RESUMO
Genome-wide association studies (GWAS) have revealed the implication of several Wnt signaling pathway components, including its effector transcription factor 7-like 2 (TCF7L2) in diabetes and other metabolic disorders. As TCF7L2 is expressed in adipocytes, we investigated its expression and function in rodent fat tissue and mature adipocytes. We found that TCF7L2 mRNA expression in C57BL/6 mouse epididymal fat tissue was up-regulated by feeding but down-regulated by intraperitoneal insulin injection. In high-fat diet (HFD) fed mice, db/db mice and Zucker (fa/fa) rats, epididymal fat TCF7L2 mRNA levels were lower than the corresponding controls. Treating rat adipocytes with 100nM insulin repressed TCF7L2 mRNA and protein levels, associated with the repression of leptin mRNA level. The treatment with 1nM insulin, however, stimulated TCF7L2 and leptin mRNA levels. This stimulation could be attenuated by iCRT14, an inhibitor of ß-catenin/TCF-responsive transcription. Wnt3a stimulated leptin mRNA level, which was also blocked by iCRT14 co-treatment. Utilizing the leptin-expressing cell line HTR8 as a tool, we defined an evolutionarily conserved CREB binding motif that mediated Wnt3a activation. Although Wnt activation is known to repress the differentiation of 3T3-L1 cells towards mature adipocytes, short-term Wnt3a treatment of differentiated 3T3-L1 cells stimulated leptin mRNA levels. Thus, wnt pathway plays a dual function in adipocytes, including the well-known repressive effect on adipogenesis and the stimulation of leptin production in mature adipocytes in response to nutritional status.
Assuntos
Regulação da Expressão Gênica , Leptina/metabolismo , Via de Sinalização Wnt , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Colforsina/farmacologia , Dieta Hiperlipídica , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/sangue , Insulina/farmacologia , Leptina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Zucker , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/antagonistas & inibidores , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
The study aimed to reveal alterations in expression and methylation levels of the growth-related imprinted genes H19 and Igf2 in fetuses of diabetic mice. Diabetes was induced in female mice by intraperitoneal injection of streptozotocin. DNA and total RNA were extracted from fetuses obtained from diabetic and control dams on embryonic day (E) 14. Real-time RT-PCR analysis revealed that the mRNA expression of Igf2 in fetuses from diabetic mice was 0.65-fold of the control counterparts. Bisulfite genomic sequencing demonstrated that the methylation level of the H19-Igf2 imprint control region was 19.1% higher in diabetic fetuses than in those of control dams. In addition, the body weight of pups born to diabetic dams was 26.5% lower than that of the control group. The results indicate that maternal diabetes can affect fetal development by means of altered expression of imprinted genes. The modified genomic DNA methylation status of imprinting genes may account for the change in gene expression.
Assuntos
Metilação de DNA , Diabetes Mellitus Experimental , Feto/fisiologia , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , Gravidez em Diabéticas , RNA não Traduzido/genética , Adulto , Animais , Ilhas de CpG , Diabetes Gestacional/genética , Endonucleases/metabolismo , Feminino , Feto/anatomia & histologia , Humanos , Masculino , Camundongos , Gravidez , RNA Longo não CodificanteRESUMO
Insulin promotes early embryonic development, but whether this action affects postimplantation fetal development and alters the expression of imprinted genes remain to be determined. This study analyzed the expression and methylation levels of the growth-related imprinted genes H19 and insulin-like growth factor 2 (Igf2) in fetuses exposed to insulin before implantation. We cultured 2-cell embryos in either 0 or 0.25 microg/ml insulin until the blastocyst stage and then transferred them into pseudopregnant recipient mice. The number of embryos developing to blastocysts after insulin exposure was 16.4% higher than that of the control, and the birth body weight of the insulin-exposed group was 17.8% higher than that of the control group. Real-time reverse transcription-polymerase chain reaction analysis revealed that exposure of preimplantation embryos to insulin increased the mRNA expression of both Igf2 and H19 in embryonic day (E) 14 fetuses. Bisulfite genomic sequencing demonstrated that the methylation level of the H19-Igf2 imprint control region was 19.3% lower in insulin-exposed E14 fetuses than in controls. The present study indicates that insulin exposure during the preimplantation stage alters the expression of imprinted genes and affects fetal development.