Time schedules and experimental designs.

<p>Male Wistar rats were sham injected at least two times prior to LPA treatment to familiarize animals with the i.c.v. procedure. The open field test (OFT) and elevated plus maze (EPM) were performed under novelty (A) and habituation (B) conditions after 5 min of LPA 18∶1 infusion. For habituation, the rats were sham injected and tested in both paradigms 24 h before LPA infusion. Rats were exposed to the sample trial of the Y maze (YMT) 5 min after LPA 18∶1 administration, and performed the test trial 2 h later (C). The forced swimming test (FST) was performed after 5 min of LPA 18∶1 infusion (D). Food and water intake were evaluated at different times in 24 h food-deprived animals after 5 min of LPA 18∶1 infusion (E). c-Fos immunoreactivity (IR) was performed through perfusion after 90 min of LPA 18∶1 infusion (F).</p

Wistar rats were studied in the elevated plus maze (EPM) and open field test (OFT) under novelty and habituation conditions following LPA 18∶1 infusion at doses of 0, 0.4 and 2 µg.

<p>In the EPM, the time (s) exploring the exposed arms (A) and the total number of arm entries (B) were evaluated under novelty conditions. Similarly to the novelty conditions, the time exploring the open arms (C) and the total arm entries (D) were evaluated in animals previously habituated to the EPM (A). Locomotor activity was measured based on the number of crossings (E) and the time (%) spent in the center of the field (F) under novelty conditions. Again, both the number of crossings (G) and the percentage of time spent in the center (H) were evaluated under habituation conditions. The bars are the means ± SEM (n = 7–12 animals per group). The data were analyzed using one-way ANOVA. *p<0.05 and ***p<0.001 denote significant differences versus the vehicle-treated group, determined using Bonferroni’s <i>post-hoc</i> test.</p

c-Fos immunohistochemistry in the rat dorsal periaqueductal gray matter (PAG) following LPA 18∶1 infusion at doses of 0 and 2 µg.

<p>Analyses were performed in the dorsomedial and dorsolateral divisions of the PAG (<b>A</b>). Stereological quantification of c-Fos immunoreactive (IR) nuclei in the DPAG (<b>B</b>). Low magnification microphotographs for c-Fos immunohistochemistry are depicted for the vehicle (<b>C</b>)- and LPA 18∶1 (<b>D</b>)-treated groups. In addition, high magnification images for the vehicle (<b>E</b>)- and LPA 18∶1 (<b>F</b>)-treated groups are shown. Each point represents the total number of immunopositive nuclei per animal. The dotted lines are medians (n = 4 animals pr group). The data were analyzed using Kruskal-Wallis one-way ANOVA. *p<0.05 denotes significant differences versus the vehicle-treated group, determined using Dunn’s <i>post-hoc</i> test. The arrowheads indicate immunopositive nuclei for c-Fos.</p

Rats were studied for novelty recognition in the Y maze (YMZ).

<p>LPA 18∶1 infusion at doses of 0, 0.4 and 2 <b>µ</b>g was carried out 5 min before the sample trial, and the test trial was performed 2 h later. The percent of rats of each group that first entered the novel arm (A), the total time of novel arm exploration (B) and the total arm entries (C) in the test trial were avaluated. The bars are the means ± SEM (n = 11–12 animals per group). The data were analyzed using a Chi-square test (A) or an one-way ANOVA followed by Bonferroni’s <i>post-hoc</i> tests (B–C). *p<0.05 denote significant differences versus the vehicle-treated group. The comparison of the 2 <b>µ</b>g and the vehicle group in (B) was significant at p = 0.0558.</p

Time-course effects of i.c.v. administration of LPA 18∶1 or vehicle on food and water intake in 24 h food-deprived Wistar rats.

<p>¹ Data are means ± SEM of 8 determinations per group.</p

Wistar rats were studied in the forced swimming test (FST) following LPA 18∶1 infusion at doses of 0, 0.4 and 2 µg.

<p>The immobility time (s) was evaluated in these animals. The bars are the means ± SEM (n = 8 animals per group). The data were analyzed using one-way ANOVA. *p<0.05 and **p<0.01 denote significant differences versus the vehicle-treated group, determined using Bonferroni’s <i>post-hoc</i> test.</p

Computational and Biological Evaluation of <i>N</i>-octadecyl-<i>N</i>′-propylsulfamide, a Selective PPARα Agonist Structurally Related to <i>N</i>-acylethanolamines

<div><p>To further understand the pharmacological properties of N-oleoylethanolamine (OEA), a naturally occurring lipid that activates peroxisome proliferator-activated receptor alpha (PPARα), we designed sulfamoyl analogs based on its structure. Among the compounds tested, N-octadecyl-N′-propylsulfamide (CC7) was selected for functional comparison with OEA. The performed studies include the following computational and biological approaches: 1) molecular docking analyses; 2) molecular biology studies with PPARα; 3) pharmacological studies on feeding behavior and visceral analgesia. For the docking studies, we compared OEA and CC7 data with crystallization data obtained with the reference PPARα agonist GW409544. OEA and CC7 interacted with the ligand-binding domain of PPARα in a similar manner to GW409544. Both compounds produced similar transcriptional activation by <i>in vitro</i> assays, including the GST pull-down assay and reporter gene analysis. In addition, CC7 and OEA induced the mRNA expression of CPT1a in HpeG2 cells through PPARα and the induction was avoided with PPARα-specific siRNA. <i>In vivo</i> studies in rats showed that OEA and CC7 had anorectic and antiobesity activity and induced both lipopenia and decreases in hepatic fat content. However, different effects were observed when measuring visceral pain; OEA produced visceral analgesia whereas CC7 showed no effects. These results suggest that OEA activity on the PPARα receptor (e.g., lipid metabolism and feeding behavior) may be dissociated from other actions at alternative targets (e.g., pain) because other non cannabimimetic ligands that interact with PPARα, such as CC7, do not reproduce the full spectrum of the pharmacological activity of OEA. These results provide new opportunities for the development of specific PPARα-activating drugs focused on sulfamide derivatives with a long alkyl chain for the treatment of metabolic dysfunction.</p></div

Docking of compounds in the ligand binding domain of PPARα.

<p>Docking energy (kcal/mol) and the percentage of the occurrence of hydrogen bonds (HB) to the hydroxyl group of Tyr 464 residue.</p

mRNA expression of CPT1a in HepG2 cells.

<p>(<b>A</b>) The CPT1a mRNA expression was determined by qRT-PCR after the incubation of cells with OEA (0.1, 1, 5 and 10 μM) for 48 h. (<b>B</b>) HepG2 cells were serum deprived for 16 h and transfected with PPARα-specific siRNA or control siRNA followed by incubation with 10 μM of OEA and CC7 for 48 h. Then, the CPT1a mRNA was determined by qRT-PCR. Expression was normalized to GAPDH expression. Experiments were repeated 3 times and results are presented as x-fold induction over vehicle treated cells. Columns represent the mean ± SEM, and these data were analyzed by Student’s t test. *P<0.05, **P<0.01 and ***P<0.01 vs vehicle or control siRNA.</p

Basal and ligand-induced activities of PPARα were determined using luciferase reporter gene assays.

<p>(<b>A</b>) MCF-7 cells were transiently transfected with a reporter construct containing the human CPT1 DR1-type PPRE (CPT1-PPRE) and the indicated expression vectors for PPARα, RXRα, SRC1 (co-activator) and NCoR (co-repressor). The columns represent the mean ± SEM of at least three experiments, and the data were analyzed by Student’s t test. **P<0.01 and ***P<0.001 compared to vehicle alone. (<b>B</b>) MCF-7 cells were treated for 16 h with 10 μM of OEA, CC7, CC12 or GW7647 in either presence or absence and of 100 nM of the HDAC inhibitor trichlorostatin A (TSA). Stimulation of luciferase activity was normalized to the basal activity of PPARα-RXRα in absence of ligands. The columns represent the mean ± SEM of at least three experiments, and these data were analyzed by Student’s t test. *P<0.05, **P<0.01 and ***P<0.001 compared to the absence of TSA and the presence of vehicle.</p