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

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from BD Pharmingen (San Jose, CA). Rodamine red and fluorescein isothiocyanate- conjugated IgGs were purchased from Jackson Immunoresearch (West Grove, GA). Gene specific primers for real time PCR were purchased from IDT (Coralville, IA). DMAP and MG132 were purchased from Calbiochem (San Diego, CA). LY294002, U0126, and primary antibodies for p-IκBα kinase (IKK) α/β (Ser 180/ser181, rabbit), p-stress- activated protein kinase/Jun-N-terminal kinase (SAPK/JNK) (Thr183/Tyr185, mouse), p- AKT (protein kinase B)(Ser473, rabbit), p-extracellular signal-related kinase (ERK) 1/2 (Thr 202/Tyr204 rabbit) were purchased from Cell Signaling Technology (Beverly, MA). Pref-1 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN). All the other chemicals reagents were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated. Cell Cultures Abdominal adipose tissue was obtained from females with a body mass index (BMI) < 30 during elective surgery with approval from the Institutional Review Board at the University of North Carolina-Greensboro. SV cells were isolated and cultured as previously described with minor modification (Brown et al. 2003). To induce ~50% differentiation (e.g., 50% of cells containing visible lipid droplets), confluent cultures of SV cells were supplemented with differentiation media-1 (DM1; 97% DMEM/Ham F-12, 3% FBS, 1 µM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone, 33 µM biotin, 17 µM panthothenate, 100 nM insulin) for the first 3 days (referred to as DM1 in Fig 4.1 or AD50 in the other figures). To obtain maximum 95
differentiation, cultures of SV cells were exposed to DM1 for 6 days, which resulted in ~90% differentiation (AD90). To generate cultures that did not differentiate into adipocytes (referred to as –DM1 in Fig 4.1 or AD0 in the other figures), cultures of SV cells were supplemented with adipocyte media (AM1; 97% DMEM/Ham F-12, 3% FBS, 1 µM dexamethasone, 33 µM biotin, 17 µM panthothenate, 100 nM insulin) beginning on day 1 of differentiation until the assays were performed. For the co-culture experiment (Fig 4.5C), SV cells were initially seeded as a mono- culture in individual cell culture inserts (Falcon 0.4 µm pores cat#3090, Fisher Scientific, Norcross, GA) and suspended above 6-well Multiwell TM plates containing AM1 for 12 d (AD0). On day14, inserts containing the AD0 cultures were transferred to 6-well Multiwell TM plates and co-cultured above AD50 cultures for 8 h, thereby allowing the AD0 and AD50 cultures to communicate with one another by sharing the same medium during treatment with LPS. For the positive controls of macrophages, the U937 human monocyte line (ATCC CRL1593, Rockville, MD) was used. U937 cells were supplemented with RPMI 1640 medium containing 10% FBS and induced to differentiation by adding 10 nM phorbol 12-myristate 13-acetate (PMA) for 72 h. Fractionation of SV Cells and Adipocytes Using Density Gradient For fractionation of lipid-laden adipocytes from the non-differentiated SV cells, cultures grown in 100 mm plates (~3 million cells) were washed with ice cold HBSS/0.5mM EDTA and trypsinized with Trypsin-like enzyme. Cells were layered onto 96
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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)
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Anti-adipogenic Mechanisms of CLA T
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2005). Chapter II of this dissertat
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Figure 1. 2. Multiple mechanisms by
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In contrast, studies conducted in a
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activation. Similarly, Chen et al.
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Central Hypothesis and Specific Obj
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CHAPTER II TRANS-10, CIS-12 CLA INC
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of MEK/ERK signaling by trans-10, c
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Materials and Methods Materials All
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the cultures for additional 12 h. A
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precipitated with ethanol, dried, a
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or for 30 min with 10 µM isoproter
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either 30 µM cis-9, trans-11 CLA o
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dependent effects of CLA on the pho
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Rapamycin Blocks CLA’s Increase i
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Figure 2. 2. Trans-10, cis-12 CLA a
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Figure 2. 4. Trans-10, cis-12 CLA a
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Figure 2. 6. Trans-10, cis-12 CLA i
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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: adipocytes from WAT in inflammation
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
Page 127 and 128: 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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