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

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Using a second approach, we manipulated the degree of differentiation of the cultures (e.g., AD0, AD50, or AD90) before LPS treatment, and then measured cytokine expression (Fig 4.4), glucose uptake (Fig 4.5), PPARγ activity and phosphorylation (Fig 4.6), and NFκB (Fig 4.7) and MAPK activation (Fig 4.7) and signaling (Fig 4.8). Both of these approaches gave consistent results demonstrating that the presence of preadipocytes in the cultures was positively associated with the degree of inflammatory gene expression, implicating preadipocytes as dominant source of cytokine production that adversely affect PPARγ activity and insulin sensitivity involving NFκB and MAPK activation and signaling. Role of PPARγ in Inflammation and Insulin Resistance Despite increasing evidence of the casual link between inflammation and insulin resistance, elucidating the precise mechanism by which cytokines impair glucose uptake has proved difficult (reviewd in Rajala and Scherer 2003; Wellen and Hotamisiligil 2005). Our data highlight the importance of preadipocytes in mediating insulin resistance. One possible explanation for this observation is the suppression of adiponectin gene expression by LPS, which is exclusively excreted from adipocytes and positively associated with insulin sensitivity (Ajuwon and Spurlock 2005 a,b). In addition to its role in the modulation of glucose and lipid metabolism, adiponectin has been reported to have potent anti-suppressive properties due to its ability to induce the production of anti- inflammatory cytokines (i.e., IL-10), and to inhibit proinflammatory cytokine production (Reviewed in Gil-Campos et al. 2004). Thus, it seems reasonable to presume that LPS 121
attenuation of insulin sensitivity in AD50 model is due, at least in part, by the suppression of adiponectin expression. Consistent with this notion, LPS administration to cultures containing almost exclusively adipocytes (AD90) did not adversely affect insulin-stimulated glucose uptake (Fig 4.5) or adiponectin gene expression (data not shown). Ajuwon and Spurlock (2005a) reported direct induction of PPARγ by adiponectin, coupled with suppression of NFκB activation, suggesting mutual transcriptional activation of PPARγ and adiponectin may determine adipocyte susceptibility to inflammatory stimuli. The PPAR subfamily of nuclear receptors controls many different target genes involved in both lipid metabolism and glucose homeostasis. Loss of function, PPARγ mutations in humans cause insulin resistance (Zhang et al. 2004; Freedman et al 2005; reviewed in Hegele 2005), and activation of PPARγ by thiazolidinediones act as insulin sensitizers (reviewed in Yki-Jarvinen 2004). However, detailed mechanisms describing how inflammation suppresses PPARγ activity in human WAT are unclear. In our study, LPS suppressed ligand-induced, but not basal, PPARγ activity. Similarly, the PPARγ antagonist GW9662 decreased ligand-dependent, but not basal, PPARγ activity (our unpublished data), implicating ligand-inducible PPARγ activity is critical in regulating insulin sensitivity. One of the putative mechanisms modulating PPARγ activity is phosphorylation. It has been suggested that phosphorylation of PPARγ 1) impairs PPARγ affinity for its ligand (Shao et al. 1998), 2) controls interactions between PPARs and corepressors and/or coactivators of transcription (Guan et al. 2005), or 3) alters PPARγ binding to the 122
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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
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espectively, by the MEK inhibitor U
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Furthermore, the CLA-mediated incre
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proteins. Inhibition of NFκB activ
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Sopasakis et al. 2004) and insulin
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antibodies for anti-glyceraldehydre
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pM of human insulin in the presence
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were (accession #NM000600) sense (5
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Transfection efficiency was examine
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treatment (Brown et al. 2004), we d
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myotubes (Sinha et al. 2004; Weiger
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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 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: To determine if non-adipocytes othe
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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