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Endocrinology Vol. 147, No. 7 3318-3325
Copyright © 2006 by The Endocrine Society

Role of Phosphatidylinositol 3-Kinase{gamma} in the ß-Cell: Interactions with Glucagon-Like Peptide-1

Li-Xin Li, Patrick E. MacDonald, Diane S. Ahn, Gavin Y. Oudit, Peter H. Backx and Patricia L. Brubaker

Departments of Physiology (L.-X.L., D.S.A., G.Y.O., P.H.B., P.L.B.) and Medicine (G.Y.O., P.H.B., P.L.B.), and Heart and Stroke/Richard Lewar Centre of Excellence (G.Y.O., P.H.B.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; Department of Experimental Medical Science (P.E.M.), Lund University, S-221 84 Lund, Sweden; and Oxford Centre for Diabetes, Endocrinology, and Metabolism (P.E.M.), Oxford OX3 7LJ, United Kingdom

Address all correspondence and requests for reprints to: Dr. Patricia Brubaker, Room 3366 Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: p.brubaker{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucagon-like peptide-1 (GLP-1) increases ß-cell function and growth through protein kinase A- and phosphatidylinositol-3-kinase (PI3-K)/protein kinase B, respectively. GLP-1 acts via a G protein-coupled receptor, and PI3-K{gamma} is known to be activated by Gß{gamma}. Therefore, the role of PI3-K{gamma} in the chronic effects of GLP-1 on the ß-cell was investigated using PI3-K{gamma} knockout (KO) mice treated with the GLP-1 receptor agonist, exendin-4 (Ex4; 1 nmol/kg sc every 24 h for 14 d). In vivo, glucose and insulin responses were similar in PBS- and Ex4-treated KO and wild-type (WT) mice. However, glucose-stimulated insulin secretion was markedly impaired in islets from PBS-KO mice (P < 0.05), and this was partially normalized by chronic Ex4 treatment (P < 0.05). In contrast, insulin content was increased in PBS-KO islets, and this was paradoxically decreased by Ex4 treatment, compared with the stimulatory effect of Ex4 on WT islets (P < 0.05–0.01). Transfection of INS-1E ß-cells with small interfering RNA for PI3-K{gamma} similarly decreased glucose-stimulated insulin secretion (P < 0.01) and increased insulin content. Basal values for ß-cell mass, islet number and proliferation, glucose transporter 2, glucokinase, and insulin receptor substrate-2 were increased in PBS-KO mice (P < 0.05–0.001) and, although they were increased by Ex4 treatment of WT animals (P < 0.05), they were decreased in Ex4-KO mice (P < 0.05–0.01). These findings indicate that PI3-K{gamma} deficiency impairs insulin secretion, resulting in compensatory islet growth to maintain normoglycemia. Chronic Ex4 treatment normalizes the secretory defect, thereby relieving the pressure for expansion of ß-cell mass. These studies reveal a new role for PI3-K{gamma} as a positive regulator of insulin secretion, and reinforce the importance of GLP-1 for the maintenance of normal ß-cell function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCAGON-LIKE PEPTIDE-1 (GLP-1) is a 30-amino-acid peptide secreted by the intestinal L cell in response to nutrient ingestion (1, 2). GLP-1 is an incretin hormone that possesses a number of anti-diabetic actions, most notably, enhancement of ß-cell function through stimulation of glucose-dependent insulin synthesis and secretion (3, 4, 5), as well as inhibition of glucagon release (5, 6), gastric emptying (6, 7), and food intake (8). Importantly, these actions have been demonstrated in long-term studies in patients with type 2 diabetes, in whom treatment with GLP-1 or the long-acting GLP-1 receptor agonist, exendin-4 [Ex4; (9)] leads to reductions in glycemia, glycated hemoglobin, and body weight (10, 11).

The stimulatory effects of GLP-1 on ß-cell function are mediated through the G{alpha}s-coupled GLP-1 receptor, which increases cAMP levels upon ligand binding (12, 13, 14). Activation of both protein kinase (PK) A-dependent and PKA-independent pathways by cAMP then enhances glucose-stimulated insulin secretion through modulation of ion channel activity as well as stimulation of granule exocytosis (15). However, a number of studies have now shown that GLP-1 and Ex4 also increase ß-cell mass (BCM) in rodents through mechanisms involving stimulation of both ß-cell proliferation and islet neogenesis (12, 16, 17, 18, 19, 20). Interestingly, these growth effects of GLP-1 have recently been established to be dependent upon the pleiotropic signaling protein kinase B (PKB)/Akt pathway (16, 21, 22). Nonetheless, the mechanism by which GLP-1 receptor activation is coupled to the PKB signaling pathway remains unclear.

Phosphatidylinositol-3-kinase (PI3-K) is the major upstream activator of PKB in most cells. The family of PI3-Ks includes class I-IV isoforms, with class I being further subdivided into IA and IB (23, 24, 25). All of the class I PI3-K isozymes are characterized by their ability to produce phosphatidylinositol 3,4,5-trisphosphate, essential for the activation of PKB. However, the class IA enzymes are linked to tyrosine kinase receptor signaling, whereas the single class IB isozyme, PI3-K{gamma}, is activated by G protein-coupled receptor ligand binding. Although G protein-coupled receptors are associated with several subunits of the G protein complex, only the ß{gamma} subunit can bind to and activate PI3-K{gamma} (25, 26, 27, 28). Because PI3-K{gamma} has recently been demonstrated to be expressed in the islet (29), these findings suggested that PI3-K{gamma} may serve as one possible link between GLP-1 and PKB in the ß-cell growth and survival pathway. Alternatively, recent studies have demonstrated that PI3-K{gamma} also associates with and activates cAMP-phosphodiesterase-3B (PDE3B), leading to decreased levels of cAMP (26, 30). PDE3B is expressed in the ß-cell, where it plays a role in the regulation of insulin secretion (31, 32, 33, 34). Thus, PI3-K{gamma} may modulate ß-cell function through alterations in cAMP.

Unexpectedly, a previous study has shown that mice lacking PI3-K{gamma} have enhanced BCM, suggesting an inhibitory role for this enzyme in ß-cell growth (29). Interestingly, the mice had normal fasting insulin secretion and a normal insulin response to acutely administered Ex4, but exhibited impaired ß-cell responses to glucose both in vivo and in vitro. However, whether PI3-K{gamma} is required for the chronic growth effects of GLP-1 on the ß-cell was not examined in that study. Therefore, we have administered Ex4 for 2 wk to PI3-K{gamma} knockout (KO) mice (35, 36, 37), and have examined whether lack of PI3-K{gamma} alters the chronic effects of GLP-1 on ß-cell growth and/or function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The generation of PI3-K{gamma}-deficient mice on a 129SV/C57BL6 background and method for genotyping have been reported previously (35). Mice were housed under controlled light (12 h light, 12 h dark) and temperature conditions, and had free access to food and water. All procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the University of Toronto Animal Care Committee. Age- (9–12 wk), gender-, and weight-matched wild-type (WT; +/+), heterozygous (HT; +/–), and homozygous KO (–/–) littermates were injected with Ex4 (1 nmol/kg, ip; Bachem, Torrance, CA) or vehicle PBS at 0900 h daily for 14 d, as previously described (16). There were no significant differences in body weight between WT, HT, and KO animals after the treatment period (PBS treated: 21.9 ± 1.0, 22.5 ± 5.5, and 20.9 ± 1.4 g, respectively; and Ex4 treated: 21.5 ± 2.5, 23.2 ± 6.8, and 20.2 ± 1.0 g, respectively).

All experiments were performed at least 24 h after the last injection to ensure clearance of the Ex4 from the circulation (38). Mice were fasted for 15 and 6 h for oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT), respectively, and a blood sample (20 µl) was collected from a tail vein (t = 0 min) for measurement of glucose using the One Touch Basic glucose meter (a kind gift from Lifescan Canada, Burnaby, British Columbia, Canada). For OGTT, mice were gavaged with glucose (1.5 mg/g) and additional blood samples were collected at t = 10, 20, 30, 60, 90, and 120 min for glucose measurements, and at t = 0 and 30 min for determination of plasma insulin concentrations using a highly sensitive Insulin ELISA kit designed for small sample volumes (Crystal Chem, Chicago, IL). For ITT, mice were injected (ip) with human biosynthetic insulin (0.3 U/kg; Novo Nordisk Pharmaceutical Industries, Toronto, Ontario, Canada), and blood samples were collected at t = 0 and 30 min for glucose measurements. Insulin sensitivity was determined as the change in blood glucose over 30 min normalized for starting glycemia.

Immunological and morphometric analyses
Some mice were injected with 100 mg/kg (ip) 5-bromo-2-deoxyuridine (BrdU; Sigma Chemical Co., St. Louis, MO) 6 h before removal of pancreas (16). BCM was measured as described previously (16). Briefly, dissected pancreata were weighed, cut into six to eight pieces, fixed in Bouin’s, and paraffin-embedded, and 4-µm sections through all six to eight pieces (to make n = 1) were dewaxed, hydrated, and incubated overnight at 4 C with guinea pig anti-insulin antibody (Dako Diagnostics, Mississauga, Ontario, Canada). The sections were then incubated for 1 h with biotinylated anti-guinea pig antibody (Vector Laboratories, Burlington, Ontario, Canada), and subsequently treated for 1 h with avidin/biotin complex (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA). Sections were then stained with 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) for 10 min, washed with tap water, and counterstained with hematoxylin. ß-Cell and total pancreatic area per section were measured using a Zeiss Axioplan microscope with Axiovision software (Carl Zeiss Canada, Don Mills, Ontario, Canada). Total BCM for each pancreas was determined as the product of the total cross-sectional ß-cell area over total tissue area normalized for the weight of the pancreas before fixation. Pancreatic sections were also stained for BrdU using mouse anti-BrdU IgG (Amersham, Oakville, Ontario, Canada), followed by biotinylated antimouse IgG and 3,3'-diaminobenzidine (Sigma Chemical Co.), as previously described (16).

PI3-K{gamma} was colocalized with insulin in fresh-frozen pancreatic sections fixed with formalin, using rabbit antihuman PI3-K{gamma} (p110{gamma}, 1:50; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) and guinea pig anti-porcine insulin (1:100; DakoCytomation Inc., Mississauga, Ontario, Canada), respectively, followed by donkey antirabbit AlexaFluor-488- (Invitrogen Corp., Burlington, Ontario, Canada) and donkey anti-guinea pig Cy2 (Jackson ImmunoResearch Laboratories, West Grove, PA)-coupled secondary antibodies, respectively.

Isolated islets
Pancreatic islets of Langerhans were isolated by collagenase digestion as described (39), and groups of five islets were preincubated for 30 min in 200 µl of Krebs-Ringer bicarbonate (KRB) buffer supplemented with 10 mM HEPES, 2 g/liter BSA, and 3.3 mM glucose. Islets were then incubated at 37 C in KRB buffer containing 27 mM glucose for 60 min, the media was collected, and insulin release was quantitated by RIA (Linco Research, St. Louis, MS). For determination of islet insulin and cAMP content, groups of three to five islets of similar size were extracted (39) and insulin (Linco Research) and cAMP (Biomedical Technologies, Inc., Stoughton, MA) levels were determined by RIA.

Immunoblotting
The isolated pancreas was homogenized in lysis buffer containing 1% Triton X-100 and a mixture of protease and phosphatase inhibitors (16, 22). The protein content was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA) and equal amounts of protein (50 µg) were separated by 7–10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride filters (Bio-Rad Laboratories). After probing with specific primary antibodies [anti-p110{gamma} (1:1000; Santa Cruz Biotechnologies, Inc.), anti-insulin receptor substrate-2 (IRS-2, 1:250), anti-glucose transporter 2 (GLUT2, 1:500; Chemicon International Inc., Temecula, CA), anti-glucokinase (1:500; a kind gift from Dr. M. A. Magnuson, Vanderbilt University School of Medicine, Nashville, TN), and anti-ß-actin (1:4000; Sigma Chemical Co.)], the immunoreactive bands were visualized with horseradish peroxidase-conjugated sheep antirabbit IgG or with HRP-conjugated goat anti-rabbit IgG, as appropriate, using ECL detection (Amersham Pharmacia Biotech, Inc., Baie d’Urfe, Quebec, Canada), as described previously (16, 22).

RNA interference
INS-1E cells (passage number 50–70; a kind gift from Dr. C. Wollheim, University Medical Center, Geneva, Switzerland) were grown in RPMI 1640 medium (Life Technologies, Inc. Invitrogen Corp., Burlington, Ontario, Canada) supplemented with 10 mmol/liter HEPES, 10% heat-inactivated fetal calf serum, 2 mmol/liter L-glutamine, 1 mmol/liter sodium pyruvate, 50 µmol/liter 2-mercaptoethanol, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured at 37 C in a humidified (5% CO2, 95% air) atmosphere (40). PI3-K{gamma} specific and control small interfering RNA (siRNA) sequences were designed using the GenScript Corp. (Piscataway, NJ) siRNA target finder and subsequently synthesized by GenScript Corp. and inserted into the pRNAT-H1.1/Adeno vector (Stratagene), which coexpresses green fluorescent protein (GFP) to allow identification of transfected cells. Cells were plated in 24-well plates 3 d before transfection with 2.2 µg siRNA using lipofectamine (Invitrogen Corp.). The transfection efficiency was 25–60%, as determined by counting of GFP-expressing cells 3 d after transfection. Transfected cells were preincubated for 30 min in KRB buffer containing 10 mM HEPES (KRBH) without glucose, and then incubated for 30 min in KRBH in the presence of 3.3 or 27 mM glucose. Incubation media were collected and cells were extracted in acid-ethanol, and insulin secretion and cell content were determined by RIA (Linco Research).

Statistical analysis
All data are presented as mean ± SEM. Statistical analyses were performed using Student’s t test or ANOVA with "n-1" post hoc custom hypotheses tests, as appropriate. A P value of less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Whereas expression of PI3-K{gamma} has been demonstrated in whole-islet protein lysates from mouse and human (29), the expression of this isoform has not been investigated in pancreatic ß-cells per se. To establish the presence of PI3-K{gamma} in the murine ß-cell, pancreatic sections were immunostained for both p110{gamma} and insulin. Highly specific staining for insulin was detected in islets scattered throughout the pancreas (Fig. 1Go, A, D, and G). In contrast, diffuse staining for PI3-K{gamma} was detected throughout the pancreas, including both islets and acinar cells (Fig. 1Go, B and E). Overlay of the images indicated colocalization of PI3-K{gamma} with insulin in the ß-cells (Fig. 1Go, C and F). Direct comparison of WT and KO sections immunostained for PI3-K{gamma} revealed significant staining in WT pancreas, but background staining only in sections from KO animals (Fig. 1Go, H and I).


Figure 1
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  		FIG. 1. Immunolocalization of PI3-K{gamma} and insulin in the murine pancreas. Fresh-frozen pancreas was sectioned and fixed in formalin. A–G, Sections were costained for insulin (A and D) and the p110{gamma} subunit of PI3-K{gamma} (B and E). The overlay is shown in C and F. Primary antiserum was omitted in G (negative control). H and I, In separate studies, WT (H) and KO (I) pancreatic sections were stained in parallel for PI3-K{gamma}, and identical camera exposure times were used to collect the images (magnification, x400).

 
To confirm the results of the mouse genotyping, pancreatic lysates were examined by Western blot for the presence or absence of PI3-K{gamma} expression in WT, HT, and KO animals. As shown in Fig. 2AGo, WT pancreas was positive for PI3-K{gamma}, whereas expression was reduced in HT animals and could not be detected at all in pancreata from PI3-K{gamma} KO mice. Fasting glycemia was in the normal range for PBS-treated WT mice (5.8 ± 1.1 mM), and levels were not different in PBS-HT and -KO mice, nor were they altered by chronic Ex4 treatment in any of the animals (Fig. 2BGo). Similarly, the area-under-the-curve for the glycemic response to an OGTT was also not different in PBS- or Ex4-treated WT, HT, and PI3-K{gamma}-deficient mice (Fig. 2CGo). Insulin levels in response to oral glucose, were also not different in PBS- or Ex4-treated mice compared with WT animals (Fig. 1DGo), although the overall response was found to be muted in both groups of mice, likely due to the background strain of animals used (41). Similarly, the glycemic response to insulin in an ITT ("insulin sensitivity") was also not different between WT and KO animals, regardless of treatment (Fig. 1EGo).


Figure 2
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  		FIG. 2. Western blot for PI3-K{gamma} and responses to OGTT and ITT. WT, HT, and KO mice were injected with either PBS or Ex4 (1 nmol/kg, ip) for 14 d. Western blot of pancreatic lysates from PBS-treated WT mice for PI3-K{gamma} expression (A), fasting blood glucose concentrations (B), and glycemic responses to OGTT (1.5 mg/g) (C), shown as the area-under-the-curve for the {Delta} from fasting glycemia (PBS, open bars; Ex4, closed bars). D, Plasma insulin levels at t = 0 (open bars) and 30 min (hatched bars) after administration of oral glucose. E, Glycemic response to insulin (0.3 U/kg, ip) at t = 30 min after administration, normalized for fasting glycemia (PBS, open bars; Ex4, closed bars) (n = 5–6).

 
As circulating insulin levels in vivo are a reflection of peripheral influences on the islet, as well as of ß-cell function itself, insulin secretion from isolated islets was also examined. Glucose-stimulated (27 mM) insulin secretion (GSIS) was augmented in islets isolated from Ex4-treated WT animals compared with islets from PBS-WT mice (by 49 ± 5%, P < 0.05; Fig. 3AGo). However, in contrast to the normal insulin levels observed in vivo, the insulin response was markedly decreased in islets isolated from PBS-KO mice compared with PBS-WT animals (by 70 ± 8%, P < 0.05 vs. WT mice). This impairment in insulin secretion in the KO islets was partially normalized by chronic treatment with Ex4. Furthermore, despite the abnormal secretory response of islets from PBS-KO mice, insulin content was actually increased (by 927 ± 63%, P < 0.01; Fig. 3BGo) in islets from these mice. In addition, although chronic treatment with Ex4 increased islet insulin levels in WT mice (by 326 ± 14% P < 0.05), Ex4 actually decreased insulin content in the KO islets toward the levels seen in PBS-treated WT animals (by 67 ± 13%, P < 0.01). A similar pattern was observed for islet cAMP levels (Fig. 3CGo), which were increased in both the Ex4-WT and PBS-KO animals (by 34 ± 8%, P < 0.05 and 33 ± 12%, P = 0.06, respectively), and were normalized in islets from Ex4-treated KO mice (by 37 ± 20%, P < 0.05).


Figure 3
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  		FIG. 3. Glucose-stimulated insulin secretion and insulin and cAMP content in isolated islets. WT, HT, and KO mice were injected with either PBS (control) or Ex4 (1 nmol/kg, ip) for 14 d, after which islets were isolated. A, GSIS (at 27 mM) expressed as a percent of the paired response at 3 mM (islets from PBS-treated mice, open bars; islets from Ex4-treated animals, closed bars). Secretion by WT-PBS islets was 6.0 ± 2.5 pg/islet per 60 min at 3.3 mM glucose, and this was increased by 2.4-fold in WT-PBS islets treated with 27 mM glucose. Insulin content of isolated islets (B) and cAMP content of isolated islets (C) are shown (PBS, open bars; Ex4, closed bars). *, P < 0.05 and **, P < 0.01 vs. WT-PBS mice; #, P < 0.05 and ##, P < 0.01 vs. KO-PBS animals (n = 3–7).

 
Consistent with the findings made in islets from PI3-K{gamma} KO mice, siRNA-mediated silencing of PI3-K{gamma} in INS-1E cells (Fig. 4Go) decreased insulin secretory capacity in response to both low and high glucose (by 34 ± 3% and by 31 ± 4%, respectively, P < 0.01), as well as increasing total insulin content (by 39 ± 4%). To eliminate the effects of differences in transfection efficiency, total insulin content was also examined in cells after FACS-mediated sorting for GFP, and was found to be increased by 200 ± 15% (data not shown).


Figure 4
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  		FIG. 4. Insulin secretion and content in p110{gamma}-siRNA transfected INS-1E cells. INS-1E cells were transfected with control (GFP) and siPI3-K{gamma} constructs for 3 d. A, GSIS by transfected cells (3 mM, open bars; 27 mM, hatched bars). A representative blot of PI3-K{gamma} levels in transfected cells is shown. B, Insulin content in transfected cells. **, P < 0.01 vs. control cells at 3.3 mM glucose; ##, P < 0.01 vs. control cells at 27 mM glucose (n = 4).

 
One possible mechanism underlying the normal insulin responses in vivo in KO mice compared with the diminished release from the isolated KO islet could involve changes in the total number of ß-cells. Hence, pancreata were examined immunohistochemically to determine total BCM. BCM was 0.5 ± 0.1 mg in PBS-WT mice (Fig. 5AGo), and this was increased by 55 ± 20% (P < 0.01) in PBS-HT animals, and further increased in PBS-KO mice (by 136 ± 35%, P < 0.001 vs. PBS-WT, P < 0.01 vs. PBS-HT), demonstrating a gene dosage effect of PI3-K{gamma} deficiency to increase basal BCM. In WT mice, Ex4 treatment increased BCM by 200 ± 81% (P < 0.05). Unexpectedly, this response was completely abolished in HT animals, whereas KO mice demonstrated a significant decrease in BCM in response to chronic Ex4 treatment (by 44 ± 11%, P < 0.01).


Figure 5
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  		FIG. 5. Islet morphometry. WT, HT, and KO mice were injected with either PBS (control, open bars) or Ex4 (1 nmol/kg, ip, closed bars) for 14 d, after which the pancreas was collected, fixed, and sectioned. Immunohistochemical analysis of BCM (A), total number of islets per pancreatic section (B), percent of small islets (area less than 200 µm2) per pancreatic section (C), and islet cell proliferation (D), as determined by the number of BrdU-positive (+ve) cells. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 vs. WT-PBS mice; #, P < 0.05 and ##, P < 0.01 vs. KO-PBS mice; and $$, P < 0.01 vs. HT-PBS animals (n = 5–6).

 
To determine whether the changes in BCM were consequent to increased numbers of ß-cells and/or islets, pancreatic sections immunostained for insulin were subjected to morphometric analysis. The average number of islets per pancreatic section was increased in PBS-KO mice (by 68 ± 14%, P < 0.05) compared with PBS-WT mice (Fig 5BGo). As expected, Ex4 treatment increased islet number in WT mice (by 64 ± 12%, P < 0.05), but again, a reduction toward control levels was observed in Ex4-KO animals (P < 0.05). These changes could be accounted for almost totally by differences in the number of small islets (area less than 200 µm2; Fig. 5CGo), suggesting effects on islet neogenesis. However, examination of BrdU incorporation demonstrated that proliferation also paralleled the changes observed in BCM and islet number, with increases in both Ex4-WT and PBS-KO mice (by 150 ± 45%, P < 0.05 and by 137 ± 30%, P < 0.05, respectively) compared with PBS-treated WT mice, and normalization of proliferation after Ex4 treatment of KO animals (P < 0.01; Fig. 5DGo).

Finally, to examine potential mediators of the altered islet function and/or growth, the expression of several proteins that are expressed in islets, but not in exocrine pancreas (42, 43, 44), was determined. In keeping with the changes observed in total BCM and islet number, the levels of both GLUT2 and glucokinase were increased in PBS-KO mice compared with PBS-WT animals (by 140 ± 52% and 279 ± 102%, respectively, P < 0.05; Fig. 6Go, A and B). Chronic administration of Ex4 similarly increased the expression of these proteins in WT mice (P < 0.05), but decreased levels toward basal in KO animals. However, expression of IRS-2 was markedly increased in PBS-KO compared with PBS-WT animals (by 413 ± 52%, P < 0.01), and was increased by Ex4 treatment of WT mice (P < 0.05; Fig. 6CGo), but was normalized in the Ex4-KO animals (P < 0.05).


Figure 6
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  		FIG. 6. Pancreatic protein expression. WT, HT, and KO mice were injected with either PBS (open bars) or Ex4 (1 nmol/kg, ip, closed bars) for 14 d, after which the pancreas was collected and extracted for Western blot for analysis of the islet-specific proteins, GLUT2 (A), glucokinase (B), and IRS-2 (C). Representative blots are shown below the histograms, and all data were normalized to ß-actin. *, P < 0.05 and **, P < 0.01 vs. WT-PBS mice; #, P < 0.05 vs. KO-PBS animals (n = 5–6).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In addition to its known stimulatory effects on ß-cell function (3, 4, 5, 17), several studies have demonstrated that chronic treatment with GLP-1 stimulates ß-cell growth (12, 16, 17, 18, 19, 20). These tropic effects of GLP-1 have been demonstrated to be dependent upon the pleiotropic regulator of cell growth, PKB (16, 21, 22), suggesting a relationship between the G protein-coupled GLP-1 receptor and this enzyme. Thus, the present studies were conducted to determine whether the Gß{gamma}-dependent enzyme, PI3-K{gamma}, serves as an essential link between the GLP-1 receptor and PKB, thereby mediating the tropic actions of GLP-1 on the ß-cell. Unexpectedly, the results of this study suggest that PI3-K{gamma} does not mediate the chronic effects of GLP-1 on ß-cell growth but, rather, plays an essential role in the maintenance of ß-cell secretory responses. Hence, loss of this enzyme impaired insulin secretion from isolated islets; this occurred in association with compensatory growth of the ß-cell and was reversed by normalizing islet secretory function by chronic treatment with Ex4. When taken together, these changes may account for the normal insulin secretion and euglycemia that was observed in these animals in vivo. Consistent with this hypothesis, chronic treatment of the PI3-K{gamma} KO mice with Ex4 not only normalized this secretory defect, but also resulted in a paradoxical decrease in BCM, likely due to reduced metabolic demand on the ß-cell.

Insulin secretion in response to oral glucose was normal in the KO mice, in contrast to the results of a previous study in which a profound impairment in the insulin response to ip glucose was observed (29). These findings indicate that PI3-K{gamma} null animals retain normal incretin responses to oral glucose, and are consistent with the observation that insulin responses to Ex4 are normal in islets from these mice (29). However, the response to glucose was severely blunted in islets isolated from KO animals, and impaired glucose-stimulated insulin release from the perfused pancreas of these mice has also been reported (29). Consistent with these findings, knockdown of PI3-K{gamma} with siRNA also reduced the ability of glucose to enhance insulin release by INS-1E ß-cells. Together, these findings indicate that PI3-K{gamma} deficiency results in a specific impairment in glucose-dependent insulin secretion.

The relationship between PI3-K{gamma} and the insulin secretory pathway has not been previously examined. However, consistent with the elevated levels of cAMP found in islets from the KO mice, PI3-K{gamma} has been reported to serve an essential role in the regulation of PDE3B activity (26, 30). Nonetheless, elevated cAMP levels in the islets from KO animals did not translate into increased release of insulin. It can be speculated that loss of the interaction between PI3-K{gamma} and PDE3B may lead to an altered distribution of PDE3B, resulting in dissociation between cAMP levels and insulin secretion. Consistent with this hypothesis, previous studies have shown that overexpression of PKA-anchoring proteins causes redistribution of PKA to the cell membrane in association with increased GLP-1-stimulated insulin secretion, whereas expression of untargeted or mutant forms of these proteins decreases the effectiveness of GLP-1 (45, 46). As chronic treatment of the KO mice with Ex4 normalized the capacity of isolated islets to secrete insulin, these findings also suggest that GLP-1 may serve a role in the physical coupling of the cAMP pathway with the insulin secretory compartment.

Which molecular component of the glucose-stimulated insulin secretory pathway is affected by loss of PI3-K{gamma} remains unclear. However, as GLUT2 and glucokinase levels remained constant relative to total BCM in the KO mice (e.g. all three parameters were increased in parallel), these findings suggest that PI3-K{gamma} deficiency may modulate downstream glucose-sensing pathways in the ß-cell, such as mitochondrial metabolism, KATP or calcium channel function, and/or insulin granule exocytosis (15, 47). Dissection of the exact biochemical perturbations in this pathway will require further analysis, including electrophysiological studies of ß-cells, in both the absence and presence of PI3-K{gamma}.

BCM is elevated in PI3-K{gamma} KO mice (Ref. 29 and present study), in association with an increased number of small islets per pancreas. Parallel increases were also seen in expression of IRS-2, a critical factor for ß-cell growth (48), as well as in BrdU uptake, an indicator of cell proliferation (49). Of particular note, IRS-2 is expressed not only in ß-cells, but also in ductal epithelial cells, which are believed to be a source of islets arising by neogenesis (50). Thus, the increases observed in IRS-2 were also consistent with the finding of increased numbers of small islets in these animals. No other changes that are known to enhance BCM, such as impaired insulin tolerance (51) were found. Indeed, PI3-K{gamma} null animals demonstrate a slight increase in insulin sensitivity during prolonged exposure to insulin (29). However, compensatory increases in BCM have long been known to occur in response to perturbations in the glucoregulatory system. For example, islet insulin secretion is impaired after chronic glucose infusion in vivo (52), but euglycemia is restored through an enhancement of BCM that normalizes total insulin secretion (53, 54). Similarly, reduced insulin levels, such as in the partially pancreatectomized rat, also result in enhanced BCM leading to normalization of glycemia (55). Therefore these findings are consistent with the hypothesis that the reduced insulin release per islet in the PI3-K{gamma} KO mice results in adaptive ß-cell growth, possibly mediated by IRS-2, to match the need for insulin production (48). The finding that chronic treatment with Ex4 normalized insulin secretion in vitro, and concomitantly decreased BCM in vivo, in the KO animals, is also compatible with this notion. Interestingly, treatment of obese, prediabetic Zucker diabetic fatty rats with a GLP-1 analog for 2 wk has also been reported to decrease BCM by 27%, in association with a 72% decrease in ß-cell proliferation, whereas prolongation of the treatment into the diabetic phase increased both of these parameters (56). Although these findings were interpreted as indicating that the action of GLP-1 on BCM are glucose-dependent, an alternative explanation, based on the results of the present study, is that the GLP-1 treatment improved ß-cell function in these animals (57), thus reducing the initial pressure for expansion of BCM.

Both overexpression of PKB{alpha} and reduction of PTEN expression lead to enhanced BCM (58, 59, 60), whereas abrogation of PKB signaling reduces both basal and GLP-1-stimulated proliferation in ß-cells (22). However, neither P- nor T-PKB levels were altered in pancreas from the PI3-K{gamma}-deficient mice (data not shown), nor were they found by others to be altered in the heart of these animals (29, 37), suggesting that PI3-K{gamma} is not required for maintenance of PKB levels, at least in these tissues (25, 35). Furthermore, other class I isoforms of PI3-K have been detected in INS-1 ß-cells (61), and a very recent study has demonstrated that GLP-1 can stimulate PI3-K activity in human islet cells via activation of Rap1 (62). Transactivation of the epidermal growth factor receptor by GLP-1-mediated stimulation of src has also been demonstrated to activate PI3-K in INS-832/13 cells (63), and is implicated in GLP-1 receptor regulation of K+ channels in rat ß-cells (64). When taken together, these findings suggest that isoforms of PI3-K other than PI3-K{gamma} may mediate the activation of PKB by GLP-1.

In summary, the results of the present study indicate that PI3-K{gamma} plays an essential role in the regulation of glucose-stimulated insulin secretion from the ß-cell, and suggest that PI3-K{gamma} may serve as a novel target to modulate ß-cell function in patients with type 2 diabetes. As the impairment in insulin secretion in PI3-K{gamma} KO mice was restored by treatment with Ex4, these findings provide further evidence of the utility of GLP-1 for therapeutic use in patients with type 2 diabetes. Furthermore, as PI3-K{gamma} inhibitors are now being proposed for use in the treatment of patients with chronic inflammatory conditions, such as lupus and rheumatoid arthritis (65, 66), the results of the present study suggest caution should be exerted with respect to the potential impact of such treatment on the ß-cell.


   Acknowledgments
 
We are grateful to Dr. M. A. Magnuson for the gift of anti-glucokinase antiserum, Dr. C. Wollheim for the INS-1E cells, and Ms. S. Sarrazin (Lifescan Canada, Burnaby, British Columbia, Canada) for the One Touch glucose meters.


   Footnotes
 
This was work was supported by an operating grant (to P.L.B.) from the Canadian Diabetes Association and a grant (to P.E.M.) from the Novo Nordisk Foundation (Copenhagen). L.L. was supported by postdoctoral fellowships from the Canadian Diabetes Association and from the Banting and Best Diabetes Centre and the Department and Faculty of Medicine, University of Toronto. P.E.M. was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research and by the European Foundation for the Study of Diabetes/AstraZeneca Fellowship in Islet Biology. D.S.A. was supported by summer studentships from the Banting and Best Diabetes Centre. G.Y.O. was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research and the Heart and Stroke foundation of Canada. P.H.B. was supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario, and P.L.B. was supported by the Canada Research Chairs Program.

Disclosure: Patricia L. Brubaker is a consultant to Merck and Amgen. The remaining authors have nothing to disclose.

First Published Online March 30, 2006

Abbreviations: BCM, ß-Cell mass; BrdU, 5-bromo-2-deoxyuridine; Ex4, exendin-4; GFP, green fluorescent protein; GLP-1, glucagon-like peptide-1; GLUT2, glucose transporter 2; GSIS, glucose-stimulated insulin secretion; HT, heterozygous; IRS-2, insulin receptor substrate-2; ITT, insulin tolerance test; KO, knockout; KRB, Krebs-Ringer bicarbonate; OGTT, oral glucose tolerance test; PDE3B, phosphodiesterase-3B; PI3-K, phosphatidylinositol-3-kinase; PK, protein kinase; siRNA, small interfering RNA; WT, wild type.

Received February 8, 2006.

Accepted for publication March 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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