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1.
Nat Commun ; 11(1): 4765, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958780

RESUMO

Fatty acids (FAs) are essential nutrients, but how they are transported into cells remains unclear. Here, we show that FAs trigger caveolae-dependent CD36 internalization, which in turn delivers FAs into adipocytes. During the process, binding of FAs to CD36 activates its downstream kinase LYN, which phosphorylates DHHC5, the palmitoyl acyltransferase of CD36, at Tyr91 and inactivates it. CD36 then gets depalmitoylated by APT1 and recruits another tyrosine kinase SYK to phosphorylate JNK and VAVs to initiate endocytic uptake of FAs. Blocking CD36 internalization by inhibiting APT1, LYN or SYK abolishes CD36-dependent FA uptake. Restricting CD36 at either palmitoylated or depalmitoylated state eliminates its FA uptake activity, indicating an essential role of dynamic palmitoylation of CD36. Furthermore, blocking endocytosis by targeting LYN or SYK inhibits CD36-dependent lipid droplet growth in adipocytes and high-fat-diet induced weight gain in mice. Our study has uncovered a dynamic palmitoylation-regulated endocytic pathway to take up FAs.


Assuntos
Antígenos CD36/metabolismo , Endocitose/fisiologia , Ácidos Graxos/metabolismo , Lipoilação , Células 3T3-L1 , Aciltransferases/metabolismo , Adipócitos/metabolismo , Animais , Antígenos CD36/deficiência , Antígenos CD36/genética , Cavéolas/metabolismo , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Humanos , Gotículas Lipídicas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Obesidade/tratamento farmacológico , Fosforilação , Transdução de Sinais , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Ganho de Peso/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
2.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1386-1394, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748596

RESUMO

We used CRISPR/Cas9 to delete plin1 of 3T3-L1 preadipocyte, to observe its effect on lipolysis in adipocytes and to explore regulatory pathways. We cultured 3T3-L1 preadipocytes, and the plin1 knockout vectors were transfected by electroporation. Puromycin culture was used to screen successfully transfected adipocytes, and survival rates were observed after transfection. The optimized "cocktail" method was used to differentiate 3T3-L1 preadipocytes. The glycerol and triglyceride contents were determined by enzymatic methods. The changes in lipid droplet form and size were observed by Oil red O staining. The protein expression of PLIN1, PPARγ, Fsp27, and lipases was measured by Western blotting. RT-PCR was used to measure the expression of PLIN1 and lipases mRNA. After the adipocytes in the control group were induced to differentiate, the quantity of tiny lipid droplets was decreased, and the quantity of unilocular lipid droplets was increased and arranged in a circle around the nucleus. Compared with the control group, the volume of unilocular lipid droplets decreased, and the quantity of tiny lipid droplets increased after induction of adipocytes in the knockout group. The expression of PLIN1 mRNA and protein in the adipocytes was significantly inhibited (P<0.05); glycerol levels increased significantly (0.098 4±0.007 6), TG levels decreased significantly (0.031 0±0.005 3); mRNA and protein expression of HSL and ATGL increased (P<0.05); PPARγ and Fsp27 expression unchanged in adipocytes. The above results indicate that the knockout of plin1 enhances the lipolysis of 3T3-L1 adipocytes by exposing lipids in lipid droplets and up-regulating lipases effects.


Assuntos
Sistemas CRISPR-Cas , Lipólise , Perilipina-1 , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Técnicas de Inativação de Genes , Lipase/metabolismo , Lipólise/genética , Camundongos , Perilipina-1/genética , Perilipina-1/metabolismo
3.
Life Sci ; 258: 118240, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781072

RESUMO

As a dicarboxylic acid with the structural formula HOOCCH (OH) COOH, tartronic acid is considered as an inhibitor of the transformation of carbohydrates into fat under fat-deficient diet conditions. However, the effect of tartronic acid on lipogenesis under high-fat diet conditions has yet to be established. In this work, we investigated the regulatory role of tartronic acid in lipogenesis in 3T3-L1 adipocytes and C57BL/6J mice. The results confirmed that tartronic acid promoted weight gain (without affecting food intake) and induced adipocyte hypertrophy in epididymal white adipose tissue and lipid accumulation in the livers of high-fat diet-induced obese mice. In vitro, tartronic acid promoted 3T3-L1 adipocyte differentiation by increasing the protein expression of FABP-4, PPARγ and SREBP-1. Moreover, the contents of both acetyl-CoA and malonyl-CoA were significantly upregulated by treatment with tartronic acid, while the protein expression of CPT-1ß were inhibited. In summary, we proved that tartronic acid promotes lipogenesis by serving as substrates for fatty acid synthesis and inhibiting CPT-1ß, providing a new perspective for the study of tartronic acid.


Assuntos
Acetilcoenzima A/biossíntese , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Lipogênese/efeitos dos fármacos , Malonil Coenzima A/biossíntese , Tartronatos/farmacologia , Regulação para Cima/efeitos dos fármacos , Células 3T3-L1 , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Dieta Hiperlipídica/efeitos adversos , Lipogênese/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regulação para Cima/fisiologia
4.
Mol Cell Biol ; 40(17)2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601106

RESUMO

Transcription factors C/EBPß and C/EBPδ are induced within hours after initiation of adipogenesis in culture. They directly promote the expression of master adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα and are required for adipogenesis in vivo However, the mechanism that controls the induction of C/EBPß and C/EBPδ remains elusive. We previously showed that histone methyltransferases MLL3/MLL4 and associated PTIP are required for the induction of PPARγ and C/EBPα during adipogenesis. Here, we show MLL3/MLL4/PTIP-associated protein PAGR1 (also known as PA1) cooperates with phosphorylated CREB and ligand-activated glucocorticoid receptor to directly control the induction of C/EBPß and C/EBPδ in the early phase of adipogenesis. Deletion of Pagr1 in white and brown preadipocytes prevents the induction of C/EBPß and C/EBPδ and leads to severe defects in adipogenesis. Adipogenesis defects in PAGR1-deficient cells can be rescued by the ectopic expression of C/EBPß or PPARγ. Finally, the deletion of Pagr1 in Myf5+ precursor cells impairs brown adipose tissue and muscle development. Thus, by controlling the induction of C/EBPß and C/EBPδ, PAGR1 plays a critical role in adipogenesis.


Assuntos
Adipogenia/fisiologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Diferenciação Celular/fisiologia , Histona Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , PPAR gama/metabolismo , Ligação Proteica
5.
Food Chem ; 333: 127478, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32663752

RESUMO

Moringa oleifera Lam. (M. oleifera) leaves have long been consumed as both nutritive vegetable and popular folk medicine for hyperglycemia and hyperlipidemia in Kenya communities. In the current study, in vitro inhibition by M. oleifera leaf extract (MOLE, 90% (v/v) ethanol) of α-glucosidase and pancreatic lipase was demonstrated, followed by determination of the effects of MOLE on both glucose consumption and lipid levels (TC, TG, HDL-C and LDL-C) in 3T3-L1 cells. Potential ligands in MOLE were fast screened using affinity ultrafiltration LC-MS, and 14 and 10 components displayed certain binding affinity to α-glucosidase and pancreatic lipase, respectively. Docking studies revealed the binding energies and hydrogen bonds between potential ligands and enzymes. This study suggests that M. oleifera leaves may be a promising natural source for the prevention and treatment of hyperglycemia and hyperlipidemia as well as a functional food or other product for health care in the near future.


Assuntos
Moringa oleifera/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Células 3T3-L1 , Animais , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipolipemiantes/química , Hipolipemiantes/farmacologia , Lipase/antagonistas & inibidores , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos
6.
Mol Cell Biol ; 40(19)2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32719109

RESUMO

Recent studies have demonstrated the existence of a discrete pool of cholesterol in the plasma membranes (PM) of mammalian cells-referred to as the accessible cholesterol pool-that can be detected by the binding of modified versions of bacterial cytolysins (e.g., anthrolysin O). When the amount of accessible cholesterol in the PM exceeds a threshold level, the excess cholesterol moves to the endoplasmic reticulum (ER), where it regulates the SREBP2 pathway and undergoes esterification. We reported previously that the Aster/Gramd1 family of sterol transporters mediates nonvesicular movement of cholesterol from the PM to the ER in multiple mammalian cell types. Here, we investigated the PM pool of accessible cholesterol in cholesterol-loaded fibroblasts with a knockdown of Aster-A and in mouse macrophages from Aster-B and Aster-A/B-deficient mice. Nanoscale secondary ion mass spectrometry (NanoSIMS) analyses revealed expansion of the accessible cholesterol pool in cells lacking Aster expression. The increased accessible cholesterol pool in the PM was accompanied by reduced cholesterol movement to the ER, evidenced by increased expression of SREBP2-regulated genes. Cosedimentation experiments with liposomes revealed that the Aster-B GRAM domain binds to membranes in a cholesterol concentration-dependent manner and that the binding is facilitated by the presence of phosphatidylserine. These studies revealed that the Aster-mediated nonvesicular cholesterol transport pathway controls levels of accessible cholesterol in the PM, as well as the activity of the SREBP pathway.


Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Células 3T3-L1 , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Lipossomos/metabolismo , Macrófagos Peritoneais/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espectrometria de Massa de Íon Secundário/métodos , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
7.
Clin Sci (Lond) ; 134(12): 1537-1553, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32556103

RESUMO

Hyperuricaemia (HUA) significantly increases the risk of metabolic syndrome and is strongly associated with the increased prevalence of high serum free fatty acids (FFAs) and insulin resistance. However, the underlying mechanisms are not well established, especially the effect of uric acid (UA) on adipose tissue, a vital organ in regulating whole-body energy and FFA homeostasis. In the present study, we noticed that adipocytes from the white adipose tissue of patients with HUA were hypertrophied and had decreased UCP1 expression. To test the effects of UA on adipose tissue, we built both in vitro and in vivo HUA models and elucidated that a high level of UA could induce hypertrophy of adipocytes, inhibit their hyperplasia and reduce their beige-like characteristics. According to mRNA-sequencing analysis, UA significantly decreased the expression of leptin in adipocytes, which was closely related to fatty acid metabolism and the AMPK signalling pathway, as indicated by KEGG pathway analysis. Moreover, lowering UA using benzbromarone (a uricosuric agent) or metformin-induced activation of AMPK expression significantly attenuated UA-induced FFA metabolism impairment and adipose beiging suppression, which subsequently alleviated serum FFA elevation and insulin resistance in HUA mice. Taken together, these observations confirm that UA is involved in the aetiology of metabolic abnormalities in adipose tissue by regulating leptin-AMPK pathway, and metformin could lessen HUA-induced serum FFA elevation and insulin resistance by improving adipose tissue function via AMPK activation. Therefore, metformin could represent a novel treatment strategy for HUA-related metabolic disorders.


Assuntos
Adipócitos/patologia , Tecido Adiposo Bege/patologia , Tecido Adiposo Branco/patologia , Ácidos Graxos não Esterificados/sangue , Hiperuricemia/sangue , Hiperuricemia/tratamento farmacológico , Resistência à Insulina , Metformina/uso terapêutico , Células 3T3-L1 , Adenilato Quinase/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo Bege/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Adulto , Animais , Ativação Enzimática , Feminino , Humanos , Hipertrofia , Leptina/metabolismo , Lipogênese , Lipólise , Masculino , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transdução de Sinais , Triglicerídeos/metabolismo , Ácido Úrico/sangue
8.
Mol Biol (Mosk) ; 54(2): 233-243, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392192

RESUMO

Obesity is a major disease that causes significant complications. Inhibition of preadipocyte proliferation has the potential to prevent obesity and metabolic diseases. Melatonin is a pineal gland hormone that has various effects on cells and tissues. In this research, we investigated whether melatonin induces apoptosis in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were cultured until confluence and then treated with 0, 10, 100, and 1000 µM melatonin for 1, 3, and 5 days. A cell viability assay kit was used for determining cell viability. Cell death marker proteins were assessed by Western blot analysis using GAPDH for control. Apoptotic morphological changes with nuclei fragmentation were observed using DAPI staining. Melatonin treatment decreased the phosphorylated extracellular signal-regulated kinases (p-ERK) activation while increasing the activation of caspase-3, 8, and 9. Furthermore, melatonin not only increased Bcl-2-associated X protein (Bax) but decreased B-cell lymphoma 2 (Bcl-2) expression as dose increases from 0 to 1000 µM. The melatonin treatment also suppressed the growth of preadipocytes with increasing concentration. These effects were attenuated by luzindole, a melatonin receptor antagonist and U0126, an inhibitor of p-ERK activation. In conclusion, melatonin can induce apoptosis of 3T3-L1 preadipocytes via p-ERK decrease.


Assuntos
Adipócitos/citologia , Apoptose , Melatonina/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Caspases/metabolismo , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
9.
PLoS One ; 15(5): e0233390, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437400

RESUMO

Hypertrophy, associated with adipocyte dysfunction, causes increased pro-inflammatory adipokine, and abnormal glucose and lipid metabolism, leading to insulin resistance and obesity-related-health problems. By combining DNA microarray and genomic data analyses to predict DNA binding motifs, we identified the transcription factor Interferon Regulatory Factor 7 (IRF7) as a possible regulator of genes related to adipocyte hypertrophy. To investigate the role of IRF7 in adipocytes, we examined gene expression patterns in 3T3-L1 cells infected with a retrovirus carrying the IRF7 gene and found that enforced IRF7 expression induced the expression of monocyte chemoattractant protein-1 (MCP-1), a key initial adipokine in the chronic inflammation of obesity. CRISPR/Cas9 mediated-suppression of IRF7 significantly reduced MCP-1 mRNA. Luciferase assays, chromatin immunoprecipitation PCR analysis and gel shift assay showed that IRF7 transactivates the MCP-1 gene by binding to its proximal Interferon Stimulation Response Element (ISRE), a putative IRF7 binding motif. IRF7 knockout mice exhibited lower expression of MCP-1 in epidydimal white adipose tissue under high-fat feeding conditions, suggesting the transcription factor is physiologically important for inducing MCP-1. Taken together, our results suggest that IRF7 transactivates MCP-1 mRNA in adipocytes, and it may be involved in the adipose tissue inflammation associated with obesity.


Assuntos
Adipócitos/metabolismo , Quimiocina CCL2/genética , Fator Regulador 7 de Interferon/genética , Obesidade/genética , Células 3T3-L1 , Tecido Adiposo Branco/metabolismo , Animais , Quimiocina CCL2/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fator Regulador 7 de Interferon/metabolismo , Camundongos , Camundongos Knockout , Obesidade/metabolismo , Regiões Promotoras Genéticas
10.
Cell Prolif ; 53(6): e12831, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32441391

RESUMO

OBJECTIVES: AF4/FMR2 family member 1 (AFF1), known as a central scaffolding protein of super elongation complex (SEC), regulates gene transcription. We previously reported that AFF1 inhibited osteogenic differentiation of human mesenchymal stromal/stem cells (hMSCs). However, its role in adipogenic differentiation has not been elucidated. MATERIALS AND METHODS: hMSCs and 3T3-L1 pre-adipocytes were cultured and induced for adipogenic differentiation. Small interfering RNAs (siRNAs) were applied to deplete AFF1 while lentiviruses expressing HA-Aff1 were used for overexpression. Oil Red O staining, triglyceride (TAG) quantification, quantitative real-time PCR (qPCR), Western blot analysis, immunofluorescence staining, RNA sequencing (RNA-seq) analysis and ChIP-qPCR were performed. To evaluate the adipogenesis in vivo, BALB/c nude mice were subcutaneously injected with Aff1-overexpressed 3T3-L1 pre-adipocytes. RESULTS: AFF1 depletion leads to an enhanced adipogenesis in both hMSCs and 3T3-L1 pre-adipocytes. Overexpression of Aff1 in 3T3-L1 cells results in the reduction of adipogenic differentiation and less adipose tissue formation in vivo. Mechanistically, AFF1 binds to the promoter region of Tgm2 gene and regulates its transcription. Overexpression of Tgm2 largely rescues adipogenic differentiation of Aff1-deficient cells. CONCLUSIONS: Our data indicate that AFF1 inhibits adipogenic differentiation by regulating the transcription of TGM2.


Assuntos
Adipogenia/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Transglutaminases/genética , Células 3T3-L1 , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Nucleares/genética , Células-Tronco/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/genética , Transglutaminases/biossíntese , Transglutaminases/metabolismo
11.
Chemosphere ; 253: 126772, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32464760

RESUMO

Zeranol is an approved but controversial growth-promoting agent for livestock in North America. It is a mycotoxin metabolite secreted by the Fusarium family fungi. The regulatory bodies in this region have established the acceptable daily intake and exposure below the level would not significantly increase the health risk for humans. However, their European counterparts have yet to establish an acceptable level and do not permit the use of this agent in farm animals. Given the growth-promoting ability of zeranol, its effect on energy metabolism was investigated in the current study. Our results indicated that zeranol could induce glucose transporter type 4 (GLUT4) expression in 3T3 L1 cells at 10 µM and initiate the translocation of the glucose transporter to the membrane as assayed by confocal microscopy. The translocation was likely triggered by the increase of GLUT4 and p-Akt. The insulin signal transduction pathway of glucose translocation was analyzed by Western blot analysis. Since no increase in the phosphorylated insulin receptor substrate in zeranol-treated cells was evidenced, the increased p-Akt and GLUT4 amount should be the mechanism dictating the GLUT4 translocation. In summary, this study showed that zeranol could perturb glucose metabolism in differentiated 3T3 L1 adipocytes. Determining the growth-promoting mechanism is crucial to uncover an accepted alternative to the general public.


Assuntos
Transportador de Glucose Tipo 4/metabolismo , Reguladores de Crescimento de Planta/toxicidade , Zeranol/toxicidade , Células 3T3-L1 , Adipócitos , Animais , Antígenos CD , Metabolismo dos Carboidratos , Glucose/metabolismo , Insulina/metabolismo , Gado , Camundongos , América do Norte , Fosforilação , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
DNA Cell Biol ; 39(7): 1119-1126, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32379499

RESUMO

Sirtuin 2 (Sirt2) belongs to the NAD+-dependent deacetylase family, is more highly expressed than other family members in adipocytes, and plays crucial roles in a wide range of biological processes. However, the mechanisms underlying Sirt2 expression during adipogenesis are poorly studied. In this study, the transcriptional start site (TSS) of Sirt2 was identified and two alternative transcript variants were spliced from Sirt2. The 5'-regulatory region of Sirt2 was also characterized; no TATA-box or CCAAT-box was presented in the 5'-flanking region. Two cytosine-phosphate diester-guanine (CpG) islands were also identified between nucleotides -563 and +4. A dual-luciferase reporter assay revealed that a 178 base pair sequence upstream from the TSS (+1) was the core promoter of Sirt2. Results from a site-directed mutagenesis experiment, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay indicated Yin Yang 1 (YY1) to be a positive regulator of bovine Sirt2 in preadipocytes. YY1 is likely to suppress adipogenesis in two different ways by regulating peroxisome proliferator-activated receptor gamma expression. Our results expand the information on the regulatory network of adipogenesis, which is an important basis for improving beef quality, treating obesity, and other related diseases.


Assuntos
Adipócitos/metabolismo , Sirtuína 2/genética , Ativação Transcricional , Fator de Transcrição YY1/metabolismo , Células 3T3-L1 , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Fator de Transcrição YY1/química
13.
PLoS Genet ; 16(5): e1008823, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453789

RESUMO

The development of type 2 diabetes mellitus (T2DM) depends on interactions between genetic and environmental factors, and a better understanding of gene-diet interactions in T2DM will be useful for disease prediction and prevention. Ascorbic acid has been proposed to reduce the risk of T2DM. However, the links between ascorbic acid and metabolic consequences are not fully understood. Here, we report that glucose transporter 10 (GLUT10) maintains intracellular levels of ascorbic acid to promote adipogenesis, white adipose tissue (WAT) development and protect mice from high-fat diet (HFD)-induced metabolic dysregulation. We found genetic polymorphisms in SLC2A10 locus are suggestively associated with a T2DM intermediate phenotype in non-diabetic Han Taiwanese. Additionally, mice carrying an orthologous human Glut10G128E variant (Glut10G128E mice) with compromised GLUT10 function have reduced adipogenesis, reduced WAT development and increased susceptibility to HFD-induced metabolic dysregulation. We further demonstrate that GLUT10 is highly expressed in preadipocytes, where it regulates intracellular ascorbic acid levels and adipogenesis. In this context, GLUT10 increases ascorbic acid-dependent DNA demethylation and the expression of key adipogenic genes, Cebpa and Pparg. Together, our data show GLUT10 regulates adipogenesis via ascorbic acid-dependent DNA demethylation to benefit proper WAT development and protect mice against HFD-induced metabolic dysregulation. Our findings suggest that SLC2A10 may be an important HFD-associated susceptibility locus for T2DM.


Assuntos
Tecido Adiposo Branco/metabolismo , Ácido Ascórbico/metabolismo , Metilação de DNA , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica/efeitos adversos , Proteínas Facilitadoras de Transporte de Glucose/genética , Células 3T3-L1 , Adipogenia , Adulto , Idoso , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Metilação de DNA/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Hemoglobina A Glicada/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mutação , PPAR gama/genética
14.
Life Sci ; 251: 117609, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32272180

RESUMO

AIMS: To identify the target of an adipose specific aptamer adipo-8, predict the potential interaction between adipo-8 and its target, and investigate lipid-lowering effect of adipo-8 in vitro and in vivo. MAIN METHODS: Distinct membranous protein of 3T3-L1 adipocyte pulled-down by adipo-8 was mass-spectrometry analyzed as target candidate(s), and affinity of adipo-8 to target protein-silent adipocyte was detected to validate it. Interaction between adipo-8 and target was predicted by bioinformatic analysis, further confirmed by aptamer truncation and competitive binding assay. To investigate lipid-lowering effect of adipo-8 and mechanism behind, 250 nmol/L adipo-8 or library was incubated with 3T3-L1 adipocyte or target-protein-silent adipocyte for 24 h, and 0.01 µg/g/day adipo-8 or library was administrated to high-fat-fed male mice for 21 days. KEY FINDINGS: APMAP (Adipocyte Plasma Membrane Associated Protein) was identified as adipo-8 target, and adipo-8 affinity to adipocytes was in proportional to APMAP expression. Docking model between the stem-loop structure of adipo-8 and APMAP were predicted that adipo-8 was likely to interact with APMAP at its amino-acid 275-411 sequence. Moreover, adipo-8 could ameliorate fat deposition through interaction with APMAP in vitro, and administration of adipo-8 in high-fat-diet fed mice resulted in body weight loss and blood triglyceride decrease without liver or renal dysfunction. SIGNIFICANCE: Adipo-8 could recognize APMAP specifically and interact with its targets to ameliorate fat deposition in vitro and in vivo. Aptamer adipo-8 has potential to act as an effective and safe targeted drug for obesity and obesity related diseases.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Células 3T3-L1 , Animais , Dieta Hiperlipídica , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular
15.
DNA Cell Biol ; 39(5): 756-765, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32282232

RESUMO

Iron-sulfur (Fe-S) clusters are required for mitochondrial function. Fe-S cluster synthesis occurs in the mitochondria and iron uptake is required for mitochondrial biogenesis. However, Fe-S clusters inhibit the expression of the iron importer transferrin receptor 1 (TfR1), whereas lack of the Fe-S cluster stimulates TfR1 expression. Yet, it is unclear whether Fe-S cluster synthesis increases with mitochondria biogenesis and, in turn, whether this negatively modulates TfR1 expression. We manipulated peroxisome proliferator-activated receptor-gamma coactivator-1α expression to control mitochondrial biogenesis in a variety of cell types, including erythroid cells. We demonstrated that Fe-S cluster synthesis increases with mitochondria biogenesis but does not interfere with increasing TfR1 expression. In fact, TfR1 expression is stimulated through alternative means to meet iron requirement for mitochondria biogenesis. Furthermore, under enhanced mitochondria biogenesis, increased Fe-S cluster synthesis inhibits the function of iron-regulating protein (IRP)1 and hence stimulates the expression of 5'-aminolevulinate synthase 2 (ALAS2), a target of IRP1 and rate-limiting enzyme in erythroid heme biogenesis. Increased ALAS2 expression leads to enhanced heme production, hemoglobinization, and erythropoiesis. Therefore, our study also provides a mechanism to link mitochondrial biogenesis with erythropoiesis and has a potential therapeutic value in the treatment of blood disorders.


Assuntos
Ferro/metabolismo , Biogênese de Organelas , Enxofre/metabolismo , Células 3T3-L1 , 5-Aminolevulinato Sintetase/genética , Animais , Transporte Biológico/efeitos dos fármacos , Células Eritroides/citologia , Células Eritroides/metabolismo , Eritropoese/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Heme/biossíntese , Hemoglobinas/metabolismo , Humanos , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/farmacologia
16.
Arch Biochem Biophys ; 686: 108365, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32315651

RESUMO

Pelargonidin is a natural compound that exists widely in fruits, and exerts antioxidant, anti-atherosclerotic, anti-inflammatory, anti-hyperglycemic, and anti-diabetic activities. However, there have not been any studies concerning its anti-obesity potential to date. Therefore, we evaluated the anti-obesity potential of pelargonidin via inhibition of adipogenesis in 3T3-L1 cells. The cellular oil droplet content was decreased to 68.14%, 56.75%, and 48.39% and triglyceride accumulation decreased to 74.53%, 61.54%, and 47.86% after incubation with 5 µM, 10 µM, and 20 µM pelargonidin, respectively, when compared with DMSO group. Furthermore, pelargonidin treatment led to decrease in glucose consumption. Western blot assay illustrated that the expression of PPAR-γ was suppressed to 63.25%, 47.52%, and 21.23% after incubation with 5 µM, 10 µM, and 20 µM pelargonidin when compared with DMSO group. Then, we measured the expression of some target proteins of PPAR-γ, and found that pelargonidin decreased the expressions of HMGCR, LPL, Glut4, and A-FABP. Besides, the result of Luciferase Reporter Assay indicated that pelargonidin inhibited PPAR-γ transcription activity. These results indicated that pelargonidin exerts anti-adipogenic activity in 3T3-L1 cells through inhibition of PPAR-γ signaling pathway, and pelargonidin could be used as a potential anti-obesity agent.


Assuntos
Adipogenia/efeitos dos fármacos , Antocianinas/farmacologia , Fármacos Antiobesidade/farmacologia , PPAR gama/metabolismo , Células 3T3-L1 , Animais , Antocianinas/metabolismo , Fármacos Antiobesidade/metabolismo , Regulação para Baixo/efeitos dos fármacos , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Camundongos , Triglicerídeos/genética , Triglicerídeos/metabolismo
17.
Environ Sci Technol ; 54(10): 6262-6271, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32314580

RESUMO

Tetrabromobisphenol A (TBBPA), the most widely used brominated flame retardant, is reported to potentially possess risks in inducing obesity or obesity-related metabolic diseases. Considering the increasing environmental contamination of TBBPA analogues and their high structural similarities to the parent compound, whether they could influence adipogenesis or not remains to be elucidated. In this study, two of the most prevalent TBBPA derivatives [i.e., TBBPA bis(allyl ether) (TBBPA-BAE) and TBBPA bis(2,3-dibromopropyl ether) (TBBPA-BDBPE)] and their byproducts [i.e., TBBPA mono(allyl ether) (TBBPA-MAE) and TBBPA mono(2,3-dibromopropyl ether) (TBBPA-MDBPE)], together with TBBPA, were screened for their capacities in activating peroxisome proliferator-activated receptor-γ (PPARγ) and glucocorticoid receptor (GR), the key nuclear receptors involved in adipogenesis, and their structure-related effects on differentiation of 3T3-L1 preadipocytes were explored. The results indicated that the binding affinities of TBBPA and its analogues for the PPARγ ligand-binding domain (PPARγ-LBD) and GR, as well as their effects on PPARγ transactivation, followed the order of TBBPA > TBBPA-MAE > TBBPA-MDBPE > TBBPA-BAE, TBBPA-BDBPE. Nevertheless, TBBPA-MAE and TBBPA-MDBPE showed higher potentials in promoting adipogenesis in 3T3-L1 cells than did TBBPA, as evidenced by intracellular triglyceride contents and adipogenic biomarkers at both protein and transcriptional levels. The etherified group at position 4 of TBBPA phenolic rings was crucial in chemical-induced adipogenic effects, which was related with the recruitment of PPARγ and GR-mediated networks and some other unidentified signaling pathways. The findings on the disturbance of TBBPA analogues on adipogenesis revealed their potential risk in causing obesity and other lipid metabolism-related human health concerns.


Assuntos
Retardadores de Chama , Bifenil Polibromatos , Células 3T3-L1 , Adipogenia , Animais , Humanos , Camundongos , PPAR gama
18.
Bull Environ Contam Toxicol ; 104(6): 852-857, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32322934

RESUMO

Wastewater (WW) carry considerable amount of chemicals that could have mutagenic or cytotoxic effect from hospital discharges to aquatic environment. Our objective was to determinate the possible mutagenic and toxic effects of hospital originated WWs and effectiveness of the wastewater treatment plants (WTP) functions. In the study the mutagenic and cytotoxic potential of three hospitals and influent/effluent of a treatment plant WW collected in Istanbul and was examined using AMES, XTT, and lactate dehydrogenase (LDH) assays. Mutagenic effects were detected at both hospital discharges and advanced biological wastewater plant. We observed no cytotoxic effect in fibroblasts for LDH and XTT assays whereas high cytotoxicity for all samples was found in hepatocytes by XTT assay. According to the results even if advanced technology is used for treatment of WW, mutagenic and cytotoxic effects still remain, and the present technologies need to be further improved.


Assuntos
Mutagênicos/toxicidade , Eliminação de Resíduos Líquidos/métodos , Águas Residuárias/toxicidade , Purificação da Água/métodos , Células 3T3-L1 , Animais , Bioensaio , Sobrevivência Celular/efeitos dos fármacos , Hospitais , Camundongos , Testes de Mutagenicidade , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética
19.
Life Sci ; 248: 117474, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32112869

RESUMO

BACKGROUND/OBJECTIVES: Nicotinamide N-methyltransferase (NNMT) is a novel regulator of energy homeostasis in adipocytes. NNMT expression in adipose tissue is increased in obesity and diabetes. Knockdown of NNMT prevents mice from developing diet-induced obesity, which is closely linked to insulin resistance. An early sign of systemic insulin resistance is reduced expression of glucose transporter 4 (GLUT4) selectively in adipose tissue. Adipose tissue-specific knockout and overexpression of GLUT4 cause reciprocal changes in NNMT expression. The aim of the current study was to elucidate the mechanism that regulates NNMT expression in adipocytes. METHODS: 3T3-L1 adipocytes were cultured in media with varying glucose concentrations or activators and inhibitors of intracellular pathways. NNMT mRNA and protein levels were measured with quantitative polymerase chain reaction and Western blotting. RESULTS: Glucose deprivation of 3T3-L1 adipocytes induced a 2-fold increase in NNMT mRNA and protein expression. This effect was mimicked by inhibition of glucose transport with phloretin, and by inhibition of glycolysis with the phosphoglucose isomerase inhibitor 2-deoxyglucose. Conversely, inhibition of the pentose phosphate pathway did not affect NNMT expression. Pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin (mTOR) pathway caused an increase in NNMT levels that was similar to the effect of glucose deprivation. Activation of mTOR with MHY1485 prevented the effect of glucose deprivation on NNMT expression. Furthermore, upregulation of NNMT levels depended on functional autophagy and protein translation. CONCLUSION: Glucose availability regulates NNMT expression via an mTOR-dependent mechanism.


Assuntos
Adipócitos/efeitos dos fármacos , Transportador de Glucose Tipo 4/genética , Glucose/farmacologia , Nicotinamida N-Metiltransferase/genética , Serina-Treonina Quinases TOR/genética , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Desoxiglucose/farmacologia , Metabolismo Energético/genética , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/metabolismo , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Homeostase/genética , Camundongos , Morfolinas/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/genética , Floretina/farmacologia , Biossíntese de Proteínas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Triazinas/farmacologia
20.
Chem Biol Interact ; 322: 109059, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32171850

RESUMO

Fatty liver is the earliest and most common response of the liver to consumption of excessive alcohol. Steatosis can predispose the fatty liver to develop progressive liver damage. Chief among the many mechanisms involved in development of hepatic steatosis is dysregulation of insulin-mediated adipose tissue metabolism. Particularly, it is the enhanced adipose lipolysis-derived free fatty acids and their delivery to the liver that ultimately results in hepatic steatosis. The adipose-liver axis is modulated by hormones, particularly insulin and adiponectin. In recent studies, we demonstrated that an alcohol-induced increase in serum ghrelin levels impairs insulin secretion from pancreatic ß-cells. The consequent reduction in circulating insulin levels promotes adipose lipolysis and mobilization of fatty acids to the liver to ultimately contribute to hepatic steatosis. Because many tissues, including adipose tissue, express ghrelin receptor we hypothesized that ghrelin may directly affect energy metabolism in adipocytes. We have exciting new preliminary data which shows that treatment of premature 3T3-L1 adipocytes with ghrelin impairs adipocyte differentiation and inhibits lipid accumulation in the tissue designed to store energy in the form of fat. We further observed that ghrelin treatment of differentiated adipocytes significantly inhibited secretion of adiponectin, a hepatoprotective hormone that reduces lipid synthesis and promotes lipid oxidation. These results were corroborated by our observations of a significant increase in serum adiponectin levels in ethanol-fed rats treated with a ghrelin receptor antagonist verses the un-treated ethanol-fed rats. Interestingly, in adipocytes, ghrelin also increases secretion of interleukin-6 (IL-6) and CCL2 (chemokine [C-C motif] ligand 2), cytokines which promote hepatic inflammation and progression of liver disease. To summarize, the alcohol-induced increase in serum ghrelin levels dysregulates adipose-liver interaction and promotes hepatic steatosis by increasing the free fatty acid released from adipose for hepatic uptake, and by altering adiponectin and cytokine secretion. Taken together, our data indicates that targeting the activity of ghrelin may be a powerful treatment strategy.


Assuntos
Tecido Adiposo/metabolismo , Fígado Gorduroso Alcoólico/patologia , Grelina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Células 3T3-L1 , Adipocinas/metabolismo , Adiponectina/sangue , Adiponectina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Etanol/farmacologia , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/veterinária , Interleucina-6/metabolismo , Masculino , Camundongos , Oligopeptídeos/farmacologia , PPAR gama/metabolismo , Ratos , Ratos Wistar
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