Effect of an aldose reductase inhibitor on type IV collagen production by human endothelial cells cultured in high glucose

Summary Diabetic microangiopathy is characterized by a thickening of capillary basement membranes associated with type IV collagen accumulation. An increase in type IV collagen content of the aortic wall is also observed in macroangiopathy. In order to analyse the importance of the polyol pathway in the development of the collagen metabolism alterations seen in diabetic angiopathy and their prevention by aldose reductase inhibitors, we have studied the effects of sorbinil on the high glucose-induced stimulation of type IV collagen biosynthesis in human umbilical vein endothelial cells. Primary cultures were exposed to high glucose (16.7 mmol/l), with and without 0.11 mmol/l sorbinil, for 3 or 6 days after beginning of confluence. We measured the soluble type IV collagen secreted into the culture medium and the insoluble type IV collagen accumulated in the extracellular matrix and cells, by ELISA. We also studied [¹⁴C]proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. High glucose decreased the number of cells and increased the amount of type IV collagen in the culture supernatant and in the extracellular matrix and cell fraction. It also increased proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. Sorbinil corrected all these high glucose-induced alterations. The corrective effects of sorbinil on the proliferation and on type IV collagen metabolism of endothelial cells cultured in high glucose may be attributed to prevention of polyol pathway dysregulation.Abbreviations AGE Advanced glycation end product - AR aldose reductase - ARI aldose reductase inhibitor - BM basement membrane - EC endothelial cells - GGHG glucosyl-galactosyl-hydroxylysyl-collagen glucohydrolase - HUVEC human umbilical vein EC - HS human serum - PBS phosphate-buffered saline - PKC protein kinase C     

Growth factors, cytokines, and renal fibrosis during the course of diabetic nephropathy

Diabetic nephropathy is characterised by a progressive accumulation of extracellular matrix within the glomerular mesangium and the interstitium. The pathogenesis of this fibrotic process is still poorly understood, but in vitro and in vivo data suggest that TGF-B plays a key role. Local overproduction of TGF-B could be secondary to a synthesis of diacylglycerol, polyols, or glucosamines. It may also be secondary to an accumulation of advanced glycosylation end-products which modify the functions of neighbouring cells. Moreover, clinical as well as experimental data for TGF-B suggest that angiotensin II has a profibrotic effect; and it has been clearly demonstrated that angiotensin-converting enzyme inhibitors have a beneficial effect in patients with insulin-dependent diabetes mellitus. Other molecules such as endothelin-1, lipid peroxidation products, or IGF-1 may also play a role in this fibrotic process. Finally, heavy proteinuria secondary to glomerular lesions enhances the accumulation of extracellular matrix within the interstitium, probably through modifications of tubular cell functions, thereby inducing the release of pro-inflammatory and profibrotic molecules.     

Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology

Increased production of mitochondrial reactive oxygen species (ROS) by hyperglycemia is recognized as a major cause of the clinical complications associated with diabetes and obesity [Brownlee, M. (2001) Nature 414, 813-820]. We observed that dynamic changes in mitochondrial morphology are associated with high glucose-induced overproduction of ROS. Mitochondria undergo rapid fragmentation with a concomitant increase in ROS formation after exposure to high glucose concentrations. Neither ROS increase nor mitochondrial fragmentation was observed after incubation of cells with the nonmetabolizable stereoisomer L-glucose. However, inhibition of mitochondrial pyruvate uptake that blocked ROS increase did not prevent mitochondrial fragmentation in high glucose conditions. Importantly, we found that mitochondrial fragmentation mediated by the fission process is a necessary component for high glucose-induced respiration increase and ROS overproduction. Extended exposure to high glucose conditions, which may mimic untreated diabetic conditions, provoked a periodic and prolonged increase in ROS production concomitant with mitochondrial morphology change. Inhibition of mitochondrial fission prevented periodic fluctuation of ROS production during high glucose exposure. These results indicate that the dynamic change of mitochondrial morphology in high glucose conditions contributes to ROS overproduction and that mitochondrial fission/fusion machinery can be a previously unrecognized target to control acute and chronic production of ROS in hyperglycemia-associated disorders.     

TGFbeta1 is involved in high glucose-induced accumulation of pericellular chondroitin sulphate in human endothelial cells

High glucose-induced endothelial cell dysfunction is considered to be the main cause of the development of vascular diabetes complications. Cultured endothelial cells exposed to high glucose in vitro demonstrate a variety of alterations, including extracellular matrix (ECM) deposition, growth inhibition, and changes in cell motility. Some of these effects were shown to be mediated by the up-regulation of endothelial transforming growth factor-beta1 (TGFbeta1) secretion and activation. We investigated the influence of high glucose on human immortalized endothelial cell line ECV304. According to our data, confluent cells exposed to 30 mM glucose for 48 h secrete the increased amount of total and active TGFbeta1 ( approximately 1.4-fold), and accumulate more chondroitin sulphate (CS) in their conditioned medium, pericellular matrix, and cell layer ( approximately 1.6- to 2.0-fold). By blocking the coupling of CS chains to the core protein with p-nitrophenyl-beta-D-xyloside and by chondroitinase ABC treatment, we demonstrated that the increased accumulation of pericellular CS is accompanied by increased cell attachment to immobilized hyaluronic acid (HA), while the expression of cell surface CD44 remains unaltered. Since the exogenous TGFbeta1 affects ECV304 cells in a similar manner, and anti-TGFbeta1-neutralizing antibody cancels the effect of high glucose, we suggest the involvement of TGFbeta1 in the development of endothelial cell response to high glucose in terms of CS accumulation and cell binding to HA.     

Long-term culture in matrigel enhances the insulin secretion of fetal porcine islet-like cell clusters in vitro

To study the influence of extracellular matrix (ECM) on isolated fetal porcine islet-like cell clusters (ICC), ICC were cultured either embedded in Matrigel (MG) or growth factor-reduced Matrigel (MG-GF), or in the absence of matrix for 20 days. The insulin accumulation in the culture medium was determined every fifth day. After culture, the ICC were retrieved and used for acute insulin release experiments and subsequently subjected to either determination of DNA and insulin contents or immunohistochemistry. It was found that culture in MG and MG-GF significantly increased both the insulin accumulation in the culture medium and the insulin content/DNA compared to culture in the absence of matrix. In addition, MG culture significantly increased the insulin output with time compared with the initial 5-day value. The acute insulin release was significantly higher from the ICC cultured in the presence of matrix, whereas no acute glucose-induced insulin response could be detected. During the culture period, the ICC cultured in the absence of matrix disintegrated and formed a monolayer of cells in the culture dish, whereas the ICC cultured in matrix formed rounded structures containing a substantial number of insulin-positive cells. In some cases, cyst-like structures could be seen after culture in matrix. The data suggest that beta-cell differentiation during fetal development has a dual requirement for extracellular matrix components and growth factors.     

Polydatin ameliorates experimental diabetes-induced fibronectin through inhibiting the activation of NF-κB signaling pathway in rat glomerular mesangial cells

A number of studies have recently demonstrated the involvement of nuclear factor-kappa B (NF-κB) activation and the subsequent coordinated inflammatory responses in the pathogenesis of diabetic nephropathy (DN). Polydatin has been shown to have the ability of anti-adhesive inflammation. However, the possible protective and beneficial effects of polydatin on DN via suppressing inflammatory damage and extracellular matrix (ECM) accumulation are not fully elucidated. We found that the polydatin could inhibit the induction and activity of NF-κB, and meanwhile ameliorating ECM accumulation in streptozotocin-diabetic rats. We aimed to investigate the effect of polydatin on fibronectin (FN) protein expression, and to elucidate its potential mechanism involving the NF-κB inflammatory signaling pathway in rat glomerular mesangial cells (GMCs) cultured under high glucose. The results revealed that polydatin significantly suppressed high glucose-induced FN production, inhibited NF-κB nuclear translocation, reduced the DNA-binding activity of NF-κB, as well as decreased the protein expression of ICAM-1 and TGF-β in GMCs. These findings suggested that polydatin significantly represses high glucose-induced FN expression in rat GMCs, which may be closely related to its inhibition of the NF-κB signaling pathway. Hence, we elucidated the potential mechanisms of the anti-inflammatory effects and ECM accumulation alleviation of polydatin in GMCs of DN in vitro.     

The effects of glucose-induced oxidative stress on growth and extracellular matrix gene expression of vascular smooth muscle cells

Summary Vascular smooth muscle cell (VSMC) dysfunction plays a role in diabetic macrovasculopathy and this may include abnormalities in growth characteristics and the extracellular matrix. As the actual mechanisms by which glucose induces VSMC dysfunction remain unclear, the aim of this study was to assess the potential role of glucose-induced oxidative stress. Porcine aortic VSMCs were cultured for 10 days in either 5 mmol/l normal glucose or 25 mmol/l D-glucose (high glucose). There was evidence of oxidative stress as indicated by a 50 % increase in intracellular malondialdehyde (p < 0.05), increased mRNA expression of CuZn superoxide dismutase and Mn superoxide dismutase (by 51 % and 37 % respectively, p < 0.01) and a 50 % decrease in glutathione in 25 mmol/l D-glucose (p < 0.001). Growth was increased by 25.0 % (p < 0.01). mRNA expression of extracellular matrix proteins (collagens I, III, IV and fibronectin) was not altered by high glucose in these experimental conditions. Repletion of glutathione with N-acetyl L-cysteine (1 mmol/l) in VSMC grown in high glucose was associated with reduction in malondialdehyde and restored growth to that of normal glucose. The water soluble analogue of vitamin E, Trolox (200 μmol/l), reduced malondialdehyde concentrations, but had no effect on glutathione depletion or the increased growth rate seen with high glucose. The addition of buthionine sulphoximine (10 μmol/l) to VSMC cultured in normal glucose reduced glutathione, increased malondialdehyde and increased growth to a similar extent as that found in high glucose alone. These results suggest that thiol status, rather than lipid peroxides, is a key factor in modulating VSMC growth and that mRNA expression of extracellular matrix proteins is not increased in VSMC under conditions of glucose-induced oxidative stress. [Diabetologia (1998) 41: 1210–1219] Received: 26 November 1997 and in revised form: 28 April 1998     

Increasing uncoupling protein-2 in pancreatic beta cells does not alter glucose-induced insulin secretion but decreases production of reactive oxygen species

Aims/hypothesis Levels of uncoupling protein-2 (UCP2) are regulated in the pancreatic beta cells and an increase in the protein level has been associated with mitochondrial uncoupling and alteration in glucose-stimulated insulin secretion. However, it is not clear whether an increase in uncoupling protein-2 per se induces mitochondrial uncoupling and affects ATP generation and insulin secretion. Materials and methods Transgenic mice with beta cell-specific overexpression of the human UCP2 gene and INS-1 cells with doxycycline-inducible overproduction of the protein were generated and the consequences of increased levels of UCP2 on glucose-induced insulin secretion and on parameters reflecting mitochondrial uncoupling were determined. Results In transgenic mice, an increase in beta cell UCP2 protein concentration did not significantly modify plasma glucose and insulin levels. Glucose-induced insulin secretion and elevation in the ATP/ADP ratio were unaltered by an increase in UCP2 level. In INS-1 cells, a similar increase in UCP2 level did not modify glucose-induced insulin secretion, cytosolic ATP and ATP/ADP ratio, or glucose oxidation. Increased levels of UCP2 did not modify the mitochondrial membrane potential and oxygen consumption. Increased UCP2 levels decreased cytokine-induced production of reactive oxygen species. Conclusion/interpretation The results obtained in transgenic mice and in the beta cell line do not support the hypothesis that an increase in UCP2 protein per se uncouples the mitochondria and decreases glucose-induced insulin secretion. In contrast, the observation that increased UCP2 levels decrease cytokine-induced production of reactive oxygen species indicates a potential protective effect of the protein on beta cells, as observed in other cell types. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorised users. N. Produit-Zengaffinen and N. Davis-Lameloise contributed equally to this work.     

Role of protein kinase C-angiotensin II pathway for extracellular matrix production in cultured human mesangial cells exposed to high glucose levels

The intrarenal renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy. This study investigates the mechanisms for glucose-induced increase in angiotensin II (AII) production by human mesangial cells (MCs) in relation to protein kinase C (PKC). We also examine whether locally produced AII mediates extracellular matrix protein production in high-glucose conditions. Human MCs were cultured in 5 or 33 mmol/l glucose for 8 days, and were incubated with or without 5 mmol/l GFX, a PKC inhibitor, 0.1 micromol/l candesartan cilexetil (CC), a specific type 1 AII receptor antagonist, for another 24 h. In addition, MCs grown in 5 mmol/l glucose were incubated with 0.1 micromol/l phorbol-12,13-dibutyrate (PDBu) for 24 h. AII, TGF-beta1, fibronectin and type IV collagen in the culture media were measured by ELISA. The amount of AII secreted from MCs exposed to high-glucose levels was significantly greater (P<0.01) than that in normal glucose levels. The increase in AII production was completely prevented by GFX. The addition of PDBu mimicked the effect of glucose on AII production. The glucose-induced increases in the production of TGF-beta1, fibronectin and type IV collagen were partially, but significantly restored (P<0.01) by CC, while GFX totally abolished these effects of glucose. These results suggest that elevated glucose levels stimulate AII production via mechanisms dependent on glucose-induced PKC activation in human MCs, and that locally produced AII partly mediates the increase in mesangial matrix synthesis in high-glucose conditions.     

Nonenzymatic glycosylation and the pathogenesis of diabetic complications

Glucose chemically attaches to proteins and nucleic acids without the aid of enzymes. Initially, chemically reversible Schiff base and Amadori product adducts form in proportion to glucose concentration. Equilibrium is reached after several weeks, however, and further accumulation of these early nonenzymatic glycosylation products does not continue beyond that time. Subsequent reactions of the Amadori product slowly give rise to nonequilibrium advanced glycosylation end-products which continue to accumulate indefinitely on longer-lived molecules. Excessive formation of both types of nonenzymatic glycosylation product appears to be the common biochemical link between chronic hyperglycemia and a number of pathophysiologic processes potentially involved in the development of long-term diabetic complications. The major biological effects of excessive nonenzymatic glycosylation include: inactivation of enzymes; inhibition of regulatory molecule binding; crosslinking of glycosylated proteins and trapping of soluble proteins by glycosylated extracellular matrix (both may progress in the absence of glucose); decreased susceptibility to proteolysis; abnormalities of nucleic acid function; altered macromolecular recognition and endocytosis; and increased immunogenicity.     

Impact of mitochondrial ROS production on diabetic vascular complications

Vascular complications are the leading cause of morbidity and mortality in patients with diabetes. Four main molecular mechanisms have been implicated in glucose-mediated vascular disease. There are: glucose-induced activation of protein kinase C (PKC) isoforms; increased formation of glucose-derived advanced glycation end-products (AGE); increased glucose flux through the aldose reductase pathway; and increased production of reactive oxygen species (ROS). Here we demonstrate that hyperglycemia-induced production of ROS is abrogated by inhibitors of mitochondrial metabolism, or by overexpression of uncoupling protein-1 or manganese superoxide dismutase. Normalization of mitochondrial ROS production by each of these agents prevents glucose-induced activation of PKC, formation of AGE, and accumulation of sorbitol in bovine vascular endothelial cells. We also claim that 8-hydroxydeoxyguanosine, which represents mitochondrial oxidative damage was elevated in patients with either retinopathy, albuminuria or increased intima-media thickness of carotid arteries. These results suggest that hyperglycemia induces mitochondrial ROS production, and which can associate to the pathogenesis of diabetic vascular complications.     

Intra- and extracellular amyloid fibrils are formed in cultured pancreatic islets of transgenic mice expressing human islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is the constituent peptide of amyloid deposits found in the islets of non-insulin-dependent diabetic patients. Formation of islet amyloid is associated with a progressive destruction of insulin-producing beta cells. Factors responsible for the conversion of IAPP into insoluble amyloid fibrils are unknown. Both the amino acid sequence of human IAPP (hIAPP) and hypersecretion of hIAPP have been implicated as factors for amyloid fibril formation in man. We have generated transgenic mice using rat insulin promoter-hIAPP or rat IAPP (rIAPP) gene constructs. No fibrillar islet amyloid was detectable in vivo in these normoglycemic mice, although small amorphous perivascular accumulations of IAPP were observed in hIAPP mice only. To determine the effects of glucose on IAPP secretion and fibrillogenesis, pancreatic islets from transgenic and control mice were examined in vitro. Islet IAPP secretion and content were increased in transgenic islets compared with control islets. IAPP-immunoreactive fibrils were formed at both intra- and extracellular sites in isolated hIAPP islets cultured with glucose at 11.1 and 28 mM for only 7 days. At 28 mM glucose, fibrils were present in deep invaginations of beta cells as observed in non-insulin-dependent diabetic patients. No fibrils were present at low glucose concentrations in hIAPP islets or at any glucose concentration in rIAPP or control islets. Thus, glucose-induced expression and secretion of hIAPP in transgenic mouse islets can lead to formation of amyloid fibrils similar to that found in non-insulin-dependent diabetes mellitus.     

Mitochondrial fission mediates high glucose-induced cell death through elevated production of reactive oxygen species

AIMS: One of the main causes of cardiovascular complications in diabetes is the hyperglycaemia-induced cell injury, and mitochondrial fission has been implicated in the apoptotic process. We investigated the role of mitochondrial fission in high glucose-induced cardiovascular cell injury. METHODS AND RESULTS: We used several types of cultured mouse, rat, and bovine cells from the cardiovascular system, and evaluated mitochondrial morphology, reactive oxygen species (ROS) levels, and apoptotic parameters in sustained high glucose incubation. Adenoviral infection was used for the inhibition of the fission protein DLP1. We found that mitochondria were short and fragmented in cells incubated in sustained high glucose conditions. Under the same conditions, cellular ROS levels were high and cell death was increased. We demonstrated that the increased level of ROS causes mitochondrial permeability transition (MPT), phosphatidylserine exposure, cytochrome c release, and caspase activation in prolonged high glucose conditions. Importantly, maintaining tubular mitochondria by inhibiting mitochondrial fission in sustained high glucose conditions normalized cellular ROS levels and prevented the MPT and subsequent cell death. These results demonstrate that mitochondrial fragmentation is an upstream factor for ROS overproduction and cell death in prolonged high glucose conditions. CONCLUSION: These findings indicate that the fission-mediated fragmentation of mitochondrial tubules is causally associated with enhanced production of mitochondrial ROS and cardiovascular cell injury in hyperglycaemic conditions.     

An oxidative mechanism is involved in high glucose-induced serum protein modification causing inhibition of endothelial cell proliferation.

In order to investigate the role of hyperglycemia on the development of atherosclerosis in diabetics, the effects of high glucose-induced modification of serum factors on the proliferation of bovine carotid artery endothelial cells were studied. Dialysates of high glucose-treated serum inhibit cell growth in a time- and glucose concentration-dependent manner. With 6 weeks of pretreatment, 16.7 mM glucose causes a 47.2% inhibition in cell growth compared to 5.6 mM glucose (P < 0.001). Pretreatment of serum in the presence of reduced glutathione (0.5-1.0 mM), an antioxidant, significantly prevents the high glucose-induced inhibition without inhibiting the formation of early non-enzymatic glycosylation products. Dithiothreitol (7.5 mM) treatment after preincubation with glucose fully restores the glucose-induced inhibition. When the dialysates are fractionated according to molecular mass, the high glucose-induced inhibition is maximal in the MW fraction above 100 kDa. These data suggest that high glucose conditions facilitate the susceptibility of serum proteins to sulfhydryl oxidation forming disulfide crosslinks and this oxidative process may contribute to the inhibition of endothelial cell proliferation.     

Glucose-induced swelling in rat pancreatic alpha-cells

Pancreatic beta-cells increase in volume when exposed to elevated concentrations of extracellular glucose. This study has examined the effects of glucose on the volumes of pancreatic alpha-cells, which like beta-cells are regulated by glucose, and intestinal epithelial Caco-2 cells which are unresponsive to glucose. Cell volume changes were monitored by a video-imaging method. Increasing the extracellular glucose concentration caused a concentration-dependent increase in alpha-cell volume over the range 1-20mM. Glucose-induced swelling was not, however, observed in Caco-2 cells. The glucose-induced swelling in both alpha- and beta-cells was abolished by 0.5mM phloretin, an inhibitor of the GLUT proteins, indicating that GLUT mediated glucose transport is a pre-requisite for swelling. Glucose metabolism also appears to be essential, as islet cell swelling was not observed with 16 mM 3-O-methyl glucose. These data suggest that glucose-induced swelling may be a property exclusive to glucose-regulated cells.     

Protein kinase C and the development of diabetic vascular complications.

Hyperglycemic control in diabetes is key to preventing the development and progression of vascular complications such as retinopathy, nephropathy and neuropathy. Increased activation of the diacylglycerol (DAG)-protein kinase C (PKC) signal transduction pathway has been identified in vascular tissues from diabetic animals, and in vascular cells exposed to elevated glucose. Vascular abnormalities associated with glucose-induced PKC activation leading to increased synthesis of DAG include altered vascular blood flow, extracellular matrix deposition, basement membrane thickening, increased permeability and neovascularization. Preferential activation of the PKCbeta isoform by elevated glucose is reported to occur in a variety of vascular tissues. This has lead to the development of LY333531, a PKCbeta isoform specific inhibitor, which has shown potential in animal models to be an orally effective and nontoxic therapy able to produce significant improvements in diabetic retinopathy, nephropathy, neuropathy and cardiac dysfunction. Additionally, the antioxidant vitamin E has been identified as an inhibitor of the DAG-PKC pathway, and shows promise in reducing vascular complications in animal models of diabetes. Given the overwhelming evidence indicating a role for PKC activation in contributing to the development of diabetic vascular complications, pharmacological therapies that can modulate this pathway, particularly with PKC isoform selectivity, show great promise for treatment of vascular complications, even in the presence of hyperglycemia.     

Activation of the sphingosine kinase-signaling pathway by high glucose mediates the proinflammatory phenotype of endothelial cells

Vascular endothelial cells are key targets for hyperglycemic damage that facilitates vascular inflammation and the vasculopathy associated with diabetes mellitus. However, the mechanisms underlying this damage remain undefined. We now demonstrate that hyperglycemia induces activation of sphingosine kinase (SphK), which represents a novel signaling pathway that mediates endothelial damage under ambient high glucose conditions. SphK activity was significantly increased in aorta and heart of streptozotocin-induced diabetic rats. Interestingly, this increase in SphK activity was prevented by insulin treatment, which achieved euglycemia in the diabetic animals. Hyperglycemia-induced increase in SphK activity was also evident in endothelial cells that received long-term exposure to high glucose (22 mmol/L). Studies using a small interfering RNA strategy demonstrated that endogenous SphK1, but not SphK2, is the major isoenzyme that was activated by high glucose. In addition, an increase in SphK1 phosphorylation was detected in a protein kinase C- and extracellular signal-regulated kinase 1/2-dependent manner, which accounts for the high glucose-induced increases in SphK activity. Importantly, inhibition of SphK1 by either a chemical inhibitor (N',N'-dimethylsphingosine) or expression of a dominant-negative mutant of SphK1 (SphK(G82D)), or SphK1-specific small interfering RNA, strongly protected endothelial cells against high glucose-induced damage, as characterized by an attenuation in the expression of proinflammatory adhesion molecules, adhesion of leukocytes to endothelial cells, and nuclear factor kappaB activation. Thus, interventions that target the SphK-signaling pathway may have the potential to prevent vascular lesions under hyperglycemic conditions.     

High glucose-induced alterations of extracellular matrix of human skin fibroblasts are not dependent on TSP-1-TGFbeta1 pathway

Elevated glucose level is the main cause of extracellular matrix (ECM) derangement in various tissues in diabetes mellitus. The development of diabetic nephropathy is considered to be dependent on profibrotic cytokine, transforming growth factor-β1 (TGFβ1). Its excessive activation due to the up-regulation of thrombospondin-1 (TSP-1) in mesangial cells exposed to high glucose contributes to ECM accumulation. However, the role of TSP-1–TGFβ1 pathway in the development of glucose-induced imbalance of ECM homeostasis in skin connective tissue is not studied. We investigated the response of human skin fibroblasts to elevated glucose level (11.0 and 30.0 mM) in terms of: (1) the expression and secretion of fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1); (2) the accumulation of hyaluronic acid (HA) in pericellular matrix and in the conditioned medium; (3) TGFβ1 expression, secretion and activation; (4) TSP-1 expression and secretion. We demonstrated the up-regulation of FN and PAI-1 by elevated glucose and the stimulation of HA accumulation in both cellular compartments. However, we failed to demonstrate the increase of expression, secretion and activation of TGFβ1, and the increase of TSP-1 expression and secretion in fibroblasts exposed to high glucose. These results show that ECM derangement in skin fibroblasts due to high glucose is not determined by TGFβ1 and its activation by TSP-1.     

Islets and their antioxidant defense

Pancreatic β-cells secrete insulin in response to changes in extracellular glucose concentration. Persistent hyperglycemia during diabetes exerts toxic effects on islets by creating redox imbalance arising from overproduction of reactive oxygen species (ROS). ROS accumulation disturbs the integrity and physiological function of cellular biomolecules impairing viability and functionality of cells. Susceptibility of an organ to oxidative stress (OS) is determined by its defense mechanism and ability to repair DNA damage caused by ROS. Weak defense status of islets along with its inefficiency to repair oxidative DNA damage as compared to other tissues renders it extraordinarily sensitive to OS. Realizing the vulnerability of islet cells to oxidative damage, several efforts to boost their defense mechanism in the form of oral administration of antioxidants and overexpression of genes responsible for antioxidant enzymes have proven successful. Recently accountability for this low antioxidant defense of islets have been given by correlating it with its metabolic evolution.