An Extract of Artemisia dracunculus L. Inhibits UbiquitinProteasome Activity and Preserves Skeletal Muscle Mass
in a Murine Model of Diabetes
Heather Kirk-Ballard1, Zhong Q. Wang2, Priyanka Acharya1,2, Xian H. Zhang2, Yongmei Yu2, Gail Kilroy1,
David Ribnicky3, William T. Cefalu2, Z. Elizabeth Floyd1*
1 Ubiquitin Biology Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana, United States of America, 2 Diabetes and Nutrition Laboratory,
Pennington Biomedical Research Center, Baton Rouge, Louisiana, United States of America, 3 Department of Plant Biology and Pathology, Rutgers University, New
Brunswick, New Jersey, United States of America
Abstract
Impaired insulin signaling is a key feature of type 2 diabetes and is associated with increased ubiquitin-proteasomedependent protein degradation in skeletal muscle. An extract of Artemisia dracunculus L. (termed PMI5011) improves insulin
action by increasing insulin signaling in skeletal muscle. We sought to determine if the effect of PMI5011 on insulin signaling
extends to regulation of the ubiquitin-proteasome system. C2C12 myotubes and the KK-Ay murine model of type 2 diabetes
were used to evaluate the effect of PMI5011 on steady-state levels of ubiquitylation, proteasome activity and expression of
Atrogin-1 and MuRF-1, muscle-specific ubiquitin ligases that are upregulated with impaired insulin signaling. Our results
show that PMI5011 inhibits proteasome activity and steady-state ubiquitylation levels in vitro and in vivo. The effect of
PMI5011 is mediated by PI3K/Akt signaling and correlates with decreased expression of Atrogin-1 and MuRF-1. Under in
vitro conditions of hormonal or fatty acid-induced insulin resistance, PMI5011 improves insulin signaling and reduces
Atrogin-1 and MuRF-1 protein levels. In the KK-Ay murine model of type 2 diabetes, skeletal muscle ubiquitylation and
proteasome activity is inhibited and Atrogin-1 and MuRF-1 expression is decreased by PMI5011. PMI5011-mediated changes
in the ubiquitin-proteasome system in vivo correlate with increased phosphorylation of Akt and FoxO3a and increased
myofiber size. The changes in Atrogin-1 and MuRF-1 expression, ubiquitin-proteasome activity and myofiber size modulated
by PMI5011 in the presence of insulin resistance indicate the botanical extract PMI5011 may have therapeutic potential in
the preservation of muscle mass in type 2 diabetes.
Citation: Kirk-Ballard H, Wang ZQ, Acharya P, Zhang XH, Yu Y, et al. (2013) An Extract of Artemisia dracunculus L. Inhibits Ubiquitin-Proteasome Activity and
Preserves Skeletal Muscle Mass in a Murine Model of Diabetes. PLoS ONE 8(2): e57112. doi:10.1371/journal.pone.0057112
Editor: Cedric Moro, INSERM/UMR 1048, France
Received November 12, 2012; Accepted January 17, 2013; Published February 20, 2013
Copyright: ß 2013 Kirk-Ballard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by pilot funding (ZEF and ZQW) from the Botanical Research Center of Pennington Biomedical Research Center and The
Biotech Center of Rutgers University, which is funded by the National Center for Complementary and Alternative Medicine and the Office of Dietary Supplements
P50AT002776-01, and the American Diabetes Association (grant 1-10-BS-55, awarded to ZEF). This project used Genomics Core and Cell Biology and Imaging Core
facilities that are supported in part by Centers of Biomedical Research Excellence (NIH P20-RR021945) and Nutrition Obesity Research Centers (NIH P30-DK072476)
center grants from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: elizabeth.floyd@pbrc.edu
associated with insulin resistance can lead to loss of skeletal muscle
mass and function [3]. A relationship between type 2 diabetes and
loss of skeletal muscle mass has been clearly demonstrated in older
adults, particularly in women with type 2 diabetes [4] and in
sarcopenic muscle loss [5]. Preservation of skeletal muscle mass
and strength in this high risk population may depend on strategies
designed to diminish the skeletal muscle protein degradation
associated with type 2 diabetes.
Protein degradation in skeletal muscle is carried out primarily
by the ubiquitin-proteasome system, a complex network of
enzymes through which multiple ubiquitin molecules are covalently attached to a protein substrate, leading to degradation of the
substrate by the 26S proteasome [6]. Various models of skeletal
muscle atrophy show striking increases in components of the
ubiquitin proteasome system, particularly the muscle-specific
ubiquitin ligases Muscle Atrophy F-box protein (MAFBx, also
called Atrogin-1) and Muscle Ring Finger-1 (MuRF-1) [7].
Introduction
Insulin resistance in clinical states of metabolic syndrome and
type 2 diabetes involves multiple tissues, including liver, adipose
tissue and skeletal muscle. Specifically, skeletal muscle is the largest
contributor to whole-body glucose disposal, making defective
insulin signaling in skeletal muscle a primary feature of type 2
diabetes. Along with its role as the primary site of glucose uptake,
skeletal muscle is also the main protein reservoir in the body.
Protein levels in skeletal muscle are determined by insulinmediated dual regulation of protein synthesis and protein
degradation [1]. Impairment of insulin-stimulated phosphoinositol
3-kinase/Akt signaling is suggested to tilt the balance between
protein synthesis and degradation toward protein degradation in
skeletal muscle [2], generating amino acids that are released from
skeletal muscle to meet whole body energy needs under catabolic
conditions. If prolonged, the accelerated protein degradation
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Botanical Regulation of Ubiquitin System in Muscle
Expression of Atrogin-1 and MuRF-1 [8,9] as well as proteasome
activity [10] is regulated by insulin in skeletal muscle via the
PI3Kinase/Akt signaling pathway. The essential role of Atrogin-1
and MuRF-1 in maintaining skeletal muscle mass [11,12,13]
makes these two muscle-specific ubiquitin ligases attractive targets
for pharmacological intervention in insulin resistance and type 2
diabetes.
Botanical extracts have historically been an important source of
medically beneficial compounds [14]. Metformin, one of the most
commonly used agents in the treatment of type 2 diabetes, was
synthesized based on the antihyperglycemic properties of the
French Lilac [15].
In this regard, recent studies show that an ethanolic extract of
Artemisia dracunculus L. (Russian tarragon), termed PMI5011,
improves carbohydrate metabolism in animal models of type 2
diabetes [16]. The changes in whole body glucose levels mediated
by PMI5011 correlate with increased insulin sensitivity in primary
human skeletal muscle cells [17] and in rodent models of type 2
diabetes [18]. PMI5011 enhanced insulin signaling in skeletal
muscle is associated with increased phosphatidylinositol 3-kinase
activity and Akt phosphorylation along with increased protein
content [18]. These results suggest that the effect of PMI5011 in
skeletal muscle extends to regulation of ubiquitin-proteasome
activity. If so, PMI5011 may be therapeutically useful in the
preservation of skeletal muscle mass in insulin resistance and type
2 diabetes.
The aim of this study was to further evaluate the mechanism of
action of PMI5011 by determining the effect of PMI5011 on the
ubiquitin-proteasome system in skeletal muscle. In particular, we
focused on the effect of PMI5011 on ubiquitin-proteasome activity
and the expression of Atrogin-1 and MuRF-1 in vitro and in vivo.
Sourcing and characterization of PMI5011 extract
The PMI5011 botanical extract from Artemisia dracunculus L. was
provided by the Botanical Research Center at Pennington
Biomedical Research Center. Detailed information about quality
control, preparation and biochemical characterization of
PMI5011 has been previously reported [14,16,17,18,19,20,21].
PMI5011 was obtained from plants grown hydroponically in
greenhouses under uniform and strictly controlled conditions,
thereby standardizing the plants for their phytochemical content.
The PMI5011 extract was dissolved in DMSO for the in vitro
experiments.
Cell culture
Murine C2C12 myoblasts (American Type Culture Collection;
Manassas, VA) were cultured in DMEM, high glucose (25 mM)
with 10% fetal bovine serum (FBS), 2 mM glutamine, and
antibiotics (100 units/ml penicillin G and 100 mg/ml streptomycin). While the myoblasts grew optimally in 25 mM glucose, the
glucose concentration was lowered to 5 mM prior to differentiation to minimize any effect of the hyperglycemic conditions on
insulin sensitivity of the myotubes. To obtain fully differentiated
myotubes, the media was exchanged for DMEM, low glucose
(5 mM) with 2% horse serum, glutamine, and penicillin G/
streptomycin when the myoblasts reached confluence. The media
was replaced every 48 hours and the cells were maintained in this
medium. The myotubes were fully formed by the fourth day postinduction.
Wortmannin treatment
C2C12 myotubes were preincubated with PMI5011 (10 mg/ml)
for 16 hours prior to the addition of wortmannin (200 nM). After
a 1 hour preincubation in the absence or presence of wortmannin,
insulin (100 nM) was added. Whole cell extracts were harvested
two hours thereafter for isolation of whole cell extracts. Inhibition
of PI3K/Akt signaling by wortmannin was confirmed by loss of
Akt phosphorylation.
Materials and Methods
Ethics statement
This study was carried out in strict adherence to the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The
animal studies were approved by the Institutional Animal Care
and Use Committee of Pennington Biomedical Research Center
(protocol number 695).
Free fatty acid treatment
Palmitic acid was diluted in ethanol (100 mM) and further
diluted to a 6 mM working solution in 2% fatty acid free Bovine
Serum Albumin (BSA) in DMEM. The 6 mM solution was
sonicated and incubated at 55uC until a clear solution was
observed. The resulting solution was diluted to the final
concentration and filter-sterilized. C2C12 myotubes were incubated in the absence or presence of palmitic acid (200 mM) and
PMI5011 (10 mg/ml) for 16 hours in the induction media.
Thereafter, the media was exchanged for DMEM containing
0.3% fatty acid free BSA for 6 hours prior to insulin stimulation
(100 nM insulin). Two hours after adding insulin, the cells were
harvested for isolation of RNA and whole cell extracts.
Materials
Dulbecco’s Modified Eagle’s Media (DMEM) was purchased
from MediaTech (Manassas, Va). Fetal bovine (FBS) and horse
serums were from Hyclone (Logan, UT). The AKT and phosphoAKT (Ser473) antibodies were purchased from Cell Signaling
(Danvers, MA), FoxO3a antibody from Millipore (Billerica, MA)
and the Atrogin-1 antibody was obtained from ECM Biosciences
(Versailles, KY). The MuRF-1 and phospho-FoxO3a antibodies
were obtained from Abcam (Cambridge, MA). The IRS-1, PI3K,
and 19SRPN2 antibodies were purchased from Upstate (Lake
Placid, NY); the ubiquitin antibody from BD Pharmingen (San
Diego, CA). All TaqMan primer/probes pairs were obtained from
Applied Biosystems (Carlsbad, CA). The 20S Proteasome Activity
Assay kit was purchased from Millipore (Billerica, MA) and
proteasome substrates were purchased from Boston Biochem
(Cambridge, MA) and Bachem (Torrance, CA). Wortmannin,
dexamethasone and palmitic acid were obtained from Sigma
Aldrich (St. Louis, MO). The Ultra-Sensitive Mouse Insulin
ELISA kit was obtained from Crystal Chem (Downers Grove, IL)
and the glucose assay kit was from Cayman Chemical (Ann Arbor,
MI)
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Dexamethasone treatment
C2C12 myotubes were incubated in the absence or presence of
dexamethasone (1 mM) and PMI5011 (10 mg/ml). When added,
PMI5011 was present for 4 hours prior to adding dexamethasone.
The cells were harvested for isolation of RNA and whole cell
extracts 24 hours after adding dexamethasone.
Animal studies
KK-Ay mice are a murine model of obesity-induced insulin
resistance and diabetes causes by mutation of the yellow obese
gene Ay [22] that was previously used to establish PMI5011
regulates insulin receptor signaling in skeletal muscle [18]. Six2
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Botanical Regulation of Ubiquitin System in Muscle
Figure 1. PMI5011 regulates expression of Atrogin-1 and MuRF-1 in skeletal muscle in a PI3K/Akt dependent manner. (A) C2C12
myotubes were incubated with the indicated concentrations of PMI5011 and whole cell extracts were harvested 16 hours thereafter. The levels of
IRS-1, pAkt, 19S proteasome subunit RPN2, Atrogin-1 and MuRF-1 were assayed by western blot analysis. The fold change in Atrogin-1 and MuRF-1
expression was analyzed from three separate experiments. The fold change in Atrogin-1 and MuRF-1 protein expression compared to expression in
the absence of PMI5011 was analyzed from three independent experiments. (B) C2C12 myotubes were preincubated with PMI5011 (10 mg/ml) for
16 hours as indicated prior to the addition of wortmannin (200 nM). After a 1 hour preincubation with wortmannin, insulin (100 nM) was added as
indicated. Whole cell extracts were harvested two hours thereafter and subjected to SDS-PAGE followed by western blot analysis. Inhibition of PI3K/
Akt signaling by wortmannin was confirmed by loss of Akt phosphorylation. The fold change in Atrogin-1 and MuRF-1 protein expression in the
presence of Wortmannin relative to the corresponding (2) wortmannin conditions was analyzed from three independent experiments. * p,0.05.
doi:10.1371/journal.pone.0057112.g001
Lilly, Indianapolis, IN) was administered to a subgroup of the
control and PMI5011 mice at a dose of 1.5 U/kg and tissue was
harvested after 90 minutes in order to assay potential changes in
gene expression while maintaining skeletal muscle in an insulinstimulated state.
week-old male KK-Ay mice (n = 16) (Jackson Laboratory; Bar
Harbor, ME) were single housed in animal rooms maintained at
25uC with a 12-h light–dark cycle. The mice were fed a low-fat
diet containing 16.4 kcal% protein, 10.5 kcal% fat, and
71.3 kcal% carbohydrate (D12329; Research Diets, Inc.; New
Brunswick, NJ). At 10 weeks of age, the mice were randomly
divided into a control group (n = 8) and a PMI5011-treated group
(n = 8). The control group was fed the low fat diet ad libitum and the
PMI5011 treatment group was fed ad libitum the low-fat diet
containing 1% (w/w) PMI5011. Body weight was recorded weekly
and food intake was monitored daily. Fasting glucose and insulin
levels were measured at 0, 4, and 8 weeks on the diets.
Histological analysis of skeletal muscle
A portion of the skeletal muscle was fixed in 10% formalin and
subjected to standard Hematoxylin and Eosin (H&E) staining. The
H&E stained myofibers were scanned (NanoZoomer Digital
Pathology, Hamamatsu Corp., Bridgewater, NJ) and the crosssectional area of the myofibers was calculated from a minimum of
fifty myofibers/animal using ImageJ (Research Services Branch,
NIH, rsbweb.nih.gov/ij/) software.
Blood glucose and insulin measurements
Serum glucose levels were measured by a colorimetric
hexokinase glucose assay and serum insulin levels were assayed
via ELISA, according to the manufacturers’ instructions. Skeletal
muscle (gastrocnemious) was harvested from mice that were fasted
for 4 hours prior to euthanasia. Human insulin (Humulin, Eli
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Proteasome activity assay
Proteasome activity in C2C12 cells and skeletal muscle was
measured according to the manufacturer’s instructions (28)
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Figure 2. PMI5011 regulates expression of Atrogin-1 and MuRF-1 in two models of insulin resistance in vitro. (A) C2C12 myotubes were
incubated in the absence (DMSO) or presence of PMI5011 at the indicated concentrations for 16 hours prior to the addition of dexamethasone
(1 mM). Twenty-four hour after adding dexamethasone, whole cell extracts were harvested and subjected to SDS-PAGE followed by western blot
analysis to determine phospho-Akt, total Akt, Atrogin-1 and MuRF-1 protein levels. b-actin is included as a loading control. (B) The fold change over
control for phospho-Akt, MuRF-1 and Atrogin-1 protein levels was analyzed from three independent experiments. * p,0.05 compared to control. (C)
The C2C12 myotubes were incubated in the absence (DMSO) or PMI5011 (10 mg/ml) for 16 hours prior to adding palmitic acid (200 mM) as indicated.
Twenty-four hours later, the cells were serum-deprived for 4 hours prior to insulin-stimulation (100 nM insulin) for 2 hours. Whole cell extracts were
harvested and subjected to SDS-PAGE followed by western blot analysis of phospho-Akt, total Akt, Atrogin-1 and MuRF-1 protein levels. (D) The fold
change over control for phospho-Akt, MuRF-1 and Atrogin-1 protein levels was analyzed from three independent experiments. * p,0.05,*** p,0.001
compared to control.
doi:10.1371/journal.pone.0057112.g002
Millipore (Billerica, MA). Briefly, the cell lysates were harvested in
50 mM Tris-Cl, pH 7.4 with 25 mM KCl, 2 mM MgCl2, 0.1%
Triton X-100, 2 mM ATP, 2 mM PMSF. MgATP is included in
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the lysis buffer to maintain 26S proteasome activity. Proteasome
activity was measured by incubating 20 mg of protein per sample
of each lysate at 37uC for 60 min. Chymotrypsin-like activity was
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Botanical Regulation of Ubiquitin System in Muscle
assayed with the 7-Amino-4-methylcoumarin (AMC) labeled
peptide substrate Suc-Leu-Leu-Val-Tyr-AMC, tryspin-like activity
was assayed using Ac-Arg-Leu-Arg-AMC and caspase-like activity
using Ac-Nle-Pro-Nle-Asp-AMC. The free AMC released by
proteasome activity was quantified using a 380/460 nm filter set
(Molecular Devices, Sunnyvale, CA). Proteasome activity is
reported as Relative Fluorescence Units (RFU)/mg protein/hr.
Each sample was measured in triplicate both in the presence and
in the absence of epoxomicin (20 mM, Boston Biochem), a highly
specific 26S proteasome inhibitor, to account for any nonproteasomal degradation of the substrate. Non-proteasomal
proteolysis is reported as the protease activity occurring in the
presence of epoxomicin.
Analysis of protein expression
Skeletal muscle tissue lysates were prepared by dissecting the
muscle free of adipose tissue and homogenizing in 25 mM
HEPES, pH 7.4, 1% Igepal CA630, 137 mM NaCl, 1 mM
PMSF, 10 mg/ml aprotinin, 1 mg/ml pepstatin, 5 mg/ml leupeptin
using a PRO 200 homogenizer (PRO Scientific, Inc., Oxford,
CT). The samples were centrifuged at 14,0006g for 20 min at
4uC. Whole cell extracts were harvested from the C2C12
myotubes in 50 mM Tris-Cl, pH 7.4 with 150 mM NaCl,
1 mM EDTA, 1% Igepal CA 630, 0.5% Na-deoxycholate, 0.1%
SDS, 10 mM N-EM and protease inhibitors, and lysed via
sonication. Protein concentrations were determined using a BCA
assay (Thermo Fisher Scientific, Rockford, IL) according to the
manufacturer’s instructions. The tissue supernatants (50 mg) and
C2C12 whole cell extracts (50 mg) were resolved by SDS-PAGE
and subjected to immunoblotting using chemiluminescence
detection (Thermo Fisher Scientific, Rockford, IL) and quantified
as described [23].
Analysis of gene expression
Total RNA was purified from the skeletal muscle tissue using an
RNeasy Fibrous Tissue Minikit (Qiagen, Valencia, CA). In each
case, RNA (200 ng) was reverse transcribed using Multiscribe
Reverse Transcriptase (Applied Biosystems, Carlsbad, CA) with
random primers at 37uC for 2 hour. Real-time PCR was
performed with TaqMan chemistry using the 7900 Real-Time
PCR system and universal cycling conditions (50uC for 2 minutes;
95uC for 10 minutes; 40 cycles of 95uC for 15 seconds and 60uC
for 1 minute; followed by 95uC for 15 seconds, 60uC for
15 seconds and 95uC for 15 seconds). The results were normalized
to Cyclophilin B mRNA or 18S rRNA levels.
Figure 3. PMI5011 enhances the effect of insulin on proteasome activity and inhibits ubiquitylation in skeletal muscle.
C2C12 myotubes were incubated with 10 mg/ml PMI5011 for 16 hours.
The cells were subsequently incubated with wortmannin (200 nM) for
1 hour prior to the addition of insulin (100 nM) for 2 hours as indicated.
(A) The cells were harvested and assayed for the chymotrysin-like
protease activity of the proteasome. Proteasome activity is reported as
Relative Fluorescence Units (RFU) RFU/mg protein/hr. The data are
reported as the mean 2/+ standard deviation from triplicate
measurements and are representative of three independent experiments. a = compared to control; b = compared to related treatment (2)
wortmannin; *p,0.05, **p,0.01 (B) Whole cell extracts were also
subjected to SDS-PAGE followed by western blot analysis using an antiubiquitin antibody to assay steady-state ubiquitylation levels. The data
are representative of three independent experiments.
doi:10.1371/journal.pone.0057112.g003
Statistical analysis
Normal distribution of the data for glucose and insulin levels,
food intake and body weight was determined using the
D’Agostino-Pearson omnibus K2 normality test. Statistical significance for body weight, glucose and insulin levels was determined
using two-way mixed model ANOVA with post hoc Bonferroni
correction. Statistical significance for all other data was determined using a two-tailed t test. All statistical analysis was carried
out using GraphPad Prism 5 software (GraphPad Software, La
Jolla, CA). Variability is expressed as the mean 2/+ standard
deviation.
ubiquitin ligases, we assayed Atrogin-1 and MuRF-1 protein
expression with increasing amounts of PMI5011 in C2C12
myotubes. As shown in Figure 1A, Atrogin-1 protein levels are
decreased at concentrations of PMI5011 corresponding to
maximal stimulation of Akt phosphorylation while MuRF-1
protein levels show a slight, but significant increase in the presence
of PMI5011. Atrogin-1 levels are significantly increased and IRS-1
expression reduced at 100 mg/ml PMI5011, suggesting the
beneficial effects of PMI5011 on insulin signaling are limited to
5–10 mg/ml PMI5011 in vitro. In contrast, PMI5011 has no effect
on the expression of RPN2, a proteasome subunit that is required
for funneling substrates into the 20S catalytic core of the 26S
proteasome [24].
Results
PMI5011 regulates expression of Atrogin-1 and MuRF-1
in C2C12 myotubes
To determine if the effect of PMI5011 on insulin signaling [18]
involves regulating the expression of the Atrogin-1 and MuRF-1
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Figure 4. PMI5011 supplementation improves insulin sensitivity in vivo. KK-Ay mice were singly housed and maintained on a low fat diet
(control, N = 8) or a low fat diet containing 1% PMI5011 (w/w) (PMI5011, N = 8) for two months. (A) Food intake was measured daily and (B) body
weight has measured each week. (C) Plasma insulin and (D) glucose levels were determined at baseline, 4 and 8 weeks. (E) The index of homeostasis
model assessment of insulin resistance [HOMA-IR; insulin (mU/L) x glucose (mM)/22.5] was calculated from fasting glucose and insulin levels.
doi:10.1371/journal.pone.0057112.g004
(Figure 2A, B) with the maximal effect of PMI5011 observed at
10–30 mg/ml. The PMI5011-mediated reduction in Atrogin-1 and
MuRF-1 levels coincides with PMI5011 stimulation of Akt
phosphorylation in the Dex-treated myotubes (Figure 2A, B).
The effect of PMI5011 on Atrogin-1 and MuRF-1
expression is mediated by phosphatidylinositol 3-kinase
(PI3K) activity
The PI3K/Akt signaling pathway regulates expression of
Atrogin-1 and MuRF-1 in skeletal muscle [25,26]. To determine
if PMI5011 regulates Atrogin-1 and MuRF-1 expression via PI3K
signaling, a set of experiments was carried out using wortmanninmediated inhibition of PI3K activity. Inhibition of PI3K activity
and Akt phosphorylation increased Atrogin-1 expression in the
presence of insulin, PMI5011 or insulin and PMI5011 combined
(Figure 1B). In contrast, inhibition of PI3K increased MuRF-1
expression only in the presence of insulin and PMI5011 combined
when the wortmannin treated MuRF-1 levels were compared to
the corresponding untreated samples.
PMI5011 enhances the effect of insulin on Atrogin-1 and
MuRF-1 expression in fatty acid-induced insulin
resistance
To determine if PMI5011 regulates Atrogin-1 and MuRF-1
expression in a different in vitro model of skeletal muscle insulin
resistance, we assayed the effect of PMI5011 on Atrogin-1 and
MuRF-1 expression in the presence of palmitic acid, a fatty acid
that inhibits insulin signaling in C2C12 myotubes [30], modeling
fatty acid induced insulin resistance. To confirm inhibition of
insulin signaling by palmitic acid, we assayed the effect of palmitic
acid on phosphorylation of Akt (Figure 2C, D). As expected, Akt
is phosphorylated in response to insulin and the extent of Akt
phosphorylation is increased by PMI5011. Palmitic acid inhibits
insulin-dependent Akt phosphorylation, but this effect is reversed
in the presence of insulin and PMI5011 combined, but not
PMI5011 alone. Insulin resistance induced by palmitic acid also
modestly increased Atrogin-1 protein expression, but this increase
was reversed by the addition of insulin and PMI5011 (Figure 2C,
D). MuRF-1 protein levels were substantially increased by palmitic
acid under all conditions. The palmitic acid-mediated increase in
MuRF-1 protein was significantly inhibited when both insulin and
PMI5011 were present, but not with the addition of insulin alone
(Figure 2C, D). Consistent with the results obtained with Dex
treatment, the increase in Atrogin-1 and MuRF-1 levels in the
PMI5011 regulates Atrogin-1 and MuRF-1 expression in
hormone-induced insulin resistance
Treatment of C2C12 myotubes with the synthetic glucocorticoid dexamethasone (Dex) induces atrogin-1 mRNA expression
along with other markers of muscle atrophy [27,28,29] and
inhibits Akt phosphorylation [27], providing an in vitro model of
hormone-induced insulin resistance and muscle atrophy to assess
the effects of PMI5011 in skeletal muscle with impaired insulin
signaling. Dex treatment increases Atrogin-1 and MuRF-1 protein
levels in the absence of PMI5011 (Figure 2A, see Dexamethasone,
0 mg/ml PMI5011) when compared to the level of each protein in
the absence of Dex or PMI5011 (control, 0 mg/ml PMI5011)
(Figure 2 A, B). However, the Dex-mediated increase in Atrogin1 and MuRF-1 protein expression is inhibited by PMI5011
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Figure 5. PMI5011 regulates proteasome and non-proteasome protease activity in skeletal muscle. At the end of the study, the KK-Ay
mice were fasted for 4 hours and insulin (1.5 U/kg) or an equal volume of sterile PBS was administered by intraperitoneal injection to a subgroup
(N = 4) of the control or PMI5011 supplemented (N = 4) mice. Skeletal muscle tissue (gastrocnemious) was harvested 90 minutes thereafter. (A) Gene
expression of two proteasome subunits, PSMA5 and PSMB3, was analyzed by realtime RT-PCR. (B) The chymotrypin-like, trypsin-like and caspase-like
26S proteasome activities were assayed in a buffer containing MgATP to maintain the 26S proteasome structure. (C) Non-proteasomal protease
activity was assayed as the chymotrypsin-like, trypsin-like or caspase-like activity measured in the presence of epoxomicin (20 mM), a highly specific
proteasome inhibitor. Proteasome and nonproteasome activities are reported as RFU/mg protein/hr. The data are reported as the mean 2/+ standard
deviation (4 animals/group). Statistical significance is compared to control. *p,0.05, ** p,0.01, ***p,0.001.
doi:10.1371/journal.pone.0057112.g005
presence of palmitate over the levels of each protein in the absence
of palmitate (control -insulin, - PMI5011) (Figure 2C,D) is reduced
by PMI5011 (Figure 2C,D). Maximal reductions in Atrogin-1 and
MuRF-1 expression in the presence of palmitate coincide with an
increase in Akt phosphorylation that is mediated by insulin and
PMI5011 combined (Figure 2C, D).
We anticipated the decrease in proteasome activity would
correlate with an increase in ubiquitylated proteins since the
degradation of ubiquitin-modified proteins would be impaired and
insulin-mediated changes in proteasome activity are accompanied
by accumulation of ubiquitin-conjugated proteins [10]. However,
we observed that steady-state levels of ubiquitylation are substantially inhibited in the presence of insulin and PMI5011 combined,
but not with PMI5011 alone or in the presence of insulin alone
(Figure 3B). The effects on ubiquitylation are abrogated in the
presence of wortmannin, indicating the changes in steady-state
levels of ubiquitylation observed in the presence of insulin and
PMI5011 require activation of PI3K.
PMI5011 enhances the effect of insulin on proteasome
activity and ubiquitylation in C2C12 myotubes
We next asked if PMI5011 altered the effect of insulin on
proteasome activity and steady-state levels of ubiquitylated
proteins in vitro. As shown in Figure 3A, 26S proteasome activity
is significantly decreased in C2C12 myotubes in the presence of
insulin or PMI5011 and insulin-mediated modulation of proteasome activity is enhanced by PMI5011. Inhibition of PI3K
signaling is associated with increased proteasome activity in the
presence of insulin or insulin and PMI5011 combined, indicating
PMI5011-mediated enhancement of the effect of insulin on
proteasome activity depends on PI3K activity.
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PMI5011 regulates ubiquitin-proteasome activity and
non-proteasomal protein degradation in skeletal muscle
in vivo
To determine if our results from the in vitro model of fatty acidinduced insulin resistance can be reproduced in an in vivo model of
insulin resistance, we carried out experiments using the KK-Ay
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Botanical Regulation of Ubiquitin System in Muscle
Figure 6. PMI5011 alters ubiquitin conjugation patterns in skeletal muscle. Steady-state ubiquitylation were measured in (A) control (N = 4)
and PMI5011 supplemented (N = 4) KK-Ay mice or (B) control (N = 4) and PMI5011 supplemented (N = 4) KK-Ay mice administered insulin (1.5 U/kg IP)
at the end of the study with tissue harvested 90 minutes thereafter. Whole cell extracts were subjected to SDS-PAGE followed by western blot
analysis using an anti-ubiquitin antibody. b-actin is included as a loading control. Statistical significance is compared to insulin-treated animals in (B).
*p,0.05.
doi:10.1371/journal.pone.0057112.g006
Loss of muscle protein in a rat model of diabetes (streptozotocininduced) is associated with increased expression of genes involved
in ubiquitin-proteasome-dependent degradation, including subunits of the 26S proteasome and Atrogin-1 and MuRF-1 [7]. We
assayed the effect of PMI5011 on the mRNA levels of two of the
26S proteasome subunits that are strongly upregulated with
muscle loss, the 20S proteasome subunits alpha 5 (PSMA5) and
beta 3 (PSMB3) [7]. As shown in Figure 5A, dietary supplementation with PMI5011 leads to a small, but significant decrease in
the gene expression of PSMA5, but not PSMB3. Although
decreased expression of PSMA5, with no change in PSMB3
expression suggests specificity in the effect of PMI5011 on
proteasome subunit gene expression, PMI5011 had no effect on
the gene expression of either proteasomal subunit in the insulinstimulated muscle. Moreover, the changes in the gene expression
of each subunit with acute insulin stimulation do not parallel the
changes in proteasome activity, suggesting insulin-mediated
regulation of the gene expression of these proteasome subunits
does not influence proteasome activity. To determine the effect of
PMI5011 on proteasome activity, we assayed the three types of
protease activity that constitute proteasome activity (Figure 5B):
chymotrypsin-like, trypsin-like, and caspase-like activity. PMI5011
model of obesity-related type 2 diabetes. Characterized by severe
hyperinsulinemia, hyperglycemia, and hypertriglyceridemia [22],
the KK-Ay mouse is one of several murine models that show
obesity is linked to the development of insulin resistance [22,31].
Obesity is also associated with an increase in free fatty acids that
leads to skeletal muscle insulin resistance [32] and recent evidence
indicates that diet-induced obesity leads to skeletal muscle atrophy
[33]. To determine if the changes in skeletal muscle protein
content reported in the KK-Ay murine model of diabetes [18] are
accompanied by changes in ubiquitin-proteasome system activity,
male KK-Ay mice were randomized to PMI5011 dietary
supplementation (N = 8 each group) and skeletal muscle was
obtained after twelve weeks. The animals treated with PMI5011
had a small, but significant increase in food intake that
corresponded with a slight, but significant increase in body weight
(Figure 4A, B). At the end of eight weeks, serum glucose levels for
the PMI5011-fed animals were significantly lower than the control
animals and serum insulin levels trended downward (Figure 4C,
D). These changes indicate improved glucose disposal and are
reflected in an improved Homeostatis Model of Assessment of
Insulin Resistance (HOMA-IR) (Figure 4E), a measure of insulin
sensitivity based on the glucose and insulin levels [34].
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Figure 7. PMI5011 regulates Atrogin-1 and MuRF-1 gene and protein expression in skeletal muscle. (A, B) Skeletal muscle from KK-Ay
mice was processed for whole cells extracts and analyzed using SDS-PAGE followed by western blot analysis of phospho-Akt, total Akt, MuRF-1,
Atrogin-1, phospho-FoxO3a and total FoxO3a. b-actin and quantitation of the total protein loaded via MemCode staining are included as loading
controls. Fold change for phospho-Akt/total Akt, phospho-FoxO3a/total FoxO3a, MuRF-1/total protein and Atrogin-1/total protein is reported for
PMI5011 relative to control (A) or PMI5011 combined with insulin relative to insulin alone (B). (C, D) Atrogin-1 and MuRF-1 gene expression was
determined using realtime RT-PCR. Results are reported as the mean 2/+ standard deviation (N = 4/group). * p,0.05, *** p,0.001. Significance is
reported relative to control in (C, D).
doi:10.1371/journal.pone.0057112.g007
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PMI5011 decreases Atrogin-1 and MuRF-1 expression in
skeletal muscle in vivo
substantially reduces chymotrypsin-like and caspase-like proteasome activity without affecting trypsin-like activity. Acute exposure
to insulin reduces all three activities, but is not more effective than
dietary supplementation with PMI5011 in reducing chymotrypin
and caspase-like proteasome activity. PMI5011-mediated changes
in non-proteasomal protein degradation mirror the changes
observed with chymotrypin and caspase-like proteasome activity
(Figure 5C). In addition, PMI5011 inhibits the trypsin-like
activity of non-proteasome proteases.
Although proteasome activity is decreased, steady-state levels of
high molecular weight ubiquitin conjugates (.75 kD) are significantly (p = 0.031) lower with PMI5011 supplementation compared to the untreated animals while ubiquitin conjugates near
50 kD accumulate with PMI5011, suggesting PMI5011 alters the
specificity of proteins modified by ubiquitin without changing the
overall level of ubiquitylation (Figure 6A). The overall levels of
ubiquitylation are lowered by PMI5011 dietary supplementation
when compared to insulin, and the pattern of ubiquitin conjugate
accumulation also changes (Figure 6B), further supporting the
notion that PMI5011 alters the specificity of ubiquitin conjugation
in skeletal muscle.
PMI5011 supplementation improves insulin signaling in skeletal
muscle when assayed as increased phosphorylation of Akt
(Figure 7A). In addition, PMI5011 enhances insulin-stimulated
Akt phosphorylation (Figure 7B). The changes in Akt phosphorylation with PMI5011 supplementation correlate with reduced
expression of Atrogin-1 and MuRF-1 proteins (Figure 7A) while
MuRF-1 protein levels are also reduced by PMI5011 when
compared to insulin (Figure 7B).
The FoxO1and FoxO3a members of the FoxO class of
forkhead transcription factors are downstream targets of Akt that
regulate the gene expression of atrogin-1 and MuRF-1 [25,27]. To
determine if the effect of PMI5011 on atrogin-1 and MuRF-1
protein expression is related to regulation of FoxO phosphorylation, we measured the levels of FoxO3a and phosphorylated
FoxO3a (serine 253) in the skeletal muscle. Dietary intake of
PMI5011 significantly increases phosphorylation of FoxO3a while
the total amount of FoxO3a is decreased compared to the
untreated animals (Figure 7A). The PMI5011-mediated increase
in FoxO3a phosphorylation corresponds to decreased atrogin-1 and
MuRF-1 expression (Figure 7C) when compared to the control
animals, consistent with a role for FoxO3a in the effect of
PMI5011 on atrogin-1 and MuRF-1 protein levels. Acute insulin
treatment does not increase FoxO3a phosphorylation or significantly decrease atrogin-1 and MuRF-1 expression in skeletal muscle
(Figure 7B, D) over that observed with PMI5011 supplementation, although MuRF-1 protein levels are decreased by PMI5011
in the insulin-stimulated muscle (Figure 7B).
Skeletal muscle myofiber size is larger in PMI5011
supplemented animals
Consistent with inhibition of ubiquitylation and proteasome and
non-proteasome activity, the cross-sectional area of myofibers
from the PMI5011 treated animals was significantly larger than
the myofibers from the control animal (Figure 8A, B), indicating
muscle mass is conserved in the presence of PMI5011.
Discussion
PMI5011 is a well-characterized botanical extract from A.
dracunculus L., whose effects on carbohydrate metabolism are
comparable to the ability of known antidiabetic drugs (troglitazone
and metformin) to lower glucose and insulin levels in murine
models of diabetes and insulin resistance [16]. Studies exploring
the mechanisms underlying the insulin sensitizing effects of
PMI5011 show that PMI5011 enhances insulin signaling in
skeletal muscle as demonstrated by increased PI3K activity,
increased Akt phosphorylation, and decreased activity of protein
tyrosine phosphatase 1B (PTP-1B), which serves as a negative
regulator of insulin signaling [18]. The current study provides
additional insight into the mechanism of action of PMI5011 in
skeletal muscle by demonstrating PMI5011-mediated regulation of
the ubiquitin-proteasome system.
Herein, we show PMI5011-enhanced insulin signaling specifically inhibits chymotrypsin-like and caspase-like proteasome
activity and all three non-proteasome protease activities, reduces
steady-state ubiquitylation levels, regulates expression of the
ubiquitin ligases, Atrogin-1 and MuRF-1 and enhances myofiber
size in insulin resistant skeletal muscle. In contrast to PMI5011mediated reductions in Atrogin-1 levels in vitro in the absence of
insulin resistance (Figure 1A), MuRF-1 levels are increased by
PMI5011. PMI5011-mediated decreases in MuRF-1 expression
are apparent only in the in vitro models of insulin resistance. The
Figure 8. Skeletal muscle myofiber size is larger with dietary
intake of PMI5011. (A) H&E staining of cross-section and longitudinal
section of gastrocnemious skeletal muscle from control and PMI5011
supplemented KK-Ay mice. (B) The cross-sectional area of fifty
myofibers/animal in each group was determined using ImageJ
software. The statistical significance is reported as the mean 2/+
standard deviation, p = 0.02.
doi:10.1371/journal.pone.0057112.g008
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Botanical Regulation of Ubiquitin System in Muscle
PMI5011-mediated change in MuRF-1 expression in vivo is
comparable to the effect of insulin on MuRF-1 expression.
Together, these results suggest MuRF-1 is the more relevant
PMI5011 target in the presence of insulin resistance.
PI3K/Akt signaling regulates Atrogin-1 and MuRF-1 protein
levels by inhibiting FoxO transcription factor-mediated induction
of atrogin-1 and MuRF-1 gene expression [25,26,27]. Akt-dependent phosphorylation of FoxO1 or FoxO3a excludes the FoxO
proteins from the nucleus, either via binding of the phosphorylated
FoxO protein to 14-3-3 proteins in the cytoplasm or degradation
of the phosphorylated FoxO protein by the proteasome [reviewed
in [35]]. The PMI5011-mediated increase in Akt phosphorylation
and FoxO3a phosphorylation, coupled with reduced atrogin-1 and
MuRF-1 gene expression, suggests PMI5011 mediated reductions
in Atrogin-1 and MuRF-1 protein expression are linked to
enhanced Akt-dependent regulation of FoxO3a transcriptional
activity.
The effects of PMI5011 on Atrogin-1 and MuRF-1 expression
are not as pronounced as the PMI5011-mediated inhibition of
proteasome and non-proteasome protease activity. As the major
site of protein breakdown, proteasome activity is upregulated in
muscle loss associated with insulin resistance [2,3,7]. PMI5011
substantially reduces the chymotrypsin and caspase-like protease
activities in the absence or presence of insulin stimulation. This
indicates PMI5011 action has the potential to broadly inhibit
protein degradation in insulin resistant skeletal muscle since the
proteasomal chymotrypsin-like activity is required for generalized
protein degradation, in conjunction with either the trypsin-like or
caspase-like activities [36]. However, the proteasome does not
degrade intact myofibrillar proteins, the primary group of proteins
targeted for breakdown in skeletal muscle atrophy [37,38].
Although the myofibrillar proteins are ultimately degraded by
the proteasome, the filament proteins must be dissociated from the
myofibrillar structure for recognition by the proteasome [39]. This
task is most likely accomplished by the calpains, calciumdependent proteases that interact with the ubiquitin-proteasome
system [38,40], although initial cleavage of the filament components may also be carried out by caspases [41]. PMI5011mediated inhibition of non-proteasome chymotrypsin-like and
caspase activities is consistent with an effect of PMI5011 on
calpain and caspase proteases activities, suggesting PMI5011 acts
to reduce degradation of the myofibrillar proteins by regulating
the activity of several classes of proteases.
A potential role for PMI5011 in preventing degradation of the
myofibrillar proteins in insulin resistance is further supported by
our results showing PMI5011 regulates expression of MuRF-1 in
the presence of insulin resistance in vitro and in vivo. MuRF-1
dependent ubiquitylation of skeletal muscle proteins accounts for
the majority of ubiquitin modification in muscle atrophy and
MuRF-1directly interacts with and regulates the ubiquitylation of
several myofibrillar proteins [42].
A role for MuRF-1 is well established in muscle loss due to
insulin resistance associated with fasting or catabolic disease states.
But insulin resistance also exacerbates muscle loss associated with
aging, termed sarcopenia [43,44,45,46]. In contrast to the rapid
muscle atrophy associated with fasting or catabolic diseases,
sarcopenic muscle loss occurs gradually and is worsened by obesity
[5]. Insulin resistance associated with obesity accelerates sarcopenia by suppressing protein synthesis and stimulating skeletal
muscle protein degradation [46], even in the absence of type 2
diabetes [47]. In turn, the loss of muscle mass in sarcopenic obesity
increases the risk of developing type 2 diabetes due to decreased
glucose disposal in skeletal muscle. There are indications that
sarcopenic muscle loss is mechanistically different from rapid
muscle loss [48,49], but enhanced proteasome activity remains a
common factor in both forms of muscle loss related to insulin
resistance [49]. PMI5011-mediated enhanced insulin signaling,
coupled with decreased MuRF-1 expression, decreased 26S
proteasome activity and larger myofiber size in the obesity-related
insulin resistant animals indicates PMI5011 has therapeutic
potential for preserving muscle mass in insulin resistant skeletal
muscle, including treatment of muscle loss due to sarcopenia.
Author Contributions
Conceived and designed the experiments: HKB ZQW WTC ZEF.
Performed the experiments: HKB ZQW PA XHZ YY GK DR ZEF.
Analyzed the data: HKB ZQW PA GK DR WTC ZEF. Contributed
reagents/materials/analysis tools: ZQW DR WTC ZEF. Wrote the paper:
HKB ZQW WTC ZEF.
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