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

CLA’s suppression of insulin sensitivity and TG accumulation (Brown et al. 2003,
2004; Chung et al. 2005) in human adipocytes could be due to de-differentiation or
apoptosis, or blocking new differentiation. However, 30 µM trans-10, cis-12 CLA does
not cause apoptosis in our cultures based on: 1) NFκB activation antagonizes apoptosis
and promotes cells survival (Dixit et al. 2002); 2) caspase 3 is not activated by 30 µM
CLA for up to 72 h (unpublished data); 3) Hoechst and DAPI staining indicate similar
numbers of nuclei and no differences in nuclear condensation or fragmentation in CLA-
treated cultures compared to controls (Fig 3.8E) and (Brown et al. 2004); 4) protein and
RNA levels are not reduced after CLA treatment; and 5) CLA reduces the TG content of
adipocytes without reducing the number of adipocytes (Brown et al. 2004; Chung et al.
2005). Similarly, CLA does not appear to cause de-differentiation per se because: 1)
adipocytes still contain small lipid droplets after 3 wk of treatment, even though CLA
suppresses adipogenic gene expression and protein levels and impairs glucose uptake
within 24 h of treatment (Brown et al. 2004); 2) CLA increases adipose differentiation-
related protein (ADRP) and leptin expression (Brown et al. 2004; Chung et al.2005),
suggesting that CLA-treated cultures still contain adipocytes; and 3) Pref-1 gene
expression, which is abundant in our non-differentiated SV cells, is absent in CLA-
treated cultures containing adipocytes (unpublished data), suggesting these cells do not
de-differentiate back to preadipocytes. Finally, we do not observe new differentiation of
adipocytes after ~day 6 of differentiation. Instead, the increased TG content of the
cultures after day 6 is due primarily to increased size of the lipid droplets within
adipocytes. Thus, because our experiments began on ~ day 12 of differentiation, CLA is
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