JOURNAL OF NEUROCHEMISTRY
| 2015 | 133 | 343–351
doi: 10.1111/jnc.13057
Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Abstract Obesity is a worldwide health problem that urgently needs to be solved. Leptin is an anti-obesity hormone that activates satiety signals to the brain. Evidence to suggest that leptin resistance is involved in the development of obesity is increasing; however, the molecular mechanisms involved remain unclear. We herein demonstrated that 15-deoxyD12,14-prostaglandin J2 (15d-PGJ2) was involved in the development of leptin resistance. A treatment with 15d-PGJ2 inhibited the leptin-induced activation of signal transducer and activator of transcription 3 (STAT3) in neuronal cells (SHSY5Y-Ob-Rb cells). Furthermore, the intracerebroventricular administration of 15d-PGJ2 reversed the inhibitory effects of leptin on food intake in rats. The peroxisome proliferator-
activated receptor gamma (PPAR-c) antagonist, GW9662, slightly reversed the inhibitory effects of 15d-PGJ2 on the leptin-induced activation of STAT3 in neuronal cells. The PPAR-c agonist, rosiglitazone, also inhibited leptin-induced STAT3 phosphorylation. Therefore, the inhibitory effects of 15d-PGJ2 may be mediated through PPAR-c. On the other hand, 15d-PGJ2-induced leptin resistance may not be mediated by endoplasmic reticulum stress or suppressor of cytokine signaling 3. The results of the present study suggest that 15d-PGJ2 is a novel factor for the development of leptin resistance in obesity. Keywords: 15d-PGJ2, leptin, PPAR-c, STAT3. J. Neurochem. (2015) 133, 343–351.
Leptin is anti-obesity hormone that was first identified in 1994 by Friedman’s group (Zhang et al. 1994). Leptin is mainly secreted from adipose tissue and acts on brain hypothalamic neurons to reduce food intake (Campfield et al. 1995). Several splicing variants of the leptin receptor isoform have been identified to date. Of those, the Ob-Rb leptin receptor is the longest isoform of the leptin receptor and is considered to play an important role in regulating food intake and energy expenditure. The Ob-Rb leptin receptor is mainly expressed on the hypothalamus (Mercer et al. 1996). Leptin was previously shown to activate the Ob-Rb leptin receptor, which in turn induced JAK2-STAT3 signal transduction (Bjørbaek et al. 1997; Hosoi et al. 2002). The essential role of the Ob-Rb receptor-mediated activation of signal transducer and activator of transcription 3 (STAT3) in the anti-obesity effects of leptin have already been demonstrated (Bates et al. 2003). Thus, leptin treatments were initially expected to be useful for treating obesity. However, since most obese patients were in a state of leptin resistance, they did not adequately respond to the actions of leptin.
These findings suggested that leptin resistance was involved in the development of obesity (Friedman 2003). The fact that a deeper understanding of the mechanisms underlying leptin resistance is needed represents an active area of research (Hill et al. 2003). Several studies have proposed mechanisms for the development of leptin resistance. Suppressor of cytokine signaling 3 (SOCS3) (Bjørbaek et al. 1998), protein tyrosine phosphatase 1B (Cheng et al. 2002; Zabolotny et al. 2002), and endoplasmic reticulum (ER) stress (Hosoi et al. Received August 21, 2014; revised manuscript received January 23, 2015; accepted January 27, 2015. Address correspondence and reprint requests to Koichiro Ozawa (or) Toru Hosoi, Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. E-mails: ozawak@hiroshima-u. ac.jp (or) [email protected] Abbreviations used: 15d-PGJ2, 15-deoxy-D12,14-prostaglandin J2; ER stress, endoplasmic reticulum stress; JAK2, Janus kinase 2; PPAR-c, peroxisome proliferator-activated receptor gamma; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3.
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2008; Ozcan et al. 2009; Hosoi and Ozawa 2010) have been implicated in the development of leptin resistance. However, the mechanisms underlying leptin resistance have yet to be elucidated in detail. We investigated some novel mechanisms responsible for leptin resistance and suggested the possible involvement of 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2) in its development. 15d-PGJ2 is a member of the prostaglandin (PG) family, which comprises the dehydrated products of PGD2 (Fitzpatrick and Wynalda 1983; Kikawa et al. 1984). There is increasing evidence to suggest that 15d-PGJ2 plays a role in inhibiting inflammatory reactions. 15d-PGJ2 was previously shown to attenuate inflammation by inhibiting IjB kinase (Rossi et al. 2000). 15d-PGJ2 can also inhibit macrophageand monocyte-mediated inflammation through peroxisome proliferator-activated receptor gamma (PPAR-c), a nuclear receptor superfamily (Forman et al. 1995; Kliewer et al. 1995; Jiang et al. 1998; Ricote et al. 1998). 15d-PGJ2 has also been shown to attenuate brain inflammation by inhibiting microglia inducible nitric-oxide synthase (iNOS), tumor necrosis factor a, and interleukin (IL)-12 (Petrova et al. 1999; Drew and Chavis 2001). PPARc was previously reported to be expressed in the hypothalamus and 15d-PGJ2 attenuated lipopolysaccharide-induced febrile responses (Mouihate et al. 2004). Furthermore, 15d-PGJ2 was suggested to be involved in neurodegenerative diseases such as Alzheimer’s disease. A previous study reported that 15dPGJ2 inhibited b amyloid-induced inflammation in Alzheimer’s disease (Combs et al. 2000). Taken together, these findings suggest that 15d-PGJ2 may play an important role in regulating brain immune function (Petrova et al. 1999; Drew and Chavis 2001). It is generally accepted that hypothalamic inflammation contributes to the pathophysiological functions of obesity (Thaler et al. 2010). Long-chain saturated fatty acids have been shown to activate toll-like receptor 4 signaling and induce the expression of cytokines in the hypothalamus (Milanski et al. 2009). Furthermore, inflammation was suggested to be involved in the development of leptin resistance (Zhang et al. 2008; Milanski et al. 2009). However, the mechanisms underlying the development of leptin resistance in an inflammatory state remain unclear. Since 15d-PGJ2 is up-regulated in inflammation and may be critically involved in immune reactions (Ajmone-Cat et al. 2003), we hypothesized that 15d-PGJ2 may play a role in the development of leptin resistance. We herein described a novel mechanism for the development of obesity; i.e. 15dPGJ2-induced leptin resistance, which in turn may be linked to the development of obesity.
Materials and methods Reagents Rosiglitazone and GW9662 were obtained from Cayman Chemical (Ann Arbor, MI, USA). 15d-PGJ2 was obtained from Calbiochem
(Darmstadt, Germany) or ENZO Life Sciences (Farmingdale, NY, USA). Human leptin was obtained from Sigma (St Louis, MO, USA) or ENZO Life Sciences. Cell culture Human neuroblastoma SH-SY5Y cells (obtained from ATCC) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal calf serum at 37°C in a humidified incubator under 5% CO2 and 95% air. Generation of Ob-Rb leptin receptor-transfected cells The human Ob-Rb leptin receptor construct was a gift from Genetech Inc. (South San Francisco, CA, USA) The construct was transfected into SH-SY5Y cells using LipofectAMINE PLUS Reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions, and stable transfectants were then obtained by selection with the antibiotic G418 (Hosoi et al. 2006). Immunohistochemistry Immunohistochemistry was performed as described previously (Hosoi et al. 2014). Briefly, cells were fixed with methanol for 10 min at 20°C. After being washed with phosphate-buffered saline, the cells were incubated with 10% fetal calf serum at 37°C for 1 h, and allowed to react with an anti-p-STAT3 (diluted to 1 : 50; Cell Signaling Technology, Beverly, MA, USA) antibody at 4°C overnight. These cells were then incubated with an antirabbit immunoglobulin G antibody conjugated with Alexa 488 (1 : 2000) (Life Technologies, Grand Island, NY, USA) at 37°C for 1 h. PI (propidium iodide) staining was performed by incubating cells with PI (1 lg/mL) for 5 min. Cells were visualized using confocal laser scanning microscopy (LSM5 pascal, Carl Zeiss Co., Ltd., Oberkochen, Germany). Animals Adult male Wister rats were obtained from CLEA Japan Inc. (Tokyo, Japan). Rats were maintained in our animal facility at 22– 24°C under a constant day–night rhythm (lights on at 8:00 and off at 20:00) and given food and water ad libitum. Animal welfare and ethical statement All animal experiments were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee at Hiroshima University. Cannulation procedure Male Wistar rats were used in the present experiment. A 24-gauge stainless-steel guide cannula was implanted into the cerebral ventricle (1.0 mm caudal from the bregma, 1.5 mm right lateral side from the midline, and 3.7 mm below the dura matter) under sodium pentobarbital anesthesia (50 mg/kg, i.p.). The cannula was fixed to the cranium by dental resin and screws. After surgery, rats were treated with antibiotics to prevent infection. All rats were then individually housed and maintained on water and food ad libitum. After a recovery period of 10 days, leptin (5 lg/ rat) and/or 15d-PGJ2 (60 nmol/rat) were injected intracerebroventricularly. All drugs were injected using a 30-gauge stainlesssteel needle connected with an injection tube.
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Experimental schedule for the intracerebroventricular administration protocol On experimental day 1, each rat was treated with dimethylsulfoxide + saline and its food intake was then measured. Three days after the experiment (experimental day 4), each rat was treated with dimethylsulfoxide + leptin and its food intake was again measured. We subtracted the 24-h food intake of day 1 from that of day 4 and selected leptin-responsive rats. Four days after the experiment (experimental day 8), the selected rats were treated with 15d-PGJ2 + leptin and their food intake was again measured. Four days after the experiment (experimental day 12), each was treated with 15d-PGJ2 + saline and its food intake was measured (Figure S2). In the pilot study, we injected leptin twice into the same rat and compared each response in inhibiting food intake. Inhibitory effects on food intake were similar between the first and second injections (Figure S3). Therefore, there was no desensitization to the secondary leptin response under the present conditions. Procedure for the intracerebroventricular administration protocol On experimental days, all food in the individual cages was removed at 17:00. At 18:00, leptin and/or 15d-PGJ2 was injected through an intracerebroventricular (ICV) route. Food was then returned to the individual cages at 19:30 and food intake was measured for 24 h. Hypothalamic STAT3 analysis after the intracerebroventricular injection of leptin into mice Male mice were used in the present experiment. One week before the experiment, we made a hole in the skull 0.3 mm caudal from the bregma, 0.9 mm right lateral side from the midline, and 2.0 mm below the dura matter under sodium pentobarbital anesthesia (50 mg/kg, i.p.). After surgery, mice were treated with antibiotics to prevent infection. After a recovery period of 7 days, mice were starved overnight and 15d-PGJ2 (20 lg/mouse) and/or leptin (2.25 lg/mouse) were injected. Samples were snap-frozen in liquid nitrogen and stored at 80°C until use. Regarding western blotting, tissue samples were homogenized in a buffer containing 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 21 mM Na3VO4, 10 mM NaF, 10 lg/mL aprotinin, 10 lg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40 using Precellys 24 (Bertin Technologies, Orleans, France). Samples were centrifuged at 19 000 g for 45 min at 4°C, and the supernatants were collected. Western blot analysis Western blotting was performed as described previously (Hosoi et al. 2012). Briefly, cells were washed with ice-cold phosphatebuffered saline and lysed in buffer containing 10 mM HEPESNaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 10 lg/mL aprotinin, 10 lg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1% NP-40 for 20 min. The lysates were centrifuged at 20 630 g for 20 min at 4°C, and supernatants were collected. Samples were boiled with Laemmle buffer for 3 min, fractionated by sodium dodecylsulfate–polyacrylamide gel electrophoresis, and transferred at 4°C to nitrocellulose membranes. These membranes were then incubated with antiphospho STAT3 (Tyr705, 1 : 1000; Cell Signaling Technology), anti-STAT3 (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PPARc (1 : 1000; Santa Cruz Biotechnology), anti-
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phospho Tyrosine (1 : 1000; Upstate Biotechnology, Lake Placid, NY, USA), and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase, 1 : 1000; Chemicon, Temecula, CA, USA) antibodies, followed by an anti-horseradish peroxidase-linked antibody. Peroxidase binding was detected by chemiluminescence using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, England). Statistics Results are expressed as the mean SE. Statistical analyses were performed using the Student’s t-test, Dunnett’s test, or paired t-test. We conducted an ANOVA analysis for multiple comparisons.
Results 15d-PGJ2 inhibited leptin-induced STAT3 phosphorylation Circulating leptin is known to activate the Ob-Rb leptin receptor, which is located in hypothalamic neurons, and subsequently induces the JAK2-STAT3 signal transduction pathway (Ghilardi et al. 1996). We investigated the effects of 15d-PGJ2 on leptin-induced signal transduction using a human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-Ob-Rb cell line) (Hosoi et al. 2006). The treatment with leptin markedly induced the phosphorylation of STAT3 at Tyr705, indicating the expression of the functional receptor in the SH-SY5Y-Ob-Rb cell line. Therefore, we analyzed the effects of 15d-PGJ2 on leptin-induced STAT3 phosphorylation. The treatment with 15d-PGJ2 time- and dose-dependently inhibited by leptin-induced STAT3 phosphorylation (Fig. 1). The treatment with 15d-PGJ2 inhibited leptin-induced STAT3 phosphorylation from 30 min up to 4 h (Fig. 1a). The inhibition of 15d-PGJ2 on leptin-induced STAT3 phosphorylation was dose-dependent, with significant levels of inhibition being observed at 10 lM (Fig. 1b). No significant changes were noted in the phosphorylation status of global proteins at tyrosine residues after SH-SY5Y-Ob-Rb cells were treated with 15d-PGJ2 (Figure S1). Therefore, 15d-PGJ2 may not have perturbed global tyrosine kinase pathways. These results were further confirmed by immunohistochemistry, which revealed that leptin-induced nuclear phospho-STAT3 staining was markedly attenuated by 15d-PGJ2 (Fig. 2). Thus, these results suggested that 15d-PGJ2 specifically inhibited STAT3 signal transduction downstream of the ObRb leptin receptor. Involvement of PPAR-c in the development of leptin resistance It currently remains unknown how 15d-PGJ2 evokes leptin resistance. To address this issue, we focused on PPAR-c, which is activated by 15d-PGJ2 (Forman et al. 1995; Kliewer et al. 1995). We investigated whether the inhibitory effects of 15d-PGJ2 on the actions of leptin were
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(b) Fig. 1 15d-PGJ2 induced leptin resistance. (a) SH-SY5Y-ObRb cells were treated with 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2:10 lM) for the indicated times and then stimulated with leptin (Lep: 0.5 lg/mL) for 15 min. ***p < 0.001 (b) SH-SY5YObRb cells were treated with 15d-PGJ2 (15d-PGJ2: 0.3–10 lM) for 1 h and then stimulated with leptin (Lep: 0.5 lg/mL) for 15 min. A western blotting analysis was performed using specific antibodies for phospho-STAT3 (Tyr705) and signal transducer and activator of transcription 3 (STAT3). **p < 0.01.
mediated through PPAR-c. We initially examined its expression in the SH-SY5Y Ob-Rb cell line. Consistent with previous findings (Moreno et al. 2004; Sarruf et al. 2009), PPAR-c was expressed in SH-SY5Y Ob-Rb neuronal cells as well as in the mouse brain such as in the hypothalamus or cortex (Fig. 3a). Therefore, we examined the effects of GW9662, a specific PPAR-c antagonist, on 15d-PGJ2-induced leptin resistance. The treatment with GW9662 alone did not affect leptin-induced STAT3 phosphorylation (Fig. 3b). However, we observed the slight recovery of the 15d-PGJ2-induced inhibition of STAT3 phosphorylation by GW9662 in leptin-treated cells (Fig. 3b). These results suggested that PPAR-c may be involved in 15d-PGJ2-induced leptin resistance. To further confirm the involvement of PPAR-c in leptin resistance, we analyzed the effects of the PPAR-c agonist on the actions of leptin. We treated cells with rosiglitazone, a specific PPAR-c agonist, and analyzed leptin-induced STAT3 phosphorylation. As expected, a dose-dependent decrease
was observed in leptin-induced STAT3 phosphorylation following the treatment with rosiglitazone (Fig. 3c). These results indicated that 15d-PGJ2-induced leptin resistance was mediated through PPAR-c.
15d-PGJ2-induced leptin resistance was not dependent on SOCS3 or ER stress We next examined the possible relationship between the previously identified mechanisms underlying leptin resistance, such as SOCS3 (Bjørbaek et al. 1998) or ER stress (Hosoi et al. 2008; Ozcan et al. 2009), and 15d-PGJ2induced leptin resistance. We measured SOCS3 levels after the treatment with 15d-PGJ2. Consistent with previous findings (Bjørbaek et al. 1998), the treatment with leptin for 6 h increased SOCS3 levels, which acted as a negative feedback regulator on the actions of leptin (Bjørbaek et al. 1999) (Fig. 4). However, we did not observe an increase in SOSC3 levels after cells were treated with 15d-PGJ2 at any
Fig. 2 15d-PGJ2 inhibited leptin-induced induction of nuclear phospho-STAT3. SHSY5Y-ObRb cells were treated with 15dPGJ2 (15d-PGJ2: 10 lM) for 4 h and then stimulated with leptin (0.5 lg/mL) for 15 min. Immunofluorescence staining was performed using antibodies for phosphoSTAT3. PI: Nuclear staining. Original magnification: 963. Images were obtained with a confocal microscope and were analyzed by LSM software.
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Fig. 3 Peroxisome proliferator-activated receptor gamma (PPARc) was involved in 15d-PGJ2-induced leptin resistance. (a) PPARc was expressed in SH-SY5Y-ObRb cells, the hypothalamus, and cortex. A western blotting analysis was performed using specific antibodies for PPARc and GAPDH. (b) The PPARc antagonist restored 15d-PGJ2induced leptin resistance. SH-SY5Y-ObRb cells were pre-incubated with GW9662 (0.5 lM) for 30 min and then treated with 15d-PGJ2
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(15d-PGJ2: 30 lM) for 4 h. Leptin (0.5 lg/mL) was stimulated for 15 min. *p < 0.05 (vs. Leptin + 15d-PGJ2). (c) The PPARc agonist induced leptin resistance. SH-SY5Y-ObRb cells were treated with rosiglitazone (Rosi: 0.1–10 lM) for 1 h and then stimulated with leptin (Lep: 0.5 lg/mL) for 15 min. A western blotting analysis was performed using specific antibodies for phospho-STAT3 (Tyr705) and signal transducer and activator of transcription 3 (STAT3). ***p < 0.001
of the time points investigated (Fig. 4). Thus, 15d-PGJ2induced leptin resistance may not be mediated through the induction of SOCS3. 15d-PGJ2 also did not induce the apparent activation of ER stress, as measured by ER stressregulated genes such as GRP78 and the activation status of ER stress sensor proteins such as IRE1 (Inositol-requiring enzyme-1) or PERK (PKR-like kinase) (Fig. 5). As a positive control, the treatment with tunicamycin, an ER stress-inducing reagent that inhibits protein glycosylation, increased the activation of IRE1 and PERK as well as the induction of GRP78 in SH-SY5Y-Ob-Rb cells (Fig. 5). Therefore, these results suggested that 15d-PGJ2-induced leptin resistance was not mediated through ER stress. Therefore, 15d-PGJ2-induced leptin resistance may not be mediated through previously identified mechanisms such as SOCS3 and ER stress.
Fig. 4 15d-PGJ2 did not affect suppressor of cytokine signaling 3 (SOCS3) levels. SH-SY5Y-ObRb cells were treated with 15d-PGJ2 (15d-PGJ2: 10 lM) for the indicated times. A western blotting analysis was performed using specific antibodies for SOCS3 and GAPDH. Leptin (Lep: 0.5 lg/mL, 6 h) increased SOCS3 levels. However, 15dPGJ2 did not affect SOCS3 levels.
15d-PGJ2 reversed the inhibitory effects of leptin on food intake We investigated the physiological consequences of 15dPGJ2 on the actions of leptin. Leptin inhibits food intake by acting on the brain (Campfield et al. 1995). Therefore, we determined whether 15d-PGJ2 attenuated the leptin-
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Fig. 6 15d-PGJ2 reversed the satiety action of leptin. Leptin was administered through an ICV route to male rats. Cumulative food intakes were measured for 24 h. Leptin inhibited food intake. The treatment with 15d-PGJ2 reversed the inhibitory effects of leptin on food intake. ***p < 0.001.
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Fig. 5 15d-PGJ2 did not affect endoplasmic reticulum (ER) stress responses. (a) SH-SY5Y-ObRb cells were treated with 15d-PGJ2 (10 lM) for the indicated times. Tunicamycin (Tm: 1 lg/mL) was treated as a positive control. A western blotting analysis was performed using specific antibodies for IRE-1, PERK, and GAPDH. The treatment with Tm increased the phosphorylation of IRE1 and PERK, as indicated by a mobility shift to the upper band. On the other hand, the treatment with 15d-PGJ2 did not affect the phosphorylation of IRE1 or PERK. (b) A western blotting analysis was performed using specific antibodies for GRP78 and GAPDH. The treatment with Tm increased the induction of GRP78, whereas 15d-PGJ2 did not. A densitometric analysis of the induction of GRP78 was performed using image analysis software.
induced inhibition of food intake in vivo. To evaluate the effects of 15d-PGJ2 on the actions of leptin in inhibiting food intake, we centrally injected 15d-PGJ2 along with leptin into rats and measured their food intake. The intracerebroventricular administration of leptin alone decreased food intake within 24 h (Fig. 6). However, the treatment with 15d-PGJ2 significantly reversed the inhibitory effects of leptin on food intake (Fig. 6). These results suggested that 15d-PGJ2 inhibited the actions of leptin on food intake in vivo. Therefore, 15d-PGJ2 may be involved in the development of leptin resistance. To further confirm these results, we next injected 15d-PGJ2 intracerebroventricularly into the mouse brain and analyzed the leptin-induced phosphorylation of STAT3 in the hypothalamus. As shown in Fig. 7, leptin-induced STAT3 phosphorylation was inhibited by the administration of 15d-PGJ2.
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Fig. 7 15d-PGJ2 reduced leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in the hypothalamus. (a) 15d-PGJ2 and leptin were administered through an intracerebroventricular (ICV) route to male mice. Animals were pretreated with 15dPGJ2 prior to leptin injection. Thirty minutes after leptin treatment, hypothalamus was taken and phosphorylation level of STAT3 was analyzed by western blotting (b) A densitometric analysis of the induction of P-STAT3 was performed using image analysis software.
Discussion Evidence to suggest that chronic brain inflammation is associated with obesity is increasing. Previous studies reported that saturated fatty acids induced inflammatory responses by activating the toll-like receptor signaling pathway in the hypothalamus (Milanski et al. 2009; Moraes et al. 2009), suggesting the possible involvement of fatty acids in inflammation. Furthermore, recent findings suggest that hypothalamic inflammation also contributes to leptin
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resistance in obesity (Thaler et al. 2010). However, the mechanisms linking hypothalamic inflammation to obesity remain unclear. In the present study, we showed that 15dPGJ2, a member of the prostaglandin family, which regulates key aspects of immunity, was involved in the development of leptin resistance. We demonstrated that 15d-PGJ2 inhibited the leptin-induced activation of STAT3 and leptin-induced anorexia. 15d-PGJ2 exists in the CSF and its expression was shown to be up-regulated by brain inflammation caused by bacterial endotoxins (Mouihate et al. 2004). Chronic inflammation may contribute to the development of leptin resistance (Thaler et al. 2010). Therefore, an increase in 15d-PGJ2 levels in the brain may contribute to the development of leptin resistance. The production of 15d-PGJ2 from microglia cells was shown to be elevated under inflammatory conditions (Ajmone-Cat et al. 2003). Since chronic inflammation has been observed in the obese hypothalamus, microglia may induce the production of 15d-PGJ2, which may in turn be involved in the development of leptin resistance. 15d-PGJ2 possesses anti-inflammatory properties. For example, the expression of iNOS, gelatinase B, and scavenger receptor A genes was inhibited by 15d-PGJ2 in activated macrophages (Ricote et al. 1998). Furthermore, 15d-PGJ2 suppressed the activation of microglia by downregulating the expression of iNOS (Petrova et al. 1999). Therefore, we speculated that the chronic or sustained induction of inflammation may lead to the induction of 15d-PGJ2 by a negative feedback mechanism, which then affects leptin signals. The mechanisms underlying the development of leptin resistance have been suggested to be mediated through the induction of SOCS3 or ER stress. However, 15d-PGJ2induced leptin resistance may not be mediated through these mechanisms. We herein demonstrated that PPAR-c would be involved in 15d-PGJ2-induced leptin resistance. We found that 15d-PGJ2-induced leptin resistance was slightly attenuated by the PPAR-c antagonist, GW9662, in leptin-treated cells. Furthermore, the PPAR-c agonist, rosiglitazone, attenuated the leptin-induced activation of STAT3. Rosiglitazone is an anti-diabetic drug, which is classified as thiazolidinedione (TZD), and is used to treat diabetes caused by insulin resistance (Lehrke and Lazar 2005). However, the main side effect of TZD is weight gain (Larsen et al. 2003), the mechanism of action for which it has yet to be clarified. This finding is of particular interest because obesity is involved in the development of diabetes. One of the underlying mechanisms for the actions of the PPAR-c agonist on obesity may be attributed to peripheral effects such as the differentiation of adipocytes (Tontonoz et al. 1994). On the other hand, treatments with TZD have also been reported to increase food intake, suggesting the existence of the CNS actions of PPAR-c in regulating weight gain (Lehrke and Lazar 2005). We found that leptin signals were attenuated when neuronal cells were treated with the PPAR-c agonist. This result is
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consistent with recent findings reported by two groups (Lu et al. 2011; Ryan et al. 2011). Therefore, the PPAR-c agonist may attenuate the actions of leptin in the CNS, thereby increasing food intake. These findings indicate that PPAR-c induces leptin resistance, which may simultaneously account for the side effects of TZD. However, the physiological target molecule that activates PPAR-c currently remains unknown. The identity of the neuronal target molecule regulating the activation of PPAR-c, which is linked to leptin signals, is one of the major issues in this field of research (Myers and Burant 2011). In the present study, we showed that 15d-PGJ2 may be a target for activating PPAR-c, which is linked to leptin resistance. Thus, our results suggest that 15d-PGJ2-induced PPAR-c activation may be involved in the development of leptin resistance in the CNS. On the other hand, previous studies indicated the existence of a PPARc-independent pathway in 15d-PGJ2mediated actions (Chawla et al. 2001; Lu et al. 2013). Furthermore, 15d-PGJ2-induced leptin resistance was not completely ameliorated by the PPAR-c antagonist. Therefore, it is also possible that the inhibitory effects of 15d-PGJ2 on the actions of leptin were mediated through a PPARcindependent pathway. Obesity is a major health concern worldwide because it is accompanied by diseases such as diabetes, hypertension, and hyperlipemia. In the present study, we identified novel mechanisms for the development of leptin resistance in obesity. We found that 15d-PGJ2 was involved in the development of leptin resistance. Therefore, targeting 15dPGJ2 may represent a novel strategy for ameliorating these diseases, which will be an interesting future research subject. Moreover, our results provide a novel insight into how food intake is regulated.
Acknowledgments and conflict of interest disclosure This study was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University. Animal experiments were supported by the Institute of Laboratory Animal Science (Hiroshima University). This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; Takeda Science Foundation; Tokyo Biochemical Research Foundation; Suzuken Memorial Foundation, and the Nakajima Foundation. All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.
Supporting information Additional supporting information may be found in the online version of this article at the publisher's web-site: Figure S1. Effects of 15d-PGJ2 on protein tyrosine phosphorylation levels in SH-SY5Y-ObRb cells.
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Figure S2. Schematic schedule of intracerebroventricular injections for analyzing the in vivo effects of 15d-PGJ2 on the actions of leptin. Figure S3. Leptin was administered through an intracerebroventricular (ICV) route to male rats.
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| 2015 | 133 | 343–351
doi: 10.1111/jnc.13057
Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Abstract Obesity is a worldwide health problem that urgently needs to be solved. Leptin is an anti-obesity hormone that activates satiety signals to the brain. Evidence to suggest that leptin resistance is involved in the development of obesity is increasing; however, the molecular mechanisms involved remain unclear. We herein demonstrated that 15-deoxyD12,14-prostaglandin J2 (15d-PGJ2) was involved in the development of leptin resistance. A treatment with 15d-PGJ2 inhibited the leptin-induced activation of signal transducer and activator of transcription 3 (STAT3) in neuronal cells (SHSY5Y-Ob-Rb cells). Furthermore, the intracerebroventricular administration of 15d-PGJ2 reversed the inhibitory effects of leptin on food intake in rats. The peroxisome proliferator-
activated receptor gamma (PPAR-c) antagonist, GW9662, slightly reversed the inhibitory effects of 15d-PGJ2 on the leptin-induced activation of STAT3 in neuronal cells. The PPAR-c agonist, rosiglitazone, also inhibited leptin-induced STAT3 phosphorylation. Therefore, the inhibitory effects of 15d-PGJ2 may be mediated through PPAR-c. On the other hand, 15d-PGJ2-induced leptin resistance may not be mediated by endoplasmic reticulum stress or suppressor of cytokine signaling 3. The results of the present study suggest that 15d-PGJ2 is a novel factor for the development of leptin resistance in obesity. Keywords: 15d-PGJ2, leptin, PPAR-c, STAT3. J. Neurochem. (2015) 133, 343–351.
Leptin is anti-obesity hormone that was first identified in 1994 by Friedman’s group (Zhang et al. 1994). Leptin is mainly secreted from adipose tissue and acts on brain hypothalamic neurons to reduce food intake (Campfield et al. 1995). Several splicing variants of the leptin receptor isoform have been identified to date. Of those, the Ob-Rb leptin receptor is the longest isoform of the leptin receptor and is considered to play an important role in regulating food intake and energy expenditure. The Ob-Rb leptin receptor is mainly expressed on the hypothalamus (Mercer et al. 1996). Leptin was previously shown to activate the Ob-Rb leptin receptor, which in turn induced JAK2-STAT3 signal transduction (Bjørbaek et al. 1997; Hosoi et al. 2002). The essential role of the Ob-Rb receptor-mediated activation of signal transducer and activator of transcription 3 (STAT3) in the anti-obesity effects of leptin have already been demonstrated (Bates et al. 2003). Thus, leptin treatments were initially expected to be useful for treating obesity. However, since most obese patients were in a state of leptin resistance, they did not adequately respond to the actions of leptin.
These findings suggested that leptin resistance was involved in the development of obesity (Friedman 2003). The fact that a deeper understanding of the mechanisms underlying leptin resistance is needed represents an active area of research (Hill et al. 2003). Several studies have proposed mechanisms for the development of leptin resistance. Suppressor of cytokine signaling 3 (SOCS3) (Bjørbaek et al. 1998), protein tyrosine phosphatase 1B (Cheng et al. 2002; Zabolotny et al. 2002), and endoplasmic reticulum (ER) stress (Hosoi et al. Received August 21, 2014; revised manuscript received January 23, 2015; accepted January 27, 2015. Address correspondence and reprint requests to Koichiro Ozawa (or) Toru Hosoi, Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. E-mails: ozawak@hiroshima-u. ac.jp (or) [email protected] Abbreviations used: 15d-PGJ2, 15-deoxy-D12,14-prostaglandin J2; ER stress, endoplasmic reticulum stress; JAK2, Janus kinase 2; PPAR-c, peroxisome proliferator-activated receptor gamma; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3.
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2008; Ozcan et al. 2009; Hosoi and Ozawa 2010) have been implicated in the development of leptin resistance. However, the mechanisms underlying leptin resistance have yet to be elucidated in detail. We investigated some novel mechanisms responsible for leptin resistance and suggested the possible involvement of 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2) in its development. 15d-PGJ2 is a member of the prostaglandin (PG) family, which comprises the dehydrated products of PGD2 (Fitzpatrick and Wynalda 1983; Kikawa et al. 1984). There is increasing evidence to suggest that 15d-PGJ2 plays a role in inhibiting inflammatory reactions. 15d-PGJ2 was previously shown to attenuate inflammation by inhibiting IjB kinase (Rossi et al. 2000). 15d-PGJ2 can also inhibit macrophageand monocyte-mediated inflammation through peroxisome proliferator-activated receptor gamma (PPAR-c), a nuclear receptor superfamily (Forman et al. 1995; Kliewer et al. 1995; Jiang et al. 1998; Ricote et al. 1998). 15d-PGJ2 has also been shown to attenuate brain inflammation by inhibiting microglia inducible nitric-oxide synthase (iNOS), tumor necrosis factor a, and interleukin (IL)-12 (Petrova et al. 1999; Drew and Chavis 2001). PPARc was previously reported to be expressed in the hypothalamus and 15d-PGJ2 attenuated lipopolysaccharide-induced febrile responses (Mouihate et al. 2004). Furthermore, 15d-PGJ2 was suggested to be involved in neurodegenerative diseases such as Alzheimer’s disease. A previous study reported that 15dPGJ2 inhibited b amyloid-induced inflammation in Alzheimer’s disease (Combs et al. 2000). Taken together, these findings suggest that 15d-PGJ2 may play an important role in regulating brain immune function (Petrova et al. 1999; Drew and Chavis 2001). It is generally accepted that hypothalamic inflammation contributes to the pathophysiological functions of obesity (Thaler et al. 2010). Long-chain saturated fatty acids have been shown to activate toll-like receptor 4 signaling and induce the expression of cytokines in the hypothalamus (Milanski et al. 2009). Furthermore, inflammation was suggested to be involved in the development of leptin resistance (Zhang et al. 2008; Milanski et al. 2009). However, the mechanisms underlying the development of leptin resistance in an inflammatory state remain unclear. Since 15d-PGJ2 is up-regulated in inflammation and may be critically involved in immune reactions (Ajmone-Cat et al. 2003), we hypothesized that 15d-PGJ2 may play a role in the development of leptin resistance. We herein described a novel mechanism for the development of obesity; i.e. 15dPGJ2-induced leptin resistance, which in turn may be linked to the development of obesity.
Materials and methods Reagents Rosiglitazone and GW9662 were obtained from Cayman Chemical (Ann Arbor, MI, USA). 15d-PGJ2 was obtained from Calbiochem
(Darmstadt, Germany) or ENZO Life Sciences (Farmingdale, NY, USA). Human leptin was obtained from Sigma (St Louis, MO, USA) or ENZO Life Sciences. Cell culture Human neuroblastoma SH-SY5Y cells (obtained from ATCC) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal calf serum at 37°C in a humidified incubator under 5% CO2 and 95% air. Generation of Ob-Rb leptin receptor-transfected cells The human Ob-Rb leptin receptor construct was a gift from Genetech Inc. (South San Francisco, CA, USA) The construct was transfected into SH-SY5Y cells using LipofectAMINE PLUS Reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions, and stable transfectants were then obtained by selection with the antibiotic G418 (Hosoi et al. 2006). Immunohistochemistry Immunohistochemistry was performed as described previously (Hosoi et al. 2014). Briefly, cells were fixed with methanol for 10 min at 20°C. After being washed with phosphate-buffered saline, the cells were incubated with 10% fetal calf serum at 37°C for 1 h, and allowed to react with an anti-p-STAT3 (diluted to 1 : 50; Cell Signaling Technology, Beverly, MA, USA) antibody at 4°C overnight. These cells were then incubated with an antirabbit immunoglobulin G antibody conjugated with Alexa 488 (1 : 2000) (Life Technologies, Grand Island, NY, USA) at 37°C for 1 h. PI (propidium iodide) staining was performed by incubating cells with PI (1 lg/mL) for 5 min. Cells were visualized using confocal laser scanning microscopy (LSM5 pascal, Carl Zeiss Co., Ltd., Oberkochen, Germany). Animals Adult male Wister rats were obtained from CLEA Japan Inc. (Tokyo, Japan). Rats were maintained in our animal facility at 22– 24°C under a constant day–night rhythm (lights on at 8:00 and off at 20:00) and given food and water ad libitum. Animal welfare and ethical statement All animal experiments were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee at Hiroshima University. Cannulation procedure Male Wistar rats were used in the present experiment. A 24-gauge stainless-steel guide cannula was implanted into the cerebral ventricle (1.0 mm caudal from the bregma, 1.5 mm right lateral side from the midline, and 3.7 mm below the dura matter) under sodium pentobarbital anesthesia (50 mg/kg, i.p.). The cannula was fixed to the cranium by dental resin and screws. After surgery, rats were treated with antibiotics to prevent infection. All rats were then individually housed and maintained on water and food ad libitum. After a recovery period of 10 days, leptin (5 lg/ rat) and/or 15d-PGJ2 (60 nmol/rat) were injected intracerebroventricularly. All drugs were injected using a 30-gauge stainlesssteel needle connected with an injection tube.
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Experimental schedule for the intracerebroventricular administration protocol On experimental day 1, each rat was treated with dimethylsulfoxide + saline and its food intake was then measured. Three days after the experiment (experimental day 4), each rat was treated with dimethylsulfoxide + leptin and its food intake was again measured. We subtracted the 24-h food intake of day 1 from that of day 4 and selected leptin-responsive rats. Four days after the experiment (experimental day 8), the selected rats were treated with 15d-PGJ2 + leptin and their food intake was again measured. Four days after the experiment (experimental day 12), each was treated with 15d-PGJ2 + saline and its food intake was measured (Figure S2). In the pilot study, we injected leptin twice into the same rat and compared each response in inhibiting food intake. Inhibitory effects on food intake were similar between the first and second injections (Figure S3). Therefore, there was no desensitization to the secondary leptin response under the present conditions. Procedure for the intracerebroventricular administration protocol On experimental days, all food in the individual cages was removed at 17:00. At 18:00, leptin and/or 15d-PGJ2 was injected through an intracerebroventricular (ICV) route. Food was then returned to the individual cages at 19:30 and food intake was measured for 24 h. Hypothalamic STAT3 analysis after the intracerebroventricular injection of leptin into mice Male mice were used in the present experiment. One week before the experiment, we made a hole in the skull 0.3 mm caudal from the bregma, 0.9 mm right lateral side from the midline, and 2.0 mm below the dura matter under sodium pentobarbital anesthesia (50 mg/kg, i.p.). After surgery, mice were treated with antibiotics to prevent infection. After a recovery period of 7 days, mice were starved overnight and 15d-PGJ2 (20 lg/mouse) and/or leptin (2.25 lg/mouse) were injected. Samples were snap-frozen in liquid nitrogen and stored at 80°C until use. Regarding western blotting, tissue samples were homogenized in a buffer containing 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 21 mM Na3VO4, 10 mM NaF, 10 lg/mL aprotinin, 10 lg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40 using Precellys 24 (Bertin Technologies, Orleans, France). Samples were centrifuged at 19 000 g for 45 min at 4°C, and the supernatants were collected. Western blot analysis Western blotting was performed as described previously (Hosoi et al. 2012). Briefly, cells were washed with ice-cold phosphatebuffered saline and lysed in buffer containing 10 mM HEPESNaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 10 lg/mL aprotinin, 10 lg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1% NP-40 for 20 min. The lysates were centrifuged at 20 630 g for 20 min at 4°C, and supernatants were collected. Samples were boiled with Laemmle buffer for 3 min, fractionated by sodium dodecylsulfate–polyacrylamide gel electrophoresis, and transferred at 4°C to nitrocellulose membranes. These membranes were then incubated with antiphospho STAT3 (Tyr705, 1 : 1000; Cell Signaling Technology), anti-STAT3 (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PPARc (1 : 1000; Santa Cruz Biotechnology), anti-
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phospho Tyrosine (1 : 1000; Upstate Biotechnology, Lake Placid, NY, USA), and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase, 1 : 1000; Chemicon, Temecula, CA, USA) antibodies, followed by an anti-horseradish peroxidase-linked antibody. Peroxidase binding was detected by chemiluminescence using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, England). Statistics Results are expressed as the mean SE. Statistical analyses were performed using the Student’s t-test, Dunnett’s test, or paired t-test. We conducted an ANOVA analysis for multiple comparisons.
Results 15d-PGJ2 inhibited leptin-induced STAT3 phosphorylation Circulating leptin is known to activate the Ob-Rb leptin receptor, which is located in hypothalamic neurons, and subsequently induces the JAK2-STAT3 signal transduction pathway (Ghilardi et al. 1996). We investigated the effects of 15d-PGJ2 on leptin-induced signal transduction using a human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-Ob-Rb cell line) (Hosoi et al. 2006). The treatment with leptin markedly induced the phosphorylation of STAT3 at Tyr705, indicating the expression of the functional receptor in the SH-SY5Y-Ob-Rb cell line. Therefore, we analyzed the effects of 15d-PGJ2 on leptin-induced STAT3 phosphorylation. The treatment with 15d-PGJ2 time- and dose-dependently inhibited by leptin-induced STAT3 phosphorylation (Fig. 1). The treatment with 15d-PGJ2 inhibited leptin-induced STAT3 phosphorylation from 30 min up to 4 h (Fig. 1a). The inhibition of 15d-PGJ2 on leptin-induced STAT3 phosphorylation was dose-dependent, with significant levels of inhibition being observed at 10 lM (Fig. 1b). No significant changes were noted in the phosphorylation status of global proteins at tyrosine residues after SH-SY5Y-Ob-Rb cells were treated with 15d-PGJ2 (Figure S1). Therefore, 15d-PGJ2 may not have perturbed global tyrosine kinase pathways. These results were further confirmed by immunohistochemistry, which revealed that leptin-induced nuclear phospho-STAT3 staining was markedly attenuated by 15d-PGJ2 (Fig. 2). Thus, these results suggested that 15d-PGJ2 specifically inhibited STAT3 signal transduction downstream of the ObRb leptin receptor. Involvement of PPAR-c in the development of leptin resistance It currently remains unknown how 15d-PGJ2 evokes leptin resistance. To address this issue, we focused on PPAR-c, which is activated by 15d-PGJ2 (Forman et al. 1995; Kliewer et al. 1995). We investigated whether the inhibitory effects of 15d-PGJ2 on the actions of leptin were
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(b) Fig. 1 15d-PGJ2 induced leptin resistance. (a) SH-SY5Y-ObRb cells were treated with 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2:10 lM) for the indicated times and then stimulated with leptin (Lep: 0.5 lg/mL) for 15 min. ***p < 0.001 (b) SH-SY5YObRb cells were treated with 15d-PGJ2 (15d-PGJ2: 0.3–10 lM) for 1 h and then stimulated with leptin (Lep: 0.5 lg/mL) for 15 min. A western blotting analysis was performed using specific antibodies for phospho-STAT3 (Tyr705) and signal transducer and activator of transcription 3 (STAT3). **p < 0.01.
mediated through PPAR-c. We initially examined its expression in the SH-SY5Y Ob-Rb cell line. Consistent with previous findings (Moreno et al. 2004; Sarruf et al. 2009), PPAR-c was expressed in SH-SY5Y Ob-Rb neuronal cells as well as in the mouse brain such as in the hypothalamus or cortex (Fig. 3a). Therefore, we examined the effects of GW9662, a specific PPAR-c antagonist, on 15d-PGJ2-induced leptin resistance. The treatment with GW9662 alone did not affect leptin-induced STAT3 phosphorylation (Fig. 3b). However, we observed the slight recovery of the 15d-PGJ2-induced inhibition of STAT3 phosphorylation by GW9662 in leptin-treated cells (Fig. 3b). These results suggested that PPAR-c may be involved in 15d-PGJ2-induced leptin resistance. To further confirm the involvement of PPAR-c in leptin resistance, we analyzed the effects of the PPAR-c agonist on the actions of leptin. We treated cells with rosiglitazone, a specific PPAR-c agonist, and analyzed leptin-induced STAT3 phosphorylation. As expected, a dose-dependent decrease
was observed in leptin-induced STAT3 phosphorylation following the treatment with rosiglitazone (Fig. 3c). These results indicated that 15d-PGJ2-induced leptin resistance was mediated through PPAR-c.
15d-PGJ2-induced leptin resistance was not dependent on SOCS3 or ER stress We next examined the possible relationship between the previously identified mechanisms underlying leptin resistance, such as SOCS3 (Bjørbaek et al. 1998) or ER stress (Hosoi et al. 2008; Ozcan et al. 2009), and 15d-PGJ2induced leptin resistance. We measured SOCS3 levels after the treatment with 15d-PGJ2. Consistent with previous findings (Bjørbaek et al. 1998), the treatment with leptin for 6 h increased SOCS3 levels, which acted as a negative feedback regulator on the actions of leptin (Bjørbaek et al. 1999) (Fig. 4). However, we did not observe an increase in SOSC3 levels after cells were treated with 15d-PGJ2 at any
Fig. 2 15d-PGJ2 inhibited leptin-induced induction of nuclear phospho-STAT3. SHSY5Y-ObRb cells were treated with 15dPGJ2 (15d-PGJ2: 10 lM) for 4 h and then stimulated with leptin (0.5 lg/mL) for 15 min. Immunofluorescence staining was performed using antibodies for phosphoSTAT3. PI: Nuclear staining. Original magnification: 963. Images were obtained with a confocal microscope and were analyzed by LSM software.
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Fig. 3 Peroxisome proliferator-activated receptor gamma (PPARc) was involved in 15d-PGJ2-induced leptin resistance. (a) PPARc was expressed in SH-SY5Y-ObRb cells, the hypothalamus, and cortex. A western blotting analysis was performed using specific antibodies for PPARc and GAPDH. (b) The PPARc antagonist restored 15d-PGJ2induced leptin resistance. SH-SY5Y-ObRb cells were pre-incubated with GW9662 (0.5 lM) for 30 min and then treated with 15d-PGJ2
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(15d-PGJ2: 30 lM) for 4 h. Leptin (0.5 lg/mL) was stimulated for 15 min. *p < 0.05 (vs. Leptin + 15d-PGJ2). (c) The PPARc agonist induced leptin resistance. SH-SY5Y-ObRb cells were treated with rosiglitazone (Rosi: 0.1–10 lM) for 1 h and then stimulated with leptin (Lep: 0.5 lg/mL) for 15 min. A western blotting analysis was performed using specific antibodies for phospho-STAT3 (Tyr705) and signal transducer and activator of transcription 3 (STAT3). ***p < 0.001
of the time points investigated (Fig. 4). Thus, 15d-PGJ2induced leptin resistance may not be mediated through the induction of SOCS3. 15d-PGJ2 also did not induce the apparent activation of ER stress, as measured by ER stressregulated genes such as GRP78 and the activation status of ER stress sensor proteins such as IRE1 (Inositol-requiring enzyme-1) or PERK (PKR-like kinase) (Fig. 5). As a positive control, the treatment with tunicamycin, an ER stress-inducing reagent that inhibits protein glycosylation, increased the activation of IRE1 and PERK as well as the induction of GRP78 in SH-SY5Y-Ob-Rb cells (Fig. 5). Therefore, these results suggested that 15d-PGJ2-induced leptin resistance was not mediated through ER stress. Therefore, 15d-PGJ2-induced leptin resistance may not be mediated through previously identified mechanisms such as SOCS3 and ER stress.
Fig. 4 15d-PGJ2 did not affect suppressor of cytokine signaling 3 (SOCS3) levels. SH-SY5Y-ObRb cells were treated with 15d-PGJ2 (15d-PGJ2: 10 lM) for the indicated times. A western blotting analysis was performed using specific antibodies for SOCS3 and GAPDH. Leptin (Lep: 0.5 lg/mL, 6 h) increased SOCS3 levels. However, 15dPGJ2 did not affect SOCS3 levels.
15d-PGJ2 reversed the inhibitory effects of leptin on food intake We investigated the physiological consequences of 15dPGJ2 on the actions of leptin. Leptin inhibits food intake by acting on the brain (Campfield et al. 1995). Therefore, we determined whether 15d-PGJ2 attenuated the leptin-
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(a)
(b)
Fig. 6 15d-PGJ2 reversed the satiety action of leptin. Leptin was administered through an ICV route to male rats. Cumulative food intakes were measured for 24 h. Leptin inhibited food intake. The treatment with 15d-PGJ2 reversed the inhibitory effects of leptin on food intake. ***p < 0.001.
(a)
Fig. 5 15d-PGJ2 did not affect endoplasmic reticulum (ER) stress responses. (a) SH-SY5Y-ObRb cells were treated with 15d-PGJ2 (10 lM) for the indicated times. Tunicamycin (Tm: 1 lg/mL) was treated as a positive control. A western blotting analysis was performed using specific antibodies for IRE-1, PERK, and GAPDH. The treatment with Tm increased the phosphorylation of IRE1 and PERK, as indicated by a mobility shift to the upper band. On the other hand, the treatment with 15d-PGJ2 did not affect the phosphorylation of IRE1 or PERK. (b) A western blotting analysis was performed using specific antibodies for GRP78 and GAPDH. The treatment with Tm increased the induction of GRP78, whereas 15d-PGJ2 did not. A densitometric analysis of the induction of GRP78 was performed using image analysis software.
induced inhibition of food intake in vivo. To evaluate the effects of 15d-PGJ2 on the actions of leptin in inhibiting food intake, we centrally injected 15d-PGJ2 along with leptin into rats and measured their food intake. The intracerebroventricular administration of leptin alone decreased food intake within 24 h (Fig. 6). However, the treatment with 15d-PGJ2 significantly reversed the inhibitory effects of leptin on food intake (Fig. 6). These results suggested that 15d-PGJ2 inhibited the actions of leptin on food intake in vivo. Therefore, 15d-PGJ2 may be involved in the development of leptin resistance. To further confirm these results, we next injected 15d-PGJ2 intracerebroventricularly into the mouse brain and analyzed the leptin-induced phosphorylation of STAT3 in the hypothalamus. As shown in Fig. 7, leptin-induced STAT3 phosphorylation was inhibited by the administration of 15d-PGJ2.
(b)
Fig. 7 15d-PGJ2 reduced leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in the hypothalamus. (a) 15d-PGJ2 and leptin were administered through an intracerebroventricular (ICV) route to male mice. Animals were pretreated with 15dPGJ2 prior to leptin injection. Thirty minutes after leptin treatment, hypothalamus was taken and phosphorylation level of STAT3 was analyzed by western blotting (b) A densitometric analysis of the induction of P-STAT3 was performed using image analysis software.
Discussion Evidence to suggest that chronic brain inflammation is associated with obesity is increasing. Previous studies reported that saturated fatty acids induced inflammatory responses by activating the toll-like receptor signaling pathway in the hypothalamus (Milanski et al. 2009; Moraes et al. 2009), suggesting the possible involvement of fatty acids in inflammation. Furthermore, recent findings suggest that hypothalamic inflammation also contributes to leptin
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resistance in obesity (Thaler et al. 2010). However, the mechanisms linking hypothalamic inflammation to obesity remain unclear. In the present study, we showed that 15dPGJ2, a member of the prostaglandin family, which regulates key aspects of immunity, was involved in the development of leptin resistance. We demonstrated that 15d-PGJ2 inhibited the leptin-induced activation of STAT3 and leptin-induced anorexia. 15d-PGJ2 exists in the CSF and its expression was shown to be up-regulated by brain inflammation caused by bacterial endotoxins (Mouihate et al. 2004). Chronic inflammation may contribute to the development of leptin resistance (Thaler et al. 2010). Therefore, an increase in 15d-PGJ2 levels in the brain may contribute to the development of leptin resistance. The production of 15d-PGJ2 from microglia cells was shown to be elevated under inflammatory conditions (Ajmone-Cat et al. 2003). Since chronic inflammation has been observed in the obese hypothalamus, microglia may induce the production of 15d-PGJ2, which may in turn be involved in the development of leptin resistance. 15d-PGJ2 possesses anti-inflammatory properties. For example, the expression of iNOS, gelatinase B, and scavenger receptor A genes was inhibited by 15d-PGJ2 in activated macrophages (Ricote et al. 1998). Furthermore, 15d-PGJ2 suppressed the activation of microglia by downregulating the expression of iNOS (Petrova et al. 1999). Therefore, we speculated that the chronic or sustained induction of inflammation may lead to the induction of 15d-PGJ2 by a negative feedback mechanism, which then affects leptin signals. The mechanisms underlying the development of leptin resistance have been suggested to be mediated through the induction of SOCS3 or ER stress. However, 15d-PGJ2induced leptin resistance may not be mediated through these mechanisms. We herein demonstrated that PPAR-c would be involved in 15d-PGJ2-induced leptin resistance. We found that 15d-PGJ2-induced leptin resistance was slightly attenuated by the PPAR-c antagonist, GW9662, in leptin-treated cells. Furthermore, the PPAR-c agonist, rosiglitazone, attenuated the leptin-induced activation of STAT3. Rosiglitazone is an anti-diabetic drug, which is classified as thiazolidinedione (TZD), and is used to treat diabetes caused by insulin resistance (Lehrke and Lazar 2005). However, the main side effect of TZD is weight gain (Larsen et al. 2003), the mechanism of action for which it has yet to be clarified. This finding is of particular interest because obesity is involved in the development of diabetes. One of the underlying mechanisms for the actions of the PPAR-c agonist on obesity may be attributed to peripheral effects such as the differentiation of adipocytes (Tontonoz et al. 1994). On the other hand, treatments with TZD have also been reported to increase food intake, suggesting the existence of the CNS actions of PPAR-c in regulating weight gain (Lehrke and Lazar 2005). We found that leptin signals were attenuated when neuronal cells were treated with the PPAR-c agonist. This result is
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consistent with recent findings reported by two groups (Lu et al. 2011; Ryan et al. 2011). Therefore, the PPAR-c agonist may attenuate the actions of leptin in the CNS, thereby increasing food intake. These findings indicate that PPAR-c induces leptin resistance, which may simultaneously account for the side effects of TZD. However, the physiological target molecule that activates PPAR-c currently remains unknown. The identity of the neuronal target molecule regulating the activation of PPAR-c, which is linked to leptin signals, is one of the major issues in this field of research (Myers and Burant 2011). In the present study, we showed that 15d-PGJ2 may be a target for activating PPAR-c, which is linked to leptin resistance. Thus, our results suggest that 15d-PGJ2-induced PPAR-c activation may be involved in the development of leptin resistance in the CNS. On the other hand, previous studies indicated the existence of a PPARc-independent pathway in 15d-PGJ2mediated actions (Chawla et al. 2001; Lu et al. 2013). Furthermore, 15d-PGJ2-induced leptin resistance was not completely ameliorated by the PPAR-c antagonist. Therefore, it is also possible that the inhibitory effects of 15d-PGJ2 on the actions of leptin were mediated through a PPARcindependent pathway. Obesity is a major health concern worldwide because it is accompanied by diseases such as diabetes, hypertension, and hyperlipemia. In the present study, we identified novel mechanisms for the development of leptin resistance in obesity. We found that 15d-PGJ2 was involved in the development of leptin resistance. Therefore, targeting 15dPGJ2 may represent a novel strategy for ameliorating these diseases, which will be an interesting future research subject. Moreover, our results provide a novel insight into how food intake is regulated.
Acknowledgments and conflict of interest disclosure This study was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University. Animal experiments were supported by the Institute of Laboratory Animal Science (Hiroshima University). This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; Takeda Science Foundation; Tokyo Biochemical Research Foundation; Suzuken Memorial Foundation, and the Nakajima Foundation. All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.
Supporting information Additional supporting information may be found in the online version of this article at the publisher's web-site: Figure S1. Effects of 15d-PGJ2 on protein tyrosine phosphorylation levels in SH-SY5Y-ObRb cells.
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Figure S2. Schematic schedule of intracerebroventricular injections for analyzing the in vivo effects of 15d-PGJ2 on the actions of leptin. Figure S3. Leptin was administered through an intracerebroventricular (ICV) route to male rats.
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