzimologia , Adipócitos Brancos/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Gorduras na Dieta/farmacologia , Ácido Graxo Sintases/metabolismo , Lipase Lipoproteica/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , PPAR gama/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The incidence of obesity in the developed world is increasing at an alarming rate. Concurrent with the increase in the incidence of obesity is an increase in the incidence of type 2 diabetes. Cyclic AMP (cAMP) and cGMP are key second messengers in all cells; for example, when it comes to processes of relevance for the regulation of energy metabolism, cAMP is a key mediator in the regulation of lipolysis, glycogenolysis, gluconeogenesis and pancreatic ß cell insulin secretion. PDE3B, one of several enzymes which hydrolyze cAMP and cGMP, is expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, hypothalamic cells and ß cells. It has been shown, using PDE3 inhibitors and gene targeting approaches in cells and animals, that altered levels of PDE3B result in a number of changes in the regulation of glucose and lipid metabolism and in overall energy homeostasis. This article highlights the complexity involved in the regulation of PDE3B by hormones, and in the regulation of downstream metabolic effects by PDE3B in several interacting tissues.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Ingestão de Energia , Metabolismo Energético , Sistemas do Segundo Mensageiro , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/enzimologia , Adipócitos Brancos/metabolismo , Animais , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/fisiologia , GMP Cíclico/antagonistas & inibidores , GMP Cíclico/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/metabolismo , Ingestão de Energia/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Terapia de Alvo Molecular , Obesidade/tratamento farmacológico , Obesidade/enzimologia , Obesidade/metabolismo , Inibidores da Fosfodiesterase 3/farmacologia , Inibidores da Fosfodiesterase 3/uso terapêutico , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Sistemas do Segundo Mensageiro/efeitos dos fármacosRESUMO
Lipid droplets are universal intracellular organelles composed of a triglyceride, cholesteryl ester and retinyl ester core, surrounded by a monolayer of phospholipids and free (unesterified) cholesterol and lipid droplet-associated proteins. Core lipids are hydrolyzed by lipases to provide fatty acids, cholesterol and retinol for various cellular functions. In addition to cytosolic adipose triglyceride lipase and hormone-sensitive lipase, recent studies show the existence of other neutral lipid hydrolases that reside in the endoplasmic reticulum. In this review we highlight the role of these novel lipases including several members of the carboxylesterase family and enzymes termed arylacetamide deacetylase and KIAA1363/neutral cholesteryl ester hydrolase1/arylacetamide deacetylase-like 1. Some of these enzymes might be attractive targets for the treatment of dyslipidemias, viral infection and atherosclerosis.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Retículo Endoplasmático/enzimologia , Metabolismo dos Lipídeos , Organelas/metabolismo , Adipócitos Brancos/enzimologia , Adipócitos Brancos/metabolismo , Animais , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Isoenzimas/metabolismo , Macrófagos/enzimologia , Macrófagos/metabolismoRESUMO
Estrogen regulates fat mass and distribution and glucose metabolism. We have previously found that estrogen sulfotransferase (EST), which inactivates estrogen through sulfoconjugation, was highly expressed in adipose tissue of male mice and induced by testosterone in female mice. To determine whether inhibition of estrogen in female adipose tissue affects adipose mass and metabolism, we generated transgenic mice expressing EST via the aP2 promoter. As expected, EST expression was increased in adipose tissue as well as macrophages. Parametrial and subcutaneous inguinal adipose mass and adipocyte size were significantly reduced in EST transgenic mice, but there was no change in retroperitoneal or brown adipose tissue. EST overexpression decreased the differentiation of primary adipocytes, and this was associated with reductions in the expression of peroxisome proliferator-activated receptor-γ, fatty acid synthase, hormone-sensitive lipase, lipoprotein lipase, and leptin. Serum leptin levels were significantly lower in EST transgenic mice, whereas total and high-molecular-weight adiponectin levels were not different in transgenic and wild-type mice. Glucose uptake was blunted in parametrial adipose tissue during hyperinsulinemic-euglycemic clamp in EST transgenic mice. In contrast, hepatic insulin sensitivity was improved but muscle insulin sensitivity did not change in EST transgenic mice. These results reveal novel effects of EST on adipose tissue and glucose homeostasis in female mice.
Assuntos
Tecido Adiposo/metabolismo , Composição Corporal/fisiologia , Glucose/metabolismo , Resistência à Insulina/fisiologia , Sulfotransferases/metabolismo , Adipócitos Brancos/enzimologia , Adipócitos Brancos/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/enzimologia , Animais , Diferenciação Celular/fisiologia , Estrogênios/metabolismo , Ácido Graxo Sintases/sangue , Feminino , Técnica Clamp de Glucose , Leptina/sangue , Lipase Lipoproteica/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , PPAR gama/sangue , Distribuição Aleatória , Esterol Esterase/sangueRESUMO
ATF-2 is a member of the ATF/CREB family of transcription factors and is activated by stress-activated protein kinases, such as p38. To analyze the physiological role of ATF-2 family transcription factors, we have generated mice with mutations in Atf-2 and Cre-bpa, an Atf-2-related gene. The trans-heterozygotes of both mutants were lean and had reduced white adipose tissue (WAT). ATF-2 and CRE-BPa were required for bone morphogenetic protein 2 (BMP-2)-and p38-dependent induction of peroxisome proliferator-activated receptor gamma2 (PPARgamma2), a key transcription factor mediating adipocyte differentiation. Since stored fat supplies have been recognized as a possible target for antiobesity treatments, we tested whether inhibition of the p38-ATF-2 pathway suppresses adipocyte differentiation and leads to reduced WAT by treating mice with a p38 inhibitor for long periods of time. High-fat diet (HFD)-induced obesity was significantly reduced in mice fed the p38 inhibitor. Furthermore, the p38 inhibitor alleviated HFD-induced insulin resistance. In p38 inhibitor-treated mice, macrophage infiltration into WAT was reduced and the tumor necrosis factor alpha (TNF-alpha) levels were lower than control mice. Thus, p38 inhibitors may provide a novel antiobesity treatment.