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. 2017 Apr 26;14(1):91.
doi: 10.1186/s12974-017-0869-7.

Polyunsaturated fatty acid receptors, GPR40 and GPR120, are expressed in the hypothalamus and control energy homeostasis and inflammation

Nathalia R V Dragano  1 , Carina Solon  1 , Albina F Ramalho  1 , Rodrigo F de Moura  1 , Daniela S Razolli  1 , Elisabeth Christiansen  2 , Carlos Azevedo  2 , Trond Ulven  2 , Licio A Velloso  3   4
Affiliations

Polyunsaturated fatty acid receptors, GPR40 and GPR120, are expressed in the hypothalamus and control energy homeostasis and inflammation

Nathalia R V Dragano et al. J Neuroinflammation. .

Abstract

Background: The consumption of large amounts of dietary fats is one of the most important environmental factors contributing to the development of obesity and metabolic disorders. GPR120 and GPR40 are polyunsaturated fatty acid receptors that exert a number of systemic effects that are beneficial for metabolic and inflammatory diseases. Here, we evaluate the expression and potential role of hypothalamic GPR120 and GPR40 as targets for the treatment of obesity.

Methods: Male Swiss (6-weeks old), were fed with a high fat diet (HFD, 60% of kcal from fat) for 4 weeks. Next, mice underwent stereotaxic surgery to place an indwelling cannula into the right lateral ventricle. intracerebroventricular (icv)-cannulated mice were treated twice a day for 6 days with 2.0 μL saline or GPR40 and GPR120 agonists: GW9508, TUG1197, or TUG905 (2.0 μL, 1.0 mM). Food intake and body mass were measured during the treatment period. At the end of the experiment, the hypothalamus was collected for real-time PCR analysis.

Results: We show that both receptors are expressed in the hypothalamus; GPR120 is primarily present in microglia, whereas GPR40 is expressed in neurons. Upon intracerebroventricular treatment, GW9508, a non-specific agonist for both receptors, reduced energy efficiency and the expression of inflammatory genes in the hypothalamus. Reducing GPR120 hypothalamic expression using a lentivirus-based approach resulted in the loss of the anti-inflammatory effect of GW9508 and increased energy efficiency. Intracerebroventricular treatment with the GPR120- and GPR40-specific agonists TUG1197 and TUG905, respectively, resulted in milder effects than those produced by GW9508.

Conclusions: GPR120 and GPR40 act in concert in the hypothalamus to reduce energy efficiency and regulate the inflammation associated with obesity. The combined activation of both receptors in the hypothalamus results in better metabolic outcomes than the isolated activation of either receptor alone.

Keywords: GPR120; GPR40; Hypothalamic inflammation; Obesity.

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Figures

Fig. 1
Fig. 1
Cellular distribution of GPR120 in the hypothalamus of mice. Tissue sections (5.0 μm) were prepared from the hypothalamic region of lean Swiss mice and were evaluated by indirect immunofluorescence staining using antibodies against GPR120 (a–c, green), NPY (a, red), POMC (b, red), and mannose receptor (c, red). Nuclei were stained with DAPI (blue). In the captions, the arrows indicate cells co-expressing GPR120 and mannose receptor (c). Images are representative of three independent experiments
Fig. 2
Fig. 2
Cellular distribution of GPR40 in the hypothalamus of mice. Tissue sections (5.0 μm) were prepared from the hypothalamic region of lean Swiss mice and were evaluated by indirect immunofluorescence staining using antibodies against GPR40 (a–c, green), NPY (a, red), POMC (b, red), and Iba1 (c, red). Nuclei were stained with DAPI (blue). In the captions, the arrows indicate cells co-expressing GPR40 and NPY (a), and GPR40 and POMC (b). Images are representative of three independent experiments
Fig. 3
Fig. 3
Cellular distribution of GPR120 in the hypothalamus of mice. Tissue sections (5.0 μm) were prepared from the hypothalamic region of lean Swiss mice and were evaluated by indirect immunofluorescence staining using antibodies against GPR120 (a–b, red in A and green in B), vimentin (a, green), and IGFBP2 (b, red). Nuclei were stained with DAPI (blue). In the captions, the yellow circle indicates region with cells co-expressing GPR120 and vimentin (a). Images are representative of three independent experiments
Fig. 4
Fig. 4
Expression and activation of GPR120 and GPR40. The expression of GPR120 (a and c) and GPR40 (b and d) transcripts were evaluated by real-time PCR in samples collected from the small intestine, liver, white adipose tissue (WAT), frontal cortex, occipital cortex, hippocampus, and hypothalamus (a and b) of lean Swiss mice or in samples prepared from the mHypoA 2/29 CLU189 and BV2 cell lines (c and d). In e–g, lean Swiss mice were subjected to intracerebroventricular cannulation and treated with a single dose of GW9508 (2.0 μl, 1.0 mM); samples were collected after 90 min for the evaluation of GPR120 (E) or GPR40 (g) transcript expression by real-time PCR. In addition, protein samples were utilized in immunoprecipitation experiments using an anti-β-arrestin-2 antibody; immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted (IB) with GPR120 or β-arrestin-2 antibodies (f). In all experiments, n = 5. In C and D, *p < 0.05 vs. CLU189; in E and F, *p < 0.05 vs. vehicle
Fig. 5
Fig. 5
Intracerebroventricular treatment with GW9508 in lean mice. Five-week-old Swiss mice were included in the study and were fed on chow for 4 weeks before intracerebroventricular (icv) cannulation; after 1 week, mice were randomly selected for either vehicle (2.0 μl) or GW9508 (2.0 μl, 1.0 mM) icv treatment twice a day for 6 days (a). Body mass (b and c) and caloric intake (d) were measured during the experimental period. At the end of the experimental period, some mice were subjected to indirect calorimetry and the determination of spontaneous physical activity, resulting in the values for O2 consumption (E–F), CO2 production (g–h), energy expenditure (i–j), respiratory quotient (k), and spontaneous physical activity (l). At the end of the experimental period, the hypothalamus was dissected, and RNA was extracted for real-time PCR determination of transcript levels of TNF-α (m), IL1β (n), IL10 (o), and IL6 (p). In all experiments, n = 5; *p < 0.05 vs. vehicle
Fig. 6
Fig. 6
Intracerebroventricular treatment with GW9508 in obese mice. Five-week-old Swiss mice were included in the study and were fed on a high-fat diet (HFD) for 4 weeks before intracerebroventricular (icv) cannulation; after 1 week, mice were randomly selected for either vehicle (2.0 μl) or GW9508 (2.0 μl, 1.0 mM) icv treatment twice a day for 6 days (a). Body mass (b and c), caloric intake (d), and energy efficiency (e) were measured during the experimental period. At the end of the experimental period, some of the mice were subjected to indirect calorimetry and the determination of spontaneous physical activity, resulting in the values for O2 consumption (F), CO2 production (g), energy expenditure (h), spontaneous physical activity (i), and respiratory quotient (j). RNA samples were obtained from the brown adipose tissue for real-time PCR determination of the transcript expression of UCP1 (k), PGC1α (l), and cytochrome c (m). In addition, the hypothalamus was dissected, and RNA was extracted for real-time PCR determination of the transcript levels of TNF-α (n), IL1β (o), IL10 (p), and IL6 (q). In all experiments, n = 5; *p < 0.05 vs. vehicle
Fig. 7
Fig. 7
Lentiviral targeting of GPR120. In a, the mHypoA 2/29 CLU189 cell line was transfected with lentivirus containing five distinct (LV1–LV5) sequences of shRNA targeting GPR120 and, in addition, a scrambled control. Protein extracts were prepared and subjected to separation by SDS-PAGE, then transferred to a nitrocellulose membrane and blotted with anti-GPR120 antibody. The sequences LV1 and LV2 were utilized in in vivo experiments and were injected bilaterally into the arcuate nucleus (b). 7 days after LV1 or LV2 injection, hypothalami were dissected and used for protein extraction. Samples were subjected to separation by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted (IB) with an antibody against GPR120 (c). Swiss mice were fed for 4 weeks on a high-fat diet (HFD) and then injected with LV1 or the scrambled control construct into the arcuate nucleus bilaterally; after 10 weeks, the mice were subjected to intracerebroventricular cannulation and following a recovery period of 7 days, subjected to treatment with GW9508 (2.0 μl, 1.0 mM) twice a day for 6 days (d). Body mass (e and f) and caloric intake (g) were determined during the 10 weeks that followed LV1/scramble injection (before GW9508 treatment). Body mass (h), caloric intake (i), and energy efficiency (j) were evaluated during the 6 days that followed GW9508 treatment. At the end of the experimental period, IL10 protein expression was determined in extracts from the hypothalamus (k). In c and k, loading controls were obtained by reprobing the membranes with anti-α-tubulin antibody. In A, the results are representative of three independent experiments. In C, n = 4; *p < 0.05 vs. scramble. In E–K, n = 5; *p < 0.05 vs. scramble
Fig. 8
Fig. 8
Schematic representation of the main findings of this study. GPR120 and GPR40 are expressed in the hypothalamus of mice; GPR120 is expressed predominantly in microglia whereas GPR40 is expressed predominantly in POMC and NPY neurons. The intracerebroventricular (icv) injection of agonists in mice results in a number of phenotypic changes. TUG1197, the GPR120 agonist, acts predominantly reducing hypothalamic inflammation. TUG905, the GPR40 agonist, acts predominantly reducing body mass and increasing the expression of the anorexigenic precursor, POMC. The use of a compound that acts simultaneously on GPR120 and GPR40, GW9508, results in the best metabolic outcomes, reducing body mass, improving glucose tolerance and reducing hypothalamic inflammation
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