C. elegans as model for the study of high glucose- mediated life span reduction

Abstract

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 + or - 0.4 to 16.5 + or - 0.6 days and maximum life span from 25.9 + or - 0.4 to 23.2 + or - 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 + or - 0.6 to 20.6 + or - 0.4 days) and maximum life span (23.2 + or - 0.4 to 27.7 + or - 2.3 days). In contrast, inhibition of glyoxalase-1 by RNAi further reduced mean (16.5 + or - 0.6 to 13.9 + or - 0.7 days) and maximum life span (23.2 + or - 0.4 to 20.3 + or - 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.

FIG. 1.

Glucose concentration in C. elegans. C. elegans were cultured on agar having the glucose concentrations of 0, 10, 20, 30, 40, and 50 mmol/l. After 5 days, 100 C. elegans were harvested and the glucose concentrations in the C. elegans whole-body extracts were determined. Results are the means ± SE of three independent experiments; n.s. (not significant) describes a t test value (P > 0.05, *P < 0.05, and ***P < 0.001) comparing two groups as indicated.

FIG. 2.

Effect of high glucose conditions on C. elegans life span. Kaplan-Meier graphs of the fraction of C. elegans alive. Shown are wild-type C. elegans cultured under standard and high glucose conditions. Life span assays were performed as described in Research Designs and Methods. The results are from a representative experiment out of three independent experiments, each including 100 nematodes. Thin line = standard conditions; bold line = high glucose conditions.

FIG. 3.

Effect of high glucose conditions on glyoxalase-1 activity. Quantification of glyoxalase-1 activity in whole-body extracts of 5-day-old wild-type (WT) animals, cultured under standard and high glucose conditions, or sorbitol as control. Results are the means ± SE of three independent experiments; value from a t test (***P < 0.001) comparing wild type under standard conditions vs. wild type under high glucose conditions.

FIG. 4.

Mitochondrial MG-H1 immunoreactivity in C. elegans. Mitochondria were stained with MitoTracker Deep Red FM (A, D, G, and J), and MG-H1 was visualized by immunostaining with a Texas Red-labeled antibody directed against MG-H1 antibody (B, E, H, and K). Merged staining is shown in C, F, I, and L. Orange color indicates colocalization of MG-H1 with mitochondria. Images are shown for 15-day-old wild-type animals cultured under standard (A–C) and high glucose conditions (D–F) and 15-day-old transgenic glyoxalase-1–overexpressing animals cultured under standard (G–I) and high glucose conditions (J–L). Shown are animals from a representative experiment out of three independent experiments, each including 100 nematodes.

FIG. 5.

Effect of high glucose conditions on formation of ROS. Shown are ethidium-labeled wild-type animals cultured for 15 days under standard (A) and high glucose conditions (B), transgenic glyoxalase-1– overexpressing animals cultured for 15 days under standard (C) and high glucose conditions (D). Shown are animals from a representative experiment out of three independent experiments, each including 100 nematodes.

FIG. 6.

Quantification of MG-H1 and ROS in C. elegans. Formation of MG-H1 (A) and ROS (B) was quantified in 15-day-old wild-type (WT) animals, without and with 10 μmol/l myxothiazol (myxo) or 50 μmol/l FCCP, and in 15-day-old transgenic glyoxalase-1–overexpressing animals. Each group was cultured under standard and high glucose conditions. The data were compared across the groups using ANOVA; additional between-group comparisons were made using Fisher PLSD post hoc tests. Results are the means ± SE of three independent experiments with 100 nematodes each; value from a Fisher PLSD post hoc test (*P < 0.05 and ***P < 0.001) comparing the indicated group vs. wild type under standard conditions; value from a Fisher PLSD post hoc test (^oooP < 0.001) comparing the indicated group vs. wild type under high glucose conditions.