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

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To determine critical signaling steps involved in trans-10, cis-12 CLA-induced mTOR signaling pathway, we used several inhibitors to block upstream regulators of p70S6 kinase and S6 ribosomal proteins (Fig 2.7). Activation of P70S6 kinase and S6 kinase2 can be specifically blocked by the immunosuppressant rapamycin, a bacterial macrolide, without affecting kinases involved in mitogenic responses, resulting in attenuation of translational activation of 5’-TOP mRNA (Terada et al. 1994). In support of our concept that trans-10, cis-12 CLA-induced translational activation occurs via mTOR, phosphorylation of p70 S6 kinase and S6 ribosomal proteins by 3 h treatment with trans-10, cis-12 CLA were blocked by pretreatment with rapamycin (Fig 2.7). In addition, most inhibitors including the MEK/ERK inhibitor U0126, the G protein coupled receptor (GPCR)-Gi/o coupling inhibitor pertussis toxin (PTX), the phosphatidyl inositol 3 kinase (PI3K) inhibitor LY-294002, the protein kinase C (PKC) inhibitor calphostin C, and the cSRC kinase protein inhibitor PP2 blocked or attenuated CLA’s induction of phosphorylation of p70 S6 kinase and S6 ribosomal protein. Taken together, these data suggest that: 1) CLA-mediated activation of mTOR signaling pathway is isomer-specific, and 2) its regulation is controlled by multiple factors that could potentially impact on 40S ribosomal protein activation of 5’-TOP mRNA translation, including MEK/ERK, PKC, protein kinase B, and GPCR-Gi protein. These data implicate a potential role of the mTOR pathway as a signal integrator of CLA’s TG-lowering actions, and a potential mechanism by which trans-10, cis-12 CLA increases ADRP protein levels. 33
Rapamycin Blocks CLA’s Increase in ADRP Protein Expression Based on the isomer-specific activation of S6 ribosomal protein by CLA, we hypothesized that translational induction of ADRP by trans-10, cis-12 CLA is associated with activation of mTOR pathway. To test this hypothesis, we treated cultures for 24 h with either 30 µM trans-10, cis-12 CLA or BSA in the presence and absence of the mTOR-specific inhibitor rapamycin. As hypothesized, CLA’s induction of ADRP protein was blocked by the pretreatment with rapamycin (Fig 2.8). In contrast, neither perilipin nor caveolin-1 expression was affected by rapamycin. It was also notable that 24 h treatment with trans-10, cis-12 CLA modestly attenuated perilipin-A and perilipin-B gene expression even in the presence of rapamycin, suggesting that a rapamycin-sensitive pathway may not be necessary for a reduction of perilipin expression by trans-10, cis-12 CLA (Fig 2.8). In fact, our data support the notion that trans-10, cis-12 CLA-mediated reduction of perilipin expression is mediated by a more chronic activation of cytokine- induced MEK/ERK signaling (Fig 2.5A), which we have described previously (Brown et al. 2004). Collectively, these data suggest that the trans-10, cis-12 CLA-mediated increase in the levels of the small lipid droplet associated protein ADRP, is partly due to a rapamycin-sensitive increase in ADRP protein synthesis. 34
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 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: dependent effects of CLA on the pho
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 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 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
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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
Page 109 and 110:
differentiation, cultures of SV cel
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Immunoblotting and 4MUrea-SDS-PAGE
Page 113 and 114:
activity was measured using the Dua
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macrophages and myocytes, respectiv
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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
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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
Page 131 and 132:
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
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Concerning membrane specific recept
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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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