RESUMO
SCOPE: This study investigates the effect of epigallocatechin gallate (EGCG) on white and beige preadipocyte growth and explores the involvement of the miR-let-7a/HMGA2 pathway. METHODS AND RESULTS: 3T3-L1 and D12 cells are treated with EGCG. The effect of EGCG on cell proliferation and viability is evaluated, as well as microRNA (miRNA)-related signaling pathways. EGCG inhibits 3T3-L1 and D12 preadipocyte growth, upregulates miR-let-7a expression, and downregulates high-mobility group AT-hook 2 (HMGA2) mRNA and protein levels in a time- and dose-dependent manner. In addition, overexpression of miR-let-7a significantly inhibits the growth of 3T3-L1 and D12 cells and decreases HMGA2 mRNA and protein levels. MiR-let-7a inhibitor antagonizes the inhibitory effects of EGCG on the number and viability of 3T3-L1 and D12 cells. Furthermore, miR-let-7a inhibitor reverses the EGCG-induced increase in miR-let-7a expression levels and decrease in HMGA2 mRNA and protein levels. HMGA2 overexpression induces an increase in cell number and viability and antagonizes EGCG-suppressed cell growth and HMGA2 expression in 3T3-L1 and D12 preadipocytes. CONCLUSION: EGCG inhibits the growth of 3T3-L1 and D12 preadipocytes by modulating the miR-let-7a and HMGA2 pathways.
Assuntos
Catequina , MicroRNAs , MicroRNAs/genética , MicroRNAs/metabolismo , Chá , Transdução de Sinais , Proliferação de Células , Catequina/farmacologia , RNA MensageiroRESUMO
Green tea epigallocatechin gallate (EGCG) and microRNA (miRNA) molecules modulate obesity. Nevertheless, it is still unknown whether EGCG modulates fat cell growth via miRNA-related signaling. In this study, white preadipocytes were used to examine whether the antimitogenic effect of EGCG on fat cells is regulated by the miR-143/MAPK7 pathway. We showed that EGCG upregulated the levels of miR-143, but not miR-155, in 3T3-L1 preadipocytes. Moreover, EGCG downregulated MAPK7 mRNA and protein levels time- and dose-dependently. MAPK7 expression increased during 3T3-L1 cell proliferation. miR-143 overexpression in the absence of EGCG mimicked the effects of EGCG to suppress preadipocyte growth and MAPK7 expression, whereas knockdown of miR-143 antagonized the EGCG-altered levels of miR-143, MAPK7, and pERK1/2 and reversed the EGCG-inhibited cell growth. These findings suggest that EGCG inhibits 3T3-L1 cell growth via miR-143/MAPK7 pathway.
Assuntos
Catequina , MicroRNAs , Camundongos , Animais , Células 3T3-L1 , Chá , Catequina/farmacologia , MicroRNAs/genética , RNA MensageiroRESUMO
Billions of people have obesity-related metabolic syndromes such as diabetes and hyperlipidemia. Promoting the browning of white adipose tissue has been suggested as a potential strategy, but a drug still needs to be identified. Here, genetic deletion of activating transcription factor 3 (ATF3-/- ) in mice under a high-fat diet (HFD) resulted in obesity and insulin resistance, which was abrogated by virus-mediated ATF3 restoration. ST32da, a synthetic ATF3 inducer isolated from Salvia miltiorrhiza, promoted ATF3 expression to downregulate adipokine genes and induce adipocyte browning by suppressing the carbohydrate-responsive element-binding protein-stearoyl-CoA desaturase-1 axis. Furthermore, ST32da increased white adipose tissue browning and reduced lipogenesis in HFD-induced obese mice. The anti-obesity efficacy of oral ST32da administration was similar to that of the clinical drug orlistat. Our study identified the ATF3 inducer ST32da as a promising therapeutic drug for treating diet-induced obesity and related metabolic disorders.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Adipócitos Marrons/metabolismo , Obesidade/metabolismo , Células 3T3-L1 , Fator 3 Ativador da Transcrição/deficiência , Fator 3 Ativador da Transcrição/genética , Adipócitos Marrons/patologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Fármacos Antiobesidade/farmacologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Temperatura Corporal/fisiologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Humanos , Resistência à Insulina , Lipogênese/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/prevenção & controle , Orlistate/farmacologia , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Salvia miltiorrhiza/químicaRESUMO
This study investigated the pathways involved in EGCG modulation of insulin-like growth factor (IGF)-stimulated glucose uptake in 3T3-L1 adipocytes. EGCG inhibited IGF-I and IGF-II stimulation of adipocyte glucose uptake with dose and time dependencies. EGCG at 20µM for 2h decreased IGF-I- and IGF-II-stimulated glucose uptake by 59% and 64%, respectively. Pretreatment of adipocytes with antibody against the EGCG receptor (also known as the 67-kDa laminin receptor; 67LR), prevented the effects of EGCG on IGF-increased glucose uptake, but pretreatment with normal rabbit immunoglobulin did not. This suggests that the 67LR mediates the anti-IGF effect of EGCG on adipocyte glucose uptake. Further analysis indicated EGCG, IGF-I, and IGF-II did not alter total levels of GLUT1 or GLUT4 protein. However, EGCG prevented the IGF-increased GLUT4 levels in the plasma membrane and blocked the IGF-decreased GLUT4 levels in low-density microsomes. Neither EGCG nor its combination with IGF altered GLUT1 protein levels in the plasma membrane and low-density microsomes. EGCG also suppressed the IGF-stimulated phosphorylation of IGF signaling molecules, PKCζ/λ, but not AKT and ERK1/2, proteins. This study suggests that EGCG suppresses IGF stimulation of 3T3-L1 adipocyte glucose uptake through inhibition of the GLUT4 translocation, but not through alterations of the GLUT1 pathway.
Assuntos
Adipócitos/metabolismo , Catequina/análogos & derivados , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Chá/química , Células 3T3-L1 , Acetilcisteína/farmacologia , Adipócitos/efeitos dos fármacos , Animais , Anticorpos/metabolismo , Catequina/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Transporte Proteico , Receptores de Laminina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
SCOPE: This study investigated the pathways involved in epigallocatechin gallate (EGCG) modulation of insulin-like growth factor (IGF)-I-stimulated and IGF-II-stimulated mitogenesis in 3T3-L1 preadipocytes. METHODS AND RESULTS: We found that this process was dose and time dependent, and caused by suppression of IGF-I-stimulated and IGF-II-stimulated phosphorylation of p66Shc and mitogen-activated protein kinase (MAPK) pathway proteins, including MEK1 kinase (RAF1), extracellular signal-regulated protein kinase (ERK) kinase (MEK1), and ERK 1 and ERK 2 (ERK1/2), but not phospho-Jun-N-terminal kinase, protein kinase B, p52Shc, or p46Shc. Furthermore, EGCG inhibited the IGF-I-stimulated phosphorylation of the IGF-I receptor-beta (IGF-IR ß), the association of IGF-IR with the p66Shc protein, and the IGF-II-stimulated associations of the IGF-II receptor with G(αi-2) and p66Shc proteins, suggesting that EGCG selectively affects particular types of Shc and MAPK family members. Pretreatment with antiserum against the EGCG receptor (also known as the 67-kDa laminin receptor; 67LR), but not with an adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, prevented the inhibitory actions of EGCG on IGF-I- and IGF-II-stimulated ERK1/2 phosphorylation and subsequent preadipocyte proliferation. CONCLUSION: The results of this study suggest that EGCG mediates anti-IGF-I and anti-IGF-II signals in preadipocyte mitogenesis via the 67LR but not the AMPK pathway.
Assuntos
Adipócitos/efeitos dos fármacos , Catequina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Insulin-Like II/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Receptores de Laminina/metabolismo , Células 3T3-L1 , Monofosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Catequina/farmacologia , Imunoprecipitação , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Extratos Vegetais/farmacologia , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Chá/químicaRESUMO
Insulin and (-)-epigallocatechin gallate (EGCG) are reported to regulate obesity and fat accumulation, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated glucose uptake in 3T3-L1 and C3H10T1/2 adipocytes. EGCG inhibited insulin stimulation of adipocyte glucose uptake in a dose- and time-dependent manner. The concentration of EGCG that decreased insulin-stimulated glucose uptake by 50-60% was approximately 5-10 µM for a period of 2 h. At 10 µM, EGCG and gallic acid were more effective than (-)-epicatechin, (-)-epigallocatechin, and (-)-epicatechin 3-gallate. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and extended the findings for this study to clarify whether EGCG-induced changes in insulin-stimulated glucose uptake in adipocytes could be mediated through the 67LR. Pretreatment of adipocytes with a 67LR antibody, but not normal rabbit immunoglobulin, prevented the effects of EGCG on insulin-increased glucose uptake. This suggests that the 67LR mediates the effect of EGCG on insulin-stimulated glucose uptake in adipocytes. Moreover, pretreatment with an AMP-activated protein kinase (AMPK) inhibitor, such as compound C, but not with a glutathione (GSH) activator, such as N-acetyl-L-cysteine (NAC), blocked the antiinsulin effect of EGCG on adipocyte glucose uptake. These data suggest that EGCG exerts its anti-insulin action on adipocyte glucose uptake via the AMPK, but not the GSH, pathway. The results of this study possibly support that EGCG mediates fat content.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Catequina/análogos & derivados , Glucose/metabolismo , Insulina/farmacologia , Receptores de Laminina/fisiologia , Chá/química , Animais , Catequina/química , Catequina/isolamento & purificação , Catequina/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Células NIH 3T3RESUMO
Insulin and (-)-epigallocatechin gallate (EGCG) have been reported to regulate fat cell mitogenesis and adipogenesis, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated mitogenesis in 3T3-L1 preadipocytes. EGCG inhibited insulin stimulation of preadipocyte proliferation in a dose- and time-dependent manner. EGCG also suppressed insulin-stimulated phosphorylation of the insulin receptor-beta, insulin receptor (IR) substrates 1 and 2 (IRS1 and IRS2), and mitogen-activated protein kinase pathway proteins, RAF1, MEK1/2, and ERK1/2, but not JNK. Furthermore, EGCG inhibited the association of IR with the IRS1 and IRS2 proteins, but not with the IRS4 protein. These data suggest that EGCG selectively affects particular types of IRS and MAPK family members. Generally, EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in modulating insulin-stimulated mitogenic signaling. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and found that its expression was sensitive to growth phase, tissue type, and differentiation state. Pretreatment of preadipocytes with 67LR antiserum prevented the effects of EGCG on insulin-stimulated phosphorylation of IRS2, RAF1, and ERK1/2 and insulin-stimulated preadipocyte proliferation (cell number and bromodeoxyuridine incorporation). Moreover, EGCG tended to increase insulin-stimulated associations between the 67LR and IR, IRS1, IRS2, and IRS4 proteins. These data suggest that EGCG mediates anti-insulin signaling in preadipocyte mitogenesis via the 67LR pathway.
Assuntos
Adipócitos/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Camellia sinensis , Catequina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Insulina/metabolismo , Receptores de Laminina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Fármacos Antiobesidade/isolamento & purificação , Camellia sinensis/química , Catequina/isolamento & purificação , Catequina/farmacologia , Relação Dose-Resposta a Droga , Soros Imunes , Proteínas Substratos do Receptor de Insulina/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptores de Laminina/imunologia , Receptores de Laminina/metabolismo , Fatores de TempoRESUMO
Green tea (-)-epigallocatechin-3-gallate (EGCG) is known as to regulate obesity and fat cell activity. However, little information is known about the effects of EGCG on oxidative reactive oxygen species (ROS) of fat cells. Using 3T3-L1 preadipocytes and adipocytes, we found that EGCG increased ROS production in dose- and time-dependent manners. The concentration of EGCG that increased ROS levels by 180-500% was approximately 50 muM for a range of 8-16 h of treatment. In contrast, EGCG dose- and time-dependently decreased the amount of intracellular glutathione (GSH) levels. EGCG was more effective than (-)-epicatechin, (-)-epicatechin-3-gallate, and (-)-epigallocatechin in changing ROS and GSH levels. This suggests a catechin-specific effect. To further examine the relation of GSH to ROS as altered by EGCG, we observed that exposure of preadipocytes and adipocytes to N-acetyl-L-cysteine (a GSH precursor) blocked the EGCG-induced increases in ROS levels and decreases in GSH levels. These observations suggest a GSH-dependent effect of EGCG on ROS production. While EGCG was demonstrated to alter levels of ROS and GSH, its signaling was altered by an EGCG receptor (the so-called 67 kDa laminin receptor(67LR)) antiserum, but not by normal rabbit serum. These data suggest that EGCG mediates GSH and ROS levels via the 67LR pathway.
Assuntos
Adipócitos/química , Catequina/análogos & derivados , Glutationa/análise , Espécies Reativas de Oxigênio/análise , Receptores de Laminina/fisiologia , Chá/química , Células 3T3-L1 , Acetilcisteína/farmacologia , Adipócitos/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Catequina/farmacologia , Glutationa/antagonistas & inibidores , Camundongos , Receptores de Laminina/imunologiaRESUMO
Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle (Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5'-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3'-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH(2) terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly(1) and Thr(29) substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48-81% and 76-96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17beta-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.