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Hypoglycemic effect of catalpol on high-fat diet/streptozotocin-induced diabetic mice by increasing skeletal muscle mitochondrial biogenesis

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Catalpol, an iridoid glycoside, exists in the root of Radix Rehmanniae. Some studies have shown that catalpol has a remarkable hypoglycemic effect in the streptozotocin-induced diabetic model, but the underlying mechanism for this effect has not been fully elucidated. Because mitochondrial dysfunction plays a vital role in the pathology of diabetes and because improving mitochondrial function may offer a new approach for the treatment of diabetes, this study was designed. Catalpol was orally administered together with metformin to high-fat diet/streptozotocin (HFD/STZ)-induced diabetic mice daily for 4 weeks. Body weight (BW), fasting blood glucose (FBG) level, and glucose disposal (IPGTT) were measured during or after the treatment. The results showed a dose-dependent reduction of FBG level with no apparent changes in BW through four successive weeks of catalpol administration. Catalpol treatment substantially reduced serum total cholesterol and triglyceride levels in the diabetic mice. In addition, catalpol efficiently increased mitochondrial ATP production and reversed the decrease of mitochondrial membrane potential and mtDNA copy number in skeletal muscle tissue. Furthermore, catalpol (200 mg/kg) rescued mitochondrial ultrastructure in skeletal muscle, as detected with transmission electron microscopy. The relative mRNA level of peroxisome proliferator-activated receptor gamma co-activator 1 (PGC1) α was significantly decreased in muscle tissue of diabetic mice, while this effect was reversed by catalpol, resulting in a dose-dependent up-regulation. Taken together, we found that catalpol was capable of lowering FBG level via improving mitochondrial function in skeletal muscle of HFD/STZ-induced diabetic mice.
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Original Article
Hypoglycemic effect of catalpol on high-fat diet/streptozotocin-induced diabetic mice
by increasing skeletal muscle mitochondrial biogenesis
Xia Li1
, Zhimeng Xu1
, Zhenzhou Jiang1,2, Lixin Sun1,3, Jinzi Ji1, Jingshan Miao1, Xueji Zhang4, Xiaojie Li1,
Shan Huang1, Tao Wang1,3*, and Luyong Zhang1,5*
1
Jiangsu Center of Drug Screening, China Pharmaceutical University, Nanjing 210009, China
2
Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, Nanjing 210009, China
3
Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, China
4
Pharmacy Department, Shaanxi Cancer Hospital, Xi’an 710061, China
5
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
These authors contributed equally to this work.
*Correspondence address. Tel: þ86-25-83271043; Fax: þ86-25-83271142; E-mail: wangtao1331@126.com (T.W.)/lyzhang@cpu.edu.cn (L.Z.)
Catalpol, an iridoid glycoside, exists in the root of Radix
Rehmanniae. Some studies have shown that catalpol has a
remarkable hypoglycemic effect in the streptozotocin-
induced diabetic model, but the underlying mechanism
for this effect has not been fully elucidated. Because mito-
chondrial dysfunction plays a vital role in the pathology
of diabetes and because improving mitochondrial function
may offer a new approach for the treatment of diabetes,
this study was designed. Catalpol was orally administered
together with metformin to high-fat diet/streptozotocin
(HFD/STZ)-induced diabetic mice daily for 4 weeks. Body
weight (BW), fasting blood glucose (FBG) level, and
glucose disposal (IPGTT) were measured during or after
the treatment. The results showed a dose-dependent re-
duction of FBG level with no apparent changes in BW
through four successive weeks of catalpol administration.
Catalpol treatment substantially reduced serum total chol-
esterol and triglyceride levels in the diabetic mice. In add-
ition, catalpol efficiently increased mitochondrial ATP
production and reversed the decrease of mitochondrial
membrane potential and mtDNA copy number in skeletal
muscle tissue. Furthermore, catalpol (200 mg/kg) rescued
mitochondrial ultrastructure in skeletal muscle, as detected
with transmission electron microscopy. The relative mRNA
level of peroxisome proliferator-activated receptor gamma
co-activator 1 (PGC1) awas significantly decreased in
muscle tissue of diabetic mice, while this effect was reversed
by catalpol, resulting in a dose-dependent up-regulation.
Taken together, we found that catalpol was capable of
lowering FBG level via improving mitochondrial function
in skeletal muscle of HFD/STZ-induced diabetic mice.
Keywords catalpol; hypoglycemia; muscle; mitochondrial
biogenesis
Received: April 4, 2014 Accepted: June 3, 2014
Introduction
Currently, there is an increasing number of people suffering
from diabetic diseases resulting from a sedentary lifestyle,
the consumption of a high-caloric diet, obesity, a longer life
span, and others [1]. Diverse oral hypoglycemic drugs used
for the clinical treatment of diabetes have characteristic side-
effect profiles. However, the drugs used for the treatment of
diabetes have a number of limitations, such as adverse
effects and high rates of secondary failure. The Diabetes
Control and Complications Trial demonstrated that even the
optimal control of blood glucose could not improve these
complications, suggesting that alternative treatment strat-
egies are needed [2].
Mitochondria play a vital role in many cellular functions,
including ATP production, the biosynthesis of amino acids
and lipids, cytosolic calcium transport [3], and the amplifica-
tion of apoptotic stimuli [4]. Many diabetic patients have
metabolic disorders, such as lower glucose utilization, hyper-
lipidemia, and metabolic inflexibility which are related to
mitochondrial dysfunction. Meanwhile, mitochondrial dys-
functions, including mitochondrial loss and over-production
of oxidants, have been suggested to be involved in the devel-
opment of insulin resistance [5,6]. Therefore, the role of mito-
chondria in diabetes is of some concern. Biopsies of skeletal
muscle from subjects with type 2 diabetes (T2D) showed that
Acta Biochim Biophys Sin 2014, 46: 738 –748 | ªThe Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu065.
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citrate synthase and NADH-O
2
oxidoreductase activities were
decreased compared with those in lean controls [7]. Reduction
in subsarcolemmal mitochondria was confirmed with trans-
mission electron microscopy (TEM), which may contribute
to the pathogenesis of muscle insulin resistance in T2D [8].
Patients with insulin resistance or T2D also manifest decreased
mitochondrial density, oxidative activity, and mitochondrial
ATP synt hesis [9,10]. A study on STZ-diabetic rats showed a
persistent increase in reactive oxygen and nitrogen species
(ROS and RNS, respectively) production and a decrease in the
activities of the mitochondrial respiratory enzymes including
ubiquinol-cytochrome c oxidoreductase (complex III) and
cytochrome c oxidase (complex IV) [11]. Thus, increasing
mitochondrial mass and oxidative activity is viewed as a po-
tential therapeutic approach for the treatment of insulin resist-
ance and diabetes, and minimizes the complications of
diabetes or both [12].
Peroxisome proliferator-activated receptor gamma co-
activator 1 (PGC1) ais expressed in several tissues, espe-
cially in those with a high oxidative metabolism, such as
heart, skeletal muscle, kidney, brown fat, brain, and liver
[13]. Importantly, PGC1aregulates mitochondrial biogen-
esis and functions through interacting with co-activating
transcription factors, such as nuclear respiratory factors,
PPARs, thyroid hormone, glucocorticoid, and estrogen-
related ERRaand greceptors [14]. In patients with T2D,
muscle-biopsy studies showed that the expression of PGC1a
is reduced along with the occurrence of mitochondrial dys-
function. Similarly, PGC1a-knockdown mice were found to
have defective contractility of skeletal muscle [15]. All these
suggested that PGC1a-regulated mitochondrial biogenesis
may be a viable target for therapeutic intervention by prevent-
ing against mitochondrial dysfunction in diabetic patients.
Catalpol (Fig. 1) is an iridoid glycoside produced in the
root of Radix Rehmanniae. Some studies have shown a sub-
stantial hypoglycemic effect of catalpol in different diabetic
models [16,17]. Oral administration of catalpol resulted in
an increased expression of glucose transporter subtype 4
(GLUT4) in skeletal muscle of STZ-induced diabetic rats
[1618]. Catalpol can also ameliorate diabetic encephalop-
athy in rats [19]. In addition, catalpol has other pharmaco-
logical effects, such as neuron protection [20,21], against
H
2
O
2
-induced apoptosis [22], and anti-inflammatory proper-
ties [23,24]. Notably, intraperitoneal injection of catalpol
significantly reduced mitochondrial dysfunction, enhancing
complex I activity and mitochondrial membrane potential,
and decreased ROS generation in rotenone-induced mito-
chondrial damage and brain injury animal model [19,25,26].
In this study, catalpol was orally administered to high-fat diet/
streptozotocin (HFD/STZ)-induced diabetic mice, and it was
found that catalpol can reverse and increase muscle mitochon-
drial function and biogenesis partly by up-regulating mRNA
level of PGC1a. This study is the first report on the protective
effect of catalpol against HFD/STZ-induced muscle mito-
chondrial dysfunction through the up-regulation of PGC1ain
skeletal muscle.
Materials and Methods
Chemicals and reagents
STZ was purchased from Sangon Biotech Co., Ltd (Shanghai,
China); metformin (.99.8% purity) was made in Sino-
American Shanghai Squibb Co., Ltd (Shanghai, China);
glucose (Glu), total cholesterol (TC), and triglyceride (TG)
test kits were obtained from Whitman Biological Technology
Co., Ltd (Nanjing, China); EnlitenTM AT P assay kit f or ATP
concentration measurement was obtained from Promega
(Madison, USA); tissue mitochondria isolation kit, mitochon-
drial membrane potential (DC
m
) detection kit (JC-1 method),
and BCA protein assay kit were obtained from the Beyotime
Institute of Biotechnology (Haimen, China); catalpol (.95%
purity) was a gift from Qinghai Yangzong Pharmaceutical
Co., Ltd (Qinghai, China).
Experimental animals
C57BL/6J male mice (18 +4 g, 5 6 weeks old) were
obtained from the Medical Laboratory Animal Center of
Peking University (Beijing, China). Mice were housed five
per cage in environmentally controlled conditions main-
tained at a temperature of 22 +28C and a relative humidity
of 55% +5% with a 12 h light:12 h dark cycle (07 : 00 h
on, 19 : 00 h off ). Food and water were provided ad libitum,
except when noted otherwise before the experiments.
This study was approved by the Ethical Committee of
China Pharmaceutical University, and Laboratory Animal
Management Committee of Jiangsu Province (approval
No. 2110827). After having been adjusted to the laboratory
environment for 7 days prior to the experiments, mice were
randomly divided into six groups: Group I, control (n¼12);
Group II, HFD/STZ-model (diabetic) control (n¼12);
Group III, HFD/STZ-Met. 200 (n¼12); Group IV, HFD/
Figure 1. The chemical structure of catalpol
Hypoglycemic effect of catalpol on HFD/STZ-induced diabetic mice
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STZ-Cat. 50 (n¼12); Group V, HFD/STZ-Cat. 100 (n¼12);
Group VI, HFD/STZ-Cat. 200 (n¼12). All experimental
procedures were conducted in conformity with institutional
guidelines for the care and use of laboratory animals in
China.
Induction of diabetes in mice and drug administration
Diabetes was induced in mice by a single intraperitoneal injec-
tion of STZ at 85 mg/kg body weight (BW) dissolved in
freshly prepared 100 mM citrate buffer (pH 4.5). The STZ in-
jection was administered after 2 weeks of HFD (30% fat þ
70% standard chow; Nanjing Jiangning Qinglongshan Animal
Farms, Nanjing, China) feeding and immediately after a 16 h
fasting period. The normal controls were injected with citrate
buffer alone. During the trial, the HFD/STZ-injected mice
were orally given 5% Glu (2 ml/kg BW) 24 h after the injec-
tion to prevent initial STZ-induced hypoglycemic mortality,
and were then tested for fasting blood glucose (FBG) levels
48 h later, and those with FBG levels ranging from 12 to
18 mM were considered as HFD/STZ-diabetic mice.
The HFD/STZ-diabetic mice were orally gavaged each
day with 50, 100, or 200 mg/kg BW catalpol or 200 mg/kg
BW metformin (the doses were decided according to body
surface area method [27] compared with the doses in rats),
and both normal and diabetic control mice received no treat-
ment. All the control and experimental groups were then
gavaged with distilled water and provided with food and
water for 4 weeks. During the trial, individual BW and FBG
level were measured weekly.
At the termination of the study, mice were fasted and
then sacrificed. Blood sample was collected in a dry test
tube and allowed to coagulate at room temperature for
30 min. The blood samples were separated by centrifugation
at 2000 gfor 10 min and then used for the measurement of
serum TG and TC levels. After the mice were sacrificed,
muscle tissues were dissected, washed in ice-cold saline,
patted dry, and weighed. Muscle samples (100 mg) were
removed immediately from the same site of each mouse for
the isolation of the mitochondria. ATP content was then
detected, and mitochondrial membrane potential (DC
m
)was
measured. The same region of the gastrocnemius muscle
samples from normal control, diabetic control, and experi-
mental (200 mg/kg BW catalpol) groups were trimmed into
1mm
3
strips for mitochondrial ultrastructure examination
using TEM. The rest of the muscle tissue was harvested,
snap-frozen in liquid nitrogen, and stored at 2808C until
further analysis.
Intraperitoneal glucose tolerance test
After the last week of treatment, 0 min blood sample was im-
mediately taken from normal control, diabetic control, and
experimental mice after an overnight fasting. Without delay,
a glucose solution (2.5 g/kg BW) was administered
intraperitoneally. Blood samples were withdrawn from each
mouse at 30, 60, and 120 min interval. The blood samples
were used to estimate the level of glucose based on the man-
ufacturer’s instructions. The area under the curve (AUC)
was calculated according to the measurement results.
Skeletal muscle mitochondrial preparation
All skeletal muscle mitochondrial isolations were performed
at 48C. Approximately 100 mg of muscle tissue was dissected
from the animal and inserted in a tube placed on ice. Then,
the muscle tissue was cut into small pieces and homogenized.
Six different mitochondrial isolation preparations were made
for each experiment using the tissue mitochondria isolation
kit. Muscle mitochondrial protein concentrations were deter-
mined using the BCA protein assay kit.
Measurement of skeletal muscle mitochondrial
ATP content
The EnlitenTM ATP assay kit for ATP measurement was
used to measure muscle ATP content. The assay uses recom-
binant luciferase to catalyze the conversion of D-luciferin in
the presence of ATP and O
2
to oxyluciferin, PPi, AMP, CO
2
,
and light at the wavelength of 560 nm. As described above,
the isolated mitochondria were suspended in ATP-free water
and then vortexed freely. Then, ATP was measured accord-
ing to the manufacturer’s instruction. The samples were im-
mediately neutralized to pH 7.75 followed by a 2 s delay
after a 100 ml EnlitenTM rL/L reagent injection and a 10 s
RLU signal integration time. Light photons were measured
by a luminometer and compared with an ATP standard curve
to calculate ATP content. ATP content is expressed as mol/
mg protein for muscle samples. Muscle protein concentra-
tion was determined using the BCA protein assay kit.
Measurement of skeletal muscle mitochondrial
membrane potential (DC
m
)
JC-1 is an ideal fluorescent and cationic dye, and the
changes in JC-1 fluorescence is widely applied to analyze
DC
m
. According to the manufacturer’s manual, 100 mlof
purified mitochondria containing 100 mg of protein was
added in 96-well plates and then incubated with 900 mlof
JC-1 staining solution (5 mg/ml) at room temperature and
finally rinsed twice with JC-1 staining buffer. Changes in
JC-1 fluorescence intensity and mitochondrial JC-1 mono-
mers (
l
ex
514 nm,
l
em
529 nm) and aggregates (
l
ex
585 nm,
l
em
590 nm) were determined by an F-4500 fluorescence
spectrophotometer. Skeletal muscle mitochondrial DC
m
is
proportional to the fluorescence ratio of red (i.e. aggregates)
to green (i.e. monomers) and is expressed as OD
590
/OD
529
.
Hypoglycemic effect of catalpol on HFD/STZ-induced diabetic mice
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Determination of skeletal muscle mitochondrial DNA
copy number
A QIAamp DNA mini kit was used to extract DNA from
total muscle tissues. To quantify the amount of mitochon-
drial DNA (mtDNA) and nuclear DNA (nDNA), the real-
time quantitative polymerase chain reaction (PCR) was used
with EvaGreen fluorescent dye (Bio-Rad, Hercules, USA).
ND1 and b-actin were used to represent mtDNA and nDNA,
respectively. The following primers were used in this study:
ND1 sense primer 50-CAGCCTGACCCATAGCCATAATA
T-30; antisense primer 50-TGATTCTCCTTCTGTCAGGTC
GAA-30; nuclear
b
-actin sense primer, 50-GAGTGGTGC
TCTCTGAATAAGGTT-30, antisense primer, 50-GAAAGA
GGCAGTTCCATAGATGACA-30(Sunshine Biotechnology,
Nanjing, China). The results are expressed as the mtDNA
/nDNA ratio. The PCR was carried out in an iQTM5 Optical
System (Bio-Rad, Barcelona, Spain) based on the DCt
method. DNA (10 ng) and 1SsoFast EvaGreen Supermix
(10 ml) were mixed in a total volume of 20 ml and placed in
96-well plates. The PCR cycle included a hold for the
initial 30 s at 958Cfollowedby40cyclesof958Cfor5s
and 608C for 10 s. The threshold cycle number values of
mitochondrial and nuclear products were performed separ-
ately. The relative mtDNA content (mtDNA/nDNA) was
calculated with the following equation: relative copy
number (Rc) ¼2
DCt
, where DCt ¼Ct
b-actin
2Ct
mtDNA
[28]. Each melting temperature analysis was performed in
triplicate for each DNA sample.
Real-time PCR analysis of mRNA for PGC1agene
in skeletal muscle
Total RNA was prepared from muscle tissues using Trizol
reagent (Invitrogen, Carlsbad, USA). One microgram of
total RNA from each sample was reverse transcribed to
cDNA using PrimeScriptTM Reverse Transcriptase (Takara,
Osaka, Japan). The real-time PCR reaction mixture con-
tained 10 mlof1SsoFast EvaGreen Supermix, 1 mlof
cDNA, 7 ml of RNase/DNase-free water, and 500 nM of
each primer in a total volume of 20 ml. The thermal cycler
conditions were the same as those used in the previous
section. The relative mRNA levels of the target genes were
normalized to the level of the housekeeping gene, GAPDH,
and the iQTM5 Optical System based on the DCt method.
Primers used for PGC1aand GAPDH are as follows:
PGC1asense primer, 50-TTCTGGGTGGATTGAAGTGG
TG-30; PGC1aantisense primer, 50-TGTCAGTGCATCAA
ATGAGGGC-30; GAPDH sense primer, 50-GTCGTGGAT
CTAACGTGCC-30; and GAPDH antisense primer, 50-GAT
GCCTGCTTCACCACC-30(Sunshine Biotechnology). The
PCR results were confirmed at least three times for each
experiment.
Muscle mitochondrial ultrastructural analysis
with TEM
Muscle biopsies were trimmed into 1mm
3
strips and then
immediately fixed in 2.5% glutaraldehyde for at least 2 h, post-
fixed with 1% osmium tetroxide, dehydrated in a series of
graded alcohols, embedded in epoxy resin, and finally poly-
merized with a Leica automatic microwave electron micro-
scopic tissue processor at 608C for 24 h. Ultrathin sections
(7080 nm thick) were made on an RMC PowerTome XL
ultramicrotome (Butterfield Drive Tucson, Arizona, USA),
picked up by copper grids, stained with uranyl acetate and lead
citrate, and examined by a JEM-1010 transmission electron
microscopy (JEOL, Tokyo, Japan) at an accelerating voltage
of 80 kV. Ten digital photomicrographs were taken randomly
from each sample at 30,000 and 80,000 magnifications.
Statistical analysis
Data are presented as the mean +SEM for each group.
Statistical comparisons were performed using one-way ana-
lysis of variance (ANOVA) followed by Dunnett’s test.
Differences were considered significant when P,0.05.
Results
Effects of catalpol or metformin on BW
Figure 2shows that BW was significantly decreased in
HFD/STZ-induced diabetic mice compared with normal
controls (P,0.01). During the trial, no apparent changes
were observed in BW of the catalpol group compared with
diabetic mice (P.0.05). The administration of metformin
to diabetic mice resulted in significant increase in BW com-
pared with diabetic mice 3 weeks after treatment (P,0.05).
Effects of catalpol or metformin on FBG level
As shown in Table 1, the results demonstrated a dose-
dependent reduction of FBG levels during four successive
Figure 2. Effects of catalpol or metformin on BW in HFD/
STZ-induced mice Data were presented as mean +SE (n¼12).
One-way ANOVA was followed by Dunnett’s test. **P,0.01 vs. control
group;
#
P,0.05 vs. model group. Cat, catalpol; Met, metformin.
Hypoglycemic effect of catalpol on HFD/STZ-induced diabetic mice
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weeks of catalpol administration compared with the untreat-
ed diabetic mice (P,0.05). Notably, catalpol significantly
lowered FBG levels 2 weeks after treatment (Table 1).
Effects of catalpol or metformin on intraperitoneal
glucose tolerance test
As represented in Fig. 3A, the blood glucose levels in normal
mice peaked at 30 and 60 min after the glucose loading,
whereas there was a significantly higher glucose disposal rate
near baseline levels at 120 min. In the diabetic mice with
basal hyperglycemia, the hyperglycemia was exacerbated after
the glucose load at 30 and 60 min and did not return to base-
line levels after 120 min, indicating glucose intolerance and
impaired disposal. However, in the case of diabetic mice
treated with 200 mg/kg BW catalpol, there was a significantly
higher glucose disposal rate at 30, 60, and 120 min (P,0.01,
compared with the diabetic model group at any timepoint);
these disposal rates were similar to those of the metformin-
treated diabetic mice. The maximum rate of glucose disposal
was observed in mice dosed with 200 mg/kg BW catalpol
compared with the other two dosages. To evaluate the overall
glucose exposure, the AUC for intraperitoneal glucose toler-
ance test (IPGTT-AUC) was calculated, and a significant im-
provement in glucose exposure was noted in the diabetic mice
treated with 200 mg/kg catalpol compared with that for the
other two doses (Fig. 3B).
Effects of catalpol or metformin on serum TG and TC
Serum lipids (TG and TC) showed a significant increase in
diabetic mice (Fig. 4). The results demonstrated dose-
dependent reductions of both TG and TC levels by catalpol
treatment. Particularly, both catalpol (200 mg/kg BW) and
metformin (200 mg/kg BW) significantly (P,0.01) reduced
TG by 30% and 35% (Fig. 4A) and TC by 45% and 36%
(Fig. 4B), respectively, compared with the diabetic mice.
Table 1. Effects of catalpol or metformin on FBG level in HFD/STZ-induced mice
Group Dose (mg/kg) FBG (mM)
1 weeks 2 weeks 3 weeks 4 weeks
Control 3.57 +0.28 3.64 +0.23 3.63 +0.23 3.69 +0.23
Model 15.21+0.29** 15.50 +0.26** 15.87 +0.25** 15.89 +0.25**
Cat 50 15.02 +0.41 11.18 +0.40
#
10.85 +0.39
#
8.01 +0.41
##
100 14.46 +0.33 10.46 +0.35
#
9.3 +0.38
#
6.63 +0.40
##
200 14.37 +0.39 8.17 +0.41
##
6.54 +0.39
##
5.20 +0.40
##
Met 200 10.99 +0.34
#
7.15 +0.39
##
6.99 +0.35
##
6.32 +0.41
##
Data were presented as mean +SE (n¼12). One-way ANOVA was followed by Dunnett’s test.
**P,0.01 vs. normal control.
#
P,0.05.
##
P,0.01 vs. diabetic model group.
Cat, catalpol; Met, metformin.
Figure 3. Effects of catalpol or metformin on an IPGTT in HFD/STZ-induced mice (A) FBG levels at any timepoint for the IPGTT; (B) Area under
the curve for the IPGTT. Data were presented as mean +SE (n¼12). One-way ANOVA was followed by Dunnett’s test. **P,0.01 vs. control group;
##
P,0.01 vs. model group. Cat, catalpol; Met, metformin.
Hypoglycemic effect of catalpol on HFD/STZ-induced diabetic mice
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ATP content of isolated mitochondria in skeletal muscle
Figure 5shows that skeletal muscle mitochondrial ATP content
was reduced by 2.0 folds in the diabetic mice compared
with the normal control group (P,0.01), whereas this ef-
fect was reversed by the oral administration of catalpol in a
dose-dependent manner. Especially, the ATP production was
significantly increased by 3.0 and 2.5 folds after the admin-
istration of 200 and 100 mg/kg BW catalpol (P,0.01) when
compared with the diabetic model mice, respectively. However,
the administration of metformin showed no significant changes
compared with the diabetic model mice (P.0.05).
Mitochondrial membrane potential (DC
m
) levels
of isolated mitochondria in skeletal muscle
Figure 6shows that the DC
m
level of diabetic mice in skel-
etal muscle was reduced by 60% compared with the control
group (P,0.01), whereas this effect was reversed in a dose-
dependent manner after catalpol treatment. Especially, the
DC
m
level in skeletal muscle of diabetic mice was brought
back to near normal upon 200 mg/kg BW catalpol treatment
(Fig. 6). However, in the case of metformin administration,
there was no apparent change in the level of muscle DC
m
compared with the diabetic model mice (P.0.05).
mtDNA copy number normalized by nDNA
in skeletal muscle
Figure 7shows the copy number of mtDNA in skeletal
muscle of the control, diabetic model, and experimental mice.
The mtDNA copy number in the diabetic mice was decreased
by 30% compared with the control mice (P,0.05). There
was a significant increase in mtDNA upon catalpol treatment
Figure 4. Effects of catalpol or metformin on serum TG and TC of HFD/STZ-induced mice (A) Serum TG levels. (B) Serum TC levels. Data were
presented as mean +SE (n¼12). One-way ANOVA was followed by Dunnett’s test. **P,0.01 vs. control group;
#
P,0.05,
##
P,0.01 vs. model group.
Figure 5. ATP content of isolated mitochondria in the skeletal muscle of
HFD/STZ-induced mice Data were presented as mean +SE (n¼12).
One-way ANOVA was followed by Dunnett’s test. **P,0.01 vs. control
group;
#
P,0.05,
##
P,0.01 vs. model group; ATP, adenosine triphosphate.
Figure 6. Mitochondrial membrane potential levels of isolated mito-
chondria from the skeletal muscle of HFD/STZ-induced mice Data were
presented as mean +SE (n¼12). One-way ANOVA was followed
by Dunnett’s test. **P,0.01 vs. control group;
#
P,0.05,
##
P,0.01 vs.
model group.
Hypoglycemic effect of catalpol on HFD/STZ-induced diabetic mice
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in a dose-dependent manner compared with the diabetic
model group, especially for the 100 and 200 mg/kg BW catal-
pol groups (P,0.05). In contrast, no change was observed
in the 200 mg/kg BW metformin group compared with the
diabetic mice (P.0.05).
Mitochondrial ultrastructural analysis in skeletal
muscle with TEM
The mitochondrial ultrastructure in skeletal muscle is shown in
Fig. 8. In the control mice, the muscle mitochondrial ultra-
structure was within the normal range for size and contained
well-defined membrane and well-delineated cristae arrange-
ments (Fig. 8A,B). In contrast, the muscle mitochondrial ultra-
structure in the diabetic mice showed extensive mitochondrial
lesions, including swollen morphology, disrupted cristae ar-
rangement, and the loss of inner- and outer-membranes along
with a reduction in mitochondrial density (Fig. 8C,D). The
administration of 200 mg/kg BW catalpol rescued muscle
mitochondrial injury (Fig. 8E,F); specifically, these cells pos-
sessed intact sarcomeres and less swelling as well as an intact
mitochondrial membrane and well-organized cristae. The
ultrastructural images of the cells from the groups treated with
200 mg/kg BW catalpol were similar to that of the control
group (Fig. 8).
Relative mRNA level of PGC1ain skeletal muscle
Figure 9shows the relative mRNA level of the PGC1ain
skeletal muscle from the control and model mice. Real-time
PCR results showed that the mRNA level of PGC1awas
Figure 8. Mitochondrial ultrastructural analysis of the skeletal muscle in HFD/STZ-induced mice using TEM Representative images at 30,000 and
80,000 to compare the differences between the mitochondria of the control group (A,B), model group (C,D), and catalpol-treated (200 mg/kg) group (E,F).
Figure 7. mtDNA copy number in skeletal muscle on HFD/STZ-
induced mice (normalized to nDNA) Data were presented as mean +SE
(n¼12). One-way ANOVA was followed by Dunnett’s test. *P,0.05 vs.
control group;
#
P,0.05,
##
P,0.01 vs. model group.
Hypoglycemic effect of catalpol on HFD/STZ-induced diabetic mice
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decreased by 45% compared with the control (P,0.01).
Interestingly, we found that the mRNA level of PGC1ain the
diabetic mice was up-regulated by catalpol administration in a
dose-dependent manner. Particularly, the mRNA level of
PGC1awas increased by 1.2 folds in the skeletal muscle of
mice treated with 200 mg/kg BW catalpol compared with that
in the diabetic mice (P,0.001). There was no apparent
change as a result of metformin treatment (P.0.05).
Discussion
Nowadays, there is a multiple of literatures on whether mito-
chondrial dysfunction could play a relevant role in developing
insulin resistance and/or T2D, while the causal relationship
between them is still controversial. In this study, it is essential
for us to develop a suitable animal model of hyperglycemia,
insulin resistance, and mitochondrial dysfunction, resembling
the characteristics seen in human type 2 diabetes mellitus
(T2DM), to demonstrate the relationships between them and
to develop diabetic drugs. Single high-dose injection of STZ
could induce hypoinsulinemia, hyperglycemia, and mitochon-
drial dysfunction, resembling type 1 diabetes in human, but
setting this model was time-consuming [29 31]. In addition,
obesity, insulin resistance, and diabetes induced by HFD
alone did not lead to any impairment of mitochondrial per-
formance [3033]. In order to shorten the 3-month modeling
time, the combination of STZ injection and HFD has been
tried to develop the skeletal muscle mitochondrial dysfunction
and insulin resistance in diabetic mice, which took only 4
weeks [9,31].
In our study, the results showed that HFD/STZ diabetic
mice had a significant reduction in BW, and an obvious in-
crease in blood glucose, serum TG, and serum TC levels. No
apparent change was observed in BW upon treatment with
catalpol when compared with diabetic mice. The hypogly-
cemic action of this drug was observed in a dose-dependent
manner and was demonstrated by the higher rate of glucose
disposal (IPGTT). Using TEM, we observed that in the
catalpol-treated group (200 mg/kg BW), the skeletal muscle
mitochondrial morphology showed less damage, and the
mitochondrial density was increased. This phenomenon was
further supported by the observation that the mtDNA copy
number was increased compared with the diabetic mice.
Furthermore, the mitochondrial ATP content and the DC
m
level in the skeletal muscle were also reversed in a dose-
dependent manner by the catalpol supplementation.
Interestingly, the expression of PGC1a, which plays an essen-
tial role in improving muscle mitochondrial biogenesis and
function, in skeletal muscle was also significantly
up-regulated after four successive weeks of catalpol treatment,
which is consistent with the existing reports [14,34]. In single
STZ-treated mice, the levels of mitochondrial respiratory
chain complexes and mitochondrial function were markedly
reduced, resulting from the down-regulation of
PGC1a-mediated expression of mitochondrial genes [31].
Therefore, HFD/STZ-induced diabetic model is a useful
animal model for screening hypoglycemic drugs and studying
the corresponding mechanisms. Interestingly, mitochondrial
function in the skeletal muscle was also enhanced in the
control mice treated with catalpol (200 mg/kg BW) alone
(data not shown). On the basis of the current investigation, it
is strongly suggested that the hypoglycemic effect of catalpol
is mediated through the amelioration of muscle mitochondrial
dysfunction.
Mitochondria, which convert nutrients into energy through
cellular respiration, are the principal energy source of the cell
[35]. Mitochondrial adaptations (biogenesis and dynamics)
and functions largely affect muscle metabolism and have a
significant impact on whole-body metabolism [34]. Given
their essential function in aerobic metabolism, mitochondria
are intuitively of interest with regard to the pathophysiology
of diabetes [12]. Our results showed smaller mitochondrial
ultrastructures, reduced mitochondrial ultrastructure numbers
per unit volume (density), and significant reduction of mito-
chondrial ATP content and DC
m
levels in the skeletal muscle
of HFD/STZ-diabetic mice. Currently, T2DM is one of the
major diseases all over the world. Although lifestyle modifica-
tion is suggested as an initial therapy for T2DM, drug treat-
ment is also required in many cases. Metformin that plays a
role in glucose-lowering effect as implied by our present
study of lowered FBG and the IPGTT, is commonly used in
clinic to treat T2DM. There are various mechanisms for met-
formin that have been demonstrated, including inhibition of
complex I of the mitochondrial respiratory chain, decreasing
hepatic glucose production, increasing glucose uptake, and
stimulation of AMP-dependent protein kinase (AMPK) [36].
Figure 9. Relative mRNA levels of PGC1ain the skeletal muscle of
HFD/STZ-induced mice Data were presented as mean +SE (n¼12).
One-way ANOVA was followed by Dunnett’s test. **P,0.01 vs. control
group (n¼12);
#
P,0.05,
##
P,0.01 vs. model group.
Hypoglycemic effect of catalpol on HFD/STZ-induced diabetic mice
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Particularly, some literatures reported that treatment of T2DM
with metformin was associated with increased plasma ghrelin
concentrations [37]. Furthermore, in our study, BW gain in
the metformin-treated group was observed, which probably
was involved in increased plasma ghrelin concentrations,
resulting in the appetite stimulation. Thus, therapy targeting
mitochondria may provide a new way to treat diabetes. Our
results suggested that the treatment of diabetic mice with cat-
alpol significantly increased their aerobic capacity, as evi-
denced by their increased skeletal muscle ATP content.
Surprisingly, catalpol significantly lower FBG levels after 2
weeks. Furthermore, in the HFD/STZ diabetic model, blood
glucose is accompanied by an increase in serum TG and TC
levels, which were rescued to near normal with the treatment
of catalpol. The significant reduction in TC and TG levels
was presumably involved in increasing the fatty acid
b-oxidation by improved mitochondrial function, decreasing
the intracellular fatty acyl CoA and diacylglycerol, and pro-
moting to the uptake of glucose in skeletal muscle [9].
Overall, the possible mechanism for the relationship between
the glucose-lowering action and the anti-triglyceridemic
effect of catalpol could be correlated to ameliorating mito-
chondrial dysfunction and fatty acid b-oxidation in the skel-
etal muscle of diabetic mice.
PGC1ais a factor for most nuclear receptors and several
other transcription factors. Notably, PGC1ainduces and
coordinates gene expression; and it stimulates the thermogen-
ic program in brown fat, fiber-type switching in skeletal
muscle, and metabolic pathways linked to the fasting
response in the liver [38]. Notably, the increased PGC1a
expression in skeletal muscle results in the induction of a
wide array of genes involved in mitochondrial biogenesis and
function [3941]. Furthermore, the muscle-specific disrup-
tion of the PGC1agene in mice results in the decreased ex-
pression of mitochondrial genes, resulting in a switch from
oxidative fibers to more glycolytic fibers, and impairing their
endurance capacity [15]. So, catalpol may affect mitochon-
drial function through this transcriptional co-activator.
PGC1ais highly regulated at both the transcriptional and
post-translational levels, primarily through histone acetyl-
transferase GCN5-regulated acetylation [42] and NAD
þ
-
dependent deacetylase SIRT1-regulated acetylation [43].
The acetylation/deacetylation regulatory mechanism affects
PGC1aactivity, leading to increased mitochondrial biogen-
esis. When the balance between acetylation and deacetyla-
tion is altered from damage due to diabetes, PGC1a
acetylation, regulated by SIRT1, is at high levels, which is
consistent with its activity and mRNA levels at a low level,
resulting in mitochondrial dysfunction. This reversal of
balance could be significant for the treatment or prevention
of diabetes. In addition, it has been recently shown that
AMPK activation results in a net increase of NAD
þ
levels
with a consequent induction of SIRT1 activity or PGC1a
phosphorylation [44]. We hypothesize that the hypoglycemic
effect of catalpol was most likely due to the involvement of
the AMPK-SIRT1-PGC1a-regulated mitochondrial biogen-
esis signaling pathway in skeletal muscle. Meanwhile,
chronic metformin treatment for 14 days increased the
b-hydroxyacyl-CoA dehydrogenase activity, cytochrome c
protein content, and the PGC1acontent in the soleus
muscle, which suggest that metformin enhances the PGC1a
expression and mitochondrial biogenesis in the skeletal
muscle [45]. Therefore, we think that metformin may be a
right positive drug in our experiment. Furthermore, it has
been proposed that AMPK activation by metformin could
enhance mitochondrial biogenesis proteins including PGC1a
in cardiac ischemic cells [46,47]. However, no change was
observed on the PGC1amRNA expression levels of metfor-
min, so we suggest that PGC1ais presumably regulated at the
post-translational level primarily through AMPK phosphoryl-
ation. Studies in our laboratory showed that the effect of
metformin on HFD/STZ-induced diabetic mice in the skeletal
muscle mitochondrial function was not so significant;
however, this probably is an advantage of catalpol to play the
role in hypoglycemic effect.
Catalpol possesses a wide range of biological and pharma-
cological activity as described above. Whether oral adminis-
tration of catalpol has mitochondrial protective activity in
skeletal muscle to treat diabetes is still not clear. Our present
study showed that catalpol could ameliorate mitochondrial
dysfunction in the skeletal muscle of diabetic mice. The in-
crease of aerobic capacity and the decrease in blood glucose
levels might be related to the increase in the mRNA expres-
sion of PGC1ain skeletal muscle after catalpol treatment.
In summary, it has been indicated that stimulating PGC1a-
regulated mitochondrial biogenesis can improve mitochon-
drial function, decrease oxidative stress, and then ameliorate
insulin resistance [5,6,14,48]. Thus, catalpol, targeting mito-
chondria, provides hope for treating insulin resistance and/or
T2D.
Acknowledgement
We thank Qinghai Yangzong Pharmaceutical Co., Ltd for
generously providing catalpol as a gift.
Funding
This work was partially supported by grants from the 111
Project (111-2-07), 2011 Program for Excellent Scientific and
Technological Innovation Team of Jiangsu Higher Education
and National 12th Five-year Plan ‘Major Scientific and
Technological Special Project for Significant New Drugs
Creation’ project (No. 2012ZX09504001-001).
Hypoglycemic effect of catalpol on HFD/STZ-induced diabetic mice
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... The results of immunoblotting in the gastrocnemius of kl/kl mice suggests that NYT induced activation of 4E-BP1 in the protein synthesis system and regulated the pathway leading from FoxO1 to Atrogin1 in the protein degradation system. In addition, the ingredient or crude drug component of NYT that prevents skeletal muscle atrophy has been previously reported: extracts of Paeonia lactiflora, Citrus unshiu peel, and Glycyrrhiza radix prevent inflammation-induced muscle atrophy (Kim et al., 2016;Bae et al., 2020); Schisandra fruit, catalpol of Rehmanniae radix, and panaxatriol of Panax Ginseng stimulate muscle protein synthesis after exercise by activating PGC-1 (Kim et al., 2014;Li et al., 2014) or AKT/mTORC1 signaling pathway and muscle glucose disposal (Hou et al., 2015;Takamura et al., 2017); and atractylenolide III of Atractylodes rhizome reduce muscle wasting by attenuating disease-derived oxidative stress (Wang et al., 2019). These components may thus contribute to reduced muscle atrophy with aging. ...
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Iridoids, an important active ingredient, are widely distributed in varieties of Chinese herbal medicines and have varieties of pharmacological activities, such as anti-tumor, hypoglycemic, anti-inflammatory and so on, most of which exist in the form of glycosides in nature. However, its clinical application is limited by poor lipid solubility, low bioavailability and short half-life. It is necessary to optimize the structure of iridoids. It is hard to modify the hydroxyl groups at specific sites because iridoid glycosides are polyhydroxy compounds and very complex. In this paper, the words of ‘Iridoid glycosides’ and ‘Hydroxyl protection’ were used as the keywords, more than 200 articles from 1965 to 2021 were obtained from databases, such as CNKI, PubMed, Scifinder and so on. Finally, 60 articles were selected to summarize the hydroxyl protection of iridoid glycosides, which will provide a theoretical basis for their structural modification and stimulate their application potential in the field of drug research and development.
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Background Cognitive dysfunction is commonly observed in diabetic patients, yet, the underlying mechanisms are obscure and there are no approved drugs. Skeletal muscle is a key pathological organ in diabetes. Evidence is accumulating that skeletal muscle and brain communication are important for cognitive, and kynurenine (KYN) metabolism is one of the mediators. Purpose This study aims to elucidate the mechanism of diabetes-induced cognitive impairment (DCI) from the perspective of skeletal muscle and brain communication, and to explore the therapeutic effect of Zi Shen Wan Fang (ZSWF, a optimized prescription consists of Anemarrhenae Rhizoma (Anemarrhena asphodeloides Bge.), Phellodendri Chinensis Cortex (Phellodendron chinense Schneid.) and Cistanches Herba (Cistanche deserticola Y.C.Ma)), in order to provide new strategies for the prevention and treatment of DCI and preliminarily explore valuable drugs. Methods DCI was induced by intraperitoneal injection of streptozotocin (STZ) combined with a high-fat diet and treated with different dosage ZSWF extract by oral gavage for 8 weeks, once a day. Cognitive and skeletal muscle function was assessed, synaptic plasticity and L-type amino acid transporter (LAT1) was measured. KYN and its metabolites as well as metabolic enzymes in the hippocampus, peripheral blood and skeletal muscle were measured. Peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) and peroxisome proliferator-activated receptor α (PPARα) were measured in skeletal muscle. Results Compared with healthy mice, DCI mice not only showed decreased cognitive function and abnormal skeletal muscle function, but also showed imbalance of KYN metabolism in brain, circulating blood and skeletal muscle. Fortunately, ZSWF administration for 8 weeks notably attenuated the cognitive function, synaptic plasticity and skeletal muscle function in DCI mice. Besides, ZSWF significantly attenuated KYN metabolism in brain, circulation and skeletal muscle of DCI mice. Furthermore, ZSWF activated PGC1α-PPARα in skeletal muscle of DCI mice. Conclusions These results indicate that abnormal PGC1α-PPARα signaling in skeletal muscle mediating KYN metabolism disorder is one of the pathological mechanisms of DCI, and ZSWF can reverse diabetes-induced cognitive impairment via activating skeletal muscle PGC1α-PPARα signaling to maintain KYN metabolism homeostasis.
Article
Background Nonalcoholic fatty liver disease (NAFLD) is a kind of metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility, and there is no specific drug for its clinical treatment currently. In recent years, a large amount of literature has reported that many natural compounds extracted from traditional Chinese medicine (TCM) can improve NAFLD through various mechanisms. According to the latest reports, some emerging natural compounds have shown great potential to improve NAFLD but are seldom used clinically due to the lacking special research. Purpose This paper aims to summarize the molecular mechanisms of the potential natural compounds on improving NAFLD, thus providing a direction and basis for further research on the pathogenesis of NAFLD and the development of effective drugs for the prevention and treatment of NAFLD. Methods By searching various online databases, such as Web of Science, SciFinder, PubMed, and CNKI, NAFLD and these natural compounds were used as the keywords for detailed literature retrieval. Results The pathogenesis of NAFLD and the molecular mechanisms of the potential natural compounds on improving NAFLD have been reviewed. Conclusion Many natural compounds from traditional Chinese medicine have a good prospect in the treatment of NAFLD, which can serve as a direction for the development of anti-NAFLD drugs in the future.
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Aims Streptozotocin (STZ)-induced diabetic animal models have been widely used to study diabetic myopathy; however, non-specific cytotoxic effects of high-dose STZ have been discussed. The purpose of this study was to compare diabetic myopathy in a high-STZ model with another well-established STZ model with reduced cytotoxicity (high-fat diet (HFD) and low-dose STZ) and to identify mechanistic insights underlying diabetic myopathy in STZ models that can mimic perturbations observed in human patients with diabetic myopathy. Main methods Male C57BL6 mice were injected with a single high dose of STZ (180 mg/kg, High-STZ) or were given HFD plus low-dose STZ injection (STZ, 55 mg/kg/day, five consecutive days, HFD/STZ). We characterized diabetic myopathy by histological and immunochemical analyses and conducted gene expression analysis. Key findings The high-STZ model showed a significant reduction in tibialis anterior myofiber size along with decreased satellite cell content and downregulation of inflammation response and collagen gene expression. Interestingly, blood corticosteroid levels were significantly increased in the high-STZ model, which was possibly related to lowered inflammation response-related gene expression. Further analyses using the HFD/STZ model showed downregulation of gene expression related to mitochondrial functions accompanied by a significant decrease in ATP levels in the muscles. Significance The high-STZ model is suitable for studies regarding not only severe diabetic myopathy with excessive blood glucose but also negative impact of glucocorticoids on skeletal muscles. In contrast, the HFD/STZ model is characterized by higher immune responses and lower ATP production, which also reflects the pathologies observed in human diabetic patients.
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Type 2 diabetes is the most common type of diabetes and causes a decline in muscle quality. In this study, we investigated the effects of the root extract of Morinda officinalis (MORE) on skeletal muscle damage in mice with high-fat-diet (HFD)/streptozotocin (STZ)-induced diabetes and the expression of myogenic and biogenesis regulatory proteins in C2C12 myoblast differentiation. An in vivo model comprised C57BL/6N mice fed HFD for 8 weeks, followed by a single injection of STZ at 120 mg/kg. MORE was administered at 100 and 200 mg/kg once daily (p.o.) for 4 weeks. The changes in body weight, calorie intake, and serum levels of glucose, insulin, total cholesterol (TCHO), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), aspartate transaminase (AST), and alanine aminotransferase (ALT) were investigated in diabetic mice. The histological changes in the gastrocnemius muscle were observed by H&E staining, and then the myofiber size was measured. The expression of the myogenic (MHC, myogenin, and MyoD) and biogenesis (PGC-1α, SIRT1, NRF1, and TFAM) regulatory proteins was examined in the muscle tissues and differentiated C2C12 myoblasts by Western blot, respectively. The administration of MORE at 200 mg/kg in mice with HFD/STZ-induced diabetes significantly reduced weight gains, calorie intake, insulin resistance, and serum levels of glucose, TCHO, LDL-C, AST, and ALT. MORE administration at 100 and 200 mg/kg significantly increased serum insulin and HDL-C levels in diabetic mice. In addition, MORE significantly increased the expression of MHC, myogenin, MyoD, PGC-1α, SIRT1, NRF1, and TFAM in muscle tissues as well as increased the myofiber size in diabetic mice. In C2C12 myoblast differentiation, MORE treatment at 0.5, 1, and 2 mg/mL significantly increased the expression of myogenic and biogenesis regulatory proteins in a dose-dependent manner. MORE improves diabetes symptoms in mice with HFD/STZ-induced diabetes by improving muscle function. This suggests that MORE could be used to prevent or treat diabetes along with muscle disorders.
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Over the last century, the prevalence of type 2 diabetes has dramatically increased, reaching the status of epidemic. Because insulin resistance is considered the primary cause of type 2 diabetes, the identification of the cellular processes and gene networks that lead to an impairment of insulin action in target tissues is of crucial importance for the development of new drugs and therapeutic strategies to treat or prevent the disease. Numerous studies in humans and animal models have shown that insulin resistance is frequently associated to reduced mitochondrial mass or oxidative function in insulin sensitive tissues, leading to the hypothesis that defective overall mitochondrial activity could play a relevant role in the etiology of insulin resistance and, therefore, in type 2 diabetes. Although the causal relationship between mitochondrial dysfunction and insulin resistance is still controversial, numerous studies show that lifestyle or pharmacological interventions that improve insulin sensitivity are frequently associated to an increase in mitochondrial function and whole body energy expenditure. Therefore, increasing mitochondrial mass and oxidative activity is viewed as a potential therapeutic approach for the treatment of insulin resistance. Here, we review the current knowledge on the role of mitochondria in the pathogenesis of insulin resistance and discuss some of the potential therapeutic strategies and pharmacological targets for the treatment of insulin resistance based on the activation of mitochondrial biogenesis and the increase of mitochondrial oxidative function.
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Zhang W, Zhang X, Wang H, Guo X, Li H, Wang Y, Xu X, Tan L, Mashek MT, Zhang C, Chen Y, Mashek DG, Foretz M, Zhu C, Zhou H, Liu X, Viollet B, Wu C, Huo Y. AMP-activated protein kinase α1 protects against diet-induced insulin resistance and obesity. Diabetes 2012;61:3114–3125 The authors have formally requested to retract the above-titled article, which was published in the December 2012 print issue of Diabetes. The authors cite concerns that Figures 2F, 4G, and 6G included misgrouping errors that were not identified before the time of submission or publication. The authors apologize to the readers of Diabetes for any inconvenience this may have caused.
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AMP-activated protein kinase (AMPK) is an essential sensor of cellular energy status. Defects in the α2 catalytic subunit of AMPK (AMPKα1) are associated with metabolic syndrome. The current study investigated the role AMPKα1 in the pathogenesis of obesity and inflammation using male AMPKα1-deficent (AMPKα1(-/-)) mice and their wild-type (WT) littermates. After being fed a high-fat diet (HFD), global AMPKα1(-/-) mice gained more body weight and greater adiposity and exhibited systemic insulin resistance and metabolic dysfunction with increased severity in their adipose tissues compared with their WT littermates. Interestingly, upon HFD feeding, irradiated WT mice that received the bone marrow of AMPKα1(-/-) mice showed increased insulin resistance but not obesity, whereas irradiated AMPKα1(-/-) mice with WT bone marrow had a phenotype of metabolic dysregulation that was similar to that of global AMPKα1(-/-) mice. AMPKα1 deficiency in macrophages markedly increased the macrophage proinflammatory status. In addition, AMPKα1 knockdown enhanced adipocyte lipid accumulation and exacerbated the inflammatory response and insulin resistance. Together, these data show that AMPKα1 protects mice from diet-induced obesity and insulin resistance, demonstrating that AMPKα1 is a promising therapeutic target in the treatment of the metabolic syndrome.
Article
Here, we have investigated the effect of metformin pretreatment in the rat models of global cerebral ischemia. Cerebral ischemia which leads to brain dysfunction is one of the main causes of neurodegeneration and death worldwide. Metformin is used in clinical drug therapy protocols of diabetes. It is suggested that metformin protects cells under hypoxia and ischemia in non-neuronal contexts. Protective effects of metformin may be modulated via activating the AMP activated protein kinase (AMPK). Our results showed that induction of 30 min global cerebral I/R injury using 4-vesseles occlusion model led to significant cell death in the rat brain. Metformin pretreatment (200 mg kg/once/day, p.o., 2 weeks) attenuated apoptotic cell death and induced mitochondrial biogenesis proteins in the ischemic rats, analyzed using histological and Western blot assays. Besides, inhibition of AMPK by compound c showed that metformin resulted in apoptosis attenuation via AMPK activation. Interestingly, AMPK activation was also involved in the induction of mitochondrial biogenesis proteins using metformin, inhibition of AMPK by compound c reversed such effect, further supporting the role of AMPK upstream of mitochondrial biogenesis proteins. In summary, Metformin pretreatment is able to modulate mitochondrial biogenesis and apoptotic cell death pathways through AMPK activation in the context of global cerebral ischemia, conducting the outcome towards neuroprotection.
Article
Diabetes is a metabolic syndrome that results in chronically increased blood glucose (hyperglycaemia) due to defects either in insulin secretion consequent to the loss of beta cells in the pancreas (Type 1) or to loss of insulin sensitivity in target organs in the presence of normal insulin secretion (Type 2). Long term hyperglycaemia can lead to a number of serious health-threatening pathologies, or complications, especially in the kidney, heart, retina and peripheral nervous system. Here we summarize the current literature on the role of mitochondria in complications associated with diabetes, and the limitations and potential of rodent models to explore new modalities to limit complication severity. Prolonged hyperglycaemia results in perturbation of catabolic pathways and in an over-production of ROS by mitochondria, which in turn may play a role in the development of diabetic complications. Furthermore, current models don't offer a comprehensive recapitulation of these complications. The onset of complications associated with type 1 diabetes can be varied, even with tightly controlled blood glucose levels. The potential role of inherited, mild mitochondrial dysfunction in accelerating diabetic complications, both in Type 1 and 2 diabetes, remains unexplored.
Article
Purpose: To investigate the effect of catalpol on diabetic nephropathy in rats. Methods: Male Sprague-Dawley rats were randomly divided into two groups and fed with normal pallet diet (NPD) or high-fat diet (HFD) for 4 weeks respectively. Then the HFD-fed rats were injected with 35 mg/kg streptozotocin (STZ) for establishing diabetic model. The diabetic rats were randomly divided into five groups: model group, model plus catalpol 30, 60, 120 mg/kg groups and model plus metformin 200 mg/kg group. The NPD-fed rats were randomly divided into two groups: normal control group and normal plus catalpol 60 mg/kg control group. After administration for 10 weeks, random blood glucose (RBG), glycated serum protein (GSP), 24h urinary protein excretion (UPE), serum creatinine (Scr), blood urea nitrogen (BUN), and kidney weight index (KWI) were determined. The kidney pathological changes were evaluated by periodic acid-Schiff (PAS) staining. The concentrations of angiotensin II (Ang II), transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), fibronectin (FN), collagen type IV (Col IV) in renal cortex were determined. Real time RT-PCR was used to detect the mRNA expressions of TGF-β1 and CTGF. Results: Catalpol could significantly reduce the KWI, improve the kidney function and pathological change, decrease the tissue level of Ang II, TGF-β1, CTGF, FN, Col IV. Catalpol could also down regulate the mRNA expressions of TGF-β1 and CTGF in renal cortex. Conclusion: Catalpol may have beneficial effects against diabetic nephropathy. The mechanisms may be related to reducing the extracellular matrix accumulation by restraining the expression of TGF-β1, CTGF and Ang II.
Article
The present study was designed to examine the antihyperlipidaemic potential of iridoid glucoside isolated from Vitex negundo leaves in STZ-induced diabetic rats. The levels of cholesterol (TC), triglycerides, lipoproteins, free fatty acids, phospholipids, fatty acid composition, proinflammatory cytokines, muscle glycogen content, and glucose transporter 4 (GLUT4) expression were estimated in control and diabetic rats. Oral administration of iridoid glucoside at a dose of 50 mg/kg body weight per day to STZ-induced diabetic rats for a period of 30 days resulted in a significant reduction in plasma and tissue (liver and kidney) cholesterol, triglycerides, free fatty acids, and phospholipids. In addition, the decreased plasma levels of high-density lipoprotein-cholesterol and increased plasma levels of low density lipoprotein- and very low density lipoprotein-cholesterol in diabetic rats were restored to near normal levels following treatment with iridoid glucoside. The fatty acid composition of the liver and kidney was analyzed by gas chromatography. The altered fatty acid composition in the liver and kidney of diabetic rats was also restored upon treatment with iridoid glucoside. Moreover, the elevated plasma levels of proinflammatory cytokines and decreased levels of muscle glycogen and GLUT4 expression in the skeletal muscle of diabetic rats were reinstated to their normal levels via enhanced secretion of insulin from the remnant β cells of pancreas by the administration of iridoid glucoside. The effect produced by iridoid glucoside on various parameters was comparable with that of glibenclamide, a well-known antihyperglycemic drug.
Article
Purpose: Clinical and experimental investigations demonstrated that metformin, a widely used anti-diabetic drug, exhibits cardioprotective properties against myocardial infarction. Interestingly, metformin was previously shown to increase the expression of PGC-1α a key controller of energy metabolism in skeletal muscle, which is down-regulated in diabetic conditions. We hypothesized that chronic treatment with metformin could protect the aged, diabetic heart against ischemia-reperfusion injury (IRI) by up-regulating PGC-1α and improving the impaired functionality of diabetic mitochondria. Methods: Following 4 weeks of metformin (300 mg/kg) administered in the drinking water, 12 month-old diabetic Goto Kakizaki and non-diabetic Wistar rat hearts were assigned for infarct measurement following 35 min ischemia and 60 min reperfusion or for electron microscopy (EM) and Western blotting (WB) investigations. Results: Metformin elicited a cardioprotective effect in both non-diabetic and diabetic hearts. In contrast with the diabetic non-treated hearts, the diabetic hearts treated with metformin showed more organized and elongated mitochondria and demonstrated a significant increase in phosphorylated AMPK and in PGC-1α expression. Conclusions: In summary these results show for the first time that chronic metformin treatment augments myocardial resistance to ischemia-reperfusion injury, by an alternative mechanism in addition to the lowering of blood glucose. This consisted of a positive effect on mitochondrial structure possibly via a pathway involving AMPK activation and PGC-1α. Thus, metformin prescribed chronically to patients may lead to a basal state of cardioprotection thereby potentially limiting the occurrence of myocardial damage by cardiovascular events.