Download Depot-specific adipocyte cell lines reveal differential

Am J Physiol Endocrinol Metab 296: E315–E322, 2009.
First published November 25, 2008; doi:10.1152/ajpendo.90486.2008.
Depot-specific adipocyte cell lines reveal differential drug-induced responses
of white adipocytes—relevance for partial lipodystrophy
Julia Kovsan,1 Alexander Osnis,1 Adva Maissel,1 Livnat Mazor,1 Tanya Tarnovscki,1 Liat Hollander,1
Shira Ovadia,1 Britta Meier,2 Johannes Klein,2 Nava Bashan,1 and Assaf Rudich1,3
1
Department of Clinical Biochemistry, Ben-Gurion University, Beer-Sheva, Israel; 2Department of Internal Medicine I,
University of Lübeck, Lübeck, Germany; and 3The International Center for Health and Nutrition, Faculty of Health Sciences,
Ben-Gurion University, Beer-Sheva, Israel
Submitted 3 June 2008; accepted in final form 19 November 2008
of adipose tissue from different
anatomic locations to endocrine-metabolic regulation is a wellaccepted phenomenon supported by a large body of experimental evidence (39, 48). Although different venous drainage
of intra-abdominal (IA) fat compared with that of subcutanoues
(SC) fat (to the portal vs. systemic venous systems) may
partially underlie such differences (21), these two depots are
functionally distinct. In particular, key functions of adipose
tissue now known to regulate whole body metabolism, includ-
ing adipokine secretion and lipolysis, are distinctly regulated.
Moreover, the responses of these depots to clinically used
medications are also distinct. For example, in response to
thiazolidinedione (TZD) insulin sensitizers, SC adipose mass
increases, whereas IA fat mass may actually diminish (22, 23,
34, 47). As a potential mechanism, it has been suggested that
SC adipocytes express higher levels of PPAR␥, the nuclear
receptor target of TZDs, and respond to the drug with a greater
hyperplastic response than IA fat (31). In other cases, the
mechanisms underling differential response to medications are
unclear. Highly active antiretroviral therapy (HAART), known
to greatly improve the prognosis of HIV-positive persons,
subjects many to partial lipodystrophy, a condition in which
SC fat is differentially lost whereas IA fat is either unaffected
or even expands in mass (7, 19, 52). The associated health
consequences are severe, as affected persons develop metabolic dysregulation subjecting them to morbidity, including
type 2 diabetes and cardiovascular disease (16). The mechanisms for this differential response of adipose tissues to
HAART are unclear and may range from different accumulation of the drug to different biological response to it. Yet, since
IA fat in humans is rather inaccessible and rodents do not seem
to develop the full features of human HAART-induced partial
lipodystrophy, research in this field has been limited by the
lack of depot-specific cell-based systems.
An additional complication in investigating fat depot-differential responses of adipose tissue has been the recent realization that adipose tissue in different locations may be composed
of different cell populations, particularly in morbid states. For
example, obesity has been demonstrated to be associated with
infiltration of immune cells into adipose tissue (18, 54, 55).
This process appears to occur more pronouncedly in IA fat (5,
17), hence, it is difficult to dissect out whether functional
differences between SC and IA fat can be attributed to different
alterations of the adipocytes per se or rather to differential
alterations in cellular composition of adipose tissue and/or to
non-adipocyte cells composing the adipose tissue.
Here, we report the use of adipocyte cell lines derived from
immortalized preadipocytes from inguinal (Ing) and periepididymal (Peri) mouse fat pads, representing SC and IA fat,
respectively. We demonstrate that, in response to nelfinavir, an
HIV protease inhibitor, both cell lines equally accumulate the
drug and exhibit similar inhibition of adipogenesis. Yet, Ing
adipocytes are more sensitive than Peri adipocytes to the
Address for reprint requests and other correspondence: A. Rudich, Dept. of
Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of
the Negev, Beer-Sheva, 84103, Israel (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.
nelfinavir; lipolysis; perilipin; intra-abdominal adipocytes; subcutaneous adipocytes
A DIFFERENT CONTRIBUTION
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Kovsan J, Osnis A, Maissel A, Mazor L, Tarnovscki T, Hollander L, Ovadia S, Meier B, Klein J, Bashan N, Rudich A.
Depot-specific adipocyte cell lines reveal differential drug-induced
responses of white adipocytes—relevance for partial lipodystrophy.
Am J Physiol Endocrinol Metab 296: E315–E322, 2009. First published November 25, 2008; doi:10.1152/ajpendo.90486.2008.—Intraabdominal (IA) fat functionally differs from subcutaneous (SC)
adipose tissue, likely contributing to its stronger association with
obesity-induced morbidity and to differential response to medications.
Drug-induced partial lipodystrophy, like in response to antiretroviral
agents, is an extreme manifestation of the different response of
different fat depots, with loss of SC but not IA. Investigating depotspecific adipocyte differences is limited by the low accessibility to IA
fat and by the heterogenous cell population comprising adipose tissue.
Here, we aimed at utilizing immortalized preadipocyte cell lines from
IA (epididymal) or SC (inguinal) fat to investigate whether they
differentially respond to the HIV protease inhibitor nelfinavir. Preadipocytes were readily amenable to adipogenesis, as evidenced by
lipid accumulation, expression of adipose-specific genes, measurable
lipolysis, and insulin responsiveness. Leptin secretion was higher by
the SC line, consistent with known differences between IA and SC fat.
As previously reported, nelfinavir inhibited adipogenesis downstream
of C/EBP␤, but similarly in both cell lines. In contrast, nelfinavir’s
capacity to diminish insulin signaling, decrease leptin secretion,
enhance basal lipolysis, and decrease expression of the lipid dropletassociated protein perilipin occurred more robustly and/or at lower
nelfinavir concentrations in the SC line. This was despite similar
intracellular concentrations of nelfinavir (23.8 ⫾ 5.6 and 33.6 ⫾ 12.2
␮g/mg protein for inguinal and epididymal adipocytes, respectively,
P ⫽ 0.46). The cell lines recapitulated depot-differential effects of
nelfinavir observed in differentiated primary preadipocytes and with
whole tissue explants. Thus, we report the use of fat depot-specific
adipocyte cell lines for unraveling depot-differential responses to a
drug causing partial lipodystrophy.
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FAT DEPOT DIFFERENTIAL EFFECTS OF NELFINAVIR
induction of insulin resistance and to the increase in lipolysis,
consistent with the preferential diminution of SC fat mass in
HAART-induced lipodystrophy.
MATERIALS AND METHODS
AJP-Endocrinol Metab • VOL
RESULTS
Fat depot-specific preadipocyte cell lines. Adipocyte cell
lines derived from preadipocytes from Ing (SC) or Epi (IA) fat
depots were generated as described in MATERIALS AND METHODS
and for brown adipocytes in Refs. 13 and 24 –26. Both cell
lines had fibroblast-like morphology and did not exhibit significant lipid accumulation prior to differentiation. Upon adipocyte differentiation, both lines retained a similar capacity to
accumulate triglycerides, as detected by Oil red O staining
(Fig. 1A). To further confirm terminal differentiation of the two
cell lines, we measured protein levels of the lipid dropletassociated protein perilipin, HSL, GLUT4, and the adipogenic
transcription factor PPAR␥ (Fig. 1B). Adipogenesis induced
the expression of these typical adipocyte markers. Importantly,
PPAR␥ expression was higher in Ing than in Epi, consistent
with a higher expression of this transcription factor in the SC
fat depot (56). In addition, both cell lines exhibited hormoneregulated lipolysis (Fig. 1C): ␤-adrenergic stimulation with
isoproteranol increased glycerol release, with a fold stimulation in Epi higher than in Ing, also consistent with depot
differences between IA and SC adipocytes (47a, 48a). Importantly, insulin responsiveness could be demonstrated by insulin-mediated anti-lipolysis (Fig. 1C), insulin-stimulated phosphorylation of Akt/PKB on Ser473 (Fig. 1D), and glucose
uptake (Fig. 1E). The twofold higher expression of total Akt/
PKB in Epi compared with Ing adipocytes (P ⫽ 0.005) was
consistent with observations in human omental vs. subcuataeous abdominal adipose tissue (2). Finally, both cell lines
secreted leptin and adiponectin to the media, with higher leptin
secretion by Ing adipocytes than from Epi (P ⬍ 0.05; Table 1),
consistent with the known higher expression of leptin in SC
compared with IA fat (37, 53). Medium adiponectin concen-
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Adipocytes from immortalized Ing (SC) and Peri (IA) preadipocyte
cell lines. Two permanent cell lines were created as follows. Ing and
Peri white adipose tissues preadipocytes were isolated from 6-wk-old
mice as previously described (25). Briefly, tissues were minced and
digested with collagenase and filtered through a 100-␮m nylon mesh.
Cells in the pellet were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 25 mM glucose, 20% fetal bovine serum
(FBS), 20 mM HEPES, and 100 U/ml penicillin-streptomycin and,
after reaching 80% confluence, incubated for 24 h with the retroviral
pBabe vector encoding the SV40T antigen and puromycin resistance.
Following infection, the cells recovered in culture medium for 72 h,
and positively infected cells were selected with puromycin (1 ␮g/ml)
for 3 wk. Differentiation into adipocytes was performed as follows.
Cells were grown until 12–24 h postconfluence in DMEM supplemented with 20% FBS, 100 U/ml penicillin-streptomycin, 20 nM
insulin, 1 nM triiodothyronine (T3). Differentiation was induced by
adding 0.125 mM indomethacin, 2 ␮g/ml dexamethasone, and 0.5
mM 3-isobutylmethylxanthine to the medium for 24 h, after which,
cells were grown for an additional 6 days before being used.
Primary rat differentiated preadipocytes and whole tissue explants.
All procedures were approved in advance by the Animal Care Ethics
Committee at Ben-Gurion University. Sprague-Dawley (Harlan,
Jerusalem, Israel) rats (weight 200 –300 g) were killed by CO2
asphyxiation, and perigonadal (IA) or inguinal (SC) fat was immediately collected. Primary rat preadipocytes were obtained from the
pellet (stroma-vascular fraction) of collagenase-digested adipose tissue, obtained as previously described (43). Following the last wash,
cells were resuspended and cultured in DMEM supplemented with
10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 ␮g/ml amphotericin B. On the next day after
seeding, cells were thoroughly washed with PBS to discard nonadhering cells. Forty-eight hours postconfluence, differentiation was
induced exactly as we described for 3T3-L1 adipocytes (44), and cells
were used 7–10 days postdifferentiation induction, when exhibiting a
high (typically ⬎80%) differentiation, as assessed by light microscopy and Oil red O staining. Whole tissue explants were prepared by
mincing the tissue into small pieces of 2–3 mm, as we described for
human adipose tissue (2).
Nelfinavir treatment. Nelfinavir (Shanghai 21CEC Pharma, London, UK) stock solution (100 mM) was prepared in 100% ethanol.
Indicated medium concentrations of nelfinavir were achieved by
appropriate dilution from a 1:100 diluted stock in water. To assess
nelfinavir-induced inhibition of adipogenesis (Figs. 2, 3), indicated
concentrations of nelfinavir were added starting at the beginning of
differentiation induction and throughout until cells were used. To
assess nelfinavir-induced effects on fully differentiated adipocytes,
indicated concentrations of nelfinavir were added on day 6 for cell
lines or days 7–10 for primary preadipocytes, for 18 h in medium
without serum, supplemented with 0.5% wt/vol bovine serum albumin
(see Figs. 4 and 5). Protein recovery and MTT assays were used to
confirm normal viability (85–110% of control) following nelfinavir
treatment.
Measurements of intracellular nelfinavir concentrations. Adipocytes grown on 10-cm dishes were treated for 18 h with 30 ␮M
nelfinavir. Following three freeze-thaw cycles, cells were scraped in 1
ml of PBS, homogenized using a glass homogenizer, and frozen at
⫺70°C. For analysis, samples were thawed, vortexed, and extracted
with 5 ml of tert-butylmethyl ether for 10 min. The lower organic
layer was evaporated at 30 – 40°C under nitrogen, and the residue was
reconstituted with 300 ␮l of mobile phase and vortexed again for 1
min prior to injection. HPLC separation (Jasco PU-2089) was on C18,
5 ␮m 4.6 ⫻ 150 mm columns (Phenomenex, Torrance, CA) using a
series of gradients of acetonitrile and potassium phosphate, pH 5.6, at
a flow rate of 1.6 ml/min. Detection was by a UV-Vis detector
(Jasco-1570) set at 210 nm, based on nelfinavir standards, with a
retention time of 12.2 min.
Other assays. Oil red O staining was performed as described (51)
and quantified by solubilizing cells in DMSO followed by spectrophotometric detection as suggested by the manufacturer (Sigma).
Western blot analyses were performed exactly as previously reported
(45, 50), using the following antibodies: anti-Akt/PKB, p-Ser473-Akt/
PKB (Cell Signaling), peroxisome proliferator-activated receptor-␥
(PPAR␥), CCAAT-enhancer-binding protein-␣ (C/EBP␣) and
C/EBP␤ (Santa Cruz Biotechnology), GLUT1 and GLUT4 (Chemicon), and ␤-actin (Sigma). Anti-HSL (hormone-sensitive lipase) and
perilipin were kindly provided by Dr. Greenberg, Tufts University.
Peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were from
Amersham Biosciences. Glycerol release was assayed spectrophotometrically exactly as we previously reported (27). Leptin and adiponectin secretion to the medium were determined in media collected
at the end of the 18-h incubation with either vehicle or nelfinavir,
using commercial kits according to the manufacturer’s instructions. A
leptin mouse ELISA kit was from Ray Biotech, and a mouse/rat
adiponectin ELISA kit was from B-Bridge International. 2-Deoxyglucose uptake and glycerol release to the medium (lipolysis and insulinmediated anti-lipolysis) were performed exactly as described before
(40, 46).
Statistical analysis. The data are expressed as means ⫾ SE. Each
treatment was compared with control, and statistical significance of
differences between two groups was evaluated using Student’s t-test.
The criterion for significance was set at P ⬍ 0.05.
FAT DEPOT DIFFERENTIAL EFFECTS OF NELFINAVIR
E317
trations were within the wide concentration range reported in
3T3-L1 adipocytes (1a, 4a), and similar to serum concentrations in mice (18a). Moreover, nondifferentiated preadipocytes
do not seem to express or secrete significant amounts of
adiponectin (27a, 55a) or leptin (29a). Hence, adiponectin and
leptin secretion supports efficient adipogenesis of the two cell
lines. Collectively (and along with data presented next), the
two fat depot-specific preadipocyte cell lines could be efficiently differentiated from fibroblast-like into cells exhibiting
Table 1. Leptin and adiponectin secretion to the medium
by inguinal and epididymal adipocyte cell line in response
to nelfinavir
Inguinal
Leptin
Cont
Nelf 10 ␮M
Nelf 40 ␮M
Epididymal
pg/ml
%Decrease
pg/ml
%Decrease
302.7⫾20.0
198.9⫾38.7
43.3⫾0.2*
34.6⫾8.55
85.7⫾1.0
46.1⫾17.2†
39.3⫾21.4†
24.4⫾5.3†
21.4⫾13.5
43.2⫾7.9†
␮g/ml
Adiponectin
Cont
Nelf 10 ␮M
Nelf 40 ␮M
10.9⫾5.7
8.0⫾4.2
6.4⫾4.1
␮g/ml
26.6⫾0.8
41.4⫾14.8
15.5⫾4.3
11.4⫾2.8
9.6⫾2.5
25.1⫾3.4
36.6⫾5.7
Values are means ⫾ SE; n ⫽ 2–3 independent experiments. Nelf, nelfinavir;
Cont, control. Inguinal and epididymal adipocytes were treated with vehicle
(ethanol) or nelfinavir at indicated concentrations for 18 h. Before nelfinavir
treatment, the medium was changed and collected at the end of the 18-h
incubation period, and the amounts of leptin and adiponectin secreted to the
medium were assessed by specific ELISA assays, as detailed in materials and
methods. *P ⬍ 0.05 vs. control cells; †P ⬍ 0.05 vs. inguinal adipocytes.
AJP-Endocrinol Metab • VOL
typical characteristics of terminally-differentiated adipocytes,
while retaining several depot-specific characteristics.
Nelfinavir inhibits adipocyte differentiation, but not in a
depot-differential manner. We next used the two depot-specific
adipocyte cell lines to assess whether they can assist in unraveling depot-differential response to nelfinavir, an HIV protease
inhibitor (HPI) suggested to contribute to HAART-induced
partial lipodystrophy. By use of the murine 3T3-L1 or 3T3F442A cell lines, several mechanisms for HPI-induced lipodystrophy have been proposed. These include inhibition of
adipocyte differentiation (6, 11), insulin resistance due to either
direct inhibition of GLUT4 (35, 43) and/or by directly interfering with insulin signal transmission to Akt/PKB (3, 46), and
increased rate of basal lipolysis (triglyceride breakdown) (1,
46). We next assessed whether SC-derived adipocytes are more
sensitive to any of these effects of HPI, as such findings would
support the notion that the two cell lines retain certain depotspecific differences and may contribute to understanding of the
pathophysiology of HPI-induced partial lipodystrophy.
To assess fat depot-differential susceptibility to inhibition of
adipogenesis by nelfinavir, preadipocytes were grown to confluence, and nelfinavir was added to the medium at final
concentrations of 10 – 40 ␮M, with the initiation of differentiation 24 h postconfluence as performed in Ref. 11. These
concentrations did not significantly affect total cell viability as
assessed by total protein recovery and by MTT assay (data not
shown). As an initial terminal adipocyte differentiation marker,
we used Oil red O staining to detect accumulation of neutral
lipids and quantitated its amount using a microplate absorbance
reader, as detailed in MATERIALS AND METHODS. In response to
increasing concentrations of nelfinavir, neutral lipid accumulation was diminished significantly with nelfinavir concentra-
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Fig. 1. Characteristics of mouse inguinal and epididymal adipocyte cell lines. Preadipocytes isolated and
immortalized from inguinal (subcutaneous) or epididymal (intra-abdominal) fat pads were differentiated into adipocytes as detailed in MATERIALS AND
METHODS. A: Oil red O staining reveals equivalent
levels of triglyceride storage in both cell lines at days
0, 3, and 6 of the differentiation protocol. Shown are
representative results of 5 independent experiments.
B: total cell lysates were prepared from preadipocytes (day 0) or differentiated cells (day 6) and
blotted for perilipins, hormone-sensitive lipase
(HSL), GLUT4, and PPAR␥. Shown are representative blots of 2– 6 independent experiments. C: basal
and isoproteranol-stimulated lipolysis of differentiated adipocytes (day 6) of both cell lines was determined by measuring glycerol release into the medium, as detailed in MATERIALS AND METHODS. Insulin
treatment was performed by including insulin (100
nM) during incubation with isoproteranol (10 nM).
Each experiment was conducted ⱖ3 times. *P ⬍
0.05 vs. basal and vs. isoproteranol ⫹ insulin. D: lysates
were prepared from differentiated (day 6) adipocytes
stimulated or not with insulin (100 nM for 7 min) and
blotted for pS473-Akt/PKB or total Akt/PKB. Shown
are representative results of 3 independent experiments. E: 2-Deoxyglucose uptake was measured after a 20-min incubation with or without 100 nM
insulin. *P ⬍ 0.05 between insulin-stimulated and
unstimulated cells of the same line.
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FAT DEPOT DIFFERENTIAL EFFECTS OF NELFINAVIR
tions of 20 ␮M or more (Fig. 2). Yet, both cell lines exhibited
similar sensitivity to the inhibitory effect of nelfinavir on lipid
accumulation. Nelfinavir at 40 ␮M present during and after
induction of adipocyte differentiation almost completely prevented triglyceride accumulation in cells derived from both fat
depots. To verify this response in molecular terms and to
determine at what stage of adipocyte differentiation nelfinavir
exerted its inhibitory effects, we measured the protein levels of
several major transcription factors and adipocyte differentiation markers. Although the presence of nelfinavir during and
following differentiation did not affect the expression of
C/EBP␤, decreased expression of C/EBP␣, and more evidently
of PPAR␥, could be observed with nelfinavir concentrations 20
␮M or more (Fig. 3A). HSL, the lipid droplet-associated
protein perilipin (Fig. 3B), and GLUT4 (not shown) were used
as terminal adipocyte differentiation markers. In response to
increasing concentrations of nelfinavir, decreased expression
was observed, with a dose dependency that corresponded to the
decrease in PPAR␥ expression (Fig. 3A) and to the diminished
lipid accumulation (Fig. 2B). However, these findings, largely
confirming reports in 3T3-L1 adipocytes (11), did not display
a differential response between the Ing and Epi cell lines.
To corroborate these findings in a nonimmortalized adipose
cell system, we isolated preadipocytes from rat SC and perigonadal (PG) fat pads, as detailed in MATERIALS AND METHODS.
After confluence, preadipocytes (exhibiting fibroblast-like
α
Fig. 2. Nelfinavir similarly inhibits triglyceride accumulation during adipogenesis of inguinal and epididymal preadipocytes. Inguinal and epididymal
preadipocytes were differentiated into adipocytes in the absence or presence of
different concentrations of nelfinavir. A: 6 days after induction of differentiation, cells were stained with Oil red O, and representative images were
obtained at ⫻400 magnification by light microscopy. B: for quantification,
cells were solubilized in DMSO, absorbance was determined, and results were
expressed as percent of absorbance in control cells. Results are means ⫾ SE of
3– 6 independent experiments. *P ⬍ 0.05 vs. control values (no significant
differences were observed between the 2 cell lines in any of the nelfinavir
concentrations).
AJP-Endocrinol Metab • VOL
morphology) were subjected to a differentiation protocol in the
absence or presence of increasing concentrations of nelfinavir.
Compared with the cell lines, these differentiated primary
preadipocytes were more sensitive to nelfinavir, exhibiting
decreased triglyceride accumulation as assessed by Oil red O
staining with concentrations as low as 5 ␮M (data not shown).
This likely represents the effect of immortalization utilized to
generate the two cell lines. Nevertheless, PPAR␥ expression
was diminished with a similar dose-response in SC- or PGderived preadipocytes (Fig. 3C), consistent with the findings in
the two cell lines. Collectively, these data demonstrate that the
two cell lines recapitulated the effect of the HPI nelfinavir on
adipogenesis, demonstrating inhibition of this process downstream of C/EBP␤, without a fat depot-differential effect.
SC-derived adipocytes are more sensitive than IA-derived
adipocytes to the inhibitory effect nelfinavir exerts on insulinstimulated activation of akt. Previous studies in differentiated
3T3-L1 adipocytes demonstrated that 18-h exposure to nelfi-
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Fig. 3. Nelfinavir similarly affects the expression of transcription factors
regulating adipogenesis and terminal adipocyte differentiation markers in
inguinal and epididymal adipocytes. A and B: inguinal and epididymal preadipocytes were differentiated into adipocytes in the absence or presence of
different concentrations of nelfinavir. Six days after induction of differentiation, lysates were prepared and the protein level of transcription factors
regulating adipogenesis (A) or markers of terminal adipocyte differentiation
(B) were determined by Western blot analysis. C: primary preadipocytes were
isolated from collagenase-digested rat inguinal (subcutaneous, SC) and epididymal (perigonadal, PG) fat pads. Cells were grown to confluence and
differentiated into adipocytes in the absence or presence of different nelfinavir
concentrations. Cell lysates were prepared at day 7 and blotted for peroxisome
proliferator-activated receptor-␥ (PPAR␥), with ␤-actin serving as a loading
control. All experiments were performed ⱖ5 times, with similar results.
FAT DEPOT DIFFERENTIAL EFFECTS OF NELFINAVIR
SC-derived adipocytes are more sensitive to the lipolytic
effect of nelfinavir and to its tendency to diminish leptin
secretion despite similar intracellular concentrations of the
drug. Differentiated 3T3-L1 adipocytes exposed for 18 h to
nelfinavir exhibit an increased lipolytic rate in basal (non-␤adrenergic-stimulated) state, as assessed by glycerol release to
the medium (46). Ing adipocytes already exhibited this response when exposed to 10 ␮M nelfinavir, whereas concentrations exceeding 30 ␮M were required to observe this response in Epi (Fig. 5A). The lipolytic effect of nelfinavir in
3T3-L1 adipocytes was shown to be mediated by decreased
expression of perilipin (27). Consistently, perilipin expression
in both adipocyte cell lines was decreased by nelfinavir, but
with a differential dose effect (Fig. 5B): Ing cells already
exhibited decreased perilipin with 10 ␮M nelfinavir, corresponding to the increase in glycerol release at this concentration, whereas Epi cells required higher concentrations. To
verify that this finding truly represents the effect of nelfinavir
in adipose tissue, we incubated mouse PG and SC fat pad
explants with the drug for 18 h. Both tissues exhibited decreased perilipin expression in response to incubation with
nelfinavir; but in SC fat, perilipin decreased by 56.8 ⫾ 5.6%,
whereas PG fat by 20.5 ⫾ 4.5% (n ⫽ 5 independent experiments, P ⬍ 0.05). Collectively, SC fat tissue and adipocytes
are more sensitive to the lipolytic action of nelfinavir and to a
decrease in perilipin expression. Moreover, the lipolytic response of SC-derived adipocytes to nelfinavir occurs with
concentrations of the drug that are reported in serum of treated
patients (12, 30, 38).
Nelfinavir also displayed a differential effect on adipokine
secretion by the two cell lines (Table 1). Adiponectin secretion
tended to decrease in a dose-dependent manner but with
seemingly similar sensitivity in both cell lines. In contrast, the
decrease in leptin secretion reached a ⬎85% inhibition in Ing
but only ⬃45% in Epi (P ⬍ 0.05), consistent with previous
results using human adipose tissue fragments (9).
Given the apparent higher sensitivity of Ing adipocytes to
nelfinavir actions, we determined whether this could be a result
of the drug concentrating to higher intracellular levels in Ing
than in Epi adipocytes. Ing or Epi adipocytes were incubated
for 18-h with 30 ␮M nelfinavir. This concentration induced a
stronger inhibition of insulin-stimulated Akt/PKB phosphorylation (Fig. 4A), increased basal lipolysis to a higher level (Fig.
5A), and decreased perilipin expression more in Ing than in Epi
adipocytes (Fig. 5B). By use of HPLC analysis of cell extracts,
intracellular nelfinavir concentrations were 23.8 ⫾ 5.6 and
33.6 ⫾ 12.2 ␮g/mg protein for Ing and Epi adipocytes, respectively (P ⫽ 0.46). This result does not support the possibility
that differences in the transport, efflux, and/or metabolism of
nelfinavir between adipocytes derived from different depots are
the reason for higher sensitivity of SC fat to nelfinavir.
DISCUSSION
Fig. 4. Inguinal adipocytes are more sensitive to nelfinavir-induced inhibition
of insulin-stimulated Akt/PKB phosphorylation. Differentiated adipocytes were
treated with indicated concentrations of nelfinavir for 18 h. A: lysates were prepared, and the protein level of pS473-Akt/PKB and total Akt/PKB were determined by Western blot analysis. B: results of densitometry analysis of 5– 8
independent experiments is shown. Results are expressed as means ⫾ SE. *P ⬍
0.05 inguinal vs. epididymal cell line. C: similar experiment as in A, performed in
primary differentiated preadipocytes.
AJP-Endocrinol Metab • VOL
HAART-induced partial lipodystrophy, a manifestation of
fat depot-differential response to a xenobiotic, is an interesting
phenomenon but is not unprecedented. IA and SC adipose
tissues differ in the expression of various developmental genes
and exhibit differential functions, including in lipolytic rate,
response to hormones, and secreted products (28, 32, 56). In
obesity cellular composition differs, as IA fat is more infil-
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navir induced insulin resistance by interfering with insulinstimulated activation of Akt/PKB, but with normal signal
transmission to PI 3-kinase (3, 46). Fully differentiated Ing and
Epi adipocytes were exposed to different concentrations of
nelfinavir for 18 h, after which cells were rinsed and stimulated
for 7 min with insulin. Although no significant effect of
nelfinavir was observed on basal Akt/PKB phosphorylation,
insulin-stimulated Ser473 phosphorylation of Akt was diminished in a dose-dependent manner by nelfinavir (Fig. 4A). Yet,
Ing adipocytes were more sensitive to this effect, demonstrating
an already considerable decrease in p-Ser-Akt with 10 ␮M
nelfinavir (Fig. 4B), and significantly different from Epi adipocytes at 20 and 30 ␮M (Fig. 4, A and B). Similar results
were observed with differentiated primary rat preadipocytes: whereas in SC-derived adipocytes 30 ␮M nelfinavir
nearly fully inhibited insulin-stimulated Akt/PKB phosphorylation, adipocytes derived from the PG fat depot still retained
a measurable capacity, albeit diminished, to mount a phosphoAkt/PKB above non-insulin-stimulated cells (Fig. 4C). Thus,
as with the observations on adipogenesis, the two cell lines
recapitulated the results in primary cells, demonstrating that
SC-derived adipocytes are more sensitive than IA adipocytes to
nelfinavir-induced insulin resistance.
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FAT DEPOT DIFFERENTIAL EFFECTS OF NELFINAVIR
Fig. 5. Nelfinavir increases basal lipolysis
and decreases perilipin protein levels to a
higher extent in inguinal than in epididymal
adipocytes. A: differentiated inguinal and epididymal adipocytes were treated with the
indicated concentrations of nelfinavir for
18 h. Then cells were incubated for an additional hour in Krebs-Ringer phosphate buffer,
and glycerol release was assessed. B: cells
were lysed and analyzed by Western blot
using specific antibodies against perilipin.
Representative blots and densitometry analysis of 4 –7 independent experiments are
shown and expressed as means ⫾ SE. *P ⬍
0.05 inguinal vs. epididymal cell line.
AJP-Endocrinol Metab • VOL
33, 36). Yet, the cell lines do not represent depot-specific
adipocyte characteristics, and the novel approaches for generating differentiating preadipocyte lines have not yet been tested
in parallel on cells originating from different depots. Here, we
show that the Ing- and Epi-derived cell lines can be effectively
differentiated into adipocytes despite the use of SV40T antigen
for immortalization, a strategy proposed to interfere with
differentiation (8). An adipocyte phenotype following differentiation was based on lipid accumulation, expression of adipogenic transcription factors and adipocyte genes like PPAR␥,
GLUT4, and perilipin, measurable lipolysis, adipokine secretion, and insulin responsiveness (Figs. 1–3).
What is the evidence that the two cell lines retain fat
depot-specific characteristics? A higher expression of total
Akt/PKB in IA fat compared with SC fat has been shown in
human adipose tissue (2) and is seen here (Fig. 1D). Likewise,
higher expression and secretion of leptin have been previously
reported in SC compared with IA fat (37, 53), consistent with
the higher leptin secretion by Ing vs. Epi adipocytes (Table 1).
In addition, in response to the HPI nelfinavir, a constituent of
HAART-induced partial lipodystrophy, Ing cells were more
susceptible to become insulin resistant, diminished leptin secretion more robustly, and were more vulnerable to nelfinavir’s
lipolytic action than Epi cells. This differential response seems
to correspond to the clinical phenotype of partial lipodystorphy: Both impaired insulin responsiveness and enhanced basal
lipolysis could constitute mechanisms for decreased fat mass,
which preferentially occurs in this depot and not in IA fat.
Intriguingly, both cell lines accumulated similar concentrations
of the drug, suggesting that the depot differences could not be
attributed to altered uptake, export, or metabolism of the drug
but rather to a different biological response. Moreover, the
ability of nelfinavir to inhibit adipogenesis occurred similarly
in both cell lines, suggesting that it may not be a primary
process differentially affecting SC vs. IA fat. This finding is
consistent with a very recent study using other HPIs on primary
human preadipocytes (20). Consistently in the present study,
both the non-depot-differential effect on adipogenesis and the
depot-differential effect on insulin signaling and on lipolysis or
perilipin expression recapitulated observation in primary cells
and tissue explants (Figs. 3C and 4C).
Despite the above results supportive of the usefulness of the
two cell lines for investigating depot-specific differences in
adipocytes, like other model systems, it is clear that there are
limitations to the use of these cell lines. This was exemplified
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trated by macrophages (17), stress-sensing signaling pathways
are differentially activated (2, 42), and even the response to
excess nutrients may be depot specific, with IA fat favoring
hypertrophy whereas SC fat favors hyperplasia (10). This
depot-specific biology may also underlie the differences in the
response of IA vs. SC fat to other drugs, including TZDs (56).
Currently, investigating fat depot-differential characteristics of
adipose tissue relies mainly on direct analyses of fat tissue
explants (14) [also used in assessing differential effects of HPIs
(9)] or on in vitro differentiation of preadipocytes derived from
such samples, which retain some depot-specific characteristics
(49). Although yielding important insights, these experimental
approaches suffer from several limitations, the major one being
the low accessibility of human IA fat. This is particularly true
for obtaining “control samples” from lean, healthy people. In
addition, adipose tissue consists of a mixed population of cells.
Its composition differs between fat depots and may change
dynamically in response to nutritional and genetic factors. To
assess which depot-specific characteristics can be attributed to
the adipocytes themselves vs. nonadipocytes (the stroma-vascular fraction), the different cell populations comprising adipose tissue are separated after the tissue is disintegrated,
typically by collagenase digestion. Although suitable to many
investigations, this experimental procedure has been demonstrated to induce many functional alterations (15, 41), raising
the risk of introducing artifacts and overriding “real” differences that reflect the in vivo environment.
With these in mind, depot-specific cell lines may constitute
an added tool, even if not perfect, for studying depot-specific
characteristics of adipocytes. Immortalized cell lines are useful
in biomedical and life science research, despite the fact that
they may lose many cell-specific characteristics and in many
cases poorly represent the original cells they were derived
from. This may particularly raise concerns when the immortalized cell line is a model of naturally nondividing, terminally
differentiated cells. Nevertheless, immortalized cell lines are
widely used not only because they are accessible and relatively
easy to utilize, but also because very frequently they do
recapitulate and predict biological processes then found in
in vivo systems. In adipocyte biology, the 3T3-L1 and 3T3F442A murine cell lines are used extensively in studying
multiple fat functions, including adipogenesis, insulin signaling, lipolysis regulation, and adipokine regulation. Very recently, several additional exciting approaches to generate differentiating preadipocyte cell lines have been described (29,
FAT DEPOT DIFFERENTIAL EFFECTS OF NELFINAVIR
ACKNOWLEDGMENTS
We are thankful to Dr. Erica Hoffer, Laboratory of Toxicology and Clinical
Pharmacology, Rambam Medical Center, Haifa, Israel, for measurements of
intracellular nelfinavir concentrations.
GRANTS
This study was supported by the Israel Science Foundation (ISF 912-05, to
A. Rudich and N. Bashan) and by The Leslie and Susan Gonda (Goldschmied)
Center for Diabetes Research and Education. N. Bashan is Chair of the Fraida
Foundation in Diabetes Research.
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