Our studies reveal that SIRT6 functions as a histone deacetylase to control glucose homeostasis by inhibiting multiple glycolytic genes in a coordinated fashion (). Under conditions of normal glucose availability, SIRT6 represses expression of key enzymes, diverting pyruvate towards the mitochondrial TCA cycle for efficient ATP production. At those promoters, SIRT6 competes with the transcriptional activator Hif1α to maintain proper glucose flux towards mitochondrial respiration and preventing excessive glycolysis. Several lines of evidence support this model. First, SIRT6 deficiency causes up-regulation of glycolytic genes at the level of expression, a finding that is accompanied by increased glucose uptake and a switch towards glycolysis even under normal nutrient conditions (-). Second, SIRT6 directly binds to the promoters of these genes, and in the absence of SIRT6, H3K9 acetylation increases specifically in those promoters (). Third, we find that SIRT6 influence glycolysis as a co-repressor of Hif1α, and in the absence of SIRT6, the glycolytic phenotype can be rescued by knocking-down Hif1α in these cells (-). Thus, SIRT6 acts as a safeguard mechanism to down-modulate basal transcription of Hif1α target genes under normal nutrient conditions (Carmeliet et al., 1998
). In this context, there are two plausible scenarios. The first possibility is that SIRT6 binding to the promoters inhibits recruitment of Hif1α (accelerating its degradation). Alternatively, Hif1α could already localize to the promoters under normoglycemia, but the presence of SIRT6 would inhibit its transcriptional activity. Even though we found that SIRT6 and Hif1α can interact (), we were unable to perform ChIP assays with anti-Hif1α antibodies; therefore, we cannot rule out at present either possibility. However, recent studies have shown that, indeed, Hif1α occupies its target promoters even under normoxia (Xia et al., 2009
), supporting our second model. Notably, early studies have demonstrated that Hif1α activates transcription through recruitment of the histone acetyl-transferase p300/CBP (Arany et al., 1996
; Kallio et al., 1998
); consequently, SIRT6 might compete against recruitment of p300, maintaining histones in those promoters in a hypoacetylated state. Future studies will likely address this possibility. In the context of the Hif1α knock-down experiments, it is intriguing that despite full rescue of the metabolic phenotype (see ), only a subset of glycolytic genes were rescued (, see for instance PDK1). These results suggest that while SIRT6 regulates multiple glycolytic genes in a coordinated fashion, only few of them play a dominant role in this glycolytic switch.
An alternative explanation to our results would be that, in addition to deacetylating histones at those putative targets, SIRT6 actually regulates Hif1α itself. In this regard, we find no changes in HIF1α RNA levels in SIRT6 deficient cells (), indicating that SIRT6 does not regulate expression of this factor. A second possibility would be that SIRT6 deacetylates Hif1α, and in the absence of SIRT6, Hif1α is acetylated and stabilized. However, as shown above, we also failed to detect Hif1α acetylation in vivo,
even in SIRT6 KO cells, where total levels of Hif1α were significantly higher (Supplemental Figure 6A
); therefore, a direct effect for SIRT6 on Hif1α appears unlikely. On the other hand, we find that lack of SIRT6 increases both protein synthesis and protein stability of Hif1α. Previous studies have shown that conditions of nutrient stress and increase in lactate production can function as a positive feedback to induce both protein synthesis and stability of Hif1α (Lu et al., 2002
) (Hirota and Semenza, 2005
). Although at present other possibilities cannot be ruled out, SIRT6 deficient cells experienced both nutrient stress and increased lactate production, likely explaining the increased Hif1α levels observed in these cells.
Using high-resolution quantitative ChIP mapping of the LDHB gene, we gained further insight into the molecular mechanisms of SIRT6 silencing. Our results indicate that in wild type cells, SIRT6 binding maintains low levels of H3K9 acetylation at the LDHB promoter, thereby inhibiting transcription, despite the presence of pre-loaded RNAPII. In the absence of SIRT6, transcription is activated, as indicated by robust enrichment of Ser5 and Ser2 phosphorylated RNAPII, markers of promoter escape and transcriptional elongation, respectively. These results are intriguing, suggesting that SIRT6, a histone deacetylase, might repress transcription at a stage downstream of RNAPII recruitment. Recent studies have shown that engaged but paused polymerase plays an important role on genes that need to be rapidly activated (Core and Lis, 2008
). Changes in nutrient conditions could vary rapidly, and therefore, it is tempting to speculate that SIRT6 could repress transcription while maintaining an engaged polymerase, which in turn will allow rapid activation of transcription at these glycolytic genes upon changes in nutrient availability. Whether this represents a general epigenetic mode of regulation remains to be determined.
Recent studies have shown that SIRT6 can function as a co-repressor of NF-κB, modulating expression of NF-κB targets (Kawahara et al., 2009
). Furthermore, RelA haploinsufficiency was able to rescue the lethality of SIRT6 deficient animals. However, glucose levels in these animals remain low for the first weeks of life. Therefore, it is unlikely that NF-κB represents the initial trigger in the hypoglycemic phenotype observed. Consistent with this notion, we do not observe changes in expression of NF-κB targets in our muscle-microarray data (Supplemental Figure 4A
). Overall, these results strongly support a model where the defects in glucose metabolism observed in the absence of SIRT6 stem from its role in controlling glycolytic gene expression rather than through modulation of NF-κB targets.
Our model for SIRT6 function predicts that under conditions of nutrient stress, SIRT6 would be inactivated, triggering a Hif1α-dependent glycolytic switch, similar to what we observed in our SIRT6 deficient cells (). In this regard, we do not observe changes in total levels nor in localization of SIRT6 protein following glucose deprivation (Supplemental Figure 7
). One possibility is that SIRT6 activity is controlled at a post-transcriptional level, an alternative that is under current investigation. It is interesting that nutrient deprivation has been shown to increase levels of another mammalian sirtuin, SIRT1 (Cohen et al., 2004
; Nemoto et al., 2004
), indicating that these proteins might have evolved to function in contrasting scenarios. In this context, while SIRT1 activators have been shown to protect against metabolic diseases such as type-II diabetes, as published (Baur et al., 2006
; Lagouge et al., 2006
; Milne et al., 2007
), in the case of SIRT6, inhibition rather than activation might prove beneficial to lower blood glucose in metabolic diseases.
The increased glycolytic capacity and reduced oxidative phosphorylation we observe in SIRT6 deficient cells are reminiscent to the “Warburg effect” described by Otto Warburg several decades ago (Warburg, 1956
). Such a phenomenon describes the peculiarity that most cancer and highly proliferative cells rely on aerobic glycolysis rather than respiration for their energy and metabolic needs (Vander Heiden et al., 2009
). Consistent with our observations in SIRT6 deficient cells, recent studies indicate that aerobic glycolysis requires Hif1α as well (Lum et al., 2007
), and Hif1α confers resistance to apoptosis in cancer cells under hypoxic conditions in a GLUT-1 dependent manner (Kilic et al., 2007
). Based on this analogy, one could predict that lack of SIRT6 should provide an advantage for tumorigenic growth. In this context, SIRT6 deficient ES cells exhibit increased resistance to apoptosis when exposed to hypoxia/hypoglycemia (Supplemental Figure 6B
); however, we are currently unable to test this hypothesis in vivo,
since SIRT6 deficient animals die early in life. While it remains unclear what is the trigger that allows the switch from oxidative phosphorylation to aerobic glycolysis (Vander Heiden et al., 2009
), our results indicate that inhibition of SIRT6 might be an important player.
Previous studies in yeast, worms and flies have linked Sir2 proteins to the regulation of longevity (Finkel et al., 2009
; Longo and Kennedy, 2006
; Yu and Auwerx, 2009
). Whether such a role is conserved in mammals remains unclear. However, multiple lines of evidence indicate a critical role for some of these mammalian sirtuins in regulating metabolic homeostasis (Canto et al., 2009
). Since changes in calorie intake and metabolic balance has been previously linked to lifespan regulation in mammals (Yu and Auwerx, 2009
) (Barzilai and Bartke, 2009
), our new results with SIRT6 place this chromatin deacetylase as a potential candidate among sirtuins to influence ageing and age-related diseases. Notably, two recent articles have shown that Hif1α can modulate lifespan in C. Elegans
(Chen et al., 2009
; Mehta et al., 2009
). Whether this is the case in mammals remains unknown, however our results suggest that sirtuins and Hif1α may function in a coordinated fashion to modulate metabolic homeostasis in higher eukaryotes.
Overall, our studies have demonstrated a novel role for the histone deacetylase SIRT6 in controlling glucose homeostasis. The severe metabolic phenotypes we observed indicate that among the mammalian sirtuins, SIRT6 appears to play a dominant role in regulating energy balance. It also suggests that while a glycolytic switch might be an important acute adaptive response in situations of nutrient stress or cancer growth, chronic and sustained activation of this switch (as in the case of SIRT6 deficiency) is rather detrimental.