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Doctoral Dissertations Dissertations and Theses

July 2019

THE BIOACTIVE EFFECTS OF CHICORIC ACID AS A FUNCTIONAL

FOOD INGREDIENT
Ye Peng
University of Massachusetts Amherst

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Peng, Ye, "THE BIOACTIVE EFFECTS OF CHICORIC ACID AS A FUNCTIONAL FOOD INGREDIENT" (2019).
Doctoral Dissertations. 1588.
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THE BIOACTIVE EFFECTS OF CHICORIC ACID AS

A FUNCTIONAL FOOD INGREDIENT

A Dissertation Presented

by

YE PENG

Submitted to the Graduate School of the

University of Massachusetts Amherst in partial fulfillment

of the requirements for the degree of

DOCTOR OF PHILOSOPHY

MAY 2019

The Department of Food Science

© Copyright by Ye Peng 2019

All Rights Reserved

 THE BIOACTIVE EFFECTS OF CHICORIC ACID AS

A FUNCTIONAL FOOD INGREDIENT

A Dissertation Presented

by

YE PENG

Approved as to style and content by:

_______________________________________
Yeonhwa Park, Chair

_______________________________________
Lynne A. McLandsborough, Member

_______________________________________
Young-Cheul Kim, Member

____________________________________
Eric A. Decker, Department Head
Department of Food Science
 DEDICATIONS

Dedicated to my husband, my parents, my twin sister,

and parents-in-law.
 ACKNOWLEDGEMENTS

First of all, I would like to express the deepest gratitude to my advisor, Dr.

Yeonhwa Park, for giving me a great opportunity to pursue my Ph.D. study in her lab and

for her continuous guidance, support, and patience during the four years. Without her

kind help and encouragement, I cannot make it here. My sincere thanks also go to my

thesis committee, Dr. McLandsborough and Dr. Kim, for their great support and guidance

during my study here.

I would like to give special thanks to Dr. Decker, the department head, and Mr.

Peter Salmon, the CFS CPP of IFN Consulting, who generously gave great help, financial

support, and gracious encouragement to me.

Many thanks to all of my lab members, past and present: Dr. Yoo Kim, Dr. Xiao

Xiao, Dr. Peiyi Shen, Mr. Daniel Colmenares, Dr. Ou Wang, Dr. Phoebe Chen, Dr. Jason

Yang, Dr. Yiren Yue, Dr. Weipeng Qi, Dr. Renalison Farias Pereira, Ms. Jinning Liu, Ms.

Jiaying Wang, and Mr. Yuejia Xu for their friendship and help during the study period.

I would like to thank all of my friends in Amherst, especially Siyue Gao, Xinyi

Du, Ruojie Zhang, Hualu Zhou, Yuxi Wang, Tianxi Yang, Shuqi, Yanqi Qu, Weicang

Wang, Zipei Zhang, Zili Gao, for their help and friendship.

I want to thank my loving parents and my beautiful sister for their unwavering

support and encouragement. Finally, I graciously thank my husband, Quancai Sun for

giving me a warm home feeling wherever I am.

v
 ABSTRACT

THE BIOACTIVE EFFECTS OF CHICORIC ACID AS

A FUNCTIONAL FOOD INGREDIENT

MAY 2019

YE PENG

B.S., NORTHWEST A & F UNIVERSITY, CHINA

M.S., UNIVERSITY OF GEORGIA, ATHENS, GEORGIA

Ph.D., UNIVERSITY OF MASSACHUSETTS, AMHERST, MA

Directed by: Professor Yeonhwa Park

Chicoric acid, a hydroxycinnamic acid, has been reported to possess a variety of

health benefits, including antivirus, anti-oxidation, anti-inflammation, obesity prevention

and neuroprotection effects. However, its role on aging and glucose homeostasis are

largely unknown. Therefore, the bioactive effects of chicoric acid on aging and glucose

homeostasis were determined using Caenorhabditis elegans (C. elegans) and C2C12

myotubes, respectively. Our study showed that chicoric acid (25 and 50 µM) significantly

extended the lifespan of C. elegans and increased median survival rates. The declines of

pumping rate and locomotive activity, two indicators of aging, were delayed by chicoric

acid. Chicoric acid also enhanced resistance to oxidative stress compared to the control.

These effects were in part via AAK-2 (a homolog of AMP-activated protein kinase) and

vi
SKN-1 (a homolog of nuclear factor erythroid 2-related factor 2). Furthermore, chicoric

acid significantly enhanced glucose uptake and the phosphorylation of protein kinase B

(Akt) as an indicator of insulin signaling pathway independent of insulin in C2C12

myotubes. These effects were dependent of the phosphorylation of 5'-AMP-activated

protein kinase α (AMPKα), via increasing the AMP/ATP ratio in C2C12 myotubes. Our

study may suggest the potential of chicoric acid to be used as an anti-aging and anti-

diabetic bioactive compound.

vii
 TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ................................................................................................ v

ABSTRACT ....................................................................................................................... vi

LIST OF TABLES ............................................................................................................. xi

LIST OF FIGURES .......................................................................................................... xii

CHAPTER

1. INTRODUCTION .......................................................................................................... 1

2. LITERATURE REVIEW ............................................................................................... 3

2.1 Chicoric acid ........................................................................................................... 3

2.2 The biological effects of chicoric acid .................................................................... 4

2.2.1 Anti-viral effects of chicoric acid ................................................................. 4

2.2.2 Chicoric acid and inflammatory responses ................................................... 6

2.2.3 Chicoric acid and glucose metabolism ......................................................... 7

2.2.4 Chicoric acid and lipid metabolism & liver function.................................... 8

2.2.5 Chicoric acid and brain function ................................................................... 8

2.2.6 Anti-oxidative stress effects of chicoric acid ................................................ 9

2.2.7 Other bioactivates of chicoric acid ............................................................. 10

2.3 Suggested molecular targets of chicoric acid ........................................................ 20

2.4 Pharmacokinetic study of chicoric acid ................................................................ 22

viii
 2.5 Conclusion ............................................................................................................ 22

3. OBJECTIVES OF THE PROJECT .............................................................................. 23

4. EFFECTS OF CHICORIC ACID ON AGING AND GLUCOSE UPTAKE .............. 24

4.1 Effect of chicoric acid on aging in C. elegans and the underlying mechanisms .. 24

4.1.1 Introduction .................................................................................................. 24

4.1.2 Materials and methods .................................................................................. 25

4.1.2.1 Materials .................................................................................................. 25

4.1.2.2 C. elegans: strains and maintenance ....................................................... 26

4.1.2.3 Growth rate, body size, and moving speed determination ...................... 27

4.1.2.4 Progeny production and pumping rate determination ............................. 27

4.1.2.5 Measurement of intracellular ROS and oxidative stress resistance ........ 28

4.1.2.6 Quantitative real-time PCR ..................................................................... 28

4.1.2.7 Intracellular localization of SKN-1::GFP ............................................... 29

4.1.2.8 Western blotting ...................................................................................... 29

4.1.2.9 Statistical analysis ................................................................................... 30

4.1.3 Results ......................................................................................................... 30

4.1.3.1 Chicoric acid extended the lifespan of C. elegans .................................. 31

4.1.3.2 Effect of chicoric acid on the age-associated decline of phenotypes and

ROS levels ............................................................................................................ 33

4.1.3.3 Extended lifespan by chicoric acid required AAK-2 and SKN-1 ........... 35

ix
 4.1.3.4 Chicoric acid might extend C. elegans lifespan through upregulation of

aak-2 and skn-1 ..................................................................................................... 38

4.1.3.5 Chicoric acid enhanced oxidative stress resistance in wild type N2 worms

but not aak-2 mutants............................................................................................ 41

4.1.4 Discussion ................................................................................................... 42

4.2 Effect of chicoric acid on glucose uptake and the underlying mechanisms ......... 47

4.2.1 Introduction ................................................................................................. 47

4.2.2 Materials and methods ................................................................................ 48

4.2.2.1 Materials .................................................................................................. 48

4.2.2.2 C2C12 culture ......................................................................................... 48

4.2.2.3 Glucose uptake assay .............................................................................. 49

4.2.2.4 Cell membrane protein extraction ........................................................... 50

4.2.2.5 Transfection of shRNA-AMPKα ............................................................ 50

4.2.2.6 Western blotting ...................................................................................... 51

4.2.2.7 AMP/ATP assay ...................................................................................... 51

4.2.2.8 Glucose tolerance test (GTT)……………………………………………52

4.2.2.9 Statistical analysis ................................................................................... 52

4.2.3 Results ......................................................................................................... 53

4.2.3.1 Chicoric acid promoted glucose uptake .................................................. 53

4.2.3.2 Effects of chicoric acid on Akt activation and GLUT4 translocation in

C2C12 myotubes ................................................................................................... 58

x
 4.2.3.3 Effects of chicoric acid on the AMPKα signaling pathway in C2C12

myotubes ............................................................................................................... 59

4.2.4 Discussion ................................................................................................... 65

BIBLIOGRAPHY ............................................................................................................. 69

xi
 LIST OF TABLES

Table Page

2.1 In vitro effects of chicoric acid in the treatment of various disorders……….............11

2.2 In vivo effects of chicoric acid in the treatment of various disorders……………….16

4.1 Effect of chicoric acid on median lifespan of wild type and mutants………………..37

xii
 LIST OF FIGURES

Figure Page

2.1 Molecular structure of L-chicoric acid ………………………………….…………...4

2.2 Molecular targets of chicoric acid ……………………………………….…………21

4.1 Survival of wild-type C. elegans treated with chicoric acid …………...……………31

4.2 Effect of chicoric acid on growth rate, worm size and progeny production

of C. elegans …………………………………………………………...……………….32

4.3 Chicoric acid ameliorated the age-related decline of movement and pumping

rate…..……………………………………………………………………………………33

4.4 Effect of chicoric acid on the oxidative stress sensitivity of wild type N2

worms………………..…………………………………………………………………...34

4.5 Survival of several mutant strains treated with chicoric acid……………….……….38

4.6 Chicoric acid might extend C. elegans lifespan via the regulation of AAK-2 and

SKN-1……………………………………………………………………………………40

4.7 Chicoric acid enhanced the stress resistance of wild type worms but not aak-2

mutants ………….……………………………………………………………………….42

4.8 The graphic contents…………...…………………………………………………….46

4.9 Chicoric acid promoted glucose uptake in C2C12 myotube………...………………55

4.10 Effects of chicoric acid on glucose tolerance test (GTT)…………...……………...57

4.11 Activation of Akt and translocation of glucose transporter 4 (GLUT4) by chicoric

acid in C2C12 myotubes…………………...…………………………………………….59

4.12 Activation of AMP-activated protein kinase α (AMPKα) pathway by chicoric acid in

C2C12 myotubes…………………………………………………………………………60

xiii
4.13 Increased glucose uptake and activation of Akt by chicoric acid were abolished by

the inhibition of AMPKα………………………..……………………………………….62

4.14 Increased glucose uptake and activation of Akt by chicoric acid were abolished by

shAMPKα………………………….…………………………………………………….64

4.15 Chicoric acid enhanced AMP/ATP ratio in C2C12 myotubes…………...………...65

4.16 The graphic contents………...…………...…………………………………………68

xiv
 CHAPTER 1

INTRODUCTION

Chicoric acid is a hydroxycinnamic acid presents in the roots of a large number of

plants (63 genera and species), including many plants grown in the Mediterranean area

(1), and many of these plants have been consumed traditionally as food ingredients and

alternative medicines for some time (2, 3). Because chicoric acid is found in especially

high amounts in chicory (Cichorium intybus), purple coneflower (Echinacea purpurea),

and basil, the acid is often used as a marker for the quality check of herbal products from

these plants (4). More recently, an increasing number of publications have reported the

beneficial effects of chicoric acid in cell culture and animal studies. However, its role on

aging and glucose homeostasis are largely unknown.

Aging is defined as a progressive decline of intrinsic physiological functions,

resulting in an increased risk of death (5). Considering that aging is one of the key risk

factors associated with many chronic diseases, slowing aging is of great importance to

prevent age-associated diseases and improve the health span of life (6). A previous study

reported that chicoric acid extend lifespan in Caenorhabditis elegans (7), its effects on

aging-related phenotypes and its underlying mechanisms, however, remain to be

determined.

Type 2 diabetes mellitus (T2DM) has been recognized as a major public health

challenge throughout the world in recent decades (8, 9). Impaired glucose uptake in

muscle tissues is the primary associated with hyperglycemia in T2DM (10). Previous in

vitro studies reported that chicoric acid ameliorated insulin resistance in human hepatoma

cells (11). However, the effects of chicoric acid on muscle cells and its underlying

1
mechanism are still unclear. Here, C2C12 myotubes, derived from murine skeletal

muscle cells and a C57BL/6J mice model were used to determine the role of chicoric acid

on glucose homeostasis and its underlying mechanisms were determined.

2
 CHAPTER 2

LITERATURE REVIEW

2.1 Chicoric acid

Chicoric acid, a hydroxycinnamic acid that is a member of the phenylpropanoid

family, contains two caffeoyl units (2). Chicoric acid mainly presents in two forms: first,

the most abundant natural form of chicoric acid is L-chicoric acid (Figure 1); second, the

acid presents in the stereoisomer meso-chicoric acid (i.e., dicaffeoyl-meso-tartaric acid or

di-E-caffeoyl-(2R-3S)-(-)-tartaric acid) (1).

Chicoric acid is present in the roots of a large number of plants (63 genera and

species), including many plants grown in the Mediterranean area (1), and many of these

plants have been consumed as alternative medicines or food supplements for some time

(2, 3). Because chicoric acid is found in especially high amounts in chicory (Cichorium

intybus), purple coneflower (Echinacea purpurea), and basil, the acid is often used as a

marker for the quality check of herbal products from these plants (4). The roots of

chicory and purple coneflower are usually baked, grounded and used as a coffee

substitute in Europe (12). In Turkey, an herbal tea made from chicory has been used

historically for the treatment of diabetes, epilepsy, hemorrhoids, inflammation and

digestive disorders (13). The plant roots containing chicoric acid have been used in Asian

traditional medicine as a tonic for curing infectious diseases, inflammatory diseases, eye

diseases, and nerves injuries (13, 14). Commercial products made from purple

coneflower are currently popular alternative medicines widely used in North America for

cold and flu prevention (15). More recently, an increasing number of publications have

reported the beneficial effects of chicoric acid in cell culture and animal studies. This

3
review summarizes these health benefit studies and the underlying mechanisms of

chicoric acid.

Figure 2.1. Molecular structure of L-chicoric acid. The most abundant natural form of
chicoric acid is L-chicoric acid, i.e., (-)-chicoric acid, 2,3-dicaffeoyl-L-tartaric acid, 2,3-
O-dicaffeoyltartaric acid, 2R,3R-O-dicaffeoyltartaric acid, or di-E-caffeoyl-(2R-3R)-(-)-
tartaric acid.

2.2 The Biological effects of chicoric acid

The main benefits of chicoric acid include antivirus, anti-inflammation, glucose

and lipid homeostasis, neuroprotection, and anti-oxidation effects. Biological activities

and related mechanisms of chicoric acid from in vitro and in vivo studies are summarized

in Tables 1 and 2.

2.2.1 Anti-viral effects of chicoric acid

The first reported bioactive effect of chicoric acid is its ability to inhibit infection

with human immunodeficiency virus 1 (HIV-1) (16). Several studies reported that

chicoric acid inhibits infection with HIV-1 by deactivating HIV-1 integrase (16-23).

HIV-1 integrase is a multidomain enzyme required for the integration of viral DNA into

the host genome, a critical step in viral replication (24). The inhibition of HIV-1

integration by chicoric acid results in the stopping virus replication, leading to increased

T-lymphoblastoid cell viability (17-19, 21, 22). It has been further suggested that chicoric

4
acid decreases integrase binding site activity, including: the downregulation of HIV 3'-

end processing products (16), the occupation of HIV-1 integrase catalytic core (20), the

chelation of integrase divalent cations (17), the increase of long terminal repeat circle

formation (21), and the inhibition of integrase-mediated catalysis (18). Hu et al. (25)

reported that the mutation of HIV integrase might result in the blocking of chicoric acid

specific binding sites and King et al. (26) further reported that the mutation on glycine to

serine at position 140 (G140S) of HIV integrase reduced chicoric acid’s effects on HIV

infection, suggesting that the integrase G140S might be a target site of chicoric acid.

There are a few studies that are using new computational techniques including molecular

docking and quantitative structure-activity relationship (QSAR) analysis to identify the

binding sites of chicoric acid with HIV-1 integrase (25, 27, 28). Chicoric acid binds HIV-

1 integrase at its two arms, including the s-cis/s-cis isomer and s-cis/s-trans isomer

arrangements (27). Based on the observation that the s-cis/s-cis isomer exhibits the most

stable binding, this site was suggested to be the target of chicoric acid to inhibit HIV

integrase. Another study using QSAR analysis indicated that the poly aromatic rings of

chicoric acid are central linkers in binding to HIV-1 integrase (28). In addition to

integrase, reverse transcriptase is another potential target of chicoric acid (23, 29), as

chicoric acid downregulates reverse transcriptase of HIV-1 through the inhibition of the

trans-activator of transcription, an important protein promoting HIV-1 reverse

transcription (29).

Although chicoric acid may be a potential treatment for HIV, there are several

limitations of using naturally occurring chicoric acid as a treatment, such as poor stability

and limited cell permeability due to diacid moiety (30). To overcome these limitations,

5
chicoric acid analogs have been introduced, such as an analog of a decarboxyl compound,

while extending a caffeoyl group on 3,4,5-trihydroxycinnamoyl sidechains, which

showed improved stability and cell bioavailability (30-32). This analog also exhibited the

high potency against HIV integrase infectivity (30-32).

2.2.2 Chicoric acid and inflammatory responses

Chicoric acid has been found to ameliorate inflammation induced by

lipopolysaccharides (LPS) in both cell culture and mice. Reduced inflammation was

associated with down-regulations of nuclear factor κB (NF-κB) and tumor necrosis factor

α (TNF-α) (33-36), which are two major regulators of inflammation responses (37-39).

Several other pro-inflammatory factors – including nitric oxide synthase (iNOS),

cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), interleukin-1β (IL-1β), IL-12 and

IL-18 have been also reported to be downregulated by chicoric acid (33, 35, 40-43).

However, two relevant studies showed results inconsistent with the above. Matthias et al.

(44) reported that LPS inhibited NF-κB expression, which was reversed by chicoric acid

treatments in Jurkat E6.1 leukaemic T-cell lymphoblasts. The other reported that

echinacea extracts (containing chicoric acid) upregulated LPS-induced TNF-α in rat

alveolar macrophages (45). Inconsistencies may be due to dosage and types of LPS used

(i.e., 0.1 vs. 1 μg/ml) (34, 43, 46-48) and/or other components present in treatments (36,

49). Overall, since the pro-inflammatory factors above are related to the occurrence of

many chronic diseases, most research suggests chicoric acid may be considered a

preventive tool for inflammation-associated diseases (33, 35, 40-43), however, further

evaluation is needed.

6
2.2.3 Chicoric acid and glucose metabolism

Chicoric acid has been reported to promote glucose uptake in muscle cells and

hepatocytes, through activating the insulin receptor substrate/ phosphoinositide 3-kinase

/protein kinase B (Akt) pathway (11, 50, 51). Zhu et al.(51, 52) suggested that chicoric

acid activated Akt via the AMP-activated protein kinase α (AMPKα)-dependent

mechanism, which is a master regulator for energy homeostasis and also plays a key role

in glucose metabolism. However, the mechanism underlying how chicoric acid induces

AMPKα activation is not known currently. In addition to AMPKα, glucosidase, a

digestive enzyme for carbohydrates, was reported to be suppressed by chicory (53),

which can contribute to reduced glucose levels (54, 55). Another enzyme, protein

tyrosine phosphatase 1B (PTP1B) negatively regulates the insulin signaling pathway by

inhibiting the activity of insulin receptor kinase (56). Two studies with molecular

docking showed the molecular interactions between the allosteric site of PTP1B and

chicoric acid, which suggests chicoric acid might inhibit PTP1B and further activate the

insulin signaling pathway (57, 58).

In in vivo studies, chicoric acid (or leafy extracts of echinacea or basil) reduced

streptozotocin induced-hyperglycemia in mice (50, 52, 59, 60). In these studies, the

protection of pancreatic β-cells by chicoric acid was attributed to its regulation of

apoptosis related-genes, including c-Jun N-terminal kinase (JNK), B-cell lymphoma 2-

associated X/B-cell lymphoma 2 (Bax/Bcl-2) ratio, and pancreatic duodenal homeobox 1

(PDX-1) (61). In addition, as stated in the previous section, chicoric acid downregulates

several pro-inflammatory cytokines and mediators and these metabolic inflammatory

7
cytokines are related to the impairment of glucose homeostasis (62), indicating that

chicoric acid might improve glucose homeostasis via regulating inflammatory responses,

including those associated with regulators COX-2, mitogen-activated protein kinase

(MAPK), cAMP response element binding protein (CREB) and NF-κB (51, 52),

2.2.4 Chicoric acid and lipid metabolism & liver function

Chicoric acid has been found to reduce high-fat-diet-induced weight gain in mice

(63, 64). This was in part via the inhibition of peroxisome proliferator-activated receptor-

γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα), two critical transcription

factors in adipocyte differentiation and lipid accumulation, while increasing the secretion

of adiponectin (64). Similarly, a few studies suggested that chicoric acid protected the

liver from high-fat- or alcohol-induced fat accumulation and hepatic steatosis (63-65).

The hepatoprotective effects of chicoric acid were suggested to be due in part to a

decrease in the Bax/Bcl-2 ratio and the inhibition of fatty acid synthase (FAS) and pro-

inflammatory cytokines, including TNF-α, IL-6, COX-2 and JNK in the liver (63, 64).

2.2.5 Chicoric acid and brain function

There is a report that chicoric acid treatment ameliorated restraint stress-induced

behavioral despair and depression in mice (66). This neuro-protective effects of chicoric

acid may result from its upregulation of neurotransmitters, including noradrenaline,

dopamine, and 5-hydroxy tryptamine in the whole brain region of mice (66). Since

restraint stress is an important factor contributing to disorders and behavioral changes

(such as depression, memory loss, anxiety, and learning disability), the possible

8
alleviation of restraint stress by chicoric acid suggests the potential application of

chicoric acid for the above-mentioned brain disorders (66). Others reported that chicoric

acid protected neurons from memory impairment, amyloidogenesis, and hippocampus

shrinkage induced by LPS and D-galactose in mice (33, 34, 67). They also found that

chicoric acid inhibited the expression of amyloid β and its downstream enzyme, neuronal

β-secretase 1, both of which are known factors contributing to the disruption of neural

connectivity and neuronal death (68). Meanwhile, chicoric acid upregulated brain-derived

neurotrophic factor, which is a canonical nerve growth factor supporting the survival of

existing neurons and promoting the growth of new neurons and synapses (69). Although

limited, these findings suggest the potential protective activity of chicoric acid on

controlling the pathogenesis of neuro-degenerative diseases.

2.2.6 Anti-oxidative stress effects of chicoric acid

Oxidative stress is defined as the imbalance between the generation of reactive

oxygen species (ROS) and a physical ability of detoxification or damage restoration

associated with ROS (70). ROS could act as cellular massagers but cause physical

damages through disruptions of normal cell signaling pathways (71). Chicoric acid has

been found to have a high oxygen radical scavenging capacity, reducing the ROS level

and protecting cells from free radical-induced cytotoxicity (34, 53, 72-75). Moreover,

chicoric acid increased the generation of anti-oxidative enzymes that contribute to the

reduction of ROS levels, i.e., glutathione, glutathione peroxidase, superoxide dismutase,

chloramphenicol acetyl transferase, heme oxygenase and NAD(P)H dehydrogenase in

various cells (15, 21, 25, 43, 49, 61, 62, 71, 76, 77). The underlying mechanism of the

9
antioxidative effects of chicoric acid is attributed to the enhanced nuclear translocation of

nuclear factor erythroid 2-related factor 2 (Nrf-2) and the level of peroxisome

proliferator-activated receptor-γ coactivator α (PGC-1α) (34, 73, 75). The activation of

Nrf-2 and PGC-1α inhibit ROS-induced cytotoxicity by upregulation of antioxidant

response-related genes and promotion of the mitochondrial antioxidant defense system,

respectively (78, 79). Since oxidative stress is closely related to the development of

certain cancers and chronic diseases, all these findings suggest the potential future

application of chicoric acid for oxidative stress-associated diseases.

2.2.7 Other bioactivities of chicoric acid

Chicoric acid at relatively higher concentrations (105-315 μM) has been reported

to inhibit cancer cell growth via inhibiting cell proliferation, promoting cell apoptosis,

and deactivating telomerase through upregulation of DNA fragmentation, cleaved

caspase-9, and cleaved poly (ADP-ribose) polymerase (80).

Another study suggested that chicoric acid ameliorated ultraviolet A irradiation-

induced dermal fibroblast senescence by the inhibition of matrix metalloproteinase-3

activity (81). As dermal fibroblast senescence is a hallmark of intrinsic and ultraviolet-

mediated aging (77), this indicates the potential role of chicoric acid in aging.

10
Table 2.1 In vitro effects of chicoric acid in the treatment of various disorders.
Disorders Models Dose Duration Effects Suggested mechanisms References
(µM) (hrs)
Anti-viral H9 & MT-2 human T- 1.05- 48 ↑ infected cell viability; ↓ HIV 3'-end processing (16)
effects lymphoblastoid 10.5 ↓ HIV-1 integrase products
activity

H9 & MT-2 human T- 0.4 48 ↓ HIV-1 integrase ↓ HIV integrase catalytic (20)
lymphoblastoid activity core

MT-2 human T- 105 48 ↓ HIV-1 reverse ↓ HIV-1 envelope (29)

lymphoblastoid transcriptase activity glycoprotein 120

MT-2 human T- 0.4 72 ↑ infected cell viability; N/A (23)

lymphoblastoid ↓ HIV-1 reverse
transcriptase activity; ↓
HIV-1 reverse
transcriptase release
MT-2 human T- 25 72 ↓ HIV-1 integrase N/A (19)
lymphoblastoid replication
MAGI human cervical 100 52 ↓ HIV-1 integrase ↓ HIV-1 Tat protein (82)
epithelial carcinoma activity; synthesis
H9 & MT-2 human T- 0.1- 24 ↓ viral replication; ↑ integrase divalent (17)
lymphoblastoid 10 ↓ HIV-1 integrase cations chelation
activity
MT-4 human T- 70 2 ↓ viral replication cycle; ↓ HIV-1 envelope (83)

11
 lymphoblastoid ↓ viral entry glycoprotein 120

MT-2 human T- 4.2 72 ↑ infected cell viability; ↑ long terminal repeat (21)
lymphoblastoid ↓ HIV-1 integrase circle formation
activity
H9 & MT-2 human T- 50 1-4 ↓ HIV-1 integrase ↓ integrase binding site (22)
lymphoblastoid activity activity
H9 & MT-2 human T- 25 1 ↓ HIV-1 integrase ↓ integrase-mediated (18)
lymphoblastoid activity catalysis
Glucose L6 rat myotubes & 200 1-2 ↑ glucose uptake; N/A (11)
metabolism insulinoma-derived ISN- & ↑ insulin secretion
1 pancreatic β cells 20-
100

INS-1 pancreatic β- 105- 1-3 ↑ glucose uptake; N/A (50)

cells; L6 myotubes; 210 ↑ insulin secretion
isolated hepatocytes
(rat)
HepG2 human 100 24 ↓ insulin resistance; ↑ GLUT2 translocation; (51)
hepatoma ↓ NO & ROS ↑ Akt; ↓ MAPK; ↓ NF-
κB
HepG2 human 100 24 ↑ glucose uptake ↑ IRS-1; ↑ Akt; ↑ (52)
hepatoma AMPKα; ↑ SIRT1; ↑
GSK-3β; ↓ CREB
Inflammation RAW 264.7 mouse 4.2 4 N/A ↓ NF-κB; ↓ TNF-α (36)
responses macrophage
Macrophage N/A N/A ↓ cell inflammation ↓ TNF-α (35)
Jurkat E6.1 human 17 4 N/A ↑ NFκB (44)

12
 leukemic T-cell
lymphoblast
Mouse peripheral blood 0.5- 4 ↑ immune homeostasis ↑ IL-2; ↑ IFN-γ; ↓ IL-4 (41)
mononuclear cells 4.2
BV-2 mouse microglia 80 4 ↓ cell inflammation ↓ iNOS; ↓ COX-2; ↓ (33)
PGE2; ↓ IL-1β; ↓ TNF-α
Human umbilical vein 12.5- 24 ↓ endothelial ↑ SOD; ↑ eNOS; ↓ (42)
endothelial cells 100 dysfunction; ↓ cell Bax/Bcl-2; ↓ cleaved
apoptosis; ↑ cell caspase-3; ↓ MAPK; ↓
viability; ↓ ROS NF-κB
HT-29 human colorectal 42 12 ↓ cell inflammation ↓ NF-κB; ↓ COX-2; ↓ (43)
adenocarcinoma IL-1β; ↓ IL-18
Oxidative Human plasma 1 5 ↓ Cu (II)-catalyzed LDL N/A (84)
stress oxidation
RAW264.7 mouse 16- 20 ↓ oxidative stress ↓ PGE2; ↓ TNF-α; ↓ IL- (47)
macrophage 32 1β; ↓ NF-κB; ↓ Akt
RAW264.7 mouse N/A 20 ↓ oxidative stress & ↓ ↑ GSH; ↓ iNOS; ↓ NF- (48)
macrophage NO κB
L6 rat myotubes 5-50 1 ↓ ROS ↑ GPx; ↑ SOD; ↑ p- (73)
AMPKα; ↑ PGC-1α
RGC-5 rat retinal 0.025 24 ↑ cell viability & ↓ cleaved PARP; ↓ (67)
ganglion cells ↓ ROS cleaved caspase-3
RAW264.7 mouse 340 19 ↓ oxidative stress ↓ MyD88; ↓ iNOS; ↓ (65)
macrophage TNF-α
HepG2 human 100 24 ↓ NO; ↓ ROS ↓COX-2; ↓iNOS; ↓ NF- (51)
hepatoma κB
Daudi & Namalwa B 21- 12-48 ↓ B cell activating factor ↓ NF-κB; ↓ IκB (85)
lymphocyte; JeKo-1 105 belonging to the TNF

13
 mantle cell lymphoma, family (BAFF)
THP-1 monocytes &
HepG2 hepatoma
(human)
BV-2 mouse microglia 80 4 ↓ oxidative stress ↓ NF-κB; ↓ MAPK; ↑ (34)
Nrf2
SH-SY5Y human 50 24 ↓ oxidative stress; ↑ Nrf2; ↑ HO-1; ↑ NQO- (75)
neuroblastoma ↑ cell viability 1; ↑ CAT; ↑ GSH; ↓
TNF-α; ↓ IL-1β; ↓
malondialdehyde
Others HL-7702 human 20- 48 ↑ infected cell viability; ↓ HBV surface & (86)
hepatocytes & HepG2 200 ↓ HBV activity envelope antigen
human hepatoma
Caco-2 & HCT-116 105- 24-48 ↓ cell proliferation; ↑ DNA fragmentation; ↑ (80)
epithelial colorectal 315 ↑ cell apoptosis; cleaved caspase-9; ↑
adenocarcinoma ↓ telomerase activity cleaved PARP
(human)
HeLa cervical 0.05 24 ↓ doxorubicin-induced N/A (87)
carcinoma & MCF-7 cell death
breast carcinoma
(human)
Human skin fibroblasts 2 24 ↓ dermal fibroblasts ↓ MMP-3 (81)
senescence
3T3-L1 mouse 100 48 ↑ cell apoptosis & ↓ ↑ cleaved caspase-3; (64)
preadipocytes mitochondrial membrane ↓ Akt; ↑ MAPK; ↓ JNK
potential & ERK1/2
3T3-L1 mouse 100 24 ↑ free radical scavenging N/A (76)
preadipocytes

14
↑ - increase; ↓ - decrease; N/A, not available.
Acronyms: Akt, protein kinase B; AMPKα, AMP-activated Protein Kinase α; Bax/Bcl-2, B-cell lymphoma 2-associated X/B-cell
lymphoma 2; BSO, 1-buthionine-(S,R)-sulfoximine; CREB, cAMP response element binding binding protein; CAT, chloramphenicol
acetyl transferase; COX-2, cyclooxygenase-2; eNOS, endothelial nitric oxide synthase; ERK1/2, extracellular signal–regulated kinase
1/2; GLUT2, glucose transporter 2; GPx, glutathion peroxidase; GSH, glutathione; GSK-3β, glycogen synthase kinase 3β; HBV,
hepatitis B virus; HIV-1, human immunodeficiency virus 1; 4-HNE protein adducts, 4-hydroxynonenal-protein adducts; HO-1, heme
oxygenase; IFN-γ, interferon γ; IĸB, inhibitor of kappa B; IL-1β, interleukin 1 beta; iNOS, nitric oxide synthase; IRS-1, insulin
receptor substrate 1; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharides; MAGI, HeLa CD4+ HIV-1 LTR-β-gal cells; MAPK,
mitogen-activated protein kinase; MMP-3, matrix metalloproteinase-3; MyD88, myeloid differentiation primary response 88; NF-κB,
nuclear factor κB; NQO-1, NAD(P)H dehydrogenase; Nrf2, nuclear factor erythroid 2–related factor 2; LDL, low-density lipoprotein;
PARP, poly (ADP-ribose) polymerase; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator α; PGE2, prostaglandin E2;
ROS, reactive oxygen species; SIRT1, sirtuin 1; SOD, superoxide dismutase; Tat, trans-activator of transcription; TNF-α, tumor
necrosis factor α.

15
Table 2.2 In vivo effects of chicoric acid in the treatment of various disorders.
Diseases Species Models Dose Duration Effects Suggested References
(sex) (mg/kg/d) (days) Mechanisms
Brain Swiss albino Restraint stress 1-2 p.o. 14 ↓ behavioral ↑ norepinephrine; (66)
function mice (M) (Porsolt's swim despair; ↑ dopamine; ↑ 5-
stress & conical ↑ learning ability; hydroxy
polypropylene ↑ neurotransmitters tryptamine
tubes)
Sprague−Dawley Retinal damage 2 μg/eye 7 ↑ retinal ganglion ↓ cleaved PARP; ↓ (67)
rats & ICR mice (N-methyl-D- intravitreal cell viability cleaved caspase-3
(M) aspartate / optic injection
nerve crush)
C57BL/6J mice LPS challenges 0.5 g/L 45 ↓ memory ↓ amyloid β (Aβ1- (33)
(M) drinking impairment; ↓ 42);
water amyloidogenesis ↓ BACE1; ↓
MAPK; ↓ NF-κB
C57BL/6J mice LPS challenges 0.5 g/L 45 ↓ oxidative stress ↓ MAPK; ↓ NF- (34)
(M) drinking induced neuron κB; ↓ iNOS; ↓ IL-
water damage 1β; ↓ TNF-α; ↑
Nrf2; ↑ HO-1; ↑
NQO-1
C57BL/6J mice D-glactose 100 i.p. 56 ↓ neuron damage; ↓ amyloid β (Aβ1- (75)
(M) challenges ↓ hippocampus 42); ↑ BDNF
shrinkage
Glucose Wistar rats (M) No challenge 3-30 i.p. 4 ↑ insulin secretion; N/A (50)
metabolism ↓ hyperglycemia
Swiss mice (M) Streptozotocin 3 i.p. 2 hrs ↓ hyperglycemia N/A (59)
challenge

16
 Swiss mice (M) Streptozotocin 1-3 2 hrs ↓ hyperglycemia N/A (60)
challenge i.p.

C57BL/6J mice Streptozotocin 60 drinking 4 ↓ hyperglycemia; ↓ ↓ JNK; ↓ Bax/Bcl- (61)

(M) challenge water pancreas 2; ↑ PDX-1
apoptosis; ↑
insulin secretion
Lipid C57BL/6J mice High-fat diet 15-60 p.o. 8 ↓ body weight; ↓ ↓ TNF-α; ↓ IL-6; ↓ (64)
metabolism (M) challenge hepatic steatosis COX-2; ↓ JNK; ↓
& liver PPARγ; ↓
function C/EBPα; ↓ FAS; ↓
ALT & AST
C57BL/6J mice Hepatic steatosis 4 drinking 4 ↓ hepatic ↓ iNOS; ↓ 4-HNE (65)
(F) (alcohol) water triacylglycerols protein adducts; ↓
TNF-α; ↓ PAI-1; ↓
CD11c
C57BL/6J mice Methionine- and 10-30 p.o. 4 ↓ hepatic lipid ↓ SREBP-1c; ↑ (63)
(M) choline-deficient accumulation; ↓ Nrf2; ↑ AMPK; ↓
diet challenge lipid peroxidation; TNF-α; ↓ MCP-1;
↓ hepatic ↓ FAS; ↓ ALT &
ballooning, AST
steatosis &
inflammation
C57BL/6J mice Streptozotocin 60 drinking 28 ↓ hepatic injury ↑ glycogen; ↑ (52)
(M) challenge water glycolysis genes
(Gck, Pk & Pfk); ↑
AMPK
Inflammation Sprague-Dawley LPS challenge 20 p.o. 4 ↑ phagocytic ↑ TNF-α; ↑ IFN-γ (45)

17
 rats (M) activity; ↑ NO
Swiss albino Restraint stress 2 p.o. 14 ↑ Th1/Th2 ↑ CD28 & CD80; (88)
mice (M) (conical homeostasis; ↓ CTLA-4; ↓
polypropylene ↑ lymphocyte CD152; ↓ IL-10; ↑
tubes) proliferation & T IFN-γ; ↑ IL-2; ↑
cell population IL-12
[cluster
determinant 3 (+),
4(+) & 8(+)]
Sprague-Dawley Arthritis 8-32 p.o. 28 ↓ paw swelling; ↓ ↓ NF-κB; ↓ TNF- (40)
rats (M) (collagen) organ index of the α; ↓ COX-2
thymus and spleen

ICR mice (M) Anaphylactic 20 p.o. 2 hrs ↓ mortality rate; ↓ N/A (89)
shock histamine levels in
(compound blood serum
48/80)

M – male; F - female; ↑ - increase; ↓ - decrease; N/A, not available; p.o., Per os (oral administration); i.p., intraperitoneal injection.
Acronyms: Akt, protein kinase B; ALT, alanine aminotransferase; AMPKα, AMP-activated Protein Kinase α; AST, aspartate
aminotransferase; BACE1, neuronal β-secretase 1; Bax/Bcl-2, B-cell lymphoma 2-associated X/B-cell lymphoma 2; BDNF, brain-
derived neurotrophic factor; CD11c, integrin α X chain protein; CD28, cluster of differentiation 28; CD80, cluster of differentiation 80;
CD152, cluster of differentiation 152; C/EBPα, CCAAT/enhancer binding protein α; COX-2, cyclooxygenase-2; FAS, fatty acid
synthase; CTLA-4, cytotoxic T-lymphocyte associated antigen 4; 4-HNE protein adducts, 4-hydroxynonenal-protein adducts; HO-1,
heme oxygenase; IFN-γ, interferon γ; IL-1β, interleukin 1β; iNOS, nitric oxide synthase; JNK, c-Jun N-terminal kinase; Keap1, kelch-
like ECH-associated protein 1; LPS, lipopolysaccharides; MAPK, mitogen-activated protein kinase; MCP-1, monocyte
chemoattractant protein 1; NF-κB, nuclear factor κB; Nrf2, nuclear factor erythroid 2–related factor 2; NQO-1, NAD(P)H
dehydrogenase; PAI-1, plasminogen activator inhibitor-1; PARP, poly (ADP-ribose) polymerase; PDX-1, pancreatic duodenal

18
homeobox 1; PPAR γ, peroxisome proliferator activated receptor γ; ROS, reactive oxygen species; SREBP-1, sterol regulatory
element-binding protein 1; TNF-α, tumor necrosis factor α; Th1/Th2, T helper cells 1/2.

19
2.3 Suggested molecular targets of chicoric acid

As discussed in previous sections, chicoric acid modulates genes, transcription

factors, growth factors, enzymes, and proteins involved in important cellular processes,

such as virus infection, chemoresistance, inflammation, and glucose metabolism (Figure

2.2) (11, 42, 82, 85). Among them, it is suggested that NF-κB and TNF-α are two major

mediators of chicoric acid’s activities.

NF-κB is a protein complex involved in cellular responses to stress, free radicals,

heavy metals, and bacterial or viral infections through regulating DNA transcription,

cytokine production, and cell survival (37, 38, 90). It is known that the activity of NF-κB

is directly suppressed by binding with the inhibitor of κB (IκB) in the cytoplasm (90). In

contrast, the phosphorylation of IκB releases and activates NF-κB (90). Chen et al. (85)

reported that chicoric acid deactivates NF-κB by inhibiting the phosphorylation of IκB,

indicating that IκB may be a major regulator for chicoric acid-mediated inactivation of

NF-κB. Alternatively, NF-κB can be activated in combination with TNF-α (37). Several

studies reported that chicoric acid decreased TNF-α production (40, 47, 64, 65, 75),

which might be a contributing factor to the reduced activation of NF-κB. Chicoric acid-

related deregulations of NF-κB and TNF-α were shown to alleviate conditions such as

autoimmune disorders, restraint stress, hepatic steatosis and neuron damage known to be

involved in cellular inflammation and associated diseases (34-36, 40, 47, 63-65, 75).

20
Figure 2.2 Molecular targets of chicoric acid. Akt, protein kinase B; ALT, alanine aminotransferase; AMPKα, AMP-activated Protein
Kinase α; AST, aspartate aminotransferase; BACE1, neuronal β-secretase 1; Bax/Bcl-2, B-cell lymphoma 2-associated X/B-cell
lymphoma 2; BDNF, brain-derived neurotrophic factor; CD28, cluster of differentiation 28; CD80, cluster of differentiation 80;
CD152, cluster of differentiation 152; C/EBPα, CCAAT/enhancer binding protein α; COX-2, cyclooxygenase-2; ERK1/2,
extracellular signal–regulated kinase 1/2; FAS, fatty acid synthase; HO-1, heme oxygenase; GLUT2, glucose transporter 2; IFN-γ,
interferon γ; IL-1β, interleukin 1β; iNOS, nitric oxide synthase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein
kinase; MCP-1, monocyte chemoattractant protein 1; NF-κB, nuclear factor κB; Nrf2, nuclear factor erythroid 2–related factor 2;
NQO-1, NAD(P)H dehydrogenase; PARP, poly (ADP-ribose) polymerase; PDX-1, pancreatic duodenal homeobox 1; PPAR γ,
peroxisome proliferator activated receptor γ; SREBP-1, sterol regulatory element-binding protein 1; TNF-α, tumor necrosis factor α;
Th1/Th2, T helper cells 1/2.

21
2.4 Pharmacokinetic study of chicoric acid

Chicoric acid is known to have relatively low absorption in rats (91). The oral

administration of chicoric acid at 50 mg/kg body weight resulted in a peak plasma

concentration of chicoric acid at 1.63 ±0.25 mg/L after 4 h (91). Chicoric acid was

mainly distributed in the liver, lung and kidney after oral administration for 3 h. Chicoric

acid has a relatively long residence time and low body clearance, 18.58 ± 4.43 h and 2.80

±0.46 L/kg/h, respectively, in rats (91). All these findings help in better understanding

the bioavailability and fate of chicoric acid.

2.5 Conclusion

The prevention of early-stage chronic diseases by food bioactive seems to be a

promising strategy. Chicoric acid’s multi-bioactivities suggest it has great potential in

treating a number of metabolic disorders, including inflammatory responses, impairment

of energy homeostasis, brain dysfunction, and immune disorders. Even though there are

increasing number of studies reporting bioactivities of chicoric acid, these are still

limitations to make a concrete conclusion due to differences on models, doses, and

treatment durations used, as well as lack of pharmacokinetic study, including metabolism,

of chicoric acid in humans. The research on chicoric acid is still at an early stage, so there

are still many questions that need to be answered regarding its benefits to health. Thus,

more studies are needed to guide the development of chicoric acid-based functional food

products.

22
 CHAPTER 3

OBJECTIVES OF THE PROJECT

The long-term goal is to develop chicoric acid as a functional food bioactive for

the prevention and/or treatment for aging and type 2 diabetes. Previous studies reported

that chicoric acid extends C. elegans lifespan and promotes glucose transport in hepatic

cells (7). However, more experiments are needed to further investigate the effects of

chicoric acid on aging-related phenotypes in C. elegans and the glucose uptake in other

models. The object of this project is to determine the effect of chicoric acid on the aging-

associated phenotypes of C. elegans and glucose uptake in muscle cells. The central

hypothesis is that chicoric acid can protect C. elegans during aging as well as promote

glucose uptake in muscle cells. The rationale of the proposed research is that by

investigating the effect of chicoric acid on aging and glucose uptake as well as the

underlying mechanisms, we will be able to better understand chicoric acid as a beneficial

bioactive for the promotion of health span and prevention from type 2 diabetes.

The project aims are as follows:

Specific aim 1: Determine the effect of chicoric acid on aging and aging-related

phenotypes of C. elegans. The hypothesis is that chicoric acid extends the lifespan of C.

elegans and delays the decrease of aging-related phenotypes.

Specific aim 2: Investigate the effect of chicoric acid on glucose uptake in muscle

cells and mice. The hypothesis to be tested is that chicoric acid promotes the glucose

uptake in C2C12 myotubes and reduces the blood glucose level in mice.

23
 CHAPTER 4

EFFECTS OF CHICORIC ACID ON AGING AND

GLUCOSE UPTAKE

4.1 Effect of chicoric acid on aging in C elegans and the underlying mechanisms

4.1.1 Introduction

Aging is defined as a progressive decline of intrinsic physiological functions,

resulting in an increased risk of death (5). Considering that aging is one of the key risk

factors associated with many chronic diseases, slowing aging is of great importance to

prevent age-associated diseases and improve the health span of life (6). Currently, food-

derived bioactive compounds are drawing significant attention because of their anti-aging

effects and the modulation of aging-related pathways (92-95), which provide potentially

valuable preventive tools for aging and aging-related disorders.

Chicoric acid, one of the hydroxycinnamic acids, is a dicaffeoyl ester naturally

found in the roots of many plants, such as chicory, purple coneflower, and basil (4).

Chicoric acid is reported to have a number of bioactivities, including antioxidation, anti-

inflammation, antivirus and immune-stimulating properties through multiple molecular

pathways (18, 33, 41, 52). A previous study reported that chicoric acid extended the

lifespan in Caenorhabditis elegans (7), however, the effects of chicoric acid on aging-

related phenotypes and the mechanisms underlying these effects remain to be determined.

One recent study reported that chicoric acid regulates glucose metabolism via

AMP-activated protein kinase (AMPK) activation (52). AMPK is an energy sensor that

controls energy homeostasis through the regulation of AMP/ATP metabolism in cells (4).

24
Moreover, AMPK is considered important in the regulation of longevity as the increased

activation of AMPK extended the lifespan through dietary restriction and/or enhanced

stress resistance in C. elegans and Drosophila melanogaster (96, 97). aak-2, a homolog

of the α-catalytic subunits of mammalian AMPK, is evolutionarily conserved in both C.

elegans and mammals (98). Since AMPK has been reported to be activated by chicoric

acid (52), it is plausible to assume that chicoric acid might regulate the lifespan of C.

elegans via aak-2. In addition to aak-2, skn-1, encoding a homolog of nuclear factor

erythroid 2–related factor 2 (Nrf-2) in C. elegans, was also reported to extend lifespan by

regulating the oxidative stress response pathway (99). Therefore, the current research

focused on determining the influence of chicoric acid on aging-related phenotypes in C.

elegans and the role of AAK-2 and SKN-1 in this process.

4.1.2 Materials and methods

4.1.2.1 Materials

Chicoric acid (≥ 98%) was purchased from PufeiDe Biotech Co., Ltd (Chengdu,

Sichuan, China). Household bleach used for bleaching adult nematodes during

synchronization was obtained from Clorox company (Oakland, CA, USA).

Fluorodeoxyuridine (FUdR), ampicillin, and carbenicillin, paraquat, and 2’,7’-

dichlorodihydrofluorescein diacetate (DCFDA) were ordered from Sigma-Aldrich (St.

Louis, MO, USA). Rabbit antibodies for phospho-AMPK and α-tubulin, as well as goat

anti-rabbit IgG-horseradish peroxidase (HRP) were obtained from Cell Signaling

Technology (Danvers, MA, USA). Rabbit antibodies for phospho-AMPK and α-tubulin,

25
as well as goat anti-rabbit IgG-horseradish peroxidase (HRP), were obtained from Cell

Signaling Technology (Danvers, MA, USA).

4.1.2.2 C. elegans: strains and maintenance

The strains in the study were provided by the Caenorhabditis Genetics Center

(CGC, University of Minnesota, Minneapolis, MN, USA): wild-type Bristol N2, aak-2

(ok524) X, daf-2 (e1370) III, eat-2(ad1116) II, daf-16(mgDf50) I, skn-1(zu135) IV, age-

1(hx546), and ldIs7 (skn-1b/c::gfp; rol-6). All strains were grown and incubated at 20 °C

on nematode growth media (NGM) plates with Escherichia coli OP50, except daf-2,

which was maintained at 15 °C. The solutions including nematode growth medium

(NGM), M9 buffer, and S-complete required for C. elegans culture were prepared

according to the worm book (100). A synchronized culture was obtained as previously

described (101). The chicoric acid stock solutions with 12.5, 25, and 50 mM were

prepared using dimethyl sulfoxide (DMSO) as solvent. Stock solutions were diluted 1000

times resulting in final concentrations of chicoric acid at 12.5, 25, and 50 M. These

doses were based on a previous report on the effects of chicoric acid on life span

extension in C. elegans (7). For lifespan analysis, L4 stage nematodes were transferred in

Transwell-96 well plate (Corning Inc., NY, USA; n=10-15 worms per well, 15-18 wells/

treatment) and treated with 0.1% DMSO (control) or chicoric acid during the lifespan

study (94, 102, 103). FUdR (120 mM), to prevent eggs from hatching out, was also added

(104). The medium was changed every four days. The survivals were determined by a

gentle touch-prod with a sterilized picker under a microscope (Nikon Instruments Inc.,

Melville, NY, USA) (94, 103). Worm viability was recorded every other day until all

26
worms died. The day that the treatment began is considered as day 0. The lifespan test in

N2 was repeated for three independent trials.

4.1.2.3 Growth rate, body size, and moving speed determination

The growth rate of C. elegans was determined by quantifying the population of

worms at three different developmental stages after 48 h chicoric acid treatments. Results

were shown in percent of proportion of C. elegans in each stage (94). Body size and

moving speed of C. elegans were measured by the WormLab tracking system (Allied

Vision Technologies, Stadtroda, Germany) and analyzed by WormLab Software (MBF

Bioscience, Williston, VT, USA) as published (101). Worms were transferred to the low-

peptone NGM plates seeded with OP50 and were undisturbed for 30 min for acclimation

before tracking (94). A 1-min recording (8.12 frames per second) was captured. The size

of the worms (70-90/treatment) was measured by the WormLab Software after 48 h of

treatments. The moving speed of worms was measured every other day from day 2 to day

12.

4.1.2.4 Progeny production and pumping rate determination

After 48 h chicoric acid treatments, two L4 staged nematodes/treatment were

shifted to freshly prepared NGM plates with E. coli OP50 every day during the

reproduction period. The number of progeny hatching out of eggs was counted and

recorded daily (94). The pumping rate was obtained by quantifying pharyngeal

contraction times of nematodes using a microscope (Olympus Corporation, Tokyo, Japan)

27
for 30 sec. The test was repeated three times with 12 randomly-selected nematodes per

treatment (94).

4.1.2.5 Measurement of intracellular ROS and oxidative stress resistance

The intracellular reactive oxygen species (ROS) level was detected using

DCFDA, a fluorescent probe commonly applied for ROS detection (105). After a 72-h

treatment with DMSO (control group) or chicoric acid, day 1 adult nematodes were

washed with M9 buffer three times to remove bacteria. They were next transferred into a

96-well plate, ~60 worms/well, with or without chicoric acid treatments in 50 µL of M9

buffer, following the mixture of 50 µL DCFDA solution (200 µM) to achieve 100 µM of

the final concentration. A well with 100 µM DCFDA solution with no worms was used as

the background, and worms treated with DCFDA solution and 5 mM paraquat were used

as the positive control. The fluorescent readings were taken after 30 min of incubation at

an emission/excitation wavelength of 530 and 485 nm by an Infinite 200 PRO microplate

reader (Tecan Trading AG, Switzerland).

For oxidative stress resistance determination, day 1 adult worms were transferred

into the 96 trans-well plate, 12-15 worms per well, containing 5 mM paraquat, an

oxidative stress inducer. The oxidative stress resistance of C. elegans was determined by

counting the survival percentage of worms under the exposure of paraquat for 2, 4, and 6

days with or without chicoric acid treatment (i.e., 25 µM) (94).

4.1.2.6 Quantitative real-time PCR

RNA was extracted in each sample using Trizol under RNase-free conditions, as
28
previously described (101). cDNA was prepared by reverse transcription of mRNA using

a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific Inc,

Middletown, VA, USA). The gene expression was quantified by QuantStudio 3 real-time

PCR system using the TaqMan detection assay (Applied Biosystems, Waltham, MA,

USA). aak-2 (Ce02404259_g1), nhr-49 (Ce02412666_g1), skn-1 (Ce02407444_m1), gsr-

1 (Ce02444754_g1) and pbs-5 (Ce02426103_g1), and the internal control ama-1

(Ce02462726_m1) were ordered from Thermo Scientific (Middletown, VA, USA).

4.1.2.7 Intracellular localization of SKN-1::GFP

Transgenic strain ldIs7 (skn-1b/c::gfp; rol-6) was used to detect the intracellular

localization of GFP tagged SKN-1 protein. After two-day of chicoric acid treatment (25

µM), around 10-20 worms (L4 stage or young adults) were placed on each microscope

slide coated with 2% agarose pad and anesthetized with 10 mM NaN3 within 3 min (94).

Worms treated with NaN3 (2%) for 15 min were used as the positive control. The cellular

localizations of SKN-1 were detected by a Nikon Eclipse Ti-U (Nikon Instruments Inc.,

Melville, NY, USA). The nuclear translocation patterns of SKN-1::GFP were identified as

‘low’, ‘medium’, and ‘high’. ‘Low’ refers to worms with almost no visible nuclear

translocation; ‘medium’ refers to C. elegans with nuclear translocation in the partial

body; ‘high’ refers to worms with nuclear translocation throughout the whole body, as

shown in Figure 4.6D. Worms were counted and analyzed as percentages of nuclear

translocation including both ‘medium’ and ‘high’ translocation (103).

4.1.2.8 Western blotting

29
 The C. elegans protein extraction was obtained according to the protocol

published with minor modification (101, 106). Worms were collected and washed four

times with M9 buffer to remove bacteria. Then worms (approximately 6000 young adults

per sample) were suspended by 500 µL PBS mixed with protease and phosphatase

inhibitors (Beyotime Biotechnology, Shanghai, China). Samples were homogenized and

centrifuged (12,000 × g) at 4 °C for 10 min to harvest the supernatants. After protein

normalization, the samples with 5 × sample buffer were electrophoresed on a 10% SDS

polyacrylamide gel and transferred onto a polyvinylidine fluoride membrane (107). After

blocking with 5% skim milk, the membrane was incubated with rabbit monoclonal anti-

phospho-AMPK and rabbit anti-α-tubulin antibody. Horseradish peroxidase-conjugated

goat anti-rabbit IgG was used as the secondary antibody. Protein blots were pictured by a

Tanon 5500 image station (Shanghai, China) with ECL Substrate Kit (Bio-Rad Co.,

Hercules, CA, USA). Images were quantified with ImageJ software.

4.1.2.9 Statistical analysis

Survival curves were analyzed by Log-rank (Mantel-Cox) tests by Graph Pad

Prism version 6.0 (GraphPad Software, Inc., San Diego, CA, USA). The data of nuclear

translocation of SKN-1::GFP in Fig. 4.6D was analyzed by chi square tests by SAS

software (version 9.3, SAS Institute Inc., Cary, NC, USA). For other data, differences

between each treatment were analyzed using one-way ANOVA (Tukey’s multiple-range

test, SAS). Significance of differences was defined as P values of < 0.05.

4.1.3 Results
30
4.1.3.1 Chicoric acid extended the lifespan of C. elegans

Compared with the control, chicoric acid at 25 and 50 µM, but not 12.5 µM,

significantly extended the lifespan of wild type nematodes (P<0.0001 for both; Fig. 1).

There was no difference in lifespan between 25 and 50 µM chicoric acid treatments.

Table 1 also exhibited the extended median lifespan of wild type N2 worms with 25 and

50 µM chicoric acid treatment, compared to the control. This was consistently observed

from 3 independent experiments: (median lifespan as days) 18.7±0.3 for control,

23.3±0.9 for 25 µM chicoric acid, and 22.7±0.7 for 50 µM chicoric acid with P=0.0062

and P=0.0128 for 25 and 50 µM chicoric acid compared to the control, respectively.

Thus, 25 and 50 µM were used to determine the aging-related phenotypes.

Figure 4.1 Survival of wild-type C. elegans treated with chicoric acid. C. elegans (N2)
were treated with final concentrations of 0 (control), 12.5, 25, or 50 µM chicoric acid
starting from L4 stage (day 0). The survivals were recorded every other day until all the
worms died (n = 206–266/treatment). Log-rank (Mantel-Cox) test was used for statistical
analysis. Significant differences of median/maximum lifespan compared between the
control and the treatment groups were marked as ***P<0.001.

The physiological properties of C. elegans treated by chicoric acid after

31
synchronization were determined by growth rate, body size, and progeny (Fig. 4.2). After

48 h of treatment, 76–80% of the nematodes reached L4 stage or young adult stage, while

20–23% were at early/mid L4 stage (Fig. 4.2A). As shown in Fig. 4.2, no difference was

observed in the growth rate, worm length, worm width, or progeny number of C. elegans

treated with or without chicoric acid. The results suggest that the physiological properties

of nematodes were not affected by chicoric acid.

Figure 4.2 Effect of chicoric acid on growth rate, worm size and progeny production of
C. elegans. (A) Synchronized L1 worms were raised on NGM plates with 0, 25 and 50
µM chicoric acid. After 48 h treatments, the number of worms at three developmental
stages was counted and calculated in percent of proportion. (B) Synchronized L1 larval
worms were treated with chicoric acid and the number of progeny hatching out from day
1 to day 5 were recorded. (C) Worm length and (D) width were measured by WormLab
software after 48 h treatments from the L1 stage. Values are means ±S.E. (n = 3 plates
and ~150 worms/plate for growth rate; n = 3 plates and 2 worms/plate for progeny
production, n = 140–180 for worm size).

32
4.1.3.2 Effect of chicoric acid on the age-associated decline of phenotypes and ROS

levels

Two typical markers of aging-relative phenotypes in C. elegans are pharyngeal

pumping rate and locomotive activity (102). Thus, we next detected the effects of

chicoric acid on these two markers (Fig. 4.3). The pumping rate as expected declined

progressively with aging: 60% decline of the pumping rate in the control group from day

4 to day 12 (Fig. 4.3). Chicoric acid treatments with both 25 and 50 µM statistically

delayed the decrease of pumping rate compared to the control (Fig. 4.3A). A significant

difference was first observed on day 6, and a greater difference was found on day 12

(29.1% and 32.9% improvement, respectively, compared to the control). Aging resulted

in the decline of moving speed by 76.4% on day 12, compared with that of day 2 in the

control (Fig. 4.3B). Consistent with changes in the pumping rate, the decline in

locomotive activity of the worms was lessened by chicoric acid treatments; On day 12,

chicoric acid-treated C. elegans moved 59% and 86% faster than the control group,

respectively (Fig. 4.3B).

Figure 4.3 Chicoric acid ameliorated the age-related decline of movement and pumping
rate. (A) Synchronized L1 C. elegans were treated with chicoric acid and pharyngeal

33
pumping was counted every other day until day 12. (B) Synchronized L1 C. elegans were
treated with chicoric acid and moving speed was recorded by WormLab tracking system.
Then the average moving speed of each worm was calculated using WormLab Software.
Values are means ± S.E. n = 12 worms for pumping rate; n = 140–180 for worm speed.
Values with different letters at each point in time are significantly different (P < 0.05).

Previous studies have reported that extended lifespan is closely associated with

increased resistance towards oxidative stress (93, 94, 96). Since chicoric acid is known to

possess anti-oxidative properties, the intracellular ROS levels of C. elegans were

determined after the treatment of chicoric acid. The intracellular ROS levels in the

chicoric acid treatment groups were significantly reduced (14.6% and 19.2% for 25 and

50 µM chicoric acid with P=0.0392 and P=0.0134, respectively) compared with the

control (Fig. 4.4).

Figure 4.4 Effect of chicoric acid on the oxidative stress sensitivity of wild type N2
worms. Relative formation of reactive oxygen species (ROS) after 72 h of exposure to
chicoric acid (0, 25 or 50 μΜ). Paraquat (5 mM) was used as the positive control. Values
are means ±S.E. (n = 9; 60 worms/well). Values with different letters are significantly
different (P<0.05).

34
4.1.3.3 Extended lifespan by chicoric acid required AAK-2 and SKN-1

Several studies reported that aak-2 is an essential regulator in the aging of C.

elegans through dietary restriction and/or stress resistance (96, 108-110). aak-2 encodes a

homolog of the catalytic-α subunit of AMPK in C. elegans. The deletion of aak-2

shortens C. elegans lifespan, while the overexpression of aak-2 results in C. elegans

lifespan extension (108, 109). Since chicoric acid has been found to upregulate AMPK in

hepatocytes (52), we determined the role of AMPK homolog in chicoric acid’s effects on

lifespan extension using aak-2 loss-of function mutants. As shown in Table 4.1 and Fig

4.5A, the deletion of aak-2 abolished chicoric acid-mediated lifespan extension,

indicating the requirement of aak-2 in C. elegans-lifespan extension by this compound.

In addition to aak-2 , it is known that the insulin/IGF-1 signaling pathway is

involved in aging in nematodes (111, 112). daf-2 encodes for the insulin-like growth

factor 1 (IGF-1) receptor, which is an important regulator for aging in nematodes (113).

Mutants deficient in daf-2 exhibit longer lifespan due to an extended dauer stage (114).

One of the downstream targets of daf-2 is age-1, another gene mainly regulating the

longevity of C. elegans (113). Furthermore, DAF-2 and AGE-1 have been reported to

inhibit DAF-16, an important transcription factor that regulates C. elegans’ lifespan (94).

To determine whether chicoric acid extends lifespan through the insulin signaling

pathway, we tested daf-2, age-1, and daf-16 null mutants. The daf-2 deficient and age-1

deficient mutants treated with 25 and 50 µM chicoric acid displayed significantly

extended median lifespan compared to the respective controls (P<0.0001 for both; Table

4.1 and Fig 4.5B-C). Similarly, the median lifespan of daf-16 mutants was significantly

extended with the exposure of both 25 and 50 µM chicoric acid compared to the control;
35
14 days for the control (daf-16 mutants) vs. 18 and 20 days with chicoric acid at 25 and

50 µM, with P=0.0012 and P=0.05, respectively (Fig 4.5D). These results indicate that

chicoric acid may act independently of the insulin/IGF-1 signaling pathway in C. elegans

(113).

In addition to AMPK and the insulin signaling pathway, several other molecular

targets are known to regulate C. elegans lifespan, such as SKN-1 and EAT-2. SKN-1/Nrf-

2 transcription factor has been found to mediate C. elegans lifespan extension mainly

through the oxidative stress response pathway (115, 116). From Table 4.1 and Fig 4.5E,

skn-1 mutation abolished chicoric acid-induced lifespan extension, suggesting that skn-1

is required for chicoric acid-mediated lifespan extension. Lastly, we tested eat-2 mutant,

which has defects of pharyngeal pumping that leads to prolonged lifespan via dietary

restriction pathway (117). As shown in Table 1 and Fig. 4.5F, chicoric acid treatments

shortened the median lifespan of eat-2 deficient mutant, indicating the extended lifespan

by chicoric acid might not occur through modulation of dietary intake. Since the

observations that 25 and 50 µM chicoric acid led to similar effects on the aging and

lifespan of mutants, we used 25 µM chicoric acid for the rest of the experiments.

36
Table 4.1 Effect of chicoric acid on median lifespan of wild type and mutant C. elegans.

Chicoric acid Median lifespana Genetic

Strain
treatment (µM) (days) requirementb
N2 Control 19 -
25 23***
50 22***
aak-2 (ok524) X Control 12 Yes
25 12
50 14
daf-2 (e1370) III Control 32 No
25 36***
50 38***
age-1 (hx546) II Control 28 No
25 32***
50 32*
daf-16 (mgDf50) I Control 14 No
25 18**
50 20***
skn-1(zu135) IV Control 16 Yes
25 18
50 18
eat-2(ad1116) II Control 32 No
25 24***
50 26*
All strains were raised at 20 C, except daf-2 (e1370) III, which was raised at 15 °C.
Worms were treated with chicoric acid (25 or 50 µM) from the L4 stage (day 0).
Floxuridine (FUdR) was added with a final concentration of 120 µM. The survivals were
recorded every other day until all the worms died. a The median lifespan was referred to
the time when survival rate dropped to 50%. *, **, and *** mean significant difference
compare with their respective control at P<0.05, P<0.01 and P<0.001, respectively (n =
180–266 worms/treatment). b Significant differences were analyzed by Log-rank (Mantel-
Cox) tests. Genetic requirement was defined as “Yes” at P<0.05 or “No” at P>0.05.

37
Figure 4.5. Survival of several mutant strains treated with chicoric acid. (A-F) Loss of
function mutant strains in several genes related to lifespan regulation were treated with
final concentrations of 0 (control), 25, or 50 µM chicoric acid starting from L4 stage (day
0). The survivals were recorded every other day until all the mutants died (n = 180–266
worms/treatment). Log-rank (Mantel-Cox) test was used for statistical analysis.

4.1.3.4 Chicoric acid might extend C. elegans lifespan via the regulation of AAK-2

and SKN-1.

38
 Since the loss of aak-2 and skn-1 abolished chicoric acid-mediated lifespan

extension, we determined the role of aak-2 and skn-1 in chicoric acid’s effect on lifespan

extension. First, we found that chicoric acid upregulated the mRNA expression of aak-2

(Fig. 4.6A). Since it is known that AAK-2 can be regulated post-translationally (97, 118,

119), we measured the role of chicoric acid in phosphorylation of AAK-2 and found that

chicoric acid significantly increased the phospho-AAK-2 level (P<0.001, Fig. 4.6B and

C). We further determined the mRNA expression of a downstream target of AAK-2, nhr-

49, a homolog of peroxisome proliferator-activated receptor alpha (PPARα) (120). As

shown in Fig. 4.6A, chicoric acid treatment significantly increased expression of nhr-49

compared to the control (P=0.0066).

Chicoric acid also significantly upregulated gene expression of skn-1 (Fig. 4.6A).

Similar to AAK-2, SKN-1 can be regulated post-translationally, which can be measured

by nuclear translocation of SKN-1 in C. elegans (121). Chicoric acid treatment

significantly promoted SKN-1 translocation from the cytoplasm to the nuclei (P=0.0041,

Fig 4.6D), suggesting potential post-translational regulation of SKN-1 by chicoric acid.

Next, we further tested two SKN-1 target genes, gsr-1 (glutathione reductase) and pbs-5

(proteasome beta subunit) (122-124), which were increased by chicoric acid treatment – a

1.7-fold and 2.4-fold increase over the control, with P=0.0259 and P=0.0112,

respectively (Fig. 5A).

39
Figure 4.6 Chicoric acid might extend C. elegans lifespan via the regulation of AAK-2
and SKN-1. Synchronized L1 worms were treated with 0 or 25 µM chicoric acid for 48 h
(A). The mRNA expression of aak-2, nhr-49, skn-1, gsr-1, and pbs-5 were determined by
real-time PCR. ama-1 was used as an internal control. (B) Phospho-AAK-2 and tubulin
were determined by immunoblotting. (C) p-AAK-2/Tubulin, phospho-AAK-2/Tubulin.
40
Values are means ± S.E. (n=3), *P<0.05, **P<0.01, and ***P<0.001. (D) Intracellular
localization of SKN-1::GFP. The nuclear translocation patterns of SKN-1::GFP were
identified as ‘low’, ‘medium’ and ‘high’. (E) Nuclear translocation of SKN-1 in ldIs7;
worms treated with chicoric acid were analyzed using the chi square test, and therefore
do not show standard error bars (control, n=94; NaN3, n = 82; chicoric acid 25 μM, n =
107). A NaN3 (2%) treated group was used as the positive control. ** and *** mean
significant difference compared with the control at P<0.01 and P<0.001, respectively.

4.1.3.5 Chicoric acid enhanced oxidative stress resistance in wild type N2 worms but

not aak-2 mutants

As aak-2 has been reported to regulate oxidative stress resistance in nematodes

(119), we then evaluated the influence of chicoric acid on oxidative stress responses of

wild type worms and aak-2 mutants. After 2, 4, and 6 days of paraquat exposure, the

survival of chicoric acid-treated wild type worms was consistently improved over that of

the control (12.2%, 12.8%, and 20.8% improvement for 25 µM chicoric acid with

P<0.001, P=0.002, and P<0.001, respectively, Fig 4.7A). As shown in Fig 4.7B, the

beneficial effect of chicoric acid against oxidative stress was abolished in aak-2 mutants,

suggesting an essential role of AAK-2 in chicoric acid-enhanced oxidative stress

resistance in C. elegans.

41
Figure 4.7 Chicoric acid enhanced the stress resistance of wild type worms but not aak-2
mutants. Wild type worms and aak-2 mutants (day 1 adults) were exposed to 5 mM
paraquat with or without chicoric acid for 2, 4, and 6 days and survivals were counted.
Values are means ± S.E. (n = 3; 120-145 worms/treatment), **P<0.01 and ***P<0.001.

4.1.4 Discussion

In the current study, we found that chicoric acid at 25 and 50 µM significantly

extended the lifespan of C. elegans. These findings are consistent with a previous study

showing that chicoric acid at 25 µM or higher concentrations extended C. elegans

lifespan (7). Here, we further investigated that chicoric acid-induced lifespan extension

was accompanied by the prevention of age-associated declines of pumping rate and

42
locomotor activity as well as reduced oxidative stress sensitivity without influencing

other physiological functions. The effects of chicoric acid was determined to be mediated

in part by cellular energy sensor, AAK-2, and oxidative stress responsive transcription

factor, SKN-1, but not via the insulin signaling pathway or dietary restriction.

C. elegans lifespan extension is closely associated with enhanced stress

resistance, including oxidative stress (125). Increased oxidative stress, resulting from the

excess production of free radicals, typically ROS, accelerates the aging of C. elegans

(70). Here, we found that chicoric acid reduced ROS level in vivo and increased survivals

under an oxidative stress condition, which is consistent with previous reports that

chicoric acid reduced ROS and enhanced oxidative stress resistance in RGC-5 rat retinal

ganglion cells and SH-SY5Y human neuroblastoma (67, 75). Antioxidative compounds,

including chicoric acid, may directly act as ROS scavenger and/or indirectly activate

stress-related signaling pathways, although based on the current results, it is not clear

how chicoric acid elicits its effects on ROS.

The aak-2 gene encodes an isomer of the AMPKα in C. elegans (108). A few

studies have reported that aak-2 is intimately involved in the control of C. elegans

lifespan through the regulation of dietary restriction and/or stress resistance (96, 108-

110). Based on the significant effect by chicoric acid on eat-2 mutants, we determined

that chicoric acid elicits its protective effects via stress resistance. In particular, Lee et al.

(119) reported that aak-2 mutations are hypersensitive to paraquat-induced oxidative

stress, suggesting an important role of aak-2 to C. elegans oxidative stress responses.

This is supported by the current observation that aak-2 deficiency completely abolished

43
the chicoric acid-mediated lifespan extension and survival during the oxidative stress

conditions in C. elegans (Table 4.1 and Fig.4.5). The nhr-49 is one of the downstream

genes of aak-2 (120), and it was suggested that NHR-49 may participate in oxidative

stress responses and prevent ROS-induced toxicity in C. elegans (121). In fact, Moreno-

Arriola et al. suggested that phospho-AAK-2 can regulate oxidative metabolism through

increased transcriptional activity of nhr-49 (126). Thus, chicoric acid may extend lifespan

in part via a mechanism dependent upon aak-2 and its target, nhr-49. However, since we

were not able to determine the effects of chicoric acid on AAK-2 due to lack of available

antibodies for AAK-2 of C. elegans, it is not conclusive that chicoric acid upregulated

AAK-2 transcriptionally and/or post-translationally with the current results only.

skn-1 is also involved in chicoric acid-induced lifespan extension in C. elegans.

SKN-1 starts regulating C. elegans lifespan as early as the postembryonic stage by

activating cellular defense responses to oxidative stress (127). This was further supported

by the fact that loss of function mutants in skn-1 are sensitive to oxidative stress and the

transgene of SKN-1::GFP rescues their embryonic developmental defect (115). It was

reported that nuclear translocation of SKN-1 results in the higher cellular oxidative stress

resistance in C. elegans (121). The current study showed that chicoric acid induced

translocation of SKN-1 from the cytoplasm to nuclei with minimum enhancement of skn-

1 expression. This may suggest that chicoric acid’s effect on post-translational regulation

of SKN-1 contributes significantly to the antioxidation and lifespan benefits of chicoric

acid in C. elegans. This was further confirmed by the observation of increased expression

with chicoric acid treatment of two downstream genes of SKN-1, glutathione reductase

(gsr-1) and proteasome subunit (pbs-5), which are essential for oxidative stress tolerance
44
in C. elegans (122). However, it is not clear if chicoric acid regulates SKN-1 directly or

by regulating upstream regulators of SKN-1. In fact, AAK-2 was suggested to be the

upstream of SKN-1 for metformin-mediated lifespan extension in a previous publication

(96). Moreover, another study suggested that glycogen synthase kinase-3 (GSK-3) is the

upstream of SKN-1 contributing to the increased resistance of oxidative stress in C.

elegans (128). Thus, future experiments are needed to determine the target of chicoric

acid on enhanced oxidative stress resistance.

Moreover, both AAK-2 and SKN-1 have been reported to regulate mitochondrial

biogenesis against oxidative stress and contribute to longevity in C. elegans, suggesting

the possible effect of chicoric acid on mitochondrial function (98, 129). One study

reported that chicoric acid promotes mitochondrial biogenesis by increasing citrate

synthase activity and the deacetylation of peroxisome proliferator-activated receptor-γ

coactivator, which is directly activated by AMPKα (7). Thus, we cannot exclude the

possibility that chicoric acid extends lifespan via a mitochondrial biogenesis-mediated

mechanism.

Chicoric acid is metabolized to 2-caffeoyl-L-tartaric acid (cafraric acid) and

caffeic acid by rat liver microsomes (76). A recent study reported that chicoric acid has

low absorption in rats, with peak plasma concentration at 3.44±0.53 µM after 4 h of oral

administration of chicoric acid at 50 mg/kg body weight (91). Thus, the 25 µM of

chicoric acid used in the current study might be difficult to achieve in animals and

humans. Moreover, currently the comparability of chicoric plasma concentration between

45
C. elegans and other in vivo models is not known; thus, it is important to determine the

biological relevance of chicoric acid in humans.

In conclusion, chicoric acid significantly extended the lifespan of C. elegans in

part through regulation of aak-2 and skn-1. Because of the high conservation of these two

genes between nematodes and mammals, these findings indicate that chicoric acid may

have potential for promoting healthy aging as well as combatting age-related diseases in

humans.

Fig. 4.8 The graphic contents. Chicoric acid significantly extended the lifespan of C.
elegans and increase the oxidative stress resistance in part through regulation of aak-2
and skn-1.

46
4.2 Effect of chicoric acid on glucose uptake and the underlying mechanism

4.2.1 Introduction

Type 2 diabetes mellitus (T2DM) has been recognized as a major public health

challenge throughout the world in recent decades (8, 9). Impaired glucose uptake in

muscle tissues is the primary associated with hyperglycemia in T2DM (10). Upon insulin

signaling, protein kinase B, also known as Akt, is activated by phosphorylation that leads

to translocation of glucose transporter 4 (GLUT4) to the plasma membrane, which

subsequently promotes glucose uptake (126, 130-132). In addition to insulin signaling,

several studies indicated that AMP-activated protein kinase α (AMPKα), which is known

to regulate energy homeostasis (133, 134), also activates Akt, independent of insulin, and

results in increased glucose uptake (135, 136).

Accumulating evidence suggests that many natural bioactive components have

great potential to be used for the prevention or amelioration of T2DM (126, 137). Among

natural bioactives, chicoric acid has been recently reported to have potential anti-diabetic

effects (11, 51, 73). It is a naturally occurring dicaffeoyl ester often found in chicory

plants and basil (Fig. 1A) (138, 139). One study reported that chicoric acid promoted

glucose uptake in L6 muscle cells (11). More recently, another study revealed that

chicoric acid regulated glucose homeostasis, stimulated the AMPKα pathway, and

reversed insulin resistance in HepG2 cells (52). However, it is still not clear whether

chicoric acid enhances glucose uptake in muscle cells via an AMPKα-mediated pathway.

A better understanding of chicoric acid’s impact in promoting glucose uptake in muscle

cells would be significant given that muscle is responsible for approximately 75% of

glucose disposal in the body (140). We used C2C12 myotubes, derived from murine

47
skeletal muscle cells and a C57BL/6J mice model to determine the role of chicoric acid

on glucose homeostasis and its underlying mechanisms (141, 142).

4.2.2 Materials and methods

4.2.2.1 Materials

Chicoric acid (≥ 98%) was obtained from Santa Cruz Biotechnology, Inc. (Santa

Cruz, CA, USA). C2C12 murine skeletal myoblasts were from American Type Cell

Collection (Manassas, VA, USA). Bovine serum albumin, recombinant human insulin,

and a penicillin/streptomycin mixture were purchased from Thermo Fisher (Waltham,

MA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), Kaighn’s Modification of

Ham’s F-12 (F-12K), fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, purity ≥ 93%), and horse

serum (HS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dorsomorphin

dihydrochloride (purity≥98%), also named Compound C, was purchased from MedChem

Express (Monmouth Junction, NJ, USA). The protein content was determined using

protein DC assay kits from Bio-Rad Co. (Hercules, CA, USA). Rabbit antibodies for

phospho-Akt (Ser473), Akt (total), phospho-AMPKα, AMPKα (total), phospho-acetyl-

CoA carboxylase (ACC) (Ser79), ACC (total), mouse antibody for GLUT4, goat anti-

rabbit IgG-horseradish peroxidase (HRP), and goat anti-mouse IgG-HRP were obtained

from Cell Signaling Technology (Danvers, MA, USA). Rabbit antibodies for β-actin and

Na+/K+-ATPase were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA).

4.2.2.2 C2C12 culture

48
 C2C12 cells were cultured in a 5% CO2 atmosphere at 37°C. Myoblasts were

cultured and differentiated into multinucleated myotubes based on previous studies (143).

After 6 days of differentiation, myotubes were treated with chicoric acid (12.5, 25, and 50

µM) for 24 h. For insulin treatment, 100 nM insulin was added into media for 30 min

before the harvest of cells. A previous study reported that 20 µM Compound C inhibited

the activity of AMPKα in primary hepatocytes (144). In the current model, 10 µM

Compound C was able to inhibit AMPKα phosphorylation in C2C12 cells. Thus, 10 µM

Compound C was used in this study. An MTT-based cell viability test was used to

determine the cytotoxicity of chicoric acid (145). Briefly, C2C12 myoblasts were seeded

in 96-well plates at a density of 1 ×106 cells/mL. Cells were treated with 12.5, 25, and 50

μM of chicoric acid for 24 h. The medium was then replaced with 5 mg/mL MTT in

DMEM (without phenol red) for 4 h at 37°C. After incubation, cells were washed thrice

with phosphate-buffered saline (PBS) and formazan crystals dissolved in DMSO were

measured with microplate reader SpectraMax i3 (Sunnyvale, CA, USA) at 570 nm.

Concentrations of chicoric acid tested (0-50 µM) were not toxic to C2C12 cells as shown

in Fig. 4.9A.

4.2.2.3 Glucose uptake assay

For glucose uptake measurement, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-

yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent derivative of glucose, was used as

described previously (143). Myoblasts were seeded in the 24-well plates. After 6 days of

differentiation, myotubes were treated with various doses of chicoric acid in F-12K (5.5

mM glucose) serum free media for different time periods as shown the Figure legends.

49
C2C12 myotubes were then treated with and without 100 nM insulin for 30 min in a

Krebs-Ringer buffer that was prepared as published previously (146). Insulin (30 min)

stimulation was used to activate insulin signaling in C2C12 myotubes as previously

described (147, 148). After insulin stimulation, the 2-NBDG solution was added to each

well at a final concentration of 200 µM, and the cells were incubated at 37 °C for 30 min.

Cells were then rinsed thrice with ice cold PBS and the fluorescence was immediately

measured using the SpectraMax i3 microplate reader at excitation/emission 465/540 nm.

The cell lysate was prepared by adding 40 µl 0.1 M NaOH per well. The protein content

of each well was determined by protein DC assay kits. The fluorescence was normalized

by protein content.

4.2.2.4 Cell membrane protein extraction

Integral membrane proteins and membrane-related proteins from C2C12

myotubes were prepared using a Mem-PER Plus Membrane Protein Extraction Kit from

Thermo Fisher Scientific (Waltham, MA, USA) according to the manufacturer’s

instructions. GLUT4 translocated to membrane and total GLUT4 were determined by

immunoblotting.

4.2.2.5 Transfection of shRNA-AMPKα

Lentivirus-mediated short hairpin RNA (shRNA) targeting of the mouse

AMPKα1 gene (shAMPKα) was established by Shanghai R&S Biotechnology Co. Ltd

(Shanghai, China). Detailed information is provided in supplementary data

(Supplementary Table S1 and Figure S2). The effective target sequence

50
ACGAGTTGACCGGACATAAAA in the mouse AMPKα1 gene (GeneID:

NM_001013367.3) was selected, and a scrambled sequence:

GAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCA

GTACATTT that had no significant homology to any mouse gene sequences was used as

a negative control (shControl). Transfection of shRNA was performed based on the

manufacturer’s instructions. A virus counted with approximately 100 multiplicity of

infection (MOI) was used to infect one well of cells in a 6-well plate for 12 h in a serum-

free medium with polybrene (8 µg/mL), and the cell culture medium was then changed to

a normal medium containing 10% FBS. After 48 h incubation, cells were treated with

chicoric acid (0 or 25 µM) for 24 h. AMPKα protein levels were measured by Western

blotting.

4.2.2.6 Western blotting

C2C12 cell lysate was prepared for immunoblotting as described previously (107,

148). Protein concentrations were determined by the protein DC assay kits. β-Actin was

used as an internal control. Horseradish peroxidase-conjugated goat anti-rabbit IgG was

used as the secondary antibody. Protein blots were pictured by the Image Station

4000MM (Carestream Health, New Haven, CT, USA) with a Clarity Western ECL

Substrate Kit (Bio-Rad Co.). Images were quantified with ImageJ software (version 1.49,

National Institutes of Health, MD, USA).

4.2.2.7 AMP/ATP assay

The measurement of AMP and ATP from C2C12 myotubes was conducted using

51
a mouse AMP/ATP ELISA kit (Shanghai Fan Ke Biotechnology Co., Ltd., Shanghai,

China). Briefly, C2C12 cells were treated with chicoric acid (25 µM) for 24 h, followed

by washing with cold PBS twice at 4°C. Then cells were diluted by PBS to reach the final

concentration 1 x 107 cells/ml. The diluted cells were lysed with a lysis buffer according

to the manufacturer’s instructions. The supernatant was obtained by centrifugation for 20

min at the speed of 3000 rpm. Then the supernatant of cell lysate was taken to detect the

levels of AMP and ATP following the protocol of the ELISA kit. The absorbance of each

sample was read by the Multiskan FC microplate reader (Thermo Fisher Scientific,

Waltham, MA, USA) at 450nm after adding a stop solution for 15 min.

4.2.2.8 Glucose tolerance test (GTT)

Male C57BL/6J mice (8 week) were obtained from the Laboratory Animal

Research Center of Jiangsu University (Jiangsu, China) and maintained on a 12 h

light/dark cycle with free access to food and water for 2 weeks. Institutional guidelines

(serial No. UJS-LAER-2018042301) for animal care and use were followed, and the

animal protocol was approved by the animal ethics committee of Jiangsu University.

After adaptation, the mice were divided into two groups (6-8 mice/group). For the

treatment groups, mice received a daily oral administration of 5 mg/kg BW chicoric acid

for 5 days, based on a previous publication (59). For the control group, mice will be

administrated with saline solution orally. Mice body weight will be measured before

glucose tolerance test (GTT). On the fifth day, all mice will be fasted for 12 h following

the first measurement of blood glucose from the tail vein by a blood glucose meter (0 min,

ACCU-CHEK, Jiangsu, China). Then, all mice will be subjected to an intraperitoneal

52
GTT (2 g glucose/kg BW). The blood glucose levels will be further monitored at 15, 30,

60, and 120 min. The areas under the curves (AUCs) were calculated with Sigma Plot

(version 11.0, Systat Software, Inc, San Jose, CA, USA).

4.2.2.9 Statistical analysis

SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA) was used for

statistical analysis. One-way analysis of variance (ANOVA) was used for all data except

for Fig. 4.9B, Fig. 4.10A, Fig. 4.11B, Fig. 4.13, and Fig. 4.14, where two-way ANOVA

with least squares means statement was used. When there were no interactions between

chicoric acid and another factor (insulin or time), no letter was used in the figures. If

there were significant interactions between chicoric acid and another factor (Compound

C or shAMPK), letters were used in the figures to present the differences between each

group. The Tukey−Kramer method was applied for the multiple comparisons among the

groups. A difference was considered significant at P<0.05.

4.2.3 Results

4.2.3.1 Chicoric acid promoted glucose uptake

When overall glucose uptake was measured by using 2-NBDG from C2C12

myotubes, there were significant effects of insulin (P=0.0006) and chicoric acid (P<

0.0001) without any significant interaction between insulin and chicoric acid (Fig. 4.9A).

Glucose uptake in insulin-stimulated groups was 20%-25% higher than in non-insulin-

stimulated groups. This is consistent with previous reports that less than 30% increase of

glucose uptake after insulin stimulation with 2-NBDG (149, 150). The exposure of

53
C2C12 cells to chicoric acid at the concentrations of 25 and 50 µM resulted in significant

increases in glucose uptake regardless of insulin stimulation compared to the controls (P=

0.001 and P=0.009, respectively, Fig. 4.9B). The effect of increased glucose uptake by

chicoric acid (25 µM) peaked after a 24 h treatment, and was significantly 36% greater

than the control group (P<0.0001; Fig. 4.9C); thus, a 24 h chicoric acid treatment was

used for the subsequent experiments.

54
Figure 4.9 Chicoric acid promoted glucose uptake in C2C12 myotubes. (A)
Concentrations of chicoric acid tested (0-50 µM) were not toxic to C2C12 cells. (B)
Glucose uptake was measured from myotubes that were treated with chicoric acid (12.5,
25 or 50 µM) for 24 h, followed by a 30-min treatment with or without 100 nM of insulin.
(C) Myotubes were treated with 25 µM chicoric acid for 12 h, 24 h or 48 h in F-12K (5.5
55
mM glucose) serum free media, respectively. Since glucose uptake was induced by
chicoric acid independent of insulin, no insulin stimulation was conducted in this test.
After washing thrice, 2-NBDG solution was added to each well at a final concentration of
200 µM, and the cells were incubated at 37 °C for 30 min. Cells were then rinsed thrice
with ice cold PBS and the fluorescence was immediately measured using the SpectraMax
i3 microplate reader at excitation/emission 465/540 nm. Numbers are mean ±S.E. (n=5).
Means with different letters are significantly different at P<0.05.

We also conducted in vivo glucose tolerance tests (GTT) to determine if chicoric

acid supplementation would have the similar results. There were significant effects in

GTT for time (P< 0.0001) and chicoric acid (P< 0.0001) without any significant

interaction between time and chicoric acid (Fig. 4.10A). Chicoric acid treatment (5 mg/kg

BW for 5 days) led to a 14.5% reduction of the overall blood glucose level compared to

the control (P< 0.0001; Fig. 2A). The areas under the curves (AUCs) in GTT further

confirms that chicoric acid treatments improved glucose tolerance in mice (P= 0.0065,

Fig. 4.10B).

56
Fig. 4.10 Effects of chicoric acid on glucose tolerance test (GTT). Mice were
administrated with (0 and 5 mg/kg BW/day) chicoric acid orally for 5 days. (A) Blood
was collected from the tail vein, and glucose levels were measured at 0 min, then the
glucose solution (2 g/kg BW) was administered by intraperitoneal injection and blood
glucose level were determined at 15, 30, 60 and 120 min. (B) Area under the curve
(AUC). Values are means ±S.E. (n = 6), **P<0.01. Means with different letters are
significantly different at P< 0.05.

57
4.2.3.2 Effects of chicoric acid on Akt activation and GLUT4 translocation in C2C12

myotubes

It is known that the activation of Akt (phosphorylated-Akt, p-Akt) and GLUT4

translocation to the plasma membrane directly regulate glucose uptake in muscle cells (9,

131). Thus, we examined the effects of chicoric acid on the phosphorylation of Akt and

GLUT4 translocation from C2C12 myotubes. Fig. 4.11A and 4.11B show that the ratio of

p-Akt/Akt (as an indicator of activation of Akt) was significantly increased by insulin

(P<0.0001) and chicoric acid (P=0.0003), without any interaction between insulin and

chicoric acid. These results are consistent with the results in Fig. 4.9A that chicoric acid

promoted glucose uptake in an insulin-independent manner. From these results, we

selected 25 µM chicoric acid to determine its effect on GLUT4 translocation since this

concentration had the highest activation of Akt, a 132% increase over the control in this

model (P<0.0001; Fig. 4.11A). As shown in Fig 4.11C-E, the treatment of 25 µM

chicoric acid resulted in a 28% increase in GLUT4 translocation to the cell membrane

compared to the control (P=0.0086) without altering total GLUT4 expression. These

results suggest that chicoric acid activated Akt independently of insulin and promoted

GLUT4 translocation to the plasma membrane.

58
Figure 4.11 Activation of Akt and translocation of glucose transporter 4 (GLUT4) by
chicoric acid in C2C12 myotubes. (A) Myotubes were treated with the indicated
concentrations of chicoric acid for 24 h and/or 100 nM insulin for 30 min in F-12K
serum-free medium. Then phosphorylation of Akt and total Akt was determined by
immunoblotting. (B) p-Akt/Akt, phosphorylated protein kinase B/protein kinase B. (C)
Chicoric acid induced translocation of GLUT4. C2C12 myotubes were treated with 25
µM of chicoric acid for 24 h. Then the protein of plasma membrane and whole cells were
extracted to immunoblotting. (D) Mem-GLUT4/ Na+/K+-ATPase, GLUT4 on cell
membrane/internal reference on cell membrane. (E) Total GLUT4/β-actin, GLUT4 in
whole cells/internal reference in whole cells. Numbers are mean ±S.E. (n=3), **P<0.01.
Means with different letters are significantly different at P< 0.05.

4.2.3.3 Effects of chicoric acid on the AMPKα signaling pathway in C2C12

myotubes

Based on previous reports that chicoric acid promoted AMPKα activation in L6

myocytes (73), and AMPKα can activate Akt (135, 136), we determine if the effect of

chicoric acid (12.5 and 25 µM) on Akt was dependent upon AMPKα (Fig. 4.12). First,

59
we have confirmed that chicoric acid (25 µM) activated AMPKα (the ratio of p-

AMPKα/AMPKα) with a 131% increase over the control (Fig. 4.12B). It was further

confirmed that phosphorylation of ACC/ACC, one of main downstream targets of

AMPKα, was consistently increased by chicoric acid treatment (Fig. 4.12C).

Figure 4.12 Activation of AMP-activated protein kinase α (AMPKα) pathway by

chicoric acid in C2C12 myotubes. (A) Myotubes were treated with chicoric acid (12.5
µM or 25 µM) for 24 h in DMEM. Phosphorylation of AMPKα and ACC and the total
were determined by immunoblotting. (B) p-AMPKα/AMPKα, phosphorylated AMP-
activated protein kinase-α/AMP-activated protein kinase-α. (C) p-ACC/ACC,
60
phosphorylated acetyl-CoA carboxylase/acetyl-CoA carboxylase. Numbers are mean ±
S.E. (n=4). Means with different letters are significantly different at P<0.05.

Next, we further proved that activation of AMPKα is involved in the effects of

chicoric acid on activation of Akt by using two approaches: an AMPKα inhibitor

(Compound C) and AMPKα knockdown cells. Compound C was regarded as a selective

AMPKα inhibitor with no influence on kinases that have similar structures with AMPKα

(144). Therefore, we used Compound C first to determine whether Akt activation by

chicoric acid is mediated by an AMPKα-dependent pathway (144). Both Compound C

and chicoric acid had significant effects on Akt phosphorylation, with a significant

interaction between Compound C and chicoric acid (P= 0.049). The phosphorylation of

AMPKα by chicoric acid was inhibited after treatment with Compound C (10 µM) as

expected (Fig 4.13A-B). Chicoric acid consistently upregulated Akt phosphorylation

without Compound C (P= 0.003), while chicoric acid induced Akt activation was

abolished by Compound C (Fig. 4.13A & C).

61
Figure 4.13 Increased glucose uptake and activation of Akt by chicoric acid were
abolished by the inhibition of AMPKα. (A) Cells were treated with 10 µM compound C
for 12 h, then myotubes were treated with 25 µM chicoric acid for 24h. (B) p-
AMPKα/AMPKα, phosphorylated AMP-activated protein kinase-α/AMP-activated
protein kinase-α. (C) p-Akt/Akt, phosphorylated protein kinase B/protein kinase B.
Numbers are mean ±S.E. (n=3-5). Means with different letters are significantly different
at P<0.05.

62
 Next, we prepared AMPKα knockout cells to further confirm if chicoric acid had

significant effects on Akt phosphorylation and glucose uptake. As shown in Fig. 4.14A

and B, knocking-out AMPKα abolished the effects of chicoric acid on the activation of

Akt. Consistently, increased glucose uptake by chicoric acid was abolished in AMPKα

knock-out cells (Fig. 4.14C). These results indicate that chicoric acid increases the

phosphorylation of Akt and glucose uptake through an AMPKα-dependent pathway.

63
Figure 4.14 Increased glucose uptake and activation of Akt by chicoric acid were
abolished by shAMPKα. (A) Cells were infected with lentivirus vectors expressing
shRNA targeting mouse AMPKα1. Phosphorylation of Akt and the total Akt were
determined by immunoblotting. (B) p-Akt/Akt, phosphorylated protein kinase B/ protein
kinase B. (C) Glucose levels were determined in C2C12 myotubes treated with 0 or 25
µM chicoric acid for 24 h. Numbers are mean ±S.E. (n=3-5). Means with different letters
are significantly different at P<0.05.

64
 In aerobic respiration, an increase in AMP level has been reported to directly

induce AMPKα activation (151). Thus, we further determined if AMPKα activation by

chicoric acid is attributed to the enhanced AMP/ATP ratio. Figure 4.15 showed that the

AMP/ATP ratio was increased by 133% in the 25 µM chicoric acid treatment group

compared with the control (P<0.001), which suggests that chicoric acid activates AMPKα

via regulation of the AMP/ATP ratio.

Figure 4.15 Chicoric acid enhanced AMP/ATP ratio in C2C12 myotubes. After 24 h of
chicoric acid treatment, cells were lysed, and the supernatant lysate was taken to detect
the levels of AMP and ATP using a commercial kit. The absorbance of each sample was
determined by after incubation for 15 min. Values are means ±S.E. (n =3), ***P<0.001.

4.2.4 Discussion

Several phenolic secondary metabolites, such as chlorogenic acid and caffeic acid,

have been used as antihyperglycemic compounds in pharmaceutical formulations (11,

137). Chicoric acid, a new phenolic ester, has been reported to have a beneficial effect on

glucose transport in several studies (51, 52, 59, 73). Consistently, we demonstrated that

chicoric acid increased glucose uptake and activated Akt, particularly at 25 µM, and these

effects were dependent to AMPKα, but not insulin, in C2C12 myotubes. The

65
hypoglycemic effects of chicoric acid were further confirmed in vivo using mice at 5 mg

chicoric acid/kg BW.

One in vivo study showed that chicoric acid (3 mg/kg BW) decreased the blood

glucose level by 53% with a 120 min treatment in diabetic mice (59). Others reported that

the extract from Cichorium intybus root, containing about 15 or 30 mg chicoric acid/kg

BW, significantly decreased blood glucose level after 4 days in male Wistar rats (152).

This is consistent with the current study which found that chicoric acid can significantly

reduce glucose levels in male mice. Zhu et al. (51) reported that chicoric acid ameliorated

glucosamine-induced insulin resistance through enhanced glucose uptake in HepG2 cells.

Our current results indicate that chicoric acid increased glucose uptake, independent of

insulin, in C2C12 myotubes (51, 149, 153). However, the current results are inconsistent

with Tousch et al. (11), who found chicoric acid to increase glucose uptake only with

insulin stimulation in L6 skeletal muscle cells . The inconsistency between the current

results and that of Tousch et al. might be due to the different statistical analysis methods

used for the latter (i.e. one-way, vs. two-way ANOVA in the current study). Alternatively,

the inconsistency may derive from: using different cell lines (mice vs. rat muscle cell

lines); different determination methods for glucose uptake; or a different length of

treatment time (2-NBDG with 24 h treatment in the current study vs. [3H] deoxyglucose

with 1 h treatment in Tousch et al.).

AMPKα is a master regulator of energy homeostasis by activating proteins in

catabolism pathways while turning off biosynthetic pathways (154). In cellular

respiration, an increase in AMP level relative to ATP activates AMPKα, which further

stimulates the acute upregulation of ATP production and the downregulation of non-

66
essential energy expenditure (155). Previous evidence also suggests that AMPKα is an

important regulator of glucose transport, especially in muscle tissue by phosphorylation

of insulin receptors, independent of insulin (135). Moreover, chicoric acid was previously

reported to activate AMPKα, which was suggested to be involved in the regulation of

glucose metabolism in HepG2 cells (52). In our study, a different cell line (C2C12

myotubes) was used, and we further found that chicoric acid increased the cellular

AMP/ATP ratio, which might contribute to AMPKα activation. Prior study has suggested

that increased cellular AMP level may attributed to an increase in mitochondrial

respiration without a corresponding increase in ATP synthesis by oxidative

phosphorylation, a process described as uncoupling of oxidative phosphorylation (156). It

was reported that some caffeic acid phenethyl esters (CAPEs), the derivatives of caffeic

acid, activated AMPKα through uncoupling of oxidative phosphorylation-induced

increase in AMP level (156, 157). Since chicoric acid is structurally similar to CAPEs,

we speculate that the increased AMP/ATP ratio by chicoric acid may from a mechanism

similar to that of CAPEs. However, this still needs to be determined in future study.

Along with its hypoglycemic effect, there are reports of chicoric acid having other

functional properties, such as antioxidation, anti-inflammation, and anti-fatty liver

activities (63, 84). Chicoric acid has been considered a potent antioxidant agent due to its

effect on inhibiting the accumulation of reactive oxygen species and the generation of

inflammatory cytokines, such as nitrogen oxide, interleukin 6, and tumor necrosis factor-

α (51, 64). Others have reported that chicoric acid promotes mitochondrial biogenesis by

enhancing the citrate synthase activity and the deacetylation of peroxisome proliferator-

activated receptor-γ coactivator, which is directly activated by AMPKα (73, 154). More

67
information about such properties will help us better understand chicoric acid as a

functional food bioactive.

In conclusion, chicoric acid promoted insulin-independent glucose uptake and Akt

phosphorylation by post-translational regulation of AMPKα in C2C12 myotubes, and

improved glucose tolerance in the mice model. The current study demonstrated that

chicoric acid is a potential antidiabetic nutraceutical that may be used for the prevention

or treatment of diabetes.

Fig. 4.16 The graphic contents. Chicoric acid promoted insulin-independent glucose
uptake and Akt phosphorylation by post-translational regulation of AMPKα in C2C12
myotubes.

68
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