BBRC Biochemical and Biophysical Research Communications 336 (2005) 1028–1032 www.elsevier.com/locate/ybbrc
Possible role of PEPT1 in gastrointestinal hormone secretion Kimio Matsumura a,b, Takashi Miki a, Takahito Jhomori b, Tohru Gonoi c, Susumu Seino a,* a b
Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan Central Research Laboratory, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei, Inabe 511-0406, Japan c Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba 260-8670, Japan Received 26 August 2005 Available online 12 September 2005
Abstract Oligopeptides originating from ingested meal stimulate the secretion of various gastrointestinal hormones, but the mechanism is unknown. In this study, we show that transfection of oligopeptide transporter 1 (PEPT1) in STC-1 cells, a murine enteroendocrine cell line, evokes di-peptide-stimulated hormone secretion in a pH-dependent manner. Measurement of membrane potentials shows that PEPT1- transfected STC-1 cells are depolarized by di-peptide glycyl-glycine but not by glycine monomer. Glycyl-glycine stimulation induces a rise in the intracellular calcium concentration in PEPT1-transfected STC-1 cells. The secretion induced by glycyl-glycine in PEPT1-transfected STC-1 cells was blocked by nifedipine, a Ca2+ channel blocker, suggesting that the secretion is triggered by Ca2+ influx through L-type voltage-dependent Ca2+ channels. These data suggest that PEPT1 mediates oligopeptide-induced hormone secretion in enteroendocrine cells. Ó 2005 Elsevier Inc. All rights reserved. Keywords: STC-1 cells; Enteroendocrine cells; PEPT1; Oligopeptide sensing; Hormone secretion
Various gastrointestinal hormones including cholecystokinin (CCK) [1], glucose-dependent insulinotropic polypeptide (GIP) [2,3], and glucagon like peptide-1 (GLP-1) [3,4] are secreted in response to meal ingestion, participating in the maintenance of energy homeostasis by regulating gut motility, satiety, and insulin secretion from pancreatic b-cells [2,4,5]. Since direct administration of mixed meal in the intestinal lumen elicits secretion of gastrointestinal hormones such as CCK, GIP, and GLP-1 [6–8], enteroendocrine cells have been thought to possess a mechanism of nutrient sensing. Among the various constituents of mixed meal, orally administered protein, as well as carbohydrate and fat, is known to potently stimulate the secretion of gastrointestinal hormones [1–3]. It has been reported that oligopeptide potently induces CCK secretion in rat duodenojejunum, while polypeptides or monomeric amino acids generate only modest CCK secretion [7]. A candidate mediator of CCK secretion is the oligopeptide transporter *
Corresponding author. Fax: +81 78 382 5370. E-mail address:
[email protected] (S. Seino).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.08.259
PEPT1, which transports di-peptides, tri-peptides, and their related compounds [9,10]. PEPT1 was originally identified by expression cloning of a rabbit intestinal cDNA library in an attempt to clone the di-peptide transporter in gut epithelium. PEPT1 has 12 membrane spanning regions and is related structurally to the H+-dependent nitrate transporter CHL1 in plants [9,10]. In mammalian small intestine, PEPT1 is distributed widely in the intestinal brush border membrane and is responsible for absorption of oligopeptides arising from digestion of dietary protein [10,11]. PEPT1 is a proton/oligopeptide co-transporter, and its driving force for uphill transport requires electrochemical proton gradients. Oligopeptide uptake by this transporter is most effective under condition of extracellular pH ranging from 5.5 to 6.0. Proton uptake by PEPT1 generates electrical activity of PEPT1 heterologously expressed in Xenopus laevis oocytes [9,10]. In this study, we investigated the mechanism by which oligopeptide stimulates the secretion of gastrointestinal hormones in the gastrointestinal hormone secreting cell line STC-1, which lacks endogenous expression of PEPT1.
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Using PEPT1-transfected STC-1 cells, we examined hormone secretion, membrane potential, and intracellular calcium concentrations ([Ca2+]i) induced by oligopeptides. Thus, PEPT1 may act as a nutrient sensor in enteroendocrine cells that recognizes ingestion of meal. Materials and methods Cell culture and transfection. The STC-1 cells were cultured in DulbeccoÕs modified EagleÕs medium (DMEM) (Sigma, St. Louis, MO) containing 25 mM glucose, 15% heat-inactivated horse serum (Gibco, Rockville, MD), and 2.5% heat-inactivated fetal bovine serum (Sigma). For assay of human growth hormone secretion, STC-1 cells were cotransfected with human PEPT1 cDNA plasmid [pcDNA3.1(+) hPEPT1] and human growth hormone cDNA plasmid [pXGH (Nichols Institute, San Juan Capistrano, CA)] using Lipofectamine (Invitrogen, Carlsbad, CA) in 10 cm dish. For measurement of membrane potential, STC-1 cells were transfected with hPEPT1 and pEYFP-C1 (Enhanced yellow fluorescent protein vector: BD Biosciences, San Jose, CA), using Lipofectamine in 24-well plates. Measurement of human growth hormone. One day after transfection of human growth hormone (hGH) and PEPT1, STC-1 cells were harvested and replated into 24-well plates. Twenty-four hours after replating, the cells were washed twice and preincubated for 30 min in Hepes-balanced Krebs–Ringer bicarbonate buffer (KRH: 119 (in mM) NaCl, 4.74 KCl, 2.54 CaCl2, 1.19 MgCl2, 1.19 KH2PO4, 25 NaHCO3, and 10 Hepes, pH 7.4) containing 0.1% BSA with 2.8 mM glucose. After preincubation, the cells were incubated for 30 min with KRH containing various compounds such as 10 mM glycyl-glycine (Gly-Gly), 10 mM glycyl-sarcosine (GlySar), and 10 lM PMA (phorbol 12-myristate 13-acetate: Sigma). Released hGH was measured by ELISA kit (Roche, Mannheim, Germany). The amount of hGH secretion in each experiment was normalized by the cellular hGH content. To measure cellular hGH content, we obtained cellular lysate by sonicating non-stimulated cells in KRH. To assess the effect of nifedipine on di-peptide induced hGH secretion, the cells were preincubated with 1 lM nifedipine and stimulated with KRH containing 0.1% BSA, 2.8 mM glucose, 10 mM Gly-Gly or 10 mM Gly-Sar, and 1 lM nifedipine. Measurement of membrane potential. Membrane potential was measured by the current clamp-version of the nystatin-perforated patch–clamp technique as previously described [12]. Membrane current was clamped at 0 pA. In nystatin-perforated current–clamp recordings, the pipette solution contained 10 mM KCl, 10 mM NaCl, 70 mM K2SO4, 2 mM MgCl2, 10 mM Hepes, and 100 lg/ml nystatin (pH 7.3). Test agents were dissolved in extracellular medium and applied to a recording chamber at a flow rate of 6 ml/min. Measurement of Fura-2 fluorescence. One day after pEYFP and hPEPT1 gene transfection, STC-1 cells were harvested and plated on collagen-II (Sigma) coated slide glass. Twenty-four hours later, the cells were loaded with 5 lM Fura-2 acetoxymetyl ester (Fura-2 AM: Dojindo, Kumamoto, Japan) for 30 min in KRH containing 0.1% BSA with 2.8 mM glucose. The cells transfected PEPT1 were selected by yellow fluorescence. [Ca2+]i was assessed by the ratio of Fura-2 fluorescence intensity using dual-excitation wavelength method (340/380 nm) as previously described [13]. Fluorescence emission at 510 nm was monitored, and the ratio calculation was digitized every 10 s by a computerized image processor (Argus-50/CA: Hamamatsu Photonics, Hamamatsu, Japan).
Results and discussion Di-peptide triggers hormone secretion in PEPT1-transfected STC-1 cells To determine if PEPT1-mediated transport of di-peptide elicits hormone secretion of enteroendocrine cells, gastroin-
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testinal hormone secreting STC-1 cells were used. The STC-1 cell line was established from an intestinal tumor occurring in double transgenic mice expressing SV40 T antigen and the polyoma small T antigen gene under regulation of rat insulin II-promoter [14]. STC-1 cells secrete mainly CCK [15], but also several other gastrointestinal hormones including GLP-1 [16] and GIP [17]. However, the low transfection efficiency of STC-1 cells hampers determination of the direct effect of transfected PEPT1 on native hormone secretion from STC-1 cells induced by various stimuli. This difficulty was overcome by measuring the secretion of human growth hormone (GH) co-transfected with PEPT1. Using this method [18], we were able to monitor the secretion in only the transfected cells expressing PEPT1. Secretion at pH 5.5 indicates that both 10 mM Gly-Gly and 10 mM Gly-Sar increased hGH secretion of PEPT1-transfected STC-1 cells 3.8- and 4.0-fold, respectively (Fig. 1A, left), while in mock-transfected STC-1 cells, hGH secretion was not induced by Gly-Gly or Gly-Sar (Fig. 1A, right), suggesting that substrate uptake through PEPT1 triggers the secretion. By contrast, secretion by Gly-Gly or Gly-Sar was not detected in PEPT1-transfected STC-1 cells at pH7.4 (Fig. 1B). Considering that substrate uptake of PEPT1 is most effective at pH 5.5–6.0 [9,10], the pH dependency also suggests evocation by substrate uptake through PEPT1. To confirm that Gly-Gly induced hGH secretion is not mediated by monomeric glycine generated by hydrolysis of Gly-Gly, PEPT1-transfected STC-1 cells were stimulated with 10 mM glycine. Ten millimolar glycine did not induce hGH secretion of these STC-1 cells (Fig. 1C). These results suggest that incorporation of the substrate through PEPT1 is required to trigger di-peptide-induced hormone secretion of STC-1 cells. Di-peptide evokes membrane depolarization in STC-1 cells through PEPT1 PEPT1 is an oligopeptide transporter that co-transports proton and oligopeptide, and has been reported to induce membrane depolarization in X. laevis oocytes [9,10]. We investigated to determine if PEPT1-mediated transport of di-peptide triggers membrane depolarization of STC-1 cells. The resting membrane potential of STC-1 cells was approximately 70 mV, which was increased in a dose-dependent manner by subsequent stimulation with 10, 1, and 0.1 mM Gly-Gly at pH 5.5 (Fig. 2A). In mock-transfected STC-1 cells, membrane potential was not changed by treatment with 10 mM Gly-Gly (Fig. 2B), suggesting that transport of Gly-Gly through PEPT1 triggers depolarization. In contrast to stimulation with Gly-Gly, 10 mM glycine monomer failed to depolarize PEPT1-transfected STC-1 cells (Fig. 2C). Such substrate specificity is consistent with the finding that PEPT1 transports oligopeptide but not monomeric amino acids [9,10]. These results indicate that oligopeptide transport through PEPT1 triggers membrane depolarization in PEPT1-transfected STC-1 cells. Oligopeptides are absorbed mainly through PEPT1 on the apical
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Fig. 1. Di-peptide induced hormone secretion by PEPT1-transfected STC1 cells. (A) Di-peptide induced hGH secretion in STC-1 cells at pH 5.5. Cells were transfected with PEPT1 (A, left) or empty vector (Mock) (A, right) together with hGH plasmid. (B) Di-peptide induced hGH secretion at pH7.4. (C) Effect of 10 mM of the monomer glycine on hGH secretion at pH 5.5. All results show released hGH expressed as percent of total hGH content. Ten micromolar PMA was used as positive control for hGH secretion. Data are means ± SEM of at least three-independent experiments (n P 12). *P < 0.01.
surface of gut epithelium [10,11]. Enteroendocrine cells have their apical surfaces oriented toward the gut lumen [1,19]. In addition, enteroendocrine cells are stimulated by nutrients in the intestinal lumen [1,19]. Thus, it is likely that oligopeptides transported through PEPT1 induce membrane depolarization in enteroendocrine cells. Di-peptide-induced hormone secretion is triggered by a rise in [Ca2+]i Since STC-1 cells express VDCCs [20–22], membrane depolarization by oligopeptide in PEPT1-transfected STC-1 cells should lead to calcium influx through the
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Fig. 2. Changes in membrane potential by di-peptide in PEPT1-transfected STC-1 cells. (A) Representative recording of membrane potential of STC-1 cell transfected with PEPT1. The cell was stimulated consecutively by 10, 1, and 0.1 mM Gly-Gly. (B) Recording of membrane potential of STC-1 cell transfected with empty vector. The cell was stimulated by 10 mM Gly-Gly and 30 mM KCl (a positive control). (C) Recording of membrane potential of STC-1 cell transfected with PEPT1 stimulated by 10 mM of the monomer glycine. 10 mM Gly-Gly was subsequently applied as positive control. Bars represent the period of stimulation. Similar results were obtained in three-independent experiments.
VDCC and subsequent hormone secretion. To clarify, the role of [Ca2+]i in dipeptide-induced hormone secretion, a change in [Ca2+]i was assessed by the ratio of Fura-2 intensity in PEPT1-transfected cells. Ten millimolar Gly-Gly induced a distinct rise in [Ca2+]i in PEPT1-transfected STC-1 cells at pH 5.5 (Fig. 3A), which did not occur in mocktransfected cells (Fig. 3B). These results indicate that uptake of Gly-Gly by PEPT1 induces the rise in [Ca2+]i In addition, stimulation by 30 mM K+increased [Ca2+]i in both PEPT1 and mock-transfected STC-1 cells, revealing
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Enteroendocrine cells have been shown to sense glucose by electrogenic sugar entry via the sodium-glucose cotransporter (SGLT) [23–25]. Ionic currents generated by co-transport of Na+ and glucose have been proposed to trigger electrical activity and exocytosis [25]. Similarly, oligopeptide uptake through PEPT1 generates ionic currents by co-transport of proton in enteroendocrine cells. Thus, SGLT and PEPT1 share a similar mechanism of hormone secretion in that both elicit electrical activity in response to nutrients. The present study shows that oligopeptide-induced proton uptake through PEPT1 triggers membrane depolarization by Ca2+ influx through VDCCs, which induces hormone secretion in STC-1 cells. The L-type VDCC is expressed in enteroendocrine cells [26], and Ca2+ influx through VDCCs has been reported to trigger hormone secretion from human enteroendocrine cells [27]. Although expression of PEPT1 has not been reported in enteroendocrine cells, the L-type VDCC is expressed in the cells [25], and Ca2+ influx through VDCCs has been reported to trigger hormone secretion from human enteroendocrine cells [26]. Thus, PEPT1 may act as a nutrient sensor in enteroendocrine cells that recognizes ingestion of meal. Acknowledgments
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Fig. 3. Di-peptide induced [Ca2+]i rise and the effect of 1 lM nifedipine on hormone secretion in PEPT1-transfected STC-1 cells. (A) A change in Fura-2 intensity of PEPT1-transfected STC-1 cells. (B) A change in Fura-2 intensity of empty vector-transfected STC-1 cells. Vertical scale bar is the ratio of (340/380 nm) Fura-2 intensity. Bar represents the period of stimulation. Similar results were obtained in three-independent experiments. (C) Secretion induced by 10 mM Gly-Gly or 10 mM Gly-Sar at pH 5.5 in the presence or absence of 1 lM nifedipine in PEPT1-transfected STC-1 cells. The cells were stimulated as described in Fig. 1. Data are means ± SEM of at least three-independent experiments (n = 8). *P < 0.01.
expression of VDCCs in STC-1 cells. We then examined the effect of nifedipine [13], a VDCC blocker, on di-peptide-induced hGH secretion. Treatment with 1 lM nifedipine significantly inhibited both Gly-Gly-induced and Gly-Sar-induced hGH secretion (Fig. 3C), suggesting that Ca2+ influx through L-type VDCCs is critical for PEPT1mediated hormone secretion.
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