How much does it cost? Teaching physiology of energy metabolism in mice using an indirect calorimetry system in a practical course for veterinary students

In endothermic mammals total energy expenditure (EE) is composed of basal metabolic rate (BMR), energy spent for muscle activity, thermoregulation, any kind of production (such as milk, meat or egg production) and the thermic effect of feeding. The BMR is predominantly determined by body mass and the surface to volume ratio of the body. The EE can be quantified either by direct or indirect calorimetry. Direct calorimetry measures the rate of heat loss from the body, whereas indirect calorimetry measures oxygen consumption and carbon dioxide production and calculates heat production from oxidative nutrient combustion. A deep and sustainable understanding of EE in animals is crucial for veterinarians in order to properly calculate and evaluate feed rations, during special circumstances such as anaesthesia or in situations with increased energy demands as commonly seen in high yielding livestock. The practical class described in this manuscript provides an experimental approach to understand how EE can be measured and calculated by indirect calorimetry. Two important factors that affect the EE of animals (the thermic effect of feeding and the effect of ambient temperature) are measured. A profound knowledge about the energy requirements of animal life and its measurement is also relevant for education in general biology, animal and human physiology and nutrition. Therefore, this teaching unit can equally well be implemented in other areas of life sciences.

Grape Polyphenols Attenuate Diet-Induced Obesity and Hepatic Steatosis in Mice in Association With Reduced Butyrate and Increased Markers of Intestinal Carbohydrate Oxidation

A Western Diet (WD) low in fiber but high in fats and sugars contributes to obesity and non-alcoholic fatty liver disease (NAFLD). Supplementation with grape polyphenols (GPs) rich in B-type proanthocyanidins (PACs) can attenuate symptoms of cardiometabolic disease and alter the gut microbiota and its metabolites. We hypothesized that GP-mediated metabolic improvements would correlate with altered microbial metabolites such as short chain fatty acids (SCFAs). To more closely mimic a WD, C57BL/6J male mice were fed a low-fiber diet high in sucrose and butterfat along with 20% sucrose water to represent sugary beverages. This WD was supplemented with 1% GPs (WD-GP) to investigate the impact of GPs on energy balance, SCFA profile, and intestinal metabolism. Compared to WD-fed mice, the WD-GP group had higher lean mass along with lower fat mass, body weight, and hepatic steatosis despite consuming more calories from sucrose water. Indirect and direct calorimetry revealed that reduced adiposity in GP-supplemented mice was likely due to their greater energy expenditure, which resulted in lower energy efficiency compared to WD-fed mice. GP-supplemented mice had higher abundance of Akkermansia muciniphila, a gut microbe reported to increase energy expenditure. Short chain fatty acid measurements in colon content revealed that GP-supplemented mice had lower concentrations of butyrate, a major energy substrate of the distal intestine, and reduced valerate, a putrefactive SCFA. GP-supplementation also resulted in a lower acetate:propionate ratio suggesting reduced hepatic lipogenesis. Considering the higher sucrose consumption and reduced butyrate levels in GP-supplemented mice, we hypothesized that enterocytes would metabolize glucose and fructose as a replacement energy source. Ileal mRNA levels of glucose transporter-2 (GLUT2, SLC2A2) were increased indicating higher glucose and fructose uptake. Expression of ketohexokinase (KHK) was increased in ileum tissue suggesting increased fructolysis. A GP-induced increase in intestinal carbohydrate oxidation was supported by: (1) increased gene expression of duodenal pyruvate dehydrogenase (PDH), (2) a decreased ratio of lactate dehydrogenase a (LDHa): LDHb in jejunum and colon tissues, and (3) decreased duodenal and colonic lactate concentrations. These data indicate that GPs protect against WD-induced obesity and hepatic steatosis by diminishing portal delivery of lipogenic butyrate and sugars due to their increased intestinal utilization.

Gut Microbiota Represent a Major Thermogenic Biomass

Evidence supports various roles for microbial metabolites in the control of multiple aspects of host energy flux including feeding behaviors, digestive efficiency, and energy expenditure, but few studies have quantified the energy utilization of the biomass of the gut microbiota itself. Because gut microbiota exist in an anoxic environment, energy flux is expected to be anaerobic; unfortunately, commonly-utilized O2/CO2 respirometry-based approaches are unable to detect anaerobic energy flux. To quantify the contribution of the gut microbial biomass to whole-animal energy flux, we examined the effect of surgical reduction of gut biomass in C57BL/6J mice via cecectomy and assessed energy expenditure using methods sensitive to anaerobic flux, including bomb and direct calorimetry. First, we determined that cecectomy caused an acceleration of weight gain over several months due to a reduction in combined total host plus microbial energy expenditure, as reflected by an increase in energy efficiency (i.e. weight gained per calorie absorbed). Second, we determined that under general anesthesia, cecectomy caused immediate changes in heat dissipation that were significantly modified by short-term pretreatment with dietary or pharmaceutical interventions known to modify the microbiome, and confirmed that these effects were undetectable by respirometry. We conclude that while the cecum only contributes approximately 1% of body mass in the mouse, this organ contributes roughly 8% of total resting energy expenditure, that this contribution is predominantly anaerobic, and that the composition and abundance of the cecal microbial contents can significantly alter its contribution to energy flux.

Estimating Physical Activity Energy Expenditure Using an Ensemble Model-Based Patch-Type Sensor Module

Chronic diseases, such as coronary artery disease and diabetes, are caused by inadequate physical activity and are the leading cause of increasing mortality and morbidity rates. Direct calorimetry by calorie production and indirect calorimetry by energy expenditure (EE) has been regarded as the best method for estimating the physical activity and EE. However, this method is inconvenient, owing to the use of an oxygen respiration measurement mask. In this study, we propose a model that estimates physical activity EE using an ensemble model that combines artificial neural networks and genetic algorithms using the data acquired from patch-type sensors. The proposed ensemble model achieved an accuracy of more than 92% (Root Mean Squared Error (RMSE) = 0.1893, R2 = 0.91, Mean Squared Error (MSE) = 0.014213, Mean Absolute Error (MAE) = 0.14020) by testing various structures through repeated experiments.

The Hexoskin physiological monitoring shirt does not impair whole-body heat loss during exercise in hot-dry conditions

Using direct calorimetry, we determined if the Hexoskin shirt (Carré Technologies Inc., Que., Canada), a wearable device for monitoring physiological strain, would compromise whole-body heat loss and exacerbate body heat storage during moderate-intensity activity in hot-dry conditions. The shirt did not impair heat dissipation and resulted in similar body heat storage when worn alone relative to a semi-nude condition (214 vs. 211 kJ) or when worn underneath a work uniform compared with a cotton undershirt (307 vs. 318 kJ).

Partitional calorimetry

For thermal physiologists, calorimetry is an important methodological tool to assess human heat balance during heat or cold exposures. A whole body direct calorimeter remains the gold standard instrument for assessing human heat balance; however, this equipment is rarely available to most researchers. A more widely accessible substitute is partitional calorimetry, a method by which all components of the conceptual heat balance equation—metabolic heat production, conduction, radiation, convection, and evaporation—are calculated separately based on fundamental properties of energy exchange. Since partitional calorimetry requires relatively inexpensive equipment (vs. direct calorimetry) and can be used over a wider range of experimental conditions (i.e., different physical activities, laboratory or field settings, clothed or seminude), it allows investigators to address a wide range of problems such as predicting human responses to thermal stress, developing climatic exposure limits and fluid replacement guidelines, estimating clothing properties, evaluating cooling/warming interventions, and identifying potential thermoregulatory dysfunction in unique populations. In this Cores of Reproducibility in Physiology (CORP) review, we summarize the fundamental principles underlying the use of partitional calorimetry, present the various methodological and arithmetic requirements, and provide typical examples of its use. Strategies to minimize estimation error of specific heat balance components, as well as the limitations of the method, are also discussed. The goal of this CORP paper is to present a standardized methodology and thus improve the accuracy and reproducibility of research employing partitional calorimetry.

Measuring Metabolic Rates

Measuring Metabolic Rates demystifies the field of metabolic rate measurement, explaining every common variation of the art, from century-old manometric methods through ingenious syringe-based techniques, direct calorimetry, aquatic respirometry, stable-isotope metabolic measurement, and every type of flow-through respirometry. Each variation is described in enough detail to allow it to be applied in practice. Special chapters are devoted to metabolic phenotyping and human metabolic measurement, including room calorimetry. Background information on different analyzer and equipment types allows users to choose the best instruments for their application. Respirometry equations—normally a topic of terror and confusion to researchers—are derived and described in enough detail to make their selection and use effortless. Tools and skills—many of them open source—that will amplify the innovative researcher’s capabilities are described. Vital topics such as manual and automated baselining, implementing multi-animal systems, common pitfalls, and the correct analysis and presentation of metabolic data are covered in enough detail to turn a respirometry neophyte into a hardened metabolic warrior, ready to take on the task of publication in peer-reviewed journals with confidence.