Volume 121, Issue 6, Pages 1417-1427 (December 2001) Antral mucosa expresses functional leptin receptors coupled to STAT-3 signaling, which is involved in the control of gastric secretions in the rat  Hélène Goïot, *, Samir Attoub, *, Stéphanie Kermorgant, *, Jean-Pierre Laigneau, *, Bernard Lardeux, ‡, Thérèse Lehy, *, Miguel J.M. Lewin, *, André Bado, *  Gastroenterology  Volume 121, Issue 6, Pages 1417-1427 (December 2001) DOI: 10.1053/gast.2001.29581 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of leptin receptors in antral mucosa. (A) RT-PCR analysis of leptin receptor isoforms from total RNA of rat antral mucosa and entire brain. Ob-Ra (347 bp); Ob-Rb (375 bp); −RT, omission of reverse transcriptase and PCR amplification with specific primers for leptin receptor isoform. (B) Binding of [125I]-rm leptin to isolated rat antral cells. Cells (107 cells per mL) were incubated for 30 minutes at 37°C with different concentrations of rm-leptin. Data are expressed as mean ± 1 SEM from 9 separate experiments. Gastroenterology 2001 121, 1417-1427DOI: (10.1053/gast.2001.29581) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Expression of STAT proteins in antral mucosa. Representative STAT immunoblots from crude proteins (20 μg) of isolated antral cells. (A and B) Representative immunoblots of STAT-1 and STAT-5b protein after treatment of isolated rat antral cells with 6.25 nmol/L leptin, respectively. Bands at 91 and 94 kilodaltons were detected and corresponded to STAT-1 and STAT-5b protein, respectively. No significant change in the amount of STAT was observed in the cytosolic and nuclear fraction after leptin treatment. (C) A representative immunoblot of STAT-3 after treatment of isolated rat antral cells with 6.25 nmol/L leptin. The 92-kilodalton STAT-3 protein decreased in the cytosolic fraction and increased in the nuclear fraction after addition of leptin. (D) Ratio between nuclear and cytosolic STAT-3 after treatment of isolated rat antral cells with 6.25 nmol/L leptin. National Institute of Health (NIH) image analysis of immunoblots was used to quantify changes in nuclear and cytosolic STAT-3 proteins. The changes are expressed as the mean ± 1 SEM. The levels of nuclear or cytosolic STAT-3 at time zero were arbitrarily set to 1 and the levels were calculated in reference to time zero. ***P < 0.01 vs. time zero. Gastroenterology 2001 121, 1417-1427DOI: (10.1053/gast.2001.29581) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Leptin regulates somatostatin and gastrin in vitro. (A) Effects of rm-leptin on somatostatin (■) and gastrin (□) secretions by isolated rat antral cells. Cells were incubated at 37°C for 120 minutes with or without different concentrations of rm-leptin. Data are expressed in picograms per 1 × 106 antral cells and represent the mean ± 1 SEM of 10 to 12 separate experiments *P < 0.05; **P < 0.01; ***P < 0.001 vs. saline. (B, insert) Effect of rm-leptin on gastrin expression in isolated rat antral cells. Cells were incubated at 37°C with or without 6.25 nmol/L leptin at the indicated times. Antral cells lysates were hybridized with specific cRNA probes for gastrin and GAPDH. Signals were quantified by Instant Imager and normalized with GAPDH. The size of the synthesized cRNA probes is indicated on the left and the size of the protected riboprobes is shown on the right. Gastroenterology 2001 121, 1417-1427DOI: (10.1053/gast.2001.29581) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Leptin inhibits gastric acid secretion in vivo. Effect of rm-leptin on basal acid output in gastric fistula rats. Rats were injected intravenously with saline or rm-leptin (0.2, 2, or 20 nmol/kg per hour). Gastric juice was collected every 20 minutes and acid concentration was determined. Results are expressed as acid output in μEq per 20 minutes, and represent means ± SEM (n = 6 rats, 3 determinations in each rat and for each dose of rm-leptin). *P< 0.05; **P < 0.01; ***P < 0.001 vs. saline. Gastroenterology 2001 121, 1417-1427DOI: (10.1053/gast.2001.29581) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of leptin on plasma leptin levels, antral leptin content, and STAT-3 in vivo. (A) Time course of plasma immunoreactive leptin after intravenous injection of rm-leptin in rats starved for 18 hours. Data represent the mean ± 1 SEM of 6 rats for each dose. The columns correspond to plasma leptin levels, 60 minutes after intravenous injection of 20 nmol/kg (white) or intraperitoneal injection of 10 nmol/kg (black). They represent the mean ± 1 SEM of 6 rats for each column. (B) Antral leptin content, 60 minutes after intravenous injection of 20 nmol/kg leptin or intraperitoneal injection of 10 nmol/kg rm-leptin. Data represent the mean ± 1 SEM of 6 rats for each column. **P < 0.01 vs. saline. (C) A representative immunoblot of antral mucosa STAT-3 after intraperitoneal injection of 10 nmol/kg rm-leptin in rats starved for 18 hours. Twenty micrograms of nuclear or cytosolic proteins was loaded in each lane. The 92-kilodalton STAT-3 protein decreased in the cytosolic fraction and increased in the nuclear fraction after treatment with rm-leptin. Gastroenterology 2001 121, 1417-1427DOI: (10.1053/gast.2001.29581) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Leptin inhibits plasma gastrin and gastrin expression in vivo. Rats that have been starved for 18 hours were injected with vehicle or intraperitoneal rm-leptin (1 or 10 nmol/kg). Two or 6 hours after leptin injection, blood was collected and the antral mucosa was scraped. (A) Effect of 10 nmol/kg rm-leptin on plasma gastrin levels in rats. Data are expressed in pg/mL and represent the mean ± 1 SEM of 12 rats. (B) A representative Northern blot autoradiogram of gastrin and GAPDH mRNA from total RNA extracts from antrum of vehicle- or rm-leptin-treated rats. (C) Quantitative analysis of gastrin mRNA levels in antral mucosa was calculated. Data represent the mean ± 1 SEM of 9 separate experiments. **P < 0.01; ***P < 0.001 vs. vehicle. Gastroenterology 2001 121, 1417-1427DOI: (10.1053/gast.2001.29581) Copyright © 2001 American Gastroenterological Association Terms and Conditions