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SKN-1 coordination of stress adaptation, metabolism, and resource allocation in Caenorhabditis elegans

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SKN-1 coordination of stress adaptation, metabolism, and resource allocation in Caenorhabditis elegans

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SKN-1 COORDINATION OF STRESS ADAPATATION,
METABOLISM, AND RESOURCE ALLOCATION IN
CAENORHABDITIS ELEGANS

by
Dana Ann Lynn

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
MOLECULAR BIOLOGY

August 2016

ii
Acknowledgements
First of all, I would like to give a huge thanks to my current advisor, Dr. Sean Curran, for
everything he has done for me during my time at USC. Not only did he put his faith in me and
allow me to join his amazing lab when I was in need of finding a new home, he helped mold me
into who I am today. Without his expert guidance, unwavering support, and thoughtful advice I
would not have been able to accomplish what I have during my graduate career. His undying
mentorship, creativity, candor, positivity, love for pranks, and his excitement for science and
challenges has made it an absolute pleasure to work with him. I would also like to thank my
former advisor, Dr. Xuelin Wu, for helping my transition into graduate school by personally
training me, teaching me all about plants and avocados, and introducing me to lots of new and
wonderful kinds of food. She is a one-of-a-kind mentor in that she is always so patient, kind,
generous, has a great sense of humor, is wickedly intelligent, and has the best scientific hands I
have ever seen. Both of you have made me the scientist and the person I am today, and I am
forever in your debt. Thank you doesn’t even begin to describe the gratitude I have for both of
your time and efforts in training me to be a better person and scientist.
I would also like to thank my committee members: Drs. Matt Dean, Steve Finkel,
Christian Pike, and John Tower. Your constructive feedback on my scientific progress and
advice on my career have been immensely helpful over the years and I am very grateful for your
time and efforts. In the last two years, I feel like I have gained another mentor, Dr. Eileen
Crimmins. She has been a joy to interact with scientifically and during the Training Grant
program and recently she has also been extremely helpful in my job search. Thank you all for
guiding me to where I need to be, I am so thankful to have such supportive faculty throughout
my graduate studies.
iii
One of the biggest thanks I owe is to my family. To my wonderfully amazing parents, I
couldn’t have gotten this far without your love, support, and encouragement. You two are some
of the smartest, most hardworking individuals I know and you’ve instilled in me your work
ethics and determination to get the job done the right way (the first time), and for that I am
forever grateful. I also owe a lot to my older sister, Erica, who paved the way and showed me
that graduate school is worth all the hard work, sweat, and tears. I also want to give a shout out
to Erica’s husband, Michael, who has always had a fascination in my studies and with whom I
have had many fantastic scientific conversations. I would also like to thank my future family,
which includes my fiancé Kevin and his parents Tom and Jeanne. Kevin has been my rock
throughout the majority of my Ph.D. studies and I could not be more thankful for your doses of
sanity and fun when I most need them, your logical way of approaching problems, your caring
nature, and unlimited hug supply. I have the utmost respect for you and I cannot wait to start the
next chapter of our lives together. And to Tom and Jeanne, I couldn’t have asked for a better
“second family.” You two are some of the most caring and self-less individuals I have ever met
and I feel blessed to be welcomed into your family.
I also want to acknowledge my wonderful lab mates, past and present. Anna, you have
been like an older sister to me. From our first meeting at MCB Recruitment when we went on an
adventure to have my first ever In ‘N’ Out experience to becoming lab mates later that year, you
have always been so supportive, patient, and helpful. Jackie, from roommates to lab mates, you
have always been there for me, scientifically and as a friend. Thank you so much for making my
transition out to Los Angeles bright and sparkly and full of pandas. Hans, thank you for not only
being my bench mate, but also for being so helpful with experiments (especially lifespans) and
for being my source of sanity and sarcasm during classes and meetings. An, thank you for being
iv
my partner in crime when it comes to playing pranks, teaching me about drums and basses,
staying super late in lab as well as all of our collaborative studies. I couldn’t ask for a more
perfect pairing when it comes to co-authoring projects: we have worked so well together and
always make our best efforts to make each other’s lives easier when possible, and for that I am
truly grateful. Brett, thank you for bringing your amazing humor to the Curran lab. I don’t know
what I will do without your giraffe dances or the intense Midwest sports rivalries that we have.
Shanshan, thank you for your patient support and immense help during my transition into the
Curran lab. I appreciate your technical expertise as well as your guidance in planning ahead in
order to be as productive as possible. Akshat and Ajay, thank you for your helpful scientific
insights and questions as well as for teaching me how to play cricket. Jeremy, thank you for
being a wonderful student to teach and train and for always jamming out to classic rock with me.
Your creativity for Halloween costumes and gel making skills will never be forgotten. Alvin,
thank you for all of your help as I wrap up my studies here at USC. You have made lifespans and
long days of imaging much more entertaining with your beautiful singing voice and M.J. dance
moves. And thank you to our newest Curran lab member, Amy, for always bringing your fun,
bright, and creative attitude to the lab. And lastly, a huge thank you to Lori for all of her support,
from day to day tasks to helping with huge experiments it would be so much harder without you.
Not to mention all those late night food runs and just being a wonderful friend and person to
work with.

I wish all of you the best in your future endeavors.

v
Table of Contents
Acknowledgements ii
List of Tables and Figures vii
Abstract xi
Abbreviations xii
Chapter 1: Introduction 1

1.1 Significance
1.2 Introduction
1.3 Consequences of Diet Choice
1.4 SKN-1/Nrf2-Dependent Regulation of Dietary Stress
1.5 Metabolic Coordination
1.6 Human Implications
1.7 Figures
1.8 References

Chapter 2: SKN-1 and Nrf2 couples proline catabolism with 34
lipid metabolism during nutrient deprivation

2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.4 Results
2.5 Discussion
2.6 Figures
2.7 References

Chapter 3: Omega-3 and -6 fatty acids allocate somatic and 68
germline lipids to ensure fitness during nutrient
and oxidative stress in Caenorhabditis elegans

3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.4 Results
3.5 Discussion
3.6 Figures
3.7 References

Chapter 4: Acute starvation impacts epigenetic modifications 121
and lipid homeostasis in C. elegans
vi

4.1 Abstract
4.2 Introduction
4.3 Materials and Methods
4.4 Results
4.5 Discussion
4.6 Figures
4.7 References

Chapter 5: Concluding perspectives 138

vii
List of Tables and Figures
Chapter 1
Figure 1 Integration of diet into C. elegans phenotypes 15
Table 1 List of C. elegans genes and their human orthologs 16

Chapter 2
Figure 1 Mutation of alh-6 enhances fat mobilization and the 50
expression of FAO genes during starvation

Figure 2 SKN-1 coordinates proline and lipid metabolism 51
during starvation of C. elegans

Figure 3 Constitutive activation of SKN-1 protects animals 52
from HCD-induced fat accumulation

Figure 4 Conserved regulation of Nrf2 activity and FAO 53
genes by Aldh4a1

Figure 5 MDT-15 is a co-factor for SKN-1-mediated 54
lipid metabolism

Supplementary Figure 1 56
Analysis of lipid metabolism in alh-6 mutants
during starvation

Supplementary Figure 2 57
ROS is not involved in SKN-1 activation and lipid
metabolism in fasted alh-6 mutants

Supplementary Figure 3 58
Lipid metabolism genes and fat levels influenced
by SKN-1

Supplementary Figure 4 59
Mapping the lax225 mutation to mdt-15

Supplementary Table 1 60
Expression data of all annotated FAO genes in alh-6
mutant worms after three hours of fasting
viii

Supplementary Table 2 61
qPCR primer sequences

Supplementary Table 3 63
Starvation survival data

Chapter 3
Figure 1 SKN-1 activation mobilizes somatic fat 92
to the germline

Figure 2 Asdf is a starvation response dependent on 93
vitellogenesis

Figure 3 Oleic acid deficiency is causal for Asdf 94

Figure 4 ARA (omega-6) and EPA (omega-3) fatty acids 95
regulate Asdf

Figure 5 Asdf fuels germ cell maturation to ensure fitness 96

Supplementary Figure 1 97
ORO Asdf progression in wild-type and
SKN-1gf mutants

Supplementary Figure 2 98
Nile Red Asdf progression in wild-type and
SKN-1gf mutants

Supplementary Figure 3 99
Cohort analysis of % Asdf

Supplementary Figure 4 100
SKN-1 activation and oxidative stress induce Asdf

Supplementary Figure 5 101
Vitellogenin proteins transport lipids from the soma
to the germline in animals with Asdf

Supplementary Figure 6 102
Asdf is a specific response to nutrient deprivation

ix
Supplementary Figure 7 103
Asdf is a diet-dependent phenotype

Supplementary Figure 8 105
Oleic acid deficiency in SKN-1gf is reversed
by dietary glucose

Supplementary Figure 9 106
Oleic acid deficiency is sufficient to induce Asdf

Supplementary Figure 10 107
ARA and EPA precursors of eicosanoid
signaling molecules influence Asdf

Supplementary Figure 11 108
Variation of Asdf among natural isolates of C. elegans

Supplementary Figure 12 109
Matricide is enhanced in animals with Asdf after
24-hours of starvation

Supplementary Figure 13 110
Survival of acute exposure to H2O2 is influenced
by Asdf

Supplementary Table 1 111
Daily progeny production for wild-type and
SKN-1gf on OP50 diet

Supplementary Table 2 112
Daily progeny production for wild-type and
SKN-1gf on HT115 diet

Supplementary Table 3 113
OP50 RNAi knockdown efficiencies

Supplementary Table 4 114
mRNA expression levels of oleic acid and
eicosanoid biosynthesis pathway genes

Supplementary Table 5 115
Variation of Asdf phenotype among natural
isolates of C. elegans

x
Chapter 4
Figure 1 Synchronized larval stage-1 (L1) C. elegans have 130
altered adult lipid stores compared to animals
that hatch in the presence of food

Figure 2 Targeted RNA-interference (RNAi) screen identifies 131
DNA and histone modifiers that alter Asdf-
penetrance in SKN-1gf worms

Figure 3 Genetic mutants of the C. elegans 6mA demethylase 132
and methyltransferase have opposing effects on adult
lipid content that is dependent upon early-life starvation

Table 1 Genes tested in the RNAi screen 133

xi
Abstract

Animals must continually assess nutrient availability to develop appropriate strategies for
survival and reproductive success. It is no secret that nutritional state plays a large role in both
aging and health. Appropriate cellular energy usage is not only crucial for animal starvation
survival, but is also important for diseases such as obesity and cancer, which characteristically
have metabolic dysfunction. C. elegans are exceptionally well poised to handle bouts of
starvation as resource availability in the wild varies greatly. We recently discovered an
evolutionarily conserved pathway, regulated by the cytoprotective transcription factor SKN-
1/Nrf2, which integrates diet composition and availability with utilization for survival. These
responses have potent impact on organismal physiology and remarkably are influenced by
current and parental life history events, including choice of diet. It is important to be cognizant of
dietary intake and the impact that this can have throughout the life-history of the
nematode, Caenorhabditis elegans.

xii
Abbreviations
5mC: 5-methylcytosine
6mA: N6-methyladenosine
ALH-6: aldehyde dehydrogenase
AMP: adenosine monophosphate
AMPK: AMP-activated protein kinase
ARA: arachidonic acid
ARE: antioxidant response elements
Asdf: age-dependent somatic depletion of fat
ATP: adenosine triphosphate
Bag: bag of worms
bDR: bacterial dilution
BMI: body mass index
bZip: basic leucine zipper domain
C. elegans: Caenorhabditis elegans
ChIP: chromatin immunoprecipitation
CR: calorie restriction
Cyclo: cyclopropane fatty acid
DAPI: 4',6-diamidino-2-phenylindole
DGLA: dihomo-γ-linolenic acid
DIC: differential interference contrast
DR: dietary restriction
E. coli: Escherichia coli
xiii
EMS: ethyl methanesulfonate
EPA: eicosapentaenoic acid
ETA: eicosatetraenoic acid
F1: filial generation one
F2: filial generation two
FAO: fatty acid oxidation
GCMS: gas chromatography mass spectrometry
GFP: green fluorescent protein
h: hour
H2O2: hydrogen peroxide
HCD: high carbohydrate diet
hpf: hours postfeeding
HPLC: high pressure liquid chromatography
IPTG: isopropyl-β-D-thiogalactoside
Iso: iso-methyl branched chain fatty acid
Keap-1: kelch-like ECH-associated protein 1
L1: larval stage 1
L4: larval stage 4
MDT-15: mediator subunit 15
MUFA: monounsaturated fatty acid
NAC: N-acetyl-cysteine
NGM: nematode growth medium
NLP: neuropeptide-like protein
xiv
NMUR: neuromedin U receptor
NR: Nile Red
NRF2: nuclear factor, erythroid 2-like 2
O/N: overnight
ORO: Oil-Red-O
P5C: 1-pyrroline-5-carboxylate
PBS: phosphate buffered saline
PBST: 1X PBS + 0.01% Triton X-100
PUFA: polyunsaturated fatty acid
qPCR: quantitative polymerase chain reaction
RNAi: RNA-interference
ROS: reactive oxygen species
RPM: revolutions per minute
RT: room temperature
s: seconds
s.e.m.: standard error of the mean
SKN-1: SKiNhead 1
SKN-1gf: SKN-1 gain-of-function
TOR: target of rapamycin
VIT: vitellogenin
WDR-23: WD repeat-containing protein 23
WT: wild-type
1
CHAPTER 1: INTRODUCTION

The content of this chapter appears as submitted:
Lynn, DA and Curran SP. Chapter 17: Integration of metabolic signals.
C. elegans and its contribution to longevity and ageing research.

1.1 Significance
Over the last 25 years it has become evident that single gene mutations can result in
remarkable increases in lifespan. Of the gene mutations identified, the most potent at extending
life- and health-span are those that alter the quantity of food ingested [1] and those that disrupt
the animals’ perception of the amount of food ingested [2-5]. These mutations promote
longevity, animal health, and capacity for stress adaptation [6-9], but importantly reveal that an
intricate molecular and genetic network exists to integrate diet availability, utilization and animal
physiology [10-15].

1.2 Introduction
In order to survive, animals must be able to uptake and utilize diverse food sources from
their surrounding environment. The body’s main source of intracellular chemical energy, ATP, is
generated through the catabolism of macronutrients - carbohydrates, lipids, and proteins in that
food source. The nutritional quality of the diet is directly related to the macronutrient
composition and that formula has potent impacts on animal physiology and lifespan [16]. In most
multi-cellular organisms, food intake is not constant and animals must be able to store dietary
2
energy that can be easily mobilized when necessary. Therefore, the ability to adapt to changing
environments and food sources is of critical importance. C. elegans are bacteriovores that have
evolved the capacity to effectively utilize diverse bacterial diets in the wild for sustenance [17].
Surprisingly, worms are capable of effectively using many of these microorganisms to sustain
life and reproduce. The limiting factor it seems is the size of the bacterium as worms are passive
eaters and certain microbes, such as B. megaterium, is larger than the mouth opening. Regardless
of the bacteria ingested, C. elegans have evolved a remarkable capacity to adapt to the food
source provided. Recently, hints towards understanding the molecular mechanisms underlying
this dietary adaptation have emerged using worms harboring single gene mutations being fed the
two most commonly used bacterial diets in the laboratory (E. coli B—OP50 and E. coli K12—
HT115) [18-22]. The phenotypes that manifest from these gene mutations are variable on these
two similar E. coli diets, which provides evidence for diet-gene pairs; or genes that are essential
on one diet type but dispensable on others [20]. Although both diets are E.coli based, it is clear
that they are not nutritionally equivalent and feeding of these diets is known to differentially
affect organismal metabolism [23,24].
In general, limiting worms’ food consumption has been shown to increase their lifespans,
which is a conserved response in rodents and monkeys [25,4,26,27]. Calorie restriction (CR) is a
technique that reduces the amount of calories allowed in the diet to about 60- 70% of an ad
libitum diet [28]. However, it has become increasingly clear, across all organisms, that it is not
simply the number of calories that matters, but the composition of the diet, which has led to the
study of dietary restriction (DR) where the quality of the diet is altered [23]. A synthetic diet that
facilitates normal developmental timing, reproduction, and lifespan for worms has yet to be
synthesized, which makes DR studies difficult to design. However, C. elegans can eat a variety
3
of bacteria sources with varied dietary complexities, which provides an alternative approach to
assess diet composition on animal physiology. It is clear that different diets can affect worm
physiology, therefore it is of great interest to identify the key players that integrate these signals
and how the implications of these findings, which could only have been uncovered in C. elegans,
will impact our understanding of human aging, health and disease.

1.3 Consequences of Diet Choice
C. elegans is constantly on the lookout for possible food sources and has two pairs of
neurons that function to discern attractive odorants, the AWA and AWC [29,30]. In addition, the
main neuronal pair used to sense chemicals and pathogens that the worm wants to avoid is called
AWB [31,29]. C. elegans in the wild are exposed to many types of bacteria in its daily
adventures. Some of these bacteria can be used as food sources but some can be pathogenic. It is
important for these animals to be able to avoid pathogenic bacteria odors, which is a learned
aversive response [32-34]. Interestingly, laboratory cultured C. elegans does not actively avoid
E. coli OP50 or HT115 while studies have shown that these strains can be pathogenic over time
[35,17]; although when given the choice of a less pathogenic diet, such as B. subtilis, worms
prefer the safer food [36,37]. When worms are put onto plates with pathogenic Pseudomonas
aeruginosa PA14, for example, they are initially attracted to the bacterial lawn but after some
time, they leave the lawn [38]. It has been shown that worms will avoid non-pathogenic bacterial
lawns as well when they are specifically engineered to cause the knockdown of essential genes in
the host [39]. This avoidance behavior has been proposed to be a worm equivalent of “nausea”;
where it seems as though C. elegans can sense the occurrence of essential cellular deficiencies
[10] and as a first response, flee its current environment, assuming the sickness is from
4
something it ate. This idea is not so far-fetched as many of the bacteria C. elegans encounter in
the wild and may choose to eat, synthesize compounds that disable essential cellular pathways
such as protein synthesis and mitochondrial functions [40-42]. C. elegans can also sense
nutritional quality of their diet, as their behavior and physiology change depending on the
nutritional value of their food source [43]. Specifically, the animals will increase pharyngeal
pumping, pharyngeal muscle autophagy, and roaming behaviors when given a less-than-desirable
food source [1,44-46,17]. Therefore, when non-desirable food sources are present, nematodes
will alter their behaviors accordingly—either to attempt to find a new, less sickening food
source, or in order to maximize energy acquisition and maintain homeostasis.
Throughout the worm’s lifetime in a laboratory, its relationship with its E. coli food
source changes from predator:prey to prey:predator. As the worm ages, it is less able to grind up
the bacteria with its pharyngeal muscles, host defenses deteriorate, and the bacteria may have a
high proliferative capacity [47]. Therefore, the worm’s diet can be slightly toxic and as they age,
bacteria can block the pharynx and intestine leading to the worm’s death [48]. Although not
exactly a probiotic relationship, certain aspects of C. elegans physiology benefit from live
proliferating bacteria [49,50]. Developing larvae do not have live colonies of bacteria in their
gut, but this changes with age and similar to mammals [51], newly hatched worms can quickly
become infected with parasites present in their immediate environment [52]. In contrast, adult
worms have on the order of 10
4
bacteria living inside of them, which is ten times more than the
number of somatic cells they have in their bodies [53,54] Intriguingly, this is the same
ratiometric relationship that humans have with the microbes they host in their digestive system.
Although the immune system of C. elegans is much more rudimentary than human defense
mechanisms, there are commonalities between the two that make the nematode a good model for
5
teasing apart basic immune system function [55-57]. Additionally, multiple microbes that infect
humans can also infect the intestines of C. elegans as they eat the pathogen, which allows the
worm to be a good model system for virulence factor screens.
In the laboratory setting, C. elegans are normally fed with monoxenic bacteria cultures
that have been plated and allowed to dry on a petri dish containing nematode growth medium
(NGM) with agarose [58,59]. Although only one type of bacterial diet is customarily provided to
the worm at a time, differences between laboratories in culturing these bacterial strains can pose
problems with replicating phenotypes seen by other groups [60]. The recent appreciation that
bacteria type has an effect on animal physiology has facilitated the development of new and
exciting tools to examine diet-gene interactions. The C. elegans community canonically used an
E. coli B strain named OP50 as its standard food source. However, when performing RNA
interference (RNAi) experiments an E. coli K-12 strain named HT115 is routinely used. As it
was previously alluded to, these stains have strong influence on organismal physiology and
intriguingly, led to the discovery of diet-gene pairs [20,21,19]. One such example of a diet-
dependent phenotypes is found in the examination of worms lacking alh-6, which is a conserved
mitochondrial enzyme involved in proline catabolism, were found to have a shortened lifespan
on an OP50 diet yet a normal lifespan on the HT115 diet [20] (Figure 1). The diet-dependent
progeria phenotype was a result of deregulated mitochondrial function – morphology, diminished
ATP production, and increased ROS generation. While these mutants were identified in a
classical genetic screen based on their ability to activate the cytoprotective transcription factor
SKN-1 (discussed below) when these animals were raised on the OP50 diet [61,21,20] it is
important to note that these phenotypes would never have been discovered using RNAi screening
approaches, as this diet is a potent of suppressor of the negative physiological consequences of
6
alh-6 loss. Similarly, another diet-gene pair was discovered through the utilization of the HT115
diet during an RNAi screen for genes essential for germline development. The nuclear hormone
receptor, NHR-114, was found to play a protective role in germline stem cells maintenance by
suppressing the accumulation of division defects and ultimately sterility but only in the context
of the HT115 diet [62]. Taken together, these studies show how genes can be fundamentally
needed on one diet, yet nonessential on another and have opened a new and exciting quest to
uncover the potentially thousands of diet-gene pairs that may exist and possibly explain the
variability of aging rates in humans.

1.4 SKN-1/Nrf2-Dependent Regulation of Dietary Stress
While under the stress of starvation, animals change their metabolic programs so they can
adapt to their specific environmental conditions in an attempt to survive the famine until the next
feast arrives [63-67,2,68-75]. When starved, animals no longer have access to dietary
carbohydrates and instead must rely on intracellular lipids and proteins for fuel [76]. In order to
satisfy energy requirements, lipolysis and fatty acid oxidation are increased to break down lipids
and proteins are oxidized into amino acids. Critical to this adaptation response are mediators of
metabolic homeostasis because they are able to swiftly adjust an animal to effectively and
efficiently handle their current environment. Recently, the cytoprotective transcription factor
SKN-1 has been linked to this adaptation response, which provides an intriguing model where a
critical regulator of stress resistance has the capacity to tap into the cellular metabolic pathways
to pay for this costly response.
SKN-1 has been shown to be central to a variety of stress responses [77-82,61,21,83-88].
SKN-1 is a bZip transcription factor canonically known for defending against oxidative stress
7
but has recently accumulated fame for its roles in detoxification, immunity, proteostasis, and
metabolism [77,78,89,80,90,91,82,92-94,61,21,83- 87,95,88]. Recent work on SKN-1 identified
the first two gain-of-function (gf) alleles of skn-1, which result in the altered expression of genes
related to metabolism, starvation adaptation, growth, and reproduction [61,21]. Intriguingly,
when SKN-1gf animals are subjected to a bacterial dilution (bDR) mechanism of CR [96], which
leads to an increase in lifespan for wild-type animals, it resulted in an absence of attenuation of
longevity. In addition, the SKN-1gf animals have diminished larval stage 1 (L1) survival when
starved. When taken together, these findings suggest that constitutively active SKN-1 leads to a
perceived state of starvation even when the animals are fed ad libitum. Amazingly, depending on
the diet eaten immediately before starvation, SKN-1 and its co-regulator MDT-15 can establish
an organism’s response to food deprivation [21]. It was no surprise that MDT-15, a subunit of
the conserved transcriptional co-regulator complex called the “Mediator,” was involved in this
response as it had been previously implicated to regulate the transcription of genes involved in
fatty acid metabolism and ingestion associated stress responses [97,98]. Notably, these findings
support the importance of actual diet availability, perceived dietary status, and the genetic
pathways underlying diet sensing and utilization. The ability to trick our bodies into believing we
are nutritionally restricted while maintaining the ability to eat what we want remains a fantasy,
but perhaps SKN-1 and its co-factors are pieces of that puzzle.
Dietary stressors can come in many flavors. Society has placed particular interest on the
effects that a ‘Western Diet’ full of carbohydrates can have on an organism. In C. elegans, when
wild-type animals are fed an OP50 diet supplemented with 2% glucose, deemed a high
carbohydrate diet (HCD), they significantly induced a 250% increase in intestinal lipid stores
compared to their fat content on regular OP50 diets and this diet has obvious negative impact on
8
life- and health-span [99,100]. Remarkably, SKN-1gf animals fed this HCD did not accumulate
more stored intestinal lipids versus SKN-1gf animals on a regular OP50 diet [21]. This is
remarkable because constitutive SKN-1 activation can protect against dietary insults that would
normally cause fat accumulation. Using a Keap-1 knockdown mouse model, which induces Nrf2
(the mammalian homolog of SKN-1) activity, researchers have shown that this inhibits lipid
accumulation even when the animals are given a high-fat diet [101]. These findings support the
idea that we can genetically manipulate an organism’s physiology in response to less than ideal
diets and when combined with the fact that this lipid metabolic role of SKN-1 is also shared by
its human homolog Nrf2 [21], it makes this even more tantalizing. Ultimately, these findings
may have larger clinical implications because Nrf2 agonists, for which many have been
identified [102-107], could be useful for combating certain metabolic diseases.
Another way to induce dietary stress is through impairment of glucose metabolism,
which causes an increase in oxidative stress [100,99]. Concerning oxidative stress, both SKN-1
and Nrf2 are activated in response to compounds like H2O2, paraquat, and juglone [77,87].
There is, however, controversial data in regards to reactive oxygen species (ROS) and their
effects on physiology and signaling pathways [100,108-112]. Originally thought of as harmful,
high levels of ROS have been linked to cellular damage but it has been recently shown that when
animals are only mildly stressed, secondary messengers such as ROS can alter signaling
pathways in order to allow the organism to respond to stressors in a timely and appropriate way.
The mitochondria are primary sources and targets of ROS, which at low-levels, promotes health
and longevity through its activation of increased stress resistance factors [113,114]. This type of
adaptive response has been termed “mitohormesis” because of the stress-induced stress
resistance. Controversially, elevated levels of oxidative stressors have been linked to an
9
increased risk for certain cancers and degenerative diseases because they can cause damage to
cellular materials like DNA, proteins, and lipids. Along these lines, deregulated Nrf2 has been
linked to several aggressive types of cancer [102]. However, excessively low levels will also
leave the body more susceptible to cancers and infections because cellular protection pathways,
which include apoptosis and phagocytosis that rely on ROS signaling, become compromised
[115]. Taken together it is clear that SKN-1/Nrf2 is a central regulator of metabolic responses,
which we can manipulate, but we must maintain the ability to dial its activity up and down as
needed to ensure cellular and organismal health.

1.5 Metabolic Coordination
When there are available nutrients, a crucial governor of many anabolic processes, target
of rapamycin (TOR), let-363 in C. elegans, is activated in order to help facilitate biosynthetic
processes like protein synthesis and nutrient storage [116,117]. Intriguingly, when LET-
363/TOR is inhibited this results in lifespan extension [118- 120]. This phenomenon ties into
dietary restriction models of lifespan extension as TOR is potently suppressed during fasting and
nutrient limitation. Conditions that inhibit TOR derive in part from an imbalance between energy
usage and nutrient consumption, specifically when cells exhibit an increased AMP:ATP ratio and
coordinate the use of AMP-activated protein kinase (AMPK), which is a well-conserved sensor
of cellular energy levels [121-123,94]. In addition, these energy shortage conditions also
upregulate autophagy in order to recycle things like mitochondria, proteins, and stored glycogen
for cellular energy.
While many of the downstream effectors of metabolic adaptation have been identified,
albeit not to saturation, many of the intricacies upstream of the response have yet to be identified.
10
Adult hermaphrodites have only 302 neurons in their nervous system, yet the inner-workings are
quite complex. The chemical signaling involved in the C. elegans nervous system includes
neurotransmitters for disseminating signals across synapses and neuropeptides for cell to cell
communications [124]. A targeted screen of C. elegans carrying mutations in certain
neuropeptide-like genes, neuropeptide receptors, or G-coupled protein receptors was conducted
to assess potential differences in lifespan on an OP50 diet versus an HT115 diet [19].
Specifically, they discovered that most neuropeptide signaling pathways did not affect the
lifespan when animals were raised on the two diets; however, mutation of one gene nmur-1, did
have an effect and was one of the first described diet-gene pairs to be identified in C. elegans.
nmur-1 mutant animals lived long on the OP50-based diet but did not receive any lifespan
benefit when fed the E.coli K-12 HT115 diet. Additional roles for NMUR-1 integration of diet
and animal physiology were revealed when double mutants for both alh- 6 (discussed above) and
nmur-1 were fed an OP50 diet and were found to no longer display the aforementioned short
lifespan and mitochondrial deregulation phenotypes. This finding importantly revealed that
neuroendocrine signaling is required for maintaining an organismal response to the OP50 diet.
Therefore, NMUR-1 is integral in communicating dietary information to downstream effectors.
The NMUR-1 protein has significant homology to mammalian neuromedin U receptors
(NMURs), which are conserved across evolutionary boundaries. In vertebrate model systems,
NMU is a highly conserved neuropeptide that has key roles in many physiological processes,
including feeding and energy homeostasis [125]. Fruit flies have four NMU receptors that are
activated by pyrokinin neuropeptides [126,127]. The C. elegans genome also encodes four NMU
receptor homologs and an in silico search for the C. elegans pyrokinin-like peptide precursor
genes revealed NLP-44, as the only pyrokinin-like peptide in C. elegans [128]. The
11
neuropeptide-like protein (nlp) genes are a family of genes with currently 47 putative members
each containing high conservation amongst invertebrates [129-131]. nlp-44, through alternative
splicing, creates three pyrokinin-like peptides of which one binds to NMUR-2, but the specific
ligand for NMUR-1 remains elusive. It is also unknown if the diet itself triggers NMUR-1
activity prior to or during ingestion or if activation occurs as a consequence of dietary-related
intestinal signaling (Figure 1). The facility of C. elegans for cell and molecular biology makes
this a premiere organism to dissect the integration of this signaling pathway to organismal
physiology.
After consumption of dietary resources, it is imperative to the organism’s health to be
able to quickly and efficiently catabolize the ingested nutrients for immediate usage or storage.
However, diets are typically never comprised of a single macronutrient and therefore, animals
must be able to coordinate the metabolism of glucose, lipids, and amino acids in order to
maintain energy homeostasis. While several examples of coordination between carbohydrate and
lipid metabolism exist, it wasn’t understood until recently how and if organisms balance their use
of stored amino acids and lipids during starvation. Worms with disrupted mitochondrial proline
catabolism change the expression of lipid catabolism genes in a SKN-1 dependent manner when
they are undergoing starvation [21]. This finding revealed that when amino acid catabolism is
impaired, lipid utilization is upregulated to compensate and that SKN-1 can mediate this
response. This is the first evidence for how these two metabolic pathways harmonize and
maintain homeostasis throughout stressful conditions like starvation. Importantly, Nrf2
participates in this complex coordination of amino acid and lipid catabolism in human cells
revealing conservation of this essential stress response.
12
Because SKN-1 plays such a diverse role in response to stress, it was reasonable to
assume that its activity would be regulated by cofactors. With respect to finding other proteins
that help mediate SKN-1’s metabolic roles, co-immunoprecipitation studies along with Yeast 2-
Hybrid analysis showed a direct biochemical interaction of MXL-3 and PGAM-5 with SKN-1
[61]. MXL-3 is a basic helix-loop-helix transcription factor that has been more recently shown,
along with HLH-30, to be regulators of fat that link nutrient availability to lysosomal lipolysis
[132]. Especially now that both transcription factors are implicated in lipid metabolism, further
research into the SKN-1 and MXL-3 interaction is of great interest. The binding with PGAM-5,
which is a mitochondrial outer membrane protein, was of particular interest as this interaction
may act to recruit SKN-1 to the mitochondria in order to readily sense the organelle’s function,
stress levels, energy outputs, or metabolic status. This idea that a transcription factor could be
sequestered at a particular organelle and potentially released when deregulation of that organelle
is sensed is a very provocative idea. In support of this notion, SKN-1 was also found to reside on
the ER membrane and respond to unfolded proteins. The fact that Nrf2 has also been identified
on the mitochondria membrane in human cell culture further supports the necessity to explore the
role of these factors cytologically in the coordination of metabolic responses.

1.6 Human Implications
There have been many recent discoveries made in C. elegans that show coordination of
diet and the animals’ physiology. These findings are of particular importance to our
understanding of human physiology and when combined with the fact that these pathways
identified in worms are remarkably well conserved in humans (Table 1), supports the continued
and even increased use of C. elegans as a model for studying human disease. Regulating and
13
maintaining cellular homeostasis not only involves nutrient and energy sensing, but the animal
must be able to prevent the buildup of toxic metabolic byproducts. Many human diseases, like
cancer, obesity, and diabetes, have underlying metabolic dysfunctions [133,134]. In some cases,
particular diets can act as therapies or as accelerants for these diseases [135,136]. For instance,
obesity and type-2 diabetes can manifest due to a person’s long-term dietary choices. Diabetes
mellitus affects hundreds of millions of people worldwide and the number of people affected is
steadily increasing each year. Unfortunately, the World Health Organization projects diabetes to
be the 7th major cause of death by the year 2050 [137]. Diabetes and non-alcoholic fatty liver
disease are hallmarked by impaired glucose and insulin homeostasis which can damage tissues
and cells, impair cellular function though formation of advanced glycosylation end (AGE)
products, and generate oxidative stress through the overproduction of reactive oxygen species
(ROS) [138]. Pharmacological maintenance of insulin is one approach for people with defects in
the production of insulin but importantly, many aspects of this disease can be ameliorated by
diet. For example, low sugar and diabetic “friendly” meals are readily available to consumers.
Another risk factor for developing diabetes is obesity as 44% of diabetes cases are
attributed to the patient being overweight or obese. Unfortunately, obesity affects one-third of
the U.S. population. Unhealthy body composition is influenced by a combination (and
sometimes synergy) of genetic, environmental, and dietary factors. Changes in diet, exercise, and
recently bariatric surgery are the current prescriptions to reverse metabolic syndrome [139].
These however are not universally effective and better/more efficient treatments would be
welcomed. Lastly, it is becoming increasingly clear that the life-history of our parental and even
grandparental generations can significantly impact the physiology of subsequent generations.
This epigenetic predisposition has been documented across multiple organisms [140-142]. Our
14
understanding of the interconnectivity of these major factors and the exploitation of the facile
genetic, molecular and cellular manipulation of C. elegans [143] will be instrumental in
developing new strategies to combat this ever increasing epidemic. The pioneering work of
defining diet-gene pairs is the first step towards this goal and C. elegans remains the best model
to continue this line of discovery. Although the active role SKN-1 plays in protecting against
dietary induced obesity may seem attractive, as previously stated, too much of a good thing can
actually be bad as, strong correlations have been drawn between stabilized and unregulated Nrf2
and certain cancer incidences [102]. Similar to the relationship between ROS and mitohormesis,
it seems as though some activation of SKN- 1 (and perhaps even Nrf2) is necessary and even
good for the organism - regardless of the presence of stress - yet too much or too little can cause
homeostatic imbalance. Through our discoveries in the worm that are derived from directed high
throughput genetic and chemical screens and even the surprises uncovered serendipitously, it is
clear that the use of C. elegans to uncover the complex regulatory mechanisms that underlie diet
and organism physiology will undoubtedly have a continued and profound impact on our
understanding of human physiology.

15
1.7 Figures

Figure 1. Integration of diet into C. elegans phenotypes
Sensory neurons along with metabolic regulators may induce differential cellular phenotypes
depending on the diet eaten, however, whether the signal is a direct response to a specific diet or
if it is a host regulatory mechanism remains unknown (?).

16

Table 1. List of C. elegans genes and their human orthologs
1
Descriptions provided by WormBase Version: WS249.

17
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34
CHAPTER 2: SKN-1 AND NRF2 COUPLES PROLINE CATABOLISM
WITH LIPID METABOLISM DURING NUTRIENT DEPRIVATION

The content of this chapter appears as submitted:
Pang S*, Lynn DA*, Lo JY, Paek J, Curran SP. SKN-1 and Nrf2 couples proline catabolism with
lipid metabolism during nutrient deprivation. Nat Commun. 2014;5(5048):1–8.
*These authors contributed equally to this work.

2.1 Abstract
Mechanisms that coordinate different metabolic pathways, such as glucose and lipid,
have been recognized. However, a potential interaction between amino acid and lipid metabolism
remains largely elusive. Here we show that during starvation of Caenorhabditis elegans, proline
catabolism is coupled with lipid metabolism by SKN-1. Mutation of alh-6, a conserved proline
catabolic enzyme, accelerates fat mobilization, enhances the expression of genes involved in
fatty acid oxidation and reduces survival in response to fasting. This metabolic coordination is
mediated by the activation of the transcription factor SKN-1/Nrf2, possibly due to the
accumulation of the alh-6 substrate P5C, and also requires the transcriptional co-regulator MDT-
15. Constitutive activation of SKN-1 induces a similar transcriptional response, which protects
animals from fat accumulation when fed a high carbohydrate diet. In human cells, an orthologous
alh-6 enzyme, ALDH4A1, is also linked to the activity of Nrf2, the human orthologue of SKN-1,
and regulates the expression of lipid metabolic genes. Our findings identify a link between
proline catabolism and lipid metabolism, and uncover a physiological role for SKN-1 in
metabolism.
35
2.2 Introduction
Animals maintain energy homeostasis through the coordinated metabolism of available
intracellular nutrients, including glucose, lipids and amino acids. To do so, animals employ
complex but elegant molecular mechanisms to integrate the metabolism of these nutrients. In
mice for example, under well-fed conditions, the liver X receptor integrates hepatic glucose
metabolism and lipogenesis by acting as a glucose sensor (1). Glucose-mediated ChREBP
activation in adipose tissue activates fatty acid synthesis, thus connecting glucose and lipid
metabolism (2). Although mechanisms such as these linking glucose and lipid metabolism have
been well recognized, it remains largely elusive whether and how the metabolism of amino acids
and lipids, two major nutrients for fasting responses, are coordinated.
During periods of nutrient deprivation, stored lipids and amino acids are used instead of
dietary glucose to satisfy organismal energy requirements. Lipids are mobilized as an energy
resource through lipolysis and fatty acid oxidation (FAO). Meanwhile, amino acids, another
important energy resource, can either be directly oxidized or converted to glucose, and then
oxidized by organs with an obligatory glucose requirement (3). Based on the universal
importance of these metabolic pathways during starvation, it is possible that their metabolism
may be coupled together during fasting, and that this link would be well conserved.
SKN-1 is the worm homologue of the mammalian transcription factor Nrf2, both of
which share a conserved cytoprotective function in the response to cellular electrophiles (4). In
addition, SKN-1 is a well-known longevity factor that is activated in many long-lived mutant
backgrounds with altered metabolic homeostasis and is indispensable for the lifespan extension
of those mutants (5-8). Recently, we reported that gain-of-function mutations in skn-1 lead to a
36
starvation-like status in C. elegans and induce the expression of several metabolic genes (9);
however, the full extent to which SKN-1 participates in organismal physiology and metabolism
remains unknown.
C. elegans is an established model for studying conserved pathways that govern lipid
metabolism (10-13). In this study, by using a C. elegans strain with a mutation in a conserved
proline catabolic gene, we investigate the role of proline catabolism in the organismal response
to fasting and discover that proline catabolism is coupled with fasting lipid utilization by the
transcription factor SKN-1/Nrf2.

2.3 Materials and Methods
2.3.1 C. elegans growth conditions and strains
C. elegans were cultured using standard techniques at 20°C (29). The following strains were
used: wild-type N2 Bristol, SPC207: skn-1 (lax120), SPC227: skn-1 (lax188), SPC321: alh-6
(lax105), CL2166: gst4-p::gfp, SPC276: skn-1 (lax188); mdt-15 (lax225); gst4-p::gfp, VC1772:
skn-1 (ok2315) IV/nTi[qIs51] (IV; V) and XA7702: mdt-15 (tm2182). Double or triple mutants
were generated by standard genetic techniques.

2.3.2 Starvation assay
For starvation, synchronized L1 animals were added to nematode growth medium (NGM) plates
seeded with indicated bacteria. After 2 days at 20°C, L4 animals were collected, washed with M9
buffer at least three times and then subjected to fasting in M9 liquid with shaking for indicated
time before collection for further analysis. Starvation survival assay was performed as previously
described (30). Briefly, gravid worms that did not experience starvation for at least two
37
generations were used for egg preparation. After 24 h, synchronized L1 animals were
resuspended in M9 at a concentration of two worms per microliter. Starvation culture was mixed
by constant rocking. Every 2 days, a portion of animals was recovered on normal OP50-seeded
NGM plates. Animals that resumed development were considered to be surviving.

2.3.3 Nile Red staining
Nile Red staining was performed as previously described (31). Briefly, animals of indicated
genotypes were collected, fixed in 40% isopropanol at room temperature for 3 min and stained in
3 mg/ml Nile Red working solution in dark for 2 h. Worms were then washed with M9 for at
least 30 min, mounted on slides and imaged under the green fluorescent protein channel of
microscope Zeiss Axio Imager with Zen software package. Fluorescent density was measured
using ImageJ software. Approximately ten animals from each experiment (n) were used to
calculate the fluorescent density.

2.3.4 Oil-Red-O staining
Animals of indicated genotypes were collected and fixed in 1% formaldehyde in PBS for 10 min.
Next, samples were frozen and thawed three times with dry ice/ethanol bath. Worms were
washed with PBS three times before staining with freshly prepared Oil-Red-O working solution.
Worms were stained while rotating for 30 min, washed again with PBS for 15 min, mounted on
slides and imaged under a bright-field illumination.

38
2.3.5 RNAi treatment
HT115 bacteria containing specific double stranded RNA-expression plasmids were seeded on
NGM plates containing 5 mM isopropyl-β-D-thiogalactoside and 50 mg/ml carbenicillin. RNAi
was induced at room temperature for 24 h (32). Synchronized L1 animals were added to those
plates to knockdown indicated genes.

2.3.6 Quantitative reverse transcription-PCR
Quantitative reverse transcription–PCR was performed as previously described (14). Briefly,
worms of the indicated genotype and stages were collected, washed in M9 buffer and then
homogenized in Trizol reagent (Life Technologies). RNA was extracted according to the
manufacturer’s protocol. DNA contamination was digested with DNase I (New England Biolabs)
and subsequently RNA was reverse-transcribed to complementary DNA by using the
SuperScript III First-Strand Synthesis System (Life Technologies). Quantitative PCR was
performed by using SYBR Green (BioRad). The expression levels of snb-1 and actin were used
to normalize samples in worms and human cells, respectively. Primer sequences listed in
Supplementary Table 2.

2.3.7 Human cell culture
293T cells were cultured in DMEM supplemented with 10% fetal bovine serum. At 50–70%
confluence, cells were transfected with control, Nrf2/Nfe2l2(s9492) or aldh4a1(s16484) Silencer
Select (Life Technologies) siRNA by using Lipofectamine RNAiMax (Life Technologies). After
24 h, cells were washed and collected in Trizol reagent (Invitrogen) for further RNA extraction.
39
For examining the Nrf2 target genes in aldh4a1 siRNA experiment, cells were collected 48 h
after transfection.

2.3.8 Statistical analysis
Data are presented as mean±s.e.m. Data were analyzed by using unpaired Student’s t-test.
P<0.05 was considered as significant.

2.4 Results
2.4.1 Loss of alh-6 accelerates lipid mobilization during fasting
In a previous study, we identified mutations in the C. elegans gene alh-6, an
evolutionarily conserved mitochondrial enzyme involved in the catabolism of proline
(Supplementary Figure 1a) (14). In this study, we asked whether mutation of alh-6 would affect
lipid homeostasis. We first compared the fat content between wild-type and alh-6 mutant worms.
When fed the standard Escherichia coli OP50 diet ad libitum, wild-type and alh-6 mutant worms
store similar levels of intestinal fat as measured by Nile Red staining (Figure 1a, b) or Oil-Red-O
staining (Supplementary Figure 1b). However, within a short 3 h exposure to starvation, alh-
6 mutants rapidly mobilized intestinal lipids as compared with wild-type worms, which had yet
to measurably use these nutrient stores (Figure 1a, b and Supplementary Figure 1b). Following a
18 h long-term starvation period, alh-6 mutants continued the hypermobilization of intestinal fat
when compared with wild-type animals, which at this time point had also significantly depleted
stored lipids (Figure 1a, b). Thus, alh-6 mutants further enhance the mobilization of stored fat in
response to food deprivation. This data indicates that alh-6 regulates lipid mobilization during
40
starvation and implies that proline catabolism is coupled with lipid metabolism in response to
nutrient depletion.
We next examined the expression of genes involved in lipid metabolism. The expression
levels of pod-2/ACC1 and fasn-1/FASN, the key enzymes in fatty acid synthesis, were
comparably inhibited in wild-type and alh-6 mutant worms in response to fasting
(Supplementary Figure 1c). However, the expression of the fasting-induced lipase-1 (fil-1), a key
lipolytic enzyme responsible for the C. elegans starvation response (15), was significantly
induced in alh-6 mutants during starvation above the measured increase in starved wild-type
animals (Figure 1c). Under fasted conditions, fat is used through mitochondrial and peroxisomal
FAO (16-19). We tested the expression of all annotated FAO enzymes in animals starved for 3 h
(Supplementary Tables 1 and 2) (20). Consistent with the enhanced fat mobilization, alh-
6 mutants exhibited increased expression of several FAO enzymes, specifically under fasted
(Figure 1d and Supplementary Table 1) but not well-fed conditions (Figure 1e). These enzymes
constitute several main steps of mitochondrial and peroxisomal FAO pathways (20). Thus,
fasted alh-6 mutants enhance lipid mobilization characterized by increased expression of genes
involved in lipolysis and FAO but not de novo lipogenesis.
We identified several alh-6-sensitive FAO enzymes that were upregulated to enhance the
wild-type fasting response (Supplementary Figure 1d). In addition, we also discovered an
increase in the expression of a subset of FAO genes that results in the derepression of targets that
are inhibited in starved wild-type animals (Supplementary Figure 1e) (20). These data indicate
that not all FAO enzymes are universally used under all starvation conditions, but rather some
may specifically respond to the fasted state in the context of the alh-6 mutant background.
41
As mutations in alh-6 cause premature ageing in a diet-dependent manner (14), we asked
whether the enhanced lipid mobilization phenotype identified above was also dependent on diet.
Remarkably, the rapid depletion of intestinal lipid stores was abrogated when alh-6 mutants were
raised on another common C. elegans diet, the E. coli K-12 strain HT115. Specifically, on this
dietary regimen, alh-6 mutants exhibited comparable levels of fat mobilization in response to
fasting (Figure 1f, g) and showed no significant changes in the expression of FAO genes
(Supplementary Figure 1f) when compared with wild-type controls. Thus, the diet ingested
before starvation establishes an organism’s metabolic adaptation program during food
deprivation.
We also asked whether mutation of alh-6 affected animal survival during fasting. We
found that alh-6 mutants display significantly reduced animal survival in response to starvation
(Figure 1h and Supplementary Table 3), further indicating that alh-6 is an important regulator of
fasting adaptation. Intriguingly, the reduced survival of alh-6 mutant worms during fasting is not
dependent on the diet before starvation (Figure 1i and Supplementary Table 3), suggesting the
role of alh-6 for survival during acute and long-term fasting are different.

2.4.2 SKN-1 mediates lipid metabolism responses in alh-6 mutants
The increased expression of FAO genes in fasted alh-6 mutants indicates the existence of
a transcriptional response that monitors and responds to perturbations in cellular proline
metabolism. A role for the transcription factor SKN-1 has been documented under conditions of
oxidative stress (4) and lifespan extension (5-8), where nutrient availability is either perceived as
reduced or is actually reduced. Furthermore, we recently found that gain-of-function mutations in
skn-1 induce a starvation-like state (9). As such, we proposed that a SKN-1-mediated
42
transcriptional program could mechanistically link proline and fatty acid metabolism. Under
well-fed conditions, the expression of the SKN-1 transcriptional activity reporter gst-4p::GFP
was similar between juvenile wild-type and alh-6 mutant worms (Figure 2a). However, when
starved, the SKN-1 reporter was dramatically activated in alh-6 mutants but not in wild-type
controls (Figure 2a). Furthermore, loss of SKN-1 function through null mutation substantially
reduced the fasting-dependent activation of the SKN-1 reporter in alh-6 mutants (Figure 2b). We
conclude that alh-6mutants activate SKN-1 during food deprivation.
We next asked whether SKN-1 mediated the enhanced mobilization of stored lipids in
fasted alh-6 mutants. We found that seven out of nine FAO genes with increased expression in
the fasted alh-6 mutants were no longer upregulated in the absence of SKN-1 (Figure 2c). The
expression of two FAO genes were still activated independently of SKN-1 in the alh-6 mutants
during fasting (Figure 2d), indicating the existence of other compensatory pathway(s) that
function in parallel to SKN-1. Most importantly, a loss-of-function mutation in skn-1 abrogated
the enhanced depletion of intestinal lipid stores observed in alh-6 mutant worms after fasting
(Figure 2e, f), indicating an essential role for SKN-1 in mediating this fasting metabolic
response. However, mutation of skn-1 could not significantly reverse the reduced starvation
survival rate of alh-6 mutant worms (Figure 2g), further indicating the mechanistic differences
between the lipid metabolism and survival responses in fasted alh-6 mutants.
We previously reported that alh-6 mutations were capable of activating the SKN-1
reporter under fed conditions, but only after day 3 of the adult reproductive period (14). Despite
activation of SKN-1 at this time point in adult life, these alh-6 mutants did not induce a similar
transcriptional change in FAO genes (Supplementary Figure 2a) and do not reduce levels of
stored fat (Supplementary Figure 2b). These findings indicate a phenotypic difference between
43
the same SKN-1-inducing mutation under different physiologic contexts, which suggests that the
SKN-1-mediated lipid response represents a specific metabolic response to the alh-6 mutation
during starvation, and not merely an indirect side effect of global SKN-1 activation.
In our previous study, we also reported that accumulation of the alh-6 substrate P5C and
the subsequent generation of mitochondrial reactive oxygen species (ROS), such as hydrogen
peroxide, are responsible for the premature ageing phenotype observed in adult alh-6 mutants
(14). Treatment with the antioxidant N-acetylcysteine (NAC) completely abrogated the shortened
lifespan of alh-6 mutants. As such, we evaluated a role for mitochondrial ROS in alh-6-mediated
fasting lipid responses following NAC treatment. We found that although NAC treatment
blocked the SKN-1 reporter activation induced by exposure to arsenite, an inducer of oxidative
stress, NAC had no effect on the SKN-1 activation observed in fasted alh-6 mutants
(Supplementary Figure 2c). Moreover, NAC-treated alh-6 mutant worms still exhibited
accelerated fat mobilization in response to fasting (Supplementary Figure 2d). These data
suggest that mitochondrial oxidative stress is not involved in SKN-1 activation and the enhanced
lipid metabolism observed in fasted alh-6 worms. As SKN-1 can respond to multiple types of
cellular stress, it is possible that additional, non-oxidative stress signals caused by P5C
accumulation are responsible for the observed SKN-1 activation and lipid changes.

2.4.3 SKN-1 protects against diet-induced fat accumulation
Subsequently, we tested whether skn-1 gain-of-function mutations could induce a similar
transcriptional response, and more importantly, if they result in a change in stored lipids. We
discovered that well-fed skn-1 gain-of-function mutant worms upregulated a large number of
FAO genes (Figure 3a), which is consistent with our previous observation that ad libitum-
44
fed skn-1 gain-of-function animals behave as if they are starved (9). Intriguingly, some FAO
genes were found to be downregulated in skn-1 gain-of-function mutants as compared with wild-
type controls (Figure 3b), further supporting the idea that unique FAO enzymes are differentially
mobilized in response to particular metabolic stresses. Although, there was a larger set of lipid
metabolism genes altered in the skn-1 gain-of-function mutants, there was a clear overlap with
the genes increased in the alh-6 mutants during fasting (Supplementary Figure 3a). This gene
expression pattern indicates a SKN-1-dependent pathway for inducing an organism-level
metabolic response that is defined by the activation of fatty acid utilization pathways in both
the skn-1 gain-of-function mutants and SKN-1-activating alh-6 mutants under conditions of
fasting. We then measured the fat content of those gain-of-function mutant worms. Although
transcriptionally poised for increased oxidation of stored fat, well-fed skn-1 gain-of-function
mutant animals exhibited relatively similar levels of fat content compared with well-fed wild-
type controls as measured by Nile Red staining (Figure 3c, d), and a minor decrease of fat as
revealed by Oil-Red-O staining (Supplementary Figure 3b). We hypothesized that the induction
of FAO enzymatic activity in mutants with constitutive SKN-1 activation might only
significantly impact lipid homeostasis, at the organismal level, under conditions of metabolic
stress. We thus examined the function of constitutively activated SKN-1 in animals fed a high
carbohydrate diet (HCD) (21), which serves as model that mimics the diet-induced obesity
observed in mammals. We found that addition of 2% glucose to the standard diet could
significantly induce a 250% increase in stored intestinal fat in wild-type C. elegans, as compared
with worms feeding on a normal diet (Figure 3c, d and Supplementary Figure 3b). Strikingly,
when skn-1 gain-of-function mutants were fed the HCD, they did not manifest this increased
lipid phenotype (Figure 3c, d and Supplementary Figure 3b). These data suggest that constitutive
45
SKN-1 activation can transcriptionally predispose animals to successfully cope with dietary
insults, and that this adaptive capacity is capable of suppressing the lipid accumulation
phenotype resulting from a HCD.

2.4.4 Aldh4a1 and Nrf2 regulate FAO genes in human cells
We next examined the possible conservation of the alh-6/skn-1 pathway in human cells.
We first asked whether Nrf2, the human orthologue of SKN-1, also regulated the expression of
FAO genes in human cells. Although Nrf2 activity has been linked to cancer cell metabolism and
lipid biosynthesis in rodents (22), its role in regulating FAO has not been established. We found
that RNA interference (RNAi)-mediated knockdown of Nrf2 inhibited the expression of
canonical Nrf2 target genes (Figure 4a) and also several FAO genes in 293T cells (Figure 4b),
indicating that Nrf2 is a regulator of FAO genes in human cells. Next, we performed small
interfering RNA (siRNA) knockdown of aldh4a1, the human orthologue of worm alh-6, and
examined the effects on gene expression. Remarkably, aldh4a1 RNAi not only induced the
expression of Nrf2 targets (Figure 4c), which is indicative of Nrf2 activation, but also induced
the expression of a subset of FAO genes (Figure 4d). These data implicate that the SKN-1/Nrf2-
mediated regulatory axis between proline and lipid metabolism has functional conservation from
invertebrates to humans.

2.4.5 MDT-15 co-regulates lipid metabolism with SKN-1
In light of the multitude of responses that are influenced by SKN-1/Nrf2, we predicted
that the SKN-1/Nrf2 lipid metabolism response we identified would require additional
transcriptional co-regulators. To identify possible co-regulators of SKN-1 in modulating lipid
46
metabolism, we first screened an RNAi library targeting all annotated transcriptional regulators
and DNA-binding proteins in C. elegans, looking for suppression of the SKN-1 reporter
activation observed in the skn-1 gain-of-function mutants (9). We discovered that mdt-15 was
required for SKN-1 reporter activation, as RNAi targeting mdt-15 significantly abolished the
reporter activation (Figure 5a). Moreover, in a complementary approach, we performed a
classical ethyl methanesulfonate (EMS) mutagenesis screen for suppressors of the SKN-1
reporter activation in the skn-1 gain-of-function mutant background. We isolated a single
complementation group that mapped to the center of LGIII and identified a Gly to Glu mutation
in MDT-15 (Figure 5b and Supplementary Figure 4a). MDT-15 is a transcriptional regulator of
lipid metabolism (23) and has been found to physically interact with SKN-1 (24). We then
subsequently tested the role for MDT-15 in SKN-1-mediated lipid metabolism by examining the
effect of mdt-15 RNAi on lipid gene expression in the skn-1 gain-of-function mutants. These
mutants also display enhanced expression of FAO genes when raised on the control RNAi
bacteria HT115 (Figure 5c). However, it is notable that the gene expression changes observed are
not identical to those when animals were fed the OP50 E. coli B diet (Figure 3a, b), further
supporting the diet-dependent response of SKN-1 function in lipid metabolism. RNAi
knockdown of mdt-15 largely abolished the effects of skn-1 gain-of-function mutation on FAO
gene expression (Figure 5c), suggesting MDT-15 is a critical cofactor for the transcription of
these targets. Moreover, in the mdt-15 mutant background, alh-6 mutant animals no longer
exhibited the increased expression of FAO genes (Figure 5d) or enhanced fat mobilization in
response to fasting (Figure 5e). Together, our results refine the molecular mechanisms by which
SKN-1 and MDT-15 cooperate to maintain lipid homeostasis and define MDT-15 as a co-
regulator of SKN-1-dependent lipid metabolism.
47
2.5 Discussion
In this study, we reveal a novel link between proline and lipid metabolism, and identify a
SKN-1/Nrf2-dependent mechanism that coordinates these two metabolic pathways (Figure 5f).
How does mutation of alh-6 lead to SKN-1 activation and lipid responses during fasting? A
possible mechanism is that accumulation of the alh-6 substrate P5C induces SKN-1 activation
and fat mobilization. A recent study in mammalian adipose cells has reported that during nutrient
withdrawal, the activation of prodh, the enzyme producing P5C, can induce lipase expression
(25). This finding supports the model for P5C as a conserved metabolic signal in activating
SKN-1 and regulating fat mobilization during starvation. Generation and utilization of animals
with mutations in or reduced expression of prodh, the P5C generating enzyme, will be valuable
for testing this theory. Although Barbato et al. (25) identified a role for ROS, this could represent
the differences between our experimental models and readouts: apoptosis and inflammation in
3T3 cells versus organismal lipid depletion, or the inherent differences in responses for specific
tissues. A more thorough understanding of the coordination of such responses will be of
significant interest for future studies.
SKN-1 is an essential transcription factor mediating cellular stress responses, such as
oxidative stress and immune defense. Recent gene profile analyses indicate that SKN-1 may also
be an important regulator of metabolism. In this study, we identify a physiological role for SKN-
1 in metabolism, coordinating proline catabolism with lipid utilization during fasting. SKN-1 is
thus a critical transcription factor that responds to diverse cellular stresses, including metabolic
stress. Disruption of alh-6-dependent proline catabolism during fasting induces changes in the
expression of several FAO genes, most of which are regulated in a SKN-1-dependent manner.
Intriguingly, six of the seven SKN-1-dependent genes we identified contain three to six
48
conserved SKN-1-binding sites in their 2 kb promoters (Supplementary Figure 3c); a SKN-1-
binding site is generally found by chance only once in the same length of the genome (4,26,27).
This data indicates that some of these FAO genes may be direct targets of SKN-1.
We find that a subset of FAO genes, whose expression is inhibited by starvation in wild-
type animals, is derepressed in fasted alh-6 mutant worms. This finding indicates physiological
differences of the collection of FAO genes in the genome. We propose that C. elegans use
unique FAO enzymes in response to distinct metabolic stress conditions: some metabolic
enzymes can have overlapping functions and/or can be activated in response to specific cellular
needs.
Another interesting finding of our study is that compromised alh-6-mediated proline
catabolism regulates lipid metabolism during fasting in a diet-dependent manner. Although the
response is triggered under a condition without food, our data suggests that dietary intake before
food deprivation could predetermine an organism’s response during starvation. The different
nutritional composition between the OP50 and HT115 diets may lead to preferential use of
specific energy substrates. We propose that, when fasted, animals previously fed an OP50 diet
may rely more on mitochondrial alh-6 proline catabolism than those fed the HT115 diet. When
alh-6 is mutated, animals fed the OP50 diet might be more stressed when exogenous nutrients
are no longer available. This condition thereby activates the lipid metabolism response through
SKN-1 and MDT-15. Intriguingly, the diet consumed before fasting can have significant effects
on mouse behavior during food deprivation (28), which suggests that dietary pre-determination
of the adaptive response to starvation is also evolutionarily conserved.
Abnormal fat accumulation induced by diet underlies multiple metabolic diseases, such
as obesity and type II diabetes. We and others show that a diet supplemented with high glucose
49
can induce massive lipid accumulation in C. elegans, indicating the possibility of using this as a
model to study diet-induced fat accumulation. In this study, we find that skn-1 gain-of-function
mutation protects animals against the increased lipid storage phenotype when fed a HCD. This
finding implicates SKN-1 as a potential target for the treatment of abnormal lipid metabolism.
Furthermore, Nrf2 can similarly regulate FAO genes in human cells revealing the evolutionary
importance of this cellular metabolic response system. Thus, studies regarding the possible use
of Nrf2 pathway agonists for regulating lipid metabolism and improving its related metabolic
diseases will be of high clinical importance.

50
2.6 Figures

Figure 1. Mutation of alh-6 enhances fat mobilization and the expression of FAO genes
during starvation
(a,b) Nile Red staining of OP50 fed wild-type and alh-6 mutants in response to fasting. The
representative images are shown in a (scale bar, 100 µm) and quantitative data are shown
in b (n=12 for 0 h of wild-type and alh-6 (lax105) mutants, n=13 for 3 h of alh-6 mutants, n=9
for other groups). (c) Expression of fil-1 in response to 3 h fasting (n=3). (d,e) Expression of
FAO genes under 3 h fasted (d) and well-fed (e) conditions (n=3). (f,g) Nile Red staining of wild
type and alh-6 mutants fed HT115 in response to 3 h fasting. The representative images are
shown in f (scale bar, 100 µm) and quantitative data are shown in g (n=8 for fed wild type, n=9
for fed alh-6 mutants and fasted wild type, n=10 for fasted alh-6 mutants). (h,i) Survival rate of
wild type and alh-6 mutant worms during starvation when fed OP50 (h) or HT115 (i) diet before
starvation. Data were presented as mean±s.e.m. (*P<0.05, **P<0.01, ***P<0.001, Student’s t-
test, versus wild-type controls under same treatment unless specifically indicated).

51

Figure 2. SKN-1 coordinates proline and lipid metabolism during starvation of C. elegans
(a) Mutation of alh-6 activates gst-4p::GFP, a SKN-1 transcriptional activity reporter, during
overnight fasting. Scale bar, 100 µm. (b) Mutation of skn-1 abolished the activation of gst-
4p::GFP. The presence of the skn-1 balancer is indicated by green fluorescent protein expression
in the pharynx as pointed out by the arrow. Scale bar, 100 µm. (c,d) The increased expression of
FAO genes in 3 h fasted alh-6 mutant is either dependent (c) or independent (d) on skn-1 (n=3).
(e,f) Nile Red staining of worms with indicated genotypes under 3 h fasted condition. The
representative images are shown in e (scale bar, 100 µm) and quantitative data are shown
in f (n=11 for wild type, n=10 for other groups). (g) Effect of skn-1 mutation on the starvation
survival rate of alh-6 mutant worms. Data were presented as mean±s.e.m. (*P<0.05, **P<0.01,
***P<0.001, Student’s t-test versus controls under same treatment unless specifically indicated).

52

Figure 3. Constitutive activation of SKN-1 protects animals from HCD-induced fat
accumulation
(a,b) Expression of FAO genes that are upregulated (a) or downregulated (b) by skn-
1(lax188) gain-of-function (gof) mutation (n=3). (c,d) Nile Red staining of wild type, skn-
1(lax120) and skn-1(lax188) gain-of-function mutants fed OP50 or OP50 plus 2% glucose. The
representative images are shown in c (scale bar, 100 µm) and quantitative data are shown
in d (n=5 for wild type fed OP50, n=10 for wild type fed OP50 plus 2% glucose, n=8 for skn-1
(lax188) fed OP50, n=7 for other groups). Data were presented as mean±s.e.m. (*P<0.05,
**P<0.01, ***P<0.001, Student’s t-test versus controls under same treatment unless specifically
indicated).

53

Figure 4. Conserved regulation of Nrf2 activity and FAO genes by Aldh4a1
(a,b) Knockdown of Nrf2 inhibits expression of its canonical target genes (a) and FAO genes (b)
(n=3 for control, n=5 for Nrf2 RNAi). (c,d) Knockdown of aldh4a1 induces expression of Nrf2
target genes (c) (n=3) and FAO genes (d) (n=6). Data were presented as mean±s.e.m. (*P<0.05,
**P<0.01, Student’s t-test versus controls).

54

Figure 5. MDT-15 is a co-factor for SKN-1-mediated lipid metabolism
(a,b) RNAi mediated knockdown (a) or point mutation (b) of mdt-15 abolishes the activation
of gst-4p::GFP in skn-1 gain-of-function (gof) mutants skn-1 (lax188). Scale bar, 100 µm. (c)
Expression of FAO genes that are regulated by skn-1 (gof)-fed HT115 bacteria containing L4440
control or mdt-15 RNAi plasmids (n=2 for skn-1 (lax188)-fed control RNAi, n=3 for other
groups). (d) The expression of alh-6-mediated FAO genes is largely dependent on mdt-15 (n=3).
(e) Fat content of mdt-15 and alh-6; mdt-15 mutants during starvation as measured by Nile Red
staining (n=13 for 0 h of mdt-15 and 16 h of alh-6; mdt-15 mutants, n=10 for 6 h of mdt-
15 and alh-6; mdt-15 mutants, n=11 for 16 h of mdt-15mutants, n=12 for 0 h of alh-6; mdt-
15 mutants). (f) Model: during fasting, mutation of the proline catabolic gene alh-6 activates
SKN-1, possibly through accumulation of metabolic intermediate P5C to mediate transcriptional
program for the induction of FAO genes, which also requires co-regulator MDT-15.
55
Constitutively activated SKN-1 induces similar transcriptional changes in FAO genes that
protect animals from diet-induced obesity. Data were presented as mean±s.e.m. (*P<0.05,
**P<0.01, ***P<0.001, Student’s t-test versus controls under same treatment unless specifically
indicated).

56

Supplementary Figure 1. Analysis of lipid metabolism in alh-6 mutants during starvation
(a) Schematic of amino acid catabolism pathways regulated by ALH-6, which encodes the C.
elegans 1-pyrroline-5- carboxylate dehydrogenase (P5CDH). (b) Oil-Red-O staining of OP50 fed
wild type and alh-6 mutants in response to three hours of fasting. Scale bar: 100um. (c)
Expression of fatty acid synthesis genes under well-fed or three hours fasting conditions (n = 3).
(d-e) alh-6 dependent FAO genes are either upregulated (d) or downregulated (e) by starvation in
wild-type worms (n = 3). (f) Expression of FAO genes under three hours fasting conditions in
worms fed HT115 bacteria (n = 3). Data are presented as mean ± SEM. (*p < 0.05, **p < 0.01,
***p < 0.001, student’s t-test, versus controls under same treatment unless specifically
indicated.)

57

Supplementary Figure 2. ROS is not involved in SKN-1 activation and lipid metabolism in
fasted alh-6 mutants
(a-b), Expression of FAO genes (n = 3) (a) and fat content as measured by Nile Red staining (n =
13 for wild type and n = 16 for alh-6 mutants) (b) in alh-6 mutants at day 3 of reproductive
period. (c) Antioxidant NAC inhibits arsenite (AS) induced SKN-1 reporter activation but not
activation when alh-6 mutants are fasted. Scale bar: 100um. (d) Fat content of NAC treated
worms during fasting as measured by Nile Red staining (n = 8 for fasting alh-6 mutants, n = 7
for other groups). Data are presented as mean ± SEM. (**p < 0.01, student’s t-test, versus wild-
type controls.)
58

Supplementary Figure 3. Lipid metabolism genes and fat levels influenced by SKN-1
(a) Comparison of FAO genes that are deregulated in starved animals with compromised amino
acid catabolism or well-fed animals with constitutively activated SKN-1. Genes that are
increased by impaired amino acid catabolism during fasting are listed in the blue box; genes
upregulated in well-fed constitutively activated SKN-1 mutants are listed in the orange box. (b)
Oil-Red-O staining of worms with indicated genotypes fed OP50 or OP50 supplemented with
2% glucose. Scale bar: 50um. (c) Predicted SKN-1 binding sites WWTDTATC were detected
within a 2 kb promoter region of each gene using Regulatory Sequence Analysis Tools (RSAT).
D: Sense strand, R: antisense strand.
59

Supplementary Figure 4. Mapping the lax225 mutation to mdt-15
(a) lax225 is an allele that suppresses the SKN-1 reporter activation in the skn-1 gain-of-function
mutant background. Through standard SNP mapping, lax225 was linked to the center of LGIII.
Further SNP mapping narrowed the genetic region between ZK121 and R01H10. 1038 RNAi
clones covering this region were tested for suppression of the SKN-1 reporter activation in the
skn-1 gain-of-function mutant background. A single RNAi clone targeting mdt-15 was
identified. (b) Sequencing of mdt-15 in lax225 mutants identified a point mutation that causes a
Gly to Glu change. Star: stop codon.

60
Supplementary Table 1. Expression data of all annotated FAO genes in alh-6 mutant
worms after three hours of fasting

61
Supplementary Table 2. qPCR primer sequences

62

63
Supplementary Table 3. Starvation survival data

1. Data represent the average from two biological replicates from each condition.

64
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CHAPTER 3: OMEGA-3 AND -6 FATTY ACIDS ALLOCATE SOMATIC
AND GERMLINE LIPIDS TO ENSURE FITNESS DURING NUTRIENT
AND OXIDATIVE STRESS IN CAENORHABDITIS ELEGANS
The content of this chapter appears as submitted:
Lynn DA, Dalton HM, Sowa JN, Wang MC, Soukas AA, Curran SP. Omega-3 and -6 fatty acids
allocate somatic and germline lipids to ensure fitness during nutrient and oxidative stress in
Caenorhabditis elegans. Proc Natl Acad Sci. 2015;112(50):15378–83.

3.1 Abstract
Animals in nature are continually challenged by periods of feast and famine as resources
inevitably fluctuate, and must allocate somatic reserves for reproduction to abate evolutionary
pressures. We identify an age-dependent lipid homeostasis pathway in Caenorhabditis elegans
that regulates the mobilization of lipids from the soma to the germline, which supports fecundity
but at the cost of survival in nutrient-poor and oxidative stress environments. This trade-off is
responsive to the levels of dietary carbohydrates and organismal oleic acid and is coupled to
activation of the cytoprotective transcription factor SKN-1 in both laboratory-derived and natural
isolates of C. elegans. The homeostatic balance of lipid stores between the somatic and germ
cells is mediated by arachidonic acid (omega-6) and eicosapentaenoic acid (omega-3) precursors
of eicosanoid signaling molecules. Our results describe a mechanism for resource reallocation
within intact animals that influences reproductive fitness at the cost of somatic resilience.

3.2 Introduction
Trade-offs between fecundity and viability fitness components are thought to drive life-
history traits when resources are limited (1). In Caenorhabditis elegans, previous studies that
removed proliferating germ cells led to an increase in somatic fat (2) and a ∼60% increase in
69
lifespan (3), which is hypothesized to result from the reallocation of germline resources to the
soma, promoting survival through enhanced proteostasis (4) and attuned metabolism (5).
Although these previous studies are compelling, the use of reproduction-deficient animals
confounds the interpretation of their results with regard to trade-off models, and raises the
question of how altered reallocation may affect intact animals. During reproduction, somatic
resources are deposited to the germline by the actions of vitellogenins (6), which assemble and
transport lipids in the form of yolk from the intestine to developing oocytes. The increased
survival of germline-defective animals and their accumulation of somatic lipids suggest that the
levels of somatic and germline lipids may influence the age-related decline of somatic cell
function in postreproductive life (5). The mechanisms that regulate the distribution of energy
resources remain elusive, however.
SKN-1 is the worm homolog of mammalian Nrf2, a cytoprotective transcription factor
that impacts multiple aspects of animal physiology (7). Early work on SKN-1 defined its
essential roles in development (8) and oxidative stress responses (9), whereas more recent work
has identified a role mediating changes in diet availability and composition (10, 11). In the
present study, we examined the SKN-1–mediated dietary adaptation pathways (10–12) of C.
elegans and uncovered a sophisticated mechanism for mobilizing somatic lipids to the germline
when animals sense stressful environments. This altruistic act by the soma impacts organismal
viability to promote fecundity during oxidative and nutrient stress conditions. The universality of
oxidative stress responses among aerobic organisms is a tantalizing source of energetic “cost” to
maintain homeostasis that can compete with resources for reproduction. As such, an
understanding of how oxidative stress responses impact reproduction, and vice versa, will likely
yield insights into how the complex regulation of survival and reproduction trade-offs depend on
70
resource reallocation (13). Here we report a SKN-1–dependent axis of regulating the distribution
of somatic and germ cell resources.

3.3 Methods
3.3.1 C. elegans and E. coli strains and culture conditions
C. elegans were cultured using standard techniques at 20°C (15) unless otherwise noted. The
following strains were used: wild-type N2 Bristol, SPC207: skn-1(lax120), SPC227: skn-
1(lax188), SPC321: alh-6(lax105), SPC303: wdr-23 (lax211), VC1772 (skn-1(ok2315)
IV/nT1[qIs51](IV;V)), BX52: fat-4(wa14);fat-1(wa9), DA453: eat-2(ad456), MQ887: isp-
1(qm150), DR1572: daf-2(e1368); natural isolates: NL7000, ED3040, ED3021, TR403, CB4856,
CB4869, RW7000, ED3049. Staged animals were obtained by washing animals each day to new
plates during the reproductive period, allowing adults to settle by gravity, dropping samples on
plates, and burning off any progeny missed in the wash steps.

E.coli strains were grown in LB supplemented with appropriate antibiotic(s) for selection. The
following strains were used: OP50 – E.coli B, ura, OP50-RNAi (described below) E.coli B,
rnc14::ΔTn10, laczγΑ::T7pol, HT115(DE3) - Derived from E. coli K12, F-, mcrA, mcrB,
IN(rrnD-rrnE)1, rnc14::Tn10 (DE3 lysogen: lacUV5 promoter –T7 polymerase). All experiments
used plates with freshly seeded E.coli, from cultures grown for 16-18 hours (h) “overnight”
(O/N) at 37°C, and inoculated from stock plates less than 1-month of age.

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3.3.2 GC-MS
Samples were grown in triplicate on OP50 with and without 2% glucose supplementation (see
below). At larval-stage 4 (L4), 7500 animals were washed three times with 1X PBS, then
pelleted at 20,000 x g and supernatant was aspirated off. Afterwards, samples were promptly
frozen at -80°C. Lipid extracts from these samples were analyzed by solid-phase
chromatography followed by GCMS as previously reported (5,16). For all measurements, at least
two biological replicates were performed, with data shown as mean ± SEM.

3.3.3 Lipid staining
Oil-Red-O (ORO) or fixed Nile Red staining 4 was conducted by washing 200-300 animals from
experimental plates synchronized by egg-prep with 1x PBS + 0.01% Triton X-100 (PBST).
Worms were washed three times with 1x PBST and allowed to settle by gravity. To permeabilize
the cuticle, worms were resuspended in 100µl 1x PBST and 600µl of 40% isopropanol was
added while samples were rocked for three minutes. Worms were spun down at 500 RPM for 30s
and 600µl was aspirated off. Then, 600µl of 60% ORO working stock solution is added and
samples are rotated at room temperature (RT) – 21.0-23.5°C for two hours. ORO working stock
was prepared as follows: a 0.5g of ORO in 100mL isopropanol is stirred O/N and on the day of
staining, is freshly diluted to 60% with water and rocked for two hours, and debris removed
through a 0.22µm-filter. Worm samples are pelleted at 500 RPM for 30s, 600µl of solution is
aspirated off, and 600µl of PBST is added. Samples are rotated for another 30 minutes at RT and
then animals were mounted on slides in the presence of DAPI (internal control for
permeabilization of animals) and imaged with a color camera (Zeiss AxioCam ERc5s) outfitted
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with DIC optics. A minimum of twenty animals were imaged in a minimum of two independent
biological replicates and results were consistent between biological replicates.

3.3.4 Assessment of Asdf capacity
ORO stained samples were processed as indicated above and images collected. Percent (%) Asdf
is quantified by counting the number of animals in a cohort that display the phenotype compared
to the number of animals that do not. A minimum of two independent experiments with 2-3
biological replicates, n=4 to 6 are performed. Although hundreds of animals are examined and
scored over all replicates, the calculated % Asdf presented in each figure and Supplemental
Figure 3 only accounts for whole and non-overlapping animals in the field of view and where the
germline and soma are clearly defined. Blind scoring of % Asdf in each sample is then
independently assessed by two individuals and their results compiled to reflect %Asdf of the
population.

3.3.5 Reproduction assays
L1 stage animals were synchronized by egg prep, rocked O/N at 20°C, and dropped onto
experimental plates the next morning. 48-hours post-feeding, ten L4 stage animals of each strain
and diet were moved to their own respective experimental plate. These animals were then
assigned a number and their reproductive output was tracked, twice daily, by moving each
animal to a fresh plate every twelve hours until reproduction ceased. To ensure accurate counts
of progeny number, each plate was assessed at least twice; 24 to 48-h after the hermaphrodite
mother was moved from the plate.

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3.3.6 RNAi OP50 strain construction
RNAi OP50 was created by replacing the WT OP50 allele of RNAIII RNase (rnc) with a
deletion allele and introducing an IPTG-inducible T7 RNA polymerase. P1 phage lysates were
prepared from strains HT115 (rnc14::ΔTn10) and CH1681(laczγΑ::T7pol camFRT). To generate
the OP50 (rnc14::ΔTn10) strain, an overnight culture of OP50 was transduced with an equal
volume of HT115 P1 lysate and plated on LB+tet+citrate plates. Positive colonies were
reselected three times on LB+tet+citrate media plates. Individual colonies were subsequently
inoculated into LB+tet and the presence of rnc14::ΔTn10 allele was confirmed by PCR. To
generate the RNAi-competent OP50 strain an overnight culture of OP50 (rnc14::ΔTn10) was
transduced with equal volume of Ch1681 P1 lysate and selected on LB+cam+citrate plates.
Positive colonies were restreaked three times onto LB+cam+citrate media plates. Individual
colonies, were subsequently inoculated into LB+tet+cam and the presence of rnc14::ΔTn10 and
laczγΑ::T7pol was confirmed by PCR.

3.3.7 RNA interference (RNAi)
An RNAse III-deficient OP50 E. coli B strain was engineered for IPTG-inducible expression of
T7 polymerase (See above). Sequence verified double stranded RNA-expression plasmids were
transformed into this strain. RNAi feeding plates were prepared using standard NGM recipe with
5mM isopropyl-β-D-thiogalactoside (IPTG) and 50 ug/ml carbenicillin. Synchronized L1
animals were added to plates to knockdown indicated genes.

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3.3.8 Lipid feeding (supplementation assays)
Fatty acid supplementation plates were made using an adapted protocol (17). In brief, 100mM
aqueous solution stocks of each supplement were first made. These were made fresh right before
pouring regular NGM plates containing 0.1% tergitol (NP40) and added to NGM media once
cooled to 55°C at the concentrations indicated. Oleic acid (#90260), Stearic acid (#10011298),
Lauric acid (#10006626), Linoleic acid (#90150), a-linolenic acid (#90210), g-linolenic acid
(#90220), trans-vaccenic acid (#15301), and DGLA (#90230) were purchased from Cayman
Chemical and AA (#A9673) and EPA (#E2011) were purchased from Sigma Aldrich.

3.3.9 Glucose supplementation
Glucose was added to NGM media, cooled to 55°C, to obtain a final concentration of 2%
glucose in the worm plates.

3.3.10 Starvation and matricide assays
Staged adult animals were washed 5 times in 1x PBS to get rid of any food, transferred to 15mL
conical tubes, and 10mL of liquid NGM (prepared just like NGM media but without agar) was
added to ~50ul of washed worms. Volumes of liquid NGM were adjusted accordingly to make
maintain worm density across experiments. Tubes were gently rotated overnight at 20°C and 24
hours later, total lipid (ORO) and the frequency of the Bag phenotype (more than one internally
hatched progeny) was assessed by microscopy.

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3.3.11 Oxidative stress assays
Staged animals were collected and washed with 1x Phosphate Buffered Saline + Triton X-100
(PBST) three times to remove any contamination from the bacterial food source. After the final
wash, the animals were allowed to settle, by gravity, and the supernatant was aspirated leaving
~100ul behind. 1mL of H2O2 solution (concentrations ranging between 2mM to 10mM) in 1x
PBST was added to each experimental tube of worms. Tubes were gently rotated at RT for 20
minutes, followed by centrifugation at 500 RPM for 30s and the worms were washed three times
in 1X PBST. After the final wash animals were dropped onto seeded NGM plates for recovery.
Recovery times (as indicated) varied between 12 and 24 hours before analysis. ORO staining and
imaging was used for fat depletion analysis while survival was recorded based on head response
from prodding with a platinum wire.

3.3.12 NAC assay
N-acetylcysteine (NAC) solution in water was added to the top of a seeded worm plate at a final
concentration of 10mM and allowed to dry in a sterile hood. The solution was made to cover the
entirety of the 300ul bacterial lawn. Synchronized animals were moved to these plates at the L1
stage, kept on this diet their whole lives, and then total lipids stained and imaged at the specified
times.

3.3.13 Heat shock assay
Synchronized L1 wild-type animals were raised on normal OP50 bacteria seeded worm plates for
72 hours at 20°C. Then, plates were transferred to 30°C for 9 hours. After that time, plates were
76
transferred back to 20°C and worms were allowed to recover O/N. At 96-hours post feeding,
total lipids were stained and imaged. Adapted from Walker et al. (18).

3.3.14 Osmotic stress assay
NGM plates were prepared with 11.67g/L of NaCl (instead of 3.0g/L, as normal) for 200mM
final concentration and were subsequently seeded with OP50 bacteria. Synchronized wild-type
animals were moved to these plates at the L1 stage, kept on these plates their whole lives, and
then total lipids stained and imaged at 144-hours post feeding. Adapted from Lamitina et al. (19).

3.3.15 Statistics
Statistical analyses were performed with GraphPad Prism 6 software. Data are presented as
mean±s.e.m. Data were analyzed by using unpaired Student’s t-test and two-way ANOVA.
P<0.05 was considered as significant.

3.3.16 References for methods
1. Ashrafi, K. Obesity and the regulation of fat metabolism. WormBook, 1-20,
doi:10.1895/wormbook.1.130.1 (2007).
2. Curran, S. & Ruvkun, G. Lifespan regulation by evolutionarily conserved genes essential for
viability. PLoS Genet 3, e56, doi:10.1371/journal.pgen.0030056 (2007).
3. Tacutu, R. et al. Prediction of C. elegans Longevity Genes by Human and Worm Longevity
Networks. PLoS One 7, e48282, doi:10.1371/journal.pone.0048282 (2012).
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4. O'Rourke, E. J., Soukas, A. A., Carr, C. E. & Ruvkun, G. C. elegans major fats are stored in
vesicles distinct from lysosome-related organelles. Cell Metab 10, 430-435,
doi:10.1016/j.cmet.2009.10.002 (2009).
5. Soukas, A. A., Kane, E. A., Carr, C. E., Melo, J. A. & Ruvkun, G. Rictor/TORC2 regulates fat
metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes & development 23,
496-511, doi:10.1101/gad.1775409 (2009).
6. Hansen, M., Flatt, T. & Aguilaniu, H. Reproduction, fat metabolism, and life span: what is the
connection? Cell Metab 17, 10-19, doi:10.1016/j.cmet.2012.12.003 (2013).
7. Khanna, A., Johnson, D. L. & Curran, S. P. Physiological roles for mafr-1 in reproduction and
lipid homeostasis. Cell reports 9, 2180-2191, doi:10.1016/j.celrep.2014.11.035 (2014).
8. Vrablik, T. L. & Watts, J. L. Polyunsaturated fatty acid derived signaling in reproduction and
development: insights from Caenorhabditis elegans and Drosophila melanogaster. Molecular
reproduction and development 80, 244-259, doi:10.1002/mrd.22167 (2013).
9. Mak, H. Y. Lipid droplets as fat storage organelles in Caenorhabditis elegans: Thematic
Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man. J Lipid Res 53, 28-
33, doi:10.1194/jlr.R021006 (2012).
10. Maier, W., Adilov, B., Regenass, M. & Alcedo, J. A neuromedin U receptor acts with the
sensory system to modulate food type-dependent effects on C. elegans lifespan. PLoS Biol 8,
e1000376, doi:10.1371/journal.pbio.1000376 [doi] (2010).
11. Pang, S. & Curran, S. P. Adaptive Capacity to Bacterial Diet Modulates Aging in C. elegans.
Cell Metab 19, 221-231, doi:10.1016/j.cmet.2013.12.005 (2014).
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12. Pang, S., Lynn, D. A., Lo, J. Y., Paek, J. & Curran, S. P. SKN-1 and Nrf2 couples proline
catabolism with lipid metabolism during nutrient deprivation. Nature communications 5, 5048,
doi:10.1038/ncomms6048 (2014).
13. Brooks, K. K., Liang, B. & Watts, J. L. The influence of bacterial diet on fat storage in C.
elegans. PLoS One 4, e7545, doi:10.1371/journal.pone.0007545 (2009).
14. Merkel, M., Velez-Carrasco, W., Hudgins, L. C. & Breslow, J. L. Compared with saturated
fatty acids, dietary monounsaturated fatty acids and carbohydrates increase atherosclerosis and
VLDL cholesterol levels in LDL receptor-deficient, but not apolipoprotein E-deficient, mice.
Proc Natl Acad Sci U S A 98, 13294-13299, doi:10.1073/pnas.231490498 (2001).
15. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71-94 (1974).
16. Perez, C. L. & Van Gilst, M. R. A 13C isotope labeling strategy reveals the influence of
insulin signaling on lipogenesis in C. elegans. Cell Metab 8, 266-274,
doi:10.1016/j.cmet.2008.08.007 (2008).
17. Deline, M. L., Vrablik, T. L. & Watts, J. L. Dietary supplementation of polyunsaturated fatty
acids in Caenorhabditis elegans. J Vis Exp, doi:10.3791/50879 (2013).
18. Walker, G. A. et al. Heat shock protein accumulation is upregulated in a long-lived mutant of
Caenorhabditis elegans. J Gerontol A Biol Sci Med Sci 56, B281-287 (2001).
19. Lamitina, T., Huang, C. G. & Strange, K. Genome-wide RNAi screening identifies protein
damage as a regulator of osmoprotective gene expression. Proc Natl Acad Sci U S A 103, 12173-
12178, doi:10.1073/pnas.0602987103 (2006).

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3.4 Results
3.4.1 Age-dependent somatic depletion of fat is induced by activated SKN-1
Over the course of an individual’s lifespan, lipids are continually mobilized to afford
organismal energy demands for growth, cellular maintenance and repair, and reproduction (14).
We first examined total fat stores by Oil-Red-O (15) (Supplementary Figure 1 a-e) and fixed
Nile Red (Supplementary Figure 2 a-d) in the standard wild-type (WT) laboratory C.
elegans strain N2-Bristol throughout reproduction, from early adulthood (72 h postfeeding)
through reproductive senescence (144 h postfeeding). (Herein, hours postfeeding refers to the
amount of time that animals have been provided with food following synchronization at larval
stage 1 via starvation from hatching.) In these animals, similar to most metazoans, somatic lipid
stores increased throughout this time (Figure 1A, B and Supplementary Figures 1 a-e, 2 a-d).
Based on the recent discovery that SKN-1 can potently influence the ability of organisms
to metabolically adapt to changes in the environment (10, 11), we next looked at total fat stores
during reproduction in SKN-1 gain-of-function (gf) mutant animals (Figure 1 C, D and
Supplementary Figures 1 a, f-m and 2 e-l) and observed the skn-1–dependent rapid depletion of
somatic, but not germline, lipid stores near the end of the reproductive period (Figure 1 C, D and
Supplementary Figure 1i, m and 2 h, l and Supplementary Table 1), a phenotype that, based on
its characteristics, we call the age-dependent somatic depletion of fat (Asdf) phenotype. We
assessed the Asdf phenotype in each cohort by quantifying the number of animals that displayed
Asdf with those that did not (Supplementary Figure 3 provides all % Asdf measurements). The
Asdf phenotype was similar in all SKN-1–activating mutants tested, which includes strains
harboring mutations in alh-6 (10, 11) (Supplementary Figure 4 a, b) or wdr-23 (16)
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(Supplementary Figure 4 c, d), whereas skn-1 RNAi suppressed Asdf in SKN-1gf mutant
animals (Figure 1 E). These data indicate that activated SKN-1 is sufficient to induce Asdf.
SKN-1 activation is correlated with increased levels of reactive oxygen species (ROS)
from endogenous sources or environmental exposure to oxidizing agents (17). Following acute
exposure to H2O2, which can activate SKN-1, WT animals rapidly (within 12 h) deplete most
somatic lipids (Figure 1 F). The Asdf response is not a generalized stress response and is specific
to oxidative stress; WT animals exposed to heat (Supplementary Figure 4 g) or osmotic
(Supplementary Figure 4 h) stress environments did not induce the lipid depletion phenotype.
Further supporting the need for skn-1 in the Asdf response, skn-1(−/−) null mutants did not
deplete somatic fat following H2O2 exposure, and heterozygous skn-1(+/−) animals showed an
intermediate response (Supplementary Figure 4 i, j). Asdf was suppressed when animals with
activated SKN-1 were treated with the antioxidant N-acetylcysteine (NAC) (Figure 1 G and
Supplementary Figure 4 k-n). Intriguingly, treatment of WT animals with NAC or skn-1 RNAi
led to excessive accumulation of somatic lipids (Supplementary Figure 4 o-r), similar to the
increased fat observed in skn-1(−/−) animals (Supplementary Figure 4 s, t) and consistent with
previous reports of lipid phenotypes in animals with reduced skn-1 (18). This finding supports
previous predictions in the life-history theory proposing that the energetic costs to maintain
organismal oxidative stress capacity over the animal’s lifetime represent a major trade-off
variable (19). Taken together, our data indicate that the somatic depletion phenotype is sensitive
to oxidative stress and requires SKN-1 (Figure 1 H).

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3.4.2 Asdf mobilizes somatic lipids during nutrient stress
Our observation that animals with Asdf retained lipids in the germline suggests that Asdf
might result from mobilization of stored somatic lipids to the reproductive system. Members of
the vitellogenin family of proteins facilitate transport of stored lipids from the intestine to
developing oocytes (20) (Figure 2 A). RNAi of all vit genes tested resulted in suppression of
Asdf (i.e., restoration of somatic lipids), indicating that vitellogenesis is required for Asdf in the
SKN-1gf mutants (Figure 2 B and Supplementary Figure 5 a-d). The presence of somatic lipids
in SKN-1gf animals was restored when vit-2, -3, or -5 was targeted by RNAi, or was even
increased with reduced expression of vit-4. As such, the age-dependent loss of lipids in the soma
in SKN-1gf animals is not simply the result of somatic utilization, but rather is a consequence of
the unidirectional mobilization of stored lipids by the vitellogenins.
SKN-1 activity is essential for the longevity response to dietary deficiencies (21), and
starvation itself can induce oxidative stress (22). Indeed, the depletion of stored lipids in WT
animals after 24 h of starvation, albeit more extreme, resembled the Asdf observed in well-fed
animals with activated SKN-1 (Figure 2 C). Consistent with the idea that the Asdf phenotype in
SKN-1gf is a response to a perceived nutritional deficiency, eat-2 mutants, which eat
significantly less food than WT animals (23), also displayed Asdf at the same time point in their
reproductive span, whereas WT animals failed to display Asdf (Figure 2 D and Supplementary
Figure 6 a-d). Asdf was not observed in daf-2/insulin-IGF1 receptor (Supplementary Figure 6 e,
f) and isp-1/mitochondrial iron sulfur protein (Supplementary Figure 6 g-j) mutants, and thus is
not universal to all longevity-promoting mutations. The Asdf phenotype observed in eat-
2 mutants was suppressed by skn-1 (Figure 2 E) and vit-2 (Figure 2F) RNAi-treated animals.
Note that Asdf is suppressed by the HT115 diet and glucose; thus, all RNAi experiments
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reported herein were performed in an OP50-background RNAi strain (Supplementary Figure 7
and Supplementary Tables 2, 3). Our findings support an intriguing model of resource
reallocation between the C. elegans soma and germline, where activation of the cytoprotective
transcription factor SKN-1 under limited food and oxidative stress leads to the mobilization of
stored lipid pools to the germline, presumably to ensure fitness.

3.4.3 Oleic acid deficiency is sufficient to induce Asdf
To understand the mechanisms underlying Asdf, we identified the specific lipid
molecules altered in the SKN-1gf mutants by HPLC/GCMS (Supplementary Figure 8 a, b). We
noted a significant reduction in C17-branched fatty acids and the monounsaturated fatty acid
(MUFA) oleic acid (C18:1 n-9) in the triglyceride fraction of the SKN-1gf mutants compared
with WT animals. Oleic acid was the sole lipid species restored to WT levels in SKN-1gf
animals when the Asdf phenotype was suppressed by dietary glucose (Supplementary Figures 7
r-u and 8 c). C. elegans can synthesize oleic acid and all polyunsaturated fatty acid (PUFA)
species from dietary or de novo synthesized C16:0 (24) (Supplementary Figures 8 d and 9 a).
fat-6 and fat-7 encode the major isoforms of the Δ9 desaturases that convert stearic acid
to oleic acid (25). We subsequently tested for a direct relationship between oleic acid and Asdf.
First, we decreased fat-6/-7 by RNAi in WT animals, which phenocopied the Asdf phenotype at
the same 144-h postfeeding time point observed in the SKN-1gf mutants (Figure 3 A). We
measured fat-6 andfat-7 mRNA in SKN-1gf and WT animals and found similar levels of
expression. Thus, the Asdf phenotype in SKN-1gf mutant animals is not due simply to reduced
expression of the transcripts (Supplementary Table 4). Next, we supplemented the OP50 diet fed
to SKN-1gf mutants with 160 µM and 320 µM oleic acid and observed a concentration-
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dependent reversal of Asdf, with 60.7% and 81.6% suppression of the Asdf phenotype,
respectively, in this population (Figure 3 B and Supplementary Figure 9 b-g).
To test the hypothesis that the suppression of Asdf by oleic acid is related to a general
increase in total lipids, we assessed the ability of additional lipid supplements to suppress Asdf.
We tested lipid species that are biosynthetic precursors to oleic acid, including C18:0 stearic acid
(Figure 3 C and Supplementary Figure 9 h) and C12:0 lauric acid (Supplementary Figure 9 i, j),
as well lipids that are further desaturated products of oleic acid, including C18:2 n-6 linoleic
acid, C18:3 n-3 α-linolenic acid, and C18:3 n-6 γ-linolenic acid. Similar to supplementation with
stearic and lauric acid, each of these supplements dramatically increased total fat in WT animals;
however, they could not suppress Asdf in SKN-1gf mutants (Supplementary Figure 9 k-p). We
also tested trans-vaccenic acid (C18:1 trans-11), a MUFA that can be desaturated by FAT-6 and
FAT-7 (26), but found that, unlike oleic acid, it was incapable of any observable suppression of
Asdf in the SKN-1gf mutants (Figure 3 D and Supplementary Figure 9 q). Taken together, these
findings suggest that a lipid deficiency, specifically in oleic acid (C18:1), is causal for the Asdf
phenotype in SKN-1gf animals as animal reproduction declines.

3.4.4 Omega-3 and -6 C20 PUFAs oppose Asdf
We were surprised to find that lipid defects in the SKN-1gf mutant animals were specific
to a single MUFA, oleic acid, and that this defect did not propagate to longer and more
unsaturated species (Supplementary Figure 8 a). However, in our assessment of the lipid
biosynthesis pathways, we uncovered a role for specific C20 omega-3 and omega-6 PUFAs in
the regulation of Asdf. Like mammals, C. elegans synthesize a variety of lipid signaling
molecules that are epoxy and hydroxyl derivatives of dihomo-γ-linolenic acid (DGLA),
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arachidonic acid (ARA), and eicosapentaenoic acid (EPA) PUFAs, which influence complex
physiological processes that maintain homeostasis (27, 28) (Figure 4 A). DGLA and
eicosatetraenoic acid (ETA) are biosynthetic precursors for ARA and EPA, respectively;
however, ARA can be further desaturated to make EPA, and thus DGLA is a precursor for both
ARA and EPA. fat-4(wa14); fat-1(wa9) double-mutant animals, which cannot generate ARA or
EPA (29), prominently displayed Asdf at the same 144-h postfeeding time point, but not early in
reproduction at 72 h postfeeding, as was observed in SKN-1gf mutant animals (Figure 4 B and
Supplementary Figure 10 a). The levels of fat-1 and fat-4 were similar in SKN-1gf and WT
animals, indicating that the Asdf phenotype is not due to a reduction in gene expression in SKN-
1gf mutant animals (Supplementary Table 4).
The foregoing data suggest that one function of C20 omega-3 and omega-6 PUFAs is to
help maintain the distribution of somatic and germline lipids, and that reduced levels of these
lipid species promote Asdf. Treatment of SKN-1gf mutants with 160 µM or 320 µM ARA
resulted in potent suppression of Asdf, by 82% and 91%, respectively (Figure 4 C and
Supplementary Figure 10 b-e). Similarly, EPA supplementation suppressed Asdf to 40% and
54% of animals at the same concentrations (Figure 4 D and Supplementary Figure 10 f, g). The
suppression of Asdf was specific to ARA and EPA; SKN-1gf mutants fed OP50 supplemented
with DGLA or ETA, even at high concentrations, still displayed Asdf (Figure 4 E, F and
Supplementary Figure 10 h-j). Taken together, these findings further support a dose-dependent
role for specific omega-6 and omega-3 PUFAs in the homeostatic balance of somatic and
germline lipid reserves.

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3.4.5 Asdf occurs in natural isolates at C. elegans
C. elegans represent a species of particularly low genetic diversity at the molecular level
(30), and recent work to isolate and document the phenotypes of the ever-expanding library of
wild C. elegans strains has revealed interesting phenotypic variation among them when cultured
under laboratory conditions (31). A dearth of ecological data has hindered a better understanding
of the relevance of this variation in the natural context, however (32). We analyzed a small
collection of wild isolates of C. elegans and examined the abundance of somatic and germline
lipids and their propensity for Asdf (Supplementary Figure 11 a-h and Table 5). None of the wild
isolates displayed Asdf at early time points in their reproductive span; however, four of the wild
isolate strains tested displayed Asdf at the same 144-h postfeeding time point as animals with
activated SKN-1, albeit with varying penetrance. NL7000 and ED3040 had the strongest Asdf
phenotype, ED3021 displayed an intermediary phenotype, and ED3049 had a weak Asdf
response in this population. RW7000, TR403, CB4856, and CB4869 were most similar to N2-
Bristol in that they did not display Asdf at any time point.
Strains NL7000 and RW7000 are isolates of the same strain of Bergerac that recently
diverged in the laboratory setting. Although derived from the same parental isolate, NL7000
displays Asdf at 144 h postfeeding, whereas RW7000 does not. Moreover, and consistent with
the idea that Asdf promotes reproductive fitness, NL7000 animals have more progeny and
remain reproductive longer than RW7000 animals (Supplementary Figure 11 i). Taken together,
our data suggest that the Asdf phenotype is present in some, but not all, wild C. elegans strains,
and that the propensity for Asdf may be correlated with reproductive success.

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3.4.6 Asdf promotes reproduction at the cost of survival
We next assessed the role of Asdf in animal physiology and the resulting impact of
deregulating Asdf capacity. During periods of scarce resources, fertile C. elegans hermaphrodites
exhibit matricide, an altruistic behavior in which fertilized eggs are held in the uterus and hatch
internally, and the resulting larvae feed on the hermaphrodite mother as a nutrient source (33).
We observed an intriguing matricide phenomenon that correlated with Asdf in SKN-1gf mutants.
When day 3 (120 h postfeeding) adult SKN-1gf mutants with early signs of Asdf were starved
for 24 h, they became filled with newly hatched larvae, phenotypically defined as bags of worms
(Bag) (Figure 5 A and Supplementary Figure 12 a-d). This is in contrast to day 1 adult (72 h
postfeeding) SKN-1gf mutant animals and day 1 or 3 adult WT animals, which have only one, if
any, internally hatched larvae after 24 h of starvation. During the 48 h separating these two
periods in reproduction, WT C. elegans accumulate lipids in their somatic tissues
(Supplementary Figures 1 b-e and 2 a-d), whereas SKN-1gf mutants mobilize somatic fat to the
germline (Supplementary Figures 1 f-m and 2 e-l). The Bag phenotype observed in day 3 adult
SKN-1gf mutants with Asdf could be a consequence of the Asdf-mediated increase in germline
lipids.
A primary function of somatic cells is to protect the germline, but this comes at the cost
of depleting somatic resources. ARA supplementation has been linked to the survival of somatic
tissues during starvation and can increase the lifespan of ad libitum-fed WT animals (34). SKN-
1gf mutants display significant resilience to H2O2 exposure in early reproductive life compared
with WT animals (Supplementary Figure 13 a, b); however, the afforded resistance to exogenous
oxidative stress in SKN-1gf mutants declines at 144 h postfeeding (Supplementary Figure 13 b).
We hypothesized that the reduction in somatic energy reserves as lipids are mobilized to the
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germline during Asdf is causal for the diminished oxidative stress resistance capacity. To test
this, we inhibited Asdf by ARA supplementation to the OP50 diet, which resulted in a marked
increase in resilience to acute H2O2 exposure in 144 h postfeeding, but not 80 h postfeeding,
SKN-1gf animals (Figure 5 B and Supplementary Figure 13 b, c). The restoration of somatic
resistance to oxidative stress was specific, because supplementation with DGLA and stearic acid
did not increase survival at either time point (Figure 5 B and Supplementary Figure 13 c).
Intriguingly, postreproductive WT animals, which no longer need to devote as many resources to
reproduction, exhibited a significantly increased survival response to acute H2O2 exposure
(Supplementary Figure 13 a).
Finally, we examined somatic stress resistance to H2O2 in the NL7000 (Asdf +) and
RW7000 (Asdf -) Bergerac strains. Although both strains had enhanced resistance at 72 h
postfeeding (Supplementary Figure 13 d), NL7000 displayed a significant loss of resilience at
144 h postfeeding, whereas RW7000 was more apt to survive acute exposure to H2O2 (Figure 5
C). These findings are consistent with an increased capacity for stress resistance that is fueled by
additional somatic resources, and regulated by specific omega PUFAs.
Taken together, our results describe a pathway for the reallocation of resources between
the soma and germ cells of an intact organism (Figure 5 D). Our findings link the availability of
somatic and germline lipids to SKN-1 responses to oxidative stress and nutrient limitation. This
reallocation impacts somatic survival during stress and reproductive output, which may have
universal implications for organisms with specialized soma and germ cells.

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3.5 Discussion
In the present study, we examined organismal age-related levels of lipids during the C.
elegans reproductive span and found a remarkable lipid reallocation phenotype between somatic
and germ cells that impacts survival and reproduction trade-offs. We used Oil-Red-O and Nile
Red staining of fixed animals, because the former allows for qualitative assessment of tissue
distribution and the latter affords more quantitative measurements, albeit with reduced spatial
resolution. We observed similar patterns of lipid distribution with either dye, but each could have
unique specificity for different lipid species (35, 36), and differences in the intensity and size of
the lipid droplets might reflect a change in the composition of lipid molecules affected.
Our discovery was facilitated by a collection of SKN-1gf mutants that we previously
characterized as having reduced lipid levels on fat-inducing diets (10, 11), perhaps owing in part
to their starvation-like behaviors, despite being fed ad libitum (12). Although resistant to acute
exposure to oxidative stress, none of the constitutively activated SKN-1 mutants have proven to
be long-lived. This finding is surprising, given that SKN-1 is a cytoprotective transcription factor
essential for mounting an appropriate stress response. The near-complete depletion of somatic
lipid reserves from the soma in the animals could explain this lack of longevity in the SKN-1gf
mutants. The eventual depletion of somatic lipids was apparent at 144 h postfeeding, but clear
differences in lipid abundance between the somatic and germline cells were obvious by 120 h
postfeeding. Our data suggest that following the peak of reproduction, somatic resources are
mobilized to the germline, but these resources are effectively “wasted” as animals enter
reproductive senescence, because postreproductive animals no longer need to devote as many
resources to reproduction. Intriguingly, SKN-1gf mutants do indeed have an extended self-
reproductive period that does require Asdf, and thus an intriguing model for the function of Asdf
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is to promote late reproductive output. Although recent reports have shown that mated C. elegans
hermaphrodites lose fat after mating, future assessment of the impact of Asdf on the fertility of
mated animals will be of great interest, considering that maximal reproductive capacity is limited
by sperm production in hermaphrodites (37, 38).
Collectively, our data support a genetic role for skn-1 in the Asdf phenotype. Further
refinement of the role SKN-1 plays in the distribution of somatic and germline lipids will be of
particular interest. This work expands the known impact of SKN-1 on organismal physiology
beyond its role as a mediator of cellular and organismal stress responses (7). One interpretation
of this study is that SKN-1 activity is restricted to the soma, which leads to loss of lipids in this
compartment; however, the fact that both SKN-1gf and eat-2 mutant animals no longer deplete
somatic lipids when vitellogenesis is impaired suggests that mobilization of lipids to the
germline is at least partially causal for the loss of somatic lipids. In addition, the supplementation
of all lipid species resulted in an increase of somatic fat in WT animals and in SKN-1gf mutants
early in reproduction, but only oleic acid, ARA, and EPA could suppress Asdf. The fact that
most fatty acid supplements did not impact Asdf but also did not increase somatic stress
resistance in the SKN-1gf mutants suggests that the depletion of somatic lipids is not simply a
result of increased utilization in the soma.
This lipid reallocation has consequences for both somatic and germline tissues. The
enhanced resistance to oxidative stress afforded in the SKN-1gf mutant animals is progressively
impaired as animals proceed through reproduction, which correlates with the temporal
progression of Asdf. Furthermore, if Asdf is suppressed, then the decline in stress resistance is
attenuated. Thus, the reallocation of lipids between the soma and the germline is physiologically
relevant, because the ultimate location where the lipids reside impacts the function of that
90
compartment. Although body mass index (BMI) has proven to be an imperfect predictor of
human metabolic disease risk (39), recent work has suggested that moderate increases in BMI
above “normal” can be protective (40). Perhaps the reduction in mortality resulting from
increased somatic reserves is the result of enhanced utilization of those stores for adaptation.
We have identified a role for C20 PUFAs in the mobilization of somatic resources to the
germline in the SKN-1gf mutant animals. Dietary supplementation with the omega-6 PUFA
ARA and the omega-3 PUFA EPA effectively suppressed Asdf, whereas that with the omega-6
PUFA DGLA did not. ARA, EPA, and DGLA are precursors of specific classes of eicosanoid
signaling molecules (41), which play multiple and complex roles in animal physiology. Our
finding that only ARA and EPA can suppress Asdf suggests that specific species of eicosanoids
could be responsible for the physiological responses that we observed. C20 PUFAs also play a
critical role in maintaining membrane fluidity (42), and thus the addition of these C20 PUFAs
could alter membrane function and signaling capacity; however, the opposing responses to
DGLA compared with ARA and EPA suggest that this is not simply a general disorganization of
the lipid bilayer (43). Nevertheless, future assessment of the phospholipid composition of
membranes, the signaling pathways that influence Asdf, and the functional consequences of
perturbing these components on Asdf capacity and resulting phenotypes will be of great interest.
Although we examined reproductive-stage adults, previous studies of germline starvation
responses in developing larvae have documented the scavenging of material from the germline to
fuel reproduction (44) and even reproductive diapause (45). The increased germline lipid stores
in Asdf+ animals could promote two non-mutually exclusive outcomes: (i) provide additional
fuel for the rapid maturation of progeny and (ii) provide adequate nutrients to escape diapause
initiation and/or maintenance. Alternatively, because SKN-1gf mutants Bag only when starved at
91
the end of the reproductive period, this phenotype could represent a time-dependent failure to
decrease ovulation in response to nutrient limitation when SKN-1 is constitutively activated.
Nevertheless, because progeny’s success is subject to the deposition of maternal factors, and
their life-history parameters are sensitive to the experiences of the parental and grandparental
generations (46–48), future studies to assess the cumulative effects of Asdf capacity on fitness in
successive generations are needed.
We analyzed a collection of natural C. elegans isolates from diverse climates that
revealed that Asdf capacity is variable in the wild (Supplementary Table 5). The
RW7000 Bergerac isolate does not display Asdf and has a diminished reproductive period and
brood compared with the recently diverged NL7000 strain, which displays Asdf at 144 h
postfeeding and has a much larger brood size and a longer self-reproductive period. The number
of SNPs between these strains is unknown, and these strains quite possibly could be significantly
divergent from each other because they are classical mutator lines, originally used for the active
transposons in their genomes. Nonetheless, in light of our finding that single gene mutations are
sufficient to induce Asdf, future assessment of the genomic differences between these two strains
and all of the wild isolates tested will be of particular interest.
Our results identify a SKN-1 and eicosanoid signaling pathway that balances somatic
lipid mobilization to developing germ cells at the cost of survival. Our study provides insight
into the trade-offs resulting from the reallocation of lipid stores within intact animals, which are
critically important during nutrient and oxidative stress (Figure 5 D). The fundamental
similarities of the C. elegans and mammalian lipid metabolism and eicosanoid biosynthesis and
signaling pathways (41) suggests that the resource reallocation pathways and resulting trade-offs
may be conserved.
92
3.6 Figures

Figure 1. SKN-1 activation mobilizes somatic fat to the germline
(A–D) Oil-Red-O staining of somatic and germline lipids in WT animals, but only germline lipids in SKN-1gf
mutants, at 144 h postfeeding. (E) skn-1 RNAi suppresses Asdf in SKN-1gf animals. (F) Asdf is induced in WT
animals by acute exposure to H2O2. (G) NAC treatment suppresses Asdf in SKN-1gf animals. (H) Cartoon of the
Asdf phenotype. Arrows indicate soma, and arrowheads indicate germ. Bar graphs accompanying each panel
indicate the percent of population scored with the Asdf phenotype (red) vs. normal lipid distribution (black) from a
minimum of two biological replicates for each genotype and condition. (Scale bars: 100 µm.)
93

Figure 2. Asdf is a starvation response dependent on vitellogenesis
(A) Cartoon representation of the vitellogenin lipid transport system from the intestine to the germline. (B) vit-
2 RNAi suppresses Asdf in SKN-1gf animals. (C) WT animals starved for 24 h deplete somatic lipids but retain a
lipid pool in the germline. (D) eat-2(ad456) mutants display Asdf at 144 h postfeeding. (E and F) skn-1 (E) and vit-
2 (F) RNAi suppresses Asdf in eat-2 mutant animals. Arrows indicate soma, and arrowheads indicate germ. Bar
graphs accompanying each panel indicate the percent of population scored with the Asdf phenotype (red) vs. normal
lipid distribution (black) from a minimum of two biological replicates for each genotype and condition. (Scale bars:
100 µm.)

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Figure 3. Oleic acid deficiency is causal for Asdf
(A) RNAi inactivation of fat-6/-7 in WT animals is sufficient to induce Asdf. (B) Dietary supplementation of oleic
acid suppresses Asdf in SKN-1gf mutant animals. (C and D) Dietary supplementation of stearic acid (C) and trans-
vaccenic acid (D) do not suppress Asdf in SKN-1gf mutant animals. Arrows indicate soma, and arrowheads indicate
germ. Bar graphs accompanying each panel indicate the percent of population scored with the Asdf phenotype (red)
vs. normal lipid distribution (black) from a minimum of two biological replicates for each genotype and condition.
(Scale bars: 100 µm.)

95

Figure 4. ARA (omega-6) and EPA (omega-3) fatty acids regulate Asdf
(A) Schematic of eicosanoid biosynthesis pathways in C. elegans. (B) ARA and EPA deficient fat-4(wa14); fat-
1(wa9) animals induce Asdf at 144 h postfeeding. (C–F) Dietary supplementation of ARA (C) or EPA (D), but not
of DGLA (E) or ETA (F), can suppress Asdf in SKN-1gf mutant animals. Arrows indicate soma, and arrowheads
indicate germ. Bar graphs accompanying each panel indicate the percent of population scored with the Asdf
phenotype (red) vs. normal lipid distribution (black) from a minimum of two biological replicates for each genotype
and condition. (Scale bars: 100 µm.)
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Figure 5. Asdf fuels germ cell maturation to ensure fitness
(A) Following 24 h of starvation, SKN-1gf mutants display an age-dependent increase in the incidence of matricide
(Bag phenotype) that coincides with Asdf and is not induced in WT animals when starved for 24 h. Blue indicates
zero to one internal progeny; red, two to four internal progeny; green, five or more internal progeny. ****P <
0.0001, ANOVA. (B) OP50 diet supplemented with ARA, but not with DGLA or stearic acid, can increase somatic
resistance to acute H2O2 exposure in SKN-1gf mutant animals at 144 h postfeeding. ****P < 0.0001, ANOVA. (C)
Somatic resistance to H2O2 in NL7000 and RW7000 Bergerac strains correlates with Asdf competency. Data are
mean ± SEM for at least 40 animals, with a minimum of two biological replicates for each genotype and condition.
****P < 0.0001, two-tailed t test. (D) Model for the mechanisms underlying somatic survival and germline
reproduction trade-offs of lipid reallocation within intact animals.
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Supplementary Figure 1. ORO Asdf progression in wild-type and SKN-1gf mutants
(a) Daily progeny production of OP50-fed wild type (blue circle) and SKN-1gf mutant (red
square) animals, *P < 0.05, **P < 0.01, two-tailed t test. Oil-Red-O staining of total lipids in (b-
e) Wild type, (f-i) SKN-1gf (lax188), and (j-m) SKN-1gf (lax120) adult animals during
reproduction.
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Supplementary Figure 2. Nile Red Asdf progression in wild-type and SKN-1gf mutants
Nile Red staining of total lipids in (a-d) Wild-type, (e-h) SKN-1gf (lax188), and (i-l) SKN- 1gf
(lax120) adult animals during reproduction. Where labeled, arrows point to somatic lipids and
arrowheads denote germline lipids in oocytes.

99

Supplementary Figure 3. Cohort analysis of % Asdf
% Asdf is quantified by counting the number of animals in a cohort that display the phenotype
compared to the number of animals that do not. Mean ± SEM, n = # of animals used from at two
of the biological replicates performed to calculate % Asdf.
100

Supplementary Figure 4. SKN-1 activation and oxidative stress induce Asdf
(a-f) SKN-1 activation is sufficient to induce Asdf as observed in (a-b) alh-6(lax105), (c-d) wdr-
23(lax211), and (e-f) acute exposure to hydrogen peroxide (H2O2), but not (g) heat shock or (h)
osmotic stress but is dependent on (i-j) skn-1. (k-p) Dietary supplementation of the antioxidant
N-acetylcysteine (NAC) suppresses Asdf in (k-l) alh-6(lax105) and (m-n) SKN-1gf (lax188) and
increases somatic fat in (o-p) wild-type. (q-r) skn-1 RNAi and (s-t) skn-1 null mutants display
increased somatic fat as compared to controls.
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Supplementary Figure 5. Vitellogenin proteins transport lipids from the soma to the
germline in animals with Asdf
(a-d) Fixed Nile Red staining of total lipids in animals treated with RNAi of the vitellogenin
family of proteins: (a) control RNAi, (b) vit-3 RNAi, (c) vit-4 RNAi, (d) vit-5 RNAi; suppresses
the somatic transfer of lipids to the germline in SKN-1gf animals with Asdf.

102

Supplementary Figure 6. Asdf is a specific response to nutrient deprivation
(a-f) Fixed Nile Red staining of mutant animals. (a-b) WT, (c-d) eat-2 mutants in early
reproduction do not display Asdf while eat-2 mutants late in reproduction do display Asdf (d).
Long-lived (e-f) daf-2(insulin receptor) do not display Asdf. (g-j) Oil-Red-O staining reveals
lack of Asdf in (g-h) wild-type and (i-j) isp-1(mitochondrial iron sulfur cluster protein) mutants.
Notes: exposure time for daf-2 mutants with fixed Nile Red fluorescence was 50% of WT as to
properly resolve structures. daf-2 mutant animals were grown at 20°C until L2/L3 stage and then
moved to 25°C, as such 144-h daf-2 animals are likely developmentally more mature than age-
matched controls.

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Supplementary Figure 7. Asdf is a diet-dependent phenotype
(a) An HT115-diet (green square) increases early daily progeny production and suppresses
extended self-reproduction observed in SKN-1gf fed an OP50-diet (red square), **P < 0.01,
104
****P < 0.0001, two-tailed t test. (b) The HT115 bacterial diet suppresses Asdf in SKN-1gf
mutant animals at 144-hours post feeding. Oil-Red-O staining of total lipids reveals that Asdf
does not occur at any time in reproductive life when an HT115-diet is fed to: Wild-type (c-f),
SKN-1gf (lax188) (g-j), or (k-n) SKN-1gf (lax120) adult animals. (o) cartoon representation of
diet switching experiments. -, not observed; +, 25-50% of population display Asdf, ++, 50-75%
of population display Asdf; +++, 75-100% of population display Asdf. (p) Asdf is fully
suppressed in OP50-fed SKN-1gf mutants if switched to an HT115 diet at 48-hours post feeding
and (q) partially suppressed when switched at 96-hours post feeding. (r) Glucose
supplementation of the OP50 bacterial diet suppresses Asdf in SKN-1gf animals. (s-t) 2%
Glucose supplementation of the OP50-diet increases somatic lipid stores in WT animals.
(u) SKN-1gf (lax188) mutants accumulate less somatic lipids on a 2% carbohydrate
supplemented OP50-diet early in reproduction. Bar graphs accompanying each panel indicate the
percent of population scored with the Asdf phenotype (red) versus normal lipid distribution
(black) from a minimum of two biological replicates for each genotype and condition (see
Supplementary Figure 3 for more detail). Scale bars = 100um.

105

Supplementary Figure 8. Oleic acid deficiency in SKN-1gf is reversed by dietary glucose
(a) GCMS analysis of total fatty acids in the triglyceride fraction of wild-type (blue) and SKN-
1gf (red) animals fed an OP50-diet, ****P < 0.0001, two-tailed t test. (b) GCMS analysis of total
fatty acids in the triglyceride fraction of wild-type (blue) and SKN-1gf (lax120) (orange) animals
fed an OP50-diet. (c) GCMS analysis of total fatty acids in the triglyceride fraction of: WT fed
OP50 (dark blue); WT fed OP50+glucose (light blue), SKN-1gf (lax188) fed OP50 (red), SKN-
1gf (lax188) fed OP50+glucose (purple), SKN-1gf (lax120) fed OP50 (orange) and SKN-1gf
(lax120) fed OP50+glucose (yellow) animals. (d) Schematic of oleic acid biosynthetic pathway
in C. elegans. Abbreviations: cyclo, cyclopropane fatty acid; iso, iso-methyl branched chain fatty
acid.
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Supplementary Figure 9. Oleic acid deficiency is sufficient to induce Asdf
(a) Schematic of lipid biosynthesis pathway in C. elegans. (b-g) Oleic acid supplementation
increases somatic lipids in wild-type and suppresses Asdf in SKN-1gf mutants in a dose-
dependent manner. Supplementation of stearic acid, lauric acid, linoleic aicd, α-linolenic acid, γ-
linolenic acid, or trans-vaccenic acid increases somatic lipids in wild-type (h,i,k,m,o,q) but does
not suppress Asdf in SKN-1gf mutants (j,l,n,p) ****, P< 0.05, **P < 0.01, two-tailed t test.. Bar
graphs accompanying each panel indicate the percent of population scored with the Asdf
phenotype (red) versus normal lipid distribution (black) from a minimum of two biological
replicates for each genotype and condition (see Supplementary Figure 3 for more detail). Scale
bars = 100um.
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Supplementary Figure 10. ARA and EPA precursors of eicosanoid signaling molecules
influence Asdf
(a) Eicosanoid-deficient fat-4(wa14); fat-1(wa9) mutant animals do not display Asdf early in
reproduction (72-hpf). (b-e) Arachadonic acid (ARA) supplementation increases somatic lipids
in WT and suppressed Asdf in SKN-1gf mutants. (f-g) Eicosapentaenoic acid (EPA)
supplementation increases somatic lipids in WT and suppressed Asdf in SKN-1gf mutants. (h)
dihomo-γ-linolenic acid (DGLA) and (i) Eicosatetraenoic acid (ETA) increases somatic lipids in
WT as compared to controls (j).
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Supplementary Figure 11. Variation of Asdf among natural isolates of C. elegans
Oil-Red-O staining of somatic and germline lipids reveals Asdf-capacity (a-d) and -deficiency
(e-h) among isolated natural isolates of C. elegans at 144-hours postfeeding. (i) Daily progeny
output of NL7000 (blue circles) and RW7000 (red squares) isolates of C. elegans Bergerac
strains, *P < 0.05, **P < 0.01, two-tailed t-test.. Bar graphs accompanying each panel indicate
the percent of population scored with the Asdf phenotype (red) versus normal lipid distribution
(black) from a minimum of two biological replicates for each genotype and condition (see
Supplementary Figure 3 for more detail). Scale bars = 100um.
109

Supplementary Figure 12. Matricide is enhanced in animals with Asdf after 24-hours of
starvation
WT animals in early (a) and late (b) reproductive life do not Bag (> 1 internally hatched worm)
after 24-hours of starvation. (c) SKN-1gf mutants, early in reproductive life do not Bag when
starved for 24-hours. (d) SKN-1gf mutants, with Asdf, late in reproductive life display a high
incidence of matricide when starved for 24-hours. Arrows denote internally hatched larvae.

110

Supplementary Figure 13. Survival of acute exposure to H2O2 is influenced by Asdf
(a) WT animals early in reproduction are sensitive to acute exposure to H2O2 and more resistant
at the end of the reproductive period. (b) SKN-1gf mutant animals are resistant to acute exposure
to H2O2 and that resistance declines later in reproduction, which correlates with Asdf. (c)
Supplementation of stearic acid or DGLA does not increase somatic resistance to H2O2 early or
late in reproduction in SKN-1gf mutants. (d) NL7000 and RW7000 Bergerac strains are resistant
to H2O2 oxidative stress early in reproduction. ****, P
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Supplementary Table 1. Daily progeny production for wild-type and SKN-1gf on OP50 diet

1. Animals displaying Bag and Pvl phenotypes or escaped the plate were censored.

112
Supplementary Table 2. Daily progeny production for wild-type and SKN-1gf on the
HT115 diet

1. Animals displaying Bag and Pvl phenotypes or escaped the plate were censored.

113
Supplementary Table 3. OP50 RNAi knockdown efficiencies

1. Gene descriptions provided by WormBase version: WS249
2. mRNA expression levels in SKN-1gf animals treated with indicated RNAi relative to
SKN-1gf animals treated with vector control RNAi; normalized to snb-1 expression as a
control, which was invariant across samples. Green and red color indicates increased and
decreased expression, respectively.
3. *, P-value<0.05; **, P-value<0.01; ****, P-value<0.0001; n.s., difference between
samples is non significant = P-value>0.05

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Supplementary Table 4. mRNA expression levels of oleic acid and eicosanoid biosynthesis
pathway genes

1. Gene descriptions provided by WormBase version: WS249
2. Fold-change in expression in SKN-1gf mutant animals relative to wild-type animals
normalized to snb-1 expression as a control, which was invariant across samples. Green
and red color indicates increased and decreased expression, respectively.
3. *, P-value<0.05; **, P-value<0.01; ****, P-value<0.0001; n.s., difference between
samples is non significant = P-value>0.05

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Supplementary Table 5. Variation of Asdf phenotype among natural isolates of C. elegans

1. Strain designation as reported to the Caenorhabditis Genetics Center (CGC)
2. Geographic location where strain was isolated (Wormbase)
3. Penetrance of Asdf phenotype. -, not observed; +, 25-50% of population display Asdf,
++, 50-75% of population display Asdf; +++, 75-100% of population display Asdf.

116
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CHAPTER 4: ACUTE STARVATION IMPACTS EPIGENETIC
MODIFICATIONS AND LIPID HOMEOSTASIS IN C. ELEGANS

4.1 Abstract
Information can be transmitted to the next generation through DNA sequence as well as
through epigenetic modifications, such as DNA methylation or chromatin modifications (1–3).
The concept of genetic memory is emerging as studies show transgenerational inheritance of
genetic imprinting based on life experiences (4,5). Previously thought to lack DNA methylation,
Caenorhabditis elegans was recently discovered to have 6mA modifications (6). Here we show
that known histone and DNA modifiers can significantly alter lipid homeostasis in
reproductively active SKN-1 gain-of-function mutant worms. These findings begin to
characterize how nutrient status and epigenetic modifications can impact organismal life-history
traits.

4.2 Introduction
Early documentation in humans of transgenerational inheritance of gene expression used
correlations between parental nutritional status and the physiological responses of their offspring
(7,8). Famines can be used to relate the mother’s food intake during pregnancy with the
offspring’s obesity risk (9,10). Non-calorically restricted siblings had significantly less risk for
obesity compared to offspring that were conceived during harsh famine conditions where the
parents were calorically restricted. These fascinating correlations along with our observations
that our SKN-1gf animals behave as if they are being starved, even when they are well-fed, (11)
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led us to start looking at non-coding genetic regulation in terms of our documented Asdf
phenotype (12) in these mutant animals.
Evidence is accumulating to support the idea that life experiences can change gene
expression, which can affect an organism’s behavior, health, and ability to cope with their
surrounding environment (13,14). Exposure to environmental and nutritional signals can be
sensed by the body and in turn can change gene expression for the organism experiencing the
stimuli and in some cases these changes can be transmitted across multiple generations of
progeny (15–17). These changes are called epigenetic modifications and can include the activity
of any DNA or chromatin-modifying proteins (18). These modifications can transmit information
that can regulate many complex phenotypes. Fortunately for worm researchers, there are many
histone modifiers that have been identified and are being studied (19–21). Typical
heterochromatin marks include H3K9me3, H3K4me2/3, and H3K27me2/3.
In terms of DNA methylation, eukaryotic research generally focuses on 5-methylcytosine
(5mC) modifications and how it is involved in epigenetics (22,23). Previously, researchers have
looked for DNA modifications in C. elegans and thought that they might have 5mC
modifications (24). However, evidence supporting this theory is lacking since detectable 5mC
modifications and homologs of the enzymes that add methyl groups to cytosine (called DNA
cytosine 5-methyltransferase 1 and 3 in mammals) are not found in the worm (25). There are,
however, other types of DNA modifications predicted to have epigenetic regulatory effects in
organisms. There can also be methylation of NH2 groups either at the fourth position of the
pyrimidine ring in cytosines (4mC) or at the sixth position of the purine ring in adenines (6mA),
however, these modifications are primarily used by prokaryotes in order for the cell to identify
self from foreign DNA (26–28). Not long ago, it was still unknown if there were other types of
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DNA modifications, like 6mA and 4mC, that were specifically involved in modifying eukaryotic
DNA. Recently, researchers have found that C. elegans actually do have 6mA DNA
modifications (6). The researchers discovered this by performing dot blot experiments as well as
ultra high performance liquid chromatography coupled with a triple-quadrupole tandem mass
spectrometry analysis. These sensitive detection techniques coupled with other experimental
evidence will be helpful to determine other eukaryotes that may utilize 6mA DNA modifications.

4.3 Materials and Methods
4.3.1 C. elegans and E. coli strains and culture conditions
C. elegans were cultured using standard techniques at 20°C. The following strains were used:
wild-type N2 Bristol, SPC227: skn-1 (lax188), VC2552: nmad-1(ok3133), and VC40319: damt-1
(gk961032). Staged animals were obtained by washing animals each day to new plates during the
reproductive period, allowing adults to settle by gravity, dropping samples on plates, and burning
off any progeny missed in the wash steps.

E. coli strains were grown in LB supplemented with appropriate antibiotic(s) for selection. The
following strains were used: OP50—E. coli B, ura; OP50-RNAi (previously described in Chapter
3)—E. coli B, rnc14::ΔTn10, laczγA::T7pol. All experiments used plates with freshly seeded E.
coli, from cultures grown for 18 hours (h) “overnight” (O/N) at 37°C, and inoculated from stock
plates less than 1-month of age.

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4.3.2 Lipid staining
Oil-Red-O (ORO) or fixed Nile Red staining was conducted by washing 200-300 animals from
experimental plates synchronized by egg-prep with 1 x PBS + 0.01% Triton X-100 (PBST).
Worms were washed three times with 1x PBST and allowed to settle by gravity. To permeabilize
the cuticle, worms were resuspended in 100µl 1x PBST and 600µl of 40% isopropanol was
added while samples rocked for three minutes. Worms were spun down at 500 RPM for 30s and
600µl was aspirated off. Then, 600µl of 60% ORO working stock solution is added and samples
are rotated at room temperature (RT)—21.0-23.5°C for two hours. ORO working stock was
prepared as follows: a 0.5g of ORO in 100mL isopropanol is stirred O/N and on the day of
staining, is freshly diluted to 60% with water and rocked for two hours, and debris removed
through a 0.22µm-filter. Worm samples are pelleted at 500 RPM for 30s, 600µl of solution is
aspirated off, and 600µl of 1x PBST is added. Samples are rotated for another 30 minutes at RT
and then animals were mounted on slides in the presence of DAPI (internal control for
permeabilization of animals) and imaged with a color camera (Zeiss AxioCam ERc5s) outfitted
with DIC optics. A minimum of of twenty animals were imaged in a minimum of two
independent biological replicates and results were consistent between biological replicates.

4.3.3 Assessment of Asdf capacity
ORO stained samples were processed as indicated above and images collected. Percent (%) Asdf
is quantified by counting the number of animals in a cohort that display the phenotype compared
to the number of animals that do not. A minimum of two independent experiments with 2-3
biological replicates, n=4-6 are performed. Although hundreds of animals are examined and
scored over all replicates, the calculated % Asdf presented in each figure only accounts for whole
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and non-overlapping animals in the field of view and where the germline and soma are clearly
defined. Blind scoring of % Asdf in each sample is then independently assessed by two
individuals and their results compiled to reflect % Asdf of the population.

4.3.4 RNA-interference (RNAi)
An RNAse III-deficient OP50 E. coli B strain was engineered for IPTG-inducible expression of
T7 polymerase. Sequence verified double stranded RNA-expression plasmids were transformed
into this strain. RNAi feeding plates were prepared using standard NGM recipe with 5mM
isopropyl-β-D-thiogalactoside (IPTG) and 50µg/ml carbenicillin. Synchronized L1 animals were
added to plates to knockdown indicated genes.

4.3.5 Statistics
Statistical analyses were performed with GraphPad Prism 6 software. Data are presented as
mean±s.e.m. Data were analyzed by using unpaired Student’s t-test and two-way ANOVA.
P<0.05 was considered as significant.

4.4 Results
4.4.1 Egg versus larval stage-1 synchronization
Previously, we have shown that nutritional status plays a role in the manifestation of the
Age-dependent somatic depletion of fat (Asdf) phenotype (12). In particular, we noted that diet-
switching experiments (even in adulthood) could drastically change the animal’s Asdf capacity,
thus indicating that there is an adult response to nutrient status. Additionally, having this
phenotype plasticity in adult populations was interesting as others have focused on the
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importance of early-life signals in complex phenotypes in the worm (29–31). Therefore, we were
curious if the widely used process of synchronizing populations of C. elegans for the start of
experiments might cause an effect on lipid compartmentalization in the worm.
This synchronization technique is widely used in worm laboratories in order to grow up
large amounts of worms that are all at the same life-stage (32,33). This technique involves acute
starvation of the larval stage-1 (L1) C. elegans right after they hatch. Researchers will isolate the
eggs from the hermaphroditic mothers and allow them to hatch in a liquid buffer that causes
them to arrest at the first larval stage since there aren’t any nutrients to grow. This overnight
hatching process lasts about 18 hours in our laboratory and we are very strict to not allow this to
fluctuate greatly, especially because we work on metabolism and do not want to alter our
experimental set-up in any small, or large, way. The animals hatch about 12.5 hours after the egg
was fertilized, but each young adult mother’s uterus typically holds 10-15 eggs that have been
ovulated and fertilized successively after each other (34). Therefore, this 18 hour overnight
starvation technique, while it is probably only forcing starvation upon the hatching larvae for
about 5.5 to 8 hours, could be affecting the animals’ physiology. Recent studies have shown that
extended L1 arrest severely affects growth rate and fecundity although these animals do produce
progeny that are very resistant to starvation and heat stress (31).
Accordingly, we began our experiments without this acute L1 starvation period and
instead dropped the isolated and unhatched eggs onto the food directly. This way, the animals are
each able to eat, grow, and develop right after hatching. Interestingly, SKN-1gf animals that are
fed right after hatching do not prominently display Asdf by both fixed Oil-Red-O (Figure 1A)
and fixed Nile Red staining techniques (data not shown). In fact, the penetrance of the phenotype
is about 15% of the population compared to the synchronized/starved SKN-1gf animals that
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display the phenotype at around 71% (Figure 1B). We were fascinated by this idea of very early-
life starvation profoundly impacting the worm’s lipid stores at a later, adult life-stage.

4.4.2 RNA-interference screen
We then designed a targeted OP50 RNA-interference (RNAi) screen for members of the
DNA and histone modification families that might significantly alter the penetrance (or absence)
of the Asdf phenotype depending if the animals were hatched under starvation conditions or fed
from hatching (Figure 2A). Utilizing current literature, we assembled a list of 23 genes to knock
down under both the starvation and fed conditions in SKN-1gf animals (Table 1) (25).
We wanted to see if there were genes that could significantly suppress Asdf penetrance in
SKN-1gf animals that had been synchronized/starved and we also wanted to see if there were
genes that could significantly induce Asdf penetrance in SKN-1gf animals that had food since
hatching. Out of the 23 genes tested, the knockdown of 6 genes resulted in significant
suppression of the Asdf phenotype in acutely starved/synchronized SKN-1gf animals (Figure
2B). These genes include: lsd-1, nmad-1, rbbp-5, set-2, spr-5, and wdr-5. Respectively, these
conditions caused Asdf penetrance in the population to drop down to: 20%, 33%,13%, 8%, 19%,
and 5%. Out of the 23 genes tested, the knockdown of only 2 genes resulted in significant
induction of the Asdf phenotype in fed from hatching SKN-1gf animals (Figure 2C). These
genes include: sir-2.1 and damt-1. Respectively, these conditions caused Asdf penetrance in the
population to increase to: 76% and 57%.
It is important to note that the knockdowns of each of these genes doesn’t alter the
animals in any noticeable way. Through imaging, we can visibly see that none of these
conditions alter the physical appearance of the animals, including their overall size, shape, and
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their internal structures. Additionally, the lipid distribution and appearance of lipid droplets
appear normal according to life-stage.

4.4.3 Characterization of 6mA modifiers in C. elegans
While all of these genes are of significant interest, the most interesting pair of genes that
are in opposition are the nmad-1 and damt-1 genes. In C. elegans, NMAD-1 functions as a DNA
demethylase and DAMT-1 is a DNA methyltransferase (6). These proteins have been found to be
responsible for the removal and transfer of 6mA DNA modifications in C. elegans, respectively.
We obtained genetic mutants that carry deletions of both genes separately and began
characterizing the two strains. VC40319 is a strain of C. elegans that carries a full deletion of
damt-1, which is the 6mA DNA methyltransferase. VC2552 is a strain of C. elegans that carries
a large but partial deletion of nmad-1, which is the 6mA DNA demethylase. Interestingly, when
VC40319 animals are egg dropped they contain less somatic fat than their siblings that are
synchronized/starved (Figure 3A). Conversely, VC2552 animals have more somatic fat when
they are egg dropped than when they are synchronized/starved (Figure 3B). Further studies to
fully characterize these animals in the context of SKN-1 activating conditions, stress responses,
and resource allocation will be necessary.

4.5 Discussion
Until recently, 6mA has been mostly studied in prokaryotes in terms of marking self
versus foreign DNA (26,28). The idea that this type of DNA methylation can transmit epigenetic
information across generations in eukaryotes is extremely exciting. There is evidence in
Chlamydomonas reinhaardtii that 6mA is enriched around the transcriptional start sites of
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actively transcribed genes and that it is in general a mark of active genes (35). While our
phenotypic results for this project are intriguing by themselves, we are curious if this principle of
6mA correlating with active genes will hold true in C. elegans. Additionally, we would like to
perform ChIP in order to correlate the sequencing results with Antioxidant Response Elements
(AREs), non-ARE SKN-1 binding sites, as well as with microarray data for SKN-1gf animals
compared to wild-type. We are also curious how this might fit into life-history traits and how
animals in the wild might respond. One possible reason for this response is that there can be huge
shifts in the variability of food in the wild. If times are hard, then perhaps there is a need to easily
disseminate that information to future generations. This might be especially helpful for C.
elegans because it takes about 72 hours from hatching as an L1 to becoming a reproductive adult
so environmental conditions and stressors may be very similar for successive generations.
Further development of this project will shed light on how parental nutrient status and acute
starvation of hatchlings can impact an animal’s lipid stores, reproductive capacity, and stress
resistance.

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4.6 Figures

Figure 1. Synchronized larval stage-1 (L1) C. elegans have altered adult lipid stores
compared to animals that hatch in the presence of food
(A) SKN-1gf L1’s that hatch in the presence of food do not display Asdf at 144-hours post-
feeding with fixed ORO staining.
(B) Quantification of Asdf penetrance in the population of C. elegans that have been fixed
and stained by ORO.
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Figure 2. Targeted RNA-interference (RNAi) screen identifies DNA and histone modifiers
that alter Asdf-penetrance in SKN-1gf worms
(A) Cartoon overview of OP50 RNAi screen conducted on SKN-1gf worms.
(B) Genetic knockdown of certain DNA and histone modifiers can suppress Asdf phenotype
in acutely starved animals. (L1 = acute starvation; eggs = fed from hatching).
(C) Genetic knockdown of certain DNA and histone modifiers can induce Asdf phenotype in
animals hatched with food. (L1 = acute starvation; eggs = fed from hatching).
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Figure 3. Genetic mutants of the C. elegans 6mA demethylase and methyltransferase have
opposing effects on adult lipid content that is dependent upon early-life starvation
exposure.
(A) VC40319 animals (damt-1 mutants) display less adult lipid stores when they are fed from
hatching.
(B) VC2552 animals (nmad-1 mutants) display less adult lipid stores when they are acutely
starved as larvae.

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Table 1. Genes tested in the RNAi screen.
Gene Name Gene Function
nmad-1 6mA DNA demethylase
damt-1 6mA DNA methyltransferase
spr-5 H3K4me2 demethylase
wdr-5 member of the H3K4 trimethylation complex
ash-2 member of the H3K4 trimethylation complex
set-2 member of the H3K4 trimethylation complex
rbr-2 H3K4me3 demethylase
rbbp-5 H3K4me3 methyltransferase
set-15 histone methyltransferase
set-9 H3K4 methyltransferase
utx-1 H3K27 demethylase
cbp-1 H4K5 histone acetyltransferase
sir-2.1 H4K16 histone deacetylase
met-1 H3K36 histone methyltransferase
set-26 H3K4 histone methyltransferase
mes-2 H3K27 histone methyltransferase
lsd-1 H3K4 histone demethylase
jmjd-2 H3K9 histone demethylase
ogt-1 histone o-glcnac
oga-1 histone o-glcnac
let-418 nucleosome remodelers
coq-3 methyltransferase
pqn-28 histone deacetylase

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CHAPTER 5: CONCLUDING PERSPECTIVES

The content of this chapter appears as submitted:
Lynn, DA and Curran SP.
The SKN-1 hunger games: May the odds be ever in your favor.
Worm. 2015;4(3):e1078959.

In our society, the phrase “I'm starving” is overused to the point that the meaning of the
word “starvation” is lost. Starvation goes beyond just hunger. Starvation causes animals to
change their metabolic program in hopes of surviving while undergoing nutrient deprivation.
Dietary carbohydrates, lipids, and proteins are catabolized respectively into pyruvate, fatty acids,
and amino acids so that they can generate ATP toward the maintenance of cellular
homeostasis.
15
When food is not available, their internal stores of fats and proteins are instead
utilized. An animal's response to such starvation conditions is indicative of how well their
storage and energy allocation pathways function. Complex molecular events are central to the
starvation response.
16-21
SKN-1 is a cytoprotective transcription factor involved in several
aspects of animal physiology. Early work on SKN-1 defined its essential roles in
development
22
and oxidative stress responses
23
while more recent work has identified a role in
responses to diet availability and the nutrient composition of that diet.
13,14
We recently identified
SKN-1/Nrf2 as an important mediator of these catabolic pathways with the ability to sense
defects in one pathway – such as mitochondrial amino acid catabolism – and enhance others –
including fatty acid oxidation – in order to maintain homeostasis. Connections between
carbohydrate and lipid metabolism are well established while associations with amino acid
metabolism are less well defined. The alh-6 pathway regulates mitochondrial proline catabolism,
is maintained by SKN-1
14
, and remarkably this pathway is evolutionarily conserved as Nrf2
139
functions in a similar capacity in human cells.
13
The alh-6 gene is half of a newly discovered
diet-gene pair; a genetic locus that is of critical importance, but only in the context of specific
diets. The ability to cope with ever-changing environments and food-sources proves to be a
difficult task. Therefore, it is of great future interest to investigate the role of SKN-1, which
canonically protects the animal from oxidative stress, and to fully understand how this may be
affected by diet. There are several possible scenarios that could be at play: altered metabolism on
these diets could perhaps lead to an increased oxidative stress state or animals might be affected
directly by the nutrients they consume (perhaps some are toxic). In either case, the induction of
the SKN-1 response is certainly energetically costly and the integration of SKN-1 into the
pathways that regulate cellular metabolism is an elegant mechanism to maintain homeostasis,
and ensure the resources to “pay” for stress adaptation are available.
In our western culture, we are facing an obesity epidemic.
24
Certain lifestyle traits
contribute to this greatly, but the culprit gaining significant strength is our choice in what we eat.
Over the past few decades, processed and pre-packaged food has become a norm. These “easy
meals” are packed with sugar and sodium not only as preservatives but also in an attempt to
make them more delicious, if not palatable.
25
A ‘western diet’ characterized in 2005 consists of a
19 percent increase in sugar as compared to what people consumed in 1970.
26
This is
unprecedented and our bodies don't know how to handle it. Recently, we discovered that well-
fed worms with constitutively active SKN-1 behave in a manner similar to animals undergoing
starvation.
21
Wild-type animals fed the nematode version of a western diet - an E. coli OP50 diet
supplemented with 2% glucose - increase their lipid stores by 250%.
13
Unlike wild-type animals,
when these SKN-1 gain-of-function animals are fed this same high-carbohydrate diet they do not
noticeably increase their lipid stores. Interestingly, the animals with activated SKN-1 are still
140
eating roughly the same as wild-type animals, which means the energy from the extra un-stored
nutrients are being utilized in some undefined way. Notably, this experiment analyzed animals
that had been fed this diet during development and fat was measured right before the onset of
reproduction, which would equivocate these results to humans fed a western diet from birth to
adolescence (age 11–13). While this was a remarkable finding, it is of great future interest to be
able to quantify these responses during reproduction and in post-reproductive animals.
Some of the earliest studies documenting epigenetic phenomenon were a result of
correlating physiological responses that tracked the quality and availability of nutrients of their
parents.
27,28
Historically famous famines have drawn interesting correlations between nutritional
status during pregnancy and the genetic inheritance of the subsequent offspring. When the
parents were calorically restricted, this caused the offspring to have an increased risk for obesity
compared to their siblings that were conceived before or after the famine years. These human
analyses corroborated the idea that when parents face harsh conditions, their offspring's fitness
can be affected epigenetically. Similarly, although no less surprising, was our discovery that the
parent's diet (E. coli strains HT115 or OP50) established their offspring's response to food
deprivation; namely we observed rapid depletion of stored fats when animals that are deficient
for ALH-6-dependent amino acid catabolism were fed the OP50 diet and then starved while
animals fed the HT115 diet were resistant to this lipid depletion.
13
Although the trigger of this
response is unknown, it is transcriptionally regulated by SKN-1 and MDT-15, a subunit of the
mediator complex.
29,30
It should be noted, that not all phenotypes are diet-dependent. For
example, alh-6 mutants display impaired survival from chronic food deprivation, suggesting
perhaps that the alh-6 amino acid catabolism pathway is more important for acute responses to
changes in available nutrients. Together, these findings highlight the importance of fully
141
characterizing the main laboratory diets currently used in C. elegans labs and setting a standard
and defined diet. Creating a synthetic diet would allow the identification of particular macro and
micronutrients that influence phenotypes seen in C. elegans.
31
Until we can standardize diets
between laboratories, it will be crucial to understand what the fundamental differences are
between the most commonly used E. coli strains, OP50 and HT115, and how these differences
might be catalyzing diet-dependent phenotypes.
32

The importance of this multigenerational affect of diet on physiology further emphasizes
the complex and potent forces that nutrition has on an organism, which can preset the metabolic
pathways it can access, through yet to be elucidated epigenetic mechanisms.
33,34
Recently, it was
shown that starvation induced the creation of endogenous small RNAs which then could be
inherited through at least 3 generations and that these small RNAs were enriched for targeting
genes involved in metabolism.
35
While further research to identify the pathways that integrate the
sensation of the starvation state and the creation of specific small RNAs is needed, the
conservation of these pathways from worm to man begs the question if a similar connection
between small RNA response is present in humans following famine.
An overarching question revolves around the uncertainty of how specific diet-gene pairs
can alter organism-level physiological phenotypes.
14
Previous reports identified the mammalian
neuromedin U receptor, nmur-1, as responsive to the differences in the lipopolysaccharide
structures of the OP50 and HT115 bacterial diets. Similarly, nmur-1 was essential for the
mitochondrial morphology, ROS production and short lifespan phenotypes of alh-6 mutant
animals when fed the OP50 diet.
36
While the breadth of differences between the HT115/K12 and
OP50/B E. coli diets is not fully defined, the HT115 diet has a much higher carbohydrate content
than the OP50 strain.
32
Besides the effects on longevity, this higher carbohydrate content can
142
result in additional physiological defects; for example, it was recently shown that gene
expression levels of RNA polymerase III regulator, mafr-1, are significantly lowered when C.
elegans are raised on HT115 diet, which in turn affects lipid homeostasis and reproductive
output.
37
Because the HT115 strain is used for all classical RNA-interference assays, it is
intriguing to think that we may have missed several genetic determinants of phenotypes screened
because they were effectively masked by the altered metabolism on this specific diet.
38-40

Recently, a new RNAi competent bacterial strain derived from the OP50 background was
generated.
41
Even though most, if not all, RNAi screens are far from being saturated –
particularly those for aging and lifespan – this reagent will prove to be an extremely powerful
tool to reprobe previously done RNAi screens to more fully identify genetic regulators of worm
physiology.
42

C. elegans remains a premiere model to study the impact of diet – both composition and
amount – on animal life-history traits. Although recent studies challenge the notion that we have
a firm understanding of the pathways that integrate nutrients and physiology, they do in fact open
exciting new directions of study. The results of these efforts will not only answer fundamental
questions in biology but in light of their exceptional level of conservation are likely to have a
secondary consequence of uncovering some of the secrets of human health as well.

143
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Abstract (if available)

Abstract Animals must continually assess nutrient availability to develop appropriate strategies for survival and reproductive success. It is no secret that nutritional state plays a large role in both aging and health. Appropriate cellular energy usage is not only crucial for animal starvation survival, but is also important for diseases such as obesity and cancer, which characteristically have metabolic dysfunction. C. elegans are exceptionally well poised to handle bouts of starvation as resource availability in the wild varies greatly. We recently discovered an evolutionarily conserved pathway, regulated by the cytoprotective transcription factor SKN-1/Nrf2, which integrates diet composition and availability with utilization for survival. These responses have potent impact on organismal physiology and remarkably are influenced by current and parental life history events, including choice of diet. It is important to be cognizant of dietary intake and the impact that this can have throughout the life-history of the nematode, Caenorhabditis elegans.

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Asset Metadata

Creator Lynn, Dana Ann (author)

Core Title SKN-1 coordination of stress adaptation, metabolism, and resource allocation in Caenorhabditis elegans

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Publication Date 07/01/2016

Defense Date 06/06/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag ALH-6,Asdf,high carbohydrate diet,Lipids,MDT-15,metabolism,Nrf2,OAI-PMH Harvest,resource allocation,SKN-1,stress response

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Curran, Sean P. (committee chair), Dean, Matt (committee member), Finkel, Steven E. (committee member), Pike, Christian (committee member), Tower, John G. (committee member)

Creator Email danalynn@usc.edu,dlynn313@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-260305

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