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

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Cell Culture of Primary Human SV Cell Primary human stromal vascular (SV) cells were obtained from subcutaneous adipose tissue of non-obese (BMI < 30) females undergoing abdominoplasty with the consent from the Institutional Review Board at the University of North Carolina at Greensboro. Isolation, culture of SV cells from adipose tissue, and differentiation into adipocytes were performed as previously described (Brown et al. 2004) except for the condition that SV cells were pooled from 3~5 independent human subjects for most experiments. Experimental treatment of cultures containing adipocytes began on ~day 12 of differentiation unless otherwise indicated. For the preparation of cultures of non- differentiated SV cells, cells were seeded at ~80% confluent. Fatty Acid Preparation Both isomers of CLA were complexed to FA-free (>98%) bovine serum albumin (BSA) at a 4:1 molar ratio using 1 mM BSA stocks. [ 3 H]-2-deoxy-glucose Uptake For the 8 and 24 h treatments, newly-differentiated cultures of adipocytes were incubated with BSA vehicle or 30 µM trans-10, cis-12 CLA in serum-free, low glucose DMEM [1,000 mg/liter D-(+)-glucose] in the presence or absence of 20 pM insulin. For the 72 h treatment, BSA vehicle or 30 µM trans-10, cis-12 CLA were added to adipocyte medium for 48 h on day 12. Then, for an additional 24 h, cultures were incubated in 1 ml of serum-free basal DMEM containing 1,000 mg/liter D-(+)-glucose with or without 20 57
pM of human insulin in the presence of BSA vehicle or 30 µM trans-10, cis-12 CLA, giving a total of 72 h of exposure to the treatments. Following the experimental treatments, [ 3 H]-2-deoxy-glucose uptake was measured as described previously (Brown et al. 2004). PM Membrane Isolation and Glut4 Levels For the detection of Glut4 translocation, plasma membrane (PM) was fractionated according to Carvalho et al. (2004). Briefly, cultures were washed once with TES buffer (20 mM Tris-HCl, 225 mM sucrose, 1 mM EDTA, pH 7.4 at 20°C) and then homogenized in ice-chilled TES using a 1 ml dounce homogenizer. The homogenate was centrifuged at 16,000 g for 20 min at 4°C and solidified surface fat was removed. The resulting pellets (crude membrane) were resuspended in TES and layered on a 1.12 M sucrose cushion in 20 mM Tris-HCl, 1 mM EDTA, and centrifuged at 100,000 g for 30 min. PMs at the interface were collected, resuspended in TES, and centrifuged at 100,000 g for 30 min. PM pellets were resuspended in TES and immunoblotted for Glut4. The abundance of Glut4 was quantified from exposed x-ray film using the KODAK image station 440 (Eastman Kodak, Rochester, NY). IL-6 and IL-8 Secretion Differentiated cultures of adipocytes and cultures of non-differentiated SV cells were serum starved for 20 h before fatty acid treatment. Fatty acid treatment was initiated <strong>by</strong> adding either BSA vehicle or 30 µM trans-10, cis-12 CLA to the medium directly. 58
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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
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IV. PREADIPOCYTES MEDIATE LPS-INDUC
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LIST OF FIGURES ix Page Figure 1.1.
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Figure 4.7. LPS-induced NFκB activ
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in animals and some humans (reviewe
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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 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: antibodies for anti-glyceraldehydre
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
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
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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.,
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Chen, C., Koh, A.J., Datta, N.S., Z
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Diradourian, C., Girard, J., and Pe
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Granlund, L., Juvet, L.K., Pedersen
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Imamura, M., Inoguchi, T., Ikuyama,
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Loscher, C.E., Draper, E., Leavy, O
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Piper, R.C., Hess, L.J., and James,
Page 161 and 162:
Sinha, S., Perdomo, G., Brown, N.F.
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Vanden Berghe, W., Vermeulen, L., D
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