Supplemental Data Insulin Signaling in α Cells Modulates ... · β-cell specific insulin receptor...

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Cell Metabolism, Volume 4 Supplemental Data Insulin Signaling in α Cells Modulates Glucagon Secretion In Vivo Dan Kawamori, Amarnath J. Kurpad, Jiang Hu, Chong Wee Liew, Judy L. Shih, Eric L. Ford, Pedro L. Herrera, Kenneth S. Polonsky, Owen P. McGuinness, and Rohit N. Kulkarni

Transcript of Supplemental Data Insulin Signaling in α Cells Modulates ... · β-cell specific insulin receptor...

Page 1: Supplemental Data Insulin Signaling in α Cells Modulates ... · β-cell specific insulin receptor knockout mice (Kulkarni et al., 1999a) as described previously (Kulkarni et al.,

Cell Metabolism, Volume 4

Supplemental Data

Insulin Signaling in α Cells Modulates Glucagon Secretion In Vivo

Dan Kawamori, Amarnath J. Kurpad, Jiang Hu, Chong Wee Liew, Judy L. Shih, Eric L. Ford, Pedro L. Herrera, Kenneth S. Polonsky, Owen P. McGuinness, and Rohit N. Kulkarni

Page 2: Supplemental Data Insulin Signaling in α Cells Modulates ... · β-cell specific insulin receptor knockout mice (Kulkarni et al., 1999a) as described previously (Kulkarni et al.,

Figure S1. Streptozotocin-Induced Insulinopenia and Hyperglycemia in αIRKO Mice Streptozotocin (STZ) was injected in 3 month-old male mice. (A) Plasma insulin and (B) blood glucose were measured in the random fed state before and after STZ treatment n=3-7. Control: empty bar; αIRKO: filled bar: Data are expressed as means ± SEM. (C) Pancreas were harvested from untreated mice (left panel) or in mice 28 days after STZ treatment (right panel) and immunostained as indicated. Magnification: X40.

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Figure S2. Induction of Glucopenia by Phloridzin Treatment (A) Six-month-old mice were treated with 0.4 g/kg body weight of phloridzin or vehicle twice a day for 2 weeks. Blood glucose was monitored at random fed state on day 0, 9 and 15. (B) Plasma insulin and (C) glucagon were measured in the random fed state on day 15. The empty square and empty bar represent controls treated with DMSO (n=4), the grey square and grey bar represent controls treated with phloridzin (n=3); the filled triangle with dashed line and filled bar represent αIRKO mice treated with DMSO (n=3), and the empty triangle with dashed line and stippled bar represent αIRKO mice treated with phloridzin (n=4). Data are expressed as means ± SEM. *, p<0.05 control with DMSO vs control with phloridzin; #, p<0.05 αIRKO with DMSO vs αIRKO with phloridzin.

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Figure S3. Ghrelin Expression in Neonatal Pancreas The specificity of the anti-ghrelin antibody was confirmed by its detection in pancreas sections from a wild type one-day old neonatal mouse by immunofluorescent staining. Ghrelin (red); nuclear staining (DAPI, blue). Magnification X40. Arrows point to ghrelin+ cells.

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Table S1. Gene Expression of Transcription Factors by PancChip Analyses Gene expression was examined in isolated islets from 3 and 10 month-old mice by PancChip analyses. Data are expressed as mean fold change (αIRKO/control). n=4 each.

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Supplemental Experimental Procedures β-cell lines - β-cell lines were established from three independent mice each from insulin receptor floxed or β-cell specific insulin receptor knockout mice (Kulkarni et al., 1999a) as described previously (Kulkarni et al., 1999b). Phloridzin treatment - Phloridzin (Apple wood, Sigma-Aldrich) was dissolved in a vehicle solution (15% DMSO, 10% ethanol, 75% saline) and injected via intraperitoneal route to 6 month-old mice at a dose of 0.4 g/kg body weight in the random fed state at 7 AM and 7 PM for 14 days. A control group received the same amount of the vehicle solution only. After treatment, blood samples were collected in the random fed state at 7 AM on day 15. Immunostaining of neonate pancreas for ghrelin - Mouse pancreas section from a one-day-old mouse (kind gift from Drs. Wataru Nishimura and Arun J. Sharma, Joslin Diabetes Center) was immunostained for ghrelin (rabbit anti-ghrelin, Phoenix Pharmaceuticals) with the nuclear stain 4’, 6-diamidino-2-phenylindole (DAPI) as a positive control. Gene chip analysis - RNA was extracted from isolated islets of young (3 months) or old (10 months) males (control and αIRKO, n=4 each), and gene expression was analyzed by the PancChip (Scearce et al., 2002). Alterations greater than 1.5 fold were considered as significant (P<0.05). Primers insulin receptor: 5’-TGCCAAGACCTTCACTTCAA-3’ (forward) 5’-CAAGGTTAGCCTCCAGCTCA-3’ (reverse) PEPCK: 5’-GTGAGGAAGTTCGTGGAAGG-3’ (forward) 5’-TCTGCTCTTGGGTGATGATG-3’ (reverse) G6Pase: 5’-TCTGTCCCGGATCTACCTTG-3’ (forward) 5’-GTAGAATCCAAGCGCGAAAC-3’ (reverse) glucokinase: 5’-GAGATGGATGTGGTGGCAAT-3’ (forward) 5’-ACCAGCTCCACATTCTGCAT-3’ (reverse) fatty acid synthase: 5’-CTCTGATCAGTGGCCTCCTC-3’ (forward) 5’-TGCTGCAGTTTGGTCTGAAC-3’ (reverse) TBP: 5’-ACCCTTCACCAATGACTCCTATG-3’ (forward) 5’-ATGATGACTGCAGCAAATCGC-3’ (reverse) mouse insulin I: 5’-CCTGTTGGTGCACTTCCTA-3’ (forward) 5’-TCTGAAGGTCCCCGGGGCT-3’ (reverse) glucagon: 5’-TGAATTTGAGAGGCATGCTG-3’ (forward) 5’-TGGTGCTCATCTCGTCAGAG-3’ (reverse) actin: 5’-AGGGCTATGCTCTCCCTCAC-3’ (forward) 5’-AAGGAAGGCTGGAAAAGAGC-3’ (reverse)

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References

Kulkarni, R. N., Bruning, J. C., Winnay, J. N., Postic, C., Magnuson, M. A., and Kahn, C. R. (1999a). Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 96, 329-339. Kulkarni, R. N., Winnay, J. N., Daniels, M., Bruning, J. C., Flier, S. N., Hanahan, D., and Kahn, C. R. (1999b). Altered function of insulin receptor substrate-1-deficient mouse islets and cultured beta-cell lines. J Clin Invest 104, R69-75. Scearce, L. M., Brestelli, J. E., McWeeney, S. K., Lee, C. S., Mazzarelli, J., Pinney, D. F., Pizarro, A., Stoeckert, C. J., Jr., Clifton, S. W., Permutt, M. A., et al. (2002). Functional genomics of the endocrine pancreas: the pancreas clone set and PancChip, new resources for diabetes research. Diabetes 51, 1997-2004.