Medium-chain triglyceride-specific appetite is regulated by the ß-oxidation of medium-chain fatty acids in the liver.

Most studies on fat appetite have focused on long-chain triglycerides (LCTs) due to their obesogenic properties. Medium-chain triglycerides (MCTs), conversely, exhibit antiobesogenic effects; however, the regulation of MCT intake remains elusive. Here, we demonstrate that mice can distinguish between MCTs and LCTs, and the specific appetite for MCTs is governed by hepatic ß-oxidation. We generated liver-specific medium-chain acyl-CoA dehydrogenase (MCAD)-deficient (MCADL-/-) mice and analyzed their preference for MCT and LCT solutions using glyceryl trioctanoate (C8-TG), glyceryl tridecanoate (C10-TG), corn oil, and lard oil in two-bottle choice tests conducted over 8 days. In addition, we used lick microstructure analyses to evaluate the palatability and appetite for MCT and LCT solutions. Finally, we measured the expression levels of genes associated with fat ingestion (Galanin, Qrfp, and Nmu) in the hypothalamus 2 h after oral gavage of fat. Compared with control mice, MCADL-/- mice exhibited a significantly reduced preference for MCT solutions, with no alteration in the preference for LCTs. Lick analysis revealed that MCADL-/- mice displayed a significantly decreased appetite for MCT solutions only while the palatability of both MCT and LCT solutions remained unaffected. Hypothalamic Galanin expression in control mice was elevated by oral gavage of C8-TG but not by LCTs, and this response was abrogated in MCADL-/- mice. In summary, our data suggest that hepatic ß-oxidation is required for MCT-specific appetite but not for LCT-specific appetite. The induction of hypothalamic galanin upon MCT ingestion, dependent on hepatic ß-oxidation, could be involved in the regulation of MCT-specific appetite.NEW & NOTEWORTHY Whether and how medium-chain triglyceride (MCT) intake is regulated remains unknown. Here, we showed that mice can discriminate between MCTs and LCTs. Hepatic ß-oxidation participates in MCT-specific appetite, and hypothalamic galanin may be one of the factors that regulate MCT intake. Because of the antiobesity effects of MCTs, studying MCT-specific appetite may help combat obesity by promoting the intake of MCTs instead of LCTs.

Application of a novel fluorescence intensity assay: identification of distinct fatty acetates as volatile compounds that bind specifically to amino acid region 149-168 of a transmembrane receptor CD36.

The cluster of differentiation 36 (CD36) is a transmembrane receptor expressed in various cells and has diverse lipid ligands. The expression of CD36 in the murine olfactory epithelium and its ability to recognize certain species of fatty aldehydes, a class of odor-active volatile compounds, have suggested a role for this receptor in the capture of specific odorants in the nasal cavity of mammals. However, the spectrum of CD36-recognizable volatile compounds is poorly understood. In this study, we employed our recently devised assay with fluorescently labeled peptides as probes (fluorescence intensity assay) and identified distinct fatty acetates as volatile compounds that bind specifically to amino acid region 149-168 of CD36 (eg dodecyl and tetradecyl acetates). The present findings demonstrate the utility of our assay for the discovery of novel CD36 ligands and support the notion that the receptor functions as a captor of volatile compounds in the mammalian olfactory system.

A single aldehyde group can serve as a structural element for recognition by transmembrane protein CD36.

Transmembrane protein CD36 is considered to bind its distinct ligands such as long-chain fatty acids primarily by recognizing their terminal carboxyl moiety. In this study, we provide evidence that long-chain fatty aldehydes, such as oleic aldehyde, can be recognized by CD36. We suggest that a single aldehyde group may also serve as one of the structural elements recognizable by CD36.

A matter of symmetry: terahertz polarization detection properties of a multi-contact photoconductive antenna evaluated by a response matrix analysis.

While terahertz time domain spectroscopy (THz-TDS) is a well-established technique, polarization sensitive measurements are challenging due to the need of broadband polarization devices. Here, we characterize our recently introduced multi-contact photoconductive detector antenna with a response matrix analysis. We show that the lead lines attached to electrodes reduce the antenna symmetry and thereby influence the properties of the response matrices. With a wire grid polarizer, we simulate a sample influencing the polarization angle and the intensity of the incident THz pulse. Evaluating the measurements with the response matrix analysis, our results show a well agreement of the adjusted and measured polarization angles and intensities over a frequency range from 0.25 to 0.8 THz.

The opioid system majorly contributes to preference for fat emulsions but not sucrose solutions in mice.

Rodents show a stronger preference for fat than sucrose, even if their diet is isocaloric. This implies that the preference mechanisms for fat and sucrose differ. To compare the contribution of the opioid system to the preference of fat and sucrose, we examined the effects of mu-, delta-, kappa-, and non-selective opioid receptor antagonists on the preference of sucrose and fat, assessed by a two-bottle choice test and a licking test, in mice naïve to sucrose and fat ingestion. Administration of non-selective and mu-selective opioid receptor antagonists more strongly inhibited the preference of fat than sucrose. While the preference of fat was reduced to the same level as water by the antagonist administration that of sucrose was still greater than water. Our results suggest that the preference of fat relies strongly on the opioid system, while that of sucrose is regulated by other mechanisms in addition to the opioid system.

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.

Binding Interaction Between Lauric Acid and Cluster of Differentiation 36 Underpinned by a Fluorescence- Intensifying Assay.

Cluster of differentiation 36 (CD36) is a scavenger receptor expressed in various vertebrate cells that contains diverse ligands, including long-chain fatty acids. This receptor has recently been suggested as a captor of specific volatile odorants (e.g., aliphatic acetates) in the mammalian nasal epithelium. This study used a fluorescence-intensifying assay to produce the first evidence that lauric acid, an odorous fatty acid, directly binds to CD36. This expansion of the repertoire of volatile ligands supports potential applications for nasal CD36. Our present findings could promote future research aimed at understanding the mechanisms of fatty acid interactions with CD36.

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.

Increased levels of extracellular dopamine in the nucleus accumbens and amygdala of rats by ingesting a low concentration of a long-chain Fatty Acid.

Changes in the extracellular concentration of dopamine (DA) in the nucleus accumbens (NAc) shell and the basolateral amygdala (BLA) resulting from the voluntary ingestion of either corn oil, mineral oil, or 1% linoleic acid diluted with mineral oil as a vehicle were measured in rats by using in vivo microdialysis after they had been trained to establish a preference for corn oil. Ingesting the mineral oil caused no significant change in DA level in the NAc shell, whereas corn oil ingestion significantly increased the DA level during 0-15 min of the test session, reaching the maximum level of 129.8 ± 6.2% compared with the baseline after 10 min. Ingesting linoleic acid also resulted in a significant increase in DA level during 0-20 min, reaching 125.9 ± 9.0% after 10 min. Similar results were obtained in the BLA. Despite its very low calorie content, a low concentration of non-esterified fatty acid increased the DA levels equivalent to those resulting from corn oil in the brain's reward system.

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.

A point of view on human fat olfaction - do fatty derivatives serve as cues for awareness of dietary fats?

Fat (triglycerides) consumption is critical for the survival of animals, including humans. Being able to smell fat can be advantageous in judging food value. However, fat has poor volatility; thus, olfaction of fat seems impossible. What about fatty acids that comprise fat? Humans smell and discriminate medium-chain fatty acids. However, no conclusive evidence has been provided for the olfactory sense of long-chain fatty acids, including essential acids such as linoleic acid (LA). Instead, humans likely perceive the presence of essential fatty acids through the olfaction of volatile compounds generated by their oxidative breakdown (e.g., hexanal and γ-decalactone). For some people, such scents are pleasing, especially when they come from fruit. Nonetheless, it remains unclear whether the olfaction of these volatiles leads to the recognition of fat per se. Nowadays, people often smell LA-borne aldehydes such as E,E-2,4-decadienal that occur appreciably, for example, from edible oils during deep frying, and are pronely captivated by their characteristic "fatty" note, which can be considered a "pseudo-perception" of fat. However, our preference for such LA-borne aldehyde odors may be a potential cause behind the modern overdose of n-6 fatty acids. This review aims to provide a view of whether and, if any, how we olfactorily perceive dietary fats and raises future purposes related to human fat olfaction, such as investigating sub-olfactory systems for detecting long-chain fatty acids.

Identification of the matriptase second CUB domain as the secondary site for interaction with hepatocyte growth factor activator inhibitor type-1.

Matriptase is a type II transmembrane serine protease comprising 855 amino acid residues. The extracellular region of matriptase comprises a noncatalytic stem domain (containing two tandem repeats of complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein (CUB) domain) and a catalytic serine protease domain. The stem domain of matriptase contains site(s) for facilitating the interaction of this protease with the endogenous inhibitor, hepatocyte growth factor activator inhibitor type-1 (HAI-1). The present study aimed to identify these site(s). Analyses using a secreted variant of recombinant matriptase comprising the entire extracellular domain (MAT), its truncated variants, and a recombinant HAI-1 variant with an entire extracellular domain (HAI-1-58K) revealed that the second CUB domain (CUB domain II, Cys(340)-Pro(452)) likely contains the site(s) of interest. We also found that MAT undergoes cleavage between Lys(379) and Val(380) within CUB domain II and that the C-terminal residues after Val(380) are responsible for facilitating the interaction with HAI-1-58K. A synthetic peptide corresponding to Val(380)-Asp(390) markedly increased the matriptase-inhibiting activity of HAI-1-58K, whereas the peptides corresponding to Val(380)-Val(389) and Phe(382)-Asp(390) had no effect. HAI-1-58K precipitated with immobilized streptavidin resins to which a synthetic peptide Val(380)-Pro(392) with a biotinylated lysine residue at its C terminus was bound, suggesting direct interaction between CUB domain II and HAI-1. These results led to the identification of the matriptase CUB domain II, which facilitates the primary inhibitory interaction between this protease and HAI-1.

The role of asparagine-linked glycosylation site on the catalytic domain of matriptase in its zymogen activation.

Matriptase is a type II transmembrane serine protease containing one potential site for asparagine-linked glycosylation (N-glycosylation) on the catalytic domain (Asn772). It has been found that the activation of matriptase zymogen occurs via a mechanism requiring its own activity and that the N-glycosylation site is critical for the activation. The present study aimed to determine the underlying reasons for the site requirement using Madin-Darby canine kidney cells stably expressing recombinant variants of rat matriptase. A full-length variant with glutamine substitution at Asn772 appeared to be unable to undergo activation because of its catalytic incompetence (i.e., decreased availability of the soluble catalytic domain and/or of the correctly folded domain). This was evidenced by the observations that (i) a recombinant catalytic domain of matriptase with glutamine substitution at the site corresponding to matriptase Asn772 [N772Q-CD-Myc(His)(6)] was not detected in the medium conditioned by transfected cells but was on the cell surface and (ii) purified N772Q-CD-Myc(His)(6) exhibited markedly reduced activity toward a peptide substrate. It is concluded that N-glycosylation site at Asn772 of matriptase is required for the zymogen activation because it plays an important role in rendering this protease catalytically competent in the cellular environment.

Assessment of direct binding interaction between CD36 and its potential lipid ligands using a peptide mimic of the receptor labeled with a fluorophore.

Cluster of differentiation 36 (CD36) is a cell-surface receptor that recognizes diverse substances. We have presented indirect evidence that a short segment of the receptor comprising amino acids 149-168 contains a site for binding of its lipid ligands (e.g., distinct fatty acids and aldehydes). However, experimental support for their direct interactions is yet to be achieved. For this, we devised a fluorescence intensity assay, where a synthetic peptide consisting of CD36 amino acids 149-168 labeled with fluorescein isothiocyanate (FITC-CD36149-168) and its variant peptides were used as positive and negative probes, respectively. First, we obtained results indicating that 1-palmitoyl-2-(5-keto-6-octenedioyl)phosphatidylcholine (an established CD36 ligand) but not 1-palmitoyl-2-arachidonyl-phosphatidylcholine (a non-ligand of the receptor) bound in a saturable and specific manner to FITC-CD36149-168. Strikingly, the assay allowed us to provide the first evidence supporting direct and specific binding between the CD36 segment and fatty aldehydes (e.g., Z-11-hexadecenal). However, this method failed to illustrate specific interactions of the segment with fatty acids, such as oleic acid. Nonetheless, our findings offer further insight into the biologically relevant ligands and the role of CD36. In addition, we suggest that this fluorescence-based technique provides a convenient means to evaluate protein (peptide)-lipid interactions.

Matriptase does not require hepatocyte growth factor activator inhibitor type-1 for activation in an epithelial cell expression model.

Matriptase is a type II transmembrane serine protease. Paradoxically, activation of this protease is thought to require its physiological inhibitor, hepatocyte growth factor activator inhibitor type-1 (HAI-1). In the present study, however, we obtained evidence in a stable transfection experiment using Madin-Darby canine kidney cells that matriptase activation does not require HAI-1.

The structural requirements of matriptase in its ectodomain release in polarized epithelial cells.

Matriptase is a type-II transmembrane serine protease abundantly expressed in polarized epithelia. The ectodomain of matriptase is released from the cell surface. In the present study, we found that the post-translational cleavage between Gly149 and Ser150 and the existence of catalytic domain are critical for the ectodomain release of matriptase in stable transfection experiments using the polarized Madin-Darby canine kidney epithelial cell line.

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.

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.

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.