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

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TG synthesis as opposed to increased oxidation. Interestingly, CLA’s suppression of glucose and fatty acid uptake was positively correlated with activation of mitogen- activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) and G protein coupled receptor (GPCR) signaling and robust secretion of the proinflammatory cytokines interleukin-6 (IL-6) and IL-8 after 24 h of treatment. However, the underlying mechanism(s) by which trans-10, cis-12 CLA triggers cytokine production and impairs glucose and fatty acid uptake is unclear. There is growing evidence linking inflammatory cytokines with the development of obesity, insulin resistance (Wellen and Hotamisligil 2003; Xu et al 2003; Weisberg et al. 2003), and atherosclerosis (reviewed in Lau et al. 2005). Adipose tissue plays a central role in this relationship given its ability to both secrete cytokines and act as a substantial target for cytokines. A diverse array of cytokines such as tumor necrosis factor-α (TNF- α), IL-6, and IL-8 have been positively associated with obesity and the development of insulin resistance in muscle and adipose tissue. IL-6 expression can be induced by many transcription factors such as nuclear factor κB (NFκB), NF-IL6 (a.k.a., C/EBPβ), activator protein-1 (AP-1), and cAMP response element (CRE)-binding protein (CREB) depending on cell type and stimulus (Akira et al. 1997; Vanden et al. 2000). Activation of NFκB is critical for fatty acid-induced IL-6 secretion and insulin resistance in myotubes in vitro (Sinha et al. 2004; Weigert et al. 2004) and muscle in vivo (Itani et al. 2002). Interestingly, several studies have shown that chronic exposure to IL-6 reduces adipogenic gene expression (Lagathu et al. 2003; 53
Sopasakis et al. 2004) and insulin signaling and glucose uptake in adipocytes (Rotter et al. 2003). NFκB is a ubiquitous transcription factor regulating the expression of genes promoting inflammation and cell survival. NFκB-dependent transcription normally begins with the phosphorylation of the inhibitory κB proteins (IκBs) and their subsequent degradation. IκB proteins are key regulators of NFκB, sequestering the NFκB dimer in the cytoplasm. Phosphorylation of IκBα by IκBα kinase (IKK) triggers its polyubiquitination and proteosomal degradation, thereby releasing NFκB, especially p50/p65, to the nucleus (reviewed in Chen et al. 2004; Shoelson et al. 2003). Following phosphorylation by MAP kinases such as ERK1/2 and p38 or MSK1 (Reviewed in Dixit and Mak 2002; Jiang et al. 2001), phospho-p50/p65 binds to the NFκB response elements (NFκB RE) of target genes, thereby inducing their transcriptional activation (reviewed in Chen and Ghosh 1999; Camp et al. 1997). CLA’s TG-lowering actions in human adipocytes are consistent with NFκB activation based on our data (Brown et al. 2003, 2004) showing that trans-10, cis-12 CLA: 1) decreases adipogenic gene expression; 2) impairs glucose and fatty acid uptake and conversion to TG; and 3) increases the expression and secretion of several proinflammatory cytokines including IL-6 and IL-8. Therefore, the aim of the present study was to examine the extent to which NFκB activation was essential for trans-10, cis- 12 CLA-induced cytokine expression and impaired glucose uptake in primary human adipocytes. Here we demonstrate for the first time that trans-10, cis-12 CLA promotes NFκB activation prior to inducing IL-6 expression and insulin resistance in cultures of 54
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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
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 and 62: espectively, by the MEK inhibitor U
Page 63 and 64: Furthermore, the CLA-mediated incre
Page 65: proteins. Inhibition of NFκB activ
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
Page 113 and 114: activity was measured using the Dua
Page 115 and 116: 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.,
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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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