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INTRODUCTION |
The release of free fatty acids
(FFA)1 from fat cells plays
an important role in the energy homeostasis of the body. This is mainly
the result of two processes occurring simultaneously in the fat cell,
lipolysis and FFA re-esterification. During lipolysis, triglyceride
molecules are hydrolyzed to free fatty acids (FFA) and glycerol, which
are released from the fat cell. Some of the FFA formed during lipolysis
can be re-esterified to triglycerides (1, 2), whereas little or no
glycerol is re-utilized by the fat cells (3). This pathway of lipolysis
and FFA re-esterification forms an important cycle for energy turnover,
allowing the fat cell to respond rapidly to changes in peripheral
requirements for FFA (for discussion see Refs. 1 and 4-6).
Two types of FFA re-esterification in fat cells can be recognized (4).
Primary re-esterification is the total amount of FFA that is
re-esterified during a given situation and reflects the triglyceride
synthesis capacity of the fat cells (7). Fractional re-esterification
is the proportion of FFA re-esterified in relation to the amount of FFA
formed by lipolysis in fat cells (4). The latter constitutes a futile
cycle, energy-rich FFA first being formed by lipolysis of triglycerides
and then synthesized to triglycerides without the achievement of any
net biochemical work.
The mechanism by which the release of FFA from fat cells is regulated
is only partly known; insulin has been shown to inhibit lipolysis in a
phosphatidylinositol 3-kinase- (PI3K) and phosphodiesterase 3B
(PDE3)-dependent manner (8) and to stimulate fractional re-esterification (9), resulting in inhibition of FFA release. However,
little is known about the signal transduction pathways involved in the
action of insulin on FFA re-esterification, although PI3K seems to be
involved (10).
Another protein with a potentially important effect on FFA release from
human fat cells is acylation-stimulating protein (ASP) (for a review
see Ref. 11). Human fat cells synthesize and secrete three proteins in
the complement family as follows: the third component of complement
(C3), factor D (adipsin), and factor B. They interact to form
C3a-des-Arg which is identical to ASP (12-16). It has been shown that
ASP is a potent stimulator of triglyceride synthesis in human skin
fibroblasts and fat cells (16-17). After interaction with a specific
membrane receptor (18), it triggers a protein kinase C (PKC) pathway
(19), resulting in two effects. First, ASP increases glucose transport
by promoting the translocation of glucose transporters from an
intracellular pool to the plasma membrane (20-22). The intracellular
glucose can be used as the backbone of the triglyceride molecule,
glycerol 3-phosphate. Second, ASP increases the activity of
diacylglycerol acyltransferase, the enzyme catalyzing the final and
rate-limiting step in triglyceride synthesis (23). These effects of ASP
are additive to and independent of those observed with insulin (20,
24). The action of ASP on FFA release from fat cells, on the other
hand, is unknown.
The first aim of the present study was to determine the effect of ASP
on FFA produced and re-esterified during lipolysis in isolated human
subcutaneous fat cells and to compare these effects with those of
insulin. The second aim was to investigate which intracellular
signaling pathways for ASP and insulin control their effects on FFA
release. One focus was on PKC and PI3K which are reported to be linked
to the known effects of ASP and insulin, respectively, in fat cells. A
second focus was on signaling downstream of PKC and PI3K. For the
latter purpose, the roles of PDE3 and PDE4 were examined. These PDE
subtypes are present in fat cells (25) where they regulate lipolysis by
modifying intracellular cyclic AMP levels. PDE3 is known to be the key
enzyme involved in the antilipolytic action of insulin (8). Our
hypothesis was that PDE3 and other PDEs might regulate hormone effects
on FFA re-esterification as well.
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MATERIALS AND METHODS |
Subjects--
The study group consisted of 36 subjects who
underwent elective open abdominal surgery for non-malignant disorders
(gallstone, hernia, and gastric banding for obesity). According to
patient history, the subjects were healthy except for the surgical
diagnosis. None was on regular medication or had undergone a recent
weight reduction program. The subjects, who had fasted overnight, were anesthetized at 8.00 a.m., as described in detail (26). Before taking the fat biopsy, only saline was administered intravenously. Immediately after the abdominal wall had been opened, a specimen of
subcutaneous fat tissue (2-5 g) was obtained from the surgical incision and immediately transported to the laboratory in saline at
37 °C.
Most patients at the hospital are at present operated on by
laparoscopic methods, making them unsuitable for fat biopsy procedures. From the few remaining subjects undergoing open surgery, it was not
possible to select for gender (10 men and 26 women), age (range: 25-68
years), or body mass index (BMI range: 21-60 kg/m2).
The study was approved by the Ethical Committee of the Karolinska
Institute at Huddinge Hospital. It was explained in detail to all
participants. All subjects gave their consent.
Preparation of ASP--
The ASP was prepared at the Royal
Victoria Hospital. It was isolated and purified from frozen human
plasma (obtained from the local blood bank), as described previously
(19). The purity of each ASP preparation was determined by ion spray
mass spectrometry (95-99% pure), and the activity was tested through
stimulation of [3H]oleic acid incorporation into
intracellular triglycerides in human skin fibroblasts (19). ASP was
frozen (
80 °C) in aliquots in phosphate-buffered saline (pH 7.2)
containing 1 mg/ml bovine serum albumin and was shipped on dry ice to
Huddinge Hospital, where all subsequent experiments took place. Insulin
was not detected in the ASP samples, using an ultra-sensitive insulin
enzyme-linked immunosorbent assay kit (Mercodia AB, Uppsala, Sweden),
which can detect insulin at a concentration as low as 1 pM.
Isolation of Fat Cells--
Fat cells were isolated from the
subcutaneous fat tissue specimen by collagenase treatment, as described
(27). Briefly, all visible blood vessels and connective tissue were
removed, and 2 g of fat tissue was cut into fragments of around
5-10 mg and incubated in 5 ml of Krebs-Ringer phosphate (KRP) buffer
(pH 7.4), containing 0.5 g/liter collagenase and 40 g/liter dialyzed bovine serum albumin, in a shaking bath at 37 °C for 1 h. The fat cells were filtered twice through a nylon cloth and subsequently washed three times with collagenase-free KRP buffer (pH 7.4).
Incubation of Fat Cells and Determination of the Release of
Glycerol and FFA--
The fat cell incubations were conducted as
described previously (28). In brief, diluted suspensions of fat cells
(5-10,000 cells/ml) were incubated for 2 h in duplicate in a
shaking bath with air as the gas phase at 37 °C in 0.2 ml of KRP
buffer (pH 7.4) containing 2.5 g/liter fatty acid-free bovine serum
albumin, 1 g/liter glucose, and 0.035 g/liter ascorbic acid in the
presence of various agents, which were added simultaneously at the
beginning of the incubation. Under the present incubation conditions,
rates of metabolic activities are constant for at least 4 h.
In the first set of experiments (number of subjects (n) = 11), fat cells were incubated with increasing concentrations of ASP
(0.1, 0.5, 1, 2.8, and 5.6 µM) with and without
norepinephrine (1 µM). The highest ASP concentration was
also incubated together with insulin (0.1 nM). In addition,
fat cells from the same subjects were incubated with increasing
concentrations of insulin (0.01, 0.1, and 1 nM) with or
without norepinephrine (1 µM). In the second set of
experiments (n = 10), fat cells were incubated with ASP (5.6 µM) or insulin (0.1 nM) in combination
with calphostin C (a selective PKC inhibitor (29) at 1, 10, and 100 nM and 1 µM). In the third set of experiments
(n = 10), fat cells were incubated with ASP (5.6 µM) or insulin (0.1 nM) in combination with
wortmannin (selective PI3K inhibitor (30)) at 0.1 µM or
enprofylline (non-selective PDE inhibitor with insignificant adenosine
interactions (31)) at 1 mM. Fat cells from 8 of these
subjects were also incubated with norepinephrine (1 µM)
and either ASP (5.6 µM) or insulin (0.1 nM)
with or without the addition of enprofylline (1 mM). In the
fourth set of experiments (n = 9), fat cells were
incubated with ASP (5.6 µM) or insulin (0.1 nM) in combination with OPC 3911 (selective PDE3 inhibitor,
at 1, 3 and 10 µM (32)) or rolipram (selective PDE4
inhibitor, at 1, 3, and 100 µM (32-33)). The total lipid
content in the incubate was measured gravimetrically after organic
extraction with heptane.
At the end of the incubation, a cell-free aliquot of the incubation
medium was removed for glycerol release determination by a
bioluminescence method (34). Another aliquot was used for the
measurement of FFA release from fat cells, using a chemiluminescence method (35). FFA oxidation in this in vitro system is
negligible (36), so the only expected outcomes for the FFA are either
release from the fat cells during lipolysis or re-esterification. The glycerol release can be used as an accurate index of the amount of FFA
released during lipolysis, as glycerol cannot be re-utilized by fat
cells to any important extent (3). Complete hydrolysis of one
triglyceride molecule generates three molecules of FFA and one molecule
of glycerol. Since there is only minor partial hydrolysis of
triglycerides in human fat cells (37), the molar amount of FFA produced
during lipolysis was calculated as 3× molar glycerol release. The
absolute rate of primary FFA esterification was calculated as described
earlier (4): primary FFA esterification = (3× molar glycerol
release) molar FFA release. Fractional FFA re-esterification,
the proportion of FFA molecules formed during lipolysis that is
re-esterified to triglycerides, was calculated as the ratio of primary
FFA esterification to total FFA formed by lipolysis (4). This ratio can
vary between 0 and 1. It is 0 when no FFA are re-esterified and 1 when
all FFA are re-esterified. As discussed in detail (38), this way of
calculating re-esterification relies on the following: (a)
there is only minor net synthesis or breakdown of partial glycerides
(mono- and diglycerides), (b) there are only minor net
changes in the small intracellular pool of FFA, and (c) FFA
are not oxidized by fat cells to any significant extent.
Assay of PDE Activity--
PDE activity was measured in fat
cells from 7 subjects, as described (39). Fat cells (400 µl) were
incubated for 10 min in duplicate in a shaking bath at 37 °C in 2 ml
(~250,000 cells/ml) KRP buffer (pH 7.4) containing 2.5 g/liter fatty
acid-free bovine serum albumin, 1 g/liter glucose, and 0.1 mg/liter
ascorbic acid with or without insulin (0.1 nM) or ASP (5.6 µM). The fat cells were homogenized at room temperature
with 10 strokes in a glass homogenizer in 1 ml of homogenization buffer
containing 50 mM TES (pH 7.5), 250 mM sucrose,
1 mM EDTA, 0.1 mM EGTA, 600 nM
okadaic acid, and 10 µg/ml each of antipain and leupeptin and 1 µg/ml of pepstatin A. The homogenates were centrifuged at 10,000 × g for 5 min at 4 °C. Fat cakes were removed. PDE
activity, which was defined as the amount of hydrolyzed cAMP per min
(39), was measured in the homogenate. Samples were incubated at
30 °C for 20 min in a total volume of 300 µl, containing 50 mM Hepes (pH 7.4), 0.1 mM EDTA, 8.3 mM MgCl2, and 0.5 µM
[3H]cAMP. OPC 3911 and rolipram were used at 10 and 30 µM, respectively, in the assays to estimate PDE3 and PDE4
activities. PDE activity was expressed per ml of fat cells.
Drugs and Chemicals--
Bovine serum albumin (fraction V),
fatty acid-free bovine serum albumin, Clostridium
histolyticum collagenase type I, glycerol kinase from
Escherichia coli (G4509), acyl-CoA synthetase (EC 6.2.1.3)
from Pseudomonas specialis, acyl-CoA oxidase from
Candida lipolytica, horseradish peroxidase (EC 1.11.1.7,
type VI; 250-330 units/mg), sodium dodecyl sulfate, ascorbate oxidase,
adenosine deaminase, Triton X-100, ATP, insulin, calphostin C,
wortmannin, TES, antipain, leupeptin, pepstatin, Hepes, and cAMP were
obtained from Sigma. ATP-monitoring reagent containing firefly
luciferase came from LKB Wallac (Turku, Finland). Enprofylline came
from AB Draco (Lund, Sweden). Okadaic acid came from Calbiochem.
Rolipram came from Biomol (Plymouth Meeting, PA).
[2,8-3H]cAMP came from Amersham Pharmacia Biotech
(Buckinghamshire, UK). OPC 3911 was obtained from Otsuka
Pharmaceuticals (Tokyo, Japan). All other chemicals were of the highest
grade of purity commercially available and were obtained from Kebo
(Stockholm, Sweden).
Statistical Analysis--
Values are given as the mean ± S.E. Analysis of variance (ANOVA) and paired t test were
used for statistical comparisons. A correction of the p
value according to Bonferroni was made when it was appropriate.
Correlations were analyzed using the linear regression method.
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RESULTS |
The Effect of ASP and Insulin on Release, Production during
Lipolysis, and Primary Esterification of FFA--
These experiments
were performed in an identical fashion on 11 subjects. As shown in Fig.
1, ASP inhibited FFA release from fat
cells incubated under basal conditions in a
concentration-dependent way (ANOVA, F = 21, p < 0.0001). The minimum effective concentration was
6.3 log mol/liter (paired t test, p < 0.05 (corrected according to Bonferroni)). At the highest ASP
concentration (5.3 log mol/liter), FFA release was inhibited by
~85%. Norepinephrine (1 µM) doubled the rate of FFA
release in comparison to basal incubation (paired t test
p < 0.01). Also in combination with norepinephrine,
ASP inhibited FFA release (ANOVA, F = 16, p < 0.0001). The minimum effective ASP concentration
for this effect was 6 log mol/liter (paired t test
(Bonferroni), p < 0.05) and at the highest ASP concentration, FFA release was inhibited by ~45%.
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Fig. 1.
Net FFA release in the absence or presence of
increasing concentrations of ASP (upper graph, 0 to
5.25 log mol/liter) or insulin (lower graph, INS, 0 to 9 log mol/liter), in the absence (open symbols)
or presence (filled symbols) of norepinephrine (NE;
6 log mol/liter). The number of subjects (n) is 11. Values are mean ± S.E. The effects of ASP and insulin were
analyzed using ANOVA (F). NA,
norepinephrine.
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Insulin inhibited basal FFA release in a
concentration-dependent fashion (Fig. 1, ANOVA,
F = 20, p < 0.0001). The minimum effective concentration for this effect was 10 log mol/liter (paired
t test (corrected according to Bonferroni),
p < 0.05). At the highest insulin concentration (9
log mol/liter), FFA release was inhibited by ~90%. In the presence
of 1 µM norepinephrine, insulin maintained its ability to
inhibit FFA release in a concentration-dependent fashion
(ANOVA, F = 20, p < 0.0001). The
minimum effective insulin concentration for this effect was 10 log
mol/liter insulin (paired t test (Bonferroni),
p < 0.05) and at the highest insulin concentration, FFA release was inhibited by ~80%.
As shown in Fig. 2, ASP slightly
inhibited the total amount of FFA produced during basal lipolysis (as
calculated from glycerol release) in a
concentration-dependent fashion (ANOVA, F = 10, p < 0.0001). The minimum effective ASP
concentration was 6.3 log mol/liter (paired t test,
p < 0.05 (Bonferroni)). At the highest ASP
concentration (5.3 log mol/liter), lipolysis-produced FFA was
inhibited by ~30%. Norepinephrine (1 µM) almost
doubled the rate of lipolysis in comparison to the basal state
(p < 0.05). Also in combination with norepinephrine,
ASP reduced the amount of FFA produced during lipolysis (ANOVA,
F = 3, p < 0.01), but the effect was
very small, i.e. lipolysis was inhibited by ~15% at the
highest ASP concentration.
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Fig. 2.
FFA produced during lipolysis in the absence
or presence of increasing concentrations of ASP (upper
graph) or insulin (lower graph, INS), with
or without the presence of norepinephrine. See legend to Fig. 1
for more details.
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Insulin markedly inhibited the FFA production during basal lipolysis in
a concentration-dependent fashion (Fig. 2, ANOVA, F = 14, p < 0.0001). The minimum
effective concentration for this effect was 10 log mol/liter insulin
(paired t test (Bonferroni), p < 0.05). At
the highest insulin concentration (9 log mol/liter), lipolysis was
inhibited by ~60%. In the presence of 1 µM
norepinephrine, insulin maintained its marked ability to inhibit
lipolysis in a concentration-dependent fashion (ANOVA,
F = 12, p < 0.0001). The minimum
effective insulin concentration for this effect was 10 log mol/liter
insulin (paired t test (Bonferroni), p < 0.05), and at the highest insulin concentration, the amount of FFA
produced during lipolysis was decreased by ~60%.
The absolute rate of primary FFA esterification in the experiments
described in Figs. 1 and 2 was calculated as the difference between
total FFA produced by lipolysis (calculated from glycerol release) and
the actual amount of FFA released from the cells. This rate was not
influenced by ASP when fat cells were incubated in the basal state. The
rates of primary esterification with and without the highest ASP
concentration were similar: 2.92 ± 0.50 versus
2.82 ± 0.43 µmol/g lipid/2 h, p = not
significant. Norepinephrine at 1 µM increased the
absolute rate of primary FFA re-esterification (paired t
test, 2.92 ± 0.50 versus 4.36 ± 0.54 µmol/g
lipid/2 h, p < 0.05). Increasing concentrations of ASP
did not change this effect of norepinephrine (paired t test,
norepinephrine versus norepinephrine + ASP (5.3 log
mol/liter): 4.36 ± 0.54 versus 4.66 ± 0.56 µmol/g lipid/2 h, p = not significant). Insulin did not influence the absolute rate of primary FFA re-esterification significantly either, in the absence or presence of norepinephrine.
The fractional FFA re-esterification, which is the proportion of FFA
re-esterified during lipolysis, increased markedly with increasing
concentrations of ASP when fat cells were incubated in the basal state
(Fig. 3, ANOVA, F = 4, p < 0.01). This stimulation was significant at 6.3
log mol/liter ASP (paired t test (Bonferroni), p < 0.01), and at the highest concentration, ASP
increased fractional FFA re-esterification by ~40%. Norepinephrine
alone at 1 µM decreased the fractional FFA
re-esterification (paired t test, p < 0.01). In the presence of norepinephrine, ASP increased the fractional FFA re-esterification in a concentration-dependent manner
(ANOVA, F = 5, p < 0.001). This
stimulation started to be significant at 6.3 log mol/liter ASP
(paired t test (Bonferroni), p < 0.05), and
at the highest ASP concentration, fractional FFA re-esterification was
increased by ~25%. Insulin also increased the fractional FFA re-esterification in a marked and concentration-dependent
fashion under basal conditions (Fig. 3, ANOVA, F = 24, p < 0.0001). The minimum significant effective
concentration for this effect was 10 log mol/liter insulin (paired
t test (Bonferroni), p < 0.05). At the
highest insulin concentration (9 log mol/liter), fractional FFA
re-esterification was increased by ~40%. In the presence of 1 µM norepinephrine, insulin maintained its ability to
stimulate fractional FFA re-esterification (ANOVA, F = 10, p < 0.0001). The minimum effective insulin
concentration for this effect was 10 log mol/liter insulin (paired
t test (Bonferroni), p < 0.05) and at the
highest insulin concentration, fractional FFA re-esterification was
increased by ~25%.
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Fig. 3.
Fractional FFA re-esterification in the
absence or presence of increasing concentrations of ASP (upper
graph) or insulin (lower graph, INS), with
or without the presence of norepinephrine. Fractional FFA
re-esterification can vary between 0 (no FFA re-esterification) and 1 (all FFA re-esterified). See legend to Fig. 1 for more details.
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Figs. 1-3 show data with increasing concentrations of ASP and insulin
up to 5.6 µM and 1 nM, respectively. The
observed effects at these concentrations did not differ in a
significant way from the effects at higher concentrations (data not shown).
Comparing the maximum effects of ASP and insulin on release, production
and fractional re-esterification of FFA, ASP, and insulin were equally
efficient in inhibiting FFA release and stimulating fractional FFA
re-esterification, whereas insulin was significantly more efficient
than ASP in inhibiting production of FFA during lipolysis (paired
t test, p < 0.01).
Additive Effects of ASP and Insulin on FFA Release--
These
experiments were performed in an identical way on 11 subjects. In Table
I, it is shown that ASP (5.6 µM) could augment the effect of insulin (0.1 nM) on basal and norepinephrine-stimulated (1 µM) FFA release from fat cells and vice versa (paired
t test, INS versus ASP + INS, ASP
versus ASP + INS; p < 0.01). ASP could also
augment the effect of insulin on basal lipolysis and the other way
around (paired t test, INS versus ASP + INS
p < 0.05; ASP versus ASP + INS,
p < 0.01). Furthermore, insulin could augment the
effect of ASP on norepinephrine-stimulated lipolysis (ASP + NE
versus ASP + NE + INS, p < 0.01), whereas
ASP did not significantly augment the effect of insulin on
norepinephrine-stimulated lipolysis. ASP together with insulin almost
completely suppressed FFA release, whereas together they only inhibited
lipolysis by 50-60%. ASP could augment the effect of insulin on basal
and norepinephrine-inhibited fractional FFA re-esterification and the
other way around (paired t test, INS versus ASP + INS, ASP versus ASP + INS; p < 0.05).
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Table I
Additive effects of ASP and insulin in human fat cells, effects on FFA
release, FFA produced during lipolysis, and fractional FFA
re-esterification
Data are mean ± S.E. ASP (5.6 µM) and insulin (INS,
0.1 mM) values were compared with values of the two
proteins together, using a paired t test.
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Studies of Intracellular Signaling--
The possible involvement
of PKC, PI3K, and PDE as mediators of the effect of ASP and insulin on
FFA release was studied by investigating the effect of blockers that
specifically inhibit the activity of these signaling enzymes. It is
evident from the experiments presented above that the effects of ASP
and insulin on FFA release were more marked in fat cells maintained in
basal conditions than in norepinephrine-stimulated fat cells. For this reason and because catecholamines markedly stimulate PDE3 activity in
human fat cells (40), all investigations of intracellular signaling
were performed in the basal state.
Effects of Blockers of PKC, PI3K, and PDE on FFA Release--
The
possible involvement of PKC as a mediator of ASP and insulin action on
FFA release was studied using the selective PKC blocker calphostin C,
which interacts with the regulatory domain of PKC (41), at different
concentrations ranging from 1 to 1000 nM. At these
concentrations calphostin inhibits the ASP action on glucose metabolism
in human cells (19). These experiments were performed in an identical
way on 10 subjects. Calphostin C alone, at 1-1000 nM,
inhibited basal FFA release (Fig. 4;
paired t test, p < 0.01). On the other
hand, calphostin C, at 1-1000 nM, did not significantly
influence the effect of ASP or insulin on FFA release (Fig. 4). Note
that ASP and insulin themselves decrease FFA release. Inhibition of the
effect of ASP or insulin would be expected to result in an increase in
FFA release as compared with the effect of ASP or insulin alone. We
also investigated the action of another PKC inhibitor,
bisindolylmaleimide (GF 109203X), which is a competitive inhibitor of
the ATP-binding site on PKC (41) at 1 and 5 µM. This
blocker did not influence ASP or the action of insulin on FFA release
in a significant way (values not shown).
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Fig. 4.
The effect of the PKC inhibitor calphostin C
(1 µM, n = 10) and
the PI3K inhibitor wortmannin (0.1 µM, n = 10) on FFA
release from fat cells, in the absence (basal) or presence of ASP
(5.6 µM) or insulin
(INS, 0.1 nM). A paired
t test was used to analyze the effect of the inhibitors.
NS, not significant. Control, no inhibitor is
present in the incubation medium. Values are means ± S.E.
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The effect of wortmannin (PI3K inhibitor) on the action of ASP and
insulin on FFA release was tested at a concentration of 0.1 µM. At this concentration wortmannin almost completely
counteracts the effect of insulin on FFA release in isolated human fat
cells (10). Wortmannin reversed the effect of insulin on FFA release almost completely (Fig. 4; paired t test, INS
versus INS + wortmannin p < 0.001). In
contrast, it did not significantly influence the effect of ASP on FFA
release (Fig. 4), indicating PI3K-dependent and
PI3K-independent effects induced by insulin and ASP, respectively.
The results of the effects of enprofylline (a non-selective PDE
inhibitor without any adenosine interactions), OPC 3911 (a selective
PDE3 inhibitor), and rolipram (a selective PDE4 inhibitor) on FFA
release are depicted in Fig. 5. The study
with enprofylline was performed in an identical fashion on 10 subjects,
and the studies with OPC 3911 and rolipram were performed on 9 subjects. Enprofylline (1 mM) and OPC 3911 (3 µM) reversed the effects of ASP and insulin on FFA
release significantly (paired t test, ASP versus
ASP + enprofylline, p < 0.001; INS versus
INS + enprofylline; p < 0.001; ASP versus
ASP + OPC 3911; p < 0.0001 and INS versus INS + OPC 3911; p < 0.0001). Enprofylline and OPC 3911 per se stimulated FFA release significantly (Fig. 5). Other
concentrations of OPC 3911 were also tested (1 and 10 µM), and it was observed that OPC 3911 counteracted the
effects of ASP and insulin on FFA release in a
concentration-dependent fashion (data not shown). Rolipram
(1 µM) did not alter the insulin effect on FFA release, whereas it slightly counteracted the effect of ASP (paired t
test, ASP versus ASP + rolipram, p < 0.01).
Higher concentrations of rolipram (3 and 100 µM) were
also tested. The same pattern of effects as at 1 µM was
observed (data not shown).
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Fig. 5.
The effect of the PDE inhibitors enprofylline
(1 mM, n = 10), OPC 3911 (3 µM, n = 9), and
rolipram (1 µM, n = 9) on FFA release in the absence or presence of ASP or
insulin. See legend to Fig. 4 for more details.
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Effects of PDE Blockers on FFA Production during
Lipolysis--
The effects of the PDE inhibitors on FFA produced
during lipolysis, as calculated from the values for glycerol release,
are shown in Table II. For practical
reasons it was not possible to perform all types of lipolysis
experiments simultaneously in all individuals. In order to compare
results from different sets of subjects, the results were expressed as
a percentage of basal (i.e. no drug added) FFA produced
during lipolysis. The mean values for basal lipolysis did not differ in
a significant way between the different sets of experiments.
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Table II
The effect of PDE blockers and norepinephrine on the action of ASP or
insulin on FFA produced during lipolysis
Data are expressed as percentage of basal FFA produced during
lipolysis. For concentrations of the inhibitors and statistical
analysis see "Results." NE, norepinephrine; INS, insulin.
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Enprofylline (1 mM) per se significantly
stimulated FFA produced during lipolysis (paired t test,
control versus enprofylline; p < 0.01). It
also counteracted the effect of ASP and insulin on lipolysis (paired
t test, ASP versus ASP + enprofylline; INS versus INS + enprofylline; p < 0.001).
OPC 3911 (3 µM) by itself increased FFA produced during
lipolysis (paired t test, control versus OPC
3911; p < 0.01), and it reversed the effect of ASP and
insulin on lipolysis (paired t test, ASP versus
ASP + OPC3911, p < 0.0001; INS versus INS + OPC3911, p < 0.001).
Rolipram (1 µM) did not influence basal FFA produced
during lipolysis nor did it counteract the effects of ASP and insulin on this process.
Norepinephrine (1 µM) stimulated FFA produced during
lipolysis to a much higher extent than observed with enprofylline and to the same extent as observed with OPC 3911 (paired t test,
NE versus enprofylline, p < 0.05). ASP or
insulin inhibited norepinephrine-stimulated lipolysis (paired
t test, NE versus NE + ASP, p < 0.05; NE versus NE + INS; p < 0.001). The
addition of enprofylline to norepinephrine did not increase lipolysis
above that observed with norepinephrine alone. On the other hand, ASP
or insulin could not reverse norepinephrine-stimulated lipolysis when
enprofylline was present.
Effects of PDE Blockers on Fractional FFA
Re-esterification--
These data are derived from the same
experiments that are presented in Fig. 5. Since the absolute rate of
re-esterification was not influenced by insulin or ASP, the
investigations only include fractional re-esterification. The results
of the effects of enprofylline, OPC 3911, and rolipram are depicted in
Fig. 6. Enprofylline (1 mM)
and OPC 3911 (3 µM) almost completely reversed the
effects of ASP and insulin on fractional re-esterification (paired
t test, ASP versus ASP + enprofylline,
p < 0.01; INS versus INS + enprofylline;
p < 0.05; ASP versus ASP + OPC3911;
p < 0.0001 and INS versus INS + OPC3911;
p < 0.05). Rolipram (1 µM) did not alter
the insulin effect on fractional FFA re-esterification, whereas it
slightly counteracted the effect of ASP (paired t test, ASP
versus ASP + rolipram, p < 0.01).
Enprofylline, OPC 3911, or rolipram had no significant effects on
fractional FFA re-esterification per se (Fig. 6).
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Fig. 6.
The effect of the PDE inhibitors enprofylline
(1 mM, n = 10), OPC 3911 (3 µM, n = 9), and
rolipram (1 µM, n = 9) on fractional FFA re-esterification in the absence or
presence of ASP or insulin. See legend to Figs. 3 and 4 for more
details.
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When the fractional FFA re-esterification values under basal conditions
or with ASP (5.6 µM) or insulin (0.1 nM) in
Fig. 1-3 were compared with those in Fig. 6, the values in Fig. 1-3
were slightly higher than in Fig. 6. The relative effects of ASP (5.6 µM) and insulin (0.1 nM), on the other hand,
did not differ significantly between the experiments in Figs. 1-3 and
6 (ANOVA, p = not significant). The subjects in Fig.
1-3 did not differ significantly from those in Fig. 6 regarding age or
BMI (ANOVA, p = not significant). No significant
correlation was found between BMI or age on the one hand and basal,
ASP-induced, or insulin-induced fractional FFA re-esterification on the
other hand. No significant gender difference was found in fractional
FFA re-esterification values.
ASP and Insulin Effects on PDE Activity--
To examine further
the involvement of PDE in ASP and insulin action, PDE activity was
measured in fat cells, following incubation with ASP or insulin (Fig.
7). These experiments were performed in
an identical way on 7 subjects. It is shown that PDE3 activity was
stimulated significantly in a concentration-dependent
fashion with either ASP or insulin (ANOVA, ASP: F = 10, p < 0.01; insulin: F = 20, p < 0.001). Insulin did not stimulate PDE4 activity.
ASP, on the other hand, increased PDE4 activity in a
concentration-dependent manner (ANOVA F = 12, p < 0.001). ASP caused a significant increase in
PDE4 activity at 5.6 µM (paired t test
(Bonferroni): p < 0.05).
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Fig. 7.
PDE3 and PDE4 activity in fat cells incubated
with ASP (1 and 5.6 µM) or insulin
(0.1 and 1 nM). Values are means ± S.E., n = 7. Control, no ASP or insulin
present.
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DISCUSSION |
The release of FFA from human fat cells is a key feature in the
regulation of the energy balance of the body. It is the net result of
FFA formed during lipolysis and fractional FFA re-esterification. In
the current study, several new mechanisms involved in the regulation of
FFA release were observed. In particular, the involvement of ASP and
the role of PDEs in FFA re-esterification were demonstrated.
Fat cell re-esterification can be measured with two different sensitive
methods in vitro as follows: by simultaneous measurement of
the release of glycerol and FFA using luminescence or by a dual radio
isotope technique (42). We used the former method because it allows the
utilization of very dilute fat cells suspensions. A number of lipolytic
or antilipolytic substances are secreted by human fat cells, such as
adenosine (43), lactate (33), prostaglandins (44), and tumor necrosis
factor 
(45). By using dilute fat cell suspensions, the possible
influence of such substances on lipolysis is avoided or minimized. For
example, we have shown, under the present incubation conditions, that
the influence of adenosine release from human fat cells on lipolysis is
negligible (46).
ASP, which stimulates triglyceride synthesis and glucose transport in
human fat cells (16-17, 20-22), was found to markedly inhibit both
basal and norepinephrine-stimulated FFA release from fat cells as well.
This effect was due to a combination of the following two processes:
ASP markedly increased the proportion of re-esterified FFA (fractional
FFA re-esterification) and to a lesser extent it inhibited FFA produced
during lipolysis. This pattern is different to that observed for
insulin, which had marked effects on both lipolysis and fractional FFA
re-esterification. When fat cells were incubated with maximally
effective concentrations of each protein, ASP inhibited FFA produced
during lipolysis to a lesser extent than insulin, whereas it stimulated
fractional FFA re-esterification to the same or even greater extent
than insulin.
We hypothesize that the effects of insulin and ASP on fractional FFA
re-esterification are not secondary to the action of the two hormones
on lipolysis. If stimulation of FFA re-esterification were secondary to
inhibition of lipolysis, insulin and ASP would also affect the absolute
rates of FFA esterification (i.e. primary esterification).
However, no effects of insulin or ASP on primary esterification were
observed. The insulin data confirm several earlier observations (9,
47-48). Only norepinephrine, which is lipolytic, was found to be
effective in stimulating the rate of primary FFA esterification. The
latter data confirm the results of previous studies with catecholamines
(7, 48). On the other hand, the PDE blockers, enprofylline and OPC
3911, were also lipolytic in this study, but they did not affect the
absolute rate of FFA re-esterification significantly (data not shown).
Furthermore, the weak inhibitory effect of ASP on lipolysis could
hardly explain its marked effect on fractional re-esterification.
Another novel observation is that ASP could markedly augment the effect
of insulin on fractional FFA re-esterification, whereas it only
marginally enhanced the antilipolytic effect. For example, insulin in
combination with ASP reduced the lipolytic rate by 50-60% and
inhibited the FFA release almost completely; the latter was probably
due to a near maximal effect of the hormone combination on fractional
FFA re-esterification. This suggests that the effects of ASP and
insulin on FFA release are additive, which above all is due to a dual
effect on fractional FFA re-esterification. The average ASP level in
humans has been reported to be about 0.06 µM in fasting
plasma increasing postprandially in fat tissue venous effluent to about
0.1 µM (49). In the present study, we observed significant in vitro effects of ASP at 0.1 and 0.5 µM, in the presence and absence of insulin, respectively.
Thus, our findings may be of physiological relevance for circulating
ASP, although some caution should be exercised in extrapolating from
the in vitro to the in vivo situation. However,
it is tempting to speculate that ASP and insulin act together in
regulating FFA metabolism, increasing the proportion of re-esterified
FFA so that FFA release is almost completely shut down in the
postprandial state. Furthermore, increased release of FFA from fat
cells in obesity and diabetes may not only relate to insulin
resistance; a blunted response to ASP could have the same consequences.
The second aim of this study was to determine the intracellular
signaling pathways that mediate the effects of ASP and insulin on
fractional FFA re-esterification and lipolysis. From earlier studies it
is known that ASP stimulates glucose transport and triglyceride
synthesis through activation of a PKC-dependent pathway in
human fibroblasts (19). Insulin, on the other hand, is known to exert
its antilipolytic effect in human fat cells by phosphorylation of PI3K
which in turn activates PDE3 (8). In addition, PI3K mediates the
insulin effect on fractional re-esterification (10). The pathways
distal to PI3K that are involved in the effect of insulin on FFA
re-esterification and in the action of ASP on FFA re-esterification and
lipolysis have been unknown until now.
In this study, PKC blockers could not counteract the effects of ASP or
insulin on fractional FFA re-esterification or lipolysis, suggesting
that the actions of the two hormones on FFA release are not mediated by
PKC. This is in contrast to earlier results regarding the action of ASP
on triglyceride synthesis and glucose transport in human fibroblasts
(19), which is counteracted by PKC blockers.
The PI3K blocker wortmannin counteracted, as expected, the effect of
insulin on FFA release by reversing the effect of insulin on fractional
FFA re-esterification and lipolysis. The effects of ASP, on the other
hand, were not affected by wortmannin. So the pathway mediating the
effect of ASP on FFA release does not involve PI3K. This strongly
supports the hypothesis that ASP and insulin follow different pathways
in their action on FFA release. The fact that the ASP receptor
protein(s) has not been identified makes further research in this area
difficult at present.
We next addressed the question whether a more distal pathway could be
common for the actions of insulin and ASP on FFA metabolism. The
interest was focused on PDE, which previously was found to mediate not
only the antilipolytic effect of insulin but also the stimulatory
effect of hormone on glucose transport in human fat cells (50). By
catalyzing the hydrolysis of cAMP and cGMP, PDEs regulate intracellular
concentrations and biological responses of these second messengers (8).
PDEs constitute a group of structurally related enzymes that belong to
at least nine related gene families (PDE 1-9), which differ in their
primary structures, affinities for cAMP and cGMP, responses to specific
effectors, and sensitivity for inhibitors and regulatory mechanisms (8, 51). Two PDEs (PDE3 and PDE4) have high affinity for cAMP and are
present in fat cells (25). PDE3 can be distinguished from PDE4 by its
high affinity for both cAMP and cGMP. Activation of PDE3 but not PDE4
plays a role in the antilipolytic effect of insulin in vivo
in human fat tissue (33, 52). In this study, enprofylline, which is a
non-selective PDE inhibitor that does not interact with adenosine,
counteracted the actions of both ASP and insulin on FFA release by
reversing their effects on fractional FFA re-esterification and
lipolysis. The selective PDE3 blocker, OPC 3911, could also counteract
these effects of ASP and insulin. The selective PDE4 blocker, rolipram,
was not able to counteract the effect of insulin on fractional FFA
re-esterification and lipolysis. Rolipram did not alter the effect of
ASP on lipolysis either, but it counteracted slightly the effect of ASP
on fractional FFA re-esterification. These results were in agreement
with the results obtained with direct measurements of PDE activity.
PDE3 activity in fat cells was stimulated with both ASP and insulin in
a concentration-dependent manner, whereas PDE4 activity was only slightly stimulated with ASP. Thus, the effects of both ASP and
insulin on FFA release appear to be mediated by PDE3. PDE4 seems to be
involved in the action of ASP as well, although to a lesser extent than PDE3.
PDE blockade, using the PDE inhibitors enprofylline and OPC 3911, stimulated basal FFA production during lipolysis. It is likely that
when lipolysis is increased, more ASP or insulin is required to reverse
this process. One could say that the PDE inhibitors reversed the
antilipolytic effect of ASP and insulin through their own lipolytic
effect and not by specifically blocking PDE. This, however, seems
unlikely. In the present study, stimulation of lipolysis with
norepinephrine to a greater extent than observed with enprofylline did
not prevent the inhibition of lipolysis by ASP or insulin. In contrast,
enprofylline did not further increase lipolysis induced by
norepinephrine alone, but ASP or insulin could not reverse lipolysis
induced by norepinephrine combined with enprofylline. Norepinephrine
does not maximally activate lipolysis in the present incubation
conditions when compared with the selective 
-adrenoreceptor agonist
isoprenaline (53), since it is a -and also an
2-adrenoreceptor agonist. Thus, the data strongly
indicate that the antilipolytic effect of ASP is mediated by PDE.
The effects of the PDE blockers on insulin- and ASP-stimulated
fractional FFA re-esterification cannot be explained by interactions with lipolysis. Although enprofylline and OPC 3911 stimulated lipolysis, they had no effect of their own on fractional FFA
re-esterification.
The source of ASP was purified plasma. It is excluded that insulin
contaminations were present in the ASP preparations. First, the ASP
preparation was pure as evidenced by ion spray mass spectrometry analysis. Second, we could not detect insulin in the ASP preparations. Third, the mechanism of action of the two hormones differed considerably.
In this study, it was for practical reasons not possible to select the
subjects for age, gender, and BMI. However, there was no apparent
effect of these factors on the actions of ASP and insulin on fractional
FFA re-esterification and lipolysis. Only subcutaneous fat cells were
used, so whether there are regional differences in the effect of ASP on
lipolysis and re-esterification remains to be investigated. There are
some differences in insulin action on lipolysis in omental and
subcutaneous fat cells (10).
In summary, the current study shows that ASP and insulin may play a
major role in regulating basal- and catecholamine-stimulated FFA
release from fat cells, through stimulatory effects on fractional primary FFA re-esterification and inhibitory effects on FFA produced during lipolysis. Insulin has marked effects on both processes, whereas
ASP mainly stimulates fractional FFA re-esterification. The effects are
mediated in both cases by PDE3. Some of the effect of ASP on fractional
FFA re-esterification could also be mediated by PDE4. This suggests not
only an important role of PDE in fat cell lipolysis but also in FFA
re-esterification. The FFA re-esterification signaling pathway
preceding PDE for insulin involves PI3K, but is not known for ASP yet,
although PKC or PI3K do not seem to be involved.
,
,
TOP
ABSTRACT
INTRODUCTION
