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Aims: The sympathetic nervous system regulates numerous aspects of mitochondrial function in the heart through activation of adrenergic receptors (ARs) on cardiomyocytes. Mounting evidence suggests that α1-ARs, particularly the α1A subtype, are cardioprotective and may mitigate the deleterious effects of chronic β-AR activation by shared endogenous ligands. The mechanisms through which α1A-ARs exert their cardioprotective effects remain unclear. Here we tested the hypothesis that α1A-ARs adaptively regulate cardiomyocyte oxidative metabolism in the uninjured and infarcted heart. Methods: We used an α1A-AR knockout mouse (α1A-KO) to characterize the effects of α1A-AR genetic deletion on mitochondrial function and metabolism in the uninjured mouse heart using high resolution respirometry, substrate-specific electron transport chain (ETC) enzyme assays, transmission electron microscopy (TEM) and proteomics. We then compared the effects of α1A- and β-AR agonist treatment on mitochondrial function in uninjured mice and mice subjected to experimental myocardial infarction. Results: We found that isolated cardiac mitochondria from α1A-KO mice had deficits in fatty acid-dependent respiration and ETC enzyme activity. TEM revealed abnormalities of mitochondrial morphology characteristic of these functional deficits. The selective α1A-AR agonist A61603 enhanced oxidative metabolism in isolated cardiac mitochondria. The β-AR agonist isoproterenol enhanced oxidative stress in vitro and this adverse effect was mitigated by A61603. A61603 enhanced ETC Complex I activity and protected contractile function following myocardial infarction. Conclusions: Collectively, these novel findings position α1A-ARs as critical regulators of cardiomyocyte metabolism in the basal state and suggest that metabolic mechanisms may underlie the protective effects of α1A-AR activation in the failing heart.

In mammalian testes, premeiotic spermatogonia respond to retinoic acid by completing an essential lengthy differentiation program before initiating meiosis. The molecular and cellular changes directing these developmental processes remain largely undefined. This wide gap in knowledge is due to two unresolved technical challenges: (1) lack of robust and reliable in vitro models to study differentiation and meiotic initiation; and (2) lack of methods to isolate large and pure populations of male germ cells at each stage of differentiation and at meiotic initiation. Here, we report a facile in vitro differentiation and meiotic initiation system that can be readily manipulated, including the use of chemical agents that cannot be safely administered to live animals. In addition, we present a transgenic mouse model enabling fluorescence-activated cell sorting-based isolation of millions of spermatogonia at specific developmental stages as well as meiotic spermatocytes.

Clustering of obesity in families suggests that cultural inheritance, genetics, and epigenetics work together to affect children's eating habits. Emerging evidence in various species including Drosophila indicates that inter/transgenerational inheritance may play a significant role in programming offspring metabolism and behavior. In the current study, taking advantage of Drosophila physiological simplicity and genetic pliability we explored whether environmental factors, such as ancestral diet, can cause metabolic disturbances and alterations in offspring feeding behavior. Here, we demonstrate that ancestral Western diet increases offspring food consumption with concomitant changes in locomotor activity, triglyceride levels, and mitochondrial density in the brain. Mechanistically, the generational differences were associated with changes in brain proteome and miRNAs. RNAi-based knockdown approach implicated miR-10-3p as a potential factor in transmitting epigenetic information across generations. Together, this study suggests that ancestral nutrition may contribute to the programming of offspring feeding behavior through alterations in miRNAs.

Parental lifestyle and eating habits are largely responsible for childhood obesity epidemic. Clustering of obesity in families suggests that cultural inheritance, genetics, and epigenetics work together to affect children's eating habits. In the current study, we explored whether epigenetic factors, such as ancestral diet, can influence offspring feeding behavior by using a fruit fly model. Here, we demonstrate that ancestral caloric overload alters offspring feeding behavior via transgenerational inheritance, along with changes in activity, triglyceride levels, and mitochondrial density in the brain. Mechanistically, we found that the generational differences are associated with changes in brain proteome and miRNA. The findings identify ancestral nutrition as a critical factor in the generational programming of feeding behavior.

Skeletal muscle is a heterogeneous and dynamic tissue that adapts to functional demands and substrate availability by modulating muscle fiber size and type. The concept of muscle fiber type relates to its contractile (slow or fast) and metabolic (glycolytic or oxidative) properties. Here, we tested whether disruptions in muscle oxidative catabolism are sufficient to prompt parallel adaptations in energetics and contractile protein composition.Mice with defective mitochondrial long-chain fatty acid oxidation (mLCFAO) in the skeletal muscle due to loss of carnitine palmitoyltransferase 2 (Cpt2Differences in bioenergetics and macronutrient utilization in response to energy demands between control muscles were intrinsic to the mitochondria, allowing for a clear distinction of muscle types. Loss of CPT2 ablated mLCFAO and resulted in mitochondrial biogenesis occurring most predominantly in oxidative muscle fibers. The metabolism-related proteomic signature of Cpt2The loss of mitochondrial long-chain fatty acid oxidation elicits an adaptive response involving conversion of oxidative muscle toward a metabolic profile that resembles a glycolytic muscle, but this is not accompanied by changes in myosin heavy chain isoforms. These data suggest that shifts in muscle catabolism are not sufficient to drive shifts in the contractile apparatus but are sufficient to drive adaptive changes in metabolic properties.

Typified by oxidative phosphorylation (OXPHOS), mitochondria catalyze a wide variety of cellular processes seemingly critical for malignant growth. As such, there is considerable interest in targeting mitochondrial metabolism in cancer. However, notwithstanding the few drugs targeting mutant dehydrogenase activity, nearly all hopeful ‘mito-therapeutics’ cannot discriminate cancerous from non-cancerous OXPHOS and thus suffer from a limited therapeutic index. The present project was based on the premise that the development of efficacious mitochondrial-targeted anti-cancer compounds requires answering two fundamental questions: 1) is mitochondrial bioenergetics in fact different between cancer and non-cancer cells? and 2) If so, what are the underlying mechanisms? Such information is particularly critical for the subset of human cancers, including acute myeloid leukemia (AML), in which alterations in mitochondrial metabolism are implicated in various aspects of cancer biology (e.g., clonal expansion and chemoresistance). Herein, we leveraged an in-house diagnostic biochemical workflow to comprehensively evaluate mitochondrial bioenergetic efficiency and capacity in various hematological cell types, with a specific focus on OXPHOS dynamics in AML. Consistent with prior reports, clonal cell expansion, characteristic of leukemia, was universally associated with a hyper-metabolic phenotype which included increases in basal and maximal glycolytic and respiratory flux. However, despite having nearly 2-fold more mitochondria per cell, clonally expanding hematopoietic stem cells, leukemic blasts, as well as chemoresistant AML were all consistently hallmarked by intrinsic limitations in oxidative ATP synthesis (i.e., OXPHOS). Remarkably, by performing experiments across a physiological span of ATP free energy (i.e, ΔGATP), we provide direct evidence that, rather than contributing to cellular ΔGATP, leukemic mitochondria are particularly poised to consume ATP. Relevant to AML biology, acute restoration of OXPHOS kinetics proved highly cytotoxic to leukemic blasts, suggesting that active OXPHOS repression supports aggressive disease dissemination in AML. Taken together, these findings argue against ATP being the primary output of mitochondria in leukemia and provide proof-of-principle that restoring, rather than disrupting, OXPHOS and/or cellular ΔGATP in cancer may represent an untapped therapeutic avenue for combatting hematological malignancy and chemoresistance.

INTRODUCTION AND OBJECTIVES:Obesity increases the prevalence of bladder dysfunction in both males and females. The bladder’s mucosal and detrusor smooth muscle layers are involved in bladder signaling and contraction, respectively. The underlying causes of obesity-induced bladder dysfunction in males and females are poorly understood. Mitochondrial dysfunction is common in the heart, liver and skeletal muscle with high fat diet. This study aims to determine the impact of long-term high fat diet (HFD) on bladder mucosal and detrusor mitochondrial respiration in male and female mice.METHODS:Adult male and female mice were fed a control (10% kcal fat) or HFD (45% kcal fat; n=7/group) for 24 weeks. Bladders were collected, weighed and separated into mucosal and detrusor layers. Tissues were cut into strips, permeabilized, and placed in an OROBOROS Oxygraphy-2K to measure respiration. Substrates (pyruvate/malate, glutamate, L-octanoylcarnitine/malate, or succinate/rotenone) were added with increasing concentra...