Maria M. Fergusson

SIRT1 Functionally Interacts with the Metabolic Regulator and Transcriptional Coactivator PGC-1α

In lower organisms, increased expression of the NAD-dependent deacetylase Sir2 augments lifespan. The mechanism through which this life extension is mediated remains incompletely understood. Here we have examined the cellular effects of overexpression of SIRT1, the closest mammalian ortholog of Sir2. In PC12 cells, increased expression of the NAD-dependent deacetylase SIRT1 reduces cellular oxygen consumption by ∼25%. We further demonstrate that SIRT1 expression can alter the transcriptional activity of the mitochondrial biogenesis coactivator PGC-1α. In addition, SIRT1 and PGC-1α directly interact and can be co-immunoprecipitated as a molecular complex. A single amino acid mutation in the putative ADP-ribosyltransferase domain of SIRT1 inhibits the interaction of SIRT1 with PGC-1α but does not effect the interaction of SIRT1 with either p53 or Foxo3a. We further show that PGC-1α is acetylated in vivo. This acetylation is augmented by treatment with the SIRT1 inhibitor nicotinamide or by expression of the transcriptional coactivator p300. Finally we demonstrate that SIRT1 catalyzes PGC-1α deacetylation both in vitro and in vivo. These results provide a direct link between the sirtuins, a family of proteins linked to lifespan determination and PGC-1α, a coactivator that regulates cellular metabolism.

Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress

Starvation and Autophagy Starvation stimulates withdrawal from the cell cycle, as well as stimulating autophagy. Are these two events connected? Lee et al. (p. 225) show a direct and nutrient-sensitive interaction between the tumor suppressor p53 and the essential autophagy gene Atg7. Further, in the absence of Atg7, the p53-dependent induction of the cyclin-dependent kinase inhibitor p21 is inhibited. This leads to Atg7-deficient cells being unable to properly withdraw from the cell cycle under starved conditions. While Atg7 deletion leads to an impairment of p53-mediated cell-cycle arrest, the Atg7-deficient cells hyperactivate p53-mediated cell-death pathways. The physiological importance of this hyperactivation is underscored by the observation that genetic blocking of p53-mediated cell death significantly extended neonatal survival of mice in which Atg7 had been deleted. When the autophagy protein tg7 is absent, nutrient withdrawal does not stop the cell cycle. Withdrawal of nutrients triggers an exit from the cell division cycle, the induction of autophagy, and eventually the activation of cell death pathways. The relation, if any, among these events is not well characterized. We found that starved mouse embryonic fibroblasts lacking the essential autophagy gene product Atg7 failed to undergo cell cycle arrest. Independent of its E1-like enzymatic activity, Atg7 could bind to the tumor suppressor p53 to regulate the transcription of the gene encoding the cell cycle inhibitor p21CDKN1A. With prolonged metabolic stress, the absence of Atg7 resulted in augmented DNA damage with increased p53-dependent apoptosis. Inhibition of the DNA damage response by deletion of the protein kinase Chk2 partially rescued postnatal lethality in Atg7−/− mice. Thus, when nutrients are limited, Atg7 regulates p53-dependent cell cycle and cell death pathways.

The Mammalian Longevity-associated Gene Product p66shc Regulates Mitochondrial Metabolism

Previous studies have determined that mice with a homozygous deletion in the adapter protein p66shc have an extended life span and that cells derived from these mice exhibit lower levels of reactive oxygen species. Here we demonstrate that a fraction of p66shc localizes to the mitochondria and that p66shc-/- fibroblasts have altered mitochondrial energetics. In particular, despite similar cytochrome content, under basal conditions, the oxygen consumption of spontaneously immortalized p66shc-/- mouse embryonic fibroblasts were lower than similarly maintained wild type cells. Differences in oxygen consumption were particularly evident under chemically uncoupled conditions, demonstrating that p66shc-/- cells have a reduction in both their resting and maximal oxidative capacity. We further demonstrate that reconstitution of p66shc expression in p66shc-/- cells increases oxygen consumption. The observed defect in oxidative capacity seen in p66shc-/- cells is partially offset by augmented levels of aerobic glycolysis. This metabolic switch is manifested by p66shc-/- cells exhibiting an increase in lactate production and a stricter requirement for extracellular glucose in order to maintain intracellular ATP levels. In addition, using an in vivo NADH photobleaching technique, we demonstrate that mitochondrial NADH metabolism is reduced in p66shc-/- cells. These results demonstrate that p66shc regulates mitochondrial oxidative capacity and suggest that p66shc may extend life span by repartitioning metabolic energy conversion away from oxidative and toward glycolytic pathways.

Measuring In Vivo Mitophagy

Alterations in mitophagy have been increasingly linked to aging and age-related diseases. There are, however, no convenient methods to analyze mitophagy in vivo. Here, we describe a transgenic mouse model in which we expressed a mitochondrial-targeted form of the fluorescent reporter Keima (mt-Keima). Keima is a coral-derived protein that exhibits both pH-dependent excitation and resistance to lysosomal proteases. Comparison of a wide range of primary cells and tissues generated from the mt-Keima mouse revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima mice to analyze how mitophagy is altered by conditions including diet, oxygen availability, Huntingtin transgene expression, the absence of macroautophagy (ATG5 or ATG7 expression), an increase in mitochondrial mutational load, the presence of metastatic tumors, and normal aging. The ability to assess mitophagy under a host of varying environmental and genetic perturbations suggests that the mt-Keima mouse should be a valuable resource.

The NAD-dependent deacetylase SIRT2 is required for programmed necrosis

Although initially viewed as unregulated, increasing evidence suggests that cellular necrosis often proceeds through a specific molecular program. In particular, death ligands such as tumour necrosis factor (TNF)-α activate necrosis by stimulating the formation of a complex containing receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3). Relatively little is known regarding how this complex formation is regulated. Here, we show that the NAD-dependent deacetylase SIRT2 binds constitutively to RIP3 and that deletion or knockdown of SIRT2 prevents formation of the RIP1–RIP3 complex in mice. Furthermore, genetic or pharmacological inhibition of SIRT2 blocks cellular necrosis induced by TNF-α. We further demonstrate that RIP1 is a critical target of SIRT2-dependent deacetylation. Using gain- and loss-of-function mutants, we demonstrate that acetylation of RIP1 lysine 530 modulates RIP1–RIP3 complex formation and TNF-α-stimulated necrosis. In the setting of ischaemia-reperfusion injury, RIP1 is deacetylated in a SIRT2-dependent fashion. Furthermore, the hearts of Sirt2−/− mice, or wild-type mice treated with a specific pharmacological inhibitor of SIRT2, show marked protection from ischaemic injury. Taken together, these results implicate SIRT2 as an important regulator of programmed necrosis and indicate that inhibitors of this deacetylase may constitute a novel approach to protect against necrotic injuries, including ischaemic stroke and myocardial infarction.

Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor

Endothelial secretion of von Willebrand factor (VWF) from intracellular organelles known as Weibel-Palade bodies (WPBs) is required for platelet adhesion to the injured vessel wall. Here we demonstrate that WPBs are often found near or within autophagosomes and that endothelial autophagosomes contain abundant VWF protein. Pharmacological inhibitors of autophagy or knockdown of the essential autophagy genes Atg5 or Atg7 inhibits the in vitro secretion of VWF. Furthermore, although mice with endothelial-specific deletion of Atg7 have normal vessel architecture and capillary density, they exhibit impaired epinephrine-stimulated VWF release, reduced levels of high–molecular weight VWF multimers and a corresponding prolongation of bleeding times. Endothelial-specific deletion of Atg5 or pharmacological inhibition of autophagic flux results in a similar in vivo alteration of hemostasis. Thus, autophagy regulates endothelial VWF secretion, and transient pharmacological inhibition of autophagic flux may be a useful strategy to prevent thrombotic events.

Wnt Signaling Regulates Hepatic Metabolism

Nutrient deprivation triggers β-catenin–dependent expression of genes encoding gluconeogenic enzymes. β-Catenin for Gluconeogenesis In the canonical Wnt signaling pathway, binding of Wnt ligands to receptors stabilizes β-catenin, leading to the initiation of gene transcription by β-catenin in the nucleus. The role of the Wnt signaling pathway has been well established in developmental processes; however, growing evidence also implicates this pathway in the regulation of metabolism. Liu et al. found that hepatic deletion of β-catenin in mice decreased gluconeogenesis (the process by which the liver produces glucose) and serum glucose concentrations. More β-catenin interacted with the transcription factor FoxO1 (which is involved in transcriptional responses to insulin signaling) in hepatocytes from starved compared to fed mice, an interaction that promoted the transcriptional activation of genes encoding gluconeogenic enzymes. In obese mice, hepatic deletion of β-catenin improved overall glucose tolerance and reduced hepatic gluconeogenesis. Thus, therapies that target β-catenin function in the liver could be used to treat metabolic diseases such as diabetes. The contribution of the Wnt pathway has been extensively characterized in embryogenesis, differentiation, and stem cell biology but not in mammalian metabolism. Here, using in vivo gain- and loss-of-function models, we demonstrate an important role for Wnt signaling in hepatic metabolism. In particular, β-catenin, the downstream mediator of canonical Wnt signaling, altered serum glucose concentrations and regulated hepatic glucose production. β-Catenin also modulated hepatic insulin signaling. Furthermore, β-catenin interacted with the transcription factor FoxO1 in livers from mice under starved conditions. The interaction of FoxO1 with β-catenin regulated the transcriptional activation of the genes encoding glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), the two rate-limiting enzymes in hepatic gluconeogenesis. Moreover, starvation induced the hepatic expression of mRNAs encoding different Wnt isoforms. In addition, nutrient deprivation appeared to favor the association of β-catenin with FoxO family members, rather than with members of the T cell factor of transcriptional activators. Notably, in a model of diet-induced obesity, hepatic deletion of β-catenin improved overall metabolic homeostasis. These observations implicate Wnt signaling in the modulation of hepatic metabolism and raise the possibility that Wnt signaling may play a similar role in the metabolic regulation of other tissues.

Oncogene-induced senescence results in marked metabolic and bioenergetic alterations

Oncogene-induced senescence (OIS) is characterized by permanent growth arrest and the acquisition of a secretory, pro-inflammatory state. Increasingly, OIS is viewed as an important barrier to tumorgenesis. Surprisingly, relatively little is known about the metabolic changes that accompany and therefore may contribute to OIS. Here, we have performed a metabolomic and bioenergetic analysis of Ras-induced senescence. Profiling approximately 300 different intracellular metabolites reveals that cells that have undergone OIS develop a unique metabolic signature that differs markedly from cells undergoing replicative senescence. A number of lipid metabolites appear uniquely increased in OIS cells, including a marked increase in the level of certain intracellular long chain fatty acids. Functional studies reveal that this alteration in the metabolome reflects substantial changes in overall lipid metabolism. In particular, Ras-induced senescent cells manifest a decline in lipid synthesis and a significant increase in fatty acid oxidation. Increased fatty acid oxidation results in an unexpectedly high rate of basal oxygen consumption in cells that have undergone OIS. Pharmacological or genetic inhibition of carnitine palmitoyltransferase 1, the rate-limiting step in mitochondrial fatty acid oxidation, restores a pre-senescent metabolic rate and, surprisingly, selectively inhibits the secretory, pro-inflammatory state that accompanies OIS. Thus, Ras-induced senescent cells demonstrate profound alterations in their metabolic and bioenergetic profiles, particularly with regards to the levels, synthesis and oxidation of free fatty acids. Furthermore, the inflammatory phenotype that accompanies OIS appears to be related to these underlying changes in cellular metabolism.

Reciprocal regulation of acetyl-CoA carboxylase 1 and senescence in human fibroblasts involves oxidant mediated p38 MAPK activation

We sought to explore the fate of the fatty acid synthesis pathway in human fibroblasts exposed to DNA damaging agents capable of inducing senescence, a state of irreversible growth arrest. Induction of premature senescence by doxorubicin or hydrogen peroxide led to a decrease in protein and mRNA levels of acetyl-CoA carboxylase 1 (ACC1), the enzyme that catalyzes the rate-limiting step in fatty-acid biosynthesis. ACC1 decay accompanied the activation of the DNA damage response (DDR), and resulted in decreased lipid synthesis. A reduction in protein and mRNA levels of ACC1 and in lipid synthesis was also observed in human primary fibroblasts that underwent replicative senescence. We also explored the consequences of inhibiting fatty acid synthesis in proliferating non-transformed cells. Using shRNA technology, we knocked down ACC1 in human fibroblasts. Interestingly, this metabolic perturbation was sufficient to arrest proliferation and trigger the appearance of several markers of the DDR and increase senescence associated β-galactosidase activity. Reactive oxygen species and p38 mitogen activated protein kinase phosphorylation participated in the induction of senescence. Similar results were obtained upon silencing of fatty acid synthase (FAS) expression. Together our results point towards a tight coordination of fatty acid synthesis and cell proliferation in human fibroblasts.We sought to explore the fate of the fatty acid synthesis pathway in human fibroblasts exposed to DNA damaging agents capable of inducing senescence, a state of irreversible growth arrest. Induction of premature senescence by doxorubicin or hydrogen peroxide led to a decrease in protein and mRNA levels of acetyl-CoA carboxylase 1 (ACC1), the enzyme that catalyzes the rate-limiting step in fatty-acid biosynthesis. ACC1 decay accompanied the activation of the DNA damage response (DDR), and resulted in decreased lipid synthesis. A reduction in protein and mRNA levels of ACC1 and in lipid synthesis was also observed in human primary fibroblasts that underwent replicative senescence. We also explored the consequences of inhibiting fatty acid synthesis in proliferating non-transformed cells. Using shRNA technology, we knocked down ACC1 in human fibroblasts. Interestingly, this metabolic perturbation was sufficient to arrest proliferation and trigger the appearance of several markers of the DDR and increase senescence associated β-galactosidase activity. Reactive oxygen species and p38 mitogen activated protein kinase phosphorylation participated in the induction of senescence. Similar results were obtained upon silencing of fatty acid synthase (FAS) expression. Together our results point towards a tight coordination of fatty acid synthesis and cell proliferation in human fibroblasts.

Retraction: The NAD-dependent deacetylase SIRT2 is required for programmed necrosis

This corrects the article DOI: 10.1038/nature11700