Saiki R et al. (2008) Aging Cell "Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q."

Saiki R, Clarke CF, Furukawa S, Larsen PL, Bixler T, Lunceford AL, Lee W, Dang P

[Aging Cell, 2008]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. C. elegans fed E. coli devoid of Q(8) have a significant life span extension when compared to C. elegans fed a standard "Q-replete"E. coli diet. Here we examine possible mechanisms for the life span extension caused by the Q-less E. coli diet. A bioassay for Q uptake shows that a water-soluble formulation of Q(10) is effectively taken up by both clk-1 mutant and wild-type nematodes, but does not reverse life span extension mediated by the Q-less E. coli diet, indicating that life span extension is not due to the absence of dietary Q per se. The enhanced longevity mediated by the Q-less E. coli diet cannot be attributed to dietary restriction, different Qn isoforms, reduced pathogenesis or slowed growth of the Q-less E. coli, and in fact requires E. coli viability. Q-less E. coli have defects in respiratory metabolism. C. elegans fed Q-replete E. coli mutants with similarly impaired respiratory metabolism due to defects in complex V also show a pronounced life span extension, although not as dramatic as those fed the respiratory deficient Q-less E. coli diet. The data suggest that feeding respiratory incompetent E. coli, whether Q-less or Q-replete, produces a robust life extension in wild-type C. elegans. We believe the fermentation-based metabolism of the E. coli diet is an important parameter of C. elegans longevity.

Ryoichi Saiki et al. (2007) International Worm Meeting "Investigation of the effect of Q-less E. coli diet on Caenorhabditis elegans life span."

Tarra Wong, Pamela L. Larsen, Ryoichi Saiki, Catherine F. Clarke, Adam L. Lunceford, Wendy Lee

[International Worm Meeting, 2007]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Q is an essential component of respiratory chain and a lipid soluble antioxidant. C. elegans fed E. coli devoid of Q₈ have a significant life span extension when compared to C. elegans fed a Q-replete E. coli diet. Extended life spans are also observed in C. elegans clk-1 mutants which have defects in Q biosynthesis and rely on a dietary supply of Q. These results suggest that the lack of Q is the important parameter affecting life span. Here we examine possible mechanisms for the life span extension caused by the Q-less E. coli diet. We show that supplementation of the Q-less E. coli diet with a water-soluble formulation of Q₁₀ (NovaSOL® Q), did not reverse the life span extension. Rescue of the clk-1 Q auxotrophy and Q₁₀ content in isolated mitochondria indicate that the Q₁₀ supplied in this manner is being assimilated. Thus the life span extension mediated by the Q-less E. coli diet cannot be attributed to the absence of Q in the diet. We show the enhanced life span mediated by the Q-less diet cannot be attributed to dietary restriction, altered isoprenoid tail lengths of the Qn isoform provided, slowed growth of the Q-less E. coli, or to reduced pathogenicity of the Q-less E. coli and in fact depends on the viability of the Q-less E. coli mutants. Q-less E. coli have defects in respiratory metabolism and fail to grow on media containing succinate as the carbon source. C. elegans fed Q-replete E. coli mutants with impaired respiratory metabolism due to defects in the ATP synthase (complex V) also show a pronounced life span extension, although feeding the Q-less E. coli diet mediates a more pronounced life span extension. Thus, respiratory deficiency of the E. coli diet is an important parameter of worm life span extension. C. elegans ttx-3 mutants, harboring defects in the gene encoding a LIM homeodomain transcription factor, cannot distinguish the Q-less from the Q-replete respiratory defective E. coli. The difference in life span extension mediated by the two respiratory deficient E. coli diets depends on food seeking behavior exhibited by N2 worms. The data suggest that the life span extension of N2 worms fed Q-less E.coli diet is caused by a combination of respiratory deficiency of the E. coli and the food seeking behavior of C. elegans. This work was supported by NIA Grant AG19777 and by the Ellison Medical Foundation.

Pamela L Larsen (2001) International C. elegans Meeting "C. elegans life span is extended by a diet of E. coli lacking coenzyme Q"

Pamela L Larsen

[International C. elegans Meeting, 2001]

As originally described, the clk-1 mutants of C. elegans develop slowly and exhibit an extended life span. Recently, the clk-1 mutants have been shown to have a defect in the biosynthesis of coenzyme Q 9 , the normal isoform synthesized by C. elegans where 9 designates the number of isoprene units in the polyisoprenoid tail. Coenzyme Q is an essential component of the mitochondrial respiratory electron transport chain, as it is required for the function of complexes I, II, and III. The clk-1 mutants lack coenzyme Q 9 and instead rely on the coenzyme Q 8 supplied by their standard diet of E. coli . These results implied that a reduction in the levels of coenzyme Q may be responsible for the slowed development and increased life span reported for the clk-1 mutants maintained on the standard Q-replete E. coli diet. Wild type worms appear to partially rely on dietary Q, as evidenced by a one-day delay in development (egg to adult) on Q-less food. To test whether a decrease in coenzyme Q could alter life span of wild type animals, wild-type L4 larvae were switched from a standard diet of Q 8 -replete E. coli to a diet lacking coenzyme Q, and were subsequently maintained on this Q-less diet throughout adulthood. Three distinct E. coli mutant strains were provided as Q-less diets, including strains lacking either the O - or C -methyltransferase enzymes of Q biosynthesis, and an E. coli mutant lacking the 4-HB:polyprenyldiphosphate transferase. A 60% increase in nematode life span was observed for the first two of these Q-less E. coli strains, suggesting that the distinct Q-biosynthetic intermediate accumulating in each of these Q-less E. coli mutants was not responsible for producing the observed life span extension. Thus at face value, consumption of Q 8 shortens wild type life span and it is this short life span that is considered normal. Alternatively, the diet/environment interaction that changes longevity could be due to different metabolic products in the Q-replete and Q-less bacteria. Mutations in two loci, daf-12 and daf-16 , suppress the life span extension of a number of previously isolated Age mutations. However, neither daf-12 nor daf-16 suppress the life span extension generated by the Q-less E. coli diet. Assuming that the life span extension resulting from the Q-less E. coli diet is a phenocopy of the clk-1 mutation, then the lack of suppression by daf-16 is consistent with previous reports. The Age mutants clk-1(qm30, qm51, and e2519 ) and daf-2(e1370 and m41 ) at the restrictive temperature, were transferred to the Q-less E. coli diet as L4 larvae, and for each of the mutants tested, further increases in life span were observed. For clk-1 mutants, the expectation had been premature death, since its larval development depends on a supply of dietary Q. However, it is likely that the clk-1 mutants cultured on Q-replete E. coli during development accumulate coenzyme Q 8 , and the life span extension observed for the adult animals is presumably due to the declining levels of Q 8 from lack of a dietary source. The increased life span extension in Age mutants fed diets lacking coenzyme Q suggest that these longevity mechanisms are additive.

Ryoichi Saiki et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Caenorhabditis elegans lifespan is profoundly modulated by its diet of E. coli: What are the roles of E. coli metabolism and coenzyme Q?"

Peter Dang, Tarra Bixler, Catherine Clarke, Pamela Larsen, Ryoichi Saiki, Adam Lunceford, Wendy Lee

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Q is an essential component of respiratory electron transport and a potent lipid soluble antioxidant. Extended lifespans are observed in C. elegans clk-1 mutants with defects in Q biosynthesis. Intriguingly, mice heterozygous for a clk-1 gene disruption also show increased lifespan. C. elegans fed E. coli devoid of Q8 have a significant life span extension when compared to C. elegans fed a standard Q-replete E. coli diet. These results initially suggested that the lack of Q is the important parameter affecting lifespan. However, recent results indicate that feeding respiratory incompetent E. coli, whether Q-replete or Q-less, produces a robust lifespan extension in C. elegans. C. elegans skn-1 mutants fed the respiratory incompetent E. coli diets also show lifespan extension, suggesting that the response is independent of a dietary restriction effect. The respiratory incompetent E. coli diet may be imposed well after the worms reach adulthood, including post-reproductive adults, indicating that lifespan extension operates independently of worm development. Curiously, C. elegans fed the Q-less E. coli diet seem to exhibit more pronounced lifespan extension than when fed respiratory incompetent E. coli that are Q-replete. C. elegans ttx-3 mutants, harboring defects in the gene encoding a LIM homeodomain transcription factor, fail to distinguish the Q-less from the Q-replete respiratory defective E. coli. The difference in life span extension mediated by the two respiratory deficient E. coli diets depends on normal food seeking behavior exhibited by N2 C. elegans. The data suggest that the life span extension of N2 worms fed Q-less E. coli diet is caused by a combination of respiratory deficiency of the E. coli and the food seeking behavior of C. elegans. This work was supported by NIA Grant AG19777, and by the Ellison Medical Foundation.

Jonassen T et al. (2003) J Biol Chem "Reproductive fitness and quinone content of Caenorhabditis elegans clk-1 mutants fed coenzyme Q isoforms of varying length."

Larsen PL, Clarke CF, Jonassen T, Davis DE

[J Biol Chem, 2003]

Caenorhabditis elegans clk-1 mutants lack coenzyme Q(9) and accumulate the biosynthetic intermediate demethoxy-Q(9). A dietary source of ubiquinone (Q) is required for larval growth and development of the gonad and germ cells. We considered that uptake of the shorter Q(8) isoform present in the Escherichia coli food may contribute to the Clk phenotypes of slowed development and reduced brood size observed when the animals are fed Q-replete E. coli. To test the effect of isoprene tail length, N2 and clk-1 animals were fed E. coli engineered to produce Q(7), Q(8), Q(9), or Q(10). Wild-type nematodes showed no change in reproductive fitness regardless of the Q(n) isoform fed. clk-1(e2519) fed the Q(9) diet showed increased egg production; however, this diet did not improve reproductive fitness of the clk-1(qm30) animals. Furthermore, animals with the more severe clk-1(qm30) allele become sterile and their progeny inviable when fed Q(7)-containing bacteria. The content of Q(7) in the mitochondria of clk-1 animals was decreased relative to Q(8), suggesting less effective transport of Q(7) to the mitochondria, impaired retention, or decreased stability. Additionally, regardless of E. coli diet, clk-1(qm30) animals contain a dysfunctional dense form of mitochondria. The gonads of clk-1(qm30) worms fed Q(7)-containing food were severely shrunken and disordered. The differential fertility of clk-1 mutant nematodes fed Q isoforms may result from changes in Q localization, altered recognition by Q-binding proteins, and/or potential defects in mitochondrial function resulting from the mutant CLK-1 polypeptide itself.

Jonassen T et al. (2002) J Biol Chem "Development and fertility in Caenorhabditis elegans clk-1 mutants depend upon transport of dietary coenzyme Q8 to mitochondria."

Marbois BN, Jonassen T, Faull KF, Clarke CF, Larsen PL

[J Biol Chem, 2002]

The Caenorhabditis elegans clk-1 mutants lack coenzyme Q, and instead accumulate the biosynthetic intermediate demethoxy-Q(9) (DMQ(9)). clk-1 animals grow to reproductive adults, albeit slowly, if supplied with Q(8)-containing Escherichia coli. However, if Q is withdrawn from the diet, clk-1 animals either arrest development as young larvae or become sterile adults depending upon the stage at the time of the withdrawal. To understand this stage-dependent response to a Q-less diet, the quinone content was determined during development of wild-type animals. The quinone content varies in the different developmental stages in wild-type fed Q(8)-replete E. coli. The amounts peak at the second larval stage, which coincides with the stage of arrest of clk-1 larvae fed a Q-less diet from hatching. Levels of the endogenously synthesized DMQ(9) are high in the clk1(qmM30)-arrested larvae and sterile adults fed Q-less food. Comparison of quinones from animals fed a Q-replete or a Q-less diet establishes that the Q(8) present is assimilated from the E. coli. Furthermore, this E. colispecific Q(8) is present in mitochondria isolated from fertile clk-1(qm30) adults fed a Q-replete diet. These results suggest that the uptake and transport of dietary Q(8) to mitochondria prevent the arrest and sterility phenotypes of clk-1 mutants and that DMQ is not functionally equivalent to Q.

Tanya Jonassen et al. (2002) West Coast Worm Meeting "Development and fertility in C. elegans clk-1 mutants depends upon transport of dietary coenzyme Q8 to mitochondria"

Beth N Marbois, Pamela L Larsen, Catherine F Clarke, Kym F Faull, Tanya Jonassen

[West Coast Worm Meeting, 2002]

The C. elegans clk-1 mutants lack coenzyme Q9 and instead accumulate the biosynthetic intermediate demethoxy-Q9 (DMQ9). clk-1 animals grow to adulthood, albeit slowly, if supplied with Q-containing E. coli. However, depending upon when Q is withdrawn from the diet, these animals either arrest development as larvae or become sterile adults. To understand this stage-dependent response to a Q-less diet, the quinone content of wild-type and clk-1(qm30) worms during development was determined. The quinone content varies in the different developmental stages in wild-type fed Q-replete E. coli. The amounts peak at the second larval stage, which coincides with the stage of arrest of clk-1 larvae fed a Q-less diet from hatching. Levels of the endogenously synthesized DMQ9 are quite high in the clk-1(qm30) arrested larvae and sterile adults derived from dauer larvae that were fed Q-less food. Analysis of quinones from mutant and wild-type animals fed a Q-less diet establishes that the Q8 present is assimilated from the E. coli. Furthermore, this E. coli-specific Q isoform is present in mitochondria isolated from fertile clk-1(qm30) adults fed a Q-replete diet. These results suggest that DMQ cannot functionally replace Q and that the uptake and transport of dietary Q8 to mitochondria prevents the arrest and sterility phenotypes of clk-1 mutants.

Jonassen T et al. (2001) Proc Natl Acad Sci U S A "A dietary source of coenzyme Q is essential for growth of long-lived Caenorhabditis elegans clk-1 mutants."

Jonassen T, Larsen PL, Clarke CF

[Proc Natl Acad Sci U S A, 2001]

Mutations in the clk-1 gene of the nematode Caenorhabditis elegans result in slowed development, sluggish adult behaviors, and an increased lifespan. CLK-1 is a mitochondrial polypeptide with sequence and functional conservation from human to yeast. Coq7p, the Saccharomyces cerevisiae homologue, is essential for ubiquinone (coenzyme Q or Q) synthesis and therefore respiration. However, based on assays of respiratory function, it has been reported that the primary defect in the C. elegans clk-1 mutants is not in Q biosynthesis. How do the clk-1 mutant worms have essentially normal rates of respiration, when biochemical studies in yeast suggest a Q deficiency? Nematodes are routinely fed Escherichia coli strains containing a rich supply of Q. To study the Q synthesized by C. elegans, we cultured worms on an E. coli mutant that lacks Q and found that clk-1 mutants display early developmental arrest from eggs, or sterility emerging from dauer stage. Provision of Q-replete E. coli rescues these defects. Lipid analysis showed that clk-1 worms lack the nematode Q(9) isoform and instead contain a large amount of a metabolite that is slightly more polar than Q(9). The clk-1 mutants also have increased levels of Q(8), the E. coli isoform, and rhodoquinone-9. These results show that the clk-1 mutations result in Q auxotrophy evident only when Q is removed from the diet, and that the aging and developmental phenotypes previously described are consistent with altered Q levels and distribution.

Tanya Jonassen et al. (2001) International C. elegans Meeting "Developmental control of coenzyme Q levels - the L2 arrest in clk-1 mutants coincides with an increased reliance on respiratory energy metabolism"

Kym Faull, Pamela L Larsen, Catherine F Clarke, Tanya Jonassen

[International C. elegans Meeting, 2001]

The developmental and long-lived phenotypes described for the C. elegans clk-1 mutants depend critically upon a dietary source of coenzyme Q. Coenzyme Q functions in cells as an essential redox-active component of both mitochondrial and plasma membrane electron transport, as an essential lipid antioxidant, and plays a role in uridine synthesis and uncoupling activity in mitochondria. When provided a diet lacking coenzyme Q, the clk-1 mutants display a growth arrest in early development from eggs, and sterility upon emerging from dauer larvae. As observed for the corresponding yeast coq7 mutants, the clk-1 mutants have a defect in coenzyme Q biosynthesis. Following growth on Q 8 -replete E. coli , the standard diet for C. elegans , the clk-1 mutants completely lack Q 9 and accumulate the biosynthetic intermediate demethoxy-Q 9 . The subscripts 8 and 9 refer to the number of isoprene units in the tail of Q synthesized by E. coli and C. elegans , respectively. The clk-1 mutants display the early developmental arrest when fed any of three distinct Q-less E.coli strains, including ubiA - , ubiE - , or ubiG - mutants. These results indicate that the clk-1 arrest phenotype results from the lack of Q and not from the distinct Q-intermediates that accumulate in these Q-less E. coli strains. Slow growing C. elegans mutants that map to genetic loci other than clk-1 , were evaluated for Q biosynthesis. This was of particular interest due to the overlap of the physical map position of the homologs of the yeast genes COQ2 and COQ5 with the genetic interval containing clk-2 . We find that clk-2 , clk-3 , and mev-1 produce coenzyme Q, as detected by HPLC/ECD assays. We find that clk-2 , clk-3 , gro-1 and gro-2 all grow to adulthood on a diet of Q-less E. coli . The very slow developing and long-lived double mutant daf-2 clk-1 is Q-defective by both assays. These data indicate that slow growth phenotype can be accounted for by decreases in the level of Q 9 only for clk-1 . We have speculated that the clk-1 mutant worms fed the standard Q-replete E. coli may compensate for their Q 9 deficiency by increasing synthesis of demethoxy- Q 9 and rhodoquinone-9 (RQ 9 ), and by increasing their uptake of Q 8 from their diet of E. coli . To investigate this in detail, we have quantified the levels of quinones in several developmental stages for wild type fed OP50 to establish the normal pattern. We then quantified the level demethoxy-Q 9 and other Q isoforms in clk-1 mutant eggs, L1 larvae, arrested L2, L4, and young adult. These results show distinct changes in quinone content during the development of the animals. These data suggest that the arrest coincides with a period of development where there is increased demand for respiratory metabolism/nucleotide synthesis.

Adam L Lunceford et al. (2005) International Worm Meeting "Investigation of the effect of dietary Q on Caenorhabditis elegans lifespan"

Satoru Furukawa, Ryoichi Saiki, Pamela L Larsen, Catherine F Clarke, Naoaki Ishii, Wendy Lee, Tarra Wong, Adam L Lunceford

[International Worm Meeting, 2005]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Q is an essential component of respiratory electron transport, and a potent lipid soluble antioxidant. C. elegans fed E. coli devoid of Q8 have a significant lifespan extension when compared to C. elegans fed a standard Q-replete E. coli diet. However, Ishiis group has shown that C. elegans fed a standard E. coli diet supplemented with a water-soluble formulation of Q10 also have an extended lifespan. Here we examine possible mechanisms for these opposing lifespan outcomes. The rates of consumption of the Q-less and Q-replete E. coli by N2 were not different, and the brood sizes remained the same, indicating the enhanced lifespan of C. elegans fed the Q-less E. coli diet does not result from dietary restriction. C. elegans fed different strains of E. coli engineered to produce Q8, Q9, or Q10 show no significant differences in lifespan, indicating that provision of Q10 instead of Q8 via E. coli does not extend lifespan. However, worms fed a Q7-replete E. coli have a significant lifespan extension relative to Q8-replete E. coli, albeit not as dramatic as those fed the Q-less bacteria. Since N2 animals assimilate and deliver Q7 and Q8 to similar extents to the mitochondria, there is no evidence that decreased uptake of Q7 produces the lifespan extension. A biological assay for uptake was performed for a water-soluble formulation of Q10 made by AQUANOVA<sym02>. C. elegans clk-1 mutants fed a Q-less diet arrest at the L2 larval stage and are sterile. However, clk-1 mutants fed the standard Q-replete E. coli diet are rescued by the dietary supply of Q8. We found that the AQUANOVA<sym02> Q10 was able to rescue the clk-1 growth arrest and sterility phenotypes of a Q-less diet. We have shown that rescue of clk-1 mutants by dietary Q was associated with its delivery to mitochondria, suggesting that the water-soluble Q10 formulation is delivered to mitochondria, although the amount incorporated into N2 adults is unknown. The increase in adult life span due to Q-less or Q7-replete E. coli or supplemented with a water-soluble formulation of Q10 warrants further investigations into the mechanisms responsible for these lifespan outcomes. This work was supported by NIA Grant AG19777 and by the Ellison Medical Foundation.