Articles in PresS. Am J Physiol Endocrinol Metab (April 9, 2013). doi:10.1152/ajpendo.00600.2012
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GLP-1(28–36) improves beta cell mass and glucose disposal in streptozotocin induced
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diabetes mice and activates cAMP-PKA-beta-catenin signaling in beta-cells in vitro
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Weijuan Shao1, Zhaoxia Wang2, Wilfred Ip1,3, Yu-Ting Chiang1,4, Xiaoquan Xiong1,Tuanyao
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Chai1,5, Catherine Xu1, Qinghua Wang2,3,4, and Tianru Jin1,3,4 *
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Div. of Advanced Diagnostics, Toronto General Research Institutes, University Health Network.
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Div. of Endocrinology and Metabolism, the Keenan Research Centre in the Li Ka Shing
Knowledge Institute, St. Michael’s Hospital. 3Institute of Medical Science, University of Toronto.
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Dept. of Physiology, University of Toronto. 5College of Life Science,
Graduate University of Chinese Academy of Sciences, P. R. China.
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Running title: GLP-1(28–36)amide activates beta-catenin
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*: Corresponding author: Tianru Jin, MaRS Centre, Toronto Medical Discovery Tower, Room
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10-354 101 College Street, Toronto, ON, Canada, M5G 1L7, Tel: 416-581-7670
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E-mail: [email protected]
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Copyright © 2013 by the American Physiological Society.
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Abstract (200 words)
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Recent studies have demonstrated that the C-terminal fragment of the incretin hormone
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glucagon-like peptide-1 (GLP-1), a nonapeptide GLP-1(28–36)amide, attenuates diabetes and
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hepatic steatosis in diet-induced obese mice. However, the effect of this nonapeptide in
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pancreatic β-cells remains largely unknown. Here, we show that in a streptozotocin-induced
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mouse diabetes model, GLP-1(28–36)amide improved glucose disposal, increased pancreatic β-
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cell mass and β-cell proliferation. In vitro investigation revealed that GLP-1(28-36)amide
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stimulates β-catenin (β-cat) Ser675 phosphorylation in both the clonal INS-1 cell line and rat
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primary pancreatic islet cells. In INS-1 cells, the stimulation was accompanied by increased
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nuclear β-cat content. GLP-1 (28-36)amide was also shown to increase cellular cAMP levels,
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PKA enzymatic activity and cAMP response element-binding protein (CREB) and cyclic AMP-
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dependent transcription factor-1 (ATF-1) phosphorylation. Furthermore, GLP-1(28–36)amide
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treatment enhanced islet insulin secretion and increased the growth of INS-1 cells, associated
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with increased cyclin D1 expression. Finally, PKA inhibition attenuated the effect of GLP-1(28–
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36)amide on β-cat Ser675 phosphorylation and cyclin D1 expression in the INS-1 cell line. We
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have thus revealed the beneficial effect of GLP-1(28–36)amide in pancreatic β-cells in vitro and
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in vivo. Our observations suggest that GLP-1(28–36)amide may exert its effect through the
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PKA/β-catenin signaling pathway.
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Introduction
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The proglucagon gene (gcg) encodes both the pancreatic hormone glucagon and the gut incretin
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hormone glucagon-like peptide-1 (GLP-1) (15, 17). The exploration of mechanisms underlying
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the function of GLP-1 and another incretin hormone gastric inhibitory polypeptide (GIP) has led
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to the development of two categories of novel therapeutic agents, namely GLP-1 analogues and
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DPP-IV inhibitors, for diabetes and potentially its complications (11, 12). In addition to target
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pancreatic β-cells, GLP-1 also exerts its function in many other organs or tissues (1, 4, 7, 40).
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The GLP-1 receptor (GLP-1R) deficient mouse line has been utilized as a powerful tool in
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studying the function of GLP-1 not only in pancreatic β-cells, but also in a number of organs that
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are importantly involved in glucose disposal and metabolic homeostasis (7). However, studies
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with this mouse model, along with investigations with other tools suggested that certain
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functions of GLP-1 may not be mediated by its canonical receptor GLP-1R, while a previously
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assumed inactive form of GLP-1, namely GLP-1(9-36)amide, has the therapeutic potential in
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certain cardiovascular disorders (2, 25).
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Both GLP-1(7-36)amide and GLP-1(7-37) are considered as active incretin hormone. For
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convenience, we will use the term GLP-1(7-36) to represent both of them hereafter. GLP-1 (7-
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36)amide and GLP-1(9-36)amide can be further cleaved by the neutral endopeptidase, NEP24.11
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(neprilysin or CD10), leading to the generation of the C-terminal nonapeptide GLP-1(28-
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36)amide (FIAWLVKGR) (13). A few recent studies have shown that GLP-1(28-36)amide is
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able to enter mouse hepatocytes in a GLP-1R independent manner, target to the mitochondria,
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and inhibit gluconeogenesis and oxidative stress (38). In a high fat diet fed mouse model, the
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administration of GLP-1(28-36)amide inhibited weight gain and attenuated diabetes and hepatic
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steatosis (37).
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The involvement of the Wnt signaling pathway in metabolic homeostasis has drawn our
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attention in the past few years (5, 8, 9, 30, 31). The major effector of the canonical Wnt signaling
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pathway (defined as Wnt pathway hereafter) is cat/TCF, formed by free β-catenin (β-cat) and a
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member of T cell factor (TCF) transcription factor family (15). Evidently, cat/TCF7L2 plays
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important roles in both the production and the function of GLP-1 (21, 24, 31, 43). GLP-1 and the
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GLP-1 receptor (GLP-1R) agonist exendin-4 have been shown to stimulate β-cat Ser675. Here,
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we have assessed the effect of GLP-1(28-36)amide both in vivo in a streptozotocin (STZ)
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induced type 1 diabetes mouse model and in vitro in rodent pancreatic β-cells. We report here
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that in vivo GLP-1(28-36)amide administration improved glucose disposal, associated with
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increased β-cell proliferation and pancreatic β-cell mass. In vitro treatment of a pancreatic β-cell
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line with GLP-1(28-36)amide stimulated the activity of both PKA and the Wnt signaling
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pathway effector β-cat, associated with the increase of cyclin D1 expression and cell growth. In
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isolated rat pancreatic islets, GLP-1(28-36)amide also synergizes with high glucose on insulin
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secretion. Our observations suggest that GLP-1(28-36)amide plays an important role in
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modulating β-cell growth and function, involving PKA and β-cat signaling cascades.
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Materials and Methods
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Cell cultures and reagents. The rat pancreatic insulin producing β-cell line INS-1 832/13
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(INS-1) and primary rat islets were cultured in RPMI 1640 supplemented with 10% fetal bovine
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serum (FBS), 1% penicillin streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM
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Hepes, and 0.05 mM 2-mercaptoethanol, as we reported previously (33). The pancreatic α-cell
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line α-TC was maintained in Dulbecco’s Minimal Essential Medium (DMEM) with 5% fetal
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bovine serum. GLP-1(28–36)amide (FIAWLVKGRamide) was provided by Biomatik
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Corporation (Wilmington, Delaware, USA). The product was >98% valid peptide, assessed by
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HPLC and mass spectrometry analyses. Tissue culture medium, fetal bovine serum, forskolin, 3-
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Isobutyl-1-methylxanthine (IBMX), as well as the PKA inhibitors KT-5720 and H89 were
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purchased from Sigma-Aldrich (Oakville, Ontario, Canada). Antibodies for β-cat (Ser 675),
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CREB (Ser133) and β-actin were obtained from Cell Signaling Technology (Beverly, MA).
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Antibodies for β-cat and cyclin D1 were purchased from Santa Cruz Biotechnology (Santa Cruz,
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CA). TxNIP antibody was the product of MBL International Corporation (Woburn, MA). 3-
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[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) was the product of Sigma-
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Aldrich (Oakville, Ontario, Canada).
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Western blotting analysis. Methods for Western blotting against either whole cell lysate
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(~30 μg protein) or the nuclear fraction (~10 μg protein), using SDS-polyacrylamide gel
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electrophoresis (SDS-PAGE, 10%) for protein separation, has been described in our previous
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studies (44). ECL Western Blotting Detection kit (Thermo Scientific, Rockford, U.S.A) was used
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for signal detection. Nuclear and cytoplamic fractions of INS-1 cells were prepared using NE-
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PER nuclear and cytoplasmic extraction reagents provided by Thermo Scientific (Rockford,
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U.S.A).
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Cell growth measurement. MTT assay was used to evaluate the cell growth as previously
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described (42). Briefly, cells of a defined cell line were seeded in 96-well plates (approximately
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5000 cells per well) with or without 50 nM GLP-1(28-36) for 48 h, followed by the addition of
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MTT. After the incubation for 2 h, the plate was read by a spectrophotometer.
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Rat islets isolation. Rat pancreatic islets were isolated by collagenase (C7565, Sigma
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Aldrich) digestion as previously described (23, 34). Briefly, rat islets were isolated from Sprague
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Dawley rats (250–350 g) by perfusion of the pancreas through the common bile duct with 10 ml
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of a collagenase solution and incubation of the excised pancreas at 37°C. The digestion was
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washed, filtered through a 355 mm mesh, and separated on a density gradient. Islets were then
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hand-picked for a given experiment.
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Insulin secretion assay. INS-1 cells seeded on a 12-well plate were washed with the KRB
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buffer (129 mMNaCl/5 mM NaHCO3/4.8 mM KCl/1.2 mM KH2PO4/1.2 mM MgSO4/2.5 mM
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CaCl2/) and then incubated with the KRB buffer containing 2.8 mM glucose and 0.1% BSA for
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30 min before the assay. Insulin secretion from INS-1 cells was performed in the KRB buffer
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containing 2.8 mM or 20 mM glucose with or without GLP-1(28-36)amide (50 nM) at 37°C in a
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humidified incubator. The supernatants were collected and detected by insulin enzyme
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immunoassay kit (EMD Millipore, Billerica, MA) according to the manufacturer’s instructions.
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For assessing insulin secretion in rat islets, ten hand-picked islets were utilized per group. The
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islets were pre-incubated for 30min in 2.8 mM glucose KRB, followed by 1 h incubation with
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either 2.8 mM or 20 mM glucose containing KRB, in the presence or absence of 50 nM GLP-
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1(28-36). The supernatant fractions were collected for insulin level measurement.
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cAMP and PKA activity assays. Cytoplasmic cAMP levels in INS-1 cells or rat islets were
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determined with the Cyclin AMP EIA kit (Cayman Chemical Company, Ann Arbor, Michigan),
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following the manufacturer’s instructions. PKA activity in the INS1 cells was measured with the
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Peptag Assay kit (Promega, Madison, WI), with some minor modifications. Briefly, cells were
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lyzed in ice-cold PKA extraction buffer containing 25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA,
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0.5 mM EGTA, 10 mM β-mercaptoethanol, and the protease inhibitor cocktail. Cell lysates were
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collected by centrifugation (13,000 rpm for 5 min at 4°C). Lysate containing 2 µg total protein
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was taken for reaction with the PepTag A1 peptide for 30 min at 37°C. The reaction was stopped
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by incubation at 95 °C for 10 min. Samples were then run on a 0.8% agarose gel for the
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separation of phosphorylated and un-phosphorylated PKA substrate PepTag A1 peptide.
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The generation of STZ induced type 1 diabetes model. Male C57BL/6 J mice (at age 7
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wks) from Charles River (St. Laurent, Québec, Canada) were housed 5 per cage under the
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conditions of ambient temperature (22°C), a light/dark cycle of 12 h with free access to food and
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water. The mice were fasted for 4 h before the injection of STZ intraperitoneally (50 mg/kg per
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day) for 5 consecutive days. Diabetic hyperglycemia was confirmed by measuring blood glucose
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using glucometer. At the beginning of the 4th week after STZ administration, GLP-1(28-
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36)amide, or exendin-4, or vehicle was injected intraperitoneally once daily for 9 weeks. All the
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animal experiments were performed in accordance with the Guide for Care and Use of
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Experimental Animals (University Health Network).
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Intraperitoneal glucose and pyruvate tolerance test. Mice were fasted overnight for
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intraperitoneal glucose tolerance test (IPGTT); or fasted for 6 h for intraperitoneal pyruvate
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tolerance test (IPPTT), as we have reported previously (32). Briefly, following the fasting,
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glucose (0.4g/kg or 2g/kg) or pyruvate (2g/kg) was intraperitoneally injected. Blood samples
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collected from tail vein at indicated time points were used for glucose measurement (Roche
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Accu-Chek).
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β-cell mass and β-cell proliferation analysis. The whole pancreas was stretched on a
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paper towel and fixed, along with the paper towel, with 4% Paraformaldehyde (PFA) overnight.
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The pancreas was then divided into six similar sized segments for paraffin embedding.
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Immunohistochemically stained pancreatic sections (12 µM) for insulin or glucagon were
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scanned at 40X maginification by NanoZoomer 2.0RS and analyzed with VisionPharm
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Integrator System for β-cell or α-cell area. The percentage of β-cell or α-cell area per whole
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pancreas area was then calculated. β- or α-cell mass was determined by multiply these
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percentage numbers with whole pancreas weight (3).
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Proliferative islet β cells were identified in the pancreatic section double stained for
insulin and BrdU, presented as percentage of double positive cells per insulin positive cells.
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Statistical analysis. All data in this study were presented as means +/- SE. Statistical
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analysis was performed with Student t-test or ANOVA with tukey’s post hoc test as appropriate.
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Significance was assumed as a P value of less than 0.05.
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Results
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GLP-1 (28-36)amide improves glucose disposal in the STZ induced mouse diabetes model.
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To assess the effect of GLP-1(28-36)amide on pancreatic β-cells in vivo, we have generated an
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STZ-induced β-cell injury type 1 diabetic mouse model. Three weeks after STZ injection, all
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mice developed hyperglycemia as determined by ambient plasma glucose levels (> 22 mM)
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using glucometer. The mice were then received daily i.p. injection of vehicle (PBS), GLP-1(28-
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36)amide (18 nmol/kg) or exendin-4 (24nmol/kg) for 9 weeks. At the beginning of the sixth
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week, IPGTT were conducted. No improvement was observed for mice receiving either GLP-
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1(28-36)amide or exendin-4 injection. At the first day of the seventh week, IPPTT were
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performed. As shown in Fig. 1A, the mice receiving GLP-1 (28-36)amide injection showed
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improved intolerance to pyruvate injection. Blood samples were collected at the beginning of the
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eighth week and ambient plasma glucose levels were measured. Although we observed a trend of
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reduced ambient plasma glucose level in mice receiving GLP-1(28-36)amide) or exendin-4
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injection, when compared with the mice with PBS injection, the difference did not reach
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statistical significance (data not shown). At the first day of the ninth week, we conducted another
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IPGTT. As shown in Fig. 1B, improved intolerance to glucose challenge was observed for mice
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receiving GLP-1(28-36)amide injection. However, when area under the curve (AUC) was
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calculated, the P value between the control PBS group and the GLP-1(28-36) group is 0.05. At
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the end of the ninth week after an overnight fasting, we measured plasma insulin and glucose
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levels. As shown in Fig. 1C and 1D, GLP-1(28-36)amide injection significantly reduced fasting
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glucose levels and increased basal insulin levels. Significant changes on glucose and insulin
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levels were not observed in this experiment with exendin-4 injection, although we did see trends
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in the improvement (Fig. 1D and 1E). This is likely due to the relatively big variation among the
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individual extendin-4 injected animals.
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GLP-1(28-36)amide improves β-cell mass and β-cell proliferation in the STZ induced
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diabetic mice. At the first day of the tenth week, the mice were injected with BrdU 24 h before
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they were sacrificed followed by the measurement of β-cell mass and β-cell BrdU incorporation.
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Fig. 2A shows representative images of pancreatic islets immunostained with the insulin
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antibody. Fig. 2B shows that when compared with the PBS injection, GLP-1(28-36)amide
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injection increased β-cell mass for nearly 2 fold. The β-cell mass of mice receiving exendin-4
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injection was also found to be increased approximately 2 fold. GLP-1(28-36)amide and exendin-
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4 injection also significantly increased the incorporation of BrdU into the pancreatic β-cells (Fig.
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2C and 2D).
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GLP-1(28-36)amide stimulates β-cat Ser675 phosphorylation and PKA activation. β-cat
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Ser675 phosphorylation has been suggested to mediate the crosstalk between cAMP/PKA and
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Wnt signaling pathways (10, 35). GLP-1(7-36)amide and the GLP-1R agonist exendin-4 were
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shown to stimulate β-cat Ser675 phosphorylation in pancreatic β-cells (21). We found that in
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INS-1 cells, 50 nM GLP-1 (28-36)amide simulated β-cat Ser675 phosphorylation (Fig. 3A). The
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stimulation occurred as early as 5 min after the treatment and sustained for at least 60 min (Fig.
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3B). Similar activation was also observed for pancreatic islets isolated from adult rats (Fig. 3B).
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When pancreatic α-cell line α-TC (Fig. 3C and 3D) and the human 293T fibroblasts (Fig. 3E)
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were tested, no or very moderate stimulation on β-cat Ser675 phosphorylation by GLP-1(28-36)
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was observed. In the α-TC cell line, forskolin treatment increased β-cat Ser675 phosphorylation
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(Fig. 3C). In α-TC cells, we have also tested the effect of GLP-1(28-36) at different dosages (1-
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100 nM), and did not observe an appreciable stimulatory effect on β-cat Ser675 phosphorylation
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(Fig. 3D). Finally, we found that in INS-1 cells, GLP-1(28-36) or forskolin treatment increased
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nuclear β-cat content (Fig. 3F More profound increase was for a protein that is approximately 75
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kDa in size, which might be an alternatively spliced isoform of β-cat (45).
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We have then examined the effect of GLP-1(28-36)amide on Ser133 phosphorylation of
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CREB, a downstream target of cAMP/PKA. As shown in Fig. 4A top panel, when INS-1 cells
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were treated with increased dosages of GLP-1(28-36)amide for 60 min, we began to see the
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detection of CREB Ser133 as the peptide concentration reached 20 nM. A doublet band that
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migrated faster than CREB Ser133 was also detected. Based on antibody data sheet, this
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represents cyclic AMP-dependent transcription factor-1 (ATF-1), which can be phosphorylated
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at Ser63 by PKA (29). The response of INS-1 cells to 50 nM GLP-1(28-36)amide on CREB
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phosphorylation rapidly occurred as early as 5 min and sustained at 60 min (Fig. 4B). The
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stimulation of GLP-1(28-36)amide (50 nM) on CREB Ser133 phosphorylation was observed in
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rat islets when the time reached 60 min, while the activation of ATF-1 started at 5 min (Fig. 4C).
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The activation was observed in much less degree in the pancreatic α-cell line α-TC (Fig. 4D and
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4E), and in the human fibroblast cell line 293T (Fig. 4F). Forskolin, however, was shown to
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stimulate CREB Ser133 and ATF-1 Ser63 phosphorylation in these two cell lines (Fig. 4D and
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4F).
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Although PKA is the canonical stimulator of CREB and ATF-1 phosphorylation, insulin has
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been shown to stimulate CREB phosphorylation in pancreatic β-cells and in other cell lineages
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(18, 20). This, along with the lack of the activation of CRE-element fused luciferase reporter
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gene expression by GLP-1(28-36)amide, reported recently by Liu and colleagues (22), drove us
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to further investigate whether GLP-1(28-36)amide indeed stimulates cytoplasmic cAMP levels
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and PKA activity. When we tested this in the INS-1 cell line, GLP-1(28-36)amide was found to
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stimulate cytoplasmic cAMP levels approximately 2 fold (Fig. 5A). As a positive control, the
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adenyl cyclase activator forskolin increased cytoplasmic cAMP levels in this cell line
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approximately 7 fold (Fig. 5A). In primary rat islets, GLP-1(28-36) and forskolin treatment
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generated a 1.4 and 2.4 fold increase of cytoplasmic cAMP levels, respectively (Fig. 5B). The
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relative lower level activation in rat islets could be due the presence of other types of cells.
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Furthermore, we found that when INS-1 cells were treated with either GLP-1(28-36)amide or
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forskolin, PKA enzymatic activity was increased (Fig. 5C), which was attenuated by the PKA
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inhibitor H89 (Fig. 5C). In addition, we found that GLP-1(28-36)amide- and forskolin-stimulated
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β-cat Ser675 phosphorylation can also be attenuated by H89 pre-treatment. The attenuation is
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accompanied with reduced expression of cyclin D1, a known downstream target of β-cat/TCF
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(Fig. 5D). We noticed that H89 alone increased β-cat Ser675 phosphorylation (Fig. 5D), which
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might be due to an off-target effect of H89.
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GLP-1(28-36)amide promotes the growth of INS-1 cells and enhances glucose induced
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insulin secretion in rat islets. To test whether GLP-1(28-36)amide increases β-cell growth, we
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performed the MTT assay. As shown in Fig. 6A, 50 nM GLP-1(28-36)amide treatment for 24 h
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significantly increased the growth of INS-1 cells. This effect of GLP-1(28-36)amide was not
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observed in the human fibroblasts 293T (Fig. 6B). We have also found that GLP-1(28-36)amide-
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mediated increase in cell growth can be blocked by the PKA inhibitor KT5720 (Fig. 6C).
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Furthermore, TxNIP is among the mediators of glucotoxicity and its expression level can be
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dramatically increased by increasing glucose levels (33). We find that when INS-1 cells were
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pre-incubated with 50 nM GLP-1(28-36)amide for 1 h, 10 mM glucose stimulated TxNIP
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expression was attenuated. The pre-treatment, however, did not attenuate 25 mM glucose
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stimulated TxNIP expression (Fig. 6D). It appears that the effect of GLP-1(28-36) on reducing
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TxNIP level is much weaker compared with forskolin or exendin-4 (33). Finally, we tested the
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effect of GLP-1(28-36)amide on insulin secretion in INS-1 cells and in islets from adult rats. In
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the INS-1 cell line, the addition of GLP-1(28-36)amide increased insulin secretion when the cells
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were grown in medium with 2.8 mM or 20 mM glucose, but the increase did not reach the
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statistical significant level. We did not see the synergic effect of high levels of glucose and GLP-
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1(28-36)amide on insulin secretion (Fig. 6E). In the rat islets, high levels of glucose and GLP-
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1(28-36)amide) were shown to stimulate insulin secretion in synergistic manner (Fig. 6F).
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Discussion
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We demonstrated in this study the beneficial effects of GLP-1(28-36)amide in the STZ-induced
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type 1 diabetes mouse model, exemplified by the improvement of glucose disposal, the elevation
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of plasma insulin level, as well as the stimulation of β-cell proliferation. Our in vitro study
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results suggest that the beneficial effects of GLP-1(28-36)amide are likely due to the activation
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of the Wnt signaling pathway effector β-cat, via its Ser675 phosphorylation, mediated through
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the activation of cAMP/PKA signaling cascade.
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The enzyme dipeptidylpeptidase IV (DPP-IV) cleaves GLP-1(7-36)amide, leading to the
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generation of GLP-1(9-36)amide, a presumably inactive form of GLP-1. Both GLP-1(7-
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36)amide and GLP-1(9-36)amide can undergo the cleavage by NEP (24.11), creating GLP-1(28-
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36)amide (5, 13) (Fig. 7A). NEP (24.11) is a widely distributed membrane-bound metalloenzyme,
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involved in processing GLP-1 (27), glucagon (39), and other hormonal and non-hormonal
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peptides (13, 39). Both DPP-IV and NEP (24.11) mediate GLP-1(7-36)amide degradation in vivo
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(28). During GLP-1 infusion in anaesthetised pig, the administration of the NEP (24.11) inhibitor
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candoxatril resulted in an increase of C-terminal immunereactivity of GLP-1 and an
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improvement of GLP-1 pharmacokinetics (28). Candoxatril administration was also shown to
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improve glucose tolerance (28). A recent study showed that AVE7688, a compound that blocks
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the activities of both angiotensin converting enzyme (ACE) and NEP, prevented the formation of
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obesity in Sprague Dawley rats fed with high fat diet, along with the improvement of glucose
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disposal (6). These observations collectively suggested a role of NEP (24.11) in facilitating the
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degradation of GLP-1(7-36)amide into GLP-1(28-36)amide, while the attenuation of this process
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may bring short- and long-term beneficial effects on glucose or metabolic homeostasis. These
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observations, however, are not in agreement with many other observations, showing that the
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presumably cleaved or “inactivated” GLP-1 molecules possess at least some beneficial
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pharmacological effects (1, 25, 26, 40).
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GLP-1(9-36)amide was initially thought to be either biologically inactive or serving as a
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weak antagonist of GLP-1R (19). A study with healthy human subjects revealed that GLP-1(9-
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36)amide has no insulinotrophic effect (41). Several beneficial effects of GLP-1(9-36)amide in
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the cardiovascular system, however, have been recognized during the past few years (1, 25, 26,
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40). Interestingly, some of the beneficial effects of GLP-1(28-36)amide were at least not
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attributed to the canonical GLP-1R (1, 2). These observations led us to speculate that although
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the cleavage of GLP-1(7-36)amide serves as a negative feedback on the insulinotropic effect of
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this incretin hormone, it may lead to the generation of biologically active products that exert long
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term beneficial effects, without a robust effect on insulin secretion. A dual receptor theory has
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been proposed by Tomas and Habener, suggesting that GLP-1 possesses the insulin-like action
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(as an insulinomimetic hormone), in addition to its insulinotrophic effect (36).
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Tomas et al. have assessed the effect of GLP-1(28-36)amide in vivo in high fat diet fed mice.
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They demonstrated that this nonapeptide inhibited weight gain and liver triglyceride
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accumulation, and improved insulin sensitivity through the attenuation of the development of
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hyperglycaemia and hyperinsulinemia. Since GLP-1(28-36)amide administration also increases
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food intake, this nonapeptide may increase energy expenditure (38). They then reported that
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GLP-1(28-36)amide suppresses glucose production and oxidative stress in primary mouse
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hepatocytes(37). This effect of GLP-1(28-36) may explain why we observed the improvement of
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glucose disposal in IPPTT in animals received GLP-1(28-36) but not in animals received
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extendin-4. Very recently, Liu and colleagues reported that GLP-1 (28-36)amide protects β-cells
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in vitro in conditions of glucolipotoxicity, which is independent of the GLP-1R (22). This
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nonapeptide targets mitochondria and improves impaired mitochondrial membrane potential,
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associated with increased cellular ATP levels, reduced caspase activation, and cell apoptosis (22).
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Although these in vitro studies suggested a potential role for this nonapeptide in β-cell function,
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the in vivo effects of GLP-1 (28-36)amide on β-cell function and glucose homeostasis remain
320
largely unknown. Here we show that in STZ induced diabetic mouse model,
321
36)amide improved glucose disposal, associated with improved pancreatic β-cell mass.In this
322
study, the stimulatory effects on β-cell mass by GLP-1(28-36) and Exendin-4 were comparable,
323
although in trend, GLP-1(28-36)amide showed a stronger effect on BrdU incorporation (Fig. 2).
324
Nevertheless, the two treatments had similar effect on fasting insulin levels (Fig. 1D). Further
325
investigations are needed to explore mechanisms underlying the stimulation of β-cell
326
proliferation by GLP-1(28-36) administration.
GLP-1(28-
327
The involvement of Wnt and β-cat signaling in the development and function of pancreatic β-
328
cells has drawn our attention recently, although disputes do exist (5, 15). Notably, β-cat and Wnt
329
signaling is not only involved in the production of GLP-1 and another incretin hormone GIP (9,
330
43), it also mediates the function of GLP-1 (21, 31). The bipartite transcription factor cat/TCF
331
has been shown to function as mediator for many hormonal and other regulatory peptides (16).
332
This can be achieved by β-cat Ser675 phosphorylation either by cAMP/PKA or insulin (10, 14,
333
46). We show in this study that GLP-1(28-36)amide is able to stimulate β-cat Ser675
334
phosphorylation, and this stimulation is associated with cytoplasmic cAMP elevation and PKA
335
activation. We hence suggest that β-cat Ser675 phosphorylation serves as a mediator for the dual
336
function of the incretin hormone GLP-1. As shown in Fig. 7B, GLP-1(7-36)amide stimulates β-
337
cat Ser 675 phosphorylation, leading to Wnt signaling pathway activation, so does the GLP-1R
338
agonist exendin-4 (21). This is likely mediated by the canonical GLP-1R receptor (21).
16
339
Following the cleavage by DPP-IV, the product GLP-1(9-36)amide exerts certain beneficial
340
effects in GLP-1R independent manner. It has been postulated that GLP-1(9-36)amide may use
341
CD36 to exert some of its effects (36). GLP-1(28-36)amide is also able to stimulate cAMP
342
elevation, PKA activation and β-cat Ser675 phosphorylation, either through a yet to be identified
343
receptor or the direct entry and targeting to mitochondria, as suggested recently by Liu and
344
colleagues (22). We are unclear whether the activation of cAMP/PKA by GLP-1(7-36)amide is
345
mainly accompanied with the insulinotropic effect, while the activation of cAMP/PKA by GLP-
346
1(28-36)amide mainly leads to the insulinomimetic effect.
347
In summary, we show in this study the beneficial effects of GLP-1(28-36)amide in the STZ
348
β-cell injury diabetic mouse model. We have also revealed the effect of GLP-1(28-36) on
349
activating PKA and β-cat Ser675 phosphorylation, events that are known to be responsible for
350
the insulinomimetic effect of GLP-1 (21). Whether GLP-1(28-36) stimulates β-cat/TCF
351
transcriptional activity in vivo is worth to be further investigated. As the methodology for
352
detecting GLP-1(28-36)amide in circulation has yet to be developed, it is still difficult to
353
determine the physiological significance of this nonapeptide. Nevertheless, our findings suggest
354
the involvement of β-cat activity in mediating the insulinomimetic effect of GLP-1, and support
355
the notion by previous investigators that this nonapeptide possesses therapeutic potential for
356
diabetes and other metabolic disorder (22, 37, 38).
357
358
359
360
17
361
Acknowledgements: The current address for TZ is College of Life Science, Graduate University
362
of Chinese Academy of Sciences A19, Yuquan Rd, Beijing, P. R. China, 100049. We thank Dr.
363
Qiang Xu from Toronto Centre of Phenogenomics for providing technical assistance in
364
measuring β-cell mass.
365
Grants. The majority of this work was supported by an operating grant from Canadian Diabetes
366
Association (CDA, OG-3-10-3040) to TJ. Other contributions: Operating grants to QW (CDA
367
and CIHR) and operating grants from Canadian Institutes of Health Research (CIHR) to TJ
368
(MOP-89987 and MOP-97790). QW was supported by CIHR New Investigator program. WS
369
has been supported by a fellowship from Banting and Best Diabetes Centre (BBDC). WI is a
370
recipient of Ontario Graduate Scholarship and a Studentship from BBDC.
371
372
Disclosure
373
No conflicts of interests, financial or otherwise, are declared by the authors.
374
375
Author contributions
376
Author contributions: W.S., Z.W., W.I., Y.C., X.X., T.C., and C.X. performed experiments.
377
W.S., Z.W., W.I., Y.C., and X.X. analyzed data. W.S., and Z.W. prepared figures; W.S., Z.W.,
378
Q.W., and J.T. interpreted results. W.S. and T.J drafted the manuscript. W.S., Z.W., W.I., Y.C.,
379
X.X., T.C., Q.W. and T.J edited the manuscript. All authors approved the final version of the
380
manuscript.
18
381
382
Figure legends
383
Figure 1. GLP-1 (28-36)amide improves glucose disposal in the STZ induced type 1 mouse
384
diabetes model. A) At the beginning of the seventh week after i.p. injection of indicated
385
reagents, Intraperitoneal Pyruvate Tolerance Test (IPPTT) was performed after 6 h fasting. B) At
386
the beginning of the ninth week after i.p. injection of indicated reagents, Intraperitoneal Glucose
387
Tolerance Test (IPGTT) (with 0.4 g /kg glucose) was performed after overnight fasting. C and D)
388
Fasting plasma glucose (C) and insulin (D) levels of the three groups of mice were determined at
389
the end of the 9th week after GLP-1(28-36)amide, exendin-4 (Ex-4), or PBS (vehicle) injections.
390
AUC, areas under the curve (mM glucoce x 120 min). n=4 for each group; *, p<0.05.
391
392
Figure 2. GLP-1(28-36)amide increases β-cell mass and increases β-cell proliferation. Nine
393
weeks after GLP-1(28-36)amide, exendin-4 (Ex-4) or the control PBS injection, mice were
394
sacrificed for insulin staining (A), the determination of β-cell mass (B), localization of BrdU
395
incorporation in β-cells (C), and the calculation of percentage of BrdU positive β-cells (D). BrdU
396
positive β-cells are indicated by arrows in Fig. 2C (n=4 for the vehicle group, n=5 for the GLP-
397
1(28-36)amide group, and n=3 for the exendin-4 (Ex-4) group; *, p<0.05).
398
399
Figure 3. GLP-1(28-36)amide stimulates β-cat Ser675 phosphorylation and nuclear β-cat
400
accumulation. INS-1 cells (A)), or rat islets (B), or α-TC cells (C, D) , or human 293T
401
fibroblasts (E) were treated indicated amount of GLP-1(28-36)amide) for 60 min, or with 50 nM
402
GLP-1(28-36)amide for indicated time period, or treated with the adenyl cyclase activator
403
forskolin (10 µM) and IBMX (10 µM) for 60 min. Whole cell lysates (WCL) were prepared for
19
404
Western blotting with the Ser675 β-cat antibody, or the β-cat antibody, or the β-actin antibody
405
(as loading controls). F) INS-1 cells were treated with forskolin/IBMX (10 µM each), or 50 nM
406
GLP-1 (28-36)amide for 1 h. Nuclear and cytoplamic fractions were prepared for Western
407
blotting, with indicated antibodies. All panels are representative blots for experiments performed
408
at least three times. Numbers on the top of each panel are fold change of β-cat Ser675, with
409
untreated cells defined as 1 fold. F, forskolin/IBMX. Histone 3 and GAPDH are markers for
410
nuclear and cytoplasmic fractions, respectively.
411
412
Figure 4. GLP-1(28-36)amide stimulates CREB Ser133 and ATF-1 Ser63 phosphorylation.
413
INS-1 cells (A, B), or rat islets (C), or α-TC cells (D, E), or human 293T fibroblasts (F) were
414
treated with indicated amount of GLP-1(28-36)amide for 60 min, or with 50 nM GLP-1(28-
415
36)amide for indicated time period, or with 10 µM forskolin and 10 µM IBMX for 60 min.
416
Whole cell lysates (WCL) were prepared for Western blotting with the Ser133 CREB antibody,
417
or CREB antibody, or the β-actin antibody (as loading controls). All panels are representative
418
blots for experiments performed at least three times. F, forskolin/IBMX.
419
420
Figure 5. GLP-1(28-36)amide increases cytoplasmic cAMP level and PKA activity. INS-1
421
cells (A) and rat islets (B) were treated with GLP-1(28-36) (50 nM) or forskolin and IBMX (10
422
µM each) for 1 h. Cells were harvested for assessing the cytoplamic cAMP levels (*, p<0.05). C)
423
Ins-1 cells were pre-treated with or without a PKA inhibitor H89 (10 µM ) for 45 min, followed
424
by GLP-1(28-36)amide or forskolin/IBMX for additional 30 min. WCL were prepared for PKA
425
assay. Two µg proteins were utilized for the PKA assay, using the Peptag A1 (A1) as the
426
substrate. . D) Ins-1 cells were pre-treated with or without H89 (10 µM) for 45 min, followed by
20
427
GLP-1(28-36) or forskolin/IBMX for additional 60 min. WCL were prepared for Western
428
blotting against indicated antibody. Numbers on top of the β-cat Ser675 blot and the Cyclin D-1
429
blot are arbitrary units of densitometric analysis, normalized with total β-cat and β-actin,
430
respectively. Fig. 6C and 6D are representative blots for experiments performed three times. In
431
panel C and D, P and N, positive and negative controls provided by the PKA kit; V, the vehicle
432
control; G, GLP-1(28-36)amide; and F, forskolin/IBMX.
433
434
Figure 6. GLP-1(28-36)amide stimulates the growth of pancreatic β-cells and synergizes
435
with high glucose on insulin secretion. INS-1 cells (A) and 293T cells (B) were treated with or
436
without GLP-1(28-36)amide (50 nM) for 48 h. Relative OD (at 570 nm) values were determined
437
following the MTT assay (*, p<0.05). C) INS-1 cells were pr-treated with or without KT5720
438
(10 µM) for 45 min, followed with or without GLP-1(28-36)amide (50 nM) treatment for 48 h.
439
Relative OD (at 570 nm) values were determined following the MTT assay (*, p<0.05). D) INS-
440
1 cells were grown in the medium with 5 mM, 10 mM, or 25mM glucose, with or without GLP-
441
1(28-36) amide for 4 or 8 h. Whole cell lysates were prepared for Western blotting against
442
indicated antibody. Ins-1 cells (E) or rat islets (F) were incubated with or without GLP-1(28-
443
36)amide for 30 min in the KRBH buffer containing 2.8 mM (low glucose, LG) or 20 mM
444
glucose (high glucose, HG). Cell supernatants were collected for determining insulin levels by
445
RIA. Data were normalized to total protein content and represented as fold change, with the
446
insulin level in LG receiving no GLP-1(28-36)amide treatment defined as 1 fold (**, p<0.01, *,
447
p<0.05).
448
21
449
Figure 7. Schematic diagram showing the activation of Wnt effector β-cat by both GLP-
450
1(7-36)amide (defined as 7-36) and GLP-1(28-36)amide (defined as 28-36). A) Amino acid
451
sequences of GLP-1(7-36)amide, (9-36)amide and (28-36)amide, as well as the cleavage sites of
452
DPP-IV and NEP 24.11. B) 7-36 and the GLP-1R agonist exendin-4 are able to stimulate β-cat
453
Ser675 phosphorylation via cAMP/PKA activation (21), leading to Wnt signaling activation.
454
This is responsible for the insulinomimetic effect of this incretin hormone, including the
455
stimulation of β-cell proliferation and the repression of glucotoxicity. Both 7-36 and 9-36 can be
456
further cleaved, generating 28-36, which is also able to stimulate β-cat Ser675 phosphorylation
457
via cAMP/PKA activation.
458
459
460
22
461
462
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25
Figure 1
A. IPPTT (7th week)
PBS
GLP-1 (28-36)
EX-4
40
100
35
80
25
AUC x 10
30
Glucose (mM)
*
90
*
20
*
70
60
50
15
40
10
30
20
5
10
0
0
30
60
90
0
120
PBS
35
40
30
35
30
25
20
*
15
PBS
GLP-1 (28-36)
EX-4
10 *
5
0
25
20
15
10
5
0
0
30
60
90
120
PBS
C. Fasting glucose levels (end of 9th week)
GLP-1(28-36)
0.25
*
15
0.2
0.15
Insulin (ng/ml)
10
5
0
0.1
0.05
0
PBS
GLP-1(28-36)
Ex-4
Ex-4
D. Fasting insulin levels (end of 9th week)
*
20
Glucose (mM)
Ex-4
P=0.05
AUC x100
Glucose (mM)
B. IPGTT (9th week)
GLP-1(28-36)
PBS
GLP-1(28-36)
Ex-4
Figure 2
A. Representative images of pancreatic islets (with the insulin staining)
PBS
GLP-1(28-36)
B. -cell mass
*
*
1
0.9
0.8
0.7
-cell mass (mg)
0.6
0.5
0.4
0.3
0.2
0.1
0
PBS
GLP-1 (28-36)
Ex-4
Ex-4
Figure 2
C. Representative images of BrdU and insulin staining
PBS
GLP-1(28-36)
D. BrdU positive cells
*
Ex-4
*
BrdU+ cell / Insulin+ cell (%)
0.250
0.200
0.150
0.100
0.050
0.000
PBS
GLP-1 (28-36)
Ex-4
Figure 3
B. Islets (WCL)
A. Ins-1 (WCL)
1.0
1.9
1.8
2.1
2.9
2.0
1.0
2.4
3.6
2.4
3.1
-cat (S675)
-cat
-cat (S675)
-cat
-Actin
0
15
5
30
0.8
0.9
1.0
5
30
60
F Time (min)
F/60 Time (min)
60
C. -TC (WCL)
1.0
0
D. -TC (WCL)
1.1
1.5
1.0
1.0
0.8
1.1
1.3
1.2
-cat (S675)
-cat (S675)
0
15
5
30
60
-cat
-cat
-Actin
-Actin
F/60 Time (min)
0
1
10
20
50
100
GLP-1 (28-36) (nM)
E. 293 (WCL)
1.0
1.0
1.4
1.2
1.2
1.2
-cat (S675)
-cat
0
5
30
60
1.0
120 F/60 Time (min)
F. Ins-1
3.00
cytoplasmic
nuclear
-cat
Histone 3
GAPDH
C
G
F
C
G
F
Arbitrary units
2.50
2.00
Control
GLP-1 (28-36)
Forskolin
1.50
1.00
0.50
0.00
Cytoplasmic
Nuclear
Figure 4
B. Ins-1 (WCL)
A. Ins-1 (WCL)
p-CREB
ATF-1
p-CREB
ATF-1
CREB
-Actin
0
1
10
20
100 GLP-1 (28-36) (nM)
50
-Actin
0
5
15
30
60
F/60 Time( min)
C. Islets (WCL)
p-CREB
ATF-1
-Actin
0
30
5
60
F/60 Time( min)
D. -TC (WCL)
E. -TC (WCL)
p-CREB
ATF-1
p-CREB
ATF-1
CREB
CREB
-Actin
-Actin
0
5
15
30
60
F/60
Time (min)
F. 293 (WCL)
p-CREB
ATF-1
-Actin
0
5
30
60
120
F/60 Time (min)
0
1
10
20
50
100 GLP-1 (28-36) (nM)
Figure 5
A
B
*
10
*
3
8
Relative cAMP content
Relative cAMP content
9
7
6
*
5
4
3
2
1
0
Control
GLP-1(28-36)
2.5
**
2
1.5
1
0.5
0
F
Control
C
GLP-1(28-36)
P-A1
A1
P
N
1
D
V
1.40
G
F
1.42
H89
1.53
H89/G H89/F
0.84
1.06
Relative density
-cat (Ser675)
-cat
1
1.61
4.28
0.98
1.41
2.67
Relative density
Cyclin D1
-Actin
Control
F
G
H89
G/H89
F/H89
F
Figure 6
A
B
* P = 0.04
1.2
1.4
1
1
0.8
Relative OD Value
1.2
Relative OD Value
0.8
0.6
0.4
0.2
0
0.6
0.4
0.2
0
Control
GLP-1(28-36)
Control
C
GLP-1(28-36)
D
p=0.05
GLP-1(28-36)
*
5
1.25
10
25
5
10
25 Glucose (mM)
TxNIP
1.00
Relative OD Value
4hr
0.75
-Actin
0.50
TxNIP
8hr
0.25
-Actin
0.00
Control
GLP-1(28-36) KT5720 GLP-1(28-36)/KT
E
F
3.5
**
3.0
6.00
**
2.5
Relative insulin secretion
Relative insulin secretion
*
7.00
2.0
1.5
1.0
0.5
5.00
*
4.00
*
3.00
2.00
1.00
0.00-
0.0
LG
LG/
GLP-1(28-36)
HG
HG/
GLP-1(28-36)
LG
LG/
GLP-1(28-36)
HG
HG/
GLP-1(28-36)
Figure 7
A
NEP 24.11(Neutral endopeptidase)
DPP-IV
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
GLP-17-36 amide
EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
FIAWLVKGR-NH2
GLP-19-36 amide
GLP-128-36 amide
B
Exendin-4
Insulinotropic effect
(7-36)
GLP-1R
GLP-1
cAMP/PKA
-cat (Ser675)
NEP24.11
DPP-IV
Wnt signaling activation
(9-36)
NEP24.11
(28-36)
?
-cell proliferation
Glucotoxicity
Insulinomimetic effect