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Home , Amylin, Leptin

Care Publish Ahead of Print, published online September 24, 2010

Leptin and act in an additive manner to activate overlapping signaling pathways in peripheral tissues; in vitro and ex vivo studies in humans

Short running title: and amylin signaling

Hyun-Seuk Moon1, PhD, John P. Chamberland1 BS, Kalliope N. Diakopoulos1 BS, Christina G. Fiorenza1 BS, Florencia Ziemke1 MD, Benjamin Schneider2 MD, Christos S. Mantzoros1,3 MD

1Division of , Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA. 2Division of Minimally Invasive Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA. 3Section of Endocrinology, Boston VA Healthcare System, Harvard Medical School, Boston, Massachusetts 02130

Correspondence should be addressed to: Christos S. Mantzoros Email: [email protected]

Submitted 19 March 2010 and accepted 18 September 2010.

Additional information for this article can be found in an online appendix at http://care.diabetesjournals.org

This is an uncopyedited electronic version of an article accepted for publication in Diabetes Care. The American Diabetes Association, publisher of Diabetes Care, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher- authenticated version will be available in a future issue of Diabetes Care in print and online at http://care.diabetesjournals.org.

Copyright American Diabetes Association, Inc., 2010 Leptin and amylin signaling

Objective - Amylin interacts with leptin to alter metabolism. We evaluated, for the first time, amylin- and/or leptin-activated signaling pathways in human peripheral tissues (hPT).

Research design and methods - Leptin and amylin signaling studies were performed in vitro in human primary (hPA) and human peripheral blood mononuclear cells (hPBMC), and ex vivo in human adipose (hAT) tissue from male vs. female, obese vs. lean, and subcutaneous (SC) vs. omental (OM) subjects.

Results - The long form of leptin (ObRb) was expressed in human tissues and cells studied in ex vivo and in vitro, respectively. Leptin and amylin alone and in combination activate STAT3, AMPK, Akt and ERK signaling pathways in hAT ex vivo, and hPA and hPBMC in vitro; all phosphorylation events were saturable at leptin and amylin concentrations of ~50 ng/ml and ~20 ng/ml respectively. The effects of leptin and amylin on STAT3 phosphorylation in hPA and hPBMC in vitro were totally abolished under endoplasmic reticulum stress and/or in the presence of a STAT3 inhibitor. Results similar to the above in vitro studies were observed in hAT studied ex vivo.

Conclusions - Leptin and amylin activate overlapping intracellular signaling pathways in humans and have additive, but not synergistic, effects in signaling pathways studied in hPT in vitro and ex vivo.

eptin is an -secreted for treatment in both animals (2) and that plays a major humans (5). L role in energy and Comprehensive pharmacological weight balance (1). Leptin activates not only studies demonstrated that concurrent amylin central networks that suppress and leptin infusion synergistically reduce (1) but also acts in the periphery to body weight and adiposity in -induced alter immune function and metabolism (2). obesity (DIO) rats (6). It has also been Leptin administration to leptin deficient demonstrated that acute amylin infusion (ob/ob) mice has been shown to reduce food amplifies central leptin signaling, and with intake and body mass and improve sustained treatment, amylin and leptin elicit resistance even before body weight is reduced synergistic weight and loss (6). Amylin- (3). Moreover, leptin improves insulin treated rats demonstrate a trend for a greater resistance in leptin deficient lipoatrophic mice number of p-STAT3-positive cells within the and humans with the and (ARC) than vehicle-treated (3). Amylin is a 37-amino controls, an effect similar to leptin’s, whereas acid hormone that is co-secreted with rats treated with both amylin and leptin have insulin from pancreatic β-cells (4). The significantly more p-STAT3-positive cells physiologic effects of amylin receptor than vehicle-, amylin-, or leptin-treated rats agonism include decreased food intake (4) (7). Furthermore, this study also demonstrated and reduction of postprandial that mRNA was significantly release in a glucose-dependent manner (5). increased by amylin/leptin co-infusion both Moreover, it has been proposed that treatment centrally and peripherally i.e. in white adipose with a combination of amylin and leptin may tissue (WAT) and (7). Moreover, it has be more effective than leptin or amylin alone been shown that peripheral leptin injection in

2 Leptin and amylin signaling

rodents increases STAT3 and MAPK Phoenix Pharmaceuticals Inc. (Burlingame, phosphorylation in liver and AT (8). No CA). FBS and FCS were provided by Gibco previous study has evaluated leptin and Life Technologies (New York, USA). BSA, amylin signaling in human peripheral tissues α-MEM, RPMI, NaHCO3, Hepes, biotin, (hPT) nor studied how amylin interacts with pantothenate, human transferrin, gentamycin, leptin to alter signaling in hPT. insulin, , T3, DTT, TUN and We performed ex vivo and in vitro were purchased from Sigma- signaling studies to clarify the role of leptin Aldrich (St. Louis, MO). and amylin in activating signaling pathways Real-time polymerase chain reaction (RT- in hPT as well as their potential interactions. PCR) - The long form of leptin receptor We first investigated in vitro leptin and (ObRb) expression was detected with RT- amylin signaling in human primary PCR. RNA was extracted with Trizol® adipocytes (hPA) and human peripheral blood (Invitrogen, Carlsbad, CA) and first-strand mononuclear cells (hPBMC), known cDNA synthesis was performed using targets of leptin action. We then performed ex Superscript III® (Invitrogen, Carlsbad, CA), vivo leptin and amylin signaling studies in according to the manufacturer’s protocol. For human (hAT) from RT-PCR, 100ng of cDNA per 25µl reaction subcutaneous (SC) vs. omental (OM), male vs. were amplified using TaqMan Gene female, and obese vs. lean subjects. Expression system (Applied Biosystems, Foster City, CA), specific primers and FAM RESEARCH DESIGN AND METHODS tagged probes set (Applied Biosystems, Subject collection - We used human adipose Foster City, CA), and the standard real-time tissue (hAT) from subjects undergoing 7500 protocol (Applied Biosystems, Foster laparoscopic adjustable gastric banding, City, CA), with an initial polymerase hernioplasty, liposuction or abdominoplasty. activation step at 95oC for 10 min and 40 The study protocol was approved by the cycles. This included a 15 sec melting step at Institutional Review Board at the Beth Israel 95oC and a 1 min annealing-elongation step at Deaconess Medical Center (BIDMC) and 60oC. The analysis of relative subjects gave written informed consent to was based on ΔCt values obtained from RT- participate. All subjects were otherwise PCR (9). healthy, had no evidence of immunologic or Ex vivo signaling study in hAT - The ex vivo endocrine disease based on physical culture was established according to the examination and routine blood tests, and had method described by Kim et al. (8). Briefly, no history of recent infection. The subjects’ five to seven grams of fresh subcutaneous age, vitals and BMI were recorded. We (SC) and/or omental (OM) hAT were placed collected tissue samples from obese (6 males into Krebs-Ringer-HEPES buffer (20mM, pH and 6 females; age, 31-54 year-old; BMI, 42- 7.4) with 2.5% BSA and 200nM adenosine at 44 kg/m2) and lean (3 males and 4 females: 37oC in the operating room and immediately age, 22-32 year-old; BMI, 21-23 kg/m2) taken to the laboratory for further analysis. In subjects. the laboratory, the tissue samples were Materials - All primary and secondary minced into pieces of approximately 1-mm in antibodies were purchased from Santa Cruz diameter, and any non-adipose tissue and non- Biotech (San Francisco, CA). Leptin human muscle tissue were removed by washing with recombinants were purchased from ProSpec- fresh buffer. The samples were aliquoted and o Tany TechnoGene Ltd. (Rehovot, Israel). Amylin incubated at 37 C either with or without human recombinants were purchased from amylin and leptin.

3 Leptin and amylin signaling

Human primary adipocytes (hPA) culture - dithiothreitol (DTT, 1mM) for 5 hr, and The hPA culture was established according to subsequently taken to leptin and/or amylin. the method described by Ribet et al. (10). Protein extraction - For total cell extracts, Briefly, SC and OM hAT samples were collected cells were suspended in a lysis obtained from lean (35-41 years old, BMI 22- buffer containing 20mM Tris-HCl (pH 7.4), 25kg/m2) and obese (34-48 years old, BMI 150mM NaCl, 5mM ethylene diamine tetra 39-50 kg/m2) males and females, respectively. acetic acid (EDTA), 0.1 mM phenylmethyl- The hAT was then digested with sulfonyl fluoride (PMSF), 0.05% aprotinin, PBS/collagenase solution (3mg collagenase and 0.1% Igepal and then incubated for 30 per gram of tissue and 1 ml PBS per 1mg min at 4°C. The suspension was centrifuged collagenase) + 3.5% free BSA and for 25min at 14,240 g and the supernatant was then filtered using filter-bottle unit (sterile saved as the total extract. Next, the pellet was funnel with double layered gauze), then the re-suspended in a lysis buffer containing solution was centrifuged at 1,200rpm for 10 50mM Hepes-NaOH (pH 7.8), 50mM KCl, minutes. The pellets were re-suspended in 300mM NaCl, 0.1mM EDTA, 1mM DTT, alpha MEM (α-MEM) supplemented with 0.1mM PMSF and 10% (v/v) glycerol. The 15mmol/L NaHCO3, 15mmol/L Hepes, suspension was mixed for 30min at 4°C and 33µmol/L biotin, 17µmol/L pantothenate, 10 centrifuged for 15min at 890g; the mg/ml human transferrin, 0.05mg/ml supernatant was saved as the nuclear extract. gentamycin, and 10% FBS and then plated Western Blotting - For Western Blotting, overnight. After confluence, α-MEM was proteins were loaded in each lane. After SDS- removed and washed once or twice with polyacrylamide gel electrophoresis, proteins HBSS. To induce adipocyte differentiation, were blotted onto nitrocellulose membranes the cells were exposed to differentiation (Schleicher & Schuell, Inc., Keene, NH). The medium containing 66nmol/L insulin, membranes were blocked for 1hr in TBS 100nmol/L cortisol, 0.2nmol/L triiodo- containing 5% nonfat dry milk and 0.1% thyronine (T3) and 1µg/ml ciglitazone. The Tween 20. Incubation with primary antibodies medium was changed every 2 days and cells was performed in TBS containing 5% nonfat were kept in culture for 28 days. dry milk for overnight and then incubated Human peripheral blood mononuclear cells with horseradish peroxidase secondary (hPBMC) culture - The hPBMC was isolated antibodies for two hour. After incubation with by density gradient sedimentation on Ficoll- antibodies, membranes were washed with Paque (Pharmacia, Uppsala, Sweden) as TBS containing 0.1% Tween-20. Enhanced described previously (11). The cells were chemiluminescence was used for detection. washed twice in PBS and re-suspended in Measurement of signal intensity on medium appropriately for cell culture, RPMI nitrocellulose membranes after Western 1640 supplemented with 25mM Hepes, 2mM Blotting with various antibodies was L-, 100μU/ml penicillin, 100μg/ml performed using Image J processing and streptomycin, and amphotericin B (2.5μg/ml). analysis software. Endoplasmic reticulum (ER) stress Statistical Analysis - Data were analyzed induction - The induction of ER stress was using one-way ANOVA followed by post-hoc established according to the method described test for multiple comparisons. Analyses were by Hagiwara et al. (12). Briefly, to induce ER carried out using SPSS (version 11.5, SPSS). stress, the cells were pre-treated with tunicamycin (TUN, 3µg/ml) and/or RESULTS

4 Leptin and amylin signaling

1. Leptin receptor expression in hAT ex vivo, and amylin activated STAT3 signaling in and in hPA and hPBMC in vitro - The long hAT ex vivo by ~4.7-fold, in hPA in vitro by form of leptin receptor (ObRb) was expressed ~5.1-fold and in hPBMC in vitro by ~4.9-fold. in human tissues and cells studied in ex vivo Moreover, nuclear translocation of STAT3 and in vitro, respectively (data not shown). was increased by leptin, and this effect was We did not observe any increase in ObRb more enhanced by co-administration of expression as a result of leptin, amylin, and/or amylin in hPA and hPBMC in vitro (data not leptin + amylin administration for 30 min shown). By contrast, increased STAT3 when compared to control. Also, we did not phosphorylation by leptin + amylin observe any difference in ObRb expression in administration was totally abolished by pre- SC vs. OM hAT, male vs. female and obese treatment with AG490, a STAT3 inhibitor, in vs. lean (data not shown) in the limited hPA (Supplemental Figure 1D) and hPBMC number of subject studied. (Supplemental Figure 1E) in vitro. 2. Leptin and amylin signaling: Dose- and Importantly, we observed an additive, but not time-response curves in hAT ex vivo, and in synergistic, effect of amylin on leptin-treated hPA and hPBMC in vitro from obese female tissues and/or cells. There was no difference subjects - Ex vivo and in vitro leptin and in STAT3 activation in experiments amylin administration significantly induced comparing SC vs. OM hAT, obese vs. lean phosphorylation of STAT3 by ~2.8-fold and male vs. female subjects (data not shown). higher than control at 30 min in hAT (Fig. 1A 4. Activation of ERK signaling by and 1B) ex vivo, and in hPA (Fig. 1C) and administration of leptin and amylin alone or hPBMC (Fig. 1D) in vitro. Importantly, all in combination in hAT ex vivo and in hPA leptin and amylin signaling pathways were and hPBMC in vitro from obese female activated in a dose-dependent manner but subjects - ERK signaling was activated in activations of all signaling pathways were leptin-treated (by ~2.3-fold) and amylin- saturable at a leptin concentration of ~50 treated (by ~2.2-fold) hAT ex vivo, and this ng/ml and amylin concentration of ~20 ng/ml. activation was further increased in response to We did not observe any difference in STAT3 co-administration of amylin and leptin by activation in SC vs. OM hAT, obese vs. lean ~3.4-fold (Supplemental Figure 2A). As and in male vs. female subjects (data not expected, ERK signaling was activated in shown). leptin- and amylin-treated hPA in vitro by 3. Activation of STAT3 signaling by ~2.9-fold and ~2.8-fold (Supplemental Figure administration of leptin and amylin alone or 2B) and in hPBMC in vitro by ~2.8-fold and in combination in hAT ex vivo and in hPA ~2.7-fold, respectively (Supplemental Figure and hPBMC in vitro from obese female 2C). Also, activation of ERK signaling in subjects - Based on the above (Fig. 1), we response to leptin and amylin co- chose a representative administration time (30 administration in hPA in vitro was increased min) and leptin (50 ng/ml) and amylin (20 by ~5.1-fold and hPBMC in vitro by ~4.8- ng/ml) concentrations. Either leptin or amylin fold but leptin + amylin-activated ERK stimulated phosphorylation of STAT3 by ~ signaling was totally blocked by pre-treatment 2.8-fold in SC and OM hAT ex vivo with an ERK inhibitor (Supplemental Figure (Supplemental Figure 1A available at 2B and 2C). We did not observe any http://care.diabetesjournals.org), by ~2.9-fold difference in ERK activation when comparing in hPA in vitro (Supplemental Figure 1B) and cells or tissues from SC vs. OM hAT, obese by ~2.8-fold in hPBMC (Supplemental Figure vs. lean and male vs. female subjects (data not 1C) in vitro. Also, co-administration of leptin shown).

5 Leptin and amylin signaling

5. Activation of Akt and AMPK signaling by obese vs. lean and male vs. female subjects administration of leptin and amylin alone or (data not shown). in combination in hAT ex vivo and in hPA and hPBMC in vitro from obese female CONCLUSIONS subjects - Leptin activated Akt and AMPK Leptin, the prototype adipocyte- signaling in hAT ex vivo by ~1.9-fold and secreted adipocytokine, regulates food intake ~3.5-fold (Supplemental Figure 3A), in hPA and body weight acting primarily in the in vitro by ~2.6-fold and ~2.8-fold (1). It may also act in the (Supplemental Figure 3B) and in hPBMC in periphery to modulate immune and metabolic vitro by ~3.0-fold and ~3.2-fold functions (2). Peripheral leptin administration (Supplemental Figure 3C). Amylin also has been demonstrated to phosphorylate activated Akt and AMPK signaling in hAT ex STAT3 and MAPK in rodent AT and liver (8). vivo by ~2.0-fold and ~3.6-fold Similar effects have not yet been explored in (Supplemental Figure 3A), in hPA in vitro by humans. Amylin, a hormone secreted from ~2.6-fold and ~2.8-fold (Supplemental Figure the , functions to decrease food 3B) and in hPBMC in vitro by ~3.0-fold and intake, decrease gastric emptying and ~3.1-fold (Supplemental Figure 3C). glucagon (4). Although amylin has Moreover, activation of Akt and AMPK been proposed to amplify leptin signaling in signaling by leptin and amylin alone in hAT the hypothalamus, thereby synergistically ex vivo as well as hPA and hPBMC in vitro effecting weight and fat loss in animal models was increased in response to leptin and (6), the molecular mechanisms underlying amylin co-administration. By contrast, these effects remain to be fully clarified. We activation of Akt and AMPK signaling in have previously demonstrated that altering leptin + amylin-stimulated cells was blocked circulating leptin levels in humans does not by pre-treatment with each inhibitor. We did alter circulating amylin levels (13). Whether not observe any difference in Akt and/or amylin’s action can increase leptin signaling AMPK activation in cells or tissues from SC in human tissues remains to be fully vs. OM hAT, obese vs. lean and from male vs. elucidated. More specifically, there have been female (data not shown). no leptin and amylin signaling studies 6. Inhibition of STAT3 signaling by ER stress evaluating hPT, including hAT and hPBMC. in hPA and hPBMC in vitro from obese Hence, we performed ex vivo and in vitro female subjects - Stimulation of the cells with signaling studies to investigate the role of leptin + amylin led to a marked and amylin and leptin in activating signaling significant increase in phosphorylation of pathways in hPT as well as their potential STAT3 in hPA by ~5.0-fold (Supplemental interaction. Figure 4A) and in hPBMC by ~4.8-fold STAT3 mediates the expression of a (Supplemental Figure 4B) but, when variety of genes in response to cell stimuli, challenged with ER stress inducers, TUN and thus plays a key role in many cellular and/or DTT, the leptin + amylin-activated processes such as cell growth and STAT3 phosphorylation was totally abolished, (8). In fact, disrupting the ability of the leptin suggesting that amylin could not overcome receptor to activate the STAT3 pathway in ER stress-induced inhibition of leptin- mice leads to severe obesity and several other activated STAT3 signaling. We did not neuroendocrine abnormalities (14). The observe any difference in ER stress-blocked STAT3 pathway is the first identified STAT3 activation in hPA and hPBMC from signaling mechanism found to be associated with the leptin receptor (8). Leptin induces

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phosphorylation of STAT3 in keratinocytes, ex vivo as well as hPA and hPBMC in vitro. endometrial cancer cells, hypothalamic cells, Since ERK is a significant downstream target murine adipocytes and mouse AT (3, 8, 15- for leptin’s physiological effects (15-16), we 16). Amylin, similar to leptin, has been shown suggest that amylin may also have a to increase the number of p-STAT3 cells in beneficial effect on human physio- and the ARC of rats (7) and this effect was . significantly greater with co-administration of AMPK is an evolutionarily conserved leptin and amylin when compared to / protein kinase central to the individual treatments (7). Based on these regulation of energy balance at both the results, we checked for the first time whether cellular and whole-body level (17). In its leptin signaling interacts with amylin in classical role as an intracellular metabolic humans and whether co-treatment of amylin stress-sensing kinase, AMPK switches on and leptin could further increase leptin- fatty acid oxidation and glucose uptake, while stimulated STAT3 signaling in hPT. We switching off hepatic gluconeogenesis, and observed that leptin and amylin increased exerts a broader role in metabolism by STAT3 signaling in hAT ex vivo and hPA and controlling appetite (17). In fact, AMPK is hPBMC in vitro, and the effect of these two involved in cellular by was similar in magnitude and AMPK phosphorylation and inhibition of peaked around the same time. Our data in acetyl-coA carboxylase (ACC), and thus peripheral tissues is consistent with rodent stimulates fatty acid oxidation (17-18). studies previously demonstrating that amylin- However, whether leptin can activate AMPK treated rats tend to have a greater number of and/or whether amylin can activate AMPK to p-STAT3-positive cells than vehicle controls induce fatty acid oxidation in hPT has not yet in hypothalami (7). Although it is important been demonstrated. We checked leptin and to study leptin’s and amylin’s effects in amylin signaling in hPT in vitro and ex vivo, human hypothalamic or other central nervous and observed that leptin and amylin increase system cells, our data suggest that amylin- AMPK activation in all cells. Moreover, we increased STAT3 signaling in leptin- observed that leptin and amylin stimulated hAT and cells may have important administration directly increase AMPK clinical implications. activation in hAT ex vivo. Since AMPK is ERK, a member of the mitogen- associated with fatty acid oxidation, which activated protein kinase (MAPK) family, is an plays a key role in the pathophysiology of additional pathway downstream of the leptin insulin resistance (17), leptin- and amylin- receptor (16). Leptin has been shown to activated AMPK signaling in hAT and other activate ERK1/2 in a time- and dose- peripheral tissues offers further support to the dependent manner in several cultured mouse notion that the use of these hormones, alone cells in vitro as well as rodent tissues in vivo or in combination, could be a viable strategy (17). The ERK pathway has been reported to in treating obesity and type II diabetes. mediate leptin’s effects on human PBMC in The Akt signaling pathway is vitro as well as rat and rat AT (8, 17). responsible for cellular processes like cell Based on these previous studies, we further growth, proliferation and glucose metabolism investigated whether amylin could activate (19). It has been shown that obesity induces ERK signaling in hPT, since no relevant data increased levels of leptin, insulin, insulin-like in humans has been published to date. We growth factor, TNF-α and IL-6, and reduces observed that amylin increases ERK levels (19). These altered factors activation alone and in leptin-stimulated hAT change Akt signaling activity, which, in turn,

7 Leptin and amylin signaling

regulates downstream targets leading to It is generally accepted that obese vs. increased cell survival and cell growth, and lean subjects have differences in leptin promoted cell cycle (19). Based on these responsiveness (3). We also expected that previous results, we further investigated there would be differences between obese vs. whether leptin and/or amylin could activate lean group in terms of leptin responsiveness. Akt signaling in hPT in vitro and ex vivo. We Despite minor differences in the timing of observed that leptin and amylin alone and in signaling activation, we did not observe any combination increase Akt phosphorylation in differences in several leptin-activated hAT ex vivo as well as hPA and hPBMC in signaling pathways from obese vs. lean vitro suggesting that leptin and amylin, alone subjects in the tissues studied. There are a or in combination, could have beneficial number of potential explanations for this effects on cell survival. observation. First, although there are We observed that amylin activates Jak2 differences between obese vs. lean subjects in signaling pathways in hAT ex vivo and hPA response to leptin accumulation in the and hPBMC in vitro (data not shown). Since medium of cultured OM and SC AT in the OBRb has the capacity to activate Jak2 (20), early differentiation stages in vitro (22), leptin we suggest that amylin-mediated intracellular and leptin mRNA levels are not different in signaling pathways ex vivo and in vitro might obese vs. lean subjects after full be activated not only through cGMP (21) but differentiation (23). The effect of leptin on also potentially through Jak2 signaling. This is not different between cultured is the first report not only on amylin obese and lean human adipocytes in vitro (24). interacting with Jak2 signaling in human These results demonstrate that there are major peripheral tissues, but also on amylin quantitative changes in leptin responsiveness signaling in human peripheral tissues. It has between obese and lean during differentiating been suggested that amylin may act through stages, but not differentiated stages, of hAT in up-regulation of the leptin receptor and that vitro. We have used differentiated adipocytes this induces STAT3 activation in hypothalami herein. Signaling interactions of leptin and in rodent in vivo (7). We did not observe any amylin in various differentiation stages difference in ObRb expression in human remain to be studied. Second, although we peripheral tissues treated with leptin, amylin studied the signaling pathways considered and leptin + amylin but the treatment time primary targets of leptin, on the basis of was only 30 min. Longer term exposure to current evidence deriving mainly from rodent amylin and leptin as well as a receptor knock- studies, we have not looked at all signaling down experiment would also be useful in pathways potentially mediating leptin’s action determining whether amylin could act through in the periphery. Moreover, there might be ObRb in human peripheral tissues. Since it is other, totally unknown, pathways activated by impossible to perform the receptor knock- leptin since there have been no prior studies down studies in humans, alternative methods on leptin signaling in human tissues. Much such as ObRb knock-down in vivo rodent or more needs to be learned in the future. Third, mouse studies may have to be performed. In it is also possible that signaling pathways any case, the lack of ObRb up-regulation in mediating leptin actions in peripheral tissues our studies excludes this potential mechanism (and resistance thereof) could be totally and suggests that most probably amylin may different from those in hypothalami and/or activate signaling pathways downstream of other areas (CNS) (25). Jak2. This original observation has to be Since it has been proposed that centrally replicated by other studies in the future. administered leptin affects insulin sensitivity

8 Leptin and amylin signaling

and metabolism in peripheral tissues, it is sensitizer to bypass ER stress-induced leptin possible that differences in CNS signaling resistance and suggests that increased ER between lean and obese are much more stress in obese people may induce leptin and important than leptin activated signaling also amylin resistance. Since in vivo leptin pathways in the periphery. Fourth, leptin and and amylin actions may differ in comparison amylin act not only in adipose tissue but also to in vitro, studies of in vivo leptin and amylin in several other metabolically active signaling in humans are needed to prove or peripheral tissues, including but not limited to disprove this hypothesis. It is currently liver and reproductive organs (26). Since impossible to perform human in vivo ER leptin signaling could differ between lean and stress studies, however, and thus specific obese in these tissues and since this could methods for ER stress induction in humans have direct clinical implications in need to be developed. metabolism, more studies, focusing on tissues We believe that our data are important other than the ones studied herein, need to be from the physiological point of view. This is performed. Finally, it is possible that the very first attempt to map the intracellular signaling pathways in humans may be signaling pathways downstream of leptin and different from those in animals. This may be amylin in human tissues. This first paper, if analogous to the different effect of leptin in followed by other similar papers, will regulating neuroendocrine response to eventually allow full characterization of starvation we have previously shown in mice signaling pathways in all peripheral tissues in (27) vs. humans (28). humans as well as the comparative evaluation ER stress has recently been shown to of human vs. animal signaling pathways. This play a role in the development of leptin new knowledge in human will resistance in the hypothalamus of rodents (13) consequently lead to the very first attempt to and it has been suggested that ER capacity is also study pathophysiology, and later directly related to leptin sensitivity (13, 29). therapeutics, in humans. Although we are This resulted in suggestions that ER stress initiating this effort with the current ground- reversal could be utilized as a strategy to breaking paper, much more needs to be done sensitize obese mice, and by extension (additional tissues, other signaling pathways, humans, to leptin. These previous studies other physiological conditions) in the future. have shown that the reduction in ER function Despite these limitations, our initial data in creates ER stress, blocks leptin action, and human tissues suggest the existence of 1) generates leptin resistance in mice, suggesting leptin tolerance (not resistance) in the studied that ER stress inhibits at an upstream step of tissues in human and 2) that unlike STAT3 phosphorylation and provides a experiments in rodents showing synergistic potential mechanism in which increased ER effect of amylin on leptin-modulated food stress antagonizes STAT3-mediated leptin intake the effects of amylin and leptin in signaling (29). However, whether activation human peripheral tissues are additive and not of ER stress interferes with leptin and/or synergistic. amylin signaling in hPT has not yet been In summary, despite minor differences demonstrated. Here, we report for the first in the timing of signaling activation in the time that TUN and also DTT pre-treatment of different tissues and cells studied, we did not human cells completely blocks leptin- and observe major differences in the magnitude of amylin-stimulated STAT3 activation in hPA STAT3 activation in response to leptin and and hPBMC in vitro. Our data demonstrate amylin administration in hAT ex vivo, and that amylin does not function as a leptin hPA and hPBMC in vitro. Also, we did not

9 Leptin and amylin signaling

observe any differences in STAT3, ERK, and Author contribution. H.S.M. and C.S.M. AMPK phosphorylation when comparing wrote this manuscript. H.S.M., J.P.C., K.N.D., male vs. female and obese vs. lean subjects. C.G.F., F.Z., B.S. and C.S.M. participated in Importantly, leptin and amylin signaling the study design, performance and pathways were saturable at a level of ~50 coordination. C.S.M. conceived and planned ng/ml (leptin) and ~20 ng/ml (amylin), the study. suggesting that no additional signaling effect can be observed at higher doses than the ACKNOWLEDGMENTS above. Although leptin and amylin in We would like to thank to Dr. Young-Bum combination increased phosphorylation of the Kim, Division of Endocrinology, Diabetes, pathways studied herein, we observed not a and Metabolism, Beth Israel Deaconess multiplicative, but rather an additive, effect of Medical Center, Harvard Medical School, for amylin on leptin-stimulated hPT ex vivo and technical assistance in the ex vivo study. in vitro. The present study adds to our recent Funding was received from the National clinical data demonstrating that leptin Institute of Diabetes and Digestive and administration does not alter circulating Kidney Diseases grants DK58785, DK79929 amylin levels (13) and provides pre-clinical and DK81913, and AG032030. The evidence that leptin and amylin administration Mantzoros Laboratory is also supported by a activate overlapping intracellular signaling discretionary grant from Beth Israel pathways in human peripheral tissues. These Deaconess Medical Center. All authors read hormones have an additive effect in hPT and approved the final manuscript. All further suggesting that leptin and amylin may authors state that there is no conflict of play an additive role in regulating obesity and interest related to this research paper. type-II diabetes in humans.

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FIGURE LEGEND Figure 1. Leptin and amylin signaling: Dose- and time-response curves in hAT ex vivo and in hPA and hPBMC in vitro from obese female subjects - Ex vivo and in vitro leptin and amylin administration in hAT (A and B), hPA (C) and hPBMC (D) were performed as described in detail in section “RESEARCH DESIGN AND METHODS”. (A-D) The tissues and cells were incubated with leptin and/or amylin (50 ng/ml of leptin and 20 ng/ml of amylin for time-response study and 30 min incubation for dose-response study). All tissue lysates (whole protein extraction) were examined by Western blot with primary p-STAT3 and STAT3 antibodies. The secondary antibody used was horseradish peroxidase-conjugated anti-mouse and anti-goat antibodies. All figures showing quantitative analysis include data from at least three independent experiments. All density values for each protein band of interest are expressed as a fold increase. All data were analyzed using one-way ANOVA followed by post-hoc test for multiple comparisons. Values are means ± SD. Means with different letters are significantly different, p<0.05.

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