Title Page
Acknowledgements
Contents
I. Introduction 10
II. Literature Review 12
1. Origin of myocyte and adipocyte from mesenchymal stem cells 12
2. In vitro models and culture conditions used in transdifferentiation studies 15
3. Transcriptional Control of Transdifferentiation 16
4. Thiazolidinediones (TZD) and Transdifferentiation of Myocytes to Adipocytes 18
5. Effect of TZD on transdifferentiation: In vivo studies 20
6. FATTY ACIDS AND TRANSDIFFERENTIATION 20
III. Materials and Methods 22
1. Primary culture of bovine satellite cells (BSC) 22
2. Gene construction 23
3. Transfection studies 23
4. Serum lipid treatment 24
5. Short chain fatty acid (SCFA) treatment 25
6. Total RNA extraction 25
7. Semi-quantitative RT-PCR analysis 25
8. Oil-Red-O staining 26
9. Statistical Analysis 26
IV. Results and Discussion 27
1. Effect of ectopic expression of adipogenic transcription factors in myoblast cells to adipocytes. 28
A) Effect of PPAR gamma and C/EBP alpha 28
B) Effect of Sterol regulatory element binding protein 1c (SREBP1c) 32
2. Effect of Serum Lipids (SL) on transdifferentiation of bovine satellite cells (BSC) to adipocytes 36
A) Treatment of SL to Bovine satellite cells 36
B) Optimization of SL concentration for BSC transdifferentiation 39
C) SL induces transdifferentiation via PPAR gamma 39
D) Optimization of media composition for transdifferentiation of BSC using Serum Lipids 42
E) Comparison of Troglitazone with the SL 44
3. Short chain fatty acids can promote in vitro transdifferentiation of bovine myogenic satellite cells to adipocytes. 46
4. Effect of Steroid Hormones on Transdifferentiation 54
SUMMARY 58
REFERENCES 59
ABSTRACT 67
Fig.1. Overview of stages in adipocyte and myocyte differentiation from mesenchymal stem cells. 14
Fig. 2. Transdifferentiation of bovine satellite cells to adipocytes by ectopic expression of adipogenic transcription factors. 30
Fig. 3. Expression of adipogenic and myogenic genes in bovine satellite cells expressing PPARγ, C/EBPα or PPARγ + C/EBPα. 31
Fig. 4. Transdifferentiation of mouse L6 cells to adipocytes by ectopic expression of adipogenic transcription factor, tSREBP1c. 33
Fig. 5. Expression of (A) adipogenic and (B) myogenic genes in mouse L6 cells expressing tSREBP1c. 34
Fig. 6. Mouse L6 cells were treated with different concentrations of tSREBP1c vector and serum lipid (SL) as a positive control. 35
Fig. 7. (A) Functional role of serum lipid on transdifferentiation of bovine myogenic satellite cells to adipocytes. 37
Fig. 8. (A) Bovine satellite cells were treated with different concentrations of SL. After 10 days of treatment cells were fixed and stained with oil red O to detect oil droplets. (B) Effect of different concentration of SL on adipogenic marker gene, GPDH. Upper panel: representative ethidium bromide-stained agarose gel showing amplified GPDH.... 40
Fig 9. Effect of adipogenic agents on oil droplet accumulation and expression of GPDH gene when treated with SL. 43
Fig. 10. Effect of troglitazone (TRO), SL and TRO + SL on oil droplet accumulation and adipogenic genes was analyzed. 45
Fig. 11. Functional role of propionate or acetate on transdifferentiation of bovine satellite cells. 47
Fig. 12. Effect of propionate (A and C) and acetate (B and D) on expression of adipogenic genes during the transdifferentiation of myogenic BSC to adipocytes were analyzed. 49
Fig. 13. Effect of propionate (A) and acetate (B) on expression of myogenic genes during the transdifferentiation of myogenic BSC to adipocytes were analyzed. 51
Fig. 14. Effect of propionate (SCFA) and cholesterol (LCFA) on expression of GPCRs during the transdifferentiation of myogenic BSC to adipocytes were analyzed. 53
Fig. 15. Effect of steroids on lipid accumulation in myotube. 56
Fig. 16. Gene expression levels of PPAR gamma (A and B) and AP2 (C) after steroid hormone treatment only(A and B), and combination of steroid hormones with TZD (C). 57