Early-life nutrition profoundly influences long-term metabolic health, and breast milk not only provides nutrients but also conveys maternal signals shaping infant metabolic development. While postpartum exercise by lactating women benefits maternal health, its impact on milk-borne signaling remains largely undefined. Small extracellular vesicles (sEVs) in breast milk are key mediators of maternal-infant communication because of their selectively packaged bioactive cargo and resistance to infant digestive enzymes and acids, enabling delivery of their cargo to peripheral tissues. Here, we show that a single session of moderate-intensity postpartum aerobic exercise robustly increases human breast milk sEV concentration, which persists for multiple post-exercise milk collections. Exercise enriches breast milk with sEVs containing regulatory metabolic cargo (proteins, miRNAs, and metabolites), which translates into enhanced mitochondrial capacity in neonatal-stage cells. These findings implicate sEVs as an exercise-responsive signaling compartment in breast milk capable of connecting postpartum maternal physical activity to beneficial infant metabolic programming. Highlights:Acute moderate-intensity exercise increases human breast milk sEV concentrationThe exercise-mediated sEV increase lasts for multiple subsequent milk expressionsExercise coordinates a multi-omic enrichment of sEVs in breast milkExercised breast milk sEVs enhance mitochondrial respiration in UC-MSCs.

Despite indisputable benefits of different exercise modes, the molecular underpinnings of their divergent responses remain unclear. We investigate post-translational modifications in human skeletal muscle following 12 weeks of high-intensity aerobic interval or resistance exercise training. High-intensity aerobic training induces acetylproteome modifications including several mitochondrial proteins, indicating post-translational regulation of energetics machinery, whereas resistance exercise training regulates phosphoproteomic modifications of contractile/cytoskeletal machinery, consistent with greater strength. Furthermore, despite similar transcriptional responses to a single acute bout of aerobic and resistance exercise, more robust phosphoproteomic and metabolomic responses occur with acute aerobic exercise, including phosphorylation of structural/contractile and membrane transport machinery, and the nascent polypeptide-associated complex-α, a regulator of protein translation. Together, our findings provide new insight on the intricate phosphoproteomic and acetylproteomic modifications in muscle that potentially explain physiological responses to different modes of chronic and acute exercise. This study is registered with ClinicalTrials.gov, numbers NCT01477164 and NCT04158375.

Introduction and Objective: Insulin resistance (IR) increases the risk for Alzheimer’s disease and other dementias. Preclinical studies show that metformin normalizes alterations in brain mitochondrial functions induced by diet-induced IR, specifically in brain regions rich in insulin receptors. Here, we assessed the effect of 10-months of metformin treatment on cognitive function, brain network connectivity, glucose uptake, and regional volume in older people with IR. Methods: Forty participants aged 60-80 years (20 male, 20 female) with abdominal obesity, BMI ≥ 25 kg/m2, and fasting blood glucose of 100-125 mg/dL were studied. Participants were randomly assigned to metformin (2500 mg/d) or placebo (n = 20 per group) for 40 weeks. Pre- and post-treatment measurements included cognition (NIH Toolbox cognitive battery), volumetric MRI, resting-state fMRI, regional glucose uptake (18FDG-PET), and insulin sensitivity (mixed-meal tolerance test; MMTT). Results: Metformin improved processing speed and working memory concurrent to similar directional changes in amygdala and hippocampus volumes. Metformin increased white matter volume in the frontal and temporal lobes. Analysis of functional connectivity showed inverse associations between IR and connectivity strength between numerous brain regions, most notably between frontal and temporal lobe structures, potentially explaining improvement of processing speed. Increased glucose uptake in brain areas rich in insulin receptors, such as the prefrontal cortex, support that metformin enhanced insulin sensitivity in brain regions involved in memory and other cognitive functions as in the whole body noted by MMTT. Conclusion: These results support the notion that metformin ameliorates IR-related alterations in regional connectivity, brain volume, and brain glucose uptake with concurrent improvement of important aspects of cognition. G. Ruegsegger: None. H. Jo: None. M.W. Pataky: None. N. Stricker: None. K. Klaus: None. V.J. Lowe: Research Support; Eli Lilly and Company, Siemens Healthcare Diagnostics. J. Port: Consultant; Clario. K. Nair: None. National Institute of Aging (R21 AG 060139 and R01 062859)

Introduction and Objective: Human studies have shown that endurance exercise training (EET) increases the size of the hippocampus, critical for maintaining cognition and enhances brain glucose uptake. We determined whether the benefits of EET on the brain are related to enhanced mitochondrial function and how they could be related to hippocampal proteome abundance and post-translational modifications (PTMs). Methods: To do this we utilized 12-week-old C57BL/6 mice assigned to either a 6-week running wheel program or to a sedentary control group. Results: In the hippocampus, citrate synthase (Fig 1a) and cytochrome c oxidase activities (Fig 1b) were higher following EET which concurrently resulted in lower lactate dehydrogenase activity (Fig 1c). Together, these results suggest improved mitochondrial functions in the hippocampus of EET mice and a shift from a glycolytic to an oxidative profile. EET increased the levels of NAD+ and NADH in the hippocampus of animals. Further, global proteome, phosphoproteome, and acetylome were measured to determine EET effect on these post-translational events. We found substantial decrease in acetylated peptides (n=164) in the hippocampus (Fig 1e) with more robust effect in EET male mice compared to the sedentary group (Fig 1d). Conclusion: Overall, our results indicate that EET affects enzyme activity levels and deacetylation of proteins in the hippocampus, concurrent to improvement of NAD+ which may explain how EET improves cognition. Disclosure A.K. Asokan: None. R.G. Leija: None. C.J. Heppelmann: None. T. Dutta: None. K. Sevits: None. K. Klaus: None. G. Ruegsegger: None. M.W. Pataky: None. K. Nair: None.

Introduction and Objective: Metformin reduces hepatic glucose production by inhibiting gluconeogenic enzymes and altering liver mitochondrial function through Complex I inhibition, and suppressing gluconeogenesis. In skeletal muscle, the largest tissue for glucose disposal, there are conflicting reports on the molecular regulation of metformin on mitochondrial respiration and aerobic exercise capacity (VO2max). The objective was to determine the effects of long-term metformin administration on muscle mitochondrial function. Methods: We conducted a 40-week double-blind placebo-controlled trial to test the effect of oral metformin administration in 40 older (age 60-85) men and women with overweight/obesity (BMI 25-38) and high fasting glucose (100-140mg/dL) on skeletal muscle mitochondrial function. An oral mixed meal tolerance test was performed following an overnight fast. Muscle biopsies were obtained before and 1hr following and meal to measure isolated mitochondrial function (Oxygraph-2K) under fasted and fed conditions. Two-way ANOVAs were used to test the effect of intervention (metformin vs placebo) and time (pre vs post). Results: 40-weeks of metformin, but not placebo, significantly reduced glucose (P=0.002), insulin (P=0.008), and c-peptide (P=0.007) AUC during a meal tolerance test and reduced HbA1c (-0.24%, P=0.03). However, no significant differences in muscle mitochondrial function (including state 2, 3, or 4 respiration, uncoupled respiration, H2O2 production, or ATP production; per tissue weight or protein content) in the fasted or post-meal states were detected following metformin or placebo. Furthermore, 40-weeks of metformin had no effect on VO2max. Conclusion: These results indicate that long-term metformin therapy in older people with insulin resistance improves whole body glucose metabolism without impacting skeletal muscle mitochondrial function or aerobic capacity. M.W. Pataky: None. K. Klaus: None. A. Prabha Kumar: None. K. Sevits: None. J.A. Jungwirth: None. K. Nair: None. National Institute of Aging (R21 AG060139)

We investigated the link between enhancement of insulin sensitivity (SI) (by hyperinsulinemic-euglycemic clamp) and muscle metabolites following 12-weeks of aerobic (high-intensity interval training, HIIT), resistance (RT), or combined (CT) exercise training in 52 lean healthy people. Muscle RNA-sequencing revealed a significant association between SI following both HIIT and RT and the branched chain amino acid (BCAA) metabolic pathway. Concurrent to increased expression and activity of branched chain ketoacid dehydrogenase enzyme, many muscle amino metabolites including BCAAs, glutamate, phenylalanine, aspartate, asparagine, methionine, and GABA increased by HIIT, supporting substantial impact of HIIT on amino acid metabolism. Short-chain C3 and C5 acylcarnitines were reduced in muscle by all three training modes, but unlike RT, both HIIT and CT increased TCA metabolites and cardiolipins, supporting greater mitochondrial activity by aerobic training. Conversely, RT and CT increased more plasma membrane phospholipids than HIIT, suggesting a resistance exercise effect on cellular membrane protection against environmental damage. Sex and age contributed modestly to the exercise-induced changes in metabolites and their association to cardiometabolic parameters. Integrated transcriptomic and metabolomic analyses suggest various clusters of genes and metabolites are involved in distinct effects of HIIT, RT, and CT. These distinct metabolic signatures of different exercise modes independently link each type of exercise training to improved SI and cardiometabolic risk. ARTICLE HIGHLIGHTS: · We aimed to understand the link between skeletal muscle metabolites and cardiometabolic health after exercise training. · Although aerobic, resistance, and combined exercise training each enhance muscle insulin sensitivity as well as other cardiometabolic parameters, they disparately alter amino and citric acid metabolites as well as lipidome, linking these metabolomic changes independently to improvement of cardiometabolic risks by each exercise training mode. · These findings reveal an important layer of the unique exercise mode-dependent changes in muscle metabolism which may eventually lead to more informed exercise prescription for improving SI.

Different exercise modes yield distinct metabolic outcomes, yet the underlying molecular mechanisms driving these exercise responses remain unclear. Here, we investigated the impact of post-translational modifications (PTM) to skeletal muscle proteins in response to 3 months of high intensity aerobic interval training (HIIT) and resistance exercise training (RT). Frozen muscle biopsy tissue was obtained from a sub-cohort of our previously published study which showed significantly enhanced insulin sensitivity following either HIIT (n=7) or RET (n=7). After digestion, peptides were isobarically labeled and either acetyl- or phospho-enriched and fractionated by high-performance liquid chromatography (HPLC). Mass spectra were measured using a Thermo Fusion Tribrid coupled to Ultimate 3000 HPCL, revealing global-, phospho-, and acetyl-proteomic responses in muscle to HIIT or RT. Reactome analysis was used to determine exercise-regulated PTM pathways. HIIT significantly (p-value <0.05, fold-change ≥ 0.3) increased the abundance of ~9% of the global proteome (444 proteins) and ~22% of the acetylproteome (504 peptides), with primarily global and acetyl modifications occurring on mitochondrial pathways. HIIT also significantly (P < 0.05) increased the abundance of the deacetylase, SIRT3, which was positively associated with mitochondrial respiration. RT significantly (p-value <0.05, fold-change ≥ 0.3) altered the abundance of ~3% of the global proteome (144 proteins) and ~7% of the phosphoproteome (817 peptides), with minimal effect on the acetylproteome (~1%). RT primarily regulated contractile function and muscle structure pathways. These results indicate that the divergent metabolic responses to HIIT or RT are related to differential PTM mechanisms that contribute to cardiometabolic outcomes. M.W. Pataky: None. C.J. Heppelmann: None. K. Sevits: None. A.K. Asokan: None. K. Klaus: None. K. Nair: None. NIH (R01AG062859); NIH (T32DK007352)

We appreciate Dr. Astrada's interest in our recent article (1). Although he raised concern around using BMI as a screening parameter for our study, BMI was not the only "defining parameter" screening criteria in our study. Fasting glucose > 110mg/dL was also a critical exclusion criterion for the study which limits variation in metabolic characteristics in our study cohort. These and other criteria for participation in the study were reported in the original manuscript (2) from which the samples in our current study (1) were obtained. Furthermore, after participants were screened and included in the study, DEXA scans provide information on body composition, which we reported in the online supplemental material. Within a given age group, we found no differences in body composition (Fat %) between treatment groups at baseline. As for the participants' daily activities, we did not collect occupation information, but an exclusion criteria for the study was participation in regular exercise (>20 minutes more than twice per week), which was reported in the original manuscript (2). Thus, all participants were not regular exercisers, further limiting baseline metabolic variability.

We assessed the effects of 12 wk of supervised high-intensity interval training (HIIT), resistance training, and combined training (CT) on skeletal muscle mitochondrial abundance and markers of fission and fusion. HIIT increased mitochondrial area and size and promoted protein changes indicative of increased mitochondrial fusion, whereas lessor effects were observed after CT and no changes were observed after RT. Furthermore, increased mitochondrial area and size after HIIT associated with improved mitochondrial respiration, cardiorespiratory fitness, and insulin sensitivity.

Substantial divergence in cardio-metabolic risk, muscle size, and performance exists between men and women. Considering the pivotal role of skeletal muscle in human physiology, we investigated and found, based on RNA sequencing (RNA-seq), that differences in the muscle transcriptome between men and women are largely related to testosterone and estradiol and much less related to genes located on the Y chromosome. We demonstrate inherent unique, sex-dependent differences in muscle transcriptional responses to aerobic, resistance, and combined exercise training in young and older cohorts. The hormonal changes with age likely explain age-related differential expression of transcripts. Furthermore, in primary human myotubes we demonstrate the profound but distinct effects of testosterone and estradiol on amino acid incorporation to multiple individual proteins with specific functions. These results clearly highlight the potential of designing exercise programs tailored specifically to men and women and have implications for people who change gender by altering their hormone profile.

Resistance exercise training (RET) is an effective countermeasure to sarcopenia, related frailty and metabolic disorders. Here, we show that an RET-induced increase in PGC-1α4 (an isoform of the transcriptional co-activator PGC-1α) expression not only promotes muscle hypertrophy but also enhances glycolysis, providing a rapid supply of ATP for muscle contractions. In human skeletal muscle, PGC-1α4 binds to the nuclear receptor PPARβ following RET, resulting in downstream effects on the expressions of key glycolytic genes. In myotubes, we show that PGC-1α4 overexpression increases anaerobic glycolysis in a PPARβ-dependent manner and promotes muscle glucose uptake and fat oxidation. In contrast, we found that an acute resistance exercise bout activates glycolysis in an AMPK-dependent manner. These results provide a mechanistic link between RET and improved glucose metabolism, offering an important therapeutic target to counteract aging and inactivity-induced metabolic diseases benefitting those who cannot exercise due to many reasons.

Insulin sensitivity is enhanced by 3 months of either aerobic (high-intensity interval training, HIIT) or resistance exercise (RET) , but mechanisms leading to improved insulin sensitivity are not identical between modes of exercise. We sought to identify common predictors of exercise training-induced insulin sensitivity. Using RNAseq we found that branched chain amino acid (BCAA) and general amino acid metabolism were the only common pathways associated with enhanced insulin sensitivity by HIIT and RET in skeletal muscle. We explored the BCAA metabolism pathway and other amino acid metabolites as related to insulin sensitivity in response to HIIT and RET. Skeletal muscle and plasma were obtained from our previously published study which showed significantly enhanced insulin sensitivity following 12-weeks of either HIIT (n=19) or RET (n=18) . Immunoblotting was used to measure the abundance and phosphorylation of the key rate limiting enzyme in BCAA metabolism, branched chain ketoacid dehydrogenase (BCKDH) , in skeletal muscle. Liquid chromatography-mass spectrometry was used to measure amino acids and their metabolites before and after exercise training. Skeletal muscle BCKDH abundance and activity (indicated by reduced phosho/total BCKDH) were significantly increased (P<0.05) by both HIIT and RET. HIIT significantly increased (P<0.05) all three BCAAs (Ile +7.7 ± 2.2, Leu +18.9 ± 5.6, Val +24.5 ± 7.3 pmol/mg muscle) , as well as multiple other amino acids and metabolites in skeletal muscle. Although we did not detect an increase in amino acids or metabolites following RET in skeletal muscle, in plasma there was a significant increase (P<0.05) in all three aromatic amino acids (Phe +5.1 ± 2.2, Trp +5.6 ± 2.5, Tyr +4.8 ± 1.7 µM) as well as Pro (13.0 ± 4.6 µM) , Ala (+29.3 ± 12.1 µM) , and Met (2.1 ± 0.5 µM) following RET. These results suggest activation of amino acid metabolism pathways, contributing to enhanced insulin sensitivity following different modes of exercise training. Disclosure M.W.Pataky: None. A.Prabha kumar: n/a. M.M.Robinson: Stock/Shareholder; GE Healthcare Systems. K.Klaus: None. S.Dasari: None. K.Nair: None. Funding National Institutes of Health (RAG09531) National Institutes of Health (T32 DK007352)

Increased life expectancy combined with the aging baby boomer generation has resulted in an unprecedented global expansion of the elderly population. The growing population of older adults and increased rate of age-related chronic illness has caused a substantial socioeconomic burden. The gradual and progressive age-related decline in hormone production and action has a detrimental impact on human health by increasing risk for chronic disease and reducing life span. This article reviews the age-related decline in hormone production, as well as age-related biochemical and body composition changes that reduce the bioavailability and actions of some hormones. The impact of hormonal changes on various chronic conditions including frailty, diabetes, cardiovascular disease, and dementia are also discussed. Hormone replacement therapy has been attempted in many clinical trials to reverse and/or prevent the hormonal decline in aging to combat the progression of age-related diseases. Unfortunately, hormone replacement therapy is not a panacea, as it often results in various adverse events that outweigh its potential health benefits. Therefore, except in some specific individual cases, hormone replacement is not recommended. Rather, positive lifestyle modifications such as regular aerobic and resistance exercise programs and/or healthy calorically restricted diet can favorably affect endocrine and metabolic functions and act as countermeasures to various age-related diseases. We provide a critical review of the available data and offer recommendations that hopefully will form the groundwork for physicians/scientists to develop and optimize new endocrine-targeted therapies and lifestyle modifications that can better address age-related decline in heath.

Extracellular vesicles (EVs) are released into blood from multiple organs and carry molecular cargo that facilitates inter-organ communication and an integrated response to physiological and pathological stimuli. Interrogation of the protein cargo of EVs is currently limited by the absence of optimal and reproducible approaches for purifying plasma EVs that are suitable for downstream proteomic analyses. We describe a size-exclusion chromatography (SEC)-based method to purify EVs from platelet-poor plasma (PPP) for proteomics profiling via high-resolution mass spectrometry (SEC-MS). The SEC-MS method identifies more proteins with higher precision than several conventional EV isolation approaches. We apply the SEC-MS method to identify the unique proteomic signatures of EVs released from platelets, adipocytes, muscle cells, and hepatocytes, with the goal of identifying tissue-specific EV markers. Furthermore, we apply the SEC-MS approach to evaluate the effects of a single bout of exercise on EV proteomic cargo in human plasma.

Aerobic exercise training upregulates the expression of the transcriptional co-activator PGC-1α, resulting in improved oxidative metabolism and increased insulin sensitivity. Resistance exercise training (RET) is also an effective therapyfor enhancing insulin sensitivity, but the mechanisms by which RET increases insulin sensitivity are poorly understood. An isoform of the PGC-1α gene, PGC-1α4, is known to promote hypertrophy and strength, hallmark adaptations of RET. However, other metabolic effects of PGC-1α4 in response to RET in skeletal muscle are unknown. PURPOSE: Toinvestigate the roles of PGC-1α4 and RET on glycolytic metabolism regulation in skeletal muscle. METHODS: Muscle biopsies were obtained from male and female subjects before and after a single resistance exercise session (RE) and before and after 12 weeks of RET in young and older subjects. Activity of key glycolytic enzymes was measured using kit assays, and mRNA expression of PGC-1α isoforms was measured by qPCR. PGC-1α isoform protein abundance was measured by immunoblotting. In other experiments using C2C12 myotubestransfected with adenovirus overexpressing PGC-1α4 or anempty vector we measured glucose uptake, glycolysis, glycolytic capacity, and the expression of key glycolytic proteins. RESULTS: We observed elevated activity of hexokinase (2.0 ± 0.3 nmol/mg/min) and phosphofructokinase (82 ± 22 nmol/mg/min) after RE (P < 0.05), and elevated hexokinase activity (0.9 ± 0.3 nmol/mg/min) after RET (P < 0.05). mRNA expression of PGC-1α4 was increased after RE (P < 0.05), but PGC-1α4 protein expression was only increased following RET. In myotubes overexpressing PGC-1α4, glucose uptake was increased by nearly 2-fold (P < 0.05), concomitant with increased glycolysis and glycolytic capacity (P < 0.05).PGC-1α4 overexpression also resulted in a robust increase in the expression of multiple glycolytic proteins (P < 0.05). CONCLUSION: These preliminary findings support the hypothesis that PGC-1α4 regulates glycolytic metabolism. Important next steps will be to 1) identify if PGC-1α4 is required for the RET effect on glycolysis, and 2) determine if RET-mediated improvements in glycolysis are critical for enhancing insulin sensitivity.

Muscle glucose metabolism is improved by either aerobic or resistance exercise (RE) training, but the mechanisms leading to improved metabolism for each of these exercise modes are not identical. Much work has been done onimproved oxidative metabolism by aerobic exercise, but far less is known about how RE impacts fuel metabolism. Here, TCA and amino acid metabolites are measured in skeletal muscle after a single bout of RE. Twenty one healthy subjects performed a session of one-legged knee extension RE that consisted of 3 sets of 10 reps at 70% of their 1 rep max. Two muscle biopsies were obtained from the vastus lateralis of each leg. From the RE leg, biopsies were obtained ≤10min post-RE (0hr PEX) and approximately 1hr post-RE (1hr PEX), respectively. From the non-RE leg, biopsies were obtained before and 1hr PEX, respectively. Muscle samples were frozen and stored until mass spectrometry analysis of metabolites was performed. A 2-fold increase in plasma lactate (P<0.05) and a 30-40% increase in muscle glycolytic enzyme activity (P<0.05) at 0hr PEX suggest increased activation of the glycolytic pathway. Also at 0hr PEX, TCA metabolites citrate and isocitrate were increased (P<0.05) compared to the pre-RE biopsy (0.35 and 0.01 mmol/μg, respectively). Further, multiple amino acid metabolites Arg, Asn, Lys, Thr, and Val were increased (P<0.05) in the REmuscle at 0hr PEX. These amino acids can enter the TCA cycle at various steps, indicating increased flux through the TCA cycle immediately after RE. By 1hr PEX, TCA intermediates malate and fumarate were reduced (P<0.05)compared to the non-RE leg (-0.65 and -0.35 mmol/μg, respectively), and multiple amino acid metabolites were also reduced (P<0.05). The decrease in these metabolites at 1hr PEX suggests increased use of metabolites for mitochondrial respiration. These results suggest activation of glycolytic and amino metabolism pathways, contributing to citric acid flux immediately following RE in order to meet energy needs in skeletal muscle. Disclosure M. W. Pataky: None. A. Prabha kumar: None. K. Klaus: None. K. Nair: None. Funding National Institutes of Health (R01AG062859)

Health benefits of aerobic exercise are indisputable and are closely related to the maintenance of mitochondrial energy homeostasis and insulin sensitivity. Flockhart et al. (2021) demonstrate, however, that a high volume of high-intensity aerobic exercise adversely affects mitochondrial function and may cause impaired glucose tolerance.

Exercise training enhances insulin sensitivity (Si) in humans. However, it is to be determined which phenotypic and muscle molecular changes underwrite this change in Si. We sought to understand the mechanisms behind increased Si following 12 weeks of high intensity aerobicinterval (HIIT), resistance (RT), or combined (CT) exercise training in 30 young and 24 old humans. Measurements included Si estimated from a two-stage hyperinsulinemic-euglycemic clamp, and a host of independent variables including VO2 peak, muscle mitochondrial respiration, Liver FWF (Fat-Water-Fraction), visceral and subcutaneous fat, and gene expression from muscle biopsies. We performed a univariate and multivariate analysis of Si vs. phenotypic changes induced by training. After adjusting for age, sex and exercise group, VO2max was positively associated with an increase in Si from pre to post exercise (p=0.032). Interestingly, within the RT group alone, positive association between VO2max and Si (p=0.05) and negative association between liver FWF and Si (p=0.034) were observed. Within the HIIT group, there was a positive association between muscular strength and Si (p=0.04). Separate multivariate models assessed the effect of muscle gene expression changes on Si while adjusting for age, training type, response to training, VO2max, and mitochondrial function. Enrichment Analysis of genes associated with Si showed upregulation of genes in Branched-Chain Amino Acid (BCAA) and Lysine metabolism, although the latter was specifically upregulated with HIIT. Products of lysine metabolism, such as alpha-aminoadipic acid have been implicated in development of insulin resistance and BCAA are involved in signaling of insulin response. A strong positive association of Si with both BCAA (FDR 0) and lysine metabolism (FDR 0.008) following HIIT and a weaker correlation between Si and BCAA metabolism (FDR 0.002) following RT indicates the role of muscle AA metabolism as a common factor contributing to enhanced Si following exercise training. Disclosure A. Prabha kumar: None. S. Dasari: None. M. W. Pataky: None. J. Crook: None. C. T. Ball: None. K. Klaus: None. I. R. Lanza: None. M. M. Robinson: Stock/Shareholder; Self; GE Healthcare. K. Nair: None. Funding National Institutes of Health (R01AG09531)