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

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containing complicated hairpins, as we predicted using a computer simulation model (RNA structure, version 4.1; data not shown). Third, Brasaemle et al. (1997) suggested that ADRP mRNA may be recruited to polysomes for protein synthesis rather than to the endoplasmic reticulum, based on a discrepancy between mRNA and protein expression levels in differentiated 3T3L1 adipocytes. Fourth, leptin, a peptide that senses lipid status of adipocytes and is a 5’TOP mRNA (Roh et al. 2003), is also increased by trans-10, cis- 12 CLA treatment (Brown et al. 2004). Based on these observations, we hypothesize that trans-10, cis-12 CLA increases ADRP protein levels by initiating translation of ADRP mRNA via activation of the mTOR/p70S6K/S6 signaling pathways that recruit ADRP mRNA into translational 40S ribosomal machinery. In summary, these data demonstrate that acute treatment (within 3 h) of the cultures with trans-10, cis-12 CLA increases basal lipolysis and the appearance of perilipin in the cytosolic fraction. Chronic treatment (> 48 h) of the cultures with trans- 10, cis-12 CLA decreases perilipin and HSL gene and protein expression, and enhances ADRP protein expression and abundance on the lipid droplet. The robust increase in ADRP protein levels by trans-10, cis-12 CLA was accompanied by phosphorylation of ERK and acute activation of the mTOR pathway, including activation of p70S6 kinase and its downstream target S6 ribsomal protein, which was abolished by co-incubation of the CLA-treated cultures with specific inhibitors of the mTOR/p70S6K/S6 pathway. Collectively, these data suggest that trans-10, cis-12 CLA promotes adipocyte delipidation, in part, by promoting lipolysis, which may result from ERK-dependent alterations in perilipin and ADRP gene expression, protein activation, and localization. 49
Furthermore, the CLA-mediated increase in ADRP protein may be due to enhanced ADRP translation induced by activation of mTOR/p70S6K/S6 signaling. 50
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CHUNG, SOONKYU, Ph. D. Mechanisms b
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MECHANISMS BY WHICH CONJUGATED LINO
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APPROVAL PAGE This Dissertation has
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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 53 and 54: Figure 2. 6. Trans-10, cis-12 CLA i
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: espectively, by the MEK inhibitor U
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 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
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activity was measured using the Dua
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macrophages and myocytes, respectiv
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cultures, insulin’s stimulation o
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AD90 cultures treated with LPS. Con
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Table 4.1. List of human-gene speci
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Figure 4.2. Primary cultures of new
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Figure 4.4 LPS induction of cytokin
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Figure 4. 6. LPS suppresses PPARγ
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Figure 4. 8. Inhibitors of NFκB at
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Discussion Adipose tissue is a sour
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To determine if non-adipocytes othe
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attenuation of insulin sensitivity
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epression of NFκB target gene expr
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EPILOGUE The “Obesity epidemic’
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Apart from our initial goal for thi
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Concerning membrane specific recept
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esearch topic I would like to exami
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Berg, A.H., Lin, Y., Lisanti, M.P.,
Page 149 and 150:
Chen, C., Koh, A.J., Datta, N.S., Z
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Diradourian, C., Girard, J., and Pe
Page 153 and 154:
Granlund, L., Juvet, L.K., Pedersen
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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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