Genetic deletion of mGluR5 prevent increased body weight in BACHD mice
First, we decided to investigate the effect of mGluR5 deletion on body weight and visceral fat in BACHD mice (Figs. 2 and 3). Figure 2A shows body weight measurements of all mice lines from 2 to 12 months of age. Figures 2B-D display graphical analyzes of the body weight at 2, 6 and 12 months of all tested groups. Our results showed that at 2 and 6 months of age, BACHD mice present the highest body weight as compared to all genotypes (Figs. 2B-D). However, at 12 months of age, our statistical analyses did not detect a body weight difference as compared to BACHD mice and WT mice (Kruskal-Wallis, p = 0.2463) (Fig. 2D). On the contrary, BACHD/mGluR5-/- exhibited a reduction in body weight as compared to BACHD mice (One-way ANOVA, 2 months, F(3,31) = 9.531, p < 0.0001, and 6 months F(3,31) = 26.14, p < 0.0001, Kruskal-Wallis, 12 months, p < 0.0001). Notably, the weight measurements of the double mutant mice were statistically equal to WT mice (One-way ANOVA 2 and 6 months, p > 0.9999; Kruskal-Wallis, 12 months p = 0.9279, Figs. 2B-D). Taken together, these data suggest that mGluR5 deletion restore body weight to control levels.
Next, we decided to verify the adiposity indices in all tested groups. For that, we performed the measurement of the visceral fat in the epididymal, mesenteric and retroperitoneal fat pad, at 6 and 12 months of age, which correspond to the initial and late phases of HD in BACHD mice 14. Notably, WT mice and BACHD mice did not present statistical differences in the adiposity index, leading us to suggest that this parameter is not affect by mHTT. At 12 months of age, the opposite result was observed as WT mice and mGluR5 KO mice present statistical differences in the adiposity index (One-way ANOVA, 12 months, F (3,21) = 20.20, p = 0.0001). Importantly, our results demonstrated that at 6 and 12 months of age, BACHD/mGluR5-/- mice presented less visceral adiposity as compared to BACHD mice (One-way ANOVA, 6 months, F (3,11) = 14.3, p = 0.0004, and 12 months F (3,21) = 20.20, p < 0.0001) (Figs. 3A and B). Thus, it is quite tempt to infer that the deletion of mGluR5 is decreasing the adiposity index in both WT and BACHD mice.
Absence of mGluR5 promotes changes in the adipokines levels in the adipose tissue of BACHD mice
Due to the importance of adipokines, such as leptin and adiponectin, in the context of energy and inflammatory balance, we investigated the concentration of these adipokines in the hypothalamus of 12-month-old mice (Fig. 4A) and also in the EAT, MAT, RPAT and VAT in all tested groups from 6 and 12 months of age (Fig. 4B-E). Our results showed that at 6 months of age, there was no significant difference in the concentration of adiponectin between all genotypes in any of the analyzed tissues (Supplementary data, Fig. 1). However at 12 months of age, we observed in the EAT from BACHD mice, a tendency of decrease of adiponectin levels as compared to WT mice (Fig. 4B). Moreover, the deletion of mGluR5 in BACHD mice was able to normalize the adiponectin to control levels (One-Way Welch ANOVA, EAT W 8.785 (3.000, 9.133), p = 0.0047).
Next, leptin levels were increased in the VAT of BACHD mice as compared to WT mice at 6 months of age (One-Way ANOVA, MAT F(3,10) = 11, P = 0.0013) (Supplementary data, Fig. 2). However, at 12 months of age the leptin levels on BACHD mice were not statistical different from WT group in any of the adipose tissue tested (Fig. 5B-E). By contrast, we found that at 12 months of age leptin levels had a tendency of decreasing in MAT and RPAT of BACHD/mGluR5-/- mice as compared to BACHD group (One-Way Welch ANOVA, MAT W 4.843 (3.000, 8.038), p = 0.0524, RPAT W 18.26 (3.000, 9.213), p = 0.0003) (Fig. 5C and D). Interestingly, in both analysis of hypothalamic adiponectin and leptin levels, we did not observe differences between double mutant and BACHD mice (Figs. 4A and 5A), which may indicate a peripheral modulation caused by the absence of mGluR5 receptor on these adipokines.
Due to the antagonistic role of adiponectin and leptin in the obesity context, we decided to analyze the ratio of adiponectin/leptin at 6 and 12 months of age in all tested groups (Supplementary data, Fig. 3 and Fig. 6). In this milieu, at 6 months of age, there was no significant difference in the adiponectin/leptin ratio between WT and BACHD mice in all analyzed adipose tissues (Supplementary data, Fig. 3). Our data showed that BACHD mice presented a decrease in the hypothalamic adiponectin/leptin ratio as compared to WT mice at 12 months of age (Fig. 6A). Moreover, adiponectin/leptin ratio in the mGluR5 KO mice is increased as compared to WT and BACHD mice in the EAT and RPAT (Fig. 6B and 6D) and increased as compared to BACHD in the VAT (Fig. 6E). Plus, at 12 months analyzing MAT, we found that the BACHD/mGluR5-/- mice exhibited higher adiponectin/leptin ratio as compared to BACHD mice (One-way ANOVA, MAT F (3,18) = 3.425, p = 0.0394) (Fig. 6C). Notably, these data pointed out a possible mechanism involving mGluR5 in the regulation of these adipokines.
The absence of mGluR5 promotes increased an anti-inflammatory profile in the double mutant mice
In order to investigate the inflammatory state of all tested groups, we decided to measure the levels of pro and anti-inflammatory cytokines. At 6 and 12 months of age comparing WT and BACHD mice, our findings did not show remarkable differences between pro and anti-inflammatory cytokines in central and periphery tissues analyzed (Supplementary data, Figs. 4–7 and Figs. 10–13). However, our findings showed that BACHD/mGluR5-/- mice presented an increase in both pro-inflammatory and anti-inflammatory cytokines as compared to BACHD and WT mice in all VAT and also in the hypothalamus at 6 and 12 months of age (Supplementary data, Figs. 4–7 and Figs. 10–13). Thus, in order to verify which cytokine, as well as the predominant immune response in the analyzed tissues, we examined ratios between IL-4/IFN and also IL-10/TNF. We found that there was no difference in all ratios between tested genotypes at 6 months of age (Supplementary Figs. 8–9). However, at 12 months of age, BACHD/mGluR5-/- mice showed a reduction in IL-4/IFN ratio as compared to WT mice in MAT (One-way ANOVA, F(3,19) = 3,416, p = 0.0384) (Fig. 7C), indicating a pro-inflammatory state in the MAT of these mice. However, this fact was not observed in the sum of the VAT or in all other tissues analyzed (Figs. 7A, B, D and E). Furthermore, in the hypothalamus of BACHD mice a higher IL-4/IFN ratio was observed as compared to WT mice (One-way ANOVA, F(3,18) = 3.687, p = 0.0314) (Fig. 7A). Regarding the ratio IL-10/TNF (Fig. 8A-E), we observed that BACHD/mGluR5-/- mice showed a higher IL-10/TNF ratio as compared to BACHD mice in the EAT, and in the sum of VAT (One-Way Welch ANOVA, EAT W 8.373 (3.000, 10.79), p = 0.0040; One-way ANOVA, VAT F(3,20) = 4.096, p = 0.0203) (Fig. 8B and E). Therefore, these set of data may indicated that absence of mGluR5 may promote a balance in the inflammatory environment in the adipose tissue of BACHD mice.