Adenosine nucleotide biosynthesis and AMPK regulate adult life span and mediate the longevity benefit of caloric restriction in flies

Abstract

A common thread among conserved life span regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of AMP biosynthetic enzymes extend Drosophila life span. The life span benefit of these mutations depends upon increased AMP:ATP and ADP:ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended life span, while AMPK RNAi reduced life span. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed life span extension. Remarkably, this simple change in diet also blocked the prolongevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult life span; provide a mechanistic link between cellular anabolism and energy sensing pathways; and indicate that dietary adenine manipulations might alter metabolism to influence animal life span.

Figure 1. Adenylosuccinate Synthetase regulates lifespan

(A and B) Heterozygous F71 P-element insertion extends lifespan of female and male adult flies (log rank, P<0.00001 in either case). (C and D) Log mortality plots of F71 heterozygous females and males. (E) Insertions of F71 and HA2022 in the 5’ untranslated region of AdSS. (F) AdSS expression is reduced in F71 and HA2022 heterozygotes. Data presented ± s.e.m. (* P<0.05, ** P<0.01, student’s t test). (G and H) Independently derived AdSS heterozygous insertions, F71 and HA2022, extend lifespan (log rank of any heterozygote compared to controls, P<0.008). (I and J) Transgenic expression of wild type AdSS in the F71 heterozygous background restores lifespan to that of controls (log rank of F71 heterozygous to transgenic, P<0.00001; log rank of transgenic to controls, P = 0.58). Also see Table S1.

Figure 2. AMP biosynthesis pathways

AMP biosynthesis occurs though two distinct pathways, de novo and salvage. Adenylosuccinate Synthetase (AdSS) catalyzes the first committed step in de novo AMP biosynthesis followed by the action of Adenylosuccinate Lyase (AdSL). AMP Deaminase (AMP deam) is the reverse of these two reactions, converting adenosine monophosphate (AMP) into inosine monophosphate (IMP). The salvage pathway converts adenine, via Adenine Phosphoribosyltransferase (Aprt), or adenosine, via Adenosine Kinase (AdenoK), to AMP. Adenylate Kinase (Adk) also produces AMP from ADP substrate molecules. Prior to committed AMP biosynthesis, AdSL and Aminoimidazolecarboxamide Formyltransferase (AICAR-ft) sequentially catalyze the final reactions of IMP synthesis. IMP is a substrate for the synthesis of both AMP and guanosine monophosphate (GMP).

Figure 3. AMP biosynthesis enzymes regulate lifespan

(A and B) Heterozygous insertional mutation of AdSL extends lifespan of adult female and male flies (log rank, P<0.00001 in either case). (C to F) Heterozygous insertions into AMP salvage pathway biosynthesis components (AdenoK and Aprt) extend female and male lifespan (log rank of any heterozygote compared to controls, P≤0.0002). (G and H) Heterozygous insertion in Adk2 increases longevity (log rank, P<0.004 in either case). (I) Heterozygous AMP deaminase insertion does not alter lifespan (log rank, P>0.8). (J and K) Heterozygous insertion in AICAR-ft does not alter female or male lifespan (log rank, P>0.8 in either case). Also see Table S1.

Figure 4. AMP biosynthesis enzymes affect adenosine nucleotide derivatives

(A–C) Food deprivation (Starved) and those heterozygous mutations of AMP biosynthesis components that increase lifespan also increase AMP and ADP, and reduce ATP concentration compared to controls. AMP deaminase or AICAR-ft mutations do not affect lifespan or adenosine nucleotide concentration. Transgenic overexpression of wild type AdSS (black hatched bar) rescues the alterations observed in F71 heterozygotes. Data presented ± s.e.m. (* Pπ.05, ** P<0.01, student’s t test). Throughout the figure hatched bars indicate genetic alterations that do not affect lifespan. (D and E) Food deprivation and heterozygous mutations of AMP biosynthesis components that increase lifespan increase AMP:ATP (D) and ADP:ATP (E) ratios compared to controls. AMP deaminase or AICAR-ft mutations do not affect lifespan, AMP:ATP or ADP:ATP ratios. Transgenic overexpression of wild type AdSS (black hatched bar) rescues the increased AMP:ATP and ADP:ATP ratios observed in F71 heterozygotes. Data presented ± s.e.m. (* P<0.05, ** P<0.01, student’s t test).

Figure 5. AMPK is activated in the heterozygous mutants and is required for their longevity benefit

(A and B) Food deprivation (Starved) and F71 heterozygous mutation increases AMPK and ACC phosphorylation. Each panel presented is from analysis of a single blot. (C and D) ImageJ quantification of increased AMPK and ACC phosphorylation, respectively. (E and F) Dominant negative AMPK (AMPK^DN) rescues F71 heterozygous lifespan extension (log rank, P>0.5). Also see Table S1.

Figure 6. Tissue specific AMPK function controls adult lifespan determination

(A) AMPK or AMPK RNAi transgenesis alters AMPK transcript levels in the expected manner (right, ImageJ quantification). (B) Tissue specific overexpression of AMPK increases AMPK phosphorylation, while AMPK RNAi reduces AMPK phosphorylation (right, ImageJ quantification). (C) Adult fat body expression of a wild-type AMPK (UAS-AMPK^wt) via the S106 RU486-inducible GAL4 driver (S106) extends lifespan (log rank, P<0.00001). (D) Muscle expression, using the myosin heavy chain GeneSwitch GAL4 driver (MHC-GS-GAL4), of AMPK^wt during adulthood extends lifespan (log rank, P<0.00001). (E) Adult fat body expression via S106 of an RNAi allele targeting AMPK reduces lifespan (log rank, P<0.00001). (F) Adult muscle expression, via MHC-GS-GAL4, of an RNAi allele targeting AMPK reduces lifespan (log rank, P<0.00001). Also see Table S1.

Figure 7. Dietary adenine supplementation blocks adult lifespan extension

(A) Dietary adenine supplementation [0.05% or 0.1% (w/v)] restores expected Mendelian ratios of homozygous AdSS and AdSL larval mutant lethality. Data presented ± s.e.m. (* P<0.05, ** P<0.01, student’s t test). (B) Adding 0.05% (w/v) adenine to the diet of adult F71 heterozygotes restores AMP:ATP ratios to control levels. Data presented ± s.e.m. (* P<0.05, ** P<0.01, student’s t test). (C) Supplementing the diet of adult F71 heterozygotes with 0.05% (w/v) adenine rescues lifespan extension, but does not alter control longevity (log rank, P>0.3). (D) Dietary adenine supplementation blocks the lifespan benefit of dietary restriction (DR) (log rank of DR compared to all other curves, P<0.00001; log rank comparing all other curves, P>0.1). Also see Table S1.