Old Enzymes, New Tricks: Sirtuins Are NAD+-Dependent De-acylases

Seven mammalian sirtuins are nicotinamide adenine dinucleotide (NAD)+-dependent deacetylases and are important modulators of energy metabolism and stress resistance. Two new studies by Du et al. (2011) and Peng et al. (2011) identify a new enzymatic activity for SIRT5, expanding the cellular repertoire of posttranslational modifications targeted by the sirtuins

SIRT1 and SIRT3 deacetylate homologous substrates: AceCS1,2 and HMGCS1,2.

SIRT1 and SIRT3 are NAD+-dependent protein deacetylases that are evolutionarily conserved across mammals. These proteins are located in the cytoplasm/nucleus and mitochondria, respectively. Previous reports demonstrated that human SIRT1 deacetylates Acetyl-CoA Synthase 1 (AceCS1) in the cytoplasm, whereas SIRT3 deacetylates the homologous Acetyl-CoA Synthase 2 (AceCS2) in the mitochondria. We recently showed that 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) is deacetylated by SIRT3 in mitochondria, and we demonstrate here that SIRT1 deacetylates the homologous 3-hydroxy-3-methylglutaryl CoA synthase 1 (HMGCS1) in the cytoplasm. This novel pattern of substrate homology between cytoplasmic SIRT1 and mitochondrial SIRT3 suggests that considering evolutionary relationships between the sirtuins and their substrates may help to identify and understand the functions and interactions of this gene family. In this perspective, we take a first step by characterizing the evolutionary history of the sirtuins and these substrate families

In vivo Determination of Mitochondrial Function using Luciferase-Expressing Caenorhabditis elegans : Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced dysfunction

As a Duke Cancer Institute member, I acknowledge support from the Duke Cancer Institute as part of the P30 Cancer Center Support Grant (Grant ID: P30 CA014236). This work was also supported by the National Institute of Environmental Health Sciences (R01-ES017540-01A2).Peer reviewedPostprin

Neuronal CRTC-1 Governs Systemic Mitochondrial Metabolism and Lifespan via a Catecholamine Signal

SummaryLow energy states delay aging in multiple species, yet mechanisms coordinating energetics and longevity across tissues remain poorly defined. The conserved energy sensor AMP-activated protein kinase (AMPK) and its corresponding phosphatase calcineurin modulate longevity via the CREB regulated transcriptional coactivator (CRTC)-1 in C. elegans. We show that CRTC-1 specifically uncouples AMPK/calcineurin-mediated effects on lifespan from pleiotropic side effects by reprogramming mitochondrial and metabolic function. This pro-longevity metabolic state is regulated cell nonautonomously by CRTC-1 in the nervous system. Neuronal CRTC-1/CREB regulates peripheral metabolism antagonistically with the functional PPARα ortholog, NHR-49, drives mitochondrial fragmentation in distal tissues, and suppresses the effects of AMPK on systemic mitochondrial metabolism and longevity via a cell-nonautonomous catecholamine signal. These results demonstrate that while both local and distal mechanisms combine to modulate aging, distal regulation overrides local contribution. Targeting central perception of energetic state is therefore a potential strategy to promote healthy aging

Deficiency of the lipid synthesis enzyme, DGAT1, extends longevity in mice

Calorie restriction results in leanness, which is linked to metabolic conditions that favor longevity. We show here that deficiency of the triglyceride synthesis enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), which promotes leanness, also extends longevity without limiting food intake. Female DGAT1-deficient mice were protected from age-related increases in body fat, tissue triglycerides, and inflammation in white adipose tissue. This protection was accompanied by increased mean and maximal life spans of ~25% and ~10%, respectively. Middle-aged Dgat1−/− mice exhibited several features associated with longevity, including decreased levels of circulating insulin growth factor 1 (IGF1) and reduced fecundity. Thus, deletion of DGAT1 in mice provides a model of leanness and extended lifespan that is independent of calorie restriction

In Vivo Optical Metabolic Imaging of Long-Chain Fatty Acid Uptake in Orthotopic Models of Triple-Negative Breast Cancer

Targeting a tumor’s metabolic dependencies is a clinically actionable therapeutic approach; however, identifying subtypes of tumors likely to respond remains difficult. The use of lipids as a nutrient source is of particular importance, especially in breast cancer. Imaging techniques offer the opportunity to quantify nutrient use in preclinical tumor models to guide development of new drugs that restrict uptake or utilization of these nutrients. We describe a fast and dynamic approach to image fatty acid uptake in vivo and demonstrate its relevance to study both tumor metabolic reprogramming directly, as well as the effectiveness of drugs targeting lipid metabolism. Specifically, we developed a quantitative optical approach to spatially and longitudinally map the kinetics of long-chain fatty acid uptake in in vivo murine models of breast cancer using a fluorescently labeled palmitate molecule, Bodipy FL c16. We chose intra-vital microscopy of mammary tumor windows to validate our approach in two orthotopic breast cancer models: a MYC-overexpressing, transgenic, triple-negative breast cancer (TNBC) model and a murine model of the 4T1 family. Following injection, Bodipy FL c16 fluorescence increased and reached its maximum after approximately 30 min, with the signal remaining stable during the 30–80 min post-injection period. We used the fluorescence at 60 min (Bodipy60), the mid-point in the plateau region, as a summary parameter to quantify Bodipy FL c16 fluorescence in subsequent experiments. Using our imaging platform, we observed a two- to four-fold decrease in fatty acid uptake in response to the downregulation of the MYC oncogene, consistent with findings from in vitro metabolic assays. In contrast, our imaging studies report an increase in fatty acid uptake with tumor aggressiveness (6NR, 4T07, and 4T1), and uptake was significantly decreased after treatment with a fatty acid transport inhibitor, perphenazine, in both normal mammary pads and in the most aggressive 4T1 tumor model. Our approach fills an important gap between in vitro assays providing rich metabolic information at static time points and imaging approaches visualizing metabolism in whole organs at a reduced resolution

Why do bilaterally symmetrical flowers orient vertically? Flower orientation influences pollinator landing behaviour

Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone deacylases that use NAD(+) as a co-substrate during amide bond hydrolysis. Several studies have described the sirtuins as sensors of the NAD(+)/NADH ratio, but it has not been formally tested for all the mammalian sirtuins in vitro. To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins. These probes included aliphatic ϵ-N-acyllysine modifications with hydrocarbon lengths ranging from formyl (C(1)) to palmitoyl (C(16)) as well as negatively charged dicarboxyl-derived modifications. In addition to the well established activities of the sirtuins, “long chain” acyllysine modifications were also shown to be prone to hydrolytic cleavage by SIRT1–3 and SIRT6, supporting recent findings. We then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit these NAD(+)-dependent deacylase activities of the sirtuins. In the commonly used 7-amino-4-methylcoumarin-coupled fluorescence-based assay, the fluorophore has significant spectral overlap with NADH and therefore cannot be used to measure inhibition by NADH. Therefore, we turned to an HPLC-MS-based assay to directly monitor the conversion of acylated peptides to their deacylated forms. All tested sirtuin deacylase activities showed sensitivity to NADH in this assay. However, the inhibitory concentrations of NADH in these assays are far greater than the predicted concentrations of NADH in cells; therefore, our data indicate that NADH is unlikely to inhibit sirtuins in vivo. These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD(+)/NADH ratio

Loss of BCAA Catabolism during Carcinogenesis Enhances mTORC1 Activity and Promotes Tumor Development and Progression

Tumors display profound changes in cellular metabolism, yet how these changes aid the development and growth of tumors is not fully understood. Here we use a multi-omic approach to examine liver carcinogenesis and regeneration, and find that progressive loss of branched-chain amino acid (BCAA) catabolism promotes tumor development and growth. In human hepatocellular carcinomas and animal models of liver cancer, suppression of BCAA catabolic enzyme expression led to BCAA accumulation in tumors, though this was not observed in regenerating liver tissues. The degree of enzyme suppression strongly correlated with tumor aggressiveness, and was an independent predictor of clinical outcome. Moreover, modulating BCAA accumulation regulated cancer cell proliferation in vitro, and tumor burden and overall survival in vivo. Dietary BCAA intake in humans also correlated with cancer mortality risk. In summary, loss of BCAA catabolism in tumors confers functional advantages, which could be exploited by therapeutic interventions in certain cancers