PROPER GLUCOSE HOMEOSTASIS is critically dependent

0013-7227/04/$15.00/0 Endocrinology 145(9):4078 4083 Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: 10.1210/en.2004-0028 Impaired Glucose-Stimulated Insulin Secretion, Enhanced Intraperitoneal Insulin Tolerance, and Increased -Cell Mass in Mice Lacking the p110 Isoform of Phosphoinositide 3-Kinase P. E. MACDONALD, J. W. JOSEPH, D. YAU, J. DIAO, Z. ASGHAR, F. DAI, G. Y. OUDIT, M. M. PATEL, P. H. BACKX, AND M. B. WHEELER University of Toronto (P.E.M., J.W.J., D.Y., J.D., Z.A., F.D., M.B.W.), Departments of Physiology and Medicine, Toronto, Canada; Lund University (P.E.M.), Department of Molecular and Cellular Physiology, Lund 221 84, Sweden; and Division of Cardiology (G.Y.O., M.M.P., P.H.B.), University Health Network and the Heart and Stroke Richard Lewar Centre of Excellence, Toronto, Canada M5S 3E2 Phosphoinositide 3-kinase (PI3 kinase) has been implicated in G protein-coupled receptor regulation of pancreatic -cell growth and glucose-stimulated insulin secretion. The G protein-activated p110 isoform of PI3 kinase was detected in insulinoma cells, mouse islets, and human islets. In 7- to 10- wk-old mice, knockout of p110 reduced the plasma insulin response to ip glucose injection and impaired first and second phase glucose-stimulated insulin secretion from pancreata perfused ex vivo. The p110 / mice responded to preinjection with the glucagon-like peptide-1 receptor agonist exendin 4, such that plasma glucose and insulin responses to ip glucose injection were not different from wild types. Mice PROPER GLUCOSE HOMEOSTASIS is critically dependent on an adequate pancreatic -cell mass that responds appropriately to increased blood glucose. Accordingly, defects in glucose-stimulated insulin secretion and an eventual decrease in -cell mass are important factors in the development and progression of type 2 diabetes (1). G protein-coupled receptors (GPCRs) such as those activated by glucagon-like peptide-1 (GLP-1) regulate -cell mass by promoting proliferation (2, 3) and preventing apoptosis (4, 5). Phosphoinositide 3-kinase (PI3 kinase) signaling is an important pathway mediating these effects (2, 6). In addition to its role in islet growth, numerous studies implicate PI3 kinase as a negative regulator of insulin secretion because its pharmacological inhibition (7, 8) and genetic reduction in mice (9) enhances secretion. PI3 kinase is comprised of both catalytic and regulatory subunits and has been extensively reviewed (10). The catalytic subunit of type 1A PI3 kinases, which are regulated by tyrosine kinase receptors, is one of three p110 isoforms (,, and ). These associate with one of five regulatory subunits; p50, p55, and p85, resulting from alternative splicing of a single gene; and p55 and p85. The only type 1B isoform Abbreviations: AUC, Area under the curve; GPCRs, G protein-coupled receptors; GLP-1, glucagon-like peptide-1; HI, human islets; p-akt, phosphorylated Akt; PI3 kinase, phosphoinositide 3-kinase. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. lacking p110 were not diabetic and were only slightly glucose intolerant (ip glucose injection) compared with wild types, in part due to enhanced responsiveness to insulin as determined by an ip insulin tolerance test. Despite severely reduced insulin secretion in these animals, the p110 / mice had greater pancreatic insulin content, and an increased -cell mass due to -cell hypertrophy. These surprising results suggest that the G protein-coupled p110 isoform of PI3 kinase is not central to the development or maintenance of sufficient -cell mass but positively regulates glucose-stimulated insulin secretion. (Endocrinology 145: 4078 4083, 2004) of PI3 kinase, called p110, associates with the p101 regulatory subunit and is activated directly by GPCRs through an interaction with G. Like other PI3 kinases, p110 phosphorylates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P] to generate PtdIns(3,4,5)P in vivo and subsequently activates numerous downstream targets including 3-phosphoinositide-dependent kinase-1, Akt, protein kinase C, and p70s6 kinase. Activity of p110 has been demonstrated in insulinoma cells (11), whereas protein expression has been shown in human, dog, rat, and mouse pancreas by immunohistochemistry (12). Although the latter report suggests no expression of the protein in pancreatic islets, we show here that islet cells do indeed express p110. In contrast to the known effects of nonselective PI3 kinase inhibition and genetic reduction of type 1A PI3 kinase activity, we find that glucose-stimulated insulin secretion is blunted in mice lacking p110. The p110 mice are not diabetic, however, demonstrating enhanced responsiveness to ip insulin and increased -cell mass. This latter finding is surprising because G protein-activated PI3 kinase activity is thought to be a positive regulator of -cell proliferation (13). Therefore, the type 1B isoform p110 has an important role in islet function that is distinct from that of the type 1A PI3 kinases. Materials and Methods Animals and in vivo experiments Animal experiments were performed in accordance with University of Toronto guidelines. The generation of p110 / mice has been 4078

MacDonald et al. Brief Communications Endocrinology, September 2004, 145(9):4078 4083 4079 described (14). At 7 10 wk, male wild-type C57/Bl6 and p110 / mice did not differ in body weight (22.8 0.6 g and 24.5 0.4 g), and the p110 / mice were not diabetic. Intraperitoneal glucose (1.5 g/kg body weight) and insulin (0.75 U/kg body weight) tolerance tests were performed as described (15) on mice fasted overnight (15 18 h) and for 2 4 h, respectively. In some ip glucose tolerance experiments, exendin 4(5 g in PBS) was injected im (gluteal) 5 min before ip glucose. Blood glucose was measured with a Lifescan Elite glucose meter (Lifescan, Toronto, Ontario, Canada), and plasma insulin was assayed using a mouse insulin ELISA kit (Crystalchem, Chicago, IL). Glucagon was assayed after ip insulin injection (1 U/kg body weight) using a mouse glucagon RIA kit (Linco Research Inc., St. Charles, MO). Catecholamines were extracted from plasma collected after a 12-h fast using alumina, eluted, and assayed using HPLC (Waters Associates, Inc., Milford, MA) coupled with electrochemical detection (ESA Inc., Bedford, MA). Perfused pancreas Mice were fasted overnight (15 18 h), and the surgical procedure was similar to that described (16). The perfusate was a modified Krebs- Ringer-2% BSA-glucose-3% dextran solution gassed with 95% O 2 /5% CO 2 to achieve a ph of 7.4. Samples were assayed for insulin by RIA with a polyclonal insulin antibody from guinea pig (17) as described previously (15). -Cell mass and pancreatic insulin content Immunostaining for insulin and glucagon were performed as described (15). Images were collected at 4 or 40 magnification and analyzed using Image Pro software (MediaCybernetics, Carlsbad, CA). Insulin stained area, as a percentage of pancreas area, was multiplied by pancreas weight to calculate -cell mass. Islet area was calculated by dividing the insulin-positive area by the islet number for each tissue section. -Cell cross-sectional area was calculated by dividing the insulin-positive area by the number of -cell nuclei (3). -Cell density was determined by dividing the number of -cell nuclei by total pancreas area. Pancreatic insulin content was determined as described (15). Pancreata were collected after pancreas perfusion and stored at 80 C. Previous experiments confirmed no difference between this protocol and insulin content measured from pancreata taken without perfusion (15). RT-PCR and Western blot RT-PCR was performed on total RNA with 30 PCR cycles of 94 C for 30 sec, 56 C for 30 sec, and 72 C for 30 sec followed by a 10-min extension at 72 C. Primers specific to the p110 transcript, based on human sequences, were: forward 5 -ACTGCCTCAAGAACGGAGAA-3 and reverse 5 -CAGGAGGAAACGGTGGTCTA-3. Western blotting was performed as described (14) on whole cell lysates. MIN6 cells (passages 35 40) from S. Seino (Chiba University, Chuo-ku, Japan) were cultured as described previously (18). Human islets were obtained from the Juvenile Diabetes Research Foundation Human Islet Distribution Program courtesy J. Lakey (University of Alberta, Edmonton, Canada). Antibodies for p110 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and for glyceraldehyde-3-phosphate dehydrogenase were from Research Diagnostics Inc. (Flanders, NJ). Blotting for Akt and phosphorylated (p)-akt (Thr308) was performed on heart tissue from wild-type and p110 / mice 0, 10, and 30 min after ip insulin injection (1 U/kg body weight) and band densities were determined using Scion Image software (Scion Corp., Frederick, MD). Antibodies for Akt and p-akt (Thr308) were from Cell Signaling Technology (Beverly, MA). Statistics Curves were compared by one-way ANOVA and Bonferroni post test. Other data were compared using the unpaired Student s t test. P 0.05 was considered significant. Results Regulation of glucose homeostasis by p110 To examine a potential regulatory role for p110 in pancreatic islets, expression of this type 1B PI3 kinase was investigated in islets and insulin-secreting cells. Expression of p110 transcripts in total RNA from MIN-6 insulinoma cells, mouse islets (MI), and human islets (HI) was detected by RT-PCR using p110 -specific primers (Fig. 1A). No PCR product was obtained in water blank controls. Whereas heart [H( / )] and pancreas [P( / )] lysates from wild-type animals were positive for p110 protein expression by Western blot, expression could not be detected in pancreas from p110 / mice [P( / )] (Fig. 1A). Also, protein expression of p110 was detected in MIN-6 and HI lysates (Fig. 1A). Because both mouse and human islets express p110, a role for this PI3 kinase isoform in glucose homeostasis was investigated in p110 / mice. Fasting blood glucose levels were not different between wild-type (5.75 0.33 mm, n 11) and p110 / (6.03 0.27 mm, n 22) mice. The blood glucose excursion after an ip glucose load was greater in p110 / mice than in wild-type mice [area under the curve (AUC) of 1214.9 51.2 vs. 1029.0 60.1 mm min, n 22 and 11, P 0.05] (Fig. 1B). Before the glucose injection, plasma insulin levels were not different between wild-type (0.21 0.02 pg/ml, n 10) and p110 / mice (0.24 0.04 pg/ml, n 10). After the ip glucose load, plasma insulin levels in the p110 / mice were significantly impaired FIG. 1.A,Top, p110 transcripts were detected in total RNA from MIN-6 insulinoma cells, mouse islets (MI), and HI. No transcript was detectable in water blank controls (H 2 O). A, Bottom, p110 protein was detected by Western blot (two membranes) in mouse heart [H( / )], mouse pancreas [P( / )], MIN-6 cells and HI. Expression of p110 was not detected in pancreas from p110 / mice [P( / )]. B, Male mice (7 10 wk) were fasted overnight and injected ip with glucose. The blood glucose excursion was measured in wild-type (black) and p110 / (open) mice. The AUC (shown to the right) was greater in the p110 / mice. C, The plasma insulin response to the ip glucose injection was blunted in the p110 mice. The AUC values are shown at the right. *,P 0.05; **, P 0.01 compared with wild type.

4080 Endocrinology, September 2004, 145(9):4078 4083 MacDonald et al. Brief Communications (Fig. 1C) and the AUC of the insulin response in the / mice (69.3 9.9 pg ml 1 min, n 9) was decreased compared with wild types (145.8 19.8 pg ml 1 min, n 10, P 0.01) (Fig. 1C). Before ip insulin tolerance tests, blood glucose levels were not different between wild-type (8.01 0.28 mm,n 13) and p110 / mice (7.99 0.30 mm,n 15). After ip injection of insulin, blood glucose was decreased to a greater degree in the / mice compared with wild types ( AUC of 357.0 35.2 vs. 235.0 28.4 mm min, n 15 and 13, P 0.05) (Fig. 2A). Insulin-stimulated Akt phosphorylation (Thr308) in heart, at 10 or 30 min after insulin injection, was not significantly different between the wild-type and p110 / mice (Fig. 2B). In wild-type mice, plasma glucagon was elevated from 112.7 20.6 to 292.4 72.8 pg/ml (n 6, P 0.05) at 45 min after ip insulin (Fig. 2C). In the p110 / mice, plasma glucagon was depressed from 128.1 15.1 to 75.2 11.2 pg/ml (n 6, P 0.05) at 10 min but was exaggerated (799.1 162.9 pg/ml, n 6, P 0.001) compared with wild types (P 0.05) at 45 min (Fig. 2C). Fasting plasma epinephrine (3.84 0.57 and 4.28 0.87 nm, n 5) and norepinephrine (54.52 6.92 and 52.10 9.47 nm, n 5) levels were not different between the p110 / and wild-type mice (Fig. 2, D and E). Responsiveness of p110 / mice to exendin 4 Because the GLP-1 receptor can couple to PI3 kinase (2), the responsiveness of p110 / mice to the degradationresistant GLP-1 receptor agonist exendin 4 was examined. The blood glucose excursion during an ip glucose tolerance test was not different between wild-type and p110 / mice (not shown) after exendin 4 preinjection (5 g in PBS). The AUC values were 514.1 50.4 mm min (n 6) and 477.8 28.5 mm min (n 11), respectively, representing a significantly (P 0.001) enhanced glucose tolerance compared with untreated mice. Accordingly, after exendin 4 preinjection the plasma insulin response to the ip glucose tolerance test was not different between wild-type (AUC, 70.4 11.2 pg ml 1 min, n 7) and p110 / (AUC, 66.7 6.3 pg ml 1 min, n 7) mice. Pancreatic insulin content and -cell mass in p110 / mice PI3 kinase has been implicated in -cell proliferation, particularly in response to GLP-1 (13). Therefore, we examined whether the reduced plasma insulin response in p110 / mice was due to reduced -cell mass. Surprisingly, -cell mass in the / mice (1.65 0.21 mg, n 6) was greater than in wild types (0.86 0.23 mg, n 5, P 0.05) (Fig. 3B). Whereas the average islet number per pancreas area was not different (1.47 0.18 islets/mm 2,n 6 and 1.64 0.19 islets/mm 2,n 5) (Fig. 3C), average islet area was increased in p110 / (3088.6 320.8 m 2,n 6) vs. wild-type mice (1836.1 351.4 m 2,n 5, P 0.05) (Fig. 3D). Accordingly, the pancreatic insulin content of p110 / mice (91.5 12.2 ng/mg protein, n 7) was significantly greater than in wild types (54.7 5.5 ng/mg protein, n 7, P 0.05) (Fig. 3E). This resulted largely from -cell hypertrophy as demonstrated by an increase in -cell cross-sectional area from 150.0 5.4 m 2 (n 5) in the wild-type mice to 196.8 12.1 m 2 (n 5, P 0.01) in the p110 / mice (Fig. 3F), and no significant change in -cell density (22.4 4.8 and 25.0 3.7 -cells/mm 2 pancreas, n 5) (Fig. 3G). FIG. 2. A, Mice were fasted for 2 4 h and injected ip with insulin. Blood glucose (shown as the change in blood glucose levels) was reduced to a greater extent in the p110 / (open) mice compared with wild types (black). The corresponding change in AUC ( AUC) is shown at the right. B, Akt phorhporylation in heart was not significantly different between wild-type ( / ) and p110 / ( / ) mice 10 or 30 min after insulin injection. Blots are shown at the left and the fold increase in p-akt signal, compared with time 0 and normalized to total Akt, is shown at the right. Counterregulatory hormones were investigated in panel C. The plasma glucagon response after injection of insulin was depressed in the p110 / mice at 10 min but was exaggerated at 45 min. Fasting plasma norepinephrine and epinephrine levels were not different in wild-type vs. p110 / mice. *, P 0.05; **, P 0.01; and ***, P 0.001 compared wild type (A) or time 0. Ex vivo insulin secretion from p110 / pancreata Because -cell mass and pancreatic insulin content were not decreased in the p110 / mice, we examined insulin secretion using an ex vivo perfused pancreas model. Basal insulin secretion in the presence of substimulatory glucose (1.4 mm) was not different between groups, but secretion in response to glucose (13.4 mm) was decreased in the p110 / mice (Fig. 3A). Notably, first-phase insulin secretion was severely blunted (AUC from 77.0 11.0 to 36.3 8.6 pg ml 1 min, n 6, P 0.05), and second-phase secretion was also decreased (AUC from 107.0 13.7 to 66.0 11.2 pg ml 1 min, n 6, P 0.05) (Fig. 3, B and C). Insulin secretion in response to 20 mm arginine was not different

MacDonald et al. Brief Communications Endocrinology, September 2004, 145(9):4078 4083 4081 FIG. 4. Pancreata of wild-type and p110 / mice were perfused ex vivo. A, Wild-type mice (black) exhibited the classic bi-phasic secretion response to a step change in glucose (1.4 13.4 mm), whereas p110 / mice (open) showed a blunted response. The calculated AUC values for first phase (5 10 min) and second phase (10 22 min) secretion for wild-type and p110 / are shown in B and C. D, Wild-type and p110 / pancreata both responded to stimulation with 20 mm arginine. The AUC for the arginine response are shown in panel E. *,P 0.05; ***, P 0.001 compared with wild type. FIG. 3. Sections of wild-type and p110 / pancreata were stained for insulin and glucagon. A, Representative images obtained at 10 magnification. B, -Cell mass was increased in p110 / mice (open) compared with wild types (black). Although islet number (per pancreas area) was not different in wild-type vs. p110 / mice (C), the average islet area was increased in p110 / mice (D). Accordingly, total pancreatic insulin content (per microgram of total protein) was also increased in the p110 / mice (E). F and G, -Cell crosssectional area was increased in the p110 / mice, whereas -cell density (per pancreas area) was unchanged compared with wild types. *, P 0.05; **, P 0.01 compared with wild-type. between the / and wild-type preparations (AUC of 116.1 16.6 and 116.4 24.6 pg ml 1 min, respectively, n 6) (Fig. 4, D and E). Discussion The p110 isoform of PI3 kinase is activated by GPCRs thorough a direct interaction with the G -subunit (10). The initial characterization of p110 / mice demonstrated abnormalities in thymocyte development, T-cell activation, neutrophil migration, and oxidative burst in response to GPCR agonists (19). These mice also exhibit increased cardiac contractility (14), protection from isoproteranol-induced cardiac hypertrophy (20), and decreased leukotreine B 4 -induced degranulation of peritoneal granulocytes (21). In insulinsecreting cells, PI3 kinase has been implicated in the ability of GLP-1 to stimulate PDX-1 binding activity and DNA synthesis (2), proliferation (13), and to suppress apoptosis (22); and nonselective PI3 kinase inhibition enhances insulin secretion (7, 8). A role for the p110 isoform of PI3 kinase in islet function and development was previously unknown. One report suggests that p110 is not expressed in islets (12), whereas another demonstrates p110 activity in insulinoma cells (11). The present study identifies p110 expression in mouse and human islets and is the first to characterize glucose homeostasis in mice lacking p110. Although the insulin response of p110 / mice is severely blunted, the lack of massive glucose intolerance and diabetes may be explained by an increased responsiveness to insulin. A similar effect on ip insulin tolerance is observed in p85 / mice (23), p50 /p55 / mice (24), or mice heterozygous for Pik3r1 that encodes p85, p50, and p55 (25). However, there is no evidence for a -cell defect in those animals. In fact, whereas islet PI3 kinase activity is reduced by 80% in the p85 / mice, insulin secretion is increased and pancreatic insulin content is reduced (9), suggesting that the type 1A and type 1B PI3 kinases play distinct roles in islet function. The observation of -cell hypertrophy in the p110 / mice may suggest defective insulin processing leading to the secretion of undetected proinsulin split products rather than defective secretion per se. However, two points argue for a true secretion defect: 1) although in vivo and ex vivo insulin responses were reduced in the p110 / mice, their total pancreatic insulin content assayed with the same antibody as the in situ measurements was increased; and 2) the ex vivo insulin secretion response to arginine was not different between the wild-type and p110 / mice.

4082 Endocrinology, September 2004, 145(9):4078 4083 MacDonald et al. Brief Communications Insulin sensitivity of peripheral tissues is up-regulated in the p85 /, p50 /p55 /, or Pik3r1 / mice (23 25). Whereas the present results demonstrate intact insulin-stimulated Akt phosphorylation in p110 / heart, more direct measurements will be required to truly assess peripheral insulin sensitivity in the p110 / mice. Impaired counterregulation may contribute to the failure of plasma glucose to return to basal levels after ip insulin injection. Defective secretion of counterregulatory hormones cannot account for this, however, because fasting plasma catecholamine levels were normal and the plasma glucagon response was exaggerated at 45 min. Although the initial depression of glucagon in the p110 / mice may delay the return of blood glucose to basal levels, this should be rapidly corrected by the increased glucagon at 45 min because glucagon stimulates hepatic glucose production with a time constant of approximately 4 min (26). It therefore seems possible that the p110 / mice do not respond to these hormones appropriately, for example by initiating hepatic glucose production in response to glucagon and epinephrine. PI3 kinase mediates the proliferative and antiapoptotic effects of the G protein-coupled GLP-1 receptor (13). However, -cell mass was increased and -cell density was unchanged in the p110 / mice. Although this suggests that the G activated p110 isoform of PI3 kinase is not a positive regulator of islet growth, further studies are required to determine whether p110 mediates -cell mass expansion in response to exogenous GLP-1, and whether other PI3 kinase isoforms may be activated directly by the GLP-1 receptor. We also cannot rule out indirect G protein activation of other PI3 kinase isoforms because recent evidence suggests that GLP-1 activates type 1A PI3 kinases that are not commonly G protein linked through the trans-activation of epidermal growth factor tyrosine kinase receptors (18, 27). Although the GLP-1 receptor agonist exendin 4 was found to increase glucose tolerance in the p110 / mice, further studies are required to determine whether a more subtle phenotype is present, such as a shift in the GLP-1 dose response or altered responses to other GPCR agonists. Although not essential for the development of -cell mass or the hypoglycemic action of exendin 4, p110 is integral for appropriate -cell responsiveness to glucose. This represents a surprising finding given the presumed role of G proteinlinked PI3 kinase activity in islet growth and the ability of PI3 kinase inhibitors to stimulate insulin secretion. The p110 isoform of PI3 kinase can activate numerous downstream targets implicated as regulators of insulin secretion. These include protein kinase C, nitric oxide synthase and, interestingly, guanine nucleotide exchange factors that regulate small G proteins such as Rac and Cdc42 (10). It is also possible that defective insulin secretion in the p110 / mice results from altered -cell development due to the lack of this PI3 kinase isoform from birth. Clearly, further studies of the metabolic and electrical function of isolated p110 / -cells, and study of the acute effects of p110, are required to elucidate the nature of defective insulin secretion in these animals. Acknowledgments The authors thank Dr. Joseph Penninger (Toronto, Canada) for the p110 / mice, and Dr. Jonathan Lakey (Edmonton, Canada) and the JDRF Human Islet Distribution Program at the University of Alberta for human islets. Also, we thank Dr. Albert Salehi (Lund, Sweden) for critical reading of the manuscript. 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