BACE2 suppression promotes β-cell survival and function in a model of type 2 diabetes induced by human islet amyloid polypeptide overexpression

Abstract

BACE2 (β-site APP-cleaving enzyme 2) is a protease expressed in the brain, but also in the pancreas, where it seems to play a physiological role. Amyloidogenic diseases, including Alzheimer's disease and type 2 diabetes (T2D), share the accumulation of abnormally folded and insoluble proteins that interfere with cell function. In T2D, islet amyloid polypeptide (IAPP) deposits have been shown to be a pathogenic key feature of the disease. The aim of the present study was to investigate the effect of BACE2 modulation on β-cell alterations in a mouse model of T2D induced by IAPP overexpression. Heterozygous mice carrying the human transcript of IAPP (hIAPP-Tg) were used as a model to study the deleterious effects of IAPP upon β-cell function. These animals showed glucose intolerance and impaired insulin secretion. When crossed with BACE2-deficient mice, the animals presented a significant improvement in glucose tolerance accompanied with an enhanced insulin secretion, as compared to hIAPP-Tg mice. BACE2 deficiency also partially reverted gene expression changes observed in islets from hIAPP-Tg mice, including a set of genes related to inflammation. Moreover, homozygous hIAPP mice presented a severe hyperglycemia and a high lethality rate from 8 weeks onwards due to a massive destruction of β-cell mass. This process was significantly reduced when crossed with the BACE2-KO model, improving the survival rate of the animals. Altogether, the absence of BACE2 ameliorates glucose tolerance defects induced by IAPP overexpression in the β-cell and promotes β-cell survival. Thus, targeting BACE2 may represent a promising therapeutic strategy to improve β-cell function in T2D.

Keywords: BACE activity; Glucose tolerance; Islet inflammation; Proliferation; Survival; Type 2 diabetes.

Fig. 1

Metabolic phenotype of transgenic animals. a–c Intraperitoneal glucose tolerance test (ipGTT) was performed at 10 weeks of age. Plasma glucose levels (mg/dL) at indicated times (a) and area under the curve (b) are shown. c Absolute plasma insulin levels at 0 and 15 min after glucose injection. Results are presented as mean ± SEM. n = 7 animals per group. a **p < 0.01 and ***p < 0.001 relative to WT; ^### p < 0.001 relative to hIAPP-TgxBACE2-KO

Fig. 2

Pancreatic islet insulin and IAPP secretion. Mouse islets were isolated from the pancreata of 12-week-old animals, cultured overnight and subjected to glucose-stimulated insulin and IAPP secretion. a % of insulin secretion respect to insulin content. b Fold increase of insulin release at high glucose (HG, 16.7 mM glucose) vs. low glucose (LG, 2.8 mM glucose). c % of IAPP secretion respect to IAPP content. b Fold increase of IAPP release at high glucose (HG, 16.7 mM glucose) vs. low glucose (LG, 2.8 mM glucose). Results are presented as mean ± SEM. n = 3 in triplicate batches of ten islets. *p < 0.05

Fig. 3

Pancreas morphometry and β-cell proliferation of transgenic animals. β-Cell mass quantified in mg as β-cell volume density, multiplied by pancreas weight (a) and profile of islet size distribution (b). c Representative images of insulin (green) and glucagon (red) immunostaining from whole pancreas slices with differentiating β-cell area between the hIAPP-Tg and the rest of the experimental groups. Nuclei stained with Hoechst (blue). Bar scale 100 µm. Six sections per animal and three animals per group. *p < 0.05 and **p < 0.01. d β-Cell proliferation was analysed in transgenic and WT islets from 12-week-old mice. The percentage of proliferating β-cells was calculated as the percentage (%) of Ki67 positive β-cells with respect to total β-cells. Islet distribution according to the percentage of proliferating β-cells is shown. e Representative images of insulin (red) and Ki67 (green) immunostaining from representative islets with differentiated proliferation between the hIAPP-Tg and the other groups analysed in c. Nuclei stained with Hoechst (blue). Yellow arrows point to Ki67-positive nuclei. Bar scale 100 µm. Six sections per animal and three animals per group. f β-Cell proliferation was analysed in hIAPP-Tg and WT islets from 20-week-old mice. The percentage of proliferating β-cells was calculated as the percentage (%) of Ki67-positive β-cells with respect to total β-cells. Islet distribution according to the percentage of proliferating β-cells is shown. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01

Fig. 4

Global gene expression profile of pancreatic islets. a Heatmap of differentially expressed genes in pancreatic islets of the four experimental groups (highest expression in red and lowest expression in blue). b Functional category analysis of modulated pathways in hIAPP-Tg pancreatic islets in relation to WT islets. The table shows representative genes of each category with the fold induction of hIAPP-Tg vs. WT, and the reversion of hIAPP-TgxBACE2-KO vs. hIAPP-Tg. Results from global gene expression profiling data and DAVID Functional Annotation Tool. n = 3 animals per group

Fig. 5

Lower inflammation profile in hIAPP-TgxBACE2-KO islets. Fold induction of mRNA expression (related to WT animals) of selected genes in pancreatic islets from the four experimental groups. Chop (a) and Tgfbi (b) gene expression were not significantly modulated in any group. Gene expression of Ccl2 inflammation marker was significantly enhanced in hIAPP-Tg islets, but the increase was reversed to control values in hIAPP-TgxBACE2-KO animals (c). Cytokine Il-1β (d) was not significantly modulated in any model, while the inflammation-related gene Txnip (e) and the macrophage activation markers Lyz1 (f) and Mpeg1 (g) were increased in hIAPP-Tg islets and reverted in hIAPP-TgxBACE2-KO islets. Tbp-1 expression was used as a housekeeping gene. h. CD68 protein expression was quantified by immunostaining in whole pancreas slices. Results are represented as CD68-positive spots inside the pancreatic islet corrected by the total number of islets. Bars are the mean of triplicates from at least three independent experiments ±SEM. *p < 0.05

Fig. 6

Homozygous hIAPP-Tg animals. a Kaplan–Meier curves for mouse survival. Hemizygous hIAPP-Tg^+/− mice were taken as controls. hIAPP-Tg^+/+xBACE2^−/− presented a higher survival rate than hIAPP-Tg^+/+BACE2^+/+. b Follow-up of fed plasma glucose levels in the three experimental groups. All curves were started with at least 20 animals per group. c Representative images of insulin (green) and glucagon (red) immunostaining from whole pancreas slices with differentiating β-cell area between the hIAPP-Tg^+/+ and hIAPP-Tg^+/+xBACE2^−/−. Nuclei stained with Hoechst (blue). Bar scale 50 µm