Intracellular protein glycosylation modulates insulin mediated lifespan in C.elegans

Abstract

O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a regulatory, nuclear and cytoplasmic post-translational glycosylation of proteins associated with age-related diseases such as Alzheimer's, Parkinson's, and type II diabetes. Global elevation of O-GlcNAc levels on intracellular proteins can induce insulin resistance, the hallmark of type II diabetes, in mammalian systems. InC. elegans, attenuation of the insulin-like signal transduction pathway increases adult lifespan of the nematode. We demonstrate that the O-GlcNAc cycling enzymes OGT and OGA, which add and remove O-GlcNAc respectively, modulate lifespan in C. elegans. Median adult lifespan is increased in an oga-1 deletion strain while median adult life span is decreased upon ogt-1 deletion. The O-GlcNAc-mediated effect on nematode lifespan is dependent on the FoxO transcription factor DAF-16. DAF-16 is a key factor in the insulin-like signal transduction pathway to regulate reproductive development, lifespan, stress tolerance, and dauer formation in C. elegans. Our data indicates that O-GlcNAc cycling selectively influences only a subset of DAF-16 mediated phenotypes, including lifespan and oxidative stress resistance. We performed an affinity purification of O-GlcNAc-modified proteins and observed that a high percentage of these proteins are regulated by insulin signaling and/or impact insulin pathway functional outcomes, suggesting that the O-GlcNAc modification may control downstream effectors to modulate insulin pathway mediated cellular processes.

Figure 6.

A conserved insulin signaling pathway in C. elegans regulates numerous functions including stress response, metabolism, dauer formation, and reproductive development by restricting the nuclear localization of the DAF-16/FoxO transcription factor upon nutrient availability [45]. Upon ligand binding (top panel), the insulin-like receptor DAF-2 activates the AGE-1 PI3 kinase that facilitates the activation of PDK-1 and AKT-1. AKT-1-mediated phosphorylation sequesters DAF-16 in the cytoplasm. In the absence of PI3K/AKT signaling (bottom panel) DAF-16 enters the nucleus and regulates the expression of target genes to mediate numerous DAF-16 dependent processes, including those listed.

Figure 1.. Elevated O-GlcNAc levels extend adult lifespan in C. elegans.

(A) The oga-1(ok1207) mutant has an extended adult lifespan while the ogt-1(ok1474) mutant has a reduced lifespan relative to wild-type animals, assayed at 20°C. Lifespan curves are based on pooled data from multiple replicates described in Table 1. (B) OGA-1 and OGT-1 regulate cellular O-GlcNAc-modified protein levels. Western blot of total animal extract from ogt-1(ok1474), wild-type, and oga-1(ok1207) animals probed with antibodies against O-GlcNAc and tubulin showing marked differences in total O-GlcNAcylated protein levels. (C) Lifespan extension in the oga-1 mutant is DAF-16-dependent, as seen in the reduced lifespan of the oga-1(ok1207); daf-16(mu86) double mutant relative to the oga-1(ok1207) single mutant. (D) Loss of O-GlcNAc modification, in the daf-2(e1370); ogt-1(ok1474) double mutant, reduces the extended lifespan of the daf-2(e1370) mutant. Control curves are re-plotted to facilitate comparisons in panels A, C, and D.

Figure 2.. Elevated O-GlcNAc modification of proteins does not increase adult lifespan further in long-lived insulin signaling pathway mutant animals.

(A and B) Lifespan of age-1(hx546) and sgk-1(ok538) mutants is dependent on protein O-GlcNAc modification as seen in age-1; ogt-1 (A) and sgk-1; ogt-1 (B) double mutants. (C and D) The adult lifespan extension associated with the oga-1(ok1207) mutant is not synergistic or additive with mutations of the long-lived insulin signaling pathway kinase age-1 (C) or sgk-1 (D). Lifespan curves (A-D) are based on pooled data from multiple replicates as described in Table 1. Control curves are re-plotted to facilitate comparisons in panels A-D.

Figure 3.. Aberrant protein O-GlcNAc cycling modestly enhances DAF-16 nuclear localization.

DAF-16::GFP (translational fusion) nuclear localization in oga-1(RNAi) and ogt-1(RNAi) animals is modestly increased relative to control (vector) RNAi animals but much less than observed in age-1(RNAi) animals (upper panel). Lower panel is the quantification of DAF-16::GFP levels in intestinal cells upon RNAi inactivation of ogt-1, oga-1, age-1, and vector control.

Figure 4.. Inactivation of O-GlcNAc cycling enzymes only affects a subset of functions regulated by the insulin-like signaling pathway.

(A) The oga-1(ok1207) mutant is resistant to oxidative stress (100mM paraquat for 9 hrs). Notably, the oxidative stress resistance in daf-2(e1370) mutant is significantly reduced in the daf-2(e1370); ogt-1(ok1474) double mutant, suggesting that protein O-GlcNAc modification contributes to the oxidative resistance of the daf-2 mutant. (B) The long-lived oga-1(ok1207) mutant is sensitive to heat stress (35°C), unlike the long-lived daf-2 (e1370) mutant. (C) oga-1 and ogt-1 mutants have normal developmental timing [from hatch to the fourth larval molt] at 20°C. The daf-2 mutant has delayed post-embryonic development, and this delay is unaffected by loss of O-GlcNAc modification in the daf-2; ogt-1 double mutant. (D) oga-1 and ogt-1 mutants generate normal numbers of offspring while the daf-2 mutant has reduced fecundity at 20°C. The reduced fecundity in the daf-2 mutant is not rescued by loss of O-GlcNAc modification in the daf-2; ogt-1 double mutant. Asterisks above bars denote statistically significant differences from wild type (* p < 0.05 and ** p < 0.01); while asterisks above solid lines connecting two genotypes denote statistically significant differences between those genotypes.

Figure 5.. Aberrant protein O-GlcNAc cycling differentially affects distinct stress response pathways in C. elegans.

(A) Images and quantification of sod-3::GFP reporter expression upon inactivation of OGA-1 and the insulin pathway effector kinase AGE-1. (B) Images and quantification of hsp-16-2::GFP reporter expression upon inactivation of OGA-1 and AGE-1. Note that inactivation of AGE-1 by RNAi induces both SOD-3 and HSP-16 expression, while OGA-1 inactivation only induces SOD-3 expression. Asterisks denote statistically significant differences from vector RNAi controls (* p < 0.05 and ** p < 0.01).