Carnosine and advanced glycation end products: a systematic review

Damaged mitochondria generate an excess of superoxide, which may mediate tissue injury in diabetes. We hypothesized that in diabetic nephropathy, advanced glycation end-products (AGEs) lead to increases in cytosolic reactive oxygen species (ROS), which facilitate the production of mitochondrial superoxide. In normoglycemic conditions, exposure of primary renal cells to AGEs, transient overexpression of the receptor for AGEs (RAGE) with an adenoviral vector, and infusion of AGEs to healthy rodents each induced renal cytosolic oxidative stress, which led to mitochondrial permeability transition and deficiency of mitochondrial complex I. Because of a lack of glucose-derived NADH, which is the substrate for complex I, these changes did not lead to excess production of mitochondrial superoxide; however, when we performed these experiments in hyperglycemic conditions in vitro or in diabetic rats, we observed significant generation of mitochondrial superoxide at the level of complex I, fueled by a sustained supply of NADH. Pharmacologic inhibition of AGE-RAGE-induced mitochondrial permeability transition in vitro abrogated production of mitochondrial superoxide; we observed a similar effect in vivo after inhibiting cytosolic ROS production with apocynin or lowering AGEs with alagebrium. Furthermore, RAGE deficiency prevented diabetes-induced increases in renal mitochondrial superoxide and renal cortical apoptosis in mice. Taken together, these studies suggest that AGE-RAGE-induced cytosolic ROS production facilitates mitochondrial superoxide production in hyperglycemic environments, providing further evidence of a role for the advanced glycation pathway in the development and progression of diabetic nephropathy.

Tumor necrosis factor (TNF) is selectively cytotoxic to some types of tumor cells in vitro and exerts antitumor activity in vivo. Reactive oxygen intermediates (ROIs) have been implicated in the direct cytotoxic activity of TNF. By using confocal microscopy, flow cytometry, and the ROI-specific probe dihydrorhodamine 123, we directly demonstrate that intracellular ROIs are formed after TNF stimulation. These ROIs are observed exclusively under conditions where cells are sensitive to the cytotoxic activity of TNF, suggesting a direct link between both phenomena. ROI scavengers, such as butylated hydroxyanisole, effectively blocked the formation of free radicals and arrested the cytotoxic response, confirming that the observed ROIs are cytocidal. The mitochondrial glutathione system scavenges the major part of the produced ROIs, an activity that could be blocked by diethyl maleate; under these conditions, TNF-induced ROIs detectable by dihydrorhodamine 123 oxidation were 5- to 20-fold higher.

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).