Unsaturated fatty acids revert diet-induced hypothalamic inflammation in obesity

Abstract

Background: In experimental models, hypothalamic inflammation is an early and determining factor in the installation and progression of obesity. Pharmacological and gene-based approaches have proven efficient in restraining inflammation and correcting the obese phenotypes. However, the role of nutrients in the modulation of hypothalamic inflammation is unknown.

Methodology/principal findings: Here we show that, in a mouse model of diet-induced obesity, partial substitution of the fatty acid component of the diet by flax seed oil (rich in C18:3) or olive oil (rich in C18:1) corrects hypothalamic inflammation, hypothalamic and whole body insulin resistance, and body adiposity. In addition, upon icv injection in obese rats, both ω3 and ω9 pure fatty acids reduce spontaneous food intake and body mass gain. These effects are accompanied by the reversal of functional and molecular hypothalamic resistance to leptin/insulin and increased POMC and CART expressions. In addition, both, ω3 and ω9 fatty acids inhibit the AMPK/ACC pathway and increase CPT1 and SCD1 expression in the hypothalamus. Finally, acute hypothalamic injection of ω3 and ω9 fatty acids activate signal transduction through the recently identified GPR120 unsaturated fatty acid receptor.

Conclusions/significance: Unsaturated fatty acids can act either as nutrients or directly in the hypothalamus, reverting diet-induced inflammation and reducing body adiposity. These data show that, in addition to pharmacological and genetic approaches, nutrients can also be attractive candidates for controlling hypothalamic inflammation in obesity.

Figure 1. Experimental protocols.

A, Four-week old male Swiss mice were fed regular chow (CT) or high-fat diet (HF) for eight weeks and then randomly divided into eight groups: CT mice, maintained for another eight weeks on the same CT diet; or HF mice which were either maintained on the same HF diet; or transferred to diets containing substitution of lard by olive or flax seed oils to final concentrations of 10, 20 or 30%, according to Table 1. At the end of the experimental period, the mice were submitted to a glucose tolerance test (GTT) and an insulin tolerance test (ITT), four days later, feeding behavior was evaluated and samples were obtained for immunobloting (IB) and real-time PCR (RT-PCR). B, Four-week old male Wistar rats were fed for eight weeks on CT or HF diets and then submitted to intracerebroventricular (icv) cannulation. Drinking response elicited by angiotensin II (AII) was tested three days after cannulation and responsive rats were treated icv with either ω3, ω9, stearic acid (SA) or diluents (Alb) for seven days. Feeding behavior and body mass were determined throughout the experimental period. At the end of the experimental period some rats were randomly selected for histology, IB and RT-PCR; the remainder of the rats was followed up for feeding behavior for an additional five days after the discontinuation of icv treatment.

Figure 2. Food intake and body mass variation.

A, Mean daily spontaneous food intake (g) of Swiss mice fed on regular chow (CT), high-fat diet (HF), flax seed- (FS) or olive oil- (OL) substituted (10, 20 or 30%) diets for eight weeks; results are depicted as daily food intake along the time (A), and as the means obtained during the whole period (A1). B, Body mass variation for each group during the whole experimental period. C-E, Body mass variation (g) during the 60-day experimental period (C-E) or during each of the four 15-day experimental periods (C1-E1) for CT and HF groups (C, C1); for the FS substituted groups (D, D1); and for the OL substituted groups (E, E1). F, Diet preference assay, lean Swiss mice were fasted for 10 h and then similar amounts of CT or HF (HF) diets were offered; the same approach was used to compare the preference for each of the FS or OL substituted diets against HF; results are presented as the relative caloric consumption of the tested diet during 12h. In all experiments, n = 5; in A and B, #p<0.05 vs. CT and *p<0.05 vs. HF; in C and F *p<0.05 vs. CT; in B, §p<0.05 vs. FS10 or OL10.

Figure 3. Metabolic parameters.

Blood glucose levels (A), and constant for glucose decay during an insulin tolerance test (Kitt) (%/min) (A1); and, blood glucose levels (B) and the area under glucose curve (AUC) (B1) during an intraperitoneal glucose tolerance test (ipGTT) were obtained at the end of an eight-week experimental period for Swiss mice fed on regular chow (CT), high-fat diet (HF), flax seed- (FS) or olive oil- (OL) substituted (10, 20 or 30%) diets. In all experiments, n = 5; #p<0.05 vs. CT and *p<0.05 vs. HF.

Figure 4. Signal transduction in the hypothalamus.

Hypothalamic total protein extracts obtained from Swiss mice fed on regular chow (CT), high-fat diet (HF), flax seed- (FS) or olive oil- (OL) substituted (10, 20 or 30%) diets for eight weeks were used in immunoblotting (IB) experiments to evaluate protein expression and/or activity. Specific antibodies against phospho-IκB-α (P-IκBα) (A), phospho-JNK (P-JNK) (B), TNF-α (C), SOCS-3 (D), iNOS (E), IL-10 (F), Caspase-3 (CASP-3) (G), BAX (H), Bcl-2 (I), phospho-ACC (P-ACC) (J), FAS (K) and CPT-1 (L) were used to identify respective protein targets. Loading was evaluated by re-probing the membranes with anti-β-actin (A, C-I, K and L), anti-JNK (B) or anti-ACC (J) antibodies. In all experiments, n = 5; #p<0.05 vs. CT and *p<0.05 vs. HF.

Figure 5. Food intake, body mass and adiposity in icv-treated rats.

Wistar rats fed on a regular chow (CT) or on a high-fat diet (HF) were icv cannulated and treated for five (A) or seven (B-F) days with diluent (albumin, Alb), ω3-, ω9-fatty acids or stearic acid (SA) and then used for determination of feeding behavior and adiposity. A, daily food intake (g) of rats treated icv with Alb (filled circles), ω3 (filled squares) or ω9 (filled triangles) fatty acids for five days; the beginning (I) and the end (II) of treatment are labeled with arrows. B, The suppression of spontaneous food intake (g) by leptin was evaluated at the end of the experimental period. C, Body mass variation (g) during the seven-day icv treatment period. D, Epididymal fat mass (g) at the end of the experimental period. E, Histological evaluation (hematoxilin-eosin staining of 5 µm sections) of epididymal fat. F, Mean adipocyte area obtained from histological sections. In all experiments, n = 5. In A, C and D, *p<0.05 vs. Alb; in B, #p<0.05 vs. Alb(−) and *p<0.05 vs. Alb(+); in F, #p<0.05 vs. CT, *<0.05 vs. HF.

Figure 6. Expression of inflammatory and apoptotic proteins in the hypothalamus of icv-treated rats.

Wistar rats fed on a regular chow (CT) or on a high-fat diet (HF) and icv cannulated were treated for seven days with diluent (albumin, Alb), ω3 or ω9 fatty acids and then used in immunoblotting (IB) and immufluorescence experiments. Specific antibodies against iNOS (A), IL-6 (B), TNF-α (C), IL-10 (D), phospho-JNK (P-JNK) (E), BAX (G), and Bcl-2 (H) were used to identify respective protein targets in hypothalamic samples. Loading was evaluated by re-probing the membranes with anti-β-actin (A-D, G and H) or anti-JNK (E). In F, 5 µm sections of the hypothalamus were labeled with an anti-F4/80 antibody. In all experiments, n = 5. In A-E, *p<0.05 vs. Alb; in A and B, #p<0.05 vs. ω3; In G and H, #p<0.05 vs. CT, *<0.05 vs. Alb.

Figure 7. Effect of icv ω3 and ω9 on hypothalamic signaling.

Wistar rats fed on a regular chow (CT) or on a high-fat diet (HF) and icv cannulated were treated for seven days with diluent (albumin, Alb), ω3 or ω9 fatty acids. In addition, in some experiments, rats were acutely treated with a single dose of either leptin (2 µl, 10⁻⁶M: A-G) or insulin (2 µl, 10⁻⁶M: H) and then used in immunoblotting (IB) experiments. Specific antibodies against phospho-JAK2 (P-JAK2) (A and D), phospho-STAT3 (P-STAT3) (B and E), phospho-Akt (P-Akt) (C, F and H), phospho-FoxO1 (P-FoxO1) (G), phospho-ACC (P-ACC) (I), FAS (J), CPT-1 (K) and SCD-1 (L) were used to identify respective protein targets in hypothalamic tissue. Loading was evaluated by re-probing the membranes with anti-β-actin (J-L), anti-JAK2 (A and D), anti-STAT3 (B and E), anti-Akt (C, F and H), anti-FoxO1 (G) or anti-ACC (I). In A-H, #p<0.05 vs. Alb (−), *p<0.05 vs. Alb (+); in I-L, #p<0.05 vs. CT, *p<0.05 vs. Alb.

Figure 8. Effect of icv ω3 and ω9 on neurotransmitter expression and thermogenesis.

Wistar rats fed on a regular chow (CT) or on a high-fat diet (HF) and icv cannulated were treated for seven days with diluent (albumin, Alb), ω3 or ω9 fatty acids and then used in real-time PCR and immunobloting experiments. Total RNA obtained from hypothalami were used in real-time PCR to amplify the mRNAs of NPY (A), MCH (B), POMC (C), and CART (D). Brown adipose tissue total protein extracts were used for evaluation of UCP-1 expression by immunoblot (E). In all experiments, n = 5. In A-D, *p<0.05 vs. Alb (−); in E, #p<0.05 vs. CT, *p<0.05 vs. Alb.

Figure 9. GPR120 signal transduction in the hypothalamus.

Five µm sections of the hypothalamus obtained from obese Wistar rats were labeled with anti-GPR120 (green) and NPY (red) antibodies, low (A) and high (B) magnifications are depicted. Icv cannulated obese Wistar rats were acutely treated with diluent (albumin, Alb), ω3 or ω9 fatty acids and then used in immunoprecipitation (IP)/immunoblotting (IB) experiments employing antibodies against GRP120 (C), β-arrestin 2 (C and D), TAK1 (E), and TAB1 (D and E). In all experiments n = 5. In C-E, *p<0.05 vs. Alb.