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

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adipose depot and species. For example, Hauner (2005) reported that human adipocytes represent ~50-70% of the cells in human WAT. In contrast, Fain et al. (2002) reported that ~70% of the protein from human WAT digests is associated with tissue matrix, and the remaining 30% was equally divided between SV cells and floating adipocytes. Interestingly, non-adipocytes from human WAT, especially from the tissue matrix, accounted for ~90% of the cytokine secretion (Fain et al. 2004). Furthermore, the more robust cytokine secretion from WAT obtained from morbidly obese subjects [e.g., body mass index (BMI) = 45] compared to obese subjects (BMI = 32) was from the non- adipocytes. In contrast, Granneman et al. (2004) reported that mature adipocytes account for only 16% of murine epididymal WAT cells. Thus, the relative abundance of adipocytes compared to non-adipocytes in human WAT has yet to be resolved, and the type and function of these non-adipocytes in human WAT are poorly understood. Based on these gaps, we first characterized the cells in our cultures, and then examined the role that the non-adipocytes play in inflammation and their impact on signaling, gene expression, and glucose metabolism in neighboring adipocytes. Initially, we characterized the cultures using DM1 during the first 3 days of differentiation (+DM1) compared to cultures not receiving DM1 (−DM1). Cultures receiving DM1 had about ~50% of their cells (Fig 4.1A) filled with lipid. In contrast, few of the cells lacking DM1 had visible lipid droplets. As shown in Fig 4.1B-D, cultures receiving DM1 abundantly expressed markers of adipocytes (e.g., PPARγ, aP2) and lesser amounts of the preadipocyte marker Pref-1 compared to cultures lacking DM1. Thus, our differentiated cultures contain both adipocytes and preadipocytes. 119
To determine if non-adipocytes other than preadipocytes were present in our cultures receiving DM1, we measured the expression levels of markers of macrophages and myocytes using CD68/MAC-1 and Myo-D, respectively. As shown in Fig 4.2, neither CD68/MAC-1 nor Myo-D mRNA were detectable in our cultures. Thus, we could find no evidence that macrophages or myocytes are present in our cultures on day 12 of differentiation. Clearly, this is not the situation in vivo, where immune cells may be recruited to WAT (Weisburg et al. 2003; Xu et al. 2003) and non-adipocytes in tissue matrix associated with adipose tissue robustly secrete cytokines (Fain et al. 2004). This discrepancy is most likely due to the loss of macrophages during growth and differentiation of the cultures. Therefore, because our model lacks of macrophages as occurs in vivo in adipose tissue, it provides a model for examining the role that preadipocytes play in inflammation and insulin resistance. To access the role of non-adipocytes in inflammation and insulin resistance, cultures of newly differentiated human adipocytes (AD50) were initially fractionated after LPS stimulation and the expression of markers of preadipocytes (e.g., AEBP-1) and adipocytes (e.g., aP2, APM-1, PPARγ) and inflammatory cytokines (e.g., IL-6, IL-8, TNF-α, IL-1B) and markers (e.g., COX-2) were measured (Fig 4.3). These data are consistent with work <strong>by</strong> Harkins et al. (2004) showing that preadipocytes produce cytokines in response to LPS to a much greater extent than adipocytes using the 3T3-L1 cell line. These data demonstrate that preadipocytes have a greater inflammatory response to LPS than adipocytes in our cultures, and are associated with attenuated expression of adiponectin and PPARγ, two markers of insulin sensitivity. 120
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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
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 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: Discussion Adipose tissue is a sour
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
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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