Qiang Tong

Hepatic gluconeogenesis is a crucial process to maintain glucose level during starvation. However, unabated glucose production in diabetic patients is a major contributor to hyperglycemia. Palmitoleic acid is a monounsaturated fatty acid (16:1n7) that is available from dietary sources. Palmitoleic acid exhibits health beneficial effects on diabetes, insulin resistance, inflammation, and metabolic syndrome. However, the mechanism by which palmitoleate reduces blood glucose is still unclear. SIRT3 is a key metabolism-regulating NAD⁺-dependent protein deacetylase. It is known that fasting elevates the expression of SIRT3 in the liver and it regulates many aspects of liver’s response to nutrient deprivation, such as fatty acid oxidation and ketone body formation. However, it is unknown whether SIRT3 also regulates gluconeogenesis. Our study revealed that palmitoleic acid reduced hepatic gluconeogenesis and the expression of SIRT3 under high-fat diet conditions. Overexpression of SIRT3 in the liver and hepatocytes enhanced gluconeogenesis. Further study revealed that SIRT3 played a role in enhancing the activities of gluconeogenic enzymes, such as PEPCK, PC, and MDH2. Therefore, our study indicated that under a high-fat diet, palmitoleic acid decreased gluconeogenesis by reducing enzymatic activities of PEPCK, PC, and MDH2 by down-regulating the expression of SIRT3.

We have previously reported that the expression of mitochondrial deacetylase SIRT3 is high in the slow oxidative muscle and that the expression of muscle SIRT3 level is increased by dietary restriction or exercise training. To explore the function of SIRT3 in skeletal muscle, we report here the establishment of a transgenic mouse model with muscle-specific expression of the murine SIRT3 short isoform (SIRT3M3). Calorimetry study revealed that the transgenic mice had increased energy expenditure and lower respiratory exchange rate (RER), indicating a shift towards lipid oxidation for fuel usage, compared to control mice. The transgenic mice exhibited better exercise performance on treadmills, running 45% further than control animals. Moreover, the transgenic mice displayed higher proportion of slow oxidative muscle fibers, with increased muscle AMPK activation and PPARδ expression, both of which are known regulators promoting type I muscle fiber specification. Surprisingly, transgenic expression of SIRT3M3 reduced muscle mass up to 30%, likely through an up-regulation of FOXO1 transcription factor and its downstream atrophy gene MuRF-1. In summary, these results suggest that SIRT3 regulates the formation of oxidative muscle fiber, improves muscle metabolic function, and reduces muscle mass, changes that mimic the effects of caloric restriction.

Sirtuin deacetylases and FOXO (Forkhead box, class O) transcription factors have important roles in many biological pathways, including cancer development. SIRT1 and SIRT2 deacetylate FOXO factors to regulate FOXO function. Because acetylation and ubiquitination both modify the ɛ-amino group of lysine residues, we investigated whether FOXO3 deacetylation by SIRT1 or SIRT2 facilitates FOXO3 ubiquitination and subsequent proteasomal degradation. We found that SIRT1 and SIRT2 promote FOXO3 poly-ubiquitination and degradation. Proteasome-inhibitor treatment prevented sirtuin-induced FOXO3 degradation, indicating that this process is proteasome dependent. In addition, we demonstrated that E3 ubiquitin ligase subunit Skp2 binds preferentially to deacetylated FOXO3. Overexpression of Skp2 caused poly-ubiquitination of FOXO3 and degradation, whereas knockdown of Skp2 increased the amount of FOXO3 protein. We also present evidence that SCF-Skp2 ubiquitinates FOXO3 directly in vitro. Furthermore, mutating four known acetylated lysine residues (K242, K259, K290 and K569) of FOXO3 into arginines to mimic deacetylated FOXO3 resulted in enhanced Skp2 binding but with inhibition of FOXO3 ubiquitination; this suggests that some or all of these four lysine residues are likely the sites for ubiquitination. In the livers of mice deficient in SIRT1, we detected increased expression of FOXO3, indicating SIRT1 regulates FOXO3 protein levels in vivo. Furthermore, we found that the elevation of SIRT1 and Skp2 expression in malignant PC3 and DU145 prostate cells is responsible for the downregulation of FOXO3 protein levels in these cells. Taken together, our data support the notion that deacetylation of FOXO3 by SIRT1 or SIRT2 facilitates Skp2-mediated FOXO3 poly-ubiquitination and proteasomal degradation.

Sirtuin family of proteins possesses NAD-dependent deacetylase and ADP ribosyltransferase activities. They are found to respond to nutrient deprivation and profoundly regulate metabolic functions. We have previously reported that caloric restriction increases the expression of one of the seven mammalian sirtuins, SIRT2, in tissues such as white adipose tissue. Because adipose tissue is a key metabolic organ playing a critical role in whole body energy homeostasis, we went on to explore the function of SIRT2 in adipose tissue. We found short-term food deprivation for 24 h, already induces SIRT2 expression in white and brown adipose tissues. Additionally, cold exposure elevates SIRT2 expression in brown adipose tissue but not in white adipose tissue. Intraperitoneal injection of a β-adrenergic agonist (isoproterenol) enhances SIRT2 expression in white adipose tissue. Retroviral expression of SIRT2 in 3T3-L1 adipocytes promotes lipolysis. SIRT2 inhibits 3T3-L1 adipocyte differentiation in low-glucose (1 g/l) or low-insulin (100 nM) condition. Mechanistically, SIRT2 suppresses adipogenesis by deacetylating FOXO1 to promote FOXO1's binding to PPARγ and subsequent repression on PPARγ transcriptional activity. Overall, our results indicate that SIRT2 responds to nutrient deprivation and energy expenditure to maintain energy homeostasis by promoting lipolysis and inhibiting adipocyte differentiation.

PU.1 transcription factor is a critical regulator of hematopoiesis and leukemogenesis. Because PU.1 interacts with transcription factors GATA-2 and C/EBPα, and both are involved in the regulation of adipogenesis, we investigated whether PU.1 plays a role in the regulation of adipocyte differentiation. Our data indicate that PU.1 is expressed in white adipose tissue. PU.1 protein can also be detected in cultured 3T3-L1 adipocytes. Forced expression of PU.1 in 3T3-L1 cells inhibits adipocyte differentiation, whereas deletion of the transactivation domain of PU.1 abolishes this effect. The inhibition of adipocyte differentiation by PU.1 is achieved, at least in part, through repression of the transcriptional activity of C/EBPα and C/EBPβ. Furthermore, GATA-2 and PU.1 have an additive inhibitory effect on C/EBP transactivation and adipogenesis. Finally, the expression of PU.1 is increased in white adipose of obese mice.

Summary: The sirtuin family of nicotinamide adenine dinucleotide‐dependant (NAD) deacetylases plays an important role in aging and metabolic regulation. In yeast, the Sir2 gene and its homolog Hst2 independently mediate the action of caloric restriction on lifespan extension. The mammalian Sir2 ortholog, SIRT1, is up‐regulated by caloric restriction and deacetylates a variety of substrates, including histones and the forkhead box O (FOXO) transcription factors. The mammalian ortholog of Hst2, SIRT2, was shown to co‐localize with microtubules and functions as α‐tubulin deacetylase. During G2/M phase, SIRT2 proteins enter nuclei and deacetylate histones. We report here that the expression of SIRT2 is elevated in the white adipose tissue and kidney of caloric‐restricted mice. Oxidative stress, such as hydrogen peroxide treatment, also increases SIRT2 expression in cells. We have demonstrated that SIRT2 binds to FOXO3a and reduces its acetylation level. SIRT2 hence increases FOXO DNA binding and elevates the expression of FOXO target genes, p27^Kip1, manganese superoxide dismutase and Bim. As a consequence, SIRT2 decreases cellular levels of reactive oxygen species. Furthermore, as Bim is a pro‐apoptotic factor, SIRT2 promotes cell death when cells are under severe stress. Therefore, mammalian SIRT2 responds to caloric restriction and oxidative stress to deacetylate FOXO transcription factors.