Title Running Title Authors - Diabetes · 2014. 7. 28. · 3 38 Abstract 39 The branched-chain...
Transcript of Title Running Title Authors - Diabetes · 2014. 7. 28. · 3 38 Abstract 39 The branched-chain...
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Title 1
Impaired Adiponectin Signaling Contributes to Disturbed Catabolism of 2
Branched-Chain Amino Acids in Diabetic Mouse 3
Running Title 4
Hypoadiponectinemia and Diabetic BCAA Catabolism 5
Authors 6
Kun Lian1, Chaosheng Du
1, Yi Liu
1, Di Zhu
1, Wenjun Yan
1, Haifeng Zhang
2, Zhibo 7
Hong1, Peilin Liu
1,4, Lijian Zhang
1, Haifeng Pei
1, Jinglong Zhang
1, Chao Gao
1, Chao 8
Xin1, Hexiang Cheng
1, Lize Xiong
3 and Ling Tao
1* 9
Affiliations 10
1Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, 11
15 Changlexi Road, Xi’an, 710032, China; 12
2Experiment Teaching Center, The Fourth Military Medical University, 169 Changlexi 13
Road, Xi’an, 710032, China; 14
3Department of Anesthesiology, Xijing Hospital, The Fourth Military Medical Uni-15
versity, 15 Changlexi Road, Xi’an, 710032, China; 16
4Department of Cardiology, The 306th Hospital of PLA, 9 Anxiangbeili Street, Bei-17
jing, 100101, China. 18
Contact Information 19
Page 1 of 50 Diabetes
Diabetes Publish Ahead of Print, published online July 28, 2014
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Ling Tao, MD, PhD 20
Department of Cardiology 21
Xijing Hospital 22
The Fourth Military Medical University 23
15 Changlexi Road, 24
Xi’an, 710032, China; 25
E-mail: [email protected]; 26
Tel: 86-29-84771024, 86-29-84775183; 27
Fax: 86-29-84771024. 28
Additional Footnotes 29
Kun Lian, Chaosheng Du and Yi Liu contributed equally to this work. 30
Word Count 31
4411 32
Number of Figures 33
5 34
Number of Supplemental Figures 35
4 36
37
Page 2 of 50Diabetes
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Abstract 38
The branched-chain amino acids (BCAA) accumulated in type 2 diabetes mellitus 39
are independent contributors to insulin resistance. The activity of branched-chain 40
α-keto acid dehydrogenase (BCKD) complex, rate-limiting enzyme in BCAA catabo-41
lism, is reduced in diabetic states, which contributes to elevated BCAA concentrations. 42
However, the mechanisms underlying decreased BCKD activity remain poorly under-43
stood. Here we demonstrate that mitochondrial phosphatase 2C (PP2Cm), a newly 44
identified BCKD phosphatase which increases BCKD activity, was significantly 45
down-regulated in ob/ob and type 2 diabetic mice. Interestingly, in adiponectin 46
knockout (APN-/-
) mice fed with high-fat-diet (HD), PP2Cm expression and BCKD 47
activity were significantly decreased, whereas BCKD kinase (BDK) which inhibits 48
BCKD activity was markedly increased. Concurrently, plasma BCAA and 49
branched-chain α-keto acids (BCKA) were significantly elevated. APN treatment 50
markedly reverted PP2Cm, BDK, BCKD activity, BCAA and BCKA levels in HD-fed 51
APN-/-
and diabetic animals. Additionally, increased BCKD activity caused by APN 52
administration was partially but significantly inhibited in PP2Cm knockout mice. Fi-53
nally, APN-mediated up-regulation of PP2Cm expression and BCKD activity were 54
abolished when adenosine monophosphate-activated protein kinase (AMPK) was in-55
hibited. Collectively, we have provided the first direct evidence that APN is a novel 56
regulator of PP2Cm and systematic BCAA levels, suggesting that targeting APN may 57
be a pharmacological approach to ameliorating BCAA catabolism in the diabetic state. 58
Key Words: BCAA; BCKD; Diabetes; APN; AMPK; PP2Cm; BDK 59
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Introduction 60
The branched-chain amino acids (BCAA) are essential amino acids such as leucine, 61
isoleucine, and valine; their homeostasis is determined largely by catabolic activities 62
in a number of organs including liver, muscle and adipose tissue (1-3). The first step 63
of BCAA catabolism generates a set of corresponding branched-chain α-keto acids 64
(BCKA), which are irreversibly decarboxylated by the branched-chain α-keto acid 65
dehydrogenase (BCKD) complex (4). As with most nutrients, maintaining of physio-66
logical level of BCAA is critical for cell metabolism and survival. However, many 67
researchers have been described that increased BCAA and BCKA levels in diabetes 68
and obesity (3; 5-8). Furthermore, BCAA and their catabolites are strongly associated 69
with insulin resistance (9-11); and elevated BCAA contributes to the development of 70
insulin resistance (10; 12). Mechanistically, elevated BCAA levels activate 71
mTOR/p70S6 kinase, resulting in an increased IRS-1 phosphorylation thereby inhib-72
iting PI3 kinase. This inhibition of PI3K in turn leads to impaired insulin signaling (13; 73
14). It is also reported that BCAA are independent predictors of insulin resistance, di-74
abetes and cardiovascular events (15-17). Therefore, it is necessary to determine the 75
mechanisms of abnormal BCAA catabolism in order to better understand their associ-76
ation with metabolic-related pathogenesis. 77
The BCKD complex is the rate-limiting enzyme in BCAA catabolism (4; 12), reg-78
ulation of BCKD activity is therefore important for maintaining the homeostasis of 79
systemic BCAA and BCKA. The complex consists of three catalytic components: a 80
heterotetrameric (α2β2) branched-chain α-keto acid decarboxylase (E1), a homo-24 81
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meric dihydrolipoyltransacylase (E2), and a homodimeric dihydrolipoamide dehy-82
drogenase (E3). The activity of BCKD complex is controlled by the reversible phos-83
phorylation of its E1a subunit(Ser293) by specific BCKD kinase (BDK) and 84
phosphatase (BDP), respectively (4; 18). Phosphorylation catalyzed by BDK inhibits 85
the enzymatic activity of the BCKD complex, whereas it becomes activated when the 86
Ser293 residue is dephosphorylated by BDP. A series of reports have demonstrated 87
that activation of BCKD was reduced in liver and adipose tissue, resulting in in-88
creased plasma BCAA and BCKA concentrations in diabetic and obese animals (6-8). 89
Moreover, alterations of metabolism can influence BCKD activity partly through 90
changes in BDK (3), suggesting that BDP might be suppressed. The mitochondrial 91
phosphatase 2C (PP2Cm) is the only identified BDP (18; 19), which specifically binds 92
the BCKD complex and induces dephosphorylation of BCKD at Ser293 in the pres-93
ence of BCKD substrates (18). Additionally, PP2Cm deficiency impairs BCAA catab-94
olism, leading to elevated plasma BCAA and BCKA concentrations (18). However, 95
the relationship between PP2Cm and reduced diabetic BCKD activity has not yet been 96
investigated. 97
Adiponectin (APN) is an adipocytokine predominantly synthesized in and secreted 98
from adipose tissue. APN helps regulating glucose and lipid metabolism (20; 21) and 99
has vascular/cardioprotective effects (22; 23). More recently, Liu and colleagues (24) 100
have reported that APN corrects altered muscle BCAA metabolism induced by a high 101
fat diet (HD). In addition, several observations have revealed that obesity and type 2 102
diabetes are associated with decreased plasma APN levels (20; 25; 26). However, the 103
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correlation between decreased APN levels and reduced BCKD activity has never been 104
investigated. More importantly, the underlying molecular mechanisms by which APN 105
mediates disturbed BCAA catabolism in diabetes are completely unknown. 106
In the present study, we employed both in vitro and in vivo experiments to identify 107
hypoadiponectinemia as a contributing factor to reduced BCKD activity in diabetes. 108
In diabetic mice, BCKD activity was reduced, and BCAA and BCKA levels were sig-109
nificantly elevated. APN treatment effectively reversed these pathological alterations, 110
which were completely abolished by inhibition of adenosine monophos-111
phate-activated protein kinase (P-AMPK) and partially but significantly attenuated by 112
knockout of PP2Cm expression. These findings lead us to conclude that impaired 113
APN signaling is an important part of the underlying mechanism for disturbed BCAA 114
catabolism in type 2 diabetes mellitus. 115
116
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Research Design and Methods 117
Animal Care and Drug Treatment. All experiments were performed in adherence to 118
the National Institutes of Health Guidelines on the Use of Laboratory Animals, and 119
were approved by the Fourth Military Medical University Committee on Animal Care. 120
Male ob/ob and wild type (WT) C57BL/6 control mice were purchased from the De-121
partment of Pathology, The Fourth Military Medical University. Male APN knockout 122
(APN-/-
) mice (22) and WT C57BL/6 mice on the same background have been previ-123
ously described. The whole-body PP2Cm knockout (PP2Cm-/-
) mice (18; 27) were 124
gifted by professor Yibin Wang of UCLA (Los Angeles, CA). 125
Mice were rendered type 2 diabetic by the following procedures. Four-week old 126
WT C57BL/6 mice were fed with a high fat diet (HD, 60% kcal% fat, Research Diets, 127
New Brunswick, NJ) for 6 weeks and intraperitoneally injected with a low-dose of 128
streptozotocin twice (28) [25 mg/kg STZ in 0.05 M sodium citrate, pH 4.5, once daily, 129
Sigma, St. Louis, MO]. Blood glucose and body weight were measured daily, and a 130
diabetic condition was confirmed at 4 weeks after STZ injection by a non-fasting 131
blood glucose level of ≥ 200 mg/dl. Fasting blood insulin was measured, intraperito-132
neal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were carried out 133
in each successful model group. 134
In some experiments, 7-week old APN-/-
and WT mice were randomized to receive 135
a normal chow diet (ND, 12% kcal from fat, control) or HD (45% kcal from lard, Re-136
search Diets, New Brunswick, NJ) for 4 weeks. Additionally, animals received differ-137
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ent treatment as following: ① ob/ob, HD-fed APN-/-
and PP2Cm-/-
mice received 138
vehicle (phosphate buffered saline, PBS) or APN (5 µg/g body wt., PeproTech, Rocky 139
Hill, NJ); ② type 2 diabetic mice received vehicle, APN (5 µg/g body wt.), AMPK 140
activator AICAR (150 µg/g body wt., Sigma, St. Louis, MO) or APN in conjunction 141
with the AMPK inhibitor compound C (20 µg/g body wt., Sigma, St. Louis, MO) once 142
daily for an additional 3 days. After 12 hours of fasting, animals were euthanized for 143
collection of tissues (liver, epididymal fat pad and gastrocnemius muscle) and 0.5 144
ml aortic blood at 10 -12 weeks of age. 145
Cell Culture and BCKA Challenge. AML12 mouse hepatocytes (ATCC, Manassas, 146
VA) were cultured in DMEM medium (Invitrogen, Carlsbad, CA) containing 10% 147
fetal bovine plasma (HyClone Waltham, MA). Experiments were carried out at 3 or 4 148
cell passages. The hepatocytes were transfected by siRNA and incubated in DMEM 149
medium (Invitrogen, Carlsbad, CA) with an additional mixture of all of the BCKA 150
(Sigma, St. Louis, MO) at 2.5 mM. Each set of cells was randomized to receive 151
treatment with 10 µg/ml APN or vehicle control. After 1 hour of treatment, cells and 152
supernatants were collected for Western blotting and BCKA analysis. 153
RNA Interference. siRNA constructs against AMPK α1 or AMPK α2 mRNA were 154
designed and purchased from Gene Pharma (Shanghai, China). The siRNA sequences 155
are as follows: 156
AMPKα1: sense 5'-GCCGACCCAAUGAUAUCAUTT3', anti-sense 157
5'-AUGAUAUCAUUGGGUCGGCTT-3'; 158
Page 8 of 50Diabetes
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AMPKα2: sense 5'-GGACAGGGAAGCCUUAAAUTT-3', anti-sense 159
5'-AUUUAAGGCUUCCCUGUCCTT-3'; 160
Scrambled siRNA: sense 5'-UUCUCCGAACGUGUCACGUTT-3', anti-sense 161
5'-ACGUGACACGUUCGGAGAATT-3'. 162
Mouse hepatocytes were transfected with siRNA by using LipofectamineTM
2000 163
(Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Efficiency of 164
gene knockdown was confirmed using Western blotting 48 hours after siRNA trans-165
fection. 166
BCAA and BCKA Analysis. Plasma and supernatant of cultured cells were collected 167
and subsequently stored at -80 °C. Determination of BCAA concentrations was per-168
formed in triplicate using a commercially available BCAA detection kit (Biovision, 169
Milpitas, CA) per the manufacturer's instructions. BCKA concentrations were deter-170
mined by HPLC as described by Loi et al.(29). 171
BCKD enzyme activity assays. Tissue extraction and assessment of BCKD activity 172
were performed as described previously (30). BCKD complex was concentrated from 173
whole tissue extracts using 9% polyethylene glycol. BCKD activity was determined 174
spectrophotometrically by measuring the rate of NADH production resulting from the 175
conversion of α-keto-isovalerate to isobu-tyryl-CoA. A unit of enzyme activity was 176
defined as 1 µmol of NADH formed per minute at 30°C. 177
Western Blot Analysis. Proteins were separated on SDS-PAGE gels, transferred to 178
PVDF (Polyvinylidene difluoride) membranes (Millipore, Billerica, MA), and incu-179
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bated overnight at 4℃ with antibodies directed against AMPKα (1:1,000,CST, Dan-180
vers, MA), AMPKα1 (1:1,000, CST, Danvers, MA), AMPKα2 (1:1,000, CST, Dan-181
vers, MA), phospho-AMPKα (Thr172, 1:1,000, CST, Danvers, MA), PP2Cm (1:1,000, 182
a gift by professor Yibin Wang of UCLA, Los Angeles, CA), BDK (1:2,000, Abcam, 183
Cambridge, MA), phospho-BCKD E1α (1:1,000, Bethyl Laboratories, Montgomery, 184
TX), BCKD E1α (1:500, Santa Cruz Biotechnology, Dallas, TX) and GAPDH 185
(1:5,000, Zhong Shan Golden Bridge Biotechnology, Beijing). After washing to re-186
move excess primary antibody, blots were incubated for 1 hour with horseradish pe-187
roxidase (HRP) conjugated secondary antibody. Binding was detected via enhanced 188
chemiluminescence (Millipore, Billerica, MA). Films were scanned with Chemi-189
DocXRS (Bio-Rad Laboratory, Hercules, CA). Densitometry was performed using 190
Lab Image software. 191
Statistical Analysis. All values in the text and figures are presented as mean ± SEM 192
of n independent experiments. All data (except densitometry) was subjected to 193
ANOVA followed by a Bonferroni correction for a post hoc t test. Densitometry was 194
analyzed using the Kruskal-Wallis test, followed by a Dunn post hoc test. Probabili-195
ties of 0.05 or less were considered statistically significant. 196
197
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Results 198
PP2Cm Is Down-regulated in ob/ob and Type 2 Diabetic Mice 199
Consistent with previous reports, plasma BCAA and BCKA levels were signifi-200
cantly increased in both ob/ob and type 2 diabetic mice when compared to WT mice 201
(Figure 1A and 1B). Liver is the primary metabolic clearing house of BCKA; the re-202
duction of BCKD activity causes increased circulating BCAA and BCKA concentra-203
tions (3). In addition, immunoreactivity of the BCKD pSer293 antibody is directly 204
correlated with BCKD activity (18); we therefore examined the activity of BCKD and 205
phosphorylation of BCKD at Ser293 in liver. We found that BCKD activity was sig-206
nificantly reduced and phosphorylation of BCKD was significantly increased in dia-207
betic animals as compared with WT mice (Figure1C, 1D, 1E and 1H). Interestingly, 208
the newly identified BCKD phosphatase, PP2Cm, was markedly decreased in diabetic 209
mice (Figure 1F and 1H), indicating that down-regulation of PP2Cm may be involved 210
in reduced BCKD activity in diabetes mellitus. 211
Considerable evidence indicates that besides liver, adipose tissue and skeletal mus-212
cle also play an important role in modulating circulating BCAA homeostasis (1; 2), 213
following experiments were thus performed. As shown in figure 1, BCKD activity 214
was markedly down-regulated in diabetic adipose tissue and skeletal muscle (Fig-215
ure1C, 1D, 1E and 1H). Additionally, PP2Cm protein levels were significantly re-216
duced in diabetic adipose tissue (Figure 1F and 1H). To our surprise, changes of 217
PP2Cm expression in skeletal muscle were not observed in diabetic animals (Figure 218
Page 11 of 50 Diabetes
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1F and 1H), indicating that skeletal muscle may have relatively low level of 219
PP2Cm-dependent BCKD activation. Since the activity of BCKD complex are in-220
creased by PP2Cm and inhibited by BDK, we than analyzed expression of BDK. Our 221
experimental results demonstrated that BDK expression was significantly increased in 222
diabetic tissues including liver, adipose tissue as well as skeletal muscle (Figure 1G 223
and 1H). These results indicate that decreased PP2Cm and increased BDK may both 224
contribute to reduced BCKD activity in diabetic liver and adipose tissue, whereas in-225
creased BDK may be the primary cause of decreased BCKD activity in diabetic skel-226
etal muscle. 227
Adiponectin Deficiency Contributes to Decreased BCKD Activity 228
It is known that APN reverts altered BCAA metabolism in muscle (24). In order to 229
determine whether APN contributes to BCKD activity, 7-week old WT and APN-/-
230
mice were fed with either ND or 45% HD for further 4 weeks, resulting in four sub-231
groups: ND WT, HD WT, ND APN-/-
and HD APN-/-
. As shown in figure 2, APN-/-
232
mice revealed the significant increase of plasma BCAA, BCKA and hepatic BCKD 233
phosphorylation levels, but reduction of BCKD activity in response to HD. No re-234
sponse was observed in WT mice with the same genetic background (Figure 2A, 2B, 235
2C, 2D, 2E and 2S). More importantly, hepatic PP2Cm was markedly decreased in 236
HD-fed APN-/-
mice (Figure 2F and 2S). We then sought to determine whether treat-237
ment with APN could significantly reverse altered BCKD activity. HD-fed APN-/-
238
mice were given recombinant APN at 5µg/g body wt. once daily for an additional 3 239
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days after the initial 4-week HD feeding period. As we expected, treatment with APN 240
completely corrected plasma BCAA and BCKA, hepatic BCKD activity, BCKD 241
phosphorylation and PP2Cm protein levels (Figure 2L, 2M, 2N, 2O, 2P, 2Q and 2T). 242
However, plasma glucose levels, glucose tolerance, insulin tolerance and cholesterol 243
concentrations did not show difference between APN and vehicle-treated mice (Figure 244
2H, 2I, 2J and 2K). Collectively, these findings demonstrate that APN deficiency is 245
responsible for down-regulated BCKD activity and PP2Cm expression. 246
Since BCKD activity is also significantly reduced in adipose tissue and skeletal 247
muscle, we thus determined the effect of APN knockout upon BCKD activity in these 248
tissues. Compared with HD-fed WT mice, HD-fed APN-/-
mice revealed greater re-249
duction of BCKD activity in adipose tissue and skeletal muscle (Figure 2C, 2D, 2E 250
and 2S), which were completely corrected by APN treatment (Figure 2N, 2O, 2P and 251
2T). Interestingly, there was no change of PP2Cm level in skeletal muscle but not in 252
adipose tissue from HD-fed APN-/-
mice (Figure 2F and 2S). APN treatment markedly 253
increased the down-regulated PP2Cm expression in adipose tissue (Figure 2Q and 2T). 254
In addition, BDK expression was significantly increased in liver, skeletal muscle and 255
adipose tissue from HD-fed APN-/-
mice (Figure 2G and 2S); and that were reverted 256
by APN treatment (Figure 2R and 2T). Collectively, these findings demonstrate for 257
the first time that APN deficiency causes down-regulated BCKD activity and PP2Cm 258
expression, and up-regulated BDK levels. 259
PP2Cm Deficiency Partially Inhibits Adiponectin-activated BCKD 260
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Data have shown that plasma APN concentration is significantly reduced in diabet-261
ic patients and animals (20; 26). Consequently, we sought to determine whether ex-262
ogenous APN administration could change BCKD activity in diabetic mice. Diabetic 263
animals were injected with vehicle or APN (5 µg/g body wt., daily) for 3 days. As il-264
lustrated in figure 3, plasma glucose levels, glucose tolerance, insulin tolerance did 265
not show difference between APN and vehicle treated mice (Figure 3A, 3B and 3C). 266
To our expectation, there were significant decrease in BCAA and BCKA levels, but 267
increase in BCKD activity in APN-treated mice when compared to those only treated 268
with vehicle (Figure 3D, 3E, 3F, 3G and 3H). In addition, APN treatment significantly 269
increased PP2Cm protein expression in diabetic liver and adipose tissue, but not in 270
skeletal muscle (Figure 3I). Injection with APN markedly reduced BDK levels in dia-271
betic animals (Figure 3J). Taken together, these results indicate that APN can increase 272
down-regulated BCKD activity and ameliorate disturbed BCAA catabolism during 273
diabetes. 274
To further determine whether PP2Cm is required by APN to mediate its effects on 275
BCKD activity, we treated PP2Cm-/-
mice with vehicle or APN. Interestingly, the pos-276
itive effects of APN on BCKD were reduced but not completely lost in PP2Cm-/-
mice. 277
Specifically, plasma BCAA and BCKA concentrations were significantly higher, but 278
BCKD activity was markedly lower in PP2Cm-/-
mice treated with APN when com-279
pared with those in APN-treated diabetic and WT control groups (Figure3D, 3E, 3F, 280
3G, 3H, S3A, S3B and S3C). These data suggested that other factors may also con-281
tribute the stimulatory effect of APN upon BCKD. Indeed, APN treatment markedly 282
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attenuated the BDK expression in PP2Cm-/-
mice compared with vehicle treated-ones 283
(Figure 3K). Taken together, these observations support the hypothesis that APN can 284
regulate PP2Cm (up-regulating)and BDK (down-regulating) in the opposite ways, 285
thus increase BCKD activity and ameliorate disturbed BCAA catabolism in diabetic 286
disease. 287
AMPK Is Necessary for Adiponectin - mediated BCKD Activation 288
Considerable evidence exists that AMPK acts as an integrator of nutritional and 289
hormonal signals that monitor systemic and cellular energy status (31). We then in-290
vestigated whether AMPK contributes to decreased BCKD activity in diabetes. Con-291
sistent with previous reports, phosphorylated AMPK was markedly down-regulated in 292
type 2 diabetic mice (Figure 4A). We treated these mice with AICAR (21) (a 293
cell-permeable activator of AMPK, 150 µg/g body wt., daily) for 3 days. As shown in 294
figure 4, AICAR treatment resulted in a significant decrease in BCAA and BCKA 295
concentrations, while augmenting BCKD activity and PP2Cm in diabetic mice liver 296
(Figure 4B, 4C, 4D, 4E, 4F and 4G). To determine whether adipose tissue and skeletal 297
muscle contribute to system BCAA homeostasis in response to AICAR treatment, the 298
following experiments were conducted. Treatment with AICAR significantly in-299
creased the activity of BCKD in diabetic adipose tissue and skeletal muscle (Figure 300
4D and 4E). Moreover, AICAR injection significantly up-regulated PP2Cm protein 301
levels in adipose tissue, but did not influent PP2Cm expression in skeletal muscle 302
from diabetic animals (Figure 4G). However, AICAR caused a significant reduction 303
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of BDK in diabetic skeletal muscle, as well as in liver and adipose tissue (Figure 4H). 304
Altogether, these results suggest that AMPK may be an endogenous regulator of 305
BCKD activity, PP2Cm and BDK expression. 306
Since AMPK activation has been recognized as a mechanism of action for APN (20; 307
32), we therefore examined whether AMPK contributes to APN-activated BCKD in 308
diabetic mice. To do so, type 2 diabetic mice were treated in three groups: vehicle, 309
APN(5 µg/g body wt., daily), and APN plus compound C (a potent AMPK inhibitor, 310
30 mins before APN injection, 20 µg/g body wt., daily) for 3 days. As per our expec-311
tations, compound C (CC) completely blocked the effect of APN on diabetic BCAA 312
and BCKA, BCKD activity, PP2Cm and BDK expression (Figure 4I, 4J, 4K, 4L, 4M, 313
4N and 4O). These findings indicate that AMPK is necessary for APN-activated 314
BCKD in vivo. 315
The remote actions of AMPK could potentially have secondary effects on BCKD. 316
Moreover, liver has an extremely high BCKD activity compared with other tissues (3; 317
33). Thus, in vitro experiments were performed in mouse hepatocytes. BCKA chal-318
lenged mouse hepatocytes were incubated with vehicle, APN (10 µg/ml), APN (10 319
µg/ml) + CC (added 30 mins before APN treatment, 20 pmol/ml), or AICAR (2 320
pmol/ml) for 1 hour. As shown in figure S4, AICAR resulted in significantly reduced 321
BCKA and phosphorylated BCKD levels, and increased PP2Cm protein expression; 322
in contrast, coincubation with APN and CC had no effect on BCKA catabolism (Fig-323
ure S4E, S4F, S4G and S4H). Taken together, these data directly demonstrate that 324
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AMPK contributes to APN-activated BCKD and BCAA catabolism in mouse hepato-325
cytes. 326
AMPKα2 Contributes to Total AMPKα Activity in Adiponectin-stimulated 327
BCKD Activation 328
AMPK exists as a heterotrimeric complex consisting of a catalytic α subunit, and 2 329
regulatory β and γ subunits. Phosphorylation of Thr 172 in AMPKα is associated with 330
activation of both the α1 and α2 subunits of AMPK (34). Thus, we investigated the 331
relative contributions of AMPKα2 and AMPKα1 to total AMPKα activity in BCKD of 332
mouse hepatocytes challenged with BCKA. Mouse hepatocytes were transfected with 333
AMPKα2 or AMPKα1-specific siRNA, and AMPKα2 or AMPKα1 levels were ex-334
amined by Western blot. As depicted in figure 5A, siRNA transfection reduced the 335
levels of AMPKα2 and AMPKα1 protein by 81% and 83%, respectively. Importantly, 336
PP2Cm expression and BCKD activity were reduced, and BCKA accumulated in the 337
AMPKα2 siRNA knockdown group (Figure 5A, 5B, 5C and 5D). 338
We further examined whether AMPKα2 is involved in APN-induced BCKD activa-339
tion and PP2Cm expression. Mouse hepatocytes transfected with either AMPKα2 or 340
AMPKα1 siRNA were challenged with BCKA and then treated with vehicle or APN341
(10 µg/ml)for 1 hour. As shown in figure 5, AMPKα2 siRNA completely blocked 342
the effects of APN on PP2Cm expression, BCKD activity and BCKA levels. This ef-343
fect was not seen with scrambled siRNA or AMPKα1 siRNA transfection (Figure 5E, 344
5F and 5G). Overall, these results directly demonstrate that AMPKα2 dele-345
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tion/inactivation modulates BCKD activity, and that the PP2Cm regulatory function 346
of APN is dependent on AMPKα2 in vitro. 347
348
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Discussion 349
Here we have made several important observations. First, we validated that PP2Cm 350
was down-regulated in diabetic mice, which may, at least in part, contribute to re-351
duced BCKD activity potentially caused by an APN deficiency. Second, APN treat-352
ment reverted down-regulated PP2Cm expression and BCKD activity, as well as ele-353
vated plasma BCAA and BCKA levels in diabetic mice. However, the increased 354
BCKD activity mediated by APN treatment was partially abolished in PP2Cm-/-
mice. 355
Third, the down-regulated BDK level was involved in APN-activated BCKD complex, 356
especially in skeletal muscle. Last, AMPK in diabetic mice was significantly impaired 357
which results in reduced BCKD activity and accumulation of BCAA and BCKA. 358
These effects could be reversed by APN injection. Importantly, APN up-regulated 359
PP2Cm expression in hepatocytes, which depends on AMPKα2. Collectively, our 360
studies have established a novel mechanism which impaired adiponectin signaling 361
contributes to diabetic BCAA catabolism. 362
BCKD complex is the most important regulatory enzyme in BCAA catabolism; the 363
activity of BCKD is regulated by a phosphorylation-dephosphorylation cycle and re-364
sponsive to alterations in various metabolic conditions (35). Studies have reported that 365
decreased BCKD activity causes down-regulation of BCAA catabolism in diabetes (6; 366
7; 36). It also has demonstrated that BDK is responsible for phosphorylation and inac-367
tivation of BCKD complex and considered a primary regulator of BCKD activation 368
(6). Diabetic and obese animals showed increased BDK expression (6; 8). Moreover, 369
alteration of metabolic status can influence BCKD activity partly via changes in BDK 370
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(3), indicating that alteration of BDP may also be important. But no information about 371
BDP is available in diabetes mellitus. Identification of a specific BDP proved elusive 372
for many years; PP2Cm was identified as the only endogenous BDP until recently (18; 373
19; 27). In the present study, we demonstrated for the first time that PP2Cm was 374
significantly decreased in liver and adipose tissue from diabetic animals, which may 375
at least partially contribute to reduction of BCKD activity and elevated plasma BCKA 376
levels. Altogether, these findings provide a new explanation for the disturbed regula-377
tion of BCAA catabolism in diabetes. 378
Hypoadiponectinemia is commonly seen in diabetes mellitus (20; 26), and APN is a 379
critical regulator in lipid and glucose metabolism (20). Moreover, Liu and colleagues 380
(24) recently demonstrated that HD-fed APN-/-
mice have significantly increased lev-381
els of muscle BCAA, which can be corrected with APN supplementation for 2 weeks. 382
However, the role of APN in systemic BCAA catabolism remains unclear. In our study, 383
APN-/-
mice fed with 45% HD for 4 weeks showed mild insulin resistance (Figure 384
S2C and S2E), but normal blood glucose levels (Figure S2A). In addition, we demon-385
strated that HD-fed APN-/-
mice had significant reduced BCKD activity and PP2Cm 386
levels, increased BDK expression, along with high plasma BCAA and BCKA concen-387
trations. Treatment with APN for 3 days significantly reversed these trends but insulin 388
resistance remained (Figure 2H-J). More importantly, APN treatment completely re-389
verted the reduction of BCKD activity and PP2Cm expression, and the elevation of 390
BDK level in diabetic models. These results suggest that hypoadiponectinemia con-391
tributes to down-regulated BCKD activity and PP2Cm expression, up-regulated BDK 392
Page 20 of 50Diabetes
21
level, as well as abnormal BCAA catabolism. This supports the notion that APN has a 393
wide range of impacts on metabolism (24). Additionally, evidence by Newgard et al. 394
(10) indicates that changes in BCAA levels contribute to the development of insulin 395
resistance. An interesting point that warrants further investigation is that the positive 396
effect of APN on BCAA may subsequently contribute to the insulin sensitizing effect 397
of APN. Lastly, BCAA levels changed acutely upon short-time APN administration, 398
whereas plasma glucose, glucose tolerance, insulin tolerance and plasma cholesterol 399
levels were unchanged (Figure 2H-2K and 3A-3C). Although the precise mechanism 400
causing the rapid BCAA regulatory response after short period APN administration 401
remains unclear, this result further support that BCAA may be useful biomarkers for 402
monitoring the early response to therapeutic interventions for metabolic disease. 403
In the present study, we observed that APN deficiency in the APN-/- mice did not 404
result in elevated BCAA levels under basal physiological condition. However, in re-405
viewing numerous previous publications utilizing this model, our observation is very 406
consistent with previous publication, showing that APN-/-
mouse does not have phe-407
notypic changes under physiological condition. However, when pathologically chal-408
lenged, such as exposure to HD (37) or myocardial ischemia (22), these animals show 409
much severer tissue injury than WT controls. These results indicate that although oth-410
er molecules present in APN-/- animals are sufficient to compensate the effect of APN 411
under physiological condition, APN plays essential role in counteracting the patho-412
logical stress, such as HD. Additionally, the observation that APN-stimulated BCKD 413
activation was partially retained and APN markedly down-regulated BDK expression 414
Page 21 of 50 Diabetes
22
in PP2Cm-deficient mice, suggesting that BDK was involved in APN-activated 415
BCKD. It has been shown in literature that thyroid hormone and sex hormones regu-416
late expression of BDK (40) and our present data also revealed that BDK was the 417
down-stream molecular which attributes to APN-mediated BCAA catabolism. Thus, 418
APN may signal both BDK and PP2Cm in opposite ways to increase BCKD activity 419
and improve BCAA catabolism in diabetes mellitus. 420
AMPK has been considered a master switch in regulating glucose and lipid metab-421
olism (38), and plays an essential role in the actions of APN (39). Nevertheless, 422
AMPK can also modulate transcription of specific genes involved in energy metabo-423
lism (31). In this study, we observed that BCKD activity was elevated by activation of 424
AMPK both in vitro and in vivo by pharmacological or genetic methods. Moreover, 425
when the AMPK inhibitor CC or siRNA was applied, the effect of APN on BCKD 426
was virtually abolished. These results might provide us with a better understanding of 427
the role of AMPK in the regulation of metabolism. 428
The possibility remains that the remote role of APN-AMPK signaling in glucose 429
and lipid metabolism could show secondary effects on BCKD activity in vivo, we thus 430
performed in vitro experiments. Because BCKD capacity mostly resides in liver (3; 431
33), mouse hepatocytes were used in present study. Here, mouse hepatocytes chal-432
lenged with BCKA were incubated with APN or AICAR helped to address the fact 433
that APN-AMPK directly up-regulated BCKD activity (Figure S4A-S4H). Interest-434
ingly, effect of APN upon BCKA occurred prior to the significant up-regulation of 435
PP2Cm levels, suggesting that APN may improve BCKA catabolism via signaling 436
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23
system in addition to up-regulation of PP2Cm. Although the detailed molecular 437
mechanisms cannot be addressed in the current study, it is possible that APN may en-438
hance BCKD/PP2Cm interaction, thus increase BCKD activity independent PP2Cm 439
expression. This interesting possibility will be directly investigated in our future study. 440
To date, it remains unclear how PP2Cm expression is regulated. Previous studies (27; 441
40) and our unpublished data have revealed that PP2Cm expression can be regulated 442
by the availability of nutrients as well as stress (i.e. ischemia and heart failure). Here 443
we demonstrated that APN was the first identified endogenous molecule that can 444
up-regulate PP2Cm level. 445
In summary, this study provides evidence that reductions of APN signaling could 446
underlie the decreased BCKD activity observed in type 2 diabetes mellitus. Our study 447
also reveals direct evidence that APN can modulate expression of hepatic PP2Cm via 448
an AMPKα2 dependent pathway. These new insights provide a better understanding 449
of the underlying regulatory mechanisms involved in diabetic BCKD activity, and 450
identify potential therapeutic targets to mitigate BCAA catabolism in metabolic dis-451
eases. 452
453
Page 23 of 50 Diabetes
24
Acknowledgments 454
This work was supported by Program for National Science Fund for Distinguished 455
Young Scholars of China(Grant No.81225001), National Key Basic Research Pro-456
gram of China (973 Program, 2013CB531204), New Century Excellent Talents in 457
University (Grant No.NCET-11-0870), National Science Funds of China(Grants No. 458
81070676 and 81170186), Program for Changjiang Scholars and Innovative Research 459
Team in University (No. PCSIRT1053) and Major Science and Technology Project of 460
China “Significant New Drug Development” (Grant No. 2012ZX09J12108-06B). 461
No potential conflicts of interests relevant to this article were reported. 462
K. L. designed methods and experiments, carried out the laboratory experiments, 463
and wrote the paper. C. S. D. performed RT-PCR analysis and analyzed data. Y. L. 464
analyzed the data and interpreted the results. D. Z. contributed to data of figure 5. W. J. 465
Y. reviewed and edited the manuscript. H. F. Z. and Z. B. H. researched data and con-466
tributed to discussion. J. L. Z., P. L. L. and H. F. P. performed animal mode and col-467
lected the samples. J. L. Z., C.G and C. X. performed BCAA and BCKA analysis. 468
H.X.C and L.Z.X contributed to discussion. L. T. defined the research theme and re-469
vised the manuscript critically. L. T. is the guarantor of this work and, as such, had 470
full access to all the data in the study and takes responsibility for the integrity of the 471
data and the accuracy of the data analysis. 472
The authors thank Dr. Xinliang Ma, Thomas Jefferson University, for help with re-473
vising the manuscript. The authors also thank Dr. Yibin Wang and Dr. Haipeng Sun, 474
Page 24 of 50Diabetes
25
University of California, Los Angeles, for the generous supply of the antibody to 475
PP2Cm and PP2Cm-/-
mice. 476
477
Page 25 of 50 Diabetes
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Figure Legends 610
Figure 1 PP2Cm Is Down-regulated in ob/ob and Type 2 Diabetic Mice 611
(A and B) Plasma BCAA and BCKA concentrations in WT, ob/ob and type 2 diabetic 612
mice. (C and D) Analysis of BCKD activity, (E) quantification of pSer293 BCKD E1α, 613
(F) PP2Cm and (G) BDK protein levels in liver, adipose tissue and skeletal muscle 614
from WT, ob/ob and type 2 diabetic mice. (H) Representative immunoblots of 615
pSer293 BCKD E1α, BCKD E1α, PP2Cm, BDK and GAPDH in WT, ob/ob and type 616
2 diabetic mice. 617
All results are presented as mean ± SEM. *Significant difference between ob/ob or 618
type 2 diabetic group versus WT group. * P < 0.05, ** P < 0.01. n = 6 - 8. L, liver; AT, 619
adipose tissue; SM, skeletal muscle. 620
621
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32
Figure 2 Adiponectin Deficiency Contributes to Decreased BCKD Activity 622
(A and B) Plasma BCAA and BCKA, (C and D) BCKD activity, (E) quantification of 623
pSer293 BCKD E1α, (F) PP2Cm and (G) BDK protein levels in liver, adipose tissue 624
and skeletal muscle from WT and APN-/-
mice fed with ND or HD. (H) Analysis of 625
blood glucose concentrations, (I) glucose tolerance tested by IPGTT, (J) insulin toler-626
ance tested by ITT, (K) blood cholesterol levels, (L and M) plasma BCAA and BCKA 627
concentrations in HD-fed APN-/-
mice after vehicle or APN treatment. (N and O) De-628
tection of BCKD activity, (P) quantitative results of pSer293 BCKD E1α, (Q) PP2Cm 629
and (R) BDK protein levels in liver, adipose tissue and skeletal muscle from HD-fed 630
APN-/-
mice injected with APN. (S) Representative Western blots of pSer293 BCKD 631
E1α, BCKD E1α, PP2Cm, BDK and GAPDH in ND or HD-fed WT and APN-/-
mice. 632
(T) Representative immunoblots of pSer293 BCKD E1α, BCKD E1α, PP2Cm, BDK 633
and GAPDH in HD-fed APN-/-
mice injected with APN or vehicle. 634
All results are presented as mean ± SEM. *Significant difference between APN-/-
and 635
WT mice fed with HD. %
Significant difference between APN and vehicle treatment. *%
636
P < 0.05, **%%
P < 0.01. n= 6 - 8. L, liver; AT, adipose tissue; SM, skeletal muscle. 637
638
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33
Figure 3 PP2Cm Deficiency Partially Inhibits Adiponectin-activated BCKD 639
(A) Analysis of blood glucose concentrations, (B) glucose tolerance tested by IPGTT 640
and (C) insulin tolerance tested by ITT in type 2 diabetic animals administered with 641
APN or vehicle. (D and E) BCAA and BCKA concentrations in plasma, (F and G) 642
BCKD activity in liver, adipose tissue and skeletal muscle from ob/ob, type 2 diabetic 643
and PP2Cm-/- mice each injected with APN or vehicle. (H) Western blot for hepatic 644
BCKD E1α phosphorylation in ob/ob, type 2 diabetic and PP2Cm-/- mice each admin-645
istered with APN. (I) PP2Cm and (J) BDK protein levels were detected by western 646
blot in liver, adipose tissue and skeletal muscle from ob/ob and type 2 diabetes mice 647
injected with APN. (K) BDK levels were assessed by Western blot in liver, adipose 648
tissue and skeletal muscle from PP2Cm-/- mice treated with APN. 649
All results are presented as mean ± SEM. %
Significant difference between APN and 650
vehicle injected group. &
Significant difference between APN treated ob/ob and 651
PP2Cm-/-
group. %&
P < 0.05, %%&&
P < 0.01. n = 5 - 8. A, APN; AT, adipose tissue; L, 652
liver; SM, skeletal muscle; V, vehicle. 653
654
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34
Figure 4 AMPK Is Necessary for Adiponectin - mediated BCKD Activation 655
(A) Expression of phospho-AMPKα at Thr172 and AMPKα levels in liver, adipose 656
tissue and skeletal muscle from type 2 diabetes mice; left, phospho-AMPKα, AMPKα 657
and GAPDH protein levels were assessed by Western blot analysis; right, 658
quantification of Western blot data. (B and C) BCAA and BCKA concentrations in 659
plasma, (D and E) BCKD activity in liver, adipose tissue and skeletal muscle from 660
type 2 diabetes mice treated with AICAR. (F) Immunoblotting of hepatic BCKD E1α 661
phosphorylation levels in type 2 diabetic mice treated with AICAR. (G) PP2Cm and 662
(H) BDK protein levels were measured by Western blot in liver, adipose tissue and 663
skeletal muscle from type 2 diabetic mice treated with AICAR or vehicle. (I and J) 664
Analysis of BCAA and BCKA in plasma, (K and L) BCKD activity in liver, adipose 665
tissue and skeletal muscle from type 2 diabetic animals treated with APN or APN plus 666
CC. (M) hepatic BCKD E1α phosphorylation levels in type 2 diabetic mice injected 667
with APN or APN plus CC. (N) PP2Cm and (O) BDK expression were detected in 668
type 2 diabetic animals treated with APN or APN plus CC. 669
All results are presented as mean ± SEM. %
Significant difference between AICAR and 670
vehicle treatment. &
Significant difference between APN and APN plus CC treat-671
ment. %&
P < 0.05, %%&&
P < 0.01. n = 5 - 8. A, APN; AT, adipose tissue; CC, com-672
pound C; L, liver; SM, skeletal muscle; V, vehicle. 673
674
Page 34 of 50Diabetes
35
Figure 5 AMPKα2 Contributes to Total AMPKα Activity in Adiponec-675
tin-stimulated BCKD Activation 676
(A) Representative immunoblots of hepatocytes lysate for pSer293 BCKD E1α, 677
BCKD E1α, AMPKα1, AMPKα2, PP2Cm and GAPDH after transfection with scram-678
ble, AMPKα1 or AMPKα2 siRNA. (B) The relative levels of PP2Cm, (C) pSer293 679
BCKD E1α were quantified and (D) BCKA concentrations were analyzed in hepato-680
cytes transfected with scramble, AMPKα1 or AMPKα2 siRNA. (E) PP2Cm protein, 681
(F) BCKD E1α phosphorylation and (G) BCKA levels in scramble, AMPKα1 or 682
AMPKα2 siRNA transfected hepatocytes treated with or without APN for 1 hour. 683
All results are presented as mean ± SEM. *Significant difference between scramble 684
siRNA and AMPKα2 siRNA group. %
Significant difference between APN and vehicle 685
treated group. % P < 0.05, **
%%P < 0.01. n = 6 - 12 wells. 686
687
688
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243x138mm (300 x 300 DPI)
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Supplemental Figure Legends
Figure S1 Adiponectin Is Reduced in Type 2 Diabetic Mice
(Related to Figure 1, Figure 3 and Figure 4 )
(A) Body weight, (B) blood glucose concentrations, (C) glucose tolerance tested by
IPGTT, (D) insulin tolerance tested by ITT and (E) fasting blood insulin levels in
normal controls and type 2 diabetic mice. (F) APN concentrations in plasma and (G)
APN mRNA levels in adipose tissue of type 2 diabetic mice and their controls.
All results are presented as mean ± SEM. *Significant difference between type 2 dia-
betic group and normal group. * P < 0.05, **P < 0.01. n = 5 - 8.
Page 41 of 50 Diabetes
Figure S2 HD-fed Adiponectin Knockout Reveals Insulin Resistance
(Related to Figure 2)
(A) Blood glucose levels, (B and C) glucose tolerance tested by IPGTT, (D and E)
insulin tolerance tested by ITT and (F) blood cholesterol levels in WT and APN-/-
mice fed with normal diet (ND) or high fat diet (45% HD).
All results are presented as mean ± SEM. *Significant difference between HD and ND
fed group. * P < 0.05, **P < 0.01. n = 6 - 8.
Page 42 of 50Diabetes
Figure S3 PP2Cm Deficiency Partially Inhibits Adiponectin-activated BCKD
(Related to Figure 3)
(A and B) Plasma BCAA and BCKA concentrations, (C) hepatic BCKD E1α phos-
phorylation levels in WT and PP2Cm-/-
mice treated with vehicle or APN for 3 days.
All results are presented as mean ± SEM. %
Significant difference between APN and
vehicle injected group. &
Significant difference between APN treated WT and
PP2Cm-/-
mice. %& P < 0.05,
%%&& P < 0.01. n = 5 - 8.
Page 43 of 50 Diabetes
Figure S4 APN up-regulates PP2Cm expression and improves BCAA catabolism
which depend on AMPK in vitro (Related to Figure 2, 3 and 4)
(A) BCKA levels, (B) BCKD phosphorylation (E1α at Ser293), (C) PP2Cm protein
and (D) PP2Cm mRNA levels in hepatocytes challenged with BCKA and treated with
APN. Measurements by western blot were made at 30 mins (blot lane 1-4, lane 2 and
4 represent APN treated group); 1 hour (blot lane 5-8, lane 6 and 8 represent APN
treated group); 3 hours (blot lane 9-12, lane 10 and 12 represent APN treated group);
24 hours (blot lane 13-16, lane 14 and 16 represent APN treated group). (E) Bio-
chemical analysis of BCKA concentrations, (F) BCKD phosphorylation, (G) PP2Cm
protein expression and (H) PP2Cm mRNA levels in BCKA challenged hepatocytes
which treated with vehicle, APN, APN + CC or AICAR. Measurements were made at
1 hour.
All results are presented as mean ± SEM. %
Significant difference between APN and
vehicle treated group. &
Significant difference between APN and APN plus CC treated
group. *
Significant difference between AICAR and vehicle treated group. %&*P <
0.05, %%&&**
P < 0.01. n = 6 - 12 wells. CC, compound C.
Page 44 of 50Diabetes
Supplemental Experimental Procedures:
Cholesterol Content Assays.
Plasma cholesterol concentrations were performed in triplicate by the Department of
Clinical Laboratory, Xijing Hospital, The Fourth Military Medical University.
Intraperitoneal Glucose Tolerance Test (IPGTT).
After a 16-hour fast, alert mice were challenged with a glucose load of 1.5g/kg, ad-
ministered via intraperitoneal injection. Tail blood was taken 0, 15, 60, and 120
minutes after the glucose load, and blood glucose levels were determined with a
OneTouch II glucose meter (Lifescan, Milpitas, CA).
Insulin Tolerance Test (ITT)
After a 16-hour fast, insulin (0.5 IU/kg, Sigma, St. Louis, MO) was administered by
intraperitoneal injection. Tail blood samples were collected at 0, 15, 30, 60, 90 and
120 minutes for the measurement of plasma glucose. Blood glucose levels were de-
tected by a OneTouch II glucose meter (Lifescan, Milpitas, CA).
Detection of Plasma Insulin Levels.
Fasting blood insulin concentrations were detected with a mouse insulin ELISA kit
(EMD Millipore, Billerica, MA) in accordance with the manufacturer’s instructions.
Determination of Plasma Total Adiponectin Concentrations.
Endogenous plasma adiponectin levels were determined with a mouse adiponectin
ELISA kit (R&D Systems, Minneapolis, MN) in accordance with the manufacturer’s
instructions.
Page 45 of 50 Diabetes
Cell Culture and BCKA Challenge.
AML12 mouse hepatocytes (ATCC, Manassas, VA) were cultured in DMEM medium
(Invitrogen, Carlsbad, CA) containing 10% fetal bovine plasma (HyClone Waltham,
MA). Experiments were carried out at 3 or 4 cell passages. After 5 hours of plas-
ma-starvation (plasma-free growth medium incubation), hepatocytes were washed
twice with PBS and incubated in DMEM medium (Invitrogen, Carlsbad, CA) which
contained an additional mixture of all of the BCKA (Sigma, St. Louis, MO) at 2.5mM.
Cell cohorts were randomized to receive one of the following treatments: vehicle
(PBS), APN (10 µg/ml), APN (10 µg/ml) + AMPK inhibitor compound C (added 30
mins before APN treatment, 20 pmol/ml), AMPK activator AICAR (2 pmol/ml). After
30 mins, 1 hour, 3 hours, or 24 hours of treatment, cells and supernatants were col-
lected for Western blotting, real-time RT-PCR and BCKA analysis.
RNA Preparation and Real-Time RT-PCR Analysis. Total RNA from mouse adi-
pocytes, liver, and cultured hepatocytes were prepared with TRIzol (Invitrogen,
Carlsbad, CA) according to the manufacturer’s instructions. Total RNA was reverse
transcribed into first-strand cDNA using the SuperScript First-Strand Synthesis Kit
(TaKaRa, Otsu, Shiga). cDNA transcripts were quantified by the Step-One Plus
RT-PCR System (Bio-Rad, Hercules, CA) using SYBR Green (TaKaRa, Otsu, Shiga).
β-actin ((TaKaRa, Otsu, Shiga) served as an endogenous control. Each reaction was
performed in triplicate and values were averaged to calculate relative expression lev-
els. Primers sequences are available upon request.
Page 46 of 50Diabetes
204x120mm (300 x 300 DPI)
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162x128mm (300 x 300 DPI)
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166x80mm (300 x 300 DPI)
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243x208mm (300 x 300 DPI)
Page 50 of 50Diabetes