Body mass, body composition and energy expenditure

At the beginning of HFD feeding, AKR/J and SWR/J mice were body weight matched. One day, after onset of HFD feeding, AKR/J mice weighed significantly more than SWR/J mice (figure 26A). During the following days, this distinct difference in body weight proceeded. After 3 days on HFD mean body mass gain was 3.3 ± 1.0 g in AKR/J, compared to 1.6 ± 0.5 g in SWR/J (figure 26B).

Figure 26: Body mass, body composition, energy intake and energy expenditure in AKR/J and SWR/J mice one day before and during first 3 days of high-fat diet feeding. After 4 weeks of adaptation to purified control diet (CD), body mass matched AKR/J and SWR/J mice were fed high-fat diet (HFD, plant-based with 48 kJ% of fat). (A) Body mass was measured daily. (B) Changes in body mass (bm), lean mass (lm) and fat mass (fm) during the first three days on high-fat diet were highlighted. Body composition was determined before and three days after start of high-fat diet feeding. (C) Energy intake was monitored daily in the feeding-drinking device. (D) Energy expenditure was measured by indirect calorimetry, shaded columns symbolize scotophase. Differences between strains were calculated using Student’s t-test and One-Way-ANOVA repeated measurement; *p < 0.05, ***p < 0.001; n=8-24.

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time on HFD [d]

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bm lm fm

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mass [g/3d]

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energy intake [kJ/24h]

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D

Body mass gain in AKR/J mice was mainly caused by an expansion of fat mass, whereas in SWR/J mice both lean and fat masses accounted for moderate body mass gain. Both mouse strains exhibited hyperphagia on HFD, with the most pronounced effect being evident after one day on HFD (figure 26C). Afterwards, elevated energy intake waned but had not reached CD levels after three days of HFD feeding. Differences between strains were only first observed one day after onset of HFD, with AKR/J mice (132 ± 16 kJ) showing higher energy intake than SWR/J mice (111 ± 12 kJ).

At the onset of HFD feeding, an instantaneous increase of energy expenditure during day and night was recorded for both strains (figure 26D). Differences in the diurnal pattern of energy expenditure between strains observed on CD (figure 21A) were maintained on HFD. AKR/J mice continued with a clear day-night-rhythm, whereas SWR/J mice showed a distinct peak in energy expenditure at the start of photophase. Therefore, despite a HFD induced increase in both strains, energy expenditure during photophase was higher in SWR/J mice than in AKR/J mice (figure 27A).

Figure 27: Measurement of energy expenditure in AKR/J and SWR/J mice one day before and during first 3 days of high-fat diet feeding. Energy expenditure was measured by indirect calorimetry in AKR/J and SWR/J mice (n=8). Daily energy expenditure (DEE) was divided in (A) photophase and (B) scotophase contribution. Based on measurement intervals with the lowest and highest oxygen consumption, respectively (C) resting metabolic rate (RMR) and (D) maximal metabolic rate (MMR) were calculated. (E) Mean of adjusted RMR, DEE and MMR of 24 h on control diet (CD) and of 3 days on high-fat diet (HFD) were calculated for both mouse strains, respectively. All energy expenditure data were adjusted to baseline regression (energy expenditure of control diet fed animals normalized to body mass). Differences between strains were calculated using Two-Way ANOVA and One-Way-ANOVA repeated measurement; *p < 0.05, **p < 0.01, ***p < 0.001, for statistics over time see appendix.

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Continuously rising energy expenditure during scotophase did not differ between strains (figure 27B). Independent of strain, RMR increased due to HFD feeding (figure 27C). AKR/J and SWR/J mice elevated RMR immediately after onset of HFD. On the following two days on HFD increasing RMR stagnated. Since the progress of RMR proceeded in parallel in both strains, elevation of RMR observed in CD fed SWR/J, compared to AKR/J mice (figure 25B), also persisted on HFD feeding. MMR was unaffected by diet change in AKR/J mice (figure 27D).

SWR/J mice increased MMR with ongoing HFD feeding, resulting in higher MMR compared to AKR/J mice on the third day on HFD. Building the mean of all three days on HFD, and comparing it to CD revealed an HFD-mediated elevation of DEE, due to an increased RMR independent in the strain (figure 27E). Higher RMR and DEE in SWR/J mice, compared to those in AKR/J mice on CD remained on HFD. MMR tended to increase more in SWR/J mice than in AKR/J mice.

3.3.2. Energy balance

To point out proximate causes of obesity, energy balance during the first three days of HFD feeding was assessed by analyzing the contributions of energy intake, energy expenditure and energy accumulation. Feeding HFD to body mass matched AKR/J and SWR/ mice led to body mass gain in both strains. Nevertheless, AKR/J mice fed HFD diet were always heavier than SWR/J mice and gained significantly more body mass, mainly due to fat mass expansion. HFD-induced hyperphagia occurred in both mouse strains, although the increased energy intake was more pronounced in AKR/J than in SWR/J mice (compare figure 26). Due to equally high assimilation efficiency on HFD for both strains (figure 33D), levels of adjusted metabolizable energy reflect those of energy intake (figure 28A). In the subset of mice used for indirect calorimetry measurements, adjusted energy expenditure before feeding HFD was significantly higher in SWR/J mice than in AKR/J mice (figure 28B). Mice of both strains increased energy expenditure continuously during HFD feeding. For a daily energy balance equation the difference of metabolizable energy and energy expenditure was calculated (figure 28C). Both mouse strains revealed a positive energy balance during high-fat diet feeding, which peaked on the first day of HFD. Adding together all three days of positive energy balance, AKR/J reached a surplus of 139.9 ± 11.5 kJ/3d and SWR/J of 104.3 ± 11.4 kJ/3d. The difference of excess energy between strains of about 36 kJ matched the body mass gain perfectly. Additionally, correlation of body mass increase and positive energy balance was highly significant (figure 28D). A simple calculation comparing energy balance and mass gain can explain differences in susceptibility to DIO in AKR/J and SWR/J mice: it is estimated that building up 1 g of fat mass corresponds to the storage of about 30 kJ (Wishnofsky 1958; Thomas 1962). Body mass gain can be divided into lean and fat mass increases. Since lean mass gain is similar in both strains, only the variance in

fat mass gain needs to be considered. The difference between strains of 1.4 g reflects approximately 42 kJ. Consequently, between AKR/J and SWR/J the difference of accumulation of fat mass (42 kJ) is almost equivalent to the difference of accumulated energy (36 kJ).

Figure 28: Metabolizable and expended energy during diet change. Food intake and energy expenditure were measured daily one day before and three days after changing diet from control to high-fat diet (HFD). Energy intake was calculated by multiplying food intake with the energy density of the respective diet. Energy intake reduced by the resorption efficiency and energy loss via urine resulted in (A) metabolizable energy. (B) Energy expenditure was measured by indirect calorimetry. (C) Energy balance is the result of the difference between metabolizable energy (Emet) and energy expenditure (DEE). Emet and DEE were adjusted to baseline regression. (D) Sum of positive energy during 3 days HFD is plotted against change of body mass within 3 days HFD. Differences between strains were calculated using One-Way-ANOVA repeated measurement; *p < 0.05, **p < 0.01.