Leptin Impairs Insulin Signaling in Rat Adipocytes

Home , Insulin receptor, Leptin receptor

Leptin Impairs Insulin Signaling in Rat Adipocytes Coralia Pe´rez,1 Carmen Ferna´ndez-Galaz,2 Teresa Ferna´ndez-Agullo´,2 Carmen Arribas,3 Antonio Andre´s,4 Manuel Ros,2 and Jose´ M. Carrascosa1

Leptin modulates glucose homeostasis by acting as an system to adapt metabolic and neuroendocrine function to insulin-sensitizing factor in most insulin target tissues. changes in the nutritional state of the organism (4). Nevertheless, insulin-dependent glucose uptake in Considerable experimental data indicate that leptin white adipose tissue decreases after in vivo treatment modulates glucose homeostasis (5). In most cases (6–8), with leptin. Moreover, elevated leptin concentrations leptin appears to act as an insulin-sensitizing factor at the inhibit insulin metabolic effects in adipocytes. Here we whole body level in rats. Nevertheless, studies on glucose studied both, direct and centrally mediated effects of uptake by different tissues after leptin treatment suggest leptin on insulin signaling in rat adipocytes. Adipocyte that the hormone exerts tissue-specific effects. Thus, mi- incubation with low leptin concentrations did not mod- croinjection of leptin into ventromedial hypothalamus ify the insulin stimulation of mitogen-activated protein kinase (MAPK). However, at elevated concentrations, increases glucose uptake in brown adipose tissue and leptin impaired insulin-stimulated MAPK activity, gly- heart and skeletal muscles but not in white adipose tissue cogen synthase kinase (GSK)3␤ phosphorylation, and (9). A subcutaneous infusion of leptin for 7 days combined insulin receptor tyrosine phosphorylation without al- with a euglycemic-hyperinsulinemic clamp induces an tering vanadate stimulation. An increase of suppressor increase of glucose uptake in brown adipose tissue and of cytokine signaling-3 protein was also observed. Cen- skeletal muscles but a decrease in white adipose tissue tral administration of leptin decreased insulin effects (6,10). on adipocyte MAPK and GSK3␤ phosphorylation. In Further evidence for the role of leptin on glucose insulin-resistant aged rats with hyperleptinemia and homeostasis was obtained in ob/ob and in lipodystrophic central leptin resistance, insulin poorly stimulated mice, which lack detectable amounts of circulating leptin. MAPK and central leptin infusion did not further dete- riorate adipocyte insulin responsiveness. Food restric- In both cases, peripheral treatment with leptin alone tion increased MAPK stimulation by insulin and (11–13) or in combination with the adipocyte-derived restored the ability of centrally infused leptin to atten- hormone adiponectin (14) reversed the characteristic dia- uate adipocyte insulin signaling in aged rats. We con- betic phenotype and insulin resistance of these animals, clude that leptin can modulate, in an inhibitory manner, and the same has been observed in lipodystrophic patients adipocyte insulin signaling by two different ways: as an under chronic leptin treatment (15). In contrast, in hyper- autocrine signal and, indirectly, through neuroendo- leptinemic db/db mice and fa/fa rats, which have muta- crine pathways. These mechanisms may be of relevance tions in the leptin receptor, and in obese rats and humans in situations of hyperleptinemia, such as aging and/or with hyperleptinemia, leptin administration does not im- obesity. Diabetes 53:347–353, 2004 prove glucose tolerance and insulin sensitivity, probably due to the existence of leptin resistance (5). The molecular bases for the metabolic effects of leptin eptin is a hormone, mainly produced by the are not completely understood. Experimental evidence adipose tissue, involved in the regulation of en- indicates that leptin acts predominantly in the central ergy balance (1–3). Compelling evidence indi- nervous system, mainly in the hypothalamus, bringing cates that leptin serves as a mediator in the about effects on appetite and in neuroendocrine pathways, L as well as on autonomic nerves, which are transmitted to cross-talk between the peripheral and central nervous the periphery (4). On the other hand, expression of the leptin receptor has been observed in peripheral tissues, From the 1Centre of Molecular Biology “Severo Ochoa,” Autonomous Univer- including pancreatic ␤-cells, liver, fat, and muscle (5), sity, Madrid, Spain; the 2Health Sciences Faculty, University Rey Juan Carlos, Alcorco´ n, Madrid, Spain; the 3Environment Sciences Faculty, Regional Centre suggesting direct effects of leptin that are independent of for Biomedical Research, University of Castilla La Mancha, Toledo, Spain; and central pathways. Although the relative importance of the 4Biochemistry Section, Faculty of Chemistry, Regional Centre for Biomed- peripheral versus central actions of leptin on metabolic ical Research, University of Castilla La Mancha, Ciudad Real, Spain. Address correspondence and reprint requests to Prof. Jose´ M. Carrascosa, effects of the hormone remains unknown, direct effects of Centro de Biologıá Molecular “Severo Ochoa,” Facultad de Ciencias, Univer- leptin on liver triglyceride content (16) and on skeletal and sidad Auto´ noma, Campus de Cantoblanco, 28049 Madrid, Spain. E-mail: cardiac muscle fatty acid oxidation (17,18) have been [email protected]. Received for publication 28 July 2003 and accepted in revised form 4 recently described. Additionally, a direct fat-depleting November 2003. effect at supraphysiological concentrations of leptin has The Centro de Biologıá Molecular is the recipient of institutional aid from the Ramo´ n Areces Foundation. C.P. is a recipient of a fellowship from the been reported (19). Mutis Program (Spain). In nonadipose insulin-target tissues, leptin prevents GSK, glycogen synthase kinase; IRS, insulin receptor substrate; MBP, triglyceride accumulation, thus contributing to the main- myelin basic protein; MAPK, mitogen-activated protein kinase; SOCS, suppressor of cytokine signaling. tenance of insulin sensitivity (20). However, in white © 2004 by the American Diabetes Association. adipose tissue, the effect of leptin on insulin sensitivity

DIABETES, VOL. 53, FEBRUARY 2004 347 LEPTIN IMPAIRS ADIPOCYTE INSULIN SIGNALING remains controversial. As mentioned above, while in vivo Assay of MAPK activity. MAPK activity was determined in anti-MAPK treatment with leptin reduces insulin-dependent glucose immunopellets using myelin basic protein (MBP) as substrate. Cytosolic extracts (ϳ500 ␮g protein) were immunoprecipitated with anti-MAPK 1/2 uptake of white adipose tissue (6,9,10), leptin incubation antibodies (UBI, Lake Placid, NY) following the manufacturer’s protocol. of isolated adipocytes has been reported to impair insulin Immunoprecipitates were incubated for 20 min with 0.5 mg/ml MBP and 50 stimulation of glucose transport and most of the metabolic ␮mol/l [␥-32P]ATP, and phospho-MBP was resolved on 12% SDS-PAGE, effects of insulin (21), to impair only the lipogenic action detected by autoradiography, and quantitated by scanning densitometry as previously described (35). Alternatively, bands were cut off of the gel and of insulin (22,23), and to not influence insulin-mediated phospho-MBP was quantitated by scintillation counting. The amount of MAPK effects (24,25). On the other hand, the effect of leptin on proteins in cytosolic extracts was determined by Western blot analysis with specific insulin-signaling events has not been studied, and the same antibody and was used to normalize the values of MBP phosphory- the data from studies in cell lines have been controversial (5). lation. Several studies have demonstrated that decreasing vis- Determination of phosphorylated GSK3. Cytosolic proteins (ϳ20 ␮g) were resolved by 10% SDS-PAGE and then transferred to nitrocellulose ceral fat by caloric restriction (26) or by its surgical membranes. Membranes were blocked in Tris-buffered saline containing 5% removal (27) prevent the onset of overall insulin resis- fat, skimmed, dry milk and incubated overnight at 4°C with anti–phospho- tance. Additionally, fat-specific GLUT4 gene inactivation GSK3␤ (Ser 9) polyclonal antibodies (Cell Signaling, Beverly, MA) at a 1:500 causes glucose intolerance and hyperinsulinemia (28), dilution. Phosphorylated GSK3 was visualized after incubation with goat anti-rabbit peroxidase-conjugated IgG (Nordic Immunologic, Tiburg, the Neth- whereas GLUT4 overexpression in fat tissue enhances erlands) using the enhanced chemiluminescence method and quantitated by whole-body insulin sensitivity and glucose tolerance in densitometric scanning. Membranes were reblotted with anti-GSK3 ␣/␤ mono- diabetic mice (29), suggesting that the insulin sensitivity of clonal antibodies (dilution 1:500; Biosource, Camarillo, CA) followed by adipose tissue might play a pivotal role in the development incubation with horse anti-mouse peroxidase-conjugated IgG (dilution 1:1,000; of whole-body insulin resistance and glucose intolerance. Vector, Burlingame, CA) to determine the relative amount of GSK3 proteins in the cytosolic extracts. Hyperleptinemia develops in most cases of obesity and Measurement of SOCS-3 and insulin receptor tyrosine phosphoryla- during aging in rodents and humans (1,30,31). Thus, it tion. Cell lysates (ϳ500 ␮g protein) were immunoprecipitated with 5 ␮g could be possible that the elevated levels of circulating SOCS-3 (H-103) or 5 ␮g insulin R␤ (C-19) polyclonal antibodies (Santa Cruz leptin in these cases might play a key role in the develop- Biotechnology, Santa Cruz, CA). Precipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and visualized by incubation with ment of insulin resistance in adipose tissue, leading to SOCS-3 (H-103) or anti-phosphotyrosine (UBI) monoclonal antibodies (dilu- overall insulin insensitivity. tion 1:500), respectively. The relative amount of protein in each extract was In the present work, we studied the effect of leptin evaluated by Western blot with insulin R␣ (N-20) antibodies (dilution 1:500; administration, in vivo and in vitro, on insulin signaling in Santa Cruz Biotechnology). isolated adipocytes from mature Wistar rats. Additionally, Leptin administration. Rats were anesthetized by inhalation of a mixture of O2,NO2, and isoflurane (Pharmacia-Upjohn, Barcelona, Spain) and placed in in aged rats that presented hyperleptinemia, we analyzed a stereotaxic frame (David Koppf, Tujunga, CA). An opening of the skull was whether a decrease in the circulating leptin concentration made with a dental drill at Ϫ1.6 mm lateral to the midline and 0.8 mm anterior after caloric restriction improved insulin signaling in adi- to bregma (32). A cannula connected to an osmotic minipump (Alzet, Palo pocytes and restored the ability of centrally administered Alto, CA) was implanted in the right lateral cerebral ventricle. Osmotic minipumps, with a releasing rate of 1 ␮l/h, were filled with rat leptin (Sigma), leptin to modulate adipocyte insulin responsiveness. which was diluted as indicated by the manufacturer. Leptin concentrations were adjusted to 0.41 ␮g/␮l (10 ␮g/day) and 0.0082 ␮g/␮l (0.2 ␮g/day) with PBS. Control rats were implanted with a minipump containing vehicle of the RESEARCH DESIGN AND METHODS same osmolarity of the leptin dilution. After 7 days, the rats were killed and Male Wistar rats (3 and 24 months old) from our in-house colony (Centre of isolated adipocytes were prepared from fat tissue as described by Molero et al. Molecular Biology, Madrid, Spain) were used throughout this study. Rats were (35). housed in climate-controlled quarters with a 12-h light cycle and fed ad libitum RNA extraction and RT-PCR. Total RNA was isolated from white adipose standard laboratory diet and water. For studies with food-restricted animals, tissue using the RN-Easy minikit (Qiagen, Hilden, Germany). Primers used for 21-month-old male Wistar rats were placed in individual cages and fed daily SOCS-3 and actin cDNA synthesis and for PCR were essentially the same as with an amount of diet equivalent to 75–80% of the normal food intake until those described previously (36). The template was denatured for 5 min at they reached a reduction of ϳ25% of their body weight. The amount of diet 94°C, followed by 30 cycles (25 cycles for actin amplification) with the provided was adjusted weekly to maintain this body weight up to age 24 following temperatures: denaturation, 1 min at 94°C; annealing, 2 min at 60°C; months (32). The animals were handled according to the laws of the European and elongation, 1 min at 72°C. Reactions were finished with a final extension Union and the guidelines of the National Institutes of Health, and the of 10 min at 72°C. The samples were electrophoresed and analyzed as experimental protocols were approved by the institutional committee of described (36). bioethics. Statistical analysis. Statistical analysis was performed using the Prophet Isolation and fractionation of adipocytes for in vitro experiments. software (BBN Systems and Technologies, Cambridge, MA). Significant dif- Adipocytes were prepared by the collagenase method using Dulbecco’s ferences between groups were determined by one-way ANOVA followed by modified Eagle’s medium containing 25 mmol/l HEPES, pH 7.4, and 4% BSA. the Duncan test for multiple comparisons or Student’s t test. Cells were washed in HEPES-buffered balanced salt solution (pH 7.4) containing 20 mmol/l HEPES, 120 mmol/l NaCl, 1.2 mmol/l MgSO4, 2 mmol/l CaCl2, 2.5 mmol/l KCl, 1.0 mmol/l NaH2PO4, 1% BSA, and 1 mmol/l sodium pyruvate RESULTS (33) and incubated in the same buffer (1 ml cells per 3 ml medium) at 37°C for 6 h in the presence or absence of the indicated concentration of rat Effect of leptin on insulin stimulation of MAPK in recombinant leptin (Sigma, St. Louis, MO). Following the treatment, cells isolated adipocytes. We studied the effect of incubation were incubated in the absence or presence of 16 nmol/l porcine insulin or 1 with different leptin concentrations over6honinsulin- mmol/l Na VO (Sigma). After 5 min, the medium was aspirated, cells were 3 4 stimulated MAPK activity in adipocytes isolated from washed in PBS, lysed in homogenization buffer, and centrifuged at 8,000g for 30 min as previously described (34,35). Infranatants (cytosolic extracts) were 3-month-old rats. Insulin stimulates MBP phosphorylation used for determination of mitogen-activated protein kinase (MAPK) activity by anti-MAPK immunoprecipitates ϳ2.8-fold in cells incu- and glycogen synthase kinase (GSK)3 phosphorylation. To evaluate insulin bated in the absence of leptin. Preincubation of adipocytes receptor phosphorylation and the amount of suppressor of cytokine signaling up to a level of 0.5 nmol/l leptin (which is slightly higher (SOCS)-3 protein, cells lysed in homogenization buffer were centrifuged at 5,000g for 5 min and the infranatants solubilized at 4°C for 30 min in the than the physiological concentration of leptin in mature presence of 1% Triton-X-100 and 1 mg/ml each of leupeptin, pepstatin, and rats) does not significantly modify the insulin effect on aprotinin. MBP phosphorylation. In contrast, preincubation of adipo-

348 DIABETES, VOL. 53, FEBRUARY 2004 C. PE´ REZ AND ASSOCIATES

FIG. 1. MAPK stimulation by insulin and vanadate: the effect of leptin. Adipocytes were preincubated with the indicated leptin concentrations for 6 h and subsequently stimulated with 16 nmol/l insulin or 1 mmol/l vanadate for 5 min. A: Insulin-dependent phosphorylation of MBP, a representative autoradiogram from four separate experiments is shown. B: Fold stimulation by insulin (mean ؎ SE). One-way ANOVA indicates P < 0.05 vs. cells incubated without leptin. C: Effect of 50 nmol/l leptin on MBP phosphorylation in* .(0.01 ؍ a significant effect of leptin (P ؍ response to insulin or vanadate, a representative autoradiogram. D: Fold stimulation by insulin (Ⅺ) or vanadate (f). Data are means ؎ SE (n 6). *P < 0.05 vs. vanadate effect in leptin-treated cells; **P < 0.01 vs. insulin and vanadate effects in cells without leptin. cytes with higher leptin concentrations (5–50 nmol/l) Effect of central administration of leptin on adipo- brings about a significant decrease in the insulin stimula- cyte insulin responsiveness. We next examined the tion of MBP phosphorylation (Fig. 1A and B). No signifi- effect of prolonged central administration of leptin on the cant differences were found in basal activities determined stimulation of MAPK and GSK3␤ phosphorylation by insu- after scintillation counting of the bands, which were 3.9 Ϯ lin in isolated adipocytes from 3-month-old rats. We have 0.6, 4.0 Ϯ 0.9, 4.3 Ϯ 0.9, and 4.1 Ϯ 0.8 fmol ⅐ 20 minϪ1 ⅐ ␮gϪ1 previously shown that intracerebroventricular administra- of immunoprecipitated protein, in adipocytes preincu- tion of a daily amount of 0.2–10 ␮g leptin during 7 days bated without leptin or with 0.5, 5, and 50 nmol/l leptin, results in a dose-dependent decrease in food intake and respectively. To investigate the molecular mechanisms body weight, without changes in circulating levels of leptin involved in leptin action on insulin signaling to MAPK, (32). Serum leptin concentrations at the end of the exper- adipocytes preincubated or not with 50 nmol/l leptin were stimulated with 1 mmol/l vanadate. In contrast to insulin, vanadate similarly stimulates the MAPK activity of adipocytes treated with or without leptin (Fig. 1C and D). Effect of leptin on insulin-dependent GSK3 phosphorylation. As is shown in Fig. 2, insulin stimulates the phosphorylation of GSK3␤ in adipocytes incubated in the absence of leptin by approximately threefold. When adipocytes are preincubated with 50 nmol/l leptin for 6 h, the stimulatory effect of insulin is significantly lower (ϳ2.1- fold). On the other hand, vanadate stimulates GSK3␤ phosphorylation ϳ2.5-fold, and preincubation of adipocytes with leptin does not decreases its stimulatory effect (Fig. 2A and B). Preincubation with leptin did not modify the basal phosphorylation of GSK3␤ (0.58 Ϯ 0.18 and 0.46 Ϯ 0.07 arbitrary units for cells incubated without or with leptin, respectively; n ϭ 5, P ϭ 0.43). Effect of leptin on SOCS-3 expression and insulin receptor tyrosine phosphorylation. As shown in Fig. 3A and B, incubation of adipocytes with 50 nmol/l leptin for 6 h significantly increases the cellular amount of SOCS-3 (ϳ50%). Moreover, in cells preincubated with leptin, insulin-dependent insulin receptor ␤-subunit phosphorylation is markedly lower than in adipocytes incubated without leptin (Fig. 3C and D). Basal insulin receptor phosphory- FIG. 2. Stimulation of GSK3␤ phosphorylation by insulin and vanadate: lation was not significantly different between cells prein- the effect of leptin. A: Representative blot from five separate experi- Ϯ Ϯ ments. B: Quantitation of p-GSK3␤. Data show the fold stimulation by cubated without or with leptin (0.67 0.05 and 0.83 0.10 insulin (Ⅺ) or vanadate (f) and are mean ؎ SE. *P < 0.01 vs. insulin arbitrary units, respectively; n ϭ 4, P ϭ 0.28). effect in cells without leptin.

DIABETES, VOL. 53, FEBRUARY 2004 349 LEPTIN IMPAIRS ADIPOCYTE INSULIN SIGNALING

FIG. 3. Effect of leptin on adipocyte SOCS-3 protein expression and IR tyrosine phosphorylation. A: A representative blot illustrating the ؎ presence of SOCS-3 and insulin receptor ␣ in cell lysates. B: Amount of SOCS-3 relative to the amount of insulin receptor-␣. Data are means and refer to the amount of cells treated without leptin. *P < 0.05 vs. cells without leptin. C: Representative autoradiogram illustrating (5 ؍ SE (n the tyrosine phosphorylation of the insulin receptor-␤ subunit. D: Insulin receptor ␤ phosphorylation. Data show the fold stimulation by insulin .P < 0.01 vs. cells treated without leptin. IR, insulin receptor* .(4 ؍ and are the means ؎ SE (n iment were 1.4 Ϯ 0.1, 1.4 Ϯ 0.4, and 1.1 Ϯ 0.3 ng/ml for rats units for rats treated with saline or 0.2 and 10 ␮g/day infused with saline or with 0.2 and 10 ␮g/day leptin, leptin, respectively; P ϭ 0.73). Figure 4 also shows that the respectively. As shown in Fig. 4, in adipocytes isolated stimulation of adipocyte GSK3␤ phosphorylation by insu- from rats infused with saline, insulin stimulates MAPK lin is decreased after the infusion of a daily dose of 0.2 ␮g activity Ͼ2.2-fold. In contrast, in fat cells from rats infused leptin, without significant changes in the basal phosphor- with two different doses of leptin, the insulin effect on ylation (0.25 Ϯ 0.07 and 0.44 Ϯ 0.06 arbitrary units, for MAPK is significantly lower (Ͻ1.4-fold). Leptin infusion saline-infused and leptin-treated animals, respectively; n ϭ for 1 week did not modify the basal MAPK activity of 4, P ϭ 0.11). adipocytes (1.3 Ϯ 0.2, 1.03 Ϯ 0.2, and 1.13 Ϯ 0.28 arbitrary Central effect of leptin on adipocyte insulin signaling in leptin-resistant aged rats: effect of food restriction. We next focused our attention on the stimulation of MAPK by insulin in adipocytes from 24-month-old rats, which show hyperleptinemia and central leptin resistance (31,32), in addition to overall (37) and adipocyte (34,35,37) insulin resistance. As shown in Fig. 5, in adipocytes from aged rats fed ad libitum and infused intracerebroventricularly with saline over 7 days, insulin elicits poor stimulation of MAPK activity (1.4-fold), which is similar to that observed previously in adipocytes from 24-month-old animals without pump implantation (35). Infusion of a daily dose of 0.2 ␮g leptin for 7 days did not modify the circulating leptin concentration (8.8 Ϯ 2.7 in saline vs. 9.0 Ϯ 3.2 ng/ml in leptin-infused rats) and did not cause further inhibition of insulin stimulation of adipocyte MAPK (Fig. 5). Basal MAPK activities were similar in leptin-treated and saline-infused animals (0.75 Ϯ 0.21 and 0.40 Ϯ 0.12 arbitrary units, respectively; n ϭ 5, P ϭ 0.4). The food restriction used in these experiments lowers serum leptin levels to 2.7 Ϯ 0.4 ng/ml, a value close to that of young rats, and improves adipocyte insulin sensitivity as demonstrated by the fact that insulin stimulates MAPK activity Ͼ1.9-fold (Fig. 5). Interestingly, it also restores the ability of intracerebroventricularly infused leptin to mod- FIG. 4. Effect of centrally infused leptin on insulin stimulation of ulate adipocyte insulin responsiveness as demonstrated by adipocyte MAPK and GSK3␤ phosphorylation. A: Representative auto- the low stimulation of MAPK activity by insulin (1.2-fold) radiograms illustrating the insulin-dependent phosphorylation of MBP and GSK3␤ in adipocytes from rats treated or not intracerebroven- in adipocytes from food-restricted old rats infused daily tricularly with leptin. B: Fold stimulation by insulin. Data are means ؎ with 0.2 ␮g leptin. As in the case of old animals fed ad SE of four to six animals per group. For MBP phosphorylation, one-way P < 0.05; libitum, this treatment did not increase serum leptin levels* .(0.01 ؍ ANOVA indicates a significant effect of leptin (P **P < 0.01 vs. saline-infused animals. i.c.v., intracerebroventricular. (1.4 Ϯ 0.2 ng/ml), and basal MAPK activities for saline-

350 DIABETES, VOL. 53, FEBRUARY 2004 C. PE´ REZ AND ASSOCIATES

DISCUSSION Since its identification as an adipocyte-derived hormone, leptin has been demonstrated to play a key role in the regulation of energy balance, modulating appetite, energy expenditure, and partitioning of fuels in the organism (4). Compelling evidence suggests that fat tissue is not only the main source of leptin but also a target of it. The physiological effects of leptin on adipose tissue can be mediated by neural circuits and/or elicited in an autocrine manner (4). Most of the effects of leptin are likely mediated by its interaction with hypothalamic leptin receptors. Neverthe- less, under conditions of pathological or experimentally induced hyperleptinemia, direct metabolic actions on several tissues, including fat tissue, have been reported (16– 19). In this work, we studied the effect of leptin on adipocyte insulin signaling. Direct action was analyzed using isolated fat cells preincubated over 6 h with 50 nmol/l leptin, conditions previously shown to inhibit insulin metabolic action in adipocytes (21). On the other hand, FIG. 5. Stimulation of MAPK by insulin in old rat adipocytes: the effect neural-mediated action was studied in adipocytes isolated of centrally infused leptin and food restriction. Leptin (0.2 ␮g/day) or saline was infused intracerebroventricularly over 7 days in 24-month- from intracerebroventricularly leptin-infused rats over 7 old rats, which were either fed ad libitum (AL) or under food restric- days, under conditions that do not increment the level of tion (FR). A: Representative autoradiogram illustrating the insulin- dependent phosphorylation of MBP. B: Quantitation of phospho-MBP. circulating leptin, precluding a direct effect of the hor- .(The figure shows the fold stimulation by insulin, and data are means ؎ mone (32 SE of six to eight animals. *P < 0.05 vs. all other groups. The data reported herein indicate that prolonged incubation of adipocytes with leptin impairs the two main infused and leptin-treated food-restricted rats were similar branches of insulin signal transduction (38), as is shown (0.85 Ϯ 0.12 and 0.93 Ϯ 0.1 arbitrary units, respectively; by the lower stimulation of both MAPK activity and n ϭ 5, P ϭ 0.65). Low insulin sensitivity in adipocytes from phosphorylation of GSK3. Our data also indicate that older rats could be due to the increased expression of leptin treatment does not impair the activation of insulin SOCS-3 mRNA observed in adipose tissue of 24-month-old signal pathways by vanadate, which stimulates insulin animals if compared with 3-month-old rats. Interestingly, receptor substrate (IRS)-1–associated phosphatidylinosi- the restoration of insulin responsiveness after food restric- tol 3-kinase in adipocytes (34) without activating insulin tion is paralleled by a decrease in SOCS-3 mRNA in receptor autophosphorylation. As stimulation of phospha- adipose tissue (Fig. 6). tidylinositol 3-kinase plays a key role in the phosphorylation and subsequent inhibition of GSK3 in response to insulin (38) as well as in the stimulation of MAPK by insulin and vanadate in adipocytes (35), our data suggest that leptin impairs insulin signaling at an early step of the signal cascade. Leptin is known to induce the expression of SOCS-3 in hypothalamic nuclei (39) and in cell lines (40,41), which is involved in a negative feedback loop to terminate leptin signaling (20). Additionally, SOCS-3 has been shown to inhibit insulin signaling in several cell lines (42–44). The data in this work demonstrate that treatment of adipocytes with 50 nmol/l leptin increases the cellular amount of SOCS-3. Different mechanisms of insulin-signaling inhibition by SOCS-3 have been proposed for different cell lines. In COS-7 fibroblasts, SOCS-3 inhibits the insulin-dependent phosphorylation of IRS-1 but not the autophosphorylation of the insulin receptor (42), suggesting that it competes with IRS-1 for binding to the insulin receptor. In contrast, in HepG2 cells, SOCS-3 associates with the insulin receptor and suppress its insulin-dependent autophosphorylation (44). Although we were unsuccessful in observing a direct association between the insulin recep- FIG. 6. SOCS-3 mRNA expression in adipose tissue: the effect of age and food restriction. A: The cDNA, amplified by RT-PCR from SOCS-3 tor and SOCS-3, our data indicate that in leptin-treated and actin, in the adipose tissue of four animals per group. B: The adipocytes, insulin-dependent autophosphorylation is quantitation analysis is shown. The amount of amplified SOCS-3 was markedly decreased. Therefore, it can be speculated that expressed relative to the amount of amplified actin, as nanograms of the increased amount of SOCS-3 could play a role in this ؍ SOCS-3 cDNA per nanogram of actin cDNA. Data are means ؎ SE (n 4). *P < 0.05 vs. young; **P < 0.01 vs. old. FR, food restricted. inhibition, such as that which occurs in HepG2 cells. The

DIABETES, VOL. 53, FEBRUARY 2004 351 LEPTIN IMPAIRS ADIPOCYTE INSULIN SIGNALING decreased insulin receptor autophosphorylation observed persistent impairment of insulin signaling, which agrees in leptin-treated adipocytes would explain the lower stim- with the poor effect of insulin on MAPK activity observed ulation by insulin of GSK3 phosphorylation and MAPK in aged rats. Central infusion of leptin has no additional activity, as well as its normal stimulation by vanadate. inhibitory effect on insulin sensitivity. This is in agreement From the former data it can be concluded that leptin, at with the presence of central leptin resistance in these rats elevated concentrations, is able to downmodulate insulin (32). Prolonged moderate caloric restriction lowers adi- signaling in adipose cells, whereas lower concentrations posity index and hyperleptinemia in aged rats if compared have no evident effect on insulin signaling. The concentra- with aged-matched animals fed ad libitum (32). As shown tion of leptin that is effective in inhibiting insulin signaling here, this improves insulin sensitivity in adipose tissue in is similar to that previously shown (21) to cause the parallel with a decrease in SOCS-3 expression, indicating inhibition of insulin stimulation of glucose transport and that the impairment of insulin signaling mediated by leptin other metabolic effects in adipocytes. Although physio- in the adipose tissue of aged rats can be reverted. As logic serum leptin concentrations are markedly lower, in reported previously (32,36), caloric restriction also im- situations of obesity and/or aging, circulating leptin levels proves central leptin sensitivity. This agrees well with the can be Ͼ3 nmol/l and the interstitial concentration of restoration of the central leptin effect on adipocyte insulin leptin surrounding adipocytes might be extremely high sensitivity, as manifested by the marked inhibition of the (45). Therefore, our data suggest that in these circum- effect of insulin on MAPK in adipocytes from leptin- stances that are characterized by hyperleptinemia and infused, food-restricted aged rats. central leptin resistance, leptin might act as an autocrine In conclusion, the data in this work demonstrate that factor that induces insulin resistance in adipose tissue. leptin can downmodulate insulin signaling in rat adipo- The data in this work also show that leptin, acting cytes in two different ways. In animals with normal physiological circulating leptin concentrations, leptin ac- through the central nervous system, modulates adipocyte tion takes place by stimulation of hypothalamic neural insulin sensitivity in 3-month-old rats, as demonstrated by circuits and the autonomic nervous system, likely prevent- the inhibition of the insulin effect on MAPK activity and ing excessive fat storage. On the other hand, at elevated GSK3␤ phosphorylation. This effect must be mediated by leptin concentrations, leptin also inhibits insulin signaling, the autonomic nervous system because the central infu- impairing insulin receptor autophosphorylation. This sion of leptin does not increase the leptin concentration in could be related to the increased expression of SOCS-3 plasma. Nevertheless, because the intracerebroventricular protein reported herein. Hypothalamic-independent mod- concentration reached after leptin infusion is unknown, it ulation of adipocyte insulin signaling could be relevant is difficult to evaluate whether physiopathological in- under physiological situations of hyperleptinemia and creases in serum leptin concentration would be able to central leptin resistance, such as aging and obesity. The induce intracerebroventricular leptin levels sufficient to data presented herein also suggest that as central leptin exert this inhibitory effect on adipocyte insulin signaling. resistance develops with aging and/or obesity, the increase Although intracerebroventricular leptin infusion causes a in fat mass leads to hyperleptinemia. This circumstance decrease in food intake and body weight (32), it is unlikely induces the expression of SOCS-3, which would block the that those changes account for the lower insulin sensitivity lipopenic effect of leptin (45) and likely inhibit insulin of adipocytes because lowering body weight by caloric signaling in fat tissue. As suggested by others (47), this restriction is associated with an improvement of insulin could represent an early step leading to the overall insulin signaling. These data agree with previous results (6,9,10), resistance characteristic of aged and/or obese rats. Inter- indicating that leptin has insulin-desensitizing effects, spe- estingly, caloric restriction restores adipocyte insulin sen- cifically in white adipose tissue. As the main role of insulin sitivity as well as the hypothalamic-dependent leptin in adipose tissue is to promote the incorporation of modulation of adipocyte insulin signaling. glucose into triglycerides, this central effect of leptin on insulin signaling might contribute to the control of fat storage in addition to the downregulation of the lipogenic ACKNOWLEDGMENTS enzymes that it induces (46). Accretion of fat tissue seems This work was supported by grants BFI2002-04030 from to be a cause of whole-body insulin resistance. In fact, Ministerio de Ciencia y Tecnologıá (Spain), PIGE 02-17 surgical removal of visceral adipose tissue has been re- from Universidad Rey Juan Carlos (Spain), and 08.6/0009/ ported to prevent insulin resistance in aging rats (27). 2001 from Comunidad de Madrid (Spain). Thus, the central action of leptin is likely playing a role in preserving the overall insulin sensitivity of the organism. REFERENCES Aging rats develop central leptin resistance (30–32,36), 1. Friedman JM, Halaas JL: Leptin and the regulation of body weight in fat tissue expansion, and compensatory hyperleptinemia. mammals. Nature 395:763–770, 1998 Our data show that this brings about an increase in SOCS-3 2. Porte D Jr, Seeley RJ, Woods SC, Baskin DG, Figlewicz DP, Schwartz MW: expression in adipose tissue. These data agree well with Obesity, diabetes and the central nervous system. Diabetologia 41:863– 881, 1998 the observed increment in SOCS-3 in leptin-treated adipo- 3. Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG: Central cytes that is reported herein. This increase in SOCS-3 is nervous system control of food intake. Nature 404:661–671, 2000 likely a mechanism to block autosuppression of adipose 4. Kahn BB, Flier JS: Obesity and insulin resistance. J Clin Invest 106:473– tissue by leptin, allowing adipocytes to meet the elevated 481, 2000 5. Ceddia RB, Koistinen HA, Zierath JR, Sweeney G: Analysis of paradoxical physiological demand of this factor, as has been previ- observations on the association between leptin and insulin resistance. ously reported (45) for other models of nongenetic obe- FASEB J 16:1163–1176, 2002 sity. Additionally, elevated levels of SOCS-3 might induce a 6. Wang J-L, Chinookoswong N, Scully S, Qi M, Shi Z-Q: Differential effects of

352 DIABETES, VOL. 53, FEBRUARY 2004 C. PE´ REZ AND ASSOCIATES

leptin in regulation of tissue glucose utilization in vivo. Endocrinology 27. Gabriely I, Ma XH, Yang XM, Atzmon G, Rajala MW, Berg AH, Scherer P, 140:2117–2124, 1999 Rossetti L, Barzilai N: Removal of visceral fat prevents insulin resistance 7. Sivitz WI, Walsh SA, Morgan DA, Thomas MJ, Haynes WG: Effects of leptin and glucose intolerance of aging: an adipokine-mediated process? Diabe- on insulin sensitivity in normal rats. Endocrinology 138:3395–3401, 1997 tes 51:2951–2958, 2002 8. Barzilai N, Wang J, Massilon D, Vuguin P, Hawkins M, Rossetti L: Leptin 28. Abel ED, Peroni OD, Kim JK, Kim Y-B, Boss O, Hadro E, Minnemann T, selectively decreases visceral adiposity and enhances insulin action. J Clin Shulman GI, Kahn BB: Adipose-selective targeting of the GLUT4 gene Invest 100:3105–3110, 1997 impairs insulin action in muscle and liver. Nature 409:729–733, 2001 9. Minokoshi Y, Haque MS, Shimazu T: Microinjection of leptin into the 29. Tozzo E, Gnudi L, Kahn BB: Amelioration of insulin resistance in strepto- ventromedial hypothalamus increases glucose uptake in peripheral tissues zotocin diabetic mice by transgenic overexpression of GLUT4 driven by an in rats. Diabetes 48:287–291, 1999 adipose-specific promoter. Endocrinology 138:1604–1611, 1997 10. Rouru J, Cusin I, Zakrzewska KE, Jeanrenaud B, Rohner-Jeanrenaud F: 30. Li H, Matheny M, Nicolson M, Tu¨ mer S, Scarpace PJ: Leptin gene Effects of intravenously infused leptin on insulin sensitivity and on the expression increases with age independent of increasing adiposity in rats. expression of uncoupling proteins in brown adipose tissue. Endocrinology Diabetes 46:2035–2039, 1997 140:3688–3692, 1999 31. Fernańdez-Galaz C, Fernańdez-Agullo´ T, Campoy F, Arribas C, Gallardo N, 11. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Andre´s A, Ros M, Carrascosa JM: Decreased leptin uptake in hypothalamic Collins F: Effects of the obese gene product on body weight regulation in nuclei with ageing in Wistar rats. J Endrocinol 171:23–32, 2001 ob/ob mice. Science 269:540–543, 1995 32. Fernańdez-Galaz C, Fernańdez-Agullo´ T, Pe´rez C, Peralta S, Arribas C, 12. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Andre´s A, Carrascosa JM, Ros M: Long-term food restriction prevents Lallone RL, Burley SK, Friedman JM: Weight-reducing effects of the plasma ageing-associated central leptin resistance in wistar rats. Diabetologia protein encoded by the obese gene. Science 269:543–546, 1995 45:997–1003, 2002 13. Shimomura L, Hammer RE, Ikemoto S, Brown MS, Goldstein JL: Leptin 33. Traxinger RR, Marshall S: Coordinated regulation of glutamine: fructose- reverses insulin resistance and diabetes mellitus in mice with congenital 6-phosphate amidotransferase activity by insulin, glucose and glutamine. lipodystrophy. Nature 401:73–76, 1999 J Biol Chem 266:10148–10154, 1991 14. Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide 34. Molero JC, Martıńez C, Andre´s A, Satru´ stegui J, Carrascosa JM: Vanadate T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, fully stimulates insulin receptor substrate-1 associated phosphatidyl ino- Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, sitol 3-kinase activity in adipocytes from young and old rats. FEBS Lett Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T: The fat-derived 425:298–304, 1998 hormone adiponectin reverses insulin resistance associated with both 35. Molero JC, Pe´rez C, Martıńez C, Villar M, Andre´s A, Fermıń Y, Carrascosa lipoatrophy and obesity. Nat Med 7:941–946, 2001 JM: Activation of MAP kinase by insulin and vanadate in adipocytes from 15. Petersen KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, Cline GW, De young and old rats. Mol Cell Endocrinol 189:77–84, 2002 Paoli AM, Taylor SI, Gorden P, Shulman GI: Leptin reverses insulin 36. Peralta S, Carrascosa JM, Gallardo N, Ros M, Arribas C: Ageing increases resistance and hepatic steatosis in patients with severe lipodystrophy. SOCS-3 expression in rat hypothalamus: effects of food restriction. Bio- J Clin Invest 109:1345–1350, 2002 chem Biophys Res Commun 296:425–428, 2002 16. Lee Y, Wang M-Y, Kakuma T, Wang Z-W, Babcock E, McCorkle K, Higa M, 37. Escriva´ F, Agote M, Rubio E, Molero JC, Pascual-Leone AM, Andre´sA, Zhou Y-T, Unger RH: Liporegulation in diet-induced obesity: the antistea- Satru´ stegui J, Carrascosa JM: In vivo insulin-dependent glucose uptake of totic role of hyperleptinemia. J Biol Chem 276:5629–5635, 2001 specific tissues is decreased during aging of mature Wistar rats. Endocri- 17. Minokoshi Y, Kim Y-B, Peroni OD, Fryer LGD, Mu¨ ller C, Carling D, Kahn nology 138:49–54, 1997 BB: Leptin stimulates fatty-acid oxidation by activating AMP-activated 38. Saltiel AR, Kahn CR: Insulin signalling and the regulation of glucose and protein kinase. Nature 415:339–343, 2002 lipid metabolism. Nature 414:799–806, 2001 18. Atkinson LL, Fischer MA, Lopaschuk GD: Leptin activates cardiac fatty 39. Bjorbaek C, Elmquist JK, Frantz JD, Shoelson SE, Flier JS: Identification of acid oxidation independent of changes in the AMP-activated protein SOCS-3 as a potential mediator of central leptin resistance. Mol Cell kinase: acetyl-CoA carboxylase-malonyl-CoA axis. J Biol Chem 277:29424– 1:619–625, 1998 29430, 2002 40. Bjorbaek C, El-Haschimi K, Frantz JD, Flier JS: The role of SOCS-3 in leptin 19. Wang Z-W, Zhou Y-T, Kakuma T, Lee Y, Higa M, Kalra SP, Dube MG, Kalra signaling and leptin resistance. J Biol Chem 274:30059–30065, 1999 PS, Unger RU: Comparing the hypothalamic and extrahypothalamic ac- 41. Banks AS, Davis SM, Bates SH, Myers MG Jr: Activation of downstream tions of endogenous hyperleptinemia. Proc Natl Acad SciUSA96:10373– signals by the long form of the leptin receptor. J Biol Chem 275:14563– 10378, 1999 14572, 2000 20. Spiegelman BM, Flier JS: Obesity and the regulation of energy balance. Cell 42. Emanuelli B, Peraldi P, Filloux C, Chavey C, Freidinger K, Hilton DJ, 104:531–543, 2001 Hotamisligil GS, Van Obberghen E: SOCS-3 inhibits insulin signaling and is 21. Mu¨ ller G, Ertl J, Gerl M, Preibisch G: Leptin impairs metabolic actions of up-regulated in response to tumor necrosis factor-␣ in the adipose tissue of insulin in isolated rat adipocytes. J Biol Chem 272:10585–10593, 1997 obese mice. J Biol Chem 276:47944–47949, 2001 22. Ceddia RB, William WN Jr, Lima FB, Curi R: Leptin inhibits insulin- 43. Rui L, Yuan M, Frantz D, Shoelson SE, White MF: SOCS-1 and SOCS-3 stimulated incorporation of glucose into lipids and stimulates glucose block insulin signaling by ubiquitin-mediated degradation of IRS1 and decarboxylation in isolated rat adipocytes. J Endocrinol 158:R7–R9, 1998 IRS2. J Biol Chem 277:42394–42398, 2002 23. Ceddia RB, William WN Jr, Lima FB, Flandin P, Curi R, Giacobino J-P: 44. Senn JJ, Klover PJ, Nowak IA, Zimmer TA, Koniaris LG, Furlanetto RW, Leptin stimulates uncoupling protein-2 mRNA expression and Krebs cycle Mooney RA: Suppressor of cytokine signaling-3 (SOCS-3): a potential activity and inhibits lipid synthesis in isolated rat white adipocytes. Eur mediator of interleukin-6 dependent insulin resistance in hepatocytes. J Biochem 267:5952–5958, 2000 J Biol Chem 278:13740–13746, 2003 24. Zierath JR, Frevert EU, Ryder JW, Berggren PO, Kahn BB: Evidence 45. Wang Z, Zhou Y-T, Kakuma T, Lee Y, Kalra SP, Kalra PS, Pan W, Unger RH: against a direct effect of leptin on glucose transport in skeletal muscle and Leptin resistance of adipocytes in obesity: role of suppressors of cytokine adipocytes. Diabetes 47:1–4, 1998 signaling. Biochem Biophys Res Commun 277:20–26, 2000 25. Ranganathan S, Ciaraldi TP, Henry RR, Mudaliar S, Kern PA: Lack of effect 46. Zhou Y-T, Wang Z-W, Higa M, Newgard CB, Unger RH: Reversing adipocyte of leptin on glucose transport, lipoprotein lipase, and insulin action in differentiation: Implications for treatment of obesity. Proc Natl Acad Sci U adipose and muscle cells. Endocrinology 139:2509–2513, 1998 SA96:2391–2395, 1999 26. Barzilai N, Banerjee S, Hawkins M, Chen W, Rossetti L: Caloric restriction 47. Smith U: Impaired (“diabetic”) insulin signaling and action occur in fat reverses hepatic insulin resistance in aging rats by decreasing visceral fat. cells long before glucose intolerance: is insulin resistance initiated in the J Clin Invest 101:1353–1361, 1998 adipose tissue? Int J Obes 26:897–904, 2002

DIABETES, VOL. 53, FEBRUARY 2004 353