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

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Absract CHAPTER IV PREADIPOCYTES MEDIATE LPS-INDUCED INFLAMMATION AND INSULIN RESISTANCE IN PRIMARY CULTURES OF NEWLY DIFFERENTIATED HUMAN ADIPOCYTES Recent data suggest that proinflammatory cytokines secreted from adipose tissue contribute to the morbidity associated with obesity. However, characterization of the cell types involved in inflammation and how these cells promote insulin resistance in human adipocytes are unclear. We simulated acute inflammation using endotoxin lipopolysaccharide (LPS) to define the roles of non-adipocytes in primary cultures of human adipocytes. LPS induction of the mRNA levels of proinflammatory cytokines (e.g., IL-6, IL-8, TNF-α, IL-1β) occurred primarily in the non-adipocyte fraction of newly differentiated human adipocytes. Non-adipocytes were characterized as preadipocytes based on their abundant mRNA levels of preadipocyte markers preadipocyte factor-1 (Pref-1) and adipocyte enhancer protein-1 (AEBP-1) and only trace levels of markers for macrophages and myocytes. The essential role of preadipocytes in inflammation was confirmed <strong>by</strong> modulating the degree of differentiation in the cultures from ~0-90%. LPS induced-proinflammatory cytokine expression and nuclear factor kappa B (NFκB) and mitogen activated protein kinase (MAPK) signaling decreased as differentiation increased. LPS-induced cytokine expression in preadipocytes was associated with 91
1) decreased adipogenic gene expression, 2) decreased ligand-induced activation of a peroxisome proliferator activated receptor (PPAR) γ reporter construct and increased phosphorylation of PPARγ, and 3) decreased insulin-stimulated glucose uptake. Collectively, these data demonstrate that LPS induces NFκB- and MAPK-dependent proinflammatory cytokine expression primarily in preadipocytes, which triggers the suppression of PPARγ activity and insulin sensitivity in human adipocytes. Introduction Obesity and its associated metabolic pathologies are the most common metabolic diseases in the U.S., affecting over 50% of the adult population. One emerging feature of obesity is the linkage between obesity and chronic inflammation characterized <strong>by</strong> increased cytokine production and acute-phase inflammatory signaling in adipose tissue (reviewed in Wellen and Hotamisligil 2005; Trayhurn 2005). Thus, white adipose tissue (WAT) is no longer considered an inert depot of stored energy, but also an active endocrine organ secreting a diverse array of proinflammatory “adipokines” such leptin, interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α, all of which have been linked to insulin resistance. However, the exact role of cells comprising adipose tissue in mediating inflammation and causing insulin resistance is still unclear. Human WAT has been reported to be composed of ~50-70% adipocytes, ~20-40% stromal vascular (SV) cells (i.e., preadipocytes, fibroblasts, nondifferentiated mesenchymal cells), and ~1-30% infiltrated macrophages (Hauner 2005). However, less 92
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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
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 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: most likely not impairing the diffe
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 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
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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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