Gestational saccharin consumption disrupts gut-brain axis glucose homeostasis control in adolescent offspring rats in a sex-dependent manner

Abstract

Background: Certain events that occur in early life, such as changes in nutrition, can promote structural and functional modifications in brain development, projecting to either short, medium, and/or long terms, resulting in metabolic programming. These effects depend on the timing, intensity, and duration of exposure, and are proposed to be the cause or contribute to chronic adult disorders. Recent studies have proposed that artificial non-nutritive sweeteners (NNS), such as saccharin, can be included as one of these developmental disruptors. Saccharin consumption during pregnancy is strongly discouraged, as it can cross through the placenta and accumulate in the fetus, potentially impacting metabolic control for life. However, the mechanisms underlying the metabolic syndrome induced by maternal NNS consumption during pregnancy are not well understood. Some studies suggest that NNS may affect sweet taste receptors in the adult's guts, leading to changes in the release of glucagon-like peptide-1 (GLP-1) and insulin. The objective of the study is to investigate whether maternal saccharin consumption during pregnancy affects the gut-brain connection, leading to alterations in insulin/GLP-1 signaling during neurodevelopment until adolescence.

Methods: Pregnant rats were administered 0.1% saccharin in drinking water throughout gestation, and the main components of the insulin/GLP-1 signaling pathway were analyzed in the plasma, small intestine and hypothalamus of the offspring after weaning. Perinatal exposure to saccharin was linked to disrupted glucose homeostasis and insulin sensitivity in both male and female offspring.

Results: We identified sex-dependent mechanisms that affected GLP-1 signaling in the intestine, associated with the expression of taste receptors and glucose transporters. These alterations affected the gut-brain axis and disrupted hypothalamic signaling associated with glucose regulation and food intake, primarily involving the GLP-1, leptin, and insulin signaling pathways.

Conclusions: These results suggest that developmental NNS exposure might contribute to the growing alteration in energy metabolism.

Keywords: Development; Gestation; Glucagon-like peptide-1; Insulin; Non-nutritive sweeteners; Saccharin; Toxicology.

Fig. 1

Glucose, insulin and GLP-1 adolescent offspring plasma levels. Glucose tolerance test (GTT; A, B, E) and insulin tolerance test (ITT; C, D, F). Glucose (G), insulin (H) and GLP-1 (I) measurements in blood of male and female adolescent offspring. Data are presented as mean ± SEM and analyzed by two-way ANOVA. (n = 10–33 for each experimental group). Significant post-hoc analysis are shown: *p < 0.05 versus male within the same perinatal conditions; #p < 0.05, ##p < 0.01, ###p < 0.001 versus control within the same sex

Fig. 2

Glucose homeostasis gene and protein expression in adolescent offspring small intestine. Study of gene expression of Glp1r (a), Tas1r3 (d), Pyy (f), Cd36 (g), Slc5a1 (i), Gcg (j) by qPCR (n = 9 for each experimental group) and protein expression of GLP1 (b), GLPR1 (c), TAS1R3 (e), CD36 (h) by Western blot (n = 6 for each experimental group) in the small intestine of male and female adolescent offspring. (k) Representative blots immunostained for each of the proteins tested. Data are presented as mean ± SEM and analyzed by two-way ANOVA. Significant post-hoc analysis is shown: *p < 0.05, ***p < 0.001 versus male within the same perinatal condition; ##p < 0.01 versus control within the same sex

Fig. 3

Adolescent offspring body weight and hypothalamic neuropeptides. Body weight of male and female adolescent offspring (a) (PND21). Gene expression evaluated by qPCR of Agrp (b), Pomc (c), Npy (d), Hcrt (e) in the hypothalamus of male and female adolescent offspring. Data are presented as mean ± SEM and analyzed by two-way ANOVA; n = 6 for each experimental group. Significant post-hoc analysis is shown: ##p < 0.01 versus control males; *p < 0.05, ***p < 0.001 versus male within the same perinatal conditions

Fig. 4

Gene expression on adolescent offspring hypothalamic glucose-related circuits. Gene expression of Glpr1 (a), Lepr (b), Insr (c), Irs1 (d), Irs2 (e), Insig1 (f), Insig2 (g) was evaluated by qPCR in the hypothalamus of male and female adolescent offspring. Data are presented as mean ± SEM and analyzed by two-way ANOVA; n = 9 for each experimental group. Significant post-hoc analysis is shown: *p < 0.05, **p < 0.01 versus male within the same perinatal conditions; #p < 0.05 versus control within the same sex

Fig. 5

Protein expression in adolescent offspring hypothalamic insulin-related circuits. Protein expression of IRS-1 (phosphorylated in tyr896, (a); phosphorylated in ser612, (b); total form, (c); phosphorylated (d) and total PI3K (e); phosphorylated (f) and total AKT (g); phosphorylated (h) and total GSK3β (i); phosphorylated (j) and total AMPKα (k) was evaluated by western blot in the hypothalamus of male and female adolescent offspring. (l) Representative blots immunostained for each of the proteins tested. Data are presented as mean ± SEM and analyzed by two-way ANOVA; n = 6 for each experimental group. Significant post-hoc analysis is shown: *p < 0.05, **p < 0.01 versus male within the same perinatal conditions; #p < 0.05, ##p < 0.01 versus control within the same sex