Apoptosis of hypothalamic neurons is believed to play an important role in the development and perpetuation of obesity. Similar to the hippocampus, the hypothalamus presents constitutive and stimulated neurogenesis, suggesting that obesity-associated hypothalamic dysfunction can be repaired. Here, we explored the hypothesis that n-3 polyunsaturated fatty acids (PUFAs) induce hypothalamic neurogenesis. Both in the diet and injected directly into the hypothalamus, PUFAs were capable of increasing hypothalamic neurogenesis to levels similar or superior to the effect of brain-derived neurotrophic factor (BDNF). Most of the neurogenic activity induced by PUFAs resulted in increased numbers of proopiomelanocortin but not NPY neurons and was accompanied by increased expression of BDNF and G-protein–coupled receptor 40 (GPR40). The inhibition of GPR40 was capable of reducing the neurogenic effect of a PUFA, while the inhibition of BDNF resulted in the reduction of global hypothalamic cell. Thus, PUFAs emerge as a potential dietary approach to correct obesity-associated hypothalamic neuronal loss.

The consumption of dietary fats is regarded as one of the most important epidemiological factors leading to the increased prevalence of obesity in the world (1,2). Owing to their energetic value, dietary fats have a direct impact on overall caloric consumption, which can, per se, favor the increase in body mass. However, studies performed over the last 10 years have unveiled additional mechanisms linking dietary fats to obesity. In rodents, saturated fatty acids induce hypothalamic inflammation through the activation of TLR4 signaling and endoplasmic reticulum stress (3,4). In the short term, the hypothalamic neurons involved in the control of energy homeostasis are affected at the functional level and develop resistance to the adipostatic signals delivered by leptin and insulin (5,6). However, over time, neurons may become permanently damaged and undergo apoptosis (7,8). In addition, studies using different neuroimaging methods suggest that obese humans also present abnormalities in the hypothalamus (8,9).

Neuronal plasticity contributes to the cyclical renewal of hypothalamic neurons and is known to play an important role in the physiological regulation of whole-body energy homeostasis (10). A part of the renewal process that warrants the continuous turnover of hypothalamic neurons depends on leptin and ciliary neurotrophic factor (CNTF), which induce neurogenesis of the cells that express neurotransmitters involved in the control of feeding and thermogenesis, as well as proteins involved in the response to leptin (11,12). However, recent studies have shown that the turnover of hypothalamic neurons is disrupted by the consumption of dietary fats (13,14). In fact, the adult neural stem cells of the mediobasal hypothalamus are damaged by the inflammatory signals induced by dietary fats, placing diet-induced hypothalamic inflammation in an upstream position related to hypothalamic neurogenesis defects in obesity (14).

The beneficial outcomes of the dietary consumption of polyunsaturated fatty acids (PUFAs) have been known for many years (15). These outcomes have been shown at the epidemiological level, such as the cardiovascular protective effect of the Mediterranean diet and the consumption of fish oil by the Eskimos (1517), and at the cellular and molecular levels as illustrated by the regulation of lymphocyte function and the activation of anti-inflammatory mediator synthesis (18,19). In a recent study, we showed that the dietary substitution of saturated fat by PUFAs reduced obesity-associated hypothalamic inflammation, resulting in increased responsiveness to leptin and body mass reduction (20). Here, we asked whether PUFAs, and particularly docosahexaenoic acid (DHA), are capable of inducing hypothalamic neurogenesis. Our results show that when administered either via diet or injection directly into the hypothalamus, PUFAs increase the hypothalamic neurogenesis, which is repressed by the inhibition of G-protein–coupled receptor 40 (GPR40).

Experimental Animals and Protocols

All experimental procedures were performed in accordance with the guidelines of the Brazilian College for Animal Experimentation and were approved by the Ethics Committee at the University of Campinas. Five-week-old male Swiss albinus mice were maintained in individual cages at 21 ± 2°C, with a 12–12 h dark–light cycle and diet and water ad libitum. The mice were submitted to six different experimental protocols. In protocol 1, the mice were fed a high-fat diet (HFD) for 8 weeks and then randomly divided into four groups that were fed either an HFD or an HFD with partial (10% or 20%) or total (30%) substitution of the predominantly saturated fat content with flaxseed oil containing ∼45% C18:3 (n-3) for another 8 weeks. In protocol 2, the mice were fed an HFD for 8 weeks and then randomly divided into four groups that for another 8 weeks were fed an HFD, an HFD with a partial substitution of the fat content corresponding to 20% of the predominantly saturated fat content with flaxseed oil containing ∼45% C18:3 (n-3), a normal-fat diet (NFD) (chow), or an NFD with 2.3% (wt/wt) supplementation with flaxseed oil containing ∼45% C18:3. During the final 10 days, the mice were treated with a solution containing 50 mg/kg of BrdU (no. B5002; Sigma-Aldrich) intraperitoneally twice a day. In protocol 3, the mice were fed an HFD for 8 weeks and then randomly divided into three groups that were treated for 10 days with a daily injection of saline (2.0 μL i.c.v.), brain-derived neurotrophic factor (BDNF) (no. B3795; Sigma-Aldrich) (10 ng in 2.0 μL i.c.v.), or DHA (no. D2534, Sigma-Aldrich) (10 ng in 2.0 μL i.c.v.); additionally, the mice were treated intraperitoneally with a solution containing 50 mg/kg BrdU twice a day and were then transferred to an NFD (chow) for another 20 days. In protocol 4, the mice were fed an HFD for 8 weeks and then randomly divided into three groups that were treated for 10 days with saline (200 μL i.p.), DHA (1.0 mg/kg in 200 μL i.p.), or a higher concentration of DHA (5.0 mg/kg in 200 μL i.p.). In protocol 5, the mice were fed an HFD for 8 weeks and then randomly divided into four groups that were treated for an additional 8 weeks. In two of these groups, the mice were transferred to an NFD with 2.3% (wt/wt) supplementation with flaxseed oil containing ∼45% C18:3 and treated with either an anti-BDNF antibody (no. sc546; Santa Cruz Biotechnology, Dallas, TX) (0.8 μg in 100 μL i.p. twice a week) or a similar volume of a preimmune serum (no. R9133; Sigma-Aldrich). In the remaining two groups, the mice were transferred to an HFD with a partial substitution of the fat content with 20% flaxseed oil containing ∼45% C18:3 and treated with an anti-BDNF antibody (0.8 μg in 100 μL i.p. twice a week) or a similar volume of a preimmune serum. In protocol 6, the mice were fed an HFD for 8 weeks and then randomly divided into four groups that were treated for 10 days with a daily injection of vehicle plus saline (2.0 μL i.c.v.), Gw1100 (100 ng in 2.0 μL inhibitor of GPR40 i.c.v.; Cayman Chemical Co., Ann Arbor, MI) plus saline, vehicle plus DHA (10 ng in 2.0 μL i.c.v.), or Gw1100 (100 ng in 2.0 μL i.c.v.) plus DHA (10 ng in 2.0 μL i.c.v.); additionally, the mice were treated intraperitoneally with a solution containing 50 mg/kg BrdU twice a day and were then transferred to an NFD (chow) for another 20 days.

Intracerebroventricular Instrumentation

In some experiments, mice were stereotaxically instrumented using a Stoelting stereotaxic apparatus to implant a cannula. The stereotaxic coordinates were as follows: anteroposterior, 0.34 mm; lateral, 1.0 mm; and depth, 2.2 mm to the lateral ventricle. The cannula efficiency was tested 1 week after cannulation by the evaluation of the drinking response elicited by the injection of 10−6 mol/L i.c.v. angiotensin II (no. A9525; Sigma-Aldrich).

Analysis of the Fatty Acid Composition in the Diets and Hypothalmi

The methyl esters of fatty acids were prepared as previously described (21) using a CGC Capillary Gas Chromatograph Agilent 6850 Series GC System equipped with a capillary column Agilent DB-23 (50% cyanopropyl-methylpolysiloxane) that was 60 m long with a 0.25-mm internal diameter and a 0.25-mm film.

Glucose Tolerance Test

After 6 h of fasting, the blood glucose was measured, and then a glucose solution (2.0 g/kg i.p.) was administered. The blood glucose was measured after 30, 60, 90, and 120 min.

Insulin Tolerance Test

After 6 h of fasting, the blood glucose was measured, after which an insulin solution (1 unit/kg i.p.) was administered. The blood glucose was measured after 5, 10, 15, 20, and 25 min.

Blood Glucose and Insulin

The blood glucose was measured using a glucometer from Abbott (Optium, Abbott Diabetes Care, Alameda, CA). The insulin level was determined using ELISA kits (no. EZRMI-13K; Millipore, Billerica, MA).

Leptin Tolerance Test

After 12 h of fasting, 10−6 mol/L i.p. leptin (no. 429705; Calbiochem, Darmstadt, Germany) was administered at 6:00 p.m., and the spontaneous food intake was measured for 12 and 24 h.

Determination of Spontaneous Activity

Spontaneous activity was determined using an automatic system from Harvard Apparatus (LE405) (Panlab-Harvard, Holliston, MA).

Immunoblotting

Hypothalamus protein extract samples were prepared as previously described (20). Doublecortin (no. 4604; Cell Signaling, Boston, MA), Bax, Bcl-2, BDNF, GPR40, and β-actin (nos. sc493; sc492, sc546, sc32905, and sc130656; Santa Cruz Biotechnology) antibodies were used to detect the target proteins. Enhanced chemiluminescence (SuperSignal West Pico, Pierce) after incubation with a horseradish peroxidase–conjugated secondary antibody was used for detection by autoradiography. The band intensities were quantified by optical densitometry (UN-Scan-it Gel 6.1, Silk Scientific, Orem, UT).

RNA Extraction and Real-Time PCR

The mRNA levels of BDNF and GPR40 were measured in the hypothalamus by real-time PCR (ABI Prism 7500 detection system, Applied Biosystems, Grand Island, NY). The intron-skipping primers were obtained from Applied Biosystems (Mn01334043_m1 and Mm00809442_s1, respectively). GAPDH (no. 4352339E; Applied Biosystems) was used as the endogenous control. Each PCR contained 40 ng reverse-transcribed RNA, 25 µL of each specific primer, Taqman Universal master mix no. 4369016, and RNase-free water to a 10 µL of final volume.

Histology and Cell Counting

The mice were perfused with 4% paraformaldehyde. The brains were immersed in 30% sucrose (wt/vol), embedded in OCT compound (Sakura Finetek, Torrance, CA), and cut into 12-µm coronal sections using a cryostat. Optimal thickness of the sections was defined after extensive testing of the method and according to previous publications (2224). The sections were washed in PBS and incubated with 2.0 N HCl for 10 min at 37°C followed by 0.1 mol/L sodium borate for 10 min at room temperature. Thereafter, the sections were incubated with a 5% goat serum (no. G9023; Sigma-Aldrich) blocking solution in Tris-phosphate–buffered saline (0.2% of Triton X-100) for 30 min at 37°C followed by an overnight incubation with 1:100 primary antibodies against BrdU, neuropeptide Y (NPY), proopiomelanocortin (POMC) (nos. sc32323, sc28943, and sc20148; Santa Cruz Biotechnology), neuronal nuclei (NeuN) (no. MAB377C3; Millipore, Temecula, CA), and nestin (no. ab93666; Abcam, Cambridge, U.K.) diluted in the same blocking solution. Then, the sections were incubated with secondary antibodies conjugated to fluorescein isothiocyanate or rhodamine (nos. sc2777 and sc2092; Santa Cruz Biotechnology) and covered and mounted with Vectashield Mounting Medium with DAPI (no. H-1200; Vector Laboratories, Burlingame, CA). The images were acquired by a confocal laser microscope (LSM780; Zeiss, Jena, Germany), using oil immersion 40× Plan Apochromatic objective. The tiling function was used to cover the whole section, and each image was collected at 1,012 × 1,012 pixels. Three consecutive sections for every three animals were used for quantification. Positive BrdU cells were considered only when colocalized with DAPI staining to indicate cell proliferation. In addition, positive BrdU cells colocalized with NeuN staining were used to indicate neurogenesis. For normalization of sections per size, the results were expressed as the total number of cells given by DAPI staining. Positive stains were counted by a blinded observer and automatically using ImageJ (http://rsbweb.nih.gov/ij/), which produced equivalent results. Moreover, ImageJ was used to calculate the colocalization of the double stain using the JACoP plugin and the numbers determined by Manders coefficient.

Statistical Analysis

The results are presented as means ± SE. After evaluation of the distribution of all the data, the results were analyzed by Student t test or by one-way ANOVA followed by Tukey test or two-way ANOVA followed by the Holm-Sidak method to determine the significance of the individual differences. The level of significance was set at P < 0.05, and the data were analyzed using the Sigma Stat 3.1 (Systat Software, Point Richmond, CA).

Dietary n-3 PUFA Reduces Body Mass and Improves Metabolic Parameters

Swiss mice were fed an HFD for 8 weeks and then randomly divided into four groups that for another 8 weeks were fed the same HFD or an HFD with partial (10% or 20%) or total (30%) substitution of the predominantly saturated fat content by flaxseed oil containing ∼45% C18:3 (n-3). The lipid composition of the flaxseed oil was determined by gas chromatography (Fig. 1A and Table 1). The experimental protocol is depicted in Fig. 1B. The n-3 substitution resulted in reduced body mass gain (Fig. 1C), increased caloric intake (Fig. 1D), improved responsiveness to leptin (Fig. 1E), increased spontaneous activity (Fig. 1F), reduced levels of fasting blood glucose (Fig. 1G), reduced area under the curve during a glucose tolerance test (Fig. 1H and I), and increased responsiveness to insulin during an insulin tolerance test (Fig. 1J and K). In addition, the consumption of an HFD with 20% substitution of the predominantly saturated fat by flaxseed oil resulted in increased incorporation of C18:3, C20:5, and C22:5 in the hypothalamus, resulting in a significant increase of total PUFA content compared with mice fed an HFD only (Table 2).

Dietary n-3 PUFA Increases Hypothalamic Cell Proliferation

In the preceding experiment, the substitution of dietary fat by 20% flaxseed oil presented the most consistent results regarding the improvement of metabolic parameters; therefore, we next evaluated Swiss mice fed an HFD initially for 8 weeks and then randomly divided into four groups that for another 8 weeks were fed an NFD (chow) containing 2.3% fat, an NFD supplemented with 20% flaxseed oil, an HFD, or an HFD supplemented with 20% flaxseed oil (Fig. 2A). The supplementation of flaxseed oil in the HFD resulted in the increased expression of hypothalamic doublecortin (Fig. 2B and C), a reduced expression of hypothalamic Bax (Fig. 2D), and a reduced Bax-to-Bcl2 ratio (Fig. 2E and F). Moreover, the flaxseed oil supplementation resulted in increased numbers of arcuate and paraventricular nuclei hypothalamic cells undergoing proliferation in mice fed the HFD (Fig. 2G–J). This was a region-specific effect, since no proliferation could be detected in other regions of the hypothalamus (not shown) and only a minor (not significant) increase of neuronal proliferation was detected in the hippocampus (Fig. 2K and L).

Intracerebroventricular DHA Increases Hypothalamic Neurogenesis

Because the increased dietary content of PUFA was capable of inducing hypothalamic cell proliferation, we decided to evaluate the impact of intracerebroventricular DHA to induce neurogenesis. Obese Swiss mice were treated intracerebroventricularly with DHA or BDNF for 10 days as shown in Fig. 3A. Despite the fact that it was a short-term treatment, DHA was capable of significantly improving insulin sensitivity as determined by an insulin tolerance test (Fig. 3B and C). However, there were no significant changes in caloric intake and body mass (not shown). Both BDNF and DHA induced cell proliferation and neurogenesis in the hypothalamus (Fig. 3D–F). Interestingly, DHA exerted a more pronounced neurogenic effect than BDNF, although the difference between the two treatments was not significant (Fig. 3F). Another interesting feature of the treatment with DHA was the presence of BrdU-positive cells originating from the lateral wall of the third ventricle (Fig. 3D [arrows]); this effect was virtually absent in the mice treated with BDNF. We evaluated the capacity of intracerebroventricularly BDNF and DHA to induce neurogenesis in other regions of the hypothalamus and brain. In the hypothalamus, no signs of neurogenesis were detected in the preoptic and lateral regions. However, intracerebroventricular DHA induced neurogenesis in the hippocampus (Fig. 3G–I). In the hypothalamus, the origin of DHA-stimulated new cells from the lateral wall of the third ventricle was strongly evident when staining for nestin (Fig. 4A).

Intracerebroventricular DHA Increases Neurogenesis of POMC but Not NPY Neurons

Using the same experimental approach as described in Fig. 3A, we evaluated the coexpression of BrdU with POMC or NPY neurons. As depicted in Fig. 4B–D, DHA induced a significant increase in POMC neurogenesis but not in NPY neurogenesis.

DHA Induces the Increased Expression of BDNF and GPR40 in the Hypothalamus

Next, we tested whether n-3 PUFA could induce the increased expression of GPR40. To this end, we first evaluated the hypothalami of mice fed according to the same protocol as shown in Fig. 2A. As depicted in Fig. 5A and B, the increased dietary content of fat, irrespective of type, induced the expression of both BDNF and GPR40. There were no differences in either protein expression when comparing mice fed the HFD with mice fed the HFD supplemented 20% with flaxseed oil. To evaluate whether the effect of the dietary fats was partially due to n-3 PUFA, we treated obese mice with DHA injected intraperitoneally for 10 days according to the protocol shown in Fig. 5C. As depicted in Fig. 5D and E, systemic DHA did not induce an increased expression of BDNF but did increase the hypothalamic expression of GPR40 in a dose-dependent fashion.

Inhibition of BDNF Reduces the Effect of Dietary n-3 PUFA to Increase Hypothalamic Neurogenesis

To evaluate the role of BDNF as a candidate mediator of the effects of n-3 PUFAs as inducers of hypothalamic neurogenesis, obese mice were fed an NFD or an HFD with 20% flaxseed oil for 8 weeks and treated (for 8 weeks) with preimmune serum or anti-BDNF antiserum (Fig. 6A). As depicted in Fig. 6B, the use of the anti-BDNF antiserum resulted in a maximum reduction of 40% of hypothalamic BDNF expression. The global count of BrdU-positive cells was reduced by 30% in the hypothalamus of mice fed continuously on an HFD and treated with the anti-BDNF antiserum (Fig. 6E). However, when only BrdU/NeuN double-positive cells were considered, the anti-BDNF treatment was capable of inhibiting neurogenesis only in mice fed on the NFD (Fig. 6F). The intracerebroventricular injection of anti-BDNF antiserum had no effect on n-3–induced hippocampal neurogenesis (not shown).

Chemical Inhibition of GPR40 Reduces the Effect of Dietary n-3 PUFA to Increase Hypothalamic Neurogenesis

GPR40 is one of the receptors for n-3 PUFAs and in brain regions other than the hypothalamus it has been related to neurogenesis (25). For evaluation of its potential role in hypothalamic neurogenesis in response to an n-3 PUFA, obese mice were treated with DHA in the presence or not of a chemical inhibitor of GPR40, GW1100, as depicted in Fig. 7A. As expected, DHA significantly increased hypothalamic cell proliferation (Fig. 7B) and neurogenesis (Fig. 7C), effects that were completely abolished by GW1100 (Fig. 7B–E). The GW1100 had no effect on DHA-induced hippocampal neurogenesis (not shown).

One of the most important problems during the treatment of obesity is the high rate of recurrence that follows an initial period of body mass reduction (2628). Even patients who undergo bariatric surgery experience body mass regain after some time (29). During the development of obesity, a continuous and progressive resetting of the hypothalamic adipostat is thought to take place, which is partially due to the development of hypothalamic resistance to leptin and insulin (30,31). At first, adipostatic hormone resistance was regarded as a molecular phenomenon; however, recent studies have shown that hypothalamic neurons may undergo apoptosis during diet-induced obesity (7,8), which provides a cellular basis for the loss of the coordinated control of caloric intake and energy expenditure.

Apparently, hypothalamic neurons involved in the control of body mass are differentially affected by obesity. In at least three studies, preferential reductions of POMC compared with NPY/AgRP neurons occurred in the hypothalami of mice with hypothalamic inflammation triggered by increased fat intake or induced by genetic approaches (7,8,14). Thus, the relative numbers of POMC neurons in the medium-basal hypothalamus appear to play an important role in the control of body adiposity. Accordingly, a recent study provided a major advance in the field using a reversible rodent model of obesity in which POMC expression was blocked in neurons of the hypothalamus. POMC reactivation during early obesity caused a complete reversal of the phenotype; however, late reactivation was insufficient to promote the normalization of the body weight (32).

If the high rate of recurrence associated with obesity is partially a result of the loss of certain hypothalamic neuronal populations, a definitive therapeutic solution for obesity may only be achieved by the restoration of the correct neural circuitries. In this context, stimulating the neurogenesis of hypothalamic neurons emerges as a potential mechanism to be explored for the development of more efficient approaches to treat obesity.

BDNF was the first factor shown to promote neurogenesis of hypothalamic neurons (33); however, hypothalamic neurogenesis associated with changes in whole-body energy homeostasis was first demonstrated in response to CNTF (11). The study was designed to investigate the mechanisms behind the clinical and experimental evidence for sustained body mass reduction after the transient use of CNTF or its analog, Axokine (34,35). CNTF promoted the neurogenesis of leptin-responsive neurons in the hypothalamus, and the sustained action of CNTF, which could last for weeks after the interruption of its use, was proposed to be due to the rewiring of the hypothalamic circuitries involved in whole-body energy homeostasis (11). In addition to BDNF and CNTF, other biological factors and drugs induce adult hypothalamic neurogenesis, such as estrogens (36), IGF-1 (37), ethanol (38), nicotine (39), and fluoxetine (40).

In addition to neurogenesis induced by different types of stimuli, the hypothalamus also presents constitutive neurogenesis (41), similar to the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus (42,43), which places this region among the few anatomical sites capable of continuously producing new neurons during adult life (43). Two recent studies have shown that tanycytes of the median eminence are the source of at least some of the newborn hypothalamic cells, and, interestingly, these cells are responsive to dietary factors (44,45).

An important aspect of diet-induced hypothalamic neurogenesis involves the simultaneous induction of the apoptosis of neurons and neurogenesis in animals fed an HFD (7,13,14); however, in opposition to an apparently beneficial compensation for apoptotic loss, the neurogenesis induced by an HFD results in increased production of NPY/AgRP neurons (13), therefore widening the imbalance between orexigenic and anorexigenic neuronal populations. A recent study has probed this issue by showing that the defect in neurogenesis associated with the consumption of an HFD is due to the activation of inflammation, specifically through inhibitor of κB kinase (14).

In the current study, we evaluated whether n-3 PUFAs are capable of inducing hypothalamic neurogenesis. Recent studies have unveiled new anti-inflammatory mechanisms linked to the beneficial effects of PUFAs in metabolic conditions (46,47). Defective neurogenesis generated by an HFD is due to the activation of hypothalamic inflammation (14); therefore, we reasoned that the anti-inflammatory properties of PUFAs could correct the defect. In fact, in a recent study we showed that PUFAs in the diet and injected directly in the hypothalamus reduce HFD-induced inflammation, resulting in body mass reduction (20).

In the first part of this study, we show that either when present in the diet or acting directly in the hypothalamus, n-3 PUFAs increase neurogenesis in the hypothalamus. This effect is accompanied by the reduction of apoptosis markers, increased responsiveness to leptin, and reduced body mass gain as previously reported (20). Moreover, the consumption a diet rich in n-3 PUFAs changes the fatty acid composition of the hypothalamus, increasing the total content of PUFAs.

When injected directly into the hypothalamus, DHA is capable of inducing more pronounced neurogenesis than the level induced by BDNF, which is an important outcome considering that, in parallel with CNTF, BDNF is one of the most potent inducers of neurogenesis in the brain, particularly in the hypothalamus (48,49). Interestingly, DHA produced a neurogenic response in the hypothalamus that affected the numbers and the distribution and subtypes of cells differently than BDNF. In contrast to previous reports concerning the neurogenesis induced by HFD, which induces the generation of newborn cells particularly from the tanycytes of the medium eminence (44), DHA but not BDNF induced the generation of new cells from the wall of the third ventricle. Additionally, unlike BDNF and, to our knowledge, all previously reported factors capable of inducing neurogenesis in the hypothalamus (11,13,14,3640), DHA induced the preferential neurogenesis of POMC neurons.

Owing to the well-known role of BDNF as an endogenous inducer of neurogenesis and because dietary PUFAs were capable of increasing BDNF, we hypothesized that BDNF could be the mediator of the neurogenic activity of PUFAs. To test this hypothesis, we immunoneutralized hypothalamic BDNF using an antiserum. Our approach resulted in up to 40% reduction of hypothalamic BDNF. This was accompanied by a global reduction of cell proliferation. However, when counting new neurons, BDNF inhibition was capable of reducing the effect of PUFAs in mice fed on the NFD only.

In the final part of the study, we asked whether GPR40 could act as the mediator of the neurogenic effects of n-3 PUFAs. GPR40 is a class A G-protein–coupled receptor expressed at the highest levels in the pancreas and several regions of the brain, including the hypothalamus (50,51). Studies have shown that free fatty acids in the brain could act through GPR40 to control memory-stimulating progenitor cell proliferation (52). However, no previous studies have explored the potential neurogenic actions of GPR40 in the hypothalamus. Therefore, we targeted hypothalamic GPR40 using a chemical inhibitor, GW1100. This is a selective inhibitor with almost no cross-reactivity with GPR120 (53). Upon inhibition of GPR40, we obtained a complete reversal of the neurogenic effect of DHA, suggesting that most of the effect of n-3 PUFAs in inducing neurogenesis depends on this receptor.

One important aspect of the neurogenic effect of n-3 PUFAs in the brain is its apparent anatomic specificity. We evaluated other regions of the hypothalamus and the brain, and the hippocampus was the only other site presenting some neurogenic activity in response to n-3 PUFAs. There are previous reports describing the neurogenic potential of PUFAs in the hippocampus (54). In fact, at least part of the beneficial effects of PUFAs in age-related brain disorders have been attributed to their capacity to induce neurogenenesis in the hippocampus (55,56). However, in the current study, the inhibitions of either BDNF or GPR40 were capable of mitigating PUFA-induced neurogenesis in the hypothalamus only.

In conclusion, dietary PUFAs are capable of inducing hypothalamic neurogenesis. Most of the effect is mediated by GPR40, and BDNF could be at least one of the effectors of this process. Importantly, the effect appears to be directed toward the POMC neuronal population, providing a dietary approach to potentially correct the imbalance in hypothalamic neuronal subpopulations, which is a hallmark of experimental obesity.

See accompanying article, p. 551.

Acknowledgments. The authors thank Erika Roman, Gerson Ferraz, and Marcio Cruz, from the University of Campinas, for technical assistance.

Funding. Support for the study was provided by Fundação de Amparo a Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico. The Laboratory of Cell Signaling belongs to the Obesity and Comorbidities Research Center and National Institute of Science and Technology–Diabetes and Obesity. H.F.C. belongs to the National Institute of Science and Technology–Photonics Applied to Cell Biology.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. L.F.R.N. performed most of the experiments, which are part of his PhD thesis. Some of the experiments were performed under supervision of other researchers; G.F.P.S., J.M., G.O.B., C.S., R.F.M., S.C.V., L.M.I.-S., and D.S.R. performed some of the experiments. H.F.C. provided the expertise in the microscopy experiments and helped write the paper. L.A.V. is the mentor of the study, planned most experiments, and wrote the paper. L.A.V. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

1.
Moussavi
N
,
Gavino
V
,
Receveur
O
.
Could the quality of dietary fat, and not just its quantity, be related to risk of obesity
?
Obesity (Silver Spring)
2008
;
16
:
7
15
[PubMed]
2.
Goris
AH
,
Westerterp
KR
.
Physical activity, fat intake and body fat
.
Physiol Behav
2008
;
94
:
164
168
[PubMed]
3.
Zhang
X
,
Zhang
G
,
Zhang
H
,
Karin
M
,
Bai
H
,
Cai
D
.
Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity
.
Cell
2008
;
135
:
61
73
[PubMed]
4.
Milanski
M
,
Degasperi
G
,
Coope
A
, et al
.
Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity
.
J Neurosci
2009
;
29
:
359
370
[PubMed]
5.
De Souza
CT
,
Araujo
EP
,
Bordin
S
, et al
.
Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus
.
Endocrinology
2005
;
146
:
4192
4199
[PubMed]
6.
El-Haschimi
K
,
Pierroz
DD
,
Hileman
SM
,
Bjørbaek
C
,
Flier
JS
.
Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity
.
J Clin Invest
2000
;
105
:
1827
1832
[PubMed]
7.
Moraes
JC
,
Coope
A
,
Morari
J
, et al
.
High-fat diet induces apoptosis of hypothalamic neurons
.
PLoS One
2009
;
4
:
e5045
[PubMed]
8.
Thaler
JP
,
Yi
CX
,
Schur
EA
, et al
.
Obesity is associated with hypothalamic injury in rodents and humans
.
J Clin Invest
2012
;
122
:
153
162
[PubMed]
9.
van de Sande-Lee
S
,
Pereira
FR
,
Cintra
DE
, et al
.
Partial reversibility of hypothalamic dysfunction and changes in brain activity after body mass reduction in obese subjects
.
Diabetes
2011
;
60
:
1699
1704
[PubMed]
10.
Dietrich
MO
,
Horvath
TL
.
Feeding signals and brain circuitry
.
Eur J Neurosci
2009
;
30
:
1688
1696
[PubMed]
11.
Kokoeva
MV
,
Yin
H
,
Flier
JS
.
Neurogenesis in the hypothalamus of adult mice: potential role in energy balance
.
Science
2005
;
310
:
679
683
[PubMed]
12.
Pinto
S
,
Roseberry
AG
,
Liu
H
, et al
.
Rapid rewiring of arcuate nucleus feeding circuits by leptin
.
Science
2004
;
304
:
110
115
[PubMed]
13.
McNay
DE
,
Briançon
N
,
Kokoeva
MV
,
Maratos-Flier
E
,
Flier
JS
.
Remodeling of the arcuate nucleus energy-balance circuit is inhibited in obese mice
.
J Clin Invest
2012
;
122
:
142
152
[PubMed]
14.
Li
J
,
Tang
Y
,
Cai
D
.
IKKβ/NF-κB disrupts adult hypothalamic neural stem cells to mediate a neurodegenerative mechanism of dietary obesity and pre-diabetes
.
Nat Cell Biol
2012
;
14
:
999
1012
[PubMed]
15.
Knoops
KT
,
de Groot
LC
,
Kromhout
D
, et al
.
Mediterranean diet, lifestyle factors, and 10-year mortality in elderly European men and women: the HALE project
.
JAMA
2004
;
292
:
1433
1439
[PubMed]
16.
Estruch
R
,
Ros
E
,
Salas-Salvadó
J
, et al.;
PREDIMED Study Investigators
.
Primary prevention of cardiovascular disease with a Mediterranean diet
.
N Engl J Med
2013
;
368
:
1279
1290
[PubMed]
17.
Bjerregaard
P
,
Mulvad
G
,
Pedersen
HS
.
Cardiovascular risk factors in Inuit of Greenland
.
Int J Epidemiol
1997
;
26
:
1182
1190
[PubMed]
18.
Fischer
S
,
Weber
PC
.
Prostaglandin I3 is formed in vivo in man after dietary eicosapentaenoic acid
.
Nature
1984
;
307
:
165
168
[PubMed]
19.
Fowler
KH
,
Chapkin
RS
,
McMurray
DN
.
Effects of purified dietary n-3 ethyl esters on murine T lymphocyte function
.
J Immunol
1993
;
151
:
5186
5197
[PubMed]
20.
Cintra
DE
,
Ropelle
ER
,
Moraes
JC
, et al
.
Unsaturated fatty acids revert diet-induced hypothalamic inflammation in obesity
.
PLoS One
2012
;
7
:
e30571
[PubMed]
21.
Hartman
L
,
Lago
RC
.
Rapid preparation of fatty acid methyl esters from lipids
.
Lab Pract
1973
;
22
:
475
476, passim
[PubMed]
22.
Kempermann
G
,
Kuhn
HG
,
Gage
FH
.
Experience-induced neurogenesis in the senescent dentate gyrus
.
J Neurosci
1998
;
18
:
3206
3212
[PubMed]
23.
Antle
MC
,
LeSauter
J
,
Silver
R
.
Neurogenesis and ontogeny of specific cell phenotypes within the hamster suprachiasmatic nucleus
.
Brain Res Dev Brain Res
2005
;
157
:
8
18
[PubMed]
24.
Wojtowicz
JM
,
Kee
N
.
BrdU assay for neurogenesis in rodents
.
Nat Protoc
2006
;
1
:
1399
1405
[PubMed]
25.
Boneva
NB
,
Yamashima
T
.
New insights into “GPR40-CREB interaction in adult neurogenesis” specific for primates
.
Hippocampus
2012
;
22
:
896
905
[PubMed]
26.
Haber
S
.
Effective treatment of obesity produces remission, not cure
.
Int J Obes
1980
;
4
:
265
267
[PubMed]
27.
Goodrick
GK
,
Foreyt
JP
.
Why treatments for obesity don’t last
.
J Am Diet Assoc
1991
;
91
:
1243
1247
[PubMed]
28.
Kraschnewski
JL
,
Boan
J
,
Esposito
J
, et al
.
Long-term weight loss maintenance in the United States
.
Int J Obes
2010
;
34
:
1644
1654
[PubMed]
29.
Kalarchian MA, Marcus MD, Courcoulas AP, Cheng Y, Levine MD, Josbeno D. Optimizing long-term weight control after bariatric surgery: a pilot study. Surg Obes Relat Dis 2012;8:710–715
30.
Münzberg
H
,
Myers
MG
 Jr
.
Molecular and anatomical determinants of central leptin resistance
.
Nat Neurosci
2005
;
8
:
566
570
[PubMed]
31.
Morton
GJ
,
Cummings
DE
,
Baskin
DG
,
Barsh
GS
,
Schwartz
MW
.
Central nervous system control of food intake and body weight
.
Nature
2006
;
443
:
289
295
[PubMed]
32.
Bumaschny
VF
,
Yamashita
M
,
Casas-Cordero
R
, et al
.
Obesity-programmed mice are rescued by early genetic intervention
.
J Clin Invest
2012
;
122
:
4203
4212
[PubMed]
33.
Pencea
V
,
Bingaman
KD
,
Wiegand
SJ
,
Luskin
MB
.
Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus
.
J Neurosci
2001
;
21
:
6706
6717
[PubMed]
34.
Ettinger
MP
,
Littlejohn
TW
,
Schwartz
SL
, et al
.
Recombinant variant of ciliary neurotrophic factor for weight loss in obese adults: a randomized, dose-ranging study
.
JAMA
2003
;
289
:
1826
1832
[PubMed]
35.
Lambert
PD
,
Anderson
KD
,
Sleeman
MW
, et al
.
Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity
.
Proc Natl Acad Sci U S A
2001
;
98
:
4652
4657
[PubMed]
36.
Fowler
CD
,
Liu
Y
,
Wang
Z
.
Estrogen and adult neurogenesis in the amygdala and hypothalamus
.
Brain Res Brain Res Rev
2008
;
57
:
342
351
[PubMed]
37.
Pérez-Martín
M
,
Cifuentes
M
,
Grondona
JM
, et al
.
IGF-I stimulates neurogenesis in the hypothalamus of adult rats
.
Eur J Neurosci
2010
;
31
:
1533
1548
[PubMed]
38.
Chang
GQ
,
Karatayev
O
,
Liang
SC
,
Barson
JR
,
Leibowitz
SF
.
Prenatal ethanol exposure stimulates neurogenesis in hypothalamic and limbic peptide systems: possible mechanism for offspring ethanol overconsumption
.
Neuroscience
2012
;
222
:
417
428
[PubMed]
39.
Chang
GQ
,
Karatayev
O
,
Leibowitz
SF
.
Prenatal exposure to nicotine stimulates neurogenesis of orexigenic peptide-expressing neurons in hypothalamus and amygdala
.
J Neurosci
2013
;
33
:
13600
13611
[PubMed]
40.
David
DJ
,
Samuels
BA
,
Rainer
Q
, et al
.
Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression
.
Neuron
2009
;
62
:
479
493
[PubMed]
41.
Kokoeva
MV
,
Yin
H
,
Flier
JS
.
Evidence for constitutive neural cell proliferation in the adult murine hypothalamus
.
J Comp Neurol
2007
;
505
:
209
220
[PubMed]
42.
Emsley
JG
,
Mitchell
BD
,
Kempermann
G
,
Macklis
JD
.
Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells
.
Prog Neurobiol
2005
;
75
:
321
341
[PubMed]
43.
Ming
GL
,
Song
H
.
Adult neurogenesis in the mammalian central nervous system
.
Annu Rev Neurosci
2005
;
28
:
223
250
[PubMed]
44.
Lee
DA
,
Bedont
JL
,
Pak
T
, et al
.
Tanycytes of the hypothalamic median eminence form a diet-responsive neurogenic niche
.
Nat Neurosci
2012
;
15
:
700
702
[PubMed]
45.
Haan
N
,
Goodman
T
,
Najdi-Samiei
A
, et al
.
Fgf10-expressing tanycytes add new neurons to the appetite/energy-balance regulating centers of the postnatal and adult hypothalamus
.
J Neurosci
2013
;
33
:
6170
6180
[PubMed]
46.
Oh
DY
,
Talukdar
S
,
Bae
EJ
, et al
.
GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects
.
Cell
2010
;
142
:
687
698
[PubMed]
47.
Li
P
,
Spann
NJ
,
Kaikkonen
MU
, et al
.
NCoR repression of LXRs restricts macrophage biosynthesis of insulin-sensitizing omega 3 fatty acids
.
Cell
2013
;
155
:
200
214
[PubMed]
48.
Noble
EE
,
Billington
CJ
,
Kotz
CM
,
Wang
C
.
The lighter side of BDNF
.
Am J Physiol Regul Integr Comp Physiol
2011
;
300
:
R1053
R1069
[PubMed]
49.
Sousa-Ferreira
L
,
de Almeida
LP
,
Cavadas
C
.
Role of hypothalamic neurogenesis in feeding regulation
.
Trends Endocrinol Metab
2014
;
25
:
80
88
[PubMed]
50.
Brown
AJ
,
Jupe
S
,
Briscoe
CP
.
A family of fatty acid binding receptors
.
DNA Cell Biol
2005
;
24
:
54
61
[PubMed]
51.
Ma
D
,
Tao
B
,
Warashina
S
, et al
.
Expression of free fatty acid receptor GPR40 in the central nervous system of adult monkeys
.
Neurosci Res
2007
;
58
:
394
401
[PubMed]
52.
Yamashima
T
.
A putative link of PUFA, GPR40 and adult-born hippocampal neurons for memory
.
Prog Neurobiol
2008
;
84
:
105
115
[PubMed]
53.
Briscoe
CP
,
Peat
AJ
,
McKeown
SC
, et al
.
Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules
.
Br J Pharmacol
2006
;
148
:
619
628
[PubMed]
54.
He
C
,
Qu
X
,
Cui
L
,
Wang
J
,
Kang
JX
.
Improved spatial learning performance of fat-1 mice is associated with enhanced neurogenesis and neuritogenesis by docosahexaenoic acid
.
Proc Natl Acad Sci U S A
2009
;
106
:
11370
11375
[PubMed]
55.
Janssen
CI
,
Zerbi
V
,
Mutsaers
MP
, et al
.
Impact of dietary n-3 polyunsaturated fatty acids on cognition, motor skills and hippocampal neurogenesis in developing C57BL/6J mice
.
J Nutr Biochem
2015
;
26
:
24
35
[PubMed]
56.
Denis
I
,
Potier
B
,
Vancassel
S
,
Heberden
C
,
Lavialle
M
.
Omega-3 fatty acids and brain resistance to ageing and stress: body of evidence and possible mechanisms
.
Ageing Res Rev
2013
;
12
:
579
594
[PubMed]