High glucose activates the alternative ACE2/Ang-(1-7)/Mas and APN/Ang IV/IRAP RAS axes in pancreatic β-cells

Abstract

The activation of the classical angiotensin (Ang)-converting enzyme (ACE)/Ang II/Ang II type 1 receptor (AT1R) axis of the renin-angiotensin system (RAS) has been associated with islet dysfunction and insulin resistance. Hyperglycaemia, hypertension and obesity, major components of metabolic syndrome, are all associated with increased systemic and tissue levels of Ang II. Whereas it is well established that Ang II, by binding to AT1R, impairs glucose-stimulated insulin secretion and insulin signaling, the contribution of alternative RAS axes to β-cell function remains to be fully elucidated. In this study, using the BRIN-BD11 rat insulinoma cell line, we i) examined the basal expression levels of components of classical and alternative RAS axes and ii) investigated the effects of normal (5.5 mM) and elevated (11, 15, 25 mM) glucose concentrations on their expression and/or enzymatic activity by means of reverse transcription quantitative PCR (RT-qPCR), immunoblot analysis and enzymatic activity assays. The results correlated with the insulin production and release. Essential components of all RAS axes were found to be expressed in the BRIN-BD11 cells. Components of the alternative RAS axes, ACE2, neutral endopeptidase 24.11, Mas receptor (Mas), aminopeptidases A (APA) and N (APN) and insulin-regulated aminopeptidase (IRAP) showed an increased expression/activity in response to high glucose. These alterations were paralleled by the glucose-dependent increase in insulin production and release. By contrast, components of the classical RAS axis, ACE, AT1R and Ang II type 2 receptor (AT2R), remained largely unaffected under these conditions. Glucose induced the activation of the alternative ACE2/Ang-(1-7)/Mas and APN/Ang IV/IRAP RAS axes simultaneously with the stimulation of insulin production/release. Our data suggest the existence of a functional link between the local RAS axis and pancreatic β-cell function; however, further studies are required to confirm this hypothesis.

Figure 1

Schematic illustration of RAS consisting of the classical Ang II/ACE/AT1R axis and AT2R, as well as the two alternative axes, ACE2/Ang-( 1 - 7 )/Mas and APN/Ang IV/IRAP. RAS, renin angiotensin system; AGT, angiotensinogen; Ang, angiotensin; AT1bR, angiotensin II type 1b receptor; AT2R, angiotensin II type 2 receptor; ACE, angiotensin-converting enzyme; NEP, neutral endopeptidase 24.11; PEP, prolyl endopeptidase; Mas, Mas receptor; APA/APN, aminopeptidases A and N; IRAP, insulin-regulated aminopeptidase.

Figure 2

Glucose increases (A) insulin secretion and the mRNA levels of (B) insulin 1 and (C) insulin 2 in a dose-dependent manner in BRIN-BD11 cells. (A) Insulin levels were quantified in the supernatants by ELISA 20 min after stimulation. Data are presented as the means + SEM of 13 independent experiments. 100% = 0.13 μg/l insulin [t-test ^** P<0.01 vs. control (Co.)]. (B) mRNA levels were quantified by quantitative PCR after 24 h of incubation. Data were evaluated and normalized to Rpl13a mRNA expression using the ΔΔ Cq method. Data are presented as box plots with medians, quartiles and an interquartile range (IQR) ± 1.5 × IQR of 5 independent experiments [Mann-Whitney ^** P<0.01 vs. control (Co.)]. ELISA, enzyme-linked immunosorbent assay.

Figure 3

BRIN-BD11 cells express essential components of the renin-angiotensin system. PCR products were visualized by agarose gel electrophoresis and RedSafe™ staining. The size of amplificates is given in base pairs (bp). AT1bR, angiotensin II type 1b receptor; AT2R, angiotensin II type 2 receptor; ACE, angiotensin-converting enzyme; NEP, neutral endopeptidase 24.11; Mas, Mas receptor; APA/APN, aminopeptidases A and N; IRAP, insulin-regulated aminopeptidase.

Figure 4

Effects of glucose on the mRNA expression of renin-angiotensin system (RAS) components. Elevated concentrations of glucose did not alter the mRNA levels of (A) angiotensin-converting enzyme (ACE), (B) angiotensin II type 1b receptor (AT1bR), or (C) angiotensin II type 2 receptor (AT2R) mRNA levels in BRIN-BD11 cells after 24 h, whereas the levels of (D) ACE2, (E) neutral endopeptidase 24.11 (NEP), (F) Mas, (G) aminopeptidase A (APA), (H) aminopeptidase N (APN) and (I) insulin-regulated aminopeptidase (IRAP) were dose-dependently increased. mRNA levels were determined by quantitative PCR. Data were evaluated and normalized to Rpl13a mRNA expression using the ΔΔ Cq method. Data are presented as box plots with medians, quartiles and an interquartile range (IQR) ± 1.5 × IQR of 5 independent experiments [Mann-Whitney ^* P<0.05, ^** P<0.01, vs. control (Co.)].

Figure 5

Effects of glucose on protein expression of renin-angiotensin system (RAS) components. Glucose increased the levels of (A) angiotensin-converting enzyme (ACE)2, (B) neutral endopeptidase 24.11 (NEP), (C) Mas receptor, (D) aminopeptidase A (APA), (E) aminopeptidase N (APN) and (F) insulin-regulated aminopeptidase (IRAP) protein in BRIN-BD11 after 24 h. Twenty micrograms of total protein (40 μg for Mas) were separated by SDS-PAGE and visualized by western blot analysis. Expression data were quantified densitometrically and normalized to actin or RPLP0. Shown is 1 representative western blot out of 4 and the related box plots with medians, quartiles and an interquartile range (IQR) ± 1.5 × IQR, respectively [Mann-Whitney ^** P<0.01, ^* P<0.05, vs. control (Co.)].

Figure 6

Effects of glucose on enzymatic activities of renin-angiotensin system (RAS) proteases. Exposure of BRIN-BD11 cells to elevated concentrations of glucose led to increased enzymatic activities of (A) angiotensin-converting enzyme (ACE)2, (B) aminopeptidase A (APA), (C) aminopeptidase N (APN) and (D) insulin-regulated aminopeptidase (IRAP) after 24 h. Protease activities were determined by the incubation of viable cells with chromogenic or fluorogenic (ACE2) substrates. The activities were calculated per one million cells. Illustrated are boxplots with medians, quartiles and an interquartile range (IQR) ± 1.5 × IQR of 4 independent experiments [Mann-Whitney ^** P<0.01, ^* P<0.05, vs. control (Co.)].