Low-gainer diet-induced obese microbiota transplanted mice exhibit increased fighting

Abstract

Weight gain variation is a great challenge in diet-induced obesity studies since low-gainer animals are of limited experimental value. The inbred C57BL/6 (B6) mice are frequently used models due to their genetic homogeneity and susceptibility to diet-induced obesity (DIO). The aim of this study is to investigate if the gut microbiota (GM) influences the fraction of low weight gainers in DIO studies. A total of 100 male B6 mice (donor population) were fed a high-fat diet for 14 weeks and divided into the study groups high gainer (HG) and low gainer (LG) based on their weight gain. Subsequently, fecal matter transplantation (FMT) was done on germ-free B6 mice with GM from HG and LG donors (FMT population). LG (13.35 ± 2.5 g) and HG (25.52 ± 2.0 g) animals were identified by the weight gain from week 1 to week 12. Interestingly, the start weight of the LG (20.36 ± 1.4 g) and HG (21.59 ± 0.7 g) groups differed significantly. Transplanting LG or HG fecal matter to germ-free mice resulted in significant differences in weight gain between HG and LG, as well as differences in serum leptin levels and epididymal fat pad weight. A clear LG-specific GM composition could not be distinguished by 16S rRNA gene amplicon sequencing. Surprisingly, significantly more fighting was recorded in LG groups of both donor populations and when transplanted to germ-free mice. The HG and LG phenotypes could be transferred to germ-free mice. The increased fighting in the LG group in both studies suggests not only that the tendency to fight can be transferred by FMT in these mice, but also that fighting should be prevented in DIO studies to minimize the number of LG animals.

FIGURE 1

Overview of the study design and timeline of Donor population and FMT studies.

FIGURE 2

The male donor population. (a) Weight gain distribution in the total donor population n = 100. (b) Total weight gain in the selected donor population study groups: High gainer (HG, n = 12), middle gainer (MG, n = 12) and low gainer (LG, n = 12). (c) Initial BW upon arrival from a breeder in the three study groups. (d) Weight of epididymal adipose tissue (EWAT) in the three study groups at study termination (16 weeks HFD). (e) Blood glucose measured at study week 14 in the three study groups.

FIGURE 3

The FMT population (N = 60) with the groups receiving GM from HG donors (n = 31) and LG donors (n = 29). Cofactors include sex (n = 34 males, n = 26 females) and Origin (born in isolators or in conventional housing unit (n = 30 ISO and n = 30 CONV). (a) Weight gain distribution in the FMT population n = 60. (b) Total mouse weight gain after 13 weeks of HFD. (c) Weight of epididymal adipose tissue (EWAT) in the males at study termination. (d) Serum leptin levels in the males at study termination. (e) Ileum IFNG at study termination.

FIGURE 4

Gut microbiota analysis in the FMT study, consisting of both males and females. (a) Beta‐diversity PCoA plot based on the unweighted UniFrac distance matrix presenting groups LG and HG at the beginning of the study before HFD (week 1). (b) Beta‐diversity PCoA plot based on the unweighted UniFrac distance matrix presenting groups LG and HG at the end of the study after 13 weeks of HFD. (c) Heatmap presenting significantly differentially abundant bacteria between HG and LG at study start before HFD. (d) Heatmap presenting significantly differentially abundant bacteria between HG and LG at the following 13 weeks of HFD.

FIGURE 5

Fighting recorded in the donor population and the FMT study and its effect on weight gain. (a) Frequency of fighting (YES or NO) in the three study groups in the donor population (n = 12). Counts from the contingency table are reported on the bars. (b) Total weight gain of the animals in selected study groups in the donor population when divided into fighters (YES, n = 11) and non‐fighters (NO, n = 25). (c) Frequency of fighting in males from the FMT study. Counts from the contingency table is reported on the bars. (d) Total weight gain of the male FMT animals when divided into fighters (YES, n = 7) and non‐fighters (NO, n = 27).