Frank K. Huynh

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

Low 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.

SnapShot: Mammalian Sirtuins

The mammalian sirtuins have emerged as critical regulators of cellular stress resistance, energy metabolism, and tumorigenesis. In some contexts, they delay the onset of age-related diseases and promote a healthy lifespan. The seven mammalian sirtuins, SIRT1-7, share a highly conserved NAD+-binding catalytic core domain although they exhibit distinct expression patterns, catalytic activities, and biological functions. This SnapShot provides an overview of these properties, with an emphasis on their relevance to aging.

Measurement of Fatty Acid Oxidation Rates in Animal Tissues and Cell Lines

While much oncological research has focused on metabolic shifts in glucose and amino acid oxidation, recent evidence suggests that fatty acid oxidation (FAO) may also play an important role in the metabolic reprogramming of cancer cells. Here, we present a simple method for measuring FAO rates using radiolabeled palmitate, common laboratory reagents, and standard supplies. This protocol is broadly applicable for measuring FAO rates in cultured cancer cells as well as in both malignant and nontransformed animal tissues.

Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH

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 (C1) to palmitoyl (C16) 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 sirtuin 4 leads to elevated glucose- and leucine-stimulated insulin levels and accelerated age-induced insulin resistance in multiple murine genetic backgrounds

Several inherited metabolic disorders are associated with an accumulation of reactive acyl-CoA metabolites that can non-enzymatically react with lysine residues to modify proteins. While the role of acetylation is well-studied, the pathophysiological relevance of more recently discovered acyl modifications, including those found in inherited metabolic disorders, warrants further investigation. We recently showed that sirtuin 4 (SIRT4) removes glutaryl, 3-hydroxy-3-methylglutaryl, 3-methylglutaryl, and 3-methylglutaconyl modifications from lysine residues. Thus, we used SIRT4 knockout mice, which can accumulate these novel post-translational modifications, as a model to investigate their physiological relevance. Since SIRT4 is localized to mitochondria and previous reports have shown SIRT4 influences metabolism, we thoroughly characterized glucose and lipid metabolism in male and female SIRT4KO mice across different genetic backgrounds. While only minor perturbations in overall lipid metabolism were observed, we found SIRT4KO mice consistently had elevated glucose- and leucine-stimulated insulin levels in vivo and developed accelerated age-induced insulin resistance. Importantly, elevated leucine-stimulated insulin levels in SIRT4KO mice were dependent upon genetic background since SIRT4KO mice on a C57BL/6NJ genetic background had elevated leucine-stimulated insulin levels but not SIRT4KO mice on the C57BL/6J background. Taken together, the data suggest that accumulation of acyl modifications on proteins in inherited metabolic disorders may contribute to the overall metabolic dysfunction seen in these patients.

SIRT3 Directs Carbon Traffic in Muscle to Promote Glucose Control

The escalating prevalence of the metabolic syndrome has coincided with the emergence of a lifestyle that replaces physical activity with overindulgence. In this setting, excess nutrients continuously bombard the major metabolic organs, leaving resident cells to cope with a steady influx of superfluous carbon fuel. The molecular consequences of energy surplus are far-reaching. Among these, growing evidence suggests carbon overload promotes lysine acetylation, a protein modification prominent in mitochondria and linked to detrimental effects on energy metabolism. Because this phenomenon of carbon stress is increasingly recognized as a key feature of aging and metabolic disease, scientists are now keenly interested in understanding the functional impacts of protein acetylation and the mechanisms that defend against them. The best-characterized countermeasures against protein hyperacetylation are mediated by a family of NAD+-dependent deacylases known as the sirtuins, which may have evolved as a stress response mechanism to offset spurious, nonenzymatic acetylation events that occur upon increasing carbon pressure (1). This family of proteins has a wide range of biological effects on disease processes associated with aging, including cancer, neurodegeneration, and metabolic syndrome. Sirtuins remove a variety of posttranslational acyl-modifications from proteins, including acetyl-lysine modifications (2), which accumulate during prolonged high-fat feeding (3,4). Interestingly, within 1 week of feeding mice a fat-rich diet, expression of the mitochondrial sirtuin, SIRT3, increases in the liver (3), possibly to protect against aberrant protein acetylation. Consistent with this prediction, hepatic protein acetylation remains low at this time point. However, after chronic high-fat feeding (13 weeks), SIRT3 expression …

Deacetylation by SIRT3 Relieves Inhibition of Mitochondrial Protein Function

Lysine acetylation is a chemical modification occurring on cellular proteins that plays important roles in protein regulation. In mitochondria, protein acetylation is regulated by the sirtuin SIRT3, which has been reported to control several metabolic pathways. While the functional relevance of protein acetylation is likely specific for proteins in different pathways, a picture is emerging for mitochondrial protein acetylation to be an important regulator of enzymatic activity. In this review, we integrate qualitative and quantitative proteomic acetylation studies and put forward the hypothesis that acetylation suppresses enzymatic activity, and that SIRT3 serves to relieve the suppression. We discuss the possible mechanisms of suppression, and the future studies needed to fully understand the biological role of acetylation and its regulation by the sirtuins.