Reduced contextually induced muscle thermogenesis in rats with calorie restriction and lower aerobic fitness but not monogenic obesity.

We have previously identified predator odor as a potent stimulus activating thermogenesis in skeletal muscle in rats. As this may prove relevant for energy balance and weight loss, the current study investigated whether skeletal muscle thermogenesis was altered with negative energy balance, obesity propensity seen in association with low intrinsic aerobic fitness, and monogenic obesity. First, weight loss subsequent to 3 wk of 50% calorie restriction suppressed the muscle thermogenic response to predator odor. Next, we compared rats bred based on artificial selection for intrinsic aerobic fitness - high- and low-capacity runners (HCR, LCR) - that display robust leanness and obesity propensity, respectively. Aerobically fit HCR showed enhanced predator odor-induced muscle thermogenesis relative to the less-fit LCR. This contrasted with the profound monogenic obesity displayed by rats homozygous for a loss of function mutation in Melanocortin 4 receptor (Mc4rK3a,4X/K314X rats), which showed no discernable deficit in thermogenesis. Taken together, these data imply that body size or obesity per se are not associated with deficient muscle thermogenesis. Rather, the physiological phenotype associated with polygenic obesity propensity may encompass pleiotropic mechanisms in the thermogenic pathway. Adaptive thermogenesis associated with weight loss also likely alters muscle thermogenic mechanisms.

Measuring Skeletal Muscle Thermogenesis in Mice and Rats.

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature. Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli. This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Skeletal muscle thermogenesis induction by exposure to predator odor.

Non-shivering thermogenesis can promote negative energy balance and weight loss. In this study, we identified a contextual stimulus that induces rapid and robust thermogenesis in skeletal muscle. Rats exposed to the odor of a natural predator (ferret) showed elevated skeletal muscle temperatures detectable as quickly as 2 min after exposure, reaching maximum thermogenesis of >1.5°C at 10-15 min. Mice exhibited a similar thermogenic response to the same odor. Ferret odor induced a significantly larger and qualitatively different response from that of novel or aversive odors, fox odor or moderate restraint stress. Exposure to predator odor increased energy expenditure, and both the thermogenic and energetic effects persisted when physical activity levels were controlled. Predator odor-induced muscle thermogenesis is subject to associative learning as exposure to a conditioned stimulus provoked a rise in muscle temperature in the absence of the odor. The ability of predator odor to induce thermogenesis is predominantly controlled by sympathetic nervous system activation of ß-adrenergic receptors, as unilateral sympathetic lumbar denervation and a peripherally acting ß-adrenergic antagonist significantly inhibited predator odor-induced muscle thermogenesis. The potential survival value of predator odor-induced changes in muscle physiology is reflected in an enhanced resistance to running fatigue. Lastly, predator odor-induced muscle thermogenesis imparts a meaningful impact on energy expenditure as daily predator odor exposure significantly enhanced weight loss with mild calorie restriction. This evidence signifies contextually provoked, centrally mediated muscle thermogenesis that meaningfully impacts energy balance.

Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells.

Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also known as SWELL1, subunit of the VRAC channel. The second goal is to determine if an increase in the number of VRAC channels at the cell membrane is responsible for an increase in chloride permeability of the membrane in two well-known cases: during staurosporine (STS)-induced apoptosis and after water balance disturbance caused by hypotonic medium. Human suspension lymphoid cells U937 were used as they are suitable for flow fluorometry and because we have recently studied their membrane chloride permeability during apoptosis. We found that surface expression of endogenous LRRC8A subunits can be quantified in living U937 cells using flow fluorometry with the Alomone Lab antibody. Further, we revealed that treatment of cells for 1 hour using STS or a hypotonic solution did not change the number of LRRC8A subunits to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis. This indicates that prolonged increase in chloride permeability of the cell membrane during apoptotic cell shrinkage or cell volume regulation under hypotonicity in U937 cells occurs without altering cell surface expression of VRAC.

Adult Brain Serotonin Deficiency Causes Hyperactivity, Circadian Disruption, and Elimination of Siestas.

UNLABELLED: Serotonin (5-HT) is a crucial neuromodulator linked to many psychiatric disorders. However, after more than 60 years of study, its role in behavior remains poorly understood, in part because of a lack of methods to target 5-HT synthesis specifically in the adult brain. Here, we have developed a genetic approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system by stereotaxic injection of an adeno-associated virus expressing Cre recombinase (AAV-Cre) into the midbrain/pons of mice carrying a loxP-conditional tryptophan hydroxylase 2 (Tph2) allele. We investigated the behavioral effects of deficient brain 5-HT synthesis and discovered a unique composite phenotype. Surprisingly, adult 5-HT deficiency did not affect anxiety-like behavior, but resulted in a robust hyperactivity phenotype in novel and home cage environments. Moreover, loss of 5-HT led to an altered pattern of circadian behavior characterized by an advance in the onset and a delay in the offset of daily activity, thus revealing a requirement for adult 5-HT in the control of daily activity patterns. Notably, after normalizing for hyperactivity, we found that the normal prolonged break in nocturnal activity (siesta), a period of rapid eye movement (REM) and non-REM sleep, was absent in all animals in which 5-HT deficiency was verified. Our findings identify adult 5-HT as a requirement for siestas, implicate adult 5-HT in sleep-wake homeostasis, and highlight the importance of our adult-specific 5-HT-synthesis-targeting approach in understanding 5-HT's role in controlling behavior. SIGNIFICANCE STATEMENT: Serotonin (5-HT) is a crucial neuromodulator, yet its role in behavior remains poorly understood, in part because of a lack of methods to target specifically adult brain 5-HT synthesis. We developed an approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system. Using this technique, we discovered that adult 5-HT deficiency led to a novel compound phenotype consisting of hyperactivity, disrupted circadian behavior patterns, and elimination of siestas, a period of increased sleep during the active phase. These findings highlight the importance of our approach in understanding 5-HT's role in behavior, especially in controlling activity levels, circadian behavior, and sleep-wake homeostasis, behaviors that are disrupted in many psychiatric disorders such as attention deficit hyperactivity disorder.