We have previously described the identification and characterization of small molecule activators of SIRT1. These compounds increase SIRT1 enzymatic activity by lowering the Km of SIRT1 for its acetylated substrates. Importantly, these drug-like molecules are efficacious in improving glucose and insulin homeostasis in multiple animal models of insulin resistance and T2DM [31, 32]. In the present study, we characterize the early molecular signaling mechanisms potentially underlying the therapeutic efficacy of SIRT1 activation by small molecules in a diet induced obesity model. To this end we employed a combination of Causal Network Modeling of transcriptional profiling data and supporting in vivo and in vitro data.
Reverse causal analysis identified CR as a statistically significant hypothesis in the SRT501 dataset comparison. To determine whether this hypothesis is also supported in the SRT1720 dataset comparison, we analyzed the underlying state changes at the probe set level. In doing so we determined that all 73 probe sets covering 54 changes in gene expression displayed conserved directionality between the two dataset comparisons (p < 10-5). Hence, these two chemically distinct small molecule activators of SIRT1 recapitulate many of the molecular events of calorie restriction in vivo and importantly work via the same molecular mechanism in vivo – SIRT1 activation, a conclusion which is supported previous studies which have shown that SRT501-resveratrol and SRT1720 increase the deacetylation of SIRT1 substrates (e.g. p53, Ppargc1a, and Foxo1a) in vivo and in vitro. Additionally, previous studies have demonstrated that SRT501[30, 31, 53] and SRT1720[32, 54], like calorie restriction [4, 8], enhance oxidative metabolism, protect against diet induced obesity, increase exercise endurance, and improve glucose and insulin homeostasis in rodent models of metabolic disease. These findings are consistent with the role of SIRT1 downstream of calorie restriction, which, as a therapeutic strategy, has proven efficacious in improving the metabolic profiles in rodents, large mammals, monkeys and humans [3–5, 55].
Reverse causal analysis also identified increased metabolism, mediated primarily by transcriptional activation of Ppar family members, as a network supported by six statistically significant hypotheses in the SRT501 dataset comparison. Furthermore, we determined that the changes in gene expression which support these hypotheses are modulated in the same direction in the SRT501 and SRT1720 treatment groups. Ppar-mediated signaling is a well documented molecular event downstream of calorie restriction [56, 57]. These nuclear hormone receptors regulate a multitude of physiological pathways including lipid metabolism, glucose homeostasis, inflammation and development [37, 58–60]. Notably, PPARα and PPARγ are respectively the molecular targets of the fibrate and thiazolidinedione (TZD) classes of drugs [61, 62]. Fibrates are commonly used for therapeutic intervention of hypertension, atherosclerosis, and dyslipidemia. TZDs, on-the-other-hand, are insulin-sensitizing reagents frequently used in the treatment of T2DM and Metabolic Syndrome. It is important to note that, while reverse causal analysis identified Ppar activation as a hypothesis downstream of small molecule activation of SIRT1, we did not observe the full range of pharmacological effects associated with fibrate or TZD treatment, such as increased peroxisome mass, body weight gain, increased adipocyte proliferation and edema. We speculate that the levels of Ppar activation and/or the subset of upregulated Ppara gene-targets generated by treatment with SRT501 or SRT1720 are likely more consistent with those observed downstream of calorie restriction.
The exact mechanism by which SRT501 and SRT1720 increase transcriptional activation of Ppars is an area for future investigation. However, it is likely due, at least in part, to deacetylation and transcriptional activation of Ppargc1α, a phenomenon supported by transcriptional data in this study. Ppargc1α has been well characterized as a co-activator for numerous nuclear hormone receptors including Ppars . Also, Ppargc1α is known to be deacetylated at multiple lysine residues by SIRT1, leading to increased Ppargc1α transcriptional activity .
Importantly, the effect of SIRT1 activation on Ppar activity is likely tissue-specific and family member-specific. For example, previous work in rodents has demonstrated that SIRT1 represses Pparγ mediated transcription, leading to increased fat mobilization and inhibition of adipogenesis . Future studies may elucidate whether Ppar activation in tissues other than liver is important for the efficacy of SIRT1 activating compounds and what the relative contribution of specific Ppar family members is in a tissue-specific context.
Closely linked to the increased metabolism and Ppar family activity is increased mitochondrial biogenesis, a phenomenon strongly associated with CR . The key node of this network is transcriptional activation of Ppargc1α, which, as mentioned previously, is deacetylated by SIRT1, leading to increased transcriptional activity of Ppargc1α . This network is also supported by the increased transcriptional activities of Pparα, Pparγ and Essrα and by decreased transcriptional activity of Nrip1. Pparα, Pparγ and Essrα can be coactivated by Ppargc1α, leading to increased expression of target genes, many of which are involved in mitochondrial respiration and biogenesis [37, 65]. Conversely, Nrip1 (RIP140) acts a transcriptional co-repressor of several nuclear hormone receptors, including Esrrα, leading to decreased expression of genes involved in oxidative metabolism and mitochondrial biogenesis .
Within the context of the liver, the increased mitochondrial biogenesis may contribute to an improved metabolic profile via increased efficiency in fatty acid oxidation and lowering of serum triglycerides, both of which have been shown to improve insulin sensitivity in peripheral tissues . Furthermore, it is certainly plausible that increased mitochondrial biogenesis in other tissues, such as skeletal muscle and adipose tissue, also contributes to therapeutic efficacy of SIRT1 activating compounds. In fact, previous reports have indicated that both resveratrol and SRT1720 increase mitochondrial function in skeletal muscle [31, 32]. This increase in mitochondrial capacity allows for efficient substrate switching between lipids, during times of fasting, and carbohydrates in response to insulin [67, 68]. Moreover, mitochondrial oxidative capacity in skeletal muscle has been shown to be a strong predictor of insulin sensitivity in humans .
Finally, SRT501 and SRT1720 mimic calorie restriction as demonstrated by the hypotheses consistent with decreased inflammation. As previously mentioned, calorie restriction is well known to inhibit inflammatory processes. This hypothesis is based upon decreases in signaling of Tnfα, Egf and NF-κB complex and upon increased transcriptional activity of the glucocorticoid receptor, Nr3c1. Tnfα is a well characterized inflammatory cytokine and has been implicated in the pathogenesis of numerous diseases, including autoimmune disorders, cancer, insulin resistance and Metabolic Syndrome [70–72]. Similarly, the NF-κB complex, which lies downstream of the Tnfα signaling cascade, is a transcriptional complex, which regulates the transcription of wide variety of target genes including inflammatory signaling factors, such as Tnfα . Egf also has an established role in inflammatory signaling, most notably implicated as a fibrogenic factor underlying the pathogenesis of COPD, cystic fibrosis and cardiovascular disease [74, 75]. Finally, the glucocorticoid receptor Nr3c1 is a central regulator of inflammatory responses, capable of transcriptionally repressing pro-inflammatory genes and of transcriptionally activating anti-inflammatory genes (reviewed in ). In summary, the predicted decrease in inflammation is comprised of a combination of upregulation of anti-inflammatory pathways and downregulation of pro-inflammatory pathways. Elucidation of the exact mechanism by which SIRT1 activation leads to these events will be an area for future investigation.
Notably, this network is supported by in vitro data, which demonstrates that both SRT501 and SRT1720 induce dose-dependent inhibition of LPS-stimulated Tnfα secretion in a cultured macrophage cell line. Also, several prior reports have implicated SIRT1 [47–49, 51, 77] and resveratrol [78–80] as negative regulators of inflammation both in vitro and in vivo.
The decreased inflammation network supports the calorie restriction model, as CR has been shown to inhibit inflammation in multiple animal models, including humans. Whether this decreased inflammation contributes to the therapeutic efficacy of our SIRT1 activating compounds remains under investigation. Certainly, there is increasing evidence that the development of insulin resistance in obese individuals involves paracrine interplay between white adipose tissue and infiltrating macrophages. At the molecular level, this pathogenic progression is characterized by an increase in secretion of inflammatory cytokines, such as TNFα, IL-6 and MCP-1, and a decrease of anti-inflammatory adipokines, such as adiponectin (reviewed in). Exacerbation of this pathogenic inflammatory response to include tissues such as the liver and vasculature can ultimately contribute to the onset of T2DM and/or Metabolic Syndrome. It has been reported that the anti-inflammatory effects of TZDs, mediated through PPARγ, are essential for the therapeutic efficacy of that drug class in treating T2DM . Hence, it is quite plausible that SIRT1 activating compounds improve glucose and insulin homeostasis, in part, by inhibiting inflammatory signaling.