SIRT3 is a member of the sirtuin family of NAD(+)-dependent deacetylases, which is localized to the mitochondria and is enriched in kidney, brown adipose tissue, heart, and other metabolically active tissues. We report here that SIRT3 responds dynamically to both exercise and nutritional signals in skeletal muscle to coordinate downstream molecular responses. We show that exercise training increases SIRT3 expression as well as associated CREB phosphorylation and PGC-1alpha up-regulation. Furthermore, we show that SIRT3 is more highly expressed in slow oxidative type I soleus muscle compared to fast type II extensor digitorum longus or gastrocnemius muscles. Additionally, we find that SIRT3 protein levels in skeletal muscle are sensitive to diet, for SIRT3 expression increases by fasting and caloric restriction, yet it is decreased by high-fat diet. Interestingly, the caloric restriction regimen also leads to phospho-activation of AMPK in muscle. Conversely in SIRT3 knockout mice, we find that the phosphorylation of both AMPK and CREB and the expression of PGC-1alpha are down regulated, suggesting that these key cellular factors may be important components of SIRT3-mediated biological signals in vivo.

A member of the sirtuin family of NAD⁺-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD⁺-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3^−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD⁺-dependent deacetylase, SIRT3.

Osteolytic lesions in multiple myeloma are caused by osteoclast-mediated bone resorption and reduced bone formation. A unique feature of myeloma is a failure of bone healing after successful treatment. We observed adipocytes on trabecular bone near the resorbed area in successfully treated patients. Normal marrow adipocytes, when cocultured with myeloma cells, were reprogrammed and produced adipokines that activate osteoclastogenesis and suppress osteoblastogenesis. These adipocytes have reduced expression of peroxisome proliferator–activated receptor γ (PPARγ) mediated by recruitment of polycomb repressive complex 2 (PRC2), which modifies PPARγ promoter methylation at trimethyl lysine-27 histone H3. We confirmed the importance of methylation in the PPARγ promoter by demonstrating that adipocyte-specific knockout of EZH2, a member of the PRC2, prevents adipocyte reprogramming and reverses bone changes in a mouse model. We validated the strong correlation between the frequency of bone lesions and the expression of EZH2 in marrow adipocytes from patients in remission. These results define a role for adipocytes in genesis of myeloma-associated bone disease and that reversal of adipocyte reprogramming has therapeutic implications.

Background: High doses of doxorubicin put cancer patients at risk for developing dilated cardiomyopathy. Previously, we showed that doxorubicin treatment decreases SIRT3 (sirtuin 3), the main mitochondrial deacetylase and increases protein acetylation in rat cardiomyocytes. Here, we hypothesize that SIRT3 expression can attenuate doxorubicin induced dilated cardiomyopathy in vivo by preventing the acetylation of mitochondrial proteins. Methods: Nontransgenic, M3-SIRT3 (truncated SIRT3; short isoform), and M1-SIRT3 (full-length SIRT3; mitochondrial localized) transgenic mice were treated with doxorubicin for 4 weeks (8 mg/kg body weight per week). Echocardiography was performed to assess cardiac structure and function and validated by immunohistochemistry and immunofluorescence (n=4–10). Mass spectrometry was performed on cardiac mitochondrial peptides in saline (n=6) and doxorubicin (n=5) treated hearts. Validation was performed in doxorubicin treated primary rat and human induced stem cell derived cardiomyocytes transduced with adenoviruses for M3-SIRT3 and M1-SIRT3 and deacetylase deficient mutants (n=4–10). Results: Echocardiography revealed that M3-SIRT3 transgenic mice were partially resistant to doxorubicin induced changes to cardiac structure and function whereas M1-SIRT3 expression prevented cardiac remodeling and dysfunction. In doxorubicin hearts, 37 unique acetylation sites on mitochondrial proteins were altered. Pathway analysis revealed these proteins are involved in energy production, fatty acid metabolism, and oxidative stress resistance. Increased M1-SIRT3 expression in primary rat and human cardiomyocytes attenuated doxorubicin-induced superoxide formation, whereas deacetylase deficient mutants were unable to prevent oxidative stress. Conclusions: Doxorubicin reduced SIRT3 expression and markedly affected the cardiac mitochondrial acetylome. Increased M1-SIRT3 expression in vivo prevented doxorubicin-induced cardiac dysfunction, suggesting that SIRT3 could be a potential therapeutic target for mitigating doxorubicin-induced dilated cardiomyopathy.

Obesity is increasing at an alarming rate worldwide, which is characterized by the excessive accumulation of triglycerides in adipocytes. Emerging evidence has demonstrated that macroautophagy and chaperone-mediated autophagy (CMA) regulate lipid mobilization and play a key role in energy balance. Sirtuin 3 (SIRT3) is an NAD⁺-dependent deacetylase, which is important in regulating macroautophagy and lipid metabolism. It is still unknown whether SIRT3 modulates macroautophagy and CMA in adipocytes. The current study found that macroautophagy was dynamically regulated during 3T3-L1 adipocyte differentiation, which coincided with SIRT3 expression. In mature adipocytes, overexpression of SIRT3 activated macroautophagy, mainly on lipid droplets (LDs), through activating the AMP-activated protein kinase (AMPK)-unc-51-like kinase 1 (ULK1) pathway, which in turn resulting in smaller LD size and reduced lipid accumulation. Moreover, SIRT3 overexpression induced the formation of perilipin-1 (PLN1)-heat shock cognate 71 kDa protein (HSC70)-lysosome-associated membrane protein 2 (LAMP2) complex, to activate CMA and cause the instability of LDs in adipocytes. In summary, we found SIRT3 is a positive regulator of macroautophagy and CMA in adipocytes, which might be a promising therapeutic target for treatment of obesity and its related metabolic dysfunction.

We have reported previously that ETS family transcription factor PU.1 is expressed in mature adipocytes of white adipose tissue. PU.1 expression is increased greatly in mouse models of genetic or diet-induced obesity. Here, we show that PU.1 expression is increased only in visceral but not subcutaneous adipose tissues of obese mice, and the adipocytes are responsible for this increase in PU.1 expression. To further address PU.1's physiological function in mature adipocytes, PU.1 was knocked down in 3T3-L1 cells using retroviral-mediated expression of PU.1-targeting shRNA. Consistent with previous findings that PU.1 regulates its target genes, such as NADPH oxidase subunits and proinflammatory cytokines in myeloid cells, the mRNA levels of proinflammatory cytokines (TNFα, IL-1β, and IL-6) and cytosolic components of NADPH oxidase (p47phox and p40phox) were downregulated significantly in PU.1-silenced adipocytes. NADPH oxidase is a main source for reactive oxygen species (ROS) generation. Indeed, silencing PU.1 suppressed NADPH oxidase activity and attenuated ROS in basal or hydrogen peroxide-treated adipocytes. Silencing PU.1 in adipocytes suppressed JNK1 activation and IRS-1 phosphorylation at Ser³⁰⁷. Consequently, PU.1 knockdown improved insulin signaling and increased glucose uptake in basal and insulin-stimulated conditions. Furthermore, knocking down PU.1 suppressed basal lipolysis but activated stimulated lipolysis. Collectively, these findings indicate that obesity induces PU.1 expression in adipocytes to upregulate the production of ROS and proinflammatory cytokines, both of which lead to JNK1 activation, insulin resistance, and dysregulation of lipolysis. Therefore, PU.1 might be a mediator for obesity-induced adipose inflammation and insulin resistance.