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Characterization of the Modes of Binding between Human Sweet Taste Receptor and Low-Molecular-Weight Sweet Compounds

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Wolfgang Meyerhof, Editor
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Abstract

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2–hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein–coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2–hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling–based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.

Introduction

The human sweet taste receptor (hT1R2–hT1R3) is a heteromeric complex composed of two subunits, T1R2 and T1R3, which are class C G protein–coupled receptors (GPCRs) [1], [2], [3]. Each subunit has a large amino-terminal domain (ATD) linked by an extracellular cysteine-rich domain (CRD) to a seven-transmembrane helical domain (TMD) [4]. hT1R2–hT1R3 responds to a wide variety of chemical substances including naturally occurring sugars (glucose, sucrose, fructose and sugar alcohols), D-amino acids (D-tryptophan and D-phenylalanine) and glycosides (stevioside and glycyrrhizin), as well as artificial chemical compounds such as sucralose, aspartame, neotame, saccharin Na, acesulfame K (AceK), and cyclamate (Fig. 1) [5]. Moreover, naturally occurring sweet proteins, such as brazzein, thaumatin, and monellin, and naturally occurring taste-modifying proteins, such as neoculin and miraculin, also bind to hT1R2–hT1R3 [6], [7], [8], [9], [10], [11]. hT1R2–hT1R3 has multiple ligand-binding sites for these various sweeteners. For example, the ATD of hT1R2 is responsible for binding to aspartame and sugar derivatives [9]. Neoculin binds the ATD of hT1R3 [12]. In contrast, cyclamate and neohesperidin dihydrochalcone (NHDC) bind the TMD of hT1R3 as agonists [13], whereas this region also serves as the allosteric binding site for saccharin and lactisole as antagonists [14].

Figure 1

Chemical structures of the small molecular sweeteners used in this study.

The structural features of the ATD of the homodimeric metabotropic glutamate type 1 receptor (mGluR1) have been identified by X-ray crystal structure analysis, and this was the first example to reveal the structure of a class C GPCR [15]. The ATD of mGluR1 comprises two lobes (LB1 and LB2) that form the glutamate-binding domain lying between LB1 and LB2. The structure of ATD exists in an equilibrium of two different conformations, and the structural change strongly depends on glutamate binding. In the ligand-free state, both LB1 and LB2 tend to show open conformations (open-open), whereas an agonist induces a closed conformation for LB1 and LB2 of one ATD, while the other remains in an open conformation. This closed-open structure is thought to contribute to the active state of mGluR1 [15].

Because hT1R2 and hT1R3 share sequence homology (24% and 23%) with mGluR1 (Fig. S1), they also share some common structural features with mGluR1 [16]. hT1R2–hT1R3 can form a heterodimer, with the open-open form representing an inactive structure and the closed-open form representing an active structure. When low-molecular-weight sweeteners are applied, hT1R2 probably exhibits a closed conformation because the ATD of hT1R2 receives aspartame and sugar derivatives [17], [18]. Not only these small sweeteners but also cyclic sulfamate derivatives such as saccharin sodium and AceK probably bind at the cleft formed by LB1 and LB2 of hT1R2 ATD; they differ from each other in their hydrophobicity, electric charge, molecular size and other parameters (Fig. 1). Naturally occurring hydrophilic sugars are generally different in chemical structure from rather hydrophobic artificial amino acid derivatives and cyclic sulfamate derivatives. Moreover, amino acid derivatives and cyclic sulfamate derivatives have charged groups, whereas sugar derivatives are neutral.

Several ligand-binding sites were proposed by a molecular modeling–based docking simulation for the sweet taste receptor [6], [8], [11], [16], [19]. Thus, the wedge site of an open form of the ATD of the T1R3 was proposed for sweet proteins [6], [8], [16], whereas the involvement of the CRD of the T1R3 was proposed for brazzein, a sweet protein [11]. On the other hand, the cavity of the closed form formed by LB1 and LB2 of either T1R2 or T1R3 [11], [16] is suggested for small sweeteners as glutamate bound in the glutamate receptor [15]. In this study, we found that the various structures of low-molecular-weight sweeteners were recognized by the sweet taste receptor hT1R2–hT1R3 through the different residues at the ligand-binding site of the ATD of T1R2. Modes of binding between hT1R2–hT1R3 and low-molecular-weight sweet chemical substances were characterized both by response profiles of cells expressing the mutated hT1R2–hT1R3 to sweeteners and by a molecular modeling–based docking simulation at the binding cleft formed by LB1 and LB2 of hT1R2. The candidate amino acid residues at the binding cleft of hT1R2 were targeted to produce mutated hT1R2, which was then heterologously expressed in cultured cells together with hT1R3 and its coupling Gα protein. Using the functional analysis of cell-based assays, we successfully determined the residues responsible for binding to each sweetener in the ligand-binding cleft of hT1R2 ATD and found that individual molecules use characteristic residues for binding. A mechanism of receptor activation is also discussed according to a molecular model of the receptor–ligand complex.

Materials and Methods

Site-directed mutagenesis of hT1R2 cDNA

cDNA fragments with point mutations in hT1R2 were synthesized by the overlap PCR method using mutated primer pairs. The following 15 residues in hT1R2 were mutated individually to Ala: S40, K65, Y103, D142, S144, S165, Y215, P277, D278, Y282, E302, S303, D307, E382, and R383. In the cases of Y103, D142, Y215, P277, and R383, each residue was also replaced with residues other than Ala (Y103F, D142R, Y215F, P277G, P277Q, P277S, R383D, R383Q, R383L, and R383H).

Calcium imaging analysis of the heterologously transfected cultured cells

cDNA fragments were subcloned into the pEAK10 vector (Edge Biosystems, Gaithersburg, MD, USA). Each hT1R2 mutant was transiently cotransfected together with hT1R3 and G16-gust44 [20] into HEK293T cells (kindly provided by Dr. Hiroaki Matsunami, Duke University), and calcium imaging analysis was carried out as described previously [12]. Briefly, transfected cells were seeded into 96-well Lumox multiwell black-wall plates (SARSTEDT AG & Co., Nümbrecht, Germany). After 40–46 hours, the cells were loaded with 5 µM of fura-2/AM (Invitrogen, Carlsbad, CA, USA) in assay buffer for 30 min at 37°C, and then washed with assay buffer, prior to incubation in 100 µl of assay buffer for more than 10 min at room temperature. The cells were stimulated with sweet tastants by adding 100 µl of 2× ligands. The intensities of fura-2 fluorescence emissions resulting from excitation at 340 and 380 nm were measured at 510 nm using a CCD camera. The images were recorded at 4 sec intervals and analyzed using MetaFluor software (Molecular Devices, Sunnyvale, CA, USA).

Construction of stable cell lines expressing the mutated human sweet taste receptor

The entire coding regions of hT1R2, hT1R3, and G16-gust44 were subcloned into the pcDNA5/FRT vector (Invitrogen) according to the procedure described previously [21]. To generate the expression plasmid for the mutated receptor, the hT1R2 cDNA fragment with the point mutation was used instead of using the wild-type (WT) hT1R2 cDNA.

Stable cell lines expressing mutant hT1R2 together with hT1R3 and G16-gust44 were generated to prepare the following hT1R2 mutants: Y103A, Y103F, D142A, S144A, S165A, P277A, P277G, P277S, P277Q, D278A, E302A, D307A, E382A, and R383H. The stable cell lines were generated using Flp-In 293 cells (Invitrogen) and the plasmid we constructed according to the manufacturer’s protocol for the Flp-In pcDNA5/FRT Complete System (Invitrogen) as described in our previous publication [21]. Hygromycin-resistant cells were collected, cultured, and used to measure the cellular responses to sweet tastants. The cells for these measurements were cultured in low-glucose (1.0 g/l) Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum.

Measurement of cellular responses by the cell-based assay

Trypsinized cells were seeded at a density of 80,000 cells per well into 96-well black-wall CellBIND surface plates (Corning, Corning, NY, USA) and 24 hours later were washed with assay buffer prior to loading with a calcium indicator dye from the FLIPR Calcium 4 Assay Kit (Molecular Devices) diluted with assay buffer. The cells were incubated for 60 min at 37°C, and measurements were made using FlexStation 3 (Molecular Devices) at 37°C. Fluorescence changes by excitation at 485 nm, emission at 525 nm, and cutoff at 515 nm were monitored at 2 s intervals, an aliquot of 100 µl of assay buffer supplemented with 2× ligands was added at 20 s, and scanning was continued for an additional 100 s. The response of each well was represented as ΔRFU (delta relative fluorescence unit) and defined as maximum fluorescence value minus minimum fluorescence value. To calculate EC50 values, plots of amplitude versus concentration were prepared in Clampfit Version 9.2 (Molecular Devices). Nonlinear regression of the plots produced the function:

equation image

where x is the ligand concentration and h is the Hill coefficient used to calculate the EC50 values for ligand–receptor interactions. When the EC50 value of the mutated receptor-expressing cells was changed more than 5 fold compared with wild type receptor, the corresponding mutation was judged to be largely affected.

Structure modeling of receptor and receptor–ligand complexes

The crystal structures of the ATD of mGluR1 solved in both inactive (glutamate-unbound) and active (glutamate-bound) forms (PDB: 1EWT and 1EWK, respectively) were used to construct the ATDs of hT1R2 and hT1R3. The structural model of the ATDs of the hT1R2 and hT1R3 heterodimer was constructed with homology modeling according to their sequence homology with mGluR1. For the active form of the heterodimer model, the closed form of mGluR1 was used for hT1R2 and the open form for hT1R3. Conversely, the open form of the crystal structure of mGluR1 was used to construct the inactive form of T1R2 and T1R3. Each heterodimeric structure was then energy-minimized with molecular mechanics using Discover 3 (Accelrys Inc., CA, USA), and the main chain was tethered at the conserved position.

Sweet small ligands were docked into the ligand-binding cleft of the hT1R2 model where glutamate is bound in the mGluR1; this was pursuant to the plausible interactions between the charged or hydrophilic groups of the ligands and the receptor that were deduced from the mutational experiments. Conformations of the ligands were then generated and energy-minimized with molecular mechanics using Discover 3. The minimized complex structures were then structure-optimized with molecular dynamics using Discover 3, and the residues were tethered beyond 12 Å from the ligands.

Results

Mutagenesis studies for screening the residues responsible for sweetener recognition

To define the binding modes of sweeteners at the cleft formed by LB1 and LB2 of hT1R2 ATD, we carried out a series of mutagenesis studies on hT1R2 ATD. First, a molecular model of hT1R2 ATD based on the ligand-binding structure of the closed form of mGluR1 was constructed. Based on the residues resided in the glutamate-binding cleft in the structure of mGluR1 ATD, 15 residues of hT1R2 were arbitrarily selected to introduce the point mutation (Fig. S1, Table S1), and 25 single hT1R2 mutants for the 15 residues were then constructed. The selected residues were almost hydrophilic, and were expected to form ionic or hydrogen bonds with the ligands. The responses to sweeteners were examined by a calcium imaging assay using HEK293T cells transiently expressing the T1R2 mutant and T1R3. Ten out of the 15 residues (Y103, D142, S144, S165, P277, D278, E302, D307, E382, and R383) were selected from the results of the 25 mutants because receptors mutated at these 10 residues retained the responsiveness and exhibited largely changed activities toward the sweeteners tested (Table S1).

As for the 10 residues, stable cell lines expressing the hT1R2 mutant and hT1R3 were constructed, and the cell-based assay was performed to determine the dose–response relationship with the half-maximal effective concentration (EC50) value for each sweetener. To validate the activity of each mutated receptor, we used an artificial sweetener cyclamate, which was recognized by the TMD of hT1R3, as positive controls [22]. Because all the hT1R2 mutant cell lines clearly responded to cyclamate, showing similar EC50 values to those expressing the WT receptor (Table 1), the mutated receptors were determined to be functionally expressed. The response profiles of the mutated receptors to the sweeteners are summarized in Table 1.

Table 1

Summary of point mutations in hT1R2–hT1R3.

Residues responsible for aspartame and D-tryptophan reception in hT1R2 ATD

The response to aspartame was completely lost in the cell lines expressing E302A, S144A, D142A and Y103A (Fig. 2A), and EC50 values largely increased in those expressing D278A, with a decrease in potency (EC50 value 8.14-fold increase versus WT, Fig. 2B). These results suggest that the residues E302, S144, D142, Y103, and D278 are crucial for aspartame reception, among which E302 and S144 have also been previously reported as important residues for aspartame recognition [17].

Figure 2

Dose-dependent responses of hT1R2/hT1R3-expressing cells to amino acid derivatives.

In contrast, only the application of d-tryptophan (d-Trp) to E302A-expressing cells elicited no response (Fig. 2C), and large increases in EC50 values were observed for D307A, D142A, D278A, S165A, Y103A, and P277A mutants (>5-fold increase versus WT) (Fig. 2D and Table 1). Although aspartame elicited no response in D142A and Y103A mutants (Fig. 2A and Table 1), d-Trp considerably reduced the response potency to these mutants within an 8-fold EC50 increase (Fig. 2D and Table 1). In the cases of S165A and P277A mutants, EC50 of d-Trp increases 5.40- and 6.31-fold, respectively (Fig. 2D), while those of aspartame were only changed (Table 1). Although the carboxylate of aspartame and d-Trp is located near S165 and R383 in their complex models, the carboxylate of d-Trp would interact with S165, but that of aspartame would be located at slightly different position not to directly interact with S165. A similar case is also the interactions of P277 with d-Trp and aspartame, in which the indole moiety is locate closer to P277 than the phenylalanine moiety is. The roles of S165 and P277 in receptor activation are thus ligand depended.

Residues responsible for saccharin Na and acesulfame K reception in hT1R2 ATD

Saccharin Na and AceK activated WT hT1R2–hT1R3 in a dose-dependent manner at lower concentrations, but the response was suppressed at higher concentrations (>3 mM and >10 mM, respectively, Figs. 3A and 3B), which has been observed and investigated in detail by Galindo-Cuspinera et al. [14]. Therefore, EC50 values for saccharin Na and AceK were estimated at the lower concentrations.

Figure 3

Dose-dependent responses of hT1R2/hT1R3-expressing cells to sulfamates.

The cellular responses to saccharin Na and AceK were lost in R383H, D142A and E382A (Figs. 3A and 3B). These results indicate that R383, D142, and E382 are crucial residues for activation by saccharin Na and AceK. The mutations E302, S144 and D278 scarcely affected the EC50 values for saccharin Na and AceK, unlike aspartame and d-Trp (Fig. 2 and Table 1). Moreover, the other mutations tested in this study were not sensitive to saccharin Na and AceK (Table 1), suggesting that the binding region for saccharin Na and AceK is limited to a region around R383 (see Discussion).

Residues responsible for sucralose reception in hT1R2 ATD

The response to sucralose was almost completely lost in D278A and Y103A (Fig. 4A). E302A, D307A, D142A, and P277A largely increased the EC50 values of sucralose and decreased the potency (Fig. 4B). Most of the crucial residues for sucralose reception (E302, D142, Y103, D278, and D307) appeared to overlap with those for d-Trp and aspartame reception (Table 1). However, unlike aspartame, the EC50 value of sucralose for S144A did not change dramatically (0.27 mM), and P277A elicited a remarkable increase of the EC50 value. These results indicate that sucralose partially shares the binding region with aspartame, but also interacts with sucralose-specific residue such as P277.

Figure 4

Dose-dependent responses of hT1R2/hT1R3-expressing cells to sucralose.

Roles of Y103 and P277 at the entry of the lobes

Six out of the 10 critical residues (D142, D278, E302, D307, E382, and R383) are acidic or basic residues that probably bind to ligands via electrostatic interactions (Table 1). Furthermore, S144 and S165 were important for the reception of the amino acid derivatives aspartame and d-Trp, respectively (Figs. 2A and 2D). We next evaluated the role of the hydrophobic residues, Y103 and P277, located across the cleft of LB1 and LB2, respectively (See Discussion). To further examine the effect of Y103 on receptor activity, the responses of stable cell lines expressing additional mutants (in which Y103 was replaced with Phe in addition to Ala) were evaluated. When sucralose was applied to Y103 mutants, the response was almost completely lost in Y103A but was only slightly reduced in Y103F (Fig. 5A). These results indicate that the aromatic ring of Y103 is specifically essential to sucralose binding.

Figure 5

Roles of Y103 and P277 for the reception of the sweeteners.

To evaluate the role of P277, the additional mutants P277G, P277Q and P277S were constructed. The P277Q mutant showed severely reduced responses to aspartame (Fig. 5B) and d-Trp (Table 1), while P277G and P277S did not (Fig. 5B). In contrast, these three mutants responded almost equally to saccharin Na and AceK (Table 1). These results suggest that P277 plays an important role in allowing the sweet taste receptor to discriminate amino acid derivatives (aspartame and d-Trp) from the other sweeteners.

Discussion

Critical residues for small molecular sweetener recognition in hT1R2 ATD

To clarify the roles of the 10 residues in small molecular sweetener recognition, we mapped them on the model of the open form of the hT1R2 ATD (Fig. 6). They were divided into four classes based on the results of a single point mutant analysis of hT1R2–hT1R3 corresponding to three chemically different types of ligands: amino acid derivatives (aspartame and d-Trp), sulfamates (saccharin Na and AceK), and a sugar analog (sucralose) (Table 1). Our data strongly suggest that the binding sites in hT1R2 ATD are quite different from each other, although all of them are recognized in the cleft of hT1R2 ATD. As shown in Figs. 7 and ​and8,8, aspartame, d-Trp, and sucralose share LB1 residues (Y103 and D142) and LB2 residues (D278, E302, and D307) for binding, but each compound also needs specific residues for individual interaction with the receptor (S144 for aspartame (Fig. 2A) and P277 for sucralose (Fig. 4B)). By contrast, these residues are not involved in binding saccharin Na and AceK, but the residues (D142, E382 and R383) located in another site of LB1 are indispensable for their binding (Figs. 6A).

Figure 6

Model of the open form of hT1R2 ATD.
Figure 7

Complex model of the sucralose-bound hT1R2 ATD.
Figure 8

Model of the aspartame-bound hT1R2 ATD.

The low-molecular-weight sweeteners bind in the cleft composed of LB1 and LB2 with a different binding mode at each characteristic residue. To examine further characteristic interactions between ligands and the 10 residues, we built ligand–hT1R2 ATD (closed form) complex models for sucralose, aspartame and saccharin Na (Figs. 7, ​,8,8, ​,9,9, Methods S1, S2, S3).

Figure 9

Complex model of the saccharin Na–bound hT1R2 ATD.

(i) Roles of Y103 at the entry of LB1 and D278 at the entry of LB2

The complex models of sucralose–hT1R2 and aspartame–hT1R2 suggested different roles of Y103 in receptor activation. The C2-H and C4-Cl of the hexose portion of sucralose bind to the aromatic ring of Y103 (Fig. 7C), and the hydroxyl groups in the hexose moiety of sucralose form hydrogen bonds with D278 (Fig. 7C). The binding of the hexose portion to Y103 in LB1 and D278 in LB2 may thus facilitate the formation of the closed form of hT1R2 ATD. The importance of these residues for binding of sucralose is consistent with the results reported by Zhang et al. [18].

Conversely, the phenol group of Y103 forms a hydrogen bond with D278 in the aspartame–hT1R2 model (Fig. 8C), stabilizing the closed form of hT1R2. The hydrogen bond appears to be important for the d-Trp-binding. However, the role of the phenol group in the aspartame-binding would be more significant, since the phenol group would interact with the carboxylate of aspartame. The phenol group of Y103 is thus important for the binding of aspartame, while the aromatic group is necessary for the binding of sucralose, as in the cases of the Y103A and Y103F mutants (Fig. 5A). On the other hand, Zhang et al. [18] suggested a contribution of a hydrogen bond between D278 and K65 to the stabilization of the closed form in the binding of sweet taste enhancers. However, a transiently expressed K65A mutant receptor did not show a significant difference from the native receptor in the binding of aspartame and sucralose (Table S1), being consistent with the results reported by Zhang et al. [18] and Liu et al. [23], in which K65 is not important for the binding of aspartame and sugar derivatives.

(ii) Roles of E302 at the center of LB2

The negatively charged E302 residue forms a salt bond with the positively charged amine group of aspartame (Fig. 8C), whereas a hydroxyl group of the pentose moiety of sucralose forms a hydrogen bond with E302 (Fig. 7C). E302 in the LB2 should thus be a crucial residue for the ligands, with hydrogen bond donors contributing to the formation of the closed form in receptor activation. In contrast, the E302 residue makes no electrostatic interaction with saccharin Na (Fig. 9C), so the contribution of this residue to receptor activation should be little, if any (Fig. 3B).

(iii) Roles of D142, E382, and R383 at the center of LB1

Because R383 forms a hydrogen bond network with D142 and E382 in the hT1R2 model, R383 plays a crucial role in the recognition of negatively charged groups of ligands (Fig. 9C). D142 or E382 may not directly interact with the negatively charged ligands but would play an important role in localizing the flexible R383 residue at a proper position for interacting with the ligands (Fig. 9C). For aspartame recognition, binding of both the carboxylate moiety to R383 in LB1 and the amino group to E302 in LB2 may facilitate the formation of the closed form of the ATD (Fig. 8C). The negatively charged group of saccharin and the cationic sodium ion attached to saccharin would play similar roles in the formation of the closed form (Fig. 9C). Liu et al. [23] showed that S40 and V66 contribute to the species specificity in the binding of aspartame. The S40 residue is located at the hydrogen bond distance to D142 and the V66 residue is close to R383 in the aspartame-bound model. The mutation of these residues would electronically and sterically affect the interaction of D142 and R383 which are important for the recognition of the carboxylate of aspartame. This is somewhat similar to the roles of S40 and V66 in the species specific recognition of aspartame.

The neutral ligand sucralose may directly interact with D142 through a hydrogen bond with the vicinal hydroxyl groups of the furanose moiety (Fig. 7C). This hydrogen bond probably leads to the formation of a hydrogen bond between R383 and E302 to facilitate receptor activation.

(iv) Role of P277 at the entry of LB2

Aspartame and saccharin do not bind P277 (Figs. 7C and ​and8C).8C). However, aspartame is located near the residue because the Gln mutant for P277 interrupts receptor activation by aspartame. In contrast, the mutation of smaller residues such as Gly and Ser does not affect activation (Fig. 7C). The smaller ligand, saccharin Na, may be located far from P277 and thus may not be influenced by the mutation (Fig. 9C). Still, P277 should be an important binding site for d-Trp, as observed in the P277A and P277Q mutants (Table 1). These results suggest that saccharin Na is located far from P277 whereas d-Trp is located close to P277. The distance between aspartame and P277 would be intermediate between those of saccharin Na and d-Trp.

The chloride at C1′ of the furanose moiety of sucralose showed favorable van der Waals contact with P277 (Fig. 7C), and the P277Q mutant caused unfavorable steric interactions with the chloride; however, the favorable hydrophobic interactions are lost in the P277G and P277S mutants (Table 1).

Characteristic features in receptor activation mechanisms of the human sweet taste receptor

As described above, the interaction at the core of LB1 and LB2 appears to be essential for reception of all the sweeteners, and the interaction at the entry of LB1 and LB2 would reinforce the formation of the closed structure of the receptor for activation. These results strongly suggest that the activation mechanism of the human sweet taste receptor is similar to that of mGluR1.

X-ray crystal structural analysis, molecular modeling, and many mutagenesis studies have revealed the existence of critical residues for ligand binding in other class C GPCRs, such as mGluRs [15], [24], [25], the GABA receptor [26], [27], the calcium sensing receptor [28], [29], and the human umami taste receptor (hT1R1–hT1R3) [30]. In comparison with previous data [31], our model of hT1R2–hT1R3 based on a mutagenesis analysis suggests that hT1R2–hT1R3 uses five acidic residues (D142, D278, E302, D307, or E382) for the recognition of its agonists; the other receptors use one or two acidic residues. These results suggest that hT1R2 ATD forms different sites of binding with specific sets of these residues to receive chemically diverse low-molecular-weight sweeteners, although their affinities for hT1R2 ATD are quite low.

It should be noted that we could not determine the binding mode of sugars such as sucrose. Sugars generally elicit the strong sweet taste, and they are the most common natural ligands for the receptor. Although it would be important to elucidate the key residues for the recognition of sugars, the cellular response to sucrose was quietly faint compared with the other sweeteners used in this study, and EC50 values of the mutated receptors to sucrose could not be accurately calculated. Further studies should be required to improve the sensitivity of the functional assay system for the human sweet taste receptor.

In this study, we defined how hT1R2–hT1R3 acquires the ability to recognize chemically diverse sweeteners. These results will not only provide insights into molecular recognition patterns of GPCRs but may also help develop novel sweeteners.

Supporting Information

Table S1

Summary of point mutations determined by a calcium imaging assay using HEK293T cells transiently expressing the T1R2 mutant and T1R3.

(DOC)

Figure S1

Sequence alignment of the ATDs of hT1R2 and rat mGluR1. The mutated residues in hT1R2 used for initial screening are shown in blue and magenta. Stable cell lines were also constructed for the residues shown in magenta. Critical ligand-binding residues in the rat mGluR1 ATD that interact with the carboxylate side chain and the α-amino acid moiety are shown in red and green, respectively.

(TIF)

Methods S1

Modeling for sucralose-T1R2ATD complex (Fig. 7A9C9C).

(DOC)

Methods S2

Modeling for aspartame-T1R2ATD complex (Fig. 8A–C).

(DOC)

Methods S3

Modeling for saccharin-T1R2ATD complex (Fig. 9A–C).

(DOC)

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This study was performed with a grant from the Research and Development Program for New Bio-industry Initiatives of the Bio-oriented Technology Research Advancement Institution. This work was also supported by the Japan Society for the Promotion of Science Research Fellowship for Young Scientists (to AK) and by Grants-in-aid for Scientific Research 21880015 (to KN), 20688015 and 21658046 (to TM) and 20380183 (to KA) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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12. Koizumi A, Nakajima K, Asakura T, Morita Y, Ito K, et al. Taste-modifying sweet protein, neoculin, is received at human T1R3 amino terminal domain. Biochem Biophys Res Commun. 2007;358:585–589. [PubMed]
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The Effect of Erythropoietin on Ischemia/Reperfusion Injury after Testicular Torsion/Detorsion: A Randomized Experimental Study

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Abstract

This study was conducted to investigate the protective effect of erythropoietin (EPO) on ischemia/reperfusion related changes after testicular torsion/detorsion. In a randomized experimental trial 30 male rats were randomly allocated into six equal groups of five rats each. Group I (orchiectomy for histopathologic examination), group II (sham operation), group III (torsion for 2 hours, and ischemia/detorsion for 24 hours, and orchiectomy); group IV (torsion for 2 hours, ischemia/detorsion for 24 hours with erythropoietin injection then orchiectomy), group V (torsion for 2 hours and detorsion and EPO injection and orchiectomy 1 week later, group VI (torsion for 2 hours/detorsion and orchiectomy 1 week later). Two groups (groups 4 and 5) received different protocols of erythropoietin administration after testicular torsion/distortion. other groups were not receiving erythropoietin. Johnsen’s spermatogenesis scoring method and Cosentino’s histologic staging method were used to assess main outcome measures of the study. After the experimentation, Johnsen’s score in EPO Groups was statistically different from the score in some groups not receiving erythropoietin. Cosentino’s score in EPO groups was statistically different from the score in all groups not receiving erythropoietin. Neovascularization, vascular necrosis, vascular congestion, edema, hemorrhage, and acute inflammation were observed in some groups. This study shows short-term protective efficacy of erythropoietin on rat testicular injury after ischemia/reperfusion.

1. Introduction

Testicular torsion is the twisting of the spermatic cord, which cuts off the blood supply to the testicle and surrounding structures. It is more common during infancy and early adolescence. It is a very painful condition, but mainly it is a surgical emergency because it may result in the loss of the affected testicle if not treated promptly. Torsion is the most common cause of testicle loss in adolescent males. Some men may be predisposed to testicular torsion as a result of inadequate connective tissue within the scrotum. However, the condition can result from trauma to the scrotum, particularly if significant swelling occurs. It may also occur after strenuous exercise or may not have an obvious cause [1].

Surgery is usually required and should be performed as soon as possible after symptoms begin. If surgery is performed within 6 hours, most testicles can be saved. Testicular torsion and also the detorsion procedure induce morphological as well as biochemical changes caused mostly by ischemia/reperfusion injury in the testicular tissue [2].

Dysfunction induced by free radicals is the major component of ischemic process due to torsion in the testis, and many studies focused on protective effects of special medications and erythropoietin [3, 4]. Many Animal studies have discovered a protective role for erythropoietin against the aftermaths of ischemia/reperfusion in various organ tissues including kidney, heart, liver, and ovary tissues in animals and nervous system [511]. Few studies have recently been conducted on testicular tissues either [1215]. However, available knowledge is limited considering the methodological variations. The aim of this study was to investigate the protective effect of erythropoietin on ischemia/reperfusion related changes after testicular torsion/detorsion.

2. Materials and Methods

A study was conducted in 2008 in Urology Department of Imam Reza University Hospital in Tabriz, Iran. Animals were 200–300 gr weighted male wistar rats that were breeded in Razi institute of Iran and kept, prior and through the investigation process, under normal and similar light, temperature, and feeding plan in the animal lab in Drug Applied Research Center, Tabriz University of medical sciences. In a randomized experimental trial 30 rats were randomly allocated into six equal groups of five rats each. Both testes of each rat were studied. The groups were treated as follows.

Group 1. Both testes of the five studied rats were orchidectomized and sent for histopathology examination.

Group 2. All five rats underwent lower abdominoscrotal incision (because a wide inguinal canal the testes move freely between abdomen and scrotum) and orchiopexy. After 24 hours both testes of the studied rats were orchidectomized and sent for histopathology examination.

Group 3. All five rats underwent lower abdominoscrotal incision. They received 720 degrees bilateral torsion for two hours (based on other studies) then detorsion and orchiopexy were done on them [12, 16]. After 24 hours both testes of the studied rats were orchidectomized and sent for histopathology examination.

Group 4. All five rats underwent lower abdominoscrotal incision. They received 720 degrees bilateral torsion for two hours then detorsion and orchiopexy were done on them. Then erythropoietin was injected and after 24 hours both testes of the studied rats were orchidectomized and sent for histopathology examination.

Group 5. All five rats underwent lower abdominoscrotal incision. They received 720 degrees bilateral torsion for two hours and then detorsion and orchiopexy were done on them. Then erythropoietin was injected, and after one week both testes of the studied rats were orchidectomized and sent for histopathology examination.

Group 6. All five rats underwent lower abdominoscrotal incision. They received 720 degrees bilateral torsion for two hours, and then detorsion and orchiopexy were done on them. After one week both testes of the studied rats were orchidectomized and sent for histopathology examination.

Prior to incisions general anesthesia was induced using 2.5 mg/kg midazolam as intraperitoneal injection. Erythropoietin was administered intravascular in a dose of 3000 u/kg in groups 4 and 5. The pathologist and evaluator were blind to the type of intervention. Spermatogenesis was assessed using Johnsen’s spermatogenesis scoring method which is based on the concept that testis damage causes a successive disappearance of the most mature germ cell type [17]. Following is the scoring grades:

score 10: complete spermatogenesis with regular tubules;
score 9: many sperms, irregular germinal epithelium;
score 8: few sperms;
score 7: no sperms, many spermatids;
score 6: few spermatids;
score 5: no sperm or spermatids;
score 4: few spermatocytes;
score 3: presence of spermatogonia;
score 2: presence of Sertoli’s cells;
score 1: no cells.

Histological assessments were based on Cosentino’s histologic staging method [18].

Data were gathered and analyzed using SPSS version 15 statistical software package. Measures of continuous scales were compared using Kruskall-Wallis test followed by Mann-Whitney U test as post hoc. Categorical variables were assessed using contingency tables and chi-squared or Fisher’s exact tests. P < 0.05 was considered a statistical significance level in primary tests. Bonferroni correction was used for qualitative parameters in multiple comparisons.

Study was approved by committee of ethics in Tabriz University of medical sciences. Code of ethics on working with lab animals was followed in this research and interdisciplinary principles and guidelines for the use of animals in research were considered.

3. Results and Discussion

Median Johnsen’s score was the highest among rats in group 5 when compared to other rats except control rats in groups 1 and 2 that had not undergone testicular torsion. Mean (SD) Johnsen’s score is compared among the groups in Figure 1. Medians of Johnsen’s score were 10, 10, 2, 3.5, 7.5, and 5 in groups 1–6, respectively.

Figure 1

Mean Johnsen’s score compared among the study groups.

In pairwise statistical assessment of comparisons, it was found that Johnsen’s score in group 5 was statistically different from the score in all groups not receiving erythropoietin. However, Johnsen’s score in group 4 was statistically different from the score in groups 1 and 2 not receiving erythropoietin. Further details are given in Table 1.

Table 1

P values for pairwise statistical comparisons of median Johnsen’s score and Cosentino’s score in groups of rats receiving erythropoietin versus control groups.

Mean (SD) Cosentino’s score is compared among the groups in Figure 2. Medians of Cosentino’s score were 1, 1, 3, 2, 2, and 3 in groups 1–6, respectively.

Figure 2

Mean Cosentino’s score compared among the study groups.

In pairwise statistical assessment of comparisons, it was found that Cosentino’s score in group 5 and 4 was statistically different from the score in all groups not receiving erythropoietin. Further details are given in Table 1.

Statistical assessment of comparisons made between erythropoietin groups and other four control groups found that the frequency distribution in vascular congestion was statistically different only when group 4 was compared with group 1 (P = 0.03); the difference in frequency distribution of neovascularization and vascular necrosis was not statistically significant for any of the comparison pairs; groups 4 and 5 had significantly different frequency of edema only with groups 1 and 2 (P = 0.001); hemorrhage frequency was different when comparing group 4 with groups 1 and 2 (P = 0.001), and also the frequency difference of hemorrhage between groups 5 and 3 was also statistically significant (P = 0.001); groups 4 and 5 had significantly different frequency distribution of acute inflammation only with groups 1 and 2 (P = 0.001) (Figure 3).

Figure 3

Relative frequency distribution of vascular congestion, edema, hemorrhage, and acute inflammation compared among study groups.

The protective effects of erythropoietin on renal and cardiovascular ischemic injuries are shown earlier [5, 11, 19]. Effect of erythropoietin on gonadal ischemia is also shown to be positive relieving ischemic injuries after ovarian torsion in rat and mouse models [6, 8, 9, 20].

In this study we assessed the effect of erythropoietin on ischemic/reperfusion changes after testicular torsion. The results of present study were indicative of efficacy of erythropoietin in relieving the changes after ischemia/reperfusion when compared with similar control groups that had not received erythropoietin. The study also found that seven days after ischemia/reperfusion both Johnsen’s score and histological grading were significantly better in groups receiving erythropoietin.

The first study on effects of erythropoietin on testes injury was published in 2005 by Dobashi et al. They investigated the effects of rat erythropoietin on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation and found that it may reduce the risk of the germ cell loss caused by cryptorchidism [21]. Later in 2007 Yazihan et al. in their study in a five-group rat study found that erythropoietin has antiapoptotic and anti-inflammatory effects following testicular torsion [14]. Two other studies on rat model showed also the positive effect of erythropoietin on ischemic testis injuries [12, 22]. It was found that erythropoietin can decrease cell damage and apoptosis. Köseoğlu et al. concludes that erythropoietin preserves the intact somniferous tubular morphology, lowers the percentage of necrotic seminiferous tubules, and reduces the histological damage [13]. One study used mouse model to assess effect of erythropoietin, but as darbepoetin α, on ischemia caused by testis torsion finding that it affects histological grading. The mentioned study assessments were done four hours after detorsion [15].The major methodological variations in studies conducted on effect of erythropoietin after gonadal ischemia include gonadal type as ovaries versus testes, animal type as rat versus mouse, drug administration form as oral versus parenteral, assessment timing, and drug administration timing and order such that nearly every study had some exclusive methodological aspects. However, regardless of methodological variations, our findings on efficacy of erythropoietin on aftermaths of gonadal ischemia/reperfusion were consistent with available literature.

4. Conclusions

Based on our findings and available knowledge, it can be inferred that erythropoietin has positive effects on gonadal ischemia/reperfusion injury. Specifically we conclude supporting short-term efficacy of erythropoietin on rat testicular injury after ischemia/reperfusion. However, available knowledge, including our findings, has not yet turned complete. Two major areas of necessary future research could be dose-response studies and studying time variance of erythropoietin efficacy in this regard.

Acknowledgment

The authors wish to express their sincere gratitude to Research Vice Chancellor of Tabriz University of Medical Sciences for financially supporting this project.

References

1. Schneck FX, Bellinger ME. Abnormalities of the testis and scrotum and their surgical management. In: Wein AJ, Kavoussi LR, Novick AC, Partin AW, Peters CA, editors. Campbell-Walsh Urology. 9th edition. Vol. 4. Philadelphia, Pa, USA: WB Saunders; 2007.
2. Saba M, Morales CR, De Lamirande E, Gagnon C. Morphological and biochemical changes following acute unilateral testicular torsion in prepubertal rats. Journal of Urology. 1997;157(3):1149–1154. [PubMed]
3. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. The New England Journal of Medicine. 1985;312(3):159–163. [PubMed]
4. Paschos N, Lykissas MG, Beris AE. The role of erythropoietin as an inhibitor of tissue ischemia. International Journal of Biological Sciences. 2008;4(3):161–168. [PMC free article] [PubMed]
5. Parsa CJ, Matsumoto A, Kim J, et al. A novel protective effect of erythropoietin in the infarcted heart. Journal of Clinical Investigation. 2003;112(7):999–1007. [PMC free article] [PubMed]
6. Ergun Y, Koc A, Dolapcioglu K, et al. The protective effect of erythropoietin and dimethylsulfoxide on ischemia-reperfusion injury in rat ovary. European Journal of Obstetrics Gynecology and Reproductive Biology. 2010;152(2):186–190. [PubMed]
7. Carelli S, Marfia G, Di Giulio AM, Ghilardi G, Gorio A. Erythropoietin: recent developments in the treatment of spinal cord injury. Neurology Research International. 2011;2011:8 pages.453179 [PMC free article] [PubMed]
8. Karaca M, Odabasoglu F, Kumtepe Y, Albayrak A, Cadirci E, Keles ON. Protective effects of erythropoietin on ischemia/reperfusion injury of rat ovary. European Journal of Obstetrics Gynecology and Reproductive Biology. 2009;144(2):157–162. [PubMed]
9. Shishavan MK, Sayyah-Melli M, Rad JS, Roshangar L. The ovario-protective effect of erythropoietin against oxidative damage associated with reperfusion following ovarian torsion in rat. American Journal of Animal and Veterinary Sciences. 2009;107(supplement 2):p. S223.
10. Liu X, Shen J, Jin Y, Duan M, Xu J. Recombinant human erythropoietin (rhEPO) preconditioning on nuclear factor-κ B (NF-κB) activation & proinflammatory cytokines induced by myocardial ischaemia-reperfusion. Indian Journal of Medical Research. 2006;124(3):343–354. [PubMed]
11. Johnson DW, Pat B, Vesey DA, Guan Z, Endre Z, Gobe GC. Delayed administration of darbepoetin or erythropoietin protects against ischemic acute renal injury and failure. Kidney International. 2006;69(10):1806–1813. [PubMed]
12. Bakan V, Çıralık H, Tolun FI, Atli Y, Mil A, Öztürk Ş. Protective effect of erythropoietin on torsion/detorsion injury in rat model. Journal of Pediatric Surgery. 2009;44(10):1988–1994. [PubMed]
13. Köseoğlu B, Yilmaz E, Ceylan K, Uzun E, Bayram I, Hizli F. The protective effect of erythropoietin infusion on testicular torsion/ detorsion: an experimental study. International Urology and Nephrology. 2009;41(1):85–91. [PubMed]
14. Yazihan N, Ataoglu H, Koku N, Erdemli E, Sargin AK. Protective role of erythropoietin during testicular torsion of the rats. World Journal of Urology. 2007;25(5):531–536. [PubMed]
15. Akcora B, Altug ME, Kontas T, Atik E. The protective effect of darbepoetin alfa on experimental testicular torsion and detorsion injury. International Journal of Urology. 2007;14(9):846–850. [PubMed]
16. Ozkan MH, Vural IM, Moralioglu S, Uma S, Sarioglu Y. Torsion/detorsion of the testis does not modify responses to nitric oxide in rat isolated penile bulb. Basic and Clinical Pharmacology and Toxicology. 2007;101(2):117–120. [PubMed]
17. Johnsen SG. Testicular biopsy score count–a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones. 1970;1(1):2–25. [PubMed]
18. Cosentino MJ, Nishida M, Rabinowitz R, Cockett ATK. Histopathology of prepubertal rat testes subjected to various durations of spermatic cord torsion. Journal of Andrology. 1986;7(1):23–31. [PubMed]
19. Foller M, Kaestner L, Straface E, Vogel J. Erythrocytes and erythropoietin. International Journal of Cell Biology. 2011;2011:2 pages.972536 [PMC free article] [PubMed]
20. Patel NSA, Sharples EJ, Cuzzocrea S, et al. Pretreatment with EPO reduces the injury and dysfunction caused by ischemia/reperfusion in the mouse kidney in vivo. Kidney International. 2004;66(3):983–989. [PubMed]
21. Dobashi M, Goda K, Maruyama H, Fujisawa M. Erythropoietin gene transfer into rat testes by in vivo electroporation may reduce the risk of germ cell loss caused by cryptorchidism. Asian Journal of Andrology. 2005;7(4):369–373. [PubMed]
22. Ergur BU, Kiray M, Pekcetin C, Bagriyanik HA, Erbil G. Protective effect of erythropoietin pretreatment in testicular ischemia-reperfusion injury in rats. Journal of Pediatric Surgery. 2008;43(4):722–728. [PubMed]

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A 47,X,+t(X;X)(p22.3;p22.3)del(X)(p11.23q11.2),Y Klinefelter Variant with Morbid Obesity

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Abstract

Klinefelter syndrome is the most common type of genetic cause of hypogonadism. This syndrome is characterized by the presence of 1 or more extra X chromosomes. Phenotype manifestations of this syndrome are small testes, fibrosis of the seminiferous tubules, inability to produce sperm, gynecomastia, tall stature, decrease of serum testosterone and increases of luteinizing hormone and follicle stimulating hormone. Most patients with Klinefelter syndrome are tall, with slender body compositions, and reports of obesity are rare. We report the case of a 35-yr-old man with hypogonadism and morbid obesity and diabetes mellitus. He had gynecomastia, small testes and penis, very sparse body hair and his body mass index was 44.85. He did not report experiencing broken voice and was able to have erections. We conducted a chromosome study. His genotype was 47,X,+t(X;X)(p22.3;p22.3)del(X)(p11.23q11.2). In this case, the patient was diagnosed as Klinefelter syndrome. He showed rare phenotypes like morbid obesity and average height and the phenotype may be caused by the karyotype and the excess number of X chromosome. Further studies of the relationship between chromosomes and phenotype are warranted.

Keywords: Klinefelter syndrome, morbid obesity, karyotype

INTRODUCTION

Klinefelter syndrome is the most common type of genetic cause of hypogonadism and occurs in approximately 1.2-1.53 per 1000 male births. This syndrome is characterized by the presence of 1 or more extra X chromosomes, and the most common karyotype is 47, XXY.1 Phenotype manifestations of this syndrome are small testes, fibrosis of the seminiferous tubules, inability to produce sperm, gynecomastia, tall stature, decrease of serum testosterone and increases of luteinizing hormone (LH) and follicle stimulating hormone (FSH). The deficiency of sex hormone influences the development of metabolic syndrome, obesity, and diabetes mellitus.2 Most patients with Klinefelter syndrome are tall, with slender body compositions, and reports of obesity are rare.3,4 Herein, we report a case of Klinefelter syndrome with diabetes mellitus and morbid obesity.

CASE REPORT

A 35-year-old man who visited the Department of Family Medicine due to obesity, was referred to the Department of Endocrinology for the evaluation of hormonal abnormalities. The patient’s height was 174 cm, his body weight was 135.8 kg, his waist circumference was 137 cm, his hip circumference was 146 cm, and his body mass index was 44.85. He was morbidly obese and had gynecomastia, small testes and penis, and very sparse body hair. He did not report experiencing broken voice and was able to have erections.

The level of complete blood cell count were in normal range, white blood cell count 9270/mcL (Neutrophil/Lymphocyte/Monocyte/Eosinophil/Basophil 62.9/20.6/6.1/8.3/0.8%), hemoglobin 12.6 g/dL and platelet 333000/mcL. Routine chemistry showed no specific abnormality other than oral glucose tolerance test, at 2 hours after test glucose was 224 mg/dL. The patient showed increased levels of serum LH (19.88 mIU/mL) and FSH 29.0 (mIU/mL) and decreased levels of serum testosterone (63.0 ng/dL) upon endocrine study. Both testes were reduced in size on testis ultrasounds and there were no sperm present upon sperm analysis. Under the suspicion of primary hypogonadism, we conducted a chromosome study and the patient was diagnosed with Klinefelter syndrome. His genotype was 47,X,+t(X;X)(p22.3;p22.3)del(X)(p11.23q11.2) (Figs. 1 and ​and2).2). He started treatment with testosterone enanthate injections and was followed up in the outpatient clinic.

Fig. 1

Karyotype of this patient.
Fig. 2

Structure of X chromosome that shows translocation and deletion.

DISCUSSION

About 80-90% of patients with Klinefelter syndrome have 47,XXY karyotype, while 10% have mosaicism. Cases of X chromosome structural changes such as the patient described here make up about 1% of Klinefelter patients.5 Morbid obesity and other characteristics in this patient are not common among Klinefelter patients, and were thought to be related to the X chromosome structural changes. In a previous study, a patient with Prader-Willi phenotype and Xq arm duplication showed Prader-Willi characteristics, even though the methylation of 15 (q11-15) was normal.3 That patient also had chromosome duplication, and we therefore conclude that Xq arm translocations and duplications are associated with morbid obesity.

The number of X chromosomes is also related to phenotype. The phenotypes and severity of symptoms in Klinefelter syndrome patients differ according to the number of X chromosomes.6

Klinefelter syndrome cases with metabolic syndrome or diabetes mellitus have been described in some previous studies, but cases of morbid obesity are rare. Studies of XXY or mosaicism patient phenotypes are, in contrast, common. Patients such as the one described in this case and X chromosome studies have been limited to occasional case reports. Further study of the relationships between chromosome and phenotype are warranted.

Footnotes

The authors have no financial conflicts of interest.

References

1. Bojesen A, Gravholt CH. Klinefelter syndrome in clinical practice. Nat Clin Pract Urol. 2007;4:192–204. [PubMed]
2. Saad F, Gooren LJ. The role of testosterone in the etiology and treatment of obesity, the metabolic syndrome, and diabetes mellitus type 2. J Obes. 2011;2011:pii: 471584. [PMC free article] [PubMed]
3. Gabbett MT, Peters GB, Carmichael JM, Darmanian AP, Collins FA. Prader-Willi syndrome phenocopy due to duplication of Xq21.1-q21.31, with array CGH of the critical region. Clin Genet. 2008;73:353–359. [PubMed]
4. Pramyothin P, Pithukpakorn M, Arakaki RF. A 47, XXY patient and Xq21.31 duplication with features of Prader-Willi syndrome: results of array-based comparative genomic hybridization. Endocrine. 2010;37:379–382. [PubMed]
5. Thomas NS, Hassold TJ. Aberrant recombination and the origin of Klinefelter syndrome. Hum Reprod Update. 2003;9:309–317. [PubMed]
6. Tartaglia N, Ayari N, Howell S, D’Epagnier C, Zeitler P. 48,XXYY, 48,XXXY and 49,XXXXY syndromes: not just variants of Klinefelter syndrome. Acta Paediatr. 2011;100:851–860. [PMC free article] [PubMed]

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Axillary silicone lymphadenopathy secondary to augmentation mammaplasty

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Abstract

We report a case involving a 45-year-old woman, who presented with an axillary mass 10 years after bilateral cosmetic augmentation mammaplasty. A lump was detected in the left axilla, and subsequent mammography and magnetic resonance imaging demonstrated intracapsular rupture of the left breast prosthesis. An excisional biopsy of the left axillary lesion and replacement of the ruptured implant was performed. Histological analysis showed that the axillary lump was lymph nodes containing large amounts of silicone. Silicone lymphadenopathy is an obscure complication of procedures involving the use of silicone. It is thought to occur following the transit of silicone droplets from breast implants to lymph nodes by macrophages and should always be considered as a differential diagnosis in patients in whom silicone prostheses are present.

Keywords: Augmentation, breast prostheses, implant, mammaplasty, silicone lymphadenopathy, ruptured breast implant

INTRODUCTION

During the last four decades, silicone has become one of the most extensively utilized materials for the manufacture of breast implants, mainly because it is non-biodegradable and elicits no or little reaction from human tissue. This wide application of implanted silicone prostheses stems from their biological stability, the long-term preservation of their physical properties, combined with minimal tissue reaction and lack of immunogenicity. In spite of that reputation, side effects associated with the utilization of silicone have been well documented in literature. One uncommon side effect of mammary augmentation is silicone lymphadenopathy, defined as the presence of silicone in a lymph node.[1] This case report describes this obscure complication of silicone breast implantation and discusses thoroughly the challenging diagnostic and therapeutic implications of this clinical enigma.

CASE REPORT

A 45-year-old woman presented to our clinic complaining of a lump, located in the left axilla. Despite having been aware of this lesion for two months, she had not sought immediate medical treatment, until she began to notice intermittent pain in her left axilla and a sensation of heaviness. She had undergone bilateral breast augmentation, using subglandular cohesive gel silicone implants of textured shell surface 10 years ago (Mentor™ – 220 cc each).

On physical examination, there was a relatively mobile, hard and non-tender mass, approximately 3cm in diameter that was located in the left axilla.

Mammography demonstrated an irregular contour of the left implant and a highly radiodense axillary lesion, which corresponded to the palpable mass [Figure 1], while a subsequent breast magnetic resonance imaging (MRI) documented the intracapsular rupture (linguini sign) of the left breast prosthesis, but did not show evidence of silicone leakage from the implants [Figures ​[Figures22 and ​and3].3]. Because the patient denied fine needle aspiration cytology (FNAC), excisional biopsy and frozen section analysis of the mass was proposed in order to confirm the benign nature of the lump. Before the excisional biopsy, the patient was reviewed as an outpatient by the plastic surgeon, who had performed the original augmentation procedure. A combined procedure involving excision biopsy of the left axillary lesion and replacement of the ruptured implant was eventually performed.

Figure 1

Mammography showing irregularity of the contour of the left breast implant and a radiodense mass in the left axilla
Figure 2

Axial magnetic resonance mammography revealing gross disorganization and collapse of the left implant with a positive ‘linguine sign’
Figure 3

Sagittal magnetic resonance mammography demonstrating the collapsed intracapsular rupture of the left implant

On gross examination, small amount of pus-like fluid was seen to surround the ruptured implant. Four enlarged lymph nodes were abundant of clear viscous material, which oozed from the cut surface of the specimen. Subsequent histological analysis identified a histiocytic infiltrate with multinucleated giant cells, vacuoles and refractive material consistent with silicone lymphadenopathy [Figures ​[Figures44 and ​and55].

Figure 4

Histological examination showing lymph node with multinucleated giant cells, vacuoles and refractive material consistent with silicone (Haematoxylin and Eosin staining ×200)
Figure 5

Higher magnifi cation photomicrograph revealing liquid silicone droplets appearing as round vacuoles of varying sizes in lymph node parenchyma (Haematoxylin and Eosin staining ×400)

Follow-up is satisfactory to date and 2 months later she remains well, with complete resolution of her initial postoperative discomfort.

DISCUSSION

Silicone has been used in surgery for over 40 years in breast augmentation. It is composed of dimethylsiloxane polymers, which can result in differing properties according to the variation in their chain lengths and cross-links. Despite its initial reputation as a biologically inert material, it has been related with numerous complications including local and systemic granulomatous inflammatory reactions affecting breast tissue, lymph nodes, joint capsules, heart, liver, and kidneys. Silicone lymphadenopathy involving axillary lymph nodes is an uncommon complication of augmentation mammaplasty.[24]

Silicone particles can migrate through tissues by two distinct mechanisms. The first, following rupture or erosion of a silicone-containing surface and secondly, through continued leakage through an intact surface. The risk of rupture or leakage increases with increasing age of the implant, the site of implantation (retroglandular), the presence of local tissue contractures or symptoms and the type of implant. The average age at rupture varies between studies, but is in the region of 10 to 13 years and it is best diagnosed by MRI. Rupture is usually a harmless complication, which only rarely progresses and becomes symptomatic. When leakage does happen, silicone can cause fibrosis and foreign body reaction, especially when combined with certain fatty acids, resulting in pain and contractures. Once silicone particles have breached the confines of their prosthesis, they may be dispersed through any fibrotic reaction to regional lymph nodes by macrophages in the reticuloendothelial system. The granulomatous reactions may present as lymphadenopathy and, when present in the axilla, malignancy of the ipsilateral breast needs to be excluded.[2,5,6]

The presence of silicone droplets in lymph nodes of patients with breast implants suggests that the transit of various elements, either synthetic or biologic, from breast tissue to lymph nodes via lymphatic channels may have a significant passive component. This passive component may be a crucial determinant in the metastatic process. Silicone migration from breast implants to lymph nodes may therefore represent a model that could be useful in understanding the passive component of metastasis in breast cancer.[7]

The clinical importance of silicone lymphadenopathy has several different facets. In patients who have had post-mastectomy reconstructive surgery using silicone gel breast implants, the differential diagnosis of regional lymph node enlargement should include metastatic breast cancer, as well as silicone lymphadenopathy. In most individuals, who have had cosmetic surgery for breast augmentation, one must also recognize the potential for adverse health effects of silicone migration to regional lymph nodes. The association between silicone breast prostheses and systemic diseases is a highly controversial issue. Till now, most epidemiologic studies, found no association between breast implants and a variety of connective tissue diseases, despite the fact that Brown et al. have published a statistically significant link between ruptured silicone gel implants and fibromyalgia, as well as other autoimmune diseases.[8] On the other hand, there are numerous reports of symptoms in women with breast implants, including myalgia, arthralgia, fatigue and sleep disorders, but there is no adequate evidence of such a relation in the literature. Furthermore, the role of silicone in the development of lymphoma deserves mention, since there are several case reports describing primary breast lymphoma in patients with silicone gel breast implants, as well as patients with coexistent silicone lymphadenopathy and lymphoma in the same lymph node.[6,7,9]

In conclusion, silicone lymphadenopathy is a rare complication of procedures involving insertion of silicone-containing prostheses. This case study highlights the fact that patients need a thorough preoperative evaluation with histologic confirmation of the non-malignant nature of regional lymphadenopathy and reinforces the need to employ a high index of clinical suspicion, in order to exclude malignancy, without leading patients to dangerous overtreatment regimes.

Footnotes

Source of Support: Nil

Conflict of Interest: None declared.

REFERENCES

1. Truong LD, Cartwright J, Jr, Goodman MD, Woznicki D. Silicone lymphadenopathy associated with augmentation mammaplasty. Morphologic features of nine cases. Am J Surg Pathol. 1988;12:484–91. [PubMed]
2. Adams ST, Cox J, Rao GS. Axillary silicone lymphadenopathy presenting with a lump and altered sensation in the breast: a case report. J Med Case Reports. 2009;3:6442. [PMC free article] [PubMed]
3. Shaaban H, Jmor S, Alvi R. Leakage and silicone lymphadenopathy with cohesive breast implant. Br J Plast Surg. 2003;56:518–9. [PubMed]
4. Blum A, Abboud W, Shajrawi I, Tatour I. Prolonged fever due to silicone granulomatosis. Isr Med Assoc J. 2007;9:121–2. [PubMed]
5. Gil T, Mettanes I, Aman B, Taran A, Shoshani O, Best LA, et al. Contralateral internal mammary silicone lymphadenopathy imitates breast cancer metastasis. Ann Plast Surg. 2009;63:39–41. [PubMed]
6. Tabatowski K, Elson CE, Johnston WW. Silicone lymphadenopathy in a patient with mammary prosthesis. Fine needle aspiration cytology, histology and analytical electron microscopy. Acta Cytol. 1990;34:10–4. [PubMed]
7. Katzin WE, Centeno JA, Feng LJ, Kiley M, Mullick FG. Pathology of lymph nodes from patients with breast implants: A histologic and spectroscopic evaluation. Am J Surg Pathol. 2005;29:506–11. [PubMed]
8. Brown SL, Pennello G, Berg WA, Soo MS, Middleton MS. Silicone gel breast implant rupture, extracapsular silicone, and health status in a population of women. J Rheumatol. 2001;28:996–1003. [PubMed]
9. Van Diest PJ, Beekman WH, Hage JJ. Pathology of silicone leakage from breast implants. J Clin Pathol. 1998;51:493–7. [PMC free article] [PubMed]

Articles from Indian Journal of Plastic Surgery : Official Publication of the Association of Plastic Surgeons of India are provided here courtesy of Medknow Publications
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Spermatic Cord Knot: A Clinical Finding in Patients with Spermatic Cord Torsion

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Copyright © 2011 A. Al-Terki and T. Al-Qaoud.
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Pertinent history taking and careful examination often taper the differentials of the acute scrotum; congruently the ability to diagnose acute spermatic cord torsion (SCT) when radiological adjuncts are not available is highly imperative. This observational study serves to present a series of 46 cases of spermatic cord torsion whereby we hypothesize the identification of a clinical knot on scrotal examination as an important clinical aid in making a decision to surgical exploration in patients with acute and subacute SCT, especially in centers where imaging resources are unavailable.

1. Introduction

Reaching the confluence between clinical findings and imaging adjuncts remains a difficult task in diagnosing spermatic cord torsion (SCT) [1]. Awaiting a radiological diagnosis of SCT in a young patient with a high index of suspicion may lead to unnecessary delay especially in patients presenting in the intermediate stage of torsion, hence rapid assessment is mandatory, and salvaging the affected testis is the ultimate goal within the time window available and the present facilities. Previous studies have demonstrated loss of the cremasteric reflex to be 99% sensitive in patients with suspected torsion [2, 3], however, in the young patient in extreme pain and discomfort, eliciting such sign can be cumbersome.

We present a series of cases, whereby upon clinical examination, SCT manifesting as a palpable cord knot distinct from the upper pole of the testes and epididymal head was observed, delineating the site of torsion of the cord: the spermatic cord knot. It is important to be able to demarcate the junction between the epididymal head and the cord where the knot will be felt. Following palpation of the testicle for lie, size, consistency, and to elicit tenderness, using a bimanual approach, the clinical knot is identified by starting at the epididymis and palpating its body up to the head, proceeding upward to palpate the spermatic cord for a semi-hard nodule, denoting the twisting of the cord (Figure 1).

Figure 1

(a) Bimanual examination demonstrating normal findings. (b) Palpation at the junction of the spermatic cord and epididymal head whereby the clinical knot can be felt.

2. Methods and Materials: Case Series

Available data from January 2009 to June 2011 on cases of acute scrotal pain presenting to our emergency department at Al-Amiri Hospital, Kuwait, was reviewed. Data on age (in years), duration of symptoms (in hours), site of pain, ultrasound use, presence of clinical knot on exam, and operative findings were extracted. The primary outcome was the presence of the spermatic cord knot on examination. Descriptive statistics of the series including frequency and percentages is presented stratified by diagnosis. Chi-squared test for trend tests was used to look for an association between age, site of torsion, the operative findings of degrees of rotation of the cord, and the primary outcome. Statistical analysis was conducted using STATA [4].

3. Results

In total, data was available on 114 patients (Table 1): 46 cases of suspected torsion (40%), 32 cases of epididymitits/orchitis (28%), 18 cases of varicocele (16%), 8 cases of inguinal hernia (7%), and 10 cases of undiagnosed pain (9%). The spermatic cord knot sign was seen amongst 40 (87% sensitivity) of the patients with SCT (Table 1), and amongst none of the other patients presenting with other diagnoses.

Table 1

Descriptive statistics of patients stratified by diagnosis.

Amongst patients with SCT, the age range was 4–32 years (mean 18.3 years). The clinical knot sign was observed mostly in patients presenting in the early stage (1 to 7 hours) of SCT (1 to 7 hours: 74%, 7 to 24 hours: 22%, >24 hours: 4%). All patients with suspected SCT were taken for surgical exploration, 44 out of 46 (96%) patients were operated based on the clinical suspicion and finding of the cord knot on examination without the need for supplementary Doppler ultrasound. Most patients were operated within 2 hours of presentation, and contralateral orchiopexy was performed simultaneously; 4 cases (8%) had an unsalvageable testis (Table 2). Ultrasound was performed for two patients whom had presented at a late stage (>24 hours). Most patients had at least a 360-degree rotation of the testicle around its axis (45 patients, 98%, Table 2). Chi-squared test for trend demonstrated a significant association between degree of rotation and presence of clinical knot sign on examination (P = 0.006), however, chi-squared tests did not show an association between age (<16 versus >16 years) and site of torsion (right versus left) with presence of the clinical knot sign on examination (P = 0.81 and P = 0.55).

Table 2

Clinical and operative findings of patients with spermatic cord torsion.

4. Discussion

Our modest series points to the potential aid the clinical knot sign adds to the emergency, pediatric, surgical, and urological staff attending to the case of acute scrotum presenting in the acute and subacute stage, when imaging is unavailable or delays action. Diagnosing SCT can be difficult, and distinction of the scrotal contents is necessary while paying particular attention to identifying the epididymis and delineating the cord from the epididymal head. A common clinical diagnostic dilemma in patients with acute scrotal pain is the inability to differentiate SCT from epididymitis and/or orchitis [5]. We demonstrated that one could help differentiate that by identifying clinical knot sign that was not present in other cases of acute scrotal pain, without delaying surgical exploration.

Earlier, MR imaging has shown specific signs that help differentiate SCT from epididymitis: the whirpool/twisting pattern and the torsion knot, which appear as swirls centered over a low-signal-intensity focus [6]. Later, a report on two cases was published demonstrating similar findings on sonography [7], whereby a central echogenic focus was seen correlating to the low-signal-intensity focus seen on MR equivalent to the torsion knot. Although previous reports have demonstrated the identification of the whirlpool pattern and torsion knot [610], previous literature has not approached this sign on clinical examination. Despite Doppler ultrasound having a high sensitivity and specificity [11] in detecting testicular torsion with blood flow patterns to help delineate torsion from inflammation (epididymitis/orchitis), and alternative techniques such as scintigraphy and MR imaging achieving even higher diagnostic accuracy [12], the use of imaging as an adjunct may only be justified in patients with a low suspicion of acute SCT. Ultrasound was used in our series as an adjunct only for 2 patients with SCT, whom presented in the late stage whereby further delay awaiting imaging would cause no further harm than already present.

Since our aim from this observational study, based on a case series, was to emphasize on a clinical finding, the spermatic cord knot, as a potential adjunct to ultrasound and imaging in centers where these facilities are unavailable, inherently our description lacks comprehensive statistical analysis. In an attempt, our results demonstrate that amongst those without a positive sign on exam, the clinical knot was still evident on surgical exploration, pointing to the difficulty that can be faced in eliciting such sign, and yet a considerably high sensitivity of 86%, and a very low specificity. However, this must be weighed against the small series presented and the fact that all patients taken for surgical exploration had underlying torsion, that is, no true negatives to serve as a numerator for a predictive value of a negative examination. As one would expect, our analysis shows a significant association between degrees of rotation of the testicle around its cord and the presence of knot on examination, however, no association was found between age and site of torsion with presence of the knot on examination.

5. Conclusion

We claim the identification of the clinical knot sign on examination helps to reassure the examining doctor of his/her suspicion of SCT in the acute and subacute stage, most importantly avoiding delay in awaiting imaging findings and decision to surgical exploration. The description of this clinical sign is particularly important to rural centers of limited resources, and in centers where Doppler and MRI studies are not readily available to aid diagnosis. However, as a result of the small number of cases, an inherent limitation of this descriptive series is our inability to reach a firm inference yet, and despite advocating the identification of this sign as a strong suspicion to proceed to scrotal exploration, a larger prospective study would enrich statistical power and serve to calculate more robust estimates of incidence, sensitivity, and specificity, and further facilitating exploration of factors associated with the spermatic cord knot while simultaneously accounting for possible confounders.

References

1. Prando D. Torsion of the spermatic cord: sonographic diagnosis. Ultrasound Quarterly. 2002;18(1):41–57. [PubMed]
2. Kadish HA, Bolte RG. A retrospective review of pediatric patients with epididymitis, testicular torsion, and torsion of testicular appendages. Pediatrics. 1998;102(1):73–76. [PubMed]
3. Rabinowitz R. The importance of the cremasteric reflex in acute scrotal swelling in children. Journal of Urology. 1984;132(1):89–90. [PubMed]
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7. Maroto A, Serres X, Torrent N, Figueras M, Hoyo D, Velayos A. Sonographic appearance of the torsion knot in spermatic cord torsion. American Journal of Roentgenology. 1992;159(5):1029–1030. [PubMed]
8. Kass EJ, Lundak B. The acute scrotum. Pediatric Clinics of North America. 1997;44(5):1251–1266. [PubMed]
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10. Mattrey RF. Magnetic resonance imaging of the scrotum. Seminars in Ultrasound CT and MRI. 1991;12(2):95–108. [PubMed]
11. Kravchick S, Cytron S, Leibovici O, et al. Color doppler sonography: its real role in the evaluation of children with highly suspected testicular torsion. European Radiology. 2001;11(6):1000–1005. [PubMed]
12. Wu HC, Sun SS, Kao A, Chuang FJ, Lin CC, Lee CC. Comparison of radionuclide imaging and ultrasonography in the differentiation of acute testicular torsion and inflammatory testicular disease. Clinical Nuclear Medicine. 2002;27(7):490–493. [PubMed]

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