Sustained Expression of Exendin-4 Does Not Perturb Glucose Homeostasis, β-Cell Mass, or Food Intake in Metallothionein-Preproexendin Transgenic Mice
2000; Elsevier BV; Volume: 275; Issue: 44 Linguagem: Inglês
10.1074/jbc.m005119200
ISSN1083-351X
AutoresLaurie L. Baggio, Feisal A. Adatia, Troels Bock, Patricia L. Brubaker, Daniel J. Drucker,
Tópico(s)Neuropeptides and Animal Physiology
ResumoActivation of glucagon-like peptide (GLP)-1 receptor signaling promotes glucose lowering via multiple mechanisms, including regulation of food intake, glucose-dependent insulin secretion, and stimulation of β-cell mass. As GLP-1 exhibits a short t 12 in vivo, the biological consequences of prolonged GLP-1 receptor signaling remains unclear. To address this question, we have now generated metallothionein promoter-preproexendin (MT-Ex) transgenic mice. MT-Ex mice process preproexendin correctly, as is made evident by detection of circulating plasma exendin-4 immunoreactivity using high pressure liquid chromatography and an exendin-4-specific radioimmunoassay. Despite elevated levels of exendin-4, fasting plasma glucose and glucose clearance following oral and intraperitoneal glucose tolerance tests are normal in MT-Ex mice. Induction of transgene expression significantly reduced glycemic excursion during both oral and intraperitoneal glucose tolerance tests (p < 0.05) and increased levels of glucose-stimulated insulin following oral glucose administration (p < 0.05). Despite evidence that exendin-4 may induce β-cell proliferation, β-cell mass and islet histology were normal in MT-Ex mice. MT-Ex mice exhibited no differences in basal food intake or body weight; however, induction of exendin-4 expression was associated with reduced short term food ingestion (p < 0.05). In contrast, short term water intake was significantly reduced in the absence of zinc in fluid-restricted MT-Ex mice (p < 0.05). These findings illustrate that sustained elevation of circulating exendin-4 is not invariably associated with changes in glucose homeostasis, increased β-cell mass, or reduction in food intake in mice in vivo. Activation of glucagon-like peptide (GLP)-1 receptor signaling promotes glucose lowering via multiple mechanisms, including regulation of food intake, glucose-dependent insulin secretion, and stimulation of β-cell mass. As GLP-1 exhibits a short t 12 in vivo, the biological consequences of prolonged GLP-1 receptor signaling remains unclear. To address this question, we have now generated metallothionein promoter-preproexendin (MT-Ex) transgenic mice. MT-Ex mice process preproexendin correctly, as is made evident by detection of circulating plasma exendin-4 immunoreactivity using high pressure liquid chromatography and an exendin-4-specific radioimmunoassay. Despite elevated levels of exendin-4, fasting plasma glucose and glucose clearance following oral and intraperitoneal glucose tolerance tests are normal in MT-Ex mice. Induction of transgene expression significantly reduced glycemic excursion during both oral and intraperitoneal glucose tolerance tests (p < 0.05) and increased levels of glucose-stimulated insulin following oral glucose administration (p < 0.05). Despite evidence that exendin-4 may induce β-cell proliferation, β-cell mass and islet histology were normal in MT-Ex mice. MT-Ex mice exhibited no differences in basal food intake or body weight; however, induction of exendin-4 expression was associated with reduced short term food ingestion (p < 0.05). In contrast, short term water intake was significantly reduced in the absence of zinc in fluid-restricted MT-Ex mice (p < 0.05). These findings illustrate that sustained elevation of circulating exendin-4 is not invariably associated with changes in glucose homeostasis, increased β-cell mass, or reduction in food intake in mice in vivo. glucagon-like peptide GLP-1 receptor metallothionein promoter-preproexendin exendin-4-like immunoreactivity high pressure liquid chromatography Glucagon-like peptide-1 (GLP-1),1 a product of the proglucagon gene, is released from gut endocrine cells and potentiates glucose-dependent insulin secretion (1Drucker D.J. Diabetes. 1998; 47: 159-169Crossref PubMed Google Scholar). GLP-1 also regulates gastric emptying, food intake, glucagon secretion, and islet proliferation and hence is currently under investigation as a therapeutic agent for the treatment of diabetes (1Drucker D.J. Diabetes. 1998; 47: 159-169Crossref PubMed Google Scholar). However, a significant limitation to potential GLP-1 therapy in diabetic subjects is the short biological half-life of this peptide (2Kieffer T.J. McIntosh C.H.S. Pederson R.A. Endocrinology. 1995; 136: 3585-3596Crossref PubMed Scopus (0) Google Scholar, 3Deacon C.F. Johnsen A.H. Holst J.J. J. Clin. Endocrinol. Metab. 1995; 80: 952-957Crossref PubMed Google Scholar, 4Deacon C.F. Nauck M.A. Toft-Nielsen M. Pridal L. Willms B. Holst J.J. Diabetes. 1995; 44: 1126-1131Crossref PubMed Google Scholar), limiting its ability to control blood glucose for an extended period of time. These considerations have prompted investigation of strategies designed to prolong the duration of GLP-1 action in vivo (5Deacon C.F. Knudsen L.B. Madsen K. Wiberg F.C. Jacobsen O. Holst J.J. Diabetologia. 1998; 41: 271-278Crossref PubMed Scopus (264) Google Scholar, 6Holst J.J. Deacon C.F. Diabetes. 1998; 47: 1663-1670Crossref PubMed Scopus (453) Google Scholar).Exendin-4, a peptide structurally related to but distinct from GLP-1 (7Chen Y.E. Drucker D.J. J. Biol. Chem. 1997; 272: 4108-4115Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) was originally purified from the venom of a Heloderma suspectum lizard (8Eng J. Kleinman W.A. Singh L. Singh G. Raufman J.P. J. Biol. Chem. 1992; 267: 7402-7405Abstract Full Text PDF PubMed Google Scholar, 9Raufman J.-P. Regul. Peptides. 1996; 61: 1-18Crossref PubMed Scopus (65) Google Scholar). Subsequent characterization of exendin-4 activity demonstrated that the lizard peptide was a potent agonist for the mammalian glucagon-like peptide-1 receptor (GLP-1R) (8Eng J. Kleinman W.A. Singh L. Singh G. Raufman J.P. J. Biol. Chem. 1992; 267: 7402-7405Abstract Full Text PDF PubMed Google Scholar, 9Raufman J.-P. Regul. Peptides. 1996; 61: 1-18Crossref PubMed Scopus (65) Google Scholar, 10Schepp W. Schmidtler J. Riedel T. Dehne K. Schusdziarra V. Holst J.J. Eng J. Raufman J.-P. Classen M. Eur. J. Pharmacol. Mol. Pharmacol. 1994; 269: 183-191Crossref PubMed Scopus (45) Google Scholar, 11Young A.A. Gedulin B.R. Bhavsar S. Bodkin N. Jodka C. Hansen B. Denaro M. Diabetes. 1999; 48: 1026-1034Crossref PubMed Scopus (388) Google Scholar). Exendin-4 exhibits a much longer in vivo half-life and prolonged duration of action (11Young A.A. Gedulin B.R. Bhavsar S. Bodkin N. Jodka C. Hansen B. Denaro M. Diabetes. 1999; 48: 1026-1034Crossref PubMed Scopus (388) Google Scholar), rendering it more potent for continuous stimulation of GLP-1 receptor signaling and sustained improvement in glucose homeostasis in vivo. Despite the structural homology of lizard exendin-4 and mammalian GLP-1, a mammalian exendin-4 gene has not yet been identified (7Chen Y.E. Drucker D.J. J. Biol. Chem. 1997; 272: 4108-4115Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 12Pohl M. Wank S.A. J. Biol. Chem. 1998; 273: 9778-9784Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).The finding that exendin-4 represents a potent GLP-1 analogue has prompted studies of exendin-4 activity in normal and diabetic rodents. Exendin-4 potentiates glucose-stimulated insulin secretion and lowers blood glucose in both rats and mice (11Young A.A. Gedulin B.R. Bhavsar S. Bodkin N. Jodka C. Hansen B. Denaro M. Diabetes. 1999; 48: 1026-1034Crossref PubMed Scopus (388) Google Scholar, 13Greig N.H. Holloway H.W. De Ore K.A. Jani D. Wang Y. Zhou J. Garant M.J. Egan J.M. Diabetologia. 1999; 42: 45-50Crossref PubMed Scopus (196) Google Scholar, 14Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1080) Google Scholar, 15Zhou J. Wang X. Pineyro M.A. Egan J.M. Diabetes. 1999; 48: 2358-2366Crossref PubMed Scopus (301) Google Scholar, 16Stoffers D.A. Kieffer T.J. Hussain M.A. Drucker D.J. Egan J.M. Bonner-Weir S. Habener J.F. Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (514) Google Scholar). Exendin-4 also inhibits food and water intake, raising the possibility that chronic exendin-4 treatment may decrease satiety and promote weight lossin vivo (17Tang-Christensen M. Larsen P.J. Goke R. Fink-Jensen A. Jessop D.S. Moller M. Sheikh S.P. Am. J. Physiol. 1996; 271: R848-R856PubMed Google Scholar, 18Szayna M. Doyle M.E. Betkey J.A. Holloway H.W. Spencer R.G. Greig N.H. Egan J.M. Endocrinology. 2000; 141: 1936-1941Crossref PubMed Scopus (283) Google Scholar). Furthermore, recent studies demonstrate that exendin-4 administration leads to induction of pancreatic endocrine cell differentiation, islet proliferation, and expansion of β-cell mass (11Young A.A. Gedulin B.R. Bhavsar S. Bodkin N. Jodka C. Hansen B. Denaro M. Diabetes. 1999; 48: 1026-1034Crossref PubMed Scopus (388) Google Scholar, 13Greig N.H. Holloway H.W. De Ore K.A. Jani D. Wang Y. Zhou J. Garant M.J. Egan J.M. Diabetologia. 1999; 42: 45-50Crossref PubMed Scopus (196) Google Scholar, 14Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1080) Google Scholar, 15Zhou J. Wang X. Pineyro M.A. Egan J.M. Diabetes. 1999; 48: 2358-2366Crossref PubMed Scopus (301) Google Scholar, 16Stoffers D.A. Kieffer T.J. Hussain M.A. Drucker D.J. Egan J.M. Bonner-Weir S. Habener J.F. Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (514) Google Scholar).Although the biological activities of exendin-4 and GLP-1 have been examined in numerous short term studies, limited information is available regarding the physiological actions of these peptides in experimental paradigms characterized by prolonged exposure to increased levels of GLP-1R agonists. To assess the feasibility and physiological effects of chronic expression of lizard exendin-4 in vivo, we have generated transgenic mice in which lizard exendin-4 expression is under the control of the mouse metallothionein I promoter. We now report the characterization and metabolic consequences of sustained exendin-4 expression in mice in vivo.RESULTSTo study the generation of MT-exendin transgenic mice, we used a 1.9-kilobase fragment (Fig. 1) containing the following: (i) 770 base pairs of the mouse metallothionein I promoter (including 5′ flanking and exon 1 sequences) (27Palmiter R.D. Norstedt G. Gelinas R.E. Hammer R.E. Brinster R.L. Science. 1983; 222: 809-814Crossref PubMed Scopus (480) Google Scholar), (ii) the 492-base pair lizard proexendin-4 cDNA (7Chen Y.E. Drucker D.J. J. Biol. Chem. 1997; 272: 4108-4115Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), and (iii) 625 base pairs of the human growth hormone gene (containing the polyadenylation signal and 3′-flanking sequences) (28Seeburg P.H. DNA. 1982; 1: 239-249Crossref PubMed Scopus (213) Google Scholar). Transgenic mice were identified by Southern blot analysis (data not shown). Male and female MT-exendin transgenic mice were viable and fertile and appeared to develop normally.Northern blot analysis detected transgene expression in several tissues, including heart, duodenum, jejunum, colon, and adipose tissue (data not shown). Tissue and plasma extracts from MT-exendin mice were analyzed by radioimmunoassay for exendin-4-like immunoreactivity (Ex-4-IR) using exendin-4 antiserum generated in our laboratory. 2D. J. Drucker and P. L. Brubaker, unpublished observations. The exendin-4 antiserum used for these studies does not cross-react with glucagon, glicentin, oxyntomodulin, gastric inhibitory polypeptide, vasoactive intestinal polypeptide, GLP-1, or GLP-2, nor does it require a free N terminus for binding. 3P. L. Brubaker, manuscript in preparation. In wild-type nontransgenic mice, basal levels of Ex-4-IR were less than 27 pg/ml. In contrast, basal plasma levels of Ex-4-IR were 434 ± 39 and 330 ± 84 pg/ml in male and female transgenic mice, respectively (Fig. 2 A), and induction of transgene expression with zinc treatment resulted in an ∼2.5-fold increase in the circulating levels of Ex-4-IR in both male and female mice (p < 0.01, Fig.2 A).Figure 2Detection of exendin-4-like immunoreactivity (exendin IR) in the plasma of transgenic mice. A, radioimmunoassay for detection of exendin-like immunoreactivity in plasma from control littermates (nontransgenic) and transgenic male (MT-Ex M) and female (MT-Ex F) mice. Mice were given either standard drinking water (–Zn) or water supplemented with 25 mm ZnSO4(+Zn) to up-regulate transgene expression. Zinc supplementation was for a period of 72 h. Values are expressed as means ± S.E. **, p < .01, transgenicversus control (nontransgenic). B, HPLC elution profile of exendin-like immunoreactivity extracted from the plasma of a 4-month-old zinc-treated MT-exendin male mouse. The elution position of synthetic exendin-4 is indicated by the arrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether preproexendin was both processed appropriately and secreted into the circulation, HPLC and radioimmunoassay analyses were used to characterize the molecular forms of circulating Ex-4-IR. The major exendin-immunoreactive peptide detected in plasma extracts from MT-exendin-4 transgenic mice eluted at the same position as synthetic exendin-4 (Fig. 2 B). Significant amounts of exendin-4-immunoreactivity eluting in the same position as synthetic exendin-4 were also detected in several tissues.3As GLP-1 receptor signaling is essential for control of blood glucose and glucose-stimulated insulin secretion (1Drucker D.J. Diabetes. 1998; 47: 159-169Crossref PubMed Google Scholar), we examined these parameters in control and MT-exendin transgenic mice. Fasting blood glucose levels were normal in MT-exendin mice under conditions of either basal or induced transgene expression (Fig.3). Despite clearly detectable levels of circulating exendin-4 immunoreactivity, blood glucose excursion and glucose-stimulated insulin was comparable in +/+ and MT-Ex transgenic mice following either oral (Fig. 3 A) or intraperitoneal (Fig. 3 C) glucose challenge. In contrast, induction of transgene expression with zinc treatment resulted in a significant reduction in glycemic excursion following oral (Fig. 3 B) and intraperitoneal (Fig. 3 D) glucose loading. The reduced glycemic excursion was associated with a significant increase in plasma levels of glucose-stimulated insulin after oral but not intraperitoneal glucose challenge (0.38 ± 0.04versus 0.21 ± 0.02 ng/ml, for insulin in Mt-Exversus control mice, respectively; Fig. 3 B).Figure 3Oral and intraperitoneal glucose tolerance and levels of plasma insulin in control and MT-exendin transgenic female mice. Values, averaged over three independent experiments, are expressed as means ± S.E.; n = 8–12 mice/group. *, p < 0.05, transgenic versuscontrol mice. A, oral glucose tolerance in control (open circles) and MT-exendin (solid squares) mice. Plasma insulin concentrations (inset) following oral glucose in control (open bar) and MT-exendin (solid bar) mice were measured in plasma obtained at the 10–20 min time point. B, oral glucose tolerance in control (open circles) and MT-exendin (solid squares) mice following treatment with 25 mm ZnSO4 to up-regulate transgene expression. Plasma insulin concentrations (inset) in control (open bar) and MT-exendin (hatched bar) mice were obtained at the 10–20 min time point following oral glucose. C, intraperitoneal glucose tolerance in control (open circles) and MT-exendin (solid squares) mice. Plasma insulin concentration (inset) were obtained at the 10–20 min time point following intraperitoneal glucose in control (open bar) and MT-exendin (solid bar) mice. D, intraperitoneal glucose tolerance in control (open circles) and MT-exendin (solid squares) mice following treatment with 25 mmZnSO4 to up-regulate transgene expression. Plasma insulin concentrations (inset) were measured in samples obtained at the 10–20 min time point following intraperitoneal glucose in control (open bar) and MT-exendin (hatched bar) mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The physiological importance of GLP-1 receptor signaling for central nervous system control of food intake and body weight remains unclear (29Seeley R.J. Woods S.C. D'Alessio D. Endocrinology. 2000; 141: 473-475Crossref PubMed Scopus (7) Google Scholar). Administration of intracerebroventricular GLP-1 or exendin-4 inhibits short term feeding, whereas repeated administration of the GLP-1 receptor antagonist exendin (9–39) increases food intake and promotes weight gain in rats (30Turton M.D. O'Shea D. Gunn I. Beak S.A. Edwards C.M.B. Meeran K. Choi S.J. Taylor G.M. Heath M.M. Lambert P.D. Wilding J.P.H. Smith D.M. Ghatei M.A. Herbert J. Bloom S.R. Nature. 1996; 379: 69-72Crossref PubMed Scopus (1563) Google Scholar, 31Meeran K. O'Shea D. Edwards C.M. Turton M.D. Heath M.M. Gunn I. Abusnana S. Rossi M. Small C.J. Goldstone A.P. Taylor G.M. Sunter D. Steere J. Choi S.J. Ghatei M.A. Bloom S.R. Endocrinology. 1999; 140: 244-250Crossref PubMed Scopus (234) Google Scholar). In contrast, mice with complete disruption of GLP-1R signaling do not exhibit defects in feeding control or body weight homeostasis (32Scrocchi L.A. Brown T.J. MacLusky N. Brubaker P.L. Auerbach A.B. Joyner A.L. Drucker D.J. Nat. Med. 1996; 2: 1254-1258Crossref PubMed Scopus (654) Google Scholar, 33Scrocchi L.A. Drucker D.J. Endocrinology. 1998; 139: 3127-3132Crossref PubMed Scopus (64) Google Scholar). Basal levels of exendin expression had no effect on short term (2 h) or long term (24 h) food intake (Fig. 4, A andB). However, up-regulation of transgene expression following zinc treatment led to a small but significant reduction in short term (2 h) food intake (0.026 ± 0.003 g/g of body weight in transgenicversus 0.034 ± 0.001 g/g of body weight in control mice; p < 0.05; Fig. 4, C andD). Basal levels of transgene expression were also associated with a significant reduction in short term (up to 2 h) water intake (Fig. 5, A andB). In contrast to recent studies demonstrating weight loss in exendin-treated rats (18Szayna M. Doyle M.E. Betkey J.A. Holloway H.W. Spencer R.G. Greig N.H. Egan J.M. Endocrinology. 2000; 141: 1936-1941Crossref PubMed Scopus (283) Google Scholar), no significant differences in body weight were observed in MT-Ex transgenic mice compared with nontransgenic littermates at 4, 8, 16, or 20 weeks of age (data not shown).Figure 4Food intake in control and MT-exendin mice. Following an overnight fast, food intake was monitored during specific time intervals (A and C) as well as cumulatively (B and D) for a total period of 24 h in control (open bars) and MT-exendin transgenic (solid or hatched bars) mice. +Zndenotes mice treated with zinc supplementation as described under “Materials and Methods.” Values are expressed as means ± S.E.; n = 6 mice/group. *, p < 0.05, transgenic versus control mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Water intake in control and MT-exendin mice. Following a 13-h period of water deprivation, water intake was monitored during specific time intervals (A andC), as well as cumulatively (B and D) for a total period of 24 h in control (open bars) and MT-exendin transgenic (solid or hatched bars) mice. +Zn denotes mice treated with zinc supplementation as described under “Materials and Methods.” Values are expressed as means ± S.E.; n = 5–7 mice/group. *, p < 0.05, transgenic versuscontrol mice. Zinc supplementation alone decreased water intake in both control and transgenic mice (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Increasing evidence suggests that both GLP-1 and exendin-4 stimulate β-cell replication and neogenesis, enhance islet size, and promote differentiation of pancreatic precursor cells into islet cells (14Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1080) Google Scholar, 15Zhou J. Wang X. Pineyro M.A. Egan J.M. Diabetes. 1999; 48: 2358-2366Crossref PubMed Scopus (301) Google Scholar, 16Stoffers D.A. Kieffer T.J. Hussain M.A. Drucker D.J. Egan J.M. Bonner-Weir S. Habener J.F. Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (514) Google Scholar,34Edvell A. Lindstrom P. Endocrinology. 1999; 140: 778-783Crossref PubMed Scopus (132) Google Scholar). To examine the effects of transgene expression on islet growth, we examined pancreata from MT-exendin transgenic mice. Islet histology and islet cell numbers appeared normal and comparable in transgenic (Fig.6 B) and +/+ control mice (Fig.6 A), with no evidence for islet neogenesis or abnormal distribution of endocrine cell types within the islets. Furthermore, quantitative analysis demonstrated no differences in β-cell mass in MT-Ex transgenic compared with +/+ control mice (Fig.6 C).Figure 6Normal islet morphology and β-cell mass in MT-exendin transgenic mice.Hematoxylin & eosin (H/E) and immunohistochemical staining for glucagon and insulin in the pancreatic islets of control (A) and MT-exendin transgenic (B) mice. Pancreata were obtained from control and transgenic animals that were given either standard drinking water (Zn–) or water supplemented with 25 mm ZnSO4 (Zn+) for 5–7 days to up-regulate transgene expression. C, β-cell mass in control (open bars) and MT-exendin transgenic (solid bars) mice. Values are expressed as means ± S.E.;n = 3–8 mice/group. All mice were maintained on water supplemented with 25 mm ZnSO4 for 5–7 days to up-regulate transgene expression.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe observation that GLP-1 exhibits a very short plasma half-life due to its rapid degradation by dipeptidyl peptidase IV (2Kieffer T.J. McIntosh C.H.S. Pederson R.A. Endocrinology. 1995; 136: 3585-3596Crossref PubMed Scopus (0) Google Scholar, 3Deacon C.F. Johnsen A.H. Holst J.J. J. Clin. Endocrinol. Metab. 1995; 80: 952-957Crossref PubMed Google Scholar) has prompted a search for DP IV-resistant GLP-1 analogues that exhibit longer durations of action and enhanced potency in vivo. Several GLP-1 analogues have now been reported that exhibit improved potency in both normal and diabetic rodents (5Deacon C.F. Knudsen L.B. Madsen K. Wiberg F.C. Jacobsen O. Holst J.J. Diabetologia. 1998; 41: 271-278Crossref PubMed Scopus (264) Google Scholar, 35Siegel E.G. Gallwitz B. Scharf G. Mentlein R. Morys-Wortmann C. Folsch U.R. Schrezenmeir J. Drescher K. Schmidt W.E. Regul. Pept. 1999; 79: 93-102Crossref PubMed Scopus (84) Google Scholar). Furthermore, fatty acid derivatives of GLP-1 may also result in enhanced albumin binding and more prolonged bioactivity in vivo (36Knudsen L.B. Nielsen P.F. Huusfeldt P.O. Johansen N.L. Madsen K. Pedersen F.Z. Thogersen H. Wilken M. Agerso H. J. Med. Chem. 2000; 43: 1664-1669Crossref PubMed Scopus (544) Google Scholar). The naturally occurring lizard exendin-4 peptide is not a substrate for DP IV and consequently exhibits a much longer half-life and greater potencyin vivo (9Raufman J.-P. Regul. Peptides. 1996; 61: 1-18Crossref PubMed Scopus (65) Google Scholar, 11Young A.A. Gedulin B.R. Bhavsar S. Bodkin N. Jodka C. Hansen B. Denaro M. Diabetes. 1999; 48: 1026-1034Crossref PubMed Scopus (388) Google Scholar, 13Greig N.H. Holloway H.W. De Ore K.A. Jani D. Wang Y. Zhou J. Garant M.J. Egan J.M. Diabetologia. 1999; 42: 45-50Crossref PubMed Scopus (196) Google Scholar).GLP-1 and exendin-4 have been administered daily to humans and diabetic rodents for periods of up to several weeks (11Young A.A. Gedulin B.R. Bhavsar S. Bodkin N. Jodka C. Hansen B. Denaro M. Diabetes. 1999; 48: 1026-1034Crossref PubMed Scopus (388) Google Scholar, 13Greig N.H. Holloway H.W. De Ore K.A. Jani D. Wang Y. Zhou J. Garant M.J. Egan J.M. Diabetologia. 1999; 42: 45-50Crossref PubMed Scopus (196) Google Scholar, 16Stoffers D.A. Kieffer T.J. Hussain M.A. Drucker D.J. Egan J.M. Bonner-Weir S. Habener J.F. Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (514) Google Scholar, 18Szayna M. Doyle M.E. Betkey J.A. Holloway H.W. Spencer R.G. Greig N.H. Egan J.M. Endocrinology. 2000; 141: 1936-1941Crossref PubMed Scopus (283) Google Scholar, 37Todd J.F. Wilding J.P. Edwards C.M. Ghatei M.A. Bloom S.R. Eur. J. Clin. Invest. 1997; 27: 533-536Crossref PubMed Scopus (99) Google Scholar, 38Wang Y. Perfetti R. 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Although studies of the molecular determinants of preproexendin-4 processing have not yet been reported, the finding of detectable levels of circulating exendin-4 in MT-exendin transgenic mice is consistent with the correct processing and secretion of the lizard preproexendin precursor in murine tissues in vivo. Furthermore, the levels of circulating bioactive exendin-4 detected in MT-exendin-4 transgenic mice are clearly much higher than plasma levels of less potent GLP-1 (1Drucker D.J. Diabetes. 1998; 47: 159-169Crossref PubMed Google Scholar) and are certainly within the range of or higher than the plasma levels of exendin-4 noted to decrease blood glucose in diabetic db/db mice (11Young A.A. Gedulin B.R. Bhavsar S. Bodkin N. Jodka C. Hansen B. Denaro M. Diabetes. 1999; 48: 1026-1034Crossref PubMed Scopus (388) Google Scholar, 40Bhavsar S. Lachappell R. Watkins J. Young A. Diabetes. 1998; 47 (:0746): A192Crossref PubMed Scopus (24) Google Scholar). 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Rev. 1999; 20: 876-913Crossref PubMed Google Scholar).Although incretins such as gastric inhibitory polypeptide and GLP-1 have been proposed as possible tre
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