Sex differences in microglial CX3CR1 signalling determine obesity susceptibility in mice

Abstract

Female mice are less susceptible to the negative metabolic consequences of high-fat diet feeding than male mice, for reasons that are incompletely understood. Here we identify sex-specific differences in hypothalamic microglial activation via the CX3CL1-CX3CR1 pathway that mediate the resistance of female mice to diet-induced obesity. Female mice fed a high-fat diet maintain CX3CL1-CX3CR1 levels while male mice show reductions in both ligand and receptor expression. Female Cx3cr1 knockout mice develop 'male-like' hypothalamic microglial accumulation and activation, accompanied by a marked increase in their susceptibility to diet-induced obesity. Conversely, increasing brain CX3CL1 levels in male mice through central pharmacological administration or virally mediated hypothalamic overexpression converts them to a 'female-like' metabolic phenotype with reduced microglial activation and body-weight gain. These data implicate sex differences in microglial activation in the modulation of energy homeostasis and identify CX3CR1 signalling as a potential therapeutic target for the treatment of obesity.

Figure 1. Female mice are protected from diet-induced obesity and hypothalamic microglial activation.

(a,b) Body-weight gain (a) and cumulative food intake (b) in male and female mice exposed to chow or HFD for 18 weeks. (c,d) Total fat mass (c) and lean mass (d) in chow-fed and HFD groups. See also Supplementary Fig. 1. (e) Intraperitoneal glucose tolerance test (2 g kg⁻¹) in male and female mice after 18 weeks on chow or HFD. (f) Glucose AUC analysis of data from e. M, males; F, females. Data are presented as mean±s.e.m. of 12 males and 8 females per group in a–f. (g,h) mRNA levels of inflammatory markers in isolated hypothalamic microglia following 3-mo chow or HFD exposure (g: males; h: females). Data are presented as % change relative to chow-fed controls. Mean±s.e.m., n=8. *P<0.05 by Student's t-test comparing within gender (i) CX3CL1 protein expression analysed by ELISA in homogenized hypothalamus dissected from male and female mice maintained on chow or HFD for 18 weeks. Data are presented as mean±s.e.m., n=8. (j,k) mRNA level of Cx3cr1 in isolated hypothalamic microglia following 3-mo chow or HFD exposure. Data are presented as per cent change relative to chow-fed controls. Mean±s.e.m., n=8. *P<0.05 by Student's t-test comparing within gender. For all panels (except g,h,j,k), data are analysed by repeated measures or two-way ANOVA followed by Bonferroni post hoc comparisons. *P<0.05, **P<0.01 and ***P<0.001.

Figure 2. Cx3cr1-deficient mice exhibit a female-specific obesity phenotype.

(a–d) Body-weight gain (a,b) and cumulative food intake (c,d) measured in Cx3cr1-HT and KO mice on chow and HFD for 18 weeks (a,c: males; b,d: females). (e–h) Total fat mass (e,f) and lean mass (g,h) measured at study end in HT and KO mice (e,g: males; f,h: females). (i–k) Average adjusted heat production during 12h light cycle (i), 12h dark cycle (j) and both photoperiods combined (total) (k) in HT and KO female mice exposed to HFD for 18 weeks. Heat production was adjusted using normalization to lean mass+0.2 × fat mass (see ‘Methods' section). (l,m) Intraperitoneal glucose tolerance test (2 g kg⁻¹) in HT and KO mice on chow and HFD for 18 weeks (l: males; m: females). (n,o) Glucose AUC analysis of data from (l,m), respectively. Data are presented as mean±s.e.m. of 12 males and 8 females per group for all panels except (i–k) where n=8 females per group. For all panels, data are analysed by repeated measures or two-way ANOVA followed by Bonferroni post hoc comparisons. *P<0.05, **P<0.01 and ***P<0.001 versus chow groups; ^#P<0.05, ^##P<0.01, ^###P<0.001 HT-HFD compared to HT chow. ^†P<0.05 and ^†††P<0.001 KO-HFD versus HT-HFD.

Figure 3. Cx3cr1-deficient female mice display HFD-induced hypothalamic microglial accumulation and activation.

(a–d,f–i) Representative images showing Iba1 immunoreactivity in the MBH of male (a–d) and female mice (f–i). 3V=third ventricle. (e,j) Quantification of Iba1-positive cells in bilateral MBH from six sections per animal. Mean±s.e.m., n=4 per group. Scale bar, 50 μm. (k,l) Representative images showing microglial morphology in the MBH of HT and KO females. Scale bar, 10 μm. (m) Quantification of total dendritic length in MBH from 10 individual microglial cells per animal. Mean±s.e.m., n=4 per group. (n) mRNA levels of inflammatory markers in isolated hypothalamic microglia following 12-week HFD exposure in HT and KO female mice. Data are presented as per cent change relative to HT-chow controls. Mean±s.e.m., n=5–7 per group. For all panels, data are analysed by two-way ANOVA followed by Bonferroni post hoc comparisons. *P<0.05.

Figure 4. Central administration and hypothalamic viral overexpression of CX3CL1 limits HFD-induced weight gain in males.

(a,b) Body-weight gain (a) and cumulative food intake (b) in HFD-fed Cx3cr1 KO and HT male mice infused ICV with CX3CL1 (500 ng per day) over 12 days. F(11,154)=3.14, P=0.0008 for genotype × time interaction using repeated measures ANOVA. Mean±s.e.m., n=6–10 per group. (c,d) Representative images showing Iba1 immunoreactivity in the MBH of KO (c) and HT male mice (d) centrally treated with CX3CL1 for 12 days. Scale bar, 50 μm. (e) Quantification of Iba1-positive cells in bilateral MBH from six sections per animal. Mean±s.e.m., n=4 per group. (f) Body-weight change in 3-month DIO WT males injected ICV daily with CX3CL1 (1 μg per day) or vehicle (saline) over 28 days. (g) Total fat mass measured 2 days before and after 3 weeks of ICV injections (day 22). (h) Cumulative food intake measured over 28 days of ICV vehicle or CX3CL1 treatment. Mean±s.e.m. of eight animals per group in (f–h). (i,j) Representative images of the MBH from mice injected unilaterally for validation with AAV-GFP (i) or AAV-CX3CR1-HA (j). Scale bar, 50 μm. (k,l) Body-weight gain (k) and cumulative food intake (l) in HFD-fed mice injected bilaterally in the MBH with AAV-GFP or AAV-CX3CL1-HA. Mean±s.e.m. of nine animals per group. For a,f,k data are analysed by repeated measures ANOVA followed by Bonferroni post hoc comparisons. *P<0.05, **P<0.01. ***P<0.001.