Fig 2: ISS-induced GLP-1 secretion in NCI-H716 cells. (A) Secretion of GLP-1 from ISS-treated NCI-H716 cells against increasing doses of ISS. Significance was determined by an unpaired t-test (one-tailed), *** p < 0.001 vs. NT. (B) siRNA-mediated decrease in GLP-1 secretion. Statistical analysis was conducted by one-way ANOVA with Bonferroni’s multiple comparison test, *** p < 0.001 vs. NT, **** p < 0.0001 vs. NT, ### p < 0.001 vs. siRNA control. (C) IP3R inhibitor (D) Inhibition of PLC-dependent processes. Significance was determined by an unpaired t-test (one-tailed), *** p < 0.001 vs. NT, ### p < 0.001 vs. vehicle. Values are expressed as the mean ± SEM, n = 4.

Fig 3: In vitro and ex vivo characterization of S. boulardii secreting GLP-1R agonists. (A) Schematic overview of the engineered S. boulardii for the production and secretion of peptides. (B) Concentration of GLP-1 (n = 4) and Exendin-4 (n = 4) secreted by S. boulardii in aerobic and anaerobic conditions. (C) Fold change of insulin secretion normalized to Sb-Empty from the isolated pancreatic islets of the wild-type C57BL/6J mice (n = 5) and (D) the GLP-1R knockout C57BL/6J mice (n = 5). 2 μM recombinant Exendin-4 was used as a positive control. All simulations were performed in duplicate. Bar plots are displayed as the mean ± SEM * p < 0.05. Panel (B) was analyzed with the dependent sample t test with Bonferroni adjustments for multiple comparisons. Panels (C) and (D) were analyzed by one-way ANOVA with a Dunnett’s post hoc test, with Sb-Empty as a reference.

Fig 4: End-point characterization of the synergistic effect of cold exposure and Sb-Exe4 in male C57BL/6 mice for 29 days. (A) Graphic simplification of the mechanistic effect on glucose homeostasis in male C57BL/6 mice orally administered with Sb-Exe4 and fed a 45% kcal high-fat diet for 29 days. (B) Fasting vena porta levels of Exendin-4 (pM) at the end of the study were quantified with radioimmunoassay (RIA). Fasting vena cava levels of (C) glucagon (pM), (D) insulin (uIU/mL), (E) GLP-1 (pM), (F) leptin (ng/mL), (G) TNF-α (pg/mL), and (H) IL-10 (pg/mL) at the end of the study quantified with a meso scale discovery assay (n = 8–9). (I) Relative mRNA of Ppara, Aco, Cd36, G6pc, and Pepck in the liver at the end of the study. Data are presented as fold change normalized to Sb-Empty (n = 9). (J) Weight (mg) of gut content was collected from the small intestine (SI) and large intestine (LI) of the mice (n = 9). (K) Abundance (log10 CFU/g feces) of Sb-Empty and Sb-Exe4 in SI (n = 9), cecum (n = 9), LI (n = 9), and feces (n = 27) in the mice. Data are presented as the mean ± SEM. P-value is written out as <0.1, *p < 0.05, **p < 0.01. Panels (B–H) and (J) were analyzed by a Wilcoxon signed-rank test and panel (I) was analyzed with a dependent sample t test. Bonferroni adjustments were used for multiple comparisons.

Fig 5: Pharmacological inhibition of VTA GLP-1Rs blocks the suppressive effects of activating NTS➔VTA projections on cocaine seeking.(A and B) Illustration of viral approach and experimental timeline wherein CAV2-Cre and a Cre-dependent AAV-expressing hM3D(Gq) were infused into the VTA and NTS, respectively, before the cocaine self-administration phase of the experiment. (C) Representative images of hM3D(Gq)-expressing NTS neurons that coexpress GLP-1. (D) Schematic depicting reinstatement test session treatment conditions. Exendin-(9-39) was infused into the VTA before activating endogenous NTS➔VTA projections with CNO. (E) Intra-VTA exendin-(9-39) (Ex-9) prevented the ability of CNO to suppress active lever presses during reinstatement test sessions [n = 8 (three female and five male rats); two-way RM ANOVA, systemic treatment × intra-VTA treatment: F1,7 = 8.775, P = 0.0210; Bonferroni’s test: vehicle (veh)/vehicle versus vehicle/CNO, *P = 0.0132; vehicle/CNO versus Ex-(9-39)/vehicle, *P = 0.0249; vehicle/CNO versus Ex-(9-39)/CNO, *P = 0.0441]. (F) Intra-VTA exendin-(9-39) had no effect on inactive lever responses (n = 8; two-way RM ANOVA, systemic treatment: F1,7 = 2.333, P = 0.1705; intra-VTA treatment: F1,7 = 0.320, P = 0.5891; systemic treatment × intra-VTA treatment: F1,7 = 0.138, P = 0.7207). Data are mean ± SEM.