Molecules implicated in glucose homeostasis are differentially

Molecules implicated in glucosehomeostasis are differentiallyexpressed in the trachea oflean and obese Zucker ratsF. Merigo,1 F. Boschi,2 C. Lasconi,1D. Benati,1 A. Sbarbati11Department of Neurological andMovement Sciences, Anatomy andHistology Section, University of Verona2Department of Computer Science,University of Verona, Italy

Abstract

Recent studies indicate that the processesmediated by the (T1R2/T1R3) glucose/sugarreceptor of gustatory cells in the tongue, andhormones like leptin and ghrelin contribute tothe regulation of glucose homeostasis. Alteredplasma levels of leptin and ghrelin are associ-ated with obesity both in humans and rodents.In the present study, we evaluated the ultra-structure of the mucosa, and the expression ofmolecules implicated in the regulation of glu-cose homeostasis (GLUT2, SGLT1, T1R3, ghre-lin and its receptor) in the trachea of an ani-mal model of obesity (Zucker rats). We foundthat the tracheal epithelium of obese animalswas characterized by the presence of poorlydifferentiated cells. Ciliated and secretory cellswere the cell lineages with greatest loss of dif-ferentiation. Severe epithelial alterations wereassociated with marked deposit of extracellu-lar matrix in the lamina propria. The expres-sion pattern of GLUT2 and SGLT1 glucosetransporters was similar in the trachea of boththe Zucker rat genotypes, whereas that ofT1R3 was reduced in ciliated cells of obeserats. A different immunolocalization for ghre-lin was also found in the trachea of obese rats.In conclusion, the tracheal morphologicalalterations in obese animals seem to compro-mise the expression of molecules involved inthe homeostasis of glucose.

Introduction

Data from recent studies has revealed thatin addition to the processes initiated by thesecretory activity of the endocrine pancreas,also those mediated by the same (T1R2/T1R3)glucose/sugar receptor of the gustatory cells inthe tongue and the enteroendocrine cells inthe intestine, contribute to the regulation ofglucose homeostasis. For example, in duode-nal epithelium, the expression of glucose

transporters (SGLT1, GLUT2) correlates withthe levels of luminal monosaccharides and isregulated by the T1R3 subunit of sweet tastereceptor and α-gustducin.1,2 T1R3 is an inte-gral membrane protein that functions as asweet receptor in the form of T1R2/T1R3 het-erodimer, and as a umami (L-amino acids)receptor in the form of T1R1/T1R3. T1Rsbelong to the family of G protein-coupledreceptors (GPCRs) which act through α-gust-ducin, phospholipase C-β (PLC β2), inositolt-riphosphate (IP3), and transient receptorpotential channel M5 (TRPM5) to inducemembrane depolarization and release ofadenosine triphosphate ATP.3,4 The receptorsand components of the sweet taste transduc-tion pathway have been implicated as playing arole in glucose sensing because they are dis-tributed in many tissues, such as the tastecells of the tongue, intestinal epithelial cells ofthe duodenum and mouse pancreatic β-cells,in which their functional roles are associatedwith sweet taste and feeding.5,6 The same mol-ecules have been also detected in differenttracts of the respiratory apparatus of humansand rodents, including the larynx,7 nasal respi-ratory epithelium,8,9 trachea,10-14 and bronchi,15

suggesting that taste transduction mecha-nisms also operate in the airways. In the tracheal epithelium, the most fre-

quent and recognizable cells in many speciesare goblet, ciliated-columnar and basal cells.But further types not defined by a unique mor-phology and name are also present. They com-prise a heterogeneous population whichincludes cylindrical cells with a thick brush oflong microvilli on the apical surface (namelybrush or tuft cells), or cells with a long, thinprocess that extends up to the lumen (namelyneuroendocrine cells). Their role is not com-pletely clear, but they are consideredchemosensory cells because they express com-ponents of the taste-signaling cascade and/orare related to the nerve endings.6 So, in thepresent work, they will be collectively referredto as solitary chemosensory cells (SCCs). The airway epithelium is exposed to a thin

layer of surface liquid (ASL), whose composi-tion and volume are strictly regulated. Amongthe substances present in ASL there is glucose,whose concentration is lower than in the bloodand is of considerable importance for mainte-nance of the mechanisms controlling glucosehomeostasis.16 Glucose diffuses into ASL main-ly via the paracellular pathway, and is removedby cellular uptake through glucose trans-porters.16 In humans and rats, the predominantglucose transporters are GLUT isoforms in theproximal airway, while SGLT1 appears to bemore active in the distal lung.17,18 In the air-ways, the physiological function of glucosetransporters is to maintain low glucose levelsin ASL, which is an essential requirement to

prevent bacterial colonization or infection inhumans and rodents.19,20 In human studies,ASL glucose concentrations were found to beelevated in respiratory diseases and to be asso-ciated with hyperglycemia, cystic fibrosis ordiabetes.21-26 In general, the chemoreceptiveepithelia respond to local sugar changes byregulating uptake through a direct effect onglucose transporter expression, or an indirecteffect involving different pathways (i.e., hor-monal, neural, paracrine, autocrine).27 In thisregard, the investigation of glucose trans-porter and glucose-sensing molecule expres-sion in diseases such as diabetes and obesitycan be helpful in understanding how airwayepithelial cells respond to altered ASL glucosehomeostasis. Recently, considerable attention has been

devoted to the role of the two hormones leptinand ghrelin in the control of blood glucosehomeostasis.28 Ghrelin, the endogenous ligandof the Growth Hormone (GH) Secretagoguetype 1a Receptor (GRLN-R), is mostly secretedby endocrine cells of the gastric mucosa in ratsand humans, but it exists, along with its recep-tor, in a wide variety of cells and tissues,including the hypothalamus, small intestine,lung, skeletal muscle, ovary, testis, pituitarygland and pancreas.29 Leptin is primarily syn-thesized and secreted by adipocytes and exertsits activity through the leptin receptor (Ob-R).Leptin and Ob-R are expressed in a variety oftissues, such as the hypothalamus, lung, olfac-tory system, pancreas and taste bud cells of thetongue.30 In rodents, deficiency of leptin or lep-tin receptor gene has generated animal modelsof obesity that have been widely used to under-stand the processes involved in the regulationof glucose homeostasis.31 Leptin deficientob/ob mice and Ob-R deficient db/db micedevelop hyperphagia, morbid obesity, infertili-ty, and reduced linear growth. Homozygous

European Journal of Histochemistry 2016; volume 60:2557

Correspondence: Dr. Flavia Merigo, Departmentof Neurological and Movement Sciences,Anatomy and Histology Section, University ofVerona, Strada Le Grazie 8, 37134 Verona, Italy. E-mail: [email protected]

Key words: Obesity; ultrastructure; ghrelin; ghre-lin receptor; sweet receptor; immunohistochem-istry.

Received for publication: 24 July 2015.Accepted for publication: 30 November 2015.

This work is licensed under a Creative CommonsAttribution-NonCommercial 4.0 InternationalLicense (CC BY-NC 4.0)

©Copyright F. Merigo et al., 2016Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2016; 60:2557doi:10.4081/ejh.2016.2557

[European Journal of Histochemistry 2016; 60:2557] [page 1]

EJH_2016_01.qxp_Hrev_master 10/03/16 10:48 Pagina 1

[page 2] [European Journal of Histochemistry 2016; 60:2557]

Zucker rats with fa/fa genotype are leptinresistant, hyperphagic and obese; the obesecondition is evident at 5 weeks of age. In theseanimals, the fa mutation in the Ob-R genecauses an amino acid substitution in theextracellular domain of Ob-R, preventing theexpression of the receptor long (active) form.Ghrelin is an orexigenic mediator which, apartfrom its role in the regulation of appetite andon growth hormone secretion, has many func-tions, including gastrointestinal, cardiovascu-lar, and immune functions.28,32 Leptin is ananorexigenic mediator that plays an importantrole in the regulation of food intake, energyexpenditure, metabolism, neuroendocrineaxis, and immune function.30

Certainly, the most extensively studied roleof ghrelin and leptin is their regulatory effecton glucose homeostasis. The literature regard-ing pharmacological treatment as well asgenetic manipulation in rodents, demonstratesthat ghrelin inhibits glucose-stimulatedinsulin secretion,28,33 while leptin preventsproinsulin synthesis.30 Due to these functions,circulating levels of ghrelin and leptin havebeen evaluated in metabolic diseases to under-stand whether dysregulation of their secretioncould have a pathophysiological significance.Increased levels of leptin have been found inoverfed and obese states,30 and increased lev-els of ghrelin in healthy humans and mice withelevated blood glucose levels.34 In contrast, lowplasma ghrelin levels are associated with obe-sity, insulin resistance, metabolic syndrome,also in association with type 2 diabetes melli-tus (T2DM) in humans, or with overfeeding,and high fat diet in rats.35,36 However, it shouldbe borne in mind that ghrelin and leptin act atboth the local level via their specific receptors(autocrine/paracrine), and the systemic(endocrine) level. Indeed, it might be expectedthat changes in circulating levels of ghrelinand leptin would reflect altered expressionand/or distribution of the locally produced hor-mones, leading to dysregulation of their path-way. Therefore, the expression of these mole-cules and receptors in peripheral organs maybe indicative of their role in glucose homeosta-sis. On this basis, the present study was con-

ducted to investigate the expression of mole-cules implicated in the regulation of glucosehomeostasis in the tracheal epithelium of ananimal model of genetic obesity. In particular,we evaluated i) the fine structure of themucosa; and ii) the expression of T1R3, α-gustducin, GLUT2, SGLT1, ghrelin, and ghrelinreceptor in the trachea of lean fa/+ and obesefa/fa Zucker rats.

Materials and MethodsAnimals Fourteen male obese (fa/fa) and 12 male

lean (fa/+) Zucker rats were purchased fromHarlan Laboratories Srl (Correzzana, MI, Italy)at 8 weeks of age. They were kept in individualcages with ad libitum access to standard ratchow and tap water under controlled tempera-ture, humidity, and light cycle conditions. Theanimals were acclimatized to the local facili-ties for at least two weeks before being usedfor the study. At the time of experimental ses-sions, their ages ranged from 10-18 weeks forboth Zucker rat genotypes; their body weightranged from 285 g to 340 g for lean, and from338 g to 498 g for obese rats. Concurrently, 10-12-week old male Wistar rats (n=6, weight150-200 g; Morini Co., Reggio Emilia, Italy),were similarly housed. Their immunohisto-chemical analyses were compared with thoseof the Zucker rats. The experimental animalprotocol was prospectively approved by theItalian Ministry of Health and by the UniversityCommission for Animal Care.

TissuesThe animals were anesthetized with isoflu-

rane (3% by inhalation) and perfused with 4%paraformaldehyde in phosphate buffer.Proximal tracheae were quickly collected. Forimmunohistochemical microscopy, tissueswere further fixed by immersion in the samefixative, rinsed and put into 30% sucroseovernight. Sections (40 µm thickness) werecut on a freezing microtome (Reichert-Jung,Vienna, Austria) and processed for bothimmunoperoxidase and immunofluorescencelabeling.

Transmission Electron Microscopy For ultrastructural examination, small tis-

sue blocks were further fixed with 2% glu-taraldehyde in phosphate buffer for 2 h at 4°C,postfixed in 1% OsO4, dehydrated and embed-ded in a mixture of Epon and Araldite (ElectronMicroscopic Sciences, Fort Washington, PA,USA). Semithin sections (2 μm thickness)were stained with toluidine blue, and exam-ined using an Olympus BX51 microscope con-nected to a digital camera (JVC CCDKY-F58)equipped with the image analysis digital sys-tem Image-Pro Plus 7.0 (Media Cybernetics,Silver Spring, MD, USA). Ultrathin sectionswere cut at a thickness of 70 nm on anUltracut-E ultramicrotome (Reichert-Jung),stained with lead citrate and observed in aMorgagni 268D transmission electron micro-scope equipped with a Megaview II camera fordigital image acquisition (FEI CompanyPhilips, Milan, Italy). The images derivingfrom toluidine blue stained sections of 6 lean

and 7 obese Zucker rats (one section per ani-mal) were randomly selected and used tomeasure diameter and area of lipid droplets(LDs) in the lamina propria, vessels and carti-lage. Three optical fields per section wereobserved at 60 x magnification. In the laminapropria, the lipid deposition was measured bytracing the borders of the LDs on the images,and by measuring the inner area. In vessels,the lipid deposition was measured as done pre-viously in the lamina propria, and also bymeasuring the area covered by the vessels.The ratio between the area of the lipid deposi-tion and the corresponding area of the vesselwas evaluated. In the cartilage, the diameter ofthe almost perfectly round in shape LDs wasevaluated. Measurements were done by usingthe software ImageJ (NIH, Bethesda, MD,USA). A statistical analysis was performedusing the t-test with the level of statistical sig-nificance set at 0.05. The statistical analysiswas conducted with Matlab 7.1 (Mathworks,Natick, MA, USA).

Primary antibodiesFor this study we used the following primary

antibodies: rabbit anti-ghrelin (PhoenixPharmaceuticals Inc., Burlingame, CA, USA,cat #H-031-31); rabbit anti-GLUT2 (also knownas -SLC2A2, LifeSpan Biosciences Inc., Seattle,WA, USA, cat #LS-C15390/25520); rabbit anti-α-gustducin (Santa Cruz Biotechnology Inc.,Heidelberg, Germany, cat #sc-395); rabbit anti-ghrelin receptor (-GRLN-R Abcam, Cambridge,UK, cat #ab85104), rabbit anti-SGLT1 (alsoknown as -SLC5A1, Abcam, cat #ab14686), rab-bit anti-T1R3 (Abcam, cat #ab74732), mouseanti-acetylated α-tubulin (Abcam, cat#ab24610).

Immunohistochemistry Peroxidase-immunohistochemistry and

immunofluorescence were performed asdescribed in Merigo et al.12 Briefly, endoge-nous peroxidase was quenched by immersionin a peroxidase blocking reagent (DAKO,Milan, Italy), for 20 min. After washing inphosphate-buffered saline (PBS) buffer, pH7.5, sections were treated with a blocking solu-tion (0.3% Triton X-100, 1% bovine serum albu-min BSA and 2% normal swine serum) for 1 h,and then incubated overnight with the primaryantibodies diluted with blocking solution. Afterwashes, sections were then reacted withbiotinylated swine anti-rabbit immunoglobu-lins (DAKO) diluted 1:400 for 1 h. Theimmunoreaction was detected using aVectastain ELITE ABC kit (Vector, Burlingame,CA, USA) and then visualized with 3.3�-diaminobenzidine tetrahydrochloride (DAKO)for 5-10 min. Control sections were prepared by preab-

sorbing the primary antibodies with the corre-

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sponding peptide (5 µg / 1 mL of antibody;Ghrelin peptide, Phoenix PharmaceuticalsInc.; Ghrelin Receptor peptide, Abcam; GLUT2peptide, Chemicon International Inc.,Temecula, CA, USA; SGLT1 peptide, Abcam), orby omitting the primary antibody. Sectionswere observed on an Olympus BX51 micro-scope. Images were analyzed and stored usingImage-Pro Plus software.For double immunofluorescent staining,

free floating sections were incubatedovernight in a mixture of mouse anti-α-tubu-lin antibody with rabbit anti-GLUT2, or -SGLT1,or -α-gustducin, or -T1R3 antibody, thenwashed and incubated in a mixture of second-ary antibodies (TRITC-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit;Jackson Laboratories, Baltimore, PA, USA;1:200) for 1 h. Control sections were preparedas described above.When the glucose transporter immunolabel-

ing was compared with that of ghrelin, thedouble-label assay was carried out sequentiallyusing a method which relied on the use of sec-ondary monovalent Fab fragments, because allprimary antibodies are raised in the samespecies.37,38 The double staining was performedas described in Merigo et al.12 Control sectionswere prepared using one of the followingmethods: i) adding the corresponding peptideto primary antibodies; or ii) replacing the sec-ond primary antibody with normal rabbitserum; or iii) exchanging the fluorophores ofthe secondary antibodies; or iv) omitting theprimary antibody; or v) changing the sequenceof secondary antibody application. Sectionswere observed using Leica confocal TCS-SP5microscope (Leica Microsystems Srl, Milan,Italy); images were acquired using LAS AF Litesoftware (Leica Microsystems) and processedusing ImageJ software for the selection of thethresholds in each channel and the merging ofthe channels.

ResultsLight and transmission electronmicroscopy analysis of trachea inlean and obese Zucker rats Using light and transmission electron

microscopy, the tracheal mucosa of Zucker ratswas observed in the following compartments:epithelium, connective tissue and cartilage.

EpitheliumIn lean Zucker rats, the epithelial cells had

an ultrastructural appearance similar to that ofnormal epithelium. All the major cell typeswere represented. The ciliated cells appearedwell preserved, showing a consistent numberof cilia and numerous mitochondria concen-

trated just beneath the ciliary basal bodies(Figure 1A-C). Lysosomes were observed inthe supranuclear region. The mucous cellsexhibited the classical aspects of secretorycells, showing an extensive, cisternal ergasto-plasm in the basal part, and numerous mucousdroplets, often coalescing, in the apical cyto-plasm (Figure 1C). Secretory serous cells werewell-represented and well-differentiated, withapical membrane-bound droplets, basalergastoplasm, numerous mitochondria, and alow nucleus/cytoplasm ratio (Figure 1 C,D).The tracheal epithelium also showed othertypes of cells, including SCCs, a heterogeneouspopulation which included brush and tuft cells

(Figure 1E). Brush cells, recognizable by abrush of microvilli on the apical surface, con-tained glycogen granules and a preponderanceof agranular reticulum in the cytoplasm(Figure 1 F,I). Clusters of different types ofSCCs were distributed throughout the epitheli-um, and easily distinguishable by the presenceof brush cells (Figure 1 G,I). A small number ofblood cells were also seen; most often theywere lymphocytes. Adjacent to the basementmembrane there were basal cells with theirtypical appearance consisting of a large nucle-us, scarce cytoplasm and few organules(Figure 1D). In obese Zucker rats, the trachea showed

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Figure 1. Light (A) and electron microscopy (B-I) showing histological and ultrastructur-al examinations of epithelium in trachea of lean Zucker rats. Histological section stainedwith toluidine blue (A). Arrow in (A) indicates lipid droplet. Asterisk indicates the cellsthat compose a cluster of solitary chemosensory cells. The boxed area on the left in panelG is shown at higher magnification in (H); the cells marked with asterisks on the right in(G) are shown at higher magnification in panel I. bc, basal cell; bm, basement membrane;brc, brush cell; cc, ciliated cell; cf, collagen fibers; hc, hyaline cartilage; e, epithelium; lp,lamina propria; mc, mucous cell; sc, secretory cell; scc, solitary chemosensory cell. Scalebars: 5 µm.



ultrastructural alterations with differentdegrees of severity. In all obese animals, theepithelium appeared to lose its regular pseu-dostratified organization over large areas, withcertain areas in which a superposition of celllayers was visible, with subsequent epithelialthickening and an increased number of cells(Figure 2A). Epithelial regions monolayered orincompletely stripped were also observed.These severe epithelial alterations were asso-ciated with marked deposits of material in thelamina propria (Figure 2B). The epitheliumwas characterized by the presence of poorlydifferentiated cells, which were consideredintermediate cells. Ciliated and secretory cellswere the cell lineages with greatest loss of dif-ferentiation. Intermediate ciliated cells hadpolymorphic aspects: they varied from cellswith few cilia but well-represented organules(i.e., mitochondria, endoplasmic reticulum,Golgi apparatus), to cells characterized by fewcilia, a decreased number of apical mitochon-dria, a hypertrophic Golgi apparatus, andnumerous lysosomes in the Golgi apparatuszone. A decrease in the number of cilia wasoften associated with an increase in the num-ber of microvilli on the apical surface.Intermediate secretory cells contained fewsecretory granules and numerous lysosomes(Figure 2 C,D). Necrotic and swollen cells witha depletion of cytoplasmic organelles were alsoobserved. In contrast, chemosensory elementswere well represented both quantitatively andqualitatively. In particular, brush cellsappeared to be mature, occasionally hyper-trophic, and rich in mitochondria, glycogengranules, and elements of smooth endoplasmicreticulum (Figure 2 D-F). The Golgi apparatus

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Table 1. Measure of the lipid deposition in lamina propria in Zucker rats.

Number Minimum lipid Maximum lipid Mean STD of lipid droplets droplet area (µ2) droplet area (µ2) lipid droplet area (µ2) lipid droplet area (µ2)

Lean 14 72.98 481.32 182.25 115.4657Obese 25 51.01 807.74 289.67 186.8022

Table 2. Measure of the lipid deposition in vessels in Zucker rat.

Mean STD Mean STD Mean STD Vessel Vessel lipid deposit lipid deposit lipid deposit/ lipid deposit area (µ2) area (µ2) area (µ2) area (µ2) vessel area ratio /vessel area ratio

Lean 149.85 83.11 56.82 33.05 37.5% 6.18%Obese 156.52 103.80 66.51 34.70 45.1% 9.59%

Table 3. Measure of the lipid droplet diameter in cartilage in Zucker rats.

Mean Minimum Maximum Mean STD Number of lipid lipid droplet lipid droplet lipid droplet lipid droplet droplet/cell diameter (µ) diameter (µ) diameter (µ) diameter (µ)Lean 5.00 0.71 8.57 2.68 2.00Obese 2.36 0.97 9.10 3.80 2.92

Figure 2. Light (A) and electron microscopy (B-I) showing histological and ultrastructur-al examinations of epithelium in trachea of obese Zucker rats. Histological sectionstained with toluidine blue (A). Arrow in (A) indicates lipid droplet. Asterisk indicatesthe cells that compose a cluster of solitary chemosensory cells. bc, basal cell; bm, base-ment membrane; brc, brush cell; cc, ciliated cell; hc, hyaline cartilage; e, epithelium; lp,lamina propria; mc, mucous cell; sc, secretory cell. Scale bars: 5 µm.



was generally well-developed. Microvilli wereof normal length and quantity, and appeared tobe associated with low-density bodies on thefree luminal side. Clusters of SCCs, closelyconnected to each other, were present (Figure2 G-I). They appeared well preserved, even intracts where the epithelium was characterizedby necrotic cells. Basal cells were found in theareas with pluristratified epithelium; theywere rare or absent in those with monostrati-fied epithelium. Many elements related to thelymphatic line were often observed.

Connective tissueAs observed at light and electron-microscop-

ic level, the lamina propria was normal inshape in the majority of lean rats. However, themucosa from 3 lean animals showed a mildlydifferent morphology due to the presence ofareas with an increased thickness of the lami-na propria (about 3-5 µm) just below the basalmembrane, but without an apparent alterationof the overlying epithelium. These alterationswere limited to small zones and coexisted withextensive traits with normal morphology. In obese rats, a common feature was the pres-

ence of a thick lamina propria, with a thicknessabout twice that observed in lean rats. It rangedfrom 8 to 14 µm over large areas of the mucosa.The matrix formed a thick layer of amorphousappearance, devoid of elements in the interior,or with branches of collagen fibers (Figure 3 A-C). The substantial deposits of extracellularmatrix were associated with severe alterationsof the overlying epithelium. Scarce leukocyteinfiltration was observed in lean rats, while asignificant infiltrate (lymphocytes, eosinophilsand mast cells) was present in the lamina pro-pria of obese rats. LDs were found in the connec-tive tissue and around the vessels; they weregenerally larger in size in obese compared withlean rats. In the lamina propria and submucosallayer, the LDs ranged in area from 72.98 to481.32 µm2 in lean, and 51.01 to 807.74 µm2 inobese animals (Table 1). In these latter, somedroplets of considerable size (30-40 µm in diam-eter) were localized in the deepest part of themucosa. The difference in LDs’ area was not sta-tistically significant (P=0.0592) in the twoZucker rat genotypes. Both in lean (Figure 3D)and obese animals (Figure 3G), the microvascu-lar structures of the connective layer were char-acterized by the presence of dense deposits thatwere sometimes visible at the level of sub-endothelial space, with aspects of endothelialactivation such as duplication and fragmenta-tion of the basement membrane of the capillar-ies. We estimated that they were present in 22%of the submucosal vessels in samples from leanrats, and 75% in those from obese rats. Thedense deposits occupied on average the 37.5% ofthe area of the vessel (36 vessels) in lean rats,versus a 45.1% observed in obese rats (48 ves-sels), as shown in Table 2. The difference in the

area covered by lipid deposits and the respectivearea of the vessels was statistically significant inthe two Zucker rat genotypes (P=0.0246). Athigh magnification, they appeared to consist of

rounded particles, homogeneously dense, mostlikely formed from liposomes associated withdenser material probably of protein nature(Figure 3H).

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Figure 3. Transmission electron microscopy showing ultrastructural characteristics of tra-chea in lean and obese Zucker rats. Amorphous material is present in lamina propria ofobese Zucker rats (A-C). Dense deposits are present in microvascular structure in lean (D)and obese (G) Zucker rats. The dense deposit in (G) is shown at higher magnification in(H). Lipid droplets are visible in hyaline cartilage of lean (E, F) and obese (I) Zucker rats.bv, blood vessel; cf, collagen fibers; dd, dense deposit; hc, hyaline cartilage; ld, lipiddroplet; lp, lamina propria. Scale bars: A,B,D,E,G,I) 5 µm; C,F,H) 1 µm.

Figure 4. GLUT2, SGLT1, and T1R3 expression in trachea of Wistar and Zucker rats. Theimmunoperoxidase staining is shown in the epithelium of lean (A,C,E), and obese Zucker(B,D,F) rats. bm, basement membrane; c, cilia; lp, lamina propria. Scale bars: 10 µm.



CartilageIn lean rats, the tracheal cartilage had no

significant structural alterations. In the cen-tral mass of the cartilage, oval and roundedchondrocytes showed a cytoplasm with abun-dant glycogen and spherical, homogeneouslydense LDs (Figure 3E). On average, in eachcell the intracellular lipid pattern consisted in5 droplets whose size rarely exceeded that ofthe nucleus. The majority of the LDs were sur-rounded by a rim of fine granules showing ablack electron density (Figure 3F). In obeserats, the size of the largest droplet was oftenbigger than the nucleus, which was either dis-placed towards the periphery of the cell, or notdetectable inside the cell (Figure 3I).Frequently, the LDs were surrounded by fineelectron-dense granules which were also scat-tered inside the drops. The quantitative analy-sis of 65 LDs in 13 cells and of 33 LDs in 14cells in lean and obese rats, respectively, isreported in Table 3. In the two Zucker rat geno-types, the difference in LDs’ diameter was sta-tistically significant (P=0.0272).

Immunohistochemical analyses of glucosesensing molecules in trachea of lean andobese Zucker ratsImmunohistochemical analyses were per-

formed using light immunohistochemistry andimmunofluorescence experiments.

GLUT2, SGLT1, T1R3, and α-gustducin expressionThe expression pattern of glucose trans-

porters (GLUT2 and SGLT1) in the trachea oflean and obese Zucker rats was similar to thatobserved in Wistar rats in our previous work.12

In particular, GLUT2 labeling was detected onthe apical surface of ciliated cells, just belowthe cilia, and in the cytoplasm of some non-cil-iated cells in both Zucker rat genotypes(Figure 4 A,B). However, not all ciliated cellsshowed GLUT2 immunoreactivity. SGLT1immunoreactivity was exclusively observedthroughout the entire cytoplasm of cells thatappeared as non-ciliated, flask- or pear-shapedcells, with a body localized on the basementmembrane and an apical process protrudinginto the lumen. These cells were regarded asSCCs. No differences between lean and obeserats were observed in SGLT1 expression(Figure 4 C and D, respectively). The expression pattern of T1R3 was dissim-

ilar in the two Zucker rat genotypes. In leanZucker rats, T1R3 expression was basicallysimilar to that observed in Wistar rats.12 T1R3immunostaining was detected as spots alongthe cilia, or on the apical surface and the baso-lateral membrane (Figure 4E). In obese Zuckerrats, T1R3 expression was either decreased orcompletely absent at the sites specified above.Here, T1R3 labeling was found in vesicles dif-

Original Paper

Figure 5. Double-confocal microscopy for α-tubulin with GLUT2, SGLT1, α-gustducinin trachea of Zucker rats. The immunofluorescence staining shows expression of α-tubu-lin (red) with GLUT2 (green; lines A, B), or SGLT1 (green; lines C, D), or α-gustducin(green; lines E, F) in the epithelium of lean (lines A, C, E) and obese (lines B, D, F)Zucker rats. Immunostaining for GLUT2 is expressed by α-tubulin immunoreactive cells.SGLT1 and α-gustducin immunoreactivities are observed in α-tubulin-negative cellsidentified as solitary chemosensory cells. Scale bars: 10 µm.



ferently distributed in the cell cytoplasm(Figure 4F). In both Zucker rat genotypes,T1R3 staining was also observed in the cyto-plasm of non-ciliated cells, similar to thatdetected in Wistar rats.12

The expression patterns of glucose trans-porters and T1R3 were further validated inboth Zucker rat genotypes by dual labeledimmunofluorescence experiments, using anti-body to acetylated α-tubulin as a cilia marker,and α-gustducin as a marker of chemoceptivecells. By laser-scanning confocal microscopy,the comparison of α-tubulin with GLUT2 andSGLT1 antibodies showed similar results inlean and obese rats. In particular, α-tubulin-positive cells were also GLUT2 positive, but thestaining was not colocalized, being that of α-tubulin on the cilia, and GLUT2 at the base ofthe cilia. However, not all α-tubulin-positivecells were also GLUT2-immunoreactive(Figure 5 A,B). Through a comparison of α-tubulin and SGLT1 antibodies, SGLT1 expres-sion was never found colocalized with α-tubu-lin, confirming that SGLT1-immunoreactivityis selective for chemosensory cells but absentin other cell types (Figure 5 C,D). In thisregard, to verify whether SCCs retain theirimmunohistochemical phenotype, we alsocompared α-tubulin with α-gustducin. Asexpected, we observed α-gustducin-immuno -reactive cells that did not express α-tubulin inboth Zucker rat genotypes (Figure 5 E,F). Thecomparison of α-tubulin and T1R3 expressionproduced different results in lean and obeseZucker rats. In lean rats, coexpression of α-tubulin and T1R3 was observed in cilia. T1R3expression was also found in the basolateralmembrane of α-tubulin-expressing cells.However, some α-tubulin-positive cells lackedT1R3-expression, and some T1R3-positive cellslacked α-tubulin expression, supporting theidea that T1R3 is expressed in different celltypes (Figure 6, line A). No specific labelingwas seen when glucose transporters and T1R3antibodies were preincubated with the corre-sponding antigen peptide, or the second pri-mary antiserum was replaced with normalserum (Figure 6, line B). In obese rats, differ-ent patterns of double labelling were found. Insome animals, α-tubulin-positive cells showedspots of T1R3 staining on the cilia and at thebase of cilia, but this labelling pattern was lessfrequent than in lean rats (Figure 6, line C). Inother obese animals, the majority of α-tubulin-positive cells lacked T1R3 coexpression.Cytoplasmic vesicles labeled for T1R3 were fre-quently observed in ciliated cells when cilialacked T1R3 expression (Figure 6, line D).

Ghrelin and GRLN-R expression inepithelial cells of the trachea Ghrelin and GRLN-R expression were investi-

gated in the tracheal epithelium of both Wistar

and Zucker rats for a better comparison of theresults. The investigation was conducted usingan anti-ghrelin antibody which recognizes theactive, acylated form of ghrelin, a 28 amino acidsequence with an octanoyl group attached toSer3. Ghrelin acylation is essential for the bind-ing of the hormone to its receptor GRLN-R and toelicit its action. The immunohistochemicalanalysis was performed using immunoperoxi-dase and single or double-labeling immunofluo-rescence. Immunostaining was completely abol-ished in parallel sections by pretreatment of theantibody with the corresponding antigen pep-tide. In normal Wistar rats, two different cellularpatterns of ghrelin expression were detected.Some cells exhibited ghrelin immunoreactivityon the apical portion that appeared with thecharacteristic brush of microvilli. These cellswere identified as SCCs and in particular asbrush cells (Figure 7A). The immunoreactivecells were solitary or in clusters of two cells.Other cells showed ghrelin immunoreactivity incytoplasmic vesicles, localized mainly in theGolgi zone. Their apical surface was devoid ofmicrovilli, and they were identified as secretorycells (Figure 7B and 7C). Ghrelin expression

was also observed in cells in proximity to thebasement membrane, probably basal cells, andinside the vessels of the lamina propria. To con-firm immunohistochemical findings and furtherdefine ghrelin localization, double immunofluo-rescence microscopy was performed using anti-acetylated α-tubulin and anti-glucose trans-porter (GLUT2 and SGLT1) antibodies. Theseexperiments showed that ghrelin staining wasnever colocalized with that of α-tubulin (Figure7 D,E), or GLUT2 (Figure 7 F,G). However, ciliat-ed cells with cilia-GLUT2 negative showed ghre-lin expression in the microvilli on their apicalsurface (Figure 7F). Ghrelin and SGLT1 expres-sion were never colocalized but ghrelin-express-ing cells were often found in close proximity toSGLT1-positive cells (Figure 7 H,I). In leanZucker rats, ghrelin immunoreactivity was sub-stantially similar to that detected in Wistar rats(Figure 7 J-N). In obese Zucker rats, ghrelinexpression was observed in the microvilli ofSCCs which exhibited a more extensive apicalsurface than those observed in controls (i.e.,hypertrophic brush cells; Figure 7 O,P). Ghrelinimmunoreactivity was also observed in themicrovilli of the apical surface of some but not

Original Paper Original Paper Original Paper

Figure 6. Double-confocal microscopy for α-tubulin with T1R3 in trachea of Zucker rats.The immunofluorescence staining shows expression of α-tubulin (red) with T1R3 (green) inthe epithelium of lean (lines A, B) and obese (lines C, D) Zucker rats. In lean rats, α-tubulinand T1R3 are colocalized in some spots on the cilia of tracheal cells (line A). T1R3 labelingis also observed in the apical cytoplasm beneath the cilia, and on the basolateral membraneof ciliated cells. Double-labeling is not observed when anti-α-tubulin antibody was omitted(line B). A reduced colabelling pattern of α-tubulin and T1R3 is observed in obese comparedwith lean rats, (line C). α-tubulin-positive cells lacking T1R3 expression, and only T1R3-positive cells are also observed (line D). Scale bars: 10 µm.



all ciliated cells (Figure 7Q). Ghrelin expressionin cytoplasmic vesicles of secretory cells wasmarkedly reduced or absent (Figure 7R). GRLN-R immunostaining was found in the apical por-tion of cells that were identified as brush cellsboth in Wistar and Zucker rats (Figure 8 A andC, respectively). Positivity was also observed inthe apical microvilli of some ciliated cells both inWistar and Zucker rats (Figure 8B).

Discussion Ultrastructure of the tracheal mucosain lean and obese Zucker rats The present study is the first, to our knowl-

edge, to provide information on the fine struc-ture of tracheal mucosa in lean fa/+ and obesefa/fa Zucker rats. In the majority of lean Zucker

rats (9 animals), the tracheal mucosa presentednon-obvious signs of structural alterations,especially concerning the differentiation of thevarious cell types that comprise the epithelium.In the remaining 3 lean animals, the trachealmucosa alternated morphologically normaltracts, with others having an increased thick-ness of the lamina propria. In obese animals,the most distinctive feature of the epithelium

Original Paper

Figure 7. Ghrelin expression in trachea of Wistar and Zucker rats. Immunoperoxidase (A, B, J-R) and immunofluorescent (C-I) stainingshows ghrelin immunoreactivity in the epithelium of Wistar (A-I), lean (J-N), and obese (O-R) Zucker rats. Double-immunofluorescent con-focal microscopy shows ghrelin immunoreactivity (red) in cells negative for α-tubulin (green; D, E), or GLUT2 (green; F, G), or SGLT1(green; H, I). Scale bars: A-I) 20 µm; J-R) 15 µm.

Figure 8. Ghrelin receptor (GRLN-R) expression in trachea of Wistar and Zucker rats. Immunoperoxidase (A, B) and immunofluorescent(C) staining shows GRLN-R immunoreactivity in the epithelium of Wistar (A), lean (B), and obese (C) Zucker rats. Scale bars: 9 µm.



was the presence of numerous cells having mor-phological characteristics of intermediate cells.Two distinct types of intermediate cells wereobserved. One is characterized by cells with fewcilia and many microvilli on the apical mem-brane surface; these were considered interme-diate ciliated cells. The other is represented bycells with few granules, scarce cytoplasm and anabundant profile of rough endoplasmic reticu-lum: they were evaluated as intermediate secre-tory cells. In epithelia with mild morphologicalalterations, differentiated and intermediatecells coexisted and maintained the normalepithelial structure. In more severe alterations,the epithelium was largely composed of inter-mediate cells, or presented aspects of cellulardepletion over large areas. Intermediate cellshave already been described in the airways, andthey were considered to be immature cells,probably pre-ciliated cells.39,40 Such an interpre-tation may be valid for our data too. The inter-mediate cells may represent attempts to repairepithelial damage by secretory and basal cells,which are regarded as progenitor cell pools,while ciliated cells are considered to be termi-nally differentiated cells, not capable of self-renewal or trans-differentiation.41,42 However,despite the presence of an intact basal cell layer,the ciliated and secretory cells did not appear toregenerate the lost cells, either because thecells degenerated at a much fast rate than theirreplacement, or because the intermediate cellswere not able to reach full differentiation.Unlike the other cells, the SCCs appeared welldifferentiated, devoid of signs of suffering andeven with aspects of hypertrophy, both in leanand obese Zucker rats. The resistance of SCCsis in agreement with findings in previous stud-ies. In particular SCCs have been described inrodents as a stable population that does notshow the turnover of the surrounding epithelialcells.43 They are terminally differentiated cells,like ciliated cells, and the majority of them aregenerated perinatally, but can be regeneratedfrom proliferative basal cells after trachealinjury.43 Here, we observed clusters of SCCs inthe tracheal epithelium of both Zucker rat geno-types. They were formed by cells with differentmorphology, thus resembling the buds of thegustatory papillae.44 We hypothesize that theyrepresent functional units, in which cells thatfunction as chemoreceptors coexist with cellsoperating as effectors; together they cooperateto respond to environmental stimuli. In thisstudy, the ultrastructural observations of themucosal and submucosal layers together withthe quantitative evaluations have shown thatmorphological abnormalities (such as thicken-ing of the basal membrane and the deposit ofLDs) in the lamina propria tend to be more pre-dominant in obese animals compared to leanrats. Moreover, the LDs deposits in the vesselsand the LDs diameter in the cartilage are signif-

icantly different in the two Zucker rat geno-types. In obese animals, the amorphous materi-al observed lying beneath the epithelium consti-tuted a layer of remarkable thickness, inter-posed between the basement membrane andthe lamina propria. In the areas in which thismatrix was of substantial thickness, the overly-ing epithelium was particularly badly damaged.The matrix was devoid of the typical cell popula-tion of lamina propria (i.e., fibroblasts, lympho-cytes, plasma cells, macrophages, leukocytes,eosinophils). An abnormal matrix may precludeall those processes generated by interactionsbetween adjacent cells, and bi-directional com-munication between the epithelium and under-lying layers.45 As regards the presence of LDs,there seems to be general agreement that lipiddeposits are a determining factor linking obesi-ty with inflammatory diseases, such as asthma,since the adipokines released by lipids areregarded as responsible for the developmentand escalation of the inflammatory response.46

In this respect, in the lamina propria of obeserats we observed a substantial number ofinflammatory cells such as eosinophils, lympho-cytes, and mast cells, which are the characteris-tic infiltrating cells in inflammatory processes.However, no specific marker of airway inflam-mation was evaluated in this study because thisaspect lays outside the scope of our work. Onthe other hand, the liposomal material inmicrovessels observed in this study was anunexpected result, which has not previouslybeen described in other pathologies. We foundthat the difference in the area covered by lipiddeposits and the respective area of the vesselswas statistically significant in the two Zuckerrat genotypes. Due to its characteristics of elec-tron-density, it is reasonable to assume that thedeposit was formed by lipoproteins. Similarly,we observed that the LDs diameter in the carti-lage was significantly different in lean andobese rats. Their presence was detected in aprevious morphological study in different mam-malian species, and described as a patterninfluenced by various factors including the ageof the subject, and the site of the cartilage.45 Thebiological significance of lipid storage inhealthy chondrocytes is unknown, but it isthought to be a deposit of material that can beused as a source of energy for the metabolicactivity of the cell, or for other metabolicrequirements of the animal.47

Taken together, our data demonstrate thatsubstantial structural changes are a character-istic pattern of the tracheal mucosa of rats withgenetic obesity.

Sweet taste signaling molecules aredifferently expressed in trachealepithelial cells of Zucker ratsOur study is the first that investigates the

expression of chemosensory markers and glu-

cose transporters in the tracheal epithelium oflean and obese Zucker rats, and provides evi-dence of their differentiated expression in thetwo Zucker rat genotypes. In obese rats, GLUT2expression was reduced in ciliated cells butwas always located just beneath the cilia in theapical pole of the cells. Immunolabeling forSGLT1 was similar to that observed in Wistarrats.12 However, our major finding is certainlythe substantial change in T1R3 expression inthe tracheal epithelium of obese rats, in whichT1R3 expression was strongly modified in cili-ated cells. Inside these cells, T1R3 expressionwas reduced on the cilia, at the base of cilia inthe apical cell pole, and on the basolateralmembrane in some animals. In others, T1R3immunoreactivity was not detected at thesesites but appeared in cytoplasmic vesicles,irregularly distributed through the cytoplasm.This is a somewhat unrelated finding becausesuch positivity was never observed in our pre-vious work.12 The impaired expression of T1R3in obese rats may be of physiological impor-tance because it could have consequences onthe chemoreceptive mechanisms linked to thespecific roles of ciliated cells. Recently, impor-tant advances have been made in clarifying therole of ciliated cells. It is currently believedthat they are likely to function as chemosenso-ry cells since components (such as bitterreceptors, α-gustducin, and TRPM5) of sweetand bitter taste transduction pathways werefound to be expressed on the cilia, with thefunction of activating pathways whichincrease intracellular Ca2+.15 These data pro-vide strong support for the hypothesis that cil-iated cells might participate in the functionaldetection of substances in the lumen, probablya protective response. Protection frompathogens and noxious compounds is providedby ciliary beat frequency (CBF) and mucocil-iary transport that are regulated by Ca2+, butthe exact mechanism of this process remainscontroversial.48 A signal known to increaseCBF is ATP-mediated activation of G protein-coupled receptors which promotes the releaseof Ca2+. Increases in cytosolic Ca2+ are associ-ated with increases in CBF, but Ca2+ signalsare most efficient in CBF regulation when pro-duced at the base of the cilia. This is the sitewhere the expression of PLCβ2 and α-gust-ducin were detected by Shah’s team,15 and thecoexpression of T1R3 and GLUT2 by us.12 Thepotential involvement of T1R3 in processesinvolving Ca2+ signaling is unlikely to occur inobese animals given the alterations observedin both T1R3 and GLUT2 expression, with obvi-ous effects on mucociliary clearance. However,it is interesting to note that GLUT2, SGLT1 andT1R3 expressions in SCCs are similar in obeseand lean animals, suggesting that, in additionto their morphological characteristics, SCCsalso preserve their immunohistochemical phe-

Original Paper



notype. SCCs are a distinct cell lineage, there-fore it is possible that chemoreceptor markers,although similar to those found in other celltypes, are differently modulated in them.

Ghrelin and GRLN-R are localizedin epithelial cells of the trachea There is now growing evidence that ghrelin

has widespread expression in many tissues,but data regarding ghrelin expression in tra-cheal epithelium are scarce. A single study infetal human trachea revealed ghrelinimmunoreactivity in basal cells.49 In the samestudy, significant expression of ghrelin wasalso observed in neuroendocrine and other celltypes of the fetal and infant human lung, sug-gesting that the developing lung may be asource of circulating ghrelin in addition, ofcourse, to the gastric mucosa.In the present study, using immunohisto-

chemical approaches, we provide the first evi-dence that ghrelin and GRLN-R are expressedin rat tracheal epithelium. Ghrelin was detect-ed in two different cell types in both Wistar andZucker rats, and was differentially expressedin the two Zucker rat genotypes. In obeseZucker rats, SCCs express immunoreactivityin the apical microvilli but cells with ghrelinexpression in cytoplasmic vesicles were notdetected. Furthermore, in some obese animals,ghrelin was also expressed in the microvilli onthe apical surface of ciliated cells. We furtherestablished the presence of GRLN-R in the api-cal microvilli of SCCs both in Wistar andZucker rats. These findings were consistentwith the ultrastructural data, which demon-strate that SCCs are well conserved in the tra-cheal epithelium of obese rats, suggesting thatobesity does not modulate chemosensoryexpression in Zucker rats, since obese fa/faand lean fa/+ rats presented similar ghrelinlocalization in brush cells. The distinct cellularlocation of ghrelin suggests that it can play dif-ferent roles in cellular processes: the localiza-tion on microvilli of SCCs represents achemosensory pattern, while that in cytoplas-mic vesicles is a secretory pattern. Ghrelinmay function as a signaling molecule which,after binding to its specific receptor, initiatesthe appropriate signal transduction throughCa2+-dependent signaling pathways, GRLN-Rbeing a G-protein-coupled receptor.29

Concerning the presence of ghrelin-immunoreactive vesicles, it is plausible toassume that they are located in the cytoplasmof secretory cells that synthesize and activateghrelin. This is in agreement with previousobservations of mouse gastric cells.50 Thereduced expression of ghrelin-positive vesiclesin obese Zucker rats compared with lean ratscould be related to the altered secretory pat-tern observed at ultrastructural level.Altogether, our results indicate that two dis-

tinct populations of ghrelin-expressing cellsmay be operative in rat trachea: SCCs mightact as sensors able to detect luminal stimuli,and secretory cells as effectors that releaseghrelin. In this context, it is plausible to postu-late that a communication route between thetwo cell types may modulate the release ofghrelin according to the stimuli provided bythe luminal contents. This hypothesis is con-sistent with similar results in the gastroin-testinal tract. In the murine stomach, theexpression of ghrelin has been shown in T1R3-immunoreactive brush cells arranged in closecontact with close-type endocrine cellsexpressing both T1R3 and ghrelin. In the samestudy, it was proposed that a chemosensoryinput, perceived through T1R3, induces ghre-lin secretion by the endocrine cells.51

Interestingly, a subsequent report demonstrat-ed that in the mouse stomach, ghrelin isexpressed in cells located in close proximity toα-gustducin-positive brush cells, and inendocrine cells.52 Here, it was supposed thatbrush cells activate the release of ghrelin bythe secretory cells; whereas, the endocrinecells, which coexpress ghrelin and gustatory G-proteins, α-gustducin and α-transducin, actsimultaneously as sensor and effector cells.The same study also provided functional evi-dence that activation of T2Rs bitter tastereceptor stimulates ghrelin secretion and /oracylation via the α-gustducin signaling path-way. Moreover, a recent report showed thathigh levels of TRPM5 mRNA correlated with ahigher number and density of chemosensorycells in the gastric mucosa of obese patients.TRPM5 and ghrelin are not colocalized butexpressed by neighboring cells, suggestingparacrine cell-cell communication betweenchemosensory cells and ghrelin-expressingcells.53 Collectively, the reported data provideclues that a link between the chemosensorysystem and ghrelin secretion and/or acylationexists in the gastrointestinal tract, althoughthe exact mechanism underlying the nutrient-dependent regulation of ghrelin release is notyet clear. In accordance with these data, a similar

autocrine/paracrine regulation can be pro-posed in the trachea. We suggest that ghrelinmay be locally produced by the epithelial tra-cheal cells, and exert a local action throughbinding to GRLN-R located in proximal SCCs.Alternatively, by analogy with what happens inthe intestine, it is also possible that secretionof ghrelin is induced by activation of T1Rs onthe apical surface of SCCs. In this case, thereduced expression of T1R3 in obese animalsmay be linked to defective signaling of secre-tion. In line with this hypothesis, in humangastric mucosa of obese patients, decreasedT1R3 mRNA was demonstrated concomitantwith an increased number and density of ghre-

lin-positive cells which, considering the lowplasma ghrelin levels found in obesity, wasinterpreted as hypoactivity of ghrelin secretingcells.53 Considering that ghrelin contributesprimarily with its anti-inflammatory propertyto attenuate the damage produced by inflam-matory mediators, such as neutrophils,chemokines, and lipopolysaccharide in vari-ous lung diseases,54 the most attractive of itsrole in ASL might be a protective, anti-inflam-matory action against cytokines and inflamma-tory products released by bacteria that colonizerespiratory fluids. This hypothesis was previ-ously posited for tracheal SCCs because theywere shown to be able to respond to quorumsensing molecules.55,56

Taken together, all the morphological abnor-malities characterizing the tracheal mucosa ofobese rats may impair their ability to maintainnormal tracheal homeostasis. In obese ani-mals, the expression of the investigated mole-cules (GLUT2, SGLT1, T1R3) is changed onlywhen they are expressed in ciliated cells, andis not affected in SCCs. SCCs and ciliated cellsbelong to different cell lineages, and thereforemay have a different susceptibility to metabol-ic factors and/or nutritional changes. The loca-tion and appearance of ghrelin-expressingcells strongly suggests that ghrelin can act inconcert with different cell types, which reflectsthe level of complexity of the digestive system.Although the role of ghrelin in the airwaysshould be further investigated, its presencecan be useful for a better understanding of thepathways regulating glucose sensing and thedesign of new therapeutic targets for restoringglucose homeostasis in airways.

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Molecules implicated in glucose homeostasis are differentially