Despite the known metabolic benefits of exercise, an integrated metabolic understanding of exercise is lacking. Here, we use in vivo steady-state isotope-labeled infusions to quantify fuel flux and oxidation during exercise in fasted, fed, and exhausted female mice, revealing several novel findings. Exercise strongly promoted glucose fluxes from liver glycogen, lactate, and glycerol, distinct from humans. Several organs spared glucose, a process that broke down in exhausted mice despite concomitant hypoglycemia. Proteolysis increased markedly, also divergent from humans. Fatty acid oxidation dominated during fasted exercise. Ketone production and oxidation rose rapidly, seemingly driven by a hepatic bottleneck caused by gluconeogenesis-induced cataplerotic stress. Altered fuel consumption was observed in organs not directly involved in muscle contraction, including the pancreas and brown fat. Several futile cycles surprisingly persisted during exercise, despite their energy cost. In sum, we provide a comprehensive, integrated, holistic, and quantitative accounting of metabolism during exercise in an intact organism.

Elevated levels of plasma branched-chain amino acids (BCAAs) have been associated with insulin resistance and type 2 diabetes since the 1960s. Pharmacological activation of branched-chain α-ketoacid dehydrogenase (BCKDH), the rate-limiting enzyme of BCAA oxidation, lowers plasma BCAAs and improves insulin sensitivity. Here we show that modulation of BCKDH in skeletal muscle, but not liver, affects fasting plasma BCAAs in male mice. However, despite lowering BCAAs, increased BCAA oxidation in skeletal muscle does not improve insulin sensitivity. Our data indicate that skeletal muscle controls plasma BCAAs, that lowering fasting plasma BCAAs is insufficient to improve insulin sensitivity and that neither skeletal muscle nor liver account for the improved insulin sensitivity seen with pharmacological activation of BCKDH. These findings suggest potential concerted contributions of multiple tissues in the modulation of BCAA metabolism to alter insulin sensitivity.

Cold-induced thermogenesis (CIT) is widely studied as a potential avenue to treat obesity, but a thorough understanding of the metabolic changes driving CIT is lacking. Here, we present a comprehensive and quantitative analysis of the metabolic response to acute cold exposure, leveraging metabolomic profiling and minimally perturbative isotope tracing studies in unanesthetized mice. During cold exposure, brown adipose tissue (BAT) primarily fueled the tricarboxylic acid (TCA) cycle with fat in fasted mice and glucose in fed mice, underscoring BAT's metabolic flexibility. BAT minimally used branched-chain amino acids or ketones, which were instead avidly consumed by muscle during cold exposure. Surprisingly, isotopic labeling analyses revealed that BAT uses glucose largely for TCA anaplerosis via pyruvate carboxylation. Finally, we find that cold-induced hepatic gluconeogenesis is critical for CIT during fasting, demonstrating a key functional role for glucose metabolism. Together, these findings provide a detailed map of the metabolic rewiring driving acute CIT.

BackgroundAging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age?MethodsWe conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan–Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions.ResultsChronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life.ConclusionAdministration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan.

Background: Epigenetic marks are responsive to a wide variety of environmental stimuli and serve as important mediators for gene transcription. A number of chromatin modifying enzymes orchestrate epigenetic responses to environmental stimuli, with a growing body of research examining how changes in metabolic substrates or co-factors alter epigenetic modifications. Scope of Review: Here, we provide a systematic review of existing evidence of metabolism-related epigenetic changes in white adipose tissue (WAT) and the liver and generate secondary hypotheses on how exercise may impact metabolism-related epigenetic marks in these tissues. Major Conclusions: Epigenetic changes contribute to the complex transcriptional responses associated with WAT lipolysis, hepatic de novo lipogenesis, and hepatic gluconeogenesis. While these metabolic responses may hypothetically be altered with acute and chronic exercise, direct testing is needed.

The intrinsic aerobic capacity of an organism is thought to play a role in aging and longevity. Maximal respiratory rate capacity, a metabolic performance measure, is one of the best predictors of cardiovascular- and all-cause mortality. Rats selectively bred for high-(HCR) vs. low-(LCR) intrinsic running-endurance capacity have up to 31% longer lifespan. We found that positive changes in indices of mitochondrial health in cardiomyocytes (respiratory reserve, maximal respiratory capacity, resistance to mitochondrial permeability transition, autophagy/mitophagy, and higher lipids-over-glucose utilization) are uniformly associated with the extended longevity in HCR vs. LCR female rats. Cross-sectional heart metabolomics revealed pathways from lipid metabolism in the heart, which were significantly enriched by a select group of strain-dependent metabolites, consistent with enhanced lipids utilization by HCR cardiomyocytes. Heart–liver–serum metabolomics further revealed shunting of lipidic substrates between the liver and heart via serum during aging. Thus, mitochondrial health in cardiomyocytes is associated with extended longevity in rats with higher intrinsic exercise capacity and, probably, these findings can be translated to other populations as predictors of outcomes of health and survival.

Mice are among the most widely used translational models of cardiovascular aging and offer a method to quickly assess lifespan changes in a controlled environment. The standard laboratory temperature (20–22 °C), however, imposes a cold stress on mice that causes an increase in sympathetic nervous system–mediated activation of brown adipose tissue (BAT) to maintain a core body temperature of 36–37 °C. Thus, while physiologic data obtained recapitulate human physiology to a certain degree, interpretations of previous research in mice may have been contaminated by a cold stress, due to housing mice below their thermoneutral zone (30 °C). The purpose of this investigation was to examine how chronic sympathetic stimulation evoked by acclimation to 20 °C might obscure interpretation of changes in autonomic modulation of heart rate (HR) and heart rate variability (HRV) that accompany advancing age. HR and HRV before and after administration of a dual-autonomic blockade were measured via in-vivo ECG in young (3 months) and aged (30 months) male C57BL/6 telemetry-implanted mice following temperature acclimation for 3 days at 30 °C or 20 °C. Mean basal and intrinsic HR of both young and aged mice became markedly reduced at 30 °C compared to 20 °C. In both age groups, HRV parameters in time, frequency, and non-linear domains displayed increased variability at 30 °C compared to 20 °C under basal conditions. Importantly, age-associated declines in HRV observed at 20 °C were ameliorated when mice were studied at their thermoneutral ambient temperature of 30 °C. Thus, an accurate understanding of autonomic modulation of cardiovascular functions in mice of advanced age requires that they are housed in a metabolically neutral environment.

Endothelial cells (ECs) are widely heterogenous depending on tissue and vascular localization. Jambusaria et al. recently demonstrated that ECs in various tissues surprisingly possess mRNA signatures of their underlying parenchyma. The mechanism underlying this observation remains unexplained, and could include mRNA contamination during cell isolation, in vivo mRNA paracrine transfer from parenchymal cells to ECs, or cell-autonomous expression of these mRNAs in ECs. Here, we use a combination of bulk RNASeq, single-cell RNASeq datasets, in situ mRNA hybridization, and most importantly ATAC-Seq of FACS-isolated nuclei, to show that cardiac ECs actively express cardiomyocyte myofibril (CMF) genes and have open chromatin at CMF gene promoters. These open chromatin sites are enriched for sites targeted by cardiac transcription factors, and closed upon expansion of ECs in culture. Together, these data demonstrate unambiguously that the expression of CMF genes in ECs is cell-autonomous, and not simply a result of technical contamination or paracrine transfers of mRNAs, and indicate that local cues in the heart in vivo unexpectedly maintain fully open chromatin in ECs at genes previously thought limited to cardiomyocytes.

Performing regular exercise is associated with numerous health benefits including a reduction in all-cause mortality. The mechanisms associated with exercise-induced health improvements are wide ranging and benefit virtually every organ system in the body. Of significance, recent evidence has suggested that some of these protective benefits may also be passed to offspring through multiple generations via alterations in gamete presentation, changes to the in-utero and offspring rearing environments, and epigenetic modifications. The purpose of this review was to systematically examine the current literature for evidence of exercise-induced epigenetic modifications in offspring. A systematic search yielded four papers that met inclusion criteria. Parental exercise interventions were associated with differential DNA methylation patterns in offspring. These shifts in methylation patterns were consistent with concurrent changes in offspring mRNA levels, protein expression, and functional measures. Many of the observed changes were related to metabolic pathways. Hence, the evidence suggests that exercise-induced epigenetic changes can be observed in offspring and may play a pivotal role among the multifactorial intergenerational-health impact of exercise.

PurposeIt has been well documented that aging is associated with a reduced heart rate (HR) and heart rate variability (HRV), a reduction in body weight (BW), and increased risk of frailty. The impact of chronic voluntary exercise coupled with aging on these parameters in older rats is unknown. We determined the effects of voluntary aerobic exercise on BW, HR, HRV and their relation to autonomic input, and frailty index (FI) in a longitudinal aging rat model.MethodsBaseline and repeated measures of experimental variables were recorded in 40 male F344 x Brown Norway rats on entry (EAge) into the study (between 15 and 25 months of age) and over time following EAge until death (on average 31 months). Individually housed rats at each of 3 EAges were randomized into two groups: 1) “Voluntary Wheel Exercise” (VWE) group had access to a running wheel, and 2) a control group that did not have access to a wheel. Body weight (BW) for both groups was recorded once per week. ECGs were recorded monthly via telemetry devices in awake, unrestrained rats. A 1.5‐hour basal recording was followed by a double autonomic blockade (0.55 mg/kg Atropine and 1.1 mg/kg Propranolol administered intraperitoneally) to obtain the intrinsic heart rate (IHR). ECGs were analyzed with Labchart, and statistical analysis of HR and HRV was performed in R and Python. FI scores [adapted from the Rat Clinical Frailty Index] were recorded at baseline and repeated 2 to 4 times throughout lifespan.ResultsThe mean heartbeat‐to‐heartbeat interval (mean NN in table) and numerous HRV parameters in the basal and intrinsic states were decreased at increasing EAge and the change over time following EAge increased as EAge increased (EAge: Time in table). VWE affected the mean intrinsic NN and numerous HRV parameters in the basal and intrinsic state (VWE in table). The effect of VWE on EAge (VWE: EAge in table) for mean intrinsic NN and several basal and intrinsic HRV parameters was also dependent on EAge, and the effect of VWE on intrinsic NN and a few HRV parameters changed over time following EAge (VWE: Time in table). A similar relationship between entry age and duration of exercise is observed with BW. Frailty index decreased with aging but was not affected by VWE.ConclusionsOur study confirms that aging alters overall heartbeat‐to‐heartbeat variability and that the voluntary wheel exercise has a marked impact on these variables. A greater exercise effect was observed in the intrinsic HR and several frequency and non‐linear domain parameters. Observed EAge: VWE interaction term suggests that age at which exercise is initiated influences its long‐term effects. Additionally, exercise duration time‐effect in a model of chronic voluntary exercise influences its effect on IHR.Support or Funding InformationFunding Information: Funded by the National Institutes of Health/National Institute on Aging Intramural Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Performing regular exercise is associated with numerous health benefits including a reduction in all‐cause mortality. The mechanisms associated with exercise‐induced health improvements are wide ranging and benefit virtually every organ system in the body. Of significance, recent evidence has suggested that some of these protective benefits may also be passed to offspring through multiple generations via alterations in gamete presentation, changes to the in‐utero and offspring rearing environments, and epigenetic modifications. The purpose of this review was to systematically examine the current literature for evidence of exercise‐induced epigenetic modifications in offspring. A systematic search yielded four papers that met inclusion criteria. Parental exercise interventions were associated with differential DNA methylation patterns in offspring. These shifts in methylation patterns were consistent with concurrent changes in offspring mRNA levels, protein expression, and functional measures. Many of the observed changes were related to metabolic pathways. Hence, the evidence suggests that exercise‐induced epigenetic changes can be observed in offspring and may play a pivotal role among the multifactorial intergenerational‐health impact of exercise.

Objective: To determine whether a novel anti-inflammatory, erythropoietin-derived synthetic peptide, ARA290, can improve healthspan and delay deteriorations in heart function.

Mitochondrial network function might be involved in healthspan by playing a central role in slowing the rate of damage accumulation and senescence. Although a direct cause-and-effect relationship between mitochondrial dysfunction and disease is well established, their specific roles in life-expectancy and -quality remains uncertain. We hypothesized that respiratory reserve and maximal respiration of cardiac muscle is affected by aging-related deficits in mitochondrial turnover resulting in a progressively damaged mitochondrial population that may shorten lifespan. This was tested in a cross-sectional study of 6, 17, and 24 months aged, low- (LCR) and high-running capacity (HCR) female rats, in which HCR exhibit a 40% increased longevity and an intrinsic 4-fold higher distance-running capacity. We found that, across ages tested, isolated HCR cardiomyocytes display higher respiratory reserve and maximal respiratory capacity than LCR, in the presence of the fatty acid palmitate (palm) with or without glucose (gluc). For example, in gluc+palm respiratory reserve differences between HCR vs. LCR were significantly higher in young ([in nmol O2/min/104 cells]: 4.2±0.3 vs. 1.8±0.2; n=47, 6 experiments, p<0.0001) than in old rats (5.7±0.9 vs. 4.8±0.7; n=24, 4 experiments). HCR cardiomyocytes showed significantly higher autophagy/mitophagy than LCR as a function of age, especially at middle-age (>36% HCR vs. LCR, n=47, 2 experiments, p<0.0001), as measured by quantitative electron microscopy, which correlated with 64% (p<0.001) lower intracellular concentration of acetyl-CoA in HCR in the presence of gluc+palm. These data show that greater mitochondrial turnover underlies higher cardiomyocyte energetic performance correlated with preferential selection of lipid oxidation with improved control of intracellular acetyl-CoA. Although aging is correlated with declines in all these positive indexes, they are better preserved in HCR vs. LCR, consistent with healthier aging and extended lifespan.

BackgroundChronic inflammation is linked to age‐associated declines in heart structure and function that contribute to increased risks for cardiovascular mortality and frailty, a state of high vulnerability to adverse health outcomes, and to reduced healthspan.ObjectiveTo determine whether a novel anti‐inflammatory, erythropoietin‐derived synthetic peptide, ARA290, can improve healthspan and delay deteriorations in heart function.Methods18 mo Fischer 344 × Brown Norway male rats (n = 50) were randomized to bi‐weekly injections of ARA290 or saline. Body weight (BW) was recorded every 2 weeks until death. Echocardiograms (ECHO), to measure ejection fraction (EF), and electrocardiograms (ECG), to measure heart rate (HR) before (BHR) and during a double autonomic blockade (IHR), were performed at 18, 22, 26, 30, and 33 mo, when a 33‐item frailty index (FI), scored on a scale of 0 to 1 (least to most frail), assessed health deficits in integument, musculoskeletal, vestibulocochlear, ocular, neurological, digestive, and respiratory systems. Linear mixed effects models assessed changes in cardiac markers over time, and joint models predicted the overall risk of death based on those changes and survival data.ResultsARA290 slowed the decline in BHR that accompanied aging (p < 0.03): between 18 and 33 mo BHR decreased from 327 to 276 beats per minute (BPM) in ARA290 and 314 to 261 BPM in the control; between 27 and 33 mo BHR plateaued in ARA290 but continued to decline in the control. IHR declined non‐linearly from 254 BPM to 240 BPM between 18 and 33 mo (p < 0.003), but did not differ by treatment. The difference between BHR and IHR (ΔHR), an index of autonomic modulation on HR, declined from 58 to 17 BPM in the control and from 73 to 38 BPM in ARA290 between 18 and 33 mo. Between 22 and 33 mo ΔHR was preserved in ARA290, but not control(p < 0.02). EF in ARA290 declined from 82.4% to 76.8% at a rate of 0.469% per month slower than the control, in which EF declined from 84.6% to 70.3% (p < 0.005). At 33 months EF was 7.5% greater in ARA290 (p < 0.004). BW decreased from 534 to 486 g between 25.5 and 33.0 months in ARA290 and from 529 to 442 g between 24.3 and 33.0 months in the control. ARA290 retarded the reduction in BW by 0.04 grams/month2 (p < 0.02). FI score was significantly lower in ARA290 (0.22) compared to the control (0.30) (p < 0.001). Joint model predictions show higher risks of death by 1.2% for a 1 BPM decrease in BHR (p < 0.0001), by 1.5% for a 1 BPM decrease in IHR (p < 0.0001), by 1.1% for a 1 BPM decrease in ΔHR (p < 0.12), and by 11.9% for a 1% decrease in EF (p < 0.0001).ConclusionsReduced frailty of ARA290 in advanced age is consistent with improved healthspan. Concurrent ARA290 preservation in autonomic modulation of HR and EF are reflected by changes in BHR, IHR, ΔHR, and EF with age. Because a unit decrease in any of these markers predicted significantly higher risks for mortality, the impact of ARA290 on cardiac aging may contribute to the observed improvement in healthspan.Support or Funding InformationResearch was supported by the National Institute of Health/National Institute on Aging Intramural Researc program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.