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
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
96 年 5 月 14 日 報告人姓名
丁詩同 服務機構
及職稱
國立台灣大學動物科學技術學系 教授
時間 會議 地點
95 年 4 月 26 日至 5 月 3 日 美國華盛頓首府
本會核定 補助文號
94-2313-B-002-024
會議 名稱
(中文)實驗生物學聯合會和美國農部區域討論會
(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
附件三
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. Ding1
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
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