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Endocrinology Vol. 141, No. 7 2328-2339
Copyright © 2000 by The Endocrine Society

ARTICLES

In Vivo Administration of Leptin Activates Signal Transduction Directly in Insulin-Sensitive Tissues: Overlapping but Distinct Pathways from Insulin1

Young-Bum Kim2, Shigeo Uotani, Dominique D. Pierroz, Jeffrey S. Flier and Barbara B. Kahn

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether leptin signal transduction is exerted directly upon insulin-sensitive tissues in vivo, we examined the ability of iv leptin to acutely stimulate phosphorylation of STAT3, STAT1, and MAPK, and activities of PI 3-kinase and Akt, in insulin-sensitive tissues of normal rats. Both leptin (1 mg/kg iv x 3 min) and insulin (10 U/kg iv x 3 min) stimulated tyrosine phosphorylation of STAT3 5.6- to 6.0-fold and of STAT1 4.0-fold in adipose tissue. Leptin tended to increase STAT3 phosphorylation in liver and muscle. Both hormones also increased MAPK phosphorylation: leptin increased it 3.2- to 3.8-fold in adipose tissue and liver, whereas insulin stimulated MAPK phosphorylation 5.0-fold in adipose tissue, 6.8-fold in liver, and 2.5-fold in muscle. Leptin was much less effective than insulin at stimulating IRS pathways. Leptin increased IRS-1-associated PI 3kinase activity in adipose tissue only 2.0-fold (P < 0.01) compared with the 10-fold effect of insulin. IRS-2-associated PI 3-kinase activity was increased 1.7-fold (P < 0.01) by leptin in liver and 6-fold by insulin. Akt phosphorylation and activity were not changed by leptin but increased with insulin. Lower concentrations of leptin (10 and 50 µg/kg) also stimulated STAT3 phosphorylation in fat. These effects appear to be direct because 3 min after leptin intracerebroventricular injection, phosphorylation of STAT3, STAT1, and MAPK were not stimulated in hypothalamus or adipose tissue. Furthermore, leptin activated STAT3 and MAPK in adipose tissue explants ex vivo and in 3T3-L1 adipocytes. Leptin did not activate STAT3 or MAPK in adipose tissue of db/db mice. Thus, leptin rapidly activates signaling pathways directly at the level of insulin sensitive tissues through the long-form leptin receptor, and these pathways overlap with, but are distinct from, those engaged by insulin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IDENTIFICATION of the ob gene through positional cloning (1) and the discovery that its encoded protein, leptin, is an adipocyte-derived hormone that is essential for the normal regulation of body weight, have permanently altered the field of metabolic physiology (2, 3, 4). Leptin acts on specific regions of the brain to regulate food intake, energy expenditure, and neuroendocrine function (5). The profound importance of leptin is demonstrated by the fact that mice which are homozygous for a mutation in the ob gene (ob/ob) (4) or in its receptor (db/db) (6), demonstrate massive obesity, a reduced basal metabolic rate, hyperglycemia, hyperinsulinemia, and reduced fertility (7). Administration of recombinant leptin to ob/ob mice reduces food intake, decreases body weight and adiposity, increases thermogenesis, ameliorates depressed body temperature, and can even restore fertility (2, 3, 4, 8). Leptin exerts its effects through specific receptors of which five isoforms have been described, arising through alternative splicing of the primary transcript (6). The leptin receptor is a member of the cytokine I receptor family of which gp 130 is a prototype. This class of receptors stimulates gene transcription via activation of cytosolic STAT proteins (9, 10). The long form of the leptin receptor (OBRb) has the capacity to activate the JAK/STAT (11, 12, 13, 14, 15, 16) and MAPK (17) pathways, stimulate tyrosine phosphorylation of IRS-1 (17), and increase transcription of fos, jun (17, 18) and suppressor of cytokine signaling 3 (SOCS3) (19). The short form (OBRa) has a limited capacity to activate JAK, but no capacity to activate STAT (11, 17) and is thought to play a role in the clearance of leptin from the circulation (20) and in the transport of leptin into the brain (21, 22).

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care and tissue harvest.
All animal studies were conducted in accord with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

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 [{gamma}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leptin markedly increases phosphorylation of STAT3 and STAT1 in adipose tissue (Fig. 1Go).
To determine whether STAT proteins can be activated in insulin target tissues in vivo after leptin and insulin injection peripherally, we examined STAT1 and STAT3 phosphorylation using antibodies specific for tyrosine phosphorylated STAT1 or STAT3. Strikingly, the phosphorylation of STAT3 increased 5.6-fold in adipose tissue of leptin-injected rats compared with saline-injected rats. Leptin also tended to stimulate STAT3 in liver and muscle but to a lesser degree (1.8-fold in liver and 1.4-fold in muscle) (Fig. 1AGo). 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. 1AGo).



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  		Figure 1. Phosphorylation of STAT3 (A) and STAT1 (B) in adipose tissue, liver, and muscle of rats injected with saline, insulin (10 U/kg), insulin plus leptin (1 mg/kg) or leptin alone and killed 3 min after injection. Proteins were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Phosphorylated STAT3 or STAT1 was visualized by immunoblotting with antibodies specific for phospho-STAT3 or phospho-STAT1. Bands were quantitated using a densitometer. Data are means ± SEM for 4 rats. ¶, Difference from saline-injected rats at P < 0.05. {ddagger}, Difference from insulin-injected rats at P < 0.058.

 
Peripheral administration of leptin or insulin to rats activated the phosphorylation of STAT1 4.0-fold in white adipose tissue compared with saline-injected rats (Fig. 1BGo). 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. 2Go)
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. 2Go). 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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  		Figure 2. Phosphorylation of MAPK in adipose tissue, liver, and muscle of rats injected with saline, insulin (10 U/kg), insulin plus leptin (1 mg/kg) or leptin alone and killed 3 min after injection. Proteins were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Phosphorylated MAPK was visualized by immunoblotting with a phospho-specific MAPK antibody. Bands were quantitated using a densitometer. Data are means ± SEM for four rats. Difference from saline-injected rats at P < 0.05. *, Difference from saline-injected rats at P < 0.01. **, Difference from saline-injected rats at P < 0.001. {dagger}, Difference from insulin-injected rats at P < 0.01. §, Difference from leptin-injected rats at P < 0.001.

 
Leptin modestly increases PI 3-kinase activity in adipose tissue and liver (Fig. 3Go)
In response to insulin, IRS-1-associated PI 3-kinase activity markedly increased 6.9-to 8.2-fold in adipose tissue, liver, and muscle (Fig. 3AGo). 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. 3AGo). PI 3-kinase activity was also measured in anti-IRS-2 immunoprecipitates from tissue lysates (Fig. 3BGo). 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. 3BGo).



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  		Figure 3. PI 3-kinase activity associated with IRS-1 (A), IRS-2 (B), or p85 (C) in adipose tissue, liver, and muscle of rats injected with saline, insulin (10 U/kg), insulin plus leptin (1 mg/kg) or leptin alone and killed 3 min after injection. PI 3-kinase activity was measured in immunoprecipitates and was quantitated using a PhosphorImager. Data are means ± SEM for four rats. ¶, Difference from saline-injected rats at P < 0.05. *, Difference from saline-injected rats at P < 0.01. **, Difference from lean saline-injected rats at P < 0.001.

 
Administration of insulin modestly increased p85-associated PI 3-kinase activity 2.1- fold in adipose tissue (P < 0.05) and tended to increase it 1.6-fold in liver (P < 0.07) and 1.4-fold in muscle compared with saline injected rats (Fig. 3CGo). 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. 4Go)
Insulin administration rapidly stimulated Akt phosphorylation by 5.9- to 12.2-fold in adipose tissue, liver, and muscle (Fig. 4AGo). 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. 4BGo). 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. 4BGo).



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  		Figure 4. Akt phosphorylation (A) and activity (B) in adipose tissue, liver, and muscle of rats injected with saline, insulin (10 U/kg), insulin plus leptin (1 mg/kg) or leptin alone and killed 3 min after injection. A, Proteins were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. The phosphorylated Akt was visualized by immunoblotting with a phospho-specific Akt antibody. Bands were quantitated using a densitometer. B, Tissue lysates were subjected to immunoprecipitation with an Akt antibody that recognizes Akt1 and Akt2. The immune pellets were assayed for kinase activity using Crosstide as substrate. Data are means ± SEM for four rats for each dose. **, Difference from saline-injected rats at P < 0.001. {dagger}, Difference from insulin-injected rats at P < 0.01.

 
A lower dose of leptin increases STAT3 phosphorylation (Fig. 5Go)
To determine whether the STAT3 phosphorylation is stimulated by a lower dose of leptin, we administrated leptin (2 µg/kg, 10 µg/kg, and 50 µg/kg) through the jugular vein in rats and removed epididymal adipose tissue 3 min later. These doses result in plasma leptin levels of 115 ± 11, 217 ± 49 and 1553 ± 242 ng/ml, respectively. We do not see phosphorylation of STAT3 in adipose tissue with 2 µg/kg of leptin administration iv. However, administration of 10 µg/kg and 50 µg/kg leptin significantly increased STAT3 phosphorylation 1.7-fold (P < 0.05) and 2.6-fold (P < 0.01) in adipose tissue compared with control rats injected with saline. These results indicate that leptin levels closer to the physiologic range may activate signaling in adipose tissue. It is possible that with longer time for diffusion into tissues, even lower concentrations could be effective.



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  		Figure 5. Phosphorylation of STAT3 in adipose tissue of rats injected with saline or leptin (2, 5, 10, or 50 µg/kg) and killed 3 min after injection. Proteins were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Phosphorylated STAT3 was visualized by immunoblotting with antibody specific for phospho-STAT3. Bands were quantitated using a densitometer. Data are means ± SEM for four rats for each group. *, Difference from saline-injected rats at P < 0.05. **, Difference from saline-injected rats at P < 0.01.

 
Leptin has different effects at 15 min on PI 3-kinase activity, STAT3, and MAPK phosphorylation (Fig. 6Go)
We also preliminarily investigated the effects of leptin on signaling in insulin-sensitive tissues 15 min after administration iv. At 15 min, insulin stimulated IRS-1-associated PI 3-kinase 5.8-fold in adipose tissue, 9.6-fold in liver and 2.5-fold muscle (Fig. 6Go). 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. 1Go), 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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  		Figure 6. IRS-1-associated PI 3-kinase activity, phosphorylation of STAT3 and MAPK in adipose tissue, liver, and muscle of rats injected with saline, insulin (10 U/kg), insulin plus leptin (1 mg/kg) or leptin alone and killed 15 min after injection. PI 3-kinase activity was measured in IRS-1 immunoprecipitates. Proteins were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Phosphorylated STAT3 or MAPK was visualized by immunoblotting with antibodies specific for phospho-STAT3 or phospho-MAPK. This representative of three immunoblots.

 
Intracerebroventricular leptin injection has no acute (3 min) effect on the phosphorylation of STAT3, STAT1 or MAPK in hypothalamus or adipose tissue (Fig. 7Go)
To understand whether the rapid effects are direct or mediated through the CNS, we injected leptin (3.5 µg/rat) icv in rats and harvested tissues 3 min later (Fig. 7Go). 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. 1Go and 2Go) 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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  		Figure 7. Phosphorylation of STAT1, STAT3, and MAPK in hypothalamus and adipose tissue after icv leptin. Tissue was removed 3 min after artificial cerebrospinal fluid (-) or leptin (+) (3.5 µg/kg) bolus icv. Proteins were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Phosphorylated STAT1, STAT3, and MAPK were visualized by immunoblotting with a phospho-specific antibodies.

 
Leptin does not activate STAT3 and MAPK in adipose tissue of db/db mice (Fig. 8Go)
Our results show a very rapid, and presumably direct effect of leptin to phosphorylate STAT3 and MAPK in adipocytes, and the question remains as to whether these effects are through the OBRb form of the leptin receptor. We measured STAT3 and MAPK in adipose tissue of db/db mice, which lack long-form leptin receptors as a negative control (6). Leptin injection in db/db mice did not result in phosphorylation of STAT3 and MAPK in adipose tissue. This result indicates that the effects of leptin to activate STAT3 and MAPK are exerted through the long-form leptin receptor.



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  		Figure 8. Phosphorylation of STAT3 and MAPK in adipose tissue of db/db mice injected with saline (-) or leptin (+) (1 mg/kg) and killed 3 min after injection. Proteins were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Phosphorylated STAT3 and MAPK were visualized by immunoblotting with a phospho-specific antibodies.

 
Leptin activates signaling pathways directly in adipose tissue explants and 3T3-L1 adipocytes (Fig. 9Go)
To further investigate whether the effects of leptin on intracellular signaling in adipose tissue are direct, we incubated adipose tissue explants ex vivo and 3T3-L1 adipocytes with leptin. In explants from normal rats, leptin rapidly stimulated MAPK phosphorylation 2.2-fold at 2.5 min (P < 0.001) and 2.0-fold at 15 min (P < 0.001) (Fig. 9AGo). 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. 7AGo. Figure 7BGo 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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  		Figure 9. Phosphorylation of MAPK in adipose tissue explants (A) and phosphorylation of STAT3 and MAPK in 3T3-L1 adipocytes (B). A, Adipose tissue explants were preincubated and stimulated without or with 100 nM leptin for 0, 3, 15, or 45 min. Explants were solubilized, proteins were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Phosphorylated MAPK was visualized by immunoblotting with an antibody specific for dually phosphorylated MAPK. Bands were quantitated by densitometry. Data are means ± SEM for three to five rats. **, Difference from basal adipose tissue explants at P < 0.001. *, Difference from basal adipose tissue explants at P < 0.01. B, Ten days after differentiation, cells were serum starved overnight and stimulated with leptin (100 nM) for 5 min. Lysate proteins were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Tyrosine phosphorylated STAT3 and dually phosphorylated MAPK were detected with specific antibodies and quantitated by densitometry. Data are means ± SEM and are representative of three independent experiments. ¶, Difference from control cells at P < 0.05.

 
It has been shown that insulin stimulates serine but not tyrosine phosphorylation of STAT3 in 3T3-L1 adipocytes (48). Leptin failed to activate serine phosphorylation of STAT3 in 3T3-L1 adipocytes, whereas insulin did (data not shown). Thus, while leptin and insulin activate some of the same intracellular signaling events, the pathways also diverge.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Leptin, the ob gene product, is synthesized in adipose tissue and plays a role in body weight homeostasis (1), reproduction (8), and possibly hematopoiesis (49). Leptin deficiency causes severe insulin resistance, which is rapidly reversed with leptin administration (2, 3, 4). The leptin receptor, a class I cytokine receptor, is produced in several alternatively spliced forms both in animals (23) and humans (49). Following binding of ligand, receptors in this cytokine family activate JAK tyrosine kinases, which rapidly phosphorylate STATs, leading to activation of gene transcription (10). Some class I cytokine receptors also activate other signaling cascades such as the MAPK and PI3K pathways (50, 51). To date, there are limited data (28, 38, 52) regarding the ability of leptin to activate signaling in peripheral tissues either 1) without overexpression of the leptin receptor and JAK, or 2) after leptin administration in vivo.

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. 9Go). 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. 9Go). 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. 8Go). 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. 6Go). 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.


   Acknowledgments
 
We thank Dr. O. Peroni for expert assistance with adipose explant studies, members of the lab of E. Maratos-Flier for assistance with icv injections and Dr. M. F. White for the IRS-1 and IRS-2 antibodies.


   Footnotes
 
1 This work was supported by National Institute of Diabetes and Digestive and Kidney Disease Grants NIH DK-43051, DK-28082 and grants from the American Diabetes Association and grant from Eli Lilly & Co. Back

2 Supported by Uehara Memorial Foundation Research Fellowship and a mentor based fellowship from the American Diabetes Association. Back

Received October 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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