Diabetes Publish Ahead of Print, published online August 25, 2010 ACE2 and diabetes Angiotensin-I Converting Enzyme type 2 (ACE2) gene therapy improves glycemic control in diabetic mice. Sharell M. Bindom, Chetan P. Hans, Huijing Xia, A. Hamid Boulares and Eric Lazartigues Department of Pharmacology & Experimental Therapeutics and Cardiovascular Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, LA. Running title: ACE2 and diabetes Correspondence: Eric Lazartigues, PhD E-mail address: [email protected] Submitted 25 May 2009 and accepted 8 July 2010 Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at http://diabetes.diabetesjournals.org. 1 Copyright American Diabetes Association, Inc., 2010 ACE2 and diabetes Objective—Several clinical studies have shown the benefits of renin-angiotensin system (RAS) blockade in the development of diabetes and a local RAS has been identified in pancreatic islets. Angiotensin-I Converting Enzyme (ACE) 2, a new component of the RAS has been identified in the pancreas but its role in β-cells function remains unknown. Using 8 and 16-week old obese diabetic (db/db) mice, we examined the ability of ACE2 to alter pancreatic β-cell function and thereby modulate hyperglycemia. Research design and methods—Both db/db and non-diabetic lean control (db/m) mice were infected with an adenovirus expressing human ACE2 (Ad-hACE2-eGFP) or the control virus (Ad-eGFP), via injection into the pancreas. Glycemia and β-cell function were assessed 1 week later, at the peak of viral expression. Results—In 8-week old db/db mice, Ad-hACE2-eGFP significantly improved fasting glycemia, enhanced intra-peritoneal (IP) glucose tolerance, increased islet insulin content and β-cell proliferation and reduced β-cell apoptosis when compared to Ad-eGFP. ACE2 over-expression had no effect on insulin sensitivity in comparison to Ad-eGFP treatment in diabetic mice. Angiotensin-(1-7) receptor blockade by D-Ala7-Ang-(1-7) prevented the ACE2-mediated improvements in IP glucose tolerance, glycemia and islet function and also impaired insulin sensitivity in both Ad-hACE2-eGFP and Ad-eGFP treated db/db mice. D-Ala7-Ang-(1-7) had no effect on db/m mice. In 16-week old diabetic mice, Ad-hACE2-eGFP treatment improved fasting blood glucose but had no effect on any of the other parameters. Conclusions—These findings identify ACE2 as a novel target for the prevention of β-cell dysfunction and apoptosis occurring in type 2 diabetes. n addition to the systemic renin- receptor (AT1R) axis of the RAS leads to a angiotensin system (RAS) that regulates cascade of events implicated in the Iblood pressure, the concept of a tissue development of β-cell dysfunction, including: RAS, modulating local organ function, is now increased islet fibrosis (6), oxidative stress well recognized. Accordingly, most organs (7,8) and inhibition of pro-insulin express a tissue RAS, capable of locally biosynthesis, first phase and glucose- producing Angiotensin (Ang)-II (1). A responsive insulin secretion (3,5,9). complete tissue RAS has been identified in Moreover, several studies have demonstrated the endocrine and exocrine pancreas and the the effectiveness of RAS blockade at expression of its various components has been improving islet morphology and function and demonstrated in the islets of Langerhans (2- reducing islet oxidative stress (3,5,8,10) (for 4). While the role of the islet RAS is not review see (11)). Recently, Angiotensin completely understood, recent data suggest Converting Enzyme (ACE) 2, a captopril- that it may be important in β-cell homeostasis insensitive ACE homologue, was identified and function. Indeed, it has been shown to be (12,13) and shown to cleave Ang-II into the involved in the regulation of glucose biologically active peptide Ang-(1-7) (13). stimulated-insulin secretion, insulin synthesis Ang-(1-7) properties are mediated by the G- (3), as well as islet blood flow (5). protein-coupled receptor Mas (14), causing Hyperactivity of the ACE/Ang-II/AT1 vasodilation, inhibition of fibrosis (15), 2 ACE2 and diabetes stimulation of PGE2 (16) and NO releases pancreas (n=8/group). The adenovirus was (17). The ACE2/Ang-(1-7)/Mas axis is delivered in five 20 μl injections along the hypothesized to act as a negative regulator for body of the pancreas (Fig. 2A). For a subset of the RAS. Recent data indicate that this animals, D-Ala7-Ang-(1-7), an Ang-(1-7) alternate pathway may play a compensatory receptor antagonist, was infused (600 role in the development of type 2 diabetes ng/kg/min/7 days) using mini-osmotic pumps mellitus (T2DM). ACE2 protein is elevated in (Durect, Cuppertino, CA) implanted the islets of Zucker fatty diabetic rats (10) and subcutaneously (SC) at the time of virus ACE2 knockout (ACE2-/y) mice exhibit injection. All procedures were approved by progressive impairments in glucose tolerance the Animal Use and Care Committee at and reduced first-phase insulin secretion (18). Louisiana State University Health Sciences Loss of first phase insulin secretion, an Center, New Orleans. indicator of pancreatic β-cell dysfunction, is Determination of ACE2 expression and considered to be one of the earliest insults in activity. To prevent protein degradation, T2DM and is evident before the onset of pancreata were first incubated in RNAlater® impaired glucose tolerance (19). Defects in stabilization solution and stored at -80 °C. insulin sensitivity, glucose tolerance and Western blotting for ACE2 expression and glucose uptake exhibited by Mas receptor ACE2 activity assays were performed as knockout mice (20) further implicate the loss described previously (21). of Ang-(1-7) signaling in the development of Measurements of physiological parameters. T2DM and metabolic syndrome. We To assess glucose metabolism in db/db mice, hypothesized that ACE2 overexpression may we performed an intra-peritoneal (IP) glucose ameliorate glucose homeostasis in diabetic tolerance test, where fasted (12 hr) animals mice and prevent the development of were weighed and a bolus IP injection of pancreatic β-cell dysfunction. Using leptin glucose (2 g/kg) was administered to receptor-deficient obese diabetic (db/db) conscious mice. Blood was drawn from the mice, we report that ACE2 overexpression catheterized tail vein and analyzed at 0, 15, reduced glycemia and increased islet insulin 30, 60 and 120 min after glucose content through Ang-(1-7)-mediated administration, using a glucometer (Accu- pathways. Our data confirm the pivotal role of check® Aviva, Roche, Mannheim, Germany). this peptide in the pancreas and establish To determine first phase insulin secretion, ACE2 as a new target for the treatment of fasted mice were anesthetized with isoflurane T2DM. and given a bolus of glucose (1g/kg IP). Blood samples (50 μl) were collected from a RESEARCH DESIGN AND METHODS catheterized carotid artery at 0, 2, 5 and 10 Animals and treatments. Three, 7 and 15- min following glucose administration. Plasma week old male db/db and non-diabetic (db/m) insulin was then measured using ELISA mice (BKS.Cg-m +/+ Leprdb/J, Jackson (Crystal Chem, Downers Grove, IL). Laboratories, Bar Harbor, ME) were infected Insulin sensitivity was analyzed following 1 with an adenovirus coding for human ACE2 hr fast and mice injected SC with human (hACE2), upstream of an enhanced green recombinant insulin (0.3 U/kg; Sigma, St fluorescent protein (eGFP) reporter gene (Ad- Louis, MO). Blood glucose was measured at hACE2-eGFP) or with the eGFP virus alone 0, 15, 30, 60 and 120 min following injection. (Ad-eGFP) (21) by direct injection (5x107 Fasting blood glucose, glucose tolerance and particle forming units (pfu) in a total volume insulin tolerance were measured prior and 7 of 100 μl of 0.9% w/v saline) into the days after adenovirus administration. The 3 ACE2 and diabetes animals were then sacrificed, the pancreas was developed using DAB. Images capture was removed and rapidly divided, with one was performed using a Nikon eclipse E600 half being fixed in 10% formalin in phosphate light microscope. All images were quantified buffered saline (PBS) and the other half using image pro plus software (Media frozen in liquid nitrogen. Cybernetics, Bethesda, MD). For a semi- Immunohistochemistry. Pancreas sections (5 quantitative assessment of islet insulin, mouse µm) were prepared from 10% formalin-fixed, ACE2 (mACE2) and hACE2, staining per paraffin-embedded tissue. For antigen islet (n=16-20/group) area was used to unmasking, sections were incubated in a quantify protein content from citrate buffer solution (100 mM citric acid, immunohistochemistry (10). For PCNA and 100 mM sodium citrate, Sigma) for 13 min at TUNEL staining, positive cells per islet were 100 °C. Following washes, sections were counted. To assess pancreatic β-cell mass, the incubated for 1 hr at room temperature in a mean density of islet insulin staining was blocking solution containing 5% bovine multiplied by the mean islet area per serum albumin in PBST. Sections were pancreatic section, adjusted for wet organ incubated with an anti-insulin primary weight per animal (six sections per organ) antibody (1:100; Abcam, Cambridge, MA) for (10). 4 hr at 4 °C, followed by incubation with Statistical analysis. Data are expressed as biotinylated anti-guinea pig secondary mean ±SEM. Data were analyzed by antibody (1:200; Vector Laboratories, Student’s t test or two-way ANOVA Burlingame, CA) for 1 hr at room (Bonferroni post hoc tests to compare temperature.