Portal de Periódicos da CAPES

Expression of Carbonic Anhydrase V in Pancreatic Beta Cells Suggests Role for Mitochondrial Carbonic Anhydrase in Insulin Secretion

1998; Elsevier BV; Volume: 273; Issue: 38 Linguagem: Inglês

10.1074/jbc.273.38.24620

1083-351X

Anna‐Kaisa Parkkila, Anna L. Scarim, Seppo Parkkila, Abdül Waheed, John A. Corbett, William S. Sly,

Bipolar Disorder and Treatment

Carbonic anhydrase V (CA-V) is a mitochondrial enzyme that provides bicarbonate for pyruvate carboxylase in liver and kidney. In the course of a survey of the tissue distribution of CA-V, we detected intense immunostaining in pancreatic islets when sections from rat and mouse pancreases were reacted with a polyclonal antibody to recombinant mouse CA-V. The distribution and large number of CA-V-positive cells in each islet suggested that they represented beta cells. Double immunofluorescence staining of tissue sections and isolated islet cells showed cellular colocalization of CA-V and insulin, confirming that beta cells contain CA-V. Western blotting of rat islets of Langerhans and primary beta cells showed 33- and 30-kDa polypeptides of precursor and mature CA-V, respectively. The CA-V expression was beta cell-specific since no CA-V immunoreaction was detected in the primary alpha cells. Immunohistochemical staining for CA-I, CA-II, CA-IV, CA-VI, and CA-IX was negative in beta cells, and Western blotting of beta cells also failed to identify any CA in beta cells except CA-V. The specific localization of CA-V in beta cells led us to hypothesize that CA-V may be functionally linked to the regulation of insulin secretion. Consistent with this hypothesis, the CA inhibitor acetazolamide was found to be a strong inhibitor of glucose-stimulated insulin secretion by isolated rat pancreatic islets. Carbonic anhydrase V (CA-V) is a mitochondrial enzyme that provides bicarbonate for pyruvate carboxylase in liver and kidney. In the course of a survey of the tissue distribution of CA-V, we detected intense immunostaining in pancreatic islets when sections from rat and mouse pancreases were reacted with a polyclonal antibody to recombinant mouse CA-V. The distribution and large number of CA-V-positive cells in each islet suggested that they represented beta cells. Double immunofluorescence staining of tissue sections and isolated islet cells showed cellular colocalization of CA-V and insulin, confirming that beta cells contain CA-V. Western blotting of rat islets of Langerhans and primary beta cells showed 33- and 30-kDa polypeptides of precursor and mature CA-V, respectively. The CA-V expression was beta cell-specific since no CA-V immunoreaction was detected in the primary alpha cells. Immunohistochemical staining for CA-I, CA-II, CA-IV, CA-VI, and CA-IX was negative in beta cells, and Western blotting of beta cells also failed to identify any CA in beta cells except CA-V. The specific localization of CA-V in beta cells led us to hypothesize that CA-V may be functionally linked to the regulation of insulin secretion. Consistent with this hypothesis, the CA inhibitor acetazolamide was found to be a strong inhibitor of glucose-stimulated insulin secretion by isolated rat pancreatic islets. carbonic anhydrases fluorescence-activated cell sorting interleukin-1. 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 α-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 (1Parkkila S. Parkkila A.-K. Scand. J. Gastroenterol. 1996; 31: 305-317Crossref PubMed Scopus (87) Google Scholar, 2Saarnio J. Parkkila S. Parkkila A.-K. Waheed A. Casey M.C. Zhou X.Y. Pastoreková S. Pastorek J. Karttunen T. Haukipuro K. Kairaluoma M.I. Sly W.S. J. Histochem. Cytochem. 1998; 46: 497-504Crossref PubMed Scopus (148) Google Scholar, 3Sly W.S. Hu P.Y. Annu. Rev. Biochem. 1995; 64: 375-401Crossref PubMed Scopus (743) Google Scholar). The mitochondrial CA, CA-V, was first isolated from guinea pig liver (4Dodgson S.J. Forster R.E. Storey B.T. Ann. N. Y. Acad. Sci. U. S. A. 1984; 429: 516-524Crossref PubMed Scopus (47) Google Scholar). The cDNA has been cloned from human (5Nagao Y. Platero J.S. Waheed A. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7623-7627Crossref PubMed Scopus (49) Google Scholar) and mouse and rat (6Nagao Y. Srinivasan M. Platero J.S. Svendrovski M. Waheed A. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10330-10334Crossref PubMed Scopus (32) Google Scholar). 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 (3Sly W.S. Hu P.Y. Annu. Rev. Biochem. 1995; 64: 375-401Crossref PubMed Scopus (743) Google Scholar, 7Dodgson S. Dodgson S.J. Tashian R.E. Gros G. Carter N.D. The Carbonic Anhydrases: Cellular Physiology and Molecular Genetics. Plenum Press, New York1991: 297-306Crossref Google Scholar, 8Hazen S.A. Waheed A. Sly W.S. LaNoue K.F. Lynch C.J. FASEB J. 1996; 10: 481-490Crossref PubMed Scopus (71) Google Scholar). In 1970, Ashcroft and Randle (9Ashcroft S.J.H. Randle P.J. Biochem. J. 1970; 119: 5-15Crossref PubMed Scopus (77) Google Scholar) 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 (10MacDonald M.J. Arch. Biochem. Biophys. 1995; 319: 128-132Crossref PubMed Scopus (49) Google Scholar, 11MacDonald M.J. J. Biol. Chem. 1995; 270: 20051-20058Abstract Full Text Full Text PDF PubMed Google Scholar). 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 (12MacDonald M.J. Chang C.M. Diabetes. 1985; 34: 246-250Crossref PubMed Scopus (37) Google Scholar). 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 (11MacDonald M.J. J. Biol. Chem. 1995; 270: 20051-20058Abstract Full Text Full Text PDF PubMed Google Scholar, 12MacDonald M.J. Chang C.M. Diabetes. 1985; 34: 246-250Crossref PubMed Scopus (37) Google Scholar, 13Panten U. Ishida H. Diabetologia. 1975; 11: 569-573Crossref PubMed Scopus (33) Google Scholar, 14Brolin S.E. Agren A. Petersson B. Acta Endocrinol. 1981; 96: 93-99Crossref PubMed Scopus (12) Google Scholar, 15Van de Winkel M. Pipeleers D. Biochem. Biophys. Res. Commun. 1983; 114: 835-842Crossref PubMed Scopus (85) Google Scholar, 16Ashcroft S.J.H. Christie M.R. Biochem. J. 1979; 184: 697-700Crossref PubMed Scopus (38) Google Scholar, 17Sener A. Malaisse-Lagae F. Dufrane S.P. Malaisse W.J. Biochem. J. 1984; 220: 433-440Crossref PubMed Scopus (69) Google Scholar). 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. Polyclonal rabbit antibody for recombinant mouse CA-V (a generous gift from Dr. David Silverman) was produced as described (18Zhu X.L. Sly W.S. J. Biol. Chem. 1990; 265: 8795-8801Abstract Full Text PDF PubMed Google Scholar). 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). 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 (19Niemelä O. Parkkila S. Ylä-Herttuala S. Villanueva J. Ruebner B. Halsted C.H. Hepatology. 1995; 22: 1208-1214Crossref PubMed Scopus (165) Google Scholar, 20Parkkila S. Parkkila A.-K. Juvonen T. Waheed A. Sly W.S. Saarnio J. Kaunisto K. Kellokumpu S. Rajaniemi H. Hepatology. 1996; 24: 1104-1108Crossref PubMed Google Scholar, 21Niemelä O. Parkkila S. Ylä-Herttuala S. Halsted C. Witztum J.L. Lanca A. Israel Y. Lab. Invest. 1994; 70: 537-546PubMed Google Scholar, 22Parkkila A.-K. Parkkila S. Rajaniemi H. J. Histochem. Cytochem. 1996; 44: 245-250Crossref PubMed Scopus (2) Google Scholar). 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. Similar studies on rat islets were done using, as the primary antibody, polyclonal antisera to CA-I, CA-II, CA-IV, and CA-VI and a monoclonal antibody to CA-IX. No immunoreactivity was seen in beta cells when islets were stained with any of these reagents. Primary rat alpha and beta cells were isolated by FACS as described previously (23Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1997; 272: 13697-13704Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). SDS-polyacrylamide gel electrophoresis was performed under reducing conditions according to Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212382) Google Scholar), and 50 μg of proteins were transferred to nitrocellulose membranes under semidry transfer conditions (Millipore Corp.). Blots were blocked overnight in TBST buffer (20 mmTris, 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. Islets, isolated from 250–300-g male Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) by collagenase digestion (23Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1997; 272: 13697-13704Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), were cultured for 18 h in the presence or absence of 100 μmacetazolamide in complete CMRL-1066 medium (CMRL-1066 medium supplemented with 2 mml-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 mmHEPES, 115 mm NaCl, 24 mm NaHCO3, 5 mm KCl, 1 mm MgCl2, and 2.5 mm CaCl2, pH 7.4) containing 3 mmd-glucose and 0.1% bovine serum albumin. Islets were counted (20 islets/200 μl of Krebs-Ringer bicarbonate buffer containing 3 mmd-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 mmd-glucose and 100 μmacetazolamide. Islets were incubated for 30 min, the incubation medium was removed, and insulin content was determined by radioimmunoassay (25Wright P.H. Makulu D.R. Vichick D. Sussman K.E. Diabetes. 1971; 20: 33-45Crossref PubMed Scopus (76) Google Scholar). 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. 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. 1 B (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. 1 C). 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.Figure 2Confocal 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 3 A shows a low magnification view of a double-immunostained entire mouse islet. The predominantyellow 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). 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. 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. 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 (26Hedeskov C.J. Physiol. Rev. 1980; 60: 442-509Crossref PubMed Scopus (340) Google Scholar). 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 (27Corbett J.A. Kwon G. Hill J.R. McDaniel M.L. Marshall S.M. Home P.D. Rizza R.A. The Diabetes Manual. 9. Elsevier Science Publishers B. V., Amsterdam1995: 265-294Google Scholar). 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 (4Dodgson S.J. Forster R.E. Storey B.T. Ann. N. Y. Acad. Sci. U. S. A. 1984; 429: 516-524Crossref PubMed Scopus (47) Google Scholar,7Dodgson S. Dodgson S.J. Tashian R.E. Gros G. Carter N.D. The Carbonic Anhydrases: Cellular Physiology and Molecular Genetics. Plenum Press, New York1991: 297-306Crossref Google Scholar, 8Hazen S.A. Waheed A. Sly W.S. LaNoue K.F. Lynch C.J. FASEB J. 1996; 10: 481-490Crossref PubMed Scopus (71) Google Scholar), 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 (12MacDonald M.J. Chang C.M. Diabetes. 1985; 34: 246-250Crossref PubMed Scopus (37) Google Scholar). Instead, a study by MacDonald (11MacDonald M.J. J. Biol. Chem. 1995; 270: 20051-20058Abstract Full Text Full Text PDF PubMed Google Scholar) 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 (16Ashcroft S.J.H. Christie M.R. Biochem. J. 1979; 184: 697-700Crossref PubMed Scopus (38) Google Scholar) showed that the cytosolic NADPH/NADP ratio is increased in glucose-stimulated islets, and MacDonald (11MacDonald M.J. J. Biol. Chem. 1995; 270: 20051-20058Abstract Full Text Full Text PDF PubMed Google Scholar) 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 (28Kennedy E.D. Rizzuto R. Theler J.M. Pralong W.F. Bastianutto C. Pozzan T. Wollheim C.B. J. Clin. Invest. 1996; 98: 2524-2538Crossref PubMed Scopus (132) Google Scholar). Elder and Lehninger (29Elder J.A. Lehninger A.L. Biochemistry. 1973; 12: 976-982Crossref PubMed Scopus (40) Google Scholar) and Balboni and Lehninger (30Balboni E. Lehninger A.L. J. Biol. Chem. 1986; 261: 3563-3570Abstract Full Text PDF PubMed Google Scholar) 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. (28Kennedy E.D. Rizzuto R. Theler J.M. Pralong W.F. Bastianutto C. Pozzan T. Wollheim C.B. J. Clin. Invest. 1996; 98: 2524-2538Crossref PubMed Scopus (132) Google Scholar) 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. We acknowledge Elizabeth Torno for editorial assistance.