Am J Physiol Endocrinol Metab 295: E85–E91, 2008.
First published April 22, 2008; doi:10.1152/ajpendo.90224.2008.
Adipose tissue inflammation and liver fat in patients with highly active
antiretroviral therapy-associated lipodystrophy
Ksenia Sevastianova,1,2 Jussi Sutinen,2,3 Katja Kannisto,4 Anders Hamsten,4 Matti Ristola,3
and Hannele Yki-Järvinen2
1
Minerva Institute for Medical Research, Helsinki; Divisions of 2Diabetes and 3Infectious Diseases, Department of Medicine,
Helsinki University Central Hospital, Helsinki, Finland; and 4Department of Medicine, Atherosclerosis Research Unit, King
Gustav V Research Institute, Karolinska Institutet, Stockholm, Sweden
Submitted 11 February 2008; accepted in final form 13 April 2008
the liver is the site of glucose and
very-low-density lipoprotein (VLDL) production. Once fatty,
the liver overproduces both glucose (35) and VLDL (1),
leading to hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and a low serum high-density lipoprotein (HDL) cholesterol concentration (20). The amount of fat in the liver is,
independent of obesity, closely associated with all features of
the metabolic syndrome: increased waist circumference, increased fasting serum glucose and triglyceride, and low serum
HDL cholesterol concentrations as well as increased blood
pressure (19).
Adipose tissue of obese subjects is characterized by increased macrophage infiltration and overexpression of inflammatory cytokines and chemokines (7, 10, 12). Liver fat has
been shown to be associated with increased gene expression of
macrophage-specific cell surface markers such as CD68 (18,
25) and an increased number of macrophages (10, 18) in
adipose tissue. Recently, this association has been shown to be
independent of obesity (18, 25). These data raise a possibility
that inflammatory changes in adipose tissue regulate liver fat or
vice versa (3, 8) or that a common etiological factor regulates
both liver fat and inflammation in adipose tissue.
In human immunodeficiency virus (HIV)-1-infected patients
with highly active antiretroviral therapy (HAART)-associated
lipodystrophy, increased gene and protein expression of inflammatory cytokines such as tumor necrosis factor (TNF)-␣,
interleukin (IL)-6, and IL-8 have been reported in lipoatrophic
abdominal subcutaneous fat (5, 23). An increased density of
macrophages, as determined by positive immunohistochemical
staining for CD68, has been described in lipoatrophic abdominal subcutaneous adipose tissue of HAART-treated patients
compared with HIV-negative subjects (15). The latter study,
however, lacked a HAART-treated HIV-1-positive control
group without lipodystrophy. Thus, it remained nebulous
whether the observed differences were due to HAART-associated lipodystrophy, antiretroviral drugs, or HIV-1 per se. Recently, in a study comparing HIV-negative, HIV-1-positive
HAART-naive and HAART-treated patients with and without
lipodystrophy, mRNA concentration of TNF-␣ in abdominal
subcutaneous adipose tissue was shown to be increased due to
HIV-1 infection itself, and changes were reinforced after the
commencement of antiretroviral treatment, but not following
the development of lipodystrophy (13).
We have previously reported that patients with HAARTassociated lipodystrophy have a significantly higher liver fat
content than nonlipodystrophic HAART-treated patients (38).
However, there are no data on whether increased liver fat in
these patients correlates with adipose tissue inflammation.
Therefore, in the present study, we examined whether mRNA
concentration of macrophage markers [CD68, integrin ␣M
(ITGAM, gene encoding macrophage antigen-1), epidermal
growth factor-like module containing, mucin-like, hormone
receptor-like (EMR)1, and a disintegrin and metalloproteinase
Address for reprint requests and other correspondence: K. Sevastianova,
Biomedicum Helsinki, Haartmaninkatu 8, Rm. C418b, FIN-00290 Helsinki,
Finland (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
human immunodeficiency virus; macrophages; cytokines
AMONG ITS MANY FUNCTIONS,
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Sevastianova K, Sutinen J, Kannisto K, Hamsten A, Ristola M,
Yki-Järvinen H. Adipose tissue inflammation and liver fat in patients
with highly active antiretroviral therapy-associated lipodystrophy.
Am J Physiol Endocrinol Metab 295: E85–E91, 2008. First published
April 22, 2008; doi:10.1152/ajpendo.90224.2008.—In this cross-sectional study, we sought to determine whether gene expression of
macrophage markers and inflammatory chemokines in lipoatrophic
subcutaneous abdominal adipose tissue and liver fat content are
increased and interrelated in human immunodeficiency virus (HIV)1-positive, highly active antiretroviral therapy (HAART)-treated patients with lipodystrophy (HAART⫹LD⫹; n ⫽ 27) compared with
those without (HAART⫹LD⫺; n ⫽ 13). The study groups were
comparable with respect to age, gender, and body mass index. The
HAART⫹LD⫹ group had twofold more intra-abdominal (P ⫽ 0.01)
and 1.5-fold less subcutaneous (P ⫽ 0.091) fat than the
HAART⫹LD⫺ group. As we have reported previously, liver fat was
10-fold higher in the HAART⫹LD⫹ compared with the
HAART⫹LD⫺ group (P ⫽ 0.00003). Inflammatory gene expression
was increased in HAART-lipodystrophy: CD68 4.5-fold (P ⫽
0.000013), tumor necrosis factor (TNF)-␣ 2-fold (P ⫽ 0.0094),
chemokine (C-C motif) ligand (CCL) 2 2.5-fold (P ⫽ 0.0024), CCL3
7-fold (P ⫽ 0.0000017), integrin ␣M (ITGAM) 3-fold (P ⫽ 0.00067),
epidermal growth factor-like module containing, mucin-like, hormone
receptor-like (EMR)1 2.5-fold (P ⫽ 0.0038), and a disintegrin and
metalloproteinase domain (ADAM)8 3.5-fold (P ⫽ 0.00057) higher in
the HAART⫹LD⫹ compared with the HAART⫹LD⫺ group.
mRNA concentration of CD68 (r ⫽ 0.37, P ⫽ 0.019), ITGAM (r ⫽
0.35, P ⫽ 0.025), CCL2 (r ⫽ 0.39, P ⫽ 0.012), and CCL3 (r ⫽ 0.54,
P ⫽ 0.0003) correlated with liver fat content. In conclusion, gene
expression of markers of macrophage infiltration and adipose tissue
inflammation is increased in lipoatrophic subcutaneous abdominal
adipose tissue of patients with HAART-associated lipodystrophy
compared with those without. CD68, ITGAM, CCL2, and CCL3
expression is significantly associated with accumulation of liver fat.
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Table 1. Primers and probes used for mRNA analyses
Gene Symbol
Gene Name
Accession No.
Assay ID ABI
B2m
CD68
TNF␣
CCL2
CCL3
ITGAM
ADAM8
EMR1
␤2-Microglobulin
CD68
Tumor necrosis factor-␣
Chemokine (C-C motif) ligand 2
Chemokine (C-C motif) ligand 3
Integrin ␣M
A disintegrin and metalloproteinase domain 8
Epidermal growth factor-like module-containing, mucin-like, hormone receptor-like 1
NM_004048
NM_001251
NM_000594
NM_002982
NM_002983
NM_000623
NM_001109
NM_001974
*
Hs00154355_m1
Hs00174128_m1
Hs00234140_m1
Hs00234142_m1
Hs00355885_m1
Hs00174246_m1
Hs00173562_m1
*In-house assay, previously published (17).
METHODS
Study subjects and design. The subjects for this cross-sectional
study were recruited from the HIV outpatient clinic of the Helsinki
University Central Hospital. They had to be treated with HAART for
at least 18 mo before enrolment. Patients classified as lipodystrophic
(HAART⫹LD⫹; n ⫽ 27) presented with self-reported symptoms of
loss of subcutaneous fat with or without enlargement of abdominal
girth, increase in breast size, or accumulation of fat in the dorsocervical region, i.e., buffalo hump. These findings were confirmed by a
single investigator before inclusion in the study. Patients without
lipodystrophy (HAART⫹LD⫺; n ⫽ 13) had received HAART without developing the aforementioned changes in body fat composition.
Pregnancy and signs, symptoms, or biochemical evidence of active
diseases other than HIV-1 were exclusion criteria. The study subjects
had participated in studies reported previously (17, 36 – 41, 49).
All patients were studied after an overnight fast. Blood samples
were taken to measure plasma/serum concentrations of glucose, insulin, lipids, free fatty acids (FFAs), and C-reactive protein (CRP). A
needle aspiration biopsy of abdominal subcutaneous adipose tissue
was taken from the same site by a single investigator under local
anesthesia as previously described (48). The fat sample was immediately frozen and stored in liquid nitrogen until analysis.
The purpose, nature, and potential risks of the study were explained
to the patients, and their written informed consent was obtained. The
study protocol was approved by the Ethics Committee of the Department of Medicine, Helsinki University Central Hospital.
Total RNA and cDNA preparation. Frozen adipose tissue (50 –150
mg) was homogenized in 2 ml of RNA STAT-60 (Tel-Test, Friendswood, TX), and total RNA was isolated according to the manufacturer’s
instructions. After DNase treatment (RNase-free DNase set; Qiagen,
Hilden, Germany), RNA was purified using the RNeasy mini kit
(Qiagen). RNA concentrations were measured using the RiboGreen
fluorescent nucleic acid stain (RNA quantification kit; Molecular
Probes, Eugene, OR). The quality of RNA was assessed by agarose
gel electrophoresis. Average yields of total RNA were 3 ⫾ 1 ␮g/100
mg of adipose tissue wet weight and did not differ between the groups
(data not shown). Isolated RNA was stored at ⫺80°C until quantification of the target mRNAs. A total of 0.1 ␮g of RNA was transcribed
into cDNA using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Paisley, UK) and oligo(dT)12-18 primers.
Quantification of human ␤2-microglobulin, CD68, TNF-␣, CCL2,
CCL3, ITGAM, EMR1, and ADAM8. TaqMan real-time semiquantitative PCR was performed according to the manufacturer’s protocol
AJP-Endocrinol Metab • VOL
using an ABI PRISM 7000 Sequence Detection System instrument
and software (PE Applied Biosystems, Foster City, CA). The selected
genes, including ␤2-microglobulin as a housekeeping gene, and the
assays used (TaqMan Gene Expression Assays; Applied Biosystems)
are listed in Table 1. Each quantification was run in duplicate.
Expression levels were quantified (arbitrary units) by generating a
six-point serially diluted standard curve (42). ␤2-Microglobulin was
taken to serve as an internal standard for mRNA expression. Differences in loading of cDNA were adjusted for by expressing mRNA
concentration of each gene relative to that of ␤2-microglobulin. There
was no difference between the groups in the mRNA concentrations of
␤2-microglobulin (data not shown).
Table 2. Characteristics of the study groups
HAART⫹LD⫹ HAART⫹LD⫺
Males/females
Age
Body weight and composition
Weight, kg
Body mass index, kg/m2
Waist-to-hip ratio
Sum of skinfolds at five body
sites, mm
Total abdominal fat, cm3
Subcutaneous abdominal fat, cm3
Intra-abdominal fat, cm3
Total body fat, %
Features of insulin resistance
Plasma glucose, mmol/l
Serum insulin, mU/l
HOMA-IR index
Serum HDL cholesterol, mmol/l
Serum triglycerides, mmol/l
Serum FFAs, ␮mol/l
Serum hs-CRP, mg/l
HIV-related characteristics
Time since HIV diagnosis, yr
Duration of HAART, yr
Most recent HIV RNA load,
log10 copies/ml
Most recent CD4⫹ T-cell count,
cells/mm3
Current NRTI, %
Current NNRTI, %
Current PI, %
P Value
22/5
43⫾2
9/4
39⫾2
NS
NS
73⫾2
23.6⫾0.6
0.98⫾0.01
69⫾4
22.4⫾1.1
0.89⫾0.3
NS
NS
0.0015
38⫾3
3,150⫾280
1,220⫾170
1,930⫾220
18⫾1
54⫾5
2,690⫾460
1,760⫾280
930⫾260
19⫾2
0.0098
NS
0.091
0.01
NS
5.6⫾0.3
5.0⫾0.1
11.0⫾1.3
6.5⫾1.1
2.0 (1.5–4.2) 1.2 (1.0–1.4)
1.1⫾0.1
1.6⫾0.1
2.8 (1.9–4.3) 1.0 (0.75–1.6)
560⫾38
470⫾55
1.5⫾0.3
0.6⫾0.2
NS
0.015
0.005
0.00002
0.00001
NS
0.038
8.5⫾0.7
3.7⫾0.2
8.7⫾1.3
3.1⫾0.4
NS
NS
1.8⫾0.2
1.6⫾0.2
NS
582⫾59
100
26
74
516⫾70
100
46
62
NS
NS
NS
NS
Data are shown as means ⫾ SE or median (25–75% percentile). HAART⫹LD⫹,
human immunodeficiency virus (HIV)-1-positive patients with highly active antiretroviral therapy (HAART)-associated lipodystrophy; HAART⫹LD⫺, HIV-1-positive
patients using HAART but without lipodystrophy; HDL, high-density lipoprotein;
FFAs, free fatty acids; hs-CRP, highly sensitive C-reactive protein; NRTI, nucleoside
analog reverse transcriptase inhibitor; NNRTI, nonnucleoside analog reverse transcriptase inhibitor; PI, protease inhibitor. Homeostasis model assessment of insulin resistance (HOMA-IR) calculated from the formula: fasting glucose (mmol/l) ⫻ fasting
insulin (mU/l)/22.5 (26). NS, not significant.
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domain (ADAM)8] and inflammatory chemokines [chemokine
(C-C motif) ligand 2 (CCL2, gene encoding monocyte chemoattractant protein-1), chemokine (C-C motif) ligand 3
(CCL3, gene encoding macrophage inflammatory protein-1␣),
and TNF-␣] are upregulated in lipoatrophic abdominal subcutaneous adipose tissue concurrently with increased liver fat
content in HIV-1-positive, HAART-treated patients with, compared with those without, lipodystrophy.
INFLAMMATION AND LIVER FAT IN HIV-LIPODYSTROPHY
necessary. Categorical variables were compared using Fisher’s exact
test. Correlation analyses were performed with Pearson productmoment correlation coefficient after logarithmic transformation when
necessary. All calculations were carried out using Lotus 1-2-3 of
Lotus SmartSuite Release 9.5 (Lotus Development; IBM, New York
City, NY) and GraphPad Prism version 3.02 (GraphPad Software, San
Diego, CA). Data are expressed as means ⫾ SE unless otherwise
stated. Two-tailed P values ⬍0.05 were considered statistically significant.
RESULTS
Patients. Characteristics of HAART⫹LD⫹ and HAART⫹LD⫺
groups are given in Table 2. The groups were comparable with
respect to age, gender, and body mass index. The HAART⫹LD⫹
group had 2-fold more intra-abdominal and 1.5-fold less subcutaneous fat than the HAART⫹LD⫺ group. Sum of means of
skinfold thicknesses taken at five body sites was significantly
smaller in the HAART⫹LD⫹ than in the HAART⫹LD⫺
group. The HAART⫹LD⫹ group was also more insulin resistant than the HAART⫹LD⫺ group, as determined by serum
insulin concentration and HOMA-IR index. HIV-1 RNA loads
and CD4⫹ T-cell counts were comparable between the groups
as were the classes of antiretroviral drugs used. In the
HAART⫹LD⫹ group, 21 patients were taking lamivudine, 18
stavudine, 6 zidovudine, 6 didanosine, 2 abacavir, 1 zalcitabine, 6 nevirapine, 4 efavirenz, 9 indinavir, 3 nelfinavir, 3
ritonavir, 3 lopinavir, and 2 ampenavir. In the HAART⫹LD⫺
group, 11 patients were taking zidovudine, 11 lamivudine, 3
stavudine, 2 didanosine, 2 nevirapine, 2 efavirenz, 4 indinavir,
Fig. 1. CD68, integrin ␣M (ITGAM), epidermal growth factor-like module-containing, mucin-like, hormone receptor-like 1 (EMR1), and a disintegrin and
metalloproteinase domain 8 (ADAM8) in human immunodeficiency virus (HIV)-1-positive patients with highly active antiretroviral therapy-associated
lipodystrophy (HAART⫹LD⫹) vs. those without lipodystrophy (HAART⫹LD⫺). *P ⬍ 0.01, **P ⬍ 0.001, and ***P ⬍ 0.0001.
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Analytical procedures. Serum free insulin concentration was determined by RIA (Phadeseph Insulin RIA; Pharmacia & Upjohn Diagnostics, Uppsala, Sweden) after precipitation with polyethylene glycol. Plasma glucose concentrations were measured using a hexokinase
method and HDL cholesterol and triglyceride concentrations with
respective enzymatic kits from Roche Diagnostics using an autoanalyzer (Roche Diagnostics Hitachi 917; Hitachi, Tokyo, Japan). The
homeostasis model assessment of insulin resistance (HOMA-IR) was
calculated from the formula: fasting glucose (mmol/l) ⫻ fasting
insulin (mU/l)/22.5 (26). Serum FFAs were measured by fluorometric
assay (27). Serum CRP was analyzed using a high-sensitivity commercial kit (Ultrasensitive CRP Kit; Orion Diagnostica, Espoo, Finland). CD4⫹ T-cell count was determined using a flowcytometric
apparatus (FACSort/FACSCalibur; Beckton-Dickinson, San José,
CA). HIV-1 RNA load was measured using Cobas HIV-1 Amplicor
Monitor version 1.5, normal or ultra sensitive (Roche Diagnostics;
Branchburg, NJ) with a detection limit of 1.7 log10 copies/ml.
Measures of body composition. Body circumferences were determined for the waist midway between the lower rib margin and the
iliac crest and, for the hip circumference, over the greater trochanters and recorded to the nearest 0.5 cm. Skinfold thickness (mean
values of triplicate measurements) was determined at five sites (triceps, biceps, iliac crest, thigh, and cheek) (47). Percentage of body fat
was determined using bioelectrical impedance analysis (BioElectrical
Impedance Analyzer System model no. BIA-101A; RJL Systems,
Detroit, MI) (24). Intra-abdominal and abdominal subcutaneous fat
were quantified by analyzing a total of 16 T1-weighted transaxial
magnetic resonance image scans as previously described (37). Liver
fat content was measured using proton magnetic resonance spectroscopy as previously described (38).
Statistical analysis. The unpaired t-test was used to compare the
differences between the groups after logarithmic transformation when
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INFLAMMATION AND LIVER FAT IN HIV-LIPODYSTROPHY
3 nelfinavir, 2 ritonavir, and 1 ampenavir, lopinavir, and
saquinavir, respectively.
Macrophage and cytokine/chemokine gene expression in subcutaneous adipose tissue. Macrophage-related genes (CD68,
ITGAM, EMR1, and ADAM8) and those encoding for inflammatory chemokines (TNF-␣, CCL2, and CCL3) were overexpressed in lipoatrophic abdominal subcutaneous adipose tissue
of the HAART⫹LD⫹ group compared with the HAART⫹LD⫺
group (Figs. 1 and 2).
Correlation between adipose tissue gene expression and
liver fat content. Liver fat content was 10-fold higher in the
HAART⫹LD⫹ group than in the HAART⫹LD⫺ group [median 5.0% (interquartile range 2.5–12.2) vs. 0.5% (0.5–1.75),
DISCUSSION
Fig. 2. Tumor necrosis factor-␣ (TNF-␣), chemokine (C-C motif) ligand 2
(CCL2), and chemokine (C-C motif) ligand 3 (CCL3) in HAART⫹LD⫹ vs.
HAART⫹LD⫺ patients. *P ⬍ 0.01 and **P ⬍ 0.00001.
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In the present study, we found that mRNA concentrations
of macrophage markers (CD68, ITGAM, EMR1, ADAM8)
and chemokines (CCL2, CCL3, TNF-␣) were significantly
increased in lipoatrophic subcutaneous adipose tissue of
HAART-treated lipodystrophic patients compared with that of
HAART-treated nonlipodystrophic patients. This extends the
preexisting data on inflammatory profiles in adipose tissue
in HAART-associated lipodystrophy. Expression of CD68,
ITGAM, CCL2, and CCL3 genes was found to be significantly
and positively correlated with liver fat content.
We compared HIV-1-positive patients with HAART-associated lipodystrophy to those on HAART, but without lipodystrophy. Although females comprised 19% of HAART⫹LD⫹
and 31% of the HAART⫹LD⫺ group, the difference was not
statistically significant (P ⫽ 0.44). Many of the patients in the
present study used medications, such as stavudine, the use of
which has recently decreased as newer agents with less metabolic side effects have become available. Nevertheless, because the groups were comparable with respect to other HIV1-related characteristics (Table 2), the differences in inflammatory gene expression and liver fat content are likely to be
associated with lipodystrophy. Whether one precedes another
or vice versa cannot be determined in a cross-sectional study.
We studied subcutaneous adipose tissue since it is ethically
unacceptable to sample visceral adipose tissue for research
purposes only. Changes in visceral fat may have been even
better correlated with liver fat as have been reported in obese
subjects (10). On the other hand, based on catheterization
studies, the liver receives most of its FFAs from subcutaneous
rather than visceral fat, even in abdominally obese subjects
(28). However, because HAART-associated lipodystrophy encompasses simultaneous loss of subcutaneous and gain of
visceral adipose tissue, it is possible that in these patients
visceral depot contributes more to hepatic FFA supply than it
does in nonlipodystrophic subjects. Furthermore, visceral fat
may be an important source of inflammatory mediators (4).
The finding of the current study, i.e., the increased expression
of inflammatory markers in subcutaneous adipose tissue of
lipodystrophic patients, is in accordance with the study of Jan
et al. (15). Lipogranulomata, characterized by the lipid-laden
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HAART⫹LD⫹ vs. HAART⫹LD⫺, P ⫽ 0.00003]. Expression of several genes showed a significant positive correlation
with the liver fat percent: Pearson’s r ⫽ 0.37, P ⫽ 0.019 for
CD68 and liver fat; r ⫽ 0.39, P ⫽ 0.012 for CCL2 and liver
fat; r ⫽ 0.54, P ⫽ 0.0003 for CCL3 and liver fat; and r ⫽ 0.35,
P ⫽ 0.025 for ITGAM and liver fat (Fig. 3). Correlation of
TNF-␣, EMR1, and ADAM8 with liver fat content remained
nonsignificant: r ⫽ 0.14, not significant (NS) for TNF-␣ and
liver fat; r ⫽ 0.16, NS for EMR1 and liver fat; and r ⫽ 0.22,
NS for ADAM8 and liver fat. HOMA-IR showed a borderline
significant positive correlation with CD68 expression (r ⫽
0.29, P ⫽ 0.066). The correlations between the previously
published serum adiponectin concentration (41) and the current
inflammatoryu genes were as follows: TNF-␣ r⫽ ⫺0.26, P ⫽
0.11; CCL2 r ⫽ ⫺0.38, P ⫽;CCL3 r ⫽ ⫺0.65, P ⬍ 0.0001;
ITGAM r ⫽ ⫺0.47, P ⫽ 0.0002; EMRI1 r ⫽ ⫺0.49, P ⫽
0.001; CD68 r ⫽ ⫺0.61, P ⫽ 0.0001; and ADAM8 r ⫽ ⫺0.58,
P ⬍ 0.0001.
INFLAMMATION AND LIVER FAT IN HIV-LIPODYSTROPHY
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macrophages encircling adipocytes, and the number of CD68positive cells, as determined by immunohistochemistry, have
also been found to be increased in individuals receiving nucleoside reverse transcriptase inhibitors compared with HIVnegative and antiretroviral treatment-naive subjects (29, 30).
However, in the latter two studies, HAART-treated lipodystrophic and nonlipodystrophic patients were not compared. The
antiretroviral drugs most frequently associated with the development of lipodystrophy (zidovudine, stavudine, protein inhibitors) are known to increase the release of proinflammatory
CCL2 and IL-6 in adipocytes in vitro (22) and incite increased
macrophage infiltration in suprailiac subcutaneous adipose
tissue in vivo in HIV-1-positive patients started on antiretroviral treatment (29, 30). In the present study, the groups were
comparable with respect to HIV-1-related parameters and the
classes of antiretroviral medication. Direct effects of individual
antiretroviral drugs or lipodystrophy itself cannot be excluded,
however.
Expression of genes encoding for macrophage markers such
as CD68 has been shown to correlate with the number of
macrophages, as determined by immunohistochemistry, in
obese human subjects (10, 18) and in rodents (43, 45). Recently, utilizing immunohistochemistry, an increased presence
of macrophages has been shown also in adipose tissue of
HAART-treated individuals with lipodystrophy (15).
Tissue macrophages are involved in several immune functions, including phagocytosis of cellular debris and foreign
material as well as triggering immune responses via cytokine
release and antigen presentation (51). The genes selected for
our study cover a broad spectrum of macrophage actions.
CD68 is a transmembrane glycoprotein particularly highly
expressed in human monocytes and tissue macrophages, participating in lectin/selectin-mediated cell adhesion and locomotion (32). EMR1 is also a transmembrane glycoprotein
present in peripheral blood mononuclear cells and presumably
involved in cell-cell interactions and activation of consecutive
messenger cascades (6). ITGAM is an integrin expressed in
monocytes and macrophages in response to chemoattractants
and involved in cell adhesion and aggregation during immune
reactions (11). ADAM8 is a transmembrane glycoprotein restricted to granulocytes and monocytic cells, playing a role in
collagen degradation in extravascular tissue. It is also involved
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in leukocyte extravasation functioning as a ligand for integrins
(50). CCL2, secreted by adipocytes and endothelial cells, is a
potent macrophage-attracting chemokine, the expression of
which is increased in obese human adipose tissue (9, 34).
Expression of CCL2 is also increased in human hepatosteatosis
(14). In rodents, CCL2 has been shown to contribute to insulin
resistance (16, 34) and to adipocyte dedifferentiation (34).
CCL3 is a monokine produced by macrophages and involved
in inflammation, acting as a chemoattractant and an activator of
polymorphonuclear leukocytes (44). TNF-␣ is a proinflammatory and lipolytic cytokine overexpressed in obesity by tissue
macrophages and able of inducing insulin resistance, at least in
rodents and human cells in vitro (33).
We recently demonstrated that gene expression of CD68 and
ITGAM in subcutaneous adipose tissue of obese subjects is
increased compared with nonobese subjects and is positively
correlated with liver fat content independent of obesity (25). In
the same study, also a correlation between CD68 and TNF-␣
expression was observed. Furthermore, we have found that
expression of CD68, CCL2, and CCL3 in subcutaneous adipose tissue of subjects with high liver fat content is increased
compared with carefully weight-matched subjects with normal
liver fat content (18). In the latter study, gene expression of
CD68 was also positively and significantly correlated with
liver fat content (18). In addition, Cancello et al. (10) have
shown that, in obese subjects, the percentage of macrophages
in omental adipose tissue is significantly associated with the
severity of steatotic and fibroinflammatory lesions in the liver.
We found a significant, positive correlation between expression of several inflammatory markers in subcutaneous adipose
tissue and liver fat content. Such correlations, perceptibly, do
not prove causality, but there are data linking adipose tissue
inflammation to liver fat content and vice versa. Kanda et al.
(16) have shown that overexpression of CCL2 in murine
adipocytes leads to enhanced macrophage infiltration into adipose tissue, insulin resistance in skeletal muscle and liver, and
an increase in circulating FFAs and to accumulation of fat in
the liver. Of note, the CCL2-overexpressing and the wild-type
mice in this experiment had similar and normal body weight. In
contrast, induction of hepatic steatosis by high-fat diet or by
transgenic manipulations leads to subacute hepatocellular inflammation as well as to hepatic and peripheral insulin resis-
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Fig. 3. The correlation between adipose tissue expression of CD68 and CCL2 with liver fat content. F, HAART⫹LD⫹ patients; E, HAART⫹LD⫺ patients.
Pearson product-moment correlation coefficient for correlation of liver fat with CD68 mRNA/␤2-microglobulin and with CCL2 mRNA/␤2-microglobulin yielded
P values of 0.019 and 0.012, respectively.
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INFLAMMATION AND LIVER FAT IN HIV-LIPODYSTROPHY
ACKNOWLEDGMENTS
We acknowledge Katja Sohlo, Anna-Maija Häkkinen, and Pentti Pölönen
for excellent technical assistance and the study participants for their invaluable
help.
GRANTS
This study was supported by grants from EVO Foundation (H. YkiJärvinen), Orion Research Foundation (K. Sevastianova), Lilly Foundation (K.
AJP-Endocrinol Metab • VOL
Sevastianova), and Biomedicum Foundation (K. Sevastianova). This work is
part of the project “Hepatic and adipose tissue functions in the metabolic
syndrome” (www.hepadip.org), which is supported by the European Commission as an Integrated Project under the 6th Framework Programme (Contract
LSHM-CT-2005-018734).
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tance (8). This pathway involves nuclear factor-␬B (NF-␬B), a
transcriptional factor mediating induction of inflammatory cytokines activated by I␬B kinase (IKK␤). Overexpression of
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Regarding the possible mechanism linking lipodystrophy
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HAART⫹LD⫹ group. Indeed, increased concentration of serum FFA, a potential consequence of adipose tissue inflammation, is known to induce insulin resistance in liver and the
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Another potential mediator between inflamed adipose tissue
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low adiponectin and high liver fat reported in this patient
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In conclusion, several features of patients with HAARTassociated lipodystrophy, such as intra-abdominal adiposity,
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(2). The present study extends this to finding of increased
expression of macrophage markers and inflammatory cytokines
and increased liver fat and their interrelations in HAARTtreated HIV-1-infected lipodystrophic subjects. Thus, despite
the clinical phenotypes opposite in terms of the amount of
subcutaneous fat, the pathogenetic mechanisms behind insulin
resistance developing in both HAART-associated lipodystrophy and “traditional” obesity appear to share similarities.
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