Ida Giardino

Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy

Three of the major biochemical pathways implicated in the pathogenesis of hyperglycemia induced vascular damage (the hexosamine pathway, the advanced glycation end product (AGE) formation pathway and the diacylglycerol (DAG)–protein kinase C (PKC) pathway) are activated by increased availability of the glycolytic metabolites glyceraldehyde-3-phosphate and fructose-6-phosphate. We have discovered that the lipid-soluble thiamine derivative benfotiamine can inhibit these three pathways, as well as hyperglycemia-associated NF-κB activation, by activating the pentose phosphate pathway enzyme transketolase, which converts glyceraldehyde-3-phosphate and fructose-6-phosphate into pentose-5-phosphates and other sugars. In retinas of diabetic animals, benfotiamine treatment inhibited these three pathways and NF-κB activation by activating transketolase, and also prevented experimental diabetic retinopathy. The ability of benfotiamine to inhibit three major pathways simultaneously might be clinically useful in preventing the development and progression of diabetic complications.

Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis.

Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglutathione, which in turn is converted to D-lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo-transfected control cells (21.80+/-0.1 vs. 0. 76+/-0.02 micromol/min/mg protein, n = 3, P < 0.001). In neo-transfected cells, 30 mM glucose incubation increased MG and D-lactate concentration approximately twofold above 5 MM (35.5+/-5.8 vs. 19.6+/-1.6, P < 0.02, n = 3, and 21.0+/-1.3 vs. 10.0+/-1.2 pmol/ 10(6) cells, n = 3, P < 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration at all, while increasing the enzymatic product D-lactate by > 10-fold (18.9+/-3.2 vs. 18.4+/- 5.8, n = 3, P = NS, and 107.1+/-9.0 vs. 9.4+/-0 pmol/10(6) cells, n = 3, P < 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58+/-0.15 vs. 0.19+/-0.03 total absorbance units, n = 3, P < 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis.

Nonenzymatic glycosylation in vitro and in bovine endothelial cells alters basic fibroblast growth factor activity. A model for intracellular glycosylation in diabetes.

Intracellular sugars are more reactive glycosylating agents than glucose. In vitro nonezymatic glycosylation of basic fibroblast growth factor (bFGF) by fructose, glucose-6-phosphate (G6P), or glyceraldehyde-3-phosphate (G3P) reduced high affinity heparin-binding activity of recombinant bFGF by 73, 77, and 89%, respectively. Mitogenic activity was reduced 40, 50, and 90%. To investigate the effects of bFGF glycosylation in GM7373 endothelial cells, we first demonstrated that GLUT-1 transporters were not downregulated by increased glucose concentration. In 30 mM glucose, the rate of glucose transport increased 11.6-fold, and the intracellular glucose concentration increased sixfold at 24 h and fivefold at 168 h. The level of total cytosolic protein modified by advanced glycosylation end-products (AGEs) was increased 13.8-fold at 168 h. Under these conditions, mitogenic activity of endothelial cell cytosol was reduced 70%. Anti-bFGF antibody completely neutralized the mitogenic activity at both 5 and 30 nM glucose, demonstrating that all the mitogenic activity was due to bFGF. Immunoblotting and ELISA showed that 30 mM glucose did not decrease detectable bFGF protein, suggesting that the marked decrease in bFGF mitogenic activity resulted from posttranslational modification of bFGF induced by elevated glucose concentration. Cytosolic AGE-bFGF was increased 6.1-fold at 168 h. These data are consistent with the hypothesis that nonenzymatic glycosylation of intracellular protein alters vascular cell function.

Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition

Accumulation of unwanted/misfolded proteins in aggregates has been observed in airways of patients with cystic fibrosis (CF), a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show how the defective CFTR results in defective autophagy and decreases the clearance of aggresomes. Defective CFTR-induced upregulation of reactive oxygen species (ROS) and tissue transglutaminase (TG2) drive the crosslinking of beclin 1, leading to sequestration of phosphatidylinositol-3-kinase (PI(3)K) complex III and accumulation of p62, which regulates aggresome formation. Both CFTR knockdown and the overexpression of green fluorescent protein (GFP)-tagged-CFTRF508del induce beclin 1 downregulation and defective autophagy in non-CF airway epithelia through the ROS–TG2 pathway. Restoration of beclin 1 and autophagy by either beclin 1 overexpression, cystamine or antioxidants rescues the localization of the beclin 1 interactome to the endoplasmic reticulum and reverts the CF airway phenotype in vitro, in vivo in Scnn1b-transgenic and CftrF508del homozygous mice, and in human CF nasal biopsies. Restoring beclin 1 or knocking down p62 rescued the trafficking of CFTRF508del to the cell surface. These data link the CFTR defect to autophagy deficiency, leading to the accumulation of protein aggregates and to lung inflammation.

BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intracellular advanced glycation endproducts in bovine endothelial cells.

Hyperglycemia rapidly induces an increase in intracellular advanced glycation end products (AGEs) in bovine endothelial cells, causing an alteration in bFGF activity (Giardino, I., D. Edelstein, and M. Brownlee. 1994. J. Clin. Invest. 94:110-117). Because sugar or sugar-adduct autoxidation is critical for AGE formation in vitro, we evaluated the role of reactive oxygen species (ROS) in intracellular AGE formation, using bovine aortic endothelial cells. 30 mM glucose increased intracellular ROS formation by 250% and lipid peroxidation by 330%, while not affecting ROS in the media. In cells depleted of glutathione, intracellular AGE accumulation increased linearly with ROS generation as measured by immunoblotting and the fluorescent probe DCFH (AGE 0.258-3.531 AU* mm/5x10(4) cells, DCF 57-149 mean AU, r = .998, P < .002). Deferoxamine, alpha-tocopherol, and dimethylsulfoxide each inhibited hyperglycemia-induced formation of both ROS and AGE. To differentiate an effect of ROS generation on AGE formation from an effect of more distal oxidative processes, GM7373 endothelial cell lines were generated that stably expressed the peroxidation-suppressing proto-oncogene bcl-2. bcl-2 had no effect on hyperglycemia-induced intracellular ROS formation. In contrast, bcl-2 expression decreased both lipid peroxidation (100% at 3 h and 29% at 168 h) and AGE formation (55% at 168 h). These data show that a ROS-dependent process plays a central role in the generation of intracellular AGEs, and that inhibition of oxidant pathways prevents intracellular AGE formation.

High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor α. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease.

Urea-induced ROS generation causes insulin resistance in mice with chronic renal failure

Although supraphysiological concentrations of urea are known to increase oxidative stress in cultured cells, it is generally thought that the elevated levels of urea in chronic renal failure patients have negligible toxicity. We previously demonstrated that ROS increase intracellular protein modification by O-linked beta-N-acetylglucosamine (O-GlcNAc), and others showed that increased modification of insulin signaling molecules by O-GlcNAc reduces insulin signal transduction. Because both oxidative stress and insulin resistance have been observed in patients with end-stage renal disease, we sought to determine the role of urea in these phenotypes. Treatment of 3T3-L1 adipocytes with urea at disease-relevant concentrations induced ROS production, caused insulin resistance, increased expression of adipokines retinol binding protein 4 (RBP4) and resistin, and increased O-GlcNAc-modified insulin signaling molecules. Investigation of a mouse model of surgically induced renal failure (uremic mice) revealed increased ROS production, modification of insulin signaling molecules by O-GlcNAc, and increased expression of RBP4 and resistin in visceral adipose tissue. Uremic mice also displayed insulin resistance and glucose intolerance, and treatment with an antioxidant SOD/catalase mimetic normalized these defects. The SOD/catalase mimetic treatment also prevented the development of insulin resistance in normal mice after urea infusion. These data suggest that therapeutic targeting of urea-induced ROS may help reduce the high morbidity and mortality caused by end-stage renal disease.

Tissue Transglutaminase Activation Modulates Inflammation in Cystic Fibrosis via PPARγ Down-Regulation

Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor γ and increase of the classic parameters of inflammation, such as TNF-α, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-γ and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.

Lysosomal accumulation of gliadin p31–43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARγ downregulation in intestinal epithelial cells and coeliac mucosa

Background An unresolved question in coeliac disease is to understand how some toxic gliadin peptides, in particular p31–43, can initiate an innate response and lead to tissue transglutaminase (TG2) upregulation in coeliac intestine and gliadin sensitive epithelial cell lines. Aim We addressed whether the epithelial uptake of p31–43 induces an intracellular pro-oxidative envoronment favouring TG2 activation and leading to the innate immune response. Methods The time course of intracellular delivery to lysosomes of p31–43, pα-2 or pα-9 gliadin peptides was analysed in T84 and Caco-2 epithelial cells. The effects of peptide challenge on oxidative stress, TG2 and peroxisome proliferator-activated receptor (PPAR)γ ubiquitination and p42/44–mitogen activated protein (MAP) kinase or tyrosine phosphorylation were investigated in cell lines and cultured coeliac disease biopsies with/without anti-oxidant treatment or TG2 gene silencing by immunoprecipitation, western blot, confocal microscopy and Fluorenscence Transfer Resonance Energy (FRET) analysis. Results After 24 h of challenge p31–43, but not pα-2 or pα-9, is still retained within LAMP1-positive perinuclear vesicles and leads to increased levels of reactive oxygen species (ROS) that inhibit TG2 ubiquitination and lead to increases of TG2 protein levels and activation. TG2 induces cross-linking, ubiquitination and proteasome degradation of PPARγ. Treatment with the antioxidant EUK-134 as well as TG2 gene silencing restored PPARγ levels and reversed all monitored signs of innate activation, as indicated by the dramatic reduction of tyrosine and p42/p44 phosphorylation. Conclusion p31–43 accumulation in lysosomes leads to epithelial activation via the ROS–TG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPARγ downregulation.

Methylglyoxal modification of mSin3A links glycolysis to angiopoietin-2 transcription.

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here, we report that in retinal Müller cells, increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding of the repressor complex to a glucose-responsive GC box in the angiopoietin-2 promoter, resulting in increased Ang-2 expression. A similar mechanism involving methylglyoxal-modification of other coregulator proteins may play a role in the pathobiology of a variety of conditions associated with changes in methylglyoxal concentration, including cancer and diabetic vascular disease.