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12-2014
The Effects of Probiotics Supplementation on
Health Using Caenorhabditis elegans as a Model
System
Miranda Klees
Clemson University, [email protected]
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Theses
THE EFFECTS OF PROBIOTICS SUPPLEMENTATION ON A HEALTH USING
CAENORHABDITIS ELEGANS AS A MODEL SYSTEM
A Thesis
Presented to
The Graduate School of
Clemson University
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Biological Sciences
by
Miranda L. Klees
December 2014
Accepted by:
Dr. Min Cao, Committee Chair
Dr. Yuqing Dong
Dr. Thomas A. Hughes
ABSTRACT
The “Western Diet,” prominent among developed nations, often refers to a
diet rich in meat proteins and refined sugars. Western society may in part be plagued with
obesity and obesity related diseases due to a diet enriched with glucose. With an
increasing glycemic index observed in Western society, it comes to no surprise that
obesity, diabetes, heart disease, and other obesity related diseases are on the rise. The
Centers for Disease Control and Prevention describes obesity as an epidemic, affecting
more than 35% of U.S. citizens. Shortened lifespan and increased susceptibility to
pathogens are associated with these diseases and linked to increased consumption of
foods high in sugar. In order to reverse these observed effects, the healthful benefits
probiotics have on immune system stimulation, restoration of shortened lifespan induced
by a high glucose diet, and the beneficial effects of probiotics in combination of
cranberry extracts in the models system Caenorhabditis elegans were investigated. An
exciting benefit of implementing C. elegans in such studies is the innate immunological
response and aging pathways homology with humans. By targeting these pathways, C.
elegans provides an economical model system that provides application in humans. Here,
we investigated the effects probiotics have on high glucose diet in C. elegans. Consistent
with previous studies, when C. elegans was supplemented with each probiotic strain
tested, lifespan extension was observed. Interestingly, the reversal of glucose induced
shortened lifespan and the extension of lifespan in combination of cranberry extracts was
strain dependent. The impact that probiotics have on the reversal of detrimental effects
associated with a high glucose diet, including protection against known pathogens and
ii
immunological response pathways targeted by probiotics, is currently under
investigation.
iii
DEDICATION
I would like to dedicate my work to my family, most importantly my parents.
Their love and support through failures and accomplishments have shaped me into the
woman I am today. The memory of my mother has been a major driving force for my
accomplishments, all the while striving to be the woman she shaped me to be. I would
also like to dedicate my work to my brothers and sisters and stepmom: Jacob, Marshall,
Tateum, Pyper, and Robyn for all of their support during my journey through graduate
school.
iv
ACKNOWLEDGEMENT
I would like to thank my advisor Dr. Min Cao for her invaluable support and
guidance in developing this project. I would also like to thank the rest of my committee
members Dr. Yuqing Dong and Dr. Thomas Hughes for their support and guidance in all
of my decisions and work progress.
I would also like to thank my lab mates for helpful discussions and experimental
advice and assistance: Daniel Pederson, Sujay Guha, Ethan Wilson, Joseph Angeloni,
Ojas Natarajan, Jessica Dinh, and Xiaxiao Wang. Lastly, I would like to thank my
undergraduate research advisor Dr. Joseph Flaherty for all of his support along the way
and sparking my interest in research that led to the pursuit of my master’s degree.
v
TABLE OF CONTENTS
Page
TITLE PAGE .................................................................................................................... i
ABSTRACT ..................................................................................................................... ii
DEDICATION ................................................................................................................ iv
ACKNOWLEDGEMENT ............................................................................................... v
LIST OF TABLES ........................................................................................................viii
LIST OF FIGURES ........................................................................................................ ix
LIST OF ABBREVIATIONS .......................................................................................... x
CHAPTER
1.
INTRODUCTION .......................................................................................... 1
Diet ............................................................................................................ 1
Obesity....................................................................................................... 2
Obesity and Microbiome ........................................................................... 3
Obesity and Immune Response ................................................................. 4
Caenorhabditis elegans as a Model System.............................................. 4
Probiotic Supplementation ........................................................................ 6
Cranberry and Application ........................................................................ 7
Significance ............................................................................................... 8
2.
MATERIALS AND METHODS .................................................................. 10
Bacterial Strains and Worm Strains ........................................................ 10
Preparation of CBE ................................................................................. 10
Preparation of Media ............................................................................... 11
Growth Curve .......................................................................................... 11
Lifespan Assay ........................................................................................ 12
3.
RESULTS ..................................................................................................... 14
Experimental Design ............................................................................... 14
Effects of D-glucose and CBE on bacterial growth ................................ 15
Effect of probiotics on lifespan ............................................................... 17
vi
Table of Contents (Continued)
Effect of D-glucose on lifespan ............................................................... 19
Probiotics reversal effects of glucose induced shortened lifespan ......... 20
Effects of probiotics in combination with CBE ...................................... 23
4.
DISCUSSION ............................................................................................... 26
5.
FUTURE DIRECTIONS .............................................................................. 31
REFERENCES .............................................................................................................. 33
vii
LIST OF TABLES
Table
Page
3-1
Lifespan extension via probiotics ................................................................ 18
3-2
Lifespan restoration via probiotics .............................................................. 22
3-3
Lifespan extension via probiotics + CBE .................................................... 24
viii
LIST OF FIGURES
Figure
Page
1-1 C. elegans life cycle ........................................................................................... 5
3-1 Experimental design diagram .......................................................................... 14
3-2 Growth curves of probiotic strains .................................................................. 16
3-3 Lifespan curves of each probiotic tested ......................................................... 17
3-4 Lifespan curve of control with glucose ........................................................... 19
3-5 Lifespan curves with and without 2% D-glucose ............................................ 21
3-6 Lifespan curves with and without 2mg/mL CBE ............................................ 25
ix
LIST OF ABBREVIATIONS
Bc ........................................................................................................ Bacillus coagulans
Bs ............................................................................................................. Bacillus subtilis
CBE ......................................................................................................Cranberry Extracts
CFU ................................................................................................. Colony Forming Unit
ddH2O .......................................................................................... Double-Distilled Water
E. coli OP50 ................................................................................... Escherichia coli OP50
FUDR ........................................................................................ 5-Fluoro-2′-deoxyuridine
IL .......................................................................................................................interleukin
LAB....................................................................................... Lactic Acid Bacilli/Bacteria
LB ...................................................................................................... Luria-Bertani Broth
Ld .............................................................................................. Lactobacillus delbrueckii
Lf ................................................................................................ Lactobacillus fermentum
Lp ............................................................................................. Lactobacillus planatarum
LPS ..................................................................................................... lipopolysaccharides
MRS ........................................................................................ Lactobacilli MRS Medium
NGM ...................................................................................... Nematode Growth Medium
NO ....................................................................................................................nitric oxide
p38 MAPK ..............................................................p38 mitogen-activated protein kinase
qRT-PCR......................................... Quantitative Real Time Polymerase Chain Reaction
spp .......................................................................................................................... species
subsp ............................................................................................................... sub-species
TNFα ...................................................................................... tumor necrosis factor alpha
x
CHAPTER ONE
INTRODUCTION
I. DIET
The “Western Diet,” prominent among developed nations, often refers to a diet
rich in meat proteins and refined sugars. Western society may in part be plagued with
obesity and obesity related diseases due to a diet enriched with glucose. An increasing
glycemic index has been observed in western society, and this increase has been linked to
obesity, diabetes, and heart disease. The Centers for Disease Control and Prevention
describes obesity as an epidemic, affecting more than 35% of U.S. citizens. Though
obesity is a complex trait influenced by diet, developmental stage, age, physical activity,
and genetics, the recent epidemic is probably reflective of environmental and lifestyle
changes, with diet being a major influence 1. Diet, from a scientific perspective, can be
described as the sum of food consumption by an organism. As humans, we have the
ability to consciously decide what foods we consume; however, proper nutrition involves
consumption of essential vitamins and minerals needed for metabolic processes. Much of
our food energy intake is consumed in the forms of carbohydrates, proteins, and fats. The
dietary decision of a healthful balance plays a major role on quality of life and health and
longevity. Therefore, diet not only affects metabolic activities and overall health, but it
also affects lifespan.
1
II. OBESITY
As previously mentioned, obesity is a complex characteristic influenced by many
factors including but not limited to diet, lifestyle, and genetics. The phenotype is
responsible for 6-10% of national healthcare costs in the United States; in addition to, a
lifetime of medical costs that are $10,000 greater in obese patients in comparison to nonobese. Researchers argue that the rise in obesity is the result of an increase in sedentary
lifestyles 2, 3 while others argue that the culprit is over-consumption and gluttony 4, 5. In
the 1960s, the average American weighed 168 pounds; today, that average has increased
nearly 15 pounds. Since the 1970s, obesity rates have doubled according to the Centers
for Disease Control and Prevention. Medications, cosmetic surgeries, and diet may be
methods for short-term treatment of obesity; however, long-term treatment involves a
lifelong commitment to healthy diet habits and increased physical activity. Still, most
people who previously suffered from obesity have relapses within 5 years of surgeries or
other methods of weight loss.
Obesity is characterized by excess body fat accumulation as a result of increased
energy intake compared to expenditure to the point that it becomes detrimental to health.
When more calories are consumed than expended, the resulting phenotype is weight gain.
Increased body fat proportionally increases the risk for chronic diseases such as diabetes,
heart disease, and cancer. Not only are these chronic diseases associated with obesity, but
bacterial diversity shifts are also observed in these patients
6, 7
. Other side effects of
obesity include immune system failures and increased sensitivity to pathogens.
2
III. OBESITY & THE MICROBIOME
The gut is home to a vast array of microorganisms that aid in digestive health,
immune stimulation, and provide a competitive environment to prevent colonization of
many harmful pathogens. With the alteration of diet associated with Western society, the
microbial community is severely impacted within the gut. The human body and
gastrointestinal tract are colonized at birth, and continues to shift based on factors, such
as breastfeeding, diet, and environmental exposure, until the microbial community
becomes stabilized around 3 years of age 8. The human gut is largely comprised of
Actinobacteria, Firmicutes, and Bacteriodetes 9. The functions of the gut microbiota
obtained throughout our lifespan are essential to the metabolism of xenobiotic
compounds, amino acids, and carbohydrates. The balance of bacteria within the gut can
be rapidly altered by dietary shifts. For example, when mice were fed a high-fat diet, the
gut microbial composition shifts were observed in 24 hours
10-13
. When altered gut
microbiota, resultant of a high-fat diet, were transferred to germ-free mice, the subjects
experienced increased adiposity
12
obesity are linked to microbiota
observed in obese subjects
10, 15-17
, consistent with the suggestion that adiposity and
14
. Specifically, a reduction of Bacteroidetes was
; however, not all studies consistently observed this
reduction 18. When obesity is induced via diet the resulted mice experienced an increase
of Eubacterium dolichum, a member of the Firmicutes phyla
Bifodobacterium, a member of the Actinobacteria phyla
17
12
and a decrease in
. Energy restriction and
increased physical activity have also been linked to changes in the gut microbiota 19, 20.
These microorganisms provide necessary enzymes involved in utilization of non-
3
digestible carbohydrates, involved in cholesterol reduction, important for biosynthesis of
vitamins K and B 21, 22, and involved in deconjugation and dehydroxylation of bile acids
which, necessary in solubilizing and absorbing lipids
23
. Factors other than diet also
played a crucial role in microbiota, including but not limited to, gut microbiota shifts in
obese subjects which were dependent on age 24.
IV. OBESITY & IMMUNE RESPONSE
A high-fat diet is associated with chronic low-grade inflammation and increased
susceptibility to infection 25. In obese patients, the innate immune system responds by
increased macrophage infiltration in adipose tissue and production of inflammatory
adipokines
26
. Increased plasma lipopolysaccharides (LPS) levels were observed with
obesity 17, 27, activating the synthesis of inflammatory cytokines TNFα. Chronic activation
of the innate immune system results in increased risk for development of obesity; dietary
compounds may in part be responsible 26.
V. CAENORHABDITIS ELEGANS AS A MODEL SYSTEM
C. elegans is a favorable model system for studying aging and immune response
for many reasons: 1) ease of maintenance, 2) homological pathways, including aging and
immunity, with humans, 3) transparent, 4) economically favorable, and 5) relatively short
lifespan. C. elegans offers a faster, efficient analysis of human related diseases while
allowing us to infer evolutionary history of immune components to study conserved traits
and obtain information about probable functions. Similar to humans, many pathogens
4
have the ability to colonize the digestive tract of C. elegans, giving rise to an ideal model
to study pathogenesis. However, many pathogens may exhibit other modes of infection in
the worm, adhering to the cuticle or producing lethal toxins
28, 29
. We are able to
determine the pathology of such pathogens by determination of a decrease in lifespan.
Though much less complex than the mammalian immune response, C. elegans
immune defenses similarly consists of an innate immune response in which it is solely
reliant. In its natural habit, the
soil-dwelling nematode feeds on
bacteria
and
has
frequent
encounters with pathogens. Most
pathogens infect the worm via
ingestion and then establish in
the intestine where the majority
of
the
damage
occurs.
In
response to invasive pathogens
the
Figure 1-1. C. elegans life cycle at 25°C (artwork from
http://www.scq.ubc.ca/genetic-studies-of-aging-andlongevity-in-model-organisms/).
worms
defenses
have
that
developed
include
a
behavioral response, physical barriers, and physiological response. As the first line of
defense, behavioral responses must be employed to reduce the number pathogen
encounters. However, exposure is inevitable and the worm must elicit physiological
response to prevent colonization of the gut during the process of feeding. The p38
mitogen-activated protein kinase (MAPK) pathway and the insulin-like receptor pathway
5
are two pathways involved in immune response, longevity, and mammalian homology.
These two pathways interact and respond to a vast array of stress conditions 30. The p38
MAPK pathway is activated in response to pathogens such as Pseudomonas aeruginosa
and Salmonella enterica
31, 32
, as well as in response to other pathogens and other
stressors such as acute dehydration. The insulin-like receptor pathway is also important to
the response to P. aeruginosa, Enterococcus faecalis, and Staphylococcus aureus 33. This
pathway also has complex interactions with other regulatory elements such as the heat
shock factor-1 (HSF-1) and the superoxide dismutase-3 (SOD-3).
C. elegans possesses a complex and interactive innate immunological response
that has been extensively studied 30, 34. The advantages of using this experimental system
as a means to study application in humans is evident through the extensive study of these
evolutionarily conserved pathways.
VI. PROBIOTICS SUPPLEMENTATION
A probiotic can be defined as any microorganism believed to provide health
benefits upon consumption. The most common types of probiotics found in our diet are
Bifidobacterium and lactic acid bacteria. Several strains of lactic acid bacteria, mainly
Lactobacillus spp. have been shown to induce longevity
35
as well as alleviating the
effects of infection caused by Vibrio parahaemolyticus, Salmonella typhimurium,
Salmonella
enteritidis,
Staphylococcus
aureus,
and
Enterococcus
faecalis35-38.
Bifidobacteria have the ability to increase lifespan in C. elegans via the p38 MAPK
pathway, a homologous innate immunological pathway with humans 39. However, the
6
pathway in which each probiotic targets may be dependent on the strain of probiotic and
the infectious strain of bacteria. Kim et al. suggested that Lactobacillus acidophilus had
minimal effects on Gram-negative bacteria Pseudomonas aeruginosa and Salmonella
enterica Typhimurium. On the contrary, L. acidophilus provided protection from
Enterococcus faecalis and Staphylococcus aureus infection while targeting the p38
MAPK pathway suggested during Gram-positive immune response in C. elegans 37. The
aforementioned probiotics and the probiotics utilized in this study are often consumed in
yogurts, fermented foods and dietary supplements. More interestingly, previous studies
suggest that probiotics have the ability to enhance the immune response in not only C.
elegans but also in a higher organism model system, mice 37, 40-42.
VII. CRANBERRY & APPLICATION
The probiotics targeted in this study are often consumed in yogurts,
fermented foods, and dietary supplements. Though probiotic supplementation is an
emerging area of research, little is known about the benefits they may have on
healthspan and lifespan, particularly when supplemented with nutraceuticals.
Previous studies suggest that probiotics have the ability to enhance the immune
response in not only C. elegans but also in mice 37, 40-42. Numerous studies have also
linked nutraceuticals to longevity and stress resistance 43, 44. Still, little is known of
the benefits of synergistic supplementation. For years, the North American cranberry
(Vaccinium macrocarpon) has been ingested to alleviate the effects of a urinary tract
infection by preventing adherence of bacteria to host’s cells
7
45
. However, there is no
evidence suggesting that cranberry is actually effective in treating or even preventing a
urinary tract infection 46. Preliminary data suggests components in cranberry may prevent
plaque formation on teeth 47, kill Helicobacter pylori (the causative agent of stomach
cancer and ulcers) 48, and even prevent tumor development 49. Research suggests that
cranberry provides many nutritional and healthful benefits, owing to its antioxidant,
antimicrobial, anti-apoptotic, anti-aging, anticancer, anti-inflammation, and enhancement
of endothelial functions
49
. Recently published research from our lab suggests that
cranberry, when studied in the soil dwelling nematode model organism C. elegans,
effectively extends lifespan and promotes healthy aging while elevating resistance to
certain stressors, including bacterial infection 44, 50.
VIII. SIGNIFICANCE
Previous research suggests that probiotics such as Lactobacillus isolates can
control and alleviate the effects of pathogenic infections; however, much still needs to be
studied. C. elegans provides an effective model of study of pathogenesis with application
in humans 35-38, 40. Another exciting benefit of implementing C. elegans in such studies is
the homology in humans in the innate immunological response pathways and aging
pathways. The optimal combination of probiotics that successfully stimulates the immune
system has yet to be studied. If we are able to determine a favorable combination of
probiotics that have the ability to reverse the effects of a high glucose diet while
promoting healthy aging and aiding in immune stimulation, we may be able to further
provide insight into the effects that a high-glucose diet has on overall health and how to
8
reverse such detrimental effects. Studies can then be carried out in a higher organism
such as mice to further confirm implication in supplementing a particular combination of
probiotics in our diet.
9
CHAPTER TWO
MATERIALS & METHODS
I. BACTERIAL STRAINS AND WORM STRAINS
L. fermentum (ATCC 9338), L. planatarum (ATCC 8014), L. delbrueckii (ATCC
7830), and B. coagulans were all acquired from VWR. B. subtilis CU1065 was a lab
strain originally acquired from Cornell University. E. coli OP50 was acquired from lab
stocks. Lactobacillus strains were cultured anaerobically in 5% CO2 on MRS agar at
37°C for 48 hours. Bacillus spp. were cultured aerobically on LB agar at 37°C overnight.
E. coli OP50 was cultured on LB+streptomycin (100μg/mL) at 37°C overnight.
Bacillus spp. and E. coli OP50 overnight cultures for lifespan assays were
inoculated in LB broth and incubated at 37°C shaking at 150 rpm. Lactobacillus strains
were inoculated in MRS broth and incubated at 37°C stationary in 5% CO2.
N2 worms were acquired from the Caenorhabditis Genetics Center (CGC).
Worms were maintained on nematode growth medium (NGM) seeded with E. coli OP50
at 25°C, and transferred to fresh plates every two days.
II. CBE PREPARATION
The cranberry extracts, HI-PAC BL DMAC 5, used in this study were obtained
from DECAS cranberry products, Inc. and were received in the powdered form. For
working stock solutions, CBE was dissolved in ddH2O at a concentration of 5% and final
concentration used was 0.2% (2mg/mL).
10
III. PREPARATION OF MEDIA
NGM for maintenance was prepared via standard protocol in 60mm plates. E. coli
OP50 was dropped on the center of the plates the night prior to transfer of worms.
NGM+glucose plates were prepared via standard protocol with the addition of 2% Dglucose final concentration in 60mm plates. Bacterial strains were dropped onto the
center of the plates 2 hours prior to worm transfer.
NGM-FUDR 35mm plates were used for lifespan assays. Five times concentrated
bacterial cultures were dropped in 100μL aliquots onto the center of plates 2 hours prior
to worm transfer. D-glucose was added with a final concentration of 2% to NGM-FUDR
plates prior to pouring and 5mL aliquots were added to each plate. For CBE testing, CBE
was dissolved in water and dropped and spread confluent onto 5mL NGM-FUDR the
night prior to start of assay.
IV. GROWTH CURVE
A single colony of each strain was inoculated from a freshly streaked plate into 5
mL MRS for Lactobacillus strains, LB for Bacillus strains, and LB+strep (100μg/mL) for
E. coli OP50. Lactobacillus spp. were grown statically overnight at 37°C in 5% CO2. E.
coli OP50 and Bacillus strains were grown under aerobic conditions overnight at 37°C.
On the next day, the overnight cultures were transferred into sterile 15 mL centrifuge
tube and centrifuged at 4000 rpm for 10 min at room temperature. Cell pellets were
resuspended in 10 mL NGM liquid medium. Aliquots of 50 μl were plated from the
following dilutions 105 and 106 onto designated agar plates. This was the CFU for T=0.
11
Resuspended cultures were transferred in 3 mL aliquots into each glass test tube, and then
desired concentrations of glucose were added to corresponding tubes.
Tubes were
incubated under aerobic conditions at 37°C. Lactobacillus tubes were kept at 37oC CO2
incubator. At each time point (T=2, 4, 12, 24, 48 hr), serial dilutions were performed and
dilutions to be plated were estimated based on cultural density. Aliquots of 50 μL were
plated on corresponding plates. CFU/mL were calculated the next day using the standard
plate count method.
V. LIFESPAN ASSAYS
Lifespan assays were carried out at 25°C. Worms were synchronized by
transferring 20 gravid worms to 60mm NGM plates seeded with E. coli OP50 2 days
prior to the start of assays. Worms were allowed to lay eggs for 5 hours, and then parent
worms were removed from plates, leaving only synchronized eggs. Worms were then
incubated until L4 stage. Overnight cultures of Bacillus spp. and E. coli OP50 were
concentrated by centrifugation and removing 80% of supernatant. Cultures were then
resuspended in remaining medium. Lactobacillus overnight cultures were concentrated
via centrifugation and removing 100% of supernatant. Cultures were resuspended in 1
mL of M9 buffer. Aliquots of 100μL were dropped on the center of NGM-FUDR 35mm
plates with and without 2% D-glucose and allowed to dry for 2 hours. Worms in L4 stage
were transferred to assay plates; 30 worms per plate. Plates were subsequently checked
daily and dead worms were counted and recorded. Day of transfer was defined as zero.
Statistical analysis was carried out through SPSS software under Kaplan-Maier lifespan
12
analysis. P-values were determined via log rank test, and P<0.05 was accepted as
statistically significant.
13
CHAPTER THREE
RESULTS
I. EXPERIMENTAL DESIGN
Previous studies showed that 2% glucose significantly reduced lifespan in C.
elegans51. Consistent with this studied, 2% D-glucose significantly decreased the lifespan
of N2 worms; therefore, 2% D-glucose was used to represent a high glucose diet.
Similarly, previous studies from our lab determined 2mg/mL was the ideal concentration
of CBE supplementation to extend lifespan and modulate the immune response
44, 50
.
Based on previous studies, a series of experiments were employed to determine the
effects that probiotics had on reversing the effects of a high glucose diet in C. elegans
and the effects probiotics have on lifespan in combination with CBE (Figure 3-1).
L4 worms
were
transferred
ControlOP50
No
2% D-glucose 2mg/mL CBE
supplements supplemented supplemented
Test-with
probiotics
2% D-glucose
No
supplements supplemented
2mg/mL CBE
supplemented
Figure 3-1. Diagram of the experimental design of lifespan assays carried out at 25°C
with different diets.
14
II. EFFECTS OF 2% D-GLUCOSE AND 2mg/mL CBE ON BACTERIAL GROWTH
Glucose induced shortened lifespan was first observed in C. elegans (Table 3-2).
Dietary restriction is a well-documented cause of lifespan extension in the worm model.
To determine if 2mg/mL CBE and/or 2% D-glucose would affect bacterial growth,
growth curves using the standard plate count method were performed. Each strain was
treated with either 2% D-glucose or 2mg/mL CBE for up to 72 hours, growth was not
significantly inhibited (Figure 3-2 A-F).
15
E. coli OP50
10
9
8
7
0
20
40
Bs
B.
60
80
Log10(CFU/mL)
Log 10(CFU/mL)
A.
9
8
7
6
0
Time (hours)
Bc
D.
9
8
7
6
0
20
40
60
80
8
7
6
5
0
Log10(CFU/mL)
Log10(CFU/mL)
F.
10
99
8
77
50
80
50
100
Time (Hours)
Lp
6
0
60
Lf
9
Time (Hours)
E.
40
Time (Hours)
Log10(CFU/mL)
Log10(CFU/mL)
C.
20
100
Time (Hours)
Ld
9
8
7
6
5
-20
30
80
Time (Hours)
Figure 3-2. Growth curves of each probiotic with and without 2% D-glucose and with
and without 2mg/mL CBE in NGM broth. A) E. coli OP50 B) B. subtilis C) B. coagulans
D) L. fermentum E) L. planatarum F) L. delbrueckii.
16
III. EFFECT OF PROBIOTICS ON LIFESPAN
Previous studies suggested that probiotic strains significantly extend lifespan in C.
elegans; however, the observable extension has also been suggested to be dose
dependent39-42,
52
. The five strains tested in this study all extended lifespan when
supplemented during L4 stage of worms (Figure 3-3, Table 3-1). B. subtilis fed worms
had one day lifespan extension compared to the control. B. coagulans fed worms had 4
days lifespan extension compared to the control, L. fermentum had 3 days lifespan
extension, L. planatarum had 1.5 days extension, and L. delbrueckii had 2 days lifespan
extension.
Figure 3-3. Probiotic strains extended lifespan of N2 worms compared to E. coli OP50 at
25°C. L. planatarum exhibited greatest lifespan extension with mean lifespan of 16.042
days.
17
Table 3-1. Effect of probiotics on lifespan of N2 worms at 25°C
Mean±SE (Days)
N
P-value
E. coli OP50
B. coagulans
L. fermentum
L. planatarum
L. delbrueckii
13.442±0.244
17.261±0.336
16.663±0.213
15.085±0.249
15.480±0.307
95
92
92
82
75
<0.05
<0.05
<0.05
<0.05
% Increase
of Lifespan
28.41%
28.45%
12.22%
15.16%
E. coli OP50
B. subtilis
12.737±0.459
14.468±0.369
99
109
<0.05
13.6%
Diet
Probiotic strains extended lifespan compared to E. coli OP50, consistent with previous
research. P-value <0.05 considered significant. Data shown are representative of one
round of lifespan assays.
18
IV. EFFECT OF D-GLUCOSE ON LIFESPAN
Consistent with previous studies, when glucose was supplemented at a
concentration of 2% into the worm diet, lifespan was significantly reduced. C. elegans’
lifespan was significantly reduced 15.7% as compared to the control strain without
glucose (Figure 3-4, Table 3-2).
Figure 3-4. Glucose significantly reduced lifespan of N2 worms under normal lab
conditions at 25°C. Lifespan was reduced by 2 days, P<0.05. Data shown are
representative of one round of lifespan assays.
19
V. PROBIOTICS REVERSAL EFFECTS OF GLUCOSE INDUCED SHORTENED
LIFESPAN
As previously mentioned, each probiotic strain extended lifespan, and glucose
was determined to reduce lifespan. When probiotics were supplemented with glucose,
reversal of the glucose induced shortened lifespan was dependent on the strain of
probiotic. Though lifespan was not fully restored, three of the five strains exhibited some
level of lifespan restoration. The control strain exhibited a 15.7% decrease in lifespan. B.
subtilis only exhibited a 5.3% decrease, B. coagulans had an 11.39% decrease, L.
fermentum had a 16.1% decrease, and L. delbrueckii had an 8.7% decrease. L.
planatarum exhibited the most dramatic decrease of 64.6% reduction of lifespan (Figure
3-5, Table 3-2).
20
A.
B.
C.
D.
E.
F.
Figure 3-5. Lifespan curves of N2 worms fed on each probiotic with or without 2% glucose supplemented into diet at 25°C.
A) Control E. coli OP50 B) B. subtilis fed worms C) B. coagulans fed worms D) L. fermentum fed worms E) L. planatarum
fed worms F) L. delbrueckii fed worms.
21
Table 3-2. Lifespan of N2 worms on high glucose diet and probiotics at 25°C
Diet
E. coli OP50
OP50+2% glucose
B. subtilis
Bs+2% glucose
B. coagulans
Bc+2% glucose
L. fermentum
Lf+2% glucose
L. planatarum
Lp+2% glucose
L. delbrueckii
Ld+2% glucose
Mean±SE (Days)
N
P-value
% Decrease
of Lifespan
13.442±0.225
11.333±0.207
13.477±0.202
12.759±0.138
17.261±0.221
15.301±0.142
16.663±0.262
13.986±0.240
15.085±0.273
5.342±0.130
15.48±0.256
14.135±0.318
95
69
86
87
92
73
92
73
82
73
75
74
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
15.70%
5.30%
11.39%
16.10%
64.60%
8.70%
N2 lifespan was shortened with all strains when supplemented with glucose. Lifespan
restoration was observed in Lactobacillus delbrueckii, Bacillus subtilis, and Bacillus
coagulans. Worms fed Lactobacillus planatarum exhibited an extreme decrease in
lifespan when supplemented with 2% D-glucose. Data shown are representative of one
round of lifespan assays.
22
VI. EFFECTS OF PROBIOTICS IN COMBINATION WITH CBE
Previous studies in our lab have determined that the optimum concentration of
cranberry supplementation was 2mg/mL. At this concentration C. elegans’ lifespan is
extended significantly without affecting bacterial growth of different strains such as
Vibrio cholerae and E. coli OP50. When CBE was supplemented at the time of exposure
to probiotics, C. elegans lifespan was extended (Figure 3-6 A-E). We can also conclude
that when C. elegans is fed probiotics in succession with CBE supplementation, lifespan
was significantly extended in comparison without CBE treatment. L. planatarum only
exhibited bacterial extension effect, and lifespan was not further extended when 2mg/mL
CBE was supplemented into the diet (Figure 3-6 A-E).
23
Table 3-3. Lifespan of N2 worms fed on probiotics and CBE diet at 25°C
Mean±SE (Days)
N
P-value
% Increase
of Lifespan
E. coli OP50
12.737±0.459
99
-
-
B. subtilis
14.468±0.369
109
<0.05
13.6%
B.s.+2mg/mL CBE
15.359±0.106
64
<0.05
20.6%
B. coagulans
15.295±0.181
88
<0.05
20.1%
B.c.+2mg/mL CBE
16.136±0.130
81
<0.05
26.7%
L. fermentum
13.44±0.511
90
<0.05
5.5%
L.f.+2mg/mL CBE
15.575±0.320
87
<0.05
22.3%
14.9±0.194
100
<0.05
17%
L.p.+2mg/mL CBE
14.693±0.300
75
<0.05
15.4%
L. delbrueckii
14.364±0.459
66
<0.05
12.8%
L.d.+2mg/mL CBE
15.742±0.304
66
<0.05
23.6%
Diet
L. planatarum
N2 lifespan was increased with all strains when supplemented with CBE except L.
planatarum. Lifespan extension was observed in Lactobacillus delbrueckii, Lactobacillus
fermentum, Bacillus subtilis, and Bacillus coagulans. Data shown are representative of
one round of lifespan assays.
24
A.
C.
B.
E.
D.
Figure 3-6. N2 lifespan curves carried out at 25°C with and without 2mg/mL CBE and probiotics. A) B. subtilis B) B.
coagulans C) L. fermentum D) L. planatarum E) L. delbrueckii.
25
CHAPTER FOUR
DISCUSSION
Over a century ago, a Russian scientist by the name of Élie Metchnikoff first
hypothesized that probiotics could beneficially modify the gut flora by replacing harmful
pathogens. Recently, manufacturers’ marketing of foods containing beneficial probiotics
has fueled the renewed interest in probiotics research. However, the consumption of
probiotics as a means to provide health benefits can be traced back to the Greeks and
Romans through the consumption of fermented food products such as cheeses and other
dairy products.
The modern hypothesis, adapted from Metchnikoff’s findings, stating that
probiotics and microbiota have major influences on human health, has further evolved
with the recent increase in consumer awareness. Consumers and researchers have
expanded the research interests to target the use of oral pill supplementation of probiotics
into diet and some have even experimented with fecal transplantations as a means to
combat digestive disorders. Of particular importance are the LAB strains. Lactobacillus
is a genus of Gram-positive facultative anaerobes or microaerophilic rod-shaped bacteria.
Their production of lactic acid from the conversion of lactose and other sugars, create an
acidic environment, prohibiting the growth of some harmful pathogens. The lower pH
also provides an environment for a balanced ecosystem in the gut. Metabolism and
metabolite production by Lactobacillus spp. are not only species dependent but also
dependent on the strain and/or subspecies.
26
In combination with previous research of beneficial supplementation of probiotics
as a means to extend lifespan in C. elegans and research suggesting a high glucose diet
shortens lifespan in C. elegans, the reversal effects of probiotics supplementation on a
high glucose diet were studied. Consistent with previous research, the probiotic strains
used in this study significantly extended lifespan when supplemented individually (Figure
2-3).
Lactobacillus planatarum are heterofermentative bacteria producing D-lactic acid,
L-lactic acid, and acetic acid through the Embden-Meyerhoff pathway and are also
known to produce hydrogen peroxide to eliminate other competitive bacteria. They are
found in fermented plant matter such as pickles and kimchi and the most common
bacteria used in silage. Supplementation of L. planatarum may provide anti-inflammatory
effects via reduction of IL-1β, IL-6, and C-reactive protein (CRP), immune response
components involved in inflammation, while increasing IL-10 53. L. planatarum has the
ability to extend lifespan in C. elegans (Figure 3-3, Table 3-1); however, when glucose is
supplemented into C. elegans diet along with L. planatarum, the shortened lifespan
induced by glucose is worsened dramatically (Figure 3-5E, Table 3-2). The exhibited
shortened lifespan could be a result of a multitude of reasons ranging from metabolites
produced by the bacteria to protective compound produced by the bacteria such as
hydrogen peroxide. Similarly with glucose, L. planatarum fed worms had a slight
reduction in lifespan when supplemented with CBE (Figure 3-6D). L. planatarum fed
worms exhibited a 17% increase in lifespan; however, when supplemented with CBE,
there was only a 15.4% increase in lifespan (Table 2-3).
27
Lactobacillus delbrueckii are homofermentative bacteria that produce lactic acid.
They are comprised of four subspecies separated by their metabolism. L. delbrueckii is
commonly found in yogurts and cheeses. Worms fed on L. delbrueckii exhibited
extension in lifespan (Table 3-1), and when supplemented with a high glucose diet, the
probiotic restored lifespan compared to the control. The control exhibited a 15.7%
decrease in lifespan when fed on a high glucose diet; however, when the worms were fed
a high glucose diet with L. delbrueckii, lifespan was only reduced 8.7%. The mechanism
for restorative properties has yet to be determined. L. delbrueckii subsp. bulgaricus
produces nitric oxide (NO) 54. NO has previously been shown to extend lifespan in C.
elegans 55. NO is responsible for mucus generation, motility, and water and electrolyte
transport. More interestingly, when L. delbrueckii lifespan extension properties were
further enhanced when supplemented with CBE, with an increase from 12.8% lifespan
extension to 23.6% lifespan extension compared to the control (Table 3-3, Figure 3-6E).
L. fermentum are heterofermentative bacteria. Although L. fermentum may be
responsible for dental caries, it can also be utilized as a probiotic. The probiotic is
commonly found in sourdough breads and other fermented plant products. Some strains
have the ability to metabolize cholesterol 56. Though this could be a possible cause of
lifespan extension in C. elegans (Table 3-1), the mechanism of lifespan extension has yet
to be studied. Though worms fed L. fermentum exhibited lifespan extension, when
supplemented with a high glucose diet, the reduction of lifespan was nearly the same as
the control worms fed on E. coli OP50 (Figure 3-5D, Table 3-2). On the other hand, when
the probiotic was supplemented with CBE, lifespan was further extended from 5.5% to
28
22.3%. Interestingly, this combination may provided insight into the combination of
probiotics and fruit extracts commonly supplemented together in the diet.
B. subtilis are spore forming obligate aerobes, commonly found in the human gut.
Because of the spore forming abilities that allow the bacteria to survive under harsh
conditions, the bacteria have become targets for probiotic supplementation in pasteurized
foods and beverages. B. subtilis have the ability to extend lifespan in C. elegans through
nitric oxide production 55. Here, B. subtilis had the ability to reverse the effects of a high
glucose diet in C. elegans, though not fully. B. subtilis fed worms exhibited only a 5.3%
decrease in lifespan when supplemented with glucose; whereas, the control strain had a
15.7% decrease in lifespan. More interestingly, when worms were fed B. subtilis in
combination with CBE, their lifespan was further increased from 13.6% to 20.6%.
B. coagulans are spore forming facultative anaerobes, and are used as a probiotic
in livestock feed. B. coagulans have the ability to extend lifespan in C. elegans (Table 31), and have some restorative properties when supplemented with a high glucose diet. In
worms fed B. coagulans and a high glucose diet, lifespan was only decrease 11.39% as
compared to the 15.7% decrease in lifespan exhibited in the control. Lifespan in worms
was also further extended when supplement in combination with CBE, extending lifespan
from 20.1% to 26.7%.
In this study, we observed the ability of probiotics to reverse the effects of a high
glucose diet in C. elegans. Owing to C. elegans’ pathway homology in humans, our
findings can provide insight into the possible reversal effects of probiotic
supplementation in humans. As previously mentioned, obesity and other diseases
29
associated with a high glucose diet have become a major issue in developed nations. The
insulin-like signaling pathway involved in aging and innate immune response in C.
elegans is among the pathways of homology in humans.
Insulin, a hormone involved in regulating metabolism and absorption of glucose
in mammals, and the homolog IGF-1 have been shown to regulate lifespan in many
organisms and is involved in other cellular processes such as stress response 57-59. The
FOXO transcription factor DAF-16 in the insulin/IGF-1 signaling pathway and decreased
activity of heat shock transcription factor HSF-1 reduced the rate of tissue aging,
resulting in increased lifespan 60, 61. The DAF-16 and HSF-1 transcription factor may be
inhibited by a high-glucose diet. A downstream gene encoding a glycerol channel aqp-1
was also involved in lifespan reduction induced by a high glucose diet 51. In mammals,
aquaporin glycerol channels are repressed by insulin 62, 63.
Our findings may also have implications in prevention and/or cure of obesity
related diseases such as type 2 diabetes, tumor formation and heart disease. Studies could
be further carried out in a higher organism such as mice. Because of the pathway
homology including insulin/IGF-1-mediated signaling (IIS) pathway, p38 kinase
pathway, and other immunological/stress response pathways between C. elegans and
humans, these findings may be of high human health importance. In addition these
findings may lead to relatively inexpensive and highly accessible prevention or treatment
for infection.
30
FUTURE DIRECTIONS
Through execution of these objectives, we were able to provide more insight into
the effects probiotics have on overall health with possible application in humans. In
addition, our findings may provide insight into future studies such as probiotics’ effect on
the microbiome. Based on our preliminary data that 1) each probiotic strain extends
lifespan significantly compared to the control, and 2) with the exception of L. planatarum
and L. fermentum, each strain restores lifespan when exposed to a high glucose diet,
though not fully, we propose to test the possible pathway involvement in this observed
protection. Because of the pathway homology including insulin/IGF-1-mediated
signaling (IIS) pathway, p38 kinase pathway, and other immunological/stress response
pathways between C. elegans and humans, we intend to further test the pathways
involved in the alleviated effects of a high glucose diet triggered by probiotic
supplementation. The transcription factor of the IIS pathway DAF-16 is required for
longevity in C. elegans 60. The DAF-16 target sod-3 encodes a superoxide dismutase
involved in stress response
64
. And the downstream gene of DAF-16, aqp-1, which
encodes for a protein in the aquaporin glycerol channel, may be involved in glucoseinduced decreased lifespan in C. elegans. Similarly in mammals, aquaporin glycerol
channels are repressed by insulin 62. We can test these pathways via qRT-PCR. If these
genes are involved in the regulation of restored lifespan, we would expect to see
increased expression in daf-16, sod-3, and aqp-1 in worms fed on probiotics compared to
E. coli OP50 and increased expression in worms fed probiotics in the presence of
31
glucose. Finally, we would expect to see down-regulation of these genes when worms are
fed our control strain E. coli OP50 with glucose.
Successful determination as to which pathways are involved in probiotic mediated
restoration of lifespan will provide insight into the mechanism with application in
humans and potentially lead to further studies in a higher organism.
32
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