Dual targeting of the antagonistic pathways mediated by Sirt1 and TXNIP as a putative approach to enhance the efficacy of anti-aging interventions

Abstract

The organism's ability to regulate oxidative stress and metabolism is well recognized as a major determinant of longevity. While much research interest in this area is directed towards the study of genes that inhibit oxidative stress and/or improve metabolism, contribution to the aging process of genes with antagonistic effects on these two pathways is still less understood. The present study investigated the respective roles of the histone deacetylase Sirt1 and the thioredoxin binding protein TXNIP, two genes with opposite effects on oxidative stress and metabolism, in mediating the action of putative anti-aging interventions. Experiments were carried out in vitro and in vivo to determine the effect of proven, limited calorie availability, and unproven, resveratrol and dehydroepiandrosterone (DHEA), on the expression of Sirt1 and TXNIP. The results indicated that limited calorie availability consistently inhibited TXNIP in cancer and in normal cells including stem cells, however, it only slightly induced Sirt1expression in cancer cells. In contrast, resveratrol had a biphasic effect, and DHEA inhibited the expression of these two genes in a tissue specific manner, both in vitro and in vivo. Whereas all the three approaches tested inhibited TXNIP through the glycolytic pathway, DHEA acted by inhibiting G6PD and resveratrol through the activation of AMPK. In light of previous reports that Sirt1 induces AMPK-mediated signaling pathway, our findings point to the possibility of a negative relationship between Sirt1 and TXNIP that, if validated, can be exploited to improve the efficacy of putative anti-aging interventions.

Keywords: DHEA; Sirt1; TXNIP; aging; metabolism; oxidative stress; stem cells.

Figure 1.. Schematic representation highlighting the opposite effects of Sirt1 and TXNIP on oxidative stress and metabolism.

Sirt1 has been shown to inhibit oxidative stress and improves glucose uptake as well as insulin secretion. In contrast, TXNIP was found to enhance cellular susceptibility to oxidative stress and inhibit glucose uptake and insulin secretion.

Figure 2.. Effects of limited glucose availability, resveratrol and DHEA on expression of TXNIP and Sirt1 in cancer cells.

Panel A. SaOS2 cells were incubated in the presence of reduced glucose levels in the culture medium for 48 hours. Expression of TXNIP and Sirt1 were determined by Western blot using specific antibodies. Antibody to β-actin was used as a loading control. Panel B. Respective expression of TXNIP and Sirt1 in response to limited glucose availability, measured by quantitative real time PCR (Q-PCR). SaOS2 cells were incubated in the presence of the indicated amounts of glucose for 48 hours, QPCR was then performed to detect expression of TXNIP and Sirt1 using specific primers. Panel C and D, the RGC (panel C) and SaOS2 (panel D) cells were subjected to treatment with increasing concentrations of resveratrol (Resv.) for 48 hours followed by Western blot as described in Panel A. Panel E, SaOS2 cells were treated with increasing amounts of DHEA and probed for expression of Sirt1 and TXNIP, β-actin was used as a loading control.

Figure 3.. Effects of limited glucose availability, resveratrol and DHEA on expression of TXNIP and Sirt1 in normal cells.

Aortic smooth muscle cells (panel A) and human embryonic stem cells (panel B) were treated as described in Figure 1 for cancer cells. Expression Sirt1 and TXNIP were analyzed by Western blot using specific antibodies. Antibody to GAPDH was used as loading control. (Glc: glucose., Resv: Resveratrol).

Figure 4.. Tissue distribution of Sirt1 and TXNIP in mice and their regulation by resveratrol and DHEA.

Mice CD1 strain were injected (i.p.) with 10 mg/Kg of either resveratrol or DHEA and after 2 days, the animals were sacrificed and organs harvested and processed by Western blot for the expression of Sirt1 and TXNIP. Staining with GAPDH was used as a loading control.

Figure 5.. Putative mechanism(s) by which DHEA inhibits TXNIP.

SaOS2 (panel A) were pre-incubated with Tamoxifen or CCP for one hour prior to addition of DHEA. After an additional incubation for 48 hours, proteins were extracted and probed by western blot for the expression of TXNIP and Sirt1. Panel B, The cells were treated with different concentrations of glucose with or without DHEA. After 48 hours in cultures, proteins were separated by electrophoresis and probed for TXNIP. Staining with β-actin in antibody was used as a loading control. Panel C, Effect of DHEA and resveratrol on G6PD activity. Protein extract (100 μg) was incubated with DHEA or resveratrol at the indicated concentrations, after 30 min at 37°C, the activity of G6PD was measured as described in the methods section. Data represent average of four determinations +/- SE.

Figure 6.. Effect of Resveratrol and DHEA on phosphorylation of AMPK.

Panel A, SaOS2 cells were treated with increasing concentrations of resveratrol for 48 hours, after what, proteins were extracted and probed either with anti-phospho-AMPK (Thr172), or with antibody to AMPK. Panel B, RGC cells were incubated with 20 or 100 μM resveratrol or DHEA 100 μM for 48 hours, and then probed for phosphor-AMPK and AMPK as described above.

Figure 7.. Regulation of TXNIP through the glycolitic pathway and its modulation by limited glucose availability (LGA), resveratrol and DHEA.

Down regulation of glucose levels is known to increase the AMP/ATP ratio which in turn activates AMPK, leading to increased serine phosphorylation of ChREBP. Phosphorylated ChREBP is unable to translocate into the nucleus and form a functional complex with Mlx that is required for TXNIP expression. In addition, reduced glucose levels would inhibit, the pentose pathway and lead to the inhibition of the phosphatase PP2A. This will also result in the accumulation of phosphorylated ChREBP in the cytoplasm and further inhibition of TXNIP expression. DHEA acts on this glycolytic pathway mainly through the inhibition of G6PD activity. Resveratrol would act through the induction of Sirt1-mediated phosphorylation of AMPK, leading to enhanced phosphorylation ChREBP and inhibition of its nuclear translocation. This mechanism sheds light on TXNIP as a common downstream target for putative anti-aging interventions that affect metabolism.