The Vagus Nerve and Spleen: Influence on White Adipose Mass and Histology of Obese and Non-obese Rats

Abstract

The vagus nerve (VN) and spleen represent a complex interface between neural and immunological functions, affecting both energy metabolism and white adipose tissue (WAT) content. Here, we evaluated whether vagal and splenic axis participates in WAT mass regulation in obese and non-obese male Wistar rats. High doses of monosodium glutamate (M; 4 g/Kg) were administered during the neonatal period to induce hypothalamic lesion and obesity (M-Obese rats). Non-obese or Control (CTL) rats received equimolar saline. At 60 days of life, M-Obese and CTL rats were randomly distributed into experimental subgroups according to the following surgical procedures: sham, subdiaphragmatic vagotomy (SV), splenectomy (SPL), and SV + SPL (n = 11 rats/group). At 150 days of life and after 12 h of fasting, rats were euthanized, blood was collected, and the plasma levels of glucose, triglycerides, cholesterol, insulin, and interleukin 10 (IL10) were analyzed. The visceral and subcutaneous WAT depots were excised, weighed, and histologically evaluated for number and size of adipocytes as well as IL10 protein expression. M-Obese rats showed higher adiposity, hyperinsulinemia, hypertriglyceridemia, and insulin resistance when compared with CTL groups (p < 0.05). In CTL and M-Obese rats, SV reduced body weight gain and triglycerides levels, diminishing adipocyte size without changes in IL10 expression in WAT (p< 0.05). The SV procedure resulted in high IL10 plasma levels in CTL rats, but not in the M-Obese group. The splenectomy prevented the SV anti-adiposity effects, as well as blocked the elevation of IL10 levels in plasma of CTL rats. In contrast, neither SV nor SPL surgeries modified the plasma levels of IL10 and IL10 protein expression in WAT from M-Obese rats. In conclusion, vagotomy promotes body weight and adiposity reduction, elevating IL10 plasma levels in non-obese animals, in a spleen-dependent manner. Under hypothalamic obesity conditions, VN ablation also reduces body weight gain and adiposity, improving insulin sensitivity without changes in IL10 protein expression in WAT or IL10 plasma levels, in a spleen-independent manner. Our findings indicate that the vagal-spleen axis influence the WAT mass in a health state, while this mechanism seems to be disturbed in hypothalamic obese animals.

Keywords: adipocyte; autonomic nervous system; hypothalamic obesity; splenectomy; vagotomy.

FIGURE 1

Experimental design. CTL animals were divided into CTL_SHAM, Sham surgery control; CTL_SPL, splenectomized control; CTL_SV, subdiaphragmatic vagotomy control; CTL_SV+SPL, subdiaphragmatic vagotomy + splenectomized control; M-Obese_SHAM, simulated surgery MSG; M-Obese_SPL, Splenectomized MSG; M-Obese_SV, subdiaphragmatic vagotomy MSG; M-Obese_SV+SPL, subdiaphragmatic vagotomy + splenectomized MSG N = number of animals. The dashed line indicates the events over time (from birth to euthanasia), with vertical arrows evidencing specific points.

FIGURE 2

M-Obese rats show intense adipocyte hypertrophy in WAT. (A) Representative photomicrographs of WAT-I and WAT-M, respectively, stained with H&E, under light microscopy, 40-fold magnification; (B,D) illustrate the average adipocyte size; (C,E) number of adipocytes per field in WAT-I and WAT-M, respectively. Adipocyte nuclei are indicated by arrows, and the deposition of fat in the cytosol is marked by the star. CTL, control; M-Obese, MSG; WAT-I, inguinal white adipose tissue; WAT-M, mesenteric white adipose tissue. Data are mean ± SEM (n = 6 rats/group). Asterisk (^∗) represent statistical differences between the groups. Student’s t-test (p < 0.05).

FIGURE 3

Subdiaphragmatic vagotomy induces a reduction in WAT-I content in non-obese (CTL) rats with splenic participation and without changes in IL10 protein expression. (A) Representative photomicrographs of the broad WAT-I, stained with H&E, magnification 40×; adipocyte nuclei (arrow) and fat deposition (asterisk). (B) Representative WB band densitometry; 50 kDa region (tubulin); 17 kDa region IL10; In graphical, data are mean ± SEM (C) IL-10 expression (n = 4 rats/group); (D) weight of WAT-I (n = 6 rats/group); (E) size of adipocytes; (F) number of adipocytes (n = 6 rats/group). Line and symbols (SPL, SV, and I) above bars show significant F effect for two-way ANOVA. Letters represent statistical difference among groups–(a) CTL_SHAM; (b) CTL_SPL; (c) CTL_SV; (d) CTL_SV+SPL in Tukey post hoc test (p < 0.05). Legend: CTL_SHAM, simulated surgery control; CTL_SPL, splenectomized control; CTL_SV, subdiaphragmatic vagotomy control; CTL_SV+SPL, subdiaphragmatic vagotomy + splenectomized control; WAT-I, white adipose tissue–inguinal; IL-10, Interleukin 10; SPL, splenectomy; SV, subdiaphragmatic vagotomy; I, interaction.

FIGURE 4

Subdiaphragmatic vagotomy induces reduction in WAT-M content in non-obese (CTL) rats with splenic participation and without changes IL10 protein expression. (A) Representative photomicrographs of the broad WAT-M, stained with H&E, magnification 40×; adipocyte nuclei (arrow) and fat deposition (asterisk). (B) Representative WB band densitometry; 50 kDa region (tubulin); 17 kDa region IL10; In graphical, data are mean ± SEM (C) IL-10 expression (n = 4 rats/group); (D) weight of WAT-I (n = 6 rats/group); (E) size of adipocytes; (F) number of adipocytes (n = 6 rats/group). Line and symbols (SPL, SV, and I) above bars show significant F effect in Two-way ANOVA. Letters represent statistical difference among groups—(a) CTL_SHAM; (b) CTL_SPL; (c) CTL_SV; (d) CTL_SV+SPL in Tukey post hoc test (p < 0.05). Legend: CTL_SHAM, simulated surgery control; CTL_SPL, splenectomized control; CTL_SV, subdiaphragmatic vagotomy control; CTL_SV+SPL, subdiaphragmatic vagotomy + splenectomized control; WAT-I, white adipose tissue—inguinal; IL-10, Interleukin 10; SPL, splenectomy; SV, subdiaphragmatic vagotomy; I, interaction.

FIGURE 5

Subdiaphragmatic vagotomy does not change WAT-I content in M-Obese rats but reduces adipocytes hypertrophy, without altering IL10 expression, regardless of spleen ablation. (A) Representative photomicrographs of the broad WAT-I, stained with H&E, magnification 40×; adipocyte nuclei (arrow) and fat deposition (asterisk). (B) Representative WB band densitometry; 50 kDa region (tubulin); 17 kDa region IL10; In graphical, data are mean ± SEM (C) IL-10 expression (n = 4 rats/group); (D) weight of WAT-I (n = 6 rats/group); (E) size of adipocytes; (F) number of adipocytes (n = 6 rats/group). Line and symbols (SPL, SV, and I) above bars show significant F effect in Two-way ANOVA. Letters represent statistical difference among groups—(a) M-Obese_SHAM; (b) M-Obese_SPL; (c) M-Obese_SV; (d) Obese_SV+SPL in Tukey post hoc test (p < 0.05). Legend: M-Obese_SHAM, simulated surgery MSG; M-Obese_SPL, splenectomized MSG; M-Obese_SV, subdiaphragmatic vagotomy MSG; M-Obese_SV+SPL, subdiaphragmatic vagotomy + splenectomized MSG; WAT-I, white adipose tissue–inguinal; IL10, Interleukin 10; SPL, splenectomy; SV, subdiaphragmatic vagotomy, I, interaction.

FIGURE 6

Subdiaphragmatic vagotomy induces a reduction in WAT-M content in M-Obese rats, which is enhanced in the absence of the spleen, without leading to changes in IL10 expression. (A) Representative photomicrographs of the broad WAT-M, stained with H&E, magnification 40×; adipocyte nuclei (arrow) and fat deposition (asterisk). (B) Representative WB band densitometry; 50 kDa region (tubulin); 17 kDa region IL10; In graphical, data are mean ± SEM (C) IL-10 expression (n = 4 rats/group); (D) weight of WAT-I (n = 6 rats/group); (E) size of adipocytes; (F) number of adipocytes (n = 6 rats/group). Line and symbols (SPL, SV, and I) above bars show significant F effect in two-way ANOVA. Letters representing statistical difference among groups—(a) M-Obese_SHAM; (b) M-Obese_SPL; (c) M-Obese_SV; (d) Obese_SV+SPL in Tukey post hoc test (p < 0.05). Legend: M-Obese_SHAM, simulated surgery MSG; M-Obese_SPL, splenectomized MSG; M-Obese_SV, subdiaphragmatic vagotomy MSG; M-Obese_SV+SPL, subdiaphragmatic vagotomy + splenectomized MSG; WAT-I, white adipose tissue–inguinal; IL-10, Interleukin 10; SPL, splenectomy; SV, subdiaphragmatic vagotomy; I, interaction.