Andreas Schlotterer

Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C. elegans: progressive mitochondrial protein modification by the glycolysis‐derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase‐1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase‐1 mRNA. The decrease in enzymatic activity promotes accumulation of MG‐derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase‐1 expression. Over‐expression of the C. elegans glyoxalase‐1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C. elegans lifespan. In contrast, knock‐down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C. elegans lifespan.

C. elegans as Model for the Study of High Glucose– Mediated Life Span Reduction

OBJECTIVE Establishing Caenorhabditis elegans as a model for glucose toxicity–mediated life span reduction. RESEARCH DESIGN AND METHODS C. elegans were maintained to achieve glucose concentrations resembling the hyperglycemic conditions in diabetic patients. The effects of high glucose on life span, glyoxalase-1 activity, advanced glycation end products (AGEs), and reactive oxygen species (ROS) formation and on mitochondrial function were studied. RESULTS High glucose conditions reduced mean life span from 18.5 ± 0.4 to 16.5 ± 0.6 days and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.4 days, independent of glucose effects on cuticle or bacterial metabolization of glucose. The formation of methylglyoxal-modified mitochondrial proteins and ROS was significantly increased by high glucose conditions and reduced by mitochondrial uncoupling and complex IIIQo inhibition. Overexpression of the methylglyoxal–detoxifying enzyme glyoxalase-1 attenuated the life-shortening effect of glucose by reducing AGE accumulation (by 65%) and ROS formation (by 50%) and restored mean (16.5 ± 0.6 to 20.6 ± 0.4 days) and maximum life span (23.2 ± 0.4 to 27.7 ± 2.3 days). In contrast, inhibition of glyoxalase-1 by RNAi further reduced mean (16.5 ± 0.6 to 13.9 ± 0.7 days) and maximum life span (23.2 ± 0.4 to 20.3 ± 1.1 days). The life span reduction by glyoxalase-1 inhibition was independent from the insulin signaling pathway because high glucose conditions also affected daf-2 knockdown animals in a similar manner. CONCLUSIONS C. elegans is a suitable model organism to study glucose toxicity, in which high glucose conditions limit the life span by increasing ROS formation and AGE modification of mitochondrial proteins in a daf-2 independent manner. Most importantly, glucose toxicity can be prevented by improving glyoxalase-1–dependent methylglyoxal detoxification or preventing mitochondrial dysfunction.

Apurinic/apyrimidinic endonuclease 1, p53, and thioredoxin are linked in control of aging in C. elegans.

Deletions in mitochondrial DNA (mtDNA) accumulate during aging. Expression of the Caenorhabditis elegans apurinic/apyrimidinic endonuclease 1 (APE1) ortholog exo‐3, involved in DNA repair, is reduced by 45% (P < 0.05) during aging of C. elegans. Suppression of exo‐3 by treatment with RNAi resulted in a threefold increase in mtDNA deletions (P < 0.05), twofold enhanced generation of reactive oxygen species (ROS) (P < 0.01), distortion of the structural integrity of the nervous system, reduction of head motility by 43% (P < 0.01) and whole animal motility by 38% (P < 0.05). Suppression of exo‐3 significantly reduced life span: mean life span decreased from 18.5 ± 0.4 to 15.4 ± 0.1 days (P < 0.001) and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.1 days (P = 0.001). Additional treatment of exo‐3‐suppressed animals with a mitochondrial uncoupler decreased ROS levels, reduced neuronal damage, and increased motility and life span. Additional suppression of the C. elegans p53 ortholog cep‐1 in exo‐3 RNAi‐treated animals similarly decreased ROS levels, preserved neuronal integrity, and increased motility and life span. In wild‐type animals, suppression of cep‐1, involved in downregulation of exo‐3, increased expression of exo‐3 without a significant effect on ROS levels, preserved neuronal integrity, and increased motility and life span. Suppression of the C. elegans thioredoxin orthologs trx‐1 and trx‐2, involved in the redox chaperone activity of exo‐3, overrides the protective effect of cep‐1 RNAi treatment on neuronal integrity, neuronal function, mean and maximum life span. These results show that APE1/EXO‐3, p53/CEP‐1, and thioredoxin affect each other and that these interactions determine aging as well as neuronal structure and function.

daf-16 /FOXO and glod-4 /glyoxalase-1 are required for the life-prolonging effect of human insulin under high glucose conditions in Caenorhabditis elegans

Aims/hypothesisThe aim of this study was to determine the protective effects of human insulin and its analogues, B28Asp human insulin (insulin aspart) and B29Lys(ε-tetradecanoyl),desB30 human insulin (insulin detemir), against glucose-induced lifespan reduction and neuronal damage in the model organism Caenorhabditis elegans and to elucidate the underlying mechanisms.MethodsNematodes were cultivated under high glucose (HG) conditions comparable with the situation in diabetic patients and treated with human insulin and its analogues. Lifespan was assessed and neuronal damage was evaluated with regard to structural and functional impairment. Additionally, the activity of glyoxalase-1 and superoxide dismutase (SOD) and the formation of reactive oxygen species (ROS) and AGEs were determined.ResultsInsulin and its analogues reversed the life-shortening effect of HG conditions and prevented the glucose-induced neuronal impairment. Insulin treatment under HG conditions was associated with reduced concentration of glucose, as well as a reduced formation of ROS and AGEs, and increased SOD activity. These effects were dependent on the Forkhead box O (FOXO) homologue abnormal dauer formation (DAF)-16. Furthermore, glyoxalase-1 activity, which was impaired under HG conditions, was restored by human insulin. This was essential for the insulin-induced lifespan extension under HG conditions, as no change in lifespan was observed following either suppression or overexpression of glyoxalase-1.Conclusions/interpretationHuman insulin and its analogues prevent the reduction in lifespan and neuronal damage caused by HG conditions. The effect of human insulin is mediated by a daf-2/insulin receptor and daf-16/FOXO-dependent pathway and is mediated by upregulation of detoxifying mechanisms.

Systemic Treatment with Erythropoietin Protects the Neurovascular Unit in a Rat Model of Retinal Neurodegeneration

Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four –week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.

The −174G>C IL-6 Gene Promoter Polymorphism and Diabetic Microvascular Complications

This study examined a possible association of the G>C polymorphism at nucleotide -174 in the promoter region of the interleukin-6 (IL-6) gene (rs1800795) with the prevalence of diabetic complications in 235 patients with type 1 and 498 patients with type 2 diabetes. Genotyping was performed using polymerase chain reaction (PCR) and subsequent cleavage by Nla III restriction endonuclease. Analyzing all diabetic patients together demonstrated that 301 patients (41.1%) carried the GG genotype, 114 (15.6%) the CC genotype, and 318 (43.3%) were heterozygous for the GC genotype. However, there was no correlation of any of the genotypes with the prevalence of diabetic nephropathy or diabetic neuropathy, but subjects with the CC genotype had a significantly higher prevalence of diabetic retinopathy compared to patients with the GC and GG genotype (p=0.016). This association was mainly lost when a logistic regression model was adjusted for diabetes duration (p=0.07). Consistently, a weak but not significant association of the polymorphism with diabetic retinopathy was observed when type 1 and type 2 diabetic patients were analyzed separately (patients with type 1 diabetes: p=0.12; patients with type 2 diabetes: p=0.09). Analogically, no association of the polymorphism was found for diabetic nephropathy or diabetic neuropathy in these groups. In conclusion these data suggest no major influence of the -174G>C variant in the promoter region of the IL-6 gene on the development of microvascular complications in patients with diabetes.

Understanding diabetic polyneuropathy and longevity: what can we learn from the nematode Caenorhabditis elegans?

Pathogenesis of late diabetic complications is influenced by a complex interplay of multiple exogenous and intrinsic factors. The well characterised nematode Caenorhabditis elegans is an ideal model to study causes of diabetic polyneuropathy because of its easily accessible nervous system. A repertoire of methods allows the assessment of both morphological and functional glucotoxic damages as well as reduction of lifespan, thereby helping to examine the influence of different pathways and mechanisms on neurodegeneration. Its insulin signalling system allows to directly visualize effects of insulin on high glucose induced neuronal damage, leading to a better understanding of diabetic polyneuropathy.

Protective Effects of Liraglutide and Linagliptin in C. elegans as a New Model for Glucose-Induced Neurodegeneration

Liraglutide and linagliptin are novel drugs for the treatment of diabetes. Antioxidative and neuroprotective effects have been described for both compounds. However, it is not yet known, whether these mechanisms are also protective against diabetic retinal neurodegeneration. We assessed the antioxidative and neuroprotective capabilities of liraglutide and linagliptin as well as the signaling pathways involved, by using C. elegans as a model for glucose-induced neurodegeneration. C. elegans were cultivated under conditions, which mimic clinical hyperglycemia, and treated with 160 μmol/l liraglutide or 13 μmol/l linagliptin. Oxidative stress was reduced by 29 or 78% and methylglyoxal-derived advanced glycation endproducts (AGEs) by 33 or 22%, respectively. This resulted in an improved neuronal function by 42 or 60% and an extended mean lifespan by 9 or 11%, respectively. Antioxidative and AGE reducing effects of liraglutide and linagliptin were not dependent on v-akt murine thymoma viral oncogene homologue 1/forkhead box O1 (AKT1/FOXO). Neuroprotection by liraglutide was AKT1/FOXO dependent, yet AKT1/FOXO independent upon linagliptin treatment. Both liraglutide and linagliptin exert neuroprotective effects in an experimental model for glucose-induced neurodegeneration, however, the signaling pathways differ in the present study. Further pharmacological intervention with these pathways may help to delay the clinical onset of diabetic retinopathy by preserving neuronal integrity.

Dicarbonyl Stress Mimics Diabetic Neurovascular Damage in the Retina

The net effect of euglycemic treatment is grossly overestimated in diabetes mellitus and retinopathy, similar to what is observed in diabetic individuals, is found in the absence of chronic hyperglycemia. Explanations of this clinical paradox include the excess generation of reactive intermediates of metabolism. Excess formation or impaired detoxification of reactive intermediates can also result in multiple posttranslational modifications with a wide range of cellular dysfunctions. The multicellular neurovascular unit represents the response element of the retina which is crucial for the development of diabetic retinopathy. Current evidence suggests that increased reactive intermediates in the retina induce (micro-)glial activation, neurodegeneration and vasoregression similar to alterations found in the diabetic retina. Reactive metabolites can be lowered by metabolic signal blockade, by an activation of detoxification pathways and by quenching. The translation of these novel findings into treatment of patients with complications is important to reduce individual suffering and financial burden for societies.Quick Summary:Increased levels of reactive intermediates, independent of blood glucose levels, are linked to damage of the neurovascular unit of the diabetic retina.

Comment on: Lin et al. (2007) SUMO4 M55V Variant Is Associated With Diabetic Nephropathy in Type 2 Diabetes: Diabetes 56:1177–1180

Lin et al. (1) have recently reported a positive association of the SUMO4 M55V variant with diabetic nephropathy in an Asian cohort of 430 patients with type 2 diabetes. The authors found a strong association of the G allele with an increased risk for diabetic nephropathy. Thus far, little is known about the physiologic role of …