Figure 1.
α-cell numbers are increased in db/db and streptozotocin (STZ)-treated mice.
(A) Immunofluorescent staining of insulin and glucagon in mice at ages shown. Glucagon-positive cells are on the rim of islets in non-diabetic mice (arrows). (B) Mean glucagon-positive area in islets over time, in non-diabetic and db/db mice. (C) Random plasma glucagon levels in non-diabetic and db/db mice. (D) Random plasma insulin levels, over time, in db/db and non-diabetic mice. (E) Immunofluorescent staining of insulin and glucagon and quantification of glucagon-positive area in islets of STZ-treated CD1 mice. (F) PCNA-positive nuclei in glucagon-positive cells in islets from four different db/db mice. (G) Immunofluorescent staining of insulin and glucagon in human pancreatic paraffin sections, demonstrating lack of stereotypy in the distribution of α and β cells in human islets.
Figure 2.
Culturing α cells with MIN6 β cells or insulin increases α-cell proliferation.
(A) αTC1 cells were co-cultured with MIN6 β cells (n = 16), αTC1 (n = 16), CHO-NEO (n = 12) and medium (n = 12) in transwell plates, as shown. Results show relative proliferation rates of α-TC1 cells. (B) and (C), glucagon and insulin levels in medium from αTC1 and MIN6 cells, assayed at the times shown. (D) αTC1 cells were treated with insulin for 5 days at the concentration shown. EC50 = 2 nM, n = 6 separate experiments. All data are expressed as means± SEM; ***p<0.0005.
Figure 3.
Insulin functions through insulin receptors (IR), not IGF-1 receptors (IGF-1R), in α cells.
(A) Immunofluorescent staining of IRβ in a pancreatic islet of a mouse. Merge of IRβ and glucagon is yellow, demonstrating co-localization in α cells (arrow). (B) Immunofluorescent staining of IGF-1R in a pancreatic islet of a non-diabetic mouse. There is no merge of IGF-1R and glucagon (no yellow), demonstrating lack of co-localization (arrow). IGF-1Rs are present on β cells. (C) Western Blot analysis of insulin receptors (IRβ) and IGF-1R in CHO cells, and α-and β-cell lines. IRβ is present in all cell types while IGF-1R is not expressed in α cells (CHO- IRβ are transfected with IR, CHO-IGF-1R are transfected with IGF-1R, CHO-K1 are non-transfected). (D) Immunofluorescence staining of phosphorylated insulin receptors (p-IRβ) in αTC1 cells in response to insulin at the times shown.
Figure 4.
IRs signal through IRS2 and AKT and activate mTOR, resulting in α-cell proliferation.
(A) Western blot analysis of insulin receptor signaling molecules in α cells. αTC1 cells were treated with increasing concentration of insulin for 10 minutes and whole cell lysate was used for western blotting. (B) Immunoprecipitation of IRS1 and IRS2 in pancreatic cells. αTC1 cells were treated with increasing concentration of insulin for 10 minutes and whole cell lysates was collected. IRS1 and IRS2 proteins were immunoprecipitated, and immunoblotted with an antibody that recognizes phosphorylated IRS1/2 (Tyr612). Protein levels of IRS1 (black arrows) and IRS2 (red arrows) in αTC1 and MIN6 cells. The densitometry represents relative expression of IRS2 protein levels. (C) αTC1 cells were treated with increasing concentration of insulin for 10 minutes and whole cell lysate was blotted for phosphorylated and total mTOR. (D) α-TC1 cells were treated for 72 hours (left panel) or 24 hours (right panel) with insulin (2 nM) +/− rapamycin (20 mg/ml). The cell proliferation was assessed by both ELISA-based relative proliferation assay (left panel: data are expressed as means ±SEM [n = 6]; ** p<0.005, *** p<0.0005) and by counting the cells in S phase (propidium iodide staining for DNA cell cycle analysis) (right panel).
Figure 5.
Glucagon receptor (GcgR) antagonism decrease α-cell proliferation.
(A) Immunofluorescent staining of GcgR on αTC1 cells. (B) Proliferative response of α cells after 72 hours (left panel) or 24 hours (right panel) of GcgR antagonist II (14 µM) +/− insulin (2 nM) treatment. The cell proliferation was assessed by both ELISA-based relative proliferation assay (left panel) and by counting the cells in S phase (propidium iodide staining for DNA cell cycle analysis) (right panel). (C) db/db mice were treated with GcgR antagonist II for up to 17 days. Blood glucose measurements in non-treated db/db (n = 4, open diamond) and treated db/db (n = 5, closed diamond) mice. (D) Fasting plasma insulin levels of db/db mice measured after 17days of GcgR antagonist treatment. For control db/db mice n = 4, treated db/db mice n = 5, non-diabetic mice n = 7. (E) Fasting plasma glucagon levels of db/db mice measured after 17days of GcgR antagonist II treatment. For control db/db mice n = 4, treated db/db mice n = 5, non-diabetic mice n = 7. (F) α-cell number in islets from db/db mice was counted (control n = 71 islets, treated n = 84 islets). (G) Immunofluorescent staining of glucagon and insulin in frozen sections of pancreata from treated and non-treated db/db mice. Data are expressed as means ±SEM (n = 6); ** p<0.005, *** p<0.0005.