Nervonic acid limits weight gain in a mouse model of diet-induced obesity

Abstract

Lipid perturbations contribute to detrimental outcomes in obesity. We previously demonstrated that nervonic acid, a C24:1 ω-9 fatty acid, predominantly acylated to sphingolipids, including ceramides, are selectively reduced in a mouse model of obesity. It is currently unknown if deficiency of nervonic acid-sphingolipid metabolites contribute to complications of obesity. Mice were fed a standard diet, a high fat diet, or these diets supplemented isocalorically with nervonic acid. The primary objective was to determine if dietary nervonic acid content alters the metabolic phenotype in mice fed a high fat diet. Furthermore, we investigated if nervonic acid alters markers of impaired fatty acid oxidation in the liver. We observed that a nervonic acid-enriched isocaloric diet reduced weight gain and adiposity in mice fed a high fat diet. The nervonic acid enrichment led to increased C24:1-ceramides and improved several metabolic parameters including blood glucose levels, and insulin and glucose tolerance. Mechanistically, nervonic acid supplementation increased PPARα and PGC1α expression and improved the acylcarnitine profile in liver. These alterations indicate improved energy metabolism through increased β-oxidation of fatty acids. Taken together, increasing dietary nervonic acid improves metabolic parameters in mice fed a high fat diet. Strategies that prevent deficiency of, or restore, nervonic acid may represent an effective strategy to treat obesity and obesity-related complications.

Keywords: ceramide; fatty acid oxidation; obesity; omega-9; sphingolipids.

FIGURE 1

Ceramides are altered in models of diet-induced obesity. (A) Ceramide composition was assessed by LC-MS/MS of liver samples derived from male C57Bl6/J mice (n = 10/group) fed a control or 60% high-fat chow, or isocaloric diets supplemented with nervonic acid (0.6%). The sum of the reported ceramide species is shown in the inset. Other NA-containing lipids were also quantified, including (B) C24:1-hexosylceramide, (C) C24:1-sphingomyelin, (D) C24:1-lysophosphatidylcholine (LPC), and (E–H) diacylglycerides (DG) (E) C16:0/C24:1, (F) C18:0/C24:1, (G) C18:1/C24:1, and (H) C18:2/C24:1. (I) Plasma C24:1-ceramides were assessed after 72 hours on the indicated diets (n = 4/group). For (A–I), two-way ANOVA did not reveal significant interactions between the diets and NA. (J) CerS2 knockdown decreased C24:1-ceramide, which is only partially restored by 1 μM NA treatment for 24 hours in HEK293 cells (n = 3), CerS2 siRNA diminished mRNA by 70% (data not shown). (J) Ceramide composition was determined from male Sprague-Dawley rats (n = 5/group) fed a low fat, a 40%, or a 60% fat containing diet. One-way ANOVA was utilized to assess differences between groups (*P < .05, **P < .01, ***P < .001)

FIGURE 2

Nervonic acid prevents diet-induced body weight gain. (A) Male C57BL/6J mice were fed a control or high-fat diet supplemented with or without nervonic acid and body weights were followed over 12 weeks. Pairwise comparisons between groups using a permutation test was performed (n = 10–12 mice/group *P < .05, ***P < .001). (B) Body composition was assessed from the same mice by NMR-MRI. One-way ANOVA was utilized to assess differences between groups (n = 10–12 mice/group ***P < .001 from control, ^§§§P < .001 from HFD, ^§P = .025 from HFD). Two-way ANOVA demonstrates a significant interaction of diet with NA for fat mass (P < .001) (C) Average food consumption was measured over 12 weeks (n = 10–12 mice/group ***P < .001). (D) Bomb calorimetry was utilized to quantify fecal caloric content (n = 10–11 mice/group *P < .05)

FIGURE 3

Nervonic acid-enrichment of a HFD leads to energy expenditure values similar to control mice. (A) VO2, (B) VCO2, (C) RER, (D) Heat, (E & F) locomotion were assessed by placing mice on indicated diets in metabolic cages. Shaded region in (E) represents the dark cycle (n = 10–12 mice/group **P < .01, ***P < .001). Two-way ANOVA demonstrates a significant interaction of diet with NA for VO2, VCO2, and heat in the light cycle (P < .05)

FIGURE 4

Nervonic acid improves glucose tolerance and insulin sensitivity in mice on a high-fat diet. (A) Fasting and (B) random-fed blood glucose, (C) fasting insulin, and (D) insulin levels after IP injection of glucose at indicated time points were measured in mice after 8 weeks on different diets. (E) GTT and (F) ITT were performed on mice after 10 and 11 weeks on diets, respectively. One-way ANOVA was utilized to assess differences between groups (n = 10–12 mice/group *P < .05, **P < .01, ***P < .001). Two-way ANOVA demonstrates a significant interaction of diet with NA for fasting blood glucose (P < .05), fasting insulin (P < .001), glucose-stimulated insulin production (P < .05), GTT (P < .01), and ITT (P < .005)

FIGURE 5

Nervonic acid enrichment improves markers of liver fatty acid oxidation. (A) RT-qPCR was utilized to assess transcript levels of PPARα, PGC1α, and SIRT1 (n = 8 mice/group *P < .05, **P < .01). (B) Short, (C) Medium, and (D) Long-chain acylcarnitine levels were assessed by LC-MS/MS (n = 10–11 mice/group *P < .05, **P < .01, ***P < .001 when compared to control, ^#P < .05, ^##P < .01, ^###P < .001 for HFD to HFD + NA comparisons). Where significant, results from two-way ANOVA indicating an interaction between the diet and NA are indicated above each acylcarnitine (^•P < .05, ^••P < .01, ^•••P < .001)