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

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For real time qPCR, 1 ug total RNA was converted into first strand cDNA using Omniscript RT kit (Qiagen Inc). qPCR was performed in a Smartcycler (Cepheid, Sunnyvale, CA) using the QuantiTect SYBR Green PCR kit (Qiagen) for 40 cycles. To account for possible variation related to cDNA input amounts or the presence of PCR inhibitors, the endogenous reference gene glycerol aldehyde-3-phosphate dehydrogenase (GAPDH) was simultaneously quantified in a separate tube for each sample. Initial real- time amplifications were examined by agarose gel electrophoresis to confirm the sizes of the products. After PCR amplification, a melting curve was generated for every PCR product to check the specificity of the PCR. Primer sequences and running conditions are summarized in Table 1. Transient Transfection and PPAR Activity For measuring PPARγ activity, primary human adipocytes were transiently transfected with a 3X PPAR responsive luciferase reporter construct pTK-PPRE3x-Luc (Kliewer et al. 1992; generously provided by Dr. Susanne Mandrup) using the Amaxa Nucleofactor (Amaxa, Cologne, Germany) according to the manufacturer’s protocol. On day 6 of differentiation, 1 x 10 6 cells from a 60 mm plate were trypsinized and resuspended in 100 µl of nucleofector solution (Amaxa) and mixed with 2 µg of pTK- PPRE3x-luc and 25 ng pRL-CMV for each sample. Electroporation was performed using the V-33 nucleofector program (Amaxa). Cells were replated in 96-well plates after 10 minutes recovery in calcium-free RPMI medium. Two hours later, cultures were supplemented with charcoal-stripped AM1 before LPS stimulation. Firefly luciferase 99
activity was measured using the Dual-Glo luciferase kit (Promega, Madison, WI) and normalized to Renilla luciferase activity from the co-transfected control pRL-CMV vector. All luciferase data are presented as a ratio of firefly luciferase to Renilla luciferase activity. Statistical Analysis Unless otherwise indicated, data are expressed as the mean ± S.E. (n = 3-6) obtained from cultures containing cells isolated from 3-4 different subjects. Data were analyzed using one-way analysis of variance (ANOVA), followed by student’s t-tests for each pair for multiple comparisons. Differences were considered significant if p < 0.05. All analyses were performed using JMP IN 4.04 (SAS Institute; Cary, NC) software. Results Primary Cultures of Newly Differentiated Adipocytes Contain Preadipocytes and Adipocytes Our normal differentiation protocol using DM1 for the first 3 days of differentiation resulted in a cell population on day 12 containing ~50% adipocytes and ~50% non- adipocytes (cells without visible lipid droplets). Based on our findings that the non- adipocyte fraction of our cultures robustly express and/or secrete cytokines (e.g., IL-6, IL-8, TNF-α) in response to trans-10, cis-12 CLA treatment (Brown et al. 2004; Chung et al. 2005), we wanted to know if preadipocytes were present in this non-adipocyte or SV fraction. To answer this question, we first cultured the cells in the absence or presence of 100
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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
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Discussion Feeding mixed CLA isomer
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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: Immunoblotting and 4MUrea-SDS-PAGE
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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