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

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Figure 2. 9. Model of trans-10, cis-12 CLA-mediated changes of morphology in adipocytes: transcriptional, translational, and post-translational regulation of lipid droplet-associated proteins. Under normal, nonlipolyic conditions, perilipin is exclusively expressed in mature adipocytes, serving as a barrier to protect lipid droplets from hydrolysis by lipases such as hormone sensitive lipase (HSL). Chronic supplementation of trans- 10, cis-12 CLA changes adipocyte morphology by promoting the development of numerous small lipid droplets coated with adipocyte differentiated related protein (ADRP). Trans-10, cis-12 CLA induces perilipin phosphorylation, which drives perilipin movement from the surface of lipid droplet to cytosol and thus increases the susceptibility to hydrolysis by lipase(s), resulting in increased basal lipolysis. As a physiological consequence of trans-10, cis-12 CLA supplementation, ADRP replaces perilipin as a lipid droplets coating protein due to CLA’s combined action on transcriptional repression of perilipin and HSL, and translational activation of ADRP in a mammalian target of rapamycin (mTOR) pathway-dependent manner. 43
Discussion Feeding mixed CLA isomers or trans-10, cis-12 CLA to humans decreases body fat (Blankson et al. 2000; Thom et al. 2001; Riserus et al. 2002), and supplementing cultures of human adipocytes with trans-10, cis-12 CLA reduces cell size and TG content (Brown et al. 2004). However, little is known about the mechanism by which CLA controls the flux of neutral lipids in and out of adipocyte lipid droplets. To our knowledge, the data presented here are the first to demonstrate that trans-10, cis-12 CLA alters adipocyte lipid droplet morphology from perilipin-associated large lipid droplets to ADRP-associated small lipid droplets. This process involves movement of perilipin to the cytosol, presumably by post translational regulation (e.g., perilipin phosphorylation), and differential regulation of lipid droplet-associated protein (ADRP, perilipin, and HSL) expression as shown in Fig 2.9. Furthermore, our data demonstrate for the first time that trans-10, cis-12 CLA induces the mTOR pathway, which could be responsible, in part, for the robust increase in the levels of ADRP protein. Alternatively, the CLA-mediated increase in perilipin movement away from large lipid droplets may provide greater access for ADRP binding on the lipid droplet surface, thereby increasing its stability. Taken together, we propose in our working model (Fig 2.9) that trans-10, cis-12 CLA increases basal lipolysis and reduces lipid droplet TG content by: 1) promoting perilipin dispersion to the cytosol, 2) downregulating perilipin gene expression, and 3) increasing the levels of ADRP protein bound to lipid droplets facilitated by migration of perilipin away from the large lipid droplets and/or by increased ADRP translation induced by the mTOR pathway. 44
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CHUNG, SOONKYU, Ph. D. Mechanisms b
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MECHANISMS BY WHICH CONJUGATED LINO
Page 5 and 6: APPROVAL PAGE This Dissertation has
Page 7 and 8: I also would like to thank The Univ
Page 9 and 10: IV. PREADIPOCYTES MEDIATE LPS-INDUC
Page 11 and 12: LIST OF FIGURES ix Page Figure 1.1.
Page 13 and 14: 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: Figure 2. 8. Trans-10, cis-12 CLA-i
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 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
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adipocytes from WAT in inflammation
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differentiation, cultures of SV cel
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Immunoblotting and 4MUrea-SDS-PAGE
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activity was measured using the Dua
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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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