The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D [hnRNP D]) binds to numerous mRNAs and influences their posttranscriptional fate. Given that many AUF1 target mRNAs encode muscle-specific factors, we investigated the function of AUF1 in skeletal muscle differentiation. In mouse C2C12 myocytes, where AUF1 levels rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RNP immunoprecipitation (RIP) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor MEF2C. By performing mRNA half-life measurements and polysome distribution analysis, we found that AUF1 associated with the 3′ untranslated region (UTR) of Mef2c mRNA and promoted MEF2C translation without affecting Mef2c mRNA stability. In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation. Importantly, lowering AUF1 delayed myogenesis, while ectopically restoring MEF2C expression levels partially rescued the impairment of myogenesis seen after reducing AUF1 levels. We propose that MEF2C is a key effector of the myogenesis program promoted by AUF1.
Calorie restriction decreases skeletal muscle apoptosis, and this phenomenon has been mechanistically linked to its protective action against sarcopenia of aging. Alterations in lipid composition of membranes have been related with the beneficial effects of calorie restriction. However, no study has been designed to date to elucidate if different dietary fat sources with calorie restriction modify apoptotic signaling in skeletal muscle. We show that a 6-month calorie restriction decreased the activity of the plasma membrane neutral sphingomyelinase, although caspase-8/10 activity was not altered, in young adult mice. Lipid hydroperoxides, Bax levels, and cytochrome c and AIF release/accumulation into the cytosol were also decreased, although caspase-9 activity was unchanged. No alterations in caspase-3 and apoptotic index (DNA fragmentation) were observed, but calorie restriction improved structural features of gastrocnemius fibers by increasing cross-sectional area and decreasing circularity of fibers in cross sections. Changing dietary fat with calorie restriction produced substantial alterations of apoptotic signaling. Fish oil augmented the protective effect of calorie restriction decreasing plasma membrane neutral sphingomyelinase, Bax levels, caspase-8/10, and −9 activities, while increasing levels of the antioxidant coenzyme Q at the plasma membrane, and potentiating the increase of cross-sectional area and the decrease of fiber circularity in cross sections. Many of these changes were not found when we used lard. Our data support that dietary fish oil with calorie restriction produces a cellular anti-apoptotic environment in skeletal muscle with a downregulation of components involved in the initial stages of apoptosis engagement, both at the plasma membrane and the mitochondria.
Apoptotic signaling; Calorie restriction; Dietary fat; Sarcopenia; Skeletal muscle
We previously reported that tumor-evoked regulatory B cells (tBregs) play an essential role in breast cancer lung metastasis by inducing TGFβ-dependent conversion of metastasis-promoting FoxP3+ Tregs. Here we show that resveratrol (RSV), a plant-derived polyphenol, at low and non-cytotoxic doses for immune cells can efficiently inhibit lung metastasis in mice. The mechanism of this process is that RSV inactivates Stat3 preventing the generation and function of tBregs, including expression of TGFβ. As a result, it frees antitumor effector immune responses by disabling tBreg-induced conversion of FoxP3+ Tregs. We propose that RSV at low doses may also benefit humans to control cancer escape-promoting tBreg/Tregs without non-specific inactivation of effector immune cells.
tBregs; Tregs; lung metastasis; resveratrol; Stat3; TGFβ
Eating a “Westernized” diet high in fat and sugar leads to weight gain and numerous health problems, including the development of type 2 diabetes mellitus (T2DM). Rodent studies have shown that resveratrol supplementation reduces blood glucose levels, preserves β-cells in islets of Langerhans, and improves insulin action. Although rodent models are helpful for understanding β-cell biology and certain aspects of T2DM pathology, they fail to reproduce the complexity of the human disease as well as that of nonhuman primates. Rhesus monkeys were fed a standard diet (SD), or a high-fat/high-sugar diet in combination with either placebo (HFS) or resveratrol (HFS+Resv) for 24 months, and pancreata were examined before overt dysglycemia occurred. Increased glucose-stimulated insulin secretion and insulin resistance occurred in both HFS and HFS+Resv diets compared with SD. Although islet size was unaffected, there was a significant decrease in β-cells and an increase in α-cells containing glucagon and glucagon-like peptide 1 with HFS diets. Islets from HFS+Resv monkeys were morphologically similar to SD. HFS diets also resulted in decreased expression of essential β-cell transcription factors forkhead box O1 (FOXO1), NKX6–1, NKX2–2, and PDX1, which did not occur with resveratrol supplementation. Similar changes were observed in human islets where the effects of resveratrol were mediated through Sirtuin 1. These findings have implications for the management of humans with insulin resistance, prediabetes, and diabetes.
Obesity is associated with a chronic, low-grade, systemic inflammation that may contribute to the development of insulin resistance and type 2 diabetes. Resveratrol, a natural compound with anti-inflammatory properties, is shown to improve glucose tolerance and insulin sensitivity in obese mice and humans. Here we tested the effect of a 2-year resveratrol administration on pro-inflammatory profile and insulin resistance caused by a high-fat, high-sugar (HFS) diet in white adipose tissue (WAT) from rhesus monkeys. Eighty mg/day of resveratrol for 12-month followed by 480 mg/day for the second year decreased adipocyte size, increased sirtuin 1 expression, decreased NF-κB activation and improved insulin sensitivity in visceral but not subcutaneous WAT from HFS-fed animals. These effects were reproduced in 3T3-L1 adipocytes cultured in media supplemented with serum from monkeys fed HFS +/− resveratrol diets. In conclusion, chronic administration of resveratrol exerts beneficial metabolic and inflammatory adaptations in visceral WAT from diet-induced obese monkeys.
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.
healthspan; inflammation; lifespan; muscle wasting; osteoporosis; sirtuins
We analyzed ultrastructural changes and markers of fission/fusion in hepatocyte mitochondria from mice submitted to 40% calorie restriction (CR) for 6 months versus ad-libitum-fed controls. To study the effects of dietary fat under CR, animals were separated into three CR groups with soybean oil (also in controls), fish oil, and lard. CR induced differential changes in hepatocyte and mitochondrial size, in the volume fraction occupied by mitochondria, and in the number of mitochondria per hepatocyte. The number of cristae per mitochondrion was significantly higher in all CR groups compared with controls. Proteins related to mitochondrial fission (Fis1 and Drp1) increased with CR, but no changes were detected in proteins involved in mitochondrial fusion (Mfn1, Mfn2, and OPA1). Although many of these changes could be attributed to CR regardless of dietary fat, changing membrane lipid composition by different fat sources did modulate the effects of CR on hepatocyte mitochondria.
Calorie restriction; Dietary fat; Hepatocyte; Mice; Mitochondrial ultrastructure; Mitochondrial fission and fusion
Caloric restriction (CR) and down-regulation of the insulin/IGF pathway are the most robust interventions known to increase longevity in lower organisms. However, little is known about the molecular adaptations induced by CR in humans. Here we report that long-term CR in humans inhibits the IGF-1/insulin pathway in skeletal muscle, a key metabolic tissue. We also demonstrate that CR-induced dramatic changes of the skeletal muscle transcriptional profile that resemble those of younger individuals. Finally, in both rats and humans CR evoked similar responses in the transcriptional profiles of skeletal muscle. This common signature consisted of three key pathways typically associated with longevity: IGF-1/insulin signaling, mitochondrial biogenesis and inflammation. Furthermore, our data identifies promising pathways for therapeutic targets to combat age-related diseases and promote health in humans.
human; caloric restriction; skeletal muscle; insulin/IGF-1 signaling
Significance: Calorie restriction (CR) is a known intervention that delays most aging processes. Most of the beneficial effects of CR are mediated by improved maintenance of mitochondrial performance in aged individuals. The control of mitochondrial biogenesis, apoptosis, and protein turnover is required for healthy aging. CR is able to induce molecular mechanisms that preserve oxidative capacity and decrease oxidative damage. Recent Advances and Critical Issues: Published data indicate that peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is activated in old animals under CR conditions compared to ad libitum counterparts, enhancing mitochondrial biogenesis. Molecular regulation of PGC-1α has recently attracted significant research interest. We discuss the master regulators of energy metabolism such as AMP-activated protein kinase and sirtuin 1 among others that have been demonstrated to activate mitochondrial biogenesis through increased PGC-1α activity at transcriptional and post-translational levels. Additionally, we describe the latest findings that explain how CR promotes mitochondrial efficiency and decreases mitochondrial-derived oxidative damage. Future Directions: Understanding the beneficial mitochondrial changes conferred by CR will aid design of therapies for age-related diseases and help slow the aging process. Given the difficulty for humans to adhere to CR, we also explore new molecules that have been proposed during the last years to mimic the CR phenotype and their potential as future therapeutics. Antioxid. Redox Signal. 19, 310–320.
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.
The prevention or delay of the onset of age-related diseases prolongs survival and improves quality of life while reducing the burden on the health care system. Activation of sirtuin 1 (SIRT1), an NAD+ deacetylase, improves metabolism and confers protection against physiological and cognitive disturbances in old age. SRT1720 is a specific SIRT1 activator that has health and lifespan benefits in adult mice fed a high-fat diet. We found extension in lifespan, delayed onset of age-related metabolic diseases, and improved general health in mice fed a standard diet after SRT1720 supplementation. Inhibition of pro-inflammatory gene expression both in the liver and muscle of SRT1720-treated animals was noted. SRT1720 lowered phosphorylation of NF-κB pathway regulators in vitro only when SIRT1 was functionally present. Combined with our previous work, the current study further supports the beneficial effects of SRT1720 on health across the lifespan in mice.
SRT1720; healthspan; standard diet; mice; longevity; SIRT1
Recent findings have shown the intrauterine environment can negatively influence long-term insulin sensitivity in the offspring. In an attempt to be pro-active, we set out to explore maternal voluntary exercise as an intervention in order to improve offspring insulin sensitivity and glucose homeostasis.
Female Sprague Dawley rats were split into sedentary and exercise groups with the exercise cohort having voluntary access to a running wheel in the cage prior to and during mating, pregnancy, and nursing. Female offspring were weaned into sedentary cages. Glucose tolerance tests and hyperinsulinemic–euglycemic clamp were performed in adult offspring to evaluate glucose regulation and insulin sensitivity.
Adult female offspring born to exercised dams had enhanced glucose disposal during glucose tolerance testing (P < 0.05) as well as increased glucose infusion rates (P < 0.01) and whole body glucose turnover rates (P < 0.05) during hyperinsulinemic–euglycemic clamp testing compared to offspring from sedentary dams. Offspring from exercised dams also had decreased insulin levels (P < 0.01) and hepatic glucose production (P < 0.05) during the clamp procedure compared to offspring born to sedentary dams. Offspring from exercised dams had increased glucose uptake in skeletal muscle (P < 0.05) and decreased heart glucose uptake (P < 0.01) compared to offspring from sedentary dams in response to insulin infusion during the clamp procedure.
Exercise during pregnancy enhances offspring insulin sensitivity and improves offspring glucose homeostasis. This can decrease offspring susceptibility to insulin resistant related disease such as type 2 diabetes mellitus. Maternal exercise could be an easy, short–term, non–pharmacological method of preventing disease in future generations.
Pregnancy; running; programming; hyperinsulinemic-euglycemic clamp; rat
For more than 20 years, the observation that impermeable oxidants can stimulate cell growth has not been satisfactorily explained. The discovery of sirtuins provides a logical answer to the puzzle. The NADH-dependent transplasma membrane electron transport system, which is stimulated by growth factors and interventions such as calorie restriction, can transfer electrons to external acceptors and protect against stress-induced apoptosis. We hypothesize that the activation of plasma membrane electron transport contributes to the cytosolic NAD+ pool required for sirtuin to activate transcription factors necessary for cell growth and survival.
Transplasma membrane; electron transport system; RedOx; Sirtuin; NAD; Cell growth.
The SIRT1 deacetylase is one of the best-studied putative mediators of some of the anti-aging effects of calorie restriction (CR), but its role in CR-dependent lifespan extension has not been demonstrated. We previously found that mice lacking both copies of SIRT1 displayed a shorter median lifespan than wild-type mice on an ad libitum diet. Here, we report that median lifespan extension in CR heterozygote SIRT1+/− mice was identical (51%) to that observed in wild-type mice, but SIRT1+/− mice displayed a higher frequency of certain pathologies. Although larger studies in additional genetic backgrounds are needed, these results provide strong initial evidence for the requirement of SIRT1 for the lifespan extension effects of CR, but suggest that its high expression is not required for CR-induced lifespan extension.
anti-aging; caloric restriction; lifespan; SIRT1
Resveratrol, a polyphenolic compound, has been shown to extend lifespan in different organisms. Emerging evidence suggests that the prolongevity effect of resveratrol depends on dietary composition. However, the mechanisms underlying the interaction of resveratrol and dietary nutrients in modulating lifespan remain elusive. Here, we investigated the effect of resveratrol on lifespan of Drosophila melanogaster fed diets differing in the concentrations of sugar, yeast extract, and palmitic acid representing carbohydrate, protein, and fat, respectively. Resveratrol at up to 200 μM in diets did not affect lifespan of wild-type female flies fed a standard, restricted or high sugar–low protein diet, but extended lifespan of females fed a low sugar–high protein diet. Resveratrol at 400 μM extended lifespan of females fed a high-fat diet. Lifespan extension by resveratrol was associated with downregulation of genes in aging-related pathways, including antioxidant peroxiredoxins, insulin-like peptides involved in insulin-like signaling and several downstream genes in Jun-kinase signaling involved in oxidative stress response. Furthermore, resveratrol increased lifespan of superoxide dismutase 1 (sod1) knockdown mutant females fed a standard or high-fat diet. No lifespan extension by resveratrol was observed in wild-type and sod1 knockdown males under the culture conditions in this study. Our results suggest that the gender-specific prolongevity effect of resveratrol is influenced by dietary composition and resveratrol promotes the survival of flies by modulating genetic pathways that can reduce cellular damage. This study reveals the context-dependent effect of resveratrol on lifespan and suggests the importance of dietary nutrients in implementation of effective aging interventions using dietary supplements.
Resveratrol; Lifespan; Dietary composition; Aging intervention; Superoxide dismutase 1; Oxidative stress
Metabolic diseases are characterized by the failure of regulatory genes or proteins to effectively orchestrate specific pathways involved in the control of many biological processes. In addition to the classical regulators, recent discoveries have shown the remarkable role of small noncoding RNAs (microRNAs [miRNAs]) in the posttranscriptional regulation of gene expression. In this regard, we have recently demonstrated that miR-33a and miR33b, intronic miRNAs located within the sterol regulatory element-binding protein (SREBP) genes, regulate lipid metabolism in concert with their host genes. Here, we show that miR-33b also cooperates with SREBP1 in regulating glucose metabolism by targeting phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), key regulatory enzymes of hepatic gluconeogenesis. Overexpression of miR-33b in human hepatic cells inhibits PCK1 and G6PC expression, leading to a significant reduction of glucose production. Importantly, hepatic SREBP1c/miR-33b levels correlate inversely with the expression of PCK1 and G6PC upon glucose infusion in rhesus monkeys. Taken together, these results suggest that miR-33b works in concert with its host gene to ensure a fine-tuned regulation of lipid and glucose homeostasis, highlighting the clinical potential of miR-33a/b as novel therapeutic targets for a range of metabolic diseases.
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.
Aging is associated with a progressive loss of muscle mass and strength and a decline in neurophysiological functions. Age-related neuromuscular junction (NMJ) plays a key role in musculoskeletal impairment that occurs with aging. However, whether changes in the NMJ precede or follow the decline of muscle mass and strength remains unresolved. Many factors such as mitochondrial dysfunction, oxidative stress, inflammation, changes in the innervation of muscle fibers, and mechanical properties of the motor units probably perform an important role in NMJ degeneration and muscle mass and strength decline in late life. This review addresses the primary events that might lead to NMJ dysfunction with aging, including studies on biomarkers, signaling pathways, and animal models. Interventions such as caloric restriction and exercise may positively affect the NMJ through this mechanism and attenuate the age-related progressive impairment in motor function.
aging; denervation; motor unit; neuromuscular junction; sarcopenia
The National Institute on Aging Interventions Testing Program (ITP) was established to evaluate agents that are hypothesized to increase life span and/or health span in genetically heterogeneous mice. Each compound is tested in parallel at three test sites. It is the goal of the ITP to publish all results, negative or positive. We report here on the results of lifelong treatment of mice, beginning at 4 months of age, with each of five agents, that is, green tea extract (GTE), curcumin, oxaloacetic acid, medium-chain triglyceride oil, and resveratrol, on the life span of genetically heterogeneous mice. Each agent was administered beginning at 4 months of age. None of these five agents had a statistically significant effect on life span of male or female mice, by log-rank test, at the concentrations tested, although a secondary analysis suggested that GTE might diminish the risk of midlife deaths in females only.
Longevity; aging; mice; diet; Interventions
Calorie restriction (CR) is a reliable anti-aging intervention that attenuates the onset of a number of age-related diseases, reduces oxidative damage, and maintains function during aging. In the current study, we assessed the effects of CR and other feeding regimens on wound healing in 7-month-old Fischer-344 rats from a larger cohort of rats that had been fed either ad libitum (AL) or 40% calorie restricted based on AL consumption. Rats were assigned to one of three diet groups that received three skin punch wounds along the dorsal interscapular region (12-mm diameter near the front limbs) of the back as follows: (1) CR (n = 8) were wounded and maintained on CR until they healed, (2) AL (n = 5) were wounded and maintained on AL until wound closure was completed, and (3) CR rats were refed (RF, n = 9) AL for 48 h prior to wounding and maintained on AL until they healed. We observed that young rats on CR healed more slowly while CR rats refed for 48 h prior to wounding healed as fast as AL fed rats, similar to a study reported in aged CR and RF mice (Reed et al. 1996). Our data suggest that CR subjects, regardless of age, fail to heal well and that provision of increased nutrition to CR subjects prior to wounding enhances the healing process.
Aging; Calorie restriction; Refeeding; Wound healing
hsa-miR-33a and hsa-miR-33b, intronic microRNAs (miRNAs) located within the sterol regulatory element-binding protein 2 and 1 genes (Srebp-2 and -1), respectively, have recently been shown to regulate lipid homeostasis in concert with their host genes. Although the functional role of miR-33a and -b has been highly investigated, the role of their passenger strands, miR-33a* and -b*, remains unclear. Here, we demonstrate that miR-33a* and -b* accumulate to steady-state levels in human, mouse, and nonhuman primate tissues and share a similar lipid metabolism target gene network as their sister strands. Analogous to miR-33, miR-33* represses key enzymes involved in cholesterol efflux (ABCA1 and NPC1), fatty acid metabolism (CROT and CPT1a), and insulin signaling (IRS2). Moreover, miR-33* also targets key transcriptional regulators of lipid metabolism, including SRC1, SRC3, NFYC, and RIP140. Importantly, inhibition of either miR-33 or miR-33* rescues target gene expression in cells overexpressing pre-miR-33. Consistent with this, overexpression of miR-33* reduces fatty acid oxidation in human hepatic cells. Altogether, these data support a regulatory role for the miRNA* species and suggest that miR-33 regulates lipid metabolism through both arms of the miR-33/miR-33* duplex.
Life extension by calorie restriction (CR) has been widely reported in a variety of species and remains on the forefront of anti-aging intervention studies. We report healthspan and survival effects of CR from a 23-year study in rhesus macaques conducted at the National Institute on Aging (NIA). CR initiated at older ages did not increase survival relative to Controls; however, CR monkeys demonstrated an improved metabolic profile and may have less oxidative stress as indicated by plasma isoprostane levels. When initiated in young monkeys, there was a trend (p=0.06) for a delay in age-associated disease onset in CR monkeys; but again, survival curves were not improved, in contrast to another study reported in the literature. This suggests that the effects of CR in a long-lived animal are complex and likely dependent on a variety of environmental, nutritional, and genetic factors.
We have previously reported that a modified Stone T-maze (STM), using escape from water as motivation, was effective in evaluating learning and memory ability in young C57/BL6 mice. Here we report on the effectiveness and sensitivity of the STM in the assessment of age-related learning and memory deficits in mice using either escape from foot shock or water as the motivational manipulations. C57BL/6Nia mice 7-, 12-, 20- and 24-mo old received 15 massed trials in the escape from foot shock motivated STM while C57BL/6Nia mice 5-, 12-, and 25-mo old were tested in the escape from water STM. Analysis of errors, the main performance variable, revealed similar results in both versions of the task with younger mice making fewer errors. Notably mice of all ages in the water-motivated version moved quickly through the maze, while all ages of mice in the shock-motivated version tended to wait for shock to be initiated to move forward. Overall, both versions of the STM appear to be sensitive to age-related changes in learning and memory and provide an alternative to other testing paradigms such as the Morris water waze which are susceptible to performance confounds which can lead to uninterpretable results.
Aging; mice; learning; memory; maze