Graduation Year

2015

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biology

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Patrick Bradshaw, Ph.D.

Committee Member

Meera Nanjundan, Ph.D.

Committee Member

Dominic D. Agostino, Ph.D.

Committee Member

Sandra Westerheide, Ph.D.

Keywords

Aging, C. elegans, Ketones, Metabolites

Abstract

Understanding how metabolites contribute to anaplerosis, antioxidant effects, and hormetic pathways during aging is fundamental to creating supplements and dietary habits that may decrease age-associated disease and decline, thus improving the quality of life in old age. In order to uncover metabolic pathways that delay aging, the effects of large sets of metabolites associated with mitochondrial function on lifespan were investigated.

Malate, the tricarboxylic acid (TCA) cycle metabolite, increased lifespan and thermotolerance in C. elegans. Addition of fumarate and succinate also extended lifespan and all three metabolites activated nuclear translocation of the cytoprotective DAF-16/FOXO transcription factor and protected from paraquat-induced oxidative stress. The increased longevity provided by malate addition did not occur in fumarase (fum-1), glyoxylate shunt (gei-7), succinate dehydrogenase flavoprotein (sdha-2), or soluble fumarate reductaseF48E8.3 RNAi knockdown worms. Therefore, to increase lifespan, malate must be first converted to fumarate, then fumarate must be reduced to succinate by soluble fumarate reductase and the mitochondrial electron transport chain complex II. Lifespan extension induced by malate depended upon the longevity regulators DAF-16 and SIR-2.1. Malate supplementation did not extend the lifespan of long-lived eat-2 mutant worms, a model of dietary restriction. Malate and fumarate addition increased oxygen consumption, but decreased ATP levels and mitochondrial membrane potential suggesting a mild uncoupling of oxidative phosphorylation.

Each of the twenty amino acids was individually supplemented to C. elegans and the effects on lifespan were determined. All amino acids except phenylalanine were found to extend lifespan at least to a small extent at one or more of the 3 concentrations tested with serine, histidine, and proline showing the largest effects. In most cases, amino acid supplementation did not extend lifespan in eat-2 worms, a model of dietary restriction or in daf-16, sir-2.1, rsks-1 (S6 kinase), or aak-2 (AMPK) longevity pathway mutants or in worms fed RNAi to skn-1, the C. elegans Nrf2 homolog. Serine and tryptophan addition further protected worms from Alzheimer’s amyloid-beta toxicity. Tryptophan and its catabolites nicotinic acid, picolinic acid, and NAD further induced a broad heat shock response. These results indicate that dietary amino acid imbalance and amino acid catabolism affect organismal longevity.

The ketone body beta-hydroxybutyrate (βHB) is a histone deacetylase (HDAC) inhibitor and has been shown to be protective in many disease models, but its effects on aging are not well studied. Therefore we determined the effect of βHB supplementation on the lifespan of C. elegans. βHB supplementation extended mean lifespan by approximately 20%. RNAi knockdown of HDACs hda-2 or hda-3 also increased lifespan and further prevented βHB-mediated lifespan extension. βHB-mediated lifespan extension required the DAF-16/FOXO and SKN-1/Nrf longevity pathways, the sirtuin SIR-2.1, and the AMP kinase subunit AAK-2. βHB did not extend lifespan in a genetic model of dietary restriction indicating that βHB is likely functioning through a similar mechanism. βHB addition also upregulated ΒHB dehydrogenase activity and increased oxygen consumption in the worms. RNAi knockdown of F55E10.6, a short chain dehydrogenase and SKN-1 target gene, prevented the increased lifespan and βHB dehydrogenase activity induced by βHB addition, suggesting that F55E10.6 functions as an inducible βHB dehydrogenase. Furthermore, βHB supplementation delayed Alzheimer's amyloid-beta toxicity and decreased Parkinson's alpha-synuclein aggregation. The results indicate that D-βHB extends lifespan through inhibiting HDACs and through the activation of conserved stress response pathways.

Aging is a progressive disease caused by the time dependent decline of an organism and is the primary risk factor for many human ailments, including heart disease, cancer, and Alzheimer’s disease. Uncovering metabolic pathways and metabolites that delay the onset of age-related decline was the primary drive of this investigation.

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