Resolvin RvD2 reduces hypothalamic inflammation and rescues mice from diet-induced obesity

Abstract

Background: Diet-induced hypothalamic inflammation is an important mechanism leading to dysfunction of neurons involved in controlling body mass. Studies have shown that polyunsaturated fats can reduce hypothalamic inflammation. Here, we evaluated the presence and function of RvD2, a resolvin produced from docosahexaenoic acid, in the hypothalamus of mice.

Methods: Male Swiss mice were fed either chow or a high-fat diet. RvD2 receptor and synthetic enzymes were evaluated by real-time PCR and immunofluorescence. RvD2 was determined by mass spectrometry. Dietary and pharmacological approaches were used to modulate the RvD2 system in the hypothalamus, and metabolic phenotype consequences were determined.

Results: All enzymes involved in the synthesis of RvD2 were detected in the hypothalamus and were modulated in response to the consumption of dietary saturated fats, leading to a reduction of hypothalamic RvD2. GPR18, the receptor for RvD2, which was detected in POMC and NPY neurons, was also modulated by dietary fats. The substitution of saturated by polyunsaturated fats in the diet resulted in increased hypothalamic RvD2, which was accompanied by reduced body mass and improved glucose tolerance. The intracerebroventricular treatment with docosahexaenoic acid resulted in increased expression of the RvD2 synthetic enzymes, increased expression of anti-inflammatory cytokines and improved metabolic phenotype. Finally, intracerebroventricular treatment with RvD2 resulted in reduced adiposity, improved glucose tolerance and increased hypothalamic expression of anti-inflammatory cytokines.

Conclusions: Thus, RvD2 is produced in the hypothalamus, and its receptor and synthetic enzymes are modulated by dietary fats. The improved metabolic outcomes of RvD2 make this substance an attractive approach to treat obesity.

Keywords: Brain; Hypothalamus; Lipid; Metabolism; Nutrient.

Fig. 1

Cellular distribution of GPR18 in the hypothalamus of mice. Five-micrometre tissue sections were prepared from the hypothalamic region of lean Swiss mice and were evaluated by indirect immunofluorescence staining using antibodies against GPR18 and NPY (a), POMC (b) or Iba1 (c). Nuclei were stained with DAPI. Insets depict low magnification micrographs of the region of interest. In captions, the colour of the arrow represents antigens detected in respective cells and yellow arrows mean that both antigens are present in the respective cell. Images are representative of three independent experiments

Fig. 2

The hypothalamic expression of proteins involved in the synthesis and action of RvD2. The schematic representation of the main enzymes involved in the synthesis and the receptor for RvD2 (a). The transcript expressions of phospholipase A2 (b), 15-lipoxigenase (c), 5-lipoxigenase (d) and GPR18 (e) were evaluated using real-time PCR in samples collected from the hypothalamus of mice fed either chow (CT) or a high-fat diet (HF) by the time specified in the graphics (b–e). To measure hypothalamic RvD2, we employed a MALDI method, with mass spectra standard of RvD2 corresponding to m/z 375 ((f), upper panel); a sample from the hypothalamus of a mice fed on HF ((f), lower panel); quantification of RvD2 in hypothalamic samples (g). In all experiments, n = 7. In b–e and g, *p < 0.05 vs. respective CT

Fig. 3

The impact of dietary substitution of saturated by unsaturated fats on the activity of the RvD2 system in the hypothalamus. Six-week-old Swiss mice were included in the study and fed on chow or HF for 16 weeks. Another group of mice was assigned to HF for 8 weeks following 8 weeks on a HF supplemented with 30% omega 3 (HFS) (a). Relative quantification of the RvD2 was made in the hypothalamus of mice fed either chow (CT), the high-fat diet (HF) or the HF supplemented with 30% omega 3 (HFS) (b). The transcript expressions of tumour necrosis factor alpha (TNFα) (c) and interleukin-1 beta (IL1β) (d) were evaluated using real-time PCR in samples collected from the hypothalamus of mice. Caloric intake (e) and body mass (f) were measured during the experimental period. At the end of the treatment, the experimental groups were subjected to a glucose tolerance test and an insulin tolerance test, and results are shown as the area under the curve (AUC) (g) and constant of glucose decay (kITT) (h). In all experiments, n = 7. In b–h, *p < 0.05 vs. CT and #p < 0.05 vs. HF

Fig. 4

The impact of DHA-injected icv on the activity of the RvD2 system in the hypothalamus. Six-week-old Swiss mice were included in the study and fed a HF diet for 4 weeks before intracerebroventricular (icv) cannulation; after 1 week, mice were randomly selected for either saline (2 μl) or different amounts of DHA (2 μl) icv treatment for 4 days; some mice were fed on chow throughout the experimental period (CT) (a). At the end of the experimental period, hypothalamic RNA was extracted and employed in real-time PCR determinations of phospholipase A2 (PLA2) (b), 15-lipoxigenase (15-LOX) (c), 5-lipoxigenase (5-LOX) (d), GPR18 (e), interleukin-6 (IL6) (f) and interleukin-10 (IL10) (g). Cumulative food intake (h) and body mass change (i) were evaluated during the experimental period. In all experiments, n = 7. *p < 0.05 vs. CT

Fig. 5

The impact of exogenous RvD2-injected icv on inflammatory and metabolic parameters in mice. Six-week-old Swiss mice were included in the study and fed a HF diet for 8 weeks before intracerebroventricular (icv) cannulation; after 1 week, mice were randomly selected for either saline (2 μl) or different amounts of RvD2 (2, 3 or 50 ng) icv treatment for 11 days (a). At the end of the experimental period, hypothalamic RNA was extracted and employed in real-time PCR determinations of GPR18 (b). Cumulative food intake (c) and body mass variation (d) were determined during the experimental period. At the end of the experimental period, the epididymal fat pad was measured (e). In addition, mice were submitted a glucose tolerance test, and results are expressed as the area under the curve (AUC) (f). Interleukin-6 (IL6) (g) and interleukin-10 (IL10) (h) transcripts were determined in samples from the hypothalamus, whereas uncoupling protein 1 (UCP1) (i) and PGC1a (j) transcripts were determined in samples from the brown adipose tissue. Some mice were subjected to indirect calorimetry, resulting in the values for O₂ consumption (k), CO₂ production (l) and respiratory quotient (m). In addition, some mice were subjected to a leptin tolerance test (LTT) and results are expressed as cumulative food intake during 12 h (n). In the experiments reported in panels a–j, n = 8; in the experiments reported in panels k–n, n = 5. *p < 0.05 vs. saline. Experiments for evaluation of 3 and 50 ng RvD2 in the hypothalamus were performed in different occasions. The differences obtained in some of the saline groups reflect interexperimental variability. For statistical analysis, group-specific saline control was considered as baseline