Phenotypic sexual dimorphism in response to dietary fat manipulation in C57BL/6J mice

Abstract

Background: Obesity and the metabolic syndrome are increasingly prevalent in society and their complications and response to treatment exhibit sexual dimorphism. Mouse models of high fat diet-induced obesity are commonly used for both mechanistic and therapeutic studies of metabolic disease and diabetes. However, the inclusion of female mammals in obesity research has not been a common practice, and has resulted in a paucity of data regarding the effect of sex on metabolic parameters and its applicability to humans.

Methods: Here we analyzed male and female C57BL/6 J mice beginning at 4 weeks of age that were placed on a low-fat diet (LFD, 10% calories from fat), a Western Diet (WD, 45% calories from fat), or a high fat diet (HFD, 60% calories from fat). Assessments of body composition, glucose homeostasis, insulin production, and energy metabolism, as well as histological analyses of pancreata were performed.

Results: Both male and female C57BL/6 J mice had similar increases in total percent body weight gain with both WD and HFD compared to LFD, however, male mice gained weight earlier upon HFD or WD feeding compared to female mice. Male mice maintained their caloric food intake while reducing their locomotor activity with either WD or HFD compared to LFD, whereas female mice increased their caloric food intake with WD feeding. Locomotor activity of female mice did not significantly change upon WD or HFD feeding, yet female mice exhibited increased energy expenditure compared to WD or HFD fed male mice. Glucose tolerance tests performed at 4, 12 and 20 weeks of dietary intervention revealed impaired glucose tolerance that was worse in male mice compared to females. Furthermore, male mice exhibited an increase in pancreatic β cell area as well as reduced insulin sensitivity after HFD feeding compared to WD or LFD, whereas female mice did not.

Conclusions: Male and female C57BL/6 J mice exhibited strikingly different responses in weight, food consumption, locomotor activity, energy expenditure and β cell adaptation upon dietary manipulation, with the latter exhibiting less striking phenotypic changes. We conclude that the nature of these responses emphasizes the need to contextualize studies of obesity pathophysiology and treatment with respect to sex.

Keywords: C57BL/6J; High fat diet; Obesity; Sexual dimorphism; Western diet.

Figure 1:. High fat dietary challenge results in faster weight gain in male mice compared to females.

Male and female C57BL6/J mice were placed on an intervention diet starting at 4 weeks of age. (A) Weekly body weights of male mice; (B) Percent total weight gain in males at 22 weeks of dietary intervention; (C) Weekly body weights of female mice; (D) Percent total weight gain in females at 22 weeks of dietary intervention; (E) Male fat mass at 4, 12 and 20 weeks of dietary intervention; (F) Lean mass at 4, 12, and 20 weeks of dietary intervention in male mice; (G) Female fat mass at 4, 12 and 20 weeks of dietary intervention; (H) Female lean mass at 4, 12, and 20 weeks of dietary intervention; Results are displayed as means ± SEM; means were compared by one-way ANOVA followed by Tukey’s posttest; *, p<0.05, n=4–8.

Figure 2:. Food intake and activity levels are decreased in males challenged with diet induced obesity.

Male and female C57BL6/J mice were placed on a dietary intervention starting at 4 weeks of age. Food intake was monitored and activity was assessed by recording the number of beam breaks occurring in the x + y- and z-axes at 8 and 16 weeks of diet. (A) Food intake of male mice at 8 weeks of dietary intervention shown in grams/12 hr cycle (left panel) and as kcal/12 hr cycle (right panel); (B) Male locomotor activity during the light and dark cycles at 8 weeks of dietary intervention (48 hours depicted); (C). Male locomotor activity at 8 weeks of dietary intervention (total beam break counts in 24 hours); (D) Food intake of female mice at 8 weeks of dietary intervention shown in grams/12 hr cycle (left panel) and as kcal/12 hr cycle (right panel); (E) Female locomotor activity during the light and dark cycles at 8 weeks of dietary intervention (48 hours depicted); (F). Female locomotor activity at 8 weeks of dietary intervention (total beam break counts in 24 hours); (G) Food intake of male mice at 16 weeks of dietary intervention shown in grams/12 hr cycle (left panel) and as kcal/12 hr cycle (right panel); (H) Male locomotor activity during the light and dark cycles at 16 weeks of dietary intervention; (I) Male locomotor activity at 16 weeks of dietary; (J) Food intake of female mice at 16 weeks of dietary intervention shown in grams/12 hr cycle (left panel) and as kcal/12 hr cycle (right panel); (K) Female locomotor activity during the light and dark cycles at 16 weeks of dietary intervention; (L) Female locomotor activity at 16 weeks of dietary intervention. Horizontal white bars denote light cycles (0700 – 1900) and dark gray horizontal bars denote dark cycles (1900 – 0700). Results are displayed as means ± SEM; Means were compared by one-way ANOVA followed by Tukey’s posttest; *, p<0.05, n=9–10.

Figure 3:. High fat dietary intervention results in a reduction in RER in both male and female mice.

Male and female C57BL6/J mice were placed on a dietary intervention starting at 4 weeks of age and were singly housed in a PhenoMaster LabMaster indirect calorimeter. (A) Calculated RERs in male mice at 8 weeks of dietary intervention over a 48-hour period; (B) Calculated RERs in female mice at 8 weeks of dietary intervention over a 48-hour period. (C) Average RER values during the light or dark cycle in male mice; (D) Average RER values during the light or dark cycle in female mice. (E) RER values in male mice at 16 weeks of dietary intervention over a 48-hour period; (F) RER values in female mice at 16 weeks of dietary intervention over a 48-hour period; (G) Average RER values during the light or dark cycle in male mice; (H) Average RER values during the light or dark cycle in female mice at 16 weeks of diet; Energy expenditure was calculated in three 24 hour cycles at 8 or 16 weeks of dietary intervention (I) Energy expenditure in male mice at 8 weeks of diet (48 hour cycle shown); (J) Energy expenditure in female mice at 8 weeks of diet (48 hour cycle shown); (K) Average EE values during the light or dark cycle in male mice at 8 weeks of diet; (L) Average EE values during the light or dark cycle in female mice at 8 weeks of diet; (J) Energy expenditure in female mice at 16 weeks of diet (48 hour cycle shown); (K) Average EE values during the light or dark cycle in male mice at 16 weeks of diet; (L) Average EE values during the light or dark cycle in female mice at 16 weeks of diet; Horizontal white x-axes bars denote light cycles (0700 – 1900) and dark gray horizontal bars denote dark cycles (1900 – 0700). Results are displayed as means ± SEM; n=10 per treatment group for RER studies; n=5 per treatment group for EE studies; Means were compared by one-way ANOVA followed by Tukey’s posttest; *, p<0.05 for indicated comparisons.

Figure 4:. High dietary feeding worsens glucose tolerance in male C57BL6 mice compared to female mice.

Male and female mice were placed on a dietary intervention starting at 4 weeks of age for 4, 12, or 20 weeks before a GTT. At the specified week of dietary intervention, mice were fasted overnight and injected intraperitoneally with 2g/kg of glucose. (A) GTT after 2 g/kg glucose bolus in male mice at 4 weeks of dietary intervention; (B) Corresponding area under the curve from GTTs of male mice. (C) GTT of female mice at 4 weeks of dietary intervention; (D) Corresponding area under the curve from GTTs of female mice; (E) GTT of male mice at 12 weeks of dietary intervention; (F) Corresponding area under the curve from GTTs. (G) GTT of female mice at 12 weeks of dietary intervention; (H) Corresponding area under the curve from GTTs. (I) GTT of male mice at 20 weeks of dietary intervention; (J) corresponding area under the curve from GTTs; (K) GTT of female mice at 20 weeks of dietary intervention; (L) Corresponding area under the curve from GTTs. Results are displayed as means ± SEM; n=6–10 per treatment group; Means were compared by one-way ANOVA followed by Tukey’s posttest; *, p<0.05 for indicated comparisons.

Figure 5:. High fat feeding worsens insulin sensitivity in male mice.

Male and female mice were placed on a dietary intervention starting at 4 weeks of age and subjected to an ITT at 22 weeks of diet. Mice were injected intraperitoneally with 0.75 U/kg insulin, and blood glucose concentrations were measured over the course of 60 minutes. (A) ITT in male mice and (B) corresponding area over the curve analysis of ITT performed in male mice; (C) ITT in female mice and (D) corresponding area over the curve analysis of ITT performed in female mice. Means were compared by one-way ANOVA followed by Tukey’s posttest. Results are displayed as means ± SEM; n=5–8 per treatment group; Means were compared by one-way ANOVA followed by Tukey’s posttest; *, p<0.05 for indicated comparisons.

Figure 6:. Male mice demonstrate increased β-cell area in response to either a WD or HFD.

Male and female mice C57BL6/J mice were placed on a dietary intervention starting at 4 weeks of age. After 22 weeks of diet, mice were euthanized and pancreata harvested and stained for insulin. Left panel: Images of whole pancreatic sections from representative mice immunostained for insulin (brown) and counterstained with hematoxylin. Scale bars, 1000 μm. Right panel: Quantitation of pancreatic β-cell area in male mice (top) and in female mice (bottom); Results are displayed as means ± SEM; n=3–5 mice per treatment group. Means were compared by one-way ANOVA; *, p<0.05 for indicated comparisons.