Title Page

Contents

Abstract 9

Chapter 1. INTRODUCTION 10

1.1. Definition and risk of obesity and T2DM 10

1.2. T2DM 11

1.3. PPP5 12

Chapter 2. Material and Methods 14

2.1. Overexpression and Purification of Recombinant PPP5 14

2.2. Half-Maximal Inhibitory Concentration (IC50) Determination 15

2.3. RNA interference 16

2.4. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) 16

2.5. Cell Culture and Differentiation Mouse 3T3-L1 pre-adipocytes 17

2.6. Glucose uptake assays 19

2.7. Western Blotting 20

2.8. Oil Red O Staining 21

2.9. Statistical Analysis 21

Chapter 3. Results 22

3.1. The path to discovering natural products that inhibit PPP5 22

3.2. Suppression of PPP5 increased obesity and diabetes marker 25

3.3. Norcantharidin derivatives 28

3.4. The cytotoxicity of NCT in 3T3-L1 30

3.5. Norcantharidin downregulated the adipogenic transcriptional Factors 31

3.6. Treatment with Norcantharidin increases p-AMPK and p-AKT levels 33

3.7. Glucose uptake is increased following NCT treatment 34

3.8. Effects of NCT on lipid accumulation of 3T3-L1 35

Chapter 4. Conclusion 37

References 39

논문요약 40

Table 1. Kinetic constants for DiFMUP hydrolysis by PPP5 23

Figure 1. Purified proteins according to the affinity tag, enzyme kinetics SDS-PAGE analysis of PPP5 using affinity chromatography column, TALON... 23

Figure 2. Knockdown Efficiency of PPP5 was determined by qRT-PCR and We transfected siRNA with PPP5 for 48h using 3T3-L1 pre-adipocyte(A).... 26

Figure 3. PPP5 Knock down using siRNA. We proceeded to knock down using siRNAs designed from the PPP5 sequence... 27

Figure 4. Knockdown of PPP5 shows anti-obesity and anti-diabetic effects. After PPP5 knock down, PPARγ marker confirmed by western blot.(A,B)... 27

Figure 5. NCT derivatives and inhibition percentages. The graph (A,B) summarizes the inhibition under the conditions of PPP5... 29

Figure 6. Cell viability assay cytotoxicity of Norcantharidin. Cytotoxicity of Norcantharidin on 3T3-L1 adipocyte. Cell viability was... 30

Figure 7. NCTs in 3T3-L1 were treated by concentration.The anti-obesity markers PPARγ and CEBPα were subsequently confirmed by western blot... 32

Figure 8. Concentration-specific treatment of NCT increases AMPK phosphorylation. Phosphorylation of AMPK, a diabetes marker, and AKT... 33

Figure 9. NCT improves glucose uptake ability in differentiated 3T3-L1. 3T3-L1 was incubated with NCT (5, 10 μM)or 100 nM insulin as a positive... 34

Figure 10. NCT inhibits lipid accumulation. Treatment of 3T3-L1 with NCTs at different concentrations (0.5, 1, 2, 5 μM) showed a concentration-... 35

Figure 11. Using the Oil Red O assay, we show that treatment of NCTs early in 3T3-L1 differentiation inhibits lipid accumulation more than treatment late... 36