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

To investigate the impact of CLA’s regulation on adipocytes under conditions of
NFκB depletion, we examined PPARγ expression and the activation of ERK1/2 and
STAT3. As shown in Fig 3.8D, depletion of NFκB p65 modestly attenuated CLA’s
suppression of PPARγ2 (also confirmed by immunostaining in Fig 3.8E) and its
activation of ERK1/2, but had no marked effect on STAT3 phosphorylation. These data
further support our hypothesis that NFκB p65 plays a central role in CLA-induced
suppression of adipogenic gene expression, activation of MEK/ERK signaling, and
subsequent secretion of cytokines.
Because CLA’s suppression of PPARγ was attenuated by knocking down NFκB
activity using siRNA, we hypothesized that silencing NFκB would block or attenuate
CLA’s suppression of insulin-stimulated glucose uptake demonstrated in Fig 3.1. To test
this hypothesis, we measured insulin-stimulated [ 3 H]-2-deoxy-glucose uptake in NFκB-
depleted cultures of adipocytes treated for 24 h with BSA vehicle or trans-10, cis-12
CLA. CLA’s suppression of glucose uptake was attenuated in siP65-transfected cultures
compared to CLA-treated cultures not receiving siP65 (Fig 3.9A). The degree of rescue
of insulin-stimulated glucose uptake (~45%) and Glut4 protein levels (~55%) by siP65
for NFκB in the CLA-treated group was nearly similar to the degree of knockdown
achieved for NFκB-p65 in this experiment (~54%; densitometry not shown) (Fig 3.9B).
Collectively, these data demonstrate for the first time that specific depletion of NFκB p65
in CLA-treated cultures partially rescued PPARγ and Glut4 protein levels and insulin-
stimulated glucose uptake. This isomer-specific, CLA-mediated activation of cytokines
and suppression of PPARγ and Glut4 protein levels shows striking similarity with other
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