During diabetes mellitus, advanced glycation end-products (AGEs) are major contributors to the development of alterations in cerebral capillaries, leading to the disruption of the blood-brain barrier (BBB). Consequently, this is often associated with an amplified oxidative stress response in microvascular endothelial cells. As a model to mimic brain microvasculature, the bEnd.3 endothelial cell line was used to investigate cell barrier function. Cells were exposed to native bovine serum albumin (BSA) or modified BSA (BSA-AGEs). In the presence or absence of the antioxidant compound, N-acetyl-cysteine, cell permeability was assessed by FITC-dextran exclusion, intracellular free radical formation was monitored with H2DCF-DA probe, and mitochondrial respiratory and redox parameters were analyzed. We report that, in the absence of alterations in cell viability, BSA-AGEs contribute to an increase in endothelial cell barrier permeability and a marked and prolonged oxidative stress response. Decreased mitochondrial oxygen consumption was associated with these alterations and may contribute to reactive oxygen species production. These results suggest the need for further research to explore therapeutic interventions to restore mitochondrial functionality in microvascular endothelial cells to improve brain homeostasis in pathological complications associated with glycation.

Advanced glycation end-products (AGEs) trigger multiple metabolic disorders in the vessel wall that may in turn lead to endothelial dysfunction. The molecular mechanisms by which AGEs generate these effects are not completely understood. Oxidative stress plays a key role in the development of deleterious effects that occur in endothelium during diabetes. Our main objectives were to further understand how AGEs contribute to reactive oxygen species (ROS) overproduction in endothelial cells and to evaluate the protective effect of an antioxidant plant extract. The human endothelial cell line EA.hy926 was treated with native or modified bovine serum albumin (respectively BSA and BSA-AGEs). To monitor free radicals formation, we used H2DCF-DA, dihydroethidium (DHE), DAF-FM-DA and MitoSOX Red dyes. To investigate potential sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial inhibitors were used. The regulation of different types of ROS by the polyphenol-rich extract from the medicinal plant Doratoxylon apetalum was also studied for a therapeutic perspective. BSA-AGEs exhibited not only less antioxidant properties than BSA, but also pro-oxidant effects. The degree of albumin glycoxidation directly influenced oxidative stress through a possible communication between NADPH oxidase and mitochondria. D. apetalum significantly decreased intracellular hydrogen peroxide and superoxide anions mainly detected by H2DCF-DA and DHE respectively. Our results suggest that BSA-AGEs promote a marked oxidative stress mediated at least by NADPH oxidase and mitochondria. D. apetalum plant extract appeared to be an effective antioxidant compound to protect endothelial cells.

Increased oxidative stress and advanced glycation end-product (AGE) formation are major contributors to the development of type 2 diabetes. Here plasma proteins e.g. albumin can undergo glycoxidation and play a key role in diabetes onset and related pathologies. However, despite recent progress linking albumin-AGE to increased oxidative stress and downstream effects, its action in metabolic organs such as the liver remains to be elucidated. The current study therefore investigated links between oxidative perturbations and biochemical/structural modifications of plasma albumin, and subsequent downstream effects in transgenic db/db mouse livers and HepG2 cells, respectively. Our data reveal increased oxidative stress biomarkers and lipid accumulation in plasma and livers of diabetic mice, together with albumin glycoxidation. Purified mouse albumin modifications resembled those typically found in diabetic patients, i.e. degree of glycation, carbonylation, AGE levels and in terms of chemical composition. Receptor for AGE expression and reactive oxygen species production were upregulated in db/db mouse livers, together with impaired proteolytic, antioxidant and mitochondrial respiratory activities. In parallel, acute exposure of HepG2 cells to glycated albumin also elicited intracellular free radical formation. Together this study demonstrates that AGE-modified albumin can trigger damaging effects on the liver, i.e. by increasing oxidative stress, attenuating antioxidant capacity, and by impairment of hepatic proteolytic and respiratory chain enzyme activities.

Diabetes and obesity are strongly associated with increased levels of circulating advanced glycation end products (AGEs) and reactive oxygen species (ROS). These two molecular phenomena affect the physiology of adipose tissue, a biological driver of the metabolic syndrome, leading to an inflammatory profile and insulin resistance, which could contribute to obesity/diabetes-associated complications, such as cardiovascular diseases. Herein, we investigated the impact of AGEs on mitochondrial bioenergetics in murine preadipocyte cells (3T3-L1) and cellular redox homeostasis. We show that incubation of preadipocytes with AGEs stimulates mitochondrial activity and respiration while inducing oxidative stress. This AGE-induced intracellular ROS production was blocked by diphenylene iodonium, an NAD(P)H oxidase inhibitor. In parallel, antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) were found to be activated upon AGE treatment. Our results suggest that AGE-induced oxidative stress is generated by NAD(P)H oxidase and leads to a cellular proliferation arrest associated with enhanced mitochondrial metabolism and biogenesis, and with increased levels of ROS-detoxifying enzymes, as well. These new data show how AGEs may be involved in hyperglycemia-induced oxidative damage in preadipocytes and their potential links to diabetes progression. © 2017 BioFactors, 43(4):577-592, 2017.

Diabetes and obesity are strongly associated with enhanced circulating advanced glycoxidation end products (AGE) and reactive oxygen species (ROS) formation. Despite the determinant role of adipose tissue in diabetes/obesity-associated disorders and the key position of mitochondria in energy metabolism and cellular ROS formation, impact of AGE on adipose tissue remains unexplored. Herein, we investigated the impact of human albumin-derived AGE products on mitochondrial bioenergetic and biogenesis modifications of murine preadipocyte cells (3T3-L1) and subsequent cellular redox homeostasis. We show that albumin-AGE stimulates mitochondrial activity and mitochondrial respiration of preadipocyte cells together with inducing an oxidative stress. This AGE-induced intracellular ROS production was strengthen in the presence of rotenone and antimycin A and blocked by diphenylene iodonium (NAD(P)H inhibitor). In parallel, antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) were found to be activated upon AGE treatment. Our results suggest that AGE-induced oxidative stress leads to a cellular proliferation arrest associated with intensification of mitochondrial metabolism, mitochondrial biogenesis and increased level of ROS-detoxifying enzymes. These ROS are generated by NAD(P)H oxidase and involved the mitochondrial electron transport chain. These new data show how AGEs may be involved in hyperglycemia-induced oxidative damage in adipocytes and its potential links to diabetes progression.

Numerous studies indicate that an increase in reactive oxygen species (ROS) significantly affects white adipose tissue biology and leads to an inflammatory profile and insulin resistance, which could contribute to obesity-associated diabetes and cardiovascular diseases. Mitochondria play a key role in adipose tissue energy metabolism and constitute the main source of cellular ROS such as H(2)O(2). Polyphenols constitute the most abundant antioxidants provided by the human diet. Indeed, they are widely distributed in fruits, vegetables and some plant-derived beverages such as coffee and tea. Thus, the biological effects of dietary polyphenols that may increase the antioxidant capacity of the body against obesity-induced oxidative stress are of high interest. Here, we studied the capacity of polyphenols to modulate the impact of oxidative stress on the mitochondria of preadipocytes, which are important cells governing the adipose tissue development for energy homeostasis. Whereas H(2)O(2) treatment induces a proliferation arrest associated with an increase in mitochondrial content in 3T3-L1 preadipocytes, preconditioning with some major dietary polyphenols totally or partially protects the cells against oxidative stress consequences. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

In a previous paper we have shown that it was possible to quantify protein solutions at very weak concentrations directly by UV-visible spectroscopy. Nevertheless the protein quantification could not be possible if there is any trace of interferents left in the solution. So it is necessary to eliminate all the interferents to make the measure at 190 and/or 277 nm possible. We have developed a method based on the use of Microcon membranes and centrifugation. Interferents could be eliminated from protein solutions after four centrifugations at 13000 g during 5 min. This procedure allowed the recovering of proteins, with 80 to 99% yield, and thus making microquantification possible. This method is particularly interesting for enzymatic solutions after a purification procedure by HPLC where very tiny quantities of protein are recovered. This protocol has been tested on three enzymes; enzymatic activity recovered after four centrifugations was quite high for PEPcase and malic enzyme (71 and 64% respectively).

Convergence for appearance of C4 photosynthetic system has likely occurred in plant evolution. There are evidences for such hypothesis based on phylogenies of specific C4 genes in monocotyledonous and dicotyledonous plants. It was also suggested that C4 photosynthesis evolved independently within grass subfamilies. In C4 plants, the enzyme that allows CO2 fixation is a Phosphoenolpyruvate Carboxylase (PEPCase) isoform called C4 PEPCase. Primers specific of maize - sorghum C4 PEPCase mRNAs and RT-PCR were used to generate partial PEPCase cDNA fragments in 23 C4 grasses. To check the C4 photosynthetic pathway evolution within grasses, fragments of about 1,200 bp were sequenced in 12 species representative of all grass subfamilies displaying C4 species: Panicoideae, Arundinoideae and Chloridoideae. All sequences displayed high homology with known grass C4 PEPCases. We reconstructed a phylogeny based on all known grass PEPCase nucleotide sequences using distances (percent divergence) and the Neighbor Joining algorithm. C4 PEPCases and analogues formed a monophyletic clade in the PEPCase phylogram while three grass C3 PEPCase groups were distinguished. Phylogenetic relationships between species, deduced from C4 PEPCase sequences, were similar to those deduced from other nuclear data: the different subfamilies and tribes were recognized, and the subfamily Chloridoideae displayed a basal position in our C4 PEPCase clade. Our data sustained that C4 PEPCase isoform should have appeared only once in the grass evolution. Thus, we can assume that all grass C4 photosynthetic pathways have derived from a common ancestor and convergent evolution hypothesis for this system inside grass family would be debatable.