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First published online 17 October 2006
doi: 10.1242/jcs.03234

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The a3 isoform of V-ATPase regulates insulin secretion from pancreatic ß-cells

¹ Department of Biochemistry, Faculty of Pharmaceutical Sciences, Doshisha Women's College, Kyotanabe 610-0395, Japan
² Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan
³ Nagahama Institute of Bio-Science and Technology, Nagahama 526-0829, Japan
⁴ Futai Special Laboratory, Microbial Chemistry Research Center, CREST, Japan Science and Technology Agency, Tokyo 141-0021, Japan

View larger version (147K): [in a new window]   Fig. 1. Localization of a subunit isoforms in the islet of Langerhans. (A-D) Serial paraffin sections of a pancreatic islet were stained with hematoxylin and eosin (A, HE), and anti-a1 (B), anti-a2 (C) and anti-a3 (D) antibodies. IL, islet of Langerhans; BV, blood vessel; Ac, acini; PD, pancreatic duct. Strong signals of the a3 isoform were detected in almost all the cells in the islet of Langerhans. Strong signals of the a2 isoform were also detected in the epithelial cells of blood vessels (A and C). Bar, 100 µm.

View larger version (161K): [in a new window]   Fig. 2. Serial paraffin sections of a pancreatic islet were stained with hematoxylin and eosin (A, HE), and anti-a3 (B), anti-insulin (C) and anti-glucagon (D) antibodies. IL, islet of Langerhans; BV, blood vessel; Ac, acini. Bar, 50 µm.

View larger version (41K): [in a new window]   Fig. 3. Localization of the a3 isoform to secretory granules in ßTC6 and ßHC9 cells. (A) ßTC6 cells grown on a slide glass were stained with antibodies against the a3 isoform (green), and insulin, lamp-2, GM130, or synaptophysin (red). (B) ßHC9 cells were stained with antibodies against the a isoforms (a1, a2 and a3, green) and insulin, lamp-2, GM130, or synaptophysin (red). Merged images are also shown. Bar, 10 µm.

View larger version (63K): [in a new window]   Fig. 4. Localization of the a3 isoform in pancreatic ß-cells. (A) The localization of a3 is shown by electron-dense silver-enhanced immunogold particles (arrows indicate examples): mt, mitochondria; SG, secretory granules. Bar, 1 µm. (B) Quantification of gold particle frequency in secretory granule (SG) or other compartments. Approximately 80% of total gold particles (450 µm2 x 6 sections, n=1193) were distributed in the dense-core granules. MVB, multi-vesicular body; mt, mitochondria.

View larger version (45K): [in a new window]   Fig. 5. Expressions and localizations of a isoforms in oc/oc pancreatic ß-cells. Pancreas (A) and islet (B) total lysates prepared from wild-type, +/oc heterozygous and oc/oc homozygous mice were subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The a subunit isoforms were detected with affinity-purified antibodies against isoforms a1, a2, a3 and a4. The immunoblots obtained with anti-actin and anti-A subunit of V-ATPase antibodies are also shown. The positions of molecular weight markers are indicated (arrowheads). (C) The localization of a subunit isoforms in oc/oc pancreatic ß-cells. Pancreatic ß-cells prepared from wild-type and oc/oc homozygous mice were stained with antibodies against the a isoforms (green) and insulin (red). Merged images are also shown. Bar, 5 µm.

View larger version (105K): [in a new window]   Fig. 6. Immunohistochemistry of the pancreatic islet of oc/oc mice with anti-a3 and anti-insulin antibodies. (A) Paraffin sections of pancreatic islets from wild-type and oc/oc mice were stained with anti-insulin and anti-a3 antibodies. The morphologies and sizes of islets in wild-type and oc/oc mice were indistinguishable. The insulin contents of ß-cells in the islets were comparable. The lack of a3 expression in oc/oc islets was also confirmed. Bar, 50 µm. (B) Electron micrographs of representative areas of ß-cells from wild-type and oc/oc mice are shown. The dense-core secretory granules (SG) are indicated. Golgi, Golgi apparatus; mt, mitochondria. Bar, 1 µm.

View larger version (54K): [in a new window]   Fig. 7. Immunoblot analysis of insulin processing. Equal amounts of islet protein prepared from either wild-type (lanes 2 and 4) or oc/oc homozygous mice (lanes 3 and 5) were subjected to 15-25% gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 100 mM dithiothreitol. Human insulin was loaded as standard (lane 1). Immunoblotting was performed using anti-insulin or anti-C-peptide antibodies. The positions of a molecular weight marker are shown, and the positions of insulin and proinsulin are indicated (arrows). The intensities of the bands were measured using the image analyzer LAS1000. `Fold' indicates the ratio of the level of insulin over proinsulin in wild-type or mutant islets.

View larger version (13K): [in a new window]   Fig. 8. Plasma insulin levels in oc/oc mice. Blood samples were collected from (A) natural-feeding (with a lactating mother and a chew diet) wild-type (+/+; n=7), heterozygous (oc/+; n=8) and oc/oc (n=7) mice, or (B) fasting (without a lactating mother or a chew diet) mice loaded with glucose [wild-type (+/+; n=10), heterozygous (oc/+; n=10), and oc/oc (n=10)] or saline [wild-type (+/+; n=7), heterozygous (oc/+; n=7), and oc/oc (n=3)] (B). Plasma insulin contents were measured by ELISA. (C) The blood glucose levels before (open bar) or after (solid bar) oral administration. The results are presented as means ± s.d. *P<0.05, compared with wild-type mice; **P<0.001, compared with wild-type mice.

View larger version (10K): [in a new window]   Fig. 9. Secretion of insulin from pancreatic islets. (A) Islets isolated from 2-week-old wild-type (black bars), heterozygous (oc/+) (gray bars) and oc/oc mice (open bars) were incubated under the basal condition for 30 minutes followed by a second 30-minute incubation in the presence of high glucose (16.7 mM) or K+ (60 mM). Immunoreactive insulin in the media was measured with anti-insulin antibodies and normalized by the protein content in the islet extracts. (B) The islets used in insulin-secretion assays were subsequently extracted with acid-ethanol. The insulin content was calculated per µg islet protein. The results are given as the mean ± s.d. (n=3). *P<0.05, **P<0.005, compared with wild-type mice.

View larger version (24K): [in a new window]   Fig. 10. Acidification of oc/oc mutant pancreatic islet cells, and insulin secretion in ßHC9 cells following treatment with bafilomycin A. (A) Wild-type and oc/oc islets were dispersed with trypsin and stained with LysoTracker. After fixation with 4% paraformaldehyde in PBS (pH 7.4), the cells were mounted for observation. Bar, 10 µm. (B) LysoTracker staining of control ßHC9 (w/o Baf) cells or ßHC9 cells after treatment for 1 hour with bafilomycin A (10 nM). Bar, 10 µm. (C) Insulin secretion of ßHC9 cells stimulated by 25 mM glucose after treatment for 1 hour with bafilomycin A (10 nM) as described under `Materials and Methods'. Means ± s.d. of triplicate observations in a single representative experiment are shown here. Broken line and open circle, control cells; solid line and filled square, cells treated with bafilomycin A.

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