INTRODUCTION

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Carbonic anhydrases (CAs)¹ have long been recognized to participate in the control of pH and ion transport by catalyzing the reversible hydration of carbon dioxide (CO₂ + H₂O  H⁺ + HCO₃). The -CA gene family includes at least seven isoenzymes (CA-I-VI and CA-IX) that provide bicarbonate ions and protons for the regulation of pH homeostasis (1-3). The mitochondrial CA, CA-V, was first isolated from guinea pig liver (4). The cDNA has been cloned from human (5) and mouse and rat (6). Physiologically, CA-V is known to provide bicarbonate ions for the first enzyme in the urea cycle, carbamoyl-phosphate synthetase I, and for the first step of gluconeogenesis, in which pyruvate carboxylase converts pyruvate into oxaloacetate (3, 7, 8).

In 1970, Ashcroft and Randle (9) demonstrated that islets of Langerhans contain mitochondrial pyruvate carboxylase activity in an amount equivalent to that observed in gluconeogenic tissues such as liver and kidney (10, 11). However, pyruvate carboxylase in islet cells does not appear to be associated with gluconeogenesis because these cells do not exhibit phosphoenolpyruvate carboxykinase activity, which is required for gluconeogenic pathways (12). It has been suggested that pyruvate carboxylase may participate in a pyruvate-malate shuttle operating across the mitochondrial membrane of pancreatic islet cells and play an important role in insulin secretion (11-17).

In the course of a survey of rodent tissues for expression of CA-V, we identified rat and mouse islet cells as cells in which CA-V is highly expressed. Since bicarbonate is known to be a substrate for pyruvate carboxylase, and it is also nonpermeable to the mitochondrial membrane, we assume that its role is to provide bicarbonate for pyruvate carboxylase in pancreatic islets. Double immunofluorescence staining and Western blot analyses of alpha and beta cells purified by fluorescence-activated cell sorting (FACS) allowed us to identify the beta cell as the islet cell type in which CA-V is expressed.

These findings led us to examine the effect of acetazolamide, a widely used CA inhibitor, on glucose-stimulated insulin release by isolated rat islets. Strong inhibition was observed, suggesting that at least one of the CA isoenzymes may be involved in the regulation of insulin secretion. Since CA-V is the only CA we could identify in beta cells, we concluded that CA-V plays some role in the regulation of insulin secretion.

	EXPERIMENTAL PROCEDURES

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Antibodies-- Polyclonal rabbit antibody for recombinant mouse CA-V (a generous gift from Dr. David Silverman) was produced as described (18). Affinity-purified anti-CA-V IgG was purified using recombinant mouse CA-V coupled to Affi-Gel 10 matrix. The anti-mouse CA-V IgG did not recognize any other CA isoenzymes tested (CA-I-IV) except CA-V (data not shown). The affinity-purified antibody was used for both immunocytochemical staining and Western blotting. Polyclonal guinea pig anti-human insulin was purchased from Linco Research, Inc. (St. Louis, MO).

Immunocytochemical Methods-- Paraffin-embedded, 4% formaldehyde-fixed tissue sections of Sprague-Dawley rat and NMRI mouse pancreases were stained using immunofluorescence and immunoperoxidase methods as described previously (19-22). Spread preparations of isolated Sprague-Dawley rat islet cells were fixed with 4% neutral-buffered formaldehyde for 20 min and subjected to double immunofluorescence staining. The staining steps included 1) pretreatment of the cells or tissue sections for 40 min with cow colostral whey, 2) incubation for 1 h in primary antibodies (1:100 diluted guinea pig anti-insulin antiserum and 2 µg of affinity-purified IgG for CA-V/microscope slide) in 0.1% bovine serum albumin/phosphate-buffered saline, and 3) incubation with 1:100 diluted fluorescein isothiocyanate-conjugated goat anti-guinea pig IgG (Sigma) and rhodamine-conjugated goat anti-rabbit IgG (Dakopatts, Glostrup, Denmark) antibodies in 0.1% bovine serum albumin/phosphate-buffered saline. During the immunostaining, the cells were permeabilized using 0.05% saponin. The immunostained specimens were viewed with a conventional light and epifluorescence microscope (Axioplan, Zeiss, Oberkochen, Germany) and a confocal laser scanning microscope (Zeiss Axiovert 135 microscope combined with an LSM 410 CLSM system). The specimens were excited with a laser beam at wavelengths of 488 nm (fluorescein isothiocyanate and insulin) and 568 nm (rhodamine and CA-V). The emission light was focused through a pinhole aperture. The full field was scanned in square image formats of 512 × 512 pixels, and built-in software was used to reconstruct the images obtained from the confocal sections.

Western Blot Analysis-- Primary rat alpha and beta cells were isolated by FACS as described previously (23). SDS-polyacrylamide gel electrophoresis was performed under reducing conditions according to Laemmli (24), and 50 µg of proteins were transferred to nitrocellulose membranes under semidry transfer conditions (Millipore Corp.). Blots were blocked overnight in TBST buffer (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20, pH 7.5) containing 5% nonfat dry milk and then incubated for 1.5 h at room temperature with anti-mouse CA-V antibody (6 µg/ml) in TBST buffer containing 1% nonfat dry milk. After incubation with the primary antibody, the blots were washed three times for 5 min with TBST buffer, followed by incubation for 45 min at room temperature with peroxidase-conjugated donkey anti-mouse IgG (1:5000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). After washing in TBST buffer, CA-V was detected by enhanced chemiluminescence reaction (Amersham Pharmacia Biotech). Blots were also stained with a polyclonal antibody to CA-I, CA-II, CA-IV, or CA-VI as the primary antibody, but no bands were detected with any of these antibodies.

Glucose-stimulated Insulin Secretion-- Islets, isolated from 250-300-g male Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) by collagenase digestion (23), were cultured for 18 h in the presence or absence of 100 µM acetazolamide in complete CMRL-1066 medium (CMRL-1066 medium supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin) under an atmosphere of 95% air and 5% CO₂ at 37 °C. Following culture, the islets were washed three times with Krebs-Ringer bicarbonate buffer (25 mM HEPES, 115 mM NaCl, 24 mM NaHCO₃, 5 mM KCl, 1 mM MgCl₂, and 2.5 mM CaCl₂, pH 7.4) containing 3 mM D-glucose and 0.1% bovine serum albumin. Islets were counted (20 islets/200 µl of Krebs-Ringer bicarbonate buffer containing 3 mM D-glucose) and preincubated in the presence or absence of 100 µM acetazolamide for 30 min at 37 °C with shaking. The preincubation solution was removed, and glucose-stimulated insulin secretion was initiated by the addition of 200 µl of fresh Krebs-Ringer bicarbonate buffer containing either 3 or 20 mM D-glucose and 100 µM acetazolamide. Islets were incubated for 30 min, the incubation medium was removed, and insulin content was determined by radioimmunoassay (25). As a control, the inhibitory effects of an 18-h incubation with interleukin-1 (IL-1; 1 unit/ml) on insulin secretion were compared with the effects of acetazolamide.

	RESULTS

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Fig. 1 shows the immunoperoxidase staining of CA-V in tissue sections of rat (panel A) and mouse (panel B) pancreases. In both species, the enzyme was predominantly expressed in islets of Langerhans. Only a weak immunoreactivity was found in the exocrine pancreas. The CA-V-positive cells represented a majority of the islet cells and were centrally located inside an individual islet. The high magnification view in Fig. 1B (inset) indicates that the immunoreaction for CA-V is granular, showing a typical mitochondrion-like staining pattern. Control immunostaining using normal rabbit serum showed no positive reaction in mouse islets (Fig. 1C). Fig. 2 shows confocal laser scanning microscopy images of the immunofluorescence reaction for CA-V in a mouse islet (panel A) and an isolated rat islet cell (panel B). The positive reaction was clearly granular, suggesting that the enzyme is located in the mitochondria of these cells.

View larger version (88K): [in this window] [in a new window]   Fig. 1.   Immunoperoxidase staining of CA-V in rat (A) and mouse (B) pancreases. The enzyme is expressed in islet cells in both species. The immunoreaction is granular, indicating a typical mitochondrion-like staining pattern (B, inset). Normal rabbit serum showed no positive immunoreaction in the endocrine cells of mouse islets of Langerhans (C). Bars = 20 µm (A-C) and 10 µm (B, inset).

View larger version (92K): [in this window] [in a new window]   Fig. 2.   Confocal laser scanning images of CA-V in a mouse pancreatic islet (A) and in an isolated rat islet cell (B) show granular immunoreaction. Bars = 5 µm.

To determine the identity of CA-V-positive cells found in islets, CA-V and insulin were immunostained simultaneously in pancreatic tissue sections and isolated rat islet cells. Fig. 3A shows a low magnification view of a double-immunostained entire mouse islet. The predominant yellow color indicates that CA-V and insulin colocalize in the same cells. Double staining of isolated rat islet cells for CA-V and insulin also indicated that CA-V is expressed in beta cells (Fig. 3, B and C).

View larger version (102K): [in this window] [in a new window]   Fig. 3.   Confocal laser scanning images of CA-V (A and B) and insulin (A and C) in a mouse pancreatic section (A) and in isolated islet cells of rat pancreas (B and C). The yellow color in the entire islet (A) indicates cellular colocalization of insulin and CA-V. The high magnification view of isolated beta cells further demonstrates that CA-V (B) and insulin (C) are expressed in the same cells. Bars = 30 µm (A) and 5 µm (B and C).

The results from Western blot analysis of rat islets and FACS-purified alpha and beta cells using anti-mouse CA-V antibody are shown in Fig. 4. The results indicate that islet cells and primary beta cells express the 33-kDa precursor and 30-kDa mature polypeptides of CA-V, whereas alpha cells did not show any immunoreaction. No other CAs were identified in beta cells, either by immunohistochemical staining for CA-I, CA-II, CA-IV, CA-VI, and CA-IX or by Western blotting using antisera to CA-I, CA-II, CA-IV, and CA-VI.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Western blots of CA-V expression in rat islets and FACS-purified alpha and beta cells. The results show that islets and isolated beta cells both contain the 33-kDa precursor and 30-kDa mature polypeptides of CA-V.

Since CA-V appears to be selectively expressed in beta cells, we studied the effects of acetazolamide, a selective carbonic anhydrase inhibitor, on glucose-stimulated insulin secretion. Treatment of rat islets with 100 µM acetazolamide results in an ~80% inhibition of glucose-stimulated insulin secretion. The inhibitory effects of acetazolamide on glucose-stimulated insulin secretion were similar in magnitude to the inhibitory actions of the cytokine IL-1, which results in a complete inhibition of insulin secretion (Fig. 5). These findings provide experimental evidence to support a role for carbonic anhydrase in the regulation of glucose-stimulated insulin secretion by isolated rat islets.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effects of acetazolamide on glucose-stimulated insulin secretion by rat islets. Rat islets, treated with 100 µM acetazolamide or 1 unit/ml IL-1, were isolated, and glucose-stimulated insulin secretion was examined as described under "Experimental Procedures." Results are the mean ± S.E. of three independent experiments containing three to four replicates/condition.

	DISCUSSION

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In this study, we have used immunocytochemistry and Western blotting to demonstrate the expression of mitochondrial CA-V in the endocrine pancreas. Immunohistochemical staining of rat and mouse pancreases showed granular mitochondrion-like positive reaction in islet cells. The large number of CA-V-positive cells in each islet and their central distribution suggested that the enzyme is located in beta cells. Indeed, double immunofluorescence staining of tissue sections and isolated islet cells for CA-V and insulin showed that CA-V is expressed in pancreatic beta cells. The specificity of the localization was confirmed using Western blotting, which revealed that islets and FACS-purified beta cells express the 33-kDa precursor and 30-kDa mature forms of CA-V, whereas no signal was detected in alpha cells. The high level of expression of mitochondrial CA-V in pancreatic beta cells and very little or no expression in other pancreatic cell types suggest that CA-V has a highly cell-specific function in mitochondria of beta cells, presumably related to their specific metabolic needs for mitochondrial bicarbonate.

Among the many factors capable of stimulating insulin secretion in beta cells, glucose is physiologically the most important. Mitochondrial oxidation of glucose, resulting in the production of ATP, is required for insulin secretion (26). We show here that selective inhibition of CA using acetazolamide results in potent inhibition of insulin secretion by isolated rat islets. We had previously shown that IL-1 inhibits secretion by a mechanism that includes beta cell expression of inducible nitric-oxide synthase and production of nitric oxide, followed by nitric oxide-mediated inhibition of islet mitochondrial function (27). The addition of the CA inhibitor acetazolamide resulted in an inhibition of insulin secretion to levels comparable in magnitude to the inhibitory actions of IL-1. The specific localization of CA-V in beta cells and the failure to find any other carbonic anhydrase in this cell type make CA-V the most likely target for this inhibition by acetazolamide. These findings provide functional evidence to support a role for CA-V in glucose-stimulated insulin secretion by isolated rat islets.

There are at least two possible mechanisms by which mitochondrial CA-V could participate in the regulation of insulin secretion. First, CA-V may provide HCO₃ for pyruvate carboxylase, which in turn participates in the pyruvate-malate shuttle operating across the mitochondrial membranes. Pyruvate carboxylase is abundantly expressed in the mitochondrial islet cells. Unlike the case in gluconeogenic tissues, where CA-V provides bicarbonate for pyruvate carboxylase to convert pyruvate to oxaloacetate for gluconeogenesis (4, 7, 8), the coupling of CA-V to pyruvate carboxylase in islets has another role. Islets contain essentially no phosphoenolpyruvate carboxykinase, which would be needed to use the product of pyruvate carboxylase to form phosphoenolpyruvate in gluconeogenesis (12). Instead, a study by MacDonald (11) demonstrated that the product of pyruvate carboxylase is important in the pyruvate-malate shuttle, which provides NADPH for normal beta cell function. Ashcroft and Christie (16) showed that the cytosolic NADPH/NADP ratio is increased in glucose-stimulated islets, and MacDonald (11) proposed that the pyruvate-malate shuttle is the major means of generating cytosolic NADPH from the metabolism of glucose. If cytosolic NADPH concentrations modulate insulin secretion, CA-V could play a strategic role in this regulation by providing bicarbonate for pyruvate carboxylase in the proposed shuttle mechanism.

A second mechanism by which CA-V could be linked to insulin secretion is in the regulation of mitochondrial calcium concentrations. Glucose is known to induce a rise in intramitochondrial calcium concentration and a smaller rise in cytosolic calcium concentration (28). Elder and Lehninger (29) and Balboni and Lehninger (30) used isolated rat liver mitochondria to demonstrate that mitochondrial CA is essential for a rapid mitochondrial uptake of calcium. The correlation of intramitochondrial calcium concentration with insulin secretion from INS-1 cells observed by Kennedy et al. (28) suggested a fundamental role for calcium ions in the energy requirements for exocytosis of insulin from beta cells. In light of the present and previous findings, it will be of great interest to examine the role of CA-V in regulating the intramitochondrial calcium concentration and the cytosolic NADPH/NADP ratio, both of which are believed to be involved in the regulation of insulin secretion by beta cells.