Diabetes Volume 64, January 2015
49
Kun Lian,1 Chaosheng Du,1 Yi Liu,1 Di Zhu,1 Wenjun Yan,1 Haifeng Zhang,2 Zhibo Hong,1 Peilin Liu,1,3
Lijian Zhang,1 Haifeng Pei,1 Jinglong Zhang,1 Chao Gao,1 Chao Xin,1 Hexiang Cheng,1 Lize Xiong,4 and
Ling Tao1
Impaired Adiponectin Signaling
Contributes to Disturbed
Catabolism of Branched-Chain
Amino Acids in Diabetic Mice
Diabetes 2015;64:49–59 | DOI: 10.2337/db14-0312
The branched-chain amino acids (BCAA) are essential
amino acids such as leucine, isoleucine, and valine; their
homeostasis is determined largely by catabolic activities in
a number of organs including liver, muscle and adipose
tissue (1–3). The first step of BCAA catabolism generates
a set of corresponding branched-chain a-keto acids
(BCKA), which are irreversibly decarboxylated by the
branched-chain a-keto acid dehydrogenase (BCKD) complex (4). As with most nutrients, maintaining of the physiological level of BCAA is critical for cell metabolism and
survival. However, many researchers have described increased BCAA and BCKA levels in diabetes and obesity
(3,5–8). Furthermore, BCAA and their catabolites are
strongly associated with insulin resistance (9–11), and
elevated BCAA contributes to the development of insulin
resistance (10,12). Mechanistically, elevated BCAA levels
activate mTOR/p70S6 kinase, resulting in an increased I
insulin receptor substrate-1 phosphorylation, thereby
inhibiting phosphatidylinositol 3-kinase. This inhibition
of phosphatidylinositol 3-kinase in turn leads to impaired
insulin signaling (13,14). It is also reported that BCAA are
independent predictors of insulin resistance, diabetes,
and cardiovascular events (15–17). Therefore, it is necessary to determine the mechanisms of abnormal BCAA
catabolism in order to better understand their association
with metabolic-related pathogenesis.
The BCKD complex is the rate-limiting enzyme in
BCAA catabolism (4,12); regulation of BCKD activity is
1Department of Cardiology, Xijing Hospital, Fourth Military Medical University,
Xi’an, China
2Experiment Teaching Center, Fourth Military Medical University, Xi’an, China
3Department of Cardiology, 306th Hospital of PLA, Beijing, China
4Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University,
Xi’an, China
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0312/-/DC1.
Corresponding author: Ling Tao, [email protected].
Received 22 February 2014 and accepted 9 July 2014.
K.L., C.D., and Y.L. contributed equally to this work.
© 2015 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
METABOLISM
The branched-chain amino acids (BCAA) accumulated
in type 2 diabetes are independent contributors to
insulin resistance. The activity of branched-chain a-keto
acid dehydrogenase (BCKD) complex, rate-limiting enzyme in BCAA catabolism, is reduced in diabetic states,
which contributes to elevated BCAA concentrations.
However, the mechanisms underlying decreased BCKD
activity remain poorly understood. Here, we demonstrate that mitochondrial phosphatase 2C (PP2Cm),
a newly identified BCKD phosphatase that increases
BCKD activity, was significantly downregulated in ob/ob
and type 2 diabetic mice. Interestingly, in adiponectin
(APN) knockout (APN2/2) mice fed with a high-fat diet
(HD), PP2Cm expression and BCKD activity were significantly decreased, whereas BCKD kinase (BDK), which
inhibits BCKD activity, was markedly increased. Concurrently, plasma BCAA and branched-chain a-keto acids
(BCKA) were significantly elevated. APN treatment markedly reverted PP2Cm, BDK, BCKD activity, and BCAA and
BCKA levels in HD-fed APN2/2 and diabetic animals.
Additionally, increased BCKD activity caused by APN
administration was partially but significantly inhibited in
PP2Cm knockout mice. Finally, APN-mediated upregulation of PP2Cm expression and BCKD activity were abolished when AMPK was inhibited. Collectively, we have
provided the first direct evidence that APN is a novel regulator of PP2Cm and systematic BCAA levels, suggesting
that targeting APN may be a pharmacological approach
to ameliorating BCAA catabolism in the diabetic state.
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Hypoadiponectinemia and Diabetic BCAA Catabolism
therefore important for maintaining the homeostasis of
systemic BCAA and BCKA. The complex consists of three
catalytic components: a heterotetrameric (a2b2) branchedchain a-keto acid decarboxylase (E1), a homo-24 meric
dihydrolipoyltransacylase (E2), and a homodimeric dihydrolipoamide dehydrogenase (E3). The activity of BCKD
complex is controlled by the reversible phosphorylation
of its E1a subunit (Ser293) by specific BCKD kinase (BDK)
and phosphatase (BDP), respectively (4,18). Phosphorylation catalyzed by BDK inhibits the enzymatic activity of the
BCKD complex, whereas it becomes activated when the
Ser293 residue is dephosphorylated by BDP. A series of
reports demonstrated that activation of BCKD was reduced
in liver and adipose tissue, resulting in increased plasma
BCAA and BCKA concentrations in diabetic and obese animals (6–8). Moreover, alterations of metabolism can influence BCKD activity partly through changes in BDK (3),
suggesting that BDP might be suppressed. The mitochondrial phosphatase 2C (PP2Cm) is the only identified BDP
(18,19), which specifically binds the BCKD complex and
induces dephosphorylation of BCKD at Ser293 in the presence of BCKD substrates (18). Additionally, PP2Cm deficiency impairs BCAA catabolism, leading to elevated plasma
BCAA and BCKA concentrations (18). However, the relationship between PP2Cm and reduced diabetic BCKD activity has not yet been investigated.
Adiponectin (APN) is an adipocytokine predominantly
synthesized in and secreted from adipose tissue. APN helps
regulating glucose and lipid metabolism (20,21) and has
vascular/cardioprotective effects (22,23). More recently,
Liu et al. (24) reported that APN corrects altered muscle
BCAA metabolism induced by a high-fat diet (HD). In addition, several observations have revealed that obesity and
type 2 diabetes are associated with decreased plasma APN
levels (20,25,26). However, the correlation between decreased APN levels and reduced BCKD activity has never
been investigated. More importantly, the underlying molecular mechanisms by which APN mediates disturbed
BCAA catabolism in diabetes are completely unknown.
In the current study, we used both in vitro and in vivo
experiments to identify hypoadiponectinemia as a contributing factor to reduced BCKD activity in diabetes. In
diabetic mice, BCKD activity was reduced, and BCAA and
BCKA levels were significantly elevated. APN treatment
effectively reversed these pathological alterations, which
were completely abolished by inhibition of AMPK and
partially but significantly attenuated by knockout of
PP2Cm expression. These findings lead us to conclude
that impaired APN signaling is an important part of the
underlying mechanism for disturbed BCAA catabolism in
type 2 diabetes.
RESEARCH DESIGN AND METHODS
Animal Care and Drug Treatment
All experiments were performed in adherence to the
National Institutes of Health Guidelines on the Use of
Laboratory Animals and were approved by the Fourth
Diabetes Volume 64, January 2015
Military Medical University Committee on Animal Care.
Male ob/ob and wild-type (WT) C57BL/6 control mice
were purchased from the Department of Pathology, the
Fourth Military Medical University. Male APN knockout
(APN2/2) mice (22) and WT C57BL/6 mice on the same
background have previously been described. The wholebody PP2Cm knockout (PP2Cm2/2) mice (18,27) were
a gift from Yibin Wang of University of California, Los
Angeles (Los Angeles, CA).
Mice were rendered type 2 diabetic by the following
procedures. Four-week-old WT C57BL/6 mice were fed
with an HD (60% of kcal from fat; Research Diets, New
Brunswick, NJ) for 6 weeks and injected with a low dose
of streptozotocin (STZ) twice (28) (25 mg/g body wt i.p.;
STZ in 0.05 mol/L sodium citrate, pH 4.5, once daily;
Sigma, St. Louis, MO). Blood glucose and body weight
were measured daily, and a diabetic condition was confirmed at 4 weeks after STZ injection by a nonfasting
blood glucose level of $200 mg/dL. Fasting blood insulin
was measured, and intraperitoneal glucose tolerance test
(IPGTT) and insulin tolerance test (ITT) were carried out
in each successful model group.
In some experiments, 7-week-old APN2/2 and WT mice
were randomized to receive a normal chow diet (ND) (12%
of kcal from fat; control) or HD (45% of kcal from lard;
Research Diets) for 4 weeks. Additionally, animals received
different treatment as follows: 1) ob/ob, HD-fed APN2/2 and
PP2Cm2/2 mice received vehicle (PBS) or APN (5 mg/g body
wt; PeproTech, Rocky Hill, NJ) and 2) type 2 diabetic mice
received vehicle, APN (5 mg/g body wt), AMPK activator
AICAR (150 mg/g body wt; Sigma, St. Louis, MO), or APN
in conjunction with the AMPK inhibitor compound C (CC)
(20 mg/g body wt; Sigma) once daily for an additional
3 days. After 12 h of fasting, animals were killed for collection of tissues (liver, epididymal fat pad, and gastrocnemius
muscle) and 0.5 mL aortic blood at 10–12 weeks of age.
Cell Culture and BCKA Challenge
AML12 mouse hepatocytes (American Type Culture Collection, Manassas, VA) were cultured in DMEM medium
(Invitrogen, Carlsbad, CA) containing 10% fetal bovine
plasma (HyClone, Waltham, MA). Experiments were carried out at three or four cell passages. The hepatocytes
were transfected by small interfering RNA (siRNA) and
incubated in DMEM medium (Invitrogen) with an additional mixture of all of the BCKA (Sigma) at 2.5 mmol/L.
Each set of cells was randomized to receive treatment with
10 mg/mL APN or control vehicle. After 1 h of treatment,
cells and supernatants were collected for Western blotting
and BCKA analysis.
RNA Interference
siRNA constructs against AMPK a1 or AMPK a2 mRNA
were designed and purchased from Gene Pharma (Shanghai, China). The siRNA sequences are as follows:
AMPKa1, sense 59-GCCGACCCAAUGAUAUCAUTT-39, antisense 59-AUGAUAUCAUUGGGUCGGCTT-39; AMPKa2,
sense 59-GGACAGGGAAGCCUUAAAUTT-39, antisense
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59-AUUUAAGGCUUCCCUGUCCTT-39; and scrambled
siRNA, sense 59-UUCUCCGAACGUGUCACGUTT-39, antisense 59-ACGUGACACGUUCGGAGAATT-39.
Mouse hepatocytes were transfected with siRNA by using
Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. Efficiency of gene knockdown was confirmed using Western blotting 48 h after siRNA transfection.
BCAA and BCKA Analysis
Plasma and supernatant of cultured cells were collected and
subsequently stored at 280°C. Determination of BCAA
concentrations was performed in triplicate using a commercially available BCAA detection kit (Biovision, Milpitas, CA)
per the manufacturer’s instructions. BCKA concentrations
were determined by HPLC as described by Loï et al. (29).
BCKD Enzyme Activity Assays
Tissue extraction and assessment of BCKD activity were
performed as previously described (30). BCKD complex
was concentrated from whole tissue extracts using 9%
polyethylene glycol. BCKD activity was determined spectrophotometrically by measuring the rate of NADH production resulting from the conversion of a-keto-isovalerate
to isobutyryl-CoA. A unit of enzyme activity was defined as
1 mmol NADH formed per minute at 30°C.
Western Blot Analysis
Proteins were separated on SDS-PAGE gels, transferred to
polyvinylidene fluoride membranes (Millipore, Billerica,
MA), and incubated overnight at 4°C with antibodies
directed against AMPKa (1:1,000; CST, Danvers, MA),
AMPKa1 (1:1,000; CST), AMPKa2 (1:1,000; CST),
phospho-AMPKa (Thr172, 1:1,000; CST), PP2Cm (1:1,000;
a gift from Yibin Wang, University of California, Los
Angeles), BDK (1:2,000; Abcam, Cambridge, MA), phosphoBCKD E1a (1:1,000; Bethyl Laboratories, Montgomery, TX),
BCKD E1a (1:500; Santa Cruz Biotechnology, Dallas, TX),
and GAPDH (1:5,000; Zhong Shan Golden Bridge Biotechnology, Beijing, China). After washing to remove excess primary antibody, blots were incubated for 1 h with horseradish
peroxidase–conjugated secondary antibody. Binding was
detected via enhanced chemiluminescence (Millipore).
Films were scanned with ChemiDocXRS (Bio-Rad Laboratory, Hercules, CA). Densitometry was performed using
Laboratory Image software.
Statistical Analysis
All data are presented as means 6 SEM of n independent
experiments. All data (except densitometry) was subjected
to ANOVA followed by a Bonferroni correction for a post
hoc t test. Densitometry was analyzed using the KruskalWallis test, followed by a Dunn post hoc test. Probabilities
of #0.05 were considered statistically significant.
RESULTS
PP2Cm Is Downregulated in ob/ob and Type 2 Diabetic
Mice
Consistent with previous reports, plasma BCAA and BCKA
levels were significantly increased in both ob/ob and type
2 diabetic mice compared with WT mice (Fig. 1A and B).
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Liver is the primary metabolic clearing house of BCKA;
the reduction of BCKD activity causes increased circulating BCAA and BCKA concentrations (3). In addition, immunoreactivity of the BCKD pSer293 antibody is directly
correlated with BCKD activity (18); we therefore examined the activity of BCKD and phosphorylation of BCKD
at Ser293 in liver. We found that BCKD activity was significantly reduced and phosphorylation of BCKD was significantly increased in diabetic animals compared with
WT mice (Fig. 1C–E and H). Interestingly, the newly identified BCKD phosphatase, PP2Cm, was markedly decreased in diabetic mice (Fig. 1F and H), indicating that
downregulation of PP2Cm may be involved in reduced
BCKD activity in diabetes.
Considerable evidence indicates that besides liver,
adipose tissue, and skeletal muscle also play an important
role in modulating circulating BCAA homeostasis (1,2);
thus, the following experiments were performed. As
shown in Fig. 1, BCKD activity was markedly downregulated in diabetic adipose tissue and skeletal muscle (Fig.
1C–E and H). Additionally, PP2Cm protein levels were
significantly reduced in diabetic adipose tissue (Fig. 1F
and H). To our surprise, changes of PP2Cm expression
in skeletal muscle were not observed in diabetic animals
(Fig. 1F and H), indicating that skeletal muscle may have
a relatively low level of PP2Cm-dependent BCKD activation. Since the activity of BCKD complex is increased by
PP2Cm and inhibited by BDK, we than analyzed expression of BDK. Our experimental results demonstrated that
BDK expression was significantly increased in diabetic
tissues, including liver and adipose tissue as well as skeletal muscle (Fig. 1G and H). These results indicate that
decreased PP2Cm and increased BDK may both contribute
to reduced BCKD activity in diabetic liver and adipose
tissue, whereas increased BDK may be the primary cause
of decreased BCKD activity in diabetic skeletal muscle.
APN Deficiency Contributes to Decreased BCKD
Activity
It is known that APN reverts altered BCAA metabolism in
muscle (24). In order to determine whether APN contributes to BCKD activity, 7-week-old WT and APN2/2 mice
were fed with either ND or 45% HD for a further 4 weeks,
resulting in four subgroups: ND WT, HD WT, ND APN2/2,
and HD APN2/2. As shown in Fig. 2, APN2/2 mice
revealed the significant increase of plasma BCAA, BCKA,
and hepatic BCKD phosphorylation levels but reduction of
BCKD activity in response to HD. No response was observed in WT mice with the same genetic background (Fig.
2A–E and Supplementary Fig. 2). More importantly, hepatic PP2Cm was markedly decreased in HD-fed APN2/2
mice (Fig. 2F and Supplementary Fig. 2). We then sought
to determine whether treatment with APN could significantly reverse altered BCKD activity. HD-fed APN2/2
mice were given recombinant APN at 5 mg/g body wt
once daily for an additional 3 days after the initial
4-week HD-feeding period. As we expected, treatment
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Hypoadiponectinemia and Diabetic BCAA Catabolism
Diabetes Volume 64, January 2015
Figure 1—PP2Cm is downregulated in ob/ob and type 2 diabetic mice. A and B: Plasma BCAA and BCKA concentrations in WT, ob/ob,
and type 2 diabetic mice. C and D: Analysis of BCKD activity. E: Quantification of pSer293 BCKD E1a. PP2Cm (F) and BDK (G) protein levels
in liver, adipose tissue, and skeletal muscle from WT, ob/ob, and type 2 diabetic mice. H: Representative immunoblots of pSer293 BCKD
E1a, BCKD E1a, PP2Cm, BDK, and GAPDH in WT, ob/ob, and type 2 diabetic mice. All results are presented as mean 6 SEM. *Significant
difference between ob/ob or type 2 diabetic group versus WT group. *P < 0.05, **P < 0.01. n = 6–8. AT, adipose tissue; AU, arbitrary unit; L,
liver; SM, skeletal muscle.
with APN completely corrected plasma BCAA and BCKA,
hepatic BCKD activity, BCKD phosphorylation, and PP2Cm
protein levels (Fig. 2L–Q and T). However, plasma glucose
levels, glucose tolerance, insulin tolerance, and cholesterol
concentrations did not show a difference between APN and
vehicle-treated mice (Fig. 2H–K). Collectively, these findings demonstrate that APN deficiency is responsible for
downregulated BCKD activity and PP2Cm expression.
Since BCKD activity is also significantly reduced in
adipose tissue and skeletal muscle, we thus determined
the effect of APN knockout upon BCKD activity in these
tissues. Compared with HD-fed WT mice, HD-fed APN2/2
mice revealed greater reduction of BCKD activity in adipose tissue and skeletal muscle (Fig. 2C–E and Supplementary Fig. 2), which were completely corrected by
APN treatment (Fig. 2N–P and T ). Interestingly, there
was no change of PP2Cm level in skeletal muscle but
not in adipose tissue from HD-fed APN2/2 mice (Fig.
2F and Supplementary Fig. 2). APN treatment markedly
increased the downregulated PP2Cm expression in adipose tissue (Fig. 2Q and T ). In addition, BDK expression
was significantly increased in liver, skeletal muscle, and
adipose tissue from HD-fed APN2/2 mice (Fig. 2G and
Supplementary Fig. 2), which was reverted by APN treatment (Fig. 2R and T). Collectively, these findings demonstrate for the first time that APN deficiency causes
downregulated BCKD activity and PP2Cm expression
and upregulated BDK levels.
PP2Cm Deficiency Partially Inhibits APN-Activated
BCKD
Data have shown that plasma APN concentration is
significantly reduced in diabetic patients and animals
(20,26). Consequently, we sought to determine whether
exogenous APN administration could change BCKD activity in diabetic mice. Diabetic animals were injected with
vehicle or APN (5 mg/g body wt, daily) for 3 days. As
illustrated in Fig. 3, plasma glucose levels, glucose tolerance, and insulin tolerance did not show a difference between APN and vehicle-treated mice (Fig. 3A–C). As we
expected, there was significant decrease in BCAA and BCKA
levels but increase in BCKD activity in APN-treated mice
compared with those only treated with vehicle (Fig. 3D–
H). In addition, APN treatment significantly increased
PP2Cm protein expression in diabetic liver and adipose
tissue but not in skeletal muscle (Fig. 3I). Injection with
APN markedly reduced BDK levels in diabetic animals
(Fig. 3J). Taken together, these results indicate that
APN can increase downregulated BCKD activity and ameliorate disturbed BCAA catabolism during diabetes.
To further determine whether PP2Cm is required by
APN to mediate its effects on BCKD activity, we treated
PP2Cm2/2 mice with vehicle or APN. Interestingly, the
positive effects of APN on BCKD were reduced but not
completely lost in PP2Cm2/2 mice. Specifically, plasma
BCAA and BCKA concentrations were significantly higher,
but BCKD activity was markedly lower in PP2Cm2/2 mice
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Figure 2—APN deficiency contributes to decreased BCKD activity. Plasma BCAA and BCKA (A and B) and BCKD (C and D) activity and
quantification of pSer293 BCKD E1a (E), PP2Cm (F), and BDK (G) protein levels in liver, adipose tissue, and skeletal muscle from WT and
APN2/2 mice fed with ND or HD. H: Analysis of blood glucose concentrations. I: Glucose tolerance tested by IPGTT. Insulin tolerance
tested by ITT (J), blood cholesterol levels (K), and plasma BCAA and BCKA concentrations (L and M) in HD-fed APN2/2 mice after vehicle
or APN treatment. Detection of BCKD activity (N and O) and quantitative results of pSer293 BCKD E1a (P), PP2Cm (Q), and BDK protein
level (R) testing in liver, adipose tissue, and skeletal muscle from HD-fed APN2/2 mice injected with APN. S: Representative Western blots
of pSer293 BCKD E1a, BCKD E1a, PP2Cm, BDK, and GAPDH in ND or HD-fed WT and APN2/2 mice. T: Representative immunoblots of
pSer293 BCKD E1a, BCKD E1a, PP2Cm, BDK, and GAPDH in HD-fed APN2/2 mice injected with APN or vehicle. All results are presented
as mean 6 SEM. *Significant difference between APN2/2 and WT mice fed with HD. %Significant difference between APN and vehicle
treatment. * and %P < 0.05, ** and %%P < 0.01. n = 6–8. AT, adipose tissue; AU, arbitrary unit; L, liver; SM, skeletal muscle.
treated with APN compared with those in APN-treated
diabetic and WT control groups (Fig. 3D–H and Supplementary Fig. 3A–C). These data suggested that other factors may also contribute the stimulatory effect of APN
upon BCKD. Indeed, APN treatment markedly attenuated
the BDK expression in PP2Cm2/2 mice compared with
vehicle-treated ones (Fig. 3K). Taken together, these observations support the hypothesis that APN can regulate
PP2Cm (upregulating) and BDK (downregulating) in the
opposite ways, thus increasing BCKD activity and ameliorating disturbed BCAA catabolism in diabetic disease.
AMPK Is Necessary for APN-Mediated BCKD
Activation
Considerable evidence exists that AMPK acts as an integrator
of nutritional and hormonal signals that monitor systemic
and cellular energy status (31). We then investigated whether
AMPK contributes to decreased BCKD activity in diabetes.
Consistent with previous reports, phosphorylated AMPK
was markedly downregulated in type 2 diabetic mice
(Fig. 4A). We treated these mice with AICAR (21)
(a cell-permeable activator of AMPK [150 mg/g body wt
daily]) for 3 days. As shown in Fig. 4, AICAR treatment
resulted in a significant decrease in BCAA and BCKA concentrations while augmenting BCKD activity and PP2Cm
in diabetic mice liver (Fig. 4B–G). To determine whether
adipose tissue and skeletal muscle contribute to system
BCAA homeostasis in response to AICAR treatment, the
following experiments were conducted. Treatment with
AICAR significantly increased the activity of BCKD in diabetic adipose tissue and skeletal muscle (Fig. 4D and E).
Moreover, AICAR injection significantly upregulated
PP2Cm protein levels in adipose tissue but did not influence PP2Cm expression in skeletal muscle from diabetic
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Hypoadiponectinemia and Diabetic BCAA Catabolism
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Figure 3—PP2Cm deficiency partially inhibits APN-activated BCKD. Analysis of blood glucose concentrations (A), glucose tolerance tested
by IPGTT (B), and insulin tolerance tested by ITT in type 2 diabetic animals administered with APN or vehicle (C). BCAA and BCKA
concentrations in plasma (D and E) and BCKD activity (F and G) in liver, adipose tissue, and skeletal muscle from ob/ob, type 2 diabetic,
and PP2Cm2/2 mice, each injected with APN or vehicle. H: Western blot for hepatic BCKD E1a phosphorylation in ob/ob, type 2 diabetic,
and PP2Cm2/2 mice, each administered with APN. PP2Cm (I) and BDK (J) protein levels were detected by Western blot in liver, adipose
tissue, and skeletal muscle from ob/ob and type 2 diabetic mice injected with APN. K: BDK levels were assessed by Western blot in liver,
adipose tissue, and skeletal muscle from PP2Cm2/2 mice treated with APN. All results are presented as mean 6 SEM. %Significant
difference between APN- and vehicle-injected group. &Significant difference between APN treated ob/ob and PP2Cm2/2 group. % and
&P < 0.05, %% and &&P < 0.01. n = 5–8. A, APN; AT, adipose tissue; AU, arbitrary unit; L, liver; SM, skeletal muscle; V, vehicle.
animals (Fig. 4G). However, AICAR caused a significant
reduction of BDK in diabetic skeletal muscle, as well as
in liver and adipose tissue (Fig. 4H). Altogether, these
results suggest that AMPK may be an endogenous regulator of BCKD activity and PP2Cm and BDK expression.
Since AMPK activation has been recognized as a mechanism of action for APN (20,32), we therefore examined
whether AMPK contributes to APN-activated BCKD in diabetic mice. To do so, type 2 diabetic mice were treated in three
groups: vehicle, APN (5 mg/g body wt daily), and APN plus
CC (a potent AMPK inhibitor [30 min before APN injection;
20 mg/g body wt daily]) for 3 days. As per our expectations,
compound C (CC) completely blocked the effect of APN on
diabetic BCAA, BCKA, and BCKD activity and PP2Cm and
BDK expression (Fig. 4I–O). These findings indicate that
AMPK is necessary for APN-activated BCKD in vivo.
The remote actions of AMPK could potentially have
secondary effects on BCKD. Moreover, liver has an
extremely high BCKD activity compared with other tissues
(3,33). Thus, in vitro experiments were performed in mouse
hepatocytes. BCKA-challenged mouse hepatocytes were incubated with vehicle, APN (10 mg/mL), APN (10 mg/mL) + CC
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Figure 4—AMPK is necessary for APN-mediated BCKD activation. A: Expression of phospho-AMPKa at Thr172 and AMPKa levels in liver,
adipose tissue, and skeletal muscle from type 2 diabetic mice. Left: Phospho-AMPKa, AMPKa, and GAPDH protein levels were assessed
by Western blot analysis. Right: Quantification of Western blot data. BCAA and BCKA concentrations in plasma (B and C) and BCKD
activity (D and E) in liver, adipose tissue, and skeletal muscle from type 2 diabetic mice treated with AICAR. F: Immunoblotting of hepatic
BCKD E1a phosphorylation levels in type 2 diabetic mice treated with AICAR. PP2Cm (G) and BDK (H) protein levels were measured by
Western blot in liver, adipose tissue, and skeletal muscle from type 2 diabetic mice treated with AICAR or vehicle. Analysis of BCAA and
BCKA in plasma (I and J) and BCKD activity (K and L) in liver, adipose tissue, and skeletal muscle from type 2 diabetic animals treated with
APN or APN plus CC. M: Hepatic BCKD E1a phosphorylation levels in type 2 diabetic mice injected with APN or APN plus CC. PP2Cm (N)
and BDK (O) expression was detected in type 2 diabetic animals treated with APN or APN plus CC. All results are presented as mean 6
SEM. *Significant difference between type 2 diabetic and normal groups. %Significant difference between AICAR and vehicle treatment.
&Significant difference between APN and APN plus CC treatment. % and &P < 0.05; **, %%, and &&P < 0.01. n = 5–8. A, APN; AT,
adipose tissue; AU, arbitrary unit; CC, compound C; L, liver; SM, skeletal muscle; V, vehicle.
(added 30 min before APN treatment [20 pmol/mL]),
or AICAR (2 pmol/mL) for 1 h. As shown in Supplementary
Fig. 4, AICAR resulted in significantly reduced BCKA and
phosphorylated BCKD levels and increased PP2Cm protein
expression; in contrast, coincubation with APN and CC had
no effect on BCKA catabolism (Supplementary Fig. 4E–H).
Taken together, these data directly demonstrate that AMPK
contributes to APN-activated BCKD and BCAA catabolism
in mouse hepatocytes.
AMPKa2 Contributes to Total AMPKa Activity in APNStimulated BCKD Activation
AMPK exists as a heterotrimeric complex consisting of a
catalytic a subunit and two regulatory b and g subunits.
Phosphorylation of Thr172 in AMPKa is associated with
activation of both the a1 and a2 subunits of AMPK (34).
Thus, we investigated the relative contributions of
AMPKa2 and AMPKa1 to total AMPKa activity in BCKD
of mouse hepatocytes challenged with BCKA. Mouse hepatocytes were transfected with AMPKa2- or AMPKa1-specific
siRNA, and AMPKa2 or AMPKa1 levels were examined by
Western blot. As depicted in Fig. 5A, siRNA transfection
reduced the levels of AMPKa2 and AMPKa1 protein by
81% and 83%, respectively. Importantly, PP2Cm expression
and BCKD activity were reduced, and BCKA accumulated in
the AMPKa2 siRNA knockdown group (Fig. 5A–D).
We further examined whether AMPKa2 is involved in
APN-induced BCKD activation and PP2Cm expression.
Mouse hepatocytes transfected with either AMPKa2 or
AMPKa1 siRNA were challenged with BCKA and then
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Hypoadiponectinemia and Diabetic BCAA Catabolism
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Figure 5—AMPKa2 contributes to total AMPKa activity in APN-stimulated BCKD activation. A: Representative immunoblots of hepatocyte
lysate for pSer293 BCKD E1a, BCKD E1a, AMPKa1, AMPKa2, PP2Cm, and GAPDH after transfection with scramble, AMPKa1, or AMPKa2
siRNA. The relative levels of PP2Cm (B) and pSer293 BCKD E1a (C) were quantified, and BCKA concentrations (D) were analyzed in
hepatocytes transfected with scramble, AMPKa1, or AMPKa2 siRNA. PP2Cm protein (E), BCKD E1a phosphorylation (F), and BCKA levels
(G) in scramble, AMPKa1, or AMPKa2 siRNA transfected hepatocytes treated with or without APN for 1 h. All results are presented as
mean 6 SEM. **Significant difference between scramble siRNA and AMPKa2 siRNA group. %Significant difference between APN- and
vehicle-treated group. %P < 0.05, ** and %%P < 0.01. n = 6–12 wells. AU, arbitrary unit.
treated with vehicle or APN (10 mg/mL) for 1 h. As shown
in Fig. 5, AMPKa2 siRNA completely blocked the effects
of APN on PP2Cm expression, BCKD activity, and BCKA
levels. This effect was not seen with scrambled siRNA or
AMPKa1 siRNA transfection (Fig. 5E, F, and G). Overall,
these results directly demonstrate that AMPKa2 deletion/
inactivation modulates BCKD activity and that the PP2Cm
regulatory function of APN is dependent on AMPKa2
in vitro.
DISCUSSION
Here, we have made several important observations. First,
we validated that PP2Cm was downregulated in diabetic
mice, which may, at least in part, contribute to reduced
BCKD activity potentially caused by an APN deficiency.
Second, APN treatment reverted downregulated PP2Cm
expression and BCKD activity, as well as elevated plasma
BCAA and BCKA levels in diabetic mice. However, the
increased BCKD activity mediated by APN treatment was
partially abolished in PP2Cm2/2 mice. Third, the downregulated BDK level was involved in APN-activated BCKD
complex, especially in skeletal muscle. Last, AMPK in diabetic mice was significantly impaired, which results in reduced BCKD activity and accumulation of BCAA and BCKA.
These effects could be reversed by APN injection. Importantly, APN upregulated PP2Cm expression in hepatocytes,
which depends on AMPKa2. Collectively, our studies have
established a novel mechanism that impaired APN signaling
contributes to diabetic BCAA catabolism.
BCKD complex is the most important regulatory
enzyme in BCAA catabolism; the activity of BCKD is
regulated by a phosphorylation-dephosphorylation cycle
and responsive to alterations in various metabolic conditions (35). Studies have reported that decreased BCKD
activity causes downregulation of BCAA catabolism in diabetes (6,7,36). It also has demonstrated that BDK is responsible for phosphorylation and inactivation of BCKD
complex and considered a primary regulator of BCKD
diabetes.diabetesjournals.org
activation (6). Diabetic and obese animals showed increased BDK expression (6,8). Moreover, alteration of
metabolic status can influence BCKD activity partly via
changes in BDK (3), indicating that alteration of BDP
may also be important. But no information about BDP is
available in diabetes. Identification of a specific BDP proved
elusive for many years; PP2Cm was identified as the only
endogenous BDP until recently (18,19,27). In the current
study, we demonstrated for the first time that PP2Cm was
significantly decreased in liver and adipose tissue from diabetic animals, which may at least partially contribute to
reduction of BCKD activity and elevated plasma BCKA levels. Altogether, these findings provide a new explanation for
the disturbed regulation of BCAA catabolism in diabetes.
Hypoadiponectinemia is commonly seen in diabetes
(20,26), and APN is a critical regulator in lipid and glucose
metabolism (20). Moreover, Liu et al. (24) recently demonstrated that HD-fed APN2/2 mice have significantly
increased levels of muscle BCAA, which can be corrected
with APN supplementation for 2 weeks. However, the role
of APN in systemic BCAA catabolism remains unclear. In our
study, APN2/2 mice fed with 45% HD for 4 weeks showed
mild insulin resistance (Supplementary Fig. 2C and E) but
normal blood glucose levels (Supplementary Fig. 2A).
In addition, we demonstrated that HD-fed APN2/2
mice had significant reduced BCKD activity and PP2Cm
levels and increased BDK expression, along with high
plasma BCAA and BCKA concentrations. Treatment with
APN for 3 days significantly reversed these trends, but
insulin resistance remained (Fig. 2H–J). More importantly, APN treatment completely reverted the reduction
of BCKD activity and PP2Cm expression and the elevation
of BDK level in diabetic models. These results suggest that
hypoadiponectinemia contributes to downregulated BCKD
activity and PP2Cm expression and upregulated BDK level,
as well as abnormal BCAA catabolism. This supports the
notion that APN has a wide range of impacts on metabolism (24).
Additionally, evidence by Newgard et al. (10) indicates
that changes in BCAA levels contribute to the development of insulin resistance. An interesting point that warrants further investigation is that the positive effect of
APN on BCAA may subsequently contribute to the insulin-sensitizing effect of APN. Lastly, BCAA levels changed
acutely upon short-time APN administration, whereas
plasma glucose, glucose tolerance, insulin tolerance, and
plasma cholesterol levels were unchanged (Figs. 2H–K and
3A–C). Although the precise mechanism causing the rapid
BCAA regulatory response after short-period APN administration remains unclear, this result further supports that
BCAAs may be useful biomarkers for monitoring the early
response to therapeutic interventions for metabolic disease.
In the current study, we observed that APN deficiency
in the APN2/2 mice did not result in elevated BCAA levels
under basal physiological condition. However, in reviewing numerous previous publications using this model, we
found that our observation is very consistent with
Lian and Associates
57
previous publications, showing that the APN2/2 mouse
does not have phenotypic changes under physiological
condition. However, when pathologically challenged,
such as by exposure to HD (37) or myocardial ischemia
(22), these animals show much severer tissue injury than
WT controls. These results indicate that although other
molecules present in APN2/2 animals are sufficient to
compensate the effect of APN under normal physiological
conditions, APN plays an essential role in counteracting
pathological stress, such as HD. Additionally, APN partly
upregulated BCKD activity and markedly downregulated
BDK expression in PP2Cm-deficient mice, suggesting that
BDK was involved in APN-activated BCKD. It has been
shown in literature that thyroid hormone and sex hormones regulate expression of BDK (38); our present data
also revealed that BDK was the downstream molecular of
APN and contributed to APN-mediated BCAA catabolism.
Thus, APN may signal both BDK and PP2Cm in opposite
ways to increase BCKD activity and improve BCAA catabolism in diabetes.
AMPK has been considered a master switch in
regulating glucose and lipid metabolism (39) and plays
an essential role in the actions of APN (40). Nevertheless,
AMPK can also modulate transcription of specific genes
involved in energy metabolism (31). In this study, we
observed that BCKD activity was elevated by activation
of AMPK both in vitro and in vivo by pharmacological or
genetic methods. Moreover, when the AMPK inhibitor CC
or siRNA was applied, the effect of APN on BCKD was
virtually abolished. These results might provide us with
a better understanding of the role of AMPK in the regulation of metabolism.
The possibility remains that the remote role of APNAMPK signaling in glucose and lipid metabolism could
show secondary effects on BCKD activity in vivo; we thus
performed in vitro experiments. Because BCKD capacity
mostly resides in liver (3,33), mouse hepatocytes were
used in present study. Here, mouse hepatocytes challenged with BCKA and incubated with APN or AICAR
helped to address the fact that APN-AMPK directly upregulated BCKD activity (Supplementary Fig. 4A–H). Interestingly, effect of APN upon BCKA occurred prior to the
significant upregulation of PP2Cm levels, suggesting that
APN may improve BCKA catabolism via a signaling system
in addition to upregulation of PP2Cm. Although the
detailed molecular mechanisms cannot be addressed in
the current study, it is possible that APN may enhance
BCKD/PP2Cm interaction, thus increasing BCKD activity–
independent PP2Cm expression. This interesting possibility
will be directly investigated in our future study. To date, it
remains unclear how PP2Cm expression is regulated. Previous studies (27,38) and our unpublished data have
revealed that PP2Cm expression can be regulated by the
availability of nutrients as well as stress (i.e., ischemia and
heart failure). Here, we demonstrated that APN was the
first identified endogenous molecule that can upregulate
PP2Cm level.
58
Hypoadiponectinemia and Diabetic BCAA Catabolism
In summary, this study provides evidence that reductions of APN signaling could underlie the decreased BCKD
activity observed in type 2 diabetes. Our study also
reveals direct evidence that APN can modulate expression
of hepatic PP2Cm via an AMPKa2-dependent pathway.
These new insights provide a better understanding of the
underlying regulatory mechanisms involved in diabetic
BCKD activity and identify potential therapeutic targets
to mitigate BCAA catabolism in metabolic diseases.
Acknowledgments. The authors thank Dr. Xinliang Ma, Thomas Jefferson
University, for help with revising the manuscript. The authors also thank Dr. Yibin
Wang and Dr. Haipeng Sun, University of California, Los Angeles, for the generous
supply of the antibody to PP2Cm and PP2Cm2/2 mice.
Funding. This work was supported by the Program for National Science Fund
for Distinguished Young Scholars of China (grant no. 81225001), National Key
Basic Research Program of China (973 Program, 2013CB531204), New Century
Excellent Talents in University (grant no. NCET-11-0870), National Science Funds
of China (grant nos. 81070676 and 81170186), Program for Changjiang Scholars
and Innovative Research Team in University (no. PCSIRT1053), and Major Science
and Technology Project of China “Significant New Drug Development” (grant no.
2012ZX09J12108-06B).
Duality of Interest. No potential conflicts of interests relevant to this
article were reported.
Author Contributions. K.L. designed methods and experiments, carried
out the laboratory experiments, and wrote the paper. C.D. performed RT-PCR
analysis and analyzed data. Y.L. analyzed data and interpreted the results. D.Z.
contributed to the data of Fig. 5. W.Y. reviewed and edited the manuscript. H.Z. and
Z.H. researched data and contributed to discussion. P.L., L.Z., and H.P. performed
diabetic animal model and collected the samples. J.Z., C.G., and C.X. performed
BCAA and BCKA analysis. H.C. and L.X. contributed to discussion. L.T. defined the
research theme and revised the manuscript critically. L.T. is the guarantor of this
work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
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