ACE2 deficiency reduces β-cell mass and impairs β-cell proliferation in obese C57BL/6 mice
Abstract
Drugs that inhibit the renin-angiotensin system (RAS) decrease the onset of type 2 diabetes (T2D). Pancreatic islets express RAS components, including angiotensin-converting enzyme 2 (ACE2), which cleaves angiotensin II (Ang II) to angiotensin-(1–7) [Ang-(1–7)]. Overexpression of ACE2 in pancreas of diabetic mice improved glucose homeostasis. The purpose of this study was to determine if deficiency of endogenous ACE2 contributes to islet dysfunction and T2D. We hypothesized that ACE2 deficiency potentiates the decline in β-cell function and augments the development of diet-induced T2D. Male Ace2+/y or Ace2−/y mice were fed a low-fat (LF) or high-fat (HF) diet for 1 or 4 mo. A subset of 1-mo HF-fed mice were infused with Sal (Sal), losartan (Los), or Ang-(1–7). At 4 mo, while both genotypes of HF-fed mice developed a similar level of insulin resistance, adaptive hyperinsulinemia was reduced in Ace2−/y vs. Ace2+/y mice. Similarly, in vivo glucose-stimulated insulin secretion (GSIS) was reduced in 1-mo HF-fed Ace2−/y compared with Ace2+/y mice, resulting in augmented hyperglycemia. The average islet area was significantly smaller in both LF- and HF-fed Ace2−/y vs. Ace2+/y mice. Additionally, β-cell mass and proliferation were reduced significantly in HF-fed Ace2−/y vs. Ace2+/y mice. Neither infusion of Los nor Ang-(1–7) was able to correct impaired in vivo GSIS of HF-fed ACE2-deficient mice. These results demonstrate a critical role for endogenous ACE2 in the adaptive β-cell hyperinsulinemic response to HF feeding through regulation of β-cell proliferation and growth.
drugs that inhibit the renin-angiotensin system (RAS) have been shown to delay the onset of type 2 diabetes (T2D) (34, 51, 58). Efficacy of RAS blockade in T2D may relate to improvements in insulin sensitivity or β-cell function during the adaptive phase of hyperinsulinemia. Several components of the RAS are present in rodent and human pancreatic islets (12, 29, 48), including the monocarboxypeptidase angiotensin-converting enzyme 2 (ACE2), which cleaves angiotensin II (Ang II) to generate angiotensin-(1–7) (Ang-1-7) (16). Local pancreatic ACE2 may control Ang II levels at islets and serve as an endogenous mechanism to limit RAS activity. Indeed, recent studies suggest that ACE2 is a positive regulator of pancreatic function (13, 23), as mice with whole body deficiency of ACE2 became progressively glucose intolerant due to impairments in insulin secretion (35). Moreover, in mice fed a high-fat (HF) diet, whole body ACE2 deficiency deteriorated islet function through a mechanism involving impairment of islet microvasculature (57). Conversely, adenoviral overexpression of ACE2 in the pancreas of db/db mice improved glycemic control through Ang-(1–7) effects at Mas receptors (6). Notably, overexpression of ACE2 in pancreas increased β-cell proliferation and reduced β-cell apoptosis in 8-wk-old db/db mice but had no effect in 16-wk-old db/db mice when glycemia was severely impaired. These findings suggest that ACE2 may play a role in the adaptive β-cell hyperinsulinemic phase of T2D.
Previously, we (8) demonstrated that plasma and tissue concentrations of Ang II are increased in glucose-intolerant mice with HF diet-induced obesity. Moreover, our previous studies suggested that deficits in ACE2 may contribute to an increase in the balance of Ang II vs. Ang-(1–7) in obese mice (22). In this study, we hypothesized that deficiency of ACE2 impairs the adaptive β-cell hyperinsulinemic response to HF diet in the progression to T2D. We first quantified plasma insulin secretion in wild-type and ACE2-deficient mice chronically fed a low-fat (LF) or HF diet. Since deficiency of ACE2 could influence pancreatic function either by increasing Ang II (and AT1R functions) or by reducing Ang-(1–7), we defined whether initial in vivo deficits in glucose-stimulated insulin secretion (GSIS) of HF-fed ACE2-deficient mice could be prevented by AT1R blockade or by Ang-(1–7). Finally, we focused on the regulation of β-cell mass in HF-fed ACE2-deficient mice as a mechanism for deficits in the adaptive hyperinsulinemic response.
MATERIALS AND METHODS
Experimental animals.
All studies were conducted according to National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Kentucky Institutional Animal Care and Use Committee. Male Ace2+/y and Ace2−/y littermates on a C57BL/6 background (2 mo of age; bred from male Ace2−/y and female Ace2+/− breeding pairs) were fed either LF (10% kcal as fat, D12450B; Research Diets, New Brunswick, NJ) or HF diets (60% kcal as fat, D12492; Research Diets) ad libitum with free access to water for 5 wk (n = 5–6 per group) or 17 wk (n = 5–11 per group). We focused on male mice in these studies since clinical trials do not indicate sexual dimorphism of T2D (17, 36, 38) and since there is no evidence that localization of ACE2 to the X chromosome results in gene dosage effects (30, 55). In a separate study, male Ace2+/y and Ace2−/y mice (2 mo of age; n = 12–16 per group) were infused via osmotic minipump (Alzet, model 2006) with losartan (Los; 25 μg·kg−1·min−1, Merck, Whitehouse Station, NJ) (31), Ang-(1–7) (0.4 μg·kg−1·min−1, Bachem, Torrence, CA) (50), or saline (Sal) and fed a HF diet for 28 days. Plasma Ang-(1-7) concentrations were quantified by ELISA using a commercial kit (Peninsula Laboratories, San Carlos, CA). At study end point, mice were anesthetized with ketamine-xylazine (100/10 mg/kg) for exsanguination and tissue harvest.
Glucose tolerance, insulin tolerance, and plasma glucose/insulin quantification.
For glucose tolerance tests, mice were fasted for 6 h, starting at 7 AM. Blood glucose concentrations were quantified using a glucometer (Freedom Freestyle Lite; Abbott Laboratories, Abbott Park, IL) immediately before and 15, 30, 60, 90, and 120 min following intraperitoneal (ip) administration of glucose (2 g/kg body wt). Insulin tolerance was assessed following a 4-h fast, starting at 7 AM, by quantifying blood glucose concentrations at 0, 30, 60, and 90 min after administration of human insulin (Regular, 0.5 U/kg body wt ip; Novo Nordisk, Princeton, NJ). Both glucose tolerance and insulin tolerance are expressed as area under the curve (AUC). Plasma insulin and glucose concentrations were quantified from blood samples (10–15 μl) collected via repeated tail vein stick in conscious mice (n = 5 mice per group) following a 6-h fast, started at 7 AM, and again after ip administration of glucose. A glucometer was used to quantify glucose concentrations in plasma samples, and plasma insulin concentrations were quantified by ELISA using a commercial kit (Crystal Chem, Downers Grove, IL). The homeostatic model of assessment of insulin resistance (HOMA-IR) and β-cell function (HOMA-β) were calculated using the following formulas, respectively: fasting plasma glucose (mmol/l) × fasting insulin (μU/ml)/22.5, and [fasting insulin (μU/ml) × 20]/[fasting plasma glucose (mmol/l) − 3.5] × 100 (33).
Glucose-stimulated insulin secretion from isolated pancreatic islets.
The method for islet isolation was adapted from published protocols (11, 47). Pancreata were perfused in vivo through the common bile duct with 5 ml of collagenase (0.6 mg/ml, collagenase P; Roche, Indianapolis, IN) immediately following exsanguination. The excised pancreata were incubated in 10 ml of collagenase at 37°C for 15–20 min, and the tissue was then mechanically separated. Islets were purified from exocrine tissue using a sucrose gradient (Histopaque 1077; Sigma-Aldrich, St. Louis, MO), allowed to recover in culture medium (RPMI, 10% FBS, 1% penicillin-streptomycin) for 48 h, and further purified from exocrine tissue and debris by handpicking with a pipette and transferring to fresh medium. Purified islets (20–25) were placed in inserts in 12-well plates (Greiner Bio One, Monroe, NC) and incubated in Krebs buffer with 3 mM glucose for 1 h at 37°C. The inserts containing the islets were then transferred to Krebs buffer with 28 mM glucose and incubated for 1 h at 37°C. Insulin concentrations in media and in islets (harvested in lysis buffer) were quantified by ELISA and normalized to protein content of islets (BCA assay; ThermoFisher, Rockford IL).
Determination of pAkt/Akt.
A subset of male Ace2+/y and Ace2−/y mice (n = 4–6 per group) infused via osmotic minipump with Los Ang-(1–7), or saline and fed a HF diet for 28 days were fasted for 4 h and injected with 10 U/kg body wt insulin 15 min prior to anesthetization and exsanguination. Liver, solei, and subcutaneous adipose tissue were dissected and frozen in liquid nitrogen. Phosphorylated Akt and total Akt were assessed using a commercial ELISA kit (Cell Signaling, Danvers, MA).
Immunohistochemistry and immunofluorescence.
Mouse pancreata were washed in PBS, fixed in 10% formalin overnight at 4°C, dehydrated in grades of ethanol, and paraffin embedded. Starting at a depth of 400 μm, five longitudinal sections (5 μm thick) were prepared every 100 μm. Sections were deparaffinized, rehydrated in alcohol, and subjected to antigen retrieval (steam; Vector Labs Antigen Retrieval Unmasking Solution). For β-cell mass and morphometric analysis, sections were incubated with rabbit anti-insulin antibody (1:100, Abcam ab63820, Cambridge, MA) followed by incubation with biotinylated anti-rabbit secondary antibody (1:200; Vector Labs, Burlingame, CA) at 40°C for 30 min. A streptavidin-based ABC system and peroxidase-based red chromagen AEC (both from Vector Labs) were used to identify the antigen-antibody reactions, after which sections were counterstained with hematoxylin. β-Cell proliferation was assessed in sections subjected to antigen retrieval in 10 mM Tris-1 mM EDTA-0.05% Tween 20 buffer with pH 9 followed by permeabilization with 0.05% Triton X-100. Sections were incubated for 20 h at 4°C with rabbit anti-Ki67 (Abcam ab15580, 1:150) and guinea pig anti-insulin (1:500, Dako A056401-2, Carpinteria, CA) in 0.1% Triton X-100 followed by incubation with goat anti-rabbit Alexa fluor 488 and goat anti-guinea pig Alexa fluor 594 in PBS for 30 min at 40°C and then mounted with DAPI. Isotype-matched IgG was used as controls, as was omission of primary and/or secondary antibodies. Images were captured with a Nikon Eclipse 80i microscope, and analysis was performed using NIS Elements software (Nikon Instruments, Japan). A commercial kit for measuring cell death by fluorescein detection of TUNEL staining was used to measure β-cell apoptosis (Roche, Indianapolis, IN).
Analysis and quantification of β-cell mass, islet size, proliferation, and apoptosis.
For determination of β-cell mass, an entire pancreas tissue section was imaged at ×4 under brightfield (5 sections per mouse, ∼120 μm apart, n = 3 mice per group). The total pancreas tissue area and the total insulin-positive (β-cell) area were selected for each image, and β-cell mass is reported as the mean over five sections of the ratio of insulin-stained area to total pancreas area per mouse. Three 3 × 3-mm fields from two sections per animal ∼500 μm apart (imaged at ×10) were used to obtain the average islet area/section (calculated from individual islet areas); data are reported as the mean islet area per mouse. β-Cell proliferation was determined by counting the number of nuclei positive for Ki67 within insulin-positive cells from individual islets (25–30 islets per section imaged at ×20; analyzed from 2 sections per mouse and 3 mice per group). Apoptosis in β-cells was assessed by counting the number of TUNEL-positive cells in sections double-stained for insulin (20 islets per section were imaged at ×20; 2 sections per mouse and 3 mice per group were used for analysis).
Statistical analysis.
Data are presented as means ± SE. Data were analyzed using two-way ANOVA with diet and genotype (or treatment where appropriate) as between-group factors. If statistical differences existed between experimental groups, the Holm-Sidac method was utilized for post hoc analyses. Values of P < 0.05 were considered to be statistically significant. All statistical analyses were performed using SigmaStat (SPSS) version 12.0.
RESULTS
Ace2−/y mice fed a standard diet have mild impairments in glucose tolerance and insulin secretion.
Ace2−/y mice had reduced body weight compared with controls at 8 wk of age (Fig. 1A, P < 0.01). Insulin tolerance was not different between genotypes (Fig. 1, B and C). However, during a glucose tolerance test, Ace2−/y mice exhibited increased plasma glucose concentrations at 15 and 30 min compared with controls (Fig. 1D, P < 0.05). Modest elevations in plasma glucose concentrations did not influence the AUC for plasma glucose concentrations (Fig. 1E). Notably, plasma insulin concentrations were significantly lower in Ace2−/y compared with Ace2+/y mice following a 6-h fast (time 0, Fig. 1F; P < 0.05) and at 30 min post-glucose administration (Fig. 1F, P < 0.05).
Fig. 1.Angiotensin-converting enzyme 2-deficient (Ace2−/y) mice fed standard diet exhibit impaired glucose tolerance associated with reductions of in vivo glucose-stimulated insulin secretion (GSIS). A: body weights of 8-wk-old Ace2+/y and Ace2−/y mice. *P < 0.01. B: insulin tolerance test. C: area under the curve (AUC). Glucose tolerance test (D) and AUC (E) in Ace2+/y and Ace2−/y mice. #P < 0.05 vs. Ace2+/y within time point. F: plasma insulin concentrations in Ace2+/y and Ace2−/y mice following a 6-h fast (time 0) and at 30 min after glucose administration. #P < 0.05 vs. Ace2+/y within time point. Data are means ± SE; n = 5–11 mice/group.
Plasma insulin concentrations are reduced in hyperglycemic Ace2−/y mice chronically fed a HF diet.
We examined effects of chronic (4 mo) HF feeding on glucose homeostasis and the adaptive hyperinsulinemic response in Ace2+/y and Ace2−/y mice. Initial body weights of Ace2−/y mice were lower than controls', resulting in a modest reduction in body weights of LF-fed Ace2−/y mice at study end point (Fig. 2A, P < 0.05). HF-fed mice of each genotype had significantly increased body weights compared with LF-fed controls, with no differences between genotypes (Fig. 2A, P < 0.001). Insulin tolerance was significantly impaired in obese mice of each genotype compared with LF-fed controls (Fig. 2, B and C, P < 0.01). Additionally, HF-fed Ace2−/y mice became insulin resistant to the same degree as control mice. Fasting plasma insulin concentrations were significantly reduced in Ace2−/y mice compared with controls as early as 1 mo of HF feeding, with marked reductions in plasma insulin concentrations in 4 mo HF-fed Ace2−/y mice (Fig. 2D, P < 0.05). Although there were no significant differences in fasting plasma glucose concentrations between HF-fed Ace2+/y and Ace2−/y mice at 4 mo (Fig. 2E), nonfasted plasma glucose concentrations were significantly elevated in HF-fed Ace2−/y mice compared with controls (Fig. 2F, P < 0.05).
Fig. 2.Ace2−/y high-fat diet (HF)-fed mice exhibit diminished adaptive hyperinsulinemia. Ace2+/y or Ace2−/y mice were fed a HF diet for 17 wk. A: body weight progression. *P < 0.001, effect of diet; #P < 0.05, effect of genotype. B and C: insulin tolerance test and corresponding AUC. *P < 0.01 vs. low-fat (LF) diet. D: fasting plasma insulin concentrations. *P < 0.01 vs. LF within genotype; #P < 0.05 vs. Ace2+/y within time point. Fasted (E) and nonfasted plasma glucose (F) in LF and HF-fed mice. *P < 0.01 vs. LF within genotype; #P < 0.05 vs. Ace2+/y within diet group. Data are means ± SE; n = 5–11 mice/group.
Ace2−/y mice have impaired in vivo GSIS after 1 mo of HF feeding.
Since plasma insulin concentrations were reduced in obese Ace2−/y mice compared with controls after 1 mo of HF feeding, we sought to determine whether β-cell dysfunction was also present at 1 mo of HF feeding in Ace2−/y mice. Both groups of mice developed hyperinsulinemia, hyperglycemia, and insulin resistance (Fig. 3, A–D;, P < 0.01). HOMA-IR was significantly increased in HF-fed Ace2+/y and Ace2−/y mice (3.5- and 2.5-fold, respectively, P < 0.001) compared with LF-fed controls, but there was no significant difference between genotypes (LF, Ace2+/y: 15.5 ± 5.7; LF, Ace2−/y: 12.8 ± 5.7; HF, Ace2+/y: 45.7 ± 4.3; HF, Ace2−/y: 36.4 ± 5.7). However, plasma insulin concentrations were significantly lower in HF-fed Ace2−/y mice following a 6-h fast (time 0, Fig. 3A; P < 0.05) and at 30 and 60 min following intraperitoneal glucose administration compared with Ace2+/y controls (Fig. 3A, P < 0.05). Moreover, plasma glucose concentrations were slightly elevated in Ace2−/y mice of both genotypes compared with wild-type controls (time 0), and this effect was significant at 30 min following glucose administration (Fig. 3B, P < 0.05). Analysis of HOMA-β indicated that β-cell function was increased in both genotypes with HF feeding (LF, Ace2+/y: 20.1 ± 2.1%; LF, Ace2−/y: 14.6 ± 2.1%; HF, Ace2+/y: 30.2 ± 1.7%; HF, Ace2−/y: 19.6 ± 2.3%; P < 0.01). However, HOMA-β was significantly decreased in HF-fed Ace2−/y mice compared with Ace2+/y mice (34 vs. 50% increase with HF feeding, respectively, P < 0.01).
Fig. 3.Ace2−/y mice have impaired in vivo GSIS after 1 mo of HF-feeding. Plasma insulin concentrations (A) and corresponding plasma glucose concentrations (B) following administration of glucose (2 g/kg body wt) in LF- and HF-fed Ace2+/y and Ace2−/y mice. *P < 0.01 vs. LF within time point; #P < 0.05 vs. Ace2+/y within time point. Insulin tolerance test (C) and corresponding AUC (D) in LF- and HF-fed Ace2+/y and Ace2−/y mice. *P < 0.01 vs. LF. E: insulin release from pancreatic islets isolated from LF- and HF-fed Ace2+/y and Ace2−/y mice (n = 20 islets per mouse) at low (3 mM) and high (28 mM) glucose concentrations. *P < 0.05 vs. LF within treatment. F: insulin content of pancreatic islets isolated from LF- and HF-fed Ace2+/y and Ace2−/y mice. Data are means ± SE; n = 5–6 mice/group.
To determine whether deficits of in vivo GSIS of Ace2−/y mice resulted from impaired insulin secretion from pancreatic islets, we quantified GSIS from isolated pancreatic islets from mice of each genotype and diet. Under high-glucose conditions, islets from HF-fed mice of each genotype exhibited significantly increased GSIS compared with islets from LF-fed mice (Fig, 3E, P < 0.05). However, there were no significant differences in insulin secretion from islets of HF-fed mice of either genotype. Furthermore, there was no difference in insulin content of islets isolated from HF-fed Ace2+/y or Ace2−/y mice (Fig. 3F).
Infusion of neither an AT1R antagonist nor of Ang-(1–7) restores in vivo deficits of GSIS in HF-fed Ace2−/y mice.
Since ACE2 cleaves Ang II to Ang-(1–7), we determined if antagonism of Ang II effects at AT1R (by infusion of Los), or infusion of Ang-(1–7) to restore plasma peptide concentrations in HF-fed (1 mo) mice with ACE2 deficiency would reverse in vivo deficits of GSIS. Infusion of Los had no effect on plasma concentrations of Ang-(1–7) in Ace2+y or Ace2−y mice (Fig. 4A). However, infusion of Ang-(1–7) significantly increased plasma Ang-(1–7) concentrations in both genotypes, with no differences between genotypes (Fig. 4A).
Fig. 4.Neither AT1R antagonism nor infusion of Ang-(1–7) restore in vivo deficits in GSIS of HF-fed Ace2−/y mice. A: infusion of Ang-(1–7) for 1 mo increased plasma Ang-(1–7) concentrations in Ace2+/y and Ace2−/y HF-fed mice vs. mice administered saline (Sal). Infusion of losartan (Los) had no effect on plasma Ang-(1–7) levels. *P < 0.05, overall effect of Ang-(1–7). B: glucose tolerance test in 1-mo HF-fed Ace2+/y and Ace2−/y mice administered Sal, Los, or Ang-(1–7). *P < 0.05, overall effect of Los. C: fasting blood glucose concentrations in mice of each genotype and treatment group. *P < 0.05, overall effect of Los; #P < 0.05, overall effect vs. Ace2+/y. D: plasma insulin concentrations quantified at 60 min following glucose administration. #P < 0.05, overall effect vs. Ace2+/y. E and F: ratio of pAkt to Akt in soleus muscle and subcutaneous (SubQ) adipose tissue, respectively, from Ace2+/y and Ace2−/y mice infused with Sal, Los, or Ang-(1–7). *P < 0.05, overall effect of Los. Data are means ± SE; n = 5–8 mice/group.
Administration of Los significantly improved glucose tolerance compared with Sal in mice of each genotype, with no differences between genotypes (Fig. 4B, P < 0.05). However, Los administration significantly decreased fasting blood glucose concentrations in Ace2+/y but not in Ace2−/y HF-fed mice (Fig. 4C, P < 0.05). Moreover, fasting blood glucose concentrations were significantly increased in Ace2−/y mice regardless of treatment group compared with control (Fig. 4C, P < 0.05). As described above (Fig. 3A), plasma insulin concentrations following in vivo glucose administration were significantly lower in HF-fed Ace2−/y compared with Ace2+/y mice (Sal groups, Fig. 4D; P < 0.05). Los administration was unable to restore plasma insulin concentrations in HF-fed Ace2−/y mice to the level of controls (Sal groups, Fig. 4D). Since Los administration improved glucose tolerance but had no effect on plasma insulin concentrations in mice of either genotype, we quantified tissue (skeletal muscle, adipose) concentrations of pAkt as an index of insulin sensitivity. Los administration significantly increased the ratio of pAkt to Akt in soleus muscle and subcutaneous adipose tissue of both genotypes compared with Sal controls, with no differences between genotypes (Fig. 4, E and F, P < 0.05).
Despite significant elevations in plasma Ang-(1–7) concentrations in mice infused with the peptide (Fig. 4A), Ang-(1–7) had no effect on glucose tolerance (Fig. 4B) or fasting blood glucose concentrations (Fig. 4C) in mice of either genotype. Moreover, infusion of Ang-(1–7) had no effect on plasma insulin concentrations in Ace2+/y or Ace2−/y HF-fed mice (Fig. 4D).
HF-fed Ace2−/y mice have reduced islet size and β-cell mass.
To define mechanisms for in vivo deficits in GSIS of HF-fed Ace2−/y mice, we quantified average islet area and β-cell area (mass) in pancreatic sections from 1-mo LF- and HF-fed mice of each genotype (representative images in Fig. 5A). In LF-fed mice, average islet area and β-cell mass were significantly decreased in Ace2−/y compared with Ace2+/y mice (Fig. 5, B and C, P < 0.05). HF feeding significantly increased β-cell mass in both genotypes (Fig. 5, A and B, P < 0.05). However, the magnitude of increase in β-cell mass was greater in Ace2+/y (44%) than in Ace2−/y (30%) mice. Moreover, average islet area and β-cell mass were significantly reduced in HF-fed Ace2−/y compared with Ace2+/y mice (Fig. 5, B and C, P < 0.05).
Fig. 5.HF-fed Ace2−/y mice have reduced islet size and β-cell mass. A: representative 3 × 3-mm fields from pancreas tissue sections immunostained with anti-insulin antibody (red) in 1-mo LF- and HF-fed Ace2+/y and Ace2−/y mice. B: average islet area in LF- and HF-fed mice of each genotype. #P < 0.01 vs. Ace2+/y within diet group. C: β-cell mass as determined by %insulin-positive (β-cell) area per total area of pancreatic sections in LF- and HF-fed mice of each genotype. *P < 0.05 vs. LF within genotype; #P < 0.05 vs. Ace2+/y within diet group. Data are means ± SE; n = 3 mice/group.
β-Cell proliferation is decreased in HF-fed Ace2−/y mice.
To define mechanisms for reductions in β-cell mass of HF-fed Ace2−/y mice, we measured β-cell proliferation by analysis of pancreatic sections from 1-mo LF- and HF-fed mice of each genotype double-stained for insulin (representative images, Fig. 6A, top) and Ki67 (respresentative images, Fig. 6A, middle). β-Cell proliferation was quantified by counting the number of insulin-positive cells (β-cells) that were positive for Ki67 (representative images, Fig. 6A, bottom). In HF-fed mice of each genotype, β-cell proliferation was significantly increased compared with LF controls (quantification of immunostaining in Fig. 6B, P < 0.001). However, the magnitude of increase in β-cell proliferation with HF feeding was more pronounced in Ace2+y (1.6-fold increase) than in Ace2−/y mice (<1-fold). β-Cell proliferation was decreased significantly in pancreatic sections from both LF- and HF-fed Ace2−/y compared with Ace2+/y mice (Fig. 6B, P < 0.01). To assess whether apoptosis contributed to decreased β-cell mass of HF-fed Ace2−/y mice, TUNEL staining was performed on pancreatic sections. Apoptosis was minimal in islets of LF or HF mice of either genotype, and there was no difference in the number of TUNEL-positive islets between groups (data not shown).
Fig. 6.β-Cell islet proliferation with HF feeding is reduced in Ace2−/y mice. A: representative immunofluorescence images in islets from HF-fed Ace2+/y and Ace2−/y mice at ×20 insulin (red), Ki67 (green), and DAPI staining of nuclei (blue, bottom). β-Cells positive for Ki67 with and without nuclei staining are illustrated in the bottom two panels. B: quantification of number of β-cells with Ki67-positive nuclei per islet in LF- and HF-fed mice of each genotype. *P < 0.001 vs. LF within genotype; #P < 0.05 vs. Ace2+/y within diet group. Data are means ± SE; n = 3 mice/group.
DISCUSSION
It has previously been reported that ACE2 deficiency is associated with altered glucose homeostasis (4, 35), but mechanisms for this effect have not been extensively investigated. Our findings provide new insight into the importance of ACE2 in the adaptive β-cell hyperinsulinemic response to insulin resistance. The onset of T2D in humans is characterized by a decrease in insulin secretion and an inability to maintain hyperinsulinemia (40). A reduction in the insulin response to glucose, considered to be an early indicator of β-cell dysfunction, is associated with the transition from normal to impaired glucose tolerance in humans and rodents (1, 53). Our results demonstrate that HF-fed Ace2−/y mice became obese and developed insulin resistance to the same degree as controls but were not able to maintain adaptive hyperinsulinemia, resulting in hyperglycemia in the fed state. In vivo GSIS deficits of HF-fed Ace2−/y mice were not the result of impaired insulin-secretory mechanisms in pancreatic islets or reduced islet insulin content. Rather, HF-fed Ace2−/y mice exhibited reductions in β-cell mass associated with impaired proliferative capacity of islets. Finally, in vivo GSIS deficits of HF-fed Ace2−/y mice could not be overcome by AT1R blockade or by Ang-(1–7) infusions, suggesting mechanisms unrelated to angiotensin peptide balancing properties of ACE2. These results suggest that ACE2 is a critical regulator of β-cell compensatory responses to HF-induced hyperinsulinemia.
Previous studies have demonstrated that mice with ACE2 deficiency have low insulin gene expression (4) and age-dependent impaired first-phase insulin secretion when fed a standard mouse diet (35). Conversely, ACE2 overexpression improved first-phase insulin secretion in vivo in db/db mice (6). Our results agree with and extend these findings by demonstrating that modest impairments in in vivo GSIS in young Ace2−/y mice contribute to failure to maintain adaptive hyperinsulinemia in the development of T2D. Ang II has been suggested to regulate insulin secretion from islet cells of various species, with conflicting reported findings of a stimulatory effect on insulin secretion from human islets (41) vs. reductions in insulin secretion from primary mouse islets (29). Our results do not support differences in insulin-secretory mechanisms from pancreatic islets of HF-fed Ace2−/y mice as contributors to the observed deficits of in vivo GSIS, since GSIS from pancreatic islets ex vivo is unchanged with ACE2 deficiency. Several studies have demonstrated that Ang II can regulate islet blood flow and thereby influence insulin release (10, 28). Thus, ACE2 deficiency may have impaired in vivo GSIS of HF-fed mice through Ang II/AT1R-mediated reductions in islet blood flow. Since administration of Los to HF-fed Ace2−/y mice did not reverse deficits of in vivo GSIS, it is unlikely that elevated systemic or local Ang II concentrations in obese mice (22) contributed to deficits of in vivo GSIS through Ang II/AT1R-mediated regulation of islet blood flow. These findings agree with a recent study in which administration of an ACE inhibitor to ACE2-deficient mice fed a Western diet did not correct β-cell defects (4). While neither Los nor Ang-(1–7) restored impaired GSIS of HF-fed Ace2−/y mice, losartan improved insulin signaling in skeletal muscle of both genotypes, and Ang-(1–7) infusion to Ace2−/y mice resulted in modest improvements in skeletal muscle insulin receptor signaling. These results suggest that AT1R blockade or Mas receptor activation may exert beneficial effects against insulin resistance in T2D.
AT1R antagonists have been shown to improve glucose tolerance and increase insulin secretion (14, 24, 43), suggesting that Ang II acting through the AT1R contributes to the pathogenesis of T2D. Consistent with previous findings (18, 24), in this study losartan administration improved glucose tolerance in both genotypes. Our results suggest that losartan reduces insulin resistance, as evidenced by increased pAkt/Akt ratios in insulin-sensitive tissues. Similarly, ACE2 activation of the Ang-(1–7)/MasR axis increased insulin secretion in vivo (6). Specifically, infusion of Ang-(1–7) (at a 4-fold lower dose) improved insulin sensitivity in nonobese ACE2-deficient mice fed a high-sucrose diet (20). Moreover, in a rat transgenic model of insulin resistance, an oral formulation of Ang-(1–7) improved insulin sensitivity and glycemia (44). Our results demonstrate that infusion of Ang-(1–7) at a dose that elevated plasma Ang-(1–7) concentrations and blunted an activated RAS (49, 50) had no effect on glucose homeostasis in HF-fed mice of either genotype. Differences in the dose or formulation of Ang-(1–7), coupled with varying models of T2D, may have contributed to diverging effects of the peptide on glucose homeostasis.
Our results demonstrate that the compensatory response to increase β-cell mass with chronic HF feeding (15, 25) was blunted in Ace2−/y mice. Compensatory β-cell mass expansion is achieved through increases in islet size (hypertrophy) or β-cell proliferation (hyperplasia) (26, 56). In humans with T2D, a curvilinear relationship has been reported between β-cell volume and fasting blood glucose concentrations (42). Our results agree with previous findings where islet vascularization and insulin staining were reduced in chronically obese ACE2-deficient mice (57). Moreover, our results extend previous findings by demonstrating insulin deficits at 5 wk of HF feeding in Ace2−/y mice that are associated with decreased β-cell mass and proliferation. Both LF- and HF-fed Ace2−/y mice exhibited significant reductions in average islet area and β-cell mass and proliferation, which may have contributed to impaired insulin secretion (35) and loss of the adaptive hyperinsulinemic response. We found that reductions in β-cell mass of HF-fed Ace2−/y mice were attributed to deficits in proliferation rather than apoptosis, contributing to an impaired adaptive hyperinsulinemic response to metabolic challenge.
Since neither an AT1R antagonist nor infusion of Ang-(1–7) could restore deficits in in vivo GSIS of HF-fed Ace2−/y mice, it is unlikely that angiotensin peptide balance contributes to effects of ACE2 deficiency in adult mice. Alternatively, effects of ACE2 deficiency to reduce β-cell adaptation to obesity in adults may be a consequence of inadequate establishment of β-cell mass in utero as a result of maternal ACE2 deficiency. Because fetal β-cell mass is primarily established during gestation, β-cell mass is sensitive to perturbations in the intrauterine environment (9). It is well documented that intrauterine growth restriction (IUGR) increases the risk for impaired glucose homeostasis or T2D in both humans and animals (39), and IUGR in rodents is associated with reduced β-cell mass in adult offspring (21, 45). Maternal ACE2 deficiency has been reported to increase concentrations of Ang II in placenta, associated with a negative impact on both gestational body weight gain and pup weight (5). Increased placental Ang II concentrations in Ace2+/− female mice used as breeders in this study may have contributed to IUGR, negatively impacting fetal β-cell mass.
In addition to Ang II, ACE2 is also capable of hydrolyzing the peptide apelin, rendering it inactive (52). Apelin binds to the APJ receptor, which is expressed in pancreatic islets and has been demonstrated to inhibit insulin secretion from mouse islets (46). Circulating levels of apelin are reported to be increased with obesity (7). Since ACE2 is the only enzyme known to regulate apelin metabolism (27), effects of apelin to decrease insulin secretion may be exacerbated in obese mice with ACE2 deficiency. Thus, it is possible that increased actions of apelin may contribute to decreased plasma insulin concentrations of ACE2-deficient mice. Our results demonstrate reduced β-cell mass with ACE2 deficiency, suggesting that possible effects of apelin on insulin secretion are in addition to effects of ACE2 deficiency to impair β-cell proliferation. To date, no studies have investigated the role of apelin in the regulation of β-cell mass.
Little is known about the nonenzymatic functions of ACE2 independent from its role to enzymatically cleave Ang II in the generation of Ang-(1–7). ACE2 shares considerable homology with the membrane protein collectrin, but collectrin lacks an active dipeptidyl carboxypeptidase catalytic domain. Both collectrin and ACE2 are reported to be downstream targets of the transcription factor hepatocyte nuclear factor-1α (HNF-1α) (19, 37), which is mutated in maturity-onset diabetes of the young (MODY) (54). Collectrin has been demonstrated to increase insulin secretion in vitro (3) through regulation of insulin exocytosis (19). However, mixed results have been reported regarding the role of collectrin to regulate cell growth of pancreatic β-cells, as overexpression of collectrin increased β-cell mass in vivo (2) but had no effect on β-cell proliferation in vitro, while whole body collectrin deletion had no effect on β-cell mass (32). Since ACE2 deficiency had no effect on insulin secretion from isolated islets but did regulate β-cell mass, these results do not support a role for the collectrin domain of ACE2 as the mechanism for impaired adaptive hyperinsulinemia.
In summary, results demonstrate a critical role for ACE2 in the adaptive hyperinsulinemia response to insulin resistance induced by HF feeding. Reduced hyperinsulinemia of HF-fed Ace2−/y mice was detrimental, as Ace2−/y mice exhibited hyperglycemia following a glucose challenge. Moreover, insulin deficits did not appear to be related to imbalances in the RAS, as neither an AT1R antagonist nor infusion of Ang-(1–7) could reverse insulin deficits of HF-fed Ace2−/y mice; rather, ACE2 deficiency reduced the adaptive response to increase β-cell mass in HF-fed mice, associated with deficits in β-cell proliferation. These results demonstrate that ACE2 is a critical regulator of β-cell proliferation and growth and may serve as a therapeutic target for T2D.
GRANTS
This work was supported by
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: R.S. and L.A.C. conception and design of research; R.S. performed experiments; R.S. analyzed data; R.S. and L.A.C. interpreted results of experiments; R.S. prepared figures; R.S. and L.A.C. drafted manuscript; R.S., F.Y., S.E.T., and L.A.C. edited and revised manuscript; R.S., F.Y., S.E.T., and L.A.C. approved final version of manuscript.
ACKNOWLEDGMENTS
We thank the skillful technical assistance of Dr. Wendy Katz for embedding and sectioning pancreas tissue.
REFERENCES
- 1. . Insufficient islet compensation to insulin resistance vs. reduced glucose effectiveness in glucose-intolerant mice. Am J Physiol Endocrinol Metab 283: E738–E744, 2002.
Link | Web of Science | Google Scholar - 2. . Tmem27: a cleaved and shed plasma membrane protein that stimulates pancreatic beta cell proliferation. Cell Metab 2: 385–397, 2005.
Crossref | PubMed | Web of Science | Google Scholar - 3. . The role of transmembrane protein 27 (TMEM27) in islet physiology and its potential use as a beta cell mass biomarker. Diabetologia 53: 1406–1414, 2010.
Crossref | PubMed | Web of Science | Google Scholar - 4. . ACE2 deficiency shifts energy metabolism towards glucose utilization. Metabolism 64: 406–415, 2014.
Crossref | PubMed | Web of Science | Google Scholar - 5. . Angiotensin-converting enzyme 2 deficiency is associated with impaired gestational weight gain and fetal growth restriction. Hypertension 58: 852–858, 2011.
Crossref | PubMed | Web of Science | Google Scholar - 6. . Angiotensin I-converting enzyme type 2 (ACE2) gene therapy improves glycemic control in diabetic mice. Diabetes 59: 2540–2548, 2010.
Crossref | PubMed | Web of Science | Google Scholar - 7. . Apelin, a newly identified adipokine up-regulated by insulin and obesity. Endocrinology 146: 1764–1771, 2005.
Crossref | PubMed | Web of Science | Google Scholar - 8. . Activation of the systemic and adipose renin-angiotensin system in rats with diet-induced obesity and hypertension. Am J Physiol Regul Integr Comp Physiol 287: R943–R949, 2004.
Link | Web of Science | Google Scholar - 9. . Regulation of pancreatic beta-cell mass. Physiol Rev 85: 1255–1270, 2005.
Link | Web of Science | Google Scholar - 10. . Angiotensin II and the endocrine pancreas: effects on islet blood flow and insulin secretion in rats. Diabetologia 41: 127–133, 1998.
Crossref | PubMed | Web of Science | Google Scholar - 11. . A practical guide to rodent islet isolation and assessment. Biol Proced Online 11: 3–31, 2009.
Crossref | PubMed | Web of Science | Google Scholar - 12. . Evidence for an intrinsic angiotensin system in the canine pancreas. J Hypertens 9: 751–759, 1991.
Crossref | PubMed | Web of Science | Google Scholar - 13. . Pancreatic angiotensin-converting enzyme 2 improves glycemia in angiotensin II-infused mice. Am J Physiol Endocrinol Metab 304: E874–E884, 2013.
Link | Web of Science | Google Scholar - 14. . Angiotensin II type 1 receptor blockade improves beta-cell function and glucose tolerance in a mouse model of type 2 diabetes. Diabetes 55: 367–374, 2006.
Crossref | PubMed | Web of Science | Google Scholar - 15. . Progression of diet-induced diabetes in C57BL6J mice involves functional dissociation of Ca2(+) channels from secretory vesicles. Diabetes 59: 1192–1201, 2010.
Crossref | PubMed | Web of Science | Google Scholar - 16. . Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417: 822–828, 2002.
Crossref | PubMed | Web of Science | Google Scholar - 17. . Predictors of progression from impaired glucose tolerance to NIDDM: an analysis of six prospective studies. Diabetes 46: 701–710, 1997.
Crossref | PubMed | Web of Science | Google Scholar - 18. . Renin-angiotensin system blockers protect pancreatic islets against diet-induced obesity and insulin resistance in mice. PLoS One 8: e67192, 2013.
Crossref | PubMed | Web of Science | Google Scholar - 19. . The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation. Cell Metab 2: 373–384, 2005.
Crossref | PubMed | Web of Science | Google Scholar - 20. . Effect of transient TCDD exposure on immortalized human trophoblast-derived cell lines. Hum Exp Toxicol 31: 550–556, 2012.
Crossref | PubMed | Web of Science | Google Scholar - 21. . In utero undernutrition impairs rat beta-cell development. Diabetologia 40: 1231–1234, 1997.
Crossref | PubMed | Web of Science | Google Scholar - 22. . Angiotensin converting enzyme 2 contributes to sex differences in the development of obesity hypertension in C57bl/6 mice. Arterioscler Thromb Vasc Biol 32: 1392–1399, 2012.
Crossref | PubMed | Web of Science | Google Scholar - 23. . High glucose activates the alternative ACE2/Ang-(1–7)/Mas and APN/Ang IV/IRAP RAS axes in pancreatic beta-cells. Int J Mol Med 32: 795–804, 2013.
Crossref | PubMed | Web of Science | Google Scholar - 24. . Claudin-3 gene silencing with siRNA suppresses ovarian tumor growth and metastasis. Proc Natl Acad Sci USA 106: 3426–3430, 2009.
Crossref | PubMed | Web of Science | Google Scholar - 25. . Dietary-fat-induced obesity in mice results in beta cell hyperplasia but not increased insulin release: evidence for specificity of impaired beta cell adaptation. Diabetologia 48: 1350–1358, 2005.
Crossref | PubMed | Web of Science | Google Scholar - 26. . Mechanisms of compensatory beta-cell growth in insulin-resistant rats: roles of Akt kinase. Diabetes 54: 2294–2304, 2005.
Crossref | PubMed | Web of Science | Google Scholar - 27. . Apelin and ACE2 in cardiovascular disease. Curr Opin Investig Drugs 11: 273–282, 2010.
PubMed | Google Scholar - 28. . Angiotensin II type 1 receptor inhibition markedly improves the blood perfusion, oxygen tension and first phase of glucose-stimulated insulin secretion in revascularised syngeneic mouse islet grafts. Diabetologia 48: 1159–1167, 2005.
Crossref | PubMed | Web of Science | Google Scholar - 29. . Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets. Diabetologia 47: 240–248, 2004.
Crossref | PubMed | Web of Science | Google Scholar - 30. . Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17beta-oestradiol-dependent and sex chromosome-independent. Biol Sex Diff 1: 6, 2010.
Crossref | PubMed | Google Scholar - 31. . Comparative effects of different modes of renin angiotensin system inhibition on hypercholesterolaemia-induced atherosclerosis. Br J Pharmacol 165: 2000–2008, 2012.
Crossref | PubMed | Web of Science | Google Scholar - 32. . Increased insulin sensitivity in mice lacking collectrin, a downstream target of HNF-1alpha. Mol Endocrinol 23: 881–892, 2009.
Crossref | PubMed | Google Scholar - 33. . Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28: 412–419, 1985.
Crossref | PubMed | Web of Science | Google Scholar - 34. . Effect of valsartan on the incidence of diabetes and cardiovascular events. N Engl J Med 362: 1477–1490, 2010.
Crossref | PubMed | Web of Science | Google Scholar - 35. . Loss of angiotensin-converting enzyme 2 leads to impaired glucose homeostasis in mice. Endocrine 34: 56–61, 2008.
Crossref | PubMed | Web of Science | Google Scholar - 36. . Effects of diet and exercise in preventing NIDDM in people with impaired glucose tolerance. The Da Qing IGT and Diabetes Study. Diabetes Care 20: 537–544, 1997.
Crossref | PubMed | Web of Science | Google Scholar - 37. . The transcription factor HNF1alpha induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs. Biochim Biophys Acta 1829: 1225–1235, 2013.
Crossref | PubMed | Web of Science | Google Scholar - 38. . Sex differences in diabetes risk and the effect of intensive lifestyle modification in the Diabetes Prevention Program. Diabetes Care 31: 1416–1421, 2008.
Crossref | PubMed | Web of Science | Google Scholar - 39. . Early-life origins of type 2 diabetes: fetal programming of the beta-cell mass. Exp Diabet Res 2011: 105076, 2011.
Crossref | PubMed | Google Scholar - 40. . The natural history of type 2 diabetes. Implications for clinical practice. Primary Care 26: 771–789, 1999.
Google Scholar - 41. . Direct regulation of insulin secretion by angiotensin II in human islets of Langerhans. Diabetologia 49: 321–331, 2006.
Crossref | PubMed | Web of Science | Google Scholar - 42. . Relationship between beta-cell mass and fasting blood glucose concentration in humans. Diabetes Care 29: 717–718, 2006.
Crossref | PubMed | Web of Science | Google Scholar - 43. . Angiotensin receptor blockade increases pancreatic insulin secretion and decreases glucose intolerance during glucose supplementation in a model of metabolic syndrome. Endocrinology 153: 1684–1695, 2012.
Crossref | PubMed | Web of Science | Google Scholar - 44. . Oral angiotensin-(1–7) prevented obesity and hepatic inflammation by inhibition of resistin/TLR4/MAPK/NF-kappaB in rats fed with high-fat diet. Peptides 46: 47–52, 2013.
Crossref | PubMed | Web of Science | Google Scholar - 45. . Intrauterine growth retardation leads to the development of type 2 diabetes in the rat. Diabetes 50: 2279–2286, 2001.
Crossref | PubMed | Web of Science | Google Scholar - 46. . The apj receptor is expressed in pancreatic islets and its ligand, apelin, inhibits insulin secretion in mice. Regul Pept 131: 12–17, 2005.
Crossref | PubMed | Google Scholar - 47. . Murine pancreatic islet isolation. J Vis Exp 2007; (7):255. doi: 10.3791/255. Epub 2007 Aug 22. 2007.
Crossref | Google Scholar - 48. . The tissue renin-angiotensin system in human pancreas. J Endocrinol 161: 317–322, 1999.
Crossref | PubMed | Web of Science | Google Scholar - 49. . Vasoprotective and atheroprotective effects of angiotensin (1–7) in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 30: 1606–1613, 2010.
Crossref | PubMed | Web of Science | Google Scholar - 50. . Angiotensin-converting enzyme 2 deficiency in whole body or bone marrow-derived cells increases atherosclerosis in low-density lipoprotein receptor−/− mice. Arterioscler Thromb Vasc Biol 31: 758–765, 2011.
Crossref | PubMed | Web of Science | Google Scholar - 51. . Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers and diabetes: a meta-analysis of placebo-controlled clinical trials. Am J Hypertens 24: 582–590, 2011.
Crossref | PubMed | Web of Science | Google Scholar - 52. . Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem 277: 14838–14843, 2002.
Crossref | PubMed | Web of Science | Google Scholar - 53. . The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest 104: 787–794, 1999.
Crossref | PubMed | Web of Science | Google Scholar - 54. .Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY3). Nature 384: 455–458, 1996.
Crossref | PubMed | Web of Science | Google Scholar - 55. . Global survey of escape from X inactivation by RNA-sequencing in mouse. Genome Res 20: 614–622, 2010.
Crossref | PubMed | Web of Science | Google Scholar - 56. . Betatrophin: a hormone that controls pancreatic beta cell proliferation. Cell 153: 747–758, 2013.
Crossref | PubMed | Web of Science | Google Scholar - 57. . Angiotensin-converting enzyme 2 deficiency aggravates glucose intolerance via impairment of islet microvascular density in mice with high-fat diet. J Diabet Res 2013: 405284, 2013.
Crossref | PubMed | Web of Science | Google Scholar - 58. . Ramipril and the development of diabetes. JAMA 286: 1882–1885, 2001.
Crossref | PubMed | Web of Science | Google Scholar
