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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: J Neurochem. 2015 Sep 29;135(5):918–931. doi: 10.1111/jnc.13298

Regulation of the orexigenic neuropeptide, enkephalin, by PPARδ and fatty acids in neurons of the hypothalamus and forebrain

Kinning Poon a, Mohammad Alam a, Olga Karatayev a, Jessica R Barson a, Sarah F Leibowitz a,*
PMCID: PMC4715552  NIHMSID: NIHMS720161  PMID: 26332891

Abstract

Ingestion of a high-fat diet composed mainly of the saturated fatty acid, palmitic (PA), and the unsaturated fatty acid, oleic (OA), stimulates transcription in the brain of the opioid neuropeptide, enkephalin (ENK), which promotes intake of substances of abuse. To understand possible underlying mechanisms, this study examined the nuclear receptors, peroxisome proliferator-activated receptors (PPARs), and tested in hypothalamic and forebrain neurons from rat embryos whether PPARs regulate endogenous ENK and the fatty acids themselves affect these PPARs and ENK. The first set of experiments demonstrated that knocking down PPARδ, but not PPARα or PPARγ, increased ENK transcription, activation of PPARδ by an agonist decreased ENK levels, and PPARδ neurons coexpressed ENK, suggesting that PPARδ negatively regulates ENK. In the second set of experiments, PA treatment of hypothalamic and forebrain neurons had no effect on PPARδ protein while stimulating ENK mRNA and protein, whereas OA increased both mRNA and protein levels of PPARδ in forebrain neurons while having no effect on ENK mRNA and increasing ENK levels. These findings show that PA has a stronger, stimulatory effect on ENK and weaker effect on PPARδ protein, whereas OA has a stronger stimulatory effect on PPARδ and weaker effect on ENK, consistent with the inhibitory effect of PPARδ on ENK. They suggest a function for PPARδ, perhaps protective in nature, in embryonic neurons exposed to fatty acids from a fat-rich diet and provide evidence for a mechanism contributing to differential effects of saturated and monounsaturated fatty acids on neurochemical systems involved in consummatory behavior.

Keywords: PPARdelta, fatty acid, hypothalamus, forebrain, enkephalin

Introduction

A diet rich in fat is known to increase caloric intake that promotes obesity (Dourmashkin et al. 2006) and also enhance the intake of substances of abuse (Krahn & Gosnell 1991, Carrillo et al. 2004, Morganstern et al. 2013). These effects of a high-fat diet (HFD) in adult animals are similarly evident with prenatal exposure to this diet, which predisposes the offspring to overconsuming not only dietary fat (Chang et al. 2008) but also drugs of abuse such as nicotine (Morganstern et al. 2013) and alcohol (Cabanes et al. 2000). Such fat-rich diets can vary in their fatty acid content, with the typical Western diet of 35% fat (Astrup et al. 2011, Last et al. 2011) comprised of 28% palmitic acid (PA), a saturated fat, plus 42% oleic acid (OA), a monounsaturated fat (Baylin et al. 2002), and the Mediterranean fat-rich diet comprised of only 13% PA plus 72% OA (Renaud et al. 1995, Willett et al. 1995). This Western diet with higher levels of PA has been associated with a higher prevalence of obesity, heart disease, and diabetes (Fung et al. 2001, Haslam & James 2005), disorders shown to be produced by intake of PA (Kien et al. 2005, Cintra et al. 2012). The Mediterranean diet, in contrast, with higher levels of OA, is believed to play a protective role against these conditions (Obici et al. 2002, Cintra et al. 2012).

Two important brain areas that are affected by these fatty acids and involved in controlling intake of a HFD are the hypothalamus, a region that regulates energy homeostasis (Williams et al. 2001), and the forebrain, which consists of the nucleus accumbens (NAc) that mediates reward processes (Olds & Milner 1954, Hyman et al. 2006) and the septal nucleus that, in addition to affecting positive reinforcement and food intake (Olds & Milner 1954, Numan & Quaranta 1990), has a role in relaying information between the limbic areas and hypothalamus (Risold & Swanson 1997). In the hypothalamus, there is evidence that PA and OA have opposite effects, with PA inducing insulin and leptin resistance (Benoit et al. 2009, Posey et al. 2009) and OA reducing food intake, glucose production, and orexigenic neuropeptide expression while stimulating anorexigenic neuropeptides (Obici et al. 2002, Jo et al. 2009, Cintra et al. 2012). In the NAc, the presence or absence of n-3 polyunsaturated fatty acids, such as linoleic acid, also has differential effects on dopamine signaling that further enhance or inhibit the rewarding aspect of dietary fat (Zimmer et al. 2002, Adachi et al. 2013). In the septal nucleus, there is evidence that ingestion of saturated fatty acids increases orexigenic neuropeptide levels (Huang et al. 2003). These findings, suggesting that fatty acids can alter the expression of neuropeptides involved in consummatory behavior, lead us to investigate possible mechanisms that may mediate this relationship and the stimulatory effect that a HFD has on a specific neuropeptide known to stimulate the intake of substances of abuse.

A commonality of these hypothalamic and forebrain regions in relation to HFD intake is that they contain a high density of neurons expressing the opioid neuropeptide, enkephalin (ENK), which stimulates the consumption of both a fat-rich diet and drugs of abuse, as shown with ENK analog or agonist injections in the hypothalamus (Chang et al. 2010), NAc (Zhang et al. 1998) or septum (Majeed et al. 1986). In adult rats, intake of a HFD consisting of 24% PA and 49% OA increases the expression of ENK in both the hypothalamus and NAc (Chang et al. 2010), and when consumed by pregnant rats, this HFD increases the expression and number of ENK neurons in the hypothalamus and NAc of both the embryos and postnatal offspring (Chang et al. 2008, Vucetic et al. 2010). With the ingestion of this HFD found to increase fatty acid levels in the hypothalamus (Posey et al. 2009, Barson et al. 2012) and whole brain (Carlson et al. 1986), it is possible that they have an important role in modulating ENK function, perhaps acting indirectly through a molecular mechanism in the neuron that is sensitive to fatty acids.

One such mechanism may involve the peroxisome proliferator-activated receptors (PPARs), a group of nuclear receptors that have three isoforms, PPARα, PPARγ, and PPARδ, and are encoded by different genes (Dreyer et al. 1993, Desvergne & Wahli 1999). These nuclear receptors are found to function differentially in regulating the metabolism of fatty acids. Whereas PPARδ and PPARα bind saturated as well as unsaturated fatty acids with high affinity (Gottlicher et al. 1992, Xu et al. 1999) and induce the oxidation of fatty acids (Escher et al. 2001), PPARγ preferentially binds polyunsaturated fats (Xu et al. 1999) and mediates the induction of lipogenesis (Tontonoz et al. 1994). Studies in the brain have shown these PPARs to exist in the hypothalamus and NAc (Moreno et al. 2004, Higashiyama et al. 2007), to colocalize with hypothalamic neuropeptides such as agouti-related protein and pro-opiomelanocortin (Sarruf et al. 2009), and to have both stimulatory and inhibitory effects on the expression of these peptides (Mally et al. 2004, Parab et al. 2007, Chikahisa et al. 2008, Festuccia et al. 2008). Also, intake of a HFD and prenatal exposure to a HFD that increases fatty acid levels have been found to stimulate both PPARα and PPARγ in peripheral tissues (Zhang et al. 2005, Kannisto et al. 2006, Zhang et al. 2009, Shamsi et al. 2014, Shen et al. 2014), with prenatal HFD exposure also shown stimulate PPARδ in the hypothalamus of postnatal offspring (Chang et al., manuscript under review). This evidence suggests that the PPARs may provide a mechanism through which the fatty acids affect the expression of ENK.

With the common effect of HFD ingestion being an increase in the expression and levels of ENK, this study used isolated primary neurons extracted on embryonic day 19 (E19) from the hypothalamus and forebrain, to determine whether: 1) the PPAR isoforms modulate the expression and levels of ENK in these neurons; and 2) the fatty acids, PA and OA, affect in these neurons, perhaps differentially, the expression and levels of PPAR in close relation to ENK. The results of this study demonstrate a close relationship between PPARδ and this opioid peptide, pointing to a molecular mechanism through which a HFD may act within the neurochemical systems of the hypothalamus and forebrain known to control consummatory behavior.

Methods

Animals

Timed-pregnant, E10 Sprague-Dawley rats were acquired from Charles River Laboratories (Hartford, CT). All experimental procedures were performed according to institutionally approved protocols as specified in the NIH Guide to the Care and Use of Animals and also with approval of the Rockefeller University Animal Care and Use Committee. The dams were individually housed in a fully accredited AAALAC facility (22°C, with a 12:12-h light-dark cycle with lights off at 12 pm). The rats were maintained ad libitum on standard lab chow (3.36 kcal/g) with 13% fat (Purina, St. Louis, MO) and sacrificed on embryonic day 19 (E19), as previously described (Poon et al. 2012). The whole hypothalamus was extracted and dissociated for plating into cell culture. Because the NAc and septal nucleus are not fully developed at this age (Altman & Bayer 1995), the entire forebrain consisting of these two developing regions was extracted.

Cell culture

Hypothalami or forebrain from E19 embryos were micro-dissected and placed in 0.05% trypsin-EDTA for 30 min at 37°C (Life Technologies, Grand Island, NY). The hypothalamus was dissected as previously described (Poon et al. 2012). For the forebrain, two coronal cuts were made anteriorly at the level of the rhinencephalon and posteriorly at the level of the preoptic area. This slice was then oriented coronally, and the forebrain was dissected as a rectangle, with the dorsal cut made ~1.0 mm from the dorsal aspect of the slice and the lateral cuts made ~1.5 mm from the medial aspect of the slice. The cells were triturated with 0.01% deoxyribonuclease, passed through a 70 μm and then 40 μm cell strainer (Fisher Scientific, Waltham, MA), and spun down. The cells (1 million / mL) were resuspended in Neurobasal Media containing B27 supplement (Life Technologies, Grand Island, NY) and cultured in a 6-well plate (BD Biosciences, Sparks, MD) or on #1.5, 18 mm round coverslips (Warner Instruments, Hamden, CT). Cells were then placed in a humidified, 5% CO2 incubator at 35°C. All plates and coverslips were coated with poly-D-lysine (Sigma-Aldrich, St. Louis, MO). Neurons were allowed to settle for 3 days for immunofluorescence imaging and 5 days for mRNA extraction. For each experiment, 4 wells per cell culture group were used for the control, and 4 wells were used for each treatment, for a total of 4 cell culture groups per experiment.

siRNA

Hypothalami and forebrain from 4 embryonic groups were dissociated for neuronal culture, and for each group, a total of 12 cultured wells were used with 4 wells each for the knockdown, siRNA non-silencing control and non-knockdown control, as previously described (Poon et al. 2013). The PPARα (forward: 5’-CCUUACCUGUGAACACGAUtt-3’; reverse: 5’-AUCGUGUUCACAGGUAAGGat-3’), PPARγ (forward: 5’-GAUUGAAGCUUAUUUAUGAtt-3’; reverse: 5’-UCAUAAAUAAGCUUCAAUCgg-3’), and PPARδ siRNA (forward: 5’-CAUGAGUUCUUGCGCAGUAtt-3’; reverse: 5’-UACUGCGCAAGAACUCAUGgg-3’) was customized from Life Technologies (Life Technologies, Grand Island, NY). The PPARs and scrambled negative control (Life Technologies, Grand Island, NY) siRNA were used at a concentration of 75 pmol / μL to transfect hypothalamic neuronal cultures using Lipofectamine RNAiMAX (Life Technologies, Grand Island, NY) for 48 hours. Knockdown efficiency of siRNAs was confirmed by qRT-PCR analysis. The siRNA itself did not affect gene expression, as the non-silencing control yielded no change in PPARs or ENK expression as compared to untreated controls. The relatively low expression levels of PPARγ in the hypothalamus failed to produce a positive knockdown of this nuclear receptor and thus was not examined in this brain region.

Fatty acid and drug preparation

OA (Sigma-Aldrich, St. Louis, MO) and PA (Nu-Chek Prep, Elysian, MN) were dissolved in ethanol to a stock concentration of 100 or 200 mM. This was then diluted into media to a final concentration of 100 or 200 uM OA or PA, as used in other cell culture studies (de Vries et al. 1997, Ulloth et al. 2003). The agonist, GW 0742 (Tocris, Minneapolis, MN), was dissolved in ethanol to make a 10 mM stock solution and was diluted into media to a final concentration of 1 μM, with the EC50 for GW 0742 in activating PPARδ activity in cell culture found to be 1 nM. Higher concentrations were not used, as those higher than 1 μM are found to start activating PPARα and PPARγ (Sznaidman et al. 2003), and those higher than 10 μM can activate other receptors, such as the vitamin D receptor (Nandhikonda et al. 2013). To ensure no effect from ethanol, neuronal treatment with diluted ethanol to final working concentrations (1000× or 2000× dilution) alone did not affect the gene expression of PPARs or ENK in hypothalamic or forebrain neurons. Neurons were treated for 24 hours with the agonist and fatty acids.

qRT-PCR

The mRNA from hypothalamic or forebrain neurons was purified using a Qiagen RNeasy kit (Qiagen, Valencia, CA), cDNA was synthesized using high capacity RNA-to-cDNA master mix (Life Technologies, Grand Island, NY), and the SYBR Green PCR core reagents kit (Life Technologies, Grand Island, NY) was used for qRT-PCR and performed as previously described (Poon et al. 2012). The levels of target gene expression were quantified relative to the level of cyclophilin-A, using the relative quantification method. Each sample was run in triplicate and included a no-template control and a negative RT control. Primers were designed with the NCBI Primer design tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to span an exon-exon gap to eliminate amplification of genomic DNA. The primers used were: PPARα forward: 5′-CCCTCGGAGAGGAGAGTTCC-3′, reverse: 5′- GCTGGAGAGAGGGTGTCTGT-3′; PPARγ forward: 5′-CTTGTGAAGGATGCAAGGGTT-3′, reverse: 5′-TCCGACAGTTAAGATCACACC-3′; PPARδ forward: 5′-CAGCCATAACGCACCCTTCA-3′, reverse: 5′-ATGCACGCTGATCTCGTTGT-3′; ENK forward: 5′-GGACTGCGCTAAATGCAGCTA-3′, reverse: 5′-GTGTGCATGCCAGGAAGTTG-3′; cyclophilin-A forward: 5′-GTGTTCTTCGACATCACGGCT-3′, reverse: 5′-CTGTCTTTGGAACTTTGTCTGCA-3′. The specificities of PCR products were confirmed by a single band of corresponding molecular weight revealed by agarose gel electrophoresis. The concentration of all target primers was 100 nM, and the CYC primer was 200 nM.

Immunofluorescence cytochemistry

Hypothalamic or forebrain neurons were dissociated and plated onto coverslips and, after 3 days in culture, were fixed and processed with primary antibodies for PPARβ and/or ENK. Primary neuronal cells were cultured on poly-D-lysine (Sigma-Aldrich, St. Louis, MO) coated, #1.5, 18 mm round glass coverslips (Warner Instruments, Hamden, CT). The cells were gently washed with 1% phosphate buffered saline (Life Technologies, Grand Island, NY), fixed with 2% paraformaldehyde, permeabilized using 0.01% triton (Shelton Scientific, Shelton, CT), and then blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO), as previously described (Poon et al. 2012). The cells were incubated with either mouse anti-ENK (1:300; Millipore, Billerica, MA), rabbit anti-PPARδ (Pierce Thermofisher Scientific, Waltham, MA) followed by goat anti-mouse Alexa-fluor-488 or chicken anti-rabbit Alexa-fluor-594 (1:400, Life Technologies, Grand Island, NY) and DAPI. Cells were washed and the coverslips attached to glass slides using Prolong gold anti-fade reagent (Life Technologies, Grand Island, NY). The cells were imaged using a Zeiss Axioplan 2 microscope (Zeiss, Thornwood, NY). The immunostaining of PPARδ was similar to that described in other studies, showing PPARδ to be detected in both the cytoplasm and neurites of some neurons as well as in the nucleus (Cristiano et al. 2005, Higashiyama et al. 2007). The 4 embryonic groups (n=4) and the 4 conditions (control, GW0742, 200 PA, and 200 OA) were tested on the same experimental day. These cells from the immunofluorescence experiments were processed and imaged at the same time using the same conditions, and the total number of cells, both fluorescent and non-fluorescent, from each condition was manually counted using ImageJ (http://imagej.nih.gov/ij/), as previously described (Poon et al. 2012). All immunofluorescence data were collected and analyzed by blind analysis. The number of double-labeled PPARδ with ENK cells was calculated relative to ENK, PPARδ or total number of cells, with similar results for all three, and thus results reported here are relative to PPARδ. To measure relative levels of peptide in fixed neurons, the immunofluorescence intensity was analyzed. After subtraction of the mean background intensity of a nearby area of comparable size, fluorescence intensity measurements were calculated as the mean intensity within the region of a neuron multiplied by its area, with units as arbitrary units (a.u.). To determine the mean fluorescence intensity and the number of independent variables or neuronal populations, the fluorescence intensity of all neurons in a specific treatment group was plotted as a frequency count histogram and fitted to a Gaussian distribution. The number of peaks that resulted in an R2 value closest to one was used. The number of fitted distributions to the histogram reflects the number of populations, the median peak of each distribution reflects the mean fluorescence intensity with higher intensities reflecting higher levels of protein, and the area under the curve reflects the percentage of neurons within that distribution. Between 2000 and 3000 cells were measured for each treatment group.

Data analysis

Statistical analyses with multiple comparisons were completed using a one-way ANOVA, with Tukey post hoc test to determine significant differences between the groups. A direct comparison between a pair of groups was made using an unpaired two-tailed Student’s t-test. The criterion for statistical significance was p < 0.05. For Gaussian fits to fluorescence intensity histograms, to determine the goodness of fit, X2 values corresponding to p < 0.05 were considered significant.

Results

PPARδ knockdown increases expression of ENK in both hypothalamic and forebrain neurons

To determine if any of the PPARs play a role in regulating the expression of the orexigenic neuropeptide, ENK, three separate experiments with dissociated hypothalamic and forebrain neurons were conducted using siRNA to knock down each of the PPARs and observe their effects on levels of ENK mRNA compared to non-knockdown controls. In hypothalamic neurons, a significant knockdown of PPARδ (−60 ± 7%; t(3) = 9.13, p < 0.01) and PPARα (−70 ± 1%; t(3) = 80.25, p < 0.001) was achieved, while knockdown of PPARγ could not be performed due to low mRNA levels (see Methods). Differential effects of the PPAR isoforms were observed in the hypothalamus (Figure 1A), with mRNA levels of ENK significantly increased by reducing the expression of PPARδ (t(3) = 4.67, p < 0.05) but unaffected by decreasing the expression of PPARα (t(3) = 1.19, p = 0.32). In forebrain neurons, significant knockdowns of PPARδ (−53 ± 1%; t(3) = 31.6, p < 0.001), PPARα (−59 ± 2%; t(3) = 28.8, p < 0.001) and PPARγ (−67 ± 2%; t(3) =, p < 0.001) were achieved. As in the hypothalamus, decreasing the expression of PPARδ in the forebrain significantly increased mRNA levels of ENK (t(3) = 7.52, p < 0.01), while ENK mRNA was unaffected by decreasing the expression of PPARα (t(3) = 1.50, p = 0.23) or PPARγ (t(3) = 1.59, p = 0.20) (Figure 1B). These results clearly distinguish PPARδ from PPARα or PPARγ as the only PPAR isoform that regulates the expression of ENK, with its knockdown increasing ENK mRNA, thus leading us to focus on PPARδ in subsequent experiments.

Figure 1.

Figure 1

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Measurement of ENK mRNA was performed after siRNA knockdown of the PPARs on dissociated hypothalamic and forebrain neurons. A. In hypothalamic neurons, knockdown of PPARδ significantly increased expression of ENK as compared to control, with no effect from PPARα knockdown. B. In forebrain neurons, knockdown of PPARδ significantly increased the expression of ENK as compared to control, with no effect from knockdown of PPARα or PPARγ. N=4 cell culture wells per group, total of 4 groups. *p < 0.05, †p < 0.01; KD = knockdown.

PPARδ colocalizes with ENK in both hypothalamic and forebrain neurons

The significant effects of PPARδ knockdown on ENK mRNA led us to further investigate the anatomical relationship between this PPAR isoform and endogenous ENK using double-labeling immunofluorescence. The results showed that the hypothalamic and forebrain neurons can be immunofluorescently labeled with PPARδ and ENK and that this nuclear receptor colocalizes with ENK in some neurons, as identified by arrows in the photomicrographs (Figures 2A and 2B). With respect to total hypothalamic neurons, 18 ± 3% of the neurons contained ENK, 20 ± 4% labeled PPARδ, and 16 ± 3% exhibited colocalization of PPARδ and ENK. With respect to total forebrain neurons, 34 ± 4% of the neurons contained ENK, 59 ± 7% labeled PPARδ, and 32 ± 4% showed colocalization of PPARδ and ENK. These results indicate that forebrain neurons have a higher percentage than hypothalamic neurons with PPARδ, ENK or PPARδ together with ENK, with their co-existence in the same neuron strengthening the idea that this nuclear receptor directly regulates endogenous expression and levels of this opioid peptide.

Figure 2.

Figure 2

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Immunofluorescence labeling of PPARδ and ENK in hypothalamic and forebrain neurons. A. Double-labeling in untreated hypothalamic neurons reveals 16 ± 3% of all neurons to colocalize PPARδ and ENK. B. Double-labeling in untreated forebrain neurons reveals 32 ± 4% of all neurons colocalize PPARδ and ENK. C. Treatment of hypothalamic neurons with the PPARδ agonist, GW0742. D. Treatment of forebrain neurons with GW0742. E. Treatment of hypothalamic neurons with 200 μM PA. F. Treatment of forebrain neurons with 200 μM PA. G. Treatment of hypothalamic neurons with 200 μM OA. H. Treatment of forebrain neurons with 200 μM OA. Red neurons are PPARδ, green neurons are ENK, blue is nuclear staining, and yellow/orange neurons are double-labeled, as shown by the arrows. Bar = 50 μm. PA = palmitic acid; OA = oleic acid. All images on the left are representative of hypothalamic neurons, and images on the right are representative of forebrain neurons.

Activation of PPARδ reduces levels of ENK in neurons of the hypothalamus and forebrain

With ENK mRNA increased by knocking down PPARδ, this experiment focused specifically on this isoform to test in hypothalamic and forebrain neurons whether activation of this nuclear receptor with the PPARδ agonist, GW 0742, does in fact influence ENK, either its expression, number of ENK neurons, or levels of ENK peptide within the neuron. Treatment of the neurons with 1 μM of this PPARδ agonist as compared to untreated controls had no significant effect on ENK mRNA in hypothalamic (t(3) = 1.57, p = 0.22) or forebrain (t(3) = 1.02, p = 0.38) neurons (Figure 3A). Also, immunofluorescence labeling of protein revealed no significant effect of GW 0742 compared to control on the percent of hypothalamic neurons that contained ENK alone (t(6) = 1.72, p = 0.14) (Figure 3B) or that co-labeled PPARδ / ENK (t(6) = 1.72, p = 0.14) (Figure 3C) as illustrated in the photomicrographs (Figure 2C vs 2A), or of forebrain neurons that contained ENK alone (t(8) = 1.38, p = 0.20) (Figure 3B) or that co-labeled PPARδ / ENK neurons (t(8) = 0.55, p = 0.60) (Figure 3C), as illustrated in the photomicrographs (Figure 2D vs 2B). However, significant effects of this agonist (1 μM) were demonstrated with further fluorescence intensity histogram analyses of the average intensity of each individual neuron (see Methods). This analysis revealed two populations of ENK neurons, with Low (L) or High (H) intensity, as indicated by the dotted line in the histogram for the hypothalamus (Figure 4) and forebrain (Figure 5). Treatment with GW 0742 of neurons in both brain regions as compared to control induced a shift in these neuronal populations, with a decrease in the percent of H intensity neurons and an increase in the L intensity neurons (Table 1), as shown in the hypothalamus (Figure 4B vs 4A) and forebrain (Figure 5B vs 5A) as a decrease in the area under the curve, showing an overall decrease in ENK intensity in individual neurons. These results with the agonist further demonstrate a close relationship between PPARδ and ENK, showing direct activation of this nuclear receptor to significantly decrease intracellular levels of ENK while having no effect on the number of ENK neurons. This evidence, together with results of the first experiment showing knockdown of PPARδ to increase ENK mRNA, supports the idea that this particular isoform of PPAR negatively regulates ENK in both hypothalamic and forebrain neurons.

Figure 3.

Figure 3

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Effects of GW0742 on ENK in hypothalamic and forebrain neurons. A. Treatment of neurons with 1 μM GW0742 revealed no significant effects on ENK expression. B. There was no significant effect of GW0742 treatment on the percent of ENK neurons from the hypothalamus or forebrain. C. Immunofluorescence labeling revealed no significant effect of GW0742 treatment on the percent of PPARδ / ENK co-labeled neurons from the hypothalamus or forebrain. The number of ENK neurons is measured relative to all neurons, and the number of PPARδ / ENK double labeled neurons is measured relative to PPARδ. N=4 cell culture wells per group, total of 4 groups.

Figure 4.

Figure 4

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Fluorescence intensity histogram analysis of hypothalamic neurons. The fluorescence intensity of ENK for each neuron was normalized by subtracting the mean background intensity of a nearby area of comparable size and multiplying the mean intensity within the region by its area, plotted as a histogram, and fitted to a probability density function. Each peak represents one population of neurons, the dotted line represents the mean intensity of that population, and the area under the curve represents the density of cells within each population and is portrayed as a percent of the total area. This intensity histogram analysis was performed for neurons in: A. Control; B. GW0742; C. 200 PA; and D. 200 OA groups. Fits to the histogram with X2 values corresponding to p < 0.05 were considered significant. This analysis revealed two populations of ENK neurons, of Low (L) and High (H) intensity, in hypothalamic neurons. PA = palmitic acid; OA = oleic acid; a.u. = arbitrary units.

Figure 5.

Figure 5

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Fluorescence intensity histogram analysis of forebrain neurons. The fluorescence intensity of ENK for each neuron was normalized by subtracting the mean background intensity of a nearby area of comparable size and multiplying the mean intensity within the region by its area, plotted as a histogram, and fitted to a probability density function. Each peak represents one population of neurons, the dotted line represents the mean intensity of that population, and the area under the curve represents the density of cells within each population and is portrayed as a percent of the total area. Intensity histogram analysis was performed for neurons in: A. Control; B. GW0742; C. 200 PA; and D. 200 OA groups. Fits to the histogram with X2 values corresponding to p < 0.05 were considered significant. This analysis revealed two populations of ENK neurons, of Low (L) and High (H) intensity, in forebrain neurons. PA = palmitic acid; OA = oleic acid; a.u. = arbitrary units.

Table 1.

Average fluorescence intensity of ENK in hypothalamic and forebrain neurons.

L (a.u.) % (L) H (a.u.) % (H)
Hypothalamus
Control 1.41 × 105 62 2.26 × 105 38
1 μM GW0742 1.46 × 105 75* 2.34 × 105 25*
200 PA 1.52 × 105 50* 2.19 × 105 50*
200 OA 1.39 × 105 42* 2.06 × 105 58*
Forebrain
Control 1.07 × 105 52 1.75 × 105 48
1 μM GW0742 9.10 × 104 61* 1.39 × 105 39*
200 PA 9.06 × 104 43* 1.50 × 105 57*
200 OA 1.05 × 105 46* 1.49 × 105 54*
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Two populations of neurons, Low (L) and High (H) intensity, corresponding to relative levels of ENK protein, were found with both Hypothalamic and forebrain neurons. The PPARδ agonist, GW0742, decreased the number of neurons containing High levels of protein, while both PA and OA increased the number of neurons containing High levels of protein.

*

compared to control.

PA and OA differentially affect expression of PPARδ and ENK in hypothalamic and forebrain neurons

With the previous experiments revealing an inverse relationship between PPARδ and ENK, this experiment investigated using qRT-PCR whether PA and OA can themselves influence ENK and PPARδ in hypothalamic and forebrain neurons. These two fatty acids can bind to PPARδ and are the main components of a fat-rich diet shown to stimulate ENK in the hypothalamus and NAc (see Introduction). In hypothalamic neurons, treatment with the saturated fatty acid, PA, compared to control induced a significant change in mRNA levels at both 100 μM (F(2, 9) = 22.60, p < 0.001) and 200 μM (F(2, 9) = 21.99, p < 0.001), reflecting a significant increase in the expression of both PPARδ and ENK (p < 0.001) (Figure 6A). A similar effect was observed with PA treatment of forebrain neurons, which caused a significant change in mRNA expression at both 100 μM (F(2, 9) = 10.21, p < 0.01) and 200 μM (F(2, 9) = 30.38, p < 0.001), again reflecting a significant increase in the expression of both PPARδ and ENK (p < 0.01) (Figure 6B). These findings reveal a strong, stimulatory effect of PA on this nuclear receptor and opioid peptide, an effect observed similarly in the hypothalamus and forebrain. However, the results obtained with the unsaturated fatty acid, OA, were very different from PA and clearly differentiated the response of two brain regions to OA. In hypothalamic neurons, treatment with OA compared to control had no impact on mRNA levels at either 100 μM (F(2, 9) = 1.52, p = 0.27) or 200 μM (F(2, 9) = 0.42, p = 0.67), reflecting no change in PPARδ or ENK mRNA at either concentration (Figure 6A). In contrast, OA treatment of forebrain neurons produced a main effect in mRNA levels at both 100 μM (F(2, 9) = 4.65, p < 0.05) and 200 μM (F(2, 9) = 9.54, p < 0.01), which reflected an increase in expression of PPARδ at both 100 uM (t(3) = 12.12, p<0.01) and 200 uM (t(3) = 4.29, p<0.05), with no change in ENK mRNA at either dose (100 μM, p = 0.32; 200 μM, p = 66) (Figure 6B). This evidence reveals differential effects of PA and OA, with PA increasing PPARδ expression along with ENK mRNA in both hypothalamic and forebrain neurons and OA increasing only PPARδ mRNA in forebrain neurons with ENK expression unaffected in both regions.

Figure 6.

Figure 6

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Fatty acid effects on mRNA levels of PPARδ and ENK. A. Treatment of cultured hypothalamic neurons with 100 or 200 μM PA significantly increased the expression of both PPARδ and ENK, while treatment with 100 and 200 μM OA had no effect on mRNA levels. B. Similar to hypothalamic neurons, treatment of cultured forebrain neurons with 100 or 200 μM PA significantly increased the expression both PPARδ and ENK. Treatment with 100 and 200 μM OA induced a small but significant increase in PPARδ expression, with no change in ENK expression. N=4 cell culture wells per group, total of 4 groups. *p < 0.05, †p < 0.01, ‡ p < 0.001; PA = palmitic acid; OA = oleic acid.

PA and OA differentially affect protein levels of PPARδ and ENK in hypothalamic and forebrain neurons

With the fatty acids found to have differential effects on mRNA levels of PPARδ and ENK in hypothalamic and forebrain neurons, this experiment used immunofluorescence histochemistry to test whether PA and OA also have differential effects on the protein levels or co-localization of PPARδ with ENK in individual neurons. Dissociated hypothalamic and forebrain neurons were treated with 200 μM of PA or OA and co-labeled for PPARδ and ENK, as illustrated in the photomicrographs (Figures 2E-2H). In hypothalamic neurons, these fatty acids had no significant effect on the percent that co-labeled PPARδ / ENK (F(2, 11) = 0.23, p = 0.80) or that contained ENK alone (F(2, 11) = 0.18, p = 0.84) or PPARδ alone (F(2, 11) = 0.04, p = 0.96). Similarly, in forebrain neurons, there was no significant effect on the percent that co-labeled PPARδ / ENK (F(2, 11) = 1.23, p = 0.33) or that contained ENK alone (F(2, 11) = 1.21, p = 0.34) or PPARδ alone (F(2, 11) = 0.89, p = 0.44). However, significant effects were revealed by the additional fluorescence intensity histogram analyses of ENK and PPARδ neurons, consistent with the changes in mRNA levels and a possible inverse relation between PPARδ and ENK. In both the hypothalamic (Figure 4) and forebrain (Figure 5) neurons, the analyses of ENK uncovered two comparable populations with L and H intensity and revealed a significant shift in these neuronal populations. Compared to untreated controls, both PA (Figures 4C and 5C) and OA (Figures 4D and 5D) in neurons of both brain regions induced an increase in the percent of ENK neurons in the H intensity population and a decrease in the L intensity population, as depicted by an increase and decrease in the area under the curve, respectively, reflecting an overall increase in intracellular levels of ENK (Table 1). A similar fluorescence intensity analysis of PPARδ neurons, in contrast, yielded different results in the two brain regions. In the hypothalamus, neither PA nor OA had any effect on the percent of PPARδ neurons in the L or H intensity populations (Table 2). However, in forebrain neurons, there were three populations of PPARδ neurons, L, Medium (M) and H. Whereas PA had no effect on the percent of forebrain neurons in any of these populations, the forebrain neurons treated with OA existed in the higher two populations of PPARδ, M and H, compared to the three populations in untreated control (Table 2). This indicates a shift to the higher intensity populations, suggesting that OA increases the amount of PPARδ protein within individual forebrain neurons, similar to the OA-induced increase in mRNA seen in the prior experiment. Together, these results demonstrate that, while the fatty acids have no effect on the colocalization of PPARδ and ENK, they differentially alter their levels in individual neurons, with PA having no effect on PPARδ protein while increasing ENK levels in hypothalamic and forebrain neurons and OA having a marked, stimulatory effect on PPARδ protein specifically in forebrain neurons.

Table 2.

Average fluorescence intensity of PPARδ in hypothalamic and forebrain neurons.

L (a.u.) % (L) M (a.u.) % (M) H (a.u.) % (H)
Hypothalamus
Control 7.79 × 104 41 — — 1.16 × 105 59
200 PA 9.24 × 104 48 — — 1.31 × 105 52
200 OA 8.43 × 104 43 — — 1.16 × 105 57
Forebrain
Control 1.57 × 105 25 2.70 × 105 43 5.67 × 105 32
200 PA 1.61 × 105 33 3.09 × 105 41 5.63 × 105 26
200 OA — — 4.76 × 105 20* 6.67 × 105 80*
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Two populations of neurons, Low (L) and High (H) intensity, corresponding to relative levels of ENK protein, were found with both Hypothalamic and forebrain neurons. Only two populations of neurons existed in hypothalamic neurons, with no change in neuronal population with either PA or OA treatment. Three populations of neurons existed in the forebrain neurons, L, Medium (M), and H, with only OA increasing the percent of number of neurons containing high levels of PPARδ.

*

compared to control.

Discussion

The fatty acids, PA and OA, are two of the most abundant fatty acids found in a Western fat-rich diet. Animal studies show that intake of a HFD mainly composed of these fatty acids stimulates the opioid neuropeptide ENK, which in turn increases ingestion of this diet and other substances of abuse, and prenatal exposure to this HFD stimulates the genesis of ENK-expressing neurons in the embryo (see Introduction). With the nuclear receptors, PPARs, known to bind fatty acids and affect transcription of different proteins, this study investigated in hypothalamic and forebrain neurons whether these receptors have a role in regulating expression and levels of ENK and whether the fatty acids themselves affect endogenous PPARδ in relation to ENK. The results demonstrate that PPARδ, but not PPARα or PPARγ, negatively affects ENK expression and levels and that PPARδ co-exists with ENK in the same neurons. They also show that the fatty acids, PA and OA, have differential effects on PPARδ and ENK. Whereas the saturated fatty acid, PA, has no effect on protein levels of PPARδ despite increased mRNA and stimulates both mRNA and protein levels of ENK, the monounsaturated fatty acid, OA, increases both protein and mRNA levels of PPARδ while having no effect on ENK mRNA and increasing ENK levels. These findings reveal an inverse relationship between PPARδ and ENK protein, as well as differential effects of the fatty acids on these two proteins, with PA more strongly affecting ENK and OA more strongly affecting PPARδ. These results suggest a function for PPARδ during development, perhaps one that is protective against fatty acid exposure, and indicate a mechanism that may mediate the differential effects of saturated and monounsaturated fatty acids on neuronal systems and behavior.

Relationship of PPARδ to ENK in hypothalamic and forebrain neurons

One focus of the present study is on the relationship of the PPAR family of nuclear receptors to ENK, a neuropeptide known to be highly sensitive to a fat-rich diet. While there are no studies to date relating PPARδ to the neuropeptides, there is evidence that PPARα and PPARγ have some function in relation to neuropeptides in the hypothalamus. In adult animals, activation of PPARα or PPARγ is shown to increase expression of the orexigenic peptide, neuropeptide Y, while decreasing expression of the anorexigenic peptide, pro-opiomelanocortin (Chikahisa et al. 2008, Kocalis et al. 2012). The results of the present study provide the first evidence that PPARδ also directly affects a specific orexigenic neuropeptide. Whereas knocking down PPARα or PPARγ is found to have no effect on ENK, knocking down PPARδ causes an increase in ENK mRNA, an effect seen in both hypothalamic and forebrain neurons. Although one report found activation of PPARγ by a short-chain fatty acid to increase ENK in immortalized PC-12 cells (Parab et al. 2007), the absence of this effect here may be due to the fact that the hypothalamic and forebrain neurons likely have much lower levels of PPARγ than immortalized cells. Our findings further demonstrate that activation of PPARδ by an agonist has the opposite effect of a PPARδ knockdown, causing a decrease in intracellular levels of ENK peptide again in both hypothalamic and forebrain neurons. The concentration of the agonist (1 μM) used in this experiment was chosen based on evidence showing higher concentrations to activate PPARα and PPARγ, as well as other receptors (Sznaidman et al. 2003, Nandhikonda et al. 2013). The failure of this agonist to affect ENK mRNA may be due to agonist clearance after 24 hours of treatment, with the decrease in ENK protein being the only long-term effect. This difference may also reflect the fact that PPARδ can function as an obligate heteromer to RXR receptors (Krey et al. 1997) and is modulated by coregulators and corepressors (Lee et al. 2003, Puigserver & Spiegelman 2003). Together, this evidence provides strong support for an inverse relationship between PPARδ and ENK, with activation of this PPAR subtype inhibiting the expression of ENK in neurons from both the hypothalamus and the forebrain.

Anatomical relationship between ENK and PPARδ

Anatomical studies demonstrate that PPARδ, although ubiquitously expressed in the brain, has relatively high levels in the NAc and septal nucleus and moderate levels in the hypothalamus (Moreno et al. 2004, Higashiyama et al. 2007), brain areas where endogenous ENK is also highly expressed (Gall & Moore 1984, Mathieu et al. 1996, Chang et al. 2010). Unpublished results from our lab in postnatal rats also show that PPARδ colocalizes with ENK in neurons of the hypothalamus and that prenatal exposure to a HFD increases their expression and colocalization (Chang et al., manuscript under review). In the present report, we also provide evidence that PPARδ colocalizes with ENK in neurons of the hypothalamus and also the forebrain that contains both the NAc and septum. Our evidence, showing that knockdown of PPARδ markedly increases ENK mRNA in these neurons while knockdown of PPARα and PPARγ has no effect on ENK, may be explained, in part, by the anatomical distribution of these other PPAR isoforms described in the literature. Studies show that PPARα is not expressed in the hypothalamus and has low levels in the forebrain (Moreno et al. 2004) and that PPARγ is expressed at low levels in the hypothalamus and only moderately in the forebrain (Moreno et al. 2004). Additionally, while yet to be anatomically related to ENK, PPARγ may differ in colocalizing with anorexigenic neuropeptides, such as α-melanocyte-stimulating hormone and pro-opiomelanocortin, in the hypothalamus in addition to agouti-related protein (Sarruf et al. 2009). Our anatomical evidence revealing the colocalization of PPARδ with ENK in neurons strengthens the idea that this nuclear receptor and neuropeptide are closely related and interact endogenously, with PPARδ negatively regulating the expression and intracellular levels of ENK.

Effect of fatty acids on ENK and PPARδ

The fatty acids, PA and OA, the two most abundant fatty acids in a HFD, may play a role in mediating the stimulatory effects of a HFD on ENK and affecting the function of PPARδ in the regulation of ENK. There are no studies in the brain directly linking fatty acids to endogenous ENK, with only two studies showing OA in the hypothalamus to inhibit the orexigenic peptide, neuropeptide Y, while stimulating the anorexigenic peptide, pro-opiomelanocortin (Obici et al. 2002, Jo et al. 2009). The present report examining these two fatty acids clearly distinguishes their actions in isolated neurons. The results obtained with PA reveal a strong, stimulatory effect on both mRNA and protein levels of ENK in neurons of the hypothalamus or forebrain, consistent with the changes induced by a HFD (see below). This is in contrast to OA, which has no impact on ENK expression in hypothalamic or forebrain neurons even though it increases ENK protein levels. These differential effects of the fatty acids on this opioid peptide may be related inversely to their differential effects on PPARδ. Whereas PA in both hypothalamic and forebrain neurons fails to stimulate protein levels of PPARδ despite an increase in mRNA, OA stimulates both protein and mRNA levels of PPARδ in forebrain neurons. While this higher level of PPARδ protein activity may then inhibit the expression of ENK, its failure to completely reverse the OA-induced increase in ENK protein may be due to other effects of this fatty acid on regulatory processes, such as a decrease in protein exocytosis and degradation (Kudo et al. 2006, Grasso & Calderon 2013). It may also reflect other proteins besides PPARδ, such as the transcription enhancer factor-1 and Yes-associated protein, which are affected by a HFD and positively or negatively regulate ENK expression and levels (Poon et al. 2013). Together, these findings with measurements of both mRNA and protein suggest that PA has a stronger effect on ENK while OA has a stronger effect on PPARδ with a lesser effect on ENK, consistent with the evidence for an inverse relationship between this nuclear receptor and opioid peptide.

Differential responsiveness of hypothalamic and forebrain neurons to oleic acid

The results of these experiments with measurements of both mRNA and intracellular levels of PPARδ and ENK generally reveal similar effects in neurons from the hypothalamus and forebrain, with these replications strengthening the conclusions of this study. In both brain regions, they demonstrate that PPARδ and ENK exist and co-exist in neurons and that PA has similar effects on ENK, suggesting that it mediates the changes induced by consumption of a fat-rich diet. What is notable is the one specific difference between these brain areas found in the effect of OA on PPARδ. This fatty acid has stimulatory effects on both the expression and protein levels of PPARδ in forebrain neurons but neither of these effects in hypothalamic neurons. One possible explanation for this is suggested by the anatomical differences detected in the distribution patterns of this nuclear receptor. Studies show that PPARδ is highly expressed in the NAc and septal nucleus but is only moderately expressed in the hypothalamus (Moreno et al. 2004). Our results are consistent with this, showing a higher percent of PPARδ neurons in the forebrain than those in the hypothalamus. Other than this anatomical difference in the effects of OA on PPARδ, our results generally show similarities between the hypothalamus and forebrain in terms of the inverse relation between PPARδ and ENK in individual neurons and the effects induced by the fatty acids on this nuclear receptor and neuropeptide.

Effect of a fat-rich diet on ENK and PPARδ

Numerous studies of ENK, a neuropeptide positively related to consummatory behavior (Gomori et al. 2003, Barson et al. 2009, Barson et al. 2010, Drews & Zimmer 2010), have shown this opioid to be particularly sensitive to a HFD known to be rich in PA and OA. Both the expression and levels of this peptide in the hypothalamus and NAc are shown to be stimulated by intake of a HFD in adult animals (Chang et al. 2010), and embryonic development of ENK-expressing neurons is increased in these areas by maternal consumption of a HFD (Chang et al. 2008, Vucetic et al. 2010). Our studies show prenatal HFD exposure to stimulate PPARδ (Chang et al., manuscript under review), studies of PPARα and PPARγ have revealed in peripheral tissues a stimulatory effect of HFD intake in adult animals and of prenatal exposure to a HFD on levels of these PPAR isoforms (Zhang et al. 2005, Kannisto et al. 2006, Zhang et al. 2009, Shamsi et al. 2014, Shen et al. 2014). Also, a stimulatory effect of chronic HFD ingestion on apoptosis has been described in the hypothalamus (Moraes et al. 2009) and hippocampus (Molteni et al. 2002) and suggested to be attributed to an increase in PPARα or PPARγ, which are known to stimulate apoptosis in other cell types (Smith et al. 2001, Kim et al. 2006). Investigations of PPARδ suggest that this isoform has very different functions, actually exerting effects that are protective in nature. This is based on evidence that a neuronal knockout of PPARδ increases susceptibility to HFD-induced obesity and increases adiposity (Kocalis et al. 2012), while oral administration of a PPARδ agonist decreases food intake and weight gain (Harrington et al. 2007). Additionally, PPARδ is heavily expressed during the embryonic period and plays an important role in neuronal maturation (Di Loreto et al. 2007, Cimini & Ceru 2008). These studies of PPARδ in relation to a fat-rich diet suggest that an increase in the activity of this PPAR isoform helps to protect against the neurotoxic effects of this diet on peptide function.

Fatty acids and their relationship to ingestive behavior

While being endogenously stimulated by ingestion of a HFD, the opioid ENK in turn is known to cause a further increase in consummatory behavior. Injection of ENK analogs into the hypothalamus and forebrain stimulates ingestion of a HFD and also drugs of abuse, such as nicotine and ethanol (Gomori et al. 2003, Barson et al. 2009, Barson et al. 2010, Drews & Zimmer 2010). In addition, saturated fatty acids like PA are generally associated with negative physiological outcomes including spontaneous overconsumption and weight gain (Wang et al. 2002), while monounsaturated fatty acids like OA are linked to more positive physiological effects (Kien et al. 2005). The results of the present study demonstrate that PA has a stronger stimulatory effect than OA on the expression and levels of ENK in both hypothalamic and forebrain neurons, suggesting that PA in a fat-rich diet plays a more prominent role, perhaps acting through ENK, in promoting this increase in consumption. This fatty acid is believed to contribute to the higher prevalence of obesity induced by the ingestion of a Western diet containing high levels of PA (Fung et al. 2001, Baylin et al. 2002, Haslam & James 2005), as compared to leaner counterparts ingesting a diet with high levels of OA and low levels of PA (Obici et al. 2002, Cintra et al. 2012). With PA and OA accounting for 70% of the fatty acids that compose a HFD and a HFD found to stimulate both PPARδ and ENK expression as well as their colocalization (Chang et al., manuscript under review), our results with the individual fatty acids suggest that the administration of PA and OA in combination would have a similar stimulatory effect as the HFD on PPARδ and ENK in the dissociated neurons. In conclusion, the present study reveals an inverse relationship between PPARδ and ENK, and differential effects of the fatty acids, PA and OA, on the expression and levels of PPARδ and ENK that may ultimately contribute to the increased consummatory behavior of both a HFD and drugs of abuse.

Acknowledgements

The authors would like to thank Hui Tin Ho for her assistance in the cell culture experiments and The Rockefeller University’s Bio-Imaging Resource Center for the use of their equipment.

Grants

This work was supported by grants from the National Institutes of Health, F32DK100058 (KP), K99AA021782 (JRB), and 1R21 AA020593 (SFL). This work was additionally supported by grant # UL1 TR000043 from the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health Clinical and Translational Science Award (CTSA) program (The Rockefeller University TTCL Resource Center).

Footnotes

Author contributions: KP conceived, designed and performed experiments, analyzed data, prepared figures and drafted manuscript; MA performed experiments and analyzed data; OK conceived of research; JRB performed experiments and edited manuscript; SFL conceived of research and edited manuscript.

Disclosures

None.

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