Effect of hyperglycemia and thiazolidinediones on cardiac angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) in db/db diabetic mice.

Type 2 diabetes mellitus is strongly associated with increased risk of cardiovascular disease. Despite availability of several antidiabetic medications, the cardiovascular outcomes remain unchanged. Activation of renin angiotensin system is one of the critical factors in development of diabetic complications. Hyperglycemia causes an increase in tissue and circulating Angiotensin II. Angiotensin converting enzyme (ACE2) and neprilysin (NEP) are angiotensin (1-7) forming enzymes. ACE2 was first cloned from heart failure patients and has cardioprotective and renoprotective properties. Combination of angiotensin receptor antagonism and NEP inhibition is a new therapeutic strategy for treatment of heart failure. The aim of this study was to investigate whether there is an alteration of cardiac NEP and ACE2 in db/db mice and to study the effect of glycemic control with rosiglitazone and pioglitazone on these enzymes. Lean control and db/db mice were fed 20 mg/Kg/day rosiglitazone and pioglitazone. Diabetic mice demonstrated hyperglycemia which was attenuated by rosiglitazone and pioglitazone treatment. Western blot revealed two bands for NEP and ACE2 in both lean and db/db mice in both the studies. Mature NEP was downregulated while fragment was upregulated in db/db mice. Expression of mature ACE2 was upregulated in db/db mice while the fragment was not affected. Both rosiglitazone and pioglitazone had no effect on cardiac NEP and ACE2 expression. There was a significant increase in cardiac ADAM 17 expression in db/db mice. Another aim was to optimize the fluorescence based enzyme activity assay for renal and cardiac ACE2 and NEP. Protease inhibitors for prolyl endopeptidase, prolyl carboxypeptidase, aminopeptidase A-M were introduced in the assay buffer. Addition of protease inhibitors improved the specificity of the renal ACE2 activity assay. Protease inhibitors however, did not affect renal NEP activity assay. This is the first study to report the presence of two immunoreactive bands for cardiac ACE2 and NEP expression. We also report the possibility of fully mature form of cardiac NEP being degraded to form fragmented form. To the best of our knowledge, this study is also the first to report an upregulation in cardiac ACE2 in db/db mice.