Conserved roles of glucose in suppressing reactive oxygen species-induced cell death and animal survival

Carbohydrate overconsumption increases blood glucose levels, which contributes to the development of various diseases including obesity and diabetes. It is generally believed that high glucose metabolism increases cellular reactive oxygen species (ROS) levels, damages insulin-secreting cells and leads to age-associated diabetic phenotypes. Here we find that in contrast, high glucose suppresses ROS production induced by paraquat in both mammalian cells and the round worm C. elegans. The role of glucose in suppressing ROS is further supported by glucose’s ability to alleviate paraquat’s toxicity on C. elegans development. Consistently, we find that the ROS-regulated transcription factor SKN-1 is inactivated by glucose. As a result, the ROS/SKN-1-dependent lifespan extension observed in paraquat-treated animals, mitochondrial respiration mutant isp-1 and germline-less mutant glp-1 are all suppressed by glucose. Our study reveals an unprecedented interaction of glucose with ROS, which could have significant impact on our current understanding of glucose- and ROS-related diseases.

Hatching rate measurement C. elegans gravid adult were transferred to NGM agar plate containing control NGM agar plate (60 x 15 mm) and those supplemented with either 1mM paraquat only, 1% of glucose only, or both. Gravid worms were allowed to lay eggs for 2 hours to obtain sufficient eggs (5 worms/plate, 3 plates for each sample). Gravid worms were then picked and removed from drug plate. Eggs were allowed to hatch and animals developed for about 4 days to reach adulthood. Adult animals were then picked on normal NGM agar plate (5 worms/plate) to lay synchronized eggs for 2 hours for hatching measurement. After egg laying, eggs were counted, then incubated at 20°C for 2 days. Hatching rate were calculated by dividing the number of live animals by the number of eggs.

Western blot
Western blot of worms crude lysate has also been described before [2]. C. elegans gravid worms (CF2189, Is001[Pskn-1::skn-1::GFP + rol-6(su1006)]) were picked to NGM agar plate (60 x 15 mm, 5 worms/plate) with or without 0.5% glucose for 2 hours to lay synchronized eggs (10 glucose plate an 10 control plate). Worms were then cultured at 20°C for 2 days to reach L4/young adult stage, then worms were washed form plates and half of the glucose-treated animals and half of the control animals added to NGM agar plate with 1mM paraquat. Other halves were cultured on regular NGM agar plate to serve as control. Worms from these treatments were then harvested 2 days later, washed extensively with ice-cold M9, then resuspended in ice-cold lysis buffer (50 mM HEPES, pH 7.4, 1 mM EGTA, 1 mM MgCl2, 100 mM KCl, 10% glycerol, 0.5% NP-40, 2mM PMSF, Roche protease Complete inhibitor cocktail and phosSTOP tablet). Worms were then sonicated 10 times, 5 second each time on ice with interval of 15 second at the power level of 30%. Crude lysate was obtained by centrifugation. Whole lysate was subjected to SDS-PAGE and transferred to PVDF membrane. Membranes were blocked in PBST (PBS with 0.1% Tween-20) containing 5% non-fat milk for 1 hour and probed with anti-GFP (Abcam, ab32146) and anti-actin antibodies (Abcam, ab14128) at 1000X dilution for 1 hour. Membrane was washed extensively with 0.1% PBST, then incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibodies diluted in 0.1%PBST at 10,000X for 30 min. membrane were then proved with enhanced chemiluminescence (ECL) chemicals developed on radiographic films.

Mammalian cell culture
NIH3T3 cells were obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were maintained in DMEM (Sigma) supplemented with 10% FBS (Gbico) and 100 µg/mL Penicillin-Streptomycin Solution (Gbico) at 37 °C in a humidified atmosphere containing 5% CO2 with frequent medium change. Cells were allowed for at least 3 passages after thawing from nitrogen tank for any assay.

Cell viability assay
Cell viability assay was evaluated by Cell Counting Kit-8 (CCK-8) (Dojin Laboratories, Japan). Cells were plated and incubated in 96-well plates at a density of 2×10 3 www.aging-us.com 2 AGING cells/well at different glucose conditions (1g/L,4g/L, 8g/L). When reaching 60-70% confluence, cells were treated with different concentrations of paraquat for 24 h. Then the plates were incubated with 10ul CCK-8 for 60 min. Finally, absorbance was measured at 450nm through SpectraMAXi3x plate reader (Molecular Devices, Austria).

Apoptosis assay
Apoptotic cells were quantified using the Annexin Vfluorescein isothiocyanate (FITC) /propidium iodide (PI) apoptosis assay (KeyGen BioTECH,China). Briefly, cells were plated in 6-well plates at a density of 5×10 4 cells/well with norma(4g/L) and high(8g/L) glucose concentration. When the confluence was 60-70%, cells were treated with 500 µM paraquat for 24 h. Subsequently, adherent cells were collected and washed twice with cold PBS. Cells were resuspended in 500 μl of manufacturer-supplied 1X binding buffer. Then, 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI) were added and incubated for 15 min in the dark at room temperature.Lastly,the ratios of apoptotic cells were monitored by CytoFLEX-S flow cytometer (Beckman Coulter, USA).

Image quantification
For quantification of signal intensity of ROS and gst-4::gfp, images taken were applied to image J software, worm were outlined and the signal inside the outline were determined by software. The intensity was obtained by dividing the signal reads by the worm areas. 10 images for each sample from 3-independent experiments were chosen randomly for quantification and normalized to the average value of control group. For SKN-1 nuclear localization quantification, due to the variation in different part of the animal body, we count how many intestine cells show observable SKN-1::GFP signal. Images for 10 worms were randomly chosen and SKN-1:GFP positive nuclei were counted and plotted in Prism software.

Statistical analysis
Statistical study was conducted by using software Prism. Bar data were examined by student's t-test, with significant difference defined by P < 0.05. Survival curves were examined by Log-rank test, with significant difference defined by P<0.05.