cell biochemistry and function Cell Biochem Funct 2014; 32: 470–475. Published online 2 June 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3039
Uptake and metabolism of D-glucose in isolated acinar and ductal cells from rat submandibular glands Sibel Cetik*, Aigun Rzajeva, Emeline Hupkens, Willy J. Malaisse and Abdullah Sener Laboratory of Experimental Hormonology, Université Libre de Bruxelles, Brussels, Belgium
The present study deals with the possible effects of selected environmental agents upon the uptake and metabolism of D-glucose in isolated acinar and ductal cells from the rat submandibular salivary gland. In acinar cells, the uptake of D-[U-14C]glucose and its non-metabolised analogue 3-O-[14C-methyl]-D-glucose was not affected significantly by phloridzin (0.1 mM) or substitution of extracellular NaCl (115 mM) by an equimolar amount of CsCl, whilst cytochalasin B (20 μM) decreased significantly such an uptake. In ductal cells, both phloridzin and cytochalasin B decreased the uptake of D-glucose and 3-O-methyl-D-glucose. Although the intracellular space was comparable in acinar and ductal cells, the catabolism of D-glucose (2.8 or 8.3 mM) was two to four times higher in ductal cells than in acinar cells. Phloridzin (0.1 mM), ouabain (1.0 mM) and cytochalasin B (20 μM) all impaired D-glucose catabolism in ductal cells. Such was also the case in ductal cells incubated in the absence of extracellular Ca2+ or in media in which NaCl was substituted by CsCl. It is proposed that the ductal cells in the rat submandibular gland are equipped with several systems mediating the insulin-sensitive, cytochalasin B-sensitive and phloridzin-sensitive transport of D-glucose across the plasma membrane. Copyright © 2014 John Wiley & Sons, Ltd. key words—isolated acinar and ductal cells; 3-O-[14C-methyl]-D-glucose or D-[U-14C]glucose handling; D-glucose metabolism
INTRODUCTION It is well known that human secretes about 1 l of saliva per day and that the resting saliva secretion is contributed mainly by the submandibular and sublingual glands. More than 100 (glyco)proteins have been identified, and some of them are secreted in the oral cavity from the human submandibular and sublingual salivary glands.1,2 The salivary gland acinar cells secrete NaCl-rich isotonic fluid, and the salivary ducts modify the ionic composition of this fluid and deliver a hypotonic saliva to the oral cavity.3 These two considerations point to the importance of the uptake of glucose and its metabolism in the acinar and ductal cells from the submandibular gland. In fact, several reports show that salivary glands express glucose transporters such as sodium–glucose co-transporter (SGLT) 1 and glucose transporter (GLUT) 1 in parotid and submandibular glands and GLUT4 in ductal cells from submandibular glands.4–9 It was recently shown that the resting salivary D-glucose turn-over rate, as calculated from the amount of glucose secreted from saliva that comes from parotid and submandibular and sublingual glands represented more than 200% min 1.10 Moreover, as the ducts represent only between 5% (parotid gland) and 20% (submandibular gland) of the gland cell volume, submandibular glands offer the opportunity to differentially study ductal and acinar cells.11,12 *Correspondence to: Sibel Cetik, Laboratory of Experimental Hormonology, Université Libre de Bruxelles, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
In the light of the latter observations, it was considered of interest to study the uptake of D-glucose and its metabolism in isolated acinar and ductal cells from the submandibular gland.
MATERIALS AND METHODS Isolation of acinar and ductal cells from the submandibular gland For the isolation of acinar and ductal cells from the submandibular gland, female Wistar rats (aged 12 to 13 weeks, obtained from Charles River Laboratory, Brussels, Belgium) were used. The method was adapted from that previously proposed.11 Briefly, submandibular glands from two rats (about 0.27 g) were isolated and digested in the presence of 10 mg of collagenase A (Roche, Mannheim) in 15 ml of Hank’s balanced salt solution calcium-free and magnesium-free media (Gibco, Invitrogen, Carlsbad, CA, USA) during twice 15 min at 37 °C. Between each incubation of 15 min, tissues were mechanically digested ten times through a syringe 20 g. The suspension was then passed through a layer of nylon strainer, which separated the submandibular cells from pieces of undigested connective tissue. After a series of washing in the previously mentioned solution in the aim to separate the tissues from the enzyme solution, acinar cells were purified by isopycnic centrifugation as described by Dehaye and Turner.11 Acinar and ductal cells were incubated during 60 min at 37 °C in a medium Received 6 March 2014 Revised 5 May 2014 Accepted 6 May 2014
D-GLUCOSE HANDLING BY ISOLATED SALIVARY CELLS
471
equilibrated against an O2/CO2 mixture (95/5, v/v). After incubation, they were suspended in the appropriate incubation bicarbonate-buffered media13 containing a HEPES-NaOH buffer (20 mM, pH 7.4) and bovine serum albumin (1.0 mg ml 1). The cell viability was always assessed by the trypan blue exclusion method, and it exceeded 80% over many preparations.
of 14CO2 and 3HOH under the same experimental conditions. The recovery of 3HOH formed during incubation was 16.8 ± 1.1% (n = 9) under our experimental conditions. The recovery of 14CO2 prepared from H14CO3 was nearly 100% under our experimental conditions (data not shown). D-[5-3H]glucose and H14CO3 were purchased from PerkinElmer, Boston, MA, USA.
3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose handling by isolated acinar and ductal cells
Protein determination
The method used for measuring the net uptake 3-O-methylD-glucose or D-glucose by isolated acinar and ductal cells was adapted from that previously described.14 Briefly, 50 μl of cell suspension (50–75 · 103 cells) was mixed with 50 μl of a bicarbonate-buffered and HEPES-buffered saltbalanced medium containing either L-[1-14C]glucose (2.0 mM) and 3HOH (5.0 μCi ml 1) for the determination of extracellular and intracellular spaces or 3-O-[14Cmethyl]-D-glucose (1.0 μCi ml 1) or D-[U-14C]glucose (1.0 μCi ml 1), each mixed, respectively, with 16.7 mM unlabelled 3-O-methyl-D-glucose or D-glucose and 3HOH (5.0 μCi ml 1). All these isotopes were purchased from PerkinElmer, Boston, MA, USA. Cells were incubated for 5 to 30 min at 37 °C. In a series of experiments, either 0.1 mM phloridzin dehydrate (Sigma, St. Louis, MO, USA) or 20 μM cytochalasin B (Sigma) was added to the incubation medium, whilst in other experiments, NaCl (115 mM) was substituted in the incubation medium by an equimolar amount of CsCl. After incubation, 0.15 ml of a mixture of dibutylphthalate and di-isononylphthalate (10:3, v/v) was added to each tube, which was then centrifuged for 3 min at 5000g. The bottom of the tube containing the cell pellet was then cut, placed in a counting vial containing 5.0 ml of scintillation fluid and, after mixing, examined for its radioactive content in double channel 14C/3H. After correction for the blank value found under the same experimental conditions in the absence of cells, the results were expressed as nl 103 cells 1. Metabolism of D-glucose in isolated acinar and ductal cells The method used for the measurement of D-[5-3H]glucose utilisation and D-[U-14C]glucose oxidation was described previously.15 In a first series of experiments, 50 μl of isolated acinar cells (74 ± 4 · 103 cells/assay; n = 9), or isolated ductal cells (58 ± 8 · 103 cells/assay; n = 9), was mixed with 50 μl of a bicarbonate-buffered medium containing either 5.6 or 16.7 mM, with tracer amounts of D-[5-3H]glucose (6.0 μCi ml 1) and D-[U-14C]glucose (0.7 μCi ml 1). After 120-min incubation at 37 °C, the metabolism of glucose was stopped by the addition of 100 μl of a citrate-NaOH buffer (400 mM, pH 4.9) containing metabolic poisons such as NaF 10 mM and KCN 5 mM. The 14CO2 and 3HOH formed during incubation were recovered over 60 min and 20 h at room temperature (22–24 °C) respectively. The results were calculated as pmol/120 min and per 103 cells after subtraction of the blank value found after incubation in the absence of cells and taking into account the recovery Copyright © 2014 John Wiley & Sons, Ltd.
The protein content of acinar and ductal cells was measured by the method of Lowry et al.16 in acinar and ductal cell homogenates (about 1.3 · 106 cells/ml H2O), using 5–25-μl homogenate and bovine serum albumin as standard (2.5 to 15 μg/assay). Statistical analysis All results are presented as mean values (±SEM) together with either the number of individual determinations (n) or degree of freedom. The statistical significance of differences between mean values was assessed by use of Student’s t-test. RESULTS Uptake of 3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose by acinar cells In the acinar cells, the 3HOH distribution space, expressed as nl/103 cells, averaged after 10, 20 and 30-min incubation respectively 3.88 ± 0.59 (n = 15), 3.42 ± 0.19 (n = 5) and 3.69 ± 0.46 (n = 16), yielding an overall mean value of 3.73 ± 0.32 nl/103 cells (n = 36). In the same experiments, the L-[1-14C]glucose distribution space averaged 1.47 ± 0.20 nl/103 cells (n = 36), representing 37.7 ± 2.7% (n = 36) of the paired 3HOH space. As judged from these data, the intracellular space averaged 2.26 ± 018 nl/103 cells (n = 36). Relative to the paired 3HOH distribution space, that of D-[U-14C]glucose (8.3 mM) averaged after 5, 15 and 20–30-min incubation respectively 59.3 ± 2.6% (n = 5), 62.4 ± 1.3% (n = 5) and 60.3 ± 3.6% (n = 20). The overall mean value for the D-[U-14C]glucose distribution space thus averaged 60.5 ± 2.4% (n = 30) of the paired 3 HOH space. The latter value was not significantly different ( p > 0.31) from the somewhat lower mean value found for the distribution space of 3-O-[14C-methyl]-D-glucose (also 8.3 mM), again expressed relative to the paired 3 HOH space, i.e. 54.9 ± 4.1% (n = 47). Relative to the paired 3HOH space, the mean apparent distribution spaces of either D-[U-14C]glucose or 3-O-[14C-methyl]D-glucose were both higher ( p < 0.003 or less) than that of L-[1-14C]glucose (Table 1). Over 30-min incubation, phloridzin (0.1 mM) did not affect significantly (p > 0.85) the distribution space of 3O-[14C-methyl]-D-glucose, which averaged 103.6 ± 19.5% (n = 9) of the mean corresponding control values recorded within the same experiments (100.0 ± 7.4%; n = 9). Likewise, as judged from the ratio between the distribution space of D-[U-14C]glucose and that of 3HOH, phloridzin also Cell Biochem Funct 2014; 32: 470–475.
472 Table 1.
s. cetik Apparent distribution spaces in acinar and ductal cells
Parameters 3
3
HOH space (nl/10 cells) 14 3 space (nl/10 cells) 3 space/ HOH space (%) 3 Intracellular space (nl/10 cells) 14 3 D-[U- C]glucose space/ HOH space (%) 14 3-O-[ C-methyl]-D-glucose 3 space/ HOH space (%) L-[1- C]glucose 14 L-[1- C]glucose
Acinar cells
Ductal cells
3.73 ± 0.32 (36) 1.47 ± 0.20 (36) 37.7 ± 2.7 (36)
3.61 ± 0.32 (36) 1.17 ± 0.13 (36) 34.8 ± 2.5 (36)
2.26 ± 0.18 (36) 60.5 ± 2.4 (30)
2.44 ± 0.27 (36) 67.8 ± 3.2 (26)
54.9 ± 4.1 (47)
56.1 ± 4.8 (41)
failed to affect significantly ( p > 0.37) the fate of the labelled hexose, the experimental results averaging 95.9 ± 4.4% (n = 9) of the mean control values recorded within the same experiments (100.0 ± 1.5%; n = 10). Based on the same ratio between the distribution space of 14 3 D-[U- C]glucose and that of HOH, cytochalasin B (20.0 μM) decreased significantly ( p < 0.03) the uptake of 14 D-[U- C]glucose, the experimental data averaging 88.6 ± 4.6% (n = 10) of the mean corresponding control values (100.0 ± 1.6%; n = 10). Such was also the case ( p < 0.025) in the cells exposed to 3-O-[14C-methyl]-Dglucose, the experimental data averaging 77.0 ± 5.0% (n = 8) of the mean corresponding control values (100.0 ± 7.2%; n = 9). The relative magnitude of the inhibitory effect of cytochalasin B was not significantly different ( p > 0.1) in the case of D-[U-14C]glucose and 3-O-[14C-methyl]-D-glucose uptake. Last, the substitution of NaCl (115 mM) by an equimolar amount of CsCl in the incubation medium was found not to affect significantly (p > 0.19) the uptake of D-[U-14C] glucose by the acinar cells. Thus, the ratio between the distribution space of D-[U-14C]glucose and that of 3HOH averaged in the presence of CsCl 93.2 ± 4.0% (n = 10) of the mean corresponding control values recorded in the usual incubation medium (100.0 ± 3.1%; n = 10). Uptake of 3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose by ductal cells Within 10 min of incubation, the 3HOH distribution space in ductal cells reached a steady value averaging 3.61 ± 0.32 nl/103 cells (n = 36). There was indeed no significant difference (p > 0.88) between the measurements made after either 10–20-min incubation (3.67 ± 0.38 nl/ 103 cells; n = 15) or 30-min incubation (3.57 ± 0.48 nl/103 cells; n = 21). Likewise, after 20–30-min incubation, the distribution space of L-[1-14C]glucose was not higher than that recorded after only 10-min incubation, with an overall mean value of 1.17 ± 0.13 nl/103 cells (n = 36), corresponding to 34.8 ± 2.5% of the paired 3HOH space. As judged from these measurements, the intracellular space averaged in the ductal cells 2.44 ± 0.27 nl/103 cells (n = 36), a value not significantly different (p > 0.58) from that found in acinar cells (2.26 ± 0.18 nl/103 cells; n = 36). The mean protein content was somewhat higher, albeit not significantly so, in acinar cells (0.77 ± 0.25 μg/103 cells; n = 4) Copyright © 2014 John Wiley & Sons, Ltd.
ET AL.
than in ductal cells (0.44 ± 0.05 μg/103 cells; n = 4), with an overall mean value of 0.61 ± 0.13 μg/103 cells (n = 8). After 5 to 60-min incubation, the paired ratio between the distribution space of 3-O-[14C-methyl]-D-glucose (8.3 mM) and that of 3HOH averaged 56.1 ± 4.8% (n = 41), a value significantly higher ( p < 0.001) than that found between the distribution space of L-[1-14C]glucose and that of 3HOH (34.8 ± 2.5%; n = 36). After 5 to 30-min incubation, the paired ratio between the distribution space of D-[U-14C]glucose (also 8.3 mM) and that of 3HOH averaged 67.8 ± 3.2% (n = 26), a value somewhat higher, albeit not significantly so ( p < 0.08) than that found for the paired ratio between the distribution space of 3-O-[14C-methyl]-D-glucose and that of 3HOH. In the latter respect, the situation found in ductal cells was comparable with that observed in acinar cells, the difference between the mean values recorded with D-[U-14C]glucose and somewhat lower mean values found with 3-O-[14C-methyl]-D-glucose only achieving statistical significance ( p < 0.05) when pooling together the two sets of data collected in both acinar and ductal cells, as duly listed in Table 1. Thus, such pooled data averaged 63.9 ± 2.0% (n = 56) in the case of D-[U-14C]glucose, as distinct ( p < 0.05) from 55.5 ± 3.1% (n = 88) in the case of 3-O-[14C-methyl]-D-glucose. The substitution of NaCl (115 mM) by CsCl decreased (p < 0.001) in the ductal cells the paired ratio between the distribution space of D-[U-14C]glucose and that of 3HOH to 58.1 ± 7.1% (n = 9) of the mean control values recorded within the same experiments (100.0 ± 7.1%; n = 9). None of the other agents tested in the present study, i.e. phloridzin (0.1 mM) or cytochalasin B (20.0 μM), affected significantly ( p > 0.17 or more) the paired ratio between the distribution space of either D-[U-14C]glucose or 3-O[14C-methyl]-D-glucose and that of 3HOH. Thus, pooling together the results recorded in ductal cells exposed to either D-glucose or its non-metabolised analogue, such a paired ratio averaged in the presence of either phloridzin 69.9 ± 9.2% (n = 8) or cytochalasin B 67.3 ± 3.5% (n = 10), as compared with a mean control value recorded within the same experiment(s) of 74.3 ± 3.5% (n = 10) The two following considerations should be duly stressed, however. First, at variance with the situation found in acinar cells, phloridzin severely decreased in the ductal cells both the 3HOH distribution space and that of either 14 14 D-[U- C]glucose or 3-O-[ C-methyl]-D-glucose to respectively 46.4 ± 9.8% (n = 14; p < 0.001) and 61.2 ± 6.5% (n = 27; p < 0.001) of the mean corresponding control values, i.e. 100.0 ± 5.9% (n = 15) and 100.0 ± 6.8% (n = 28). The relative extent of the phloridzin-induced decrease in these distribution spaces was not significantly different (p > 0.20) in the case of 3HOH and in that of D-glucose and its non-metabolised analogue. Second, in a larger set of experiments, in which the distribution spaces of either 14 14 D-[U- C]glucose or 3-O-[ C-methyl]-D-glucose were duly measured, but not that of 3HOH, cytochalasin B was found to decrease significantly ( p < 0.02) the distribution space of D-glucose and its non-metabolised analogue to 68.0 ± 8.6% (n = 22) of the mean corresponding control values (100.0 ± 10.7%; n = 23). Cell Biochem Funct 2014; 32: 470–475.
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Metabolism of D-glucose in acinar and ductal cells In the acinar cells exposed to 2.8 mM D-glucose, the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation averaged no more than 6.84 ± 0.63% (n = 32). A rise in D-glucose concentration from 2.8 to 8.3 mM increased significantly D-[5-3H]glucose conversion to 3HOH (p < 0.02) but did not affect significantly D-[U-14C]glucose conversion to 14CO2 (Table 2). The mean paired 14CO2/3HOH ratio was thus lower at 8.3 mM than at 2.8 mM D-glucose concentration. Such a difference failed, however, to achieve statistical significance ( p > 0.3). A comparable situation prevailed in ductal cells. Thus, the rise in D-glucose concentration did not affect significantly D-[U-14C]glucose oxidation, despite a significant increase in D-[5-3H]glucose utilisation ( p < 0.002). Such changes resulted in a significant decrease of the paired 14CO2/3HOH ratio ( p < 0.002). The relative magnitude of the increase in D-[5-3H]glucose conversion to 3HOH in response to the rise in D-glucose concentration from 2.8 to 8.3 mM was virtually identical ( p > 0.77) in the acinar cells (+40.5%) and ductal cells 14
3
Table 2. D-[U- C]glucose oxidation and D-[5- H]glucose utilisation by acinar and ductal cells from submandibulary glands Metabolic variable Acinar cells 2.8 mM D-glucose 8.3 mM D-glucose Ductal cells 2.8 mM D-glucose 8.3 mM D-glucose
14
CO2*
3
HOH†
CO2/HOH‡
14
10.3 ± 1.2 (37) 164.7 ± 20.7 (33) 10.3 ± 1.7 (31) 232.0 ± 35.5 (34)
3
6.84 ± 0.63(32) 4.97 ± 0.85(29)
40.2 ± 4.5 (38) 304.8 ± 16.8 (36) 13.08 ± 1.48 (34) 31.1 ± 2.8 (39) 449.3 ± 34.6 (34) 7.42 ± 0.74 (34)
14
3
*D-[U- C]glucose oxidation (expressed as D-glucose equivalent/10 cells per 120 min). 3 3 † D-[5- H]glucose utilisation (expressed as D-glucose equivalent/10 cells per 120 min). 14 3 ‡ Paired ratio between D-[U- C]glucose oxidation and D-[5- H]glucose utilisation (expressed as a percentage).
Table 3.
(+47.4%). Two differences between these two types of cells merit, however, to be underlined. First, whether at 2.8 or 8.3-mM D-glucose concentration and whether in the case of D-[5-3H]glucose utilisation or D-[U-14C]glucose oxidation, the mean values recorded in ductal cells were about twice (3HOH) or three to four times higher (14CO2) than the corresponding mean values found in acinar cells. Second, whether at 2.8 or 8.3 mM D-glucose, the paired 14 CO2/3HOH ratio was significantly higher (p < 0.04 or less) in ductal cells than in acinar cells. In ductal cells incubated for 120 min in the presence of 8.3 mM D-glucose, phloridzin (0.1 mM) decreased significantly both D-[U-14C]glucose oxidation and D-[5-3H] glucose utilisation, whilst failing to affect significantly the paired ratio between these two variables (Table 3). Ouabain (1.0 mM) decreased significantly not only D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation but also the paired 14 CO2/3HOH ratio. Expressed relative to the mean control values recorded within the same experiment(s), the measurements of D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation recorded in the presence of ouabain were significantly lower than those found in the presence of phloridzin, such not being the case for the paired 14CO2/3HOH ratio. An even more severe inhibition of D-[U-14C]glucose oxidation was observed in the nominal absence of extracellular Ca2+ and presence of EDTA (1.0 mM), in media in which 115 mM NaCl was substituted by 115 mM CsCl, and in the presence of cytochalasin B (20.0 μM). Under the latter three experimental conditions, the utilisation of D-[5-3H]glucose was also impaired, such a decrease being least pronounced upon substitution of NaCl by CsCl and most marked in the presence of cytochalasin B. The absence of extracellular Ca2+ and the presence of cytochalasin B both decreased by about 40% the paired 14 CO2/3HOH ratio, a further decrease in such a ratio to about one fifth of the corresponding control values being observed upon substitution of NaCl by CsCl (Table 3).
Effects of environmental agents on the metabolism of D-glucose (8.3 mM) in ductal cells
Metabolic variable Absolute control values Control† Phlorizin (0.1 mM)** Control† Ouabain (1.0 mM)** Control† 2+ No Ca + EDTA (1.0 mM)** Control† NaCl substituted by CsCl** Control† Cytochalasin B (20.0 μM)**
14
CO2*
25.7 ± 1.7 (82)§ 100.0 ± 4.4 (36) 73.0 ± 4.1 (36)†† 100.0 ± 5.4 (33) 49.9 ± 3.8 (32)†† 100.0 ± 6.9 (19) 28.4 ± 2.7 (19)†† 100.0 ± 6.9 (19) 13.8 ± 1.6 (19)†† 100.0 ± 8.5 (20) 8.9 ± 1.4 (16)††
3
HOH†
339.6 ± 23.1 (77)§ 100.0 ± 5.2 (36) 78.7 ± 4.8 (32)‡‡ 100.0 ± 7.3 (34) 58.7 ± 4.2 (32)†† 100.0 ± 8.7 (18) 43.7 ± 5.5 (16)†† 100.0 ± 8.7 (18) 71.9 ± 6.7 (18)*** 100.0 ± 5.8 (19) 19.4 ± 3.2 (19)††
14
CO2/HOH‡ 3
8.84 ± 0.65 (74)¶ 100.0 ± 6.4 (34) 90.0 ± 5.1 ((30)§§ 100.0 ± 7.3 (29) 75.2 ± 6.0 (30)¶¶ 100.0 ± 8.4 (18) 62.0 ± 5.7 (16)†† 100.0 ± 8.4 (18) 22.9 ± 4.7 (16)†† 100.0 ± 9.9 (196) 56.4 ± 11.3 (16)†††
14
*D-[U- C]glucose oxidation. 3 † D-[5- H]glucose utilisation. 14 3 ‡ Paired ratio between D-[U- C]glucose oxidation and D-[5- H]glucose utilisation. 3 § Results expressed as D-glucose equivalent/10 cells per 120 min. ¶ Paired ratio expressed as a percentage. **Results expressed in % of the mean control values found within the same experiment(s). †† p < 0.001; ‡‡p < 0.005; §§p > 0.22; ¶¶p < 0.009; ***p < 0.02; †††p < 0.007. Copyright © 2014 John Wiley & Sons, Ltd.
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s. cetik
DISCUSSION As indicated in the Introduction of this article, a major aim of the present study was to investigate the uptake and catabolism of D-glucose in acinar and ductal cells from the rat submandibular salivary gland. Moreover, attention was also paid to the possible effects of selected environmental agents upon such variables. In this respect, cytochalasin B was used as a GLUT inhibitor, whilst phloridzin was used as a non-specific SGLT inhibitor. The substitution of NaCl by CsCl aimed at investigating the possible co-transport of Na+ and D-glucose (or 3-O-methyl-D-glucose) by SGLT. The incorporation of ouabain in the incubation medium aimed at inhibiting the ATP-dependent Na+-K+-ATPase. Last, the omission of extracellular Ca2+ was considered as a potential tool to inhibit the activity of Ca2+-responsive mitochondrial dehydrogenases. Four major findings concerning the apparent distribution spaces of D-[U-14C]glucose and 3-O-[14C-methyl]-Dglucose in acinar and ductal cells merit to be underlined. First, pooling together the results collected in these two cell types, relative to the paired 3HOH space, that of D-[U-14C] glucose slightly but significantly exceeded that of 3-O[14C-methyl]-D-glucose. Such a difference is likely to reflect the time-related intracellular accumulation of the radioactive metabolites of D-[U-14C]glucose. The latter view is also compatible with the trend towards a greater difference between the results obtained with D-[U-14C]glucose and those obtained with 3-O-[14C-methyl]-D-glucose in ductal cells than in acinar cells. Our metabolic data indeed document the more efficient catabolism of D-glucose in ductal cells than in acinar cells. Second, in ductal cells, but not so in acinar cells, phloridzin decreased severely the distribution space of both 3HOH and either D-[U-14C]glucose or 3-O-[14C-methyl]-D-glucose, with a trend towards a more pronounced decrease in the former 3HOH space than in the latter distribution space of D-glucose or its non-metabolised analogue. These findings are consistent with the driving role of Na+ in the SGLT-mediated symport of the monovalent cation and D-glucose (or its non-metabolised analogue). Third, cytochalasin B decreased significantly the distribution space of D-[U-14C]glucose and 3-O-[14C-methyl]-D-glucose in both acinar and ductal cells. Last, in ductal cells, but not so in acinar cells, the substitution of extracellular NaCl by CsCl significantly decreased the paired ratio between the distribution space of D-[U-14C]glucose and that of 3HOH. Such a finding could conceivably be attributed, in part at least, to an impaired symport of D-glucose and Na+ as mediated by SGLT. Three sets of data relate to the possible role of Na+ cation in the uptake and catabolism of D-glucose in ductal cells. First, the already mentioned decrease caused by phloridzin in the apparent distribution spaces of both 3HOH and either 14 14 D-[U- C]glucose or 3-O-[ C-methyl]-D-glucose coincided with a sizeable decrease of both D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation but no significant decrease of the paired 14C02/3HOH ratio. Taken as a whole, these findings suggest that the inhibition of the Na+ and D-glucose Copyright © 2014 John Wiley & Sons, Ltd.
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symport, as mediated by SGLT, coincided with a decrease in D-glucose catabolism and, hence, ATP generation. The latter
decrease is conceivably attributable to a lower activity of the Na+-K+-ATPase pump, itself resulting from the impaired entry of Na+ into the ductal cells and, hence, lesser energy need required for the extrusion of Na+ cations from the same cells. Second, the severe and preferential impairment of D-[U-14C]glucose oxidation, as distinct from D-[5-3H] glucose utilisation, observed in ductal cells upon substitution of NaCl (115 mM) by an equimolar amount of CsCl again points to the interdependence of energy need and Na+ extrusion by the Na+-K+-ATPase pump as modulated, in this case, by the availability of extracellular Na+ and, hence, its influx into the ductal cells. Last, the comparable inhibitory effects (p > 0.08) of ouabain and NaCl substitution by CsCl upon D-[5-3H]glucose utilisation, but less a severe decrease of D-[U-14C]glucose oxidation caused by the cardiac glycoside as compared with the extracellular substitution of NaCl by CsCl, remain compatible with a significant contribution of the ouabain-sensitive Na+ extrusion process in the consumption of energy in the ductal cells. Incidentally, according to Mangos et al.,17 ouabain also affects sodium transport into rat parotid acinar cells. Two further sets of data should not be ignored. First, the decrease of D-[U-14C]glucose oxidation, D-[5-3H]glucose utilisation and paired ratio between the latter two variables observed in ductal cells incubated in the absence of extracellular Ca2+ is consistent with the normal activation by Ca2+ of several mitochondrial dehydrogenases.18 Second, the even more severe decrease in both D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation caused by cytochalasin B, which coincided with a significant decrease of either 14 14 D-[U- C]glucose or 3-O-[ C-methyl]-D-glucose distribution space in the ductal cells, argues in support of the view that the transport-mediated inflow of D-glucose in these cells may represent a rate-limiting step of its intracellular catabolism. In this respect, the situation found in ductal cells is tightly comparable with that found in rat pancreatic islets.19,20 For instance, the relative magnitude of the inhibitory action of cytochalasin B upon the apparent distribution space of D-glucose or its non-metabolised analogue 3-O-methyl-D-glucose is not significantly different (p > 0.6) in the ductal cells and pancreatic islets, the experimental data recorded in the presence of cytochalasin B (20.0 μM) and expressed relative to the mean corresponding control values found within the same experiments averaging respectively 68.0 ± 8.6% (n = 22) in ductal cells and 73.8 ± 4.0% (n = 14) in rat pancreatic islets.19 It was recently reported that insulin increases D-glucose uptake by ductal rings from the rat submandibular gland.21 In the light of both the latter observation and the present findings, it might be concluded that the ductal cells from this salivary gland are indeed equipped with several systems, such as GLUT1, GLUT4 and SGLT, participating in insulin-sensitive, cytochalasin B-sensitive and phloridzin-sensitive D-glucose transport across the plasma membrane. Cell Biochem Funct 2014; 32: 470–475.
D-GLUCOSE HANDLING BY ISOLATED SALIVARY CELLS
CONFLICT OF INTEREST The authors have declared that there is no conflict of interest. ACKNOWLEDGEMENTS This study was supported by the Belgian Foundation for Scientific Medical Research (grant 3.4520.07) and by a grant from the European Commission (Collaborative project VIBRANT 228933: In Vivo Imaging of Beta Cell Receptors by Applied Nano-Technology). REFERENCES 1. Gonzalez-Begne M, Lu B, Liao L, et al. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology. J Proteome Res 2011; 10: 5031–5046. 2. Stoeckelhuber M, Scherer EQ, Janssen KP, et al. The human submandibular gland: immunohistochemical analysis of SNAREs and cytoskeletal proteins. J Histochem Cytochem 2012; 60: 110–120. 3. Lee MG, Ohana E, Park HW, Yang D, Muallem S. Molecular mechanism of pancreatic and salivary gland fluid and HCO3 secretion. Physiol Rev 2012; 92: 39–74. 4. Tarpey PS, Wood IS, Shirazi-Beechey SP, Beechey RB. Amino acid sequence and the cellular location of the Na+-dependent D-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell. Biochem J 1995; 312: 293–300. 5. Balen D, Ljubojevic M, Breljak D, et al. Revised immunolocalization of the Na+/D-glucose cotransporter SGLT1 in rat organs with an improved antibody. Am J Physiol Cell Physiol 2008; 295: C475–C489. 6. Sabino-Silva R, Freitas HS, Lamers ML, Okamoto MM, Santos MF, Machado UF. Na+/glucose cotransporter SGLT1 protein in salivary glands: potential involvement in the diabetes-induced decrease in salivary flow. J Membr Biol 2009; 228: 63–69. 7. Sabino-Silva R, Alves-Wagner AB, Burgi K, et al. SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats. Am J Physiol Endocrinol Metab 2010; 99: E1028–E1037.
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8. Jurysta C, Nicaise C, Cetik S, Louchami K, Malaisse WJ, Sener A. Glucose transport by acinar cells in rat parotid glands. Cell Physiol Biochem 2012; 29: 325–330. 9. Jurysta C, Nicaise C, Giroix MH, Cetik S, Malaisse WJ, Sener A. Comparison of GLUT1, GLUT2, GLUT4 and SGLT1 mRNA expression in the salivary glands and six other organs of control, streptozotocin-induced and Goto-Kakizaki diabetic rats. Cell Physiol Biochem 2013; 31: 37–43. 10. Cetik S, Zhang Y, Hupkens E, Jurysta C, Malaisse WJ, Sener A. A tentative model for D-glucose turnover in human saliva. Arch Oral Biol 2013; 2013: 1265–1270. 11. Dehaye JP, Turner RJ. Isolation and characterization of rat submandibular intralobular ducts. Am J Physiol 1991; 261: C490–C496. 12. Turner JT, Weisman GA, Camden JM. Upregulation of P2Y2 nucleotide receptors in rat salivary gland cells during short-term culture. Am J Physiol 1997; 273: C1100–C1107. 13. Malaisse WJ, Maggetto C, Leclercq-Meyer V, Sener A. Interference of glycogenolysis with glycolysis in pancreatic islets from glucoseinfused rats. J Clin Invest 1993; 91: 432–436. 14. Ramirez R, Rasschaert J, Laghmich A, et al. Uptake of Dmannoheptulose by normal and tumoral pancreatic islet cells. Int J Mol Med 2001; 7: 631–638. 15. Malaisse WJ, Sener A. Hexose metabolism in pancreatic islets. Feedback control of D-glucose oxidation by functional events. Biochim Biophys Acta 1988; 971: 246–254. 16. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–275. 17. Mangos JA, Mcsherry NR, Boutcher F, Irwin K, Barber T. Dispersion rat parotid acinar cells. I. Morphological and functional characterisation. Am J Physiol 1975; 229: 553–559. 18. Denton RM. Regulation of mitochondrial dehydrogenases by calcium ions. Biochim Biophys Acta 2009; 1787: 1309–1316. 19. Levy J, Herchuelz A, Sener A, Malaisse-Lagae F, Malaisse WJ. Cytochalasin B-induced impairment of glucose metabolismo in islets of Langerhans. Endocrinology 1976; 98: 429–437. 20. Jijakli H, Zhang HX, Dura E, Remedos R, Sener A, Malaisse WJ. Effect of cytochalasin B and D upon insulin relase and pancreatic islet cell metabolism. Int J Mol Med 2002; 9: 163–172. 21. Cetik S, Hupkens E, Malaisse WJ, Sener A, Ristea PI. Expression and localization of glucose transporters in rodent submandibular salivary glands. Cell Physiol Biochem 2014; 33: 1149–1161.
Cell Biochem Funct 2014; 32: 470–475.
Uptake and metabolism of D-glucose in isolated acinar and ductal cells from rat submandibular glands Sibel Cetik*, Aigun Rzajeva, Emeline Hupkens, Willy J. Malaisse and Abdullah Sener Laboratory of Experimental Hormonology, Université Libre de Bruxelles, Brussels, Belgium
The present study deals with the possible effects of selected environmental agents upon the uptake and metabolism of D-glucose in isolated acinar and ductal cells from the rat submandibular salivary gland. In acinar cells, the uptake of D-[U-14C]glucose and its non-metabolised analogue 3-O-[14C-methyl]-D-glucose was not affected significantly by phloridzin (0.1 mM) or substitution of extracellular NaCl (115 mM) by an equimolar amount of CsCl, whilst cytochalasin B (20 μM) decreased significantly such an uptake. In ductal cells, both phloridzin and cytochalasin B decreased the uptake of D-glucose and 3-O-methyl-D-glucose. Although the intracellular space was comparable in acinar and ductal cells, the catabolism of D-glucose (2.8 or 8.3 mM) was two to four times higher in ductal cells than in acinar cells. Phloridzin (0.1 mM), ouabain (1.0 mM) and cytochalasin B (20 μM) all impaired D-glucose catabolism in ductal cells. Such was also the case in ductal cells incubated in the absence of extracellular Ca2+ or in media in which NaCl was substituted by CsCl. It is proposed that the ductal cells in the rat submandibular gland are equipped with several systems mediating the insulin-sensitive, cytochalasin B-sensitive and phloridzin-sensitive transport of D-glucose across the plasma membrane. Copyright © 2014 John Wiley & Sons, Ltd. key words—isolated acinar and ductal cells; 3-O-[14C-methyl]-D-glucose or D-[U-14C]glucose handling; D-glucose metabolism
INTRODUCTION It is well known that human secretes about 1 l of saliva per day and that the resting saliva secretion is contributed mainly by the submandibular and sublingual glands. More than 100 (glyco)proteins have been identified, and some of them are secreted in the oral cavity from the human submandibular and sublingual salivary glands.1,2 The salivary gland acinar cells secrete NaCl-rich isotonic fluid, and the salivary ducts modify the ionic composition of this fluid and deliver a hypotonic saliva to the oral cavity.3 These two considerations point to the importance of the uptake of glucose and its metabolism in the acinar and ductal cells from the submandibular gland. In fact, several reports show that salivary glands express glucose transporters such as sodium–glucose co-transporter (SGLT) 1 and glucose transporter (GLUT) 1 in parotid and submandibular glands and GLUT4 in ductal cells from submandibular glands.4–9 It was recently shown that the resting salivary D-glucose turn-over rate, as calculated from the amount of glucose secreted from saliva that comes from parotid and submandibular and sublingual glands represented more than 200% min 1.10 Moreover, as the ducts represent only between 5% (parotid gland) and 20% (submandibular gland) of the gland cell volume, submandibular glands offer the opportunity to differentially study ductal and acinar cells.11,12 *Correspondence to: Sibel Cetik, Laboratory of Experimental Hormonology, Université Libre de Bruxelles, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
In the light of the latter observations, it was considered of interest to study the uptake of D-glucose and its metabolism in isolated acinar and ductal cells from the submandibular gland.
MATERIALS AND METHODS Isolation of acinar and ductal cells from the submandibular gland For the isolation of acinar and ductal cells from the submandibular gland, female Wistar rats (aged 12 to 13 weeks, obtained from Charles River Laboratory, Brussels, Belgium) were used. The method was adapted from that previously proposed.11 Briefly, submandibular glands from two rats (about 0.27 g) were isolated and digested in the presence of 10 mg of collagenase A (Roche, Mannheim) in 15 ml of Hank’s balanced salt solution calcium-free and magnesium-free media (Gibco, Invitrogen, Carlsbad, CA, USA) during twice 15 min at 37 °C. Between each incubation of 15 min, tissues were mechanically digested ten times through a syringe 20 g. The suspension was then passed through a layer of nylon strainer, which separated the submandibular cells from pieces of undigested connective tissue. After a series of washing in the previously mentioned solution in the aim to separate the tissues from the enzyme solution, acinar cells were purified by isopycnic centrifugation as described by Dehaye and Turner.11 Acinar and ductal cells were incubated during 60 min at 37 °C in a medium Received 6 March 2014 Revised 5 May 2014 Accepted 6 May 2014
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471
equilibrated against an O2/CO2 mixture (95/5, v/v). After incubation, they were suspended in the appropriate incubation bicarbonate-buffered media13 containing a HEPES-NaOH buffer (20 mM, pH 7.4) and bovine serum albumin (1.0 mg ml 1). The cell viability was always assessed by the trypan blue exclusion method, and it exceeded 80% over many preparations.
of 14CO2 and 3HOH under the same experimental conditions. The recovery of 3HOH formed during incubation was 16.8 ± 1.1% (n = 9) under our experimental conditions. The recovery of 14CO2 prepared from H14CO3 was nearly 100% under our experimental conditions (data not shown). D-[5-3H]glucose and H14CO3 were purchased from PerkinElmer, Boston, MA, USA.
3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose handling by isolated acinar and ductal cells
Protein determination
The method used for measuring the net uptake 3-O-methylD-glucose or D-glucose by isolated acinar and ductal cells was adapted from that previously described.14 Briefly, 50 μl of cell suspension (50–75 · 103 cells) was mixed with 50 μl of a bicarbonate-buffered and HEPES-buffered saltbalanced medium containing either L-[1-14C]glucose (2.0 mM) and 3HOH (5.0 μCi ml 1) for the determination of extracellular and intracellular spaces or 3-O-[14Cmethyl]-D-glucose (1.0 μCi ml 1) or D-[U-14C]glucose (1.0 μCi ml 1), each mixed, respectively, with 16.7 mM unlabelled 3-O-methyl-D-glucose or D-glucose and 3HOH (5.0 μCi ml 1). All these isotopes were purchased from PerkinElmer, Boston, MA, USA. Cells were incubated for 5 to 30 min at 37 °C. In a series of experiments, either 0.1 mM phloridzin dehydrate (Sigma, St. Louis, MO, USA) or 20 μM cytochalasin B (Sigma) was added to the incubation medium, whilst in other experiments, NaCl (115 mM) was substituted in the incubation medium by an equimolar amount of CsCl. After incubation, 0.15 ml of a mixture of dibutylphthalate and di-isononylphthalate (10:3, v/v) was added to each tube, which was then centrifuged for 3 min at 5000g. The bottom of the tube containing the cell pellet was then cut, placed in a counting vial containing 5.0 ml of scintillation fluid and, after mixing, examined for its radioactive content in double channel 14C/3H. After correction for the blank value found under the same experimental conditions in the absence of cells, the results were expressed as nl 103 cells 1. Metabolism of D-glucose in isolated acinar and ductal cells The method used for the measurement of D-[5-3H]glucose utilisation and D-[U-14C]glucose oxidation was described previously.15 In a first series of experiments, 50 μl of isolated acinar cells (74 ± 4 · 103 cells/assay; n = 9), or isolated ductal cells (58 ± 8 · 103 cells/assay; n = 9), was mixed with 50 μl of a bicarbonate-buffered medium containing either 5.6 or 16.7 mM, with tracer amounts of D-[5-3H]glucose (6.0 μCi ml 1) and D-[U-14C]glucose (0.7 μCi ml 1). After 120-min incubation at 37 °C, the metabolism of glucose was stopped by the addition of 100 μl of a citrate-NaOH buffer (400 mM, pH 4.9) containing metabolic poisons such as NaF 10 mM and KCN 5 mM. The 14CO2 and 3HOH formed during incubation were recovered over 60 min and 20 h at room temperature (22–24 °C) respectively. The results were calculated as pmol/120 min and per 103 cells after subtraction of the blank value found after incubation in the absence of cells and taking into account the recovery Copyright © 2014 John Wiley & Sons, Ltd.
The protein content of acinar and ductal cells was measured by the method of Lowry et al.16 in acinar and ductal cell homogenates (about 1.3 · 106 cells/ml H2O), using 5–25-μl homogenate and bovine serum albumin as standard (2.5 to 15 μg/assay). Statistical analysis All results are presented as mean values (±SEM) together with either the number of individual determinations (n) or degree of freedom. The statistical significance of differences between mean values was assessed by use of Student’s t-test. RESULTS Uptake of 3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose by acinar cells In the acinar cells, the 3HOH distribution space, expressed as nl/103 cells, averaged after 10, 20 and 30-min incubation respectively 3.88 ± 0.59 (n = 15), 3.42 ± 0.19 (n = 5) and 3.69 ± 0.46 (n = 16), yielding an overall mean value of 3.73 ± 0.32 nl/103 cells (n = 36). In the same experiments, the L-[1-14C]glucose distribution space averaged 1.47 ± 0.20 nl/103 cells (n = 36), representing 37.7 ± 2.7% (n = 36) of the paired 3HOH space. As judged from these data, the intracellular space averaged 2.26 ± 018 nl/103 cells (n = 36). Relative to the paired 3HOH distribution space, that of D-[U-14C]glucose (8.3 mM) averaged after 5, 15 and 20–30-min incubation respectively 59.3 ± 2.6% (n = 5), 62.4 ± 1.3% (n = 5) and 60.3 ± 3.6% (n = 20). The overall mean value for the D-[U-14C]glucose distribution space thus averaged 60.5 ± 2.4% (n = 30) of the paired 3 HOH space. The latter value was not significantly different ( p > 0.31) from the somewhat lower mean value found for the distribution space of 3-O-[14C-methyl]-D-glucose (also 8.3 mM), again expressed relative to the paired 3 HOH space, i.e. 54.9 ± 4.1% (n = 47). Relative to the paired 3HOH space, the mean apparent distribution spaces of either D-[U-14C]glucose or 3-O-[14C-methyl]D-glucose were both higher ( p < 0.003 or less) than that of L-[1-14C]glucose (Table 1). Over 30-min incubation, phloridzin (0.1 mM) did not affect significantly (p > 0.85) the distribution space of 3O-[14C-methyl]-D-glucose, which averaged 103.6 ± 19.5% (n = 9) of the mean corresponding control values recorded within the same experiments (100.0 ± 7.4%; n = 9). Likewise, as judged from the ratio between the distribution space of D-[U-14C]glucose and that of 3HOH, phloridzin also Cell Biochem Funct 2014; 32: 470–475.
472 Table 1.
s. cetik Apparent distribution spaces in acinar and ductal cells
Parameters 3
3
HOH space (nl/10 cells) 14 3 space (nl/10 cells) 3 space/ HOH space (%) 3 Intracellular space (nl/10 cells) 14 3 D-[U- C]glucose space/ HOH space (%) 14 3-O-[ C-methyl]-D-glucose 3 space/ HOH space (%) L-[1- C]glucose 14 L-[1- C]glucose
Acinar cells
Ductal cells
3.73 ± 0.32 (36) 1.47 ± 0.20 (36) 37.7 ± 2.7 (36)
3.61 ± 0.32 (36) 1.17 ± 0.13 (36) 34.8 ± 2.5 (36)
2.26 ± 0.18 (36) 60.5 ± 2.4 (30)
2.44 ± 0.27 (36) 67.8 ± 3.2 (26)
54.9 ± 4.1 (47)
56.1 ± 4.8 (41)
failed to affect significantly ( p > 0.37) the fate of the labelled hexose, the experimental results averaging 95.9 ± 4.4% (n = 9) of the mean control values recorded within the same experiments (100.0 ± 1.5%; n = 10). Based on the same ratio between the distribution space of 14 3 D-[U- C]glucose and that of HOH, cytochalasin B (20.0 μM) decreased significantly ( p < 0.03) the uptake of 14 D-[U- C]glucose, the experimental data averaging 88.6 ± 4.6% (n = 10) of the mean corresponding control values (100.0 ± 1.6%; n = 10). Such was also the case ( p < 0.025) in the cells exposed to 3-O-[14C-methyl]-Dglucose, the experimental data averaging 77.0 ± 5.0% (n = 8) of the mean corresponding control values (100.0 ± 7.2%; n = 9). The relative magnitude of the inhibitory effect of cytochalasin B was not significantly different ( p > 0.1) in the case of D-[U-14C]glucose and 3-O-[14C-methyl]-D-glucose uptake. Last, the substitution of NaCl (115 mM) by an equimolar amount of CsCl in the incubation medium was found not to affect significantly (p > 0.19) the uptake of D-[U-14C] glucose by the acinar cells. Thus, the ratio between the distribution space of D-[U-14C]glucose and that of 3HOH averaged in the presence of CsCl 93.2 ± 4.0% (n = 10) of the mean corresponding control values recorded in the usual incubation medium (100.0 ± 3.1%; n = 10). Uptake of 3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose by ductal cells Within 10 min of incubation, the 3HOH distribution space in ductal cells reached a steady value averaging 3.61 ± 0.32 nl/103 cells (n = 36). There was indeed no significant difference (p > 0.88) between the measurements made after either 10–20-min incubation (3.67 ± 0.38 nl/ 103 cells; n = 15) or 30-min incubation (3.57 ± 0.48 nl/103 cells; n = 21). Likewise, after 20–30-min incubation, the distribution space of L-[1-14C]glucose was not higher than that recorded after only 10-min incubation, with an overall mean value of 1.17 ± 0.13 nl/103 cells (n = 36), corresponding to 34.8 ± 2.5% of the paired 3HOH space. As judged from these measurements, the intracellular space averaged in the ductal cells 2.44 ± 0.27 nl/103 cells (n = 36), a value not significantly different (p > 0.58) from that found in acinar cells (2.26 ± 0.18 nl/103 cells; n = 36). The mean protein content was somewhat higher, albeit not significantly so, in acinar cells (0.77 ± 0.25 μg/103 cells; n = 4) Copyright © 2014 John Wiley & Sons, Ltd.
ET AL.
than in ductal cells (0.44 ± 0.05 μg/103 cells; n = 4), with an overall mean value of 0.61 ± 0.13 μg/103 cells (n = 8). After 5 to 60-min incubation, the paired ratio between the distribution space of 3-O-[14C-methyl]-D-glucose (8.3 mM) and that of 3HOH averaged 56.1 ± 4.8% (n = 41), a value significantly higher ( p < 0.001) than that found between the distribution space of L-[1-14C]glucose and that of 3HOH (34.8 ± 2.5%; n = 36). After 5 to 30-min incubation, the paired ratio between the distribution space of D-[U-14C]glucose (also 8.3 mM) and that of 3HOH averaged 67.8 ± 3.2% (n = 26), a value somewhat higher, albeit not significantly so ( p < 0.08) than that found for the paired ratio between the distribution space of 3-O-[14C-methyl]-D-glucose and that of 3HOH. In the latter respect, the situation found in ductal cells was comparable with that observed in acinar cells, the difference between the mean values recorded with D-[U-14C]glucose and somewhat lower mean values found with 3-O-[14C-methyl]-D-glucose only achieving statistical significance ( p < 0.05) when pooling together the two sets of data collected in both acinar and ductal cells, as duly listed in Table 1. Thus, such pooled data averaged 63.9 ± 2.0% (n = 56) in the case of D-[U-14C]glucose, as distinct ( p < 0.05) from 55.5 ± 3.1% (n = 88) in the case of 3-O-[14C-methyl]-D-glucose. The substitution of NaCl (115 mM) by CsCl decreased (p < 0.001) in the ductal cells the paired ratio between the distribution space of D-[U-14C]glucose and that of 3HOH to 58.1 ± 7.1% (n = 9) of the mean control values recorded within the same experiments (100.0 ± 7.1%; n = 9). None of the other agents tested in the present study, i.e. phloridzin (0.1 mM) or cytochalasin B (20.0 μM), affected significantly ( p > 0.17 or more) the paired ratio between the distribution space of either D-[U-14C]glucose or 3-O[14C-methyl]-D-glucose and that of 3HOH. Thus, pooling together the results recorded in ductal cells exposed to either D-glucose or its non-metabolised analogue, such a paired ratio averaged in the presence of either phloridzin 69.9 ± 9.2% (n = 8) or cytochalasin B 67.3 ± 3.5% (n = 10), as compared with a mean control value recorded within the same experiment(s) of 74.3 ± 3.5% (n = 10) The two following considerations should be duly stressed, however. First, at variance with the situation found in acinar cells, phloridzin severely decreased in the ductal cells both the 3HOH distribution space and that of either 14 14 D-[U- C]glucose or 3-O-[ C-methyl]-D-glucose to respectively 46.4 ± 9.8% (n = 14; p < 0.001) and 61.2 ± 6.5% (n = 27; p < 0.001) of the mean corresponding control values, i.e. 100.0 ± 5.9% (n = 15) and 100.0 ± 6.8% (n = 28). The relative extent of the phloridzin-induced decrease in these distribution spaces was not significantly different (p > 0.20) in the case of 3HOH and in that of D-glucose and its non-metabolised analogue. Second, in a larger set of experiments, in which the distribution spaces of either 14 14 D-[U- C]glucose or 3-O-[ C-methyl]-D-glucose were duly measured, but not that of 3HOH, cytochalasin B was found to decrease significantly ( p < 0.02) the distribution space of D-glucose and its non-metabolised analogue to 68.0 ± 8.6% (n = 22) of the mean corresponding control values (100.0 ± 10.7%; n = 23). Cell Biochem Funct 2014; 32: 470–475.
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Metabolism of D-glucose in acinar and ductal cells In the acinar cells exposed to 2.8 mM D-glucose, the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation averaged no more than 6.84 ± 0.63% (n = 32). A rise in D-glucose concentration from 2.8 to 8.3 mM increased significantly D-[5-3H]glucose conversion to 3HOH (p < 0.02) but did not affect significantly D-[U-14C]glucose conversion to 14CO2 (Table 2). The mean paired 14CO2/3HOH ratio was thus lower at 8.3 mM than at 2.8 mM D-glucose concentration. Such a difference failed, however, to achieve statistical significance ( p > 0.3). A comparable situation prevailed in ductal cells. Thus, the rise in D-glucose concentration did not affect significantly D-[U-14C]glucose oxidation, despite a significant increase in D-[5-3H]glucose utilisation ( p < 0.002). Such changes resulted in a significant decrease of the paired 14CO2/3HOH ratio ( p < 0.002). The relative magnitude of the increase in D-[5-3H]glucose conversion to 3HOH in response to the rise in D-glucose concentration from 2.8 to 8.3 mM was virtually identical ( p > 0.77) in the acinar cells (+40.5%) and ductal cells 14
3
Table 2. D-[U- C]glucose oxidation and D-[5- H]glucose utilisation by acinar and ductal cells from submandibulary glands Metabolic variable Acinar cells 2.8 mM D-glucose 8.3 mM D-glucose Ductal cells 2.8 mM D-glucose 8.3 mM D-glucose
14
CO2*
3
HOH†
CO2/HOH‡
14
10.3 ± 1.2 (37) 164.7 ± 20.7 (33) 10.3 ± 1.7 (31) 232.0 ± 35.5 (34)
3
6.84 ± 0.63(32) 4.97 ± 0.85(29)
40.2 ± 4.5 (38) 304.8 ± 16.8 (36) 13.08 ± 1.48 (34) 31.1 ± 2.8 (39) 449.3 ± 34.6 (34) 7.42 ± 0.74 (34)
14
3
*D-[U- C]glucose oxidation (expressed as D-glucose equivalent/10 cells per 120 min). 3 3 † D-[5- H]glucose utilisation (expressed as D-glucose equivalent/10 cells per 120 min). 14 3 ‡ Paired ratio between D-[U- C]glucose oxidation and D-[5- H]glucose utilisation (expressed as a percentage).
Table 3.
(+47.4%). Two differences between these two types of cells merit, however, to be underlined. First, whether at 2.8 or 8.3-mM D-glucose concentration and whether in the case of D-[5-3H]glucose utilisation or D-[U-14C]glucose oxidation, the mean values recorded in ductal cells were about twice (3HOH) or three to four times higher (14CO2) than the corresponding mean values found in acinar cells. Second, whether at 2.8 or 8.3 mM D-glucose, the paired 14 CO2/3HOH ratio was significantly higher (p < 0.04 or less) in ductal cells than in acinar cells. In ductal cells incubated for 120 min in the presence of 8.3 mM D-glucose, phloridzin (0.1 mM) decreased significantly both D-[U-14C]glucose oxidation and D-[5-3H] glucose utilisation, whilst failing to affect significantly the paired ratio between these two variables (Table 3). Ouabain (1.0 mM) decreased significantly not only D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation but also the paired 14 CO2/3HOH ratio. Expressed relative to the mean control values recorded within the same experiment(s), the measurements of D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation recorded in the presence of ouabain were significantly lower than those found in the presence of phloridzin, such not being the case for the paired 14CO2/3HOH ratio. An even more severe inhibition of D-[U-14C]glucose oxidation was observed in the nominal absence of extracellular Ca2+ and presence of EDTA (1.0 mM), in media in which 115 mM NaCl was substituted by 115 mM CsCl, and in the presence of cytochalasin B (20.0 μM). Under the latter three experimental conditions, the utilisation of D-[5-3H]glucose was also impaired, such a decrease being least pronounced upon substitution of NaCl by CsCl and most marked in the presence of cytochalasin B. The absence of extracellular Ca2+ and the presence of cytochalasin B both decreased by about 40% the paired 14 CO2/3HOH ratio, a further decrease in such a ratio to about one fifth of the corresponding control values being observed upon substitution of NaCl by CsCl (Table 3).
Effects of environmental agents on the metabolism of D-glucose (8.3 mM) in ductal cells
Metabolic variable Absolute control values Control† Phlorizin (0.1 mM)** Control† Ouabain (1.0 mM)** Control† 2+ No Ca + EDTA (1.0 mM)** Control† NaCl substituted by CsCl** Control† Cytochalasin B (20.0 μM)**
14
CO2*
25.7 ± 1.7 (82)§ 100.0 ± 4.4 (36) 73.0 ± 4.1 (36)†† 100.0 ± 5.4 (33) 49.9 ± 3.8 (32)†† 100.0 ± 6.9 (19) 28.4 ± 2.7 (19)†† 100.0 ± 6.9 (19) 13.8 ± 1.6 (19)†† 100.0 ± 8.5 (20) 8.9 ± 1.4 (16)††
3
HOH†
339.6 ± 23.1 (77)§ 100.0 ± 5.2 (36) 78.7 ± 4.8 (32)‡‡ 100.0 ± 7.3 (34) 58.7 ± 4.2 (32)†† 100.0 ± 8.7 (18) 43.7 ± 5.5 (16)†† 100.0 ± 8.7 (18) 71.9 ± 6.7 (18)*** 100.0 ± 5.8 (19) 19.4 ± 3.2 (19)††
14
CO2/HOH‡ 3
8.84 ± 0.65 (74)¶ 100.0 ± 6.4 (34) 90.0 ± 5.1 ((30)§§ 100.0 ± 7.3 (29) 75.2 ± 6.0 (30)¶¶ 100.0 ± 8.4 (18) 62.0 ± 5.7 (16)†† 100.0 ± 8.4 (18) 22.9 ± 4.7 (16)†† 100.0 ± 9.9 (196) 56.4 ± 11.3 (16)†††
14
*D-[U- C]glucose oxidation. 3 † D-[5- H]glucose utilisation. 14 3 ‡ Paired ratio between D-[U- C]glucose oxidation and D-[5- H]glucose utilisation. 3 § Results expressed as D-glucose equivalent/10 cells per 120 min. ¶ Paired ratio expressed as a percentage. **Results expressed in % of the mean control values found within the same experiment(s). †† p < 0.001; ‡‡p < 0.005; §§p > 0.22; ¶¶p < 0.009; ***p < 0.02; †††p < 0.007. Copyright © 2014 John Wiley & Sons, Ltd.
Cell Biochem Funct 2014; 32: 470–475.
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s. cetik
DISCUSSION As indicated in the Introduction of this article, a major aim of the present study was to investigate the uptake and catabolism of D-glucose in acinar and ductal cells from the rat submandibular salivary gland. Moreover, attention was also paid to the possible effects of selected environmental agents upon such variables. In this respect, cytochalasin B was used as a GLUT inhibitor, whilst phloridzin was used as a non-specific SGLT inhibitor. The substitution of NaCl by CsCl aimed at investigating the possible co-transport of Na+ and D-glucose (or 3-O-methyl-D-glucose) by SGLT. The incorporation of ouabain in the incubation medium aimed at inhibiting the ATP-dependent Na+-K+-ATPase. Last, the omission of extracellular Ca2+ was considered as a potential tool to inhibit the activity of Ca2+-responsive mitochondrial dehydrogenases. Four major findings concerning the apparent distribution spaces of D-[U-14C]glucose and 3-O-[14C-methyl]-Dglucose in acinar and ductal cells merit to be underlined. First, pooling together the results collected in these two cell types, relative to the paired 3HOH space, that of D-[U-14C] glucose slightly but significantly exceeded that of 3-O[14C-methyl]-D-glucose. Such a difference is likely to reflect the time-related intracellular accumulation of the radioactive metabolites of D-[U-14C]glucose. The latter view is also compatible with the trend towards a greater difference between the results obtained with D-[U-14C]glucose and those obtained with 3-O-[14C-methyl]-D-glucose in ductal cells than in acinar cells. Our metabolic data indeed document the more efficient catabolism of D-glucose in ductal cells than in acinar cells. Second, in ductal cells, but not so in acinar cells, phloridzin decreased severely the distribution space of both 3HOH and either D-[U-14C]glucose or 3-O-[14C-methyl]-D-glucose, with a trend towards a more pronounced decrease in the former 3HOH space than in the latter distribution space of D-glucose or its non-metabolised analogue. These findings are consistent with the driving role of Na+ in the SGLT-mediated symport of the monovalent cation and D-glucose (or its non-metabolised analogue). Third, cytochalasin B decreased significantly the distribution space of D-[U-14C]glucose and 3-O-[14C-methyl]-D-glucose in both acinar and ductal cells. Last, in ductal cells, but not so in acinar cells, the substitution of extracellular NaCl by CsCl significantly decreased the paired ratio between the distribution space of D-[U-14C]glucose and that of 3HOH. Such a finding could conceivably be attributed, in part at least, to an impaired symport of D-glucose and Na+ as mediated by SGLT. Three sets of data relate to the possible role of Na+ cation in the uptake and catabolism of D-glucose in ductal cells. First, the already mentioned decrease caused by phloridzin in the apparent distribution spaces of both 3HOH and either 14 14 D-[U- C]glucose or 3-O-[ C-methyl]-D-glucose coincided with a sizeable decrease of both D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation but no significant decrease of the paired 14C02/3HOH ratio. Taken as a whole, these findings suggest that the inhibition of the Na+ and D-glucose Copyright © 2014 John Wiley & Sons, Ltd.
ET AL.
symport, as mediated by SGLT, coincided with a decrease in D-glucose catabolism and, hence, ATP generation. The latter
decrease is conceivably attributable to a lower activity of the Na+-K+-ATPase pump, itself resulting from the impaired entry of Na+ into the ductal cells and, hence, lesser energy need required for the extrusion of Na+ cations from the same cells. Second, the severe and preferential impairment of D-[U-14C]glucose oxidation, as distinct from D-[5-3H] glucose utilisation, observed in ductal cells upon substitution of NaCl (115 mM) by an equimolar amount of CsCl again points to the interdependence of energy need and Na+ extrusion by the Na+-K+-ATPase pump as modulated, in this case, by the availability of extracellular Na+ and, hence, its influx into the ductal cells. Last, the comparable inhibitory effects (p > 0.08) of ouabain and NaCl substitution by CsCl upon D-[5-3H]glucose utilisation, but less a severe decrease of D-[U-14C]glucose oxidation caused by the cardiac glycoside as compared with the extracellular substitution of NaCl by CsCl, remain compatible with a significant contribution of the ouabain-sensitive Na+ extrusion process in the consumption of energy in the ductal cells. Incidentally, according to Mangos et al.,17 ouabain also affects sodium transport into rat parotid acinar cells. Two further sets of data should not be ignored. First, the decrease of D-[U-14C]glucose oxidation, D-[5-3H]glucose utilisation and paired ratio between the latter two variables observed in ductal cells incubated in the absence of extracellular Ca2+ is consistent with the normal activation by Ca2+ of several mitochondrial dehydrogenases.18 Second, the even more severe decrease in both D-[U-14C]glucose oxidation and D-[5-3H]glucose utilisation caused by cytochalasin B, which coincided with a significant decrease of either 14 14 D-[U- C]glucose or 3-O-[ C-methyl]-D-glucose distribution space in the ductal cells, argues in support of the view that the transport-mediated inflow of D-glucose in these cells may represent a rate-limiting step of its intracellular catabolism. In this respect, the situation found in ductal cells is tightly comparable with that found in rat pancreatic islets.19,20 For instance, the relative magnitude of the inhibitory action of cytochalasin B upon the apparent distribution space of D-glucose or its non-metabolised analogue 3-O-methyl-D-glucose is not significantly different (p > 0.6) in the ductal cells and pancreatic islets, the experimental data recorded in the presence of cytochalasin B (20.0 μM) and expressed relative to the mean corresponding control values found within the same experiments averaging respectively 68.0 ± 8.6% (n = 22) in ductal cells and 73.8 ± 4.0% (n = 14) in rat pancreatic islets.19 It was recently reported that insulin increases D-glucose uptake by ductal rings from the rat submandibular gland.21 In the light of both the latter observation and the present findings, it might be concluded that the ductal cells from this salivary gland are indeed equipped with several systems, such as GLUT1, GLUT4 and SGLT, participating in insulin-sensitive, cytochalasin B-sensitive and phloridzin-sensitive D-glucose transport across the plasma membrane. Cell Biochem Funct 2014; 32: 470–475.
D-GLUCOSE HANDLING BY ISOLATED SALIVARY CELLS
CONFLICT OF INTEREST The authors have declared that there is no conflict of interest. ACKNOWLEDGEMENTS This study was supported by the Belgian Foundation for Scientific Medical Research (grant 3.4520.07) and by a grant from the European Commission (Collaborative project VIBRANT 228933: In Vivo Imaging of Beta Cell Receptors by Applied Nano-Technology). REFERENCES 1. Gonzalez-Begne M, Lu B, Liao L, et al. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology. J Proteome Res 2011; 10: 5031–5046. 2. Stoeckelhuber M, Scherer EQ, Janssen KP, et al. The human submandibular gland: immunohistochemical analysis of SNAREs and cytoskeletal proteins. J Histochem Cytochem 2012; 60: 110–120. 3. Lee MG, Ohana E, Park HW, Yang D, Muallem S. Molecular mechanism of pancreatic and salivary gland fluid and HCO3 secretion. Physiol Rev 2012; 92: 39–74. 4. Tarpey PS, Wood IS, Shirazi-Beechey SP, Beechey RB. Amino acid sequence and the cellular location of the Na+-dependent D-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell. Biochem J 1995; 312: 293–300. 5. Balen D, Ljubojevic M, Breljak D, et al. Revised immunolocalization of the Na+/D-glucose cotransporter SGLT1 in rat organs with an improved antibody. Am J Physiol Cell Physiol 2008; 295: C475–C489. 6. Sabino-Silva R, Freitas HS, Lamers ML, Okamoto MM, Santos MF, Machado UF. Na+/glucose cotransporter SGLT1 protein in salivary glands: potential involvement in the diabetes-induced decrease in salivary flow. J Membr Biol 2009; 228: 63–69. 7. Sabino-Silva R, Alves-Wagner AB, Burgi K, et al. SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats. Am J Physiol Endocrinol Metab 2010; 99: E1028–E1037.
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