Nav1.8-expressing neurons control daily oscillations of food intake, body weight and gut microbiota in mice

Recent evidence suggests a role of sensory neurons expressing the sodium channel Nav1.8 on the energy homeostasis control. Using a murine diphtheria toxin ablation strategy and ad libitum and time-restricted feeding regimens of control or high-fat high-sugar diets, here we further explore the function of these neurons on food intake and on the regulation of gastrointestinal elements transmitting immune and nutrient sensing. The Nav1.8+ neuron ablation increases food intake in ad libitum and time-restricted feeding, and exacerbates daily body weight variations. Mice lacking Nav1.8+ neurons show impaired prandial regulation of gut hormone secretion and gut microbiota composition, and altered intestinal immunity. Our study demonstrates that Nav1.8+ neurons are required to control food intake and daily body weight changes, as well as to maintain physiological enteroendocrine and immune responses and the rhythmicity of the gut microbiota, which highlights the potential of Nav1.8+ neurons to restore energy balance in metabolic disorders.


Genotype
Primer
Fecal sample collection after 12h of fasting (ZT12) and 6h (ZT18) and 12h (ZT24) of refeeding vi.Daily food intake every 6h in the last 24h of the ad libitum feeding vii.Daily body weight variations every 6h in the last 24h of the ad libitum feeding viii.Fecal sample collection every 6h in the last 24h of the ad libitum feeding ix.Energy metabolism-related hormone secretion.Fasting glycemia x.Intestinal immunity characterization xi.Gene expression (brain, gut and adipose tissue)

a
Body weight (Bw) follow-up and Bw gain of control mice (Cre -) and Nav1.8-cre/DTA mice (Cre + ) fed ad libitum with either control diet (CD) or high fat high sugar diet (HFHSD) for 5 weeks.12h-body weight gain (positive values) and body weight loss (negative values) every 2 days in: b Control mice fed CD or HFHSD during the light restricted feeding (LRF).c Nav1.8cre/DTA mice fed CD or HFHSD during LRF.d Control mice fed CD or HFHSD during DRF.e Nav1.8-cre/DTA mice fed CD or HFHSD along DRF.Data are represented as scatter plots for individual values with mean follow-up curve or as scatter plots indicating individual values ± SEM (Cre -CD and Cre -HFHSD, n = 10 mice; Cre + CD, n = 6 mice; Cre + HFHSD, n = 5-7 mice.Mice fed CD are depicted by solid yellow (Cre -) or empty orange (Cre -) circles, mice fed HFHSD are depicted by solid blue (Cre -) or empty blue (Cre -) circles; shadings in light yellow and grey represent LRF and DRF regimes, respectively.(a): Two-way ANOVA with genotype (Cre -or Cre + ) and diet (CD/HFHSD) as between-subject factor; (b-e): Two-way ANOVA with diet (CD or HFHSD) and day of the LRF or DRF as between-subject factors.Bonferroni's post hoc test.a ## p < 0.01 and ### p < 0.001 vs Cre -HFHSD and * p < 0.05 vs Cre -CD; (b-e) * p < 0.05, ** p < 0.01 and *** p < 0.001 vs either Cre -CD or Cre + CD Supplementary Figure 4: Body weight changes under 24-hour free access to food conditions during 3 weeks a Body weight (Bw) follow-up along the last 3 weeks of the experiment when control mice (Cre -) and mice lacking Nav1.8+ neurons (Cre + ) on either control diet (CD) or high-fat high-sugar diet (HFHSD) were switched from dark-restricted feeding (DRF) to ad libitum feeding (ad-lib).b Impact of the ad libitum feeding reverting the effects of DRF on Bw: from the end of the ad-lib feeding and the DRF (ΔBw).Results are represented as scatter plots indicating individual values with mean ± SEM. (Cre -CD and Cre -HFHSD, n = 5-10 mice; Cre + CD, n = 5-6 mice; Cre + HFHSD, n = 2-5 mice).Mice fed CD are depicted by solid yellow (Cre -) or empty orange (Cre -) circles, mice fed HFHSD are depicted by solid blue (Cre -) or empty blue (Cre -) circles; shading in grey represents DRF regime.Two-way ANOVA with genotype (Cre -or Cre + ) and diet (CD or HFHSD) as between-subject factors followed by Bonferroni's post hoc test.the small intestine.c Weight of the spleen, scale bar: 0.5 cm.d Percentage of M1, M2, and memory and effector T cells in the spleen.e Vasoactive intestinal peptide (VIP) concentration in ileum.Results are shown in a heatmap in which color intensity represents the relative gene expression (a) or the relative abundance of immune cells expressed as a percentage (b, d), and missing values are indicated as "X" (a, b, d); or shown as scatter plots indicating individual values with mean ± SEM (c, e) (Cre -n = 9-10 mice and Cre + n = 5 mice).Mice fed CD are depicted by solid yellow (Cre -) or empty orange (Cre -) circles.All measurements were analyzed using Student's t test except for (c), where mRNA levels of DefA, Lyz1 and Ki67 in ileum and DefA and Reg3g in colon were analyzed with the Mann-Whitney U test.**p < 0.01.Supplementary Figure 6 (related to Figure 6 and 7).Gene expression of hypothalamic markers involved in food intake control and plasma levels of hormones involved in regulating glycemia In mice lacking Nav1.8+ neurons (Cre + ) and their control littermates (Cre -) fed control diet (CD) we analyzed at the end of the experiment: a mRNA levels of different genes including the precursor of GLP-1 (Proglucagon) and peptide tyrosine tyrosine (Pyy) in ileum and colon, and food intake-related peptides and receptors in the hypothalamus , Cart, Agrp, Npy2r, and Ghsr).The heatmap shows the gene expression variations, represented by fold-change relative to control littermates; color intensity depicts the relative gene expression, and missing values are indicated as "X".b, c Plasma insulin and glucagon levels in 4-h fasted f mice and 15 min after an oral gavage of the nutrient mixed solution (ensure, EN).Results are represented as scatter plots indicating individual values with mean ± SEM. (Cre -n = 6-10 mice and Cre + n = 5 mice).Mice fed CD are depicted by solid yellow (Cre -) or empty orange (Cre -) circles.(a) Student´s t test.(b, c): Two-way ANOVA with genotype (Cre -or Cre + ) and prandial condition (fasting/ensure, F/EN) as between-subject factor.

Table 2 :
Anti-mouse antibodies against immune markers to quantify immune cells in the spleen and intestinal epithelium and lamina propria by flow cytometry.

Table 3 :
Genes, primer pair sequences and conditions of the qPCR reactions All primer pairs were supplied by Isogen Life Science except primers for Glp1r amplification, which were supplied by Sigma.