Can theobromine stimulate SIRT1 (silent information regulator 1) and reduce Diabetes-associated oxidative stress and renal fibrosis?

Abstract

Hyperglycemia-induced oxidative stress causes an accumulation of extracellular matrix markers (ECM) that can progress to fibrosis and end stage renal disease, which is characteristic of diabetic nephropathy (DN). As part of our thematic project (2008/57560-0), we have observed that treatment with cocoa enriched in polyphenols as well as treatment with cocoa with a low polyphenol content were both able to prevent oxidative stress and ECM in diabetic rats as well as human mesangial cells exposed to high glucose. Moreover, the activity of NAD+-dependent deacetylases such as silent information regulator 1 (Sirt1) in mesangial cells exposed to high levels of glucose was reduced, and this was reversed after exogenous administration of either cocoa or theobromine (identified as the main non-polyphenolic compound in cocoa). The aims of this study are as follows: 1. To investigate the influence of theobromine in oxidative stress and indices of renal damage such as ECM accumulation and proteinuria in vivo. 2. To test the contribution of Sirt1 and its mechanisms in mediating the potential effects of cocoa and theobromine in alleviating diabetic kidney damage both in diabetic rats as well as in vitro in mesangial cells exposed to high levels of glucose. Diabetes mellitus (DM) will be induced by streptozotocin in spontaneously hypertensive rats (SHR) 12 weeks of age. Diabetic and control animals will receive (or not receive) treatments with cocoa or theobromine for 16 weeks. A human mesangial cell line will be grown under normoglucose (5.6 mM) and high glucose (30 mM) conditions in the presence and absence of theobromine or cocoa and inhibitors of Sirt1 activity or AMPK or CaM-KK or PARP-1 or activators of AMPK. An analysis of oxidative stress will be done using reactive oxygen species (ROS) detection, NADPH oxidase-induced superoxide quantification, and nitrotyrosine Western blot analysis. ECM markers such as collagen IV, fibronectin, and mesangial matrix expansion will be assessed via immunohostochemistry, Western blot, and PAS staining, respectively. Mesangial apoptosis will also be assessed by caspase-3 activity quantification. The role of cocoa and theobromine on Sirt1 in diabetes will be approached via quantification of Sirt1 activity and NAD+ levels by ELISA in the presence of AMPK and PARP-1 inhibitors. The mechanisms of Sirt1 mediating the possible effects of cocoa and theobromine on oxidative stress, apoptosis, and fibrosis will be approached via immunoprecipitation of acetylated Foxo3 or Foxo4 or acetylated p53 as well as acetylated Smad3 followed by Western blot for Sirt1. The identification of Sirt1 as a mechanism of action of cocoa-enriched polyphenol or theobromine to improve the indices of renal damage such as oxidative stress and fibrosis in diabetic kidney disease represents a pathophysiological basis for the use of such maneuvers to prevent the progression of DN. (AU)