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J Biol Chem, Vol. 275, Issue 15, 11348-11354, April 14, 2000

Leptin Induces Insulin-like Signaling That Antagonizes cAMP Elevation by Glucagon in Hepatocytes^*

Allan Z. Zhao^§, Michi M. Shinohara, Daming Huang, Masami Shimizu, Hagit Eldar-Finkelman^¶, Edwin G. Krebs, Joseph A. Beavo, and Karin E. Bornfeldt^**

From the Departments of  Pharmacology and  Pathology, University of Washington, Seattle, Washington 98195

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although many effects of leptin are mediated through the central nervous system, leptin can regulate metabolism through a direct action on peripheral tissues, such as fat and liver. We show here that leptin, at physiological concentrations, acts through an intracellular signaling pathway similar to that activated by insulin in isolated primary rat hepatocytes. This pathway involves stimulation of phosphatidylinositol 3-kinase (PI3K) binding to insulin receptor substrate-1 and insulin receptor substrate-2, activation of PI3K and protein kinase B (AKT), and PI3K-dependent activation of cyclic nucleotide phosphodiesterase 3B, a cAMP-degrading enzyme. One important function of this signaling pathway is to reduce levels of cAMP, because leptin-mediated activation of both protein kinase B and phosphodiesterase 3B is most marked following elevation of cAMP by glucagon, and because leptin suppresses glucagon-induced cAMP elevation in a PI3K-dependent manner. There is little or no expression of the long form leptin receptor in primary rat hepatocytes, and these signaling events are probably mediated through the short forms of the leptin receptor. Thus, leptin, like insulin, induces an intracellular signaling pathway in hepatocytes that culminates in cAMP degradation and an antagonism of the actions of glucagon.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Leptin (OB)¹ is a 16-kDa protein secreted primarily from adipocytes (1-3). Rodents that are defective in leptin synthesis, the ob/ob mice, or leptin receptor function, the db/db mice, Zucker fa/fa rats, and Koletsky rats, are obese and develop hyperinsulinemia and insulin resistance similar to the metabolic abnormalities associated with type 2 diabetes. Leptin suppresses food intake (4-7) and increases thermogenesis (8) and metabolic rate (9). These responses appear to be mediated mainly through the central nervous system, because they can be achieved by intracerebroventricular injections of leptin (10, 11). Leptin has also been shown to have a wide repertoire of peripheral effects, some of which are mediated through the central nervous system, and others that are induced through a direct action on target tissues. The latter include direct inhibition of insulin secretion and gene expression in pancreatic -cells, stimulation of fatty acid oxidation in adipocytes, and stimulation of angiogenesis and T-cell proliferation (12-16).

Molecular cloning of the leptin receptors (OB-R) has revealed that they are single membrane-spanning receptors with homology to members of the cytokine receptor superfamily (10, 17). The different leptin receptors arise from alternative splicing, and although the extracellular domains of these splice variants are identical, differences are apparent in the intracellular signaling domain. The splice variants containing transmembrane domains can be divided into two groups: one group that has short 32-97 amino acid residue intracellular domains (OB-Ra, -Rc, -Rd, -Rf, and a unique form expressed in hematopoietic cells, termed Rg here) and another group (OB-Rb) that has a long 302-residue intracellular domain (10, 18-20). OB-Rb (the long form of the leptin receptor) is mainly expressed in hypothalamus, whereas OR-Ra, -Rc, -Rd, and -Rf (collectively called the short forms) are expressed in a variety of tissues throughout the body (21). Both the long and short forms of the leptin receptor signal through the Janus kinases, which are tyrosine protein kinases, but only the long form (OB-Rb) has been shown to be capable of activating transcription factors of the signal transducers and activators of transcription family (10, 21, 22).

Evidence that leptin can mimic some of the anabolic actions of insulin in liver and other tissues is rapidly accumulating. Accordingly, leptin increases glucose uptake in skeletal muscle and brown adipose tissue in vivo (11, 23), normalizes blood glucose levels in diabetic rats (24), and increases glucose uptake in a myotube cell line in vitro (25). Peripheral leptin injections have been found to lower hepatic glucose production by decreasing both glycogenolysis in fasted mice and ob/ob mice that lack endogenous leptin (26). Leptin also markedly enhances the inhibitory effects of insulin on glycogenolysis and hepatic glucose production in liver in vivo (27) and in hepatocytes in vitro (28). Overexpression of leptin in rats reduces hepatic glycogen loss during fasting, most likely by reducing glycogenolysis (29), and leptin increases glycogen synthesis in perfused mouse liver (30). Some of the effects of leptin described above appear to be mediated by the central nervous system (11, 31). However, two recent studies on perfused rat liver and isolated rat primary hepatocytes demonstrate a direct suppressive effect of high concentrations of leptin on phosphorylase and glycogenolysis (28, 32).

Because liver is one of the major regulators of blood glucose, and as such, a target in the treatment of type 2 diabetes, we decided to investigate direct effects of physiological concentrations of leptin on isolated primary hepatocytes. We focused our studies on signaling pathways likely to result in suppression of hepatic glucose production and glycogenolysis, namely phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB) (33-35), and a cAMP-degrading phosphodiesterase, PDE3B (36, 37). Here we show that physiological levels (1-5 nM) of leptin indeed have direct effects on primary rat hepatocytes, leading to activation of PI3K and PDE3B and subsequent antagonism of glucagon-mediated cAMP elevation. In addition, we demonstrate for the first time that leptin, like insulin, is capable of activating PKB under conditions where cAMP is elevated.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hormones, Antibodies, and Peptides-- Recombinant mouse leptin was obtained from Peprotech Inc. (Rocky Hill, NJ), recombinant human insulin, and glucagon were obtained from Sigma. Protein kinase inhibitor peptide, a peptide inhibitor of cAMP-dependent protein kinase (TTYADFIASGRTGRRNAIHD), and Crosstide (GRPRTSSFAEG) were synthesized at the peptide synthesis facility, Howard Hughes Medical Institute, Seattle, WA or purchased from Upstate Biotechnology Inc. (Lake Placid, NY). The IRS-1 C-terminal antibody was purchased from Transduction Laboratories (Lexington, KY). The anti-IRS-2 antibody, the PKB antibody, and PKB assay kit were purchased from Upstate Biotechnology Inc. A rabbit polyclonal antibody against leptin receptors was generated against a hybrid protein containing a stretch of the extracellular domain of the mouse leptin receptor (amino acids 634-784) fused to the coding sequences of glutathione S-transferase. The glutathione S-transferase-OB-R fusion protein was expressed in bacteria and purified to homogeneity before injection into rabbits for immunization. The resultant polyclonal antibody recognizes all known splice variants of leptin receptors.

Cell Cultures-- Male Harlan Sprague-Dawley rats (150-200 g) were used for isolation of primary hepatocytes. Hepatocytes were prepared using the two step collagenase perfusion method, as described previously (38, 39). After isolation, the cells were washed three times in William's E medium with 5% fetal bovine serum, 10 mM HEPES (pH 7.4) (Life Technologies, Inc.), 2 mM L-glutamine (Sigma), 100 nM dexamethasone (Sigma), 6.25 µg/ml transferrin, 6.25 µg/ml selenious acid, 1 µM insulin (Beckton-Dickinson, Bedford, MA), and 5.35 µg/ml linoleic acid (Sigma). The cells were centrifuged at 50 × g for 2 min between washes. Cell viability, as estimated by trypan blue exclusion, was routinely above 80% following this procedure. The cells were plated on collagen type I-coated plates in the above medium at 8-10 million cells/100-mm dish or at 0.5 million cells/well in 12-well plates for cAMP measurements. The cells were allowed to adhere onto the culture dishes for 1.5 h (80-90% of the viable cells attach during this time). The cells were washed two times with serum-free William's E medium and then incubated in serum-free William's E medium with 1.25 mg/ml bovine serum albumin (Sigma) and 25 nM dexamethasone without selenious acid, transferrin, and insulin. The cells were stimulated and harvested 18-24 h after the medium change, as described below.

Measurement of PKB Activity-- Cells in 100-mm dishes were stimulated with leptin and insulin in the presence and absence of glucagon and then lysed in 1 ml of PDE3B lysis buffer containing 50 mM NaF, 150 mM NaCl, 10 mM NaSO₄, 2 mM EDTA, 25 mM Tris (pH 7.4), 100 µM Na₃VO₄, 100 nM calyculin, 2 µg/ml pepstatin A, 10 µM benzamidine, and 0.5% Lubrol. The cell lysates were scraped and briefly sonicated for 10 s using a Braun-Sonic 2000 sonicator at 50% output. The samples were then centrifuged for 2 min at 4000 × g in a microfuge. Protein concentrations in the supernatants were quantitated using the BCA^® protein assay (Pierce).

For each assay, 4 µg of an anti-pleckstrin homology domain PKB peptide sheep antibody (Upstate Biotechnology Inc.) was preincubated with protein G-Sepharose in 0.5 ml of extraction buffer overnight at 4 °C. After washing the protein G-Sepharose complex, 1 mg of protein extract was added to the complex, and the incubation was continued for 90 min. The samples were then assayed for PKB activities using a PKB assay kit from Upstate Biotechnology Inc. The assays were initiated by the addition of assay mix containing [-³²P]ATP. The final concentrations in the assay were 12.5 mM -glycerophosphate (pH 7.4), 10 mM MOPS (pH 7.2), 1.33 mM EGTA (pH 8.0), 0.5 mM dithiothreitol, 0.5 mM Na₃VO₄, 15 mM MgCl₂, 100 µM ATP, 10 µM protein kinase inhibitor, and 0.1 mM Crosstide. After an incubation for 10 min at 30 °C with shaking, the addition of 40 µl of trichloroacetic acid terminated the reactions, and 40 µl of the reaction mix was then spotted onto P-81 phosphocellulose paper (Whatman, Hillsboro, OR). The P-81 papers were washed three times in 150 mM phosphoric acid and once in methanol. The radioactivity associated with the papers was then measured in 2 ml of Ecolume scintillation fluid (ICN Biomedicals, Inc., Irvine, CA).

To investigate if PKB is phosphorylated following insulin or leptin stimulation, we utilized an antibody that specifically detects PKB phosphorylation on Ser-473 (New England Biolabs, Inc., Beverly, MA) in Western blot analysis. The same protein samples were also run on parallel gels and probed for total PKB expression using a rabbit anti-PKB antibody generated against the pleckstrin homology domain (Upstate Biotechnology, Inc.).

Immunoprecipitation of IRS-1 and IRS-2-- Expression of both IRS-1 and IRS-2 in primary rat hepatocytes was detected using Western blot analyses. Immunoprecipitation of IRS-1 and IRS-2 was carried out by incubating 1 mg of protein of hepatocyte cell lysate with IRS-1 or IRS-2 antibodies overnight. Following precipitation of the immunocomplexes with protein A-Sepharose, the beads were washed three times in the PDE3B lysis buffer as described above.

PI3K Assays-- PI3K activity was immunoprecipitated using antibodies directed against tyrosine-phosphorylated proteins (PY20, Transduction Laboratories), IRS-1 or IRS-2. The PI3K assay and subsequent thin-layer chromatography were carried out according to a standard protocol with phosphatidylinositol (Avanti, Pelham, AL) as a substrate in the presence of [-³²P]ATP as described previously (12).

Cyclic AMP Assays-- Immediately after hormonal treatments, the hepatocytes were lysed and incubated in 0.5 ml of ice-cold 5% trichloroacetic acid overnight. Five µl of the lysates were neutralized in 20 µl of 0.1 M Tris to a final pH of 7. The cAMP assay was then carried out using a radioimmunoassay kit from NEN Life Science Products.

PDE3B Assays-- Primary hepatocytes were treated with leptin or insulin in the presence or absence of glucagon for the indicated periods of time. Immunoprecipitation of PDE3B as well as the assay of PDE3B activity was carried out according to a protocol published previously, using 1 µM cAMP as a substrate (40). The PDE3B activity was normalized to the quantity of immunoprecipitated PDE3B as shown on Western blots and was expressed as pmol hydrolyzed cAMP/min/density unit of the PDE3B band.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolated Hepatocytes Express the Short Forms of the Leptin Receptor-- To study the direct effects of leptin and insulin on hepatocytes, we chose a culture system that allows investigation of differentiated primary hepatocytes with preserved physiologically relevant signaling pathways. For example, insulin does not activate the mitogen-activated protein kinases (extracellular signal-regulated kinases) in these cells (data not shown) or fetal primary hepatocytes (41), whereas many hepatoma cell lines show a marked activation of extracellular signal-regulated kinases following insulin treatment (42).

Initial studies were carried out to identify the major leptin receptor in these primary rat hepatocytes. We generated an anti-OB-R antibody that recognizes all known variants of leptin receptors. Western blot analysis using this antibody revealed both the long form (OB-Rb) and short forms of the leptin receptor in samples prepared from hypothalamus (Fig. 1). However, we detected specific immunoreactive bands only near 100 kDa, representing the short forms of leptin receptors, in isolated hepatocytes (Fig. 1). Notably, we did not see OB-Rb in the rat primary hepatocytes even with extensive exposures. Although we cannot completely rule out the existence of very low levels of OB-Rb, the molecular signaling events discussed below are most likely mediated through the short forms of the leptin receptor.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Expression of the short forms but not the long form of leptin receptors in rat primary hepatocytes. A rabbit polyclonal antibody was developed against a stretch of extracellular region common to all known leptin receptor splice variants. Protein extracts from rat primary hepatocytes or hypothalamus (~50 µg each) were applied to SDS gels. The arrows indicate the expression of the long Ob-Rb form and the short forms of leptin receptors in hypothalamus. Note that the long form (Rb) is absent in hepatocytes. Hyp., hypothalamus; Hep., hepatocytes.

Leptin Activates PI3K and PKB-- Our previous studies, as well as studies by others, have shown that leptin activates PI3K in cell types such as C2C12 myotubes and pancreatic -cells (12, 25, 43). To measure activation of PI3K in the primary hepatocytes, proteins phosphorylated on tyrosine residues were immunoprecipitated, and PI3K activity in the immunoprecipitates was measured. Both leptin and insulin activated PI3K more than 3-fold in hepatocytes, as shown in Fig. 2. Glucagon, a hormone that stimulates hepatic glucose production and glycogenolysis, also activated PI-3 kinase but did not affect the activation of PI3K by leptin or insulin (Fig. 2). To gain further understanding of the mechanism of action of leptin to activate PI3K, we examined the association of the p85 subunit of PI3K with IRS-1 and IRS-2 and the PI3K activity associated with IRS-1 and -2. The primary rat hepatocytes expressed significant amounts of both IRS-1 and IRS-2, as judged by Western blot analysis (data not shown). We immunoprecipitated either IRS-1 or IRS-2 and analyzed the PI3K activity as well as the level of p85 in the immunoprecipitates. Both insulin and leptin were found to stimulate the association of p85 with IRS-1 as well as IRS-2 and to increase PI3K activities in both immunoprecipitates (Fig. 3). Glucagon did not affect the binding of p85 to IRS-1 or IRS-2 in the presence of insulin or leptin (data not shown).

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Leptin (Lept) activates PI3K in primary hepatocytes. Rat primary hepatocytes were stimulated with 10 nM insulin or 2 nM leptin in the absence or presence of 10 nM glucagon. PI3K activity was measured following immunoprecipitation of tyrosine-phosphorylated proteins using a monoclonal antibody (PY20) and subsequent separation of the lipid products on thin layer chromatography plates (upper panel). The phosphatidylinositol 3-phosphate (PIP) product was quantitated using a densitometer and NIH Image 1.6 software (lower panel). The results in the lower panel are shown as mean ± S.E. The experiment was repeated three times with similar results. C, control; Ins, insulin.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Leptin induces association of PI3K activity with IRS-1 and -2. Rat primary hepatocytes were stimulated with 10 nM insulin or 2 nM leptin (Lept) and then lysed. IRS-1 and -2 were immunoprecipitated by using anti-IRS-1 or anti-IRS-2 antibodies, and the immunoprecipitation pellets were divided in two parts for PI3K activity assay (upper panel) and Western blot assays (lower panels). Like insulin, leptin induced the association of p85 and PI3K activity with both IRS-1 and IRS-2. C, control; Ins, insulin; IP, immunoprecipitation.

The activation of PKB has recently been shown to be dependent on PI3K (34, 44, 45). To investigate if leptin activates PKB in primary hepatocytes, PKB was immunoprecipitated, and its activity measured using in vitro kinase assays. In the absence of glucagon, insulin had an ~2.5-fold stimulatory effect, whereas leptin had no obvious effect on PKB activity (Fig. 4A). However, in the presence of glucagon, leptin (2 and 5 nM) significantly stimulated PKB activity, and insulin also displayed greater stimulatory effect on PKB in the presence of glucagon (Fig. 4A). We next utilized an antibody that detects PKB phosphorylation on Ser-473, one of the two phosphorylation sites required for the full activation of PKB. Stimulation of the hepatocytes with either insulin or leptin resulted in a weak or undetectable phosphorylation of PKB on Ser-473 (Fig. 4B). However, phosphorylation of PKB on Ser-473 by both insulin and leptin was greatly enhanced in the presence of glucagon (Fig. 4B). We also took advantage of the fact that phosphorylated active PKB migrates slower than nonphosphorylated inactive PKB on SDS gels. As shown by Fig. 4B, both insulin and leptin caused a band-shift of PKB in the presence of glucagon, whereas no band-shift was observed in the absence of glucagon. The abilities of insulin and leptin to stimulate phosphorylation of Ser-473 and to cause a band-shift of PKB following exposure of the cells to glucagon are consistent with the activity profile of PKB (Fig. 4A).

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Leptin activates PKB in the presence of glucagon in primary hepatocytes. Rat primary hepatocytes were stimulated with or without 10 nM glucagon for 15 min and then with insulin or leptin for an additional 30 min. The concentrations were 10 nM for insulin and 2 or 5 nM for leptin (Lept). PKB was immunoprecipitated from 1 mg of protein using an anti-PKB antibody (Upstate Biotechnology Inc.), and PKB kinase activity was subsequently measured as phosphorylation of Crosstide during a 10-min incubation at 30 °C (A). The results are shown as mean ± S.E. The experiment was repeated six times with similar results. In B, PKB was detected by Western blot analysis, using an antibody that detects PKB phosphorylation on Ser-473 (New England Biolabs, Inc.; upper panel) and an antibody that detects total PKB (Upstate Biotechnology, Inc.; lower panel). PH, pleckstrin homology; C, control; Ins, insulin.

Leptin Activates PDE3B and Reduces cAMP Levels-- Activation of PDE3B is a PI3K-dependent process (12, 36). To investigate if PDE3B is activated by leptin in primary hepatocytes, PDE3B activity from hepatocytes stimulated with leptin or insulin in the presence or absence of 10 nM glucagon was measured. As shown in Fig. 5A, both leptin and insulin mildly activate PDE3B in the absence of glucagon (the stimulatory effect ranging from 50 to 80%). Glucagon (10 nM) by itself also had a stimulatory effect (~ 80%) on PDE3B activity (Fig. 5A). In the presence of glucagon, insulin (10 nM) further stimulated PDE3B activity ~50% relative to that in glucagon-treated cells, whereas physiological concentrations of leptin (2 or 5 nM) activated the PDE3B more than 2-fold (Fig. 5A). This activation was maintained for at least 1 h following hormone treatment (data not shown). We also tested the effects of PI3K inhibitors, wortmannin (20 nM) and LY294002 (2 µM), on the activation of PDE3B in the primary hepatocytes. As shown in Fig. 5B, both inhibitors eliminated the stimulatory effect of insulin and leptin on PDE3B activity. These results indicate that activation of PDE3B by either insulin or leptin is dependent on PI3K.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Leptin activates PDE3B in primary hepatocytes. A, the cells were stimulated with or without 10 nM glucagon for 10 min and then with 2 or 5 nM leptin or 10 nM insulin for an additional 30 min. PDE3B activity was measured, using 1 µM cAMP as substrate, after immunoprecipitation of PDE3B and a 10-min incubation at 30 °C. The results are shown as mean ± S.E. The experiment was repeated six times with similar results. B, PI3K inhibitors, wortmannin or LY294002, blocked the activation of PDE3B by insulin or leptin.

Because PDE3B is a cAMP-hydrolyzing PDE, an activation of PDE3B would be expected to lead to the reduction of cAMP levels. To investigate if this is indeed the case, cells were incubated with or without 10 nM glucagon for 10 min and then stimulated with insulin or leptin for 15 min. Glucagon increased cAMP levels approximately 10-fold (compare Fig. 6, A and B). Insulin and leptin did not affect basal cAMP levels (Fig. 6A) but significantly reduced glucagon-stimulated cAMP levels by ~50% (Fig. 6B). A specific inhibitor of PDE3, milrinone, at 10 µM (IC₅₀ = 0.3 µM) blocked the reduction of cAMP levels induced by leptin and insulin (Fig. 6B). The PI3K inhibitor wortmannin (20 nM) also completely reversed the inhibitory effect of insulin or leptin on cAMP (Fig. 6B). Thus, the ability of insulin and leptin to reduce glucagon-stimulated cAMP accumulation is because of a PI3K-dependent activation of PDE3B.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Both leptin and insulin reduce glucagon-stimulated cyclic AMP formation through PI3K-dependent activation of PDE3B. Primary hepatocytes were preincubated in the absence (A) or presence (B) of 10 nM glucagon for 5 min. Five minutes after the addition of glucagon, 2 nM leptin or 10 nM insulin were added to the cell culture medium, and the cells were harvested 15 min later. Preincubation of the hepatocytes with a specific PDE3 inhibitor, milrinone (10 µM), or a PI3K inhibitor, wortmannin (20 nM), blocked the leptin and insulin effects. The measurement of cAMP was carried out using a radioimmunoassay kit from NEN Life Science Products. The results are shown as mean ± S.E. Each sample was analyzed in triplicates.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Leptin Induces Insulin-like Signaling in Isolated Hepatocytes through the Short Forms of the Leptin Receptor-- We show here that physiological concentrations of leptin (1-5 nM) induce intracellular signaling events through a direct action in isolated differentiated rat hepatocytes. Further, the signaling pathway induced is a well known insulin-stimulated pathway that includes IRS-1 and IRS-2, PI3K, PKB, and PDE3B. The present study thus supports the concept that important physiologic actions of leptin are mediated through endocrine actions directly on target tissues and that not all effects of leptin are mediated through the central nervous system. This concept is further supported by the presence of short forms of leptin receptors capable of inducing signaling events in hepatocytes and a myotube cell line (43, 46). By designing a polyclonal antibody that recognizes all known splice variants of the leptin receptor we show expression of both the long form, OB-Rb, and short forms in the hypothalamus, whereas expression in isolated hepatocytes is limited to the short forms of the leptin receptor. The lack of OB-Rb in hepatocytes is consistent with previous reports using reverse transcriptase-polymerase chain reaction and RNase protection assays of whole liver (19, 47) and the fact that in most peripheral tissues OB-Rb is not expressed, whereas the short forms are expressed ubiquitously (10, 21). Instead, the main leptin receptors in liver are OB-Ra and OB-Rf (19, 47). Thus, it is unlikely that the signaling events observed here are mediated through the OB-Rb form of the leptin receptor. Recent studies have shown that in cells expressing short forms of OB-R, Janus kinase 2 and IRS-1, leptin can induce tyrosine phosphorylation of IRS-1 through the short form of the leptin receptor (48). Our results assign one signaling pathway to the short forms of the leptin receptor, i.e. activation of PI3K (through IRS-1/IRS-2), PKB, and PDE3B.

PI3K, PKB, and PDE3B Are Activated by Both Leptin and Insulin in Hepatocytes-- The fact that leptin can induce activation of PI3K through IRS in different cell types has been reported previously. However, the present study extends these studies to include primary hepatocytes, a cell type relevant to blood glucose control under normal conditions and in type 2 diabetes. The finding that leptin activates PI3K in primary hepatocytes is consistent with reports of leptin-induced activation of PI3K in a myotube cell line and pancreatic -cells (12, 25, 43). The activation of PI3K by leptin seems to occur by activation of Janus kinase and subsequent tyrosine phosphorylation of the insulin receptor substrates IRS-1 and IRS-2 (43, 48) and recruitment of the p85 subunit of PI3K to IRS-1 and -2 (this study; 43, 49-51).

The present study demonstrates for the first time that leptin, like insulin, is capable of activating PKB. Because PKB activity is regulated by PI3K (34, 44, 45), its activation is consistent with the increased activity of PI3K after leptin stimulation. However, the activation of PKB by leptin requires the presence of a cAMP-elevating hormone (glucagon). Thus, activation of PI3K does not appear to be sufficient to mediate leptin-induced activation of PKB. The cAMP-dependent protein kinase (PKA) has been reported to activate PKB in some cell types (52). The synergistic action of leptin and cAMP on PKB does not seem to be because of a direct phosphorylation of PKB by PKA, because PKA does not phosphorylate PKB with high enough stoichiometry in vivo (52). One possibility is that PKA, when activated by cAMP, may inhibit a phosphatase in hepatocytes (53) and other cells (52) and that this phosphatase, in turn, affects PKB activity. Such a mechanism could enhance the effects of leptin on PKB activity.

It has been known for many years that insulin activates PDE3B in insulin-sensitive tissues (54) through a PI3K-dependent mechanism (36). We show here that leptin, like insulin, activates PDE3B in primary hepatocytes and that the activation is additive or synergistic to that of glucagon. This finding is consistent with previous results that show activation of PDE3B by leptin in -cells (12). Recent studies have shown that PKB is required for insulin-induced PDE3B activation through a direct phosphorylation of Ser-273 of mouse PDE3B by PKB in adipocytes (55, 56). The activation of PDE3B in primary hepatocytes by leptin is dependent on PI3K and can be completely blocked by low concentrations of the PI3K inhibitors wortmannin and LY294002. Thus, because leptin can activate PI3K, PKB, and PDE3B in primary hepatocytes, it is likely that PI3K and subsequent activation of PKB, at least in part, mediate the leptin effect on PDE3B activity. The additive or synergistic effect of leptin and glucagon on PDE3B activity may be because of the synergistic effects of these hormones at the level of PKB as well as a direct phosphorylation of PDE3B by PKA (37).

It should be noted that preincubation of some cells with leptin reduces the subsequent stimulatory effects of insulin on tyrosine phosphorylation of IRS-1, on PI3K activation (46), and its inhibitory effect on PKA activity (13). Because these signaling molecules are regulated in a similar manner by leptin and insulin, it is possible that the antagonistic effects of leptin on insulin signaling are explained by a down-regulation or modulation of this shared intracellular signal transduction pathway much in the same way as cells are desensitized to a second dose of insulin following preincubation with insulin. It is also possible that leptin induces additional antagonistic signaling pathways.

Taken together, we show that leptin stimulates PI3K recruitment to IRS-1 and IRS-2 and subsequent activation of PI3K, PKB, and PDE3B through a direct action on hepatocytes. The same signaling pathway is activated by insulin and modulated by glucagon.

The Leptin-induced Signaling Constitutes a Negative Feedback Loop on Glucagon-stimulated cAMP Levels in Hepatocytes-- Many metabolic processes in hepatocytes are regulated in an opposite manner by insulin and glucagon or other cAMP-elevating agents, such as epinephrine. The principal net effect is that insulin suppresses hepatic glucose production, whereas glucagon accelerates it (57). It has been shown that the antagonistic action of insulin on the effects of glucagon and other cAMP-elevating agents is, at least in part, because of the activation of the cAMP-hydrolyzing PDE3B and a subsequent reduction of cAMP levels (36). We show here that leptin, like insulin, suppresses the ability of glucagon to elevate cAMP levels in primary hepatocytes. This leptin effect is mediated through a PI3K-dependent activation of PDE3B, as has previously been shown for insulin (37). Interestingly, the effects of leptin on PKB and PDE3B are additive or synergistic to those of glucagon. It has previously been shown that although glucagon accelerates hepatic glucose production, glucagon can also potentiate the ability of insulin to suppress hepatic glucose production through a direct hepatic mechanism (58, 59). Thus, many effects of insulin in isolated hepatocytes or perfused liver are accentuated in the presence of glucagon. This can be seen at the level of release of glucose (60) and the activation of glycogen synthase, phosphorylase (61), and PDE3B (37). In this context it has been proposed that the major direct action of insulin in the liver is to suppress the effects of glucagon and that this direct suppressive effect is greater in the presence of elevated glucagon (62). Our results show that not only are the effects of insulin greater in the presence of glucagon but that the effects of leptin on isolated hepatocytes are also enhanced under conditions where glucagon is elevated.

Glucagon is secreted from -cells in the pancreas and its release is regulated by circulating levels of glucose and amino acids. After a meal, plasma levels of both glucagon and insulin are elevated because glucagon release is stimulated by ingested amino acids and because a small rise in blood glucose levels results in only a small drop of glucagon levels and a large increase in insulin levels (57). We propose that the enhanced action of insulin in the presence of glucagon on PKB and PDE3B and the subsequent degradation of cAMP serves as a negative feedback mechanism on cAMP levels and hepatic glucose metabolism. Because leptin levels are increased following food intake (63), this negative feedback on hepatic signaling may well be of physiologic relevance for the metabolic effects of leptin. This feedback loop may serve to reduce the actions of glucagon on liver glucose production and perhaps other glucagon-mediated processes.

	ACKNOWLEDGEMENTS

We thank Drs. Nelson Fausto, Irina Kirillova, and Robert Pierce at the Department of Pathology, and Drs. Jaspreet S. Sidhu and Curtis Omiecinski at the Department of Environmental Health, University of Washington, for help and advice in establishing the primary hepatocyte cell culture system. The technical assistance of Lucy Suzuki during part of this work is gratefully acknowledged.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK-21723 (to J. A. B.) and DK-42528 (to E. G. K.), a Pilot and Feasibility Award from the Diabetes Endocrinology Research Center at the University of Washington funded by National Institutes of Health Project Grant P30 DK17047, a Career Development Award from the American Diabetes Association, and the Marian E. Smith Junior Faculty Research Award (to K. E. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Current address: Dept. of Cell Biology and Physiology, University of Pittsburgh, S-309 BSTWR, 3500 Terrace St., Pittsburgh, PA 15261.

^¶ Current address: Dept. of Medicine, Harvard Medical School, Boston, MA 02115.

^** To whom correspondence should be addressed: Dept. of Pathology, Box 357470, University of Washington School of Medicine, Seattle, WA 98195-7470. Tel.: (206) 543-1681; Fax: (206) 543-3644; E-mail: bornf@u.washington.edu.

	ABBREVIATIONS

The abbreviations used are: OB, leptin; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PDE3B, phosphodiesterase 3B; IRS, insulin receptor substrate; MOPS, 4-morpholinepropanesulfonic acid; OB-R, leptin receptor; PKA, protein kinase A.

	REFERENCES

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