- Open Access
Dietary fats promote functional and structural changes in the median eminence blood/spinal fluid interface—the protective role for BDNF
© The Author(s). 2018
Received: 9 May 2017
Accepted: 20 December 2017
Published: 9 January 2018
The consumption of large amounts of dietary fats activates an inflammatory response in the hypothalamus, damaging key neurons involved in the regulation of caloric intake and energy expenditure. It is currently unknown why the mediobasal hypothalamus is the main target of diet-induced brain inflammation. We hypothesized that dietary fats can damage the median eminence blood/spinal fluid interface.
Swiss mice were fed on a high-fat diet, and molecular and structural studies were performed employing real-time PCR, immunoblot, immunofluorescence, transmission electron microscopy, and metabolic measurements.
The consumption of a high fat diet was sufficient to increase the expression of inflammatory cytokines and brain-derived neurotrophic factor in the median eminence, preceding changes in other circumventricular regions. In addition, it led to an early loss of the structural organization of the median eminence β1-tanycytes. This was accompanied by an increase in the hypothalamic expression of brain-derived neurotrophic factor. The immunoneutralization of brain-derived neurotrophic factor worsened diet-induced functional damage of the median eminence blood/spinal fluid interface, increased diet-induced hypothalamic inflammation, and increased body mass gain.
The median eminence/spinal fluid interface is affected at the functional and structural levels early after introduction of a high-fat diet. Brain-derived neurotrophic factor provides an early protection against damage, which is lost upon a persisting consumption of large amounts of dietary fats.
Body mass stability relies on a complex interaction between neurons that sense the energy status of the body and effector neurons that coordinate food intake and energy expenditure . Most energy status-sensing neurons are located in the mediobasal hypothalamus and are set to respond to circulating hormones and nutrients that indicate the short- and long-term fluctuations in whole-body energy stores [1, 2]. In order to reach energy-sensing neurons, circulating factors must cross the median eminence (ME)/spinal fluid interface (SFI) [3, 4]. Recent studies have provided strong evidence to support an important role for ME tanycytes as gatekeepers that control the access of circulating factors to mediobasal neurons [5, 6].
In diet-induced obesity, long-chain saturated fats trigger a TLR4- and endoplasmic reticulum stress-dependent inflammatory response in the hypothalamus resulting in severe damage to the neurons that control food intake and energy expenditure [7–10]. In the short run, the damage inflicted to hypothalamic neurons results in leptin and insulin resistance and is reversible [11, 12]. However, upon prolonged exposure to dietary fats, neurons may undergo apoptosis and the reversibility of the obese status becomes unlikely [12–14]. Interestingly, studies have shown that the consumption of dietary fats predominantly affects the hypothalamus, sparing other brain regions [7, 11, 14]. In fact, hypothalamic inflammation in response to the consumption of dietary fats is a very premature phenomenon, which can be detected as early as 1 day after the introduction of a high-fat diet [14, 15]. Here, we first hypothesized that the ME-SFI is particularly sensitive to dietary fats, leading to an early exposure of the neurons of the mediobasal hypothalamus to potentially damaging circulating factors; to explore this hypothesis, we microdissected the main periventricular BBB regions, i.e., ME, vascular organ of lamina terminalis (OVLT), subfornical organ (SFO), and subcomissural organ (SCO), and evaluated the impact of the consumption of large amounts of dietary fats on the expression of structural and inflammatory markers; we also evaluated the expression of brain-derived neurotrophic factor (BDNF), which has an important trophic action in the blood-brain barrier (BBB) . These experiments demonstrated that the ME presented the earliest changes in the expression of inflammatory markers and BDNF. Next, we performed a series of structural studies to determine the temporal evolution of changes in the integrity of the ME-BBB in response to the consumption of large amounts of dietary fats. As early as 1 week, the consumption of dietary fats led to changes in the integrity and architecture of the region of the ME-SFI. Finally, we evaluated the impact of the modulation of BDNF in the progression of diet-induced obesity and the integrity of the ME-SFI. We show that reducing BDNF increases diet-induced body mass gain and enhances the functional and structural disarrangements in the ME-SFI zone.
Macronutrient composition of the diets
Chow (Nuvilab CR1) (g)
High-fat diet (g)
Protein (casein 85%)
Dextrinized corn starch
Mineral mix (AIN-93)
Vitamin mix (AIN-93)
mRNA extraction and real-time PCR
The brains were rapidly excised, rinsed in a saline solution, frozen on dry ice, and stored at − 80 °C. Initially, thick (40 μm) brain sections were prepared in a cryostat (LEICA® CM1520) to determine the correct locations of the target regions. Once identified, the target regions were submitted to laser capture microdissection using a LCM from PALM Robot Microbean (Carl-Zeiss, Gottingen, Germany). The anatomical landmarks of the distinct regions were always identified using the coordinates as described in the Paxinos Atlas of Stereotaxic Coordinates (Elsevier Science 2004). Specimens were immediately submitted to RNA extraction using the RNeasy Plus Micro Kit® (Qiagen Sciences, Germantown, MD, USA) according to the manufacturer’s protocol. Because of the low levels of starting material, cDNA synthesis was conducted with total RNA obtained at extraction and using the Reverse Transcription High Capacity Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). In another protocol, the brain was excised and the hypothalamus was removed and frozen at − 80 °C. Samples were homogenized in TRIzol® (Invitrogen, São Paulo, Brazil) using a tissue homogenizer (Polytron-Aggregate, Kinematica, Littau/Luzern, Switzerland). Total RNA was isolated according to the manufacturer’s guidelines and quantified using NanoDrop® (Synerge MX, BioTek®, Winooski, VT, USA). The integrity of RNA was evaluated by agarose gel electrophoresis. Complementary DNA was prepared using 2 μg of total RNA and reverse transcriptase. Subsequently (for both protocols), the cDNA was diluted depending on the concentration needed for efficient amplification of genes of interest. Real-time PCR reactions were performed using the TaqMan® TM system (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control of the reaction, serving to normalize the expression of genes of interest in different samples. The genes analyzed were GLAST (Mm00600697_m1), GLUT1 (Mm00441480_m1), CAVEOLIN1 (Mm00483057_m1), TNFα (Mm00443258_m1), IL1β (Mm00434228_m1), IL6 (Mm00446190_m1), IL10 (Mm01288386_m1), BDNF (Mm01334043_m1), and CLAUDIN5 (MmPT5833394738.g) for the first protocol, plus GFAP (Mm01253033_m1), IGFBP2 (Mm00805581), NGFR (Mm.PT.58.10419996), and TrkB (Mm.PT.5842284287) added for the second protocol. For the relative quantification of genes under study, real-time PCR reactions were performed in triplicate from 3 μl TaqMan Universal PCR Master Mix 2X, 0.25 μl of primers and probe solution, 2.75 μl water, and 4.0 μl cDNA. The cycling conditions were 50 °C for 2 min and 95 °C for 10 min and 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The values of relative gene expression were obtained by analyzing the results in the programme 7500 System SDS software (Applied Biosystems).
Blood-brain barrier leakage analysis
Male mice were anesthetized, and fluorescein isothiocyanate dextran (FITC-dextran—2000 kDa, 100 μl, 50 mg/ml, in saline, Sigma-Aldrich, St. Louis, MO) was injected intravenously in the inferior vena cava vein. After 1 min, the brains were removed, rinsed with saline, and fixed in 4% paraformaldehyde for 24 h; thereafter, the specimens were cryoprotected in 30% sucrose for 48 h and then submitted to coronal sectioning (20 μm thickness) using the cryostat (Leica Microsystems, CM1860, Buffalo Grove, USA). Fluorescent images of the sections were obtained using a confocal fluorescence microscope (Leica) always employing the same parameters for acquiring images (laser 488, wavelength = 405, %laser = 20%, gain = 1015, offset = − 0.3799). Regional FITC-dextran fluorescent intensity was measured by ImageJ (National Institute of Health, Bethesda, MD). Optical density was measured by applying the following formula: (right fluorescent intensity − left fluorescent intensity)/left fluorescent intensity × 100.
Mice were perfused with 4% paraformaldehyde, and the whole brain was removed and submitted to 24-h fixation in 4% paraformaldehyde. Thereafter, the brain was rinsed in 1× PBS and cryoprotected in 30% sucrose for 48 h and then submitted to coronal sectioning (20 μm thickness) using the cryostat (LEICA Microsystems®, CM1860, Buffalo Grove, IL, USA). Sections were rinsed in 0.1 M PBS (pH 7.4) and blocked for 1 h at room temperature in a blocking solution (5% normal goat serum, 0.2% Tween 80® in PBS). The slides were incubated overnight at 4 °C with either anti-IGFBP2 (sc-365368, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), anti-FGF10 (ABN44, 1:200, Merck Millipore, Temecula, CA, USA), anti-vimentin (sc-373717, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GFAP (ab7260, 1:1000, ABCAM, Cambridge, UK), anti-BDNF (sc-546, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), or anti-TrkB (sc-8316, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA) in a blocking solution (1% bovine serum albumin), then for 1 h with goat Anti-Mouse FITC® (sc-2010, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA) and goat Anti-rabbit Cy3® (ab6941, ABCAM, 1:500, ABCAM, Cambridge, UK) in a blocking solution, and the nuclei were stained with (TO-PRO®-3 Iodide (642/661), T3605, 1:1000, Life Technologies, Carlsbad, CA, EUA) in PBS. The specificity of the antibodies have been tested either by others [17–20] or by us, by performing immunodepletion experiments. Negative controls were performed by omitting the first antibody. In all experiments, 3–4 mice were analyzed in each group; at least four distinct sections from each mouse hypothalamus were evaluated. The sections were examined by fluorescence microscopy using a confocal microscope LEICA TCS SP5 II (Leica). Regional fluorescent intensity was measured by ImageJ (National Institute of Health, Bethesda, MD). Optical density was measured by applying the following formula: (right fluorescent intensity − left fluorescent intensity)/left fluorescent intensity × 100. All fluorescence images were quantified, and the means ± SD are presented in Additional files 1, 2, 3, 4, 5, and 6: Tables S1–S6.
Mice were perfused with Karnowsky reagent, and the whole brain was removed and prepared for transmission electron microscopy analysis as previously described .
Body composition by PET/CT
Body composition was obtained by positron emission tomography/CT scan (Albira/Bruker, Billerica, MA, USA) as previously described .
Results are presented as the means ± standard deviation (SD). For statistical analysis, first, we applied the Levene test to check the homogeneity of variances. For the comparison of means between two groups, we used Student’s t test for independent samples. The significance level to reject the null hypothesis was set at p < 0.05. One-way or two-way ANOVA was used to evaluate the experiments with treatment with anti-BDNF. The level of significance was set at p < 0.05. The data were analyzed using the GraphPad Prism®.
The median eminence is the circumventricular organ affected the earliest in response to the consumption of a HFD
Mice fed chow or HFD for 1 or 2 weeks were used in experiments aimed at microdissecting the circumventricular organs, OVLT (Fig. 1b), SFO (Fig. 1c), SCO (Fig. 1d), and ME (Fig. 1e). The microdissected specimens were used in real-time PCR experiments for determining the expression of transcripts encoding structural proteins of the BBB, GLAST, GLUT1, and caveolin-1 (Fig. 1f); the transcripts encoding cytokines, IL1β, IL6, IL10, and TNFα (Fig. 1g); and the transcript encoding BDNF (Fig. 1h). The earliest changes induced by the consumption of a HFD occurred after 1 week were as follows: GLAST was increased in OVLT, SFO, and ME and caveolin-1 was increased only in ME (Fig. 1f); IL1β and TNFα (Fig. 1g) as well as BDNF (Fig. 1h) were increased only in ME. Interestingly, after 2 weeks on a HFD, the expression of BDNF was normalized in ME, whereas in all the remaining regions evaluated, its expression increased (Fig. 1h). Because major changes occurred in ME, we measured transcript expression of claudin-5, a tight junction protein. As shown in Additional file 7: Figure S1, in ME, claudin-5 expression was reduced after 1 week on a HFD, returning to normal levels after 2 weeks and reducing again after 4 weeks.
The consumption of a HFD leads to an early loss of integrity in the ME-BBB
The consumption of a HFD affects the expression of proteins of the BDNF system in the region of the ME-SFI
The immunoneutralization of BDNF increases diet-induced body mass gain, intensifies hypothalamic inflammation, and intensifies the structural disorganization of the ME-SFI region
The immunoneutralization of BDNF increases body mass gain and worsens diet-induced hypothalamic inflammation in obesity-resistant mice
The main objective of this study was to evaluate the impact of the consumption of a HFD on the structural organization and integrity of the ME-SFI. Studies have shown that the consumption of large portions of dietary fat can trigger an inflammatory response in the hypothalamus leading to an impaired capacity of hypothalamic neurons to control caloric intake and energy expenditure, resulting in a progressive increase of body adiposity (this theme was revised previously ). Interestingly, there is certain specificity in diet-induced inflammation in the central nervous system since regions other than the hypothalamus either are not affected or, if so, are affected in much lower magnitude, and later than the hypothalamus [11, 14, 27].
Fatty acids present in the bloodstream are not freely diffusible into the brain . Under certain conditions, they rely on specific transport systems present in the structures of the BBB [29, 30]. The appropriate function and distribution of these transport systems is of major importance in brain development and physiology throughout life because most fatty acids that constitute the central nervous system phospholipids cannot be synthetized de novo in the brain and thus must be imported from the periphery [28, 30]. Regarding the hypothalamus, its function as a nutrient sensor [31, 32] may explain why the ME-BBB presents some degree of permissiveness to certain nutrients, including fatty acids [31, 32]. In fact, a number of studies have explored the roles of fatty acids to control the function of hypothalamic neurons, implying that under physiological conditions, they are readily available . Nevertheless, despite the fact that under physiological conditions and under the consumption of diets containing nutritionally adequate amounts of fat (such as in regular chow for rodents), there is some leakiness of the ME-BBB to fatty acids; the increased consumption of dietary fats may be capable of further increasing the BBB permeability to the point that the composition of fatty acids in the hypothalamus can be modified by dietary approaches [33, 34]. With these concepts in mind, in the first part of the study, we asked if the consumption of large portions of dietary fats would act differently in the distinct CVOs to promote changes in the expression of proteins involved in the structure of the BBB, cytokines, and BDNF. For that, we microdissected OVLT, SFO, SCO, and ME and evaluated the expression of target transcripts by real-time PCR. Except for GLAST that was increased in OVLT, SFO, and ME after 1 week on HFD, all the other transcripts undergoing rapid (1 week) induction by the dietary approach were increased in ME only. There were increases of transcripts encoding proteins of the BBB (GLAST and caveolin-1), cytokines (IL1β and TNFα), and BDNF. Thus, we concluded that the ME is particularly and rapidly responsive to the presence of high amounts of fatty acids in the diet, a fact that may contribute to a diet-induced dysfunction of the BBB at this anatomical location. It is noteworthy that changes in the expression of both inflammatory markers and BBB-related transcripts occurred in a biphasic manner. This is in consensus with the studies that evaluated details of diet-induced hypothalamic inflammation [14, 15, 35]. Particularly, in the study by Dalvi and coworkers , the changes in the hypothalamus appear to go through a temporary recovery from the effects of the HFD, perhaps due to astrocyte reactivity.
It is well documented that consumption of HFDs can also induce a systemic sub-clinical inflammatory status, which is regarded as an important mediator of whole-body insulin resistance . In this context, it could be argued that the changes in the ME expression of transcripts encoding for inflammatory and BBB proteins could be secondary to diet-induced systemic inflammation. Despite the fact that we did not test this particular question, the timing of events occurring in the ME following the introduction of the HFD precedes by several weeks the emergence of experimental diet-induced systemic inflammation , and is in accordance with previous studies evaluating diet-induced hypothalamic inflammation [14, 38].
Next, we evaluated the impact of the consumption of a HFD on the permeability of the BBB in the ME and mediobasal hypothalamus. For that, mice were injected with FITC-dextran, and fluorescence was inspected by confocal microscopy and measured using an ImageJ software. As suspected, there was a rapid increase in the permeability of the ME-BBB, which peaked after 1 week on a HFD, reducing gradually after 2 and 4 weeks. Recent studies have made great advances in the field by identifying ME tanycytes as gatekeepers for nutrients and other peripheral signals that modulate the function of hypothalamic neurons [6, 39]. Tanycytes are specialized glial cells of the hypothalamus that lay in the walls and floor of the third ventricle and act as physical barriers modulating the permeability of fenestrated endothelial cells in the ME . Using transmission electron microscopy, we demonstrated that the cells in the transition between the floor and walls of the third ventricle undergo a major structural disarrangement as early as 1 week after the introduction of the HFD. No previous study has reported such phenomenon; however, it coincides with the very early induction of hypothalamic inflammation by dietary fats [14, 15].
Because the transmission electron microscopy alone is not sufficient to prove that the ME cellular disarrangement promoted by the HFD is in fact due to changes in tanycytes, we performed a series of experiments with confocal microscopy to evaluate the expression and distribution of different proteins involved in the organization of the BBB. We explored the expressions of vimentin, FGF10, and particularly IGFBP2, which is regarded as the most specific marker of β1-tanycytes [4, 39, 40]. We also evaluated the expressions of GLUT1, VEGF and occludin [4, 39, 40], which were not shown in this study because the findings were similar to the results obtained with vimentin, FGF10 and IGFBP2. In general, the immunofluorescence studies revealed that the consumption of a HFD induced a rapid change in the organization and spatial distribution of tanycytes along the floor and wall of the third ventricle and in the transition between the ME and the arcuate nucleus. These changes are particularly evident in the FGF10 labeling in Figs. 4 and 5. The linear distribution was lost, and many cells appeared in a non-organized disposition scattered throughout the mediobasal hypothalamus.
An important finding of this study was that upon persistent consumption of the HFD, the cells expressing IGFBP2 began, after 4 weeks, to express GFAP as well. GFAP is a marker of astrocytes . Studies have shown that, in parallel with microglia , astrocytes play an important role in the induction of the hypothalamic inflammatory response in diet-induced obesity [42–44]. Moreover, it has recently been shown that tanycytes can differentiate into astrocytes [45, 46]. Thus, it can be proposed that under prolonged exposure to large portions of dietary fats, tanycytes differentiate into astrocytes, which can mediate at least part of the inflammatory signals that damage the hypothalamic neurons.
In the final part of the study, we explored the hypothesis that BDNF could act as a protective factor against diet-induced ME and hypothalamic damage. Studies have shown that BDNF can attenuate BBB disruption under different conditions [16, 47, 48]. Moreover, acting in the hypothalamus, and particularly through the receptor TrkB, BDNF can attenuate diet-induced obesity through a mechanism related to MC4R signaling . Hypothalamic BDNF can also control other metabolic functions, such as hepatic glucose output and cardiovascular activity [50, 51]. Interestingly, a recent report showed that BDNF is an important determinant of induced pluripotent steam cell (iPSC) differentiation into POMC neurons , which suggests that BDNF could play an important role in the putative recovery of the damaged hypothalamus during body mass loss, in the treatment of obesity. Here, we showed that BDNF transcript levels undergo a rapid oscillation following the introduction of a HFD—initially increasing, at 1 week, and then progressively falling to levels that are even lower than those of control after 4 weeks on the HFD. Moreover, the distribution of BDNF and TrkB undergo drastic changes over time during the consumption of a HFD. In lean mice, BDNF is present mostly in the cytoplasm of cells nearby the walls of the third ventricle and shows no co-localization with IGFBP2. Upon consumption of a HFD, BDNF expression occurs predominantly in the perinuclear region of cells scattered throughout the mediobasal hypothalamus; moreover, some cells co-express BDNF and IGFBP2. The immunoneutralization of BDNF in mice fed a HFD resulted in a major phenotypic change, increasing body mass gain, increasing hypothalamic inflammation, and accentuating the already anomalous distribution of IGFBP2-expressing cells. In addition, the immunoneutralization of BDNF was capable of transforming diet-induced obesity-resistant mice in obese-prone mice, which was accompanied by increased expression of inflammatory cytokines in the hypothalamus and increased leakage of the ME-BBB. Taken together the results of our study and previous studies evaluating the effects of BDNF in the hypothalamus, we believe that the immunoneutralization of BDNF reduces whole-body energy expenditure impacting on a number of metabolic functions.
Despite the number of approaches employed to provide a broad view of the main phenomenon described in this study, there are two limitations that should be considered: (i) some of the quantifications were restricted to transcript amount, which may not be always similar to protein amount; (ii) the study did not explore the direct vs. indirect effects of BDNF, since it is known that BDNF has beneficial effects on the metabolic phenotype after HFD feeding and the effects herein described could be a consequence of that and not a direct effect of BDNF on the hypothalamus.
This is the first report showing that the consumption of large amounts of dietary fats can damage the ME-SFI zone at the functional and structural levels. Moreover, we show that the ME responds to the presence of dietary fats much faster than other CVOs, which may contribute to the anatomical specificity of diet-induced inflammation of the brain. Finally, we identify BDNF as an endogenous ME-BBB protective factor, suggesting that its rapid oscillation (increase followed by an abrupt fall) shortly after the introduction of the dietary fats can play a role in the fragility of the ME.
We thank Dr. Iscia Lopes-Cendes and Andre S. Vieira for sharing their expertise in laser microdissection. We thank Erika Roman, Gerson Ferraz, and Marcio Cruz from the University of Campinas for the technical assistance. The support for this 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 the National Institute of Science and Technology—Diabetes and Obesity.
Support for this study was provided by Fundação de Amparo a Pesquisa do Estado de São Paulo. The Laboratory of Cell Signaling belongs to the Obesity and Comorbidities Research Center and the National Institute of Science and Technology—Diabetes and Obesity.
Availability of data and materials
All raw data used in this manuscript are available on request.
LAV, EPA, and AFR designed the studies. NRD, CS, JM, MF, and BB performed the various experiments. RB performed the transmission electron microscopy experiments. All authors contributed to the writing and editing of the manuscript, and all authors read and approved the final manuscript. AFR designed and performed the experiments and wrote the manuscript; NRD, CS, JM, MF, BB, and RB performed the experiments and acquired and analyzed the data; LAV and EPA designed the research studies, analyzed the data, and wrote the manuscript. All authors reviewed the manuscript.
The investigation was conducted in accordance with the principles and procedures described by the National Institutes of Health Guidelines for the Care and Use of Experimental Animals and was previously approved by the University of Campinas Ethical Committee (ID 2574-1).
Consent for publication
The authors declare that they have no competing interests.
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