MSG exposure in early life enhances Lee index, increases adiposity, and reduces water consumption and body weight in male and female rats on PND 60

To assess the impact of MSG exposure on growth and metabolic parameters, we first analyzed the Lee Index, food and water intake, and body weight gain.

A two-way ANOVA of the Lee Index revealed no significant sex × MSG interaction at PND 30 (F(1, 60) = 3.93, p > .05) or PND 60 (F(1, 60) = 0.56, p > .05). At PND 60, however, significant main effects of sex (F(1, 60) = 6.27, p < .05) and MSG (F(1, 60) = 29.40, p < .0001) were observed, indicating increased adiposity in MSG-exposed rats (Figs. 1b, c).

Repeated-measures ANOVA of cumulative food intake showed a significant time × MSG interaction in males (F(11, 154) = 11.18, p < .0001), but not in females (F(11, 154) = 1.83, p > .05). Post hoc tests revealed reduced food intake in MSG-treated males from PND 24 (p < .001) to PND 57 (p < .05) (Figs. 1d, e).

Repeated-measures ANOVA of cumulative water intake indicated significant time × MSG interactions in both males (F(11, 154) = 13.71, p < .0001) and females (F(11, 154) = 43.25, p < .0001). MSG-treated rats consumed less water than controls across multiple time points (Figs. 1f, g).

Repeated-measures ANOVA of body weight revealed significant time × MSG interactions in males (F(13, 390) = 40.17, p < .0001) and females (F(13, 390) = 5.62, p < .001), with MSG-treated rats consistently weighing less than controls (Figs. 1h, i).

Two-way ANOVAs of adipose tissue weights revealed no significant sex × MSG interactions for BAT, MAT, gWAT, or iWAT. However, a significant main effect of MSG was found for MAT, gWAT, and iWAT in both sexes, and for BAT in females (Table S1).

MSG exposure and social-SPS, both individually and combined, reduce food intake on eating behavior for male and female rats

To evaluate the effects of MSG and social-SPS on eating behavior, food intake was measured before and after exposure to social-SPS.

Before social-SPS exposure, a two-way ANOVA revealed a significant sex × MSG interaction (F(1, 60) = 10.76, p < .01). Male rats showed higher food intake than females in both saline (p < .0001) and MSG-treated groups (p < .05). MSG-treated males exhibited reduced food intake than controls (p < .001), whereas MSG exposure did not alter food intake in females (Fig. 2a).

Fig. 2

Effect of MSG on eating behavior in male and female rats a. Effects of social-SPS and MSG on eating behavior in male b and female c rats, anxiety index in male d and female e, open arms time (%) in male f and female g rats and open arms entries (%) in male h and female i rats. Results represent the mean ± S.E.M. of 16 a or 8 b–i rats per group. *p < .05; **p < .01; ***p < .001; ****p < .0001 when compared with saline a or saline control b–i group, @p < .05 when compared with male MSG group a, #### p < .0001 when compared with male saline group a. Data were analyzed by two-way ANOVA followed by the Sidak’s post-test. MSG means monosodium glutamate, social-SPS means social-single prolonged stress

Seven days after social-SPS exposure, two-way ANOVAs revealed significant social-SPS × MSG interactions in both males (F(1, 28) = 5.69, p < .05) and females (F(1, 28) = 6.60, p < .05). Social-SPS reduced food intake in saline-treated males (p < .05) and females (p < .01). In MSG-treated rats, both MSG and social-SPS exposure reduced food intake in males (p < .0001) and females (p < .001) (Figs. 2b, c).

MSG exposure and social-SPS individually induce anxiety-like phenotype in female rats

To investigate the effects of MSG and social-SPS on anxiety-related behavior of male and female rats, we analyzed the anxiety index and open-arm parameters in the elevated plus maze.

In males, two-way ANOVA revealed no significant MSG × social-SPS interaction (F(1, 28) = 0.45, p > .05), but a main effect of social-SPS was observed (F(1, 28) = 5.56, p < .05). Anxiety index values were similar across male groups (Fig. 2d).

In females, a significant MSG × social-SPS interaction was found (F(1, 28) = 13.93, p < .001). Both MSG (p < .05) and social-SPS (p < .01) increased the anxiety index compared to controls, with no additive effect (Fig. 2e).

Analysis of OAT% revealed no significant interaction in males (F(1, 28) = 1.51, p > .05), but a significant MSG × social-SPS interaction in females (F(1, 28) = 10.96, p < .01). Social-SPS (p < .05) and MSG (p < .05) reduced OAT% in females compared to controls (Figs. 2f, g).

For OAE%, no significant interaction was observed in males (F(1, 28) = 0.09, p > .05), whereas females showed a significant MSG × social-SPS interaction (F(1, 28) = 17.03, p < .001). Female rats exposed to MSG or social-SPS spent less time in open arms compared to controls (p < .05) (Figs. 2h, i).

MSG exposure and social-SPS, both individually and combined, reduce time in the center but do not alter other locomotor parameters of male and female rats

To assess MSG and social-SPS effects on locomotor activity, we analyzed center time, crossings, rearings, distance, and speed in male and female rats in the SLA apparatus.

In males, two-way ANOVA revealed a significant MSG × social-SPS interaction for center time (F(1,28) = 15.07, p < .001). Social-SPS (p < .0001), MSG (p < .0001), and MSG + social-SPS (p < .0001) reduced center time compared to control (Table 1). Two-way ANOVAs revealed no significant MSG × social-SPS interactions for crossings, rearings, distance, or speed. However, significant main effects of MSG were observed for crossings (F(1, 28) = 47.31, p < .0001), rearings (F(1, 28) = 9.37, p < .01), distance (F(1, 28) = 12.59, p < .01), and speed (F(1, 28) = 15.58, p < .001).

In females, two-way ANOVA revealed a significant MSG × social-SPS interaction for center time (F(1, 28) = 8.48, p < .01), along with main effects of MSG (F(1, 28) = 22.72, p < .0001) and social-SPS (F(1, 28) = 11.40, p < .01). Social-SPS (p < .001), MSG (p < .0001), and MSG + social-SPS (p < .0001) reduced center time compared to controls (Table 2). No significant interactions were found for crossings, rearings, distance, or speed, but MSG exerted significant main effects on all parameters (crossings: F(1, 28) = 62.38, p < .0001; rearings: F(1, 28) = 26.82, p < .0001; distance: F(1, 28) = 58.56, p < .0001; speed: F(1, 28) = 76.95, p < .0001).

MSG exposure and social-SPS, both individually and combined, reduce Ob-R, GHS-R1, and D2R levels in the hypothalamus of male rats

To investigate the impact of MSG and social-SPS on hypothalamic receptor expression in males, we analyzed Ob-R, GHS-R1, D1R, and D2R protein levels.

Two-way ANOVAs revealed significant MSG × social-SPS interactions for GHS-R1 (F(1, 16) = 7.93, p < .05), Ob-R (F(1, 16) = 22.69, p < .001), and D2R (F(1, 16) = 8.38, p < .05). Post hoc comparisons indicated that social-SPS (p < .01), MSG (p < .01), and combined MSG + social-SPS (p < .001) exposure significantly reduced hypothalamic GHS-R1 levels compared to controls (Fig. 3a). Similarly, Ob-R levels were decreased in social-SPS (p < .001), MSG (p < .0001), and MSG + social-SPS (p < .01) groups relative to controls (Fig. 3b). D2R levels were also reduced in social-SPS (p < .05), MSG (p < .01), and MSG + social-SPS (p < .01) groups compared to controls (Fig. 3c).

Fig. 3

Effects of social-SPS and MSG on the levels of GHS-R1 a, Ob-R b, D2R c, and D1R d in the hypothalamus of male rats. Results represent the mean ± S.E.M. of 5 rats per group. *p < .05; **p < .01; ***p < .001; ****p < .0001 when compared with saline control group. Data were analyzed by two-way ANOVA followed by the Sidak’s post-test. MSG means monosodium glutamate, social-SPS means social-single prolonged stress, GHS-R1 means Ghrelin receptor 1, Ob-R means leptin receptor, D2R means dopamine receptor 2, D1R means dopamine receptor 1

For D1R, no significant interaction was observed (F(1, 16) = 0.03, p > .05), but a main effect of MSG was detected (F(1, 16) = 6.69, p < .05). All groups showed similar hypothalamic D1R levels, with MSG exposure alone accounting for the reduction (Fig. 3d).

Pearson’s correlation analyses in males revealed significant positive associations between food intake and D2R levels (Fig. 4a), between GHS-R1 and D2R levels (Fig. 4b), and between Ob-R and D2R levels (Fig. 4c). In contrast, a significant negative correlation was found between food intake and anxiety index (Fig. 4d).

Fig. 4

Effects of social-SPS and MSG on the levels of GHS-R1 a, Ob-R b, D2R c, and D1R d in thehypothalamus of female rats. Results represent the mean ± S.E.M. of 5 rats per group. *p < .05; **p < .01;when compared with saline control group. Data were analyzed by two-way ANOVA followed by theSidak’s post-test. MSG means monosodium glutamate, social-SPS means social-single prolonged stress,GHS-R1 means Ghrelin receptor 1, Ob-R means leptin receptor, D2R means dopamine receptor 2, D1Rmeans dopamine receptor 1

MSG exposure and social-SPS, both individually and combined, reduce Ob-R and D1R levels in the hypothalamus of female rats

To assess the effects of MSG and social-SPS on hypothalamic receptor expression in females, we measured the protein levels of Ob-R, GHS-R1, D1R, and D2R.

Two-way ANOVAs revealed significant MSG × social-SPS interactions for Ob-R (F(1, 16) = 9.26, p < .01) and D1R (F(1, 16) = 8.78, p < .01). Post hoc analyses showed that social-SPS (p < .05), MSG (p < .01), and MSG + social-SPS (p < .05) exposure significantly reduced Ob-R levels compared to controls (Fig. 5b). Similarly, D1R levels were decreased in social-SPS (p < .01), MSG (p < .05), and MSG + social-SPS (p < .05) groups relative to controls (Fig. 5d).

Fig. 5

Pearson’s correlation test between hypothalamic levels of D2R and food intake a, D2R and GHS-R1 b, D2R and Ob-R c, and between anxiety index and food intake d in male rats. In female rats, correlation between anxiety index and food intake e, D1R levels and anxiety levels f, and D1R and food intake g. Each point represents one individual animal. GHS-R1 means Ghrelin receptor 1, Ob-R means leptin receptor, D2R means dopamine receptor 2, D1R means dopamine receptor 1

For GHS-R1 and D2R, no significant interactions were observed (GHS-R1: F(1, 16) = 0.87, p > .05; D2R: F(1, 16) = 0.26, p > .05). All groups demonstrated comparable hypothalamic levels of GHS-R1 (Fig. 5a) and D2R (Fig. 5c).

Pearson’s correlation analyses in females revealed significant negative associations between food intake and anxiety index (Fig. 4e), and between anxiety index and D1R levels (Fig. 4f). Additionally, a positive correlation was found between food intake and D1R levels (Fig. 4g).