Glucose-dependent insulinotropic polypeptide regulates dipeptide absorption in mouse jejunum

Abstract

Glucose-dependent insulinotropic polypeptide (GIP) secreted from jejunal mucosal K cells augments insulin secretion and plays a critical role in the pathogenesis of obesity and Type 2 diabetes mellitus. In recent studies, we have shown GIP directly activates Na-glucose cotransporter-1 (SGLT1) and enhances glucose absorption in mouse jejunum. It is not known whether GIP would also regulate other intestinal nutrient absorptive processes. The present study investigated the effect of GIP on proton-peptide cotransporter-1 (PepT1) that mediates di- and tripeptide absorption as well as peptidomimetic drugs. Immunohistochemistry studies localized both GIP receptor (GIPR) and PepT1 proteins on the basolateral and apical membranes of normal mouse jejunum, respectively. Anti-GIPR antibody detected 50-, 55-, 65-, and 70-kDa proteins, whereas anti-PepT1 detected a 70-kDa proteins in mucosal homogenates of mouse jejunum. RT-PCR analyses established the expression of GIPR- and PepT1-specific mRNA in mucosal cells of mouse jejunum. Absorption of Gly-Sar (a nondigestible dipeptide) measured under voltage-clamp conditions revealed that the imposed mucosal H⁺ gradient-enhanced Gly-Sar absorption as an evidence for the presence of PepT1-mediated H⁺:Gly-Sar cotransport on the apical membranes of mouse jejunum. H⁺:Gly-Sar absorption was completely inhibited by cephalexin (a competitive inhibitor of PepT1) and was activated by GIP. The GIP-activated Gly-Sar absorption was completely inhibited by RP-cAMP (a cAMP antagonist). In contrast to GIP, the ileal L cell secreting glucagon-like peptide-1 (GLP-1) did not affect the H⁺:Gly-Sar absorption in mouse jejunum. We conclude from these observations that GIP, but not GLP-1, directly activates PepT1 activity by a cAMP-dependent signaling pathway in jejunum.

hormones, genetics, and environmental factors contribute to obesity and comorbidities such as Type 2 diabetes, hypertension, and metabolic syndrome (11, 13). The release into the circulation of incretin hormones glucose-dependent insulinotropic polypeptide (GIP) from jejunal K cells and glucagon-like peptide-1 (GLP-1) from ileal L cells are stimulated by the ingestion of a meal (12, 37). GIP and GLP-1 affect blood glucose levels by enhancing insulin secretion from pancreatic β-cells (25).

After digestion, fatty acids and monosaccharides have been shown to stimulate GIP and GLP-1 secretion (8, 14, 18). Previous studies have shown that Na-glucose cotransporter (SGLT1)-mediated glucose absorption and GIP stimulate one another (33). Recent studies have shown that GIP but not GLP-1 further stimulates nutrient absorption by direct action on jejunal epithelia creating a cycle of increased release and stimulation that eventually leads to incretin resistance, nutrient overabsorption, and obesity (6, 12, 33). Similar to glucose absorption, peptide absorption may also be regulated by GIP. However, the absorption of peptides and amino acids and its physiological relationship to GIP have not been investigated.

In addition to fat and carbohydrate, protein is also a major nutrient. Protein digestion products include small peptides (di- and tripeptides) and free amino acids (1). The assimilation of amino acids is crucial for the health and maintenance of bodily functions, since they serve as the building blocks of proteins and precursors for several important metabolites (36). Peptides and amino acids are absorbed by distinct apical membrane transporters in the intestine (1). Several apical Na-amino acid cotransporters have been shown to mediate different amino acids, whereas peptides have been shown transported by an electrogenic proton H⁺-peptide cotransporter (10). Molecular studies have identified the H⁺-peptide cotransporter as peptide transporter-1 (PepT1) in the apical membranes of intestinal mucosa (3, 22, 32). PepT1, a member of the SLC15A family, is expressed throughout the small intestine with its highest expression in the jejunum (10). In addition to mediating peptides, PepT1 also transports peptidomimetic drugs such as cephalexin (2).

In general, compared with fats and carbohydrates, high-protein meals have been shown to stimulate GIP release to a lesser degree (37). High-protein meals stimulate GIP secretion, which can occur from individual amino acid absorption (14, 18, 33, 35). Increased blood amino acid levels have been reported in patients prior to the onset of diabetes (21, 35, 36). Although intestine has the ability to effectively absorb both peptides and amino acids, peptide absorption has been shown to have kinetic advantage over amino acid absorptive processes (1). Therefore, it is not known whether in addition to amino acid transporters, activated PepT1 also contributed to increased blood amino acid levels in patients prior to onset of diabetes. We hypothesized that, similar to SGLT1, GIP might also directly activate PepT1. Therefore, the present study was initiated to investigate whether GIP would directly activate PepT1 in mouse jejunum. The results presented in this study demonstrate that GIP increases PepT1 activity and that GIP regulates PepT1 activity through the cAMP-dependent signal transduction pathway.

METHODS

Animals.

Male wild-type (C57/B6; 6–8 wk old, Jackson Labs) and GIP receptor (GIPR) knockout mice (6–8 wk old; kindly provided by Dr. M. Michael Wolfe, Case Western University, Cleveland, OH) were given standard chow and tap water ad libitum. Experimental protocols performed in this study were approved by the Boston University Institutional Animal Care and Use Committee.

Ussing chamber studies.

One pair of 2-cm segments of nonstripped proximal jejunum (just distal to the ligament of Treitz) was removed from overnight-fasted anesthetized mice for each experiment. The tissue was opened along the mesenteric border and mounted under voltage-clamp conditions in Ussing-type chambers with 0.3-cm² openings (EasyMount, Warner Instruments, New Haven, CT). Both sides of the tissue were bathed with 5 ml of Ringer solution (in mM: 115 NaCl, 25 NaHCO₃, 2.4 K₂HPO₄, 0.4 KH₂PO₄, 1.2 CaCl₂, 1.2 MgCl₂; pH 7.4). Bathing solutions maintained at 37°C were continuously gassed with 5% CO₂-95% O₂. Following a 20-min equilibration, 10 mM Gly-Sar (a nonhydrolyzable dipeptide) mixed with a trace of [³H]Gly-Sar was added to the mucosal bath, whereas 10 mM mannitol was added simultaneously to the serosal bath to maintain isoosmolarity. Mucosal-to-serosal (m-s) and serosal-to-mucosal (s-m) Gly-Sar fluxes, transepithelial potential difference (PD), and short-circuit current (I_sc; a measure of electrogenic transport process) were measured for every 15 min both in the presence and absence of incretins and inhibitors. Net Gly-Sar absorption was calculated by subtracting s-m fluxes from m-s fluxes of tissue pairs that matched on the basis of differences in basal conductance (G) of less than 10%. Basal G was calculated from the basal PD and I_sc using Ohm's law.

Treatments.

GIP and GLP-1 (Bachem, 500 μM each), when applied, were added to the serosal bathing solution. RP-cAMP (50 nM, adenosine 3′,5′-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt; Calbiochem) was used as a cAMP inhibitor and when applied was added to the serosal bathing solution. Amiloride (1 mM; Sigma) in certain experiments was applied to the mucosal bath to inhibit Na-H exchange.

Immunohistochemistry.

The proximal jejunum was dissected from mice was fixed in ice-cold 4% paraformaldehyde at 4°C for 2 h and then embedded into paraffin. Blocks were cut into 5-μm sections with a standard microtome. Slides were placed in 60°C oven for 20 min and then were deparaffinized by standard xylene-ethanol methods. Slides were placed in a decloaking chamber (Biocare Medical); antigen was retrieved by use of Rodent Decloaker at 95°C for 35 min and cooldown of 85°C for 10 min and then transferred to distilled water.

Slides were stained with an automated Intellipath autostainer (Biocare Medical). Slides were first blocked with peroxidase-1 and then with Rodent Block M (30 min), and washed with Tris-buffered saline-Tween 20 (TBST) before, after, and in between each step. Primary antibodies PepT1 (E3, 1:100; Santa Cruz Biotechnology) or GIPR antibody (K17, 1:100; Santa Cruz Biotechnology) were added at room temperature for 2 h. The slides washed with TBST were treated with secondary antibodies [goat horseradish peroxidase (HRP) for PepT1, 15 min; mouse-on-mouse HRP, for GIPR; 30 min]. A tertiary antibody was also used for the PepT1 (goat HRP; 15 min).

Finally, all slides were washed with TBST, diaminobenzidine (5 min), twice with deionized water, and hematoxylin (1 min) and rinsed with TBST and deionized water. The dehydrated (70% ethanol-xylene substitute) slides were air dried and then mounted. Photographs were taken with an Olympus upright light microscope.

Reverse transcriptase-polymerase chain reaction.

RT-PCR analyses were performed to identify PepT1- and GIPR-specific mRNA expression in mouse proximal jejunum. Total RNA was isolated from pancreas, liver, and scraped mucosa of proximal jejunum by use of a RNA isolation kit (Invitrogen). RT-PCR was performed with total-RNA using One Step RT-PCR kit (Qiagen). Sense (5′-CTTGGAGCCACCACAATGG-3′) and antisense (5′-ACAGAATTCATTGACCACGATGA-3′) primers that amplify a 97-bp segment were used to identify PepT1-specific mRNA expression (24). Sense (5′-TCACCTTTCAAGGATGCCCC-3′) and antisense (5′-TCGTCAGGGACAGGGAGTAG-3′) primers that amplify 452 bp (codons 154–605 of GIPR) were used to identify GIPR-specific mRNA expression. The reaction conditions for the reactions are as follows: 50° for 30 min (reverse transcription), 95° for 15 min, and then 40 cycles of denaturation at 94° for 30 s, annealing at 60° (for PepT1) or 68° (for GIPR) for 30 s and elongation at 72° for 60 s, concluding with a final elongation at 72° for 10 min. The veracity of RT-PCR products were confirmed by sequence analyses.

Western blotting.

Scraped jejunal mucosa was homogenized in RIPA buffer (Boston BioProducts, Boston, MA) containing Complete protease inhibitor (Roche Applied Science, Indianapolis, IN). Following 15-min incubation on ice, the homogenate was centrifuged at 14,000 rpm for 5 min at 4°C, and 10 μl of supernatant (50 μg protein) collected from the homogenates was resolved on SDS-PAGE and transferred to nitrocellulose membranes (PerkinElmer, Waltham, MA). The blot blocked 2 h with 5% nonfat milk-PBS-Tween 20 was incubated overnight with primary antibody [anti-PepT1 (1:200); anti-GIPR (1:200); mouse anti-β-actin (1:2,000; BD Biosciences, San Jose, CA)] at room temperature for 2 h. Following washes in TBST (3 × 5 min), the blot was incubated with appropriate HRP-conjugated secondary antibody (anti-goat for PepT1; anti-rabbit for GIPR; anti-mouse for β-actin). Immune complexes were detected on film by enhanced chemiluminescence (Super SignalWest Pico).

Statistical analyses.

The values presented represent means ± SE of four tissue pairs obtained from four different mouse proximal jejuna. Statistical analyses were performed by unpaired or paired Student's t-test by using Graph Pad. A P < 0.05 is considered statistically significant.

RESULTS

RT-PCR was performed to establish that PepT1 and GIPR are located in the villus cells of mouse jejunum. RT-PCR amplification of a 97-bp fragment (Fig. 1A) and a 452-bp fragment (Fig. 1B) establishes that both PepT1- and GIPR-specific mRNA are expressed in mucosa of jejunum, respectively. GIPR mRNA (Fig. 1B) is expressed in pancreas, but not in liver (data not shown).

Fig. 1.

mRNA expression of proton-peptide cotransporter-1 (PepT1) and glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in mouse proximal jejunum. PepT1-specific 97-bp (A) and GIPR-specific 452-bp (B) fragments amplified by RT-PCR analyses as described in methods show the presence of RNA for both proteins in the epithelial cells of proximal jejunum. RNA from jejunum, liver, and pancreas are used with primers for GIPR and PepT1. A: primers for PepT1 were used to generate a 97-bp band in jejunum mucosa and also in liver as a positive control. B: GIPR is also present in jejunum but also in pancreas (positive control).

To determine whether RNA is translated into protein, Western blot analyses were performed to identify whether PepT1 and GIPR are expressed as single- or multiple-size proteins in mucosal cells of jejunum. As shown in Fig. 2A, anti-PepT1 antibody detected a 70-kDa protein in mucosal cell homogenate of jejunum, which is consistent with earlier observations (30). In contrast to PepT1 antibody, anti-GIPR antibody detected multiple-size (50, 55, 60, and 70 kDa) proteins in mucosal homogenates of jejunum, whereas it detected only a 50-kDa protein in the homogenates of pancreas (Fig. 2B). In contrast, anti-GIPR antibody did not detect any proteins in liver homogenate (data not shown). These observations indicate that PepT1 expresses a single-size protein, whereas GIPR expresses multiple-size proteins that may represent different levels of phosphorylation and/or glycosylation in the mucosa of mouse jejunum.

Fig. 2.

Protein expression of PepT1 and GIPR in mouse proximal jejunum. A: anti-PepT1 antibody detects a 70-kDa protein in normal mouse jejunal mucosal homogenate. B: anti-GIPR antibody detects 50-, 55-, 65-, and 70-kDa proteins in normal jejunum. The antibody also detects a 55-kDa band in normal pancreas (positive control). Western blots presented represent typical blots selected from 3 different normal mouse mucosal homogenates.

After determining that PepT1 as well as GIPR are expressed in the mucosa in normal mouse jejunum, immunohistochemistry was performed to determine the membrane-specific localization of PepT1 and GIPR proteins. PepT1 antibody localizes PepT1 proteins primarily on the apical membranes of villus cells, whereas its expression diminishes down the villus and is absent in crypt cells (Fig. 3A). GIPR antibody on the other hand localizes GIPR proteins only on the basolateral membranes of villus, but not crypt cells (Fig. 3, C and E). IgG control reveals that neither PepT1-antibody (Fig. 3B) nor GIPR antibody (Fig. 3, D and F) nonspecifically binds to epithelial cells of mouse jejunum. For clarity, results presented in Fig. 3, C and D, were presented in higher magnification in Fig. 3, E and F, respectively. These observations indicate that PepT1 and GIPR proteins are localized on the apical and basolateral membranes of villus epithelial cells of jejunum, respectively.

Fig. 3.

Immunohistochemistry localization of GIPR and PepT1 protein in normal mouse. A: anti-PepT1 antibodies show extensive expression along the apical membranes of villus cells and absent in crypt cells. B: IgG control for the PepT1 antibody lacks any nonspecific binding. C: anti-GIPR antibodies also localize GIPR proteins on the basolateral surface of the epithelial cells of proximal jejunum. D: IgG controls for the GIPR antibody lack any nonspecific binding. A through D are magnification ×40; E and F are higher magnifications of the same villi from C and D, respectively (magnification ×60).

The PepT1 activity was assessed by measuring Gly-Sar absorption in normal mouse jejunum. In these studies Gly-Sar absorption was measured as a function of short-circuit current (I_sc) (Fig. 4A) and by direct m-s unidirectional [³H]Gly-Sar fluxes (Fig. 4B). Gly-Sar fluxes were measured both under basal and mucosal-to-cytosolic acidic pH gradient (i.e., transapical H⁺ gradient) conditions. Transapical H⁺ gradient was achieved by either mucosal acidic Ringer solution (pH 6.0) or a normal Ringer solution supplemented with 0.5 mM NH₄Cl that increases cytosolic pH. Both in the presence and in the absence of a transapical H⁺ gradient, mucosal Gly-Sar addition markedly increased the I_sc (Fig. 4A). This observation is consistent with Gly-Sar absorption that is mediated by PepT1 via a proton gradient-driven electrogenic transport process (i.e., H⁺:Gly-Sar cotransport). Near complete inhibition by mucosal cephalexin (a competitive inhibitor of PepT1) indicates that I_sc enhanced by Gly-Sar in the presence and in the absence of pH gradients are attributed to PepT1-mediated Gly-Sar absorption (Fig. 4A). In contrast to mucosal Gly-Sar, serosal Gly-Sar did not alter I_sc (data not shown). As shown in Fig. 4B, imposition of a mucosal-to-cytoplasmic acidic pH gradient enhanced the Gly-Sar absorption by 2.0-fold, whereas the cephalexin-insensitive fraction of Gly-Sar absorption, likely owing to diffusion, remained constant in both basal and pH gradient conditions. These observations establish that cephalexin-sensitive, H⁺ gradient-driven electrogenic unidirectional (from mucosa to serosa) Gly-Sar absorption mediated by PepT1 is present in normal mouse jejunum.

Fig. 4.

Effect of mucosal-to-cytosolic acidic pH gradient on Gly-Sar absorption in mouse proximal jejunum. Either mucosal-to-cytosolic acidic pH gradient was generated by adding Ringer solution (i.e., pH 6.0) to the mucosal bath, or cytosolic alkaline pH was generated by adding 0.5 mM NH₄Cl to the mucosal bath. Gly-Sar absorption was measured both as a function of short-circuit current (I_sc; A) and as a direct measurement using [³H]Gly-Sar trace (B). A: Gly-Sar absorption (10 mM) as a function of I_sc was measured both in the absence (○), and in the presence of pH gradients generated by either mucosal acidic Ringer solution (▲) or NH₄Cl (⧫). B: direct measurement of Gly-Sar absorption was also measured in the absence and in the presence of pH gradient generated by no pH gradient (neutral-neutral), mucosal acidic pH (acidic-neutral) and cytosolic alkaline pH (neutral-alkaline). Gly-Sar was also measured in the presence of cephalexin (Ceph; 10 mM) added to the mucosal bath. Results presented represent means ± SE from 4 tissue pairs from 4 mice. #P < 0.05 for 5 time points for a pH gradient generated by mucosal acidic pH compared with control (Cont) and also for the first of those 3 time points for the internal pH-altered gradient (before viability was lost); and £P < 0.05 compared with control; $P < 0.05 for cephalexin-treated time points compared with peak dipeptide absorption values and *P < 0.05 compared with each flux from each condition.

It should be noted that in the presence of an enhanced inward H⁺ gradient, Gly-Sar absorption was 2.0-fold greater than control (Fig. 4B), the Gly-Sar-enhanced I_sc was substantially higher in the presence of a pH gradient than that in the absence of pH gradient (Fig. 4A). In addition, the pH gradient generated by using mucosal NH₄Cl (to alkalinize the cell) seems to eventually affect the tissue viability and limit the extent of transport at the later time points (Fig. 4A). Therefore, all further characterization of Gly-Sar absorption was performed in the absence of pH gradient.

In Fig. 5, the effect of GIP and GLP-1 were examined on Gly-Sar-enhanced I_sc and Gly-Sar absorption. GIP significantly enhanced both the Gly-Sar-enhanced I_sc and Gly-Sar absorption (Fig. 5). Since GLP-1 has been shown to modulate nutrient absorption in ileum, we also examined the effect of GLP-1 on Gly-Sar absorption in jejunum. In contrast to GIP, the addition of GLP-1 did not alter either Gly-Sar-enhanced I_sc or Gly-Sar absorption in jejunum (Fig. 5). These observations indicate that GIP, but not GLP-1, augmented PepT1-mediated Gly-Sar absorption in mouse jejunum.

Fig. 5.

Effect of incretins on Gly-Sar absorption in mouse proximal jejunum. Gly-Sar absorption was measured as function of I_sc (A) and direct measurement by use of [³H]Gly-Sar (B). Gly-Sar absorption was measured in the presence and absence of serosal incretins (0.5 μM of either GIP or GLP-1). Results presented represent means ± SE from 4 tissue pairs from 4 mice. #P < 0.05 compared with normal control; *P < 0.05 compared with normal control.

Under basal conditions, the apical Na-H exchanger isoform-3 (NHE3) maintains the mucosal acidic pH microclimate that provides the favorable mucosal-to-cytosol H⁺ gradient required for H⁺-peptide cotransport (20). We sought to determine whether GIP-enhanced Gly-Sar absorption occurred by direct activation of PepT1 and/or NHE3 activation by GIP with secondary activation of PepT1 by an enhanced H⁺ gradient. To differentiate these possibilities, the effect of GIP on Gly-Sar absorption was examined in the presence of amiloride (a NHE inhibitor, 1 mM) applied to the mucosal bathing solution. Despite the presence of mucosal amiloride, GIP significantly enhanced both Gly-Sar-enhanced I_sc (Fig. 6A) and Gly-Sar absorption (Fig. 6B). Although mucosal amiloride significantly inhibited Gly-Sar absorption in the absence of GIP (Fig. 6B), amiloride did not block further enhancement of I_sc by Gly-Sar (Fig. 6A). These observations indicated that GIP increased Gly-Sar absorption directly in jejunum.

Fig. 6.

Effect of amiloride on GIP-enhanced Gly-Sar absorption in mouse proximal jejunum. Gly-Sar absorption was measured as function of I_sc. A: 45 min following the mucosal addition of Gly-Sar, 1 mM amiloride was added to mucosal bath. Following amiloride addition, 0.5 μM GIP was added to serosal bath. I_sc was calculated for every 15 min, whereas direct Gly-Sar absorption was measured every 45 min. GIP-treated jejunum (●) was compared with control (○). B: direct measurement of Gly-Sar absorption using trace of [³H]Gly-Sar was also measured in the absence (control, open bar) and in the presence of amiloride (Amil, shaded bar) and then in absence (no GIP; Normal; hatched bar) or the presence of GIP (+GIP; Exp; solid bar). In both the normal and the experimental control (Gly-Sar only) and the amiloride (Amil; Gly-Sar + amiloride), the conditions are the same. Only in the normal condition, there is no GIP eventually added (no GIP, hatched bar) compared with the experimental condition (+GIP, solid bar) B: results presented represent means ± SE from 4 tissue pairs from 4 rat jejunum. #P < 0.05 compared with control; *P < 0.05 compared with control; $P < 0.05 compared with plus amiloride.

Increased intracellular cAMP was shown as the mechanism for GIP-enhanced glucose absorption via SGLT1 in jejunum (33). To determine whether GIP-enhanced Gly-Sar absorption observed in the present study is regulated by cAMP-dependent pathways, the effect of Rp-cAMP (a competitive inhibitor of cAMP-dependent protein kinase) on GIP-mediated increases in PepT1 activity was examined. In Fig. 7, both Gly-Sar-enhanced I_sc and Gly-Sar absorption were completely inhibited in the presence of Rp-cAMP. In contrast, basal Gly-Sar-enhanced I_sc and Gly-Sar absorption were not inhibited by Rp-cAMP. These observations indicated that cAMP-dependent signaling pathways modulated the GIP-enhanced Gly-Sar absorption mediated by PepT1 in mouse jejunum.

Fig. 7.

Effect of cAMP inhibitor (RP-cAMP) on GIP-enhanced Gly-Sar absorption in mouse proximal jejunum. Gly-Sar absorption was measured as function of I_sc (A) and direct measurement using trace of [³H]Gly-Sar (B). At 45 min following the addition of Gly-Sar, I_sc and Gly-Sar absorption were measured either in the presence (Experiment) or absence (Normal) of GIP. I_sc and Gly-Sar absorption in both normal and experiment conditions were also examined in the presence of RP-cAMP. I_sc was measured for every 15 min, whereas Gly-Sar absorption was measured for every 45 min. GIP-treated jejunum (●) was compared with control (○). Results presented represent means ± SE from 4 tissue pairs from 4 rat jejunum. #P < 0.05 compared with respective control; +P < 0.05 compared with peak I_sc (i.e., at 15 min); £P < 0.05 compared with normal control; *P < 0.05 compared with experiment control.

DISCUSSION

Nutrient absorption per se, but not the blood nutrient levels, has been shown responsible for the release of the incretin hormones GIP and GLP-1 that regulate blood glucose levels through insulin secretion (39). The decrease of GIP serum levels or the restoration of GIPR function have been suggested as potential causes for the disappearance of diabetic syndromes in obese patients who underwent bariatric surgery (4, 29, 31). In addition to inducing insulin release among other functions, GIP and GLP-1 have also been shown to increase nutrient absorption (6, 12). However, the mechanism of GIP- and GLP-1-enhanced nutrient absorption is not known. In recent studies, we have shown that GIP directly activated the SGLT1-mediated glucose absorption in mouse jejunum (33). It is not known whether, in addition to SGLT1, GIP also activates other nutrient transporters. The present study demonstrates that GIP secreted from jejunal K cells but not GLP-1 secreted from ileal L cells directly activates PepT1 mediated H⁺-dependent peptide (Gly-Sar) absorption through a cAMP-dependent signaling pathway in mouse jejunum. This conclusion is supported by the following observations: 1) both GIPR and PepT1 proteins are localized on villus epithelial cells (Figs. 1–3); 2) the imposed mucosal-to-cytosolic acidic pH gradient significantly enhanced the Gly-Sar absorption (Fig. 4); 3) GIP, but not GLP-1, significantly stimulated the H⁺ gradient-driven Gly-Sar absorption (Fig. 5); 4) GIP directly activated the PepT1 protein in villus cells (Fig. 6), and 5) cephalexin and RP-cAMP completely inhibited the GIP-stimulated Gly-Sar absorption (Fig. 7).

The observations presented in this study demonstrate that GIP directly binding to its receptor (GIPR) activates PepT1-mediated peptide absorption in jejunal villus cells. The conclusion is supported by the demonstration that both GIPR and PepT1 are localized on the villus cells of mouse jejunum (Figs. 1–3), and that GIP enhances H⁺ gradient-driven cephalexin-sensitive Gly-Sar absorption in the jejunum (Fig. 4). The villus cell localization of PepT1 is consistent with an earlier report (7). Although previous studies have localized GIPR in entire intestine (34), this is the first study to localize GIPR expression on villus cells in jejunum (Figs. 1–3). Although GIPR cDNA expressed a 55-kDa protein in an in vitro expression system (15), multiple GIPR bands (50, 55, 60, and 70 kDa) were detected in mouse jejunum (Fig. 2A). Although the 50-kDa protein detected might be a degraded product, the high-molecular-size GIPR protein detected might represent either glycosylated and/or phosphorylated forms of GIPR. Since GIPR splice variants have been suggested to regulate different functions in endothelial and pancreatic β-cells (17, 40), it is possible that the different sizes of GIPR detected might also be encoded by various splice variants in mouse jejunum. It is also possible that one or more detected GIPR variants might also be expressed in neurons, since GIPR has also been localized in the enteric nervous system (5). Since both GIPR and PepT1 are colocalized, it is likely that the direct activation of epithelial GIPR- and PepT1-mediated peptide absorption might occur in a coordinated fashion in villus cells. However, on the other hand, the possibility of indirect neuronal GIPR-regulated activation of PepT1 cannot be ruled out, since GIPR is expressed in the enteric nervous system (5) and this study utilized nonstripped jejunum for Gly-Sar flux measurements.

This study demonstrates that PepT1-mediated peptide absorption could be characterized under voltage-clamp conditions in intact mouse jejunum. The peptide uptake has been characterized as H⁺ dependent (i.e., H⁺-peptide cotransport) and electrogenic in apical membrane vesicles (28, 33), whereas in vitro-expressed PepT1 has been shown to mediate H⁺ gradient-dependent electrogenic peptide absorption in Xenopus oocytes (23). The demonstration of increased I_sc by mucosal Gly-Sar and enhanced Gly-Sar absorption by imposed mucosal-to-cytosolic acidic pH gradient indicate that Gly-Sar absorption is mediated through an electrogenic and H⁺-dependent process in mouse jejunum, respectively (Figs. 4 and 5). The cephalexin inhibition further established that the electrogenic H⁺ gradient-driven Gly-Sar absorption is mediated via PepT1 in mouse jejunum (Fig. 4). In addition, PepT1 is localized on the apical membranes of surface, but not crypt, epithelial cells of jejunum (Figs. 1–3). Thus this study established that the properties of PepT1 are successfully characterized under voltage-clamp conditions in mouse jejunum.

In general, nutrient absorption induces GIP secretion and regulates blood glucose levels by inducing insulin secretion from pancreatic β-cells (6). Reduction of plasma GIP levels may contribute to the disappearance of diabetes symptoms in obese/diabetic patients after bariatric surgery (29, 31). However, on the basis of our recent demonstration that GIP directly activated the SGLT1-mediated glucose absorption, we proposed that the absence and/or the decreased GIP activation of excess nutrient absorption might be responsible for the disappearance of diabetes symptoms in patients after bariatric surgery (29, 31). This conclusion is also supported by the present observation that GIP activated PepT1-mediated Gly-Sar absorption in mouse jejunum (Fig. 4). Thus this study establishes that, in addition to inducing insulin secretion, GIP also plays critical role in activating nutrient transport activity in jejunum. Although this study characterized only PepT1, GIP might also activate amino acid transporters, since amino acid concentrations have been shown increased in obese patients preceding the onset of diabetes (21, 37). It may be of interest, which requires an extensive study to identify whether GIP activates a specific or entire amino acid transport systems in jejunum.

This study also demonstrates that the GIP-activated PepT1-mediated Gly-Sar absorption is regulated by the cAMP-dependent signaling transduction pathway. The conclusion is supported by the observations that Rp-cAMP completely inhibited both GIP-activated Gly-Sar-dependent I_sc and Gly-Sar absorption in jejunum (Fig. 7). GIP enhances cellular cAMP levels in the jejunal mucosa (33). The GIP-stimulated insulin secretion in pancreatic β-cells and lipid absorption in adipocytes is regulated by the cAMP-mediated pathway (16, 38). The involvement of the cAMP-activated pathway has been shown for GIP-activated, PepT1-mediated dipeptide absorption in an intestinal epithelia cell line (27). Preliminary signaling studies have also determined that GIP increases PepT1 trafficking in a mucosal model of intestinal absorption in IEC6 cells by activating the cAMP pathway (9). These observations indicate that GIP signals through the cAMP-dependent pathway to augment and promote PepT1-mediated dipeptide absorption.

In summary, this study demonstrates that PepT1-mediated peptide absorption can be successfully characterized as an electrogenic and H⁺-dependent transport process under voltage-clamp conditions in mouse jejunum. In addition, this study also demonstrates for the first time that GIP directly activates PepT1-mediated Gly-Sar absorption through the cAMP-dependent signaling pathway. On the basis of these observations, we conclude that, in addition to stimulating insulin secretion, GIP also enhances nutrient absorption by directly activating the transport processes. Since GIPR-knockout mice fed a high-calorie diet have been shown not to gain weight (19, 26), a potential target for the treatment of obesity and diabetic symptoms would be to control GIP secretion and/or to modulate GIPR affinity. Nevertheless, these observations suggest that GIP plays a central role not only in insulin secretion but also in nutrient absorption that leads to obesity and its associated diseases such as diabetes.

GRANTS

Support from the Boston University Clinical and Translational Science Institute grant UL1-TR000157 was used for this project and also grant support for V. M. Rajendran by NIH/NIDDK DK-018777.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

S.D.C., J.H.S., and S.K.S. conception and design of research; S.D.C. and L.I.J. performed experiments; S.D.C. and L.I.J. analyzed data; S.D.C., J.H.S., V.M.R., L.I.J., and S.K.S. interpreted results of experiments; S.D.C., V.M.R., and L.I.J. prepared figures; S.D.C., V.M.R., and L.I.J. drafted manuscript; S.D.C., J.H.S., V.M.R., L.I.J., and S.K.S. edited and revised manuscript; S.D.C., J.H.S., V.M.R., L.I.J., and S.K.S. approved final version of manuscript.