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Home , Cholecystokinin, Leptin, Pepsin

Duodenal Stimulates Secretion Evidence of a Positive Leptin-Cholecystokinin Feedback Loop Sandra Guilmeau, Marion Buyse, Annick Tsocas, Jean Pierre Laigneau, and Andre´ Bado

Some of the actions of leptin depend on cholecystokinin gp130 family of receptors. It occurs in several (CCK). However, it is unknown whether leptin modu- isoforms resulting from the alternative splicing of the db lates the release of CCK. Here, we demonstrate in vitro leptin gene (2,3). It is currently thought that the that leptin induces the phosphorylation of extracellular long isoform, Ob-Rb, can activate the signal transducers signal–related kinase (ERK)-1/2 and increases and activators of transcription (STAT) pathways, whereas ؍ CCK release (EC50 0.23 nmol/l) in CCK-secreting both Ob-Rb and the short isoform (Ob-Ra) can transduce STC-1 cells. We showed that duodenal juice contains signals through receptor substrates and through leptin that circulates free and bound to macromole- cules, suggesting that leptin has a lumenal action on the mitogen-activated kinase (MAPK) pathways (7). intestine. In vivo in the rat, duodenal infusion of leptin The signals that arise from the upper gastrointestinal increased plasma CCK at levels comparable to those tract upon feeding are transmitted to the by the induced by feeding. Moreover, -induced increases . These signals are key components in the in plasma CCK were markedly reduced in obese fa/fa control of meal-induced satiety. Cholecystokinin (CCK) is , whereas the mobilization of the gastric leptin pool secreted from duodenal endocrine I cells and typically was similar in lean and obese Zucker rats. The release of functions as one of these short-term satiety signals (8,9). CCK by leptin presumably generates a positive feedback Interestingly, the leptin-induced inhibition of intake loop. Indeed, the blockade of CCK receptors reversed (10) and the stimulation of pancreatic exocrine secretions the meal reduction of the leptin pool and the meal-increased plasma insulin, consistent with the pre- (11) can be blocked by a CCK-1 . These vious concept of an entero-insular axis. Collectively, data suggest that endogenous CCK is involved in these these data support a novel mode of action of leptin effects, operating through CCK-1 receptors. However, it is where leptin and CCK may potentiate their own effects not currently known whether leptin directly modulates the by cross-stimulating their secretion. The impairment of release of CCK. this leptin-CCK loop may have pathological implications Leptin is also produced by the stomach (12–14) and is related to and diabetes. Diabetes 52:1664–1672, mainly secreted into the gastric juice after CCK in rats 2003 (12,15) and after or vagal stimulation in humans (14,16). Some of the stomach-derived leptin is not fully degraded by , indicating that it reaches the intestine in an active form and thus can initiate biological eptin, the ob gene product, was initially reported processes controlling functions of the intestinal tract. to be produced by adipose cells (1). It is released Indeed, lumenal leptin increases the activity of the brush into the bloodstream and transported across the border proton-dependent transporter, PepT1, which en- Lblood-brain barrier into the , where hances the intestinal absorption of oligopeptides (17). This it activates specific leptin receptors (2,3) and regulates raises the possibility that leptin could also modulate the energy by altering energy intake and expen- secretory activity of intestinal endocrine cells, provided diture (4–6). Leptin regulates food intake by mechanisms that leptin is present in the intestinal juice. involving cross-talk between hypothalamic leptin recep- In this study, we examined the in vitro effects of tors and various involved in the control of recombinant leptin on the release of CCK and investigated feeding. The (Ob-R) is a member of the the intracellular mechanisms of leptin action in CCK- secreting STC-1 cells. We hypothesized that leptin that From the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) reaches the is able to modulate the release of Unite´ 410, Faculte´deMe´decine Xavier Bichat, Paris, France. CCK. To test this hypothesis, we determined whether Address correspondence and reprint requests to Andre Bado, INSERM leptin was present in the duodenal juice and then exam- Unite´ 410, Faculte´deMe´decine Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France. E-mail: [email protected]. ined the in vivo effect of direct duodenal delivery of leptin Received for publication 15 November 2002 and accepted in revised form on plasma CCK concentrations in the rat duodenal- 4 April 2003. CCK, cholecystokinin; ERK, extracellular signal–related kinase; ERK-P, perfused model. We questioned the physiological rele- phospho-ERK; INSERM, the Institut National de la Sante´ et de la Recherche vance of the data by analyzing the in vivo kinetic patterns Me´dicale; IR, immunoreactive; MAPK, mitogen-activated protein kinase; MEK, of plasma CCK and insulin concentrations and by analyz- MAPK kinase; PI3-K, phosphoinositide 3-kinase; RIA, radioimmunoassay; STAT, signal transducers and activators of transcription. ing the leptin content of the stomach after food intake in © 2003 by the American Diabetes Association. Zucker rats. Finally, we investigated the in vivo effects of

1664 DIABETES, VOL. 52, JULY 2003 S. GUILMEAU AND ASSOCIATES

CCK receptor antagonists on feeding-induced changes in MAPK (pTpY185/187) phosphospecific (Biosource Europe, Nivelles, Belgium) these parameters. diluted 1:1,000. The same membranes were then stripped and immunoblotted with an ERK-1 antiserum (Santa Cruz Biotechnology) diluted 1:500 to deter- mine total MAPK proteins. The immune complexes were detected by en- hanced chemiluminescence (Pierce). Immunoblots were quantified using the RESEARCH DESIGN AND METHODS NIH image analysis (Scion Corporation), and the results were expressed as a culture. STC-1 cells (a gift from Dr. J. Abello, the Institut National de la ratio of phospho-ERK (ERK-P) to total ERK. Sante´ et de la Recherche Me´dicale [INSERM] U-45, Lyon, France) are derived In vitro CCK secretory studies. STC-1 cells were seeded on 12-well culture from intestinal endocrine tumor cell lines developed from mice expressing the plates (5 ϫ 104 cells/well). When the cells reached 80% confluence, the transgene for the rat insulin promoter linked to the simian virus 40 large T medium was removed and the monolayer cultures of STC-1 cells were washed antigen and the polyoma virus small t antigen (18). STC-1 cells between twice with PBS. Then, culture medium supplemented with murine leptin or passage 15 and 35 were grown in RPMI-1640 plus (Sigma, St Louis, was added to the cells and incubated at 37°C in 95% CO for 1 h. The MO), supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 2 supernatants were collected and frozen at Ϫ20°C. The cells contents were 100 ␮g/ml streptomycin (Life Technologies, Grand Island, NY) in a humidified extracted in 2 mol/l CH COOHϪ20 mmol/l HCl, sonicated, boiled for 10 min, atmosphere containing 95% O and 5% CO at 37°C. 3 2 2 adjusted to pH 7 with 1 mol/l NH , and frozen at Ϫ20°C. CCK was determined RT-PCR analysis of leptin receptors. Total RNA was extracted from STC-1 3 in supernatants and cell extracts by radioimmunoassay (RIA). cells with the Trizol reagent (Invitrogen, Carlsbad, CA). Briefly, the first-strand In another set of experiments, the STC-1 cells were preincubated for 30 min cDNA was synthesized from 2 ␮g total RNA and was reverse-transcribed with at 37°C with 10 ␮mol/l PD98059, a selective inhibitor of MEK activity, or 10 200 units of reverse transcriptase using the Superscript II kit (Invitrogen) ␮ according to the manufacturer’s recommendations. The following oligonucle- mol/l wortmannin, a PI3-K inhibitor, before addition of vehicle (control) or otides primers were synthesized by Sigma Genosys (Cambridgeshire, U.K.): leptin. One hour later, the supernatants were removed and used for CCK the forward primer for Ob-Rb was 5Ј-ATGAAGTGGCTTAGAATCCCTTCG-3Ј determination by RIA. and the reverse primer was 5Ј-ATATCACTGATTCTGCATCCTG-3Ј. The prim- Animals. Male Wistar rats weighing 260–280 g and male 6-week-old obese ers used for the ␤-actin gene were as follows forward 5Ј-CGAGAAGATGAC (fa/fa) and lean (Fa/fa) Zucker rats (Iffa Credo, Les Oncins, L’Arbresle, CCAGATCATG-3Ј and reverse 5Ј-AGTGATCTCCTTCTGCATCCTG-3Ј. Samples France) were caged under standard laboratory conditions with tap water and were denatured by heating at 95°C for 3 min. PCR was then carried out under regular food provided ad libitum, in a 12-h/12-h light/dark cycle at a temper- the conditions previously described (14). PCR products were separated by ature of 21–23°C. The animals were treated in accordance with European electrophoresis in a 2% agarose gel. The gel was stained with ethidium Committee Standards concerning the care and use of laboratory animals. bromide and viewed under ultraviolet illumination. The expected sizes of the Determination of leptin in duodenal juice and size exclusion chroma- PCR products were 375 bp for Ob-Rb and 606 bp for ␤-actin. tography. Rats fed ad libitum or rats that had been deprived of food for 24 h Western blot analysis. For total protein extraction, STC-1 cell pellets were were anesthetized, and an outflow catheter was surgically implanted 1 cm homogenized at 4°C in lysis buffer supplemented with 0.1 mg/ml phenylmeth- below the ligament of Treitz for the collection of duodenal juice for 60 min ␮ ␮ ylsulfonyl fluoride, 100 mol/l aprotinin, and 100 mmol/l NaVO4. The homog- after intravenous (through the femoral vein) injection of saline or 30 g/kg enates were centrifuged at 15,000g for 30 min at 4°C, and the supernatants carbachol (Sigma, St. Louis, MO). The pH was measured, and leptin was de- were collected for Western blot analysis. Protein concentration was quantified termined by RIA. A sample of duodenal juice or murine leptin was submitted using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). to size exclusion chromatography using Superdex 200 column (16/60) (Phar- Briefly, solubilized proteins were resolved by electrophoresis on 7.5% macia Biotech, Freiburg, Germany). The column was equilibrated with PBS SDS-PAGE gels. The resolved proteins were transferred onto nitrocellulose containing 0.1% BSA and 0.01% sodium azide at 4°Cataflow rate of 2 ml/min. membranes and subjected to immunoblot analysis with a polyclonal Ob-Rb The applied sample volume was 5 ml, and fractions of 2 ml were collected and antiserum raised in rabbit against a COOH-terminal of the leptin stored at Ϫ20°C until leptin RIA. The column was adjusted with commercially receptor (amino 890–903) specific to the Ob-Rb isoform (OBR 12-A; available calibration proteins kits (Pharmacia Biotech). Biotrend Chemikalien, Cologne, Germany) diluted 1:750. After a 1-h incuba- Duodenal perfusion studies. Duodenal perfusion studies were carried out tion of the membranes with anti-rabbit horseradish peroxidase–conjugated on rats that had been deprived of food for 24 h, with water available ad (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1,000, the libitum. The rats were anesthetized with ethylurethane (1.2 g/kg, intramuscu- immune complexes were detected by enhanced chemiluminescence (Pierce, lar) (Prolabo, Paris, France), and after laparotomy, a transpyloric inflow Rockford, IL). cannula was inserted into the duodenum and an outflow cannula was inserted Immunocytochemistry. STC-1 cells were seeded into 4-well Lab-Tek II glass ϳ1 cm below the ligament of Treitz. The duodenal segment was perfused at a slides (Nalge Nunc International, Naperville, IL). After 2 days of incubation at flow rate of 4 ml every 15 min with a Krebs-Ringer buffer consisting of the

37°C with 95% O2:5% CO2, the medium was removed and the cells were following (in mmol/l): 0.5 MgCl2, 4.5 KCl, 120 NaCl, 0.7 NaHPO4, 1.5 NaH2PO4, washed twice in cold PBS. We then added a formalin fixative (4% paraformal- 1.2 CaCl2, 15 NaHCO3, and 10 adjusted to pH 7.5. The solution was dehyde) and incubated the cells for 10 min. Cells were then digested with 0.1% maintained at 37°C by a water-jacket before its entry into the duodenal in 0.01N HCl, pH 2.25, for 10 min to retrieve the antigen. Before segment. After a 15-min stabilization period, vehicle containing BSA as a immunoreaction, endogenous peroxidase activity was removed by adding nonspecific protein or murine leptin (1–100 nmol/l) was added in the perfusion

0.3% H2O2 and incubating for 5 min. The cells were incubated overnight at 4°C solution. Blood samples were collected through a carotid catheter in tubes with a rabbit polyclonal Ob-Rb antiserum (OBR 12-A) or polyclonal Ob-R containing EDTA as previously described (11) and were centrifuged at 10,000g

antiserum (TP283) raised in rabbit against an NH2-terminal peptide of leptin for 3 min. Plasma was removed and speedVac concentrated after an ethanol receptor (amino acids 25–208) (Clinisciences, Montrouge, France) or with a extraction and stored at Ϫ20°C until CCK RIA. rabbit anti-CCK8 antibody (Sigma), each of which was diluted 1:100. Slides Effects of feeding alone or in association with CCK receptor antago- were then incubated1hatroom temperature with a pig anti-rabbit antibody nists on stomach leptin, plasma insulin, and CCK levels. Male Wistar or followed by a rabbit peroxidase-antiperoxidase complex Z0113 (Dako, Carpin- Zucker rats were habituated to eat their pellet diets between 10:00 A.M. and teria, CA), each diluted 1:100. Immunohistochemical staining was performed 6:00 P.M. when food was withdrawn until the next morning, but water was using 3Ј,3Ј-diaminobenzidine hydrochloride as the peroxidase chromogen available ad libitum. (Sigma). The day of the experiment starting in the morning (10:00 A.M.), the animals Controls. No immunostaining was observed in STC-1 cells when under the were allowed to feed on preweighed standard laboratory pellet for following conditions: 1) normal rabbit serum used in place of immune rabbit different time periods. At each period, food was weighed and intake was serum and 2) prior incubation of Ob-Rb antiserum (OBR 12-A) with 20 ␮g determined. The rats were anesthetized, blood was collected from the control peptide (OBR 12-P) per milliliter of diluted antiserum for 3–4hat abdominal aorta and centrifuged, and plasma was removed and stored at room temperature. Ϫ20°C until RIA of CCK and insulin (RIA kits; Linco Research, St. Charles, Extracellular signal–related kinase (ERK) phosphorylation. Overnight, MO). The stomach was removed and opened. Its contents were collected and serum-starved cultures of STC-1 cells were first incubated for 30 min at 37°C centrifuged, and the supernatants were removed and stored at Ϫ20°C until with vehicle, with 10 ␮mol/l PD98059 (a selective inhibitor of MAPK kinase assayed for leptin in gastric juice by RIA. The fundic mucosa was scrapped off, [MEK] activity), or with 1–10 ␮mol/l wortmannin (a phosphoinositide 3-kinase weighed, homogenized, and centrifuged 10,000g for 10 min as previously [PI3-K] inhibitor). Then, vehicle (control), 100 nmol/l mouse leptin (R&D described. The supernatants were used for the determination of stomach Systems, Minneapolis, MN), or bombesin (Sigma) was added to the cells and leptin. further incubated for 1 h. Solubilized extract protein samples (80 ␮g) were For the antagonist studies, 10 min before food intake, the animals were resolved by electrophoresis on 12.5% SDS-PAGE gels as described. The blots intraperitoneally injected with vehicle (control) or 1 mg/kg L364718 (a gift were probed overnight at 4°C with rabbit polyclonal : anti–ERK-1/2 from Professor B. Roques, INSERM 266, Paris, France) or YM022 (a gift from

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FIG. 1. A: Expression of leptin receptors on STC-1 cells. Total RNA extracted from STC-1 cells was assayed for Ob-Rb by RT- PCR as described in RESEARCH DESIGN AND METHODS. Agarose gel electrophoresis of PCR products was generated with primers specific for the long isoform (Ob-Rb) of the leptin receptor gene. Lane 1: marker DNA ladder; lane 2: STC-1 cells; lane 3: negative control in which the reverse transcriptase was omitted; lane 4: ␤-actin. The expected sizes of the PCR products were 375 bp for Ob-Rb and 606 bp for ␤-actin (arrows). B: Western blot analysis of STC-1 cell extracts at passage 15 (lane 1), 25 (lane 2), and 35 (lane 3), with a specific anti–Ob-Rb anti- body (OBR 12-A). A representative immu- noblot is shown for the leptin receptor, showing two IR proteins, 130 and 170 kDa in size. Note that the amount of leptin recep- tor protein did not change from passage 15–35. C and D: STC-1 cells were fixed in 4% paraformaldehyde. Immunostaining of lep- tin receptors was performed with two dif- ferent rabbit polyclonal antibodies: TP 283 (C), which recognizes all Ob-R isoforms, and OBR 12-A (D), an antiserum specific for the Ob-Rb isoform. Arrows indicate strong signals on STC-1 cell membranes.

Yamanouchi Pharmaceutical, Tokyo), CCK-1 and CCK-2 receptor antagonists, CCK (data not shown), which is consistent with the results respectively, in saline containing 1% DMSO. After 15 min of food intake, of previous studies (20). The mean cellular CCK content plasma insulin, plasma CCK, stomach leptin content, and leptin release were determined. was 1,308 Ϯ 101.5 pmol/l (n ϭ 16), and the basal level of RIA of CCK. CCK was determined according to the procedure described by CCK release over 1 h (24.9 Ϯ 6.2 pmol/l, n ϭ 16) accounted Rehfeld (19). Briefly, the COOH-terminal anti-CCK antibody (a gift from for 1.9% of the total CCK content of the cells. Professor J. Rehfeld, Copenhagen, Denmark) was incubated at 4°C for 4 days Consistent with the results of previous studies (21,22), with CCK-8 (Sigma) or with plasma samples and [125I]CCK-8 (Amersham Pharmacia Biotech, Piscataway, NJ) in RIA buffer consisting of 20 mmol/l the bombesin (used as a positive control) barbital buffer, 0.6 mmol/l thiomersal, and 0.11% BSA vol/vol (pH 8.4). Bound stimulated the release of CCK in a concentration-depen- and free fractions were separated by absorbing the free [125I]CCK-8 onto dent manner; 100 nmol/l stimulated an increase of 229.0 Ϯ active dextran T70-coated charcoal (4 and 40 g/l, respectively) in RIA buffer 29.9% from the basal levels (P Ͻ 0.05 vs. basal) (Fig. 2A). containing 10% filtered horse serum. Radioactivity in the bound fraction was measured with a gamma counter. Under these conditions, the detection limit The addition of leptin to STC-1 cells also induced a was 0.5 pg CCK. concentration-dependent increase in CCK release. Stimu- Statistical analysis. The results are expressed as means Ϯ SE. They were lation began to occur at a leptin concentration of 0.01 compared by one-way ANOVA, followed by a Tukey-Kramer multiple compar- nmol/l (114 Ϯ 10.9% of basal) and became significant at a isons test if significant results were obtained. leptin concentration of 0.1 nmol/l (130.6 Ϯ 9.2% of basal; Ͻ Ϯ RESULTS P 0.05). Maximal leptin stimulation (188.1 19.2% of basal, P Ͻ 0.01) was achieved with 10 nmol/l leptin, and no STC-1 cells express leptin receptors. RT-PCR and further increase was observed with 100 nmol/l leptin. This Western blot analysis were used to study the expression maximal effect corresponded to 80% of that induced by of the leptin receptors in CCK-producing STC-1 cells. A bombesin (100 nmol/l). The concentration of leptin pro- 375-bp product, corresponding to positions 2401–2776 of ducing a half-maximal stimulation (EC50) of CCK release Ob-Rb, was detected (Fig. 1A). After cDNA sequencing, Ϯ this product was found to be 100% identical to the mouse was 0.23 0.08 nmol/l. The leptin-stimulated CCK release Ob-Rb gene transcript. was associated with a significant decrease in total cellular Immunoblotting of STC-1 protein extracts with an anti– CCK content; a 40% decrease was observed with 10 nmol/l Ob-Rb antibody detected two immunoreactive (IR) bands leptin (Fig. 2B). with relative molecular masses of 130- and 170-kDa (Fig. Leptin induces ERK phosphorylation and PD98059 1B). The prominent 130-kDa IR band corresponded to the inhibits leptin-stimulated CCK release. Immunoblot- long isoform of the leptin receptor based on the predicted ting of STC-1 cell extracts with an anti-ERK antiserum molecular weight, and the 170-kDa band probably corre- detected two IR bands 42 and 44 kDa in size, correspond- sponded to a glycosylated form of Ob-Rb. Immunocyto- ing to two closely related MAPK proteins, ERK-2 and chemical studies (Fig. 1C and D) using an antibody that ERK-1, respectively (Fig. 3). The addition of leptin to the recognizes all leptin receptor isoforms showed leptin cells induced a rapid and transient increase in phosphor- receptor immunoreactivity diffusely distributed in the cy- ylation of p42 and p44 (Fig. 3). toplasm with strong staining on the membranes of some Whether the activation of ERK proteins is linked to the cells (Fig. 1C). The use of an antiserum specific for Ob-Rb leptin-stimulated secretion of CCK from STC-1 cells was gave a similar distribution of leptin receptors in STC-1 investigated. Pretreatment of STC-1 cells with an inhibitor cells (Fig. 1D). of MEK activity (PD98059) or a PI3-K inhibitor (wortman- Leptin stimulates the release of CCK from STC-1 nin, 10 ␮mol/l) did not affect the basal CCK secretion by cells. Immunostaining of the STC-1 cells with a specific STC-1 cells. However, the leptin-stimulated CCK release anti-CCK antibody showed that 90% of the cells contained was completely blocked by 10 ␮mol/l PD98059 and unaf-

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FIG. 2. Leptin stimulates CCK secretion and reduces the CCK content of STC-1 cells. CCK was determined in both the culture medium (A) and the STC-1 cells (B) after a 1-h incubation with murine leptin or -bombesin. Each point represents means ؎ SE of 16 separate experi ments, each performed in triplicate. Results are expressed as the percent of the mean of control CCK. *P < 0.05 vs. control. fected by wortmannin at concentrations up to 10 ␮mol/l (Fig. 3B). These data indicate that the activation of ERK FIG. 3. A: Leptin stimulates ERK-1/2 phosphorylation in STC-1 cells. An immunoblot of total MAPK and phosphorylated ERK-1/2 after incuba- proteins contributes to the stimulatory effect of leptin on tion of STC-1 cells with 100 nmol/l leptin or bombesin (BN) is shown. the release of CCK from STC-1 cells. (Insert: A representative immunoblot of three separate experiments is Duodenal leptin increases plasma CCK levels in vivo. shown). Immunoblots were quantified using the NIH image analysis (Scion Corporation), and the results were expressed as the ratio of To verify our hypothesis that gastric leptin that enters the ERK-P to total ERK. B: MEK inhibitor abolished the leptin stimulation duodenum modulates intestinal biological processes, we of CCK release. STC-1 cells were treated for 30 min with 10 ␮mol/l PD98059 (PD), 10 ␮mol/l wortmannin (Wort.), or vehicle (CTRL) examined the occurrence of leptin in the duodenal juice. before the addition of 100 nmol/l leptin or vehicle for 1 h. CCK The duodenal juice contained high amounts of leptin-IR concentrations were determined in the supernatants. Each column -proteins (Fig. 4A). In basal conditions, these amounts represents means ؎ 1 SE of eight separate experiments, each per Ϯ formed in triplicate. Results are expressed as a percentage of the mean were higher in rats fed ad libitum than in fasted rats (146 of basal CCK release. ***P < 0.001 vs. control; #P < 0.01 vs. leptin 9 ng leptin/ml [n ϭ 7] vs. 92 Ϯ 11 ng leptin/ml [n ϭ 14]; P Ͻ alone (vehicle). 0.05). Furthermore, intravenous injection of the cholin- ergic muscarinic carbachol resulted in a significant We then examined the effect of delivering leptin directly increase in duodenal leptin-IR in fasted rats. The elution into the duodenum on plasma CCK concentrations. The profile of the basal duodenal juice after size exclusion basal plasma CCK levels were higher in ad libitum–fed rats chromatography showed two leptin IR peaks: peak I (0.9 Ϯ 0.2 pmol/l, n ϭ 7) than in fasted rats (0.42 Ϯ 0.1 corresponded to leptin bound to macromolecules and pmol/l). Intraduodenal infusion of leptin induced a con- peak II corresponded to murine leptin and eluted at the centration-dependent increase of basal CCK levels in expected molecular weight of 16 kDa (Fig. 4B). The fasted rats (Fig. 4C). Leptin (1 nmol/l) did not significantly molecular weight of peak I ranged from the void volume at modify plasma CCK levels. However, a significant increase 430 to 232 kDa with a maximum at 300 kDa. These data was observed for 10 nmol/l leptin (2.1 Ϯ 0.1 pmol/l at 15 indicate that leptin circulates in the duodenum juice both min). A further increase was observed with 100 nmol/l free and bound to high–molecular weight proteins. leptin with values of 5.01 Ϯ 1.2 pmol/l after 15 min and a

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FIG. 4. Intraduodenal leptin in- creases plasma CCK levels in vivo. A: Leptin immunoreactivity in 1-h collected duodenal fluid of rats fed fasted for 24 h ,(7 ؍ ad libitum (n -and intravenously in ,(14 ؍ n) ؍ jected with 30 ␮g/kg carbachol (n 8) (*P < 0.05; **P < 0.01 vs. fasted rats). B: Size exclusion profile of collected duodenal juice fraction- ated on a Superdex 200 (16/60) column. Leptin amount was deter- mined in each eluted fraction by RIA. The peak I corresponded to leptin bound to high–molecular weight protein macromolecules, and peak II corresponded to free leptin. Ve, elution volume. C: Changes in plasma CCK levels dur- ing intraduodenal infusion of vehi- cle containing BSA (used as a nonspecific protein [0 nmol/l]) or leptin (10 and 100 nmol/l). Results are expressed as plasma CCK lev- els in picomoles per liter, and each ؎ point corresponds to the means SE of 8–12 rats in each group. The arrow indicates the start of in- traduodenal infusion. *P < 0.05; **P < 0.01 vs. control (0 nmol/l). peak value of 6.02 Ϯ 1.8 pmol/l (P Ͻ 0.01) 30 min after centration of lean Fa/fa rats was not significantly different infusion; after 60 min, values then progressively decreased from that of obese fa/fa rats (0.36 Ϯ 0.08 vs. 0.29 Ϯ 0.1 to 1.25 Ϯ 0.6 pmol/l after 60 min (Fig. 4C). pmol/l) (Fig. 6B). In lean Fa/fa rats, feeding increased Feeding decreases the gastric pool of leptin and plasma CCK concentrations by up to eightfold, whereas in increases plasma CCK levels. To study the physiological obese fa/fa rats, the plasma CCK concentrations increased relevance of these results, we analyzed the kinetic pat- less dramatically in response to a meal (50%, P Ͻ 0.01 vs. terns of plasma CCK and insulin concentrations in parallel lean Fa/fa). It should be noted that there was no significant with changes in stomach leptin content and release after difference between the amounts of food consumed by lean food intake. Fa/fa rats and obese fa/fa rats (2.2 Ϯ 0.2 vs. 2.7 Ϯ 0.5 g). Feeding induced a rapid (as early as 15 min) and CCK receptor antagonists abolish feeding-induced dramatic decrease (70%, P Ͻ 0.01 vs. control) in stomach changes in stomach leptin and plasma insulin. To content of leptin and a parallel 3.5-fold increase in leptin determine whether a feedback loop exists between endog- output in the gastric juice (Fig. 5A). This effect was enous CCK and gastric leptin in physiological conditions, associated with a significant increase in the plasma insulin we investigated the effects of CCK receptor antagonists. concentration (positive control) that peaked after 15 min The prior injection of L364718 or YM022 inhibited the (7.8 Ϯ 1.1 vs. 0.9 Ϯ 0.3 ng/ml; P Ͻ 0.01 vs. control). In feeding-induced increase in plasma insulin concentration addition, the basal plasma CCK concentration also in- by 68% (P Ͻ 0.001 vs. control) (Fig. 7A) and did not affect creased upon feeding, reaching a peak of 4.42 Ϯ 0.86 postprandial plasma CCK concentrations (Fig. 7B). In pmol/l after 30 min and then slightly decreasing to a value these conditions, a complete reversion of the reduction of of 3.6 Ϯ 0.73 pmol/l after 60 min (Fig. 5B). the stomach content of leptin and the increase in gastric These kinetic patterns were further analyzed in the leptin release after food intake was observed (Fig. 7C and Zucker rat. In lean Fa/fa and in obese fa/fa rats, feeding D). Moreover, a trend toward increased food intake was markedly decreased the gastric leptin content (50% at 15 observed after CCK receptor antagonists (2.7 Ϯ 0.4 g for min, P Ͻ 0.01 vs. control) (Fig. 6A) and increased the YM022; 2.3 Ϯ 0.5 g for L364718 vs. 1.6 Ϯ 0.4 g for control). amount of leptin secreted into the gastric juice (data not These data indicate that endogenous CCK via activation of shown). However, the plasma CCK response to a meal was CCK receptors is involved in the control of gastric leptin as impaired in obese fa/fa rats. The basal plasma CCK con- well as insulin secretion.

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FIG. 6. Feeding decreases stomach leptin stores in Zucker rats. A: Changes in fundic mucosa leptin in male Zucker rats that were fasted overnight and fed for the indicated time periods. Data are expressed as B: Plasma CCK response to food intake in .9 ؍ the mean ؎ 1SEofn for 9 ؍ male Zucker rats. Data are expressed as the mean ؎ 1SEofn lean Fa/fa and obese fa/fa rats. *P < 0.05; **P < 0.01 vs. time 0; ##P < 0.01 vs. lean Fa/fa..

FIG. 5. Feeding decreases stomach leptin stores and increases plasma from STC-1 cells when the ERK activators MEK-1/2 were CCK and insulin levels. A: Changes in fundic mucosa leptin and in the pharmacologically inhibited. A similar MAPK-dependent amount of leptin released in the gastric juice in Wistar rats that were fasted overnight and fed for the indicated time periods. Data are pathway has been reported for bombesin-induced in- B: Plasma CCK and insulin creases in CCK release in the same model (22). The .8 ؍ expressed as the mean ؎ 1SEofn response to food intake in Wistar rats. Results are expressed as plasma CCK levels in picomoles per liter, and each point corresponds to the downstream events that follow the activation of MAPK means ؎ SE of eight rats in each group. *P < 0.05; **P < 0.01 vs. time and lead to CCK secretion are unknown. STC-1 cells 0. express the three synaptic core complex proteins syn- taxin-1, synaptosomal-associated protein-25 (SNAP-25), DISCUSSION and vesicle-associated membrane protein 2, which plays a In this report, we provide evidence that the long isoform of key role in vesicle exocytosis (23). Moreover, the activa- the leptin receptor (Ob-Rb) is expressed in CCK-producing tion of MAPK is associated with an increase in the STC-1 cells. These leptin receptors were diffusely distrib- phosphorylation of synapsin I in neuronal preparations uted in the cytoplasm and were detected on the plasma (24,25). Therefore, it is possible that, in our conditions, membrane of STC-1 cells. The expression of leptin recep- leptin facilitates the exocytosis of secretory vesicles con- tors in STC-1 cells makes this cell line a suitable model for taining CCK; however, this remains to be demonstrated. the investigation of leptin . Thus, we demon- Because these STC-1 cells do not closely represent strated that the incubation of STC-1 cells with leptin duodenal I cells, the in vivo relevance of these data was increases the amount of CCK released and decreases the analyzed in the rat. In particular, we verified our hypoth- total CCK cell content, suggesting that a preformed pool of esis that some of the leptin originating from the stomach CCK was mobilized. reaches the duodenum in an active form, thereby modu- It is now well established that only the Ob-Rb isoform lating the release of CCK. This result implies, however, activates the STAT pathways, whereas both the long that leptin is present in the duodenal juice. Thus, we (Ob-Rb) and short (Ob-Ra) isoforms can transduce signals demonstrated that duodenal juice contains high amounts through substrates and the MAPK cas- of leptin that circulates both free and bound to high– cade. Our data indicate that MAPK-dependent pathways molecular weight proteins. These leptin-binding proteins are actually operating in SCT-1 cells for the leptin-stimu- have been shown to be the soluble leptin receptor in the lated secretion of CCK. Indeed, leptin induces a rapid blood (26). Although the precise nature of the binding phosphorylation of ERK-1/2 proteins, in agreement with protein interacting with leptin in the duodenal juice re- the failure of leptin to affect the amount of CCK released quires further elucidation, it is possible that the ratio of

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FIG. 7. Endogenous CCK decreases stomach leptin pool and increases plasma insulin. Effects of L364718 and YM022 on plasma insulin (A), plasma CCK (B), stomach leptin content (C), and the amount of leptin released in gastric juice (D) after 15 min of food intake. These antagonists were injected intra- peritoneally 10 min before start of food intake. Data are ex- pressed as the mean ؎ 1 SE for eight rats in each group. Data were analyzed by a Tukey-Kramer multiple comparisons test after a significant ANOVA. **P < 0.01; ***P < 0.001 vs. control (CTRL); #P < 0.05, ##P < 0.01 vs. vehicle, P < 0.01 vs. vehicle (Veh.). free to bound leptin may be affected by the metabolic leptin pathways are required for full plasma CCK response status. Thus, this unknown leptin-binding protein may to a meal. However, whether leptin in duodenal fluid have physiological implications in terms of the bioavail- directly affects meal-induced CCK release requires further ability of leptin and might help us to understand the action studies. of leptin on the intestinal . The presence of The in vivo effective dose of leptin in this study is leptin immunoreactivity in duodenal juice, along with the consistent with that detected in the duodenal juice in rats previous data reporting the expression of the leptin recep- fed ad libitum (9.2 nmol/l) compared with 5.5 nmol/l in tor on the apical side of the (17,27), make it fasted rats under basal conditions. These data strongly likely that leptin exerts a lumenal action on the intestinal suggest the involvement of meal regulatory processes in epithelium. the changes of leptin in duodenal fluid in these animals. As expected, the direct infusion of leptin into the This led us to conclude that, in physiological conditions, duodenum increased plasma CCK at levels comparable to the rapid mobilization of the gastric leptin pool that enters those induced by feeding, suggesting that leptin regulates the intestine is involved in the release of CCK from the the secretory activity of the endocrine I cells. In response duodenal endocrine I cells. It is noteworthy that the to food intake, both lean Fa/fa and obese fa/fa rats amount of leptin in basal duodenal juice is greater than the exhibited a similar decrease in gastric leptin content and amount presumed to originate from the stomach. This an increase in the amount of leptin released in the gastric intriguing but interesting finding can be tentatively ex- juice. This result suggests that the meal-regulated path- plained by a contribution from other leptin-producing sites ways of gastric leptin are functional in these rats and may that remain to be identified. In addition to gastric secre- also indicate that similar amounts of released gastric tions, the duodenum receives hepato-biliary and exocrine leptin reach the duodenum in both groups of rats. How- pancreatic secretions. Whether these secretions contain ever, the plasma CCK response to a meal is markedly leptin that can contribute to the increase in leptin concen- reduced in obese fa/fa rats compared with lean Fa/fa rats. trations in the duodenal juice is currently under investiga- The genetically obese fa/fa rats carry a missense mutation tion. that results in an amino substitution at position 269 The data presented here may have physiological impli- (Gln 3 Pro) within the extracellular domain of the leptin cations. Previous studies have shown that CCK-1 receptor receptor (28) and leads to a strong decrease of function of antagonists can prevent the peripheral leptin-induced in- the receptor (29). Therefore, a decreased functional effi- hibition of food intake (10) and the stimulation of pancre- ciency of the leptin receptor may account for the loss of atic exocrine secretions (11), which suggests that CCK is sensitivity of fa/fa rats to meal-stimulated plasma CCK. involved. However, whether leptin modulates the release Similar data showing a reduced CCK release in the hypo- of CCK is unknown. Our findings that duodenal leptin thalamus of fa/fa rats in response to a meal have been increases the amount of CCK released provide a basis for reported (30). Taken together, it seems that functional the explanation of these CCK-dependent effects of leptin.

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Whereas these data support a model in which meal- 9. Woods SC, Schwartz MW, Baskin DG, Seeley RJ: Food intake and the induced leptin release into duodenal fluid contributes to regulation of body weight. Annu Rev Psychol 51:255–277, 2000 10. Barrachina MD, Martinez V, Wang L, Wei JY, Tache Y: Synergistic prandial CCK release, the contribution of this effect, interaction between leptin and cholecystokinin to reduce short-term food relative to that of known CCK secretagogues (e.g., in- intake in lean mice. Proc Natl Acad SciUSA94:10455–10460, 1997 gested nutrients), remains unknown and requires addi- 11. Guilmeau S, Nagain-Domaine C, Buyse M, Tsocas A, Roze C, Bado A: tional study. Conversely, the presence of leptin circulating Modulation of exocrine pancreatic secretion by leptin through CCK(1)- receptors and afferent vagal fibres in the rat. Eur J Pharmacol 447:99–107, in the duodenum, which also contains hepato-biliary se- 2002 cretions, could be important for the elimination of biliary 12. Bado A, Levasseur S, Attoub S, Kermorgant S, Laigneau JP, Bortoluzzi MN, cholesterol by regulating the enterohepatic circulation of Moizo L, Lehy T, Guerre-Millo M, Le Marchand-Brustel Y, Lewin MJ: The salts, as recently reported (31). stomach is a source of leptin. Nature 394:790–793, 1998 The release of CCK by leptin presumably generates a 13. Cinti S, Matteis RD, Pico C, Ceresi E, Obrador A, Maffeis C, Oliver J, Palou A: Secretory granules of endocrine and chief cells of human stomach positive feedback loop, because, as we have previously mucosa contain leptin. Int J Obes Relat Metab Disord 24:789–793, 2000 reported, CCK stimulates the release of gastric leptin (12). 14. Sobhani I, Bado A, Vissuzaine C, Buyse M, Kermorgant S, Laigneau JP, This is likely because the blockade of CCK receptors Attoub S, Lehy T, Henin D, Mignon M, Lewin MJ: Leptin secretion and completely prevents the decrease in stomach leptin con- leptin receptor in the human stomach. Gut 47:178–183, 2000 15. Attoub S, Levasseur S, Buyse M, Goiot H, Laigneau JP, Moizo L, Hervatin tent as well as the rise in plasma insulin concentrations in F, Le Marchand-Brustel Y, Lewin JM, Bado A: Physiological role of response to a meal. These effects are associated with cholecystokinin B/ receptor in leptin secretion. trends toward increased food intake, suggesting that dur- 140:4406–4410, 1999 ing normal , this positive feedback loop may be 16. Sobhani I, Buyse M, Goiot H, Weber N, Laigneau JP, Henin D, Soul JC, operational in the reduction of food intake. These data Bado A: Vagal stimulation rapidly increases leptin secretion in human stomach. 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