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J. Biol. Chem., Vol. 282, Issue 34, 24538-24546, August 24, 2007

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Glucose-induced Cytosolic pH Changes in -Cells and Insulin Secretion Are Not Causally Related

STUDIES IN ISLETS LACKING THE NA⁺/H⁺ EXCHANGER NHE1^*

Patrick Stiernet¹, Myriam Nenquin, Pierre Moulin, Jean-Christophe Jonas², and Jean-Claude Henquin³

From the Units of Endocrinology and Metabolism and Pathology, University of Louvain Faculty of Medicine, UCL 55.30, B-1200 Brussels, Belgium

Received for publication, April 4, 2007 , and in revised form, June 27, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The contribution of Na⁺/H⁺ exchange (achieved by NHE proteins) to the regulation of -cell cytosolic pH_c, and the role of pH_c changes in glucose-induced insulin secretion are disputed and were examined here. Using real-time PCR, we identified plasmalemmal NHE1 and intracellular NHE7 as the two most abundant NHE isoforms in mouse islets. We, therefore, compared insulin secretion, cytosolic free Ca²⁺ ([Ca²⁺]_c) and pH_c in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO^–₃/CO₂ or HEPES/NaOH buffers. PCR and functional approaches showed that NHE1 mutant islets do not express compensatory pH-regulating mechanisms. NHE1 played a greater role than HCO^–₃-dependent mechanisms in the correction of an acidification imposed by a pulse of NH₄Cl. In contrast, basal pH_c (in low glucose) and the alkalinization produced by high glucose were independent of NHE1. Dimethylamiloride, a classic blocker of Na⁺/H⁺ exchange, did not affect pH_c but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca²⁺]_c and insulin secretion in HCO^–₃ and HEPES buffer, although pH_c changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca²⁺]_c is clamped at an elevated level by KCl was also unrelated to pH_c and pH_c changes. All effects of glucose on [Ca²⁺]_c and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects -cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pH_c produced by glucose involve HCO^–₃-dependent mechanisms. Variations in -cell pH_c are not causally related to changes in insulin secretion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Normal glucose homeostasis requires precise regulation of insulin secretion, a complex process that pancreatic -cells achieve through changes in their metabolism (1–4). Current models ascribe the control of insulin secretion by glucose to two hierarchical signaling pathways (5). The triggering pathway involves closure of ATP-sensitive K⁺ channels (K_ATP channels),⁴ membrane depolarization, Ca²⁺ influx through voltage-dependent Ca²⁺ channels, and a rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_c), which is the indispensable triggering signal for exocytosis of insulin granules (4–7). Simultaneously, a still incompletely understood amplifying pathway is activated, which augments the amount of released insulin without increasing [Ca²⁺]_c further (5, 8–10).

Glucose induces changes in -cell cytosolic pH (pH_c), the mechanisms and role of which in stimulus-secretion coupling are still debated. Except for two studies reporting no effect of glucose on pH_c in mouse -cells (11) or an acidification in rat islets (12), there is a large consensus that high glucose increases pH_c in mouse islets (13–17), rat islets (18, 19) and insulin-secreting cell lines (20, 21). In contrast, there is no agreement on the mechanisms implicated in this alkalinization. Activation of unidentified exchangers (15), Na⁺/H⁺ exchangers (14, 19, 20) or both (18) has been suggested using pharmacologically based experiments.

Even more confusing is the issue of a possible role of these pH_c changes in insulin secretion. Studies in which changes in -cell pH_c were imposed by manipulation of pH and ionic composition of the extracellular medium or by exposure to weak bases or acids have led to the contradictory proposals that alkalinization augments (14, 22) or inhibits (23) insulin secretion, and that acidification increases it (24–26). Other studies, relying on amiloride derivatives to inhibit Na⁺/H⁺ exchange (27, 28) and cause -cell acidification, also concluded that low pH_c is beneficial to glucose-induced insulin secretion (19, 24–26). Glucose-induced priming of insulin secretion (i.e. augmentation of the response to a second stimulation), which is thought to be mediated by the amplifying pathway (29), has been linked to-cell acidification (26, 30). These proposals are at odds with the fact that glucose increases islet pH_c and with the correlative evidence that only those fuels which increase -cell pH_c amplify insulin secretion (31). It is thus unclear whether a Na⁺/H⁺ exchanger is important for glucose-induced pH_c changes in -cells, and whether these changes play a role in the stimulation of insulin secretion, through one or both pathways. These were the questions addressed in the present study.

To date, ten isoforms of Na⁺/H⁺ exchangers (NHE1 to NHE10), encoded by the Slc9 family of genes, have been identified (32–34). We recently showed that NHE1 is strongly expressed in rat islets and is present in mouse islets (35). Here, we first compared the expression of NHE isoforms in mouse islets. Having found that NHE1 is, by far, the most abundant among the plasma membrane isoforms, we next studied Slc9A1^swe/swe mutant mice bearing a spontaneous inactivating mutation of NHE1 (36) to test the role of Na⁺/H⁺ exchange in -cell pH_c regulation under basal conditions and glucose stimulation. Finally, we measured pH_c, [Ca²⁺]_c and insulin secretion in control and NHE1 mutant islets perifused with HCO^–₃-containing and HCO^–₃-free solutions to assess the possible role of pH_c in glucose stimulation of insulin secretion through the triggering and amplifying pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Solutions and Reagents—A bicarbonate-buffered medium (HCO^–₃ medium) and a HEPES-buffered medium (HEPES medium) were used. The HCO^–₃ medium contained 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 24 mM NaHCO₃, and 1 mg/ml bovine serum albumin and was gassed with O₂/CO₂ (94%:6%) to maintain pH at 7.4. The HEPES medium contained 135 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES, and 1 mg/ml bovine serum albumin and was gassed with O₂. Its pH was adjusted to 7.4 with NaOH. When the concentration of KCl was increased to 30 mM, that of NaCl was decreased accordingly. Diazoxide was a gift from Schering-Plough, Brussels. Fura PE3-AM and BCECF-AM were obtained from Molecular Probes. Cariporide was bought from Allichem, LLC, Baltimore, MD. 5-N,N-dimethylamiloride (DMA), 5-(N-ethyl-N-isopropyl) amiloride (EIPA), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and all others reagents were from Sigma. Stock solutions of DMA, EIPA and cariporide (50 mM) and DIDS (150 mM) were prepared in Me₂SO, and aliquots were added to test solutions. The solvent alone had no effects.

Animals—C57BL/6.SJL^+/swe (swe: slow wave epilepsy) mice were purchased from the Jackson Laboratory (Bar Harbor, MI). These heterozygous mice were mated, and genotyping of the pups was performed as previously described (36). Homozygous NHE1 mutant mice (Slc9A1^swe/swe, NHE1 mutant) and wild-type littermates (Slc9A1^+/+, control) were studied at 7–9 weeks of age.

Preparations—Islets were aseptically isolated by collagenase digestion of the pancreas of NHE1 mutant and control mice (37). After hand selection, the islets were cultured for about 18 h at 37 °C in RPMI 1640 medium containing 10 mM glucose, 10% heat-inactivated fetal bovine serum, 100 international units/ml penicillin and 100 µg/ml streptomycin.

RNA Extraction and Real-time Quantitative PCR—Total RNA was extracted, quantified, and reverse-transcribed into cDNA as previously described (38) using random hexamers and 200 units of M-MLV Reverse Transcriptase RNase H^– Point Mutant (Promega, Madison, WI). The sense and antisense primers were chosen in the coding region of gene mRNA sequences, and their specificity was checked by BLAST search in the GenBank^TM data bank (Table 1). Real-time PCR was performed with the iCycler iQ Real-Time PCR detection system (Bio-Rad) using the fluorescent dye SYBR Green I to monitor DNA amplification. The reaction was performed in duplicate, in a 25-µl reaction volume containing cDNA (2–20 ng of total RNA equivalents), 300 nM primers (Table 1), 12.5 µl iQ superMIX (Bio-Rad), and water to volume. TATA box-binding protein (TBP) was used as a reporter gene. Under these conditions, PCR efficiencies for amplification of NHE isoforms and TBP were similar. For all NHE isoforms, real-time PCR was performed in parallel in islets and control tissues mRNA (Table 1), with positive amplification in controls. After amplification, the specificity of PCR products was checked by agarose gel electrophoresis and analysis of the melting curve. The threshold cycle (Ct) was determined using iCycler iQ software 3.0. After correction of the Cts for differences in cDNA input in the PCR, the Ct values were calculated in every sample for each NHE isoform as follows: Ct_NHEx – Ct_TBP. The NHEx over TBP mRNA ratios can be estimated by the formula 2^–Ct (39).

Measurements of Insulin Secretion and Islet or Pancreas Insulin Content—Batches of 20 cultured islets were transferred into chambers of a perifusion system (41). The effluent medium was collected at 42-s or 2-min intervals and saved for measurement of insulin by radioimmunoassay using rat insulin as a standard and ethanol to precipitate bound insulin (42). A small volume of concentrated NaHCO₃ or neutralized HEPES solution was added to all samples of experiments performed in HEPES medium or HCO^–₃ medium, respectively, to achieve an identical final ionic composition. The standard curve was also prepared in a medium containing both HCO^–₃ and HEPES. At the end of the experiments, the islets were recovered from the chambers, counted, and transferred in acid-ethanol for insulin extraction and measurement. The insulin content of the whole pancreas of NHE1 mutant and control mice was determined after extraction by homogenization and sonication of the tissue in acid ethanol.

Morphological Studies—The pancreas of control and NHE1 mutant mice was fixed in formalin for 24 h and embedded in paraffin. Glucagon cells were immunostained as described elsewhere (43) using a mouse monoclonal antibody (GLU-001, NovoBiolabs, Bagsvaerd, Denmark). Stained pancreas slides were digitized through a Zeiss microscope coupled to a DAGE-MTI CCD camera (Michigan City, IN). Fields containing islets (5–11 per slide) were captured through a x10 objective (0.727 x 0.758 µm/pixel). Images were analyzed with a Zeiss KS400 imaging system. Islets were delineated by the user, and glucagon cells were identified by their gray level.

Presentation of Results—Results are presented as means ± S.E. for the indicated number of animals or experiments. The statistical significance of differences between control and NHE1 mutant mice was evaluated by unpaired Student's t test. The effect of various drugs on insulin secretion was assessed by analysis of variance followed by a Dunnett's test for multiple comparisons with controls.

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Expression of NHE isoforms in control and NHE1 mutant mouse islets. Real-time analysis of NHE isoforms was performed after RT-PCR of mouse islets mRNA. The Ct were calculated as CtNHEx – CtTBP after correction for the total RNA equivalent used (2 or 20 ng). The threshold cycle for TBP using 2 ng of mRNA equivalent was about 28.5 for control and NHE1 mutant. Although primers and conditions of PCR amplification used for the 10 isoforms were different, an approximate comparison between the expression level of NHE1 and other isoforms is given by 2Ct(NHEx)-Ct(NHE1) resulting in a difference of 50, 450, and 1800-fold with NHE5, NHE2, and NHE4, respectively. Values are means ± S.E. for four preparations. *, p < 0.05 between NHE1 mutant and control mice. ND, not detected after 50 cycles of amplification.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Because NHE1 was, by far, the most abundant plasma membrane isoform in normal islets, mice with an inactivating mutation of NHE1 (36) could be a useful model to study the role of the Na⁺/H⁺ exchanger in insulin secretion. As expected, no NHE1 was detected in NHE1 mutant mouse islets, which expressed the other isoforms as in controls, with only two exceptions. The plasma membrane isoforms NHE2 and 4 were more expressed in NHE1 mutant than control islets, but this expression remained very low compared with that of NHE1 in control islets, and lower than that of NHE5 in both types of islets (Fig. 1).

Characteristics of Control and NHE1 Mutant Mice—As previously reported, NHE1 mutant mice (Slc9A1^swe/swe) exhibited ataxia in the hind limbs and seizures, and grew more slowly than controls (36). Although many died of their neurological problems a few weeks after weaning, we found it possible to prolong the lifespan of the mice by providing food and water close to the floor of the cages. The in vitro experiments using isolated islets were performed with control and NHE1 mutant mice of a mean age of 54 days (range 45–63). Table 2 compares NHE1 mutant and age-matched control mice. The body weight of NHE1 mutant mice was 30% smaller. Their blood glucose was not different, but their plasma insulin was lower. The insulin content of the pancreas was lower, but not different from controls when expressed relative to the mass of pancreas or to body weight, indicating that there is no specific impact of the lack of NHE1 on -cell development. Microscopic examination of the pancreas of NHE1 mutant mice showed that the morphological organization of the islets was normal, with a regular to oval shape and peripheral localization of glucagon cells. However, the average diameter of the islets was smaller (142 ± 7 µm, n = 63) than in control mice (193 ± 8 µm, n = 76, p < 0.007), which may explain the 33% lower insulin content of the NHE1 mutant islets that we used for in vitro experiments (Table 2).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Role of NHE1-exchanger in islet pHc regulation. The NH4Cl prepulse technique was used to study how islet cells correct the cytosolic acidification following removal of NH4Cl from the extracellular medium. Control islets are shown by thin lines and NHE1 mutant islets by thick lines. The experiments were performed in a HCO–3 medium (A–D) or a HEPES medium (E–F) containing 3 mM glucose (G 3), with NH4Cl (20 mM) added and removed as indicated. DMA (40 µM), or DIDS (200 µM) were added for 10 min (B, C, F). In D, both drugs were added together for 10 min before DIDS was omitted while DMA remained present for another 10 min. Values are means ± S.E. for 31–58 islets (from 5 to 10 preparations).

To ascertain that the lack of NHE1 was not compensated for by other pH_c-regulating mechanisms, we compared the ability of control and NHE1 mutant islets to correct an intracellular acidification produced by a prepulse of NH₄Cl (Fig. 2). The islets were perifused with a medium in which both -dependent mechanisms and Na⁺/H⁺ exchangers can function, or with a HEPES medium in which only Na⁺/H⁺ exchangers can operate. In the HCO^–₃ medium, the correction of the acidification was rapid in control islets and delayed in NHE1 mutant islets (Fig. 2A). Dimethylamiloride (DMA), an inhibitor of Na⁺/H⁺ exchangers (27, 28), had no effect on the correction of pH_c in NHE1 mutant islets but impaired the correction in control islets, as did the mutation of NHE1 in test islets (Fig. 2B). In contrast, DIDS, an inhibitor of exchangers (45, 46), blocked the pH_c correction in NHE1 mutant islets but had only little effect in control islets (Fig. 2C) because of the operation of Na⁺/H⁺ exchange. Thus, the simultaneous addition of DIDS and DMA completely prevented pH_c correction in control islets (Fig. 2D) as did DIDS alone in NHE1 mutant islets (Fig. 2C). In HEPES medium, when HCO^–₃-dependent pH regulation is inoperative, the correction of acidification by control islets was slightly slower than in the HCO^–₃ medium, and no correction occurred in NHE1 mutant islets (Fig. 2E). That the correction observed in control islets is mediated by the Na⁺/H⁺ exchange is shown by its suppression with DMA (Fig. 2F). EIPA and cariporide, two other inhibitors of Na⁺/H⁺ exchange (27) produced similar effects to DMA in controls and were ineffective in NHE1 mutant islets (data not shown). Overall, these results confirm that both Na⁺/H⁺ and HCO^–₃-dependent mechanisms contribute to the regulation of pH_c in normal -cells (15), show that no mechanism has compensated for the lack of NHE1 in mutant islets, and indicate that Na⁺/H⁺ exchange is more important than HCO^–₃-dependent mechanisms for correction of an imposed acidification.

Effects of Na⁺/H⁺ Exchange Blockers on Insulin Secretion—Because DMA and the genetic loss of NHE1 produced similar effects on pH_c, one could argue that the pharmacological approach is adequate to study the impact of Na⁺/H⁺ exchange and pH_c on -cell secretory function. The following experiments show that this is not the case. When control islets were perifused with a HCO^–₃ medium containing 15 mM glucose, DMA produced a small, rapid, and reversible decrease in pH_c in 50% (19/36) of the islets (Fig. 3A) and a consistent, delayed, irreversible increase in insulin secretion (Fig. 3B). As anticipated, DMA did not affect pH_c in NHE1 mutant islets (Fig. 3A) but, most unexpectedly, increased insulin secretion as in controls (Fig. 3B). In a second series of experiments, we compared the effects of 3 inhibitors of Na⁺/H⁺ exchange. Although they had virtually no effect on pH_c under these conditions (data not shown), DMA and EIPA markedly potentiated glucose-induced insulin secretion (p < 0.01), while the trend observed with cariporide did not reach statistical significance. Again the effect of the three drugs was similar in control and NHE1 mutant islets (Fig. 3C). Taken together, these results indicate that the increase in insulin secretion produced by these inhibitors is unrelated to a change in pH_c but is caused by an action of the drugs on a target other than the Na⁺/H⁺ exchanger. DMA and related substances cannot reliably be used to evaluate the influence of pH_c on insulin secretion.

Influence of pH_c on Glucose-induced Insulin Secretion—In HCO^–₃ medium, pH_c of control islets decreased slowly when the glucose concentration remained low (3 mM), and increased when the glucose concentration was raised to 15 mM (Fig. 4A) (15). The stimulation by high glucose also produced typical changes in [Ca²⁺]_c, characterized by a small initial decrease below basal values, followed by a sharp peak and oscillations during the second phase (Fig. 4B). Glucose also induced a biphasic increase in insulin secretion (Fig. 4C). In HEPES medium, basal pH_c decreased more rapidly and no increase occurred upon stimulation by high glucose (Fig. 4A). In contrast, the increases in [Ca²⁺]_c and insulin secretion were similar to those in HCO^–₃ medium, with only a small lag in the first phase (Fig. 4, B and C).

When NHE1 mutant islets were perifused with a HCO^–₃ medium, basal pH_c slightly decreased in 3 mM glucose and increased in response to 15 mM glucose (Fig. 4D). This shows that NHE1 is not responsible for the alkalinization induced by high glucose. Glucose-induced [Ca²⁺]_c and insulin secretion changes in NHE1 mutant islets followed a similar biphasic pattern as in control islets (Fig. 4, E and F). In HEPES medium, basal pH_c was lower than in HCO^–₃ medium, and high glucose produced an additional marked decrease to reach a very low pH of 6.3 (Fig. 4D). This decrease in pH_c is due to Ca²⁺ influx as shown by its abrogation by diazoxide or chelation of extracellular CaCl₂ with EGTA (data not shown) (15). In HEPES medium, the increase in [Ca²⁺]_c produced by glucose was not only delayed, but strongly inhibited, with only small and irregular oscillations during the second phase (Fig. 4E). There was, however, no evidence for desynchronization of these [Ca²⁺]_c oscillations between different regions of the islets (data not shown). Paradoxically, glucose-induced insulin secretion by NHE1 mutant islets was similar in HEPES and HCO^–₃ media during the 50 min of stimulation (Fig. 4F). However, when the period of glucose stimulation in HEPES medium was longer, the insulin secretory rate steadily decreased in NHE1 mutant islets (data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Effects of inhibitors of Na+/H+ exchange on pHc and insulin secretion in control and NHE1 mutant mouse islets. pHc (A) and insulin secretion (B) were measured in control islets shown by thin lines and circles, and NHE1 mutant islets shown by thick lines and squares. The glucose concentration in the perifusion medium was kept at 15 mM, and 40 µM DMA was added between 15 and 45 min, except in the series shown by open symbols in B. Values are means ± S.E. for 28–34 islets (from 5 to 6 preparations) in pHc measurements and, for 5 experiments of insulin secretion. C, shows another experimental series, similar to B, in which the effects of 40 µM DMA, EIPA, or cariporide on insulin secretion were compared. The data show the difference () between the average rate of secretion during the last minutes of drug application (35–45 min) and the last minutes preceding drug addition (5–15 min). Values are means ± S.E. for 4–5 experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Roles of NHE1-exchanger in glucose-induced pHc (A, D, G), [Ca2+]c (B, E, H), and insulin secretion (C, F, I) changes in mouse islets. A–F, control or NHE1 mutant islets were stimulated by an increase in the glucose concentration from 3 to 15 mM (G 3 G 15) in a HCO–3 medium (thin lines and open circles or squares) or a HEPES medium (thick lines and filled circles or squares). The dotted lines in A and D, correspond to islets maintained in a medium containing 3 mM glucose throughout. G–I, show the integrated responses during the last 20 min in the HCO–3 or HEPES media for control and NHE1 mutant islets. Values are means ± S.E. for 27–46 islets (from 5 to 8 preparations) in pHc and [Ca2+]c measurements and, for 5 experiments of insulin secretion.

Influence of pH_c on the Amplifying Pathway of Insulin Secretion—The amplifying pathway of glucose-induced insulin secretion can be investigated by holding K_ATP channels open with diazoxide and depolarizing -cells with 30 mM KCl in the presence of low or high glucose (8). Fig. 5, A, C, and E show the results obtained with control islets perifused with a HCO^–₃ medium. pH_c was slightly higher in 15 than 1 mM glucose and barely affected by 30 mM KCl (Fig. 5A). [Ca²⁺]_c increased similarly in 1 and 15 mM glucose, following a biphasic pattern with a sharp first peak and a flat second phase (Fig. 5C). Although [Ca²⁺]_c was similar at both glucose concentrations, insulin secretion was 3-fold larger at high than low glucose (Fig. 5E), which corresponds to the amplifying action of glucose. When control islets were stimulated with KCl in HEPES medium, pH_c similarly decreased and [Ca²⁺]_c similarly increased in 1 and 15 mM glucose (Fig. 5, B and D), but insulin secretion was much larger in 15 mM glucose (Fig. 5F). A similar amplification of insulin secretion by glucose (2.6- versus 3.2-fold) thus occurred in the two buffers although pH_c was markedly different.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Role of pHc in the amplifying pathway of insulin secretion in control mouse islets. A and B, show pHc, C and D, show [Ca2+]c, and E and F show insulin secretion, respectively. Islets were stimulated by an increase in extracellular KCl from 4.8 to 30 mM as indicated, in the presence of 1 mM glucose (thin lines and open circles) or 15 mM glucose (thick lines and filled circles). The perifusion medium contained 100 µM diazoxide (Dz) to hold KATP channels open and was buffered with HCO–3 or HEPES as indicated. Values are means ± S.E. for 21–35 islets (from 4 to 6 preparations) in pHc and [Ca2+]c measurements and, for 6 experiments of insulin secretion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NHE1 and Islet Cell pH_c Regulation—Na⁺/H⁺ exchange is achieved by the NHE membrane proteins, encoded by the family of Slc9A genes. Out of the ten known isoforms of NHE (32–34), only NHE3 was not detected at the mRNA level in mouse islets. The two most abundantly expressed isoforms were the ubiquitous NHE1 and NHE7. It is possible that some of the other, weakly expressed, isoforms are restricted to non-endocrine cells (e.g. endothelial cells) of the islets. In other cell types, NHE7 is intracellular and mainly catalyzes K⁺/H⁺ exchange (49). Its function in islet cells is unknown but, by analogy, a role in the intravesicular processing of polypeptide precursors is plausible (33). NHE1 (encoded by Slc9A1) was the most expressed among the isoforms associated with the plasma membrane (NHE1-NHE5 and NHE10). Using radioactive RT-PCR, we recently showed that NHE1 is strongly expressed, whereas NHE2 and NHE5 are weakly expressed, and NHE3 and NHE4 are not detected in rat islets (35).

Slc9A1^swe/swe mutant mice bear a spontaneous point mutation, which introduces an aberrant stop codon within the coding region, and do not produce the NHE1 protein (36). We confirm (35) that the corresponding mRNA is absent from their islets and show that no significant compensatory expression of another isoform occurred. Our observations that DMA had no effect on pH_c in islets from NHE1 mutant mice established that these islet cells really lack functionally significant Na⁺/H⁺ exchanger in their plasma membrane.

Under basal conditions (3 mM glucose), the lack of NHE1 did not affect islet pH_c in medium but resulted in a marked lowering of pH_c in HEPES medium. Basal pH_c also rapidly decreased in control islets perifused with a HEPES medium, despite the presence of NHE1. This shows that HCO^–₃-dependent mechanisms are more important than Na⁺/H⁺ exchange for basal pH_c regulation in islet cells. In contrast, NHE1 significantly contributed to normal pH_c restoration after an acid load (NH₄Cl prepulse technique). This contribution was evidenced by the slower increase in pH_c in NHE1 mutant than control islets in HCO^–₃ medium, and by the ability of control islets, in contrast to NHE1 mutant islets, to correct pH_c in HEPES medium. HCO ^–₃-dependent mechanisms proved less important for this correction of an acid pH_c, as shown by the smaller impact of DIDS or HCO^–₃ omission than of NHE1 inhibition by DMA in control islets. However, when NHE1 is lacking or inhibited, these mechanisms ensure a slow pH_c correction.

Upon stimulation by high glucose, pH_c similarly increased in NHE1 mutant and control islets. These observations conclusively establish that -cell alkalinization by glucose is not mediated by Na⁺/H⁺ exchange as sometimes proposed (14, 19, 20, 44). The failure of glucose to increase islet pH_c in HEPES medium points to the implication of a HCO^–₃-dependent mechanism, presumably of a HCO^–₃/Cl^– exchanger because this alkalinization is also inhibited by DIDS (15). However, the exact mechanism, in particular the identity of the implicated exchangers, remains to be determined. Out of the numerous HCO^–₃/Cl^– exchangers (50), only one Na⁺-dependent isoform (SLC4A10) has been identified in islet cells (51), but the presence of other isoforms has not been excluded. An intervention of NHE1 was detectable when glucose stimulation occurred in HEPES medium. Under these conditions, high glucose produced a major decrease in pH_c in NHE1 mutant islets but only had a marginal acidifying effect in control islets. These results and those of the experiments based on the NH₄Cl prepulse, therefore, indicate that Na⁺/H⁺ exchange is mainly involved in a protection of islet cells against marked cytosolic acidification.

NHE1 and Insulin Secretion—Blockers of NHE1, such as amiloride and DMA (27, 28) have previously been used to study various facets of -cell function, in addition to pH_c regulation. They have been reported to decrease ⁸⁶Rb efflux from preloaded islets (an indication of K⁺ channel closure) (19), to increase glucose-induced electrical activity in -cells (24), and to augment insulin secretion (15, 19, 20, 26, 30). These effects were attributed to blockade of Na⁺/H⁺ exchange, leading to cytosolic acidification and, eventually, closure of K_ATP channels either directly (19, 25) or via improved glucose metabolism (26). However, the decrease in pH_c by DMA, which is admittedly significant in HEPES medium, is minimal in HCO^–₃ medium. Moreover, we show here that DMA and EIPA also increased insulin secretion from NHE1 mutant islets without changing pH_c. In fact, studies of excised patches of cardiomyocyte (52) or -cell (53) membranes have shown that micromolar concentrations of amiloride and derivatives can directly inhibit K_ATP channels by interacting with the pore. Our results do not rule out the possibility that changes in pH_c can influence K_ATP channels in -cells (54, 55), but preclude the use of amiloride derivatives to test the role of Na⁺/H⁺ exchange itself and of pH_c changes in insulin secretion. We therefore used two other approaches: the NHE1 mutant islets and the direct comparison of insulin secretion and pH_c changes in HCO^–₃ and HEPES media.

Compared with control islets, glucose-induced insulin secretion was not impaired in NHE1 mutant islets. The Na⁺/H⁺ exchanger is thus dispensable for stimulus-secretion coupling at least when the experiments are performed in a physiological HCO^–₃ medium.

-Cell pH_c and Insulin Secretion—Most proposals that changes in -cell pH_c participate in stimulus-secretion coupling are based on experiments testing islets or cell lines in artificial HCO^–₃-free medium and imposing large pH_c changes by drugs (14, 19, 20, 23, 26, 30), by acidifying or alkalinizing agents (14, 22, 26, 30, 56), or by modifications of extracellular pH (22, 26, 30, 56). These large, forced excursions of pH_c (above 7.2 and below 6.8) may influence the secretory process, but the underlying mechanisms are unlikely to be physiologically relevant. It has been suggested that the optimal -cell pH_c for nutrient induced insulin secretion ranges between 6.4–6.8 (26). Only one of our experiments might suggest that low pH_c is favorable for insulin secretion. In NHE1 mutant islets, glucose-induced insulin secretion was similar in HEPES medium (islet pH_c 6.3) and HCO^–₃ medium (islet pH_c 7.0) although [Ca²⁺]_c was significantly lower in HEPES medium, and thus seemingly having a greater efficacy on exocytosis at lower pH_c. However, this difference in [Ca²⁺]_c is probably apparent only, and attributable to the interference of low pH with the measurement of [Ca²⁺]_c (47, 48). When this confounding factor was taken into account, no beneficial influence of low pH_c on glucose-induced insulin secretion was detectable. -Cell pH_c admittedly decreases upon stimulation in HEPES medium, and even becomes very low when Na⁺/H⁺ exchange is pharmacologically inhibited under these conditions. However, this situation is completely artificial, glucose increasing -cell pH_c in physiological HCO^–₃ medium.

An increase in islet pH_c has also been implicated in stimulus-secretion coupling and suggested to augment (14, 22) or impair (23, 30) glucose-induced insulin secretion. Our results show that the rise in pH_c that high glucose produces in HCO^–₃ medium is not important for the secretory response. Thus, the absence of such an increase in pH_c in HEPES medium was not accompanied by a significant alteration of glucose-induced insulin secretion. Previous observations that all metabolized agents capable of increasing insulin secretion through the amplifying pathway were also causing an increase in islet pH_c led us to propose that -cell alkalinization might be implicated in the amplifying process (31). The present results do not support the hypothesis. Thus, the experiments in which [Ca²⁺]_c was increased and clamped by depolarizing the islets with KCl in the presence of diazoxide convincingly showed that the amplification of insulin secretion by high glucose is independent of pH_c or changes in pH_c.

Conclusions—We have shown that NHE1 (plasma membrane isoform) and NHE7 (intracellular isoform) are the most expressed Na⁺/H⁺ exchangers in mouse islets. NHE1 contributes to correction of an imposed acidification of -cells, but is not implicated in the increase in pH_c produced by high glucose. This alkalinization is mediated by HCO^–₃-dependent mechanisms but is not causally related to insulin secretion. Overall our study provides evidence against the hypotheses that either a decrease or an increase in pH_c is important for glucose-induced insulin secretion via the triggering or the amplifying pathways.

	FOOTNOTES

 
^* This work was supported in part by the Fonds de la Recherche Scientifique Médicale (Grant 3.4552.04), the Belgian Science Policy (Grants PAI 5/17 and PAI 6/40), and the Direction de la Recherche Scientifique of the French Community of Belgium (Grant ARC 05/10-328). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Aspirant of the Fonds National de la Recherche Scientifique, Brussels.

² Senior Research Associate of the Fonds National de la Recherche Scientifique, Brussels.

³ To whom correspondence should be addressed: Unité d'Endocrinologie et Métabolisme, UCL 55.30, Ave. Hippocrate 55, B-1200 Brussels, Belgium. Tel.: 32-2-7645529; Fax: 32-2-7645532; E-mail: henquin{at}endo.ucl.ac.be.

⁴ The abbreviations used are: K_ATP, ATP-sensitive K⁺; [Ca²⁺]_c, cytosolic Ca²⁺ concentration; pH_c, cytosolic pH; DMA, 5-N,N-dimethylamiloride; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; TBP, TATA-box binding protein.

	ACKNOWLEDGMENTS

 
We thank F. Knockaert for technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Newgard, C. B., Lu, D., Jensen, M. V., Schissler, J., Boucher, A., Burgess, S., and Sherry, A. D. (2002) Diabetes 51, Suppl. 3, S389–S393[Abstract/Free Full Text]
2. MacDonald, M. J., Fahien, L. A., Brown, L. J., Hasan, N. M., Buss, J. D., and Kendrick, M. A. (2005) Am. J. Physiol. Endocrinol. Metab. 288, E1–E15[Abstract/Free Full Text]
3. Matschinsky, F. M., Magnuson, M. A., Zelent, D., Jetton, T. L., Doliba, N., Han, Y., Taub, R., and Grimsby, J. (2006) Diabetes 55, 1–12[Abstract/Free Full Text]
4. Fridlyand, L. E., Ma, L., and Philipson, L. H. (2005) Am. J. Physiol. Endocrinol. Metab. 289, E839–E848[Abstract/Free Full Text]
5. Henquin, J. C., Ravier, M. A., Nenquin, M., Jonas, J. C., and Gilon, P. (2003) Eur. J. Clin. Investig. 33, 742–750[CrossRef][Medline] [Order article via Infotrieve]
6. Gilon, P., Ravier, M. A., Jonas, J. C., and Henquin, J. C. (2002) Diabetes 51, Suppl. 1, S144–S151[Abstract/Free Full Text]
7. MacDonald, P. E., Joseph, J. W., and Rorsman, P. (2005) Philos. Trans. R. Soc. Lond B Biol. Sci. 360, 2211–2225[Abstract/Free Full Text]
8. Henquin, J. C. (2000) Diabetes 49, 1751–1760[Abstract]
9. Aizawa, T., Sato, Y., and Komatsu, M. (2002) Diabetes 51, Suppl. 1, S96–S98[Abstract/Free Full Text]
10. Straub, S. G., and Sharp, G. W. (2002) Diabetes Metab. Res. Rev. 18, 451–463[CrossRef][Medline] [Order article via Infotrieve]
11. Grapengiesser, E., Gylfe, E., and Hellman, B. (1989) Biochim. Biophys. Acta 1014, 219–224[Medline] [Order article via Infotrieve]
12. Pace, C. S., and Tarvin, J. T. (1986) Metabolism 35, 176–181[CrossRef][Medline] [Order article via Infotrieve]
13. Lindstrom, P., and Sehlin, J. (1984) Biochem. J. 218, 887–892[Medline] [Order article via Infotrieve]
14. Juntti-Berggren, L., Arkhammar, P., Nilsson, T., Rorsman, P., and Berggren, P. O. (1991) J. Biol. Chem. 266, 23537–23541[Abstract/Free Full Text]
15. Shepherd, R. M., and Henquin, J. C. (1995) J. Biol. Chem. 270, 7915–7921[Abstract/Free Full Text]
16. Salgado, A., Silva, A. M., Santos, R. M., and Rosario, L. M. (1996) J. Biol. Chem. 271, 8738–8746[Abstract/Free Full Text]
17. Stiernet, P., Guiot, Y., Gilon, P., and Henquin, J. C. (2006) J. Biol. Chem. 281, 22142–22151[Abstract/Free Full Text]
18. Deleers, M., Lebrun, P., and Malaisse, W. J. (1985) Horm. Metab. Res. 17, 391–395[Medline] [Order article via Infotrieve]
19. Lebrun, P., van Ganse, E., Juvent, M., Deleers, M., and Herchuelz, A. (1986) Biochim. Biophys. Acta 886, 448–456[Medline] [Order article via Infotrieve]
20. Lynch, A. M., Meats, J. E., Best, L., and Tomlinson, S. (1989) Biochim. Biophys. Acta 1012, 166–170[Medline] [Order article via Infotrieve]
21. Doliba, N. M., Wehrli, S. L., Vatamaniuk, M. Z., Qin, W., Buettger, C. W., Collins, H. W., and Matschinsky, F. M. (2007) Am. J. Physiol. Endocrinol. Metab. 292, E1507–E1519[Abstract/Free Full Text]
22. Lindstrom, P., and Sehlin, J. (1986) Biochem. J. 239, 199–204[Medline] [Order article via Infotrieve]
23. Nabe, K., Fujimoto, S., Shimodahira, M., Kominato, R., Nishi, Y., Funakoshi, S., Mukai, E., Yamada, Y., Seino, Y., and Inagaki, N. (2006) Endocrinology 147, 2717–2727[Abstract/Free Full Text]
24. Pace, C. S., and Tarvin, J. T. (1983) J. Membr. Biol. 73, 39–49[CrossRef][Medline] [Order article via Infotrieve]
25. Best, L., Yates, A. P., Gordon, C., and Tomlinson, S. (1988) Biochem. Pharmacol. 37, 4611–4615[CrossRef][Medline] [Order article via Infotrieve]
26. Gunawardana, S. C., Rocheleau, J. V., Head, W. S., and Piston, D. W. (2004) BMC. Endocr. Disord. 4, 1[CrossRef][Medline] [Order article via Infotrieve]
27. Masereel, B., Pochet, L., and Laeckmann, D. (2003) Eur. J. Med. Chem. 38, 547–554[CrossRef][Medline] [Order article via Infotrieve]
28. Harris, C., and Fliegel, L. (1999) Int. J. Mol. Med. 3, 315–321[Medline] [Order article via Infotrieve]
29. Taguchi, N., Aizawa, T., Sato, Y., Ishihara, F., and Hashizume, K. (1995) Endocrinology 136, 3942–3948[Abstract]
30. Gunawardana, S. C., and Sharp, G. W. (2002) Diabetes 51, 105–113[Abstract/Free Full Text]
31. Bertrand, G., Ishiyama, N., Nenquin, M., Ravier, M. A., and Henquin, J. C. (2002) J. Biol. Chem. 277, 32883–32891[Abstract/Free Full Text]
32. Orlowski, J., and Grinstein, S. (2004) Pflugers Archiv.-Eur. J. Physiol. 447, 549–565[CrossRef][Medline] [Order article via Infotrieve]
33. Nakamura, N., Tanaka, S., Teko, Y., Mitsui, K., and Kanazawa, H. (2005) J. Biol. Chem. 280, 1561–1572[Abstract/Free Full Text]
34. Wang, D., King, S. M., Quill, T. A., Doolittle, L. K., and Garbers, D. L. (2003) Nat. Cell Biol. 5, 1117–1122[CrossRef][Medline] [Order article via Infotrieve]
35. Moulin, P., Guiot, Y., Jonas, J. C., Rahier, J., Devuyst, O., and Henquin, J. C. (2007) J. Mol. Endocrinol. 38, 409–422[Abstract/Free Full Text]
36. Cox, G. A., Lutz, C. M., Yang, C. L., Biemesderfer, D., Bronson, R. T., Fu, A., Aronson, P. S., Noebels, J. L., and Frankel, W. N. (1997) Cell 91, 139–148[CrossRef][Medline] [Order article via Infotrieve]
37. Jonas, J. C., Gilon, P., and Henquin, J. C. (1998) Diabetes 47, 1266–1273[Abstract]
38. Jonas, J. C., Sharma, A., Hasenkamp, W., Ilkova, H., Patane, G., Laybutt, R., Bonner-Weir, S., and Weir, G. C. (1999) J. Biol. Chem. 274, 14112–14121[Abstract/Free Full Text]
39. Livak, K. J., and Schmittgen, T. D. (2001) Methods 25, 402–408[CrossRef][Medline] [Order article via Infotrieve]
40. Gilon, P., and Henquin, J. C. (1992) J. Biol. Chem. 267, 20713–20720[Abstract/Free Full Text]
41. Henquin, J. C., Nenquin, M., Stiernet, P., and Ahren, B. (2006) Diabetes 55, 441–451[Abstract/Free Full Text]
42. Heding, L. G. (1972) Diabetologia 8, 260–266[CrossRef][Medline] [Order article via Infotrieve]
43. Sempoux, C., Guiot, Y., Dahan, K., Moulin, P., Stevens, M., Lambot, V., de Lonlay, P., Fournet, J. C., Junien, C., Jaubert, F., Nihoul-Fekete, C., Saudubray, J. M., and Rahier, J. (2003) Diabetes 52, 784–794[Abstract/Free Full Text]
44. Trebilcock, R., Lynch, A., Tomlinson, S., and Best, L. (1990) Mol. Cell. Endocrinol. 71, 21–25[CrossRef][Medline] [Order article via Infotrieve]
45. Cabantchik, Z. I., and Greger, R. (1992) Am. J. Physiol. 262, C803–C827[Medline] [Order article via Infotrieve]
46. Romero, M. F., and Boron, W. F. (1999) Annu. Rev. Physiol. 61, 699–723[CrossRef][Medline] [Order article via Infotrieve]
47. Martinez-Zaguilan, R., Gurule, M. W., and Lynch, R. M. (1996) Am. J. Physiol. 270, C1438–C1446[Medline] [Order article via Infotrieve]
48. Batlle, D. C., Peces, R., LaPointe, M. S., Ye, M., and Daugirdas, J. T. (1993) Am. J. Physiol. 264, C932–C943[Medline] [Order article via Infotrieve]
49. Numata, M., and Orlowski, J. (2001) J. Biol. Chem. 276, 17387–17394[Abstract/Free Full Text]
50. Romero, M. F., Fulton, C. M., and Boron, W. F. (2004) Pflugers Archiv.-Eur. J. Physiol. 447, 495–509[CrossRef][Medline] [Order article via Infotrieve]
51. Wang, C. Z., Yano, H., Nagashima, K., and Seino, S. (2000) J. Biol. Chem. 275, 35486–35490[Abstract/Free Full Text]
52. Bollensdorff, C., Zimmer, T., and Benndorf, K. (2001) Naunyn Schmiedebergs Arch. Pharmacol. 364, 351–358[CrossRef][Medline] [Order article via Infotrieve]
53. Rustenbeck, I., Herrmann, C., Ratzka, P., and Hasselblatt, A. (1997) Naunyn Schmiedebergs Arch. Pharmacol. 356, 410–417[CrossRef][Medline] [Order article via Infotrieve]
54. Carroll, P. B., Li, M. X., Rojas, E., and Atwater, I. (1988) FEBS Lett. 234, 208–212[CrossRef][Medline] [Order article via Infotrieve]
55. Misler, S., Gillis, K., and Tabcharani, J. (1989) J. Membr. Biol. 109, 135–143[CrossRef][Medline] [Order article via Infotrieve]
56. Smith, J. S., and Pace, C. S. (1983) Diabetes 32, 61–66[Abstract]

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