Nav1.8 neurons are involved in limiting acute phase responses to dietary fat

Abstract

Objective and methods: Metabolic viscera and their vasculature are richly innervated by peripheral sensory neurons. Here, we examined the metabolic and inflammatory profiles of mice with selective ablation of all Na_v1.8-expressing primary afferent neurons.

Results: While mice lacking sensory neurons displayed no differences in body weight, food intake, energy expenditure, or body composition compared to controls on chow diet, ablated mice developed an exaggerated inflammatory response to high-fat feeding characterized by bouts of weight loss, splenomegaly, elevated circulating interleukin-6 and hepatic serum amyloid A expression. This phenotype appeared to be directly mediated by the ingestion of saturated lipids.

Conclusions: These data demonstrate that the Na_v1.8-expressing afferent neurons are not essential for energy balance but are required for limiting the acute phase response caused by an obesogenic diet.

Keywords: Deafferentation; Diphtheria toxin; Energy homeostasis; Inflammation; Nodose ganglion; Obesity.

Graphical abstract

Figure 1

Ablation of vagal and spinal Na_v1.8-expressing neurons using conditional expression of diphtheria toxin. (A–D) In situ hybridization of NG and DRG with Na_v1.8 antisense riboprobe in control and ablated mice. Na_v1.8 mRNA signals represented by silver grains can be seen in many neurons of the NG and DRG of control mice, but not at all in ablated mice. Note also that sensory ganglia appear visibly smaller in ablated animals. (E–F) Isolectin B4 (IsB4) binding is commonly used as a surface marker of non-peptidergic C-fibers. No IsB4-positive cell bodies were retained in ablated mice, thus confirming the absence of C-fiber neurons.

Figure 2

Metabolic characterization of ablated mice. (A) Body composition and body weight measured in age-matched animals were identical in control (blue) and ablated (red) mice fed on a chow diet, before entering TSE metabolic chambers (n = 7/group). (B) Cumulative food intake in metabolic chambers for control littermates (blue) and ablated mice (red bars). All mice were switched to a 42% high-fat diet at 48 h. (C–D) Respiratory exchange rate and oxygen consumption during the light and dark phase, before (open circles) and after high-fat diet exposure (filled circles). Metabolic rate parameters were identical in control (blue) and ablated (red) animals. (E) Total ambulatory movements (x, y, z directions) were not significantly different between genotypes. Nonetheless, we observed a trend towards lowered activity in ablated mice (red) after high-fat diet exposure. (F) Core body temperature monitored using telemetry demonstrated that ablated mice (red) normally regulated their temperature, even in response to dietary challenges including overnight fasting and high-fat diet exposure. In particular, fasting was associated with reduced core temperature in both groups. Conversely, temperature during the light phase was slightly raised in both groups after high-fat diet exposure (n = 6 control and n = 7 ablated).

Figure 3

Characterization of meal patterns and postprandial neuronal activation. (A–F) Meal parameters of ablated (red) and controls (blue) measured in metabolic chambers during the dark phase (n = 7/group). Asterisks denote significant differences between chow- (open circles) and high-fat diet-fed mice (filled circles) (two-way ANOVA, with Bonferroni correction; *, p < 0.05; **, p < 0.01). (G) Immunohistochemistry for c-Fos in the dorsovagal complex of control (top image) and ablated mice (bottom image) 1 h 30 min after a large fatty meal. (H, I) The total number of c-Fos positive neurons in the dorsovagal complex per section was identical in both groups and positively correlated with the stomach weight (n = 7 control and n = 6 ablated). Error bars indicate SEM. Abbreviations: AP, area postrema; DMV, dorsal motor nucleus of the vagus nerve; cc, central canal; NTS, nucleus of the solitary tract.

Figure 4

Ablated mice developed an acute phase response during chronic high-fat feeding. (A) Weekly weight gain was recorded after the start of the high-fat diet in control littermates (blue, n = 19) and ablated model mice (red, n = 21). Note that the error bars (left) indicating standard deviation is skewed toward the lowest values for the ablated group. Individual body weight curves indicate that a subset of ablated mice showed unexpected weight loss (gray lines). (B) Splenomegaly was observed in ablated mice after 9 weeks on the high-fat diet (p < 0.05, Student's t-test, unpaired). To varying degrees, the spleen of ablated mice was often visibly bigger than in control animals. (C) After 9 weeks on high-fat diet, the spleen of control littermates and ablated mice was stained with hematoxylin and eosin. In ablated mice with the most severe splenomegaly, both leukopenia and red pulp enlargement were evident. (D) Plasma levels of interleukin-6 (IL-6), IL-1β, and tumor necrosis alpha (TNF-α) measured after 9 weeks of high fat feeding (p < 0.05, Student's t-test, unpaired). The dotted line indicates the limit of detection (n = 12 controls and n = 8 ablated). (E) Expression of acute phase reactant Saa-1 in livers of ablated mice (red bars, n = 16) and their control littermates (blue bars, n = 24) (p < 0.05, Student's t-test, unpaired). Error bars indicate SEM.

Figure 5

Saturated fat-induced cute phase response in ablated mice. (A, B) Plasma levels of triglyceride or non-esterified fatty acids after gavage of lard oil (open symbols) in control (blue) versus ablated animals (red). Kinetics of the appearance of triglycerides and non-esterified fatty acids in the blood stream after lard oil gavage is similar in both groups (n = 6/group). (C) Expression of Saa-1 mRNA in livers of lard-oil fed ablated mice (red bars; n = 10, saline group; n = 17 lard oil group) and their control littermates (white bars; n = 10, saline group; n = 11 lard oil group). (D) Plasma levels of IL-6 measured 3 h after lard oil or vehicle in ablated mice (red bars; n = 8, saline group; and n = 16, lard oil group) and their control littermates (white bars; n = 8, saline group; and n = 9, lard oil group). Asterisks denote significant differences when compared with littermate controls (two-way ANOVA, with Bonferroni correction; **, p < 0.01). Error bars indicate SEM.