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Am J Physiol Endocrinol Metab 292: E1599-E1606, 2007. First published February 6, 2007; doi:10.1152/ajpendo.00429.2006
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Role of phosphatidylinositol-3 kinase-{gamma} in the actions of glucagon-like peptide-2 on the murine small intestine

Younes Anini,1,4,5 Angelo Izzo,1 Gavin Y. Oudit,1,2,3 Peter H. Backx,1,2,3 and Patricia L. Brubaker1,2

Departments of 1Physiology and 2Medicine and the 3Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, Ontario; 4Ottawa Health Research Institute, Ottawa, Ontario; and 5Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada

Submitted 18 August 2006 ; accepted in final form 27 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Glucagon-like peptide-2 (GLP-2) enhances intestinal growth and function through a cAMP-linked G protein-coupled receptor (GPCR) expressed in the mucosal layer and enteric nervous system. Because the type 1B {gamma}-isoform of phosphatidylinositol 3-kinase (PI3-K) is activated by GPCRs, we determined whether this enzyme plays a role in the intestinal actions of GLP-2 by using PI3-K{gamma} knockout (KO) mice. Wild-type (WT), heterozygous, and KO mice were treated with vehicle or 1 µg Gly2-GLP-2 (a long-acting analog) twice daily for 10 days and analyzed for changes in intestinal growth, motility, and cAMP production. Basal small intestinal wet weight was increased in KO mice in association with enhanced crypt-villus height and crypt cell proliferation (P < 0.05–0.01). However, the GLP-2-induced changes in these parameters were not different between KO and WT animals. GLP-2 treatment also enhanced the number of mucous cells in the intestinal epithelium, but this effect was lost in the PI3-K{gamma} KO mice. Both basal and GLP-2-induced suppression of intestinal transit were normal in KO mice. In contrast, the ability of GLP-2 to stimulate cAMP levels in isolated muscle strips was abrogated by loss of PI3-K{gamma}, despite the expression of GLP-2 receptor mRNA transcripts in this tissue. Together, the results of this study demonstrate a role for PI3-K{gamma} in basal but not GLP-2-induced small intestinal mucosal growth. However, PI3-K{gamma} is important for the enhancement of mucous cell number by GLP-2 and in the ability of the GLP-2 receptor to couple to cAMP in the enteric nervous system.

adenosine 3',5'-cyclic monophosphate; intestinal growth; intestinal motility

GLUCAGON-LIKE PEPTIDE-2 (GLP-2) is a gut-derived hormone that stimulates intestinal crypt cell proliferation and inhibits enterocyte apoptosis, resulting in increased crypt-villus height and small bowel mass. GLP-2 also enhances nutrient digestive and absorptive capacity, barrier function and blood flow, and decreases intestinal motility, all of which contribute to improved intestinal function (3, 7, 9, 11, 16, 18, 21). Numerous studies in animal models of intestinal disease have also demonstrated protective effects of GLP-2 on intestinal structure and function (5, 6, 26, 30, 36, 37), raising the possibility that GLP-2 may be a useful adjunctive therapy in disorders associated with intestinal epithelial damage and/or nutrient malabsorption. Indeed, several of the beneficial effects of GLP-2 in rodents have now been recapitulated in humans with short bowel syndrome such that administration of either GLP-2 or a long-acting analog, Gly2-GLP-2, improves intestinal nutrient absorption and nutritional status (24). These studies have led to clinical trials for the use of GLP-2 in patients with short bowel syndrome and highlight the importance of understanding the mechanisms that underlie the biological actions of GLP-2 in the intestine.

The GLP-2 receptor (GLP-2R) is a member of the class II glucagon/vasoactive intestinal peptide (VIP) superfamily of G protein-coupled receptors that is restricted in its expression to the gastrointestinal tract and hypothalamus (31). Within the intestine, the GLP-2R has been localized to mucosal enteroendocrine (21, 42) and subepithelial myofibroblast cells (32), as well as to enteric neurons situated in the muscularis layer (4, 21). Work in our laboratory (40) has previously utilized a heterogeneous rat mucosal cell culture model to demonstrate that activation of the GLP-2R leads to production of cAMP and PKA-dependent stimulation of proliferation. Furthermore, our group (18) has recently demonstrated that GLP-2 induces insulin-like growth factor-1 (IGF-1) synthesis and secretion in the intestine and that the tropic actions of GLP-2 in the mucosa require the class 1A phosphatidylinositol 3-kinase (PI3-K)/Akt activator IGF-1. Acute treatment with GLP-2 also activates Akt in intestinal epithelial cells in vivo (11, 17). Consistent with these findings, studies in the colonic Caco-2 cell line have suggested that the class 1A isoforms of PI3-K are required for GLP-2-induced proliferation (23). Together, these findings suggest that signaling by the GLP-2R within the mucosa leads to activation of PKA, release of IGF-1, and induction of proliferation in the crypt cells via stimulation of PI3-K/Akt. In contrast, little is known about the signaling pathways utilized by the GLP-2R in the enteric nervous system (ENS). We have previously demonstrated that GLP-2 enhances cAMP levels in isolated muscle strips (37), and the GLP-2R has been localized to VIP- and endothelial nitric oxide synthase (eNOS)-expressing neurons in the myenteric and submucous plexi (4, 21). The role of the PI3-K/Akt pathway in GLP-2-mediated signaling in these cells has not been elucidated. Nonetheless, because the class 1B isoform of PI3-K, PI3-K{gamma}, is known to be activated by the beta{gamma}-subunit of G protein-coupled receptors (8, 38), we hypothesized that PI3-K{gamma} may play a role in the actions of GLP-2 in the ENS. We presently report, using mice lacking the p110{gamma} catalytic subunit of PI3-K{gamma} (13, 27, 35), that PI3-K{gamma} is required for basal but not GLP-2-stimulated growth of the intestinal mucosa and is essential for the enhancement of cAMP levels by GLP-2 in the ENS.


   METHODS
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 METHODS
RESULTS
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Animals. PI3-K{gamma} null mice were originally developed by knockout (KO) of the p110{gamma} subunit (13). Heterozygous (HT) p110{gamma} null mice were crossed to obtain wild-type (WT), HT, and knockout (KO) animals. Mice were born at the expected Mendelian ratio and appeared healthy. Genotyping was performed as previously described (27). Age- (8–12 wk) and sex-matched littermates were injected (subcutaneously, q12h) with vehicle (PBS) or 1 µg of Gly2-GLP-2 [a long-acting GLP-2 analog (39); Peptidec Technologies, Pierrefonds, QC, Canada] for 10 days (9, 16, 18). Mice were then anesthetized with halothane and weighed, and the small intestine and colon were cleaned of luminal contents and weighed using an analytical balance. One- to two-cm segments of small intestine were then collected for further analysis. All animal experiments were approved by the University of Toronto Animal Care Committee.

Morphometric and immunochemical analyses. Jejunal segments were cut into three to four pieces (to make n = 1), fixed in formalin, paraffin embedded, and then sectioned. The length of longitudinally oriented crypt-villus units (an average of 8 per mouse, to make n = 1) was measured on hematoxylin- and eosin-stained jejunal sections; muscle thickness was measured in 10 different areas of each section (to make n = 1; Refs. 9, 16, 18, 37). Some slides were processed using the Rapid Mucin Stain kit per the manufacturer's instructions (Polysciences, Warrington, PA), and the number of goblet cells was counted in an average of 18 villi per mouse and normalized to the number of positive cells per 100 µm. Cell proliferation was determined by immunostaining for the proliferative marker Ki-67 (18, 37) using the rat anti-mouse TEC-3 antibody (DAKO, Glostrup, Denmark). For each mouse, an average of 29 well-oriented and intact half crypts were analyzed such that 20 cells were counted per crypt, with the cell at the base of the crypt designated as position 1. Proliferation was determined by counting the number of labeled cells relative to total cells for every cell position (18, 37). All microscopy was performed using a Zeiss image analysis system (Carl Zeiss Canada, Don Mills, ON, Canada), and all measurements were performed in a blinded fashion.

RT-PCR. Total RNA was extracted from intestinal tissues, as well as from mechanically dissected muscle strips and residual mucosa, using an RNeasy kit (Qiagen, Mississauga, ON, Canada) and was analyzed by RT-PCR using a One-Step RT-PCR kit (Qiagen) with primers for mouse PI3-K{gamma} or for GLP-2R, proglucagon, and 18S RNA as previously reported (18, 37), followed by SDS-PAGE and visualization of the bands with ethidium bromide.

Western blot analysis. Tissue segments were homogenized in RIPA buffer containing a cocktail of protease inhibitors (Roche Diagnostics, Laval, QC, Canada), and the protein concentration was determined using the Bradford method (Bio-Rad Laboratories, Mississauga, ON, Canada). SDS-PAGE and immunoblotting were conducted using standard protocols (27). In brief, membranes were incubated overnight with an antiserum against p110{gamma} (Santa Cruz Biotechnology) or cleaved (active) and uncleaved (inactive) caspase-3 (Santa Cruz Biotechnology). Blotting membranes were stripped and reprobed for {alpha}-tubulin using a monoclonal antibody (Sigma Chemical, St. Louis, MO). The immunoreactions were revealed by chemiluminescence with the use of a kit from Perkin-Elmer (Boston, MA) and were captured on X-ray film, and densitometry was performed using the Syngene imaging system (Perkin-Elmer).

GLP-1 analysis. Ileal and colonic segments were homogenized in 1 N HCl containing 5% formic acid, 1% trifluoroacetic acid, and 1% NaCl, and the peptides were extracted using C18 SepPak cartridges (Waters, Milford, MA) as previously described (1, 9). GLP-1 content was determined by radioimmunoassay (1), and protein content by Bradford assay (Bio-Rad Laboratories).

Transit studies. Overnight-fasted WT and KO mice were injected intraperitoneally with vehicle (PBS) or Gly2-GLP-2 (1 µg/g body wt; this dose was selected based on results of preliminary dose-response studies). Five minutes later, the mice were orally gavaged (10 ml/kg) with a suspension of 5% activated charcoal in 10% gum Arabic (15). Twenty minutes after gavage, animals were anesthetized with halothane, the small intestine was collected and placed under constant tension, and the distance traveled by the charcoal was assessed visually using a ruler.

Ex vivo muscle strip analyses. As previously reported (37), 2-cm muscle strips from freshly collected jejunal segments were incubated in culture medium containing 100 µM 3-isobutyl-1-methylxanthine (IBMX) for 30 min with or without 100 µM forskolin or 100 pM Gly2-GLP-2. Muscle strips were then homogenized in –20°C ethanol, and the supernatant was assayed for cAMP (Biomedical Technologies, Stoughton, MA) and protein content (Bradford assay; Bio-Rad Laboratories).

Statistical analyses. Data were analyzed using two-way ANOVA followed by one-way ANOVA or Student's t-test, as appropriate, using SAS software (Statistical Analysis Systems, Cary, NC). Significance was assumed at P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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Expression of p110{gamma}. To determine the role of PI3-K{gamma} in the intestinal actions of GLP-2, we bred heterozygous PI3-K{gamma} null mice to obtain WT, HT, and KO animals. The results of the initial genotyping were confirmed by RT-PCR and Western blot analysis of jejunal sections from WT and KO mice. Although mRNA transcripts and protein for the 110{gamma} subunit of PI3-K{gamma} were consistently found in the small intestine of WT mice, neither was detectable in intestines from KO animals (Fig. 1, A and B). Further analysis demonstrated the presence of PI3-K{gamma} in both the muscle and the mucosal layers of the jejunum. The enteroendocrine transcript proglucagon was used as an internal control in these studies and was detected only in the mucosal layer, as expected (Fig. 1C).


Figure 1
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  		Fig. 1. Phosphatidylinositol 3-kinase {gamma} (PI3-K{gamma}) expression in wild-type (WT) and knockout (KO) mice. A: RT-PCR for p110{gamma} was performed using total jejunal RNA from WT and KO mice, and the products were separated by gel electrophoresis and detected by ethidium bromide staining. B, blank lane; L, molecular weight ladder. B: total jejunal protein was analyzed by immunoblotting for p110{gamma}, followed by chemiluminescent detection. C: RT-PCR for p110{gamma} (using a distinct set of primers) was performed using total RNA from isolated jejunal muscle strips and the residual mucosa, and the products were separated by gel electrophoresis and detected by ethidium bromide staining. ProG, proglucagon (positive control); –, water (negative) control.

 
Effects on the intestinal mucosa. There were no differences in body weight between age- and sex-matched WT, HT, and KO mice, either before or after treatment with Gly2-GLP-2 for 10 days. Small intestinal weight normalized to body weight was higher in PBS-treated KO mice compared with both PBS-WT and PBS-HT animals, by 32 and 23%, respectively (P < 0.05–0.01; Fig. 2), indicating a gene-dosage effect of PI3-K{gamma} on small intestinal weight. GLP-2 treatment increased small intestinal weight in all three groups of animals, by 25, 34, and 18% in WT, HT, and KO mice, respectively (P < 0.05–0.01); however, no differences among these responses could be detected. In contrast to the small intestine, no significant differences in colonic weight were observed among the different groups of PBS-treated mice, and there was no detectable effect of GLP-2 on colonic weight in any of the animals (Fig. 2).


Figure 2
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  		Fig. 2. Intestinal weights in PI3-K WT, heterozygous (HT) and KO mice treated chronically with glucagon-like peptide-2 (GLP-2). Small and large intestinal weights were normalized to body weight in age- and sex-matched WT (n = 4), HT (n = 5), and KO mice (n = 7) injected subcutaneously with vehicle (PBS) or 1 µg of Gly2-GLP-2 twice daily for 10 days. *P < 0.05; **P < 0.01; $P < 0.05; $$P < 0.01.

 
Determination of jejunal crypt-villus height demonstrated that, as found for intestinal weight, PBS-treated KO animals had significantly longer villi compared with PBS-treated WT mice (by 41%, P < 0.01; Fig. 3). Nonetheless, GLP-2 treatment increased crypt-villus height by a similar extent in WT and KO animals (by 29 and 22%, respectively, P < 0.05). No changes in colonic crypt depth between animals with different genotypes and among treatment groups were detected (not shown).


Figure 3
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  		Fig. 3. Crypt-villus heights in WT and KO mice treated chronically with GLP-2. Crypt-villus heights were measured in jejunal sections from age- and sex-matched WT (n = 4) and KO mice (n = 7) injected subcutaneously with vehicle (PBS) or 1 µg of Gly2-GLP-2 twice daily for 10 days. *P < 0.05; $$P < 0.01.

 
Immunohistochemical analysis of Ki-67 staining in jejunal crypts demonstrated highest levels of proliferation, in all mice, in cell positions 5–8 (Fig. 4, A and B), the putative "clonogenic stem cell region" of the crypt (29). However, differences among the different groups of animals were observed in positions 11–20, which correspond to the "rapidly proliferating transit zone" (29). PBS-treated KO mice were found to have greater numbers of proliferating cells in this region of the crypt compared with PBS-treated WT animals (by 50%, P < 0.05; Fig. 4C). Again, however, the effect of GLP-2 was not found to be significantly different between WT and KO mice (increased by 74 and 39%, respectively; P < 0.01).


Figure 4
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  		Fig. 4. Crypt cell proliferation in WT and KO mice treated chronically with GLP-2. Jejunal sections from age- and sex-matched WT (n = 4) and KO mice (n = 7) injected subcutaneously with vehicle (PBS) or 1 µg of Gly2-GLP-2 twice daily for 10 days were immunostained for Ki-67. A and B: distribution profiles of immunopositive cells along the crypt in WT (A) and KO mice (B), with the cell at the midpoint at the base of the crypt defined as position 1. C: area under the curve (AUC) for immunopositive cells between positions 11 and 20. $P < 0.05; **P < 0.01.

 
The number of detectable TdT-mediated dUTP nick end labeling-positive apoptotic cells in the villus tip was extremely low (<1 cell per 30 cell positions), precluding accurate quantification of the numbers of apoptotic cells (data not shown). However, Western blot analysis for cleaved caspase-3 levels revealed markedly lower levels of activated caspase-3 in PBS-KO compared with PBS-WT animals (Fig. 5). Furthermore, GLP-2 treatment suppressed caspase-3 activation in WT animals but had no further effect in KO mice.


Figure 5
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  		Fig. 5. Apoptosis in WT and KO mice treated chronically with GLP-2. Cleaved (20- and 17-kDa bands) and uncleaved caspase-3 levels (32-kDa band) were determined by immunoblotting of jejunal sections from WT (n = 4) and KO mice (n = 7) injected subcutaneously with vehicle (PBS) or 1 µg of Gly2-GLP-2 twice daily for 10 days. Cleaved caspase levels were normalized to those of uncleaved caspase.

 
The intestinal villus epithelium is known to contain several differentiated cell types, including absorptive, goblet, and endocrine cells (29). To determine whether lineage differentiation was affected by either PI3-K{gamma} KO or GLP-2 treatment, we examined both goblet and the GLP-1-expressing enteroendocrine L cells. Histological staining for mucin revealed no significant difference in the number of goblet cells in PBS-KO compared with PBS-WT mice, although a trend toward an increase was noted (Fig. 6A). However, chronic treatment with GLP-2 increased goblet cell number by 130% (P < 0.05) in WT animals, and this effect was completely abolished in KO mice. Similarly, no differences between WT and KO animals were found for the intestinal concentrations of GLP-1 (Fig. 6B), a proglucagon-derived peptide that is cosynthesized and cosecreted with GLP-2 (14). Previous studies have demonstrated that the intestinal levels of the proglucagon-derived peptides do not change in response to chronic administration of GLP-2 (9).


Figure 6
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  		Fig. 6. Differentiated intestinal cells in WT and KO mice treated chronically with GLP-2. A: age- and sex-matched WT (n = 4) and KO mice (n = 7) were injected subcutaneously with vehicle (PBS) or 1 µg of Gly2-GLP-2 twice daily for 10 days. Mucin-expressing goblet cells in jejunal sections were detected by histological staining, and the number of positive cells per 100 µm was determined. Representative sections from a PBS-WT and a GLP-2-WT mouse are shown. *P < 0.05. B: GLP-1 content of ileal and colonic segments from WT (n = 6) and KO mice (n = 6) was determined by RIA and normalized to the protein content of the tissue peptide extract.

 
Effects on the intestinal muscularis. Morphometric analysis of the thickness of the muscle layer did not reveal any significant differences between PBS-treated WT and KO mice (Fig. 7A). Furthermore, chronic GLP-2 treatment increased muscle thickness to a similar extent in both WT and KO animals, by 94 and 38%, respectively (P < 0.01 for KO mice). Similarly, the basal rate of intestinal transit of a bulk solid was not different between WT and KO mice, and GLP-2-induced suppression of transit was similar in both groups of animals (by 40%, P < 0.05–0.01; Fig. 7B).


Figure 7
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  		Fig. 7. Intestinal muscle thickness and transit in WT and KO mice treated chronically or acutely with GLP-2, respectively. Age- and sex-matched WT (n = 4) and KO mice (n = 7) were injected subcutaneously with vehicle (PBS) or 1 µg of Gly2-GLP-2 twice daily for 10 days. A: the thickness of the muscularis layer (longitudinal and circular muscle) was determined in jejunal sections. **P < 0.01. B: intestinal transit was determined by gastric gavage of a charcoal solution into WT (n = 3–5) and KO mice (n = 5) pretreated intraperitoneally with vehicle (PBS) or 1 µg/g body wt Gly2-GLP-2, and transit was assessed visually 20 min later. *P < 0.05; **P < 0.01.

 
Finally, to examine GLP-2R signaling in the muscle layer, isolated muscle strips were acutely treated with vehicle, forskolin, or GLP-2 and analyzed for changes in cAMP concentrations. Basal levels of cAMP were not different between WT and KO mice (Fig. 8A). Forskolin treatment increased cAMP levels in both WT and KO mouse muscle strips, by greater than 2,000% (P < 0.001), and there was no difference between these maximal responses. GLP-2 treatment also significantly stimulated cAMP production in WT muscle strips, by 98% (P < 0.01). In contrast, the effect of GLP-2 on cAMP production was completely abolished in muscle strips isolated from KO mice. RT-PCR analysis for GLP-2R mRNA transcripts established that the lack of effect of GLP-2 on cAMP in the KO muscle strips was not due to lack of expression of the GLP-2R in this tissue (Fig. 8B).


Figure 8
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  		Fig. 8. cAMP production in intestinal muscle strips from WT and KO mice treated acutely with GLP-2. A: jejunal muscle strips were isolated from WT (n = 8) and KO mice (n = 10) and incubated for 30 min in medium containing 100 µM IBMX without (control) or with 100 µM forskolin or 100 pM GLP-2 and were then extracted in ethanol. cAMP levels were determined by RIA, and protein was determined by Bradford assay. **P < 0.01; ***P < 0.001. B: RT-PCR for the GLP-2 receptor (GLP2R) and 18S RNA (loading control) in total RNA extracted from muscle strips of WT and KO mice. L, molecular weight ladder; –, water (negative) control; +, intact mouse colon (positive control).

 

   DISCUSSION
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Our understanding of GLP-2R signaling in the intestine has been complicated by the demonstration that the receptor is expressed in multiple cell types in several different compartments of the intestine (4, 21, 32, 42). Previous studies have indicated a role for IGF-1 in the tropic effects of GLP-2 in the mucosa, thereby linking PKA and the class 1A isoforms of PI3-K to crypt cell proliferation (11, 17, 18, 23, 34, 40). However, little is known about the pathways activated by the GLP-2R in the ENS. The results of the present study confirm that, like the GLP-2R, PI3-K{gamma}, a class 1B isoform of PI3-K, is localized in both the mucosal and the muscle layers of the gut wall. Interestingly, PI3-K{gamma} was not found to be required for GLP-2-induction of small intestinal growth, although a role for the enzyme in GLP-2-induced lineage differentiation and suppression of apoptosis was demonstrated. Furthermore, somewhat unexpectedly, PI3-K{gamma} was found to play an essential role in the ability of GLP-2 to stimulate cAMP synthesis in the ENS.

PI3-K{gamma} KO mice were found to have increased basal small intestinal growth, as indicated by enhanced small intestinal wet weight, crypt-villus height, and crypt cell proliferation, as well as decreased apoptosis. Interestingly, no such changes were observed in the large intestine, demonstrating a tissue-specific role for PI3-K{gamma} in suppressing basal growth of the small intestinal mucosa. Although previous studies have shown that loss of PI3-K{gamma} actually increases apoptosis in neutrophils (41), PI3-K{gamma} null mice have also been shown to exhibit enhanced basal growth and proliferation of the pancreatic beta-cell, likely as an adaptive response to impaired beta-cell function (27). Numerous factors are known to regulate small intestinal growth, including luminal nutrients, as well as nutrient-stimulated endocrine hormones, such as GLP-2 and IGF-1 (10, 28, 37). Although the levels of GLP-1, a peptide hormone that is cosynthesized and cosecreted with GLP-2 (14), were normal in the PI3-K{gamma} null mice, further studies are clearly required to determine whether adaptive or PI3-K{gamma}-mediated changes in other intestinal hormones have occurred in these animals. Finally, delayed transit of nutrients through the gut can also enhance small intestinal growth (20). However, PI3-K{gamma} KO animals were found to have normal transit times compared with WT mice, suggesting that this cannot account for the enhanced basal small intestinal growth in these animals.

Despite increased basal growth of the small intestine in PI3-K{gamma} KO mice, the ability of GLP-2 to induce intestinal growth was neither impaired nor enhanced, stimulating normal increases in small intestinal wet weight, crypt-villus height, and cell proliferation. These findings are consistent with our current model of GLP-2 action in the intestinal mucosa whereby activation of the GLP-2R leads to PKA-dependent stimulation of IGF-1 synthesis and secretion, resulting in class 1A PI3-K/Akt-dependent stimulation of crypt cell proliferation through the IGF-1 receptor (11, 17, 18, 23, 34, 40). However, unexpectedly, GLP-2 was also found to enhance the number of mucous cells in the intestinal epithelium, and this effect was lost in the PI3-K{gamma} KO mice. Although previous studies have indicated that GLP-2 treatment does not alter the number of small intestinal mucous progenitor cells in a clonogenic assay (4), there have been no reports to date on the actual number of these cells in vivo following chronic administration of GLP-2. Nonetheless, GLP-2 treatment increases the number of Musashi-1-expressing stem cells in both the lower crypt, where the actual stem cells reside, and in the upper crypt, which gives rise to the differentiated cells of the epithelium (18, 29), suggesting that GLP-2 does have the potential to affect lineage differentiation. Thus, when taken together, the results of these studies on the intestinal mucosa indicate a role for PI3-K{gamma} in basal growth and GLP-2-stimulated mucous cell differentiation but not in GLP-2-induced growth of the small intestine. How these differential responses of the gut epithelium to GLP-2 are mediated remains to be determined but likely relates to findings that the GLP-2R is expressed in diverse cell types in the mucosa, including both enteroendocrine cells and subepithelial myofibroblasts (21, 42).

In contrast to the basal changes observed in the mucosal layer of KO mice, no changes in basal muscularis thickness, small intestinal transit time, or muscle cAMP levels were observed in PI3-K{gamma} KO animals. Furthermore, although these studies demonstrated that GLP-2 can suppress intestinal transit in fed mice, in contrast to the results of studies in rats (7), GLP-2-induced suppression of intestinal transit was found to be normal in KO animals. In contrast, loss of PI3-K{gamma} was associated with a complete abrogation of the ability of GLP-2 to stimulate cAMP production in isolated muscle strips. Given that treatment with forskolin/IBMX increased cAMP levels to an equivalent extent in WT and KO animals, there did not appear to be any inherent defect in adenylyl cyclase activity in these animals. Furthermore, although PI3-K{gamma} has been reported to stimulate phosphodiesterase 3B activity through protein-protein interaction, thereby increasing the degradation of cAMP (33), this did not appear to play an important role in modulating basal or forskolin-stimulated cAMP levels in the muscle strip preparation. Furthermore, the lack of effect of GLP-2 on cAMP could not be accounted for by loss of expression of the GLP-2R, because mRNA transcripts for the receptor were readily detectable in muscle strips collected from KO animals. Nonetheless, it cannot be discounted that cAMP levels were altered in small, discrete populations of cells or within subcellular compartments (25), thereby permitting the effects of GLP-2 to suppress intestinal transit without detectable differences in cAMP levels. These findings therefore suggest a novel role for PI3-K{gamma} in the regulation of cAMP-dependent GLP-2R signaling in the ENS. Interestingly, PI3-K{gamma} has been reported to bind to several proteins in addition to phosphodiesterase 3B, including the atypical isoform of PKC, PKC{zeta} (19, 22). Indeed, relaxin binding to its cognate G protein-coupled (RXFP1) receptor induces PI3-K{gamma}-dependent activation of PKC{zeta}, resulting in enhancement of cAMP levels (22). Such a mechanism of action of the ENS GLP-2R would therefore result in loss of ligand-induced cAMP production in the KO mice. Confirmation of this hypothesis awaits the development of appropriate ENS cell models to study neuron-specific GLP-2R signaling in more detail. Finally, it remains to be determined whether the lack of induction of cAMP by GLP-2 in the ENS of KO mice is also coupled to alterations in intestinal blood flow, another biological action that has been linked to GLP-2 signaling in this tissue (21).

In summary, therefore, loss of PI3-K{gamma} signaling is associated with enhanced basal intestinal growth and loss of GLP-2-induced cAMP production in the ENS but has no effect on the intestinotropic effects of GLP-2. Given that PI3-K{gamma} inhibitors are currently being explored for the treatment of chronic inflammation, such as in lupus or rheumatoid arthritis (2, 12), our findings suggest the possibility of using such agents to enhance small intestinal growth in patients with intestinal insufficiency.


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This study was supported by an operating grant (to P. L. Brubaker) from the Canadian Institutes of Health Research (CIHR). Y. Anini was supported by postdoctoral fellowships from the Canadian Diabetes Association and from the Banting and Best Diabetes Centre and the Faculty of Medicine, University of Toronto. G. Y. Oudit was supported by postdoctoral fellowships from the CIHR and the Heart and Stroke Foundation of Canada. P. H. Backx was supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario. P. L. Brubaker was supported by the Canada Research Chairs program.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. L. Brubaker, Dept. of Physiology, Rm. 3366, Medical Sciences Bldg., Univ. of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8 Canada (e-mail: p.brubaker{at}utoronto.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 REFERENCES
 

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