Copyright ©The Histochemical Society, Inc.

Immunohistochemical Localization of Monoamine Oxidase Type B in Pancreatic Islets of the Rat

Yu-Hong Huang, Akio Ito and Ryohachi Arai

Department of Anatomy, Shiga University of Medical Science, Otsu, Shiga, Japan (Y-HH,RA), and Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Fukuoka, Japan (AI)

Correspondence to: Dr. R. Arai, Department of Anatomy, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan. E-mail: rarai{at}belle.shiga-med.ac.jp


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Monoamine oxidase (MAO) is regarded as a mitochondrial enzyme. This enzyme localizes on the outer membrane of mitochondria. There are two kinds of MAO isozymes, MAO type A (MAOA) and type B (MAOB). Previous studies have shown that MAOB activity is found in the pancreatic islets. This activity in the islets is increased by the fasting-induced decrease of plasma glucose level. Islet B cells contain monoamines in their secretory granules. These monoamines inhibit the secretion of insulin from the B cells. MAOB is active in degrading monoamines. Therefore, MAOB may influence the insulin-secretory process by regulating the stores of monoamines in the B cells. However, it has not been determined whether MAOB is localized on B cells or other cell types of the islets. In the present study, we used both double-labeling immunofluorescence histochemical and electron microscopic immunohistochemical methods to examine the subcellular localization of MAOB in rat pancreatic islets. MAOB was found in the mitochondrial outer membranes of glucagon-secreting cells (A cells), insulin-secreting cells (B cells), and some pancreatic polypeptide (PP)-secreting cells (PP cells), but no MAOB was found in somatostatin-secreting cells (D cells), nor in certain other PP cells. There were two kinds of mitochondria in pancreatic islet B cells: one contains MAOB on their outer membranes, but a substantial proportion of them lack this enzyme. Our findings indicate that pancreatic islet B cells contain MAOB on their mitochondrial outer membranes, and this enzyme may be involved in the regulation of monoamine levels and insulin secretion in the B cells.

(J Histochem Cytochem 53:1149–1158, 2005)

Key Words: MAO • glucagon • insulin • somatostatin • pancreatic polypeptide • rat • pancreas • mitochondria • double-labeling • immunofluorescence • histochemistry • electron microscopic • immunohistochemistry


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
MONOAMINE OXIDASES ARE ENZYMES that degrade biogenic monoamines (Berry et al. 1994Go; Shih et al. 1999Go; Abell and Kwan 2001Go). There are two types of monoamine oxidase enzymes, and both are separate gene products (Ito et al. 1988Go; Derry et al. 1989Go; Lan et al. 1989Go; Levy et al. 1989Go; Kuwahara et al. 1990Go). Monoamine oxidase type A (MAOA) has a higher affinity for the substrates serotonin and noradrenaline and is irreversibly inhibited by the inhibitor clorgyline, whereas monoamine oxidase type B (MAOB) has a higher affinity for the substrate ß-phenylethylamine and is irreversibly inhibited by the inhibitor deprenyl (Knoll and Magyar 1972Go; Johnston 1968Go). Dopamine (DA) is a common substrate of both MAOA and MAOB (Fowler and Benedetti 1983Go).

The pancreatic islet comprises numerous cell types that synthesize and secrete distinct peptide hormones (Erlandsen 1980Go; Reddy and Elliott 1988Go). Four major cell types are recognized in pancreatic islets of many mammalian species (including rat): A cells, which contain glucagon; B cells, which contain insulin; D cells, which contain somatostatin; and PP cells, which contain pancreatic polypeptide (PP) (Erlandsen 1980Go; Reddy and Elliott 1988Go). Biochemical studies have shown that MAOA and MAOB activities are present in homogenates of the pancreatic islets of rat, mouse, and golden hamster, with a greater predominance of MAOB activity (Feldman et al. 1980Go; Lenzen et al. 1987Go; Stenstrom and Lundquist 1990Go). In human pancreatic islets, there is an almost equal level of protein expression of MAOA and MAOB (Pizzinat et al. 1999Go). Immunohistochemical studies have shown that in guinea pig, MAOB-labeled cells appear to be evenly distributed throughout the islets (Kirchgessner and Pintar 1991Go), although in human, MAOA-positive cells are homogeneously distributed in the islets, and MAOB-positive cells are found in the periphery of the islets (Rodriguez et al. 2000Go). To our knowledge, which cell types of the pancreatic islets contain MAOA or MAOB remains to be elucidated. Furthermore, their subcellular localization has not been examined.

Recently, we raised and characterized a rabbit antiserum against bovine MAOB, which cross-reacts with rat MAOB (Sagara and Ito 1982Go; Arai et al. 2002Go). Using this antiserum, we have now performed double-labeling immunofluorescence histochemistry and electron microscopic immunohistochemistry to examine the cellular and subcellular localization of MAOB in the rat pancreatic islets.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Animals
Male Sprague-Dawley rats (n=6, 180–200 g) were obtained from Japan SLC (Hamamatsu, Japan). All experiments were carried out according to the Guidelines for Animal Experimentation at Shiga University of Medical Science. All efforts were made to minimize both the number of animals used and their suffering.

Primary Antibodies
Sheep antibody against human glucagon (4660-0930, Biogenesis; Pool, England), guinea pig antibody against porcine insulin (A0564, DAKO; Carpinteria, CA), rat antibody against synthetic somatostatin (MAB354, Chemicon; Temecula, CA), and guinea pig antibody against rat PP (4040-01, Lincoresearch; Charles, MO) were used. These antibodies have been previously characterized (Brar et al. 1989Go; Dun et al. 1994Go; Elayat et al. 1995Go; Zhang et al. 2000Go).

MAOB purified from bovine liver mitochondria was used to generate a rabbit anti-MAOB antiserum (Sagara and Ito 1982Go). This antiserum has been previously shown to immunoprecipitate MAOB of the rat liver (Sagara and Ito 1982Go). Immunohistochemical analysis has shown that this antiserum stains MAOB-containing cells but not MAOA-containing cells in the rat brain, indicating that the antiserum specifically recognizes rat MAOB (Arai et al. 2002Go).

Double-labeling Immunofluorescence Histochemistry
Three rats were anesthetized with sodium pentobarbital (60 mg/kg body weight, IP) and perfused through the ascending aorta with 50 ml of 0.01 M PBS (pH 7.4, room temperature), followed by 300 ml of a fixative solution containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4, 4C). The pancreases were dissected and placed in the same fixative solution for 24 hr at 4C and then immersed in PB containing 15% sucrose for 24 hr at 4C. Thirty-µm thick sections were cut using a cryostat (Cryocut 1800, Reichert-Jung; Heidelberg, Germany) and collected in PBS.

Double labeling of MAOB and each of four islet hormones was performed as follows. Sections were incubated in the following solutions: (a) 0.3% Triton X-100 in PBS for 1 hr at room temperature; (b) 5% normal donkey serum in PBS for 1 hr at room temperature; (c) a mixture of primary antisera:rabbit antiserum against MAOB (1:20,000 in PBS) combined with one of the four antisera:sheep anti-glucagon antiserum (1:200), guinea pig anti-insulin antiserum (1:20,000), rat anti-somatostatin antiserum (1:1,000), and guinea pig anti-PP antiserum (1:12,000), the mixture containing 1% normal donkey serum for 48 hr at 4C; and (d) a mixture of secondary antisera—Cy3-conjugated donkey anti-rabbit IgG (AP182C, Chemicon, 1:200 in PBS) combined with one of the three antisera:fluorescein-conjugated donkey anti-sheep IgG (AP184F, Chemicon, 1:200), fluorescein-conjugated donkey anti-guinea pig IgG (AP193F, Chemicon, 1:200), and fluorescein-conjugated donkey anti-rat IgG (AP189F, Chemicon, 1:200) for 2 hr at room temperature. After each incubation step, the sections were rinsed in PBS for 30 min at room temperature, mounted on glass slides, and cover slipped with a medium containing 90% glycerol and 0.1% p-phenylenediamine in PBS. Finally, MAOB was labeled with Cy3 (red), and glucagon, insulin, somatostatin, and pancreatic polypeptide were labeled with fluorescein (green). The stained sections were observed under a confocal laser scanning microscope (LSM410, Zeiss; Jena, Germany) with appropriate excitation laser beams and emission filters (for Cy3, excitation at 543 nm, emission at >570 nm; for fluorescein, excitation at 488 nm, emission at 510–525 nm). The Cy3 and fluorescence images were stored in memory, and then the superimposition of the two images was made and stored. These images were transferred to a Macintosh computer (Apple Computer; Cupertino, CA) equipped with Photoshop software (Adobe System; San Jose, CA) and printed on a color printer (Pictrography 3500, Fujifilm; Tokyo, Japan).

Electron Microscopic Immunohistochemistry
Three rats were anesthetized with sodium pentobarbital (60 mg/kg body weight, IP) and perfused through the ascending aorta with 50 ml of 0.01 M PBS (room temperature), followed by 300 ml of a fixative solution containing 4% paraformaldehyde, 0.3% glutaraldehyde, and 0.2% picric acid in 0.1 M PB (4C). The pancreases were dissected and placed in another fixative solution containing 4% paraformaldehyde and 0.2% picric acid in PB for 24 hr at 4C. Fifty-µm thick sections were cut using a microslicer (Dosaka; Kyoto, Japan) and collected in PBS.

For MAOB staining, an immunoperoxidase technique using avidin-biotin-peroxidase complex (Hsu et al. 1981Go) was performed. Sections were incubated in the following solutions: (a) 0.001% trypsin (type III, Sigma; St Louis, MO) in PBS for 5 min at room temperature; (b) 5% normal goat serum in PBS for 1 hr at room temperature; (c) rabbit anti-MAOB antiserum (1:10,000 in PBS) with 1% normal goat serum for 48 hr at 4C; (d) biotinylated goat anti-rabbit IgG (BA-1000, Vector; Burlingame, CA, 1:1,000 in PBS) for 2 hr at room temperature; (e) avidin-biotin-peroxidase complex (PK-4000, Vector, 1:1,000 in PBS) for 2 hr at room temperature; and (f) 0.025% 3,3'-diaminobenzidine (Dojindo; Kumamoto, Japan), 0.6% nickel ammonium sulfate (Nacalai Tesque; Kyoto, Japan), and 0.0075% hydrogen peroxide (H2O2) in 0.05 M trizma hydrochloride (Tris-HCl) buffer (pH 7.6) for 5 min at room temperature. The stained sections were fixed with 1% osmium tetroxide (Nacalai Tesque) in PB for 1 hr at 4C, dehydrated, and flat-embedded in epoxy resin (Luveak-812, Nacalai Tesque). Small areas containing the pancreatic islets were trimmed from the embedded section and cut on an ultramicrotome (Ultracut UCT, Leica; Heidelberg, Germany). Ultrathin sections were collected on grids (200 Cu, VECO; Eerbeek, Holland), stained with 2% uranyl acetate (Merck; Darmstadt, Germany) for 20 min and lead stain solution (Sigma-Aldrich Japan; Tokyo, Japan) for 5 min at room temperature, and examined with a transmission electron microscope (H-7100TE, Hitachi; Tokyo, Japan). Cell types of pancreatic islets were identified by the characters of their granules (Larsson et al. 1976Go; Roth et al. 1981Go; Weaver et al. 1986Go).


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Double-labeling Immunofluorescence Histochemistry
Figure 1 and Figure 2 show the cellular localization of MAOB in the pancreatic islet. All of the A cells that were labeled for glucagon (total number counted = 39, number of islets examined = 3) were also stained for MAOB (Figures 1A, 1C, and 1D). All of the B cells that were labeled for insulin (total number counted = 153, number of islets examined = 4) were also stained for MAOB (Figures 1B, 1D, and 1F). All of the D cells that were labeled for somatostatin (total number counted = 18, number of islets examined = 3) were negative for MAOB (Figures 2A, 2C, and 2D). Fifteen percent of PP cells that were labeled for PP (total number counted = 48, number of islets examined = 12) were positive for MAOB, but 85% of PP cells were negative for MAOB (Figures 2B, 2D, and 2F).



View larger version (37K):
[in this window]
[in a new window]
 
Figure 1

Immunohistochemical examination of monoamine oxidase type B (MAOB) localization in A cells and B cells of the rat pancreatic islet. Sections were processed for a double-labeling immunofluorescence method in combination of anti-MAOB (Cy3) with either anti-glucagon (fluorescein) or anti-insulin (fluorescein), and then observed with a confocal laser scanning microscope. (a,b) MAOB staining (red). (c) Glucagon staining (green). (d) Insulin staining (green). (e) Superimposition of the images in a and c. (f) Superimposition of the images in b and d. Note that all of A cells that contain glucagon are also stained for MAOB (a,c,e), and all of B cells are also stained for MAOB (b,d,f). Original magnification x 100. Bar = 10 µm.

 


View larger version (38K):
[in this window]
[in a new window]
 
Figure 2

Immunohistochemical examination of monoamine oxidase type B (MAOB) localization in D cells and pancreatic polypeptide (PP) cells of the rat pancreatic islet. Sections were processed for a double-labeling immunofluorescence method in combination of anti-MAOB (Cy3) with either anti-somatostatin (fluorescein) or anti-PP (fluorescein), and then observed with a confocal laser scanning microscope. (a,b) MAOB staining (red). (c) Somatostatin staining (green). (d) PP staining (green). (e) Superimposition of the images in a and c. (f) Superimposition of the images in b and d. Note that all of D cells that contain somatostatin are negative for MAOB (a,c,e). Some PP cells that contain pancreatic polypeptide are negative for MAOB (b,d,f), but other PP cells are positive for MAOB. Original magnification x 100. Bar = 10 µm.

 
Electron Microscopic Immunohistochemistry
Figures 35 show the subcellular localization of MAOB in the pancreatic islet.



View larger version (150K):
[in this window]
[in a new window]
 
Figure 3

Subcellular localization of monoamine oxidase type B (MAOB) in pancreatic islet A cells. Sections were stained for MAOB with an immunoperoxidase technique and then processed for electron microscopy. Note that MAOB immunoreactivity is found on mitochondrial outer membranes (arrows) of A cells (N, nucleus), which contain electron-dense granules (arrowheads) that are round in shape and enclosed by small clear space. No MAOB immunoreactivity was found in acinar cells. Original magnification x 7200. Bar = 1 µm.

 


View larger version (103K):
[in this window]
[in a new window]
 
Figure 5

Subcellular localization of monoamine oxidase type B (MAOB) in pancreatic islet pancreatic polypeptide (PP) cells. Sections were stained for MAOB with an immunoperoxidase technique and then processed for electron microscopy. Note that MAOB immunoreactivity is found on mitochondrial outer membranes (arrows) of PP cells (N, nucleus), which contain electron-dense granules (arrowheads) that are smaller in size and slightly elongated in shape. Original magnification x 7200. Bar = 1 µm.

 


View larger version (129K):
[in this window]
[in a new window]
 
Figure 4

Subcellular localization of monoamine oxidase type B (MAOB) in pancreatic islet B cells. Sections were stained for MAOB with an immunoperoxidase technique and then processed for electron microscopy. Note that MAOB immunoreactivity is found on mitochondrial outer membranes (arrows) of B cells (N, nucleus), which contain electron-dense granules that are mainly round (arrowhead) and occasionally irregular (double arrowhead) in shape and enclosed by large clear space. Some mitochondria (crossed arrows) of B cells are negative for MAOB. Original magnification: a x 7,200; b x 15,000. Bar = 0.5 µm.

 
MAOB immunoreactivity is found on mitochondrial outer membranes in A cells, which contain electron-dense granules that are round in shape and enclosed by a smaller clear space (Figure 3); in B cells, which contain electron-dense granules that are mainly round and occasionally irregular in shape and enclosed by a larger clear space (Figures 4A and 4B); and in PP cells, which contain electron-dense granules that are smaller in size and slightly elongated in shape (Figure 5).


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
We have found that MAOB is localized on mitochondrial outer membranes of almost all of both A cells and B cells and 15% of PP cells of the rat pancreatic islets. Virtually all D cells lack MAOB. The present study is the first to determine the cell types that contain MAOB and the subcellular localization of the enzyme in the pancreatic islets. These results are in general agreement with previously reported situations in pancreatic islets from rat, mouse, guinea pig, rabbit, golden hamster, and human populations (Aleyassine and Gardiner 1975Go; Feldman and Chapman 1975Go; Feldman and Henderson 1978Go; Lewinsohn et al. 1978Go,1980Go; Lenzen et al. 1983Go,1987Go; Egashira and Waddell 1984Go; Stenstrom and Lundquist 1990Go; Kirchgessner and Pintar 1991Go; Saura et al. 1992Go; Adeghate and Donath 1993Go; Panagiotidis et al. 1993Go; Pizzinat et al. 1999Go), which showed that both MAOA and MAOB were expressed in pancreas. However, we are not in agreement with Rodriguez et al. (2000)Go, who studied MAO localization in human pancreas at light microscopic level. They reported that MAOA was observed in approximately 50% of human islet cells and probably corresponded to B cells, whereas MAOB was less abundant and restricted to the periphery of the islet. Therefore, they conjectured that MAOB-positive cells might correspond to A cells. The reasons may lie in a difference in tissues, antibodies, and methods. They used human tissue, mouse anti-human MAOA/MAOB, and peroxidase-anti-peroxidase technique. On the contrary, we use rat tissue, rabbit anti-bovine MAOB, and both double-labeling immunofluorescence histochemical and electron microscopic immunohistochemical methods.

In our research, not all mitochondria exhibited a positive reaction, as judged by the formation of the electron dense precipitate. There were two kinds of mitochondria in pancreatic islet B cells: one contained MAOB on their outer membranes, but a substantial proportion of them lacked this enzyme. This finding may be attributable to one or more of the following factors (Shannon et al. 1974Go; Muller and Lage 1977Go): (a) different physiologic regions or states of the outer membranes, in which case the observed activity represents the true distribution of active MAOB; (b) uneven fixation that destroys some active enzyme sites; or (c) artificial, which was made by cutting. Further research is needed to understand the exact reasons for these differences.

MAO is synthesized in cytoplasmic polysome and inserted into the mitochondrial outer membrane (Berry et al. 1994Go). It appears that this enzyme is probably localized on both sides of the outer mitochondrial membrane (Russell et al. 1979Go). By radiochemical assay, Russell et al. (1979)Go determined MAO activity in human liver and brain-cortex nonsynaptosomal and synaptosomal mitochondria suspension. They found that MAO immunochemically accessible ß-phenethylamine–oxidizing activity was situated predominantly on the outer surface, the immunochemically accessible 5-hydroxytrptamine oxidizing activity was situated predominantly on the inner surface, and the tyramine-oxidizing activity was distributed on both sides of the mitochondrial outer membrane.

MAO has the ability to deaminate certain amines. Its substrate is relatively wide, including all primary, secondary, and tertiary monoamines (Berry et al. 1994Go). MAO plays a vital role in the metabolism of biogenic and xenobiotic amines in the central nervous system and peripheral tissues (Bach et al. 1988Go). It is the main degradative enzyme of monoamine hormones and amine neurotransmitters, such as epinephrine, norepinephrine, 5-hydroxytryptamine (5-HT), and DA (Rodriguez et al. 2000Go). The major function of MAO is the rapid inactivation of free monoamines, rather than the regulation of the content of total monoamines (Gey and Pletscher 1961Go). In addition, it plays a protective role for the body from the effects of other amines through the degradation of potentially toxic dietary amines and the inactivation of certain endogenous neurotransmitter substrates (Ryder et al. 1979Go; Thorpe et al. 1987Go).

In mammals, MAO has been identified in all cell types with the exception of the erythrocyte (Berry et al. 1994Go). Biochemical and histochemical results indicate large differences of MAO characteristics among animal species and in varying tissues. The specific localization of the two forms of MAO within different body tissues is of biological and clinical significance (Stenstrom and Lundquist 1990Go). The expression of MAO in pancreatic islets and that MAO inhibitors (clorgyline, deprenyl, pargyline, tranylcypromine, and amezinium) inhibit glucose-induced insulin secretion from isolated pancreatic islets (Lenzen et al. 1983Go) suggest that this enzyme may play an important regulatory role in the endocrine pancreas.

Previous studies have indicated that monoamines, such as 5-HT, DA, epinephrine, and norepinephrine, can inhibit glucose-mediated insulin secretion in human, golden hamster, rat, and mouse pancreatic islet B cells (Porte and Williams 1966Go; Feldman and Lebovitz 1970Go; Feldman et al. 1971Go; Aleyassine and Gardiner 1975Go; Lindstrom and Sehlin 1983Go). The catecholamines, epinephrine and norepinephrine, can regulate insulin secretion directly (Porte et al. 1976Go). When there is an increase in these sympathetic neurotransmitters, the secretion of insulin is inhibited initially. The initial decrease has been shown to be related to the activation of {alpha}2-adrenoceptoers on the B cells (Nakaki et al. 1980Go; Pizzinat et al. 1999Go). Other monoamines, such as 5-HT and DA, alter insulin secretion by acting as extracellular or intracellular agents. As extracellular agents, 5-HT and DA influence islet responses by stimulating cellular receptors directly or indirectly and by enhancing the release of norepinephrine from islet sympathetic terminals. As intracellular regulators, both 5-HT and DA have been localized within the islet B cells (Owman et al. 1973Go). Increased intracellular synthesis of these monoamines has been shown to decrease insulin output in response to standard stimuli (Porte et al. 1976Go)

In different mammalian species, pancreatic islet B cells have the ability to harbor biogenic monoamines, such as DA, 5-HT, and catecholamine, in their secretory granules (Cegrell 1967Go; Ekholm et al. 1971Go; Owman et al. 1973Go; Ericson et al. 1977Go; Pizzinat et al. 1999Go). MAO is the main degradative enzyme of monoamine hormones and amine neurotransmitters and might therefore have a regulatory influence on insulin secretion through its regulation of monoamine stores in the islet B cells (Stenstrom and Lundquist 1990Go). Panagiotidis et al. (1993)Go observed that, after an overnight fast in adult lean mice, islet MAO activity was increased and plasma level of glucose was markedly decreased. The data strongly suggest that glucose negatively modulates islet MAO activity. This, in turn, may affect insulin secretion through its effects on the monoamine content (Panagiotidis et al. 1993Go). However, the exact role of MAO in the regulation of granule-located monoamines levels has not yet been elucidated.

Furthermore, MAO might also act directly on the insulin-secretory mechanisms, because H2O2 evolution induced by islet MAO activity can affect the redox states of the B-cells' glutathione system, the balance of which is known to influence glucose-induced insulin secretion (Panagiotidis et al. 1993Go; Pizzinat et al. 1999Go). However, the exact mechanism of MAO regulating insulin release directly is poorly understood.

The hormone insulin is stored in secretory granules and released from the pancreatic B cells by exocytosis (Wollheim et al. 1996Go). In glucose-stimulated B cells, both the cytosolic Ca2+ concentration ([Ca2+]c) rises and the insulin secretion are biphasic with a transient first phase and a sustained second phase (Bergsten 1995Go; Kennedy et al. 1996Go). The universal intracellular second messenger Ca2+ is the crucial trigger for the exocytosis of insulin (Wollheim 2000Go), the process by which the insulin-containing secretory granules fuse with the plasma membrane (Maechler and Wollheim 2000Go). In the consensus model of glucose-stimulated insulin secretion (Maechler and Wollheim 1999Go,2000Go), mitochondrial metabolism increase the cytosolic ATP/ADP ratio. This lead to closure of ATP-sensitive potassium channels and depolarization of the plasma membrane. Subsequently, the [Ca2+]c is raised by the opening of voltage-sensitive Ca2+ channels. The increase in [Ca2+]c trigger insulin exocytosis. Nevertheless, the Ca2+ signal alone is not sufficient for the full development of biphasic insulin secretion. A mitochondrial messenger must therefore exist that is distinct from ATP. In 1999, Maechler and Wollheim identified this as glutamate. They show that glucose generates glutamate from B-cell mitochondria. A membrane-permeant glutamate analog sensitizes the glucose-evoked secretory response, acting downstream of mitochondrial metabolism. In permeabilized cells, under conditions of fixed [Ca2+]c, added glutamate directly stimulates insulin exocytosis independently of mitochondrial function. These results demonstrate that glutamate acts as an intracellular messenger that couples glucose metabolism to insulin secretion (Maechler and Wollheim 1999Go).

MAO has the ability to deaminate certain amines. In the process of deamination, H2O2 and ammonia are generated (Berry et al. 1994Go). H2O2 alters mitochondrial activation and insulin secretion in pancreatic B cells (Maechler et al. 1999Go). Exposure of rat islets to H2O2 result in a retarded and sustained increase of [Ca2+]c (Maechler et al. 1999Go; Nakazaki et al. 2000Go), a transient increase in insulin release (Maechler et al. 1999Go). [Ca2+]c is the crucial trigger for the exocytosis of insulin (Wollheim 2000Go); therefore, the elevated basal insulin release is the consequence of an increase in [Ca2+]c (Maechler et al. 1999Go). Ammonia can be bound to the {alpha}-carbon atom of {alpha}-ketoglutarate, and glutamate is formed. This reversible reaction is catalyzed by glutamate dehydrogenase (Fisher 1985Go; Salway 1999Go). Glutamate uptake by B-cell granules would participate in the second sustained phase of insulin secretion (Maechler and Wollheim 2000Go). All this may be interpreted as evidence of the physiological role of MAO in the islet B cells. Up to now, the exact role of MAO in the regulation of monoamines levels and the mechanism of insulin release has not yet been elucidated and requires further investigation.


    Footnotes
 
Received for publication February 20, 2005; accepted April 13, 2005


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Abell CW, Kwan SW (2001) Molecular characterization of monoamine oxidases A and B. Prog Nucleic Acid Res Mol Biol 65:129–156[Medline]

Adeghate E, Donath T (1993) Fixation and tissues preparation method for the localization of monoamine oxidase enzyme activity in lung and in control and transplanted pancreas. Biogenic Amines 10:67–71

Aleyassine H, Gardiner RJ (1975) Dual action of antidepressant drugs (MAO inhibitors) on insulin release. Endocrinology 96:702–710[Abstract]

Arai R, Karasawa N, Kurokawa K, Kanai H, Horiike K, Ito A (2002) Differential subcellular location of mitochondria in rat serotonergic neurons depends on the presence and the absence of monoamine oxidase type B. Neuroscience 114:825–835[CrossRef][Medline]

Bach AWJ, Lan NC, Johnson DL, Abell CW, Bembenek ME, Kwan S-W, Seeburg PH, et al. (1988) cDNA cloning of human liver monoamine oxidase A and B: molecular basis of differences in enzymatic properties. Proc Natl Acad Sci USA 85:4934–4938[Abstract/Free Full Text]

Bergsten P (1995) Slow and fast oscillations of cytoplasmic Ca2+ in pancreatic islets correspond to pulsatile insulin release. Am J Physiol 268:E282–287[Medline]

Berry MD, Juorio AV, Paterson IA (1994) The functional role of monoamine oxidase A and B in the mammalian central nervous system. Prog Neurobiol 42:375–391[CrossRef][Medline]

Brar AK, Brinster RL, Frohman LA (1989) Immunohistochemical analysis of human growth hormone-releasing hormone gene expression in transgenic mice. Endocrinology 125:801–809[Abstract]

Cegrell L (1967) Monoamine-containing cells in the fetal and newborn guinea-pig pancreas. Life Sci 6:1647–1652[CrossRef][Medline]

Derry JM, Lan NC, Shih JC, Barnard EA, Barnard PJ (1989) Localization of monoamine oxidase A and B genes on the mouse X chromosome. Nucleic Acids Res 17:8403[Medline]

Dun NJ, Dun SL, Wong RKS, Forstermann U (1994) Colocalization of nitric oxide synthase and somatostatin immunoreactivity in rat dentate hilar neurons. Proc Natl Acad Sci USA 91:2955–2959[Abstract/Free Full Text]

Egashira T, Waddell WJ (1984) Histochemical localization of monoamine oxidase in whole-body, freeze-dried sections of mice. Histochem J 16:919–929[CrossRef][Medline]

Ekholm R, Ericson LE, Lundquist I (1971) Monoamines in the pancreatic islets of the mouse. Diabetologia 7:339–348[CrossRef][Medline]

Elayat AA, el-Naggar MM, Tahir M (1995) An immunocytochemical and morphometric study of the rat pancreatic islets. J Anat 186:629–637[Medline]

Ericson LE, Hakanson R, Lundquist I (1977) Accumulation of dopamine in mouse pancreatic B-cells following injection of L-DOPA. Localization to secretory granules and inhibition of insulin secretion. Diabetologia 13:117–124[CrossRef][Medline]

Erlandsen SL (1980) Types of pancreatic islet cells and their immunocytochemical identification. Monogr Pathol 21:140–155[Medline]

Feldman JM, Boyd AE 3rd, Lebovitz HE (1971) Structural determinants of catecholamine action on in vitro insulin release. J Pharmacol Exp Ther 176:611–621[Medline]

Feldman JM, Chapman B (1975) Characterization of pancreatic islet monoamine oxidase. Metabolism 24:581–588[CrossRef][Medline]

Feldman JM, Henderson JH (1978) Monoamine oxidase, catechol-o-methyltransferase, and norepinephrine levels in mice with the hereditary obese-hyperglycemic syndrome. Diabetes 27:389–395[Medline]

Feldman JM, Lebovitz HE (1970) Serotonin inhibition of in vitro insulin release from golden hamster pancreas. Endocrinology 86:66–70[Medline]

Feldman JM, White-Owen C, Klatt C (1980) Golden hamster pancreatic islets: a tissue rich in monoamine oxidase. Endocrinology 107:1504–1511[Abstract]

Fisher HF (1985) L-glutamate dehydrogenase from bovine liver. Methods Enzymol 113:16–27[Medline]

Fowler CJ, Benedetti MS (1983) The metabolism of dopamine by both forms of monoamine oxidase in the rat brain and its inhibition by cimoxatone. J Neurochem 40:1534–1541[Medline]

Gey KF, Pletscher A (1961) Activity of monoamine oxidase in relation to the 5-hydroxytryptanine and norepinephrine content of the rat brain. J Neurochem 6:239–243[Medline]

Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577–580[Abstract]

Ito A, Kuwahara T, Inadome S, Sagara Y (1988) Molecular cloning of a cDNA for rat liver monoamine oxidase B. Biochem Biophys Res Commun 157:970–976[CrossRef][Medline]

Johnston JP (1968) Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem Pharmacol 17:1285–1297[CrossRef][Medline]

Kennedy ED, Rizzuto R, Theler JM, Pralong WF, Bastianutto C, Pozzan T, Wollheim CB (1996) Glucose-stimulated insulin secretion correlates with changes in mitochondrial and cytosolic Ca2+ in aequorin-expressing INS-1 cells. J Clin Invest 98:2524–2538[Abstract/Free Full Text]

Kirchgessner AL, Pintar JE (1991) Guinea pig pancreatic ganglia: projections, transmitter content, and the type-specific localization of monoamine oxidase. J Comp Neurol 305:613–631[CrossRef][Medline]

Knoll J, Magyar K (1972) Some puzzling pharmacological effects of monoamine oxidase inhibitors. Adv Biochem Psychopharmacol 5:393–408[Medline]

Kuwahara T, Takamoto S, Ito A (1990) Primary structure of rat monoamine oxidase A deduced from cDNA and its expression in rat tissues. Agric Biol Chem 54:253–257[Medline]

Lan NC, Heinzmann C, Gal A, Klisak I, Orth U, Lai E, Grimsby J, et al. (1989) Human monoamine oxidase A and B genes map to Xp 11.23 and are deleted in a patient with Norrie disease. Genomics 4:552–559[CrossRef][Medline]

Larsson LI, Sundler F, Hakanson R (1976) Pancreatic polypeptide-A postulated new hormone: identification of its cellular storage site by light and electron microscopic immunocytochemistry. Diabetologia 12:211–226[CrossRef][Medline]

Lenzen S, Freisinger-Treichel M, Panten U (1987) Monoamine oxidase in rat and bovine endocrine tissues. J Neurochem 49:1183–1190[Medline]

Lenzen S, Nahrstedt H, Panten U (1983) Monoamine oxidase in pancreatic islets, exocrine pancreas, and liver from rats. Characterization with clorgyline, deprenyl, pargyline, tranylcypromine, and amezinium. Naunyn-Schmiedebergs Arch Pharmacol 324:190–195[CrossRef][Medline]

Levy ER, Powell JF, Buckle VJ, Hsu YP, Breakefield XO, Craig IW (1989) Localization of human monoamine oxidase-A gene to Xp11.23-11.4 by in situ hybridization: implications for Norrie disease. Genomics 5:368–370[CrossRef][Medline]

Lewinsohn R, Bohm KH, Glover V, Sandler M (1978) A benzylamine oxidase distinct from monoamine oxidase B—widespread distribution in man and rat. Biochem Pharmacol 27:1857–1863[CrossRef][Medline]

Lewinsohn R, Glover V, Sandler M (1980) Development of benzylamine oxidase and monoamine oxidase A and B in man. Biochem Pharmacol 29:1221–1230[CrossRef][Medline]

Lindstrom P, Sehlin J (1983) Opposite effects of 5-hydroxytryptophan and 5-hydroxytrypramine on the function of microdissected ob/ob-mouse pancreatic islets. Diabetologia 24:52–57[Medline]

Maechler P, Jornot L, Wollheim CB (1999) Hydrogen peroxide alters mitochondrial activation and insulin secretion in pancreatic beta cells. J Biol Chem 274:27905–27913[Abstract/Free Full Text]

Maechler P, Wollheim CB (1999) Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis. Nature 402:685–689[CrossRef][Medline]

Maechler P, Wollheim CB (2000) Mitochondrial signals in glucose-stimulated insulin secretion in the beta cell. J Physiol 529:49–56[Abstract/Free Full Text]

Muller J, Lage CD (1977) Ultracytochemical demonstration of monoamine oxidase activity in nervous and non-nervous tissues of the rat. J Histochem Cytochem 25:337–348[Abstract]

Nakaki T, Nakadate T, Kato R (1980) {alpha}2-adrenoceptors modulating insulin release from isolated pancreatic islets. Naunyn Schmiedebergs Arch Pharmacol 313:151–153[CrossRef][Medline]

Nakazaki M, Kakei M, Yaekura K, Koriyama N, Morimitsu S, Ichinari K, Yada T, et al. (2000) Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic ß–cells. Cell Struct Funct 25:187–193[CrossRef][Medline]

Owman C, Hakanson R, Sundler F (1973) Occurrence and function of amines in endocrine cells producing polypeptide hormones. Fed Proc 32:1785–1791[Medline]

Panagiotidis G, Lindstrom P, Stenstrom A, Lundquist I (1993) Glucose modulation of islet monoamine oxidase activity in lean and obese hyperglycemic mice. Metabolism 42:1398–1404[CrossRef][Medline]

Pizzinat N, Chan SLF, Remaury A, Morgan NG, Parini A (1999) Characterization of monoamine oxidase isoforms in human islets of Langerhans. Life Sci 65:441–448[CrossRef][Medline]

Porte D Jr, Smith PH, Ensinck JW (1976) Neurohumoral regulation of the pancreatic islet A and B cells. Metabolism 25 (suppl l):1453–1456[Medline]

Porte D Jr, Williams RH (1966) Inhibition of insulin release by norepinephrine in man. Science 152:1248–1250[Medline]

Reddy S, Elliott RB (1988) Ontogenic development of peptide hormones in the mammalian fetal pancreas. Experientia 44:1–9[CrossRef][Medline]

Rodriguez MJ, Saura J, Finch CC, Mahy N, Billett EE (2000) Localization of monoamine oxidase A and B in human pancreas, thyroid, and adrenal glands. J Histochem Cytochem 48:147–151[Abstract/Free Full Text]

Roth J, Ravazzola M, Bendayan M, Orci L (1981) Application of the protein A-gold technique for electron microscopic demonstration of polypeptide hormones. Endocrinology 108:247–253[Abstract]

Russell SM, Davey J, Marey RJ (1979) The vectorial orientation of human monoamine oxidase in the mitochondria outer membrane. Biochem J 181:7–14[Medline]

Ryder TA, Mackenzie ML, Pryse-Davies J, Glover V, Lewinsohn R, Sandler M (1979) A coupled peroxidatic oxidation technique for the histochemical localization of monoamine oxidase A and B and benzylamine oxidase. Histochemistry 62:93–100[CrossRef][Medline]

Sagara Y, Ito A (1982) In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane. Biochem Biophys Res Commun 109:1102–1107[Medline]

Salway JG (1999) Metabolism at a glance. In Salway JG, ed. The Ornithine Cycle for the Production of Urea: ‘The Urea Cycle’. Oxford, Blackwell Science, 40–41

Saura J, Kettler R, Prada MD, Richards JG (1992) Quantitative enzyme radioautography with 3H-Ro 41-1049 and 3H-Ro 19-6327 in vitro: localization and abundance of MAO-A and MAO-B in rat CNS, peripheral organs, and human brain. J Neurosci 12:1977–1999[Abstract]

Shannon WA Jr, Wasserkrug HL, Seligman AM (1974) The ultrastructural localization of monoamine oxidase (MAO) with tryptamine and a new tetrazolium salt, 2-(29-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT). J Histochem Cytochem 22:170–182[Medline]

Shih JC, Chen K, Ridd MJ (1999) Monoamine oxidase: from genes to behavior. Annu Rev Neurosci 22:197–217[CrossRef][Medline]

Stenstrom A, Lundquist I (1990) Monoamine oxidase (MAO) A and B in pancreatic islets from the mouse. Biogenic Amines 7:547–555

Thorpe LW, Westlund KN, Kochersperger LM, Abell CW, Denney RM (1987) Immunocytochemical localization of monoamine oxidases A and B in human peripheral tissues and brain. J Histochem Cytochem 35:23–32[Abstract]

Weaver C, Sorenson RL, Kaung HL (1986) An immunohistochemical, ultrastructural, and physiologic study of pancreatic islets from copper-deficient, penicillamine-treated rats. Diabetes 35:13–19[Abstract]

Wollheim CB (2000) Beta-cell mitochondrial in the regulation of insulin secretion: a new culprit in type {alpha} diabetes. Diabetologia 43:265–277[CrossRef][Medline]

Wollheim CB, Lang J, Regazzi R (1996) The exocytotic process of insulin secretion and its regulation by Ca2+ and G-proteins. Diabetes Rev 4:276–297

Zhang W, Efanov A, Yang SN, Fried G, Kolare S, Brown H, Zaitsev S, et al. (2000) Munc-18 Associates with syntaxin and serves as a negative regulator of exocytosis in the pancreatic ß-cell. J Biol Chem 275:41521–41527[Abstract/Free Full Text]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
jhc.5A6658.2005v1
53/9/1149    most recent
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Huang, Y.-H.
Articles by Arai, R.
Articles citing this Article
PubMed
PubMed Citation
Articles by Huang, Y.-H.
Articles by Arai, R.


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]