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0344228578
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note
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A BALB/c mouse mGPDH gene containing exons 4, 5, and 6 was cloned and a neomycin resistance gene under transcriptional control of the mouse phosphoglycerate kinase-1 promoter (PGK-neo) was inserted within exon 5. The 8.1 kb (5′) and 1.7 kb (3′) of genomic DNA flanking PGK-neo was ligated into a pMCDT-A plasmid (Gibco-BRL) that produces diphtheria toxin A fragment for negative selection. The completed targeting vector was linearized and electroporated into TT2 embryonic stem cells. The cells were cultured with G418. The choice of targeted clones was based on the shift of a genomic fragment by Pvu II digestion from 3.4 to 3.1 kb, as determined by Southern (DNA) blot. Targeted embryonic stem cells were injected into blastocytes from C57BL/6J mice to sequentially obtain chimeric, heterozygous, and homozygous offspring. All the mice used were males from heterozygous breeding pairs. All islets were from 16-to 20-week-old mice.
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15
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0016207304
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Antibody to mGPDH was raised against the COOH-terminal sequence Lys-Thr-Ala-Glu-Glu-Asn-Leu-Asp-Arg-Arg-Val-Pro-Ile-Pro-Val-Asp-Arg-Ser-Cys-Gly- Gly-Leu. Islets were isolated by collagenase digestion and homogenated in 0.23 M mannitol, 0.07 M sucrose, and 5 mM Hepes (pH 7.5). Activities of mGPDH were measured as described [R. S. Gardner, Anal. Biochem. 59, 272 (1974)].
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Gardner, R.S.1
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0344228606
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note
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-1- islets had normal cellular architecture and contained approximately the same amounts of insulin and glucagon.
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17
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0345090706
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note
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For the glucose tolerance test, glucose (1.5 mg/g body weight) was injected into the peritoneum. Blood samples were drawn from tail veins.
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18
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0344228579
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note
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3, 10 mM Hepes (pH 7.4), and 0.1% bovine serum albumin at 37°C. Basal glucose concentration was 2.8 mM, and AOA was added to buffer during preincubation (30 min) and throughout the incubation period unless otherwise stated.
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19
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0344228577
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note
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Islet homogenates were centrifuged at 600g for 5 min and the supernatant was centrifuged at 5500g for 10 min to sediment the mitochondrial fraction. The resulting supernatant was used as the cytosolic fraction.
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20
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0344228576
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note
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Perifusion experiments were done with 30 islets per chamber at 37°C with a flow rate of 0.6 ml/min.
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0345090679
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note
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2, and 10 mM Hepes (pH 7.4) at 37°C. Fluorescence was excited with light emitted from a xenon lamp (TILL Photonics, Germany), collected through interference filters (Olympus), and detected using a photomultiplier (NT5783; Hamamatsu Photonics, Japan). NAD(P)H was measured by autofluorescence excited at 360 nm and filtered at 470 nm.
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24
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0030926003
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For measurement of mitochondrial membrane potential, islets were loaded with Rh123 (10 μg/ml, Sigma) at 37°C for 10 min. The fluorescence was excited at 490 nm and filtered at 530 nm as described [P. Maechler et al., EMBO J. 16, 3833 (1997)].
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(1997)
EMBO J.
, vol.16
, pp. 3833
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Maechler, P.1
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m, islets were loaded with 10 μM Rhod 2/acetoxymethylester (Molecular Probes) at 37°C for 1 hour and further incubated for 3 to 5 hours to eliminate the dye from cytosol [G. Hajnóczky et al., Cell 82, 415 (1995); D. R. Trollinger et al., Biochem. Biophys. Res. Commun. 236, 738 (1997)]. The fluorescence was excited at 480 nm and filtered at 500 nm.
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(1995)
Cell
, vol.82
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Hajnóczky, G.1
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m, islets were loaded with 10 μM Rhod 2/acetoxymethylester (Molecular Probes) at 37°C for 1 hour and further incubated for 3 to 5 hours to eliminate the dye from cytosol [G. Hajnóczky et al., Cell 82, 415 (1995); D. R. Trollinger et al., Biochem. Biophys. Res. Commun. 236, 738 (1997)]. The fluorescence was excited at 480 nm and filtered at 500 nm.
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(1997)
Biochem. Biophys. Res. Commun.
, vol.236
, pp. 738
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Trollinger, D.R.1
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0345037662
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note
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c, islets were loaded with 15 μM Fura 2/acetoxymethylester (Molecular Probes) at 37°C for 1 hour. The fluorescence was excited alternately at 340 and 380 nm and filtered at 500 nm.
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N. Hamakawa and T. Yada, Cell Calcium 17, 21 (1995); F. M. Ashcroft et al., Nature 312, 446 (1984).
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Cell Calcium
, vol.17
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Hamakawa, N.1
Yada, T.2
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N. Hamakawa and T. Yada, Cell Calcium 17, 21 (1995); F. M. Ashcroft et al., Nature 312, 446 (1984).
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(1984)
Nature
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Ashcroft, F.M.1
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unpublished data
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K. Eto et al., unpublished data.
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K, E.1
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0344174913
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note
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Results are presented as means ± SE. The significance of the differences between groups was determined using one-way analysis of variance.
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0344174914
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note
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We thank C. B. Wollheim and F. M. Ashcroft for invaluable discussion, Y. Kagawa, H. Sakura, M. Noda, and K. Yasuda for critical reading of the manuscript, and N. Takeda, M. Nagai, H. Yajima, C. Sato, H. Chiyonobu, and J. Taka for support. Supported by a grant-in-aid for creative basic research (10NP0201) from the Ministry of Education, Science, Sports, and Culture of Japan (T.K.) and by CREST of the Japan Science and Technology Corporation (H.K.).
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