Why should we allow to determine tremendous experimental flaws by the whole energy expenditure obese mouse scientific community using indirect mouse calorimetry (imc) while an alternative direct mouse calorimetry (dmc) is developed?
*Vincent Van Ginneken
Anatomy And Physiology, Blue Green Technologies, Netherlands
Vincent Van Ginneken
Anatomy And Physiology, Blue Green Technologies
Published on: 2017-02-20
In the Speakman editorial1, I would like to highlight on the opening sentence of this editorial: “Inference in Science depends on us having the right tools to measure with sufficient accuracy and precision the phenomena we are attempting to understand”. In studying the issue of obesity and its burden of chronic degenerative diseases among else type 2 diabetes2, we should open- minded without prejudice take into consideration the scientific goals we hope to achieve and that is measuring the energy expenditure of mouse models: more specific the Basal Metabolic Rate (BMR). This is an important parameter in energy expenditure of rodent models because it is a reflection of the minimal energy costs to stay a life and thus gives fundamental information to prescribe dietary energy needs of obese vs. lean subjects. Mouse models are important in Biomedical- and Life Sciences as outlined in the review3. Recently it has been stated that the genetically engineered mouse models for contemporary Obesity and/or Type-2 diabetes (T2DM) will in most cases deliver heavily metabolically abnormal animals due to changed gut micro-biotic with increased anaerobic CO2 production mainly due to food components like fructose and a high-fat diet4,5. Recent studies demonstrated that diet-induced obesity was linked to changes in the gut microbial ecology, resulting in an increased capacity of the distal gut (colon) microbiota to promote host adiposity due to an increased energy harvest from the diet. Food ingredients like fructose improve metabolic alterations associated with obesity, including: a). dyslipidaemia; b). impaired gut permeability; c). endotoxemia, d). inflammation due to a changed innate immune system resulting in the pathogenesis of metabolic disorders including T2DM, in increasing amounts of obese subjects (humans and/or mouse). An example are the genetically engineered mouse models for contemporary obesity and/or IR/T2DM6 which rely heavily on metabolically abnormal animals. Recent data implicating individual and group variation in the gut microbiota in obesity and diabetes raise important questions about transforming aerobic gas exchange into HP because 99% of gut bacteria are anaerobic and they outnumber eukaryotic cells in the body by ~10-fold4,5. This raises important questions about the allowance to use indirect calorimetry transforming aerobic gas exchange into Heat Production based on a conversion factor the Respiratory-Quotient (RQ-value: CO2/O2). A new perspective in this discussion (if nutritional intervention and its food ingredients are the cause for this generally accepted phenomenon of a CO2 overproduction in mouse models with metabolic disorders (obese, IR/T2DM)4,5, came from transplantation experiments of caecal microbiota from lean and obese mice in the gut of germ-free mice which definitely demonstrated that altered gut microbiota composition is a cause and not a consequence of obesity or altered dietary habits. High fat diet induced obese mouse models have due a changed gut microbiota for which recent data indicated 99% of gut bacteria are anaerobic and they outnumber eukaryotic cells in the body by ~10-fold according to recent studies4. Thus, an increased CO2 production of gut bacteria will result in tremendous experimental flaws by calculating the energy expenditure (RMR) or BMR based on many assumptions related to the conversion factor on gaseous exchange via the Respiratory Quotient (RQ)= [CO2]/[O2] to calculate back the indirect calorimetric measurements towards heat production. The most ideal situation would be a combination of both techniques of simultaneously direct- and indirect- mouse calorimetry together as depicted in Figure 1.
The first opening of this editorial reminding- in several articles by John Speakman almost convulsively held to the defense of the indirect mouse calorimetric measurements7,8 while the restrictions were quite clearly marked and a large discrepancy between indirect calorimetry and direct calorimetry has been measured in the studies9,10,11. In the editorial commented by Speakman1, it seems a concession has been done in the direction of direct calorimetry by the statement that both techniques have their own limitations. The so-called limitations which are mentioned in this editorial1 related to direct calorimetry such as: a). temperature difference of 2?C; b). implantation of telemetry transmitters, and c). no commercial direct calorimeters available, are all debatable.
The urgent compelling need to swap on a global scale from Indirect Mouse Calorimetry (IMC) towards Direct Mouse Calorimetry (DMC) in determining the Basal Metabolic Rate (BMR) has recently been described3. To determine the BMR of rodent models, studies have to be performed over a period of at least 24 hours under constant experimental conditions to eliminate handling stress. This can only be accomplished by a flow-through system, which requires high technology skills in order to reach acceptable signal to noise levels. In the review3, illustrated with our initial measurements of the basal metabolic rate by direct calorimetry in a "Swiss" albino mouse strain- we showed in a Calvet-type twin detection flow-through calorimeter that the determination of the BMR is technically possible by direct calorimetry by Pelletier elements and electrical calibration via a Joule effect. The flux-meter element consists of a ring of several thermocouples in series. The corresponding thermopile of high thermal conductivity surrounds the experimental space within the calorimetric block. The radial arrangement of the thermopiles around the stainless steel 1 liter calorimetric chamber guarantees an almost complete integration of the heat. The calibration of the calorimetric detectors is a key parameter and has to be performed very carefully by a mounted resistor in top of the calorimetric chamber. The system has a 1-liter stainless steel calorimetric chamber, a constant air flow of 1 liter per minute without affecting the stability of the baseline, a signal resolution of 50μW and a drift of 1.5mW per 24 h. The BMR of a single mouse of around 40g was ≈10Mw/g which corresponded to the value mentioned in literature of several wild mouse strains3. The most ideal situation would be to use this “state of the art” innovative technical tool for direct mouse calorimetry simultaneously with the common indirect calorimetry as depicted in Figure 1 in order to assist the research area of energy expenditure of mouse calorimetry to detect and measure the experimental flaws made by indirect calorimetry in order to calculate conversion factors. Because the mice commercial market for obesity research, life sciences and pharmaceuticals serves such huge fundamental-scientific-and commercial interests (Figure 2) at least conversion factors should be measured by simultaneously direct- and indirect calorimetry. Measuring the BMR in obese mouse models solely based on indirect calorimetry would give scientifically unreliable data.
- Speakman JR. Should we abandon indirect calorimetry as a tool to diagnose energy expenditure? Not yet. Perhaps not ever. Commentary on Burnett and Grobe. Mol Metab (2014) 3: 342-344.
- Ginneken V, Verheij E, Vries E, et al. The discovery of two novel biomarkers in a high-fat diet C56bl6 obese mouse model for non-adipose tissue: A comprehensive LC-MS study at hind limb, heart, carcass muscle, liver, brain, blood plasma and food composition following a lipidomics LCMS-based approach. Cell Mol Med (2016) 2: 3.
- Ginneken VV. The Urgent Compelling Need to Swap on a Global Scale from Indirect Mouse Calorimetry (IMC) Towards Direct Mouse Calorimetry (DMC) in Determining the Basal Metabolic Rate (BMR): A Principle of Proof Study. Anat Physiol (2016) 6: 223.
- Kaiyala KL, Ramsay DS. Review: Direct animal calorimetry, the underused gold standard for quantifying the fire of life. Comp Biochem Physiol (2011) 158: 252-264.
- Kaiyala KJ, Schwarts MW. Toward a More Complete (and Less Controversial) Understanding of Energy Expenditure and Its Role in Obesity Pathogenesis. Diabetes (2011) 60: 17-23.
- Rees DA, Alcolado JC. Animal models of diabetes mellitus. Diabet Med (2005) 22: 359-370.
- Tschöp MH, Speakman JR, Arch JRS, et al. A guide to analysis of mouse energy metabolism. Nature Methods (2012) 9: 57-63.
- Speakman JR. Measuring energy metabolism in the mouse-theoretical, practical, and analytical considerations. Front Physiol (2013) 4: 34.
- Walsberg GE, Hoffman TC. Direct calorimetry reveals large errors in respirometric estimates of energy expenditure. J Exp Biol (2005) 208: 1035-1043.
- Burnett CM, Grobe JL. Direct calorimetry identifies deficiencies in respirometry for the determination of resting metabolic rate in C57Bl/6 and FVB mice. Am J Physiol Endocrinol Metab. (2013) 305: e916-e924.
- Burnett CM, Grobe JL. Dietary effects on resting metabolic rate in C57BL/6 mice are differentially detected by indirect (O2/CO2 respirometry) and direct calorimetry. Mol Met (2004) 3: 460-464.
- EU-EXP-Animal. Fifth Report on the Statistics on the Number of Animals used for Experimental and other Scientific Purposes in the Member States of the European Union, Commission of the European Communities. 2007.