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

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CLA’s suppression of insulin sensitivity and TG accumulation (Brown et al. 2003, 2004; Chung et al. 2005) in human adipocytes could be due to de-differentiation or apoptosis, or blocking new differentiation. However, 30 µM trans-10, cis-12 CLA does not cause apoptosis in our cultures based on: 1) NFκB activation antagonizes apoptosis and promotes cells survival (Dixit et al. 2002); 2) caspase 3 is not activated by 30 µM CLA for up to 72 h (unpublished data); 3) Hoechst and DAPI staining indicate similar numbers of nuclei and no differences in nuclear condensation or fragmentation in CLA- treated cultures compared to controls (Fig 3.8E) and (Brown et al. 2004); 4) protein and RNA levels are not reduced after CLA treatment; and 5) CLA reduces the TG content of adipocytes without reducing the number of adipocytes (Brown et al. 2004; Chung et al. 2005). Similarly, CLA does not appear to cause de-differentiation per se because: 1) adipocytes still contain small lipid droplets after 3 wk of treatment, even though CLA suppresses adipogenic gene expression and protein levels and impairs glucose uptake within 24 h of treatment (Brown et al. 2004); 2) CLA increases adipose differentiation- related protein (ADRP) and leptin expression (Brown et al. 2004; Chung et al.2005), suggesting that CLA-treated cultures still contain adipocytes; and 3) Pref-1 gene expression, which is abundant in our non-differentiated SV cells, is absent in CLA- treated cultures containing adipocytes (unpublished data), suggesting these cells do not de-differentiate back to preadipocytes. Finally, we do not observe new differentiation of adipocytes after ~day 6 of differentiation. Instead, the increased TG content of the cultures after day 6 is due primarily to increased size of the lipid droplets within adipocytes. Thus, because our experiments began on ~ day 12 of differentiation, CLA is 89
most likely not impairing the differentiation of new adipocytes. Instead, we postulate that CLA is causing delipidation, primarily by blocking de novo TG synthesis (Brown et al. 2003, 2004), and to a lesser extent, by increasing lipolysis (Chung et al. 2005). In summary, our in vitro data demonstrate that a physiological level of trans-10, cis-12 CLA activates NFκB- and ERK1/2-dependent cytokine production, which together suppress PPARγ and Glut4 levels, and lead to impaired glucose uptake. Studies are currently underway examining: 1) how CLA regulates PPARγ and the expression of its target genes; 2) the specific signaling role of SV cells and adipocytes in mediating the TG-lowering actions of CLA; and 3) the CLA-induced, upstream signal that activates NFκB and ERK1/2. 90
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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
Page 51 and 52: 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: humans (Riserus et al. 2004a), and
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 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
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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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