Stephen J. Gardell

The Failing Heart Relies on Ketone Bodies as a Fuel

Background— Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure. Methods and Results— Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of &bgr;-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified. Conclusions— These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.

Rotenone induces reductive stress and triacylglycerol deposition in C2C12 cells.

Environmental rotenone is associated with Parkinsons disease due to its inhibitory property to the complex I of mitochondrial respiration chain. Although environmental pollution has been postulated as a causal factor for the increasing prevalence of obesity, the role of rotenone in the pathogenesis of obesity has not been studied. We employed muscle-derived cell C2C12 as a model and shotgun lipidomics as a tool for lipid analysis and found that treatment with rotenone led to the profound deposition of intracellular triacylglycerol (TAG) in a time- and dose-dependent fashion. The TAG deposition resulted from complex I inhibition. Further studies revealed that rotenone induced mitochondrial stress as shown by decreased mitochondrial oxygen consumption rate, increased NADH/NAD+ ratio (i.e., reductive stress) and mitochondrial metabolites. We demonstrated that rotenone activated fatty acid de novo synthesis and TAG synthesis and ultimately resulted in intracellular TAG deposition. These studies suggested that increased mitochondrial stresses might be an underlying mechanism responsible for TAG accumulation manifest in obesity.

Comparison of tissue harvest protocols for the quantitation of acylcarnitines in mouse heart and liver by mass spectrometry

Profiling of acylcarnitines (ACs) in tissues and biological fluids by mass spectrometry is a powerful approach to examine the impact of genetic, pharmacological, and environmental factors on intermediary metabolism. The AC pool exhibits rapid changes in composition and abundance in response to altered cellular fuel catabolism. However, the mercurial nature of the AC pool makes it prone to spurious influences arising from experimental variables related to tissue harvesting and processing. We evaluated the impact of various strategies to anesthetize, sedate, or euthanize (A/S/E) mice which included Nembutal, Beuthanasia, isoflurane, ketamine/xylazine (Ket/Xy) and CO2/cervical dislocation (CD) on the tissue AC profiles. ACs extracted from heart and liver were derivatized to methyl esters and quantitated by mass spectrometry. Marked differences were seen in the tissue AC profiles, especially in heart, depending upon the choice of the A/S/E strategy. Most importantly, a uniform A/S/E protocol must be employed. While tissue AC profiles in situ cannot be unambiguously defined, use of Nembutal appears to be superior to other A/S/E strategies especially when assessing the AC levels in the heart. We also showed that it is preferable to expeditiously harvest and flash-freeze tissues to avoid procedure-related perturbation of the AC profile. A more protracted tissue harvest, when recovering numerous tissues from the same animal, can alter the AC pool. In conclusion, this investigation provides key guidance for harvesting heart and liver from mice in order to minimize the procedure-associated change of the AC pool which can mask the influence of the intended experimental variables.

Skeletal muscle overexpression of nicotinamide phosphoribosyl transferase in mice coupled with voluntary exercise augments exercise endurance

Objective Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway that produces nicotinamide adenine dinucleotide (NAD+), an essential co-substrate regulating a myriad of signaling pathways. We produced a mouse that overexpressed NAMPT in skeletal muscle (NamptTg) and hypothesized that NamptTg mice would have increased oxidative capacity, endurance performance, and mitochondrial gene expression, and would be rescued from metabolic abnormalities that developed with high fat diet (HFD) feeding. Methods Insulin sensitivity (hyperinsulinemic-euglycemic clamp) was assessed in NamptTg and WT mice fed very high fat diet (VHFD, 60% by kcal) or chow diet (CD). The aerobic capacity (VO2max) and endurance performance of NamptTg and WT mice before and after 7 weeks of voluntary exercise training (running wheel in home cage) or sedentary conditions (no running wheel) were measured. Skeletal muscle mitochondrial gene expression was also measured in exercised and sedentary mice and in mice fed HFD (45% by kcal) or low fat diet (LFD, 10% by kcal). Results NAMPT enzyme activity in skeletal muscle was 7-fold higher in NamptTg mice versus WT mice. There was a concomitant 1.6-fold elevation of skeletal muscle NAD+. NamptTg mice fed VHFD were partially protected against body weight gain, but not against insulin resistance. Notably, voluntary exercise training elicited a 3-fold higher exercise endurance in NamptTg versus WT mice. Mitochondrial gene expression was higher in NamptTg mice compared to WT mice, especially when fed HFD. Mitochondrial gene expression was higher in exercised NamptTg mice than in sedentary WT mice. Conclusions Our studies have unveiled a fascinating interaction between elevated NAMPT activity in skeletal muscle and voluntary exercise that was manifest as a striking improvement in exercise endurance.

Cardiovascular Drug Discovery in the Academic Setting: Building Infrastructure, Harnessing Strengths, and Seeking Synergies

The flow of innovative, effective, and safe new drugs from pharmaceutical laboratories for the treatment and prevention of cardiovascular disease has slowed to a trickle. While the need for breakthrough cardiovascular disease drugs is still paramount, the incentive to develop these agents has been blunted by burgeoning clinical development costs coupled with a heightened risk of failure due to the unprecedented nature of the emerging drug targets and increasingly challenging regulatory environment. A fuller understanding of the drug targets and employing novel biomarker strategies in clinical trials should serve to mitigate the risk. In any event, these current challenges have evoked changing trends in the pharmaceutical industry, which have created an opportunity for non-profit biomedical research institutions to play a pivotal partnering role in early stage drug discovery. The obvious strengths of academic research institutions is the breadth of their scientific programs and the ability and motivation to “go deep” to identify and characterize new target pathways that unlock the specific mysteries of cardiovascular diseases—leading to a bounty of novel therapeutic targets and prescient biomarkers. However, success in the drug discovery arena within the academic environment is contingent upon assembling the requisite infrastructure, annexing the talent to interrogate and validate the drug targets, and building translational bridges with pharmaceutical organizations and patient-oriented researchers.

Impaired Mitochondrial Energetics Characterize Poor Early Recovery of Muscle Mass Following Hind Limb Unloading in Old Mice

Abstract The progression of age-related sarcopenia can be accelerated by impaired recovery of muscle mass following periods of disuse due to illness or immobilization. However, the mechanisms underlying poor recovery of aged muscle following disuse remain to be delineated. Recent evidence suggests that mitochondrial energetics play an important role in regulation of muscle mass. Here, we report that 22- to 24-month-old mice with low muscle mass and low glucose clearance rate also display poor early recovery of muscle mass following 10 days of hind limb unloading. We used unbiased and targeted approaches to identify changes in energy metabolism gene expression, metabolite pools and mitochondrial phenotype, and show for the first time that persistent mitochondrial dysfunction, dysregulated fatty acid β-oxidation, and elevated H2O2 emission occur concomitantly with poor early recovery of muscle mass following a period of disuse in old mice. Importantly, this is linked to more severe whole-body insulin resistance, as determined by insulin tolerance test. The findings suggest that muscle fuel metabolism and mitochondrial energetics could be a focus for mining therapeutic targets to improve recovery of muscle mass following periods of disuse in older animals.

Elevated Nicotinamide Phosphoribosyl Transferase in Skeletal Muscle Augments Exercise Performance and Mitochondrial Respiratory Capacity Following Exercise Training

Mice overexpressing NAMPT in skeletal muscle (NamptTg mice) develop higher exercise endurance and maximal aerobic capacity (VO2max) following voluntary exercise training compared to wild-type (WT) mice. Here, we aimed to investigate the mechanisms underlying by determining skeletal muscle mitochondrial respiratory capacity in NamptTg and WT mice. Body weight and body composition, tissue weight (gastrocnemius, quadriceps, soleus, heart, liver, and epididymal white adipose tissue), skeletal muscle and liver glycogen content, VO2max, skeletal muscle mitochondrial respiratory capacity (measured by high-resolution respirometry), skeletal muscle gene expression (measured by microarray and qPCR), and skeletal muscle protein content (measured by Western blot) were determined following 6 weeks of voluntary exercise training (access to running wheel) in 13-week-old male NamptTg (exercised NamptTg) mice and WT (exercised WT) mice. Daily running distance and running time during the voluntary exercise training protocol were recorded. Daily running distance (p = 0.51) and running time (p = 0.85) were not significantly different between exercised NamptTg mice and exercised WT mice. VO2max was higher in exercised NamptTg mice compared to exercised WT mice (p = 0.02). Body weight (p = 0.92), fat mass (p = 0.49), lean mass (p = 0.91), tissue weight (all p > 0.05), and skeletal muscle (p = 0.72) and liver (p = 0.94) glycogen content were not significantly different between exercised NamptTg mice and exercised WT mice. Complex I oxidative phosphorylation (OXPHOS) respiratory capacity supported by fatty acid substrates (p < 0.01), maximal (complex I+II) OXPHOS respiratory capacity supported by glycolytic (p = 0.02) and fatty acid (p < 0.01) substrates, and maximal uncoupled respiratory capacity supported by fatty acid substrates (p < 0.01) was higher in exercised NamptTg mice compared to exercised WT mice. Transcriptomic analyses revealed differential expression for genes involved in oxidative metabolism in exercised NamptTg mice compared to exercised WT mice, specifically, enrichment for the gene set related to the SIRT3-mediated signaling pathway. SIRT3 protein content correlated with NAMPT protein content (r = 0.61, p = 0.04). In conclusion, NamptTg mice develop higher exercise capacity following voluntary exercise training compared to WT mice, which is paralleled by higher mitochondrial respiratory capacity in skeletal muscle. The changes in SIRT3 targets suggest that these effects are due to remodeling of mitochondrial function.

Metabolite profile and mitochondrial energetics characterize poor early recovery of muscle mass following hind limb unloading in old mice

The progression of age-related sarcopenia can be accelerated by impaired recovery of muscle mass following periods of disuse due to illness or immobilization. The molecular underpinnings of poor recovery of aging muscle following disuse remain largely unknown. However, recent evidence suggests that mitochondrial energetics may play an important role. Here, we report that 22-24 month old mice with low muscle mass and insulin resistance display poor early recovery of muscle mass following 10 days of hind limb unloading. We took an unbiased approach to identify changes in energy metabolism gene expression and metabolite pools and show for the first time that persistent mitochondrial dysfunction, dysregulated fatty acid β-oxidation and elevated H2O2 emission underlie poor early recovery of muscle mass following a period of disuse in old mice. Importantly, this is linked to more severe whole-body insulin resistance. The findings suggest that muscle fuel metabolism and mitochondrial energetics should be a focus for mining therapeutic targets to improve recovery of muscle mass following periods of disuse in older animals.

A novel clinical approach to evaluating changes in fat oxidation in healthy, overnight-fasted subjects

BackgroundObesity is characterized by impaired fat oxidation, and, therefore, the development of therapeutics designed to enhance fat oxidation for treating obesity is a growing focus in drug discovery. Evaluating such agents, however, is challenging in a clinical setting; requisite overnight fasting prior to a study visit increases whole body fat oxidation in lean, healthy subjects, making incremental increases potentially difficult to detect.MethodsWe recruited 16 lean/overweight participants and eight obese participants to evaluate novel methodology designed to optimally detect increases in fat oxidation. Carbohydrate intake was increased for three days leading up to the overnight test visit and evaluated as either a standalone strategy to suppress overnight fasting-related increases in fat oxidation or was combined with overnight administration of IV glucose in a subset of lean/overweight subjects. Indirect calorimetry was performed the following morning to examine the degree to which these interventions were able to diminish fasting related increases in fat oxidation. Fat oxidation was then subsequently stimulated by challenging patients with a lipid infusion to determine the importance of the preceding interventions on the ability to detect statistically significant increases in fat oxidation.DiscussionIn lean/overweight subjects, suppression of fasting-related increases in fat oxidation via increased dietary carbohydrate intake was not necessary to detect subsequent meaningful increases in fat oxidation rates following Intralipid infusion; however, a statistically significant decrease in respiratory quotient following Intralipid infusion could only be observed when the infusion was preceded with high carbohydrate dietary intake. Glucose infusion, which further suppressed fasting-related fat oxidation, in turn led to more dramatic increases in Intralipid-driven fat oxidation. Both lean/overweight and obese subjects could demonstrate statistically significant increases in fat oxidation following Intralipid infusion, a finding which supports the inclusion of obese subjects in similar future clinical studies. Lastly, serum levels of β-hydroxybutyrate correlated strongly with fat oxidation rates, as well as respiratory quotient, suggesting potential as a surrogate biomarker of fax oxidation. Taken together, we demonstrate the utility of novel methodology to facilitate measuring changes in fat oxidation in lean/overweight and obese human volunteers in a clinical setting similar to that needed to test drugs designed to increase fat oxidation.