豬脂肪細胞結合素及其受體功能表現調控機制之探討(2/3)

行政院國家科學委員會專題研究計畫 期中進度報告

猪脂肪細胞結合素及其受體功能表現調控機制之探討(2/3)

期中進度報告(精簡版)

中 華 民 國 96 年 06 月 22 日

Running Head: PORCINE ADIPONECTIN AND ADIPONECTIN RECEPTORS

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Key Words: Adiponectin, Adiponectin receptor, Insulin, Pigs.

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Insulin regulates the expression of adiponectin and adiponectin receptors

in mature porcine adipocytes

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Bing Hsien Liu1_{, Ya Chin Wang}1_{, Shinn Chih Wu}1,2_{, Harry John Mersmann}1_{, Winston}

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Teng Kuei Cheng1,2 _{and Shih Torng Ding}1,2

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1_{Department of Animal Science and Technology, National Taiwan University, Taipei}

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106, Taiwan 9

2_{Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan}

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Corresponding Author : 11

Shih-Torng Ding, Department of Animal Science and Technology / Institute of 12

Biotechnology, National Taiwan University 13

50, Lane 155, Kee-Long Rd. Sec. 3, Taipei 106, Taiwan 14 Phone: +886953610078 15 Fax: +886227324070 16 Email = [email protected] 17 18

Abstract

19

Adiponectin is an adipocyte-derived hormone that can improve 20

insulin-sensitivity. Its functions in regulating glucose utilization and fatty acid 21

metabolism in mammals are mediated by two subtypes of adiponectin receptors 22

(AdipoR1 and AdipoR2). This study was conducted to determine the effect of 23

insulin on the expression of adiponectin and its receptors. The expression of both 24

AdipoR mRNAs was increased in the liver and s. c. adipose tissue of fasted pigs 25

compared with fed pigs. The data also showed that the expression of either AdipoR1 26

and AdipoR2 mRNA in muscle and visceral adipose tissue was not different between 27

the fasted and fed conditions. We demonstrated that in the presence of 10 nM insulin, 28

addition of 1 uM of insulin or rosiglitazone [a peroxisome proliferator - activated 29

receptor γ (PPARγ) agonist] had no effect on the expression of adiponectin and 30

AdipoR genes in differentiated porcine adipocytes. However, the addition of 1 μM 31

insulin plus 1 μM rosiglitazone significantly increased the AdipoR2 mRNA in 32

well-differentiated porcine adipocytes. Using the phosphatidylinositol 3 - kinase 33

inhibitor (PI3K inhibitor, LY294002), we found that insulin inhibited the expression 34

of AdipoR2 through the PI3K pathway and this inhibition can be blocked by addition 35

of rosiglitazone. When porcine adipocytes were cultured without insulin, 36

supplementation with 10 nM insulin inhibited the expression of AdipoR2 and this 37

inhibition effect can also be blocked by addition of rosiglitazone. Therefore, these 38

data suggest that a PPARγ agonist increases expression of AdipoR2 and that insulin 39

inhibits the expression of AdipoR2 through the PI3K pathway. 40

Introduction

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Adiponectin is an adipocyte-produced protein hormone circulating in the blood 42

(1-4). Administration of adiponectin to mice decreases plasma glucose, free fatty 43

acids, and triglycerides, but increases muscle fatty acid oxidation and induces weight 44

loss (5). The function of adiponectin is carried out through the activation of 45

AMP-activated protein kinase (4). Adiponectin-deficient mice are mildly insulin 46

resistant and glucose intolerant when fed a standard diet (6, 7). Furthermore, 47

decreased circulating adiponectin concentrations are associated with insulin resistance, 48

obesity, and type diabetes Ⅱ (8, 9). Therefore, adiponectin may modify the function 49

of insulin in mammals. 50

Thiazolidinediones (a class of type diabetes drugs)Ⅱ , ligands for peroxisome 51

proliferator-activated receptor γ (PPARγ) increase adiponectin expression and plasma 52

adiponectin concentration in rodents (8, 10). Thiazolidinediones also increase the 53

expression of adiponectin in type Ⅱ diabetes mellitus and obese patients (11, 12). 54

Dual activation of PPARα and γ increases serum adiponectin concentration in adipose 55

tissue of obese diabetic KKAy mice (13). Furthermore, there is a functional 56

PPAR-response element (PPRE) in the promoter region of the adiponectin gene (14). 57

Thus, adiponectin should be regulated by PPARγ and its ligands. 58

Yamauchi et al. (15) first cloned the cDNA encoding adiponectin receptors 1 59

(AdipoR1) and 2 of human and mouse. These two adiponectin receptors contain 7 60

transmembrane domains, but they are structurally and functionally distinct from 61

G-protein-coupled receptors (15). Both AdipoR1 and AdipoR2 can mediate the 62

function of adiponectin and the expression of these two receptors is regulated by 63

PPARγ ligands or a combination of ligands for PPARα and PPARγ in obese patients 64

and mice(11-13). In order to clarify the regulation of gene expression of adiponectin 65

receptors in pigs, we have cloned the full length cDNA from porcine AdipoR genes 66

(16); and in this study, we determined the insulin regulation of AdipoR1 and AdipoR2 67

expression in adipocytes. We also investigated the interaction of a PPARγ ligand 68

and insulin on the expression of adiponectin and AdipoR genes in porcine adipocytes. 69

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Materials and Methods

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Fasting and feeding animals. The animal protocol was approved by the 72

Experimental Animal Care and Use Committee at National Taiwan University. Four 73

male and four female crossbred pigs (Sus domesticus; sows were predominantly 74

Landrace-Yorkshire crossbreds mated to a Duroc boar) were weaned at 28 d of age 75

and fed a commercial diet (a corn, soy-based diet containing 18% crude protein and 76

4% fat) ad libitum and raised to 60 d of age for the experiment. In order to 77

determine the effect of nutritional conditions on the expression of adiponectin and its 78

receptors, fed pigs were euthanized by electrical stunning coupled with 79

exsanguination at 10:00 h after feeding at 8:00 h, whereas the fasted group was killed 80

after a 24 h fast. Liver, longissimus muscle, visceral adipose tissue of the belly and 81

dorsal subcutaneous (s.c.) adipose tissue were dissected and frozen in liquid N2 and

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stored at -70℃ for RNA extraction. The average body weight of pigs for both 83

treatments was 20.4 + 0.66 kg when euthanized. Total RNA was extracted for 84

detecting AdipoR1, AdipoR2, and β-actin gene expression by Northern analysis 85

Isolation of porcine stromal vascular (S/V) cells. Porcine adipose tissue 86

samples were digested and S/V cells were isolated and cultured as previously 87

described (17, 18). In brief, adipose tissue from 9 d old crossbred pigs was removed 88

from the dorsal s.c. depot in the neck, shoulder, and back regions. The slices of 89

adipose tissue were digested with collagenase (Sigma C6885; Sigma, St Louis, MO, 90

USA) in sterile Krebs Ringer bicarbonate buffer at 37 for 90 min℃ . The S/V cell 91

fraction was isolated by centrifugation at 800 x g for 10 min and the pellet was 92

washed three times by resuspension with DMEM/F12 medium (Sigma D8900) 93

supplemented with NaHCO3, 100 U penicillin/mL, 100 mg streptomycin/mL, 1.5

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μg/mL amphotericin B and 10% fetal bovine serum. Before the last washing step, 95

the S/V cell fraction was treated with red blood cell lysing buffer (155 mM NH4Cl,

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5.7 mM K2HPO4, 0.1 mM EDTA at pH 7.3) to remove red blood cells which may

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reduce adhesion of S/V cells. The washed S/V cells were resuspended in 98

DMEM/F12 containing 10% fetal bovine serum and plated at a concentration of 5 x 99

104 cells/cm2. The S/V cells were then cultured at 37 in air containing 5℃ % CO2 for

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48 h to let the cells fully attach to the dish. 101

Cell culture and differentiation of porcine adipocytes. After 48 h of initial 102

incubation for proliferation (defined as day 0), the medium was removed and replaced 103

by serum-free, hormone-supplemented differentiation medium (DMEM/F12 104

containing NaHCO3, 25 mM glucose, 1 μM bovine insulin, 10 µg transferrin/mL, 2

105

mM L-glutamine, 33 μM biotin, 17 μM pantothenate, 100 nM dexamethasone, 1 nM 106

triiodothyronine, 100 U penicillin/mL, 100 mg streptomycin/mL, 1.5 μg/mL 107

amphotericin B and 1 μM rosiglitazone) for 3 d to induce adipogenesis. The 108

medium was then changed to differentiation medium without rosiglitazone. The 109

medium was replaced every 3 d. After 9 d, up to 90-95% of the attached cells were 110

differentiated to cells with visible lipid droplets. For studying the expression of 111

genes during porcine adipocyte differentiation, porcine S/V cells were induced to 112

differentiation and on the indicated days (0, 3, 6, 9), total RNA was extracted for 113

detecting adiponectin, AdipoR1, AdipoR2, and β-actin mRNA by Northern analysis. 114

The results are the means of 3 independent experiments with S/V cells isolated from 3 115

different pigs. 116

Effect of rosiglitazone and insulin on AdipoRs in porcine adipocytes. To study 117

the effect of insulin on the expression of adiponectin and AdipoRs, differentiated 118

adipocytes were washed with phosphate-buffered saline and then cultured in low 119

glucose DMEM/F12 (DMEM, Sigma D5523 : nutrient mixture F12, Sigma N6760 = 120

1 : 1, with a final glucose concentration of 7.78 mM) with 10 nM insulin for 6 hrs, and 121

then 1 μM insulin or 1 μM rosiglitazone or insulin + rosiglitazone were added for 2 , 122

12, or 24 hrs. Total RNA was extracted to detect adiponectin, AdipoR1, AdipoR2, 123

and β-actin mRNA by Northern analysis. The results are the means of 4 independent 124

experiments using cells isolated from 4 different pigs. 125

The involvement insulin signal pathways on regulation expression of AdipoRs 126

in porcine adipocytes. Porcine S/V cells were differentiated for 9 days and then 127

differentiated adipocytes were cultured in low glucose (7.78 mM) DMEM/F12 with 128

10 nM insulin for 6 h. After 6 h, 1 μM insulin［concentration was reported to be 129

effective, (19)］ + 1 μM rosiglitazone (20) were added to some cells and incubation 130

was continued for 24 h. Also at 6 h, the mitogen-activated protein kinase (MAPK) 131

inhibitor, PD98059 at 25 μM (21) or the phosphatidylinositol 3 - kinase (PI3K) 132

inhibitor, LY294002 at 10 μM (19) were added along with 1 μM insulin + 1 μM 133

rosiglitazone; incubation was continued for 24 h. Total RNA was extracted for 134

determining AdipoR1, AdipoR2, and β-actin gene expression by Northern analysis. 135

The results are the means of 4 independent experiments using cells isolated from 4 136

different pigs. 137

Insulin, rosiglitazone, and PI3K inhibitor treatments in porcine adipocytes. 138

Porcine S/V cells were differentiated for 9 days and then differentiated adipocytes 139

were cultured in low glucose (7.78 mM) DMEM/F12 without 10 nM insulin for 6 hrs, 140

then 10 nM insulin, 1 μM insulin, 1 μM rosiglitazone, or 10 μM LY294002 were 141

added to study the effect of low insulin, high insulin concentrations and insulin 142

sensitizer on the expression of AdopoRs. Total RNA was extracted for determining 143

adiponectin, AdipoR2, and β-actin gene expression by Northern analysis. The 144

results are the means of 4 independent experiments using cells isolated from 4 145

different pigs. 146

Northern analysis. Total RNA was extracted by the guanidinium - phenol - 147

chloroform extraction method (22). The integrity of RNA was determined by 148

examination of the 18S and 28S ribosomal RNA bands after electrophoresis. The 149

RNA was quantified by spectrophotometry at 260 nm and stored at -70 .℃ Total 150

RNA (10 µg of each sample) was electrophoresed and transferred to nylon membranes 151

for Northern analysis following the procedure described by Liu et al (23). The 152

porcine adiponectin, AdipoR1, AdipoR2 and β-actin probe sequences were previously 153

described (16, 18) and labeled by P32dC with PCR amplification. Hybridization 154

blotting images were quantified using a Typhon 9200 phosphorimage scanner and 155

ImageQuant TL v2005 software (GE). The densitometric value for an individual 156

transcript in a sample lane was normalized to the densitometric value for the β-actin 157

mRNA in the same lane. 158

Statistical analysis. Data were presented as mean ± S.E.M. Statistical 159

analysis were using an ANOVA procedure to determine the major effects of insulin, 160

rosiglitazone, and insulin signaling pathway blockers. Duncan’s new multiple-range 161

test was used to evaluate differences among means (SAS Inst., Inc., Cary, NC). A 162

significant difference was indicated at P ≤ 0.05. 163

164

Results and Discussion

165

Fasting increased AdipoR genes expression in s.c. adipose tissue and liver. In 166

porcine s.c. adipose tissue and liver, the AdipoR1 and AdipoR2 mRNAs increased 167

(P<0.05) after a 24 h fast, but there was no effect of fasting on the expression of 168

either receptor in the visceral adipose tissue and muscle (Fig. 1A and 1B). The data 169

suggest differential regulation of AdipoR1 and AdipoR2 by nutritional status in 170

various tissues. Previously, we indicated that the expression of AdipoR2, but 171

AdipoR1 and adiponectin was increased in the s.c. adipose tissue after an 8 h fast 172

(16). In rodents, similar to the current study in pigs, both AdipoR1 and AdipoR2 173

gene expression are up-regulated after 48 h fasting in the liver (24). The reason 174

that fasting increased the expression of AdipoR genes in mouse muscle (24), but not 175

in pig muscle is not known. We speculate that the 48 h fast in the mouse is more 176

extreme than the 24 h fast in the pig; this difference may also contribute to the 177

observed species difference. Others have showed that serum adiponectin remained 178

stable concentrations during 72 h of fasting in normal- and over-weight humans (25). 179

Thus, hormonal regulation of the feeding / fasting status on adiponectin function 180

may mainly act through regulating the expression of AdipoRs. In summary, hepatic 181

and s.c. adipose tissue AdipoR genes, but not visceral adipose tissue and muscle 182

AdipoR genes, were responsive to the feeding / fasting status in pigs. 183

Expression of adiponectin and AdipoR genes during porcine adipocyte 184

differentiation. Expression of both adiponectin and AdipoR2 increased during 185

adipocyte differentiation, whereas expression of AdipoR1 increased during the initial 186

3 d with no further increase during the latter stages of differentiation (Fig. 2). The 187

data suggest that either AdipoR2 is more sensitive to the hormones present in the 188

adipocyte cell culture medium than AdipoR1 or the AdipoR2 is a more important 189

receptor mediating adiponectin function in the differentiated adipocyte. A recent 190

study indicates that adiponectin increases PPARγ2 expression in porcine adipocytes 191

(26), suggesting that adiponectin may involve in regulating the PPARγ2 function 192

during adipogenesis. Furthermore, overexpression of adiponectin not only 193

promotes adipogenesis by prolonging and enhancing the key adipogenic 194

transcription factors, ie., PPARγ, CCAAT/enhancer binding protein α (C/EBPα), and 195

adipocyte determination and differentiation factor 1/sterol-regulatory element 196

binding protein 1c (ADD1/SREBP1c), but also stimulates glucose uptake through 197

increasing glucose transporter 4 gene expression in 3T3-L1 adipocytes (27). 198

Therefore, a greater expression of the adiponectin gene in the latter stages of porcine 199

adipocyte differentiation may function to enhance or maintain adipocyte morphology 200

through the regulation of PPARγ and other adiponectin target genes. 201

Fu et al. (27) found that the expression of both AdipoR genes were 202

down-regulated in adiponectin-overexpressing adipocytes, suggesting that 203

adiponectinmay act in an autocrine or paracrine fashion to regulate the function of 204

its receptors in adipose tissue. A similar study also found that adiponectin 205

downregulated its own production and the expression of its AdipoR2 receptor in 206

transgenic mice (28). The receptor down-regulation may decrease the adiponectin 207

responses to slow the adipogenic progression; however, perhaps receptor 208

down-regulation only occurs when adiponectin concentration reaches extreme levels, 209

as in the overexpressing cells and mice. 210

Effects of insulin and rosiglitazone on expression of adiponectin and AdipoRs. 211

Treatment with a high concentration of insulin for 2 to 24 hr did not change the 212

expression of adiponectin or the AdipoRs (Fig. 3). In contrast, several studies 213

indicate that insulin inhibits the expression of adiponectin and AdipoR genes in 214

adipocytes of humans and rodents (13, 19, 29). We cannot exclude the possibility 215

that the 10 nM insulin concentration used in the pre-incubation medium was great 216

enough to suppress the expression of adiponectin and AdipoR genes so that the 217

higher insulin concentration (1 μM) used to test for insulin effects had no additional 218

effect. 219

The PPARγ agonist, rosiglitazone alone did not increase the expression of 220

adiponectin or the AdipoRs (Fig. 3). However, the combination of a high insulin 221

concentration (1 μM) plus rosiglitazone (1 μM) increased expression of adiponectin 222

and AdipoR2 in porcine adipocytes. In 3T3-L1 adipocytes cultured with DMEM + 223

10% FBS, rosiglitazone does not affect the expression of adiponectin, even though 224

there is a PPAR response element (PPRE) in the adiponectin promoter region (30). 225

Rosiglitazone does increase plasma adiponectin concentration in humans and mice in 226

vivo (30, 31). The authors suggest that an adequate insulin concentration may be 227

necessary to observe the rosiglitazone-stimulated increase in the expression of the 228

adiponectin gene (30). Recent studies also showed that AdipoR2, but not AdipoR1, 229

was increased by rosiglitazone treatment in mouse primary adipocytes, and HepG2 230

hepatocytes cultured with FBS (13, 20). 231

In order to clarify the relationship between insulin and the PPARγ agonist, we 232

used insulin signal pathway inhibitors to block phosphatidylinositol 3-kinase (PI3K) 233

or mitogen-activated protein kinase (MAPK). Expression of AdipoR2, but not 234

AdipoR1 mRNA was increased in the presence of 1 μM insulin + 1 μM rosiglitazone 235

(Fig. 4), confirming data in Fig. 3B, C. Addition of the MAPK inhibitor, PD98059 236

had no effect on expression of AdipoR1 or AdipR2 mRNA. However, the AdipoR2 237

mRNA was increased when the PI3K signal pathway was inhibited by addition of 238

LY294002, suggesting that insulin may decrease the expression of AdipoR2 through 239

the PI3K pathway. Tsuchida et al. (24) recently demonstrated that insulin 240

suppressed the expression of AdipoRs via the PI3K/Foxo1-dependent pathway in 241

rodent hepatocytes and myocytes. Although we did not observe that insulin alone 242

decreased AdipoR2 mRNA expression, the increase in AdipoR2 mRNA when the 243

PI3K pathway was inhibited by addition of LY294002, suggests that insulin has at 244

least a partial suppressive effect via the PI3K pathway. In addition, studies also 245

showed evidence of a direct insulin-induced inhibition of AMPK activity through 246

PI3K/Akt pathway in the heart (32), which is a possible mechanism by which the 247

expression of AdipoRs are inhibited. 248

In order to exclude the possibility that 10 nM insulin, used in most experiments 249

to maintain cell viability, suppressed adiponectin or AdipoRs gene expression, we 250

designed another experiment to clarify the interaction of insulin and a PPARγ agonist 251

on adiponectin and AdipoRs gene expression. After treating with insulin free 252

medium for 6 h, addition of 10 nM insulin reduced both adiponectin and AdipoR2 253

(Fig. 5A) mRNAs, indicating a minute amount of insulin suppresses the expression 254

of adiponectin and AdipoR2 genes. In pigs, the normal concentration of insulin is 255

maintained at 106 ± 12.5 pM and increased to 850 ± 137.5 pM 30 mins post-prandial 256

(33). Hence, supplementation with 10 nM or 1 μM insulin in porcine adipocyte 257

culture is much higher than concentrations in vivo. In mammals, the concentration 258

of feeding / fasting insulin could not affect the abundance of adiponectin (16, 25). 259

Insulin may regulate the ratio of high molecular weight adiponectin (active form) or 260

the expression of AdipoRs to modify the adiponectin function. This high insulin 261

concentration is similar to the conditions in type II diabetes or other 262

hyperinsulinemia-induced metabolism syndromes. 263

Adiponectin overexpressed mice have been shown to be able to improve insulin 264

resistance (34), whereas adiponectin deficient mice were shown to be mildly insulin 265

resistant and glucose intolerant when fed a standard diet, and severe insulin resistant 266

triggered by high fat diet (6, 7). Mice lacking adiponectin show decreased hepatic 267

insulin sensitivity and reduced responsiveness to PPARγ agonists (35). In addition, 268

several SNPs on adiponectin and AdipoR genes and other environmental factors such 269

as obesity appears to be hypoadiponectinemic and associated with type II diabetes 270

(36). Hence, regulation of adiponectin function may have therapeutic potential for 271

treating type II diabetes mellitus and obesity. 272

Because insulin decreased the expression of adiponectin and AdipoR2, we 273

cultured well-differentiated adipocytes in medium without insulin for 6 h. Addition 274

of the PPARγ agonist, rosiglitazone to these cells for 24 h significantly increased 275

adiponectin mRNA expression, but not AdipoR2 mRNA (Fig. 5B). It should be 276

noted that in the presence of 10 nM insulin, rosiglitazone had no effect on the 277

expression of AdipoR2 (Fig. 3C). This result confirmed data from our first 278

experiment and was similar to that reported in differentiated human myotubes (37), 279

indicating that the PPARγ agonist may improve insulin sensitivity by increasing the 280

serum adiponectin level, but had no effect on its receptors. Recently research also 281

showed that administrating TZD for type 2 diabetes mellitus to improve insulin 282

sensitivity and increase adiponectin levels did not affect the expression of AdipoR1 283

and AdipoR2 in muscle and adipose tissue (38). Although rosiglitazone may 284

require the presence of insulin to increase the expression of adiponectin and 285

AdipoR2 genes (Fig. 3), the effect of rosiglitazone might be in reversing the 286

inhibitory effect of insulin. It was interesting that LY294002 promoted the 287

expression of both adiponectin and AdipoR2 mRNAs more strongly than insulin 288

alone, rosiglitazone alone or rosiglitazone plus insulin (Fig. 5). Hence, these 289

findings suggest that insulin suppresses the expression of adiponectin and AdipoR2 290

mRNAs through PI3K pathway. When PI3K pathway blocked, both rosiglitazone 291

and insulin could increase the expression of adiponectin and AdipoR2 mRNAs 292

through a yet to be determined mechanism. 293

In conclusion, the expression of the adiponectin and AdipoR2 mRNAs is more 294

strongly inhibited by insulin and stimulated by the PPARγ agonist, rosiglitazone than 295

the AdipoR1 mRNA. The PPARγ agonist effects depend on the presence of insulin, 296

perhaps act in a permissive manner. The insulin effect mimicking the type II 297

diabetes was mediated through the PI3K pathway as indicated by use of the PI3K 298

inhibitor, LY294002. Therefore, understanding of interaction between insulin, 299

PPARγ and expression of adiponectin and its receptors will provide mechanisms that 300

may lead to control of adipose fat deposition and to the treatment of type II diabetes. 301

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J, Kasuga M, Accili D, Tobe K, Ueki K, Nagai R, Kadowaki T.

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Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors 391

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26. Ajuwon KM, Spurlock ME. Adiponectin inhibits LPS-induced NF-kappaB

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activation and IL-6 production and increases PPARgamma2 expression in 397

adipocytes. Am J Physiol Regul Integr Comp Physiol 2005;288:R1220-1225. 398

27. Fu Y, Luo N, Klein RL, Garvey WT. Adiponectin promotes adipocyte

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differentiation, insulin sensitivity, and lipid accumulation. J Lipid Res 400

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28. Bauche IB, Ait El Mkadem S, Rezsohazy R, Funahashi T, Maeda N,

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Miranda LM, Brichard SM. Adiponectin downregulates its own production

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29. Korner A, Wabitsch M, Seidel B, Fischer-Posovszky P, Berthold A,

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Stumvoll M, Bluher M, Kratzsch J, Kiess W. Adiponectin expression in

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humans is dependent on differentiation of adipocytes and down-regulated by 408

humoral serum components of high molecular weight. Biochem Biophys Res 409

Commun 2005;337:540-550. 410

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M, Berg AH, O'Rahilly S, Savage DB, Chatterjee K, Weiss S, Larson PJ,

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Gottesdiener KM, Gertz BJ, Charron MJ, Scherer PE, Moller DE.

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Induction of adipocyte complement-related protein of 30 kilodaltons by 414

PPARgamma agonists: a potential mechanism of insulin sensitization. 415

Endocrinology 2002;143:998-1007. 416

31. Kralisch S, Klein J, Bluher M, Paschke R, Stumvoll M, Fasshauer M.

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2005;6:863-872. 419

32. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR. Akt activity

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negatively regulates phosphorylation of AMP-activated protein kinase in the 421

heart. J Biol Chem 2003;278:39422-39427. 422

33. Rayner DV. The relationships between glucose absorption and insulin

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secretion and the migrating myoelectric complex in the pig. Exp Physiol 424

1991;76:67-76. 425

34. Combs TP, Pajvani UB, Berg AH, Lin Y, Jelicks LA, Laplante M,

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Nawrocki AR, Rajala MW, Parlow AF, Cheeseboro L, Ding YY, Russell

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RG, Lindemann D, Hartley A, Baker GR, Obici S, Deshaies Y, Ludgate M,

428

Rossetti L, Scherer PE. A transgenic mouse with a deletion in the

429

collagenous domain of adiponectin displays elevated circulating adiponectin 430

and improved insulin sensitivity. Endocrinology 2004;145:367-383. 431

35. Nawrocki AR, Rajala MW, Tomas E, Pajvani UB, Saha AK, Trumbauer

432

ME, Pang Z, Chen AS, Ruderman NB, Chen H, Rossetti L, Scherer PE.

433

Mice lacking adiponectin show decreased hepatic insulin sensitivity and 434

reduced responsiveness to peroxisome proliferator-activated receptor gamma 435

agonists. J Biol Chem 2006;281:2654-2660. 436

36. Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K.

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Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the 438

metabolic syndrome. J Clin Invest 2006;116:1784-1792. 439

37. Kaltenbach S, Staiger H, Weisser M, Haas C, Stumvoll M, Machicao F,

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Haring HU. Adiponectin receptor gene expression in human skeletal muscle

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cells is not regulated by fibrates and thiazolidinediones. Int J Obes (Lond) 442

2005;29:760-765. 443

38. Li W, Tonelli J, Kishore P, Owen R, Goodman E, Scherer PE, Hawkins

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MA. Insulin-Sensitizing Effects of Thiazolidinediones Are Not Linked to

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Adiponectin Receptor Expression in Human Fat or Muscle. Am J Physiol 446

Endocrinol Metab 2007;292:E1301-E1307. 447

448 449

Fig 1. The effect of fasting on AdipoR1 and AdipoR2 gene expression in pigs. 450

Samples of liver, longissimus muscle, visceral adipose tissue (VAT) and subcutaneous 451

adipose tissue (AT) were taken 2 hrs after feeding (Fed) or 24 hrs after feeding 452

(Fasted). The total RNA from each tissue of each pig (10 μg) was electrophoresed 453

and transferred to a nylon membrane. The membranes were hybridized with cDNA 454

probes for adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), and 455

β-actin. The mRNA abundance was determined by phosphorimage technology and 456

the densitometeric value for each gene was normalized to β-actin. The data 457

represent the means of 4 crossbred pigs (2 males and 2 females) per treatment. Data 458

were analyzed with ANOVA and Duncan’s new multiple range test was used to 459

evaluate the differences from the fed AT value (set to 100). Each bar represents the 460

mean ± S.E.M.; *, P ≤ 0.05; **, P ≤ 0.01. 461

462

Fig 2. Differentiation-dependent adiponectin, and AdipoR genes expression in 463

porcine adipocytes. Porcine S/V cells were differentiated and on the indicated days 464

(0, 3, 6, 9), total RNA was extracted for detecting adiponectin, AdipoR1, AdipoR2, 465

and β-actin gene expression by Northern analysis. The results are the means of 3 466

independent experiments with S/V cells isolated from 3 different pigs. The average 467

of AdipoR1 day 0 data was set to 100 and other data were expressed as relative 468

abundance to this value. Each point represents the mean ± S.E.M. and mRNA 469

values were normalized to β-actin. Means without a common letter differ 470

significantly, P ≤ 0.05. 471

472

Fig 3. The effect of rosiglitazone and insulin on adiponectin, and AdipoR genes 473

expression in porcine adipocytes. Porcine S/V cells were differentiated for 9 days, 474

as indicated in Fig 2. The 9 days differentiated adipocytes were then cultured in low 475

glucose (7.78 mM) DMEM/F12 with 10 nM insulin for 6 hrs, and then 1 μM insulin 476

or 1 μM rosiglitazone (Rosi) or insulin + Rosi were added for 2 , 12or 24 hrs. Total 477

RNA was extracted to detect adiponectin (panel A), AdipoR1 (panel B), AdipoR2 478

(panel C), and β-actin gene expression by Northern analysis, as indicated in Fig 2. 479

The results are the means of 4 independent experiments using cells isolated from 4 480

different pigs. The average of the 2 h control medium data was set to 100 and other 481

data were expressed on relative abundance to the 2 h data. Each point represents the 482

mean ± S.E.M.; *, P ≤ 0.05. 483

484

Fig 4. The effect of an insulin signal pathway inhibitor on rosiglitazone-mediated 485

induction of expression of AdipoR genes in porcine adipocytes. Porcine S/V cells 486

were differentiated for 9 days and then differentiated adipocytes were cultured in low 487

glucose (7.78 mM) DMEM/F12 with 10 nM insulin for 6 h. Control cells were 488

incubated in this medium for an additional 24 h. After 6 h, 1 μM insulin (I) plus 1 489

μM rosiglitazone (R) were added to some cells and incubation was continued for 24 h. 490

Also at 6 h, the MAPK inhibitor, PD98059 at 25 μM (PD) or the PI3K inhibitor, 491

LY294002 at 10 μM (LY) were added along with I + R; incubation was continued for 492

24 h. Total RNA was extracted for detecting AdipoR1, AdipoR2, and β-actin gene 493

expression by Northern analysis as indicated in Fig 2. The results are the means of 4 494

independent experiments using cells isolated from 4 different pigs and the average of 495

the 24 h control medium data was set to 100 with other data expressed as relative 496

abundance to the control value. Each bar represents the mean ± S.E.M. and mRNA 497

values were normalized to β-actin. Means without a common letter differ 498

significantly, P ≤ 0.05. 499

500

Fig 5. The effects of insulin and rosiglitazone on the expression of adiponectin and 501

AdipoR genes in porcine adipocytes. Porcine S/V cells were differentiated for 9 502

days and then differentiated adipocytes were cultured in low glucose (7.78 mM) 503

DMEM/F12 without 10 nM insulin for 6 hrs, then 10 nM insulin (10 nM I), 1 μM 504

insulin (I), 1 μM rosiglitazone (Rosi), or 10 μM LY294002 (LY) were added to 505

separate plates for 24 hrs. Total RNA was extracted for detecting adiponectin, 506

AdipoR2, and β-actin gene expression by Northern analysis as indicated in Fig 2. 507

The results are the means of 4 independent experiments using cells isolated from 4 508

different pigs and the average of 24 hrs control medium data was set to 100 with other 509

data expressed as relative abundance to the control value. Each bar represents the 510

mean ± S.E.M. and mRNA values were normalized to β-actin. Means without a 511

common letter differ significantly, P ≤ 0.05. 512

Table 1. List of the Northern analysis probes 513

Genes1 _Primers2 _{Source of primers Size, bp} S 5‘-GCTCAGGATGCTGTTGTTGG-3‘

Adiponectin

(AY589691) A 5‘-TGGTGGAGGCTCTGAGTTGG-3‘ Pig 768

S 5‘-AGGTACCAGCCAGATGTCTT-3‘ AdipoR1

(AY578142) A 5‘-CTCTTCCTCTCACTTCAGCA-3‘ Human 1220 S 5‘-AAAGGCTTGGGTATCCCATG-3‘

AdipoR2

(AY606803) A 5‘-CTCCTCTGGTACTGGCATCA-3‘ Human 1391 S 5'-GTGGGCCGCTCTAGGCACCA-3'

β-actin

(AF054837) A 5'-CGGTTGGCCTTAGGGTTCAGGGGGG-3' Mouse 245

514

1 _{GenBank accession number is indicated parenthetically.}

515 516 2 _{S: sense; A: antisense.} 517 518 519

A

AT VAT Muscle Liver

AdipoR1 mRNA abundance,

arbit ray units 0 20 40 60 80 100 120 140 160 180 Feeding Fasting

*

*

B

AT VAT Muscle Liver

AdipoR2 mRNA abundance, arbitray unit s 0 20 40 60 80 100 120 140 160 Feeding Fasting

**

531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 Adiponectin 548 AdipoR1 549 AdipoR2 550 β-actin 551 Fig 2. 552 553 554 555 556 557

0 day

3 day

6 day

9 day

R

e

lative m

R

NA abundanc

e,

arbitray unit

s

b

b

b

b

d

de

e

_e

c

a

A

2h

12h

24h

_Control

Insulin

Rosi

Insulin+Rosi

*

B

2 h

12 h

24 h

AdipoR1 mRNA abundance, arbitray units

60 80 100 120 140 160 180 200

_Control

Insulin

Rosi

Insulin+Rosi

C

2 h

12 h

24 h

AdipoR2 mRNA abundanc

e, ar bitray units 40 60 80 100 120 140 160 180 200

_Control

Insulin

Rosi

Insulin+Rosi

*

AdipoR1

AdipoR2

AdipoRs relative mRNA abundance, arbitray units

0 50 100 150 200 250

Control 24h

In + Rosi 24h

In + Rosi + LY 24h

In + Rosi + PD 24h

a

b

b

c

A d ip o n e c tin A d ip o R 1

A d ip o R 2

Relative mRNA abunda

nce, arbitray units

0 5 0 1 0 0 1 5 0 2 0 0 c o n tr o l 1 0 n M I 1 u M I L y 1 u M I + L y a b b c c a b a c c b

A d ip o n e c tin A d ip o R 1

A d ip o R 2

Relative mRNA abundance, arbitray units

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 c o n tr o l R o s i R o s i+ L Y I+ R o s i I+ R o s i+ L Y a b b a b a b a d c d b c 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 Fig 5. 590

A

B

行政院國家科學委員會補助國內專家學者出席國際學術會議報告

94-2313-B-002-024

(英文)Experimental Biology 2007 and USDA NCC0097 發表

論文 題目

(中文)猪 PPAR d 在脂肪細胞分化的功能

( 英 文 )

The functionality of porcine peroxisomal proliferator

activated receptor delta in adipocyte differentiation

報告內容應包括下列各項：

一、 參加會議經過

April 26 Flew to Washington DC, USA. Stay in Red Roof Inn, 500 H Street NW, Washington DC 20001.

April 27 Attended the meeting for USDA regional meeting NCC0097, Presented a talk on two topics: The function of PPARg and PPARd in pigs and The expression of genes in porcine adipose tissue under the treatment of porcine serum amyloid protein A. The fee for the meeting is 70 US dollars. Went to the dinner function with the scientists with common interests in adipocyte biology. The dinner cost for 67 US dollars. April 28. Attended the Keynote speech by Two scientist both named Tony on the discovery of tyrosine kinases and its function in regulating physiological process and gene expression in ASBMB Biochemistry session. Attended the poster section on lipid metabolism. Attended the evening session on enzyme expression regulation.

April 29. Attended the Keynote speech on Phosphoinisitide molecules and the genes involved in making the enzymes. What we learn from yeast model. Also attend Lipid metabolism. Went to Symposium on animal model for human nutrition 1 delivered by DH Baker and 2 delivered by Spurlock on adipocyte models. Also went to a

biochemistry teaching for cultivating future strong biochemist. The speakers were too good. But picked up some points, including bring research into classroom, set the expectation high, recognize the students, enthusiasm in research is contagous, and interactive teaching to enhance student learning. We also posted our poster. The full length of the report is attached at the end of the report.

April 30. Attended the ASBMB Merck Award for the talk on PEPC Kinase by R.W. Hanson. I also attended a section on role of nuclear receptors in metabolic syndrome in the morning. Went to poster section for genetics and metabolic approaches to obesity and proteionics: proteomics and bioinformatics. Went to Symposium on lipid

metabolism and transport and also lipid signaling track.

May 1. I attended the Nutrition section and mostly the poster and two keynote speeches. Two areas are very important, one was on companion animal nutrition and the other was on aging. There were lots of research on the feed limitation on aging and well-being of animals. Two specific experiments on monkey long term

restriction on feeding improve the health condition and longevity.

May 2. Attended the FASEB Excellence in Science Award before we took off to Columbus, Ohio for an international exchange program discussion.

表 Y04

二、 與會心得

This year I started to get the feeling of how well we have done researchwise. We have demonstrated a very specific pathway of regulating nutrition metabolism and such a finding is world-class. We need to collect the most recent progress of the research in order to know where we are and what to do to compete with international scientists. This meeting always has a lot of activities going on. Lots of science to learn, but too little time available. I have also got to interact with lots of scientist in my field of research. That help me develop the sense of where we are and our competitiveness.

三、 考察參觀活動(無是項活動者省略)

四、 建議

The areas of nutritional science research are evolving very fast during the past few years. This Meeting collects a broad spectrum of research progress which is

important for the researchers in Taiwan. I would suggest that we should encourage researchers to go and joint this meeting to get updated research progress report and to improve our research quality.

五、 攜回資料名稱及內容

Experimental Biology 2007, Conference information and scientific program in a CD format. All the station reports from the USDA regional meeting NCR-97.

六、 其他

The full article of our presentation.

Ectopic expression of porcine peroxisome-proliferator-activated receptor delta regulates adipogenesis in myoblasts

Y. H.Yu1 _{and S. T. Ding}1

Department of Animal Science and Technology, National Taiwan University, 50, Lane 155, Kee-Long Rd. Sec. 3, Taipei 106, Taiwan 1

ABSTRACT

It is well known that peroxisome-proliferator-activated receptor γ (PPAR γ) plays a critical role in regulating adipogenesis. In rodents, PPAR δ is expressed before PPAR γ during adipocyte differentiation. Thus, the interaction between PPAR δ and PPAR γ during adipogenesis needs to be elucidated. The current experiment was designed to study the interaction of porcine PPAR δ and PPAR γ in mouse myoblast cells. Inhibition of myogenesis was observed in myoblasts expressing porcine PPAR δ, similar to myoblast expressing PPAR γ. Treatment of myoblasts expressing PPAR δ with ligands for both PPAR δ and γ enhanced lipogenesis to a greater extent than treatment with a PPAR γ ligand alone. The ability to transdifferentiate myoblasts into adipocytes was decreased in myoblasts co-expressing PPAR δ with either wild-type or mutated PPAR γ (serine 112 was mutated to alanine) compared to myoblasts expressing either type of PPAR δ alone. Adipose transdifferentiation in myoblasts co-expressing PPAR δ and mutated PPAR γ was greater than in myoblasts co-expressing PPAR δ and wild-type PPAR γ. Our results suggest that PPAR δ has two different roles in regulating adipogenesis, ie., suppression of myogenesis to enhance transdifferentiation of myoblasts into adipocytes and interaction with PPAR γ to modify adipogenesis. Therefore, PPAR δ may have a significant role in adipogenesis.

Key Words: Adipocyte differentiation, Peroxisome proliferator-activated receptor δ, Peroxisome

proliferator-activated receptor γ.

INTRODUCTION

In rodent, peroxisome-proliferator-activated receptor δ (PPAR δ) is wildly expressed in several tissues, including adipose tissue, intestine, skeletal muscle, lung and heart. The

INTRODUCTION

In rodent, peroxisome-proliferator-activated receptor δ (PPAR δ) is wildly expressed in several tissues, including adipose tissue, intestine, skeletal muscle, lung and heart. The expression of PPAR δ in proliferating preadipocytes is undetectable and increases gradually during adipocyte differentiation (Amri et al., 1995). Preadipocyte overexpressing PPAR δ with long chain fatty acids promotes adipogenesis (Bastie et al., 2000). Ectopic expression of PPAR δ in fibroblasts with long chain fatty acids alone do not induce adipogenesis but stimulation in the presence of PPAR γ ligand (Bastie et al., 1999). Therefore, PPAR δ seems to have a facilitating role in adipogenesis.

The information of porcine PPAR δ is still poorly understood, especially in functional study. In previous studies, we have demonstrated that ectopic expression of porcine PPAR γ induces adipogenesis in myoblasts (Yu et al., 2006). The expression of PPAR δ is earlier than PPAR γ during adipocyte differentiation in rodent adipocytes (Amri et al., 1995). We hypothesize that a relationship between PPAR δ and PPAR γ in regulating adipocyte differentiation. In this study, we created C2C12 myoblasts expressing porcine PPAR δ, or co-expressing PPAR δ with either wild-type or mutated PPAR γ (serine 112 was mutated to alanine). Transfected myoblasts with porcine PPAR δ stimulated adipogenesis after addition of both PPAR δ and PPAR γ ligands, whereas a decreased lipid accumulation was observed in myoblasts co-expressing PPARs compared with expressing PPAR γ alone.

MATERIALS AND METHODS

Stably transformed cells with PPAR δ or PPAR γ and induction of myoblast transdifferentiation

The porcine PPAR δ cDNA was cloned from porcine adipose tissue. The PCR products were cloned into a mammalian expression vector and transfected into C2C12 myoblasts by lipofection. To establish expression of both porcine PPAR δ and PPAR γ cell models, C2C12 myoblasts containing either wild-type PPAR γ or mutated PPAR γ were also transfected with porcine PPAR δ. Myoblasts stably expressing PPAR δ were established by puromycin selection. After drug selection, the cells were cultured without selection medium and allowed to propagate to 80% confluence in DMEM with 10% FBS. Confluent cells were then cultured in adipogenic differentiation medium [DMEM containing 10% fetal bovine serum, 1μM dexamethasone, and 5μg/mL insulin] and with or without 1 μM rosiglitazone, a PPAR γ ligand and 1 μM L165041, a PPAR δ ligand. After 10 days of culture, total RNA was purified to determine gene expression.

Northern blot and statistical analysis

The RNA was separatedby electrophoresis and blotted to nylon membranes. The membrane was prehybridized at 42 ℃ and then hybridized withisotope labeled complementary DNA probes. Hybridization results were quantified by phosphor-image analysis. The densitometric value for an individual transcript in a sample lane was normalized to the densitometric value for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same lane. The treatment effects were analyzed using an ANOVA procedure to determine the main effects of the form of PPAR δ and PPAR γ in presence or absence of its ligands. Duncan’s new multiple range test was

used to evaluate differences among means (SAS Inst. Inc., Cary, NC). A significant difference indicates that P value is not greater than 0.05.

RESULTS AND DISCUSSION

The presence or absence of rosiglitazone and L165041 in adipogenic medium had no effect on myogenesis (Figure 1B, C and D). The myotube formation was inhibited when myoblast expressing PPAR δ compared with transfection of empty vector cells (Figure 1A vs. E). Similar results were observed in our previous studies, myoblasts containing PPAR γ had an ability to interfere in myocyte differentiation (Yu et al., 2006). After exposure of rosiglitazone to the adipogenic differentiation medium for 10 days, lipid-droplets were visualized in myoblasts expressing PPAR δ but absence in addition L165041 in adipogenic differentiation medium (Figure 1). It was well known that ligands for PPARs can activate more than one receptor isoform, hence adipocyte differentiation was increased in medium containing rosiglitazone even if absence of L165041 (Figure 1F). The maximum of lipid accumulation was observed in addition of both PPAR ligands (Figure 1H). This result suggests indirectly that PPAR γ with its ligand has crucial potential in modulating adipocyte differentiation. In loss of function study, it has been demonstrated that lipid accumulation and adipogenic marker genes are decreased in PPAR δ -null adipocytes (Matsusue et al., 2004). In our results, myoblats expressing PPAR δ with PPAR γ and PPAR δ ligands enhanced adipogenesis. It implies that PPAR δ appeared to accelerate adipogenesis. The downstream gene of PPAR γ, adipocyte fatty acid binding protein (aP2) mRNA was highly expressed in myoblasts containing PPAR δ in the presence of rosiglitazone. However, addition of PPAR δ and PPAR γ ligands in adipogenic medium had a greater stimulation of aP2 expression compared with presence of single PPAR ligand. For lipoprotein lipase (LPL) mRNA, it was also increased in the same condition. It has been known that expression of aP2 and LPL are regulated by PPAR γ. Deducing from our results, we hypothesize that high aP2 and LPL transcripts were attributed to PPAR γ function and ectopic PPAR δ modulated PPAR γ expression by binding its peroxisome proliferator response element. A late myogenic marker gene, myogenin was decreased in cells transfected with PPAR δ and both of ligands had no effect on myogenesis (Figure 3). Similar results were observed in another myogenic marker gene, myogenic regulatory factor 4 (MRF4). The suppression of myogenic marker genes in myoblasts expressing PPAR δ was consistent with ectopic expression of PPAR γ in myocytes. These results demonstrated that adipogenesis related transcription factors have the capability of impairing myogenesis. Furthermore, mRNA for aP2 and LPL were expressed at a low level in myoblasts containing either wild-type PPAR γ or mutated PPAR γ and PPAR δ compared with expressing either wild-type PPAR γ or mutated PPAR γ alone (Figure 4). This phenomenon was also found in preadipocyte expressing PPAR δ and PPAR γ. The over-expression of PPAR δ indeed can suppress PPAR γ-mediated adipogenesis (Shi et al., 2002). However, reduction of myogenic genes expression was enhanced in C2C12 myoblasts containing both PPARs (Figure 5). Thus, PPAR δ and PPAR appeared to have a synergic effect in the inhibition of myogenesis.

CONCULSION

In the current study, we demonstrated that PPAR δ has the ability to promote transdifferentiation of myoblasts into adipocytes and interact with PPAR γ to modify adipogenesis. Therefore, PPAR δ may have a significant role in adipogenesis.

REFERENCES

Amri, E., F. Bonino, G. Ailhaud, N. A. Abumrad, and P. A. Grimaldi. 1995. Cloning of a protein that mediates transcriptional effects of fatty acids in preadipocytes. J. Biol. Chem. 270： 2367-2371.

Bastie, C., D. Holst, D. Gaillard, C. Jehl-Pietri, and P. A. Grimaldi. 1999. Expression of peroxisome proliferators-activated receptor PPARδ promotes induction of PPARγ and adipocyte differentiation in 3T3C2 fibroblasts. J. Biol. Chem. 274：21920-21925.

Bastie, C., S. Luquet, D. Holst, C. Jehl-Pietri, and P. A. Grimaldi. 2000. Alterations of peroxisome proliferators-activated receptor δ activity affect fatty acid-controlled adipose differentiation. J. Biol. Chem. 275：38768-38773.

Liu, B. H., C. F. Kuo, Y. C. Wang, and S. T. Ding. 2005. Effect of docosahexaenoic acid and arachidonic acid on the expression of adipocyte determination and differentiation-dependent factor 1 in differentiating porcine adipocytes. J. Anim. Sci. 83：1516-1525.

Matsusue, K., J. M. Peters, and F. J. Gonzalez. 2004. PPARβ/δ potentiates PPARγ-stimulated adipocyte differentiation. FASEB J. 18：1477-1479.

Shi, Y., M. Hon, and R. M. Evans. 2002. The peroxisome proliferator-activated receptor δ, an integrator of transcriptional repression and nuclear receptor signaling. Proc. Natl. Acad. Sci. USA 99：2613-2618.

Yu, Y. H., B. H. Liu, H. J. Mersmann, and S. T. Ding. 2006. Porcine peroxisome proliferator-activated receptor gamma induces transdifferentiation of myocytes into adipocytes. J. Anim. Sci. 84：2655-2665.

Figure 1. Ligand-induced morphological alterations and accumulation of lipid droplets.

Microscographs of C2C12 myocyte with empty vector (A-D), C2C12 expressing porcine peroxisome proliferator-activated receptor δ (PPAR δ; E-H) are shown. Cells were maintained in adipogenic medium (Dulbecco’s modified Eagle medium/dexamethasone/insulin/10% fetal bovine serum) ± 1 μM rosiglitazone (B and F), 1 μM L165041 (C and G) and both of ligands (D and H) to

d 10 postconfluence. Magnification was 60.

Figure 2. Expression of adipogenic marker genes. After confluence, C2C12 myocytes with empty

vector (Empty vector) and C2C12 expressing porcine peroxisome proliferator-activated receptor δ (PPAR δ) were cultured for 10 d. The expression of adipocyte-specific genes [adipocyte fatty acid-binding protein (aP2) and lipoprotein lipase (LPL)] was determined and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The bars indicate the means ± SE for cells from 3 independent replicates (n = 3). ND = not detected. a–c Means without a common letter differ, P < 0.05.

Figure 3. Expression of myogenic marker genes. After confluence, C2C12 myocyte with empty

vector (Empty vector) and C2C12 expressing porcine peroxisome proliferator-activated receptor δ (PPAR δ) were cultured for 10 d. The expression of myogenic genes [Myogenin and myogenic regulatory factor-4 (MRF4)] was determined and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The bars indicate the means ± SE for cells from 3 independent replicates

(n = 3). a–c Means without a common letter differ, P < 0.05.

Figure 4. Expression of adipogenic marker genes. After confluence, C2C12 expressing wild-type porcine PPAR γ (WT-PPAR γ), C2C12 expressing mutated porcine PPAR γ (MU-PPAR γ), C2C12 expressing wild-type porcine PPAR γ and PPAR δ (WT-PPAR γ PPAR δ) and C2C12 expressing mutated porcine PPAR γ and PPAR δ (MU-PPAR γ PPAR δ) were cultured for 10 d. The expression of adipocyte-specific genes [adipocyte fatty acid-binding protein (aP2) and lipoprotein lipase (LPL)] was determined and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The bars indicate the means ± SE for cells from 3 independent replicates (n = 3). a–c Means without a common letter differ, P < 0.05.

Figure 5. Expression of myogenic marker genes. After confluence, C2C12 expressing wild-type

porcine PPAR γ (WT-PPAR γ), C2C12 expressing mutated porcine PPAR γ (MU-PPAR γ), C2C12 expressing wild-type porcine PPAR γ and PPAR δ (WT-PPAR γ PPAR δ) and C2C12 expressing mutated porcine PPAR γ and PPAR δ (MU-PPAR γ PPAR δ) were cultured for 10 d. The expression of myogenic genes [Myogenin and myogenic regulatory factor-4 (MRF4)] was determined and

normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The bars indicate the means ± SE for cells from 3 independent replicates (n = 3). ND = not detected. a–c Means without a common letter differ, P < 0.05.

行政院國家科學委員會補助國內專家學者出席國際學術會議報告

94-2313-B-002-024

(英文)Experimental Biology 2007 and USDA NCC0097 發表

論文 題目

(中文)猪 PPAR d 在脂肪細胞分化的功能

( 英 文 )

The functionality of porcine peroxisomal proliferator

activated receptor delta in adipocyte differentiation

報告內容應包括下列各項：

一、 參加會議經過

April 26 Flew to Washington DC, USA. Stay in Red Roof Inn, 500 H Street NW, Washington DC 20001.

April 27 Attended the meeting for USDA regional meeting NCC0097, Presented a talk on two topics: The function of PPARg and PPARd in pigs and The expression of genes in porcine adipose tissue under the treatment of porcine serum amyloid protein A. The fee for the meeting is 70 US dollars. Went to the dinner function with the scientists with common interests in adipocyte biology. The dinner cost for 67 US dollars. April 28. Attended the Keynote speech by Two scientist both named Tony on the discovery of tyrosine kinases and its function in regulating physiological process and gene expression in ASBMB Biochemistry session. Attended the poster section on lipid metabolism. Attended the evening session on enzyme expression regulation.

April 29. Attended the Keynote speech on Phosphoinisitide molecules and the genes involved in making the enzymes. What we learn from yeast model. Also attend Lipid metabolism. Went to Symposium on animal model for human nutrition 1 delivered by DH Baker and 2 delivered by Spurlock on adipocyte models. Also went to a

biochemistry teaching for cultivating future strong biochemist. The speakers were too good. But picked up some points, including bring research into classroom, set the expectation high, recognize the students, enthusiasm in research is contagous, and interactive teaching to enhance student learning. We also posted our poster. The full length of the report is attached at the end of the report.

April 30. Attended the ASBMB Merck Award for the talk on PEPC Kinase by R.W. Hanson. I also attended a section on role of nuclear receptors in metabolic syndrome in the morning. Went to poster section for genetics and metabolic approaches to obesity and proteionics: proteomics and bioinformatics. Went to Symposium on lipid

metabolism and transport and also lipid signaling track.

May 1. I attended the Nutrition section and mostly the poster and two keynote speeches. Two areas are very important, one was on companion animal nutrition and the other was on aging. There were lots of research on the feed limitation on aging and well-being of animals. Two specific experiments on monkey long term

restriction on feeding improve the health condition and longevity.

May 2. Attended the FASEB Excellence in Science Award before we took off to Columbus, Ohio for an international exchange program discussion.

表 Y04

二、 與會心得

This year I started to get the feeling of how well we have done researchwise. We have demonstrated a very specific pathway of regulating nutrition metabolism and such a finding is world-class. We need to collect the most recent progress of the research in order to know where we are and what to do to compete with international scientists. This meeting always has a lot of activities going on. Lots of science to learn, but too little time available. I have also got to interact with lots of scientist in my field of research. That help me develop the sense of where we are and our competitiveness.

三、 考察參觀活動(無是項活動者省略)

四、 建議

The areas of nutritional science research are evolving very fast during the past few years. This Meeting collects a broad spectrum of research progress which is

important for the researchers in Taiwan. I would suggest that we should encourage researchers to go and joint this meeting to get updated research progress report and to improve our research quality.

五、 攜回資料名稱及內容

Experimental Biology 2007, Conference information and scientific program in a CD format. All the station reports from the USDA regional meeting NCR-97.

六、 其他

The full article of our presentation.

Ectopic expression of porcine peroxisome-proliferator-activated receptor delta regulates adipogenesis in myoblasts

Y. H.Yu1 _{and S. T. Ding}1

Department of Animal Science and Technology, National Taiwan University, 50, Lane 155, Kee-Long Rd. Sec. 3, Taipei 106, Taiwan 1

ABSTRACT

It is well known that peroxisome-proliferator-activated receptor γ (PPAR γ) plays a critical role in regulating adipogenesis. In rodents, PPAR δ is expressed before PPAR γ during adipocyte differentiation. Thus, the interaction between PPAR δ and PPAR γ during adipogenesis needs to be elucidated. The current experiment was designed to study the interaction of porcine PPAR δ and PPAR γ in mouse myoblast cells. Inhibition of myogenesis was observed in myoblasts expressing porcine PPAR δ, similar to myoblast expressing PPAR γ. Treatment of myoblasts expressing PPAR δ with ligands for both PPAR δ and γ enhanced lipogenesis to a greater extent than treatment with a PPAR γ ligand alone. The ability to transdifferentiate myoblasts into adipocytes was decreased in myoblasts co-expressing PPAR δ with either wild-type or mutated PPAR γ (serine 112 was mutated to alanine) compared to myoblasts expressing either type of PPAR δ alone. Adipose transdifferentiation in myoblasts co-expressing PPAR δ and mutated PPAR γ was greater than in myoblasts co-expressing PPAR δ and wild-type PPAR γ. Our results suggest that PPAR δ has two different roles in regulating adipogenesis, ie., suppression of myogenesis to enhance transdifferentiation of myoblasts into adipocytes and interaction with PPAR γ to modify adipogenesis. Therefore, PPAR δ may have a significant role in adipogenesis.

Key Words: Adipocyte differentiation, Peroxisome proliferator-activated receptor δ, Peroxisome

proliferator-activated receptor γ.

INTRODUCTION

In rodent, peroxisome-proliferator-activated receptor δ (PPAR δ) is wildly expressed in several tissues, including adipose tissue, intestine, skeletal muscle, lung and heart. The