A role of CD36 in the perception of an oxidised phospholipid species in mice.

CD36 is a broadly expressed transmembrane protein that engages multiple ligands, including polar lipids. This protein is thought to even contribute to the chemosensory detection of long-chain fatty acids in the oral cavity of rodents. In this study, we assessed whether animals consciously perceive a ligand of CD36, 1-(palmitoyl)-2-(5-keto-6-octanedioyl)phosphatidylcholine (KOdiA-PC), and if so, whether CD36 is involved in sensing the oxidised phospholipid species. We found that mice avoided or hesitated to ingest fluids containing KOdiA-PC, suggesting a conscious perception of the lipid in the animals. We assessed the involvement and role of CD36 in the KOdiA-PC perception by comparing the behavioural responses of wild-type and CD36-deficient mice to the test fluids, and provided evidence that the protein could play a role in sensing a lower level of the lipid. We also found that transection of the olfactory nerve of wild-type mice resulted in an inability to perceive KOdiA-PC, suggesting the significance of olfactory system in the lipid sensing. Our findings, coupled with the recent finding of CD36 expression in the mouse olfactory epithelium, led us to predict that the site of CD36 action in the KOdiA-PC sensing plausibly lies within the nasal cavity of the animal.

Mechanisms Involved in Guiding the Preference for Fat Emulsion Differ Depending on the Concentration.

High-fat foods tend to be palatable and can cause addiction in mice via a reinforcing effect. However, mice showed preference for low fat concentrations that do not elicit a reinforcing effect in a two-bottle choice test with water as the alternative. This behavior indicates the possibility that the mechanism underlying fat palatability may differ depending on the dietary fat content. To address this issue, we examined the influences of the opioid system and olfactory and gustatory transductions on the intake and reinforcing effects of various concentrations of a dietary fat emulsion (Intralipid). We found that the intake and reinforcing effects of fat emulsion were reduced by the administration of an opioid receptor antagonist (naltrexone). Furthermore, the action of naltrexone was only observed at higher concentrations of fat emulsion. The intake and the reinforcing effects of fat emulsion were also reduced by olfactory and glossopharyngeal nerve transections (designated ONX and GLX, respectively). In contrast to naltrexone, the effects of ONX and GLX were mainly observed at lower concentrations of fat emulsion. These results imply that the opioid system seems to have a greater role in determining the palatability of high-fat foods unlike the contribution of olfactory and glossopharyngeal nerves.

Expression of CD36 by Olfactory Receptor Cells and Its Abundance on the Epithelial Surface in Mice.

CD36 is a transmembrane protein that is involved in the recognition of certain amphiphilic molecules such as polar lipids in various tissues and body fluids. So far, CD36 homologues in insects have been demonstrated to be present on the surface of olfactory dendrites and to participate in the perception of exogenous compounds. However, little is known about the relationship between CD36 and mammalian olfaction. Indeed, the detection of only CD36 mRNA in the mouse olfactory epithelium has been reported to date. In the present study, to provide potential pieces of evidence for the involvement of CD36 in mammalian olfactory perception, we extensively investigated the localisation of this protein in the mouse olfactory mucosa. In situ hybridisation analysis using antisense oligonucleotides to CD36 mRNA detected aggregated signals within the deeper epithelial layer of olfactory mucosa. The mRNA signals were also detected consistently in the superficial layer of the olfactory epithelium, which is occupied by supporting cells. Immunostaining with an anti-CD36 polyclonal antibody revealed that CD36 localises in the somata and dendrites of distinct olfactory receptor cells and that it occurs abundantly on the olfactory epithelial surface. However, immunoreactive CD36 was rarely detectable in the nerve bundles running in the lamina propria of olfactory mucosa, the axons forming the olfactory nerve layer in the outermost layer of the bulb and axon terminals in the glomeruli. We also obtained electron microscopic evidence for the association of CD36 protein with olfactory cilia. Altogether, we suggest that CD36 plays a role in the mammalian olfaction. In addition, signals for CD36 protein were also detected on or around the microvilli of olfactory supporting cells and the cilia of nasal respiratory epithelium, suggesting a role for this protein other than olfaction in the nasal cavity.

The opioid system contributes to the acquisition of reinforcement for dietary fat but is not required for its maintenance.

The opioid system plays an important role in ingestive behavior, especially with regard to palatable high-fat or sweetened foods. In the present study, we investigated the role of the opioid system in the regulation of ingestive behavior in mice with regard to dietary fat intake, reinforcement, and particularly the processes involved in development of these behavior types. Subcutaneous administration of the non-selective opioid receptor antagonist naltrexone (0.5 or 2.0mg/kg body weight [BW]) reduced the spontaneous intake of fat emulsion (Intralipid). We investigated the effect of naltrexone on reinforcement by using an operant behavioral paradigm under a progressive ratio schedule in which the number of lever presses required to obtain a test sample increased progressively. Mice showed stronger reinforcement by Intralipid as a function of concentration. However, naltrexone (0.5 or 2.0mg/kg BW) did not affect reinforcement at any concentration of Intralipid in mice that had repeatedly ingested Intralipid before testing was carried out. Intralipid ingestion also induced conditioned place preference (CPP), which is another evaluation index of reinforcement. High-dose naltrexone (2.0mg/kg BW) administration during CPP conditioning suppressed the reinforcement induced by Intralipid ingestion, although the drug administration (0.5 or 2.0mg/kg BW) during CPP testing did not affect reinforced behavior. These results suggest that the amount of fat ingestion and reinforcement for fat ingestion are separately regulated by the opioid system. Furthermore, our results indicate that the opioid system plays an important role in acquiring reinforcement for fat but is not required for maintenance of learned reinforcement.

Further validation of unsaturated long-chain fatty acids as inhibitors for oxidized low-density lipoprotein binding to CD36 via assays with synthetic CD36 peptide-cross-linked plates.

We recently obtained evidence that unsaturated long-chain fatty acids (LCFAs) (e.g. oleic acid) inhibit binding of oxidized low-density lipoproteins (oxLDLs) to CD36. In the present study, we validated this prediction by examining inhibition by unsaturated LCFAs of Alexa-fluor-labeled oxLDL binding to multiwell plates onto which a synthetic CD36 peptide is covalently immobilized via thiol-maleimide coupling.

Unsaturated long-chain fatty acids inhibit the binding of oxidized low-density lipoproteins to a model CD36.

Transmembrane protein CD36 binds multiple ligands, including oxidized low-density lipoproteins (oxLDLs) and long-chain fatty acids (LCFAs). Our aim was to determine whether LCFAs compete with oxLDLs for binding to CD36. We addressed this issue by examining the inhibitory effect of LCFAs against the binding of Alexa-fluor-labeled oxLDLs (AFL-oxLDL) to a synthetic peptide representing the oxLDL-binding site on CD36 (3S-CD36150₋168). All of the unsaturated LCFAs tested, inhibited the binding of AFL-oxLDL to 3S-CD36150₋168, albeit to varying degrees. For instance, the concentrations required for 50% inhibition of binding for oleic, linoleic, and α-linolenic acids were 0.25, 0.97, and 1.2 mM, respectively. None of the saturated LCFAs tested (e.g. stearic acid) exhibited inhibitory effects. These results suggest that at least unsaturated LCFAs can compete with oxLDLs for binding to CD36. The study also provides information on the structural requirements of LCFAs for inhibition of oxLDLs-CD36 binding.

Behavioral palatability of dietary fatty acids correlates with the intracellular calcium ion levels induced by the fatty acids in GPR120-expressing cells.

We recently reported that G-protein-coupled receptor 120 (GPR120) is expressed on taste buds, and that rodents showed preference for long-chain fatty acids (LCFA) at a low concentration. We also showed that the LCFA (1% linoleic acid) increased the extracellular dopamine (DA) level in the nucleus accumbens (NAc), which participates in reward behavior. However, the mechanism underlying the involvement of the GPR120-agonistic activity of LCFA in the palatability of dietary fat remains elusive. Therefore, we examined the association between the GPR120-agonistic activity and palatability of LCFA. First, we measured Ca(2+) signaling in HEK293 cells stably expressing GPR120 under stimulation by various LCFAs. We then assessed the palatability of the various LCFAs by testing the licking behavior in mice and measured the changes in the NAc-DA level by in vivo microdialysis. Consequently, 14- to 22-carbon unsaturated LCFAs showed strong GPR120-agonistic activity. Additionally, mice displayed high licking response to unsaturated 16- and 18-carbon LCFAs, and unsaturated 18-carbon LCFA significantly increased the DA level. The licking rate and the LCFA-dependent increase in DA level also correlated well with the GPR120- agonistic activity. These findings demonstrate that chemoreception of LCFA by GPR120 is involved in the recognition and palatability of dietary fat.

Assessment of key amino-acid residues of CD36 in specific binding interaction with an oxidized low-density lipoprotein.

CD36 binds oxidized low-density lipoprotein (oxLDL). A synthetic peptide comprising amino-acid residues 149-168 of mouse CD36 was recently found to bind fluorescence-labeled oxLDL particles. Based on our oxLDL-binding analysis of various synthetic CD36 peptides, we suggest that not only hydrophilic residues (e.g., Lys164 and Lys166) but also hydrophobic ones (e.g., Phe153, Leu158, and Leu161) are critical to binding.

A synthetic peptide-based assay system for detecting binding between CD36 and an oxidized low-density lipoprotein.

CD36 is an integral membrane protein that mediates the cellular uptake of oxidized low-density lipoprotein (oxLDL) through recognition of the oxidized glycerophospholipids (oxPLs) formed during LDL oxidation. We aimed to devise an assay system to detect binding between CD36 and oxLDL/oxPL without using recombinant proteins. A peptide corresponding to amino-acid residues 149-168 of mouse CD36 with biotin at its N-terminus (named biotin-CD36(149-168)) and variants of it were synthesized and immobilized onto streptavidin-coated plates. oxLDL labeled with Alexa-Fluor-488 bound specifically and saturably to immobilized biotin-CD36(149-168), but poorly or not at all to the variants, such as that with a scrambled amino-acid sequence. The binding of fluorescence-labeled oxLDL to biotin-CD36(149-168) was inhibited efficiently by an oxPL species, but not by a nonoxidized glycerophospholipid. This assay system using biotin-CD36(149-168) provides a convenient means not only of characterizing binding profiles between CD36 and oxLDL/oxPL but also of finding competitors for the binding.

Dietary fat ingestion activates ß-endorphin neurons in the hypothalamus.

The opioid system regulates food choice, consumption, and reinforcement processes, especially for palatable meals such as fatty food. ß-Endorphin is known as an endogenous opioid peptide produced in neurons of the hypothalamus. In this study, we found that Intralipid (fat emulsion) ingestion increased c-fos expression in ß-endorphin neurons. However, intragastric infusion of Intralipid only slightly increased c-fos expression 2h after infusion. Further, dissection of glossopharyngeal nerve, innervating posterior tongue taste buds, partially but significantly decreased the Intralipid-induced c-fos expression. These results indicate that mainly the orosensory stimulation from fat may activate ß-endorphin neurons, thereby promoting ß-endorphin release.

Effect of an intraduodenal injection of fat on the activities of the adrenal efferent sympathetic nerve and the gastric efferent parasympathetic nerve in urethane-anesthetized rats.

Nutrient information from the gastrointestinal tract to the brain plays a critical role in the regulation of appetite and energy homeostasis. The autonomic nervous system controls the functions of several tissues to regulate the energy homeostasis of the whole body. Autonomic nerve activity is influenced by environmental or exogenous changes in even a single tissue. In the present study, we investigated the effect of an intraduodenal injection of fat on the activities of the autonomic nerves innervating the adrenal gland and stomach in urethane-anesthetized rats. An intraduodenal injection of corn oil suppressed adrenal efferent sympathetic nerve activity (ASNA) and stimulated gastric efferent vagal nerve activity (GVNA). A lipase inhibitor, epsilon-polylysine, coinjected with corn oil completely suppressed the corn oil-induced changes in ASNA and GVNA. Further, an intraduodenal injection of fatty acid (linoleic acid) moderately suppressed ASNA and significantly stimulated GVNA; these results indicate that fat may affect autonomic nerve activity partly through the chemoreception of free fatty acids (FFAs), which are produced during the hydrolysis of fat (corn oil) by a pancreatic lipase, in the intestinal lumen. Furthermore, an intraduodenal injection of an intravenous fat emulsion with the same pH and osmotic pressure as the body fluid affected ASNA and GVNA in a similar manner as corn oil. These results suggest that intraduodenal fat suppresses ASNA and stimulates GVNA partly via the chemoreception of FFAs-the degradation products of fats-in the intestinal lumen.

Intragastric infusion of glucose enhances the rewarding effect of sorbitol fatty acid ester ingestion as measured by conditioned place preference in mice.

We investigated substances that induce a rewarding effect during the postingestive process using the conditioned place preference (CPP) test. Although mice showed high affinity for a low-energy fat substitute--sorbitol fatty acid esters and low-concentration linoleic acid solution--they did not exhibit a place preference toward a voluntary intake of fat substitute in the CPP test. However, during a conditioning session of CPP that involved intragastric administration of corn oil immediately before the intake of the fat substitute, the test mice displayed a place preference. Similarly, intragastric administration of glucose, galactose, and dextrin also induced CPP; however, fructose, mannose, and a nonmetabolized carbohydrate did not. These results suggest that administration of corn oil and glucose has the same postingestive effect with regard to inducing CPP and that the structural specificity of carbohydrates influences the postingestive effect.

Assessing palatability of long-chain fatty acids from the licking behavior of BALB/c mice.

Dietary oils such as corn oil, olive oil, and canola oil, which primarily contain triacylglycerol and small quantities of fatty acids, are highly palatable to animals. In a previous study, we examined the short-term (60 s) licking behavior of mice and observed that they exhibited a high licking response to a low concentration of fatty acid (linoleic acid), which is comparable to that observed for pure corn oil. This finding suggests that fatty acids contribute to the palatability of dietary oils. In order to supplement our knowledge of the fundamental features of fatty acid palatability in the oral cavity, we assessed the licking behavior of BALB/c mice to investigate the palatability of various types of long-chain fatty acids. The mice showed high licking responses to 1% unsaturated 16- and 18-carbon fatty acids (palmitoleic acid, 16:1; oleic acid, 18:1; linoleic acid, 18:2; and linolenic acid, 18:3), low licking responses to 16- and 20-carbon fatty acids (palmitic acid, 16:0 and arachidonic acid, 20:4), and no significant response to saturated fatty acids (stearic acid, 18:0 and arachidic acid, 20:0) or fatty acid derivatives (methyl linoleate and linole alcohol). Additionally, there were differences in the palatability of 18-carbon unsaturated fatty acids at very low concentrations. At fatty acid concentrations of 0.04% and 0.0625%, the mice showed significant preference for linoleic acid and linolenic acid, but not oleic acid, when compared with mineral oil. These results suggest that mice show high licking responses to 16- and 18-carbon unsaturated long-chain fatty acids at low concentrations. Further, we suggest that sensitivity to fatty acids is affected by the saturated state of the fatty acid, carbon chain length, and terminal carboxyl group.

Preference for dietary fat induced by release of beta-endorphin in rats.

AIMS: To determine whether beta-endorphin contributes to the ingestion of and preference for dietary oil, we examined the relationship between the dynamics of beta-endorphin, before and after the ingestion of corn oil, and the intake volume of corn oil. MAIN METHODS: Rats were offered 5% corn oil for 20 min for 5 consecutive days so they could acquire a preference for corn oil. On day 6, seven groups of rats were presented with the oil for defined time periods, and we measured the beta-endorphin levels in the serum and cerebrospinal fluid (CSF) before and after the presentation of corn oil as well as the consumed volume of corn oil at defined time points. KEY FINDINGS: Beta-endorphin levels in serum and CSF were significantly increased 15 min after the ingestion of corn oil, followed by a rapid decrease and maintenance at the basal level throughout the rest of the experimental period. The intake of corn oil was the lowest in the time period of 15-30 min, when the beta-endorphin level reached a peak value. The intake volume of corn oil might be inversely correlated with beta-endorphin levels in serum and CSF. The pretreatment of naloxone, an antagonist of the opioid receptor, decreased the initial licking rate for corn oil and increased the latency for corn oil in the licking test. SIGNIFICANCE: The beta-endorphin was rapidly released after oil ingestion, which contributed to the hedonic preference and ingestive behavior for fat.

Colocalization of GPR120 with phospholipase-Cbeta2 and alpha-gustducin in the taste bud cells in mice.

A recent study has demonstrated that the G-protein coupled receptor GPR120 is expressed in the taste bud cells in rats. In this study, we have identified the types of taste cell that express GPR120 in C57/BL6 mice. Double immunostaining for GPR120 and the markers of type II taste cells (phospholipase-Cbeta2 and alpha-gustducin) revealed that the majority of the GPR120-positive taste cells are type II taste cells. In contrast, it was observed that GPR120 was rarely colocalized with the marker of type III cells (neuronal cell adhesion molecule). These results suggested that GPR120 is mainly expressed in the type II taste cells and might function as a sensor for dietary fat.

Contribution of gustation to the palatability of linoleic acid.

We investigated the palatability of a low concentration of linoleic acid (LA) in short-term two-bottle choice tests and licking tests. To examine the contribution of gustation, mice were rendered anosmic with olfactory nerve transection surgery and test solutions were prepared using mineral oil (saturated long-chain hydrocarbon) to minimize textural effects. In the two-bottle choice tests between various pairs of different concentrations of corn oil and LA, both anosmic and the sham-operated mice constantly preferred a higher concentration of corn oil and LA. In the licking tests, the initial licking rate for 1% LA was higher than that for mineral oil in anosmic mice. In accordance with the results of the two-bottle choice test, the initial licking rate for corn oil and LA increased in a concentration-dependent manner in both anosmic and sham-operated mice in the licking test, and reached its peak at 100% corn oil and 1% LA. A preference comparison between 1% LA and 100% corn oil showed that anosmic mice preferred 1% LA over 100% corn oil. These results suggest that mice could recognize dietary fat and fatty acid solutions in the oral cavity without any olfactory or textural cues and the fatty acid recognition on their tongues might provide a pivotal cue to how dietary fat is recognized in the oral cavity.

Mercaptoacetate inhibition of fatty acid beta-oxidation attenuates the oral acceptance of fat in BALB/c mice.

We investigated the effect of beta-oxidation inhibition on the fat ingestive behavior of BALB/c mice. Intraperitoneal administration to mice of mercaptoacetate, an inhibitor of fatty acid oxidation, significantly suppressed intake of corn oil but not intake of sucrose solution or laboratory chow. To further examine the effect of mercaptoacetate on the acceptability of corn oil in the oral cavity, we examined short-term licking behavior. Mercaptoacetate significantly and specifically decreased the number of licks of corn oil within a 60-s period but did not affect those of a sucrose solution, a monosodium glutamate solution, or mineral oil. In contrast, the administration of 2-deoxyglucose, an inhibitor of glucose metabolism, did not affect the intake or short-term licking counts of any of the tasted solutions. These findings suggest that fat metabolism is involved in the mechanism underlying the oral acceptance of fat as an energy source.

Zerumbone suppresses phorbol ester-induced expression of multiple scavenger receptor genes in THP-1 human monocytic cells.

Unregulated uptake of oxidized low-density lipoproteins (ox-LDL) via macrophage scavenger receptors (SRs), such as lectin-like ox-LDL receptor-1 (LOX-1), is a key event in atherosclerosis. In the present study, we used differentiated Caco-2 cells as a model of the human small intestine to evaluate the suppressive effects of 16 traditional food items selected from Okinawa on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced LOX-1 mRNA expression in THP-1 human monocyte-like cells. Three Zingiberaceae plants, Curcuma aromatica Salisbury, Curcuma longa L., and Zingiber zerumbet Smith, markedly suppressed that expression. When added to the apical sides of Caco-2 monolayers, zerumbone, a sesquiterpene from Z. zerumbet Smith, was found to permeate into the basolateral medium as an intact structure in a time-dependent manner. alpha-Humulene, a structural analog of zerumbone lacking the alpha,beta-unsaturated carbonyl group, did not suppress LOX-1 mRNA expression, indicating that its electrophilic moiety might play pivotal roles in its activities. Further, zerumbone attenuated the expression of SR-A, SR-PSOX, and CD36, but not that of CD68 or CLA-1, leading to a blockade of DiI-acLDL uptake, while it also inhibited the transcriptional activities of activator protein-1 and nuclear factor-kappaB. Together, our results indicate that zerumbone is a potential phytochemical for regulating atherosclerosis with reasonable action mechanisms.

Suppressive effects of demethylated metabolites of nobiletin on phorbol ester-induced expression of scavenger receptor genes in THP-1 human monocytic cells.

Unregulated uptake of oxidized low-density lipoproteins (ox-LDL) via macrophage scavenger receptors (SRs) is a key event in atherosclerosis. We previously reported that nobiletin (NOB), a citrus polymethoxylated flavone, markedly reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced SRs and adhesion molecules mRNA expression and blockade of DiI-acLDL uptake in THP-1 human monocyte-like cells. In this study, we examined the effects of NOB metabolites, 3'-hydroxy-5,6,7,8,4'-pentamethoxyflavone (3'-demethyl-NOB), 4'-hydroxy-5,6,7,8,3'-pentamethoxyflavone (4'-demethyl-NOB) and 3', 4'-dihydroxy-5,6,7,8,-tetramethoxyflavone (3', 4'-didemethyl-NOB) and NOB analog, tangeretin, on SRs and adhesion molecules mRNA expression. 3'-Demethyl-NOB significantly suppressed CD36 expression, moreover, 4'-demethyl- and 3', 4'-didemethyl-NOB significantly suppressed TPA-induced expression of SR-A and LOX-1. Further, the suppressive effects of 4'-demethyl- and 3', 4'-didemethyl-NOB on the expression of CD36 mRNA were greater extent than parent NOB. The inhibitory effects of the metabolites toward TPA-induced SR mRNA expression are partly associated with the suppression of AP-1 and NF-kappaB transcriptional activities. Together, our results suggest that metabolites of NOB, such as 4'-demethyl- and 3', 4'-didemethyl-NOB, have comparable or higher potentials to attenuate SR expression than NOB.

Nobiletin, a citrus flavonoid, suppresses phorbol ester-induced expression of multiple scavenger receptor genes in THP-1 human monocytic cells.

Unregulated uptake of oxidized low-density lipoproteins (ox-LDL) via macrophage scavenger receptors (SRs) such as lectin-like ox-LDL receptor-1 (LOX-1) is a key event in atherosclerosis. In this study, we examined the effects of five selected food phytochemicals on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced LOX-1 mRNA expression in THP-1 human monocyte-like cells. Nobiletin, a citrus polymethoxylated flavone, markedly reduced it in dose- and time-dependent manners. It also suppressed the phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK) 1/2, and c-Jun (Ser-63), thereby inhibiting the transcriptional activity of activator protein-1. Further nobiletin attenuated expression of SR-A, SR-PSOX, CD36, and CD68, but not CLA-1, mRNA, leading to the blockade of DiI-acLDL uptake. Together, our results suggest that nobiletin is a promising phytochemical for regulating atherosclerosis with reasonable action mechanisms.