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Activation of GPR40 induces hypothalamic neurogenesis through p38-dependent mechanisms

AbstractHypothalamic adult neurogenesis provides the basis for renewal of neurons involved in the regulation of whole-body energy status. In addition to hormones, cytokines and growth factors, components of the diet, particularly fatty acids, have been shown to stimulate hypothalamic neurogenesis; however, the mechanisms behind this action are unknown. Here, we hypothesized that GPR40 (FFAR1), the receptor for medium and long chain unsaturated fatty acids, could mediate at least part of the neurogenic activity in the hypothalamus. We show that a GPR40 ligand increased hypothalamic cell proliferation and survival in adult mice. In postnatal generated neurospheres, acting in synergy with brain-derived neurotrophic factor (BDNF) and interleukin 6, GPR40 activation increased the expression of doublecortin during the early differentiation phase and of the mature neuronal marker, microtubule-associated protein 2 (MAP2), during the late differentiation phase. In Neuro-2a proliferative cell-line GPR40 activation increased BDNF expression and p38 activation. The chemical inhibition of p38 abolished GPR40 effect in inducing neurogenesis markers in neurospheres, whereas BDNF immunoneutralization inhibited GPR40-induced cell proliferation in the hypothalamus of adult mice. Thus, GPR40 acts through p38 and BDNF to induce hypothalamic neurogenesis. This study provides mechanistic advance in the understating of how a fatty acid receptor regulates adult hypothalamic neurogenesis.

IntroductionNeurons of the mediobasal hypothalamus play central roles in the homeostatic control of food intake and energy expenditure1,2. They are responsive to hormones, neural signals, and nutrients that indicate the energy stores in the body; as long as orexigenic and anorexigenic responses are preserved, body mass stability is sustained over time3,4,5. However, a number of environmental and genetic factors can affect the function and viability of hypothalamic neurons, changes that result in abnormal regulation of body mass6,7,8,9.In aging and obesity, hypothalamic neurons are damaged by inflammation; they undergo abnormal function and eventually apoptosis10,11,12,13,14,15. This phenomenon generates an imbalance in neuronal orexigenic and anorexigenic subpopulations and further contributes to the progression of increased adiposity and metabolic complications10,11,12,13,14,15. If neuronal loss is prevented by modifications in the diet or inhibition of hypothalamic inflammation, whole body energy homeostasis is restored8,15. However, upon long-lasting exposure to the damaging effects of obesity and aging, neuronal loss may be reverted only by the generation of new neurons16,17.Physiological adult hypothalamic neurogenesis occurs at a much lower rate than neurogenesis in the subventricular and subgranular zones, the most important anatomical sources of newborn neurons in adulthood18. Nevertheless, under certain types of stimuli, hypothalamic neurogenesis can increase substantially and impact whole body energy homeostasis9,12,17. This phenomenon occurs for stimuli provided by growth factors, such as ciliary neurotrophic factor (CNTF), fibroblast growth factor 10 (FGF10), and brain-derived neurotrophic factor (BDNF)17,19,20, as well as hormones, such as leptin, insulin, and estradiol12,21,22.Diet can also modulate hypothalamic neurogenesis; as compared to solid diet, liquid diet results in decreased cell proliferation in the hypothalamus of adult rodents23. Nutrients can impact either positively or negatively the generation of new neurons depending on the type of nutrient that is employed12,24. Mono- and polyunsaturated fatty acids (MUFAs and PUFAs, respectively) act through distinct pathways in the hypothalamus by controlling food intake, energy expenditure, systemic glucose metabolism, and inflammation25,26,27,28,29. One of the putative mechanisms behind the beneficial effects of MUFAs and PUFAs in the hypothalamus is neurogenesis30,31. We have previously shown that one PUFA, docosahexaenoic acid (DHA), induces hypothalamic neurogenesis via the activation of medium- and long-chain fatty acid receptor, GPR4030, which is also regarded as a mediator of the neurogenic effect of PUFAs in the hippocampus32. However, the mechanisms that drive GPR40-dependent induction of hypothalamic neurogenesis are currently unknown. Here, employing potent synthetic agonists of GPR40 in living animals, neuronal cell culture, and neurospheres, we demonstrated that BDNF and p38 are important components of the system mediating GPR40-induced hypothalamic neurogenesis.ResultsGPR40 modulated proliferation and survival of hypothalamic adult neural precursor cellsTo address the putative involvement of GPR40 activation on adult hypothalamic neurogenesis, we determined the rates of cellular proliferation in 8-week old C57BL/6J mice treated with GW9508 or vehicle. Both experimental groups were injected via intracerebroventricular (icv) and intraperitoneal (ip) routes with the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) and euthanized 24 h or 28 days later (Fig. 1A). The phenotype of BrdU-positive cells was characterized by colocalization with the neural precursor cell markers Sox2 and vimentin (Fig. 1B). As shown in Fig. 1C, in the hypothalamic ventricular zone (HVZ), GW9508-treated mice presented increased numbers of BrdU-labeled cells compared to wild-type mice, data that indicate increased proliferation of precursor cells. In addition, in order to estimate cell survival, we determined the number of newly generated cells that persisted four weeks after BrdU incorporation; as shown in Fig. 1D, there was an increase in the number of BrdU-labeled cells in the GW9508 treated animals in both the HVZ and parenchyma (PA).Figure 1GPR40 modulates cell proliferation and survival in the hypothalamus of adult mice. C57BL/6J mice received a 7-day repeated treatment of GW9508 or vehicle, and BrdU, were sacrificed 24 h or 28 days after the last BrdU injection by transcardial perfusion and their brains were processed for immunohistochemistry (A). The co-labeling of BrdU/vimentin and BrdU/sox2 positive cells indicates the neural precursor phenotype of newborn cells after 24 h of BrdU injections (B). Panel B also shows representative images of BrdU-positive cells in the hypothalamic ventricular zone (HVZ) of vehicle and GW9508 treated mice after 24 h. The GW9508 treated mice showed increased number of BrdU immunopositive cells in the in the HVZ (C). Immunolabeling for BrdU-positive cells present in the hypothalamus 28 days after the last BrdU administration reveals higher survival of newborn cells in both the HVZ and parenchyma (PA) of GW9508 treated mice (D). White arrows indicate either BrdU, vimentin and sox2 immunopositive cells in the HVZ. Scale bars = 50 μm (B). The effect of GPR40 over adult NPC proliferation was also assessed ex vivo. Cell proliferation was estimated by quantifying the number of primary neurospheres generated after 13 days exposure to GPR40 agonists (GW9508 and TUG905) and antagonist (GW1100) (E). Phase-contrast image of hypothalamic neurosphere generated from adult hypothalamic NPC cells and cultured with growth factors in non-adhesive conditions (F). Neurospheres obtained from control group showed high mRNA expression of NPC markers and hypothalamus-related genes (G). GPR40 activation increased the number of generated neurospheres, while its inhibition reduced NPC proliferation (H). Scale bar = 100 μm (F). Data are presented as means ± SEM. N = 5–7 per group (C, D) 1 (G) and 2–5 preparations (H). *p 
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