In order to investigate the possible link between PMCA (plasma-membrane Ca2+-ATPase) activity and D-glucose catabolism in insulin-producing cells, BRIN-BD11 cells were transfected with two isoforms of PMCA2. Transfection of insulin-producing BRIN-BD11 cells with PMCA2yb and PMCA2wb was documented by RT-PCR (reverse transcription-PCR), Western blot analysis, indirect immunofluorescence microscopy and 45Ca2+ uptake by microsomes. In the transfected cells, the overexpression of PMCA coincided with three major anomalies of D-glucose metabolism, namely a lower rate of D-[5-3H]glucose utilization prevailing at a low extracellular concentration of D-glucose (1.1 mM), a low ratio between D-[U-14C]oxidation and D-[5-3H]glucose utilization prevailing at a high extracellular glucose concentration (16.7 mM), and a high ratio between the net generation of 14C-labelled acidic metabolites and amino acids and that of 3H2O from D-[5-3H]glucose. These anomalies resulted in a decreased estimated rate of ATP generation (linked to the catabolism of the hexose) and a lowered ATP cell content, whether at low or high extracellular D-glucose concentrations. The net uptake of 45Ca2+ by intact cells was also decreased in the transfected cells, but to a greater extent than can apparently be attributed to the change in the ATP-generation rate. These findings document the relevance of PMCA activity to both D-glucose metabolism and Ca2+ handling in insulin-producing cells, with emphasis on the key role of both cytosolic and mitochondrial Ca2+ concentrations in the regulation of D-glucose catabolism. They also reveal that overexpression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and consumption.

INTRODUCTION

The PMCA (plasma-membrane Ca2+-ATPase) is responsible in part for the ejection of Ca2+ from all eukaryotic cells [1]. Glucose, the major physiological stimulus for insulin release, initiates the voltage-dependent entry of Ca2+ into pancreatic islet β-cells [2]. This regulatory effect of glucose on insulin secretion involves the intracellular metabolism of the sugar by oxidative glycolysis and an increase in the ATP/ADP ratio [35]. A rise in intracellular Ca2+ is the key trigger of insulin secretion [47]. The maintenance of normal Ca2+ homeostasis during sustained cellular activity involves, among other things, PMCA and the Na+/Ca2+ exchanger [8,9].

One approach to studying the physiological function of PMCA isoforms is to develop systems which would allow modulation of their expression. Successful overexpression of different isoforms has been achieved in different systems [1012]. Overexpression of an isoform of PMCA2, PMCA2wb, has been achieved previously in BRIN-BD11 cells, an insulin-secreting cell line established by electrofusion of RINm5F cells with NEDH rat pancreatic islet cells [13,14]. BRIN-BD11 cells were selected for such a purpose because they display an improved metabolic and secretory response to D-glucose when compared to the difference otherwise found between normal and tumoral islet cells [14,15]. In response to D-glucose, overexpressing cells showed a markedly reduced rise in [Ca2+]i (intracellular Ca2+ concentration) and suppression of [Ca2+]i oscillations, despite increased D-glucose metabolism and insulin release [13].

The present study extends such investigations by examining BRIN-BD11 cells transfected with the two PMCA2 isoforms PMCA2wb or PMCA2y by exploring the feedback control of D-glucose catabolism resulting from changes in Ca2+ handling, with emphasis on the measurement of ATP cell content, the rate of ATP generation attributable to the catabolism of D-glucose and the relationship between the latter rate and the net uptake of 45Ca in control and transfected cells. The results reveal that overexpression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and utilization.

MATERIALS AND METHODS

Materials

RPMI 1640 medium, L-glutamine, penicillin, streptomycin, G418, fetal calf serum, Ca2+- and Mg2+-free HBSS (Hanks balanced salt solution), cell-dissociation buffer, PBS, NotL and AlwNI were purchased from GibcoBRL. SgrAI and KpnI were from Boehringer Mannheim. Oligomycin, thapsigargin and calmodulin were from Calbiochem, and ouabain was purchased from ICN Biomedicals.

Construction of the expression vectors

Pancreatic islet isolation, total RNA preparation, reverse transcription, PCR, cloning and DNA sequencing of PCR products were performed as described previously [16]. In order to construct full-length PMCA2wb and PMCA2yb cDNAs, three different fragments were amplified by PCR based on the rat PMCA2 cDNA sequences [17]. Fragment (1) contains the ATG starting codon and a 93 bp exon (for PMCA2yb) or 135 bp exon (for PMCA2wb) at site A (Figure 1). Fragments (2) and (3) are the same for both PMCA2yb and PMCA2wb, and fragment (3) contains the TAG stop codon. These fragments were subcloned into the pCR®-Blunt vector (Invitrogen), DNA sequenced and digested with NotI/AlwNL, AlwNI/SgrAI and SgrAI/KpnI respectively. The complete full-length clones corresponding to the alternatively spliced PMCA isoforms PMCA2yb or PMCA2wb were obtained as follows. The 5′-end 1087 bp NotI/AlwNl fragment containing the alternatively spliced region site A, the 1107 bp AlwNI/SgrAI central portion fragment and the 1542 bp SgrAI/KpnI 3′-end fragment were ligated to each other. The final full-length PMCA2wb construct was subcloned into the multicloning site of the pcDNA3.1 (−) mammalian expression vector (Invitrogen) digested by NotI and KpnI. The positive clones were verified by restriction-enzyme mapping and DNA sequencing.

Scheme summarizing the splice variants of the PMCA2 isoforms found in insulin-producing cells

Figure 1
Scheme summarizing the splice variants of the PMCA2 isoforms found in insulin-producing cells

cDNA organization of PMCAs is shown at the top. The putative transmembrane segments 1–10 are indicated by black bars. Exons involved in the alternative splicing at sites A, B and C are represented by grey boxes. Flanking sequences that are conserved are represented by white boxes.

Figure 1
Scheme summarizing the splice variants of the PMCA2 isoforms found in insulin-producing cells

cDNA organization of PMCAs is shown at the top. The putative transmembrane segments 1–10 are indicated by black bars. Exons involved in the alternative splicing at sites A, B and C are represented by grey boxes. Flanking sequences that are conserved are represented by white boxes.

Culturing of BRIN-BD11 cells and stable transfection

Cells were grown at 37°C in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 i.u./ml penicillin and 100 μg/ml streptomycin, and equilibrated against 5% CO2 and 95% air. The D-glucose concentration of the culture medium was 11.1 mM unless otherwise stated. Cells were gently washed with Ca2+- and Mg2+-free HBSS (5 ml per T75 tissue-culture flask) for 30 s at room temperature (20°C) before being detached from the tissue-culture flask by incubation with cell-dissociation buffer (5 ml per T75 tissue-culture flask) for 1–2 min and the cells were used immediately. To establish cell lines that stably express rat PMCA2yb or PMCA2wb, 5 μg/ml of either of the PMCA constructs was transfected into BRIN-BD11 cells using Lipofectamine™ (GibcoBRL) following the manufacturer's instructions. The neomycin-resistant colonies were selected with 250 μg/ml G418 in RPMI 1640 medium containing 10% (v/v) fetal calf serum. Colonies were picked and grown as individual cell lines in the presence of 250 μg/ml G418. Each cell line was examined to confirm the expression of PMCA.

Western blotting

Western blotting was performed as described previously [13].

Indirect immunofluorescence microscopy

The cells were plated on to coverslips and analysed by indirect immunofluorescence microscopy 48–60 h after plating. The cells were washed with TBS (Tris-buffered saline) [20 mM Tris and 137 mM NaCl (pH 7.2)], fixed for 20 min at 4°C in 4% (w/v) formaldehyde (pH 7.4) and washed with TBS. The cells were permeabilized in a solution containing 0.01% Triton X-100, 197 μM MgCl2, 19.5 μM DTT (dithiothreitol) and 10% (v/v) glycerol (pH 7.4) at 4°C, washed twice with TBS and incubated in blocking buffer containing 1% horse serum (Vector Laboratories) in TBS for 20 min at room temperature. The coverslips were overlaid with a primary antibody raised against PMCA2 [rabbit anti-(Ca2+-ATPase isoform 2) antibody, 1:1000 dilution; SWant, Bellinzona, Switzerland] in TBS with 1% BSA buffer for 1 h at room temperature. The control cells were incubated in TBS with 1% BSA buffer without the primary antibody and washed three times with TBS. The cells were treated with a secondary antibody [Alexa Fluor® 594-conjugated goat anti-rabbit IgG (H+L) antibody, 1:400 dilution; Molecular Probes] in TBS with 1% BSA for 45 min at room temperature and washed four times with TBS. The cells were incubated with 300 nM DAPI (4′,6-diamidino-2-phenylindole) solution (Molecular Probes) and washed twice with TBS. The coverslips were mounted using Vectashield® (Vector Laboratories) and the cells were observed with a Axioplan microscope (Zeiss) equipped with a microscope illuminator with HBO 100 W or XBO 100 W and a ×40 objective lens, and photographed with a Dual-mode cooled CCD Camera C4880 (Hamamatsu).

Isolation of microsomes from cultured BRIN-BD11 cells

Crude microsomal membranes were prepared following a method described previously [18] with modifications. Cells (5×106–10×106 cells/ml) were suspended in a hypotonic solution [10 mM Tris/HCl buffer (pH 7.5) containing 1 mM MgCl2, 0.1 mM PMSF, 4 μg/ml aprotinin, 1 μg/ml leupeptin and 2 mM DTT]. The cells were swollen for 15 min on ice and then homogenized with 40 strokes of a Dounce homogenizer. The homogenate was diluted with an equal volume of 10 mM Tris/HCl (pH 7.5), 0.5 M sucrose, 0.3 M KCl and 2 mM DTT, homogenized again with 20 strokes of a Dounce homogenizer and centrifuged at 5000 g for 15 min at 4°C. KCl (0.6 M final concentration) was added to the supernatant and, in order to remove calmodulin, an excess of EDTA (1.5 mM) was also added. The suspension was centrifuged at 50 000 rev./min for 1 h at 4°C (Beckman 50 Ti rotor) to sediment the microsomal fraction. The final pellet was resuspended in a solution containing 10 mM Tris/HCl (pH 7.5), 0.25 M sucrose, 0.15 M KCl, 2 mM DTT and 20 μM CaCl2 at a protein concentration of 1–3 mg/ml, aliquoted, frozen immediately in liquid nitrogen and stored at −70°C. Protein concentration was determined using the Bio-Rad Dc Protein Assay (Bio-Rad) with BSA used as a standard.

Ca2+ uptake in microsomes

Calcium influx into microsomal vesicles was measured at 37°C by rapid filtration through filters using a method described previously [19] with some modifications. The filters were pre-soaked in 150 mM KCl and 1 mM CaCl2 to reduce the background. Microsomal proteins (20 μg) were resuspended in the uptake buffer containing 25 mM TES/triethanolamine (pH 7.2), 100 mM KCl, 5 mM NaN3, 4 μg/ml oligomycin, 0.5 mM ouabain, 7 mM MgCl2, 40 mM KH2PO4, 1 mM EGTA and sufficient CaCl2 [mixed with 1 μCi/ml 45Ca2+ (PerkinElmer)] to obtain the desired free [Ca2+] using the Max Chelator program (http://maxchelator.stanford.edu/). Thapsigargin (200 nM) was also included to inhibit the activity of the SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) pumps. Immediately after the addition of 45Ca2+, the reaction was started by the addition of 6 mM ATP. The incubation time was 10 min, and the transport activity was determined in the absence or presence of 240 nM calmodulin. At stated timepoints, 100 μl of the mixture was rapidly filtered through nitrocellulose filters (0.45 μm pore size; Millipore) which had been washed three times with ice-cold 150 mM KCl and 1 mM CaCl2. The filters were then counted using a Beckman scintillation counter.

Measurement of glucose metabolism

The methods used to measure D-[5-3H]glucose metabolism, D-[U-14C]glucose oxidation and the generation of 14C-labelled acidic metabolites (e.g. lactic acid) and 14C-labelled amino acids from D-[U-14C]glucose were performed as described previously [2022].

ATP measurement

BRIN-BD11 cells (4×106 cells/ml) were incubated at 37°C for 90 min in bicarbonate-buffered medium [23] containing BSA (5 mg/ml) and 1.1 mM or 16.7 mM D-glucose. The incubation was halted by the addition of PCA (perchloric acid) to a final concentration of 3% (v/v). The tubes were then mixed by sonication (3×10 s) on ice and centrifuged at 15000 g for 10 min at 4°C. An aliquot of the supernatant was mixed with an equal volume of 0.5 M KOH/0.2 M imidazole solution and left at −70°C for at least 1 h. Then the extracts were thawed on ice and centrifuged at 15000 g for 10 min at 4°C. The supernatants were transferred into new tubes and an aliquot of each tube was diluted 10 times with a buffer containing 5 mM MgCl2, 50 mM KCl and 0.1 M imidazole (pH 7.75). The ATP content was determined by the luciferin–luciferase method using the ATP Bioluminescence assay described previously [24] with modifications. Briefly, 0.5 ml of the diluted extract was mixed with 0.5 ml of the luciferin–luciferase reagent from the ATP Bioluminescence Assay Kit CLSII (Roche). Bioluminescence was measured on an LKB Wallace 250 luminometer (LKB-Pharmacia, Uppsala, Sweden). The main benefits of using this method are linearity of the ATP concentration between 1 and 9 nM and within-day precision for repeated assays (n=6) yielding a coefficient of variation of ≤5.7%. The recovery of a standard after the whole extraction procedure was 62.9±1.1% (n=8).

DNA measurement

DNA content was measured by a fluorimetric assay following the method described by Labarca and Paigen [25].

Washed cells were ultrasonicated in 950 μl of 50 mM phosphate buffer (pH 7.40) containing 2 M NaCl. After addition of 50 μl of the fluorochrome reagent bisbenzimidazole (20 μg/ml in PBS) (Hoechst 33258, Sigma), the relative fluorescence intensity was measured using a spectrophotofluorimeter with λexcitation=356 nm and λemission=448 nm. Standard curves were prepared from calf thymus DNA type 1 (Sigma), and the standard concentration was exactly determined by measurement of the absorbance (A) at 260 nm. Samples and standards were assayed in duplicate.

45Ca uptake by intact cells

The medium used for incubation of the BRIN-BD11 cells at 37°C was a Krebs–Ringer bicarbonate-buffered solution (pH 7.4) containing 10 mM Hepes/NaOH, 115 mM NaCl, 1 mM CaCl2, 5 mM KCl and l nM MgCl2. The medium was gassed with ambient air and contained either 1.1 or 11.1 mM D-glucose. The method used for the measurement of 45Ca2+ uptake was performed as described previously [26]. In brief, BRIN-BD11 cells were pre-incubated in 1 ml of a non-radiaoctive medium for 30 min at 37°C and then incubated for 90 min at 37°C in 1 ml of the same medium containing 45Ca2+ (10 μCi/ml) in addition. At the end of the incubation, the cells were separated from the incubation medium using a combined lanthanum and oil technique as described previously [27].

Presentation of results

All results are presented as mean values (±S.E.M.) together with either the number of individual determinations (n) or d.f. (degree of freedom). The statistical significance of differences between mean values was assessed by ANOVA followed by Tukey's post test and/or by the Student's t test.

RESULTS

Generation and selection of the BRIN-BD11 stable cell line expressing the PMCA pump

The full-length clones for PMCA2 pump isoforms PMCA2yb and PMCA2wb contain the 93 or 135 bp exon at site A respectively, but not the 227 bp exon at site C. These two isoforms were constructed from PCR-amplified cDNA. Stably transfected BRIN-BD11 cell colonies were chosen and grown as individual cell lines in the presence of G418. Quantitative RT-PCR (reverse transcription-PCR) and Western blot analysis confirmed the overexpression of PCMA2wb [13] and PCMA2yb (Figure 2).

RT-PCR (reverse transcription-PCR) amplification and Western blot analysis of PMCA2yb in control and PMCA2yb transfected BRIN-BD11 cells

Figure 2
RT-PCR (reverse transcription-PCR) amplification and Western blot analysis of PMCA2yb in control and PMCA2yb transfected BRIN-BD11 cells

(A) RT-PCR amplification of PMCA2yb after 32 cycles yielded bands of 300 and 589 bp for PMCA2 and β-actin (used as a control) respectively. Lane 1, control RT-PCR (non-transfected BRIN-BD11 cells). Lane 2, BRIN-BD11 cells transfected with PMCA2yb clone 1. Lane 3, BRIN-BD11 cells transfected with PMCA2yb clone 2. Lane 4, BRIN-BD11 cells transfected with PMCA2yb clone 5. The PCR products were separated by agarose-gel electrophoresis and stained with ethidium bromide. MW, 1 kb DNA ladder marker (bp). (B) Membrane proteins (100 μg) were separated by SDS/PAGE (7.5% gels). The gel was electroblotted on to nitrocellulose membrane, which was then cut and incubated with primary antibodies [rabbit anti-(Ca2+-ATPase isoform 2) antibody, 1:1000 dilution]. The bound antibody was detected with a horseradish-peroxidase-labelled secondary antibody following the manufacturer's instructions. Lane 1, control non-transfected BRIN-BD11 cells. Lane 2, BRIN-BD11 cells transfected with PMCA2yb clone 1. Lane 3, BRIN-BD11 cells transfected with PMCA2yb clone 2. Lane 4, BRIN-BD11 cells transfected with PMCA2yb clone 5. M, molecular mass marker [Kaleidoscope Prestained Standard (Bio-Rad)].

Figure 2
RT-PCR (reverse transcription-PCR) amplification and Western blot analysis of PMCA2yb in control and PMCA2yb transfected BRIN-BD11 cells

(A) RT-PCR amplification of PMCA2yb after 32 cycles yielded bands of 300 and 589 bp for PMCA2 and β-actin (used as a control) respectively. Lane 1, control RT-PCR (non-transfected BRIN-BD11 cells). Lane 2, BRIN-BD11 cells transfected with PMCA2yb clone 1. Lane 3, BRIN-BD11 cells transfected with PMCA2yb clone 2. Lane 4, BRIN-BD11 cells transfected with PMCA2yb clone 5. The PCR products were separated by agarose-gel electrophoresis and stained with ethidium bromide. MW, 1 kb DNA ladder marker (bp). (B) Membrane proteins (100 μg) were separated by SDS/PAGE (7.5% gels). The gel was electroblotted on to nitrocellulose membrane, which was then cut and incubated with primary antibodies [rabbit anti-(Ca2+-ATPase isoform 2) antibody, 1:1000 dilution]. The bound antibody was detected with a horseradish-peroxidase-labelled secondary antibody following the manufacturer's instructions. Lane 1, control non-transfected BRIN-BD11 cells. Lane 2, BRIN-BD11 cells transfected with PMCA2yb clone 1. Lane 3, BRIN-BD11 cells transfected with PMCA2yb clone 2. Lane 4, BRIN-BD11 cells transfected with PMCA2yb clone 5. M, molecular mass marker [Kaleidoscope Prestained Standard (Bio-Rad)].

The overexpression of PMCA2yb and PMCA2wb proteins at the level of the plasma membrane in transfected cells was also visualized by immunofluorescence using a rabbit anti-(Ca2+-ATPase isoform 2) antibody, with PMCA2 and nuclei stained red and blue respectively. The results showed that higher levels of the Ca2+ pump were expressed in the PMCA2yb- and PMCA2wb-transfected cells compared with the control BRIN-BD11 cells (Figure 3). A pattern of peripheral distribution of stained material was quite obvious in the cells overexpressing PMCA2yb and PMCA2wb, suggesting that a major portion of the overexpressed protein was associated with the plasma membrane. In control cells, minimal staining of the endogenous proteins was observed. These experiments support the view that the largest fraction of the overexpressed PMCA2 pump was targeted correctly to the plasma membrane.

Immunolocalization of the overexpressed PMCA2 proteins

Figure 3
Immunolocalization of the overexpressed PMCA2 proteins

Immunofluorescence was performed detailed in the Materials and Methods section, using rabbit anti-(Ca2+-ATPase isoform 2) antibody raised against PMCA2, and the PMCA2 protein (red) and nuclei (blue) are shown. Untransfected control BRIN-BD11 cells not incubated with the primary antibody are also shown (Ctrl BRIN). NT BRIN, non-transfected BRIN-BD11 cells; PMCA2wb, BRIN-BD11 transfected with PMCA2wb; PMCA2yb, BRIN-BD11 cells transfected with PMCA2yb.

Figure 3
Immunolocalization of the overexpressed PMCA2 proteins

Immunofluorescence was performed detailed in the Materials and Methods section, using rabbit anti-(Ca2+-ATPase isoform 2) antibody raised against PMCA2, and the PMCA2 protein (red) and nuclei (blue) are shown. Untransfected control BRIN-BD11 cells not incubated with the primary antibody are also shown (Ctrl BRIN). NT BRIN, non-transfected BRIN-BD11 cells; PMCA2wb, BRIN-BD11 transfected with PMCA2wb; PMCA2yb, BRIN-BD11 cells transfected with PMCA2yb.

Ca2+ uptake by microsomes

Additional support for the correct targeting of PMCA2 resulted from the assay of PMCA activity in isolated microsomes, as judged by ATP-dependent Ca2+ uptake. In these experiments, an excess concentration of thapsigargin was used to ensure complete inhibition of SERCA pump activity without affecting the calmodulin-dependent Ca2+ uptake mediated by the PMCA.

As shown in Table 1, the PMCA activity was higher (P<0.05 or less) in transfected cells than in the control BRIN-BD11 cells, whether in the absence or presence of calmodulin. The enzymic activity in the PMCA2wb-transfected cells exceeded that in the PMCA2yb-transfected cells (P<0.01 or less) both in the absence or presence of calmodulin. Calmodulin increased the mean value for 45Ca uptake by the microsomes (P<0.05 or less; paired comparison). The relative extent of such an increase was not significantly different between control BRIN-BD11 and transfected cells, with an overall mean value of 84.1±7.4% (n=3; P<0.01). The expressed isoforms PMCA2yb and PMCA2wb thus displayed characteristic dependencies on calmodulin for activation.

Table 1
Ca2+-dependent ATPase activity in microsomes from control and PMCA2-transfected cells
Ca2+ uptake (nmol/mg protein per 10 min)
Calmodulin0 nM240 nM
BRIN-BD11 0.513±0.143 (4) 0.885±0.130 (4) 
BRIN-BD11 transfected with PMCA2yb 1.537±0.166 (4) 3.041±0.276 (4) 
BRIN-BD11 transfected with PMCA2wb 2.585±0.213 (8) 4.705±0.147 (4) 
Ca2+ uptake (nmol/mg protein per 10 min)
Calmodulin0 nM240 nM
BRIN-BD11 0.513±0.143 (4) 0.885±0.130 (4) 
BRIN-BD11 transfected with PMCA2yb 1.537±0.166 (4) 3.041±0.276 (4) 
BRIN-BD11 transfected with PMCA2wb 2.585±0.213 (8) 4.705±0.147 (4) 

D-Glucose metabolism

In both the control cells and in the transfected BRIN-BD11 cells, the rise in D-glucose concentration significantly enhanced (P<0.01 or less) the generation of 3H2O from D-[5-3H]glucose and that of 14C-amino acids and 14C-acidic metabolites (e.g. lactic acid) from D-[U-14C]glucose, whereas it failed to significantly affect the production of 14CO2 from D-[U-14C]glucose (Table 2). Hence, in these tumoral cells, the 14CO2/3H2O ratio was always lower (P<0.001) at 16.7 mM D-glucose than at 1.1 mM D-glucose.

Table 2
D-[U-14C]Glucose and D-[5-3H]glucose metabolism in control and PMCA2 gene-transfected BRIN-BD11 cells

All results are expressed as pmol of D-glucose equivalent per ng of DNA after 90 min incubation. 1.1 and 16.7 are the concentrations of D-glucose used (in mM). Values in parentheses indicate the number of individual determinations (n) performed.

Non-transfected BRIN-BD11BRIN-BD11 transfected with PMCA2ybBRIN-BD11 transfected with PMCA2wb
Parameters1.116.71.116.71.116.7
D-[U-14C]Glucose oxidation 1.54±0.07 (27) 1.82±0.12 (28) 1.11±0.08 mM (41) 1.35±0.10 (39) 1.37±0.09 (37) 1.46±0.10 (38) 
D-[5-3H]Glucose utilization 9.66±0.11 (28) 19.30±0.58 (28) 6.23±0.27 (42) 16.47±0.77 (39) 8.74±0.51 (41) 22.72±1.21 (41) 
14CO2/3H2O ratio (%) 15.92±0.73 (27) 9.48±0.60 (28) 17.43±0.91 (41) 8.89±0.66 (34) 15.13±0.43 (36) 6.47±0.23 (39) 
14C-Amino acids 0.95±0.04 (28) 1.33±0.03 (21) 0.81±0.02 (41) 1.33±0.04 (21) 1.08±0.02 (42) 1.44±0.03 (40) 
14C-Acidic metabolites 1.04±0.08 (27) 1.51±0.14 (28) 1.25±0.04 (41) 2.44±0.09 (42) 2.17±0.03 (41) 3.32±0.08 (39) 
Non-transfected BRIN-BD11BRIN-BD11 transfected with PMCA2ybBRIN-BD11 transfected with PMCA2wb
Parameters1.116.71.116.71.116.7
D-[U-14C]Glucose oxidation 1.54±0.07 (27) 1.82±0.12 (28) 1.11±0.08 mM (41) 1.35±0.10 (39) 1.37±0.09 (37) 1.46±0.10 (38) 
D-[5-3H]Glucose utilization 9.66±0.11 (28) 19.30±0.58 (28) 6.23±0.27 (42) 16.47±0.77 (39) 8.74±0.51 (41) 22.72±1.21 (41) 
14CO2/3H2O ratio (%) 15.92±0.73 (27) 9.48±0.60 (28) 17.43±0.91 (41) 8.89±0.66 (34) 15.13±0.43 (36) 6.47±0.23 (39) 
14C-Amino acids 0.95±0.04 (28) 1.33±0.03 (21) 0.81±0.02 (41) 1.33±0.04 (21) 1.08±0.02 (42) 1.44±0.03 (40) 
14C-Acidic metabolites 1.04±0.08 (27) 1.51±0.14 (28) 1.25±0.04 (41) 2.44±0.09 (42) 2.17±0.03 (41) 3.32±0.08 (39) 

The utilization of D-[5-3H]glucose and oxidation of D-[U-14C]glucose were, on occasion, significantly lower in transfected cells than in control BRIN-BD11 cells. However, the most substantial difference between the control and transfected cells was in the higher generation of 14C-acidic metabolites in the latter cells than in the former ones. Whether incubated with 1.1 mM D-glucose or 16.7 mM D-glucose, the net production of 14C-acidic metabolites was indeed significantly higher (P<0.02 or less) in BRIN-BD11 cells transfected with the PMCA2yb isoform than in control cells. At both 1.1 mM and 16.7 mM D-glucose, it was further increased (P<0.001) in the PMCA2wb-transfected cells. In this respect, there was thus a close parallel between the generation of 14C-acidic metabolites (Table 2) and Ca2+-dependent ATPase activity (Table 1).

Three further differences between the control cells and the transfected BRIN-BD11 cells should be mentioned. First, the rise in D-glucose concentration enhanced D-[5-3H]glucose utilization in PMCA2yb-transfected cells (multiplication factor: 2.664±0.168; d.f.=79) and PMCA2wb-transfected cells (multiplication factor: 2.600±0.205; d.f.=80) to a greater relative extent (P<0.02 or less) than in control BRIN-BD11 cells (multiplication factor: 1.998±0.064; d.f.=54). Secondly, whether incubated at 1.1 or 16.7 mM D-glucose, the mean generation of 14C-labelled acidic metabolites, expressed relative to D-[5-3H]glucose utilization, was always higher in the transfected cells than in the control cells, with the generation in the transfected cells averaging 198.3±10.8% (P<0.005) of the mean corresponding value found at the same D-glucose concentration in the control untransfected BRIN-BD11 cells. When the net generation of both 14C-labelled acidic metabolites and amino acids were taken into account, the latter average percentage was 159.7±7.9% and, as such, remained significantly higher than unity (P<0.005). Thirdly, the rise in glucose concentration decreased the ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization to a greater absolute or relative extent in PMCA2-transfected cells than in control BRIN-BD11 cells. Such a difference achieved statistical significance (P<0.05) in the PMCA2wb-transfected cells, but not in the PMCA2yb-transfected cells, once again paralleling the changes in Ca2+-dependent ATPase activity.

Cell ATP content

Table 3 provides the absolute values for the ATP content of control BRIN-BD11 cells and PMCA2yb- or PMCA2wb-transfected cells measured after incubation for 90 min in the presence of either 1.1 mM or 16.7 mM D-glucose. In the three cell types, the ATP content was lower (P<0.02 or less) at 16.7 mM D-glucose than at 1.1 mM D-glucose. Thus at 16.7 mM D-glucose, the average values were 87.6±2.7% (n=9) 90.2±2.3% (n=27) 87.8±1.8% (n=27) (for control BRIN-BD11 cells, PMCA2yb-transfected cells and PMCA2wb-transfected cells respectively) of the corresponding mean values found within the same experiment(s) and same cell type at 1.1 mM D-glucose (i.e. 100.0±1.2%, n=8; 100.0±2.9%, n=27 and 100.0±2.3%, n=27). Whether at concentrations of 1.1 mM or 16.7 mM D-glucose, the ATP content of the PMCA2yb and PMCA2wb-transfected cells was significantly lower (P<0.005 or less) than that recorded at the same hexose concentration and within the same experiment(s) in control BRIN-BD11 cells. Relative to the latter control values, the results for the transfected cells were not significantly different at 1.1 mM or 16.7 mM D-glucose and in PMCA2yb- or PMCA2wb-transfected cells, with an overall mean value of 81.3±1.5% (n=108).

Table 3
ATP content in BRIN-BDII non-transfected or transfected cells

All results are expressed as pmol ATP/μg of DNA. Values in parentheses indicate the number of individual determinations (n) performed.

ATP content
Cells1.1 mM D-Glucose16.7 mM D-Glucose
BRIN-BD11 606±67 (8) 541±49 (9) 
BRIN-BD11 transfected with PMCA2yb 489±27 (27) 432±13 (27) 
BRIN-BD11 transfected with PMCA2wb 500±29 (27) 438±25 (27) 
ATP content
Cells1.1 mM D-Glucose16.7 mM D-Glucose
BRIN-BD11 606±67 (8) 541±49 (9) 
BRIN-BD11 transfected with PMCA2yb 489±27 (27) 432±13 (27) 
BRIN-BD11 transfected with PMCA2wb 500±29 (27) 438±25 (27) 

45Ca uptake by intact cells

The measurements of 45Ca net uptake taken at low and high D-glucose concentrations (1.1 mM or 16.7 mM D-glucose) are summarized in Table 4. In all cell types, the net uptake of 45Ca was significantly higher (P<0.005 or less) at high, rather than low, hexose concentration. At the two concentrations of D-glucose, the net uptake of 45Ca was lower (P<0.001) in transfected cells than in the control BRIN-BD11 cells.

Table 4
Effect of D-glucose on 45Ca net uptake by BRIN-BD11 non-transfected or transfected cells

All results are expressed as fmol per 103 cells after 90 min incubation. Values in parentheses indicate the number of individual determinations (n) performed.

Cells1.1 mM D-glucose16.7 mM D-glucose
BRIN-BD11 696±23 (12) 814±24 (12) 
BRIN-BD11 transfected with PMCA2yb 495±19 (30) 591±24 (30) 
BRIN-BD11 transfected with PMCA2wb 487±11 (30) 563±11 (30) 
Cells1.1 mM D-glucose16.7 mM D-glucose
BRIN-BD11 696±23 (12) 814±24 (12) 
BRIN-BD11 transfected with PMCA2yb 495±19 (30) 591±24 (30) 
BRIN-BD11 transfected with PMCA2wb 487±11 (30) 563±11 (30) 

Figure 4 illustrates the relationship between 45Ca net uptake and the estimated rate of ATP generation. The latter rate was calculated from the results listed in Table 2 as follows. The ATP generation rate was taken as the sum of (i) the rate of D-[U-14C]glucose oxidation (multiplied by 36) and (ii) the differences between the rate of D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation (multiplied by 2). Figure 4 shows that, in all three cell types, a given increase in ATP generation (resulting from a rise in D-glucose concentration) coincided with a comparable increase in the net uptake of 45Ca. However, for any given ATP generation rate, the net uptake of 45Ca was lower in transfected cells than in the control untransfected BRIN-BD11 cells. Such a difference appeared to be most pronounced in the PMCA2wb-transfected cells, in which the activity of PMCA2 was also the highest (see Table 1).

Mean values for 45Ca net uptake and the estimated ATP generation rate in control BRIN-BD11 cells, PMCA2yb-transfected cells or PMCA2wb-transfected cells incubated in the presence of 1.1 mM D-glucose (○) or 16.7 mM D-glucose (●)

Figure 4
Mean values for 45Ca net uptake and the estimated ATP generation rate in control BRIN-BD11 cells, PMCA2yb-transfected cells or PMCA2wb-transfected cells incubated in the presence of 1.1 mM D-glucose (○) or 16.7 mM D-glucose (●)
Figure 4
Mean values for 45Ca net uptake and the estimated ATP generation rate in control BRIN-BD11 cells, PMCA2yb-transfected cells or PMCA2wb-transfected cells incubated in the presence of 1.1 mM D-glucose (○) or 16.7 mM D-glucose (●)

DISCUSSION

The present study shows that in BRIN-BD11 cells transfected with either PMCA2yb or PMCA2wb, the estimated ATP yield resulting from the catabolism of D-glucose is decreased, which coincides with a lower ATP content and lower 45Ca net uptake in intact transfected cells. The latter cationic anomaly is likely to be directly caused by overexpression of PMCA2 in the transfected cells. It coincides with a lesser increase in cytosolic Ca2+ concentration in these cells when exposed to insulin secretagogues [13].

The alteration of the ATP content in the transfected cells is likely to be attributable mainly to the increased consumption of ATP caused by enhanced activity of PMCA2, rather than to any major change in D-glucose catabolism, at least in the PMCA 2wb-transfected cells.

The metabolism of D-glucose in both the control and transfected BRIN-BD11 cells displayed the typical features of a Crabtree effect, namely the inhibition of cellular respiration at high concentration of D-glucose, as documented previously in the tumoral insulin-producing cells of the RINm5F line [28].

In the BRIN-BD11 cells overexpressing PMCA2, changes in D-glucose metabolism, when present, are probably attributable, at least in part, to the modulation by mitochondrial Ca2+ of the activity of FAD-linked glycerophosphate dehydrogenase [29,30]. Such a proposal is supported by three sets of findings. First, the rise in glucose concentration indeed decreased the ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization in PMCA2-transfected cells to a greater absolute or relative extent than in control BRIN-BD11 cells. Secondly, the anaerobic modality of glycolysis, as judged by the generation of 14C-acidic metabolites from D-[U-14C]glucose, was indeed higher in PMCA2-transfected cells than in control BRIN-BD11 cells. In these two cases, the hierarchy in mean values paralleled that of microsomal Ca2+-dependent ATPase activity. Thirdly, the generation of 14C-acidic metabolites, considered alone or together with that of 14C-amino acids, as judged from the production of 3H2O from D-[5-3H]glucose, represented a greater fraction of total glycolytic flux in the PMCA2-transfected cells than in the control BRIN-BD11 cells.

In conclusion, the present study extends the concept of a feedback control by cationic variables of D-glucose catabolism to changes in PMCA activity in insulin-producing cells [20]. It documents alterations in both glycolysis and mitochondrial oxidative events in cells overexpressing PMCA, with such alterations impeding the rate of ATP generation, attributable to catabolism of the hexose to match the accelerated consumption of ATP caused by increased activity of this Ca2+ pump.

Abbreviations

     
  • [Ca2+]i

    intracellular Ca2+ concentration

  •  
  • DAPI 4′

    6-diamidino-2-phenylindole

  •  
  • d.f.

    degree of freedom

  •  
  • DTT

    dithiothreitol

  •  
  • HBSS

    Hanks balanced salt solution

  •  
  • PMCA

    plasma-membrane Ca2+-ATPase

  •  
  • SERCA

    sarcoplasmic/endoplasmic reticulum Ca2+-ATPase

  •  
  • TBS

    Tris-buffered saline

This study was supported by the Belgian Foundation for Scientific Medical Research (grants 3.4562.00 and 3.4520.07).

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