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

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trans-10, cis-12 CLA-mediated reduction of human adipocyte TG content is associated with differential localization and expression of lipid droplet-associated proteins. This process involves both the translational control of ADRP through activation of mTOR/p70S6K/S6 signaling and transcriptional control of perilipin A. Introduction Conjugated linoleic acid (CLA) refers to a group of dienoic derivatives of linoleic acid. The two primary isomers of CLA found in ruminant meats and milk products and commercial preparations are cis-9, trans-11 CLA and trans-10, cis-12 CLA. CLA isomers have potential anticancer (Ha et al. 1997; Belury 2002) and antiobesity properties (reviewed in Evans et al. 2002; Brown et al. 2003). Concerning the isomer specificity of CLA and obesity, numerous animal studies have demonstrated that trans-10, cis-12 CLA prevents the development of adiposity (Park et al. 1997, 1999a, b; Delany et al. 1999, Ostrowska 1999). Similarly, we have shown that trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, inhibits differentiation of human preadipocytes into adipocytes and reduces the triglyceride (TG) content of mature or newly differentiated human adipocytes (Brown et al. 2001, 2003, 2004). However, the molecular mechanism(s) and physiological consequences of CLA supplementation are unclear, especially in humans. Recently, we reported that trans-10, cis-12 CLA treatment of cultures of human stromal vascular (SV) cells containing newly differentiated adipocytes caused delipidation by activating mitogen-activated protein kinase kinase/extracellular signal related kinase (MEK/ERK) signaling (Brown et al. 2004). This relatively rapid activation 19
of MEK/ERK signaling by trans-10, cis-12 CLA was followed by a decrease in the mRNA levels of peroxisome proliferator-activated receptor gamma (PPARγ) and many of its downstream adipogenic target genes, including perilipin, glucose transporter 4 (GLUT4), adipocyte-specific fatty acid binding protein (aP2), lipoprotein lipase (LPL), and adiponectin. These CLA-mediated alterations were accompanied by decreased glucose and fatty acid uptake, leading to decreased cellular TG content (e.g., delipidation) of the cultures, and increased the number of cells containing small lipid droplets. Given the important role of lipid droplet-associated proteins such as perilipin and adipose differentiation-related protein (ADRP) in facilitating lipid deposition or hydrolysis of fatty acids from lipid droplets, we hypothesized that these proteins were intimately involved in CLA’s delipidation of adipocytes. The perilipins, exclusively found at the outer surface of lipid storage droplets in adipocytes and steroidogenic cells, provide a protective protein coat on the lipid droplet surface that shields stored TG against the basal (i.e., non-hormonally stimulated) lipolytic actions of cellular lipases, and serves as a cofactor for catecholamine-induce lipolysis (reviewed in Londos et al. 1999). Increased expression of perilipin increases TG storage (Brasaemle et al. 2000a; Souza et al. 1998), whereas tumor necrosis factor-alpha (TNF-α) stimulates lipolysis partly by terminating perilipin gene expression, leading to decreased TG storage (Souza et al. 1998). Perilipin ablation reduces fat mass, increases basal lipolysis, and alters lipid droplet morphology, including reducing adipocyte size (Tansey et al. 2001; Martinez- Botas et al. 2000). Protein kinase A (PKA)-mediated phosphorylation of hormone sensitive lipase (HSL) and perilipin promotes HSL movement to the lipid droplet and 20
Page 1 and 2: CHUNG, SOONKYU, Ph. D. Mechanisms b
Page 3 and 4: 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: CHAPTER II TRANS-10, CIS-12 CLA INC
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 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
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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-
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Based on our working model (Fig 3.1
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mRNA levels of TNF-α (Fig. 3). How
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humans (Riserus et al. 2004a), and
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most likely not impairing the diffe
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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’
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,
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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