Fig 1.
AMPK is deleted selectively in pancreatic α-cells and enteroendocrine L-cells in iGlu AMPKdKO mice.
(A) Immunofluorescent staining of pancreatic islets using P-AMPKα1/α2 (red) and proglucagon (green) antibody in iGluAMPKdKO mice versus WT littermates, (B) percentage of α-cells co-staining for AMPK and glucagon. (C) Immunofluorescent staining of ileum slices staining with the above antibodies to quantify AMPK knock-down in enteroendocrine cells (D), n = 3 mice/genotype (all male). Body weight was measured weekly in (E) male and (F) female iGluAMPKdKO and WT mice, weeks 4–20. N = 9–11 mice/genotype, **P<0.01, ***P<0.001, by unpaired Student’s t-test. Data are expressed as means ± SEM.
Fig 2.
AMPKα1 and -α2 deletion in proglucagon expressing cells improves glucose tolerance after oral administration of glucose in male mice but is unchanged after glucose injection.
(A,B) Glycaemia was measured in male and female iGluAMPKdKO and WT mice when fed ad libitum. n = 6–9 mice/genotype/sex. (C,D) Glucose was administered using oral gavage (1 g/kg) after mice were fasted overnight and blood glucose levels measured at 0, 15, 30, 45, 60, 90 and 120 min. after glucose administration in 10 week old male and female mice. (E, F) Oral glucose tolerance tests repeated in 15 week old male and female iGluAMPKdKO and WT mice. Area under the curve (AUC) is displayed at the top right of panels. (G,H) IPGTTs were performed on 20 week old male and female mice. n = 5–9 mice/sex/genotype, *P<0.05, **P<0.01, by 2-way ANOVA. Data are expressed as means ± SEM.
Fig 3.
AMPKα1 and -α2 deletion has no or minor effects on glycaemia after insulin injection, or on circulating insulin levels.
(A,B) Intraperitoneal insulin tolerance tests (ITTs) were performed on 10 week old male and female mice after mice after fasting for 5 h. Blood glucose levels were measured 0, 15, 30, 45 and 60 min. after insulin injection. (C,D) ITTs were repeated on 15 week old male and female mice, n = 5–9 mice/sex/genotype. **P<0.01, by 2-way ANOVA. (E,F) Plasma insulin levels were measured in iGluAMPKdKO and WT mice when fed as normal or after being fasted overnight and (G) at 0, 15 and 30 min. after glucose injection. n = 3 mice/genotype, mixed male and female. Data expressed as means ± SEM.
Fig 4.
L-cell mass and GLP-1 secretion are enhanced in iGluAMPKdKO mice.
(A) Immunohistochemical analysis of an ileum section from an iGluAMPKdKO mouse; red staining represents proglucagon (GLP-1) staining in an enteroendocrine cell, dotted lines represent magnified section. (B) Percentage of proglucagon staining cells in the ileum. (C) L-cell count/area of gut, P = 0.06 by unpaired Student’s t-test. (D) Approximate endothelial cell count for the whole gut section; n = 3 mice/genotype (all male). (E,G) GLP-1 levels in the plasma of fed or fasted male and (F,H) female mice, n = 3 mice/sex/genotype. *P<0.05, **P<0.01, ***P<0.001 by unpaired Student’s t-test. Data are expressed as means ± SEM.
Fig 5.
AMPKα1,α2KO has no effect on α-cell mass or glucagon secretion in male mice.
(A) Immunofluorescent staining of pancreatic sections using guinea pig anti-insulin (1:200; green) or rabbit anti-glucagon (1:100; red) antibodies from iGluAMPKdKO or WT mice. (B) Alpha:beta cell ratio, (C) % alpha-cell mass, (D) % beta-cell mass; n = 3 mice/genotype (all male), (E) glucagon secretion, measured from 12 size-matched islets incubated in 0.5, 3 or 10 mmol/l glucose and measured using radioimmunoassay, (F,G) levels of glucagon in the plasma of iGluAMPKdKO and WT mice when fed or fasted overnight. n = 6 mice/genotype, *P<0.05, by Student’s t-test; ****P<0.0001 by 2-way ANOVA. Data are expressed as means ± SEM.