Identification of body fat mass as a major determinant of metabolic rate in mice - PubMed (original) (raw)

. 2010 Jul;59(7):1657-66.

doi: 10.2337/db09-1582. Epub 2010 Apr 22.

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Identification of body fat mass as a major determinant of metabolic rate in mice

Karl J Kaiyala et al. Diabetes. 2010 Jul.

Abstract

Objective: Analysis of energy expenditure (EE) in mice is essential to obesity research. Since EE varies with body mass, comparisons between lean and obese mice are confounded unless EE is normalized to account for body mass differences. We 1) assessed the validity of ratio-based EE normalization involving division of EE by either total body mass (TBM) or lean body mass (LBM), 2) compared the independent contributions of LBM and fat mass (FM) to EE, and 3) investigated whether leptin contributes to the link between FM and EE.

Research design and methods: We used regression modeling of calorimetry and body composition data in 137 mice to estimate the independent contributions of LBM and FM to EE. Subcutaneous administration of leptin or vehicle to 28 obese ob/ob mice and 32 fasting wild-type mice was used to determine if FM affects EE via a leptin-dependent mechanism.

Results: Division of EE by either TBM or LBM is confounded by body mass variation. The contribution of FM to EE is comparable to that of LBM in normal mice (expressed per gram of tissue) but is absent in leptin-deficient ob/ob mice. When leptin is administered at physiological doses, the plasma leptin concentration supplants FM as an independent determinant of EE in both ob/ob mice and normal mice rendered leptin-deficient by fasting.

Conclusions: The contribution of FM to EE is substantially greater than predicted from the metabolic cost of adipose tissue per se, and the mechanism underlying this effect is leptin dependent. Regression-based approaches that account for variation in both FM and LBM are recommended for normalization of EE in mice.

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Figures

FIG. 1.

FIG. 1.

Limitations inherent in the use of traditional ratio methods for normalizing EE to TBM or LBM in mice fed either standard food (○, n = 89) or a HFD (●, n = 48). Both average (A and B) and minimum (G and H) EE vary as linear functions of TBM or LBM. Because the regression lines characterizing these relationships have positive intercepts, normalizing EE as a simple ratio of total or lean body mass (EE/TBM or EE/LBM) yield negative nonlinear associations between the normalized values and body mass compartments (D, E, J, and K). Consequently, the normalized EE values decrease with increasing body mass irrespective of whether or not the heavier mice actually have lower standardized EE values when EE is standardized to mass in a way that demonstrably controls for the influence of mass variation. (See

research design and methods

for the mathematical premise underlying this analysis). C and I depict the positive association between EE and FM across studies, while F and L demonstrate that the relationship between EE and FM remains highly significant even after accounting for the contribution of LBM to each trait, indicating that FM predicts EE independently of LBM in these mice. Interpretation: Within diet groups, commonly used ratio normalization methods spuriously assign a more efficient (lower) metabolic rate phenotype to larger animals. In addition, between diet groups, these ratio-based normalization methods favor assignment of an elevated metabolic rate phenotype to the HFD-fed heavier mice (and this bias is magnified when LBM is used in the ratio) because the increase of EE is disproportionate to the increase of LBM, as would be predicted if FM exerts an independent positive effect on EE.

FIG. 2.

FIG. 2.

Relationship between EE and FM residuals (the components of EE and FM that are not explained by LBM) within eight subgroups defined by project number and genotype. ○, Chow-fed mice; ■, HFD-fed mice. The top two rows show the relationship between average EE and FM residuals depicted in Fig. 1_F_ stratified by project and genotype, and the bottom two rows do likewise for the minimum EE and FM residuals depicted in Fig. 1_L_. The LBM-adjusted EE versus LBM-adjusted FM associations are positive and significant in all subgroups except for those involving project 3, which entailed small sample sizes (n = 8 chow, n = 8 HFD). Multiple regression models for EE as a function of LBM, FM, sex, diet, activity, and membership in each subgroup defined by project and genotype revealed that FM is a quantitatively important and highly significant determinant of murine EE (see text). Interpretation: FM is strongly associated with EE, even after controlling for the relationship between each of these traits and LBM.

FIG. 3.

FIG. 3.

Bivariate analyses indicate that caloric EE is positively related to LBM and to FM in ob/ob mice. Bivariate associations of average 24-h EE, LBM, FM, and plasma leptin levels in 28 ob/ob mice that were studied first at baseline and then again during continuous subcutaneous infusion of either saline (n = 7) or a dose of leptin intended to achieve physiological replacement (n = 21) (range of plasma leptin achieved is shown in D–F). A: EE was positively correlated with LBM in both the absence and presence of leptin administration, although the slope of this relationship was steeper in the latter setting, consistent with a leptin-mediated increase of metabolic cost per unit LBM. B: Highly significant co-variation between LBM and FM occurred in both the presence and absence of leptin, which confounds the analysis of relationships between EE and tissue compartments and hence illustrates the need for proper statistical control. C: EE was positively associated with FM before leptin replacement (but the effect was not significant after statistical adjustment; Table 2). D: EE varied directly with plasma leptin levels, but this association did not reach significance (this association became highly significant when multiple regression analysis was used to account for variation in tissue compartment masses; Table 2). E: As expected, leptin treatment was negatively related to the change from the baseline study in FM such that higher leptin concentrations were associated with greater FM loss. F: Although mean LBM increased among ob/ob mice receiving subcutaneous leptin, higher plasma leptin levels were associated with a tendency to limit LBM gain. Interpretation: Basic bivariate analyses indicate that the slope of EE on LBM was higher in animals receiving leptin, consistent with an effect of leptin to augment energy expenditure in this tissue (confirmed in Table 2). EE was positively related to plasma leptin levels, consistent with an effect of leptin to augment metabolic rate (confirmed in Table 2). S.C., subcutaneous.

FIG. 4.

FIG. 4.

Leptin replacement in the physiological range determines caloric EE in fasting mice. A: Scatterplots of average EE values recorded during the last 24-h period of a 36-h fast versus body mass compartments in wild-type mice receiving continuous subcutaneous infusion of either saline (n = 8) or a physiological dose of leptin (n = 24). B: Association of average EE values with leptin concentrations in plasma obtained from the same mice. Interpretation: Bivariate analyses indicates that EE in fasted WT mice receiving leptin replacement in the physiological range is more determined by the plasma leptin level than by LBM or FM (confirmed in Table 4).

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