A Multi-Ingredient Supplement Protects against Obesity and Infertility in Western Diet-Fed Mice.

The Western diet (WD) predisposes to bodyweight gain and obesity and is linked to mitochondrial dysfunction, oxidative damage, inflammation, and multisystem disease, even affecting the reproductive organs, fertility, and pregnancy outcomes. In this study, we investigated the effects of multi-ingredient supplementation (MIS) with antioxidants, phytonutrients, and vitamins ('Fertility Enhancer'; FE) on white adipose tissue (WAT) expansion, nonalcoholic fatty liver disease (NAFLD), and infertility in WD-fed C57BL/6J mice. Five-month-old male (M) and female (F) mice were fed a low-fat diet (LF) or a high fat/sucrose WD (HF) for six weeks, followed by six weeks of LF (3.64 kcal/g), HF (4.56 kcal/g), or HF combined with FE (4.50 kcal/g). A sub-set of animals were sacrificed at 12 weeks, while the remainder were harem-mated in a 1:2 male-to-female ratio, and singly housed during the gestational period. Two-way, factorial ANOVA analysis revealed a main effect of diet on bodyweight (BW), total body fat, % body fat, white adipose tissue mass, and liver lipid content (all p < 0.001), driven by the anti-obesogenic effects of the 'Fertility Enhancer'. Similarly, a main effect of diet was found on PGC1-α mRNA levels (p < 0.05) and mitochondrial protein content (p < 0.001) in perigonadal WAT, with PGC1-α induction and higher complex II and complex III expression in FE vs. HF animals. Copulatory plug counts were higher in FE vs. HE couples (30% vs. 6%), resulting in more litters (4 vs. 0) and higher copulatory success (67% vs. 0%). Although the trends of all histology outcomes were suggestive of a benefit from the FE diet, only the number of atretic follicles and testicular mass were significant. Ovarian IL-1ß mRNA induction was significantly attenuated in the FE group (p < 0.05 vs. HF) with CASP1 attenuation trending lower (p = 0.09 vs. HF), which is indicative of anti-inflammatory benefits of the 'Fertility Enhancer.' We conclude that supplementation with specific phytonutrients, antioxidants, and vitamins may have utility as an adjunctive therapy for weight management, fatty liver disease, and infertility in overweight and obese couples.

Nutritional co-therapy with 1,3-butanediol and multi-ingredient antioxidants enhances autophagic clearance in Pompe disease.

Alglucosidase alpha is an orphan drug approved for enzyme replacement therapy (ERT) in Pompe disease (PD); however, its efficacy is limited in skeletal muscle because of a partial blockage of autophagic flux that hinders intracellular trafficking and enzyme delivery. Adjunctive therapies that enhance autophagic flux and protect mitochondrial integrity may alleviate autophagic blockage and oxidative stress and thereby improve ERT efficacy in PD. In this study, we compared the benefits of ERT combined with a ketogenic diet (ERT-KETO), daily administration of an oral ketone precursor (1,3-butanediol; ERT-BD), a multi-ingredient antioxidant diet (ERT-MITO; CoQ10, α-lipoic acid, vitamin E, beetroot extract, HMB, creatine, and citrulline), or co-therapy with the ketone precursor and multi-ingredient antioxidants (ERT-BD-MITO) on skeletal muscle pathology in GAA-KO mice. We found that two months of 1,3-BD administration raised circulatory ketone levels to ≥1.2 mM, attenuated autophagic buildup in type 2 muscle fibers, and preserved muscle strength and function in ERT-treated GAA-KO mice. Collectively, ERT-BD was more effective vs. standard ERT and ERT-KETO in terms of autophagic clearance, dampening of oxidative stress, and muscle maintenance. However, the addition of multi-ingredient antioxidants (ERT-BD-MITO) provided the most consistent benefits across all outcome measures and normalized mitochondrial protein expression in GAA-KO mice. We therefore conclude that nutritional co-therapy with 1,3-butanediol and multi-ingredient antioxidants may provide an alternative to ketogenic diets for inducing ketosis and enhancing autophagic flux in PD patients.

A Novel Multi-Ingredient Supplement Activates a Browning Program in White Adipose Tissue and Mitigates Weight Gain in High-Fat Diet-Fed Mice.

We investigated the effects of a novel multi-ingredient supplement comprised of polyphenol antioxidants and compounds known to facilitate mitochondrial function and metabolic enhancement (ME) in a mouse model of obesity. In this study, 6-week-old male C57/BL6J mice were placed on a high-fat diet (HFD; ~60% fat) for 6 weeks, with subsequent allocation into experimentalgroups for 4 weeks: HFD control, HFD + ME10 (10 components), HFD + ME7 (7 components), HFD + ME10 + EX, HFD + EX (where '+EX' animals exercised 3 days/week), and chow-fed control. After the intervention, HFD control animals had significantly greater body weight and fat mass. Despite the continuation of HFD, animals supplemented with multi-ingredient ME or who performed exercise training showed an attenuation of fat mass and preservation of lean body mass, which was further enhanced when combined (ME+EX). ME supplementation stimulated the upregulation of white and brown adipose tissue mRNA transcripts associated with mitochondrial biogenesis, browning, fatty acid transport, and fat metabolism. In WAT depots, this was mirrored by mitochodrial oxidative phosphorylation (OXPHOS) protein expression, and increased in vivo fat oxidation measured via CLAMS. ME supplementation also decreased systemic and local inflammation markers. Herein, we demonstrated that novel multi-ingredient nutritional supplements induced significant fat loss independent of physical activity while preserving muscle mass in obese mice. Mechanistically, these MEs appear to act by inducing a browning program in white adipose tissue and decreasing other pathophysiological impairments associated with obesity, including mitochondrial respiration alterations induced by HFD.

A Five-Ingredient Nutritional Supplement and Home-Based Resistance Exercise Improve Lean Mass and Strength in Free-Living Elderly.

Old age is associated with lower physical activity levels, suboptimal protein intake, and desensitization to anabolic stimuli, predisposing for age-related muscle loss (sarcopenia). Although resistance exercise (RE) and protein supplementation partially protect against sarcopenia under controlled conditions, the efficacy of home-based, unsupervised RE (HBRE) and multi-ingredient supplementation (MIS) is largely unknown. In this randomized, placebo-controlled and double-blind trial, we examined the effects of HBRE/MIS on muscle mass, strength, and function in free-living, older men. Thirty-two sedentary men underwent twelve weeks of home-based resistance band training (3 d/week), in combination with daily intake of a novel five-nutrient supplement ('Muscle5'; M5, n = 16, 77.4 ± 2.8 y) containing whey, micellar casein, creatine, vitamin D, and omega-3 fatty acids, or an isocaloric/isonitrogenous placebo (PLA; n = 16, 74.4 ± 1.3 y), containing collagen and sunflower oil. Appendicular and total lean mass (ASM; +3%, TLM; +2%), lean mass to fat ratios (ASM/% body fat; +6%, TLM/% body fat; +5%), maximal strength (grip; +8%, leg press; +17%), and function (5-Times Sit-to-Stand time; -9%) were significantly improved in the M5 group following HBRE/MIS therapy (pre vs. post tests; p < 0.05). Fast-twitch muscle fiber cross-sectional areas of the quadriceps muscle were also significantly increased in the M5 group post intervention (Type IIa; +30.9%, Type IIx, +28.5%, p < 0.05). Sub-group analysis indicated even greater gains in total lean mass in sarcopenic individuals following HBRE/MIS therapy (TLM; +1.65 kg/+3.4%, p < 0.05). We conclude that the Muscle5 supplement is a safe, well-tolerated, and effective complement to low-intensity, home-based resistance exercise and improves lean mass, strength, and overall muscle quality in old age.

Correction: Lifelong aerobic exercise protects against inflammaging and cancer.

[This corrects the article DOI: 10.1371/journal.pone.0210863.].

Bone marrow-derived mitochondrial DNA has limited capacity for inter-tissue transfer in vivo.

Studies have shown that mitochondrial DNA (mtDNA) can be exchanged between tissues; however, the mechanism(s) behind this phenomenon remain unclear. Exosomes and other extracellular vesicles (EVs) including microvesicles (MV) have been shown to contain mtDNA. EVs can be derived from a number of tissues; however, the source and relative proportion of EVs containing mtDNA remains unknown. We sampled whole blood and the EV fractions (exosome-enriched, MV-enriched, and apoptotic body-enriched) as well as several tissues (epithelial-cheek and urine sediment), connective (fibroblasts), and skeletal muscle in two subjects who received allogenic bone marrow transplants. Next generation sequencing of the mtDNA confirmed that all EV fractions contained mtDNA and most was derived from the donor, confirming that most of the EV fractions in the serum are bone marrow/blood cell-derived. Even after exposure to the donor mtDNA in EV fractions (and potentially free in the plasma) for years, there was little to no transfer of the donor mtDNA to the host mtDNA fraction in epithelial, connective, or skeletal muscle tissues. These data call into question the potential therapeutic use of bone marrow transplant or EV-based delivery systems for mtDNA-based disorders and establish bone marrow as the primary source of most of the mtDNA enriched EVs in serum.

Lifelong aerobic exercise protects against inflammaging and cancer.

Biological aging is associated with progressive damage accumulation, loss of organ reserves, and systemic inflammation ('inflammaging'), which predispose for a wide spectrum of chronic diseases, including several types of cancer. In contrast, aerobic exercise training (AET) reduces inflammation, lowers all-cause mortality, and enhances both health and lifespan. In this study, we examined the benefits of early-onset, lifelong AET on predictors of health, inflammation, and cancer incidence in a naturally aging mouse model (C57BL/J6). Lifelong, voluntary wheel-running (O-AET; 26-month-old) prevented age-related declines in aerobic fitness and motor coordination vs. age-matched, sedentary controls (O-SED). AET also provided partial protection against sarcopenia, dynapenia, testicular atrophy, and overall organ pathology, hence augmenting the 'physiologic reserve' of lifelong runners. Systemic inflammation, as evidenced by a chronic elevation in 17 of 18 pro- and anti-inflammatory cytokines and chemokines (P < 0.05 O-SED vs. 2-month-old Y-CON), was potently mitigated by lifelong AET (P < 0.05 O-AET vs. O-SED), including master regulators of the cytokine cascade and cancer progression (IL-1ß, TNF-α, and IL-6). In addition, circulating SPARC, previously known to be upregulated in metabolic disease, was elevated in old, sedentary mice, but was normalized to young control levels in lifelong runners. Remarkably, malignant tumours were also completely absent in the O-AET group, whereas they were present in the brain (pituitary), liver, spleen, and intestines of sedentary mice. Collectively, our results indicate that early-onset, lifelong running dampens inflammaging, protects against multiple cancer types, and extends healthspan of naturally-aged mice.

Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice.

BACKGROUND: Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. RESULTS: Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increases lifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We report evidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 is canonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53 translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response to endurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNA mutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigate premature mortality. CONCLUSIONS: Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome and present mitochondrially targeted p53 as a novel therapeutic modality for diseases of mitochondrial etiology.

Elevated mitochondrial oxidative stress impairs metabolic adaptations to exercise in skeletal muscle.

Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in Sod2 (+/-) mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training Sod2 (+/-) mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised Sod2 (+/-) mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.

Supplementation with α-lipoic acid, CoQ10, and vitamin E augments running performance and mitochondrial function in female mice.

Antioxidant supplements are widely consumed by the general public; however, their effects of on exercise performance are controversial. The aim of this study was to examine the effects of an antioxidant cocktail (α-lipoic acid, vitamin E and coenzyme Q10) on exercise performance, muscle function and training adaptations in mice. C57Bl/J6 mice were placed on antioxidant supplement or placebo-control diets (n = 36/group) and divided into trained (8 wks treadmill running) (n = 12/group) and untrained groups (n = 24/group). Antioxidant supplementation had no effect on the running performance of trained mice nor did it affect training adaptations; however, untrained female mice that received antioxidants performed significantly better than placebo-control mice (p ≤ 0.05). Furthermore, antioxidant-supplemented females (untrained) showed elevated respiratory capacity in freshly excised muscle fibers (quadriceps femoris) (p ≤ 0.05), reduced oxidative damage to muscle proteins (p ≤ 0.05), and increased expression of mitochondrial proteins (p ≤ 0.05) compared to placebo-controls. These changes were attributed to increased expression of proliferator-activated receptor gamma coactivator 1α (PGC-1α) (p ≤ 0.05) via activation of AMP-activated protein kinase (AMPK) (p ≤ 0.05) by antioxidant supplementation. Overall, these results indicate that this antioxidant supplement exerts gender specific effects; augmenting performance and mitochondrial function in untrained females, but does not attenuate training adaptations.

Aerobic training as an adjunctive therapy to enzyme replacement in Pompe disease.

BACKGROUND: Aerobic exercise may be used in conjunction with enzyme replacement therapy (ERT) to attenuate cardiovascular deconditioning, skeletal muscle wasting, and loss of motor function in Pompe disease (glycogen storage disease type II; GSDII), but the effects on lysosomal glycogen content and macroautophagy have not been defined to date. PURPOSE: The main objectives of this study were to determine if acute aerobic exercise enhances 24-h uptake of recombinant human enzyme (rhGAA; Myozyme® [aim 1]) and if endurance training improves disease pathology when combined with ERT [aim 2] in Pompe mice. METHODS: For the first aim in our study, Pompe mutant mice (6(neo)/6(neo)) were grouped into ERT (Myozyme® injection only [40 mg/kg]) and ERT+EX (Myozyme® injection followed by 90 min treadmill exercise) cohorts, and enzyme uptake was assessed in the heart and quadriceps 24h post injection. For the second aim of our study, mutant mice were randomized into control, endurance-trained, enzyme-treated, or combination therapy groups. Exercised animals underwent 14 weeks of progressive treadmill training with or without biweekly Myozyme® injections (40 mg/kg) and tissues were harvested 1 week post last treatment. RESULTS: Myozyme® uptake (GAA activity) was not improved in ERT+EX over ERT alone at 24-h post injection. Endurance exercise training, with or without ERT, improved aerobic capacity and normalized grip strength, motor function, and lean mass (P<0.05), but did not reduce glycogen content or normalize macroautophagy beyond traditional enzyme replacement therapy. CONCLUSIONS: Endurance training is beneficial as an adjunctive therapy to ERT in Pompe disease, although it works by mechanisms independent of a reduction in glycogen content.

Maternal antioxidants prevent ß-cell apoptosis and promote formation of dual hormone-expressing endocrine cells in male offspring following fetal and neonatal nicotine exposure.

BACKGROUND: Fetal and neonatal nicotine exposure causes ß-cell oxidative stress and apoptosis in neonates, leading to adult-onset dysglycemia. The aim of the present study was to determine whether an antioxidant intervention could prevent nicotine-induced ß-cell loss. METHODS: Nulliparous female Wistar rats received daily subcutaneous injections of either saline or nicotine bitartrate (1.0 mg/kg per day) for 2 weeks prior to mating until weaning. Nicotine-exposed dams received either normal chow or diet containing antioxidants (1000 IU/kg vitamin E, 0.25% w/w coenzyme Q10, and 0.1% w/w α-lipoic acid) during mating, pregnancy, and lactation; saline-exposed dams received normal chow. Pancreatic tissue was collected from male offspring at 3 weeks of age to measure ß-cell fraction, apoptosis, proliferation, and the presence of cells coexpressing insulin and glucagon. RESULTS: The birth weight of offspring born to nicotine-exposed dams was significantly reduced in those receiving dietary antioxidants compared with those fed normal chow. Most interestingly, the antioxidant intervention to nicotine-exposed dams prevented the ß-cell loss and apoptosis observed in nicotine-exposed male offspring whose mothers did not receive antioxidants. Male pups born to nicotine-treated mothers receiving antioxidants also had a tendency for increased ß-cell proliferation and a significant increase in islets containing insulin/glucagon bihormonal cells compared with the other two treatment groups. CONCLUSION: The present study demonstrates that exposure to maternal antioxidants protects developing ß-cells from the damaging effects of nicotine, thus preserving ß-cell mass.

Effects of creatine and exercise on skeletal muscle of FRG1-transgenic mice.

BACKGROUND: The FRG1-transgenic mouse displays muscle dysfunction and atrophy reminiscent of fascioscapulohumeral muscular dystrophy (FSHD) and could provide a model to determine potential therapeutic interventions. METHODS: To determine if FRG1 mice benefit from treatments that improve muscle mass and function, mice were treated with creatine alone (Cr) or in combination with treadmill exercise (CrEX). RESULTS: The CrEx treatment increased quadriceps weight, mitochondrial content (cytochome c oxidase (COX) activity, COX subunit one and four protein), and induced greater improvements in grip strength and rotarod fall speed. While Cr increased COX subunits one and four protein, no effect on muscle mass or performance was found. Since Cr resulted in no functional improvements, the benefits of CrEx may be mediated by exercise; however, the potential synergistic action of the combined treatment cannot be excluded. CONCLUSION: Treatment with CrEx attenuates atrophy and muscle dysfunction associated with FRG1 overexpression. These data suggest exercise and creatine supplementation may benefit individuals with FSHD.

Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice.

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities.

Exercise increases mitochondrial PGC-1alpha content and promotes nuclear-mitochondrial cross-talk to coordinate mitochondrial biogenesis.

Endurance exercise is known to induce metabolic adaptations in skeletal muscle via activation of the transcriptional co-activator peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α). PGC-1α regulates mitochondrial biogenesis via regulating transcription of nuclear-encoded mitochondrial genes. Recently, PGC-1α has been shown to reside in mitochondria; however, the physiological consequences of mitochondrial PGC-1α remain unknown. We sought to delineate if an acute bout of endurance exercise can mediate an increase in mitochondrial PGC-1α content where it may co-activate mitochondrial transcription factor A to promote mtDNA transcription. C57Bl/6J mice (n = 12/group; ♀ = ♂) were randomly assigned to sedentary (SED), forced-endurance (END) exercise (15 m/min for 90 min), or forced endurance +3 h of recovery (END+3h) group. The END group was sacrificed immediately after exercise, whereas the SED and END+3h groups were euthanized 3 h after acute exercise. Acute exercise coordinately increased the mRNA expression of nuclear and mitochondrial DNA-encoded mitochondrial transcripts. Nuclear and mitochondrial abundance of PGC-1α in END and END+3h groups was significantly higher versus SED mice. In mitochondria, PGC-1α is in a complex with mitochondrial transcription factor A at mtDNA D-loop, and this interaction was positively modulated by exercise, similar to the increased binding of PGC-1α at the NRF-1 promoter. We conclude that in response to acute altered energy demands, PGC-1α re-localizes into nuclear and mitochondrial compartments where it functions as a transcriptional co-activator for both nuclear and mitochondrial DNA transcription factors. These results suggest that PGC-1α may dynamically facilitate nuclear-mitochondrial DNA cross-talk to promote net mitochondrial biogenesis.

Effect of combining rosiglitazone with either metformin or insulin on ß-cell mass and function in an animal model of Type 2 diabetes characterized by reduced ß-cell mass at birth.

BACKGROUND: Interventions that preserve or increase ß-cell mass may also prevent Type 2 diabetes. Rosiglitazone alone, as well as in combination with metformin, prevents diabetes in people with high, yet non-diabetic glucose levels. These effects may be mediated through changes in ß-cell mass. In the present study, the effect of combining rosiglitazone with metformin and/or insulin on ß-cell mass and glucose levels was examined in a rat model of Type 2 diabetes. METHODS: Diabetes-prone pups were randomized to receive rosiglitazone alone or in combination with metformin and/or insulin starting at 4 weeks of age. ß-Cell mass and glucose homeostasis were examined in adulthood. RESULTS: Rosiglitazone treatment reduced insulin resistance and partially restored ß-cell mass in animals with reduced ß-cell mass at birth. The addition of metformin to rosiglitazone decreased insulin resistance and reduced weight gain, but had no additional effect on ß-cell mass. Conversely, the addition of insulin had no additional effect on these outcomes. Although the combination of rosiglitazone and metformin did not affect ß-cell mass at 26 weeks of age, it did result in reduced body weight and insulin resistance. CONCLUSION: The results of the present study suggest that the addition of metformin to rosiglitazone improves the metabolic profile through an effect on insulin resistance and not ß-cell mass.

miRNA in the regulation of skeletal muscle adaptation to acute endurance exercise in C57Bl/6J male mice.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNA species involved in post-transcriptional gene regulation. In vitro studies have identified a small number of skeletal muscle-specific miRNAs which play a crucial role in myoblast proliferation and differentiation. In skeletal muscle, an acute bout of endurance exercise results in the up-regulation of transcriptional networks that regulate mitochondrial biogenesis, glucose and fatty acid metabolism, and skeletal muscle remodelling. The purpose of this study was to assess the expressional profile of targeted miRNA species following an acute bout of endurance exercise and to determine relationships with previously established endurance exercise responsive transcriptional networks. C57Bl/6J wild-type male mice (N = 7/group) were randomly assigned to either sedentary or forced-endurance exercise (treadmill run @ 15 m/min for 90 min) group. The endurance exercise group was sacrificed three hours following a single bout of exercise. The expression of miR- 181, 1, 133, 23, and 107, all of which have been predicted to regulate transcription factors and co-activators involved in the adaptive response to exercise, was measured in quadriceps femoris muscle. Endurance exercise significantly increased the expression of miR-181, miR-1, and miR-107 by 37%, 40%, and 56%, respectively, and reduced miR-23 expression by 84% (P