Figure 1.
Gross characteristics of Gcgr−/− mice.
(A) Weight pattern from weaning to 12 months. Data were from 48 WT, 111 heterozygous, and 52 Gcgr−/− mice. Body weight was measured periodically over time. (B) Abdominal visceral fat of 12-month WT, heterozygous, and Gcgr−/− mice. (C) Representative pancreas of WT, heterozygous, and Gcgr−/− mice at 12 months. Shown are pancreata after being fixed overnight in 10% formalin. (D) Pancreas appearance on MRI of an age- and sex-matched unrelated subject and the patient with homozygous inactivating P86S mutation of glucagon receptor. The pancreas contour was highlighted with a white line. HZ, heterozygous.
Table 1.
Gross characteristics and pancreatic neuroendocrine tumors (PNETs) of Gcgr−/− mice at 10-12 months.
Figure 2.
Hyperplastic and dysplastic α cells in Gcgr−/− mice at 5-7 months.
(A-H) Hematoxylin and eosin (H&E) staining of pancreas of WT and Gcgr−/− mice. (A and B) WT pancreas at low-power (4x) and an islet from A shown in 20x (B). (C) Gcgr−/− pancreas at low-power (4x). (D–H) Five areas of C shown in 20x (D–F) and 40x (G and H). (D) A neogenetic islet (arrow) arising from an exocrine duct; (E) part of a large islet with trapped exocrine ducts (thick arrows), acinar cells (thin arrow), and blood island (arrowhead); (F) singlet endocrine cells and irregularly-shaped islets; and (G and H) intra-islet clearances. The cells around the clearance assume a trabecular growth pattern in H. (I–X) Immunofluorescent staining of pancreas of WT and Gcgr−/− mice. (I and J) PCNA (purple) labeling of WT (I) and Gcgr−/− (J) islet cells. Cells were costained with antibodies for glucagon (green). Inset, Gcgr−/− islet cells costained with antibodies for insulin (green) and PCNA (purple). (K–M) Neogenetic α cells (green) from large (K) and small (L and M) exocrine ducts with formation of a small islet (M). (N–P) Co-production of glucagon (green) and insulin (red) in some Gcgr−/− islet cells. Orange or yellow indicates overlap. (Q–T) WT (Q) or Gcgr−/− (R–T) pancreata were costained for glucagon (green) and PDX-1 (purple or pink). PDX-1 was not present in neogenetic α cells (R) but in some α cells in larger islets (S and T). (U and V) WT (U) or Gcgr−/− (V) pancreata were costained for insulin (green) and PDX-1 (purple or pink). PDX-1 was only present in β cells in WT pancreas but also present in cells without insulin in Gcgr−/− islets. (W and X) Hormonal expression profile of cells around the intra-islet clearances of Gcgr−/− pancreata. The cells without a trabecular growth pattern mostly only express glucagon (*). The cells with a trabecular growth pattern express only glucagon or co-express glucagon and insulin (**). Blue, counterstained with Hoechst 33342 to highlight nuclei. White scale bar, 250 µm; red scale bar, 40 µm. The scale bar in N applies to N–V.
Figure 3.
Pancreatic neuroendocrine tumors (PNETs) and dysplastic endocrine cells in Gcgr−/− mice at 10-12 months.
(A–P) PNETs in Gcgr−/− mice. (A) A PNET (arrow) inside the tail of a fixed whole pancreas from a 12-month Gcgr−/− mouse. (B and C) H&E staining of the tumor in A (4x and 20x, respectively). (D) Immunostaining for chromogranin A (CGA, green) of the tumor in A. (E–L) PNETs from 10-12-month Gcgr−/− mice were immunostained for glucagon (Glu) (E, green), somatostatin (SST) (F, green), insulin (Ins) (G and K, red), pancreatic polypeptide (PP) (H and L, green), Ki-67 (I, pink), and cyclin D1 (J, pink). (M) H&E staining of liver with metastatic PNET foci. (N) Focal fat replacement of exocrine pancreas with preservation of dysplastic islets. (O and P) A PNET co-expressing insulin (O, red) and glucagon (P, green). (Q-T) Dysplastic endocrine cells from 12-month Gcgr−/− mice immunostained for islet hormones. (Q and R) Pancreas from Gcgr−/− mice was doubled stained for insulin (red) and glucagon (green). Orange or yellow denotes overlap of red and green fluorescence. (S and T) Pancreas from Gcgr−/− mice was stained for pancreatic polypeptide (S) or somataostatin (T) (both green). Blue represents nuclei in fluorescent images. White scale bar, 250 µm; red scale bar, 125 µm. The scale bar in E applies to E–P except for I and J.
Figure 4.
The Gcgr−/− mice recapitulate all stages of PNETs in a human patient with a homozygous inactivating mutation of glucagon receptor.
(A–E, G–K) H&E-stained pancreatic sections. (F) A posterior PNET revealed after the pancreas was flipped after euthanasia. (L) Abdominal MRI of the patient. (A–F) From 12-month-old Gcgr−/− mice. (G–L) From the 60-year-old patient. Dysplastic islets with stromal component (A, G) or with blood islands (B, H), microadenomas about 300 µm (C, I), 600 µm (D, J), or 1 mm (E, K), and gross tumors (F, L) are all present. Scale bar, 250 µm until otherwise indicated. Panel L is from reference 18.
Figure 5.
Abnormal menin expression and angiogenesis in Gcgr−/− endocrine pancreas.
Pancreatic sections from 12-month-old WT or Gcgr−/− mice were immunostained for menin (red), vascular endothelial growth factor (VEGF, red), and VEGF receptor Flk-1 (green). All slides were counterstained with Hoechst 33342 for highlighting nuclei (blue). Insets, dysplatic islets and adenomas in Gcgr−/− pancreas were costained for menin (red) and glucagon (green). Yellow or orange indicates colocalization of menin and glucagon. Bar, 20 µm.