Volume 138, Issue 5, Pages 1966-1975.e1 (May 2010) Glucose-Dependent Insulinotropic Polypeptide Is Expressed in Pancreatic Islet α-Cells and Promotes Insulin Secretion  Yukihiro Fujita, Rhonda D. Wideman, Ali Asadi, Gary K. Yang, Robert Baker, Travis Webber, Tianjiao Zhang, Rennian Wang, Ziliang Ao, Garth L. Warnock, Yin Nam Kwok, Timothy J. Kieffer  Gastroenterology  Volume 138, Issue 5, Pages 1966-1975.e1 (May 2010) DOI: 10.1053/j.gastro.2010.01.049 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 GIP immunoreactivity is present in murine pancreatic islets. Mouse pancreas sections were immunostained for GIP using 4 different antibodies (Abs 1–4) and fluorescently labeled secondary antibodies with or without preadsorption with blocking peptide. Scale bars, 50 μm. Gastroenterology 2010 138, 1966-1975.e1DOI: (10.1053/j.gastro.2010.01.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 GIP mRNA is present in murine pancreatic islets. (A) Sequential in situ hybridization (dark purple) and immunostaining for GIP (red; Ab 3) and nuclei (DAPI, blue) was performed in paraffin-sectioned mouse duodenum. A representative K-cell is shown. Scale bar, 25 μm. (B) Reverse-transcription PCR for GIP and insulin (ins) in an enteroendocrine cell line (STC-1) and isolated mouse islets. rt, reverse transcriptase. In situ hybridization was performed using a (C) GIP probe or (D) no probe in cryosectioned mouse pancreas. Scale bar, 50 μm. Gastroenterology 2010 138, 1966-1975.e1DOI: (10.1053/j.gastro.2010.01.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Pancreatic GIP expression is localized to α-cells. (A) Immunohistochemistry for GIP (red; Ab 3) and glucagon or insulin (green) was performed in pancreas sections from multiple species. Merged images are shown. Low-level GIP immunoreactivity sometimes was observed in insulin-positive cells in the python (top panel). In all species tested, GIP immunoreactivity colocalized with glucagon immunoreactivity (lower panel). Scale bars, 25 μm. (B) Colocalization of glucagon (green) and GIP (red) in a human α-cell. Orthogonal views through the same cell show some colocalization of GIP and glucagon (arrowhead) and also discrete areas of GIP immunoreactivity without glucagon immunoreactivity (arrows). Scale bar, 2 μm. Gastroenterology 2010 138, 1966-1975.e1DOI: (10.1053/j.gastro.2010.01.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 ProGIP is processed differentially in intestinal K-cells and islet α-cells. (A) Immunohistochemistry for GIP was performed in mouse pancreas sections. GIP immunoreactivity is detectable in wild-type and PC2−/− mouse islets (PC2 KO) using a side view antibody for GIP (GIP Ab 3) but only in PC2−/− islets using a C-terminal antibody (GIP Ab 5). Scale bars, 25 μm. (B) Organization of mouse proGIP. Sites at which PC1/3 and/or PC2 can cleave proGIP are indicated with arrowheads and hatched vertical lines. Key amino acid residues at processing sites are noted, and C-terminal ends of fully processed GIP1–30 and GIP1–42 are marked with a vertical line. Regions toward which Ab 3 (red) and Ab 5 (green) are directed are indicated. N- and C-terminal regions of proGIP are shown in white. Gastroenterology 2010 138, 1966-1975.e1DOI: (10.1053/j.gastro.2010.01.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Expression of GIP in human fetal pancreas. Human fetal pancreas sections were immunostained for insulin (green), and GIP (Ab 3), or GIPR. (A) At 10 weeks of fetal age, intense GIP immunostaining was sparse but largely coincident with insulin immunoreactivity; (C) by 15 weeks, GIP immunoreactivity was more prevalent and largely restricted to non–insulin-positive areas. (B and D) GIPR immunoreactivity was abundant at both 10 and 15 weeks of fetal age, although coexpression with insulin was more predominant at 10 weeks. Scale bars, 25 μm. Gastroenterology 2010 138, 1966-1975.e1DOI: (10.1053/j.gastro.2010.01.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Isolated islets secrete bioactive GIP. (A) A representative GIP1–30 standard curve for a GIP radioimmunoassay that detects both GIP1–30 and GIP1–42 (n = 3). (B) Islet and duodenum GIP content determined using the radioimmunoassay in panel A. Data are normalized for total protein content (n = 5 rats; ***P < .001 vs control). (C) A representative GIP bioassay standard curve constructed using GIP1–30 standards (n = 3). In human embryonic kidney–GIPR-Luc cells, GIPR activation causes cAMP release and activation of a cAMP response element, which is coupled to luciferase production. (D) Bioactive GIP secretion from isolated islets during incubation with media containing 3 mmol/L glucose (basal) or 3 mmol/L glucose with 15 mmol/L arginine (stimulated). n = 2–4. Gastroenterology 2010 138, 1966-1975.e1DOI: (10.1053/j.gastro.2010.01.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Islet GIP is insulinotropic. (A) Mouse pancreata were perfused with 16.67 mmol/L glucose alone (control; ctrl) or in combination with a gradient of GIP1–42 or GIP1–30NH2. Insulin release is expressed as a percentage of basal (glucose only) or as the area under the curve (auc, inset; arbitrary units). *,#P < .05 vs glucose; n = 4–5. (B) Ten IE or (C) 50 IE aliquots of mouse islets were pre-incubated for 48 hours in control media or in media containing (B) Ab 4 (gip ab) or GIP1–42 or a (C) GIPR antibody (gipr ab). Glucose-stimulated insulin secretion was assessed in media containing 3 mmol/L (KRB3) or 20 mmol/L (KRB20) glucose. n = 3–11. **P < .01, ***P < .001 vs respective KRB3; #P < .05, ###P < .001 vs control in KRB20. Gastroenterology 2010 138, 1966-1975.e1DOI: (10.1053/j.gastro.2010.01.049) Copyright © 2010 AGA Institute Terms and Conditions