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

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secretion was not detectable (data not shown, < 0.5 pg/ml). The mRNA levels of IL-6 and IL-8 in adipose tissue explants treated with CLA were also higher compared to those receiving the BSA vehicle (data not shown). To determine the extent to which CLA’s induction of IL-6 and TNF-α gene expression was isomer-specific, differentiated cultures of adipocytes were treated with either BSA vehicle, 30 µM cis-9, trans-11 CLA, or 30 µM trans-10, cis-12 CLA for 3 h and IL-6 and TNF-α mRNA levels were measured. As a positive control, another set of cultures was treated with 100 ng/ml of human recombinant TNF-α for 30 min. As seen in Fig 3.3B, trans-10, cis-12 CLA, but not cis-9, trans-11 CLA or BSA, induced IL-6 and TNF-α gene expression in cultures of differentiated adipocytes (Fig 3.3B) as well as in cultures of non-differentiated SV cells (data not shown). Collectively, these data demonstrate for the first time that cells isolated from human adipose tissue secrete proinflammatory cytokines ex vivo when treated with trans-10, cis-12 CLA. Trans-10, Cis-12 CLA Promotes NFκB Activation- We hypothesized that trans-10, cis-12 CLA treatment would lead to NFκB activation in cultures of newly-differentiated adipocytes. This is supported by the following evidence:1) both IL-6 and TNF-α possess a nuclear factor κB response element (κBRE) (Chen et al. 1999); 2) transient TNF-α gene expression is positively linked to NFκB activation (Bouwmeester et al. 2004; Ruan et al. 2002); 3) ERK1/2 phosphorylation of nuclear p50/p65 is important for its activation (Jiang et al. 2001); 4) NFκB-mediated IL-6 and TNF-α production are associated with insulin resistance in 65
myotubes (Sinha et al. 2004; Weigert et al. 2004; Jove et al. 2005); and 5) human adipose tissue is a target for IL-6-mediated insulin resistance (Rotter et al. 2003). To test this hypothesis, cultures were treated with either BSA vehicle or 30 µM trans-10, cis-12 CLA and IκBα protein level was measured by immunoblotting. In support of our hypothesis, IκBα was lower in cultures treated for 3 h with trans-10, cis-12 CLA than in the controls (Fig 3.4A), implying NFκB is activated by CLA. CLA-mediated NFκB activation appeared to be isomer-specific as evidenced by phosphorylation and subsequent degradation of IκBα and ERK1/2 phosphorylation in trans-10, cis-12 CLA-treated cultures compared to cultures treated with cis-9, trans-11 CLA or BSA (Fig 3.4B). In contrast to TNF-α treatment, trans-10, cis-12 CLA had no effect on IκBβ degradation. To determine the upstream regulator of IκBα phosphorylation by CLA, cultures treated with either BSA vehicle or 30 µM trans-10, cis-12 CLA were immunostained with an antibody that recognizes active phospho-IKKα/β (Ser176/180, Ser177/181). Trans-10, cis-12 CLA treatment for 2 h increased the phosphorylation of IKKα/β compared to the vehicle controls (Fig 3.4C), suggesting that trans-10, cis-12 CLA activates the IKKs-NFκB-IκBα cascade in differentiated cultures of human adipocytes. To determine the specificity of CLA induced-NFκB DNA binding among five NFκB families (i.e., p50, p52, p65, c-Rel, Rel B) (reviewed in Chen et al. 2004), differentiated cultures of adipocytes were treated for 3 h with BSA vehicle or 30 µM trans-10, cis-12 CLA. Subsequently, nuclear extracts were added to 96 well plates to which an oligonucleotide containing an NFκB consensus binding site (5'GGGACTTTCC3') had been immobilized as provided in the Trans-AM NFκB Kit 66
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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
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 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: treatment (Brown et al. 2004), we d
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
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
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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,
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Sinha, S., Perdomo, G., Brown, N.F.
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Vanden Berghe, W., Vermeulen, L., D
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