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Virginia Pardo Marqués, Águeda González-Rodríguez, Ángela Martínez Valverde
media containing Dulbecco's modified Eagle's medium 3. RESULTS AND DISCUSSION
and Ham's F-12 medium (1:1) with 10% FBS,
supplemented with 2 mm glutamine, 100 U/ml penicillin, RESULTS
100 µg/ml streptomycin and 1 mM sodium pyruvate
(attachment media) and maintained for 24 h before 3.1. Insulin signaling is opposite modulated in human
treatments. The generation and characterization of hepatocytes pretreated with conditioned medium from
immortalized mouse hepatocyte cell line has been macrophages stimulated with palmitate or oleate
previously described (31). Cells were grown in DMEM
plus 10% heat inactivated FBS, 100 U/ml penicillin, 100 The liver is composed primarily of hepatocytes, but
µg/ml streptomycin and 2 mM glutamine. also contains blood and lymph vessels, nerves and immune
cells. Since all these cell types can potentially respond to a
2.6. Preparation of protein extracts and Western blot high fat-mediated inflammatory environment in vivo, we
performed a cell culture-based approach to investigate the
To obtain total cell lysates, attached cells were scraped specific cross-talk between macrophages and hepatocytes
off and incubated for 10 min on ice with lysis buffer (25 in the context of fatty acid overload. For this goal, RAW
mM HEPES, 2.5 nM EDTA, 0.1% Triton X-100, 1 mM 264.7 macrophages were treated with palmitate, a typical
PMSF and 5 µg/ml leupeptin). After protein content saturated FFA found in western diets, for 24 h. Then,
determination with Bradford reagent, total protein were culture media (conditioned medium) was removed.
boiled in Laemmli sample buffer and submitted to 8-15% Conditioned medium was hereafter called CM-P (collected
SDS-PAGE. Proteins were transferred to Immunoblot from RAW 264.7 cells treated with palmitate) or CM-B
PVDF membrane and, after blocking with 3% BSA or 5% (from RAW 264.7 cells treated only with BSA as a
non-fat dry milk, membranes were incubated overnight control). We also obtained conditioned medium from
with several antibodies as indicated. Immunoreactive RAW 264.7 cells treated with oleate, a well-known
bands were visualized using the ECL Western blotting nontoxic monounsaturated FFA. This conditioned medium
protocol. Densitometric analysis of the bands was was hereafter called CM-O (collected from RAW 264.7
performed using Image J software. The anti-phospho- cells treated with oleate). Once the conditioned media
PERK (Thr 980) (#3179), anti-phospho-eIF2a (Ser 51) were prepared, they were added for 24 h to human
(#9721), anti-phospho-JNK (#9251), anti-phospho-STAT3 hepatocytes and after that these cells were stimulated with
(#9131) antibodies were from Cell Signaling Technology 10 nM insulin for 10 min. As depicted in Figure 1, insulin
(Danvers, MA). The anti-JNK (sc-571), anti-IR ß (sc-711), induced IR tyrosine phosphorylation and Akt
anti-eIF2a (sc-11386), anti-CHOP (sc-7351), anti- phosphorylation at both residues were enhanced in
phospho-p38 (sc-17852), anti-p38 (sc-9212), anti-IRS1 hepatocytes preincubated with CM-O as compared to
(sc-559), anti-phospho-IR (sc-25103), anti-phospho-Akt control hepatocytes preincubated with control CM-B.
(Thr 308) (sc-16646), anti-phospho-Akt (Ser 473) (sc- Conversely, IR tyrosine and Akt serine/threonine
7985), anti-Akt (sc-8312) and anti-PTP1B (sc-1718) phosphorylations were reduced in presence of CM-P.
antibodies were from Santa Cruz Biotechnology (Santa Moreover, protein levels of IR and IRS1 decreased only in
Cruz, CA). Anti-a-tubulin (T5168) was from Sigma hepatocytes treated with CM-P. Altogether, these results
Aldrich (St. Louis, MO). indicate opposite effects of palmitate and oleate in
triggering a cross-talk between macrophages and
2.7. RNA isolation and qPCR hepatocytes with relevant effects in insulin signaling.
Total RNA was isolated using Trizol reagent and was 3.2 Differential effects of palmitate and oleate in levels of
reverse transcribed using a SuperScriptTM III First-Strand proinflammatory cytokines expressed by macrophages
Synthesis System for qPCR following manufacturer’s
indications. qPCR was performed with an ABI 7900 In order to confirm the differential effects of oleate and
sequence detector using the SyBr Green method and d(N)6 palmitate in a physiological context, we isolated and
random hexamer with primers purchased from Invitrogen. cultured Kupffer cells from C57/BL6 mice and these
PCR thermocycling parameters were 95°C for 10 minutes, resident macrophages were stimulated with oleate or
40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. palmitate for 24 h. The purity of Kupffer cells was
Each sample was run in triplicate and normalized to 18s checked by the analysis of CD68 and F4/80 mRNA levels
RNA. Fold changes were determined using the ??Ct (Figure 2).
method. Primer sequences are available upon request.
In an attempt to characterize the molecules released by
2.8. Statistical analysis macrophages to the CM, we assessed the expression of
proinflammatory cytokines characteristic of M1
Data are presented as mean ± SEM, and were macrophage polarization by real-time PCR. Treatment of
compared by using the Bonferroni ANOVA test. All Kupffer cells with palmitate, but not oleate, increased IL6,
statistical analyses were performed using the IBM SPSS IL1ß, TNFa and MCP1 mRNA levels. On the other hand,
Statistics 21.0 (SPSS Inc. IBM, Armonk, NY) software treatment with oleate significantly up-regulated mRNA
with 2-sided tests. Differences were considered levels of M2 polarization markers (IL10, Mcr1, Mgl1 and
statistically significant at p<0.05. arginase 1) (Figure 3).
202 @Real Academia Nacional de Farmacia. Spain
