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Division of Endocrinology and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Barbara B. Kahn, M.D., Diabetes Unit, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, Massachusetts 02215. E-mail:bkahn{at}caregroup.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OBRb is highly expressed in the hypothalamus, the primary site where leptin is thought to act (23). Leptin has been shown to activate STAT3 in the hypothalamus presumably via a JAK kinase (16). Tyrosine-phosphorylated STAT is translocated to the nucleus where it is thought to bind to specific DNA sequences and activate genes important for energy homeostasis. No such activation was observed in other tissues in which the short form of the receptor predominates (16). However, other data suggest that leptin may exert direct effects at the level of gene expression or cellular function on nonhypothalamic target tissues including hematopoietic cells, T cells, the endocrine pancreas, the pituitary, the ovary, adipocytes, skeletal muscle, and hepatocytes either through the short or long forms of the receptor (24, 25, 26, 27, 28, 29, 30, 31, 32). Direct administration of leptin activates STAT1 in cultured brown adipocytes and in white adipose tissue explants and clearly these effects do not involve the hypothalamus (28). Therefore, it remains unresolved whether, and by what mechanisms, a component of the important metabolic effects of leptin could be exerted directly at the level of peripheral tissues, as opposed to indirectly though the central nervous system.
Because the most common alteration in energy balance, obesity, is tightly associated with insulin resistance, it has been proposed that leptin may play a role in carbohydrate metabolism and insulin action. This notion was supported by early investigations of the effect of exogenous leptin in ob/ob mice. Within hours of leptin administration, a marked decrease in both plasma insulin and glucose concentrations occurred, and this preceded any changes in food intake or body weight (2, 3, 33, 34, 35). With prolonged leptin administration, the decline in plasma insulin and glucose was greater in leptin-treated mice than in pair-fed control mice (4, 34). A recent study showed that leptin administration reversed severe insulin resistance and hyperglycemia in mice lacking white adipose tissue, whereas food restriction had no effect (36). Taken together, these data strongly support the hypothesis that leptin improves in vivo insulin action independent of its effect to decrease food intake.
Importantly, whether the effect of leptin on insulin action is exerted directly at the insulin target tissues or indirectly via the central nervous system is unknown. In addition, the underlying molecular mechanisms remain unknown, and studies are conflicting regarding the effect of leptin on insulin-stimulated signal transduction. Some data suggest that leptin can impair the early steps of insulin signaling including autophosphorylation of the insulin receptor and tyrosine phosphorylation of IRS-1 in certain cell types such as rat-1 fibroblasts and hepatocytes (32, 37). Other studies demonstrate that leptin can mimic effects of insulin such as stimulation of glucose transport and glycogen synthesis in C2C12 myotubes (38) and that these effects may be mediated by stimulation of PI 3-kinase, although unlike insulin, this does not involve IRS-1 (39). In isolated muscle or adipocytes, short-term incubation with leptin does not stimulate glucose transport or lipogenesis (40, 41). Thus, the relationship between leptin and insulin action on metabolism and the signaling pathways involved are currently unclear.
The present study was designed to investigate the rapid and potentially direct effects of leptin on signal transduction in peripheral tissues and to determine whether insulin and leptin share common intracellular signal transduction pathways. Here we show that 3 min after leptin injection iv in normal rats there is increased phosphorylation of STAT3 and STAT1 in adipose tissue and phosphorylation of MAPK in adipose tissue and liver. In addition, IRS-1-associated PI 3-kinase activity in adipose tissue and IRS-2-associated PI 3-kinase activity in liver are modestly increased in leptin-injected rats. Leptin intracerebroventricular (icv) injection does not elicit the phosphorylation of STAT3, STAT1, and MAPK this rapidly in either hypothalamus or adipose tissue. Furthermore, incubation of 3T3-L1 adipocytes or adipose tissue explants with leptin activates signaling directly. The signaling effects of leptin are not seen in db/db mice. Our data suggest that leptin has rapid effects to activate signaling pathways directly at the level of insulin sensitive tissues, and these effects overlap with, but are distinct from, those engaged by insulin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1) Peripheral administration study. Male Sprague Dawley rats, 6 wk of age, were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). The rats were fed standard chow (PMI Feeds, Inc., St. Louis, MO) and water ad libitum for 2 wk. They were housed under controlled temperature at 24 C and 12-h light, 12-h dark cycle with light from 0630 to 1830 h. On the day of the experiment, rats (8 weeks of age) were anesthetized by inhalation of methoxyflurane (Mallinckrodt Veterinary, Inc., Mundelein, IL) after an overnight fast. Thereafter, a bolus injection of insulin (10 U/kg), insulin (10 U/kg) plus leptin (1 mg/kg) or leptin (1 mg/kg) was administered through the jugular vein and 3 min or 15 min later gastrocnemius, liver, and epididymal fat tissue were rapidly removed, frozen in liquid nitrogen, and stored at -80 C until analysis. To investigate effects of lower doses of leptin (2 µg/kg, 10 µg/kg and 50 µg/kg), rats were injected iv, and the epididymal fat was harvested at 3 min as described above.
2) Intracerebroventricular administration study. Catheters were inserted under anesthesia (100 mg/kg ketamine hydrochloride and 6 mg/kg Xylazine, ip) into the lateral ventricle (1.0 mm posterior to bregma, 1.6 mm lateral to the midline, and 4.0 mm ventral to the surface of the dura) before rats were shipped from Taconic to our animal facility. The animals were treated with 20 mg/kg Keflin at the time of surgery and one daily injection of 20 mg/kg Keflin for 3 days immediately following the surgery. Animals were allowed to recover for at least 2 weeks before icv injection. Cannulation placement was confirmed by demonstration of increased thirst after administration of angiotensin (50 ng). Animals were handled on a daily basis for cannula maintenance. A bolus injection of leptin (3.5 µg/rat) was administered icv and 3 min later epididymal fat tissue and hypothalamus were rapidly removed as described previously (42).
3) db/db mice study. Male db/db mice, 12 weeks of age, were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were fed and housed as described above. After an overnight fast, mice (14 weeks of age) were injected with leptin (1 mg/kg) through the inferior cava vein and 3 min later epididymal fat tissue was rapidly removed.
Preparation of tissue lysates
Fifty milligrams of tissues were homogenized using a polytron at
half maximum speed (15,000 rpm) for 1 min on ice in 500 µl buffer A
(20 mM Tris pH 7.5, 5 mM EDTA, 10
mM
Na4P2O7,
100 mM NaF, 2 mM
Na3VO4) containing 1%
NP-40, 1 mM PMSF, 10 µg/ml aprotinin and 10 µg/ml
leupeptin. Tissue lysates were solubilized by continuous stirring for
1 h at 4 C, and centrifuged for 10 min at 14,000 x
g. The supernatants were stored at -80 C until
analysis.
Determinations of STAT3, STAT1, MAPK, and Akt
phosphorylation
One hundred to 300 µg of tissue lysate protein per lane was
resolved by SDS-PAGE (8% gel) and transferred to nitrocellulose
membranes (Schleicher & Schuell, Inc., Keene, NH). The
nitrocellulose membranes were blocked with 5% nonfat dry milk for
1 h at room temperature, incubated with either phospho-specific
STAT3 (Tyr705) polyclonal antibody (New England Biolabs, Inc., Beverly, MA), phospho-specific STAT1 (Tyr701) polyclonal
antibody (Upstate Biotechnology, Inc., Lake Placid,
NY), active-MAPK polyclonal antibody (Promega Corp.,
Madison, WI) with dually phosphorylated The/Glu region (pTEpY) derived
from the active form of MAP kinase enzymes or phospho-specific Akt
(Ser473) polyclonal antibody (New England Biolabs, Inc.)
in 1% nonfat dry milk overnight at 4 C. The membranes were washed with
Tris-buffered saline (TBS) containing 0.05% Tween 20 for 30 min,
incubated with horseradish peroxidase secondary antibody (1:2000
dilution; Amersham Pharmacia Biotech, Arlington Heights,
IL) for 1 h and washed with TBS containing 0.05% Tween 20 for 30
min. The bands were visualized using the enhanced chemiluminescence
system (Amersham Pharmacia Biotech) and quantified by
densitometry (Molecular Dynamics, Inc., Sunnyvale,
CA).
Determination of PI 3-kinase activity
Tissue lysates (0.5–1.0 mg protein) were subjected to
immunoprecipitation with 5 µl IRS-1 polyclonal antibody, 5 µl IRS-2
monoclonal antibody (gifts from Dr. Morris White, Joslin Diabetes
Center) and 3 µg of a polyclonal antibody against the p85 subunit of
PI 3-kinase (Upstate Biotechnology, Inc.) coupled to
protein A-Sepharose (Sigma, St. Louis, MO). The immune
complex was washed as described (43) and resuspended in 50 ml Tris-NaCl
buffer (10 mM Tris, pH 7.5; 100 mM NaCl; 1
mM EDTA; 100 mM
Na3VO4). PI 3-kinase
activity was measured as previously reported (43). The radioactivity in
the spots corresponding to PI 3-phosphate was quantitated using
PhosphorImager and Image Quant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Determination of Akt/PKB activity
Tissue lysates (500 µg protein) were subjected to
immunoprecipitation for 4 h at 4 C with 4 µg of a polyclonal
antibody (Upstate Biotechnology, Inc.), which recognizes
both Akt 1 and Akt 2, coupled to protein G-Sepharose beads
(Amersham Pharmacia Biotechnology, Piscataway, NJ).
Immune pellets were washed three times with buffer A containing 1%
NP-40 and 2 times with 50 mM Tris, pH 7.5; 10
mM MgCl2; and 1 mM DTT.
The beads were resuspended in 50 µl of kinase mixture (50
mM Tris, pH 7.5; 10 mM
MgCl2; 1 mM DTT; 5 µM
ATP; 1 µM protein kinase inhibitor; 30 µM
Crosstide (Upstate Biotechnology, Inc.); and 2
µCi [
-32P]ATP) (44) and incubated at 30 C
for 30 min. Forty microliters of samples were spotted onto
phosphocellulose p81 paper (Whatman, Clifton, NJ), and
washed four times with 75 mM orthophosphoric acid and 1
time with acetone. Radioactivity of the paper was determined by
scintillation counting (45).
Determination of plasma leptin levels
Plasma leptin levels were determined by RIA (Linco Research, Inc., St. Louis, MO).
Adipose tissue explants and culture of 3T3-L1 adipocytes
Epididymal fat pads from Sprague Dawley rats in the postprandial
state were removed and minced into approximately 1-mm diameter pieces.
These adipose tissue explants were preincubated at 37 C for 30 min with
Krebs-Ringer-HEPES buffer (20 mM, pH 7.4) with 2.5% BSA
and 200 nM adenosine, and without or with 100
nM leptin for 0, 3, 15, or 45 min. Explants were
solubilized for analysis of phosphorylation of signaling proteins.
3T3-L1 preadipocytes were grown in DMEM with 10% FCS, 50 U/ml penicillin, and 50 µg streptomycin at 37 C, 5% CO2. Two days after confluence, differentiation was induced with 0.5 mM 3-isobutyl-1-methylxanthine, 0.25 µM dexamethasone (Sigma, St. Louis, MO), and 1 µg/ml insulin (Eli Lilly & Co., Indianapolis, IN) for 3 days. Cells were used for experiments 10–12 days after induction of differentiation. After overnight starvation, cells were stimulated with 100 nM leptin for 5 min, harvested, and solubilized for analysis of phosphorylation of signaling proteins.
Statistical analysis
Data are presented as mean ± SEM. Statistical
analyses were performed using the Stat View program (Abacus Concepts,
Inc., Berkeley, CA). Statistical significance among the groups was
tested with factorial ANOVA or unpaired Student’s t
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).).
Insulin also stimulated STAT3 tyrosine phosphorylation 6.0-fold in
adipose tissue, and tended to increase it 1.4-fold in liver and
1.3-fold in muscle (Fig. 1A).
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). The effect of leptin to stimulate the
phosphorylation of STAT1 in adipose tissue tended to be additive with
the effect of insulin (P < 0.058). However, no effects
on STAT1 phosphorylation in liver or muscle were seen after injection
of leptin or insulin.
Leptin increases phosphorylation of MAPK in adipose tissue and
liver (Fig. 2)
Cytokines and growth factors transduce signals to the nucleus by
activating a cascade involving Ras and MAPK (46). It has previously
been shown that OBRb can also lead to activation of the MAPK pathway
(17). We measured MAPK phosphorylation after leptin or insulin
administration in vivo for 3 min. Leptin resulted in rapid
phosphorylation of MAPK 3.2-fold in adipose tissue and 3.8-fold in
liver with no effect in muscle (Fig. 2). Insulin increased MAPK
phosphorylation 5.0-fold in adipose tissue, 6.8-fold in liver, and
2.5-fold in muscle. The phosphorylation of MAPK in adipose tissue by
leptin and insulin was additive. No additivity was seen in liver
or muscle.
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)). In contrast, leptin stimulated IRS-1-associated PI 3-kinase
activity 2.0-fold in adipose tissue (P < 0.01) and
tended to increase it 1.3-fold in liver and 1.5-fold in muscle. No
additive effect was observed between leptin and insulin (Fig. 3A). PI
3-kinase activity was also measured in anti-IRS-2 immunoprecipitates
from tissue lysates (Fig. 3B). At 3 min after insulin injection,
IRS-2-associated PI 3kinase activity was stimulated 2.4- to
4.8-fold above basal in adipose tissue, liver, and muscle. In
leptin-injected rats, IRS-2-associated PI 3-kinase activity increased
1.7-fold in liver (P < 0.01) but was reduced 28% in
muscle (P < 0.05). There were no significant
differences in insulin-plus-leptin-injected rats compared with insulin
alone (Fig. 3B).
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). Leptin
tended to increase p85-associated PI 3-kinase activity in adipose
tissue and liver, although the variability in response made it not
reach statistical significance. Because of the possibility that PI
3-kinase activity associated with other phosphoproteins could be
involved in the leptin signaling pathway, we also measured IRS-3-,
STAT3-, JAK2-, and Gab-1-associated PI 3-kinase activities in insulin
sensitive tissues. There were no detectable effects of leptin
administration in vivo on PI 3-kinase activity associated
with these proteins (not shown).
Leptin does not affect Akt phosphorylation and activity (Fig. 4)
Insulin administration rapidly stimulated Akt phosphorylation by
5.9- to 12.2-fold in adipose tissue, liver, and muscle (Fig. 4A). In
parallel, Akt activity measured by an immune complex assay, was
increased 4.0- to 12.8-fold in the same tissues after insulin injection
(Fig. 4B). Akt phosphorylation and activation in liver and muscle were
unaltered by leptin injection, although there was a tendency for a
slight increase in activity in adipose tissue. In muscle, Akt activity
was decreased 20% (P < 0.01) in insulin plus leptin
injected rats compared with rats injected with insulin alone (Fig. 4B).
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)
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)). Insulin
and leptin together increased IRS-1-associated PI 3-kinase activity
above the effects of insulin alone by 69% in liver and 46% in muscle,
but there was no additive effect in adipose tissue. Leptin alone had no
effect on IRS-1-associated PI 3-kinase activity in any tissue. Similar
to the 3 min results (Fig. 1), at 15 min STAT3 phosphorylation in
response to leptin or insulin is sustained in adipose tissue, but there
is no significant effect of either hormone on STAT3 in liver. The
phosphorylation of MAPK in adipose tissue is increased approximately
2.0-fold with insulin stimulation and approximately 3.0-fold with
leptin. The effect of insulin and leptin is similar to leptin alone.
The effects of insulin and leptin on MAPK phosphorylation in muscle and
liver at 3 min after injection are transient and are no longer present
at 15 min. Our data show an evolution over time of the effects of
leptin on signaling with some additivity with insulin in a
tissue-specific fashion in insulin-sensitive tissues.
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)). This dose suppresses food
intake (47) and maximally activates STAT3 phosphorylation in
hypothalamus 30 min after injection (El-Haschimi, K., and
J. S. Flier, unpublished data). There was no effect of a 3-min
exposure to icv leptin on phosphorylation of STAT1, STAT3, or MAPK in
the hypothalamus. There was also no consistent effect on STAT1, STAT3,
or MAPK phosphorylation in adipose tissue. For adipose tissue STAT3,
the quantitation was: saline control 100 ± 14 (n = 6) and
leptin 110 ± 16% of control (n = 8, p = NS). This
indicates that the rapid effects that we see with iv administration of
leptin on phosphorylation of STAT1, STAT3, and MAPK in adipose tissue
(Figs. 1 and 2) could be direct and are not mediated through the CNS.
While clearly activation of STAT3 occurs in hypothalamus with longer
exposure to leptin (16), our data indicate that leptin may also have
rapid effects to activate signaling pathways directly at the level of
peripheral tissues before effects occur that are mediated by the
hypothalamus.
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)
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)). This
activation was sustained at 1.8-fold after 45-min exposure to
leptin (P < 0.01). There was no change in the
basal level of MAPK phosphorylation over this time as shown in the
first two lanes of the autoradiogram at the top of Fig. 7A. Figure 7B
shows that, in 3T3-L1 adipocytes, leptin rapidly (5 min) induced
tyrosine phosphorylation of STAT3 3.0-fold and of MAPK 2.7-fold
(P < 0.05). These results indicate that leptin can
activate signaling directly in adipocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we investigated the effects of leptin on signal
transduction in insulin-sensitive tissues in vivo, with
particular focus on whether leptin has the capacity to signal directly
at the level of peripheral tissues, as opposed to indirectly via the
central nervous system. We observed that, 3 min after iv injection of
leptin, phosphorylation of STAT3 and STAT1 in adipose tissue and
phosphorylation of MAPK in adipose tissue and liver are significantly
increased, although the activity of PI 3-kinase in adipose tissue and
liver is only slightly increased. Importantly, 3 min after icv leptin
injection we do not see activation of STAT3, STAT1, or MAPK in the
hypothalamus or adipose tissue, suggesting that at this early time
point, leptin can stimulate signaling directly at the level of
peripheral tissues. Further support for a direct effect is the fact
that incubation of white adipose tissue explants with leptin stimulates
MAPK phosphorylation (Fig. 9). Because these explants contain cell
types other than adipocytes, we also incubated 3T3-L1 adipocytes with
leptin and demonstrated rapid activation of MAPK and STAT3
phosphorylation (Fig. 9). The rapid signaling effects of iv leptin
appear to be mediated by the long-form leptin receptor because they are
not seen in adipose tissue from db/db mice, which have only
short-form leptin receptors (Fig. 8). Thus, our data suggest that
leptin can rapidly activate signaling pathways directly in insulin
sensitive tissues through the OBRb.
Originally, it was thought that the hypothalamus was the only tissue expressing OBRb. However, recent evidence at the level of messenger RNA expression and cellular function suggest that peripheral organs including adipose tissue also express OBRb (11, 25, 27, 28). The OBRa, unlike OBRb, appears incapable of activating STATs, although it can mediate tyrosine phosphorylation of JAK2 (17). Thus, our data showing leptin-induced STAT3 phosphorylation in adipose tissue and cultured adipocytes (as well as the lack of leptin effect in db/db adipose tissue) support the notion that adipocytes express OBRb. These results are in agreement with a recent observation that leptin stimulates STAT1 gel shift in brown and white adipose tissue of normal rats but not Zucker fa/fa rats (28).
A previous in vivo study implicated only STAT3 in hypothalamic leptin signaling, whereas experiments involving transfected cells provide evidence for the activation of other STAT isoforms, depending on the cellular model (11, 13, 15, 16, 53). The nature of the STAT isoforms required for transducing the leptin signal is still controversial and may depend on the cellular context and the concomitant presence of other stimuli in vivo. Here we show that the tyrosine phosphorylation not only of STAT3, but also of STAT1, is robustly activated in white adipose tissue by leptin administration iv. These results agree with a recent study showing that leptin administration iv induces STAT1 gel shift in nuclear extracts of adipose tissue (28). The current study is the first demonstration of leptin-induced STAT3 activation in adipose tissue.
To further distinguish whether the effects of leptin on STAT3, STAT1, and MAPK phosphorylation are direct or are mediated via the CNS, we examined leptin signaling in the hypothalamus and adipose tissue at the same early time point after icv leptin administration in vivo. There was no activation of STAT3 or MAPK in the hypothalamus or in white adipose tissue at 3 min after icv administration of leptin. This is in contrast to the activation of these pathways that we observe in adipose tissue after iv administration of leptin. We also performed preliminary studies in which we created unilateral surgical sympathectomy of white adipose tissue in normal rats. One week later, activation of leptin signaling in vivo was preserved in sympathectomized fat (data not shown). This further supports the evidence that the observed early effects of leptin to activate signaling events in peripheral tissues are exerted directly at the level of the target tissue. These data agree with the findings of Siegrist-Kaiser et al. (28) that STAT1 was activated in BAT only after iv injection of leptin (90 min) and not after icv injection.
Although leptin has an insulin-sensitizing effect, which is evident
from the rapid reduction of glucose and insulin levels in
leptin-deficient, insulin-resistant ob/ob mice after leptin
administration (4) and the enhanced insulin-stimulated glucose disposal
in normal rats infused with leptin (54), in the present study we find
that leptin and insulin elicit overlapping but distinct signaling
pathways. Notably, iv injection of leptin results in only a small
activation of IRS-1-associated PI 3-kinase activity in adipose tissue
and IRS-2-associated PI 3-kinase activity in liver and no activation of
Akt in adipose tissue, muscle or liver compared with the large effects
of insulin on these signaling steps. Leptin tends to increase
p85-associated PI 3-kinase activity in adipose tissue and liver and
STAT3 phosphorylation in liver. The effects of leptin and insulin on
MAPK activation are additive suggesting that the two hormones may
affect MAPK via distinct upstream signals. By 15 min after bolus
injection of leptin or insulin, the effect of insulin on MAP kinase
activation in adipose tissue is waning (
2-fold above control),
whereas the effect of leptin is sustained at approximately 3-fold above
control (Fig. 6). Hence, the kinetics of the effects of leptin and
insulin also appear to differ and the effects of leptin may be more
sustained. We studied primarily the 3-min time point when major
pathways in insulin signaling approach maximal levels to determine
whether leptin mimics or modifies insulin-induced signal
transduction.
Interestingly, the marked effect of leptin iv on MAPK phosphorylation occurs with minimal activation of STATs in liver. This may be explained by recent data indicating that phosphorylation of different tyrosyl residues may mediate leptin’s effects on the MAPK and STAT pathways (Myers, M., Joslin Diabetes Center, Boston, MA, personal communication). There may be tissue specific factors such as specific phosphatases which differentially dephosphorylate specific residues in the leptin receptor. Alternatively, some leptin signaling may occur though the short form of the receptor (17).
Further evidence for differences in the signaling characteristics of the two hormones are seen in the STAT effects. Although both leptin and insulin stimulate tyrosine phosphorylation of STAT3, leptin fails to serine phosphorylate STAT3 (YBK and BBK unpublished data). In contrast, insulin also serine phosphorylates STAT3 in adipocytes (48). Because tyrosine phosphorylation of STAT3 is necessary for dimerization and DNA binding and serine phosphorylation enhances the efficiency of transcription (55, 56), this could have implications for the efficiency of leptin to activate transcription of certain genes. Taken together, these observations indicate that while leptin and insulin activate some of the same intracellular signaling events, the pathways also diverge.
The implications of the rapid leptin-induced signaling in peripheral tissues for the metabolic actions of leptin remains a critical question and data to date are conflicting. Observations has been made that leptin could stimulate glucose transport in cultured muscle cells (38) and inhibit insulin-stimulated glucose transport in rat adipocytes (57). However, other data with varying lengths of leptin exposure in either cultured muscle or adipose cell lines or primary muscle and adipocytes show no effects (40, 41). In vivo treatment of normal rats with leptin for 5 h or 5 days results in increased glucose uptake in skeletal muscle and brown adipose tissue (BAT) and not in white adipose tissue (58, 59). Denervation blocks the effect in glycolytic but not in oxidative muscle, indicating that leptin may have both direct effects and indirect effects mediated by the CNS and that the nature of leptin’s effects may be tissue specific and muscle fiber type specific. Furthermore, leptin’s effects on glucose uptake in vivo may be secondary to other alterations such as changes in gene expression or fatty acid metabolism.
Leptin has been shown to have direct effects on the expression of genes involved in metabolism in adipocytes, pancreatic islets, and liver. Leptin induces expression of malic enzyme and lipoprotein lipase in cultured brown adipocytes (28) and blunts the expression and activity of acetyl CoA carboxylase, the rate limiting step in long-chain fatty acid synthesis, in a cultured adipocyte cell line (30). The latter observation coupled with the effects of leptin to directly regulate multiple genes involved in fatty acid metabolism in islet cells (60) suggest that a key biologic consequence of leptin-induced signaling may be enhancement of fatty acid oxidation via regulation of gene expression through the STAT and MAPK pathways. In support of this, leptin has been shown to enhance fatty acid oxidation and inhibit fatty acid esterification directly in muscle in vitro (29). This effect could result in increased insulin sensitivity because increased triglyceride stores in muscle are associated with insulin resistance (61).
Leptin also has an autocrine effect on its own secretion from white adipocytes (62). Studies will need to determine whether the activation of STATs or MAPK is involved in this effect. One of the few other direct biologic effects of leptin that have been demonstrated in insulin target tissues is the stimulation of lipolysis in adipose explants (28). This effect is opposite to that of insulin, again underscoring the presence of both similarities and differences in the actions of leptin and insulin. The hepatic effects of leptin administration are complicated because leptin enhances the ability of insulin to inhibit hepatic glucose production but at the same time increases gluconeogenesis and expression of PEPCK and glucose 6-phosphatase (63), which are suppressed by insulin. While these effects appear to be mediated through CNS (63), similar alterations in gene expression have been shown in a liver cell line (32), indicating that there may be both central and local actions of leptin on hepatic glucose metabolism.
Although our data show leptin directly activates phosphorylation of STAT3 and MAPK in adipose tissue, the question remained as to whether these effects are through the leptin receptor. To date, leptin has not been shown to bind to any receptors other than the leptin receptor subtypes. In addition, the leptin receptor has been shown to homodimerize but not to heterodimerize (13, 15). However, to control for the possibility that some effects of leptin could be through another receptor, we performed the same experiments in db/db mice that have a mutation in the leptin receptor gene resulting in absence of the long form of the receptor. As expected, we could not see activation of signaling in response to leptin in adipose tissue from db/db mouse. This result suggests that the leptin-induced signaling that we observe in peripheral tissues is through the long-form leptin receptor.
In conclusion, we have provided evidence for rapid direct effects of leptin administration in vivo on intracellular signaling pathways in insulin target tissues. Our data suggest that the insulin-sensitizing effects of leptin involve convergence or synergism between distinct but overlapping leptin-activated and insulin-activated signal transduction pathways.
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Acknowledgments |
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Footnotes |
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2 Supported by Uehara Memorial Foundation Research Fellowship and a
mentor based fellowship from the American Diabetes Association.
Received October 19, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 D. M. Penn, L. C. Jordan, E. W. Kelso, J. E. Davenport, and R. B. S. Harris Effects of central or peripheral leptin administration on norepinephrine turnover in defined fat depots Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2006; 291(6): R1613 - R1621. [Abstract] [Full Text] [PDF]
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 S. Shetty, E. R.M. Eckhardt, S. R. Post, and D. R. van der Westhuyzen Phosphatidylinositol-3-Kinase Regulates Scavenger Receptor Class B Type I Subcellular Localization and Selective Lipid Uptake in Hepatocytes Arterioscler Thromb Vasc Biol, September 1, 2006; 26(9): 2125 - 2131. [Abstract] [Full Text] [PDF]
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J. H. Lee, J. L. Chan, E. Sourlas, V. Raptopoulos, and C. S. Mantzoros Recombinant Methionyl Human Leptin Therapy in Replacement Doses Improves Insulin Resistance and Metabolic Profile in Patients with Lipoatrophy and Metabolic Syndrome Induced by the Highly Active Antiretroviral Therapy J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2605 - 2611. [Abstract] [Full Text] [PDF]
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 D. Lavens, T. Montoye, J. Piessevaux, L. Zabeau, J. Vandekerckhove, K. Gevaert, W. Becker, S. Eyckerman, and J. Tavernier A complex interaction pattern of CIS and SOCS2 with the leptin receptor J. Cell Sci., June 1, 2006; 119(11): 2214 - 2224. [Abstract] [Full Text] [PDF]
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W. Huang, N. Dedousis, A. Bandi, G. D. Lopaschuk, and R. M. O'Doherty Liver Triglyceride Secretion and Lipid Oxidative Metabolism Are Rapidly Altered by Leptin in Vivo Endocrinology, March 1, 2006; 147(3): 1480 - 1487. [Abstract] [Full Text] [PDF]
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M. Gartsbein, A. Alt, K. Hashimoto, K. Nakajima, T. Kuroki, and T. Tennenbaum The role of protein kinase C {delta} activation and STAT3 Ser727 phosphorylation in insulin-induced keratinocyte proliferation J. Cell Sci., February 1, 2006; 119(3): 470 - 481. [Abstract] [Full Text] [PDF]
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 N. Mehebik, A.-M. Jaubert, D. Sabourault, Y. Giudicelli, and C. Ribiere Leptin-induced nitric oxide production in white adipocytes is mediated through PKA and MAP kinase activation Am J Physiol Cell Physiol, August 1, 2005; 289(2): C379 - C387. [Abstract] [Full Text] [PDF]
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C. R. Rooks, D. M. Penn, E. Kelso, R. R. Bowers, T. J. Bartness, and R. B. S. Harris Sympathetic denervation does not prevent a reduction in fat pad size of rats or mice treated with peripherally administered leptin Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2005; 289(1): R92 - R102. [Abstract] [Full Text] [PDF]
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 J. W. Mastaitis, E. Wurmbach, H. Cheng, S. C. Sealfon, and C. V. Mobbs Acute Induction of Gene Expression in Brain and Liver by Insulin-Induced Hypoglycemia Diabetes, April 1, 2005; 54(4): 952 - 958. [Abstract] [Full Text] [PDF]
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J. L. Chan, S. J. Moschos, J. Bullen, K. Heist, X. Li, Y.-B. Kim, B. B. Kahn, and C. S. Mantzoros Recombinant Methionyl Human Leptin Administration Activates Signal Transducer and Activator of Transcription 3 Signaling in Peripheral Blood Mononuclear Cells in Vivo and Regulates Soluble Tumor Necrosis Factor-{alpha} Receptor Levels in Humans with Relative Leptin Deficiency J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1625 - 1631. [Abstract] [Full Text] [PDF]
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R. Gutierrez-Juarez, S. Obici, and L. Rossetti Melanocortin-independent Effects of Leptin on Hepatic Glucose Fluxes J. Biol. Chem., November 26, 2004; 279(48): 49704 - 49715. [Abstract] [Full Text] [PDF]
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C. Duan, M. Li, and L. Rui SH2-B Promotes Insulin Receptor Substrate 1 (IRS1)- and IRS2-mediated Activation of the Phosphatidylinositol 3-Kinase Pathway in Response to Leptin J. Biol. Chem., October 15, 2004; 279(42): 43684 - 43691. [Abstract] [Full Text] [PDF]
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N. T. Lam, J. T. Lewis, A. T. Cheung, C. T. Luk, J. Tse, J. Wang, M. Bryer-Ash, J. K. Kolls, and T. J. Kieffer Leptin Increases Hepatic Insulin Sensitivity and Protein Tyrosine Phosphatase 1B Expression Mol. Endocrinol., June 1, 2004; 18(6): 1333 - 1345. [Abstract] [Full Text] [PDF]
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W. Huang, N. Dedousis, B. A. Bhatt, and R. M. O'Doherty Impaired Activation of Phosphatidylinositol 3-Kinase by Leptin Is a Novel Mechanism of Hepatic Leptin Resistance in Diet-induced Obesity J. Biol. Chem., May 21, 2004; 279(21): 21695 - 21700. [Abstract] [Full Text] [PDF]
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G. S. Fraley, J. M. Scarlett, I. Shimada, D. N. Teklemichael, B. V. Acohido, D. K. Clifton, and R. A. Steiner Effects of Diabetes and Insulin on the Expression of Galanin-Like Peptide in the Hypothalamus of the Rat Diabetes, May 1, 2004; 53(5): 1237 - 1242. [Abstract] [Full Text] [PDF]
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 K. Tsuda, I. Nishio, C. Vecchione, and G. Lembo Insulin, Leptin, and Membrane Microviscosity in Blood Pressure Regulation * Response: Insulin-Leptin Interplay May Differ Among Tissues Hypertension, April 1, 2004; 43(4): e15 - e16. [Full Text] [PDF]
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Y.-H. Yu and H. Zhu Chronological changes in metabolism and functions of cultured adipocytes: a hypothesis for cell aging in mature adipocytes Am J Physiol Endocrinol Metab, March 1, 2004; 286(3): E402 - E410. [Abstract] [Full Text]
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 Y. Tabe, M. Konopleva, M. F. Munsell, F. C. Marini, C. Zompetta, T. McQueen, T. Tsao, S. Zhao, S. Pierce, J. Igari, et al. PML-RAR{alpha} is associated with leptin-receptor induction: the role of mesenchymal stem cell-derived adipocytes in APL cell survival Blood, March 1, 2004; 103(5): 1815 - 1822. [Abstract] [Full Text] [PDF]
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 C. Bjorbaek and B. B. Kahn Leptin Signaling in the Central Nervous System and the Periphery Recent Prog. Horm. Res., January 1, 2004; 59(1): 305 - 331. [Abstract] [Full Text]
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 M. J. Roth, D. J. Baer, P. S. Albert, T. W. Castonguay, J. F. Dorgan, S. M. Dawsey, E. D. Brown, T. J. Hartman, W. S. Campbell, C. A. Giffen, et al. Relationship Between Serum Leptin Levels and Alcohol Consumption in a Controlled Feeding and Alcohol Ingestion Study J Natl Cancer Inst, November 19, 2003; 95(22): 1722 - 1725. [Abstract] [Full Text] [PDF]
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J.-N. Huan, J. Li, Y. Han, K. Chen, N. Wu, and A. Z. Zhao Adipocyte-selective Reduction of the Leptin Receptors Induced by Antisense RNA Leads to Increased Adiposity, Dyslipidemia, and Insulin Resistance J. Biol. Chem., November 14, 2003; 278(46): 45638 - 45650. [Abstract] [Full Text] [PDF]
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 V. Y Polotsky, J. Li, N. M Punjabi, A. E Rubin, P. L Smith, A. R Schwartz, and C. P O'Donnell Intermittent Hypoxia Increases Insulin Resistance in Genetically Obese Mice J. Physiol., October 1, 2003; 552(1): 253 - 264. [Abstract] [Full Text] [PDF]
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C. Vecchione, A. Aretini, A. Maffei, G. Marino, G. Selvetella, R. Poulet, V. Trimarco, G. Frati, and G. Lembo Cooperation Between Insulin and Leptin in the Modulation of Vascular Tone Hypertension, August 1, 2003; 42(2): 166 - 170. [Abstract] [Full Text] [PDF]
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M. K. S. Leow, C. L. Addy, and C. S. Mantzoros Human Immunodeficiency Virus/Highly Active Antiretroviral Therapy-Associated Metabolic Syndrome: Clinical Presentation, Pathophysiology, and Therapeutic Strategies J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 1961 - 1976. [Full Text] [PDF]
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 K. D. Niswender, B. Gallis, J. E. Blevins, M. A. Corson, M. W. Schwartz, and D. G. Baskin Immunocytochemical Detection of Phosphatidylinositol 3-kinase Activation by Insulin and Leptin J. Histochem. Cytochem., March 1, 2003; 51(3): 275 - 283. [Abstract] [Full Text] [PDF]
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R. B.S. Harris, T. D. Mitchell, and S. Hebert Leptin-Induced Changes in Body Composition in High Fat-Fed Mice Experimental Biology and Medicine, January 1, 2003; 228(1): 24 - 32. [Abstract] [Full Text] [PDF]
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M. Z. Strowski, M. Kohler, H. Y. Chen, M. E. Trumbauer, Z. Li, D. Szalkowski, S. Gopal-Truter, J. K. Fisher, J. M. Schaeffer, A. D. Blake, et al. Somatostatin Receptor Subtype 5 Regulates Insulin Secretion and Glucose Homeostasis Mol. Endocrinol., January 1, 2003; 17(1): 93 - 106. [Abstract] [Full Text] [PDF]
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H. Ge, L. Huang, T. Pourbahrami, and C. Li Generation of Soluble Leptin Receptor by Ectodomain Shedding of Membrane-spanning Receptors in Vitro and in Vivo J. Biol. Chem., November 22, 2002; 277(48): 45898 - 45903. [Abstract] [Full Text] [PDF]
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A. M. Madiehe, S. Hebert, T. D. Mitchell, and R. B. S. Harris Strain-Dependent Stimulation of Growth in Leptin-Treated Obese db/db Mice Endocrinology, October 1, 2002; 143(10): 3875 - 3883. [Abstract] [Full Text] [PDF]
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L. L. Atkinson, M. A. Fischer, and G. D. Lopaschuk Leptin Activates Cardiac Fatty Acid Oxidation Independent of Changes in the AMP-activated Protein Kinase-Acetyl-CoA Carboxylase-Malonyl-CoA Axis J. Biol. Chem., August 9, 2002; 277(33): 29424 - 29430. [Abstract] [Full Text] [PDF]
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R. B. CEDDIA, H. A. KOISTINEN, J. R. ZIERATH, and G. SWEENEY Analysis of paradoxical observations on the association between leptin and insulin resistance FASEB J, August 1, 2002; 16(10): 1163 - 1176. [Abstract] [Full Text] [PDF]
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 P. Chavatte-Palmer, Y. Heyman, C. Richard, P. Monget, D. LeBourhis, G. Kann, Y. Chilliard, X. Vignon, and J.P. Renard Clinical, Hormonal, and Hematologic Characteristics of Bovine Calves Derived from Nuclei from Somatic Cells Biol Reprod, June 1, 2002; 66(6): 1596 - 1603. [Abstract] [Full Text] [PDF]
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K. Kawamura, N. Sato, J. Fukuda, H. Kodama, J. Kumagai, H. Tanikawa, A. Nakamura, and T. Tanaka Leptin Promotes the Development of Mouse Preimplantation Embryos in Vitro Endocrinology, May 1, 2002; 143(5): 1922 - 1931. [Abstract] [Full Text] [PDF]
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G. Matarese, V. Sanna, R. I. Lechler, N. Sarvetnick, S. Fontana, S. Zappacosta, and A. La Cava Leptin Accelerates Autoimmune Diabetes in Female NOD Mice Diabetes, May 1, 2002; 51(5): 1356 - 1361. [Abstract] [Full Text] [PDF]
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C.-Y. Lin, D. A. Higginbotham, R. L. Judd, and B. D. White Central leptin increases insulin sensitivity in streptozotocin-induced diabetic rats Am J Physiol Endocrinol Metab, May 1, 2002; 282(5): E1084 - E1091. [Abstract] [Full Text] [PDF]
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F. Machinal-Quelin, M. N. Dieudonne, M. C. Leneveu, R. Pecquery, and Y. Giudicelli Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways Am J Physiol Cell Physiol, April 1, 2002; 282(4): C853 - C863. [Abstract] [Full Text] [PDF]
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C. Vecchione, A. Maffei, S. Colella, A. Aretini, R. Poulet, G. Frati, M. T. Gentile, L. Fratta, V. Trimarco, B. Trimarco, et al. Leptin Effect on Endothelial Nitric Oxide Is Mediated Through Akt-Endothelial Nitric Oxide Synthase Phosphorylation Pathway Diabetes, January 1, 2002; 51(1): 168 - 173. [Abstract] [Full Text] [PDF]
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F. A. Khan, P. B. Goforth, M. Zhang, and L. S. Satin Insulin Activates ATP-Sensitive K+ Channels in Pancreatic {beta}-Cells Through a Phosphatidylinositol 3-Kinase-Dependent Pathway Diabetes, October 1, 2001; 50(10): 2192 - 2198. [Abstract] [Full Text]
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H. Yamashita, J. Shao, T. Ishizuka, P. J. Klepcyk, P. Muhlenkamp, L. Qiao, N. Hoggard, and J. E. Friedman Leptin Administration Prevents Spontaneous Gestational Diabetes in Heterozygous Leprdb/+ Mice: Effects on Placental Leptin and Fetal Growth Endocrinology, July 1, 2001; 142(7): 2888 - 2897. [Abstract] [Full Text] [PDF]
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F. LARCHER, M. DEL RIO, F. SERRANO, J. C. SEGOVIA, A. RAMIREZ, A. MEANA, A. PAGE, J. L. ABAD, M. A. GONZALEZ, J. BUEREN, et al. A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts FASEB J, July 1, 2001; 15(9): 1529 - 1538. [Abstract] [Full Text] [PDF]
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G. Fruhbeck, J. Gomez-Ambrosi, F. J. Muruzabal, and M. A. Burrell The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation Am J Physiol Endocrinol Metab, June 1, 2001; 280(6): E827 - E847. [Abstract] [Full Text] [PDF]
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L. O’Rourke, S. J. Yeaman, and P. R. Shepherd Insulin and Leptin Acutely Regulate Cholesterol Ester Metabolism in Macrophages by Novel Signaling Pathways Diabetes, May 1, 2001; 50(5): 955 - 961. [Abstract] [Full Text]
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C. Bjorbak, R. M. Buchholz, S. M. Davis, S. H. Bates, D. D. Pierroz, H. Gu, B. G. Neel, M. G. Myers Jr., and J. S. Flier Divergent Roles of SHP-2 in ERK Activation by Leptin Receptors J. Biol. Chem., February 9, 2001; 276(7): 4747 - 4755. [Abstract] [Full Text] [PDF]
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L. Huang, Z. Wang, and C. Li Modulation of Circulating Leptin Levels by Its Soluble Receptor J. Biol. Chem., February 23, 2001; 276(9): 6343 - 6349. [Abstract] [Full Text] [PDF]
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, Difference from insulin-injected rats at P <
0.058.
, Difference from insulin-injected rats at
P < 0.01. §, Difference from
leptin-injected rats at P < 0.001.
