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Apical CD36 immunolocalization in human and porcine taste buds from circumvallate and foliate papillae
Acta Histochemica 113 (2011) 839–843
Contents lists available at ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Short communication
Apical CD36 immunolocalization in human and porcine taste buds from circumvallate and foliate papillae Peter J. Simons a,∗ , J. Alain Kummer b,c , Joost J.F.P. Luiken d , Louis Boon a a
Department of Cell Biology, Bioceros BV, Yalelaan 46, 3584 CM Utrecht, The Netherlands Department of Pathology, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Department of Pathology, St. Antonius Hospital, Koekoekslaan 1, 3534 CM Nieuwegein, The Netherlands d Department of Molecular Genetics, Maastricht University, Universiteitssingel 50, 6200 MD Maastricht, The Netherlands b c
a r t i c l e
i n f o
Article history: Received 14 August 2010 Accepted 27 August 2010
Keywords: CD36 Orosensory receptor Long chain fatty acids Taste buds Dietary fat preference Obesity Human
a b s t r a c t CD36 is the receptor for long chain fatty acids (LCFA), and is expressed in lingual taste cells from rodents. In these animals, CD36 has been proposed to play an important role in oral detection of LCFA, and subsequently, determines their dietary fat preference. Humans also seem to detect LCFA in the oral cavity, however, information on the molecular mechanism of this human orosensory LCFA recognition is currently lacking. The aim of our study was to investigate whether CD36 is also expressed in lingual human and porcine taste buds cells. Using fluorescence immunohistochemistry, apical CD36 expression was revealed in human and porcine taste bud cells from circumvallate and foliate papillae. These data suggest CD36 as the putative orosensory receptor for dietary LCFA in human, and, therefore, may be involved in our preference for fatty foods. © 2010 Elsevier GmbH. All rights reserved.
Introduction CD36 or fatty acid translocase is an integral membrane ditopic glycoprotein, and its amino acid sequence predicts a large hydrophobic pocket between two short cytoplasmic tails (Greenwalt et al., 1992). The C-terminal cytoplasmic tail has been shown to be associated with Src kinase family members, suggesting an involvement of CD36 in cell signaling. CD36 is expressed on numerous cell types (e.g. macrophages, enterocytes, hepatocytes, endothelial cells, myocytes, adipocytes, and platelets), and is described to be a receptor for multiple ligands including long chain fatty acids (LCFA), high-density lipoprotein, oxidized low-density lipoprotein, glycated collagen type IV, thrombospondin-1, fibrillar -amyloid, advanced glycated end products, chondroitin sulfate, apoptotic cells, and Plasmodium falciparum-infected erythrocytes (Husemann et al., 2002). Recent investigations (Fukuwatari et al., 1997; Zhang et al., 2003; Laugerette et al., 2005) have illustrated that lingual rat and mouse taste bud cells also express CD36 at the apical side. Furthermore, Laugerette et al. (2005) elegantly showed this CD36 as a possible oral sensor for LCFA in mice. They and others (Sclafani
∗ Corresponding author. E-mail address: [email protected] (P.J. Simons). 0065-1281/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2010.08.006
et al., 2007) demonstrated that CD36+/+ wild type mice consumed significantly more lipid-enriched (LCFA or triglycerides) solutions and solid diets than CD36−/− null mice. However, consumption of sucrose and aversion for quinine remained similar in both mouse models. Such a fat preference is not species-specific since it was already demonstrated in rats (Tsuruta et al., 1999). Laugerette et al. (2005) also showed that pancreatobiliary excretions were enhanced in esophagus-ligated rats and mice after deposition of purified LCFA onto the tongue. In addition, LCFA-mediated induction of pancreatobiliary excretions found in CD36+/+ wild-type mice was fully abolished in CD36−/− null mice. The above described data suggest that rodent CD36-expressing lingual taste cells could interact with – possibly dietary – LCFA, which in turn directs the preference for lipid-rich foods and the cephalic phase of digestive excretions. In Western society, with an access to a diet of virtually any composition and a free-choice condition, humans consume approximately 40% of food energy as fat (Mela, 1995). Moreover, obese individuals seem to exhibit a higher dietary fat preference compared to lean individuals (Drewnowski et al., 1985; Mela and Sacchetti, 1991). In humans, oral lipid-rich food stimulation – without food swallowing and smelling – rapidly enhances serum triglyceride levels, implicating incoming oral fat in the release of stored fat (Mattes, 2001a,b, 2002). Noteworthy, fatty foods may consist of >0.5% free FA (Smith et al., 1986). Humans also show
840
P.J. Simons et al. / Acta Histochemica 113 (2011) 839–843
The specimens from post-mortem human tongue, which were dissected within 8 h from three subjects (a 78 year-old female, an 84 year-old male, and a male of unknown age), containing circumvallate and foliate papillae, were obtained from a commercial tissue bank (Analytical Biological Services Inc., Wilmington, USA) and from the Department of Pathology/University Medical Centre Utrecht Biobank, and used in accordance with the hospital scientific committee regulations and the code “Proper Secondary Use of Human Tissue” as installed by the Federation of Biomedical Scientific Societies (www.federa.org). Immunohistochemistry The specimens of tongue were embedded in OCT medium (Sakura Finetek Europe BV, Zoeterwoude, The Netherlands), snapfrozen in liquid nitrogen, and stored at −150 ◦ C. Serial 9–10 m cryosections were cut and mounted onto slides, air dried, and fixed in acetone for 10 min at room temperature (RT). The slides were then incubated with:
Fig. 1. Representative light microscopic images of hematoxylin/eosin-stained human (A) and porcine (B) taste buds in circumvallate papillae. Taste buds (TB; white dashed-lined ovals) are embedded in a non-sensory squamous epithelial (NSE) layer. White triangles indicate apical taste pore areas, and C indicates the cleft in the oral cavity. Note: A few pycnotic nuclei were observed in human taste buds suggesting some degree of autolysis. Scale bar = 20 m.
the ability to detect trace amounts of purified LCFA on the tongue (Chalé-Rush et al., 2007). These observations suggest the existence of a human orosensory LCFA detection system, which might be similar to that described in rodents. However, the role of oral CD36 as a putative LCFA receptor in humans is currently not known. In this study, we immunohistochemically examined the expression of CD36 in lingual taste buds from human circumvallate and foliate papillae. Taste buds from porcine tongue were also investigated, because of the pig’s (Sus scrofa) high genomic homology with human. Furthermore, pigs have been described as a major mammalian model for human biomedical studies because of their similarity in physiology, in organ development and disease progression. Our data clearly demonstrate that the putative LCFArecognizing CD36 receptor is also apically expressed in human and porcine taste bud cells from lingual gustatory papillae, suggesting its role in orosensory perception of dietary lipids in mammals. Materials and methods
A. CD36 expression: 10 g/mL mouse anti-human CD36 monoclonal antibody (Mab) clone CB38, also known as clone NL07, (isotype mouse IgM; BD Biosciences, Alphen aan den Rijn, The Netherlands) in PBS/1% BSA (Sigma–Aldrich, Zwijndrecht, The Netherlands) for 1.5 h at RT. To corroborate the specificity of CD36 staining in human taste buds, 10 g/mL (≈11 nM) anti-CD36 Mab clone CB38/NL07 was adsorbed in an excess of 50 g/mL (≈342 nM) soluble recombinant human CD36:Fc chimeric protein (R&D Systems, Minneapolis, MN, USA) in PBS/1% BSA during 30 min at RT prior to incubation on slides. 10 g/mL mouse IgM isotype control Mab clone G155-228 (BD Biosciences) was run in parallel as a negative control. After incubation, cryosections were gently rinsed in PBS and incubated with 1:200 diluted Alexa Fluor® 488-labelled goat anti-mouse IgM antibodies (Invitrogen Ltd., Paisley, UK) in PBS/1% BSA for 1 h at RT. B. Alpha-gustducin and NCAM/CD56 expression: 2 g/mL affinitypurified rabbit IgG anti-␣-gustducin I-20 polyclonal antibody (directed against a peptide corresponding to amino acids 93–112 mapping within a highly divergent domain of rat ␣-gustducin, and known to cross-react with mouse, hamster and human ␣-gustducin (Boughter et al., 1997; Santa Cruz Biotechnology, Santa Cruz, USA) or 10 g/mL mouse anti-human NCAM/CD56 Mab clone B159 (isotype mouse IgG1; BD Biosciences) in PBS/1% BSA for 1.5 h at RT. Respectively, anti-␣-gustducin polyclonal antibody was omitted, or 10 g/mL mouse IgG1 isotype control Mab clone MOPC-21 (BD Biosciences) were run in parallel as negative controls. After incubation, cryosections were gently rinsed in PBS and incubated with 1:200 diluted phycoerythrinconjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) or with 1:200 phycoerythrin-conjugated goat anti-mouse IgG antibodies (Jackson ImmunoResearch) in PBS/1% BSA for 1 h at RT. After another rinse step in PBS, all cryosections were coverslipped with ProLong® Gold anti-fade reagent (Invitrogen) prior to microscopic examination.
Tongue tissue specimens Results and discussion Necropsy normal porcine tongue tissue specimens, which were dissected within 15 min from two 24 weeks-old specific pathogenfree female crossbred Great Yorkshire/Large White × Dutch Landrace pigs (weighing around 120 kg), containing circumvallate and foliate papillae, were obtained from Van Beek SPF Varkens BV, Lelystad, The Netherlands.
CD36 expression in human taste buds Human taste buds are onion-shaped organs embedded in the full thickness of the non-sensory stratified squamous epithelium of the tongue, and are easily recognized using routine
P.J. Simons et al. / Acta Histochemica 113 (2011) 839–843
841
Fig. 2. Apical CD36 expression in human taste bud cells from circumvallate and foliate papillae. (A) Fluorescence microscopic image of a circumvallate papilla after incubation with soluble human CD36:Fc chimeric protein pre-adsorbed anti-human CD36 Mab. (B) CD36 expression in taste bud cells from circumvallate papilla (same area as section A) after incubation with anti-human CD36 Mab. (C) Image of a foliate papilla after incubation with soluble human CD36:Fc chimeric protein pre-adsorbed anti-human CD36 Mab. (D) CD36 expression in taste bud cells from foliate papilla (same area as section C) after incubation with anti-human CD36 Mab. White dashed-lined ovals indicate the location of taste buds embedded in the epithelial layer, white triangles indicate apical taste pore area, and asterisks indicate papillae clefts. Scale bar = 20 m.
histological staining methods (see Fig. 1A) or phase contrast microscopy. To determine whether these human taste buds are expressing CD36, an immunohistochemical analysis was performed using the anti-CD36 specific Mab, clone CB38/NL07. This Mab was originally raised against human CD36-expressing platelet extracts (Alessio et al., 1991), and its specificity has been extensively characterized (Gruarin et al., 2000a,b). These investigations demonstrated that clone CB38/NL07 recognizes a conformational epitope on mature human CD36 that is acquired only during the late steps of intracellular transport (Golgi apparatus) and cell surface expression. As shown in Fig. 2B and D, human taste bud cells from both circumvallate and foliate gustatory papillae clearly exhibited mature CD36 expression, typically at the apical side. Despite the fact that tested tissue specimens from all examined donors showed autolytic signs (Fig. 1A) – post-mortem cryopreservation was performed within 8 h – virtually all taste buds demonstrated CD36 positive staining. However, CD36 expression levels varied from one taste bud to the other, most likely because spherical taste buds were cut at different levels. In some cases, the taste pore or pit area showed an extremely high density of CD36 molecules, which indicated CD36 expression on taste cell’s microvilli protruding into the oral cavity/cleft. This CD36 expression was specific in human taste buds, since no staining was observed after the anti-CD36 Mab clone CB38/NL07 was pre-adsorbed with soluble recombinant CD36 proteins (Fig. 2A and C). In addition, a negative isotype control antibody was devoid of any staining (data not shown) in human taste buds cells. In an attempt to delineate the subtype(s) of CD36 expressing human taste bud cells, we also examined ␣-gustducin (marker for type II taste cells) and NCAM/CD56 (marker for type III taste cells) expression (Finger, 2005). Unfortunately, and in contrast to above-described CD36 expression, specific antibodies against ␣-gustducin and NCAM/CD56 failed to show any immunoreactivity in human taste bud cells (data not shown). We suspect degradation of ␣-gustducin and NCAM/CD56 proteins in our
autopsy tissue specimens, because the antibodies against these proteins did show immunoreactivity in the human Caco-2 cell line obtained from American Type Culture Collection (Le Gall et al., 2007) and in human peripheral blood natural killer cells respectively (data not shown). It has been previously shown that CD36 proteins degrade slowly and maintain immunoreactive in multiple human tissue specimens during a 24-h post-mortem interval (Maleszewski et al., 2007). Finally, non-sensory stratified squamous epithelial cells (Fig. 2B and D) and non-gustarory filliform papillae (data not shown) demonstrated no CD36 expression. Taste buds from fungiform papillae were not included in our study. CD36 expression in porcine taste buds As found with human taste buds, porcine taste bud cells also expressed mature CD36, most remarkably at the apical side (Fig. 3). Anti-human CD36 Mab clone CB38/NL07 has been previously described to cross-react with porcine CD36 (Delgado et al., 2003). As in human taste buds, CD36 staining intensity was also variable in these ‘fresh’ (i.e. 15 min after death) porcine taste buds. CD36 expression was discerned in almost all taste buds embedded in epithelial layers of circumvallate and foliate papillae obtained of pigs. Occasionally, the taste pore area exhibited very intense CD36 staining, which suggested CD36 expression on microvilli of taste cells. No staining was observed (data not shown), when porcine taste buds cells were exposed to isotype negative control antibodies. Lingual CD36 as the putative oral LCFA-sensing receptor in humans In our view, the recent discovery of CD36 in lingual taste bud cells in inbred and outbred rodents, together with CD36 expression in lingual taste bud cells from unrelated humans and crossbred pigs in our Short Communication, substantiates the idea that CD36
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variations have been identified in humans (Rac´ et al., 2007). In attempt to address the global fight against obesity and overweight, it will be challenging to examine whether these CD36 genetic variations influence our ability to sense dietary LCFA, and subsequently, our fatty food preference.
Acknowledgements The authors thank Mr. Martijn van Osch and Mr. Willem van Wolferen for their excellent technical assistance, Mr. Rob Bleumink for use of cryomicrotome, fluorescence microscope and digital image software, and Prof. Dr. Jan Glatz for fruitful discussions.
References
Fig. 3. Apical CD36 expression in porcine taste bud cells from circumvallate and foliate papillae. (A) CD36 expression in taste bud cells from a circumvallate papilla after incubation with anti-human CD36 Mab. (B) CD36 expression in taste bud cells from a foliate papilla after incubation with anti-human CD36 Mab. White dashedlined ovals indicate the location of taste buds embedded in the epithelial layer, white triangles indicate apical taste pore area, and asterisks indicate papillae clefts. Scale bar = 20 m.
might function as a putative orosensory receptor of dietary lipids in possibly all mammals. As lingual tissues are very vulnerable to post-mortem degradation, our examined human taste buds from autopsies demonstrated some autolytic morphology. Nonetheless, CD36 expression did not seem to be affected, and was unequivocally observed in human taste buds. Future investigations using ‘fresh’ surgical specimens will be needed to determine the subtype(s) of these CD36-expressing human taste bud cells. Furthermore, alternative candidate oral receptors for LCFA – like delayed rectifying K+ channels, GPR40, and GPR120 (Gilbertson et al., 1997; Matsumura et al., 2007, 2009; Cartoni et al., 2010) – need to be examined in human taste buds. Detailed in vitro mechanistic studies (El-Yassimi et al., 2008) have revealed that LCFA were able to increase free intracellular calcium ([Ca2+ ]i ), inositol 1,4,5-triphosphate levels, and phosphorylation of Src kinases in purified mouse CD36-expressing taste cells. Increasing [Ca2+ ]i was observed to be a critical step to release neurotransmitters, i.e. nor-adrenalin and serotonin, from these mouse CD36-expressing taste cells. Furthermore, selective bilateral transsection of gustatory nerves in CD36+/+ wild-type mice decreased the preference for LCFA-enriched solutions. LCFA deposition on the tongue of CD36+/+ wild-type mice also activated brain stem gustatory areas connected to these nerves, but not in CD36−/− null mice (Gaillard et al., 2008). The molecular mechanism of lingual CD36 in dietary LCFA detection in humans, however, remains to be investigated. Interestingly, a number of CD36 genetic
Alessio M, Ghigo D, Garbarino G, Geuna M, Malavasi F. Analysis of the human CD36 leucocyte differentiation antigen by means of the monoclonal antibody NL07. Cell Immunol 1991;137:487–500. Boughter Jr JD, Pumplin DW, Yu C, Christy RC, Smith DV. Differential expression of alpha-gustducin in taste bud populations of the rat and hamster. J Neurosci 1997;17:2852–8. Cartoni C, Yasumatsu K, Ohkuri T, Shigemura N, Yoshida R, Godinot N, et al., Ninomiya Y. Taste preference for fatty acids is mediated by GPR40 and GPR120. J Neurosci 2010;30:8376–82. Chalé-Rush A, Burgess JR, Mattes RD. Evidence for human orosensory (taste?) sensitivity to free fatty acids. Chem Senses 2007;32:423–31. Delgado AV, Alexander SL, McManus AT, Pusateri AE. Antibodies against human cell receptors, CD36, CD41a, and CD62P crossreact with porcine platelets. Cytometry B Clin Cytom 2003;56:62–7. Drewnowski A, Brunzell JD, Sande K, Iverius PH, Greenwood MR. Sweet tooth reconsidered: taste responsiveness in human obesity. Physiol Behav 1985;35:617–22. El-Yassimi A, Hichami A, Besnard P, Khan NA. Linoleic acid induces calcium signaling. Src kinase phosphorylation, and neurotransmitter release in mouse CD36-positive gustatory cells. J Biol Chem 2008;283:12949–59. Finger TE. Cell types and lineages in taste buds. Chem Senses 2005;30:54–5. Fukuwatari T, Kawada T, Tsuruta M, Hiraoka T, Iwanaga T, Sugimoto E, et al. Expression of the putative membrane fatty acid transporter (FAT) in taste buds of the circumvallate papillae in rats. FEBS Lett 1997;414:461–4. Gaillard D, Laugerette F, Darcel N, El-Yassimi A, Passilly-Degrace P, Hichami A, et al. The gustatory pathway is involved in CD36mediated orosensory perception of long-chain fatty acids in the mouse. FASEB J 2008;22:1458–68. Gilbertson TA, Fontenot DT, Liu L, Zhang H, Monroe WT. Fatty acid modulation of K+ channels in taste receptor cells: gustatory cues for dietary fat. Am J Physiol 1997;272:C1203–10. Greenwalt DE, Lipsky RH, Ockenhouse CF, Ikeda H, Tandon NN, Jamieson GA. Membrane glycoprotein CD36: a review of its roles in adherence, signal transduction, and transfusion medicine. Blood 1992;80:1105–15. Gruarin P, De Monte L, Alessio M. CD36 folding revealed by conformational epitope expression is essential for cytoadherence of Plasmodium falciparum-infected red blood cells. Parasite Immunol 2000a;22:349–60. Gruarin P, Thorne RF, Dorahy DJ, Burns GF, Sitia R, Alessio M. CD36 is a ditopic glycoprotein with the N-terminal domain implicated in intracellular transport. Biochem Biophys Res Commun 2000b;275:446–54.
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Husemann J, Loike JD, Anankov R, Febbraio M, Silverstein SC. Scavenger receptors in neurobiology and neuropathology: their role on microglia and other cells of the nervous system. Glia 2002;40:195–205. Laugerette F, Passilly-Degrace P, Patris B, Niot I, Febbraio M, Montmayeur JP, et al. CD36 involvement in orosensory detection of dietary lipids, spontaneous fat preference, and digestive secretions. J Clin Invest 2005;115:3177–84. Le Gall M, Tobin V, Stolarczyk E, Dalet V, Leturque A, BrotLaroche E. Sugar sensing by enterocytes combines polarity, membrane bound detectors and sugar metabolism. J Cell Physiol 2007;213:834–43. Maleszewski J, Lu J, Fox-Talbot K, Halushka MK. Robust immunohistochemical staining of several classes of proteins in tissues subjected to autolysis. J Histochem Cytochem 2007;55:597–606. Matsumura S, Mizushige T, Yoneda T, Iwanaga T, Tsuzuki S, Inoue K, et al. GPR expression in the rat taste bud relating to fatty acid sensing. Biomed Res 2007;28:49–55. Matsumura S, Eguchi A, Mizushige T, Kitabayashi N, Tsuzuki S, Inoue K, et al. Colocalization of GPR120 with phospholipase-Cbeta2 and alpha-gustducin in the taste bud cells in mice. Neurosci Lett 2009;450:186–90. Mattes RD. The taste of fat elevates postprandial triacylglycerol. Physiol Behav 2001a;74:343–8. Mattes RD. Oral exposure to butter, but not fat replacers elevates postprandial triacylglycerol concentration in humans. J Nutr 2001b;131:1491–6.
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Mattes RD. Oral fat exposure increases the first phase triacylglycerol concentration due to release of stored lipid in humans. J Nutr 2002;132:3656–62. Mela DJ, Sacchetti DA. Sensory preferences for fats: relationships with diet and body composition. Am J Clin Nutr 1991;53:908–15. Mela DJ. Understanding fat preference and consumption: applications of behavioural sciences to a nutritional problem. Proc Nutr Soc 1995;54:453–64. Rac´ ME, Safranow K, Poncyljusz W. Molecular basis of human CD36 gene mutations. Mol Med 2007;13:288–96. Sclafani A, Ackroff K, Abumrad NA. CD36 gene deletion reduces fat preference and intake but not post-oral fat conditioning in mice. Am J Physiol Regul Integr Comp Physiol 2007;293:R1823– 32. Smith LM, Clifford AJ, Hamblin CL, Creveling RK. Changes in physical and chemical properties of shortenings used for commercial deep-fat frying. J Am Oil Chem Soc 1986;63:1017– 23. Tsuruta M, Kawada T, Fukuwatari T, Fushiki T. The orosensory recognition of long-chain fatty acids in rats. Physiol Behav 1999;66:285–8. Zhang X, Fitzsimmons RL, Cleland LG, Ey PL, Zannettino AC, Farmer EA, et al. CD36/fatty acid translocase in rats: distribution, isolation from hepatocytes, and comparison with the scavenger receptor SR-B1. Lab Invest 2003;83: 317–32.
Contents lists available at ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Short communication
Apical CD36 immunolocalization in human and porcine taste buds from circumvallate and foliate papillae Peter J. Simons a,∗ , J. Alain Kummer b,c , Joost J.F.P. Luiken d , Louis Boon a a
Department of Cell Biology, Bioceros BV, Yalelaan 46, 3584 CM Utrecht, The Netherlands Department of Pathology, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Department of Pathology, St. Antonius Hospital, Koekoekslaan 1, 3534 CM Nieuwegein, The Netherlands d Department of Molecular Genetics, Maastricht University, Universiteitssingel 50, 6200 MD Maastricht, The Netherlands b c
a r t i c l e
i n f o
Article history: Received 14 August 2010 Accepted 27 August 2010
Keywords: CD36 Orosensory receptor Long chain fatty acids Taste buds Dietary fat preference Obesity Human
a b s t r a c t CD36 is the receptor for long chain fatty acids (LCFA), and is expressed in lingual taste cells from rodents. In these animals, CD36 has been proposed to play an important role in oral detection of LCFA, and subsequently, determines their dietary fat preference. Humans also seem to detect LCFA in the oral cavity, however, information on the molecular mechanism of this human orosensory LCFA recognition is currently lacking. The aim of our study was to investigate whether CD36 is also expressed in lingual human and porcine taste buds cells. Using fluorescence immunohistochemistry, apical CD36 expression was revealed in human and porcine taste bud cells from circumvallate and foliate papillae. These data suggest CD36 as the putative orosensory receptor for dietary LCFA in human, and, therefore, may be involved in our preference for fatty foods. © 2010 Elsevier GmbH. All rights reserved.
Introduction CD36 or fatty acid translocase is an integral membrane ditopic glycoprotein, and its amino acid sequence predicts a large hydrophobic pocket between two short cytoplasmic tails (Greenwalt et al., 1992). The C-terminal cytoplasmic tail has been shown to be associated with Src kinase family members, suggesting an involvement of CD36 in cell signaling. CD36 is expressed on numerous cell types (e.g. macrophages, enterocytes, hepatocytes, endothelial cells, myocytes, adipocytes, and platelets), and is described to be a receptor for multiple ligands including long chain fatty acids (LCFA), high-density lipoprotein, oxidized low-density lipoprotein, glycated collagen type IV, thrombospondin-1, fibrillar -amyloid, advanced glycated end products, chondroitin sulfate, apoptotic cells, and Plasmodium falciparum-infected erythrocytes (Husemann et al., 2002). Recent investigations (Fukuwatari et al., 1997; Zhang et al., 2003; Laugerette et al., 2005) have illustrated that lingual rat and mouse taste bud cells also express CD36 at the apical side. Furthermore, Laugerette et al. (2005) elegantly showed this CD36 as a possible oral sensor for LCFA in mice. They and others (Sclafani
∗ Corresponding author. E-mail address: [email protected] (P.J. Simons). 0065-1281/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2010.08.006
et al., 2007) demonstrated that CD36+/+ wild type mice consumed significantly more lipid-enriched (LCFA or triglycerides) solutions and solid diets than CD36−/− null mice. However, consumption of sucrose and aversion for quinine remained similar in both mouse models. Such a fat preference is not species-specific since it was already demonstrated in rats (Tsuruta et al., 1999). Laugerette et al. (2005) also showed that pancreatobiliary excretions were enhanced in esophagus-ligated rats and mice after deposition of purified LCFA onto the tongue. In addition, LCFA-mediated induction of pancreatobiliary excretions found in CD36+/+ wild-type mice was fully abolished in CD36−/− null mice. The above described data suggest that rodent CD36-expressing lingual taste cells could interact with – possibly dietary – LCFA, which in turn directs the preference for lipid-rich foods and the cephalic phase of digestive excretions. In Western society, with an access to a diet of virtually any composition and a free-choice condition, humans consume approximately 40% of food energy as fat (Mela, 1995). Moreover, obese individuals seem to exhibit a higher dietary fat preference compared to lean individuals (Drewnowski et al., 1985; Mela and Sacchetti, 1991). In humans, oral lipid-rich food stimulation – without food swallowing and smelling – rapidly enhances serum triglyceride levels, implicating incoming oral fat in the release of stored fat (Mattes, 2001a,b, 2002). Noteworthy, fatty foods may consist of >0.5% free FA (Smith et al., 1986). Humans also show
840
P.J. Simons et al. / Acta Histochemica 113 (2011) 839–843
The specimens from post-mortem human tongue, which were dissected within 8 h from three subjects (a 78 year-old female, an 84 year-old male, and a male of unknown age), containing circumvallate and foliate papillae, were obtained from a commercial tissue bank (Analytical Biological Services Inc., Wilmington, USA) and from the Department of Pathology/University Medical Centre Utrecht Biobank, and used in accordance with the hospital scientific committee regulations and the code “Proper Secondary Use of Human Tissue” as installed by the Federation of Biomedical Scientific Societies (www.federa.org). Immunohistochemistry The specimens of tongue were embedded in OCT medium (Sakura Finetek Europe BV, Zoeterwoude, The Netherlands), snapfrozen in liquid nitrogen, and stored at −150 ◦ C. Serial 9–10 m cryosections were cut and mounted onto slides, air dried, and fixed in acetone for 10 min at room temperature (RT). The slides were then incubated with:
Fig. 1. Representative light microscopic images of hematoxylin/eosin-stained human (A) and porcine (B) taste buds in circumvallate papillae. Taste buds (TB; white dashed-lined ovals) are embedded in a non-sensory squamous epithelial (NSE) layer. White triangles indicate apical taste pore areas, and C indicates the cleft in the oral cavity. Note: A few pycnotic nuclei were observed in human taste buds suggesting some degree of autolysis. Scale bar = 20 m.
the ability to detect trace amounts of purified LCFA on the tongue (Chalé-Rush et al., 2007). These observations suggest the existence of a human orosensory LCFA detection system, which might be similar to that described in rodents. However, the role of oral CD36 as a putative LCFA receptor in humans is currently not known. In this study, we immunohistochemically examined the expression of CD36 in lingual taste buds from human circumvallate and foliate papillae. Taste buds from porcine tongue were also investigated, because of the pig’s (Sus scrofa) high genomic homology with human. Furthermore, pigs have been described as a major mammalian model for human biomedical studies because of their similarity in physiology, in organ development and disease progression. Our data clearly demonstrate that the putative LCFArecognizing CD36 receptor is also apically expressed in human and porcine taste bud cells from lingual gustatory papillae, suggesting its role in orosensory perception of dietary lipids in mammals. Materials and methods
A. CD36 expression: 10 g/mL mouse anti-human CD36 monoclonal antibody (Mab) clone CB38, also known as clone NL07, (isotype mouse IgM; BD Biosciences, Alphen aan den Rijn, The Netherlands) in PBS/1% BSA (Sigma–Aldrich, Zwijndrecht, The Netherlands) for 1.5 h at RT. To corroborate the specificity of CD36 staining in human taste buds, 10 g/mL (≈11 nM) anti-CD36 Mab clone CB38/NL07 was adsorbed in an excess of 50 g/mL (≈342 nM) soluble recombinant human CD36:Fc chimeric protein (R&D Systems, Minneapolis, MN, USA) in PBS/1% BSA during 30 min at RT prior to incubation on slides. 10 g/mL mouse IgM isotype control Mab clone G155-228 (BD Biosciences) was run in parallel as a negative control. After incubation, cryosections were gently rinsed in PBS and incubated with 1:200 diluted Alexa Fluor® 488-labelled goat anti-mouse IgM antibodies (Invitrogen Ltd., Paisley, UK) in PBS/1% BSA for 1 h at RT. B. Alpha-gustducin and NCAM/CD56 expression: 2 g/mL affinitypurified rabbit IgG anti-␣-gustducin I-20 polyclonal antibody (directed against a peptide corresponding to amino acids 93–112 mapping within a highly divergent domain of rat ␣-gustducin, and known to cross-react with mouse, hamster and human ␣-gustducin (Boughter et al., 1997; Santa Cruz Biotechnology, Santa Cruz, USA) or 10 g/mL mouse anti-human NCAM/CD56 Mab clone B159 (isotype mouse IgG1; BD Biosciences) in PBS/1% BSA for 1.5 h at RT. Respectively, anti-␣-gustducin polyclonal antibody was omitted, or 10 g/mL mouse IgG1 isotype control Mab clone MOPC-21 (BD Biosciences) were run in parallel as negative controls. After incubation, cryosections were gently rinsed in PBS and incubated with 1:200 diluted phycoerythrinconjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) or with 1:200 phycoerythrin-conjugated goat anti-mouse IgG antibodies (Jackson ImmunoResearch) in PBS/1% BSA for 1 h at RT. After another rinse step in PBS, all cryosections were coverslipped with ProLong® Gold anti-fade reagent (Invitrogen) prior to microscopic examination.
Tongue tissue specimens Results and discussion Necropsy normal porcine tongue tissue specimens, which were dissected within 15 min from two 24 weeks-old specific pathogenfree female crossbred Great Yorkshire/Large White × Dutch Landrace pigs (weighing around 120 kg), containing circumvallate and foliate papillae, were obtained from Van Beek SPF Varkens BV, Lelystad, The Netherlands.
CD36 expression in human taste buds Human taste buds are onion-shaped organs embedded in the full thickness of the non-sensory stratified squamous epithelium of the tongue, and are easily recognized using routine
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Fig. 2. Apical CD36 expression in human taste bud cells from circumvallate and foliate papillae. (A) Fluorescence microscopic image of a circumvallate papilla after incubation with soluble human CD36:Fc chimeric protein pre-adsorbed anti-human CD36 Mab. (B) CD36 expression in taste bud cells from circumvallate papilla (same area as section A) after incubation with anti-human CD36 Mab. (C) Image of a foliate papilla after incubation with soluble human CD36:Fc chimeric protein pre-adsorbed anti-human CD36 Mab. (D) CD36 expression in taste bud cells from foliate papilla (same area as section C) after incubation with anti-human CD36 Mab. White dashed-lined ovals indicate the location of taste buds embedded in the epithelial layer, white triangles indicate apical taste pore area, and asterisks indicate papillae clefts. Scale bar = 20 m.
histological staining methods (see Fig. 1A) or phase contrast microscopy. To determine whether these human taste buds are expressing CD36, an immunohistochemical analysis was performed using the anti-CD36 specific Mab, clone CB38/NL07. This Mab was originally raised against human CD36-expressing platelet extracts (Alessio et al., 1991), and its specificity has been extensively characterized (Gruarin et al., 2000a,b). These investigations demonstrated that clone CB38/NL07 recognizes a conformational epitope on mature human CD36 that is acquired only during the late steps of intracellular transport (Golgi apparatus) and cell surface expression. As shown in Fig. 2B and D, human taste bud cells from both circumvallate and foliate gustatory papillae clearly exhibited mature CD36 expression, typically at the apical side. Despite the fact that tested tissue specimens from all examined donors showed autolytic signs (Fig. 1A) – post-mortem cryopreservation was performed within 8 h – virtually all taste buds demonstrated CD36 positive staining. However, CD36 expression levels varied from one taste bud to the other, most likely because spherical taste buds were cut at different levels. In some cases, the taste pore or pit area showed an extremely high density of CD36 molecules, which indicated CD36 expression on taste cell’s microvilli protruding into the oral cavity/cleft. This CD36 expression was specific in human taste buds, since no staining was observed after the anti-CD36 Mab clone CB38/NL07 was pre-adsorbed with soluble recombinant CD36 proteins (Fig. 2A and C). In addition, a negative isotype control antibody was devoid of any staining (data not shown) in human taste buds cells. In an attempt to delineate the subtype(s) of CD36 expressing human taste bud cells, we also examined ␣-gustducin (marker for type II taste cells) and NCAM/CD56 (marker for type III taste cells) expression (Finger, 2005). Unfortunately, and in contrast to above-described CD36 expression, specific antibodies against ␣-gustducin and NCAM/CD56 failed to show any immunoreactivity in human taste bud cells (data not shown). We suspect degradation of ␣-gustducin and NCAM/CD56 proteins in our
autopsy tissue specimens, because the antibodies against these proteins did show immunoreactivity in the human Caco-2 cell line obtained from American Type Culture Collection (Le Gall et al., 2007) and in human peripheral blood natural killer cells respectively (data not shown). It has been previously shown that CD36 proteins degrade slowly and maintain immunoreactive in multiple human tissue specimens during a 24-h post-mortem interval (Maleszewski et al., 2007). Finally, non-sensory stratified squamous epithelial cells (Fig. 2B and D) and non-gustarory filliform papillae (data not shown) demonstrated no CD36 expression. Taste buds from fungiform papillae were not included in our study. CD36 expression in porcine taste buds As found with human taste buds, porcine taste bud cells also expressed mature CD36, most remarkably at the apical side (Fig. 3). Anti-human CD36 Mab clone CB38/NL07 has been previously described to cross-react with porcine CD36 (Delgado et al., 2003). As in human taste buds, CD36 staining intensity was also variable in these ‘fresh’ (i.e. 15 min after death) porcine taste buds. CD36 expression was discerned in almost all taste buds embedded in epithelial layers of circumvallate and foliate papillae obtained of pigs. Occasionally, the taste pore area exhibited very intense CD36 staining, which suggested CD36 expression on microvilli of taste cells. No staining was observed (data not shown), when porcine taste buds cells were exposed to isotype negative control antibodies. Lingual CD36 as the putative oral LCFA-sensing receptor in humans In our view, the recent discovery of CD36 in lingual taste bud cells in inbred and outbred rodents, together with CD36 expression in lingual taste bud cells from unrelated humans and crossbred pigs in our Short Communication, substantiates the idea that CD36
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variations have been identified in humans (Rac´ et al., 2007). In attempt to address the global fight against obesity and overweight, it will be challenging to examine whether these CD36 genetic variations influence our ability to sense dietary LCFA, and subsequently, our fatty food preference.
Acknowledgements The authors thank Mr. Martijn van Osch and Mr. Willem van Wolferen for their excellent technical assistance, Mr. Rob Bleumink for use of cryomicrotome, fluorescence microscope and digital image software, and Prof. Dr. Jan Glatz for fruitful discussions.
References
Fig. 3. Apical CD36 expression in porcine taste bud cells from circumvallate and foliate papillae. (A) CD36 expression in taste bud cells from a circumvallate papilla after incubation with anti-human CD36 Mab. (B) CD36 expression in taste bud cells from a foliate papilla after incubation with anti-human CD36 Mab. White dashedlined ovals indicate the location of taste buds embedded in the epithelial layer, white triangles indicate apical taste pore area, and asterisks indicate papillae clefts. Scale bar = 20 m.
might function as a putative orosensory receptor of dietary lipids in possibly all mammals. As lingual tissues are very vulnerable to post-mortem degradation, our examined human taste buds from autopsies demonstrated some autolytic morphology. Nonetheless, CD36 expression did not seem to be affected, and was unequivocally observed in human taste buds. Future investigations using ‘fresh’ surgical specimens will be needed to determine the subtype(s) of these CD36-expressing human taste bud cells. Furthermore, alternative candidate oral receptors for LCFA – like delayed rectifying K+ channels, GPR40, and GPR120 (Gilbertson et al., 1997; Matsumura et al., 2007, 2009; Cartoni et al., 2010) – need to be examined in human taste buds. Detailed in vitro mechanistic studies (El-Yassimi et al., 2008) have revealed that LCFA were able to increase free intracellular calcium ([Ca2+ ]i ), inositol 1,4,5-triphosphate levels, and phosphorylation of Src kinases in purified mouse CD36-expressing taste cells. Increasing [Ca2+ ]i was observed to be a critical step to release neurotransmitters, i.e. nor-adrenalin and serotonin, from these mouse CD36-expressing taste cells. Furthermore, selective bilateral transsection of gustatory nerves in CD36+/+ wild-type mice decreased the preference for LCFA-enriched solutions. LCFA deposition on the tongue of CD36+/+ wild-type mice also activated brain stem gustatory areas connected to these nerves, but not in CD36−/− null mice (Gaillard et al., 2008). The molecular mechanism of lingual CD36 in dietary LCFA detection in humans, however, remains to be investigated. Interestingly, a number of CD36 genetic
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