Submandibular gland and caries susceptibility in the obeseZucker rat

Mahmood S. Mozaffari1, Rafik Abdelsayed

2, Ibrahim Zakhary

1, Mohammed El-Salanty

1, Jun Yao Liu

1,

Hereward Wimborne1, Ahmed El-Marakby

1

1Department of Oral Biology, Medical College of Georgia School of Dentistry, Augusta, Georgia; 2Department of Oral Health andDiagnostic Sciences, Medical College of Georgia School of Dentistry, Augusta, Georgia

BACKGROUND: Obesity is a prevalent disorder char-

acterized as marked insulin resistance and low grade

inflammation. We tested the hypothesis that obesity

upregulates inflammatory markers in the submandibular

gland in association with derangements of its architec-

ture and pre-disposition to caries in obese Zucker rats

(OZR). We also examined the potential impact of chro-

mium picolinate (Cr(Pic)3), a nutritional supplement

suggested to improve glycemic control, on the afore-

mentioned parameters.

DESIGN: Male OZR were treated with diets lacking and

containing 5 or 10 mg ⁄ kg chromium (as Cr(Pic)3) from

6 weeks to about 6 months of age; lean Zucker rats (LZR)

served as controls. Thereafter, glycemic status, salivary

tissue architecture, and the levels of several inflamma-

tory markers were determined in association with caries

susceptibility.

RESULTS: OZR showed reduced insulin sensitivity,

increased ratio of phospho-nuclear factor-kappa B

(NF-jB) to total NF-jB, and increased intercellular

adhesion molecule-1 level but similar histological features

compared to LZR. Importantly, compared to LZR, OZR

displayed rampant caries and a tendency for reduced

dentin mineral density. Treatment of OZR with Cr(Pic)3

attenuated upregulation of these proinflammatory indi-

cators in association with reduced severity of caries

without improving insulin sensitivity.

CONCLUSIONS: Obesity promotes proinflammatory

changes within the submandibular gland, without affect-

ing glandular architecture, in association with rampant

caries; Cr(Pic)3 treatment provided some protective

effects.

J Oral Pathol Med (2011) 40: 194–200

Keywords: caries; cell adhesion molecules; chromium picolinate;

NF-jB; submandibular gland; Zucker rat

Introduction

Obesity increases the risk for the development ofsystemic disorders such as type 2 diabetes and cardio-vascular diseases (1, 2). Surprisingly, however, informa-tion on the impact of obesity on oral health is sparse. Asobesity represents a state of low grade inflammation (3,4), it could potentially influence oral health as exempli-fied by the close correlation of obesity with periodontaldisease and other chronic inflammatory diseases (e.g.,type 2 diabetes) (2, 5, 6). Indeed, reduction in salivaryflow rate and increased caries susceptibility have beenreported for the Prader–Willi Syndrome (PWS); PWS isan example of a monogenic disorder (i.e., abnormalitiesof chromosome 15) resulting in marked obesity (7).Given the limitations of clinical studies, the impact ofobesity on oral health in relation to salivary glands canbe better established with the use of appropriate animalmodels of the disease.

The obese Zucker rat (OZR) lacks the leptin receptorbecause of an autosomal recessive mutation of thefa-gene. As a result, the animal is hyperphagic andbecomes markedly obese compared to its lean control.Other characteristic features of the OZR are peripheralinsulin resistance, hyperinsulinemia, and glucose intol-erance (8, 9). Utilizing this animal model, we tested thehypothesis that obesity causes derangements of thesubmandibular gland architecture in associated withincreased expression of the key proinflammatory tran-scription factor, nuclear factor-kappa B (NF-jB), andits downstream mediators (e.g., vascular adhesion mol-ecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). These studies were carried out in thecontext of assessment of carious lesions as a surrogatemarker of oral health status. In addition, we alsoexamined the impact of chronic chromium picolinate(Cr(Pic)3) treatment of the OZR on aforementionedparameters. This nutritional supplement has gainedpopularity because of its purported beneficial influenceon glycemic control (10–12). Further, a recent studysuggests that Cr(Pic)3 attenuates the plasma levels ofseveral inflammatory markers (13). Accordingly, we

Correspondence: Dr. Mahmood S. Mozaffari, Department of OralBiology; CL2134, Medical College of Georgia, Augusta 30912-1128,Georgia. Tel: 706 721 3181, Fax: 706 721 6276, E-mail: Mmozaffa@mail.mcg.eduAccepted for publication September 8, 2010

J Oral Pathol Med (2011) 40: 194–200

ª 2010 John Wiley & Sons A/S Æ All rights reserved

wileyonlinelibrary.com/journal/jop

doi: 10.1111/j.1600-0714.2010.00965.x

Journal of

Oral Pathology & Medicine

postulated that chronic Cr(Pic)3 treatment will reduceinsulin resistance and the expression of proinflammatoryfactors in association with preservation of salivary glandarchitecture and reduced caries susceptibility.

Materials and methods

Six-week-old male lean and obese Zucker rats (i.e., LZRand OZR, respectively) were obtained from HarlanLaboratories (Indianapolis, IN, USA) and were fed theregular non-cariogenic rodent diet (Harlan Teklad diet8604). Additional OZR were fed Cr(Pic)3-enriched 8604diet providing chromium at a dose of either 5 or10 mg ⁄ kg diet (i.e., OZR; 5 Cr and OZR; 10 Cr; HarlanTeklad diet numbers 07602 and 07603, respectively)(14–16), providing chromium at a dose of 0.19 and0.41 mg ⁄ kg ⁄ day, respectively. The animals (n = 7–9animals ⁄ group) had free access to food and waterthroughout the study. The use of animals in this studywas in accordance with the NIH guide on HumaneTreatment of Laboratory Animals.

At about 6 months of age, fasting plasma glucose(Beckman Glucose Analyzer, Beckman InstrumentsInc., Brea, CA, USA) and insulin (125I radio-immuno-assay kit; MP Biomedicals, LLC, Solon, OH, USA) weremeasured and used to calculate the quantitative insulinsensitivity check index (QUICKI) as follows: 1 ⁄ [logfasting insulin (lU ⁄ml) + log fasting glycemia (mg ⁄ dl)](17). Thereafter, the animals were sacrificed and thesubmandibular glands procured. The right submandib-ular gland was immediately frozen in liquid nitrogen forWestern blot analysis; the left submandibular gland wasplaced in buffered formalin for histopathological exam-ination as described previously (16, 18).

For Western blotting, the salivary gland tissue waspulverized and placed in 1· RIPA buffer (MilliporeCorp., Billerica, MA, USA) composed of 10 ll ⁄mlProtease Inhibitor Cocktail (Sigma-Aldrich, St. Louis,MO, USA), 20 mM of sodium orthovanadate, 10 mMof sodium fluoride, 1 mM of sodium pyrophosphatetetra-basic decahydrate. Protein concentration wasmeasured using Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA). Standard protocols wereused for Western blot analysis as described previously(19), utilizing primary antibodies against NF-jB,phospho-NF-jB, VCAM-1 (Santa Cruz BiotechnologyInc., Santa Cruz, CA, USA), and ICAM-1 (goat IgG;R&D Systems, Minneapolis, MN, USA); secondaryantibodies included goat anti-rabbit (Cell Signaling,Danvers, MA, USA) and donkey anti-goat (Santa Cruz,CA, USA); protein levels were corrected for b-actin andexpressed as percent of the LZR group.

For caries detection, five hemi-mandibles of eachgroup were dissected and clinically examined for theexistence of coronal cavities. The hemi-mandibles werethen scanned in a micro-computerized tomographysystem (micro-CT; Skyscan 1172; Skyscan, Aartlesaar,Belgium) at an image pixel size of 30 lm. Reconstruc-tion was carried out using a Skyscan Nrecon program.Data sets were loaded into Skyscan CT-analyzer soft-ware for the measurement of mineral density of dentin

and the evaluation of radiological evidence of cariouslesions; the average of three standardized regions ofinterest, one at each molar tooth, was measured as wellas the average of two regions of interest in a recognizedcoronal cavity (20, 21).

StatisticsData were analyzed by the analysis of variance(ANOVA). Duncan’s post hoc test was used to establishsignificance between mean values (P < 0.05). All dataare reported as means ± SEM.

ResultsEffects of obesityThe OZR gained more weight than their lean counter-parts (Fig. 1A). Salivary gland tissue weight was lowerin the OZR than the LZR (Fig. 1B). The OZR displayedmarked elevation in plasma insulin concentrationaccompanied by a mild increase in plasma glucose level

Figure 1 Bar graphs show body weight (A), submandibular glandtissue weight (B), and QUICKI values (C) of lean Zucker rats (LZR),obese Zucker rats (OZR), and OZR treated with chromium picolinate-enriched diets providing chromium at 5 and 10 mg ⁄ kg of diet (OZR; 5Cr and OZR; 10 Cr, respectively). Data are average ± SEM of 7–9animals ⁄ group. *P < 0.05 compared to other groups. QUICKI,quantitative insulin sensitivity check index.

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compared to the LZR group [(insulin: 584.5 ± 31.8 vs.148.5 ± 10.1 pM; P < 0.05) and (glucose: 9.2 ± 0.4vs. 7.3 ± 0.3 mM; P < 0.05)]. As a result, the QUICKIvalue, an index of insulin sensitivity, was significantlylower for the OZR than the LZR (Fig. 1C).Histologic evaluation of hematoxylin–eosin (H&E)-

stained tissue revealed well-preserved lobular morphol-ogy with intact, uniform aggregates of parenchymalsecretory units interspersed by evenly spaced intercalatedducts in both LZR and OZR groups (Fig. 2, panels A–B). Cytologic and nuclear details were uniform in shapeand size and staining characteristics. Trichrome-stainedtissue sections showed normal lobular architecture with-out intralobular fibrosis or collagen deposition (Fig. 2,panels E–F). Surrounding some of the striated andexcretory ducts, thin perilobular fibrous tissue is noted.Tissue sections stained with Oil-Red-O revealed fewscattered positive intra- and extracellular fatty dropletdeposits in LZR; the OZR showed more fatty dropletsper same unit area of examination (Fig. 2, panels I–J).Figure 3 shows expression profile of proinflammatory

mediators in the salivary tissue of experimental animals.The phospho-NF-jB ⁄ total NF-jB ratio was greater inthe OZR than LZR (Fig. 3A). Similarly, ICAM-1 levelwas greater in salivary tissue of the OZR than their leancounterparts (Fig. 3B). However, VCAM-1 expressionwas not affected (Fig. 3C).

On clinical examination, the LZR showed minimal orno caries (Fig. 4A) while the untreated OZR groupshowed rampant caries ranging from simple class onecavity to badly decayed teeth involving the entire crown(Fig. 4B). Micro-CT shows no signs of coronal cavitiesin the LZR group (Fig. 4E) as opposed to the OZRgroup with the clear evidence of carious lesions(Fig. 4F). Also, three dimensional reconstructs showedevidence of decay at various stages in OZR compared toLZR as evidenced by badly decayed teeth (Fig. 4J vs.4I).

Dentin mineral density (g ⁄ cm3) was lower, albeit non-significantly, in the OZR group compared to the LZRgroup (0.56 ± 0.06 vs. 0.66 ± 0.05).

Effects of Cr(Pic)3 treatmentTreatment of OZR with the nutritional supplement,Cr(Pic)3, did not affect body weight or salivary glandweight compared to their untreated counterparts(Fig. 1A–B). Also, plasma glucose concentration(9.7 ± 0.4 and 9.8 ± 0.3 mM for OZR; 5 Cr andOZR; 10 Cr, respectively), plasma insulin concentration(508.9 ± 45.6 and 555.6 ± 69.0 pM for OZR; 5 Cr andOZR; 10 Cr groups, respectively), or the QUICKI valuewere similar among the two Cr(Pic)3-treated OZRgroups and the untreated OZR group (Fig. 1C andabove, under the effects of obesity).

LZR

A B C D

E F G H

I J K L

OZR OZR; 5 Cr OZR; 10 Cr

Figure 2 Panels show representative hematoxylin–eosin-stained (A–D; ·100), Masson’s trichrome-stained (E–H; ·100), and Oil-Red-O-stained(I–L, ·200) sections of the submandibular glands from experimental groups. Arrows point to oil droplets in panels I–L.

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The Cr(Pic)3-treated OZR showed histologicalfeatures generally resembling their untreated counter-parts (Fig. 2). Nonetheless, tissue sections from OZR;10 Cr showed a benign expansion of the secretory acinarunits, spacing out the ductal structures (Fig. 2D), butwith preserved lobular morphology and glandularmicroanatomy. The proliferative acinar units wereuniform with respect to cellular and nuclear details.

A noted finding was the observation that dietaryCr(Pic)3 treatment of the OZR was associatedwith reversal of the upregulation of the phospho-NF-jB ⁄ total NF-jB ratio and ICAM-1 (Fig. 3A–B).Also noteworthy was the finding that Cr(Pic)3-treatedOZR groups showed carious lesions that were interme-

diate of those noted for LZR and untreated OZR(Fig. 4). However, dentin mineral density (g ⁄ cm3) wassimilar between the Cr(Pic)3-treated OZR [(OZR; 5 Cr:0.61 ± 0.05) and (OZR; 10 Cr: 0.60 ± 0.10)] and theiruntreated counterparts (0.56 ± 0.06).

DiscussionEffects of obesityThis study shows upregulation of a key proinflamma-tory transcription factor, NF-jB, and increased expres-sion of ICAM-1 in the submandibular gland of the OZRthan LZR. However, histological features of the salivarytissue were generally similar between the OZR com-pared to the LZR control group. Importantly, com-pared to LZR, molar teeth of OZR displayed prominentcarious lesions. To our knowledge, this is the first studythat has examined the impact of obesity on salivarygland structure in the context of key inflammatorymediators and caries susceptibility.

Adipose tissue is now considered as a major endocrineorgan producing a myriad of proinflammatory factorsamong others. The association of obesity with inflam-mation was first proposed by the seminal studies ofHotamisligi et al. (3) which established tumor necrosisfactor-a (TNF-a) as a key factor linking inflammation,obesity, and insulin resistance. Subsequent studiesdemonstrated that binding of TNF-a with the TNFreceptor results in the mobilization of complex signalingpathways, one arm of which leads to serine phosphor-ylation of insulin receptor substrate-1, culminating inthe inhibition of insulin signaling (22). Another conse-quence of TNF-a signaling is the activation ofIKKb ⁄NF-jB pathways. Aside from being a majorintracellular pathway regulating insulin resistance,upregulation of NF-jB culminates in the expression ofproinflammatory adhesion molecules such as ICAM-1and VCAM-1 (23). Thus, obesity represents a proin-flammatory state with elevations in plasma concentra-tions of a myriad of inflammatory mediators ⁄markerssuch as IL-6, C-reactive protein, and TNF-a (2, 6, 23).Interestingly, it is also believed that macrophages whichreside within adipose tissue (and derived from bloodmonocytes) are a source of proinflammatory factors andthat they could modulate the secretory capacity ofadipocytes. Indeed, the mononuclear cells (derived frommonocytes) of obese subjects have been shown toexpress increased amounts of proinflammatory cyto-kines and related factors (23). These cells also haveincreased binding capacity for NF-jB and increasedintracellular expression of p65, the major proteincomponent of NF-jB (23).

Consistent with our hypothesis that obesity upregu-lates inflammatory changes, salivary gland of OZRdisplayed the upregulation of NF-jB in association withincreased expression of ICAM-1 compared to their leancounterparts. This finding is in general agreement withother reports indicating that OZR display increased theexpression of a number of proinflammatory markers inother tissues (e.g., cyclooxygenase-2 in renal microvas-culature and whole kidney) (16, 24). By contrast,

A

B

C

Figure 3 Bar graphs show the phospho-NF-jB ⁄ total NF-jB ratio(A), levels of ICAM-1 (B), and VCAM-1 (C) in the submandibulargland of experimental groups as described in Figure 1; the ICAM-1and VCAM-1 values are normalized to b-actin and expressed aspercent of the LZR group. Also shown are representative blots foreach group. *P < 0.05 compared to other groups. VCAM-1, vascularadhesion molecule-1; LZR, lean Zucker rat; ICAM-1, intercellularadhesion molecule-1.

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VCAM-1 expression was similar between the OZR andthe LZR which may relate to its determination in wholesalivary tissue rather than in salivary tissue micro-vasculature. Nonetheless, despite promotion of aninflammatory state, histopathological examination ofH&E-stained salivary gland tissue did not reveal differ-ential changes between OZR and their lean counterparts.Ectopic lipid accumulation is advanced as a potential

mechanism for target organ dysfunction ⁄ damage inobesity ⁄ diabetes (i.e., lipotoxicity mechanism) (25).Interestingly, however, despite mild differences, Oil-Red-O staining did not reveal marked accumulation offatty droplets within salivary gland tissue of OZRcompared to their lean counterparts. In this context,recent studies have suggested that periorgan fat depo-sition, as occurs in OZR, can serve as a potentialsource for locally released chemokines ⁄ cytokines withdetrimental effects on the involved organ. Indeed, anadverse effect of pericardiac fat deposition has beensuggested to play a major pathogenic role in cardiacdysfunction of fat-fed rats (26). It remains to beestablished whether a similar mechanism contributes tothe abnormalities of salivary gland (e.g., encapsulatedorgans) in obesity.

The most noted finding of the study relates torampant caries in the OZR than LZR. Clearly, cariesdevelopment is the culmination of the interactionsamong host factors, microorganisms, substrate, andtooth surface over time. As LZR and OZR wereprovided with the same non-cariogenic diet, it is unlikelythat dietary-related factors contributed to the increasedcaries in the OZR. Further, to our knowledge, there isno information available regarding the status of oralmicroflora or salivary gland secretory products in OZRwhich precludes attributing their differential contribu-tions to caries susceptibility in this animal model.Nonetheless, it is tempting to speculate that chronicmild–moderate upregulation of proinflammatoryNF-jB signaling on salivary gland could adverselyimpact salivary function thereby pre-disposing toincreased caries susceptibility. In addition, the smallersalivary gland size could also adversely impact thecontribution of salivary secretions to protection againstcaries. This contention is supported by several lines ofevidence. First, the size of salivary gland is a determi-nant of the salivary flow and protein secretion ratesunder baseline, unstimulated, condition (27). Second, asignificant correlation exists between demineralization

LZR

A B C D

E F G H

I J K L

OZR OZR; 5 Cr OZR; 10 Cr

Figure 4 Hemi-mandible of OZR (panel B) displaying prominent caries compared to that of LZR (panel A) or the OZR; 5 Cr (panel C) and OZR;10 Cr (panel D). On the other hand, panels E–H show micro-CT images of experimental groups showing class I carious lesions (arrows). Finally,panels I–L show three dimensional reconstructs of hemi-mandibles of experimental groups showing badly decayed tooth in untreated OZR (panelJ). OZR, obese Zucker rat; LZR, lean Zucker rat.

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parameters (mineral loss, lesion depth, and mineraldistributions) in experimental root caries and unstimu-lated saliva flow rate (28). Indeed, dentin mineraldensity of molar teeth tended to be lower for OZR thanLZR, an aspect that may also relate, in part, to theprogression of carious lesion into dentin. Third, devel-opment of carious lesions is negatively related to theunstimulated saliva flow rate (29). In addition, theabnormal glycemic status of the OZR could alsocontribute to caries susceptibility. Indeed, previousstudies suggest that the concentration of glucose inparotid saliva is elevated after glucose ⁄ food intake inindividuals with glucose intolerance and diabetes mell-itus (30). This is an important consideration because adirect relation has been established between salivaryglucose concentration and decayed-missing-filled surfacescores in type 1 diabetic children (31). Thus, whileacknowledging differences in the composition of humanand rodent saliva (32), the OZR may prove to be avaluable animal model for investigation of the impactand mechanisms of obesity ⁄ type 2 diabetes on oralhealth.

Effects of Cr(Pic)3 treatmentAnother notable finding of this study relates to the lackof a beneficial influence of chronic Cr(Pic)3 treatment onbody weight or insulin sensitivity of OZR; the chromiumintake of animals in this study was several fold higherthan those consumed by human subjects (14, 16).Nonetheless, the treatment was associated with attenu-ated upregulation of both NF-jB and ICAM-1, therebysuggesting an uncoupling between the effect of theformulation on glycemic control and inflammatorymarkers (33). In this context, it is noteworthy that arecent study shows downregulation of inflammatorymarkers such as IL-6 in plasma of Cr(Pic)3-treated rats(13). Interestingly, the Cr(Pic)3-induced attenuation ofinflammatory markers was associated with reducedseverity of carious lesions in OZR, although therelevance and importance of this association remainsto be established.

Conclusion

While the relation between changes in salivary glandinflammatory markers and presence of caries lesions inthe OZR remains to be established, our collectiveobservations are suggestive of a major adverse impactof obesity on oral health. Interestingly, the nutritionalsupplement, Cr(Pic)3, reversed the upregulation ofsubmandibular gland inflammatory markers in associa-tion with reduction in the severity of caries withoutinfluencing either body weight or glycemic control inOZR.

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Acknowledgements

This study was supported by a grant from the National Institutes of Health

(1R21AT003012-01A2). We thank Dr. David Pashley (MCG School of

Dentistry) for his review of this submission and Nutrition 21 for the

generous gift of chromium picolinate. The authors have no competing

interests.

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