Elena Fitzenberger

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 (10mM) 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 10mM 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.

The polyphenol quercetin protects the mev‐1 mutant of Caenorhabditis elegans from glucose‐induced reduction of survival under heat‐stress depending on SIR‐2.1, DAF‐12, and proteasomal activity

SCOPE Hyperglycemia is a hallmark of diabetes mellitus but slighter increases of blood glucose levels are observed also during ageing. Using the Caenorhabditis elegans mev-1 mutant, we identified molecular mechanisms underlying the protection from glucose toxicity by the polyphenol quercetin. METHODS AND RESULTS We fed C. elegans mev-1 mutants on a liquid medium supplemented with 10 mM glucose, which resulted in a reduced survival at 37°C. The polyphenol quercetin (1 μM) was able to prevent glucose-induced lifespan reduction completely. RNA interference revealed that the sirtuin SIR-2.1, the nuclear hormone receptor DAF-12, and its putative co-activator MDT-15 were critical for the quercetin effects. Moreover, RNA interference for key factors of proteostasis reduced survival, which was not further affected by glucose or quercetin, suggesting that those proteins are a target for both substances. Besides unfolded protein response, proper functionality of the proteasome was shown to be crucial for the survival enhancing effects of quercetin and the polyphenol was finally demonstrated to activate proteasomal degradation. CONCLUSION Our studies demonstrate that lowest concentrations of quercetin prevent a glucose-induced reduction of survival. SIR-2.1, DAF-12, and MDT-15 were identified as targets that activate unfolded protein response and proteasomal degradation to limit the accumulation of functionally restricted proteins.

Carnitine protects the nematode Caenorhabditis elegans from glucose-induced reduction of survival depending on the nuclear hormone receptor DAF-12.

Besides its function in transport of fatty acids into mitochondria in order to provide substrates for β-oxidation, carnitine has been shown to affect also glucose metabolism and to inhibit several mechanisms associated with diabetic complications. In the present study we used the mev-1 mutant of the nematode Caenorhabditis elegans fed on a high glucose concentration in liquid media as a diabetes model and tested the effects of carnitine supplementation on their survival under heat-stress. Carnitine at 100 μM completely prevented the survival reduction that was caused by the application of 10 mM glucose. RNA-interference for sir-2.1, a candidate genes mediating the effects of carnitine revealed no contribution of the sirtuin for the rescue of survival. Under daf-12 RNAi rescue of survival by carnitine was abolished. RNA-interference for γ-butyrobetaine hydroxylase 2, encoding the key enzyme for carnitine biosynthesis did neither increase glucose toxicity nor prevent the rescue of survival by carnitine, suggesting that the effects of carnitine supplementation on carnitine levels were significant. Finally, it was demonstrated that neither the amount of lysosomes nor the proteasomal activity were increased by carnitine, excluding that protein degradation pathways, such as autophagy or proteasomal degradation, are involved in the protective carnitine effects. In conclusion, carnitine supplementation prevents the reduction of survival caused by glucose in C. elegans in dependence on a nuclear hormone receptor which displays high homologies to the vertebrate peroxisomal proliferator activated receptors.

The zinc matrix metalloproteinase ZMP-2 increases survival of Caenorhabditis elegans through interference with lipoprotein absorption

Matrix metalloproteinases are zinc-dependent endopeptidases conserved throughout the animal kingdom which primarily degrade components of the extracellular matrix. In the nematode Caenorhabditis elegans, the zinc matrix metalloproteinase (ZMP-2) was demonstrated to increase resistance versus heat and bacterial pathogens. Here, we show that the survival reducing activities caused by the knockdown of zmp-2 in C. elegans essentially requires the presence of vitellogenin-6, a protein homologous to mammalian apolipoprotein B, and RME-2, a receptor mediating endocytosis of cholesterol particles. Measurements of reactive oxygen species inside and outside C. elegans revealed that knockdown of zmp-2 causes a prooxidative extracellular mileu which is a prerequisite for the reduction of survival. Interestingly, RNAi for the foxo transcription factor daf-16 completely prevented those survival reducing effects of zmp-2 RNAi, and RNAi in mutants of the steroid signalling pathway revealed that DAF-16 acts by inhibition of DAF-9 and DAF-12. In conclusion, our study demonstrates survival reducing activities caused by the functional loss of ZMP-2 in C. elegans. Those effects are mediated by the transport of oxidized cholesterol adducts which then trigger the inhibition of DAF-9 and DAF-12 through the activation of DAF-16.