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New therapeutic targets for the treatment of insulin resistance…
(PKC)-mediated pathways (5, 6). In addition, cytokines
down-regulate early key mediators of the insulin signaling
cascade such as the insulin receptor (IR) or insulin
receptor substrate 1 (IRS1) (7, 8), as well as increase the
expression of negative modulators of this pathway such as
the protein tyrosine phosphatase 1B (PTP1B) (9, 10). On
the other hand, circulating FFAs, which are usually
increased in insulin resistant states (11-13), also activate
these proinflammatory pathways boosting the defects in
peripheral insulin actions (14-16).
In addition to the inflammatory effects, the activation
of endoplasmic reticulum (ER) stress-mediated signaling
pathways by FFAs has been linked to obesity-associated
immunometabolic dysregulation and insulin resistance (17,
18). ER stress is sensed by three main proteins: X-box
binding protein 1 (X-BP1), PRKR-like endoplasmic
reticulum kinase (PERK) and activating transcription
factor-6 (ATF6), which cooperate to mitigate ER stress by
reducing protein translation, stabilizing proteins by
chaperones and activating ER-associated protein
degradation (19). Chemical chaperones
(tauroursodeoxycholic acid (TUDCA) and 4-
phenylbutryate) that alleviate ER stress through protein
stabilization, improve systemic glucose homeostasis,
increase glucose uptake in adipose and skeletal muscle and
reduce hepatic glucose production (20). Moreover, a study
in obese subjects has revealed that treatment with TUDCA
increases insulin sensitivity in liver and skeletal muscle by
approximately 30% compared to the placebo therapy (21).
Several studies have reported that besides adipocytes,
adipose tissue resident macrophages, which migrate and
accumulate in WAT, have a relevant role in obesity-
induced chronic inflammation (22-24) through their
polarization toward the M1-like state (25). Although the
importance of macrophages in the molecular mechanisms
triggering insulin resistance in skeletal muscle and adipose
tissue has been explored (25-27), it remains unclear
whether the inflammatory milieu from hepatic
macrophages impact insulin signaling in hepatocytes. On
that basis, in this study we have investigated for the first
time the effect of Kupffer cells activated by two distinct
FFAs, palmitate (saturated) and oleate (unsaturated), on
stress-mediated pathways, lipoapoptosis and the insulin
signaling cascade in hepatocytes.
2. MATERIALS AND METHODS
2.1. Reagents
Fetal bovine serum (FBS) (#10270) and culture media
DMEM (#41966-029) were obtained from Invitrogen (Life
Technologies, Gran Island, NY). TRIzol reagent (#T9424),
sodium palmitate (#P9767), sodium oleate (#O7501),
bovine serum albumin (BSA) (#A6003), fatty acid free-
BSA endotoxin free (#A8806) and insulin (#I0516) were
from Sigma Aldrich (St. Louis, MO). Bradford reagent,
acrylamide, immunobloting PVDF membrane and
Immobilon Western Chemiluminescent HRP Substrate
were purchased from Bio-Rad (Hercules, CA).
@Real Academia Nacional de Farmacia. Spain
2.2. Free fatty acid preparation
2.5 mM free fatty acid (FFA) stock solutions were
prepared by modification of Spector method (28). Briefly,
cold sodium palmitate or sodium oleate were dissolved in
0.1M NaOH by heating at 70°C while 0.5 mM BSA
solution was prepared by dissolving fatty acid free-BSA in
NaCl 0.9% by heating at 50°C (at maximum). Once BSA
and FFA solutions were completely dissolved, palmitate
and oleate solutions were diluted 10-times in the BSA
solution and mixed by pipetting to achieve a final molar
ratio of 5:1. Control BSA was prepared by adding the
same amount of 0.1M NaOH into 0.5 mM BSA solution.
All preparations were filtered, aliquoted and stored at –
20°C.
2.3. Culture of RAW 264.7 murine macrophages
Murine RAW 264.7 macrophage cell line, kindly
provided by Dr. Tarín (CNIC, Madrid, Spain), were
cultured in RPMI supplemented with 10% heat inactivated
FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2
mM glutamine. Confluent macrophages were treated with
BSA or FFAs solutions (750 µM conjugated oleate/BSA
or 750 µM conjugated palmitate/BSA) for 24 h to obtain
the corresponding conditioned media (CM) (27). CM were
centrifuged to remove dead cells and directly added
(without dilution) to hepatocytes for several time periods.
2.4. Isolation and culture of Kupffer cells
Kupffer cells were obtained from livers of 3 months-
old male C57/BL6 mice. All animal experimentation has
been conducted according to the Guide of the care and use
of laboratory animals (National Academy of Sciences,
USA). For Kupffer cells (KC) isolation, the supernatant
from the first centrifugation of the hepatocyte isolation
protocol was collected and centrifuged twice at 50 x g for
5 min to discard the pellet with the remaining hepatocytes.
The latest supernatant was centrifuged at 500 x g for 5 min
at 4ºC and the pellet containing the KC was resuspended in
attachment media. Cells were mixed by inversion with
50% percoll and centrifuged at 1.000 x g for 30 min
without acceleration or brake at room temperature. Finally,
KC pellet was washed with PBS 1X, centrifuged twice at
500 x g for 10 min at 4ºC to wash out the residual percoll
solution and cells were resuspended in RPMI
supplemented with 10% heat inactivated FBS, 100 U/ml
penicillin, 100 µg/ml streptomycin and 2 mM glutamine.
Cells were then plated on 12 well-plates and maintained
for 24 h before treatments. Conditioned media was
prepared as described in RAW 264.7 cells.
2.5. Hepatocyte cell culture.
Human hepatocytes were isolated by the two-step
collagenase procedure from non-tumor areas of liver
biopsies from patients submitted to a surgical resection for
liver tumours after obtaining patients' written consent (29).
Primary mouse hepatocytes were isolated from non-fasting
male C57BL/6 mice (10-12-week-old) by perfusion with
collagenase as described (30). Cells were seeded on 6
well-plate (Corning, New York, NY) and cultured in
201