Environmental and genetic preconditioning for long-term anoxia responses requires AMPK in Caenorhabditis elegans

Abstract

Background: Preconditioning environments or therapeutics, to suppress the cellular damage associated with severe oxygen deprivation, is of interest to our understanding of diseases associated with oxygen deprivation. Wildtype C. elegans exposed to anoxia enter into a state of suspended animation in which energy-requiring processes reversibly arrest. C. elegans at all developmental stages survive 24-hours of anoxia exposure however, the ability of adult hermaphrodites to survive three days of anoxia significantly decreases. Mutations in the insulin-like signaling receptor (daf-2) and LIN-12/Notch (glp-1) lead to an enhanced long-term anoxia survival phenotype.

Methodology/principal findings: In this study we show that the combined growth environment of 25°C and a diet of HT115 E. coli will precondition adult hermaphrodites to survive long-term anoxia; many of these survivors have normal movement after anoxia treatment. Animals fed the drug metformin, which induces a dietary-restriction like state in animals and activates AMPK in mammalian cell culture, have a higher survival rate when exposed to long-term anoxia. Mutations in genes encoding components of AMPK (aak-2, aakb-1, aakb-2, aakg-2) suppress the environmentally and genetically induced long-term anoxia survival phenotype. We further determine that there is a correlation between the animals that survive long-term anoxia and increased levels of carminic acid staining, which is a fluorescent dye that incorporates in with carbohydrates such as glycogen.

Conclusions/significance: We conclude that small changes in growth conditions such as increased temperature and food source can influence the physiology of the animal thus affecting the responses to stress such as anoxia. Furthermore, this supports the idea that metformin should be further investigated as a therapeutic tool for treatment of oxygen-deprived tissues. Finally, the capacity for an animal to survive long bouts of severe oxygen deprivation is likely dependent on specific subunits of the heterotrimeric protein AMPK and energy stores such as carbohydrates.

Figure 1. Temperature and E. coli food source preconditions for long-term anoxia survival.

N2 adult hermaphrodites were raised at either 25°C or 20°C, fed either the OP50 or HT115 E. coli strain, exposed to either three days (A) or four days of anoxia (B). The survivors were examined for an unimpaired or impaired phenotype. The plates that contained antibiotics and temperature in which animals were grown on is noted as such. Animals grown at 25°C have a higher survival rate in comparison to those grown at 20°C (p<.05, Student's T-test) (A, B). Animals grown at 25°C and fed HT115 E. coli strain, in comparison to those raised at 25°C and fed OP50 E. coli, have a significantly higher unimpaired phenotype after long-term anoxia exposure (A). Line denotes statistically significant groups (α = .05, SNK multiple range test) (A, B). Food switching experiments involve the feeding of one food source during development and then transferred to the other food source prior to three or four days of anoxia exposure (C, D); survivors were examined for an unimpaired or impaired phenotype. Animals fed OP50 during development and then transferred to the HT115 strain have a significantly higher unimpaired phenotype in comparison to those animals fed HT115 during development and then transferred to OP50 prior to anoxia exposure or to those animals only fed OP50. (C). Animals that were raised on HT115 or transferred to HT115 had a significantly higher survival rate then those only raised on OP50. Line denotes statistically significant groups, α = .05, SNK multiple range test (C, D). For all experiments, the total number of animals assayed is N>175 from four independent experiments; error bar represents standard deviation.

Figure 2. Animals fed heat-killed HT115 have an impaired phenotype after anoxia treatment.

N2 adult hermaphrodites were fed either the OP50 or HT115 E. coli strain, raised at 25°C, transferred to heat-killed OP50 or HT115 and exposed to either three days (A) or four days of anoxia (B). Food switching experiments involve the feeding of one food source (live bacteria) during development and then transferred to the other food source (head killed bacteria) prior to three or four days of anoxia exposure. Transfer of animal to heat killed HT115 resulted in a significant decrease in the unimpaired phenotype (*). For all experiments, the total number of animals assayed is N>168 from four independent experiments; error bar represents standard deviation. (p<.05, Student's T-test).

Figure 3. Reduction in aak-2 or daf-16 function suppresses the long-term anoxia survival preconditioning induced by temperature and E. coli food source.

Adult hermaphrodites, of specified genotype or RNAi experiment, were raised at 25°C (HT115 E. coli food source) and exposed to either three days (A) or four days (B) of anoxia; survivors were examined for an unimpaired or impaired phenotype. Line denotes groups with a significant decrease in the number of animals with an unimpaired phenotype in comparison to N2 control (p<. 05, Dunnett's Multiple Range test) (A). The aak-2 mutants exposed to four days of anoxia had a significant decrease in survival rate in comparison to control (B). Line denotes groups with a significant decreae in the number of animals with an unimpaired phenotype in comparison to N2 control (p<. 05, Dunnett's Multiple Range test). For all experiments, the total number of animals assayed is N>180 from four independent experiments; error bar represents standard deviation.

Figure 4. Suppression of daf-2(e1370) enhanced long-term anoxia phenotype.

Adult hermaphrodites of the given genotype were fed HT115 E. coli strain or specified RNAi food and exposed to either three days (A) or four days of anoxia (B). The survivors were examined for an unimpaired or impaired phenotype. Line denotes groups with a significant decrease in the number of animals with an unimpaired phenotype in comparison to daf-2(e1370) (p<.05 Student's paired one tailed-T-test). For all experiments, the total number of animals assayed is N>180 from four independent experiments; error bar represents standard deviation.

Figure 5. Suppression of glp-1(e2141) enhanced long-term anoxia phenotype.

Adult hermaphrodites of the given genotype were fed HT115 E. coli strain or specified RNAi food and exposed to either three days (A) or four days of anoxia (B). The survivors were examined for an unimpaired or impaired phenotype. Line denotes groups with a significant decrease in the number of animals with an unimpaired phenotype in comparison to glp-1(e2141) (p<.05 Student's paired one tailed-T-test). For all experiments, the total number of animals assayed is N>180 from four independent experiments; error bar represents standard deviation.

Figure 6. Wild-type animals fed metformin survive long-term anoxia.

N2 adult hermaphrodites were raised in the specified conditions from L1 larvae to one-day old adult and then exposed to three days of anoxia. The survivors were examined for an unimpaired or impaired phenotype. Line indicates a statistically significant increase in overall survival compared to animals grown at 20°C on OP50 bacteria in the absence of 50 mM Metformin (p<.05 Student's paired one-tailed T-test). The long-term anoxia survival rate of aak-2(gt33) fed metformin was significantly less in comparison to wildtype animals fed metformin (*). For all experiments the total number of animals assays is N>180 from four independent experiments; error bars indicate standard deviation.

Figure 7. Animals with an enhanced anoxia survival phenotype have increased levels of carminic acid staining.

Animals were fed carminic acid, a fluorescent derivative of glucose that incorporates into glycogen and trehalose, to detect carbohydrates in the intestine of C. elegans. Images shown include DIC, fluorescent and merged image (A); merged images are show for (B, C). Animals grown at 25°C have increased level of carminic acid which decreases after exposure to long-term anoxia (A). Animals switched to an HT115 diet have an increased level of carminic acid (B). Animals fed metformin, regardless of temperature, have increased level of carminic acid (C). aak-2(RNAi) and aakg-2(RNAi) suppresses the high level of carminic acid detected in N2 animals raised at 25°C (D). Animals shown are representative of >25 animals assayed for each experiment. Scale bar, 20 µm.

Figure 8. The daf-2(e1370) and glp-1(e2141) animals have increased levels of carminic acid staining that can be suppressed by specific components of AMPK.

Animals of specified genotype or RNAi experiment were fed carminic acid to detect carbohydrate levels in the intestine. Merged images (DIC and fluorescence) of anterior region of the animal are shown. Images shown are representative of >25 number of animals assayed for each experiment. Scale bar, 20 µm.