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New therapeutic targets for the treatment of insulin resistance…

Figure 1. Insulin signaling is differentially modulated in human hepatocytes pretreated with CM-O or CM-P. Conditioned medium
from RAW 264.7 macrophages treated with BSA (CM-B), oleate (CM-O) or palmitate (CM-P) was added to human primary hepatocytes
for 24 h. Then, cells were stimulated with 10 nM insulin for 10 min. Total protein was analyzed by Western blot using the indicated
antibodies. Representative blots are shown. (n=4 independent experiments performed in duplicate).

Figure 2. Purity of Kupffer cells. Kupffer cells and hepatocytes were isolated from mice as described in the Experimental Procedures
and mRNA levels of CD68 and F4/80 were determined. CD68 and F4/80 were undetectable in hepatocytes (n=3 independent experiments
performed in duplicate).

Figure 3. Expression of cytokines and markers of inflammation from Kupffer cells. Primary Kupffer cells were treated with BSA
(B), oleate (O) or palmitate (P) for 24 h. TNFa, IL-6, IL-1ß, MCP1, IL-10, arginase 1, Mcr1 and Mgl1 mRNA levels were analyzed by

qRT-PCR. Results are expressed as fold increase relative to BSA condition (1) and are mean ± SEM (n=3), *p<0.05, **p<0.01, O or P,

respectively, vs B.

3.3. Conditioned medium from Kupffer cells stimulated or CMK-P, respectively). As a control, hepatocytes were
with palmitate activates stress-mediated signaling
pathways stimulated with CM from Kupffer cells loaded with BSA

Next, we confirmed data on proinflammatory cytokines (CMK-B). As shown in Figure 4, phosphorylations of
by analyzing the activation of stress kinases in primary STAT3, p38 MAPK, JNK, PERK and eIF2a were
mouse hepatocytes treated with CM from Kupffer
macrophages stimulated with oleate or palmitate (CMK-O observed exclusively in hepatocytes treated with CMK-P.

@Real Academia Nacional de Farmacia. Spain 203