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INTRODUCTION |
Adenoviral transfer of the leptin gene into normal rats causes
rapid loss of all visible body fat within 7 days (1). Unlike the
ketotic fat loss in starvation or insulin deficiency, in which the
fatty acids (FA)1 and
glycerol are released proportionately from the adipocytes, hyperleptinemic fat loss is unaccompanied by elevations in plasma free
fatty acid (FFA) levels or ketones or by ketonuria (2). One possible
explanation for the nonketotic fat loss is that the FAs are oxidized
inside the adipocytes. This idea has received further support from the
demonstration that the expression of two major enzymes of long chain FA
oxidation, acyl CoA oxidase (ACO), and carnitine palmitoyl
transferase-1 (CPT-1) are strikingly increased in the adipocytes of
hyperleptinemic rats during the disappearance of their fat (3, 4). This
finding implies that experimentally induced hyperleptinemia can convert
adipocytes from fat-storing cells into fat-burning cells.
Most workers in the field believe that leptin acts largely, if not
exclusively, via centers in the hypothalamus, suppressing appetite by
inhibiting orexic factors such as neuropeptide Y (5) and by increasing
thermogenesis via sympathetic innervation of brown adipose tissue (6).
It seemed possible, therefore, that the disappearance of the fat of
white adipocytes might be the result of leptin-induced, adrenergically
mediated activation of lipolysis. Yet there is evidence consistent with
the possibility that, at least at high concentrations, leptin can act
directly on tissues independently of hypothalamic mediation. First,
leptin receptors (OB-R), including the full-length isoform, OB-Rb, are expressed in white adipocytes (10). Second, leptin has been shown
in vitro to reduce the expression of lipogenic enzymes in preadipocytes (8) and to increase glycerol release from mature adipocytes (7). Third, if the fat depletion caused by hyperleptinemia is, in fact, caused by norepinephrine through stimulation of
sympathetic centers in the hypothalamus, it would be accompanied by a
concomitant increase in plasma FFA levels (9), which did not occur in
the hyperleptinemic rats. For these reasons, we suspected that the fat
loss of hyperleptinemia involved a novel type of leptin-mediated lipolysis that was independent of catecholamines. The following study
was designed to test this possibility.
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MATERIALS AND METHODS |
Adipocyte Isolation and Culture--
Isolation of adipocytes
from lean (+/+) and obese (fa/fa) Zucker rats was
performed as described previously (9). Briefly, minced epididymal fat
pads were digested at 37 °C for 2-3 h in a buffer containing type
II collagenase (1 mg/ml), albumin (3.5%), and glucose (0.55 mM). The digestion mixture was swirled and poured through
100-µm nylon mesh into 50-ml conical polypropylene tubes. Cells were
washed three times with Krebs-Ringer bicarbonate buffer (pH 7.4)
containing 5% albumin and cultured for 0, 3, 6 and 24 h at
37 °C in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum, antibiotics (penicillin and streptomycin) and with or
without recombinant leptin (kindly provided by Dr. Gayle Yamamoto of
Zymogenetics, Inc., Seattle, WA) or norepinephrine.
Reverse Transcriptase PCR--
mRNA was semiquantified by
reverse transcriptase-PCR. Total RNA from adipocytes was extracted
using TRIzol Reagent. After treating with RNase-DNase I, reverse
transcription was carried out using 1 µg of total RNA. First strand
cDNA was PCR-amplified with sequences specific for leptin
(5'-GGAGGAATCCCTGCTCCAGC-3' and 5'-CTTCTCCTGAGGATACCTGG-3'), ACO
(5'-GCCCTCAGCTATGGTATTAC-3' and 5'-AGGAACTGCTCTCACAATGC-3'), CPT-1
(5'-TATGTGAGGATGCTGCTTCC-3' and 5'-CTCGGAGAGCTAAGCTTGTC-3'), FAS
(5'-GGTTGATGGCTCACACACCT-3' and 5'-TCAACTCACTCGAGGCTCAG-3'), and
PPAR
(5'-AAGCCATCTTCACGATGCTG-3' and
5'-TCAGAGGTCCCTGAACA-GTG-3'). The conditions of PCR
were as follows: denaturation for 45 s at 92 °C, annealing for
45 s at 55 °C, and elongation for 1 min at 72 °C with 30 cycles. The PCR products were subjected to electrophoresis on 1.2%
agarose gel and were quantified by Southern blot analysis by means of
gene-specific 32P-labeled probes for leptin
(5'-CGGATACCGACTGCGTGTGTGAAATGTCAT-3'), ACO
(5'-GCCTGCACTTTCTTCAGCCATCTTCAACGA-3'), CPT-1
(5'-ACTCTGGTTGGAATCTGA-CTGGGTGGGATT-3'), FAS
(5'-AAGAAGCATATGGCTTCAGCTTCAG-CCTCA-3'), and
PPAR
(5'-ACTCGGTCTTCTTGATGACCTGCAC-GAGCT-3'). The
Molecular Imager (Bio-Rad) was used for quanti-fication. As a
control for RNA quality and quantity,
-actin mRNA was amplified.
Glycerol and FFA Assays--
Glycerol in the medium was measured
by the method of McGowen and co-workers (10). For glycerol assay, 200 µl of medium was deproteinated with 20 µl of 5 M
perchloric acid. After deproteination, samples were neutralized with 10 M KOH (pH 9.5) and placed on ice for 30 min. Samples were
centrifuged for 10 min at 4 °C to remove KClO4
precipitate, and glycerol was measured in the supernatant. To measure
FFA, 500 µl of medium was extracted with 500 µl of chloroform,
dried under N2, and measured using the method of Shimizu et al. (11).
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RESULTS |
Effect of Leptin on Leptin mRNA--
A dramatic early effect
of adenovirus-induced hyperleptinemia in vivo is the
disappearance of leptin mRNA from the adipocytes (4). To establish
the in vitro biologic activity of the recombinant leptin, we
cultured adipocytes in 20 ng/ml of recombinant leptin, which
approximates the levels in hyperleptinemic rats. Leptin mRNA had
declined significantly (p < 0.05) by 14, 39 and 50%
of normal in 3, 6, and 24 h, respectively (Fig.
1A). In adipocytes from
fa/fa rats, there was no effect on leptin mRNA (Fig.
1B). These results establish a direct action of leptin on
mature adipocytes.

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Fig. 1.
The effect of 20 ng/ml of recombinant leptin
(upper panels) and 100 nM norepinephrine
(lower panels) on leptin mRNA in isolated
adipocytes of (A and C) normal
+/+ and (B and D)
obese fa/fa ZDF rats. A representative blot is
displayed. *, p < 0.05 (n = 3).
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Norepinephrine at 100 nM concentration reduced leptin
mRNA by 30% (p < 0.05) in the adipocytes of
+/+ rats (Fig. 1C), confirming earlier
observations in 3T3-L1 cells (12). Norepinephrine did not reduce leptin
mRNA in the adipocytes of fa/fa (Fig.
1D).
Effect of Leptin on mRNA of Fatty Acid
Synthetase--
Adenovirus-induced hyperleptinemia causes
down-regulation of the mRNA of the lipogenic enzymes, acetyl CoA
carboxylase and FAS. To determine whether leptin can directly
down-regulate expression of the FAS gene, we measured its mRNA at
3, 6, and 24 h after culture of adipocytes in 20 ng/ml of
recombinant leptin. FAS expression was reduced at all time points
(p < 0.05) and at 24 h was only 10% of controls
(Fig. 2A). This was by far the
most dramatic of the direct effects of leptin observed in this study.
There was no effect on FAS expression in fa/fa adipocytes
(Fig. 2B). Norepinephrine at 100 nM
concentration had no effect on FAS mRNA of adipocytes from either
the wild-type (+/+) or fa/fa ZDF rats (Fig. 2,
C and D).

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Fig. 2.
The effect of 20 ng/ml of recombinant leptin
(upper panels) and 100 nM norepinephrine
(lower panels) on mRNA of fatty acid synthetase in
adipocytes isolated from (A and C)
normal +/+ and (B and
D) obese fa/fa ZDF rats. A
representative blot is displayed. *, p < 0.05 (n = 3).
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Effect of Leptin on mRNA of PPAR
, CPT-1, and ACO mRNA in
Adipocytes--
We had observed previously that adenovirus-induced
hyperleptinemia dramatically up-regulates the in vivo
expression of the enzymes of FA oxidation, of ACO and CPT-1 in
adipocytes and of their transcription factor PPAR
, whereas their
triacyl glycerol virtually disappears (4). To determine whether this
was a direct effect of hyperleptinemia upon the adipocytes, isolated
adipocytes of normal (+/+) ZDF rats were cultured in recombinant leptin
at a concentration of 20 ng/ml. After 6 h in culture PPAR
mRNA had increased 50% (p < 0.05), whereas CPT-1
and ACO mRNA increased ~2-fold compared with controls
(p < 0.05) (Fig.
3A). In similarly treated
adipocytes isolated from obese fa/fa ZDF rats, there was no
effect on the mRNA of any of the foregoing genes (Fig.
3B).

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Fig. 3.
The effects of 20 ng/ml of recombinant leptin
on mRNA of PPAR , ACO, and CPT-1 of
adipocytes isolated from normal +/+
(A) and obese fa/fa ZDF rats
(B). A representative blot is displayed. *,
p < 0.05 (n = 3).
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The presence of 100 nM norepinephrine alone had no effect
on PPAR
mRNA and caused only a minimal increase in ACO and CPT-1 mRNA (not significant) (data not shown). Norepinephrine could not,
therefore, have mediated the in vivo effects of leptin on the expression of these enzymes.
Comparative Lipolytic Effects of Leptin and
Norepinephrine--
The foregoing direct effects of leptin on the
expression of enzymes of fatty acid oxidation and synthesis were
consistent with the hypothesis that the nonketotic fat loss of
adenovirus-induced hyperleptinemia was the consequence of increased
oxidation of FA within adipocytes, coupled with a reduction in
lipogenesis. To determine whether the direct action of leptin involves
a novel type of lipolytic action that differs from that of
norepinephrine, we compared the release of glycerol and FFA from normal
rat adipocytes cultured for 6 h in 20 ng/ml of leptin, in 100 nM norepinephrine, or in buffer alone. As shown in Fig.
4A, norepinephrine and leptin each significantly increased glycerol release from adipocytes isolated
from +/+ normal ZDF rats (p < 0.05),
confirming the observations of Siegrist-Kaiser et al. (7).
However, whereas norepinephrine elicited the expected rise in FFA,
leptin did not increase FFA release. In adipocytes of fa/fa
rats, by contrast, leptin caused no increase in either glycerol or FA,
whereas norepinephrine elicited a robust increase in both (Fig.
4B).

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Fig. 4.
Comparative effects of 20 ng/ml of
recombinant leptin ( ) and 100 nM norepinephrine ( ) on
the release of glycerol and FFA from the isolated adipocytes of normal
+/+ (A) and obese fa/fa
ZDF rats (B). *, p < 0.05 versus controls ( ; n = 5).
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Dose-Response Characteristics of Leptin-induced Lipolysis--
The
foregoing effects of leptin on lipolysis were observed at the
unphysiologically high concentration of 20 ng/ml. To determine whether
the effect on lipolysis was operative at more physiologic levels of
leptin, a dose-response study was done (Fig.
5). Five ng/ml of leptin was the lowest
concentration that stimulated release of glycerol (p < 0.05). This concentration is at the upper end of the range of plasma
leptin levels of rodents and humans (13, 14). There was no effect of
leptin on glycerol release from fa/fa adipocytes at any of
the concentrations employed.

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Fig. 5.
Leptin dose-response curve for glycerol
release from isolated adipocytes of normal +/+ ( )
and fa/fa ( ) ZDF rats (n = 3). Incubation time was 24 h.
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DISCUSSION |
These results provide evidence for multiple direct actions of
leptin on mature adipocytes in vitro. Previously, leptin had been shown to reduce expression of acetyl CoA carboxylase and FAS in
3T3-L1 cells (8) and to increase glycerol release from normal
adipocytes (7). In the present study, the expression of CPT-1 was
increased ~2-fold above the base-line level by 20 ng/ml of leptin,
whereas that of the lipogenic enzyme, FAS, was lowered by ~90%. It
is possible that the up-regulation of ACO and CPT-1 mRNA was
mediated by the increase in expression of their transcription factor,
PPAR-
, which rose 50%. The mechanism by which leptin up-regulates
PPAR
is not known.
The changes in mRNA induced by recombinant leptin in isolated
adipocytes correspond qualitatively with all those observed previously
in vivo in adipocytes of rats with adenovirus-induced hyperleptinemia (4). Because norepinephrine failed to induce any of
these changes in the cultured adipocytes, it appears that they are the
consequences of direct action of leptin on adipocytes rather than of
norepinephrine. It should be stressed, however, that direct effects of
leptin on adipocytes were at a concentration at 20 ng/ml, which is far
above the physiologic levels in normal humans or rodents. A small but
significant increase in glycerol was observed in rat adipocytes in
response to 5 ng/ml (p < 0.05), which is in the upper
end of the physiologic range (13, 14). However, Siegrist-Kaiser
et al. (7) reported an effect at 1.8 ng/ml on glycerol
release, which is within the physiologic range. Irrespective of the
physiologic relevance of these direct effects of leptin upon
adipocytes, they do suggest that the pharmacologic strategy of reducing
adipocyte fat content by means of direct lipolytic actions of
supraphysiologic levels of leptin may be useful (4).
In the earlier reports (7, 15) indicating that leptin increases
glycerol release from rodent adipocytes, FFA had not been measured. The
finding here that leptin-induced glycerol release is unaccompanied by
FFA release explains the in vivo observation that fat loss
occurring during adenovirus-induced hyperleptinemia is unaccompanied by
a rise in plasma FFA, ketonemia, and ketonuria (2). This and the
up-regulation of ACO and CPT-1 support the idea that the FFA are
oxidized inside the adipocytes rather than exported to the liver for
oxidation to ketoacids. This may provide a valuable therapeutic
advantage for leptin treatment of massive obesity, because it permits
the rapid removal of fat without the ketoacidosis and hyperuricemia
that otherwise complicate rapid weight loss induced by diet restriction.
The mechanism of leptin-induced lipolysis remains to be elucidated.
Clearly it requires a functional leptin receptor, because it was
completely absent in adipocytes of fa/fa ZDF rats
with defective OB-R, in which norepinephrine elicited a relatively normal lipolytic response. Leptin signal is believed to be transduced via the STAT/JAK pathway (16). It will be of interest to determine whether leptin increases cAMP and whether, like norepinephrine, it
activates the hormone-sensitive lipase (17).