CHUNG, SOONKYU, Ph. D. Mechanisms by Which Conjugated ...

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Figure 2. 5. Trans-10, cis-12 CLA differentially affects gene and protein expression of lipid droplet-associated proteins. Cultures of SV cells containing newly differentiated adipocytes were treated for 3 days with either BSA (B) or 30 µM trans-10, cis-12 CLA (10) or cis-9, trans-11 CLA (9). Cells were harvested on day 3 based on the data in Fig 2.3, indicating that between days 2 and 4, adipose differentiation-related protein (ADRP) protein increased, and perilipin and hormone sensitive lipase (HSL) proteins decreased. A: Total cell extracts were immunoblotted for adipose differentiated-related protein (ADRP), perilipin, and HSL. Actin was used as a loading control for the Western blots. Results shown are representative of three separate experiments using cells isolated from different human subjects each time. ERK phosphorylation was measured using a phosphospecific antibody for ERK. B: Total RNA was harvested using Tri-reagent and mRNA expression levels of ADRP, perilipin, and HSL were analyzed using real time quantitative (q) PCR. TATA binding protein (TBP) was used as a control for real time qPCR. Means (+ SEM, n=3 for ADRP, n=2 for perilipin and HSL) not sharing a common superscript differ significantly (p
Figure 2. 6. Trans-10, cis-12 CLA induces mammalian target of rapamycin (mTOR) signaling. A: Cultures of SV cells containing newly differentiated adipocytes were treated for 0, 1, 3, 6, 12, 24, 48, or 72 h with either a bovine serum albumin vehicle (BSA) or 30 µM trans-10, cis-12 CLA (10,12 CLA). Total cell extracts were immunoblotted using phospho-specific or non-phosphospecific antibodies targeting the S6 ribosomal protein (S6). B: Cultures grown on coverslips were treated for 3 h with either a bovine serum albumin vehicle (BSA), or 30 µM trans-10, cis-12 CLA (10,12 CLA). A 30 min, 20% fetal bovine serum (FBS) treatment was used as a positive control. Following treatment, p-S6 was visualized <strong>by</strong> immunostaining using rabbit anti-pS6 ribosomal protein and FITC-conjugated anti rabbit IgG. C: Cultures were treated for 3 h with either a bovine serum albumin vehicle (BSA), 30 µM cis-9, trans-11 CLA (9,11), or 30 µM trans-10, cis-12 CLA (10,12). A 10 min human insulin (100 nM) treatment or 30 min TNF-α (100 ng/ml) treatment was used as a positive control. Total cell extracts were immunoblotted using phospho-specific antibodies targeting protein kinase B (Akt), mTOR, p70S6K, S6, eukaryotic initiation factor 4E (eIF4E), or MNK1. Results shown are representative of two independent experiments. 40
Page 1 and 2: CHUNG, SOONKYU, Ph. D. Mechanisms b
Page 3 and 4: MECHANISMS BY WHICH CONJUGATED LINO
Page 5 and 6: APPROVAL PAGE This Dissertation has
Page 7 and 8: I also would like to thank The Univ
Page 9 and 10: IV. PREADIPOCYTES MEDIATE LPS-INDUC
Page 11 and 12: LIST OF FIGURES ix Page Figure 1.1.
Page 13 and 14: Figure 4.7. LPS-induced NFκB activ
Page 15 and 16: in animals and some humans (reviewe
Page 17 and 18: CLA is potentially effective in: 1)
Page 19 and 20: Anti-adipogenic Mechanisms of CLA T
Page 21 and 22: 2005). Chapter II of this dissertat
Page 23 and 24: Figure 1. 2. Multiple mechanisms by
Page 25 and 26: In contrast, studies conducted in a
Page 27 and 28: activation. Similarly, Chen et al.
Page 29 and 30: Central Hypothesis and Specific Obj
Page 31 and 32: CHAPTER II TRANS-10, CIS-12 CLA INC
Page 33 and 34: of MEK/ERK signaling by trans-10, c
Page 35 and 36: Materials and Methods Materials All
Page 37 and 38: the cultures for additional 12 h. A
Page 39 and 40: precipitated with ethanol, dried, a
Page 41 and 42: or for 30 min with 10 µM isoproter
Page 43 and 44: either 30 µM cis-9, trans-11 CLA o
Page 45 and 46: dependent effects of CLA on the pho
Page 47 and 48: Rapamycin Blocks CLA’s Increase i
Page 49 and 50: Figure 2. 2. Trans-10, cis-12 CLA a
Page 51: Figure 2. 4. Trans-10, cis-12 CLA a
Page 55 and 56: Figure 2. 8. Trans-10, cis-12 CLA-i
Page 57 and 58: Discussion Feeding mixed CLA isomer
Page 59 and 60: in lipolysis may occur via an ERK-d
Page 61 and 62: espectively, by the MEK inhibitor U
Page 63 and 64: Furthermore, the CLA-mediated incre
Page 65 and 66: proteins. Inhibition of NFκB activ
Page 67 and 68: Sopasakis et al. 2004) and insulin
Page 69 and 70: antibodies for anti-glyceraldehydre
Page 71 and 72: pM of human insulin in the presence
Page 73 and 74: were (accession #NM000600) sense (5
Page 75 and 76: Transfection efficiency was examine
Page 77 and 78: treatment (Brown et al. 2004), we d
Page 79 and 80: myotubes (Sinha et al. 2004; Weiger
Page 81 and 82: controls was found in cytosol (Fig
Page 83 and 84: Depletion of NFκB p65 by RNAi Atte
Page 85 and 86: eports of cytokine-mediated insulin
Page 87 and 88: Figure 3. 2. Trans-10, cis-12 CLA i
Page 89 and 90: Figure 3. 4. Trans-10, cis-12 CLA a
Page 91 and 92: Figure 3. 6. Trans-10, cis-12 CLA-i
Page 93 and 94: Figure 3. 8. Specific depletion of
Page 95 and 96: Figure 3. 10. Working model: Trans-
Page 97 and 98: Based on our working model (Fig 3.1
Page 99 and 100: mRNA levels of TNF-α (Fig. 3). How
Page 101 and 102: humans (Riserus et al. 2004a), and
Page 103 and 104:
most likely not impairing the diffe
Page 105 and 106:
1) decreased adipogenic gene expres
Page 107 and 108:
adipocytes from WAT in inflammation
Page 109 and 110:
differentiation, cultures of SV cel
Page 111 and 112:
Immunoblotting and 4MUrea-SDS-PAGE
Page 113 and 114:
activity was measured using the Dua
Page 115 and 116:
macrophages and myocytes, respectiv
Page 117 and 118:
cultures, insulin’s stimulation o
Page 119 and 120:
AD90 cultures treated with LPS. Con
Page 121 and 122:
Table 4.1. List of human-gene speci
Page 123 and 124:
Figure 4.2. Primary cultures of new
Page 125 and 126:
Figure 4.4 LPS induction of cytokin
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Figure 4. 6. LPS suppresses PPARγ
Page 129 and 130:
Figure 4. 8. Inhibitors of NFκB at
Page 131 and 132:
Discussion Adipose tissue is a sour
Page 133 and 134:
To determine if non-adipocytes othe
Page 135 and 136:
attenuation of insulin sensitivity
Page 137 and 138:
epression of NFκB target gene expr
Page 139 and 140:
EPILOGUE The “Obesity epidemic’
Page 141 and 142:
Apart from our initial goal for thi
Page 143 and 144:
Concerning membrane specific recept
Page 145 and 146:
esearch topic I would like to exami
Page 147 and 148:
Berg, A.H., Lin, Y., Lisanti, M.P.,
Page 149 and 150:
Chen, C., Koh, A.J., Datta, N.S., Z
Page 151 and 152:
Diradourian, C., Girard, J., and Pe
Page 153 and 154:
Granlund, L., Juvet, L.K., Pedersen
Page 155 and 156:
Imamura, M., Inoguchi, T., Ikuyama,
Page 157 and 158:
Loscher, C.E., Draper, E., Leavy, O
Page 159 and 160:
Piper, R.C., Hess, L.J., and James,
Page 161 and 162:
Sinha, S., Perdomo, G., Brown, N.F.
Page 163 and 164:
Vanden Berghe, W., Vermeulen, L., D
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