Quercetin Induces
Mitochondrial Biogenesis
The regeneration
of mitochondria by regulated biogenesis plays an important homeostatic
role in cells and tissues and furthermore may provide an adaptive
mechanism in certain diseases such as sepsis. The heme oxygenase
(HO-1)/carbon monoxide (CO) system is an inducible cytoprotective
mechanism in mammalian cells. Natural
antioxidants can provide therapeutic benefit, in part, by inducing the
HO-1/CO system. This study focused on the mechanism by which
the natural antioxidant quercetin can
induce mitochondrial biogenesis in HepG2 cells.
We found that quercetin treatment induced
expression of mitochondrial biogenesis activators (PGC-1α, NRF-1, TFAM),
mitochondrial DNA (mtDNA), and proteins (COX IV) in HepG2 cells. The HO
inhibitor SnPP and the CO scavenger hemoglobin reversed the effects of
quercetin on mitochondrial biogenesis in HepG2 cells. The
stimulatory effects of quercetin on mitochondrial biogenesis could be
recapitulated in vivo in liver tissue and antagonized by SnPP. Finally,
quercetin conferred an anti-inflammatory effect
in the liver of mice treated with LPS and prevented impairment of
mitochondrial biogenesis by LPS in vivo. These salutary effects
of quercetin in vivo were also antagonized by SnPP.
Thus, our results suggest that quercetin
enhances mitochondrial biogenesis mainly via the HO-1/CO system in vitro
and in vivo. The beneficial effects of quercetin may provide a
therapeutic basis in inflammatory diseases and sepsis.
1. Introduction--Mitochondrial
biogenesis plays an important role in cell survival and repair [1–3].
Increased oxidative damage and inflammation can cause mitochondrial damage
that may lead to serious acute and chronic pathologies such as multiorgan
failure, neurodegeneration, and cardiovascular disease [3–6].
Mitochondrial biogenesis can enhance
cellular function and survival in vivo and in vitro and promote cellular
recovery from damage caused by adverse environmental, pathophysiological,
and/or infectious agents [7, 8].
--Mitochondrial biogenesis
is regulated by a complex network of factors. The peroxisome proliferator-activated
receptor gamma coactivator (PGC) family of transcription co-activators
(e.g., PGC-1α) coactivate nuclear respiratory factor 2 (NRF-2/GA-Binding
protein-A) and nuclear respiratory factor-1 (NRF-1) [1, 9]. PGC-1α and
NRF-1 activate mitochondrial transcription factor A (TFAM) that is
responsible for transcribing nuclear encoded mitochondrial proteins,
including structural proteins as well as proteins involved in
mitochondrial DNA (mtDNA) transcription, translation, and repair [1, 2,
8–11].---Quercetin is a naturally occurring
flavonoid which has a broad spectrum of bioactive effects.
Among these, quercetin can impact mitochondrial biogenesis by modulating
enzymes and transcription factors in the inflammatory signaling cascade
[10, 12]. Previous studies have shown that
quercetin can increase messenger RNA (mRNA) for PGC-1α, the cytosolic
deacetylase SirtI, and cytochrome concentration in soleus muscles [13].
Quercetin, a potent phenolic antioxidant, can also modulate mitochondrial
biogenesis by reducing ROS production in various cell types
[14, 15]. Mitochondrial ROS can perturb cellular oxidant/antioxidant
balance and participate in redox signaling. Oxidative stress-related ROS
production can stimulate adaptive responses, such as Nrf2 translocation
and binding to antioxidant response element (AREs) motifs in protective
phase II antioxidant genes including heme oxygenase-1 (Hmox1). However,
increased ROS production can cause mitochondrial dysfunction and cell
death [9, 16]. Polyphenol antioxidants can
prevent ROS-induced cellular damage by scavenging free radicals.
The process of excess ROS elimination and
mitochondrial biogenesis is connected with innate cellular antioxidant
defense mechanisms.---Heme
oxygenase-1 (HO-1) is an important antioxidant enzyme that catalyses the
rate-limiting step in heme-degradation. HO-1 induction protects against
prooxidant heme release induced by many agents like LPS, cytokines, and
ROS. Degradation of heme results in production of biliverdin,
iron, and CO which have important physiological effects. Biliverdin is
converted to the potent endogenous antioxidant bilirubin by NADPH:
biliverdin reductase. Humans with HO-1
deficiency exhibit severe medical conditions such as anemia, leukocytosis,
and hyperlipidemia, while animal models with HO-1 deficiency are
susceptible to endotoxemia and chronic hypoxia [17, 18]. HO-1
deficient endothelial cells display increased injury in the presence of
oxidative challenge, suggesting that the HO-1
pathway is a key cytoprotective mechanism against oxidative stress which
contributes to cellular homeostasis [11, 17–19].----------Endogenous
CO contributes to the protective effects of HO-1 by modulation of the
inflammatory response. CO binds to cytochrome oxidase resulting in
increased mitochondrial ROS production, which enhances mitochondrial
biogenesis. Limited bioavailability of CO
by hemoglobin treatment triggers cell death with a concomitant decline in
ATP production, and mitochondrial generation of ATP significantly declined
when CO availability was limited. These results suggest that
CO, an enzymatic byproduct of HO-1 activity, is responsible for the
function of HO-1 and that the HO-1/CO system may preserve mitochondrial
biogenesis [17–20].--In the current study we demonstrate the role of the
HO-1/CO system in mediating mitochondrial biogenesis induced by the
antioxidant quercetin in HepG2 cells. An understanding of the mechanisms
underlying mitochondrial biogenesis may facilitate the development of
therapeutics in diseases involving mitochondrial dysfunction (e.g.,
sepsis, and metabolic syndrome).--2. Materials and Methods-2.1. Reagents--Quercetin,
Hemoglobin (Hb), and bacterial lipopolysaccharide (LPS, from Escherichia
coli 055:B5) were purchased from Sigma-Aldrich (St Louis, MO). Tin protoporphyrin-IX (SnPP)
was from Porphyrin Products Inc. (Logan, UT). Antibodies against β-actin
were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA), and antibodies to
cytochrome oxidase subunit IV (COX) IV and α-tubulin were purchased from
Cell Signaling (Danvers, MA). Antibody against HO-1 was
purchased from Assay Designs (Ann Arbor, MI). All other chemicals were
purchased from Sigma-Aldrich.
2.2. Cell Culture
and Quercetin Treatment--HepG2 cells were purchased from ATCC (Manassas, VA). Cells were cultured in DMEM
media supplemented with 10% fetal bovine serum, 100 U/mL
penicillin, and 100 mg/mL
streptomycin (Gibco, NY). Cells were maintained in a humidified incubator
at 37°C under an atmosphere of 5% CO2. For quercetin treatment, HepG2
cells (4 × 104 cells/well) were grown on 6-well plates overnight and
quercetin was administered at various doses (5–25 μM) and times (3–24 hrs).-2.3.
Animals--All experiments with mice were approved by the Animal Care
Committee of the University of
Ulsan. Seven week-old male
C57BL/6 mice were purchased from ORIENT (Pusan, Korea). The mice were maintained
under specific pathogen-free conditions at 18–24°C and 40–70% humidity,
with a 12 h light-dark cycle,
and food and drinking water were available ad libitum.---C57BL/6 mice were
treated with an intraperitoneal (i.p.)
injection of quercetin (50 mg/kg)
dissolved in 0.5% DMSO/PBS
solution for seven alternate days. The control group of mice received the
same amount of 0.5% DMSO/PBS solution. In some experiments, SnPP (50 μmol/kg)
was administered intraperitoneally (i.p.) to mice before quercetin
injection. SnPP was dissolved in 0.1 N
NaOH and diluted with PBS (pH 7.4). To study sepsis in mice, twenty-four
hours after the final injection of quercetin, mice received an injection
of LPS (10 mg/kg, i.p.). At 24 h
after LPS injection, mice were sacrificed under anesthesia and liver
tissue was harvested for RNA, mtDNA, and protein measurements.-2.4.
Reverse Transcription Polymerase Chain Reaction (RT-PCR)--
Total RNA of HepG2
was extracted using Trizol reagent (Invitrogen, CA). Two micrograms of
total RNA were used for reverse transcription polymerase chain reaction (RT-PCR)
analysis using oligo-dT primers (Qiagen, CA) and M-MLV reverse
transcriptase (Promega, WI) according to the manufacturer’s
instructions. The forward and reverse primers used in the present study
are shown in Table 1. PCR products were electrophoresed on 1.5% agarose
gel and visualized by ethidium bromide staining. GAPDH cDNA level was used
as an internal control.
tab1--Table 1:
Gene primers used in this study.--2.5. Western Blotting Analysis-
Cells were
harvested in lysis buffer [25 mMTris-HCl
(pH 7.5), 137 mM NaCl, 2.7 mM
KCl, 1% Triton X-100] containing protease and phosphatase inhibitors
cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentration was
measured with BCA protein assay reagent (Pierce, Rockford, IL). Equal amounts of proteins
were separated using SDS-PAGE and transferred to polyvinylidene difluoride
membranes (Thermo Scientific, Rockford, IL). Membranes were blocked with
5% skim milk in PBS containing 0.1% Tween 20 (PBS-T) for 1 h
and then incubated with the specified antibodies. Signals were detected
using the ECL detection system (Thermo Scientific, Rockford, IL,
USA).--2.6. Quantitative Real-Time PCR Analysis of mt DNA Content--Genomic
DNA (containing both mitochondrial and nuclear DNA) was isolated from
cells using a Blood and Cell Culture DNA Mini Kit (Qiagen, Valencia, CA) according to the
manufacturer’s instructions. mtDNA was determined by SYBR green
quantitative PCR (qPCR). The following primers for mtDNA were used: Human
Complex II (succinate-ubiquinone oxidoreductase): forward primer
5′-CAAACCTACGCCAAAATCCA-3′ reverse primer 5′-GAAATGAATGAGCCTACAGA-3′.
Mouse cytochrome b (Mus musculus domesticus mitochondrion): forward primer
5′-CCACTTCATCTTACCATTTA-3′ reverse primer 5′-ATCTGCATCTGAGTTTAATC-3′. The
following primers for nuclear DNA were used: human β-actin: forward primer
5′-TCACCCACACTGTGCCCATCTACGA-3′ reverse primer
5′-CAGCGGAACCGCTCATTGCCAATGG-3′ and mouse 18 S
rRNA: forward primer 5′-GGGAGCCTGAGAAACGGC-3′ reverse primer 5′-
GGTCGGGAGTGGGTAATTT-3′. Reactions were performed with SYBR Green qPCR
Master Mix (2X; USB production, Affymetrix) on an ABI 7500 Fast Real-Time
PCR System (Applied Biosystems, Carlsbad, CA).-2.7. Statistical
Analysis----Multiple mean values were compared using analysis of variance
(ANOVA) with GraphPad Prism. Values presented are mean ± SD. ANOVA using
-tests was applied to compare the mean of each group with that of the
control group. A was considered to be statistically significant.--3.
Results 3.1. Quercetin Induces the Expression
of Activators and Mitochondrial Proteins Associated with Mitochondrial
Biogenesis--We determined the potential of quercetin to induce
mitochondrial biogenesis by analyzing the mRNA expression levels of major
regulators of mitochondrial biogenesis (i.e., PCG-1α, NRF-1, and TFAM).
Treatment of HepG2 cells with quercetin (15 μM)
significantly increased the levels of PGC-1α, NRF-1, and TFAM mRNA in a
time-dependent and dose-dependent manner (Figures 1(a) and 1(b)).
Quercetin treatment also stimulated the
expression of the major mitochondrial protein
COX
IV in a time- and dose-dependent manner (Figures 1(c) and 1(d)). Since
increases of mtDNA
copy number also represent an index of mitochondrial biogenesis,
we measured the amount of mtDNA in HepG2 cells treated with quercetin
(Figures 1(e) and 1(f)). Quercetin increased mtDNA copy number with an
apparent maximum at a quercetin dose of 15 μM
for 3 hrs.-fig1 Figure 1: Time-
and dose-dependent increases of mitochondrial biogenesis in HepG2 cells by
quercetin treatment. (a–f) HepG2 cells (4 × 105 cells/well) were exposed
for indicated times (0, 0.5, 1, 3, 5 h)
to various concentrations (0, 5, 10, 15, 25 μM)
of quercetin. (a, b) mRNA expressions of markers of mitochondrial
biogenesis (PGC-1, NRF-1, and TFAM) were determined by reverse
transcription PCR. GAPDH served as the standard. (c, d) Expression of COX
IV protein was determined by Western blot analysis. β-actin served as the
standard. (e, f) Expression of mitochondrial DNA (mtDNA) content was
quantified by real-time PCR. Relative amount of mtDNA and nuclear DNA (nDNA)
contents were compared. Results are expressed as mean ± SE of three
independent experiments, and representative data are shown. * compared
with untreated control group. HepG2 cells (4 × 105 cells/well) were
exposed to quercetin 15 μM,
quercetin and SnPP (a), and Hb (b) for 3 h
as described in panels (a) and (b). mRNA expressions of markers of
mitochondrial biogenesis (PGC-1, NRF-1, and TFAM) were determined by
reverse transcription (RT) PCR. Results are expressed as mean ± SE of
three independent experiments, and representative data are shown. *
compared with untreated control group.--3.2. Quercetin Induces
Mitochondrial Biogenesis via Expression of the HO-1/CO System in HepG2
Cells--
Quercetin has previously been shown to induce HO-1 expression in various
cell types, which may account in part for the cytoprotective,
antiapoptotic, antioxidant, and anti-inflammatory effects of this compound
[19, 21–23]. In the current study, we examined whether quercetin can
induce the expression of HO-1 at the RNA or protein level in HepG2 cells.
As shown in Figure 2(a), an increase in HO-1
mRNA and protein was detected at various times and doses of quercetin. The
maximal effect of quercetin on HO-1 mRNA and protein expression was
observed after treatment with 15 μM
for 3 h
(Figure 2(a), left and middle). When HepG2 cells were treated with
different concentrations of quercetin (5–25 μM)
for 3 hrs,
the maximum induction of HO-1 protein was detected at 15 μM
(Figure 2(a), right). Thus, the increases of HO-1 expression achieved with
quercetin treatment were consistent with previous reports
[24–26].
fig2---Figure 2:
Induction of mitochondrial biogenesis by quercetin is regulated by
activation of HO-1. (a) Expression levels of HO-1 mRNA and protein were
determined after HepG2 cells were exposed for indicated times (0, 0.5, 1,
3, 5 h) and with the indicated
concentrations (0, 5, 10, 15, 25 μM)
of quercetin. Expressions of HO-1 mRNA and protein were determined by
RT-PCR and Western blotting. GAPDH and β-actin served as the standards,
respectively. (b–d) HepG2 cells were exposed to 15 μM
of quercetin for 3 h with or
without 20 μM of SnPP. (b)
Expressions of PGC-1, NRF-1, and TFAM mRNA were determined by RT-PCR. (c)
Expression of COXIV protein was determined by Western blotting. (d)
Expression of mtDNA content was quantified by real-time PCR. Relative
amounts of mtDNA and nDNA contents were compared. Results are expressed as
mean ± SE of three independent experiments, and representative data are
shown. * compared with untreated control group; † compared with cells
treated with quercetin alone.---
It
is also known that CO generated by HO-1 can activate mitochondrial
biogenesis [27, 28].
Therefore, we hypothesized that the activation of mitochondrial biogenesis
by quercetin also involves the activation of the HO-1/CO system in HepG2
cells. To investigate whether HO-1/CO is involved in quercetin-induced
mitochondrial biogenesis, the competitive HO inhibitor tin-protoporphyrin-IX
(SnPP) and hemoglobin (Hb), a CO scavenger, were employed with or without
addition of quercetin, and expression levels of PGC-1α, NRF-1, and TFAM
mRNA were evaluated. As shown in Figure 2(b), treatment of HepG2 cells
with SnPP and quercetin resulted in reduced levels of PGC-1α, NRF-1, and
TFAM mRNA expression levels compared with cells treated with quercetin
alone. Quercetin-induced COX IV expression was also inhibited by SnPP
treatment (Figure 2(c)). Likewise, the increase in mtDNA by quercetin was
suppressed by SnPP treatment (Figure 2(d)). To evaluate the involvement of
CO in quercetin-induced mitochondrial biogenesis, HepG2 cells incubated
with quercetin were cotreated with Hb (Figure 3). The Hb treatment
inhibited the increases of PGC-1α, NRF-1, and TFAM mRNA expression induced
by quercetin in HepG2 cells (Figure 3(a)). Hb treatment decreased the
expression of COX IV protein induced by quercetin (Figure 3(b)).
Furthermore, the induction of mtDNA levels by quercetin was inhibited by
treatment with Hb (Figure 3(c)).--fig3-Figure 3:
Quercetin induction of mitochondrial biogenesis
requires CO. (a–c) HepG2 cells were exposed to 15 μM
of quercetin for 3 h with or
without 20 μg/mL of Hb. (a)
Expression of PGC-1, NRF-1, and TFAM mRNA was determined by RT-PCR. (b)
Expression of COX IV protein was determined by Western blotting. β-actin
served as the standard. (c) Expression of mitochondrial DNA (mtDNA)
content was quantified by real-time PCR. Relative amounts of mtDNA and
nDNA contents were compared. Results are expressed as mean ± SE of three
independent experiments, and representative data are shown. * compared
with untreated control group; † compared with cells treated with quercetin
alone.--To examine the role of HO-1 in quercetin-induced mitochondrial
biogenesis in vivo, quercetin was injected intraperitoneally in C57BL/6
mice for 7 alternate days. Mice were treated with SnPP prior to quercetin
injection. In accordance with the results observed in HepG2 cells,
quercetin increased the expression of PGC-1α, NRF-1, and TFAM mRNA, COX IV
expression, and mtDNA in vivo (Figure 4). The cotreatment with SnPP
inhibited quercetin-induced PGC-1α, NRF-1, and TFAM mRNA, COX IV
expression, and mtDNA (Figure 4). Thus, these
results suggest that HO-1/CO system is required for quercetin-induced
mitochondrial biogenesis in vitro and in vivo.-fig4-Figure 4:
Quercetin induces mitochondrial biogenesis through the induction of
HO-1/CO system in vivo. (a–c) C57BL/6 Mice were injected intraperitoneally
(i.p.) with quercetin (50 mg/kg)
for 7 alternate days, with or without SnPP (50 μmol/kg)
prior to injection with the addition of quercetin. Liver tissues were
excised and analyzed for mitochondrial biogenesis in mice. Experimental
analyses were performed with liver tissue. (a) Expressions of PGC-1,
NRF-1, and TFAM in mRNA were determined by RT-PCR. 18 S
rRNA served as the standard. (b) Expressions of COX IV protein were
determined by Western blotting. α-tubulin served as the standard. (c)
Expression of mtDNA content was quantified by real time PCR. Relative
amounts of mtDNA and nDNA contents were compared. Results are expressed as
mean ± SE of three independent experiments ( /group), and representative
data are shown. * compared with the uninjected control group; † compared
with LPS injected mice group.-3.3. Quercetin
Restores LPS-Damaged Mitochondrial Integrity via HO-1/CO Induction-Finally,
we examined whether quercetin could contribute to cellular protection
against LPS-induced mitochondrial damage in a HO-1/CO-dependent manner.
LPS treatment increased the expression PGC-1α, NRF-1, and TFAM mRNA in
mouse liver. Moreover, quercetin treatment further increased the
expression of PGC-1α, NRF-1, and TFAM mRNA after LPS treatment (Figure
5(a)). However, LPS treatment clearly diminished hepatic COX IV and mtDNA
content (Figures 5(b) and 5(c)). In contrast, quercetin protected against
the loss of COX IV and mtDNA content in LPS-treated animals. SnPP
antagonized the protective effects of quercetin with respect to hepatic
PGC-1α, NRF-1, and TFAM mRNA expression, COX IV expression, and mtDNA
content in this model (Figures 5(a), 5(b), and 5(c)). LPS caused increases
in the hepatic expression of TNFα, IL-1β, and IL-6 mRNA. Quercetin
administration inhibited the LPS-dependent induction of TNFα, IL-1β, and IL-6. This effect of
quercetin was in turn inhibited by SnPP injection (Figure 5(d)). These
results suggest that quercetin restores mitochondrial integrity from LPS
damage via activating the HO-1/CO system.
fig5
Figure 5:
Quercetin restores mitochondrial biogenesis in LPS-treated mice in a
HO-dependent fashion. (a to d) C57BL/6 mice were injected with quercetin
(50 mg/kg) for 7 alternate days,
with or without SnPP (50 μmol/kg)
prior to injection with quercetin, and then challenged for 24 hours with
i.p. injection of LPS (10 mg/kg).
(a) Expressions of PGC-1, NRF-1, and TFAM in mRNA were determined by
RT-PCR. 18 S rRNA served as the
standard. (b) Expression of COX IV protein was determined by Western blot
analysis. α-tubulin served as the standard. (c) Expression of mtDNA
content was quantified by real-time PCR. Relative amounts of mtDNA and
nDNA contents were compared. (d) Expression of TNFα, IL-1β, and IL-6 mRNA was
quantified by real-time PCR. 18 S
rRNA served as the standard. Results are expressed as mean ± SE of three
independent experiments ( /group), and representative data are shown. *
compared with the uninjected control group; † compared with LPS injected
mice group; # compared with mice in the LPS + quercetin group.-4.
Discussion--Mitochondrial biogenesis has been
the focus of extensive studies due to its beneficial effects in many
health conditions related to performance, diabetes, neurodegeneration, the
cardiovascular system, cancer, and infection.
Death resulting from multiple organ failure (MOF)
during severe sepsis and septic shock has been related to mitochondrial
damage. Rescue of mice from lethal Staphylococcus aureus sepsis
and protection against cardiomyocyte apoptosis have been linked to
mitochondrial biogenesis induction [27, 28].----Quercetin is a
polyphenolic compound that exerts several potent bioactivities including
antiproliferative, anti-inflammatory, antioxidant, and immune system
effects. Recent in vitro and in vivo experiments have shown that the
salutary effects of quercetin may involve activation of mitochondrial
biogenesis [11, 14, 15]. Previous research
has shown positive effects of quercetin on endurance and health
maintenance [4, 13, 29, 30]. These benefits may involve the antioxidant,
anti-inflammatory, and psychostimulant effects of quercetin, as well as
effects on mitochondrial biogenesis. Because abnormalities that
contribute to impaired health or development of metabolic disorders are
linked to mitochondrial dysfunction, the stimulation of mitochondrial
biogenesis by quercetin may represent the most important bioactivity of
this compound [1, 2, 10, 12, 31].--Our
results demonstrate that quercetin can enhance the expression of PGC-1α, a
master regulator of the transcriptional network that regulates
mitochondrial biogenesis, in HepG2 cells. PGC-1α is responsible
for activating the transcription of genes involved in oxidative
phosphorylation and mtDNA replication. NRF-1 and NRF-2, which are
transcription factors acting on nuclear genes coding for proteins
necessary for the mitochondrial respiratory chain or for mtDNA
transcription and replication, are also activated by PGC-1α. PGC-1α and
NRFs coactivate the expression of TFAM, which is important for regulation
and maintenance of mtDNA copy number [1, 2, 10]. Our results also showed
increased expression of PGC-1-related transcription factors associated
with mitochondrial biogenesis in HepG2 cells treated with quercetin
(Figure 1). Similarly, the mitochondrial respiratory chain consisting of
four membrane-bound complexes (Complex I–IV)
involved in ATP synthesis and transfer of electrons formed by NADH or
FADH2 is an indicator of mitochondrial biogenesis [32].
Increases in cytochrome concentration typically occur in conjunction with
similar increases in other mitochondrial enzymes of the electron transport
chain and enzymes in the tricarboxylic acid cycle and β-oxidation pathway,
that lead to an overall increase in mitochondrial capacity [13, 32].
Previous reports of quercetin-induced increases in mitochondrial
biogenesis are consistent with the increases in COX IV protein expression
observed in our study (Figures 1–3). The critical effects of quercetin on
mitochondrial biogenesis in vitro have been demonstrated at a dose of 15 μM
quercetin. To study the effects of quercetin in vivo, animals were treated
with a 50 mg/kg dose of
quercetin. According to Ruiz et al. [33],
treatment of mice with up to 3000 mg/kg
quercetin did not cause any toxicity.
Among the doses of quercetin (25, 50 and 100 mg/kg)
tested in mice, we have found that 50 and
100 mg/kg
doses of quercetin have a significant effect on mitochondrial biogenesis.
However, one of the limitations of the current study is that the effect of
oral administration of quercetin has not been tested. Further studies
would be needed to determine the therapeutic benefit of oral quercetin in
these systems.---Recent reports have also
shown that the HO-1/CO system can stimulate mitochondrial biogenesis which
may account in part for the cytoprotective effects of this system
[27, 28]. Recent research has elucidated the role of HO-1 and CO in
cellular defense mechanisms against oxidative damage. Quercetin has gained
much attention because of its ability to confer cytoprotective effects
through induction of HO-1 in various cell lines and primary hepatocytes
[9, 11, 13, 17–20, 34]. CO, an enzymatic byproduct of HO-1, can mediate
the cytoprotective effects of HO-1 activity. Our recent work has shown
that endoplasmic reticulum (ER) stress caused significant decline of CO
bioavailability that reduced mitochondrial ATP generation [9, 13, 17, 18,
35]. Similarly, in our current study, we have
shown that the deleterious effect on mitochondria due to LPS
administration was restored by quercetin. The beneficial
effects of quercetin in the LPS model were in turn abrogated by SnPP.
Thus, it is likely that quercetin induces mitochondrial biogenesis via the
HO-1/CO system in HepG2 cell lines [18, 28, 35].--High
glucose produces a high concentration of ROS that induces cellular
dysfunction. Previous research in the human hematoma cell line,
HepG2, has shown that hyperglycemia
elicits detrimental changes in liver cells [5, 36].
Increased oxidative damage caused either by an overproduction of free
radicals and ROS or by an impairment of the endogenous antioxidant defense
system is well studied in epithelial cells and HepG2 cells [16, 19, 36].
Prevention of lung oxidative damage in acute lung injury/acute respiratory
distress syndrome by quercetin has been shown to involve increases in HO-1
production [19]. In conclusion, we demonstrated that quercetin enhances
cell survival against oxidative stress through an HO-1/CO-dependent
increase in mitochondrial biogenesis. The antioxidant and mitochondrial
biogenesis properties of quercetin may be helpful in developing
therapeutic strategies to enhance cell survival during oxidative stress
imposed by environmental and dietary factors
***************************************************************************
Liposome and How they Work
Liposomes were discovered in 1961
when Alec D. Bangham of the Agricultural Research Council's Institute of Animal Physiology
in Cambridge, England was evaluating the effect of phospholipids on blood
clotting. Dr. Bangham noticed
that when he put
water in a flask containing a phospholipid film, the water forced the
molecules to arrange themselves into what he later discovered were
microscopic closed vesicles composed of a bilayered phospholipid membrane
surrounding water. Phospholipids form closed, fluid-filled spheres when
mixed with water in part because the molecules are amphipathic having a
hydrophobic tail and a hydrophilic or polar head. Two fatty acid chains,
each containing from 10 to 24 carbon atoms, make up the hydrophobic tail
of most naturally occurring phospholipid molecules.
Phosphoric acid bound to any of
several water-soluble molecules composes the hydrophilic head.
When phospholipids are mixed with water in a
high enough concentration, the hydrophobic tails spontaneously herd
together to exclude water, whereas the hydrophilic heads bind to water.
The result is a bilayer in which the fatty
acid tails point into the membrane's interior and the polar head point
outward. As the liposome forms,
any water-soluble molecules that
have been added to the water are incorporated into the aqueous spaces in
the interior of the spheres and lipid-soluble molecules added to the
solvent are incorporated into the lipid bilayer.
Ostro, 256 (1) Sci. AMER. 103-11
(1987).
The separation between the bilayers
is determined by the balance between the repulsive forces between the
layers, probably mainly electrostatic interactions between headgroups and
hydration forces of the head-groups, and attractive forces which are
consistent with the expected van der Waal's forces between the layers. The
equilibrium distance between the bilayers of pure egg phosphatidyl choline
in water is 2.75 nm. Tyrrell et al., 457 BIOCHIMICA
ETBIOPHYSICAACTA259-302 (1976). When
solutes are sequestered into liposomes, the rate at which they leak out
depends on both the nature of the solute and the composition of the
liposome.
By modification of the composition of the lipid bilayers it is possible to
reduce the leakage of particular molecules. --Depending on the number of
lipid layers, size, surface charge, lipid composition and methods of
preparation, various types of liposomes have been utilized.
Multilamellar lipid vesicles (MLV)
were first described by Bangham et al., (13 J. MOL. BIOL. 238-52 (1965)).
A wide variety of phospholipids form MLV on hydration.
MLV are composed of a number of bimolecular lamellar interspersed with an
aqueous medium.
U. S. Patent No. 4,485,054. --Unilamellar
vesicles consist of a single spherical lipid bilayer entrapping aqueous
solution.
-According to their size they are referred to as small unilamellar
vesicles (SUV) with a diameter of 200 to 500 A; and large unilamellar
vesicles (LUV) with a diameter of 1000 to 10,000 A. -The small lipid
vesicles are restricted in terms of the aqueous space for encapsulation,
and thus they have a very low encapsulation efficiency for water soluble
biologically active components. -The large
unilamellar vesicles, on the other hand, encapsulate a high percentage of
the initial aqueous phase and thus they can have a high encapsulation
efficiency. -A variety of liposomal products are known to
enhance uptake or facilitate delivery of various products. For example,
the parental and topical uses of liposomal carriers were
reported to protect a drug against hostile
environments and to provide controlled release of the drug while
circulating in the blood or after immobilization at a target tissue such
as the skin.
Recipe—take
whatever you want to liposme –use 1 table or 15 grams and use the
equivalent in volume with sunflower lecithin powder or more as you add to
the mix—take water and add to it to equal level or just slightly over the
top of the mix—blend til all is completely covered and surrounded and
smooth –use 1.4-1.2 tsp increments til you adjust to the effect and then
increase if need be or decrease
Recipes- when you are making
any form of lipase you would use small quantities and utilize
herbs-vitamins-extracts-or any form of supplement
Adrenals- B 5-1tsp-creatine
1 tsp- Vitamin A ( retinol palmitate) 100,000IU-rhodiola -1 tsp ( if you
use an extract then use a tsp ) ginseng( Siberian ) same as the rhodiola---and
then add 45 grams of sunflower lecithin –blend all the components together
till they are mixed- then when this is finish then add water or any other
solution to the powder –about ¼ inch above the mix-and blend for about 5
minutes or til there is a completely fusion or saturation-then use 2-3
grams 2-3 times a day
Liver- Ascorbic Acid 1-2
tsp- Milk Thistle Powder 1 tsp- Dandelion Powder 1 tsp- you can then from
here you can add other things –shizandra berry powder 1 tsp and rosemary
1 tsp- ( with the herbs if you have extracted with a solvent or water then
again ise just a tsp of these – you can increase or decrease as your
tolerance and 3 tablespoons of the sunflower lecithin—and mix then again
add water till it is slightly over the mix and then blend –use 2-3 grams
4-5 times a day
Brain- add to this B1- 1 tsp
( powder) B12 ( if you use a liquid 1 tsp) folic acid powder 1 tsp-piracetam
1 tsp- rosemary powder 1 tsp-sage powder 1 tsp- thyme 1 tsp –you can
modify these herbs or vitamins with other things or other nootropics-add 3
tablespoons of sunflower lecithin –mix them all in the blender –when mixed
add water or a solution again just above the mix ¼ inch above –blend til
smooth or saturated or completely absorbed—then ustilize 2-3 grams and
consume this 1-2 times a day
Depression- take 1 tsp of
cocoa powder-1 tsp of vanilla-1 tsp of niacinamide- 1 tsp of taurine-1 tsp
of Siberian ginseng and 3 tablespoons of sunflower lecithin ---mix
together til mixed and then again when the mix is complete add water to
the mix ¼ above the mix and then blend til absorbed-use 2-3 grams as
needed
With these recipes
you can substitute-you can decrease or increase the volume in the
mix-never take anything prescribed medically with any of these –there will
be an interaction with prescriptions--
***********************************************************************
Mitochondria are essential to all higher forms of life.
Every animal and plant depends on these
small intracellular structures. Mitochondria have multiple tasks: Since
they generate most of the cell's biochemical energy, they are referred to
as the powerhouses of the cell.
In addition, they are responsible for producing and breaking down amino
acids and fats. They also regulate cellular death, called apoptosis.--As
a result, the spectrum of diseases that are linked to mitochondrial
defects is wide, ranging from severe muscular
and nervous disorders to neurodegenerative diseases as well as all
symptoms of aging.--"It was by pure chance that we discovered
this completely new control mechanism of mitochondrial function," says
first author Deniz Senyilmaz, who works in Aurelio Teleman's group at the
German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ). In
collaboration with colleagues from Cambridge, Teleman and his team had
planned to investigate the metabolism of long-chain fatty acids. For this
purpose, the researchers bred flies whose cells were unable to produce
stearic acid, a fatty acid that is composed of 18 carbon atoms.
Animals with this defect did not develop beyond
the pupal stage and were not viable afterwards.--Teleman and
his team were curious to find out why this happened. They then discovered
a highly complex biological control mechanism that regulates the fusion --
as well as fragmentation -- of mitochondria and, hence, the performance of
these organelles.--The key element in this
control mechanism is the transferrin receptor,
which binds stearic acid. "For the
first time in biological research, we have found out that stearic acid,
which up until now has been believed to be
simply a metabolic product, also has signaling function," says Teleman.
The researchers demonstrated that mitochondrial control via stearic acid
works not only in flies but also in the HeLa human cancer cell line.--When
the researchers added stearic acid to fly food, the animals' mitochondria
fused; when they kept fatty acid levels low, the organelles fragmented.
"If using stearic acid as a food additive
improves the performance of normal mitochondria, then it might do the same
in pathogenically dysfunctional mitochondria,"
Teleman
explained, describing their experimental approach.--The researchers
studied flies that exhibit Parkinson's-like symptoms resulting from a
mitochondrial defect in the PINK and Parkin proteins and are recognized as
a model system for studying this neurodegenerative disease.
When the affected animals were fed stearic acid
with their food, their motor skills and energy balance improved and they
survived for much longer.--"This opens up the fascinating
possibility of using a food additive to
alleviate symptoms in patients with mitochondrial disease,"
says Teleman. "However, this still is a dream of the future, because we do
not yet know whether human cells respond in the same way as fly cells do
to increased quantities of stearic acid in the diet. Our diet naturally
contains much more stearic acid than fly food does. Therefore, a further
increase might not make any more difference."--Story Source-The
above post is reprinted from
materials provided by
German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ).
Journal Reference-Deniz Senyilmaz, Sam Virtue, Xiaojun Xu, Chong
Yew Tan, Julian L. Griffin, Aubry K. Miller, Antonio Vidal-Puig, Aurelio
A. Teleman. Regulation of mitochondrial morphology and function by
stearoylation of TFR1. Nature, 2015; DOI:
10.1038/nature14601 -----German Cancer
Research Center
(Deutsches Krebsforschungszentrum, DKFZ). "Fatty acid increases
performance of cellular powerhouse: Fundamentally new biological signaling
pathway discovered." ScienceDaily. ScienceDaily, 28 July 2015. <www.sciencedaily.com/releases/2015/07/150728101220.htm>.
***********************************************************************
Leucine Modulates
Mitochondrial Biogenesis
and SIRT1-AMPK Signaling in C2C12 Myotubes
Chunzi Liang,1
Benjamin J. Curry,2 Patricia L. Brown,1 and Michael B. Zemel3
1Department of
Nutrition, University of
Tennessee, Knoxville, 1215 W. Cumberland Avenue,
229 Jessie Harris
Building, Knoxville, TN
37996-1920, USA
2Ension, Inc.,
11020 Solway School Road, Suite 108, Knoxville, TN 37931,
USA
3NuSirt Biopharma,
11020 Solway School Road, Suite 109, Knoxville, TN 37931,
USA
Received 19 June
2014; Accepted 17 September 2014; Published 7 October 2014
Academic Editor:
Robert B. Rucker
Previous studies
from this laboratory demonstrate that dietary
leucine protects against high fat diet-induced mitochondrial impairments
and stimulates mitochondrial biogenesis and energy partitioning from
adipocytes to muscle cells through SIRT1-mediated mechanisms.
Moreover, β-hydroxy-β-methyl butyrate (HMB),
a metabolite of leucine, has been reported to activate AMPK
synergistically with resveratrol in C2C12 myotubes. Therefore,
we hypothesize that leucine-induced activation of SIRT1 and AMPK is the
central event that links the upregulated mitochondrial biogenesis and
fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated
with leucine (0.5 mM), alanine
(0.5 mM), valine (0.5 mM),
EX527 (SIRT1 inhibitor, 25 μM),
and Compound C (AMPK inhibitor, 25 μM)
alone or in combination to determine the roles of AMPK and SIRT1 in
leucine-modulation of energy metabolism.
Leucine significantly increased mitochondrial content, mitochondrial
biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity
and gene expression, and AMPK phosphorylation in C2C12 myotubes
compared to the controls, while EX527 and Compound C markedly attenuated
these effects. Furthermore, leucine
treatment for 24 hours resulted in time-dependent increases in cellular
NAD+, SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding
that of AMPK, indicating that leucine activation of SIRT1, rather than
AMPK, is the primary event.
1. Introduction---
Impaired
mitochondrial function in skeletal muscle is one of the major predisposing
factors to metabolic diseases, such as insulin resistance, type 2
diabetes, and cardiovascular diseases [1]. Indeed, lower mitochondrial
content and decreased expression of oxidative enzymes are observed in
patients with type 2 diabetes [2]. SIRT1 and
AMP-activated protein kinase (AMPK) are known to promote mitochondrial
biogenesis and oxidative capacity and prevent the mitochondrial
dysfunction in skeletal muscle [3, 4].
SIRT1, a nicotinamide adenine dinucleotide- (NAD+-) dependent deacetylase,
is the key enzyme that mediates caloric restriction- (CR-) induced
longevity in mammals [5]. By
sensing intracellular NAD+/NADH ratio, SIRT1 regulates target gene
expression via changing acetylation status of histones of transcriptional
factors, such as peroxisome proliferator-activated receptor gamma
coactivator 1-alpha (PGC-1α), tumor suppressor p53 (p53), nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-κB), and forkhead box
O3 (FOXO3) [6], resulting in modulation of
wide range of cellular fundamental processes, including DNA repairing,
energy metabolism, and cell apoptosis [7, 8]. Overexpression
and activation of SIRT1 protect against high fat diet- (HFD-) induced
metabolic abnormalities in mice, such as insulin resistance, glucose
intolerance, and liver steatosis, without extending their lifespan [9,
10]. Therefore, small molecules that could activate SIRT1 and mimic the CR
impacts have drawn considerable attention.---
Leucine, a
branched-chain amino acid (BCAA), plays a distinct role in energy
metabolism in addition to its pivotal function in protein synthesis [11,
12]. For example, leucine promotes energy
partitioning from adipocytes to muscle cells, leading to decreased lipid
storage in adipocytes and increased fat utilization in muscle cells
[13]. Leucine administration increases
insulin sensitivity and glucose tolerance by promoting glucose uptake and
fatty acid oxidation in skeletal muscle in
HFD-fed
mice [14–17]. In fact these
effects are mediated partially through SIRT1-dependent pathway, as Sirt1
knockout significantly attenuates these effects [18, 19].
Further, recent data indicate that leucine can
directly activate SIRT1 by promoting the enzyme affinity for its
substrates and NAD+ [18], resulting in elevated mitochondrial biogenesis
and fatty acid oxidation in both adipocytes and myotubes [20,
21].--HMB, a minor metabolite of leucine,
has been reported to stimulate AMPK phosphorylation synergistically with
metformin, resulting in significant increases in insulin sensitivity and
glucose tolerance in mice [22]. Similar to SIRT1, AMPK is an
evolutionary conserved enzyme and acts as an energy status sensor via
intracellular AMP or AMP/ATP ratio in eukaryotes [3]. In response to
nutrient restriction, activated AMPK promotes a cell catabolic shift with
increased ATP production to rescue the cellular fuel crisis [23].
Furthermore, phosphorylated AMPK is highly associated with SIRT1
activation in both in vivo and in vitro studies [5, 24], and part of these
two enzymes signaling pathways are overlapped [25].
These findings
provide a mechanistic framework for leucine-modulation of mitochondrial
biogenesis [21, 26]. We hypothesize that
leucine activation of SIRT1 and AMPK is the major event that regulates
fatty acid oxidation and mitochondrial biogenesis in skeletal muscle.
Accordingly, we examined the effects of leucine, valine (branched-chain
amino acid control), and alanine (nonbranched chain amino acid control) on
mitochondrial content, mitochondrial biogenesis-related gene expression,
SIRT1 activity, and AMPK phosphorylation in C2C12 myotubes. In addition,
we used EX-527 (SIRT1 selective inhibitor) and Compound C (specific AMPK
inhibitor) to probe the roles of each enzyme in leucine-modulation of
energy metabolism in C2C12 myotubes.
2. Materials and
Methods
2.1. Cell Culture
C2C12 myoblast
cells were seeded at a density of 1.2 106 cells per well in 6-well plates
and incubated in Dulbecco’s modified eagle medium (DMEM) containing 4.5 g/L
D-glucose, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin at
37°C and 5% CO2. After the cells reach 90% confluence, the medium was
switched to a standard differentiation medium (DMEM supplemented with 2%
horse serum and 1% penicillin-streptomycin) for 2 to 4 days. The
differentiation medium was changed every other day to allow 90% of the
cells to fully form myotubes (3–5 days later) before additional treatments
began.--The dosages of reagents were 0.5 mM
for leucine, 0.5 mM for alanine,
0.5 mM for valine, 100 nM
for resveratrol, 25 μM for
EX527, 25 μM for Compound C, and
50 μM for AICAR. The incubation
lengths were from 1 to 48 hours as indicated in the figure legend. 2.2.
RNA Extraction and Quantitative Real-Time PCR (RT-PCR) Analyses----Total
RNA was extracted from C2C12 myotubes using Ambion Totally RNA Isolation
Kit (Ambion, Inc., Austin, TX, USA) according to the manufacturers’
instructions. The RNA content was determined using NanoDrop ND-1000
Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). RNA
quality was assessed by the 260 nm/280 nm
ratio (1.8–2.0) and 260 nm/230 nm
ratio (2.0). The mRNA expression of selected genes related to
mitochondrial biogenesis, including Sirt1, Sirt3, PGC-1α, cytochrome c
oxidase subunit 5b (Cox5b), heat shock cognate protein 1 (Hspd 1), and
Cox2, was analyzed using a TaqMan Universal PCR Master Mix kit (Applied
Biosystem) according to the manufacturers’ instructions. The primers and
probes sets were obtained from Applied Biosystems TaqMan Gene Expression
Assays primers and probe set collection. The quantitative RT-PCR reactions
were carried out in 96-well format using ABI 7300HT instrument (Applied
Biosystem) according to the instructions. Mouse 18S ribosomal RNA was used
as the housekeeping gene. Data for each gene was normalized to 18S and
presented as a ratio to the transcript of interest to 18S.-2.3. SIRT1
Activity Measurement--SIRT1 Fluorometric Drug Discovery Kit (BML-AK555,
ENZO Life Science International, Inc., PA, USA) was used to measure SIRT1
activity in C2C12 myotubes, following the manufacturer instruction. In
this assay, SIRT1 activity is determined by the degree of deacetylation of
a standardized substrate that contains an acetylated lysine residue. This
Fluor de Lys substrate is a peptide containing amino acids 379–382 of
human p53 (Arg-His-Lys-Lys [Ac]) and serves as a direct target for SIRT1.
SIRT1 activity is proportionally related to the degree of deacetylation of
Lys-382 and the corresponding fluorescence signal changes.
Cell lysates were
harvested by homogenizing cells in RIPA buffer (Sigma-Aldrich, MO, USA),
which contains protease inhibitor cocktail (MP Biomedicals LLC, Solon, OH,
USA) (100 : 1 v/v).
After 5 seconds of ultrasonication on ice, the cell lysates were
centrifuged at 12,000 ×g for 5
minutes. The supernatant was used for SIRT1 activity assessment and other
experiments. According to the protocol, 5 μL
of cell lysate was used for the endogenous SIRT1 activity detection.
Samples were incubated in a phosphate-buffered saline solution with
peptide substrate (25 μM) and
NAD+ (500 μM) at 37°C on a
horizontal shaker for 45 minutes. The deacetylation reaction was stopped
with the addition of the stop solution (2 mM
nicotinamide) and developer that binds to the deacetylated lysine to form
a fluorophore. Following 10 minutes of incubation at 37°C, fluorescence
intensity was measured using Glomax Multi Detection System (Promega, WI, USA), with excitation and
emission wavelengths of 360 nm
and 450 nm, respectively.
Resveratrol (100 mM) and suramin
sodium (25 mM) were used as
positive and negative controls, respectively. To normalize the data of
SIRT1 activity, concentrations of the sample cellular protein were
measured via BCA-assay (Thermo Scientific Inc, Waltham, MA, USA). Data for each sample
SIRT1 activity is presented as a ratio to the protein content.
2.4. Cellular NAD+
NAD+ was measured
in C2C12 myotubes using a colorimetric assay (Cayman Chemical Company, Ann
Arbor, MI, USA) that uses an alcohol dehydrogenase reaction to reduce NAD+
in cell lysates to NADH and the NADH is used to reduce a tretrazolium salt
substrate (WST-1) to formazan. Formazan absorbance, measured at 450 nm,
is proportional to the NAD+ in the cell lysate.
2.5. Fatty Acid
Oxidation
Cellular fatty
acid oxidation was measured using [3H]-palmitate, as described in our
previous studies [13]. C2C12 cells in 12-well plates were washed with 2 mL
of cold PBS solution twice and incubated in 1 mL
of Hank’s basic salt solution containing 0.5 mg/mL
BSA, 22 μM-unlabeled palmitate
plus 5 μM [3H]-palmitate (32.4 mCi/μm)
for 2 hours. All of the reaction solutions were collected from each well,
and then 200 μL of 10%
trichloroacetic acid and 70 μL 6 N NaOH
were added in the solution.
Mixtures were then removed from each well and placed in corresponding
poly-prep chromatography columns with 1.5 mL
Dowex-1 overnight. The 3H2O that passed through the column was collected
into a scintillation vial, and radioactivity was measured with a liquid
scintillation counter. The protein level of each well was measured using
BCA-assay (Thermo Scientific Inc., Waltham, MA, USA) and was used to
normalize the palmitate oxidation data.
2.6. Western
Blotting
Primary antibodies
for total AMPK, phospho-AMPK (Thr172), total ACC (Acetyl-CoA Carboxylase),
and phospho-ACC (Ser79) were obtained from Cell Signaling Technology Inc.
(Danvers, MA, USA). Horseradish peroxidase- (HRP-)
conjugated goat anti-rabbit secondary antibody was obtained from Thermo
Scientific Inc. (Waltham, MA, USA).
Following the
indicated treatments, C2C12 myotubes were washed twice with ice cold PBS,
and the total cell lysates were prepared using RIPA buffer plus protease/phosphatase
inhibitor cocktails with 100 : 1 : 1
(v/v/v, ratio) (Sigma-Aldrich). Following a 10-minute centrifugation at
14,000 ×g, the supernatants were
collected for the determination of protein content using BCA assay kit
(Thermo Scientific Inc., Waltham, MA, USA) and western blotting. Equal
amounts of total cell lysates (20 μg)
were loaded to 10% SDS-PAGE (10 cm
× 10 cm, Criterion precast gel,
Bio-Rad Laboratories, Hercules, CA, USA) and transferred to PVDF membrane
(polyvinylidene difluoride membrane) (Bio-Rad, Hercules, CA). The membrane
was incubated in 25 mL blocking
buffer (1 TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk) for 1 hour at
room temperature. Then the membrane was incubated in TBST containing 5%
dry milk with primary antibody (1 : 1000)
with gentle agitation at 4°C overnight, washed three times with TBST, and
incubated with TBST containing rabbit HRP-conjugated secondary antibody (1 : 5000)
for 1 hour at RT. Bound antibodies were visualized by chemiluminescence (ECL
Western Blotting Substrate, Thermo Scientific) and membranes were exposed
to X-ray films (Phenix Research Product, Candler, NC) for protein band
detection. The films were scanned using an HP Scanjet 39070 (Palo Alto, CA 94304) and stored as tagged
image file format (TIFF) at 300 dpi.
The protein bands were quantified by densitometry using BioRad ChemiDoc
instrumentation and software of Image Lab 4.0 (Bio-Rad Laboratories).
2.7. Measurement
of Mitochondrial Contents
Mitochondrial
abundance in C2C12 myotubes was assessed by 10-N-nonyl acridine orange (NAO)
dye (Life Sciences, PA, USA) according to manufacturer’s instruction.
After desired treatment, cells in 96-well plates were treated with 10 M
NAO dye, following 2-hour incubation at 37°C in the dark. NAO is not
fluorescent, but it can be oxidized into the fluorescent-NAO by oxidative
species and accumulated in mitochondrial membrane. The absorbance in each
well was measured at 570 nm
wavelengths (Promega, WI, USA) and normalized with cellular
protein level. The image of mitochondria was taken using a Nikon Eclipse
Ti-E Ti-E Fluorescence microscope (Nikon Metrology, Inc., US) equipped
with an automated stage and a 20x objective. A 3 × 3 large image scan was
taken in each of 5 random fields by multichannel capture (channel 1:
excitation/emission = 488/517, channel 2: excitation/emission = 550/567 nm).
2.8. Statistical
Analysis
Data is presented
as means ± standard deviation (SD). Levene’s test was used to determine
homogeneity of variance among groups using SPSS 21.0 statistical software
(IBM, Armonk, NY) and where necessary natural log
transformation was performed before analysis. Multiple comparisons were
analyzed by one-way analysis of variance (ANOVA) using least significant
difference when equal variance was assumed, and Games-Howell test was used
when equal variance was not assumed. The independent sample t-test was
used to compare two conditions. Differences were considered statistically
significant at .
3. Results -3.1.
Leucine Treatment Induced the Mitochondrial Biogenesis in C2C12 Myotubes
Leucine significantly increased mitochondria content in C2C12s
compared to alanine and valine () (Figure 1(a)). These effects were
accompanied by increases in mRNA levels of PGC-1α (198%, ) and SIRT3
(167%, ) (Figure 1(b)). SIRT1 activity () and fatty acid oxidation () in
the C2C12 myotubes were significantly elevated by leucine compared to the
control groups (Figures 1(c) and 1(d), resp.).
fig1
Figure 1:
Leucine treatment induces mitochondrial
biogenesis and SIRT1 enzymatic activity in C2C12 myotubes. (a)
Mitochondrial content was quantitated with NAO dye (10 μM)
48 hours after treatment with leucine (0.5 mM),
alanine (0.5 mM), and valine
(0.5 mM); (b) mRNA expression
levels of PGC-1α and Sirt3 with the same treatments were evaluated by
quantitative RT-PCR. The relative mRNA expression was normalized to 18S
and expressed as dark bars for PGC-1α and grey bars for Sirt3. (c)
Cellular SIRT1 activity and (d) palmitate oxidation were measured after
treatment for 48 hours. The results were normalized to cellular protein
level for each sample. Data are mean ± SE (). Significantly different from
controls with .3.2. SIRT1 Is Required for Leucine-Induced Mitochondria
Biogenesis in C2C12 Myotubes--We used a selective SIRT1 inhibitor (EX527)
to determine the role of SIRT1 in leucine-induced mitochondrial
biogenesis. Leucine increased mitochondrial
biogenesis as demonstrated by significant increases in mitochondrial
content (), palmitate oxidation () and expression of mitochondrial
biogenesis-related gene markers PGC-1α(), SIRT3 (), and COX5b
() (Figures 2(a), 2(c) and 2(d), dark panel), and these effects were
markedly attenuated by EX527 administration (Figures 2(a), 2(c) and 2(d),
grey panel). Comparing the relative Sirt1 expression, leucine and
resveratrol (positive control) markedly increased Sirt1 mRNA level (); the
SIRT1 inhibitor (EX527) plus leucine treatment () revealed the same
pattern (Figure 2(b)).--fig2-Figure 2:
Leucine improves mitochondrial biogenesis in C2C12 myotubes in a
SIRT1-dependent manner. (a) Mitochondrial content was measured
using NAO (10 μM) dye after
48-hour leucine (dark bars), leucine plus SIRT1 inhibitor (EX527 25 μM;
grey bars) for 48 hours in C2C12 myotubes. (b, c) SIRT1 activity and
mitochondrial biogenesis- related genes (PGC-1α, Sirt3, and COX5b) mRNA
levels were measured after the same treatments. The relative SIRT1
activity was normalized to cellular protein level, and mRNA level was
normalized to housekeeper gene 18S. (b) Dark bars are DMSO control, grey
bars are EX527. (c) Dark bars are PGC-1α, grey bars are Sirt3; striped
bars are COX5b. (d) Palmitate oxidation level was detected after the same
treatment, and the results were normalized to cellular protein for each
sample. Data are mean ± SE (). Different letters indicate significant
differences within a given variable. Dark bars are DMSO control and grey
bars are EX527. Significantly different from controls, and significantly
different from control and EX527 groups with .
3.3.
Leucine Stimulates Phosphorylation of AMPK
in a SIRT1-Dependent Manner--Six hours of
leucine treatment resulted in a 3-fold increase in AMPK phosphorylation in
the C2C12 myotubes, which was significantly different from
baseline, valine, and alanine. Consistent with this observation,
phosphorylation of ACC, a downstream target enzyme of AMPK, was also
elevated by leucine compared to the controls () (Figure 3(a)), while EX527
treatment resulted in corresponding suppression of AMPK phosphorylation ()
(Figure 3(b)), indicating the necessity of SIRT1 for leucine-induced AMPK
activation.
fig3--Figure 3:
Leucine-induced phosphorylation of AMPK and ACC requires SIRT1 in C2C12
myotubes. (a) C2C12 myotubes were serum starved overnight and treated with
leucine (0.5 mM), alanine (0.5 mM),
valine (0.5 mM), and DMSO for 6
hours. The cell lysates were assessed by western blotting analysis with
specific antibodies against phosphor-AMPKα (Thr 172), phosphor-ACC (Ser
79), total AMPKα (Thr 172), and beta-actin. Integrated density values for
the p-AMPK and p-ACC were normalized to total-AMPK band density and
represented as dark or gray bars. (b) C2C12 myotubes were treated with
0.2% FBS medium overnight and then treated with leucine (0.5 mM),
resveratrol (100 nM), and
leucine plus EX527 (25 μM) for 6
hours. Whole cell lysates were prepared and detected by western blotting
with specific antibodies against phosphor-AMPKα, AMPKα, and beta-actin.
Integrated density value for phosphor-AMPK was normalized to total-AMPK.
Significantly different from controls with .--3.4. Leucine Stimulates
SIRT1 Activity, Phosphorylation of AMPK, and Cellular NAD+ in a
Time-Dependent Manner
To determine the
interplay between SIRT1 and AMPK, we measured the cellular NAD+ level,
SIRT1 activity, and phosphorylation of AMPK at time points: 0, 1, 4, 6,
12, and 24 hours by leucine treatment in C2C12 myotubes. Leucine increased
SIRT1 activity at 1 (), 12 () and 24 hours () compared to the baseline
(Figure 4(a)). However, no change was observed for the NAD+ content and p-AMPK
level during the first 4 hours. NAD+ level was elevated almost twofold
higher at time points 4 () and 24 hours () compared to baseline level and
otherwise remained low (Figure 4(b)); the levels of p-AMPK were markedly
increased and stayed high from 4 to 24 hours () (Figure 4(c)).
fig4
Figure 4: Leucine
stimulates SIRT1 activity, AMPK phosphorylation, and cellular NAD+ in a
time-dependent manner. C2C12 myotubes were serum starved overnight and
treated with leucine (0.5 mM).
Cell lysate was collected and analyzed for cellular SIRT1 activity,
western blotting of p-AMPK and cellular NAD+ levels at indicated certain
time points. (a) SIRT1 activity. (b) Cellular NAD+. Both SIRT1 activity
and NAD+ level were normalized to cellular protein for each sample. (c)
Phosphorylation level of AMPK was detected using western blotting
following the same time course in C2C12 cells, with resveratrol serving as
positive control. Data are mean ± SE (). Different letters indicate
significant differences between dark or gray bars. Significantly different
from point 0, and significantly different from time point 1.
3.5. Leucine-Induced
Mitochondrial Biogenesis in C2C12 Myotubes Requires AMPK
We next examined
whether AMPK also mediates leucine’s impacts on mitochondrial biogenesis
in C2C12 myotubes. As shown in Figure 5, leucine treatment markedly
increased the mitochondrial component genes expression (Figure 5(a), dark
columns), Hspd1 () and COX2 (). Similar effects were found for genes
encoding mitochondrial biogenesis regulatory proteins, [PGC-1α (), Sirt1
()] and component proteins [Cox5b ()], (Figure 5(b), dark columns), while
Compound C treatment markedly impaired all these inductions (Figure 5,
grey panels).
fig5
Figure 5: Leucine-induced
mitochondrial biogenesis in C2C12 myotubes requires AMPK. (a) C2C12
myotubes were treated with leucine (0.5 mM),
AICAR (20 μM), and Compound C
(25 μM) for 24 hours. mtDNA
levels of the cells were analyzed by the mitochondrial markers gene
expression, Hspd1 and COX2, using real-time PCR. (b) Sirt1 and
mitochondrial biogenesis related mRNA level of PGC-1α and COX5b were
evaluated also by RT-PCR after treating with leucine and Compound C for 24
hours was measured; all the mRNA levels were normalized to 18S
housekeeping gene. Data are mean ± SE (). Dark bars are vehicle control;
grey bars are Compound C. Significantly different from controls.
Significant Compound C effects.
4. Discussion--These
data indicate that leucine stimulates significant muscular metabolic
changes, including SIRT1 activation, AMPK phosphorylation, and
mitochondrial biogenesis in C2C12 myotubes. These changes may contribute
to leucine’s beneficial effects on energy metabolism and insulin
sensitivity in both animal and human models [17, 19, 27,
28].--A previous clinical trial has shown that high dairy intake (rich in
BCAAs) induces significant suppression of
reactive oxygen species (ROS) and inflammatory stress, indicated by
decreased plasma tumor necrosis factor alpha (TNF-α), interleukin 6
(IL-6), and monocyte chemoattractant protein-1 (MCP-1) levels [19].
Doubling leucine intake in mice has been
found to reverse multiple HFD-induced metabolic abnormalities, including
glucose intolerance, hepatic steatosis, and inflammation [11].
These effects are accompanied by corresponding increases in mitochondrial
oxidative capacity and mitochondrial content. Since mitochondrial
dysfunction and mitochondrial content loss are directly linked to the
development of metabolic disorders [1, 29],
increased mitochondrial biogenesis appears to rescue part of these
obesity-related abnormalities [30].--Consistent
with our previous studies [13], here we show that 0.5 mM
leucine treatment, which is comparable to the plasma leucine concentration
achieved by a leucine-rich diet [31], can markedly increase mitochondrial
content, mitochondrial biogenesis-related gene expression, and fatty acid
oxidation in C2C12 myotubes,
compared to valine and alanine.
The data herein
demonstrate that the improvement of fatty acid oxidation and mitochondrial
content by leucine is accompanied by increased SIRT1 activity in C2C12
cells. SIRT1 has been demonstrated to play significant roles in leucine’s
effects on energy metabolism. In Macotela’s study [11], leucine restores
HFD-reduced hepatic NAD+ and SIRT1 expression back to normal levels.
Similarly, Li et al. demonstrate that leucine increases SIRT1 expression
and decreases acetylation level of PGC-1α, resulting in attenuation of HFD-induced
mitochondrial dysfunction, insulin resistance, and obesity in mice [32].
Furthermore, Sun and Zemel found that leucine induces mitochondrial
biogenesis in muscle cells by stimulating the expression of PGC-1α and
NRF-1 via a SIRT1-dependent pathway [26]. These findings, along with the
observations reported here, are in agreement with our recent work that
leucine could activate SIRT1 enzyme through allosteric interaction in
adipocytes and myotubes [25].
To establish
whether or not SIRT1 is required for leucine-induced mitochondrial
biogenesis, EX527, a selective SIRT1 enzyme inhibitor, was used to treat
the cells in combination with leucine. EX527 significantly attenuated
leucine-induced mitochondrial content, mitochondrial biogenesis-related
genes expression, and fatty acid oxidation in C2C12 myotubes. The
observations reported here are consistent with Price’s work,in which SIRT1
knockout completely blocked resveratrol-induced mitochondrial biogenesis
and β-oxidation in skeletal muscle [33], further supporting the essential
roles of SIRT1. However, the leucine-induced Sirt1 gene expression was not
affected by EX527, possibly due to the unique inhibition mechanism of
EX527 on SIRT1 catalytic activity [34].
We also found that
AMPK phosphorylation, which is elevated in response to metabolic stress
[35], was also increased by leucine in C2C12 myotubes. This change might
help to explain the increased fatty acid oxidation in the cells.
Similarly, in mice, leucine supplementation has been reported to activate
AMPK synergistically with resveratrol and metformin, resulting in
increased insulin sensitivity and glucose tolerance [22]. On the other
hand, Compound C, an inhibitor of AMPK, markedly blocked leucine’s effects
on mitochondrial biogenesis, indicating that like SIRT1 elevated
mitochondrial biogenesis and fatty acid oxidation by leucine requires AMPK
in C2C12 myotubes.
Notably, we found
that leucine-induced AMPK phosphorylation was markedly blocked by EX527,
suggesting that AMPK might serve as a downstream target of SIRT1. In
support of this concept, Price et al. reported that SIRT1 activation is
required for AMPK phosphorylation and improvement of mitochondrial
function via deacetylation and activation of LKB1, a primary upstream
kinase of AMPK [33, 36], while Park et al. found that resveratrol
activates SIRT1 via an indirect pathway involving calmodulin-dependent
protein kinase kinase β (Camkkβ) and AMPK activation [36]. Currently
available evidences suggest that AMPK and SIRT1 display mutual
interactions with each other; AMPK could activate SIRT1 by increasing
cellular NAD+ level via promoting expression of nicotinamide
phosphoribosyltransferase (Nampt), a rate-limiting enzyme in NAD+
biosynthesis; however, SIRT1 can also directly deacetylate and activate
LKB1, resulting in the activation and phosphorylation of AMPK [37].
Our time-course
data suggest that SIRT1 may be the initial target of leucine. SIRT1
activity was increased within the first hour of leucine treatment, while
cellular NAD+ and p-AMPK levels remained unchanged. Considering that the
increased Sirt1 mRNA and SIRT1 activity level occurred at some time after
the leucine treatment for 24 hours, it is possible that SIRT1 activity is
elevated by leucine first, and then activation of AMPK is a subsequent
event, which may be responsible for the further SIRT1 activation at the
later time points.
Our data may also
reflect dose-dependent effects of leucine treatment. For example,
high-dose leucine infusion and supplementation have been shown to induce
insulin resistance and glucose intolerance in both human and animal models
[38, 39], possibly via activation of mammalian target of rapamycin- (mTOR-)
insulin receptor substrate 1 (IRS-1) signaling pathways [40]. In contrast,
modest increases in leucine intake, sufficient to induce plasma leucine
elevations to ~0.5 mM,
significantly reduced obesity-related oxidative and inflammatory stress,
resulting in improvement of insulin sensitivity in humans [19]. Similarly,
Vaughan et al. found that leucine in the 0.1–0.5 mM
range induces a dose-dependent increases of PGC-1α expression, leading to
significant elevated mitochondrial density and oxidative capacity in
skeletal muscle cells [17]. Consistent with these evidences, we found
comparable levels of leucine promoted mitochondrial biogenesis and fatty
acid oxidation in C2C12 myotubes.
There are several
limitations to this study. One of them is the use of the Fleur de Lys
assay to measure SIRT1 activity. Studies have challenged the validity of
the assay, as some of them have found that sirtuin-activating compounds (STACs)
only increased SIRT1 activity by using fluorophore-tagged substrates but
not the matching nontagged peptides, which also might explain why the
activation can be found exclusively in vitro but not in vivo [41, 42].
According to Gertz et al., the fluorophore can act synergistically with
STACs to promote binding between substrates and SIRT1 enzyme [43].
Furthermore, evidence suggests that resveratrol-induced SIRT1 activation
is actually mediated through an indirect signaling pathway involved in
cAMP phosphodiesterases
(PDE) and AMPK in vivo [36]. However, Hubbard et al. recently provided
more evidences to support the allosteric binding and activation theory
between STACs and SIRT1. They found that specific hydrophobic motifs in
SIRT1 substrates and a single amino acid (Glu230) in SIRT1 enzyme mediate
the structure change during the deacetylation [44]. As a highly
hydrophobic amino acid, leucine might directly activate SIRT1 through
conformation change. Indeed, recent evidence demonstrates that leucine
exerts direct effects on SIRT1 kinetics by decreasing 50% km for NAD+ and
substrates. With the presence of leucine and HMB, lower concentration of
resveratrol is required for the activation of SIRT1 [45]. Therefore,
further experiments using fluorophore-free substrates to measure the SIRT1
activity are needed to elucidate the exact pathways of leucine-activated
SIRT1. A second limitation is lack of data assessing the cellular
acetylation status of LKB1 and PGC-1α, as well as Nampt phosphorylation
and expression.
In summary, with
the present work, we demonstrate that leucine improves mitochondrial
biogenesis and fatty acid oxidation in C2C12 myotubes through SIRT1 and
AMPK-dependent pathway, with secondary activation of AMPK mediated by
SIRT1 (Figure 6).
239750.fig.006
Figure 6: Proposed
mechanism of leucine-induced mitochondrial biogenesis. In C2C12 myotubes,
leucine treatment leads to activation of SIRT1. SIRT1 then deacetylates
and activates LKB1, which subsequently induces AMPK phosphorylation and
activation. In turn, activated AMPK could promote SIRT1 activation via
intracellular NAD+ level by changing expression and activity of Nampt.
Activated AMPK and SIRT1 further activate PGC-1α via phosphorylation and
deacetylation, resulting in elevated mitochondrial biogenesis and
oxidative function.
Conflict of
Interests
The authors
declare that there is no conflict of interests regarding the publication
of this paper.
Acknowledgments
The authors thank
Drs. Ling Zhao and Antje Bruckbauer for technical support in cell culture
and SIRT1 activity measurement.
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