As NAD(+) is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38-both NAD(+) consumers-increases NAD(+) bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD(+) precursor with the ability to increase NAD(+) levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD(+) levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.

The biological effects of urolithins remain poorly characterized, despite wide-spread human exposure via the dietary consumption of their metabolic precursors, the ellagitannins, which are found in the pomegranate fruit, as well as in nuts and berries. We identified urolithin A (UA) as a first-in-class natural compound that induces mitophagy both in vitro and in vivo following oral consumption. In C. elegans, UA prevented the accumulation of dysfunctional mitochondria with age and extended lifespan. Likewise, UA prolonged normal activity during aging in C. elegans, including mobility and pharyngeal pumping, while maintaining mitochondrial respiratory capacity. These effects translated to rodents, where UA improved exercise capacity in two different mouse models of age-related decline of muscle function, as well as in young rats. Our findings highlight the health benefits of urolithin A and its potential application in strategies to improve mitochondrial and muscle function.

No abstract

Urolithin A (UA) is a natural compound produced by gut bacteria from ingested ellagitannins (ETs) and ellagic acid (EA), complex polyphenols abundant in foods such as pomegranate, berries, and nuts. UA was discovered 40 years ago, but only recently has its impact on aging and disease been explored. UA enhances cellular health by increasing mitophagy and mitochondrial function and reducing detrimental inflammation. Several preclinical studies show how UA protects against aging and age-related conditions affecting muscle, brain, joints, and other organs. In humans, benefits of UA supplementation in the muscle are supported by recent clinical trials in elderly people. Here, we review the state of the art of UA's biology and its translational potential as a nutritional intervention in humans. Urolithin A: A Natural Gut Microbiome-Derived Metabolite UA belongs to the family of urolithins, characterized by a chemical structure containing an α-benzo-coumarin scaffold (Figure 1). Urolithins are produced in the colon following the microbiome-mediated transformation of the natural polyphenols ETs and EA, which are contained in dietary products, such as pomegranates, strawberries, raspberries, and walnuts [1-3]. (Figure 1 and Box 1).First identified as an EA metabolite in rats in 1980 [4], similar gut microbiome (see Glossary) conversion of ETs to UA was later demonstrated across many species, including flies and mice [1] . A pioneering study also showed the production of UA from ETs by the human gut microbiota [5], making UA the most common urolithin species produced in nature. Two clinical studies then measured UA in human plasma after consumption of pomegranate [6], berries, and nuts [7]. Interestingly, the conversion of dietary precursors to UA does not occur in all individuals. The process is variable [8] and takes place in only approximately 40% of the human elderly population [9]. Being a 'UA producer' requires an appropriate gut microbiome and varies with age, health status, and dietary intake [10].Backed by growing interest in nutritional interventions to address the ever-increasing health problems of an aging population [11,12], several research groups started to study the role and relevance of direct supplementation with UA instead of with UA precursors. This review outlines the most relevant in vivo preclinical studies that show positive impacts of UA on health conditions due to natural aging and on progressive diseases linked to aging. It describes the molecular mechanisms that explain how UA can counter the hallmarks of aging. Finally, this review explores the translational relevance and potential applications of UA as a nutritional intervention in humans.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy, and despite advances in genetic and pharmacological disease-modifying treatments, its management remains a major challenge. Mitochondrial dysfunction contributes to DMD, yet the mechanisms by which this occurs remain elusive. Our data in experimental models and patients with DMD show that reduced expression of genes involved in mitochondrial autophagy, or mitophagy, contributes to mitochondrial dysfunction. Mitophagy markers were reduced in skeletal muscle and in muscle stem cells (MuSCs) of a mouse model of DMD. Administration of the mitophagy activator urolithin A (UA) rescued mitophagy in DMD worms and mice and in primary myoblasts from patients with DMD, increased skeletal muscle respiratory capacity, and improved MuSCs’ regenerative ability, resulting in the recovery of muscle function and increased survival in DMD mouse models. These data indicate that restoration of mitophagy alleviates symptoms of DMD and suggest that UA may have potential therapeutic applications for muscular dystrophies.

The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin ␣9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tubelike structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro. The blood and lymphatic vascular systems are anatomically and histologically closely related but distinct, and do not anastomose except at the level of the main lymphatic ducts. The blood vascular system supplies nutrients and oxygen to the body while the lymphatic vasculature is crucial for immune cell traffic and the uptake of extracellular fluid. The formation of new blood vessels from pre-existing blood vessels (angiogenesis) occurs in physiological (eg, postnatal growth, inflammation, and tissue repair) and pathological (eg, tumor recruitment of blood vessels) settings. Lymphangiogenesis is the formation of new lymphatic vessels from pre-existing lymphatic capillaries, and occurs in similar settings. Blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) are quiescent cells that line the lumina of blood and lymphatic vessels.The lymphatic system has traditionally been overshadowed by the greater emphasis placed on the blood vascular system. This has been due in part to the absence of suitable markers that distinguish lymphatic from blood vascular endothelium. In the past few years, this limitation has been overcome. Lymphatic markers include LYVE-1, a lymphatic endothelial receptor for the extracellular matrix/lymphatic fluid glycosaminoglycan hyaluronan; 1 Prox-1, a homeobox gene product involved in regulating early lymphatic development; 2 podoplanin, a glomerular podocyte membrane mucoprotein; 3 and vascular endothelial growth factor re...

␤TC-tet cells are conditionally immortalized pancreatic ␤fere either with the intrinsic mechanism of growth arrest cells which can confer long-term correction of hyperglycepresent in the ␤TC-tet cells or with their normal glucose mia when transplanted in syngeneic streptozocin diabetic dose-dependent insulin secretory activity. Furthermore, mice. The use of these cells for control of type I diabetes Bcl-2 expressing ␤TC-tet cells retained their capacity to in humans will require their encapsulation and transplansecrete insulin under mild hypoxia. Finally, transplantation tation in non-native sites where relative hypoxia and cytoof these cells under the kidney capsule of streptozocin diakines may threaten their survival. In this study we genetibetic C3H mice corrected hyperglycemia for several cally engineered ␤TC-tet cells with the anti-apoptotic gene months. These results demonstrate that the murine ␤TC-Bcl-2 using new lentiviral vectors and showed that it protet cell line can be genetically modified to improve its resisttected this cell line against apoptosis induced by hypoxia, ance against different stress-induced apoptosis while prestaurosporine and a mixture of cytokines (IL-1␤, IFN-␥ and serving its normal physiological function. These modified TNF-␣). We further demonstrated that Bcl-2 expression cells represent an improved source for cell transplantation permitted growth at higher cell density and with shorter therapy of type I diabetes. doubling time. Expression of Bcl-2, however, did not inter-

Stimulating angiogenesis by gene transfer approaches offers the hope of treating tissue ischemia which is untreatable by currently practiced techniques of vessel grafting and bypass surgery. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) are potent angiogenic molecules, making them ideal candidates for novel gene transfer protocols designed to promote new blood vessel growth. In this study, an ex vivo gene therapy approach utilizing cell encapsulation was employed to deliver VEGF and FGF-2 in a continuous and localized manner. C(2)C(12) myoblasts were genetically engineered to secrete VEGF(121), VEGF(165) and FGF-2. These cell lines were encapsulated in hollow microporous polymer membranes for transplantation in vivo. Therapeutic efficacy was evaluated in a model of acute skin flap ischemia. Capsules were positioned under the distal, ischemic region of the flap. Control flaps showed 50% necrosis at 1 week. Capsules releasing either form of VEGF had no effect on flap survival, but induced a modest increase in distal vascular supply. Delivery of FGF-2 significantly improved flap survival, reducing necrosis to 34.2% (P < 0.001). Flap vascularization was significantly increased by FGF-2 (P < 0.01), with numerous vessels, many of which had a large lumen diameter, growing in the proximity of the implanted capsules. These results demonstrate that FGF-2, delivered from encapsulated cells, is more efficacious than either VEGF(121) or VEGF(165) in treating acute skin ischemia and improving skin flap survival. Furthermore, these data attest to the applicability of cell encapsulation for the delivery of angiogenic factors for the treatment and prevention of tissue ischemia