Methylglyoxal-modified collagen promotes myofibroblast differentiation

Fibrosis is a frequent complication of diabetes mellitus in many organs and tissues but the mechanism of how diabetes-induced glycation of extracellular matrix proteins impacts the formation of fibrotic lesions is not defined. As fibrosis is mediated by myofibroblasts, we investigated the effect of collagen glycation on the conversion of human cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO). Cells cultured on MGO-treated collagen exhibited increased activity of the α-smooth muscle actin promoter and enhanced expression of α-smooth muscle actin, ED-A fibronectin and cadherin, which are markers for myofibroblasts. In cells remodeling floating or stress-relaxed collagen gels, MGO treatment promoted more contraction (p < 0.025) than vehicle controls, which was MGO dose-dependent. Transwell assays showed that cell migration was increased by MGO-treated collagen (p < 0.025). In shear-force detachment assays, cells on MGO-treated collagen were less adherent than untreated collagen, and the formation of high affinity, β1 integrin-dependent adhesions was inhibited. MGO-collagen-induced expression of SMA was dependent on TGF-β but not on Rho kinase. We conclude that collagen glycation augments the formation and migration of myofibroblasts, critical processes in the development of fibrosis in diabetes.     

Microvessel vascular smooth muscle cells contribute to collagen type I deposition through ERK1/2 MAP kinase, alphavbeta3-integrin, and TGF-beta1 in response to ANG II and high glucose

This study determines that vascular smooth muscle cell (VSMC) signaling through extracellular signal-regulated kinase (ERK) 1/2-mitogen-activated protein (MAP) kinase, alphavbeta(3)-integrin, and transforming growth factor (TGF)-beta1 dictates collagen type I network induction in mesenteric resistance arteries (MRA) from type 1 diabetic (streptozotocin) or hypertensive (HT; ANG II) mice. Isolated MRA were subjected to a pressure-passive-diameter relationship. To delineate cell types and mechanisms, cultured VSMC were prepared from MRA and stimulated with ANG II (100 nM) and high glucose (HG, 22 mM). Pressure-passive-diameter relationship reduction was associated with increased collagen type I deposition in MRA from HT and diabetic mice compared with control. Treatment of HT and diabetic mice with neutralizing TGF-beta1 antibody reduced MRA stiffness and collagen type I deposition. Cultured VSMC stimulated with HG or ANG II for 5 min increased ERK1/2-MAP kinase phosphorylation, whereas a 48-h stimulation induced latent TGF-beta1, alphavbeta(3)-integrin, and collagen type 1 release in the conditioned media. TGF-beta1 bioactivity and Smad2 phosphorylation were alphavbeta(3)-integrin-dependent, since beta(3)-integrin antibody and alphavbeta(3)-integrin inhibitor (SB-223245, 10 microM) significantly prevented TGF-beta1 bioactivity and Smad2 phosphorylation. Pretreatment of VSMC with ERK1/2-MAP kinase inhibitor (U-0126, 1 microM) reduced alphavbeta(3)-integrin, TGF-beta1, and collagen type 1 content. Additionally, alphavbeta(3)-integrin antibody, SB-223245, TGF-beta1-small-intefering RNA (siRNA), and Smad2-siRNA (40 nM) prevented collagen type I network formation in response to ANG II and HG. Together, these data provide evidence that resistance artery fibrosis in type 1 diabetes and hypertension is a consequence of abnormal collagen type I release by VSMC and involves ERK1/2, alphavbeta(3)-integrin, and TGF-beta1 signaling. This pathway could be a potential target for overcoming small artery complications in diabetes and hypertension.     

Accelerated re-epithelialization in beta3-integrin-deficient- mice is associated with enhanced TGF-beta1 signaling

The upregulation of TGF-beta1 and integrin expression during wound healing has implicated these molecules in this process, but their precise regulation and roles remain unclear. Here we report that, notably, mice lacking beta(3)-integrins show enhanced wound healing with re-epithelialization complete several days earlier than in wild-type mice. We show that this effect is the result of an increase in TGF-beta1 and enhanced dermal fibroblast infiltration into wounds of beta(3)-null mice. Specifically, beta(3)-integrin deficiency is associated with elevated TGF-beta receptor I and receptor II expression, reduced Smad3 levels, sustained Smad2 and Smad4 nuclear localization and enhanced TGF-beta1-mediated dermal fibroblast migration. These data indicate that alpha(v)beta(3)-integrin can suppress TGF-beta1-mediated signaling, thereby controlling the rate of wound healing, and highlight a new mechanism for TGF-beta1 regulation by beta(3)-integrins.     

Advanced glycation end-products diminish tendon collagen fiber sliding

Connective tissue aging and diabetes related comorbidity are associated with compromised tissue function, increased susceptibility to injury, and reduced healing capacity. This has been partly attributed to collagen cross-linking by advanced glycation end-products (AGEs) that accumulate with both age and disease. While such cross-links are believed to alter the physical properties of collagen structures and tissue behavior, existing data relating AGEs to tendon mechanics is contradictory. In this study, we utilized a rat tail tendon model to quantify the micro-mechanical repercussion of AGEs at the collagen fiber-level. Individual tendon fascicles were incubated with methylglyoxal (MGO), a naturally occurring metabolite known to form AGEs. After incubation in MGO solution or buffer only, tendons were stretched on the stage of a multiphoton confocal microscope and individual collagen fiber stretch and relative fiber sliding were quantified. Treatment by MGO yielded increased fluorescence and elevated denaturation temperatures as found in normally aged tissue, confirming formation of AGEs and related cross-links. No apparent ultrastructural changes were noted in transmission electron micrographs of cross-linked fibrils. MGO treatment strongly reduced tissue stress relaxation (p < 0.01), with concomitantly increased tissue yield stress (p < 0.01) and ultimate failure stress (p = 0.036). MGO did not affect tangential modulus in the linear part of the stress–strain curve (p = 0.46). Microscopic analysis of collagen fiber kinematics yielded striking results, with MGO treatment drastically reducing fiber-sliding (p < 0.01) with a compensatory increase in fiber-stretch (p < 0.01). We thus conclude that the main mechanical effect of AGEs is a loss of tissue viscoelasticity driven by matrix-level loss of fiber–fiber sliding. This has potentially important implications to tissue damage accumulation, mechanically regulated cell signaling, and matrix remodeling. It further highlights the importance of assessing viscoelasticity – not only elastic response – when considering age-related changes in the tendon matrix and connective tissue in general.     

Smad3 mediates the TGF-beta-induced contraction of type I collagen gels by mouse embryo fibroblasts

TGF-beta signals through TGF-beta receptors and Smad proteins. TGF-beta also augments fibroblast-mediated collagen gel contraction, an in vitro model of connective tissue remodeling. To investigate the importance of Smad2 or Smad3 in this augmentation process, embryo-derived fibroblasts from mice lacking expression of Smad2 or Smad3 genes were cast into native type I collagen gels. Fibroblast-populated gels were then released into 0.2% FCS-DMEM alone or with recombinant human TGF-beta1, beta2, beta3, or recombinant rat PDGF-BB. Gel contraction was determined using an image analyzer. All three isoforms of TGF-beta significantly augmented contraction of collagen gels mediated by fibroblasts with genotypes of Smad2 knockout (S2KO), Smad2 wildtype (S2WT), and Smad3 wildtype (S3WT), but not Smad3 knockout (S3KO) mice. PDGF-BB augmented collagen gel contraction by all fibroblast types. These results suggest that expression of Smad3 but not Smad2 may be critical in TGF-beta augmentation of fibroblast-mediated collagen gel contraction. Thus, the Smad3 gene could be a target for blocking contraction of fibrotic tissue induced by TGF-beta.     

Transforming growth factor-beta switches the pattern of integrins expressed in MG-63 human osteosarcoma cells and causes a selective loss of cell adhesion to laminin

Transforming growth factor-beta (TGF-beta) induces a marked decrease in adhesion of MG-63 human osteosarcoma cells to laminin-coated surfaces, but does not significantly alter adhesion to fibronectin- or collagen-coated surfaces. We provide evidence that this effect is due to a switch in the repertoire of cell adhesion receptors in response to TGF-beta. MG-63 cells express high levels of alpha 3 beta 1-integrin, which is a polyspecific laminin/collagen/fibronectin receptor, and low levels of alpha 2 beta 1- and alpha 5 beta 1-integrins, which are collagen and fibronectin receptors, respectively. No other integrins of the beta 1-class could be detected in MG-63 cells. Treatment with TGF-beta 1 induces a marked (approximately 60%) decrease in the level of expression of alpha 3-integrin subunit mRNA and protein and a concomitant 8-fold increase in alpha 2-subunit expression. These responses become maximal 7-12 h after addition of TGF-beta 1 to the cells. Expression of alpha 5- and beta 1-integrin subunits also increases in response to TGF-beta 1, but to a lesser extent than alpha 2-subunit expression. Thus, as a result of TGF-beta action, the alpha 2 beta 1-collagen and alpha 5 beta 1-fibronectin receptors replace the alpha 3 beta 1-laminin/collagen/fibronectin receptor as the predominant integrins of the beta 1-class in MG-63 cells. These results suggest that one of the effects of TGF-beta is to modify the adhesive behavior of certain tumor cells by changing the binding specificity of the complement of integrins that they express.     

Abrogation of transforming growth factor-beta signaling by SMAD7 inhibits collagen gel contraction of human dermal fibroblasts

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.     

Transforming growth factor-beta1, transforming growth factor-beta2, and transforming growth factor-beta3 enhance ovarian cancer metastatic potential by inducing a Smad3-dependent epithelial-to-mesenchymal transition

Transforming growth factor-beta (TGF-beta) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-beta/Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-beta1, TGF-beta2, and TGF-beta3 induced pro-matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-to-mesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-beta-mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-beta1, TGF-beta2, and TGF-beta3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-beta-stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-beta/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential.     

Methylglyoxal-induced modification of arginine residues decreases the activity of NADPH-generating enzymes

Inadequate control of plasma and cellular glucose and ketone levels in diabetes is associated with increased generation of reactive aldehydes, including methylglyoxal (MGO). These aldehydes react with protein side chains to form advanced glycation end-products (AGEs). Arg residues are particularly susceptible to MGO glycation and are essential for binding NADP⁺ in several enzymes that generate NADPH, a coenzyme for many critical metabolic and antioxidant enzymes. In most animal cells, NADPH is produced predominantly by glucose-6-phosphate dehydrogenase (G6PD) in the oxidative phase of the pentose phosphate pathway and, to a lesser extent, by isocitrate dehydrogenase (IDH) and malic enzyme (ME). In this study, the activities of isolated G6PD, IDH, and ME were inhibited by MGO (0–2.5 mM, 2–3 h, 37 °C), in a dose- and time-dependent manner, with G6PD and IDH more sensitive to modification than ME. Significant inhibition of these two enzymes occurred with MGO levels ≥500 μM. Incubation with radiolabeled MGO (0–500 µM, 0–3 h, 37 °C) demonstrated dose- and time-dependent adduction to G6PD and IDH. HPLC analysis provided evidence for AGE formation and particularly the hydroimidazolones MG-H1 and MG-H2 from Arg residues, with corresponding loss of parent Arg residues. Peptide mass mapping studies confirmed hydroimidazolone formation on multiple peptides in G6PD and IDH, including those critical for NADP⁺ binding, and substrate binding, in the case of IDH. These results suggest that modification of NADPH-producing enzymes by reactive aldehydes may result in alterations to the cellular redox environment, potentially predisposing cells to further damage by oxidants and reactive aldehydes.     

Matrix density alters zyxin phosphorylation,which limits peripheral process formation and extension in endothelial cells invading 3D collagen matrices

This study was designed to determine the optimal conditions required for known pro-angiogenic stimuli to elicit successful endothelial sprouting responses. We used an established, quantifiable model of endothelial cell (EC) sprout initiation where ECs were tested for invasion in low (1 mg/mL) and high density (5 mg/mL) 3D collagen matrices. Sphingosine 1-phosphate (S1P) alone, or S1P combined with stromal derived factor-1α (SDF) and phorbol ester (TPA), elicited robust sprouting responses. The ability of these factors to stimulate sprouting was more effective in higher density collagen matrices. S1P stimulation resulted in a significant increase in invasion distance, and with the exception of treatment groups containing phorbol ester, invasion distance was longer in 1 mg/mL compared to 5 mg/mL collagen matrices. Closer examination of cell morphology revealed that increasing matrix density and supplementing with SDF and TPA enhanced the formation of multicellular structures more closely resembling capillaries. TPA enhanced the frequency and size of lumen formation and correlated with a robust increase in phosphorylation of p42/p44 Erk kinase, while S1P and SDF did not. Also, a higher number of significantly longer extended processes formed in 5 mg/mL compared to 1 mg/mL collagen matrices. Because collagen matrices at higher density have been reported to be stiffer, we tested for changes in the mechanosensitive protein, zyxin. Interestingly, zyxin phosphorylation levels inversely correlated with matrix density, while levels of total zyxin did not change significantly. Immunofluorescence and localization studies revealed that total zyxin was distributed evenly throughout invading structures, while phosphorylated zyxin was slightly more intense in extended peripheral processes. Silencing zyxin expression increased extended process length and number of processes, while increasing zyxin levels decreased extended process length. Altogether these data indicate that ECs integrate signals from multiple exogenous factors, including changes in matrix density, to accomplish successful sprouting responses. We show here for the first time that zyxin limited the formation and extension of fine peripheral processes used by ECs for matrix interrogation, providing a molecular explanation for altered EC responses to high and low density collagen matrices.     

Deleterious effects of reactive aldehydes and glycated proteins on macrophage proteasomal function: Possible links between diabetes and atherosclerosis

People with diabetes experience chronic hyperglycemia and are at a high risk of developing atherosclerosis and microvascular disease. Reactions of glucose, or aldehydes derived from glucose (e.g. methylglyoxal, glyoxal, or glycolaldehyde), with proteins result in glycation that ultimately yield advanced glycation end products (AGE). AGE are present at elevated levels in plasma and atherosclerotic lesions from people with diabetes, and previous in vitro studies have postulated that the presence of these materials is deleterious to cell function. This accumulation of AGE and glycated proteins within cells may arise from either increased formation and/or ineffective removal by cellular proteolytic systems, such as the proteasomes, the major multi-enzyme complex that removes proteins within cells. In this study it is shown that whilst high glucose concentrations fail to modify proteasome enzyme activities in J774A.1 macrophage-like cell extracts, reactive aldehydes enhanced proteasomal enzyme activities. In contrast BSA, pre-treated with high glucose for 8 weeks, inhibited both the chymotrypsin-like and caspase-like activities. BSA glycated using methylglyoxal or glycolaldehyde, also inhibited proteasomal activity though to differing extents. This suppression of proteasome activity by glycated proteins may result in further intracellular accumulation of glycated proteins with subsequent deleterious effects on cellular function.     

Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function

During cardiac remodeling, cardiac fibroblasts (CF) are influenced by increased levels of interleukin-1α (IL-1α) and transforming growth factor-β1 (TGFβ1). The present study investigated the interaction between these two important cytokines on function of human CF and their differentiation to myofibroblasts (CMF). CF were isolated from human atrial appendage and exposed to IL-1α and/or TGFβ1 (both 0.1 ng/ml). mRNA expression levels of selected genes were determined after 6–24 h by real-time RT-PCR, while protein levels were analyzed at 24–48 h by ELISA or western blot. Activation of canonical signaling pathways (NFκB, Smad3, p38 MAPK) was determined by western blotting. Differentiation to CMF was examined by collagen gel contraction assays. Exposure of CF to IL-1α alone enhanced levels of IL-6, IL-8, matrix metalloproteinase-3 (MMP3) and collagen III (COL3A1), but reduced the CMF markers α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF/CCN2). By contrast, TGFβ1 alone had minor effects on IL-6, IL-8 and MMP3 levels, but significantly increased levels of the CMF markers αSMA, CTGF, COL1A1 and COL3A1. Co-stimulation with both IL-1α and TGFβ1 increased MMP3 expression synergistically. Furthermore, while TGFβ1 had no effect on IL-1α-induced IL-6 or IL-8 levels, co-stimulation inhibited the TGFβ1-induced increase in αSMA and blocked the gel contraction caused by TGFβ1. Combining IL-1α and TGFβ1 had no apparent effect on their canonical signaling pathways. In conclusion, IL-1α and TGFβ1 act synergistically to stimulate MMP3 expression in CF. Moreover, IL-1α has a dominant inhibitory effect on the phenotypic switch of CF to CMF induced by TGFβ1.     

Chronic ingestion of a Western diet increases O-linked-β-N-acetylglucosamine (O-GlcNAc) protein modification in the rat heart

AimsProtein O-GlcNAcylation is both a nutrient sensing and cellular stress response that mediates signal transduction in the heart. Chronically elevated O-GlcNAc has been associated with the development of cardiac dysfunction at both the cellular and organ levels in obesity, insulin resistance and diabetes. Development of these pathologies is often attributed to diets high in saturated fat and sugar (a “Western” diet; WES) but a role for O-GlcNAc in diet-induced cardiac dysfunction has not been established. The aims of this study were to examine the effect of chronic consumption of WES on cardiac O-GlcNAcylation and investigate associations of O-GlcNAc with cardiac function and markers of cellular stress.Main methodsYoung male rats received either a control diet (CON; n = 9) or WES (n = 8) diet for 52 weeks.Key findingsThere was no evidence of cardiac dysfunction, advanced glycation endproduct (AGE) accumulation, pathological cardiac hypertrophy, calcium handling impairment, fibrosis or endoplasmic reticulum stress in WES hearts. However, cardiac O-GlcNAc protein, particularly in the higher molecular weight range, was significantly higher in WES hearts compared to CON (P < 0.05). Protein levels of the enzymes that regulate O-GlcNAc attachment were not different between groups; thus, the increased O-GlcNAcylation in WES hearts appears to be due to increased nutrient availability rather than enzymatic regulation of cellular stress.SignificanceThese data suggest that diets high in saturated fat and sugar may contribute to the adverse effects of metabolic syndrome and diabetes by an O-GlcNAc-mediated process and that this may occur in the absence of overt cellular stress.     

Transdifferentiation-dependent expression of alpha-SMA in hepatic stellate cells does not involve TGF-beta pathways leading to coinduction of collagen type I and thrombospondin-2.

Hepatic stellate cells (HSC) cultured on plastic spontaneously transdifferentiate to a myofibroblast-like cell type (MFB). This model system of hepatic fibrogenesis is characterized by phenotypic changes of the cells and increased matrix synthesis. Here, we analyzed if transdifferentiation-dependent induction of ECM components, e.g., collagen type I and thrombospondin-2 (TSP-2), and phenotypic changes are coregulated events and if both processes are mediated via TGF-beta pathway(s). Blocking the TGF-beta-dependent p38 MAPK pathway in HSC with the specific inhibitor SB203580 strongly reduces collagen I and TSP-2 mRNA expression without inhibiting upregulation of the typical MFB-marker, alpha-smooth-muscle actin (alpha-SMA). Similarly, interference with the Smad2/3/4 pathway using dexamethasone also heavily decreased expression of collagen type I and TSP-2 whereas transdifferentiation of HSC to the typical morphology of MFB with loss of fat droplets and increasing alpha-SMA was unchanged. Further, p38 MAPK mediated induction of collagen I and TSP-2 expression by TGF-beta1 was still achieved in the presence of dexamethasone, showing that dexamethasone does not block p38 while it delays Smad2 phosphorylation and antagonizes stimulation of a Smad3/Smad4 dependent TGF-beta reporter construct. Interestingly, in contrast to SB203580 and dexamethasone, overexpression of the TGF-beta antagonist Smad7 reduced ECM expression and simultaneously inhibited morphologic transdifferentiation, indicating that Smad7 fulfills additional features in HSC. In conclusion, our data show that phenotypic changes of transdifferentiating HSC and induction of matrix synthesis are independent processes, the latter being stimulated by both, Smad dependent and MAPK dependent TGF-beta signaling.     

Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

Impairment of the proteasome is crucial for glucose-induced lifespan reduction in the mev-1 mutant of Caenorhabditis elegans

Hyperglycemia is a hallmark of diabetes that is associated with diabetic complications and a reduction of lifespan. Using the mev-1 mutant of the nematode Caenorhabditis elegans we here tried to identify molecular mechanisms underlying the lifespan reducing effects of glucose. The lowest glucose concentration tested (10 mM) caused a significant lifespan reduction at 37 °C and was used to assess effects on mitochondrial efficiency, formation of protein carbonyls and levels of methylglyoxal, a precursor of advanced glycation end products (AGEs). RNA-interference (RNAi) served the identification of targets for glucose-induced damage. Levels of protein carbonyls and AGEs remained unaffected by 10 mM glucose. Levels of reactive oxygen species inside mitochondria were increased but their scavenging by ascorbic acid did not influence lifespan reduction by glucose. Mitochondrial efficiency was reduced by glucose as concluded from a lowered P/O-ratio. A reduced lifespan of mev-1 that was unaffected by the addition of glucose resulted from RNAi of key players of mitochondrial unfolded protein response. Besides increased accumulation of misfolded proteins, reduced proteasomal degradation caused the same phenotype as was evidenced by RNAi for UBQ-1 or UBA-1. Accumulation of functionally impaired proteins, e.g. in mitochondria, underlies the lifespan reducing effects of glucose. Our study provides evidence for a crucial importance of the proteostasis network for lifespan regulation which is impaired by glucose.