Purpose of the study: Obesity is one of the major global health issues driven by a combination of increased calorie intake and decreased physical activity and it is strongly influenced by our genetic background [1]. Endocannabinoid signaling through the cannabinoid receptor type-1 (CB1R) plays a key role in energy balance regulation and its physiological functions are influenced by the diet [2]. We used the Diet-induced obesity (DIO) rat model to analyze the differences in the epigenetic regulation of CB1R gene expression between rats placed on a high-fat diet (HFD) becoming obese, compared to their diet resistant (DR) counterparts [3]. Moreover, in a subset of obese human subjects and controls, we assessed DNA methylation level at CB1R gene promoter and also genotyped two Single nucleotide polymorphisms (SNPs) within this gene (rs806368 and rs6454674) already associated with obesity [4]. Methods: Male Sprague Dawley rats were fed with High Fat diet. After 5 weeks, one group of rats showed higher body weight (DIO) compared to one part of the animals, DR rats. A subset of animals was sacrificed at week 5, whereas the other part was maintained on diet up to week 21. Biological material (e.g., total RNA and DNA) was isolated from hypothalamus (HY) and PBMCs. mRNA abundances and gene promoter DNA methylation were assessed by qRT-PCR and pyrosequencing, respectively. In obese human subjects and controls, TaqMan SNP Genotyping Assays (Life Technologies) were used for genotyping the CB1R SNPs and pyrosequencing to assess DNA methylation at gene promoter. Results: At week 5, the average body weight of DIO rats began to be significantly higher compared to DR rats (DIO: 465.7±12.7; DR: 406.0±5.7; p<0.01) and this variation is further increased after week 21 (DIO: 721.8±25.3; DR: 593.8±9.7; p<0.001). Gene expression analysis in the HY revealed a significant decrease of CB1R mRNA level in DR respect to DIO after 5 weeks (DIO: 1.00±0.04; DR: 0.54±0.11; p<0.01), whereas no changes were observed in rat sacrificed after 21 weeks. Epigenetic studies showed a selective and consistent significant increase in DNA methylation at the 3nd CpG site in DR rats (2.01±0.23) respect to DIO (1.29±0.07; p<0.05). No significant changes in DNAmethylation levels were instead observed in human obese subjects when compared to controls in any CpG sites under study. However, rs6454674 showed significant associations (p<0.05) with obesity. Conclusions: We here provide evidence of selective and time dependent transcriptional regulation of CB1R gene in DIO rats when compared to DR. The alterations in genes regulation would contribute to develop overweight in rats possibly to the hedonic impact of palatable food in DIO rats when compared to DIOR. Studying how environmental factors, as in this animal model, might induce obesity would be of help to understand which changes occur in central circuits. Further studies are required to provide the gene expression regulation and DNA methylation patterns of other possible genes involved in the development of obesity in target tissues to better understand the molecular basis relevant in the resistance to DIO. References [1] Mutch, D.M., Clement, K., 2006. Unraveling the genetics of human obesity. PLoS Genetics 2, e188. [2] Matias, I., Di Marzo, V., 2007. Endocannabinoids and the control of energy balance. Trends Endocrinol Metab. 18(1), 27−37. [3] Sclafani, A., Springer, D., 1976. Dietary obesity in adult rats: similarities to hypothalamic and human obesity syndromes. Physiol Behav 17(3), 461–471. [4] Benzinou, M., Ch`evre, J.C., Ward, K.J., Lecoeur, C., Dina, C., Lobbens, S., Durand, E., Delplanque, J., Horber, F.F., Heude, B., Balkau, B., Borch-Johnsen, K., Jørgensen, T., Hansen, T., Pedersen, O., Meyre, D., Froguel, P., 2008. Endocannabinoid receptor 1 gene variations increase risk for obesity and modulate body mass index in European populations. Hum Mol Genet 17(13), 1916−21.

Genetic and epigenetic regulation of cannabinoid receptor type-1 in obesity

M. V. Micioni Di Bonaventura;C. Cifani;
2015

Abstract

Purpose of the study: Obesity is one of the major global health issues driven by a combination of increased calorie intake and decreased physical activity and it is strongly influenced by our genetic background [1]. Endocannabinoid signaling through the cannabinoid receptor type-1 (CB1R) plays a key role in energy balance regulation and its physiological functions are influenced by the diet [2]. We used the Diet-induced obesity (DIO) rat model to analyze the differences in the epigenetic regulation of CB1R gene expression between rats placed on a high-fat diet (HFD) becoming obese, compared to their diet resistant (DR) counterparts [3]. Moreover, in a subset of obese human subjects and controls, we assessed DNA methylation level at CB1R gene promoter and also genotyped two Single nucleotide polymorphisms (SNPs) within this gene (rs806368 and rs6454674) already associated with obesity [4]. Methods: Male Sprague Dawley rats were fed with High Fat diet. After 5 weeks, one group of rats showed higher body weight (DIO) compared to one part of the animals, DR rats. A subset of animals was sacrificed at week 5, whereas the other part was maintained on diet up to week 21. Biological material (e.g., total RNA and DNA) was isolated from hypothalamus (HY) and PBMCs. mRNA abundances and gene promoter DNA methylation were assessed by qRT-PCR and pyrosequencing, respectively. In obese human subjects and controls, TaqMan SNP Genotyping Assays (Life Technologies) were used for genotyping the CB1R SNPs and pyrosequencing to assess DNA methylation at gene promoter. Results: At week 5, the average body weight of DIO rats began to be significantly higher compared to DR rats (DIO: 465.7±12.7; DR: 406.0±5.7; p<0.01) and this variation is further increased after week 21 (DIO: 721.8±25.3; DR: 593.8±9.7; p<0.001). Gene expression analysis in the HY revealed a significant decrease of CB1R mRNA level in DR respect to DIO after 5 weeks (DIO: 1.00±0.04; DR: 0.54±0.11; p<0.01), whereas no changes were observed in rat sacrificed after 21 weeks. Epigenetic studies showed a selective and consistent significant increase in DNA methylation at the 3nd CpG site in DR rats (2.01±0.23) respect to DIO (1.29±0.07; p<0.05). No significant changes in DNAmethylation levels were instead observed in human obese subjects when compared to controls in any CpG sites under study. However, rs6454674 showed significant associations (p<0.05) with obesity. Conclusions: We here provide evidence of selective and time dependent transcriptional regulation of CB1R gene in DIO rats when compared to DR. The alterations in genes regulation would contribute to develop overweight in rats possibly to the hedonic impact of palatable food in DIO rats when compared to DIOR. Studying how environmental factors, as in this animal model, might induce obesity would be of help to understand which changes occur in central circuits. Further studies are required to provide the gene expression regulation and DNA methylation patterns of other possible genes involved in the development of obesity in target tissues to better understand the molecular basis relevant in the resistance to DIO. References [1] Mutch, D.M., Clement, K., 2006. Unraveling the genetics of human obesity. PLoS Genetics 2, e188. [2] Matias, I., Di Marzo, V., 2007. Endocannabinoids and the control of energy balance. Trends Endocrinol Metab. 18(1), 27−37. [3] Sclafani, A., Springer, D., 1976. Dietary obesity in adult rats: similarities to hypothalamic and human obesity syndromes. Physiol Behav 17(3), 461–471. [4] Benzinou, M., Ch`evre, J.C., Ward, K.J., Lecoeur, C., Dina, C., Lobbens, S., Durand, E., Delplanque, J., Horber, F.F., Heude, B., Balkau, B., Borch-Johnsen, K., Jørgensen, T., Hansen, T., Pedersen, O., Meyre, D., Froguel, P., 2008. Endocannabinoid receptor 1 gene variations increase risk for obesity and modulate body mass index in European populations. Hum Mol Genet 17(13), 1916−21.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11581/405309
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