Final Report Summary - SIRT1 AND DIABETES (IDENTIFICATION OF THE GENES REGULATED BY THE SIRT1 HISTONE DEACETYLASE AND THEIR CONTRIBUTION IN THE PATHOGENESIS OF TYPE 2 DIABETES AND OBESITY)
Obesity, whose incidence has increased dramatically in recent decades, is a major risk factor for other chronic diseases such as insulin resistance and type 2 diabetes. Because of the serious social problem posed, it is necessary to increase our knowledge of these diseases and to develop new and more effective therapeutic approaches. To design new effective therapies, in addition to the identification of new genes involved in the pathogenesis of this disease, a better knowledge of the molecular regulation of diabetogenic genes is necessary. Gene regulation at transcriptional level has been described to be directly correlated, among other factors, with alterations in chromatin structure by covalent modifications of NH2-terminal end of histones, mainly through acetylation. This MIRG Project has allowed us to start and set up our research, focused on the study of the pathophysiological causes of obesity, insulin resistance and type 2 diabetes. Specifically, we are centered in determining how epigenetic alterations (especially changes in chromatin structure) in glucose metabolism key tissues (such as liver and/or adipose tissue) can lead to the development of these diseases. To do that, we have developed two approaches:
In this first part, which constitutes the main objective of the project, we proceeded to study the effect of chromatin deacetylation by members of the family of Sirtuins (mainly SIRT1) in the pathogenesis of obesity and insulin resistance. As we already described in the previous report of this project, we have also developed the ChIPSeq technology (chromatin immunoprecipitation (ChIP) associated with massive sequencing (Seq)) in liver samples from animals that have developed an obese phenotype and insulin resistance. Specifically, we carried out ChIP experiments by using specific antibodies against SIRT1 in liver lysates from SIRT1 knockout mice and control mice and transgenic overexpressing SIRT1 which were provided with a high lipid content diet (HFD) as a model of obesity and type 2 diabetes. With the resulting immunoprecipitated DNA fragments, we performed massive sequencing and alignment with the entire mouse genome. This allowed us to identify hotspots that will indicate specific SIRT1 reaction with endogenous genes, suggesting potential target genes for this histone deacetylase.
Due to its intrinsic HDAC activity, SIRT1 is involved in promoting the formation of heterochromatin and, therefore, increasing the presence of changes in repression/gene silencing chromatin markers in its target genes (such as histone 3 trimethylation in lysines 9 and 27 (H3K9me3 and H3K27me3, respectively)). In contrast, in active gene regions we will observe a decrease in SIRT1 and increased euchromatin/gene activation markers (such as histone 3 trimethylation in lysine 4 (H3K4me3) and, in particular, SIRT1 has been found to specifically deacetylate the histone 4 lysine 16 residue when it is acetylated (H4K16Ac)). Thus, during this project, we conducted ChIPSeq experiments using antibodies against chromatin modifications, allowing us to confirm the specificity of candidate target genes obtained after the SIRT1ChIPSeq. Therefore, in addition to the ChIPSeq of SIRT1, we also have performed ChIPSeq experiments with antibodies against H3K9me3, H3K27me3, H3K4me3 and H4K16Ac in liver samples from SIRT1 knockout mice and control mice and transgenic overexpressing SIRT1, which were provided with a HFD as a model of obesity and type 2 diabetes. Those genes showing a high signal SIRT1, enrichment in H3K9me3 and H3K27me3 signal and a marked decrease in H4K16Ac and H3K4me3, are the ones we focused to validate biologically. All these results are allowing us to determine the effect of chromatin remodeling mediated by SIRT1 in the development of obesity, insulin resistance and type 2 diabetes. Thus, SIRT1 transgenic mice fed a HFD showed alterations in genes involved in regulation of oxidative stress, drug metabolism, arachidonic acid metabolism, immune response and PPAR signaling pathway, among others. By contrast, control animals in a HFD showed marked alterations in genes regulating biosynthetic process of macromolecules, cell motility and transcription factor activity. Therefore, the (dys) regulation in the control of these metabolic pathways may be involved in the onset and development of obesity and type 2 diabetes.
In this first part, which constitutes the main objective of the project, we proceeded to study the effect of chromatin deacetylation by members of the family of Sirtuins (mainly SIRT1) in the pathogenesis of obesity and insulin resistance. As we already described in the previous report of this project, we have also developed the ChIPSeq technology (chromatin immunoprecipitation (ChIP) associated with massive sequencing (Seq)) in liver samples from animals that have developed an obese phenotype and insulin resistance. Specifically, we carried out ChIP experiments by using specific antibodies against SIRT1 in liver lysates from SIRT1 knockout mice and control mice and transgenic overexpressing SIRT1 which were provided with a high lipid content diet (HFD) as a model of obesity and type 2 diabetes. With the resulting immunoprecipitated DNA fragments, we performed massive sequencing and alignment with the entire mouse genome. This allowed us to identify hotspots that will indicate specific SIRT1 reaction with endogenous genes, suggesting potential target genes for this histone deacetylase.
Due to its intrinsic HDAC activity, SIRT1 is involved in promoting the formation of heterochromatin and, therefore, increasing the presence of changes in repression/gene silencing chromatin markers in its target genes (such as histone 3 trimethylation in lysines 9 and 27 (H3K9me3 and H3K27me3, respectively)). In contrast, in active gene regions we will observe a decrease in SIRT1 and increased euchromatin/gene activation markers (such as histone 3 trimethylation in lysine 4 (H3K4me3) and, in particular, SIRT1 has been found to specifically deacetylate the histone 4 lysine 16 residue when it is acetylated (H4K16Ac)). Thus, during this project, we conducted ChIPSeq experiments using antibodies against chromatin modifications, allowing us to confirm the specificity of candidate target genes obtained after the SIRT1ChIPSeq. Therefore, in addition to the ChIPSeq of SIRT1, we also have performed ChIPSeq experiments with antibodies against H3K9me3, H3K27me3, H3K4me3 and H4K16Ac in liver samples from SIRT1 knockout mice and control mice and transgenic overexpressing SIRT1, which were provided with a HFD as a model of obesity and type 2 diabetes. Those genes showing a high signal SIRT1, enrichment in H3K9me3 and H3K27me3 signal and a marked decrease in H4K16Ac and H3K4me3, are the ones we focused to validate biologically. All these results are allowing us to determine the effect of chromatin remodeling mediated by SIRT1 in the development of obesity, insulin resistance and type 2 diabetes. Thus, SIRT1 transgenic mice fed a HFD showed alterations in genes involved in regulation of oxidative stress, drug metabolism, arachidonic acid metabolism, immune response and PPAR signaling pathway, among others. By contrast, control animals in a HFD showed marked alterations in genes regulating biosynthetic process of macromolecules, cell motility and transcription factor activity. Therefore, the (dys) regulation in the control of these metabolic pathways may be involved in the onset and development of obesity and type 2 diabetes.
