Ana P. Gomes

SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function

Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD(+) levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.

Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging

Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD(+) and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD(+) levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.

Metformin improves healthspan and lifespan in mice

Metformin is a drug commonly prescribed to treat patients with type 2 diabetes. Here we show that long-term treatment with metformin (0.1% w/w in diet) starting at middle age extends healthspan and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with metformin mimics some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced LDL and cholesterol levels without a decrease in caloric intake. At a molecular level, metformin increases AMP-activated protein kinase activity and increases antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation. Our results indicate that these actions may contribute to the beneficial effects of metformin on healthspan and lifespan. These findings are in agreement with current epidemiological data and raise the possibility of metformin-based interventions to promote healthy aging.

SRT1720 improves survival and healthspan of obese mice

Sirt1 is an NAD+-dependent deacetylase that extends lifespan in lower organisms and improves metabolism and delays the onset of age-related diseases in mammals. Here we show that SRT1720, a synthetic compound that was identified for its ability to activate Sirt1 in vitro, extends both mean and maximum lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, enhanced locomotor activity and normalization of gene expression profiles and markers of inflammation and apoptosis, all in the absence of any observable toxicity. Using a conditional SIRT1 knockout mouse and specific gene knockdowns we show SRT1720 affects mitochondrial respiration in a Sirt1- and PGC-1α-dependent manner. These findings indicate that SRT1720 has long-term benefits and demonstrate for the first time the feasibility of designing novel molecules that are safe and effective in promoting longevity and preventing multiple age-related diseases in mammals.

Flavonoid Apigenin Is an Inhibitor of the NAD+ase CD38: Implications for Cellular NAD+ Metabolism, Protein Acetylation, and Treatment of Metabolic Syndrome

Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD+ metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD+ levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD+ase in mammals. Moreover, CD38 knockout mice have higher NAD+ levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD+ levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD+ levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD+ levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD+-dependent pathways.

SRT2104 extends survival of male mice on a standard diet and preserves bone and muscle mass

Increased expression of SIRT1 extends the lifespan of lower organisms and delays the onset of age‐related diseases in mammals. Here, we show that SRT2104, a synthetic small molecule activator of SIRT1, extends both mean and maximal lifespan of mice fed a standard diet. This is accompanied by improvements in health, including enhanced motor coordination, performance, bone mineral density, and insulin sensitivity associated with higher mitochondrial content and decreased inflammation. Short‐term SRT2104 treatment preserves bone and muscle mass in an experimental model of atrophy. These results demonstrate it is possible to design a small molecule that can slow aging and delay multiple age‐related diseases in mammals, supporting the therapeutic potential of SIRT1 activators in humans.

Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis

Berberine (BBR) has recently been shown to improve insulin sensitivity in rodent models of insulin resistance. Although this effect was explained partly through an observed activation of AMP-activated protein kinase (AMPK), the upstream and downstream mediators of this phenotype were not explored. Here, we show that BBR supplementation reverts mitochondrial dysfunction induced by High Fat Diet (HFD) and hyperglycemia in skeletal muscle, in part due to an increase in mitochondrial biogenesis. Furthermore, we observe that the prevention of mitochondrial dysfunction by BBR, the increase in mitochondrial biogenesis, as well as BBR-induced AMPK activation, are blocked in cells in which SIRT1 has been knocked-down. Taken together, these data reveal an important role for SIRT1 and mitochondrial biogenesis in the preventive effects of BBR on diet-induced insulin resistance.

The Sirt1 activator SRT3025 provides atheroprotection in Apoe−/− mice by reducing hepatic Pcsk9 secretion and enhancing Ldlr expression

Aims The deacetylase sirtuin 1 (Sirt1) exerts beneficial effects on lipid metabolism, but its roles in plasma LDL-cholesterol regulation and atherosclerosis are controversial. Thus, we applied the pharmacological Sirt1 activator SRT3025 in a mouse model of atherosclerosis and in hepatocyte culture. Methods and results Apolipoprotein E-deficient (Apoe−/−) mice were fed a high-cholesterol diet (1.25% w/w) supplemented with SRT3025 (3.18 g kg−1 diet) for 12 weeks. In vitro, the drug activated wild-type Sirt1 protein, but not the activation-resistant Sirt1 mutant; in vivo, it increased deacetylation of hepatic p65 and skeletal muscle Foxo1. SRT3025 treatment decreased plasma levels of LDL-cholesterol and total cholesterol and reduced atherosclerosis. Drug treatment did not change mRNA expression of hepatic LDL receptor (Ldlr) and proprotein convertase subtilisin/kexin type 9 (Pcsk9), but increased their protein expression indicating post-translational effects. Consistent with hepatocyte Ldlr and Pcsk9 accumulation, we found reduced plasma levels of Pcsk9 after pharmacological Sirt1 activation. In vitro administration of SRT3025 to cultured AML12 hepatocytes attenuated Pcsk9 secretion and its binding to Ldlr, thereby reducing Pcsk9-mediated Ldlr degradation and increasing Ldlr expression and LDL uptake. Co-administration of exogenous Pcsk9 with SRT3025 blunted these effects. Sirt1 activation with SRT3025 in Ldlr−/− mice reduced neither plasma Pcsk9, nor LDL-cholesterol levels, nor atherosclerosis. Conclusion We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice. Pharmacological activation of Sirt1 appears promising to be tested in patients for its effects on plasma Pcsk9, LDL-cholesterol, and atherosclerosis.

Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers

Significance Serine supports a number of anabolic processes, including protein, lipid, and nucleic acid synthesis. Cells can either import serine or synthesize it de novo. Recently, overexpression of 3-phosphoglycerate dehydrogenase (PHGDH), the gene encoding the first committed step of serine synthesis, via focal amplification and other mechanisms, has been identified in human cancers. Cancer cell lines that overexpress PHGDH are uniquely sensitive to PHGDH knockdown whereas lines that express little PHGDH are insensitive, suggesting that PHGDH may be a clinically interesting target. Here, we report the discovery of a specific small molecule inhibitor of PHGDH, which enables preclinical evaluation of PHGDH as a target in cancer and provides a tool to study the biology of de novo serine synthesis. Cancer cells reprogram their metabolism to promote growth and proliferation. The genetic evidence pointing to the importance of the amino acid serine in tumorigenesis is striking. The gene encoding the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first committed step of serine biosynthesis, is overexpressed in tumors and cancer cell lines via focal amplification and nuclear factor erythroid-2-related factor 2 (NRF2)-mediated up-regulation. PHGDH-overexpressing cells are exquisitely sensitive to genetic ablation of the pathway. Here, we report the discovery of a selective small molecule inhibitor of PHGDH, CBR-5884, identified by screening a library of 800,000 drug-like compounds. CBR-5884 inhibited de novo serine synthesis in cancer cells and was selectively toxic to cancer cell lines with high serine biosynthetic activity. Biochemical characterization of the inhibitor revealed that it was a noncompetitive inhibitor that showed a time-dependent onset of inhibition and disrupted the oligomerization state of PHGDH. The identification of a small molecule inhibitor of PHGDH not only enables thorough preclinical evaluation of PHGDH as a target in cancers, but also provides a tool with which to study serine metabolism.

Berberine reverts hepatic mitochondrial dysfunction in high-fat fed rats: A possible role for SirT3 activation

Berberine is an isoquinoline alkaloid with anti-diabetic properties. Despite the central role of liver and thus hepatic mitochondria in whole-body metabolism, berberine effects on hepatic mitochondrial function in an obesity model are still unknown. Here, we demonstrate that berberine treatment recovers mitochondrial efficiency when altered by a high-fat feeding. Mitochondria isolated from the liver of high-fat fed rats exhibited decreased capacity to accumulate calcium and impaired oxidative phosphorylation (OXPHOS) capacity, as shown by impaired mitochondrial membrane potential, oxygen consumption and cellular ATP levels. Interestingly, the recovery of mitochondrial function by berberine was associated with an increased activity of the mitochondrial sirtuin 3 (SirT3). In conclusion, berberine potent protective effects against metabolic syndrome may rely on increasing mitochondrial SirT3 activity, normalizing mitochondrial function and preventing a state of energetic deficit caused by impaired OXPHOS.