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The melanocortin system is crucial to regulation of energy homeostasis. The melanocortin receptor type 4 (MC4R) modulates insulin signaling via effects on c-Jun N-terminal kinase (JNK). The melanocortin agonist NDP-MSH dose-dependently inhibited JNK activity in HEK293 cells stably expressing the human MC4R; effects were reversed by melanocortin receptor antagonist. NDP-MSH time- and dose-dependently inhibited IRS-1ser307 phosphorylation, effects also reversed by a specific melanocortin receptor antagonist. NDP-MSH augmented insulin-stimulated AKT phosphorylation in vitro. The melanocortin agonist melanotan II increased insulin-stimulated AKT phosphorylation in the rat hypothalamus in vivo. NDP-MSH increased insulin-stimulated glucose uptake in hypothalamic GT1-1 cells. The current study shows that the melanocortinergic system interacts with insulin signaling via novel effects on JNK activity.
In the hypothalamus, melanocortin signaling is a key component of energy homeostasis. The melanocortinergic system consists of the prohormone proopiomelanocortin (POMC), its derivative melanocortin peptides (α-MSH, β-MSH, γ-MSH), adrenocorticotropic hormone (ACTH), and the endogenous melanocortin peptide antagonists, agouti and agouti-related peptide (AgRP). In hypothalamic control of caloric intake, the relevant components of the melanocortin system include POMC-derived peptides, the antagonist AgRP, and melanocortin receptor types 3 and 4 (MC3R and MC4R) [9, 12].
Melanocortin receptors belong to a superfamily of seven transmembrane G-protein coupled receptors. Conventional understanding of melanocortin receptor signaling involves the cyclic AMP transduction pathway via a Gs protein and adenylyl cyclase [1, 13]. MC3R signaling has also been associated with changes in intracellular Ca2+ and inositol trisphosphate levels and with the protein kinase C pathway [20, 26]. Previous studies have demonstrated that MC3R and MC4R activation can also stimulate extracellular signal-regulated kinases (ERK) activation, implying that multiple signal pathways may exist beyond the traditional cAMP pathway [6, 7, 10, 33].
Growth factors, hormones, cytokines and environmental stressors such as ultraviolet radiation have the capacity to activate stress-activated protein/mitogen-activated kinase pathways (SAP/MAP). Protein kinases in these groups include extracellular signal-regulated kinases (ERK-1, -2 and -5), the four p38 isoforms (α, β, γ and δ) and the c-Jun-N-terminal kinase isoforms (JNK-1, -2 and -3). Numerous studies have demonstrated that SAP/MAP pathways regulate cellular proliferation, apoptosis and cellular differentiation, and members of these protein kinase families and upstream kinases have been implicated in a variety of human diseases [22, 30, 34]. Although initially identified by their ability to phosphorylate c-Jun in response to UV-irradiation, JNKs are now recognized to have a central role in obesity and obesity-related insulin resistance [15, 31]. In the present study, interactions between the MC4R and insulin signaling pathways were demonstrated via alterations in JNK activity. Mediation of JNK activity by the melanocortinergic system provides a mechanism by which insulin signaling may be regulated within the hypothalamus.
NDP-MSH (α-MSH analogue, [Nle4, D-Phe7] α-MSH), MT II, and SHU9119 were purchased from Bachem (King of Prussia, PA).β-actin antibody and FLAG-M2 antibody-conjugated agarose were purchased from Sigma-Aldrich (St. Louis, MO). Phospho-AKT (Thr308) antibody and GST-c-Jun (1–89) were obtained from Cell Signaling Technologies (Beverly, MA). Rabbit HA antibodies were purchased from BD Clontech (Palo Alto, CA). Anti-phospho-IRS-1(Ser 307) was purchased from Upstate (Lake Placid, NY). HA-IRS-1 was provided by Dr. Liangyou Rui (Department of Molecular & Integrative Physiology, University of Michigan). FLAG-JNK1 vector was provided by Dr. Deepak Nihalani (Department of Internal Medicine, University of Michigan). Secondary IgG HRP antibody was purchased from Santa Cruz Biotechnology. Anisomycin and SP600125 were from Calbiochem (San Diego, CA).
HEK293 cells, stably transfected with the coding region of human MC4R gene expressed in pcDNA3.1 (Invitrogen, Carlsbad, CA), were used for this study. This cell line has previously been characterized [36, 37]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) with 100 U/ml penicillin and 100 U/ml streptomycin. GT1-1 cells (a generous gift from Dr. Richard I. Weiner, University of California-San Francisco) were cultured in DMEM with 10% FBS and 100 U/ml penicillin and 100 U/ml streptomycin. Cells were plated on 100 mm dishes and maintained at 37°C in a water-saturated atmosphere of 95% O2 and 5% CO2.
The JNK immunocomplex kinase assay was performed as previously described [5, 24]. Plasmid (1μg/well) encoding Flag-JNK1 was transfected into HEK293 cells expressing MC4R in six well plates. Forty hours after transfection, cells were exposed to serum-free medium overnight. After treatment, cells were lysed in PK buffer [50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1mM Na3VO4, 50 mM NaF, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail (Sigma)]. Equal amounts of lysate proteins from each well were incubated with M2 monoclonal antibody conjugated to agarose for 18 hr at 4°C. Beads were washed twice with PK lysis buffer and twice with kinase buffer (25 mM HEPES, pH 7.4, 20 mM MgCl2, 0.5 mM EGTA, 12.5 mM β-glycerophosphate, 0.1 mM orthovanadate, 0.5 mM NaF). The complex was incubated for 30 min at 30°C in 30 μl of kinase buffer containing 20 μM ATP, 5μCi [γ-32P ]ATP, and 2μg GST-c-Jun (1–89). The reaction was terminated by adding 10 μl 4 × SDS loading buffer and heating at 80°C for 5 min. The reaction mixture was subjected to SDS-PAGE electrophoresis, transferred to polyvinylidene membrane and exposed to X-ray film.
HEK293 cells expressing MC4R were cultured in six well plates. Cells were transfected with plasmid (1μg/well) encoding Flag-JNK1 and HA-IRS-1(1μg/well). Forty hours after transfection, cells were exposed to serum-free medium for 16 hours. After treatment, cell lysates were used to assay for phospho-IRS-1ser307 by Western blotting.
Protein samples from cell lysates (15–20 μg) were subjected to electrophoretic separation on a 10% polyacrylamide gel (BioRad) and then transferred onto Immobilon™-P PVDF membrane (Millipore Corp., Bedford, MA). Blots were blocked at room temperature for 1 h in 5% milk in TBS-Tween 20 (0.05%) and then incubated overnight in primary antibody diluted 1/1000. Membranes were washed three times in TBS-Tween (0.05%) and then incubated for 1 h with secondary antibody, diluted 1/5000 in 5% TBS-Tween (0.05%). Detection was performed using Lumi-Light Blotting Substrate (Roche, Indianapolis, IN).
AKT, a downstream signaling molecule of PI3K, was detected by Western blot analysis using phospho-AKT antibody.
The hypothalamic neuronal cell line GT1-1 was maintained in DMEM supplemented with 10% fetal bovine serum. Cells were cultured for 24 hours, then media was changed to serum-free DMEM containing 5 mM glucose and 1% bovine serum albumin for 2 hr at 37°C.
Before glucose transport measurements, cells were washed with KRH buffer (20 mM HEPES (pH 7.4) 1.25 mM MgSO4, 1.25 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, and 0.1% bovine serum albumin). Glucose transport was determined using KRH buffer containing 0.1 mM 2-deoxyglucose and 0.5 μCi of 2-[1,2-3H]-deoxy-D-glucose (Sigma, St. Louis, MO). Nonspecific uptake, measured in the presence of 10 μM cytochalasin B, was subtracted from measured values. Glucose transport experiments were terminated after 10 min by aspiration and three washes with ice-cold phosphate-buffered saline. Cells were lysed in 0.1% SDS in phosphate-buffered saline and sonicated. Radioactivity was determined by scintillation counting [23, 24].
Sprague Dawley rats with a body weight of 280–300g were used. Rats were anaesthetized with ketamine/xylazine and placed on a stereotaxic device with the incisor bar 3.3 mm below the interaural line according to Paxinos and Watson . A stainless steel 26 gauge guide cannula was implanted into the third ventricle using the following stereotaxic coordinates: 2.2 mm posterior to the bregma, 8.2 mm ventral to the surface of the skull and directly along the midline. The cannula was anchored to the skull with screws and dental cement. An internal cannula (17 mm) was placed into the guide cannula to maintain patency. Rats were allowed to recover for 1 week. Guide cannula patency was assessed by injection of 10 ng angiotensin II in 5 μl of saline. Cannulas were considered patent if rats consumed at least 5 ml of water within 1 hr of injection. Rats with correct third ventricle cannulation were used five days later.
The rats were injected with either MT II (50 ng/rat), insulin (1.3 mU), both MT II and insulin (in 5 μl of saline), or saline into the third ventricle through a guide cannula [8, 27]. The length of the injection needle is 17 mm. The rats were sacrificed after 20 minutes. Hypothalamus was rapidly dissected, placed into a 1.5-ml microcentrifuge tube with 250 μl ice-cold lysis buffer, and homogenized using a Dounce homogenizer. Homogenates were centrifuged and supernatants were used to measure phospho-AKT by Western blot analysis.
Experiments were performed at least three times. For Western blots, analysis of densitometry was performed using Kodak 1D 3.6 software (Eastman Kodak, New Haven, CT). Data was analyzed using Graphpad Prism 4.0 (Graphpad Software, San Diego, CA), and expressed as mean ± SEM. Differences were analyzed by unpaired two-tailed Student’s t test. A value of p < 0.05 was taken as significant.
HEK293 cells expressing MC4R were transfected with a Flag-JNK vector. The melanocortin agonist NDP-MSH (0.1 μM) inhibited both basal and insulin-stimulated (0.1 μM) JNK activity (Figure 1). The inhibition of insulin-stimulated JNK activity by NDP-MSH was dose-dependent (Figure 2). The melanocortin receptor antagonist, SHU9119, reversed the inhibitory effect of NDP-MSH on JNK activity (Figure 2). These results demonstrate that NDP-MSH-induced inhibition of JNK activity is a MC4R-mediated effect.
It has been reported that JNK activation increases IRS-1ser307 phosphorylation . HA-IRS-1 and Flag-JNK vector were transfected into HEK293 cells expressing MC4R. NDP-MSH inhibited IRS-1ser307 phosphorylation, reaching maximum at 20 and 30 minutes, and returning to control values at 60 minutes (Figure 3). NDP-MSH inhibition of IRS-1ser307 phosphorylation was dose-dependent over a range of 0.1 nM to 1 μM (Figure 4). The melanocortin receptor antagonist SHU9119 dose-dependently reversed NDP-MSH-induced inhibition of IRS-1ser307 phosphorylation, indicating that the effect of NDP-MSH on IRS-1ser307 phosphorylation is mediated by MC4R (Figure 5).
The above results are consistent with an interpretation that NDP-MSH inhibits JNK activity and IRS-1ser307 phosphorylation by activating MC4R. To examine the effects of JNK on IRS-1ser307 phosphorylation, the JNK activator, anisomycin, and the JNK inhibitor, SP600025, were utilized. NDP-MSH decreased anisomycin-stimulated IRS-1ser307 phosphorylation (Figure 6A). Inhibition of JNK by SP600125 decreased basal IRS-1ser307 phosphorylation in HEK293 cells expressing MC4R (Figure 6B), implying that direct JNK inhibition decreases IRS-1ser307 phosphorylation. The inhibitory effects of SP600025 were additive to those of NDP-MSH (Figure 6B).
MC4R activation inhibits IRS-1ser307 phosphorylation, implying that MC4R activation modulates insulin signaling. In order to further examine the effects of MC4R activation on insulin signaling, the effect of NDP-MSH on insulin-stimulated AKT phosphorylation was tested. NDP-MSH promoted insulin-stimulated AKT phosphorylation in HEK cells expressing MC4R (Figure 7A). Similar results were observed in GT1-1 cells (Figure 7B). GT1-1 cells are derived from hypothalamus, and endogenously express MC4R [7, 19].
MT II and DNP-MSH are analogues of MSH. MT II has been widely used in vivo because of its high potency in vivo and ability to across the blood-brain barrier. They have the same downstream signaling mechanisms as the parent compound. Intracerebroventricular injection of insulin (1.5 mU) significantly increased AKT phosphorylation in rat hypothalamus. The melanocortin agonist, MT II (50ng), alone did not increase AKT phosphorylation, whereas administration of both insulin and MT II significantly enhanced AKT phosphorylation relative to insulin alone (Figure 8).
The effects of MC4R activation on insulin-stimulated glucose uptake were next investigated. Insulin (10−7M) significantly increased glucose uptake in GT1-1 cells. NDP-MSH (10−7M) alone did not affect basal glucose uptake, but the combination of NDP-MSH and insulin significantly increased glucose uptake relative to the effects of insulin alone (Figure 9).
The melanocortinergic signaling system has important biological functions beyond feeding behavior. For example, MC3R and MC4R mediate inflammatory responses and regulate apoptosis [7, 14, 21]. The range of recognized biological actions is reflected in a diversity of signaling pathways. The current study demonstrates that melanocortin signaling regulates the activity of c-Jun NH2-terminal kinases, and in turn, has effects on IRS-1 and AKT phosphorylation. NDP-MSH inhibited JNK activity in cells expressing human MC4R. NDP-MSH inhibited phosphorylation of IRS-1ser307, effects that were dose-dependent, reversible by the antagonist SHU9119, and mimicked by direct JNK activation or inhibition. JNKs phosphorylate c-Jun at Ser63 and Ser73 as well as other transcription factors . A variety of proliferative signals, cytokines and cellular stressors activate JNK-dependent pathways, but mediation of JNK activity by the melanocortinergic system has not been previously reported. Members of this protein kinase family have been implicated in human diseases, and this linkage has motivated the development of a growing number of small inhibitory molecules.
JNKs have been reported to have a role in obesity and obesity-related insulin resistance [15, 16]. In obesity-induced insulin resistance or type 2 diabetes, a defect in insulin signaling lies distal to the insulin receptor. Binding of insulin to the insulin receptor results in insulin receptor substrate (IRS-1) phosphorylation on tyrosine residues; tyrosine-phosphorylated IRS-1 recruits and activates various effectors that contain phospho-tyrosine binding domains . In the context of obesity and systemic insulin resistance, IRS-1 may be phosphorylated at Ser307. Serine307 phosphorylation of IRS-1 interferes with its association with the insulin receptor, reduces tyrosine phosphorylation of IRS-1 in response to insulin and thereby suppresses downstream signaling and insulin action [2, 3, 17, 29]. JNKs can phosphorylate IRS-1 on Ser307, and in this way, may contribute to the development of insulin resistance [2, 3]. JNK also mediates feedback inhibition of insulin signaling. Insulin stimulates JNK activity; JNK promotes IRS-1 Ser307 phosphorylation and attenuates tyrosine phosphorylation. Inhibiting JNK enhances insulin signal transduction and glucose uptake. In this manner, insulin-activated JNK may act as a negative feedback regulator for insulin signaling[3, 23]. JNK activity is increased in the hypothalamus of rats with Western diet-induced obesity . In mouse models, suppression of JNK results in decreased adiposity .
During regulation of energy homeostasis, melanocortin receptors interact with other factors, such as leptin, cholecystokinin and neuropeptide Y [9, 25, 32]. Insulin is an important satiety factor, but direct regulation of insulin signaling by melanocortin peptides has not previously been reported. Banno et al reported that central administration of melanocortin agonist MT II improved insulin tolerance and increased the number of small-sized adipocytes in diet-induced obese rats, implying that melanocortin receptors may interact with insulin . In the current study, MC4R activation increased insulin-stimulated AKT phosphorylation in vitro and in vivo and glucose uptake in vitro. NDP-MSH alone did not stimulate AKT phosphorylation, and did not significantly increase glucose uptake. The details of this interaction remain to be elucidated.
In conclusion, the present studies demonstrate that MC4R agonist enhances insulin signaling with a mechanism that involves inhibition of JNK activity, resulting in decrease of IRS-1ser307 phosphorylation and increase in AKT phosphorylation.
This work was supported by NIH grant DK054032 and 5R37DK043225-17
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