β-Cell-targeted Overexpression of Phosphodiesterase 3B in Mice Causes Impaired Insulin Secretion, Glucose Intolerance, and Deranged Islet Morphology

Abstract

The second messenger cAMP mediates potentiation of glucose-stimulated insulin release. Use of inhibitors of cAMP-hydrolyzing phosphodiesterase (PDE) 3 and overexpression of PDE3B in vitro have demonstrated a regulatory role for this enzyme in insulin secretion. In this work, the physiological significance of PDE3B-mediated degradation of cAMP for the regulation of insulin secretion in vivo and glucose homeostasis was investigated in transgenic mice overexpressing PDE3B in pancreatic β-cells. A 2-fold overexpression of PDE3B protein and activity blunted the insulin response to intravenous glucose, resulting in reduced glucose disposal. The effects were “dose”-dependent because mice overexpressing PDE3B 7-fold failed to increase insulin in response to glucose and hence exhibited pronounced glucose intolerance. Also, the insulin secretory response to intravenous glucagon-like peptide 1 was reduced in vivo. Similarly, islets stimulated in vitro exhibited reduced insulin secretory capacity in response to glucose and glucagon-like peptide 1. Perifusion experiments revealed that the reduction specifically affected the first phase of glucose-stimulated insulin secretion. Furthermore, morphological examinations demonstrated deranged islet cytoarchitecture. In conclusion, these results are consistent with an essential role for PDE3B in cAMP-mediated regulation of insulin release and glucose homeostasis. The second messenger cAMP mediates potentiation of glucose-stimulated insulin release. Use of inhibitors of cAMP-hydrolyzing phosphodiesterase (PDE) 3 and overexpression of PDE3B in vitro have demonstrated a regulatory role for this enzyme in insulin secretion. In this work, the physiological significance of PDE3B-mediated degradation of cAMP for the regulation of insulin secretion in vivo and glucose homeostasis was investigated in transgenic mice overexpressing PDE3B in pancreatic β-cells. A 2-fold overexpression of PDE3B protein and activity blunted the insulin response to intravenous glucose, resulting in reduced glucose disposal. The effects were “dose”-dependent because mice overexpressing PDE3B 7-fold failed to increase insulin in response to glucose and hence exhibited pronounced glucose intolerance. Also, the insulin secretory response to intravenous glucagon-like peptide 1 was reduced in vivo. Similarly, islets stimulated in vitro exhibited reduced insulin secretory capacity in response to glucose and glucagon-like peptide 1. Perifusion experiments revealed that the reduction specifically affected the first phase of glucose-stimulated insulin secretion. Furthermore, morphological examinations demonstrated deranged islet cytoarchitecture. In conclusion, these results are consistent with an essential role for PDE3B in cAMP-mediated regulation of insulin release and glucose homeostasis. A vital part of the signaling network responsible for stimulus-secretion coupling in pancreatic β-cells involves cAMP as second messenger. In particular, several physiological enhancers of glucose-stimulated insulin release, such as pituitary adenylate cyclase-activating polypeptide and glucagon-like peptide (GLP) 1The abbreviations used are: GLP, glucagon-like peptide; PDE, phosphodiesterase; GLUT2, glucose transporter 2; PKA, protein kinase A; RIP, rat insulin promoter; PBS, phosphate-buffered saline; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. 1, act mainly through increased formation of cAMP (1Gromada J. Holst J.J. Rorsman P. Pflugers Arch. 1998; 435: 583-594Crossref PubMed Scopus (221) Google Scholar, 2Ahrén B. Bioessays. 1998; 20: 642-651Crossref PubMed Scopus (115) Google Scholar, 3Filipsson K. Kvist-Reimer M. Ahren B. Diabetes. 2001; 50: 1959-1969Crossref PubMed Scopus (66) Google Scholar) and, at least in part, its subsequent activation of protein kinase A (PKA). However, the exact mechanisms by which cAMP mediates the potentiating effect on insulin secretion remain to be established. Also, glucose itself has been reported to stimulate cAMP formation in β-cells. The significance, however, of glucose-induced increase in cAMP has been debated (4Howell S.L. Jones P.M. Persaud S.J. Diabetologia. 1994; 37: S30-S35Crossref PubMed Scopus (51) Google Scholar, 5Wollheim C.B. Maechler P. Diabetes. 2002; 51: S37-S42Crossref PubMed Google Scholar) and full understanding of its contribution to insulin release has not been reached. Utilization of inhibitors of PKA has been shown not to impair glucose-stimulated release of insulin (6Harris T.E. Persaud S.J. Jones P.M. Biochem. Biophys. Res. Commun. 1997; 232: 648-651Crossref PubMed Scopus (71) Google Scholar, 7Persaud S.J. Jones P.M. Howell S.L. Biochem. Biophys. Res. Commun. 1990; 173: 833-839Crossref PubMed Scopus (59) Google Scholar). It has therefore been suggested that cAMP plays a minor role in nutrient-stimulated insulin secretion. Recently, newly described targets for cAMP, e.g. the family of exchange proteins directly activated by cAMP (Epac), were shown to participate in PKA-independent β-cell signaling and may prove to mediate previously unknown secretion stimulatory effects of cAMP (8Ozaki N. Shibasaki T. Kashima Y. Miki T. Takahashi K. Ueno H. Sunaga Y. Yano H. Matsuura Y. Iwanaga T. Takai Y. Seino S. Nat. Cell Biol. 2000; 2: 805-811Crossref PubMed Scopus (401) Google Scholar, 9Kang G. Chepurny O.G. Holz G.G. J. Physiol. 2001; 536: 375-385Crossref PubMed Scopus (173) Google Scholar, 10Eliasson L. Ma X. Renström E. Barg S. Berggren P.O. Galvanovskis J. Gromada J. Jing X. Lundquist I. Salehi A. Sewing S. Rorsman P. J. Gen. Physiol. 2003; 121: 181-197Crossref PubMed Scopus (217) Google Scholar). The significance of accurate regulation of cAMP levels is illustrated by the large number of gene products known to generate, target, and degrade the cyclic nucleotide (reviewed in Ref. 11Steinberg S.F. Brunton L.L. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 751-773Crossref PubMed Scopus (320) Google Scholar). Degradation of cAMP is achieved through phosphodiesterase (PDE)-catalyzed hydrolysis, which balances the activity of adenylyl cyclase, and thereby contributes to the control of appropriate levels of cAMP within the cell. Inhibitors of PDE, some of which are in clinical use, are well known cAMP elevating tools. One effect of PDE inhibition is augmented insulin secretion (12Siegel E.G. Wollheim C.B. Kikuchi M. Renold A.E. Sharp G.W. J. Clin. Invest. 1980; 65: 233-241Crossref PubMed Scopus (39) Google Scholar, 13El-Metwally M. Shafiee-Nick R. Pyne N.J. Furman B.L. Eur. J. Pharmacol. 1997; 324: 227-232Crossref PubMed Scopus (24) Google Scholar, 14Parker J.C. VanVolkenburg M.A. Nardone N.A. Hargrove D.M. Andrews K.M. Biochem. Biophys. Res. Commun. 1997; 236: 665-669Crossref PubMed Scopus (30) Google Scholar). PDEs comprise a superfamily of enzymes with 11 known members (PDE1–11), each containing multiple isoforms and splice variants (15Soderling S.H. Beavo J.A. Curr. Opin. Cell Biol. 2000; 12: 174-179Crossref PubMed Scopus (651) Google Scholar, 16Francis S.H. Turko I.V. Corbin J.D. Prog. Nucleic Acids Res. Mol. Biol. 2001; 65: 1-52Crossref PubMed Google Scholar). The isozymes are structurally related but differ both functionally, e.g. with regard to regulation and substrate specificity, and in tissue distribution. PDE3B, which is one of two enzymes forming the PDE3 family, has been shown to reside particularly in tissues of importance for energy homeostasis (17Degerman E. Rahn L ström T. Stenson Holst L. Göransson O. Ahmad F. Shakur Y. Kenan Y. Manganiello V. Le Roith D. Olefsky J.M. Taylor S. Diabetes Mellitus: A Fundamental and Clinical Text. Lippincott Williams & Wilkins, Philadelphia, PA2000: 284-289Google Scholar). A critical role of PDE3B in insulin-induced inhibition of adipose tissue lipolysis is well established. Also, a role in inhibition of hepatic glycogenolysis has been described (17Degerman E. Rahn L ström T. Stenson Holst L. Göransson O. Ahmad F. Shakur Y. Kenan Y. Manganiello V. Le Roith D. Olefsky J.M. Taylor S. Diabetes Mellitus: A Fundamental and Clinical Text. Lippincott Williams & Wilkins, Philadelphia, PA2000: 284-289Google Scholar). Besides these tissues, β-cells of the endocrine pancreas express this particular isoform. In β-cells, PDE1 and PDE4 enzymes are expressed in addition to PDE3B (18Han P. Werber J. Surana M. Fleischer N. Michaeli T. J. Biol. Chem. 1999; 274: 22337-22344Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 19Ahmad M. Abdel-Wahab Y.H. Tate R. Flatt P.R. Pyne N.J. Furman B.L. Br. J. Pharmacol. 2000; 129: 1228-1234Crossref PubMed Scopus (28) Google Scholar, 20Zhao A.Z. Zhao H. Teague J. Fujimoto W. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3223-3228Crossref PubMed Scopus (126) Google Scholar), but the differential roles of these enzymes remain to be clarified. Although all three have been implicated in insulin release, the firmest evidence of an important role has been obtained by manipulating the activity of PDE3B (20Zhao A.Z. Zhao H. Teague J. Fujimoto W. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3223-3228Crossref PubMed Scopus (126) Google Scholar, 21Härndahl L. Jing X.J. Ivarsson R. Degerman E. Ahrén B. Manganiello V.C. Renström E. Stenson Holst L. J. Biol. Chem. 2002; 277: 37446-37455Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar); when inhibited, rat islet insulin secretion was shown to be enhanced and when increased by overexpression, insulin secretion was reduced (21Härndahl L. Jing X.J. Ivarsson R. Degerman E. Ahrén B. Manganiello V.C. Renström E. Stenson Holst L. J. Biol. Chem. 2002; 277: 37446-37455Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). To study the physiological role of PDE3B in cAMP-mediated insulin release, we have generated transgenic mice with β-cell-targeted overexpression of the enzyme. Here we show that a modest degree of PDE3B overexpression impairs the insulin response to intravenous loads of glucose and GLP-1 and that mainly the first phase of insulin secretion is impaired. Furthermore, the islet dysfunction in these mice is associated with glucose intolerance and histological examination reveals deranged islet cytoarchitecture similar as in models of type 2 diabetes. The effects appear to be “dose”-dependent, because glucose homeostasis was further impaired in a second transgenic line exhibiting a more pronounced overexpression of PDE3B. Our findings highlight the important physiological role of cAMP in the control of insulin secretion. Generation of PDE3B Transgenic (RIP-PDE3B) Mice—A transgene construct for β-cell-specific expression was achieved by placing a FLAG epitope-equipped cDNA for mouse PDE3B (described in Ref. 22Shakur Y. Takeda K. Kenan Y. Yu Z.X. Rena G. Brandt D. Houslay M.D. Degerman E. Ferrans V.J. Manganiello V.C. J. Biol. Chem. 2000; 275: 38749-38761Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) under control of the rat insulin promoter (RIP) 2. To this end the pcDNA3/mPDE3B-Flag vector (22Shakur Y. Takeda K. Kenan Y. Yu Z.X. Rena G. Brandt D. Houslay M.D. Degerman E. Ferrans V.J. Manganiello V.C. J. Biol. Chem. 2000; 275: 38749-38761Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) was modified by subcloning a 689-bp fragment of RIP2 (nucleotides 244–933) into the KpnI/BamHI restriction sites upstream of mPDE3B cDNA (sitting in the XhoI site). A 958-bp genomic fragment of the human growth hormone gene (nucleotides 1193–2151) was then subcloned into the XbaI/ApaI restriction sites downstream of PDE3B cDNA in the same vector. After excision of the whole construct (6.3 by into mice was the Transgenic of the mice to J.A. 2002; PubMed Scopus Google Scholar). Transgenic mice were by of a to of the mouse PDE3B and a to the FLAG resulting in a transgenic mice have been used the and have been as have been a and in an with and The all was was used to one cDNA 1 of the The cDNA was the same as described were in of TES, 1 and with and 1 were to were to and were with in a of and for 1 were with PDE3B (21Härndahl L. Jing X.J. Ivarsson R. Degerman E. Ahrén B. Manganiello V.C. Renström E. Stenson Holst L. J. Biol. Chem. 2002; 277: 37446-37455Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) for were and a PDE were as and PDE activity was in as described F. Manganiello V. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (173) Google Scholar) in the of 3 To increase the for PDE3 in the the PDE4 was when were at for In for 3 were with an of containing and a of and After of a was the and then 1 of was into the The was were 1, and The were in and on After was and at of was as described in experiments GLP-1 glucose in with 1 GLP-1 was In insulin for were with insulin and were at and the and were in and in PBS, in containing and on were and on were in and in were on and further were in PBS, containing and were at with the GLUT2, After in with with for and to were on the and for 1 at were in and then in were in to activity and with as was then to the and for by with for was with The of was with of of at by of was in was in were with a To islet were The pancreatic in each were at three levels with a of each with were at each of all islets for insulin within each were and the islet were was used for of the islet and islets were by a (21Härndahl L. Jing X.J. Ivarsson R. Degerman E. Ahrén B. Manganiello V.C. Renström E. Stenson Holst L. J. Biol. Chem. 2002; 277: 37446-37455Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) modified and M. Diabetes. PubMed Scopus Google Scholar). After in at glucose, of 3 islets were to and in of containing glucose at the with addition of GLP-1 for 1 at was then for of the of insulin of islets was by of 3 islets were in of in and on was and were at Perifusion of insulin release was in vitro the as described by G. Lundquist I. Ahren B. Physiol. PubMed Scopus (24) Google Scholar). In islets were for 1 at in was used the and at of islets were two of and at a of containing After the was to containing glucose, and to containing glucose and of was to were and for insulin and were by not were by the glucose glucose are as the of the number of significance was PDE3B in Mice—A cDNA construct was used to mice with a β-cell-specific overexpression of PDE3B. 11 mice were and for islet PDE3B revealed of overexpression, and in all not on these results and of islet PDE3 activity we two transgenic One a increase in islet PDE3B expression and activity with in islets of the line the increase is of transgene expression were in tissues, as both by and not and PDE3 activity of the two appear with regard to as to and with a of in the of the mice not differ transgenic and mice activity in tissues transgenic and mice adipose and were used to PDE3 are as the of three in a of mice at the of and were and to of for insulin and glucose were each glucose was the glucose, in a and in to and the of β-cell-targeted overexpression of PDE3B on insulin release, intravenous glucose were experiments revealed insulin secretory response and glucose in but not transgenic for further of mice were mice and of In glucose in glucose was not differ control mice In glucose was increased in mice for insulin of glucose but in of the transgenic with After intravenous of glucose the of insulin 1 was in both of transgenic mice with In mice the insulin response was of that in to in to in insulin levels at not A of insulin secretory response at 1 glucose was in mice as to the increase in insulin in to in to in a of the reduced insulin glucose was impaired in both and increased glucose levels were in of mice at and the glucose and in and in and In the in glucose at was with and the was reduced with end glucose levels in mice glucose the of of both similar results and we not of not and transgenic mice with regard to insulin secretory response glucose and that the described effects are for glucose glucose in mice and mice at the of were and an intravenous of glucose insulin and glucose levels at the were experiments with intravenous of were in mice at of are as the mice in each To the physiological insulin secretory response of mice to GLP-1 GLP-1 was with glucose to insulin in response to glucose these mice to glucose in with GLP-1 However, the insulin in mice was of that of in mice in mice this of insulin not to increase glucose disposal. glucose was in and in PDE3 enzyme activity in not levels To the a expression of the transgene specifically in have signaling and an we insulin experiments in the at 1 of glucose in the transgenic mice and control that insulin is not reduced in the were of the in vivo experiments and the secretory capacity was in with of 3 to large in the secretory response to glucose was reduced in islets mice in with islets insulin 3 islets of the PDE3 with glucose at glucose-stimulated insulin secretion. In the islets of transgenic mice addition of of the insulin secretory response to The response to GLP-1 in the of glucose a reduction in islets of mice with insulin 3 islets addition of the response was results were obtained in islets mice not The of secretory was with of islet insulin such islets were in of 3 insulin was increased in islets the mice with mice for islet protein the insulin was in islets of and the two of transgenic mice in study the insulin secretion in mice we experiments on in was a reduction in first phase insulin secretion with the of glucose, the secretory response of the transgenic islets was of as with for islets and The reduction was at all the first phase for the under the a reduction in first phase insulin release of in and of islets in reduced insulin response to glucose in transgenic as with islets not of PDE3B in islets and to islet for and was revealed that the transgenic mice increased islet with mice The increase was more pronounced in of of transgenic exhibited increased of islets with insulin with some islet insulin with control mice In of some islets more and insulin expression as well as more insulin large islet insulin and and and for insulin in and insulin with in for insulin and several more in the and with the for and more in and with in A is for and in is for To study the of in to the β-cells, for and insulin was in islets of control the were at the of the islets a of islet were in islets The of islets with this was in in type of islet has been in several models of impaired glucose M. 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S. S. J. T. H. Sharp G.W. Diabetes. 2002; 51: PubMed Google Scholar). have shown that PDE3B is and that in with the as was suggested experiments with β-cells with cAMP (21Härndahl L. Jing X.J. Ivarsson R. Degerman E. Ahrén B. Manganiello V.C. Renström E. Stenson Holst L. J. Biol. Chem. 2002; 277: 37446-37455Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), is to that PDE3B of cAMP directly in The reported of cAMP to 2 (8Ozaki N. Shibasaki T. Kashima Y. Miki T. Takahashi K. Ueno H. Sunaga Y. Yano H. Matsuura Y. Iwanaga T. Takai Y. Seino S. Nat. Cell Biol. 2000; 2: 805-811Crossref PubMed Scopus (401) Google Scholar) and nucleotide exchange L. Ma X. Renström E. Barg S. Berggren P.O. Galvanovskis J. Gromada J. Jing X. Lundquist I. Salehi A. Sewing S. Rorsman P. J. Gen. Physiol. 2003; 121: 181-197Crossref PubMed Scopus (217) Google Scholar, M. A. M. V. S. L. J. Diabetes. 2002; 51: PubMed Scopus Google Scholar), proteins that are in PKA-independent and suggested to with the such a have been to the role of PDE3B in this of the in regulatory mechanisms cAMP effects of cAMP be to at The for cAMP in by to proteins one of cAMP directly impair of In such a was suggested to the of an PKA-independent in mice L. Ma X. Renström E. Barg S. Berggren P.O. Galvanovskis J. Gromada J. Jing X. Lundquist I. Salehi A. Sewing S. Rorsman P. J. Gen. Physiol. 2003; 121: 181-197Crossref PubMed Scopus (217) Google Scholar). of cAMP by to adenylyl activity critical The known potentiating effect for pituitary adenylate cyclase-activating polypeptide on insulin secretion involves increased cAMP formation and was reported that mice the pituitary adenylate cyclase-activating reduced islet cAMP, insulin secretory response to glucose, and glucose intolerance F. K. G. N. R. J. Ahrén B. P. J. Clin. Invest. 2000; PubMed Scopus Google Scholar). In the study we in transgenic that increased degradation of cAMP by a phosphodiesterase may the insulin secretory In we show that β-cell-specific overexpression of PDE3B results in reduced insulin release and deranged islet cytoarchitecture associated with glucose intolerance. a role for β-cell PDE3B in energy homeostasis. expression of β-cell PDE3B in the PDE3B gene to the of as well as at of type 2 diabetes. In expression of PDE3B has been in adipose tissue with P. P. J. J. J. Clin. PubMed Scopus Google Scholar) and a study a coupling of a in of the human PDE3B gene to E. and L. of and by of PDE3 inhibitors has been 1999; Scopus Google Scholar). on the findings of the to further that of PDE3 inhibitors specifically β-cell PDE3B be and for