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Glucagon-like peptides-1 from phylogenetically ancient fish show
potent anti-diabetic activities by acting as dual GLP1R and GCGR
agonists
Galyna V. Graham, J. Michael Conlon*, Yasser H. Abdel-Wahab, Peter R. Flatt
SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, Ulster
University, Cromore Road, Coleraine, Northern Ireland BT52 1SA, UK
*Corresponding author.
Prof. J. M. Conlon
School of Biomedical Sciences
Ulster University,
Cromore Road,
Coleraine,
Northern Ireland BT52 1SA, UK
E-mail address: [email protected] (J.M. Conlon)
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Highlights
GLP-1 peptides from lamprey, dogfish, ratfish, paddlefish, bowfin and trout
stimulated insulin release from clonal β-cells and mouse islets.
Insulinotropic activity was attenuated by a GLP1R antagonist and activity of
lamprey and paddlefish GLP-1 was attenuated by a GCGR antagonist.
All peptides stimulated cAMP production in CHL cells transfected with
GLP1R.
Lamprey and paddlefish GLP-1 stimulated cAMP production in HEK293 cells
transfected with GCGR
Administration of all peptides improved glucose tolerance in mice with lamprey
and paddlefish GLP-1 as effective as human GLP-1 .
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Abstract
Glucagon-like peptides-1 (GLP-1)from phylogenetically ancient fish (lamprey, dogfish,
ratfish, paddlefish and bowfin) and from a teleost, the rainbow trout produced
concentration-dependent stimulations of insulin release from clonal β-cells and isolated
mouse islets. Lamprey and paddlefish GLP-1 were the most potent and effective.
Incubation of BRIN-BD11 cells with GLP-1 receptor (GLP1R) antagonist, exendin-
4(9-39) attenuated insulinotropic activity of all peptides whereas glucagon receptor
(GCGR) antagonist [des-His1,Pro4,Glu9] glucagon amide significantly decreased the
activities of lamprey and paddlefish GLP-1 only. The GIP receptor antagonist GIP (6-
30) Cex-K40[Pal] attenuated the activity of bowfin GLP-1. All peptides (1 μM)
produced significant increases in cAMP concentration in CHL cells transfected with
GLP1R but only lamprey and paddlefish GLP-1 stimulated cAMP production in
HEK293 cells transfected with GCGR. Intraperitoneal administration of lamprey and
paddlefish GLP-1 (25nmol/kg body weight) in mice produced significant decreases in
blood glucose and increased insulin concentrations comparable to the effects of human
GLP-1. Lamprey and paddlefish GLP-1 display potent insulinotropic activity in vitro
and glucose-lowering activity in vivo that is mediated through GLP1R and GCGR so
that these peptides may constitute templates for design of new antidiabetic drugs.
Key words: GLP-1; Glucagon; GIP; Insulinotropic; Antihyperglycaemic, Lamprey,
Paddlefish
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Introduction
Glucagon-like peptide-1 (GLP-1) was first identified from the nucleotide
sequence of a cDNA encoding preproglucagon in a Brockmann body (principal islet)
cDNA library from an anglerfish Lophius americanus (Lund et al., 1982). Shortly
afterwards, cDNAs encoding preproglucagon from several mammalian species
including the human (Bell et al., 1983) were characterized but the incretin activity of
GLP-1 was not demonstrated until it was realized that the active forms of the peptide in
mammals were GLP-1(7-37) and GLP-1(7-36)amide which resemble anglerfish GLP-1
and glucagon more closely (Mojsov et al., 1987). In humans, the effects of GLP-1 and
glucagon are mediated through respective interactions with the receptors GLP1R and
GCGR belonging to the class B-1 (secretin receptor-like) G protein-coupled receptors
that are highly specific for their native ligands (Fredriksson et al., 2003). In teleost fish,
the Gcgr receptor also binds glucagon and not GLP-1 (Chow et al., 2004) but the GLP-
1-specific receptor has been lost, being replaced by a receptor that has dual selectivity
for both GLP-1 and glucagon (Oren et al., 2016). In mammals, GLP-1 is
multifunctional. As well as stimulating post-prandial insulin release, the peptide inhibits
glucagon secretion, decreases food intake, delays gastric emptying, protects β- cells
from apoptosis and has beneficial effects on cognitive impairment and cardiac function
(Drucker, 2018). In contrast, GLP-1 is not an incretin hormone in teleost fish and has a
glucagon-like effects on metabolism stimulating glycogenolysis, gluconeogenesis and
lipolysis (Plisetskaya and Mommsen, 1996; Mommsen, 2000).
GLP-1 peptides have been isolated from the Brockmann bodies of a wide range
of teleost species and cDNAs genes encoding proglucagon-derived peptides from
several teleosts have been characterized [reviewed in (Irwin, 2005; Irwin and Mojsov,
2018)]. GLP-1 peptides have also been purified from a range of phylogenetically more
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ancient fishes: the agnathan sea lamprey Petromyzon marinus [Petromyzontiformes]
(Conlon et al., 1993); the cartilaginous fishes European common dogfish Scyliorinus
canicula [Elasmobranchii] (Conlon et al., 1994) and Pacific ratfish Hydrolagus colliei
[Holcephali] (Conlon et al.,1987); and the ray-finned fishes North American paddlefish
Polyodon spathula [Acipenseriformes] (Nguyen et al., 1994), alligator gar Lepisosteus
spatula [Lepisosteiformes] (Pollock et al., 1988), and bowfin Amia calva [Amiiformes]
(Conlon et al., 1993). Characterization of a cDNA from the intestine of P. marinus
demonstrated that the sea lamprey proglucagon gene encodes the three glucagon-related
peptides (glucagon, GLP-1 and GLP-2) analogous to those encoded by the proglucagon
genes of tetrapods. It has been suggested that the glucagon sequence first arose in
evolution approximately 1000 million years ago (MYA) while GLP-1 and GLP-2
diverged from each other approximately 700 MYA (Irwin et al, 1999).
The biological activities of GLP-1 peptides from phylogenetically ancient fishes
and their interaction with mammalian GLP-1, glucagon, and glucose-dependent
insulinotropic peptide (GIP) receptors have not been investigated in detail.
Consequently, the aim of the present study is to evaluate the in vitro insulinotropic
properties of synthetic replicates of GLP-1 peptides from the sea lamprey, dogfish,
ratfish, paddlefish, and bowfin using established rat and human insulin-producing cell
lines and isolated mouse islets. Their acute glucose-lowering and insulin stimulating
actions in vivo were assessed in overnight-fasted NIH Swiss mice. Their activities are
compared with those of human GLP-1 and GLP-1 from a more highly derived teleost
fish, the rainbow trout Onchorynchus mykiss (Irwin and Wong, 1995).
Although drugs based upon the structure of human GLP-1, such as liraglutide
and semaglutide, are widely used in clinical practice, patients with Type 2 diabetes
mellitus (T2DM) often cannot be treated effectively with a single pharmaceutical agent
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(Bailey 2018). The therapeutic potential of unimolecular dual-agonist peptides based
upon the primary structures of human GLP-1 and glucagon that regulate multiple
metabolic pathways is becoming increasingly apparent and such peptides that target the
GLP1R and the GCGR are currently in development (reviewed in (Irwin and Flatt
2015, Brandt et al., 2018). A major advantage of administration of dual-agonist
peptides over their constituent monotherapies is their improved actions on blood
glucose control, appetite, suppression, weight loss and hyperlipidemia/
hypercholestrolemia (Sánchez-Garrido et al., 2017, Brandt et al., 2018). Consequently,
the longer term objective of the study was assess possible therapeutic advantages of the
fish GLP-1 peptides as agents for treatment of patients with T2DM compared with
mono-agonist peptides based upon the structure of human GLP-1.
Materials & methods
2.1. Peptides
All peptides were supplied in crude form by EZBiolab Inc. (Carmel, IN, USA).
The peptides were purified to > 98% homogeneity by reversed-phase HPLC on a (2.2
cm x 25 cm) Vydac 218TP1022 (C-18) column (Grace, Deerfield, IL, USA). The
concentration of acetonitrile in the eluting solvent was raised from 21% to 56 % over
60 min using a linear gradient. Absorbance was measured at 214 nm and the flow rate
was 6 ml/min. The identities of the peptides were confirmed by matrix-assisted laser
desorption ionization time-of-flight mass (MALDI-TOF) spectrometry using a Voyager
DE PRO instrument (Applied Biosystems, Foster City, USA) as previously described
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(Conlon et al., 2018). The primary structures and molecular masses of the peptides used
in this study are shown in Table 1.
Table 1. Primary structures of the fish GLP-1 peptides investigated in this study.
Peptide Amino acid sequenceCalculated molecular mass
Observed molecular mass
Lamprey GLP-1 HADGTFTNDMTSYLDAKAARDFVSWLARSDKS 3577.92 3576.49
Dogfish GLP-1 HAEGTYTSDVDSLSDYFKAKRFVDSLKSY 3330.64 3330.14
Ratfish GLP-1 HADGIYTSDVASLTDYLKSKRFVESLSNYNRKQNDRRM 4479.90 4480.22
Paddlefish GLP-1 HADGTYTSDASSFLQEQAARDFISWLKKGQ 3358.50 3357.91
Bowfin GLP-1 YADAPYISDVYSYLQDQVAKKWLKSGQDRRE 3694.11 3695.36
Trout GLP-1 HADGTYTSDVSTYLQDQAAKDFVSWLKSGRA 3418.68 3420.76
2.2. In vitro insulin release studies using BRIN-BD11 and 1.1B4 cells
The abilities of the purified synthetic peptides (0.01 nM - 3 µM; n = 8) to
stimulate the rate of release of insulin from BRIN-BD11 rat clonal β-cells
(McClenaghan et al., 1996) and 1.1B4 human-derived pancreatic β-cells (McCluskey et
al. 2011) was determined as previously described (Graham et al., 2018). In a second
series of experiments, the effects of 1 µM concentrations of the (A) GLP-1 receptor
antagonist, exendin-4(9-39) (Thorens et al., 1993), (B) glucagon receptor antagonist,
[desHis1,Pro4,Glu9] glucagon amide (O’Harte et al., 2013), and (C) GIP receptor
antagonist, GIP(6-30)Cex-K40[Pal] (Pathak et al., 2015) on the insulin-releasing activity
of the peptides (0.1 µM) were studied by incubating BRIN-BD11 cells for 20 min at 37
˚C in Krebs-Ringer Bicarbonate (KRB) buffer, pH 7.4 supplemented with 5.6 mM
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glucose as previously described (Graham et al., 2018). All samples were stored at −20
°C prior to measurement of insulin release by radioimmunoassay (Flatt and Bailey,
1981).
2.3. Insulin release studies using isolated mouse islets
Freshly harvested pancreatic islets from adult, male National Institutes of Health
(NIH) Swiss mice (Harlan Ltd, Bicester, UK) were cultured for 48 h at 37 °C in an
atmosphere of 5% CO2 and 95% air as previously described (Ojo et al., 2016). The
islets were incubated with peptides (10 nM and 1µM; n =4) and alanine (10 mM; n =4))
for 1 h at 37˚C in KRB buffer supplemented with 16.7 mM glucose. Insulin release was
measured by radioimmunoassay and the insulin content was measured by extraction
with acid-ethanol as previously described (Ojo et al., 2016).
2.4. Effects on cAMP production.
Chinese hamster lung (CHL) cells transfected with the human GLP-1 receptor
(GLP1R) (Thorens et al., 1993) and human embryonic kidney (HEK293) cells
transfected with the human glucagon receptor (GCGR) (Ikegami et al., 2001) were
cultured and seeded as previously described (Graham et al., 2018). The cells were pre-
incubated for 40 minutes with KRB buffer supplemented with 0.1% (w/v) bovine serum
albumin and 1.1 mmol/l glucose. A test solution containing KRB buffer supplemented
with 0.1% (w/v) bovine serum albumin, 5.6 mmol/l glucose, 200 μM 3-isobutyl-1-
methylxanthine (IBMX) and peptides (10 nM and 1µM) were added to the cells. After
incubating for 20 min the medium was removed and cells were lysed before
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measurement of cAMP using a Parameter cAMP assay kit (R&D Systems, Abingdon,
UK) according to the manufacturer’s recommended protocol.
2.5. Insulin release studies using CRISPR/Cas9-engineered INS-1 cells
Wild-type INS-1 832/3 rat clonal pancreatic β-cells and CRISPR/Cas9-
engineered cells with knock-out of either the GLP-1 receptor (GLP1R KO) or the GIP
receptor (GIPR KO) (Naylor et al., 2016) were cultured and incubated as described for
BRIN-BD11 cells with the addition of 1 mM sodium pyruvate and 50 mM 2-
mercaptoethanol to the medium (Graham et al. 2018). The cells were incubated with 10
nM and 1 μM concentrations of GLP-1, glucagon, GIP, and fish peptides for 20 min
37 °C in KRB buffer supplemented with 5.6 mM glucose and insulin release was
measured by radioimmunoassay.
2.6. In vivo insulin release studies
All experiments involving animals were performed in accordance with the UK
Animals (Scientific Procedures) Act 1986 and EU Directive 2010/63EU and approved
by Ulster University Animal Ethics Review Committee. Acute in vivo studies were
carried out using 8-10 week-old male National Institutes of Health (NIH) Swiss mice (n
= 6) maintained in environmentally controlled rooms (12:12 light/darkness cycle, 22 ±
2 oC) with free access to a standard rodent diet (10% fat, 30% protein, 60%
carbohydrate: total energy 12.99 kJ/g; Trouw Nutrition, Northwich, UK) and water.
After an overnight fast, glucose (18 mmol/kg body weight), either alone or together
with a GLP-1 peptide (25 nmol/kg body weight), was administrated intraperitoneally to
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the animals as previously described (Owolabi et al., 2016). Blood samples were
collected from a tail vein at the time points shown in Fig. 7 and glucose concentrations
were measured using a Bayer Counter glucose meter. Peptide administration had no
apparent adverse behavioural effects on the animals.
2.7. Statistical analysis
Data were compared using one-way ANOVA followed by Bonferroni post-hoc
test and unpaired Student’s t test (non-parametric, with two-tailed P values and 95%
confidence interval) using GraphPad PRISM (Version 5.0 San Diego, California). Area
under the curve (AUC) analysis was performed using the trapezoidal rule with baseline
correction. Data are presented as mean ± S.E.M where the comparison was considered
to be significantly different if P < 0.05.
3. Results
3.1. In vitro insulin-releasing activity of fish peptides
In the presence of 5.6 mM glucose alone, the rate of insulin release from BRIN-
BD11 rat clonal β-cells was 1.02 ± 0.02 ng/106cells/20 min and this rate increased to
2.4 ± 0.2 ng/106cells/20 min during incubation with 0.1 µM human GLP-1 and to 1.4 ±
0.2 ng/106cells/20min during incubation with 0.1 µM glucagon. Incubation of BRIN-
BD11 cells with GLP-1 peptides from all fish species produced concentration-
dependent increases in the rate of insulin release (Fig. 1). As shown in Table 2, lamprey
and paddlefish GLP-1 displayed the lowest threshold concentrations (minimum
concentration producing a significant increase in insulin release) and the greatest
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responses at 3 µM concentration, These values were not significantly different from
those of human GLP-1.
Table 2. Effects of fish GLP-1 peptides, human GLP-1 and glucagon on the rate of
insulin release from BRIN-BD11 cells.
Peptide Threshold concentration Effect at 3µM (ng/106/20min)
Control - 1.0 ± 0.02Lamprey GLP-1 30 pM 3.9 ± 0.1Dogfish GLP-1 3 nM 1.8 ± 0.1Ratfish GLP-1 1 nM 2.6 ± 0.1
Paddlefish GLP-1 10 pM 3.7 ± 0.2Bowfin GLP-1 0.3 nM 2.3 ± 0.2Trout GLP-1 Human GLP-1
30 pM 10 pM
2.4 ± 0.1 3.6 ± 0.2
Human glucagon 10 nM 2.7 ± 0.1
Control refers to the rate of insulin release at 5.6 mM glucose alone. The threshold
concentration is the minimum concentration of the peptide producing a significant (p <
0.05) increase in the rate of insulin release. The values are mean ± SEM for n = 8. The
values are obtained from the results of the incubations shown in Fig.1.
In the presence of 16.7 mM glucose alone, the rate of insulin release from
1.1B4 human clonal β-cells was 0.08 ± 0.01 ng/106cells/20min rising significantly to
0.13 ± 0.01 ng/106 cells/20 min and to 0.24 ± 0.02 ng/106cells/20min during incubations
with 10 nM and 1 µM human GLP-1 respectively. As shown in Fig. 2, incubations
with GLP-1 from all fish species produced a significant increase in insulin release at 1
μM concentration and GLP-1 peptides from lamprey, paddlefish and trout produced a
significant increase at 10 nM concentration. Consistent with the data obtained with
BRIN-BD11 cells, the lamprey and paddlefish peptides were the most effective with the
responses produced by lamprey GLP-1 at both concentrations (0.23 ± 0.01
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ng/106cells/20min at 10 nM and 0.31 ± 0.02 ng/106cells/20min at 1 μM) being
significantly greater than those produced by GLP-1.
The rate of insulin release from isolated mouse islets in the presence of 16.7
mM glucose alone during a 60 min incubation was 9.7 ± 1.3 % of the total insulin
content of the islets rising to 16.7 ± 1.4 % in the presence of 10 nM GLP-1 and to 20.7
± 2.5 % in the presence of 1 µM GLP-1. As shown in Fig. 3, GLP-1 from all species
except dogfish and ratfish produced a significant increase in insulin release at both 10
nM and 1 µM concentrations. Ratfish GLP-1 was effective at 1 µM but dogfish GLP-
1 did not produce a significant increase at either concentration.
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Fig. 1. Effects of increasing concentrations of GLP-1 from (A) lamprey, (B) dogfish,
(C) ratfish, (D) paddlefish, (E) trout, (F) bowfin, and (G) human and (H) human
glucagon on insulin release from BRIN-BD11 rat clonal β-cells. Values are mean ±
S.E.M., n = 8. *P < 0.05, **P < 0.01, ***P < 0.001 compared to 5.6 mM glucose alone.
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Fig. 2 . Effects of fish GLP-1 peptides at 10 nM and 1 µM concentrations on insulin
release from 1.1 B4 human clonal β-cells. Values are mean ± S.E.M., n = 8 *P < 0.05,
*P < 0.01, ***P < 0.001 compared to 16.7 mM glucose alone. ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 compared with the response to human GLP-1.
Fig. 3. Effects of fish GLP-1 peptides at 10 nM and 1 µM concentrations on insulin
release from pancreatic islets isolated from NIH Swiss mice. The values are mean ±
SEM for n=4; *P < 0.05, **P < 0.01, ***P < 0.001 compared to 16.7 mM glucose
alone.
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3.2. Receptor antagonist studies
The insulin releasing activities of human GLP-1 and all fish GLP-1 peptides
were significantly attenuated when BRIN-BD11 cells were co-incubated with the GLP-
1 receptor antagonist, exendin-4(9-39) (Fig. 4A). The activity of human glucagon and
GIP were not significantly affected. The insulin releasing activities of glucagon and
lamprey GLP-1 (P < 0.001) and paddlefish GLP-1 (P < 0.001) were also significantly
decreased in the presence of the glucagon receptor antagonist [des-His1,Pro4,Glu9]
glucagon amide (Fig. 4B) whereas the actions of GLP-1, GIP, and the other fish GLP-1
peptides were unaffected. Human GIP (P < 0.001) and bowfin GLP-1 (P < 0.05) were
the only peptides whose insulin-releasing effects were significantly attenuated by co-
incubation with the GIP receptor antagonist GIP(6-30)Cex-K40 [Pal] (Fig. 4C).
3.3. Effect on fish GLP-1 peptides on cAMP production
cAMP production in CHL cells transfected with the human GLP-1 receptor
(GLP1R) was significantly stimulated by incubation with human GLP-1 and the GLP-1
peptides from lamprey, paddlefish and trout at both 10 nM and 1 µM concentrations
(Fig. 5A). In the case of GLP-1 from dogfish, ratfish and bowfin a significant increase
in cAMP production was achieved only at 1 µM concentration. Human glucagon and
GLP-1 from lamprey and paddlefish, at both 10 nM and 1 µM concentrations, were the
only peptides that produced a significant increase cAMP production in HEK293 cells
transfected with the human glucagon receptor (GCGR) (Fig. 5B).
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Fig. 4. Effects of (A) GLP-1 receptor antagonist, exendin-4(9-39), (B) glucagon
receptor antagonist [desHis1,Pro4,Glu9]glucagon amide, and (C) GIP receptor antagonist
GIP (6-30) Cex-K40[Pal], on the ability of fish GLP-1 peptides (100 nM) to stimulate
insulin release from BRIN-BD11 cells. Values are mean ± S.E.M., n = 8 **P < 0.01,
***P < 0.001 compared with 16.7 mM glucose alone. ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 compared with the effect in the presence of antagonist.
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Fig. 5. Effects of fish GLP-1 peptides (10 nM and 1 µM) on intracellular cAMP
production in (A) GLP1R transfected CHL cells, and (B) GCGR transfected HEK293
cells. Values are mean ± SEM for n = 4. *P < 0.05, **P < 0.01 and ***P < 0.001
compared with 5.6 glucose + IBMX alone.
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3.4. Insulin release studies using CRISPR/Cas9-engineered INS-1 cells
Incubation of all fish GLP-1 peptides, as well as human GLP-1, glucagon, and
GIP, at concentrations of 10 nM and 1 μM with wild-type INS-1 cells produced a
significant increase in the rate of release of insulin compared with the rate in the
presence of 5.6 mM glucose only (Fig. 6A). The increases in rate in response to GLP-1
and the GLP-1 from lamprey and trout were completely abolished in the GLP1R KO
cells and the responses to the other fish peptides were significantly attenuated (Fig.
6B). In contrast, the increases in rate in response to human (P < 0.001) and bowfin (P <
0.05) GIP were significantly reduced in the GIPR KO cells but the stimulatory
responses to glucagon, GLP, and the other fish GLP-1 peptides were maintained (Fig.
6C).
3.5. In vivo insulin release studies.
Blood glucose concentrations in overnight fasted NIH Swiss mice were
significantly lower after receiving intraperitoneal administration of glucose (18 mmol/
kg body weight) together with all GLP-1 peptides (25 nmol/kg body weight) except
dogfish GLP-1 compared with mice receiving intraperitoneal glucose only (Figs. 7A
and C). The integrated responses showed significant differences for lamprey GLP-1 (P
< 0.001), ratfish GLP-1 (P < 0.05), paddlefish GLP-1(P < 0.001), trout GLP-1 (P <
0.001), and bowfin GLP-1 (P < 0.05) compared with the glucose control. The integrated
responses to GLP-1 peptides from lamprey, paddlefish and trout were not significantly
different from the response to human GLP-1 (Fig 7E). The concentrations of plasma
insulin were significantly greater at the 15 and 30 min time points after administration
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of GLP-1 from lamprey, ratfish, paddlefish and trout and after 30 min in the case of
bowfin GLP-1 compared with control animals receiving glucose alone (Fig. 7B and D).
The integrated insulin responses to all peptides except dogfish GLP-1 were not
significantly different from that of human GLP-1 (Fig. 7F).
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Fig. 6. Effects of fish GLP-1 peptides (10 nM and 1 μM) on the rate of insulin release
from (A) wild-type INS-1 cells, (B) CRISPR/Cas9-engineered GLP1R KO cells, and
(C) CRISPR/Cas9-engineered GIPR KO cells. Values are mean ± S.E.M., n = 8, *P <
0.05, **P < 0.01 and ***P < 0.001 compared with 5.6 mM glucose alone. Δ P < 0.05,
ΔΔ P < 0.01 and ΔΔΔP < 0.001 compared with responses in wild-type INS-1 cells.
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Fig. 7. Effects of acute administration of fish GLP-1 peptides (25 nmol/kg body weight)
on plasma glucose (panels A and C) and plasma insulin (panels B and D)
concentrations in mice after intraperitoneal injection of glucose (18 mmol/kg body
weight). The integrated responses (area under the curve AUC) are shown in panels E
and F. The values are mean ± SEM for n = 6. **P < 0.01 and ***P < 0.001 compared to
glucose alone; ΔP < 0.05, ΔΔP < 0.01 and ΔΔΔP < 0.001 compared to human GLP-1.
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4. Discussion
The present study has demonstrated that GLP-1 peptides from phylogenetically
ancient fish and a teleost differ appreciably in potency and effectiveness in terms of
their abilities to stimulate insulin release from rodent and human cells in vitro and
lower blood glucose concentrations in mice in vivo. The peptides from sea lamprey, a
representative of the Agnatha or jawless fishes whose line of evolution diverged from
that leading to gnathostomes at least 550 MYA, and paddlefish, an extant representative
of a group of primitive Actinopterygian (ray-finned) fish whose line of evolution
diverged from the sturgeons in the Cretaceous, were the most potent and effective with
activities generally comparable to human GLP-1. However, these GLP-1 peptides differ
from the human peptide by functioning as dual agonists at the GLP-1 and glucagon
receptors. The primary structures of the GLP-1 peptides investigated in this study are
compared with those of human GLP-1, glucagon, and the potent and therapeutically
valuable GLP-1 receptor agonist, exendin-4 (Brown et al, 2018) in Fig. 8.
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GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRa
Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSa
Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGIP YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ Lamprey GLP-1 HADGTFTNDMTSYLDAKAARDFVSWLARSDKS Dogfish GLP-1 HAEGTYTSDVDSLSDYFKAKRFVDSLKSYRatfish GLP-1 HADGIYTSDVASLTDYLKSKRFVESLSNYNRKQNDRRMPaddlefish GLP-1 HADGTYTSDASSFLQEQAARDFISWLKKGQ Bowfin GLP-1 YADAPYISDVYSYLQDQVAKKWLKSGQDRRETrout GLP-1 HADGTYTSDVSTYLQDQAAKDFVSWLKSGRA
Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRa
Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSa
GIP YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ Lamprey GLP-1 HADGTFTNDMTSYLDAKAARDFVSWLARSDKS Dogfish GLP-1 HAEGTYTSDVDSLSDYFKAKRFVDSLKSYRatfish GLP-1 HADGIYTSDVASLTDYLKSKRFVESLSNYNRKQNDRRMPaddlefish GLP-1 HADGTYTSDASSFLQEQAARDFISWLKKGQ Bowfin GLP-1 YADAPYISDVYSYLQDQVAKKWLKSGQDRRETrout GLP-1 HADGTYTSDVSTYLQDQAAKDFVSWLKSGRA
GIP YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRa
Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSa
Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT Lamprey GLP-1 HADGTFTNDMTSYLDAKAARDFVSWLARSDKS Dogfish GLP-1 HAEGTYTSDVDSLSDYFKAKRFVDSLKSYRatfish GLP-1 HADGIYTSDVASLTDYLKSKRFVESLSNYNRKQNDRRMPaddlefish GLP-1 HADGTYTSDASSFLQEQAARDFISWLKKGQ Bowfin GLP-1 YADAPYISDVYSYLQDQVAKKWLKSGQDRRETrout GLP-1 HADGTYTSDVSTYLQDQAAKDFVSWLKSGRA
Fig. 8. A comparison of the primary structures of human GLP-1, glucagon, and GIP
with the corresponding primary structures of exendin-4 and the fish GLP-1 peptides.
Sequence similarities to the human peptides are highlighted in grey. a denotes C-
terminal α-amidation.
Structure-activity studies of human GLP-1 involving a complete alanine scan
showed that the amino acids His1, Gly4, Phe5, Thr7, and Asp9 in the N-terminal domain
play an important role in binding and activation of the rat Glp1r, and Phe22 and Ile23 in
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the C-terminal domain are of critical importance in maintaining the conformation of the
molecule that is recognized by the receptor (Adelhorst et al., 1994; Gallwitz et al.,
1994). In addition, it is known that the Ala18, Ala19, Lys20, and Leu26 residues of GLP-1
interact with the GLP-1 receptors as revealed by the crystal structure of GLP-1 bound
to the large N-terminal extracellular domain of the receptor (Underwood et al., 2010). It
is speculated that the higher potency and efficacy of GLP-1 from lamprey, paddlefish,
and trout in the mammalian test systems compared with the peptides from the other
species may be explained, at least in part, by the fact that they are the only peptides that
contain the Ala18 and Ala19 residues found in human GLP-1. The lower potency peptides
from dogfish and ratfish contain a basic Lys residue at position 18 and bowfin GLP-1
contains a Val residue (Fig. 8). The metabolic effects of dogfish and ratfish GLP-1 in
their species of origin have not been studied but dogfish GLP-1 has been shown to
stimulate the activity of the rectal gland of North Pacific spiny dogfish Squalus
suckleyi, increasing chloride secretion rates above baseline by approximately 16-fold
(Deck et al., 2017). Recent studies have shown that the proglucagon genes of the
holocephalan fish Callorhinchus milii (elephant shark) and the elasmobranch
Rhincodon typus (whale shark) encode a GLP-3 in addition to glucagon, GLP-1, and
GLP-2 (Irwin and Mojsov, 2018). Although only a single GLP-1 was identified in
extracts of the pancreata of the dogfish (Conlon et al., 1994) and ratfish (Conlon et al.,
1997), it is possible that both GLP-1 and GLP-3 are synthesized and released from the
intestines of these species.
The insulinotropic activity of all fish peptides in BRIN-BD11 cells was
attenuated by the GLP1R antagonist, exendin-4(9-39) and all peptides stimulated
cAMP production in CHL cells transfected with GLP1R demonstrating that they are
capable of activating the GLP-1 receptor in mammalin test systems. However, the
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insulinotropic activity of lamprey GLP-1 and paddlefish GLP-1 was significantly
decreased by the GCGR antagonist, [desHis1,Pro4,Glu9]glucagon amide suggesting that
these peptides may act as dual agonists activating both GLP-1 and glucagon receptors.
Consistent with this observation, the activity of lamprey GLP-1 was abolished in INS-1
GLP1R knockout cells in a similar manner as human GLP-1 (Fig. 6). A comparison of
the primary structures of lamprey/paddlefish GLP-1 (Fig. 8) with glucagon does not
provide any obvious clues in terms of the presence or absence of particular amino acid
residues as to why only these peptides and not the other fish GLP-1 peptides are
glucagon receptor agonists. Rather it must be assumed that only lamprey/paddlefish
GLP-1 are able to adopt a stable conformation that is recognized by the receptor.
Glucagon is predominantly disordered in aqueous solution but forms a stable, extended
helix when bound to its receptor (Siu et al., 2013). The conformations of the fish GLP-1
peptides have yet to be determined experimentally but application of the AGADIR
program, an algorithm based on the helix/coil transition theory which predicts the
helical behaviour of monomeric peptides (Muñoz and Serrano, 1994), indicates that
lamprey and paddlefish GLP-1 have a strong propensity to adopt a stable α-helical
conformation between residues 9-28 whereas the predicted helices adopted by the
dogfish, ratfish, and bowfin peptides are much less stable and extended. The GLP1R
and GCGR are highly specific for their cognate ligands. The present study has indicated
that this exquisite specificity arose relatively late in evolution, possibly not until after
emergence of tetrapods.
The bowfin, the sole extant representative of the Amiiformes, occupies an
important position in phylogeny as the surviving representatives of a group of primitive
ray-finned fishes from which present-day teleosts may have evolved (Carroll, 1988).
Bowfin GLP-1 contains several amino acid substitutions compared with human GLP-1
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that are not seen in the other fish peptides such as Gly4 → Ala, Phe22 → Trp, Ile23 →
Leu, and Leu26 → Gly (Fig.8). In particular, bowfin GLP-1, in common with GIP,
contains an N-terminal Tyr residue and a Thr7 residue that is substituted by Ile.
Evidence has been obtained using a chimeric GLP-1/GIP peptides that amino acids at
these sites (His/Tyr1 and Thr/Ile7) are responsible for the selective interaction toward
GLP-1 and GIP receptors (Moon et al., 2010). It has been proposed that His1 of GLP-1
interacts with Asn302 in extracellular loop 2 of GLP1R and that Thr7 of GLP-1 has close
contact with a binding pocket formed by Ile196 in transmembrane helix 2 and Leu232, and
Met233 in extracellular loop 1 of GLP1R (Moon et al., 2012). Despite the low overall
sequence similarity between bowfin GLP-1 and GIP (Fig. 8), the presence of Tyr1 and
Ile7 in the bowfin peptide may account for the fact that the GIP receptor antagonist,
GIP(6-30) Cex-K40[Pal] produced a small but significant attenuation of the
insulinotropic response to bowfin GLP-1 in BRIN-BD11 cells (Fig. 4) and the activity
of the peptide was reduced in CRISPR/Cas9-engineered INS-1 cells with knock-out of
the GIP receptor compared the response with wild-type INS-1 cells (Fig.6).
Bowfin GLP displayed relatively weak insulinotropic activity compared with human
GLP-1 in the mammalian systems employed in this study. Similarly, in a piscine test
system, bowfin GLP-1 stimulated glycogenolysis in isolated hepatocytes from the
copper rockfish Sebastes caurinus (Teleostei) but was 3-fold less effective and 23-fold
less potent than human GLP-1 (Conlon et al., 1993). It was speculated that selective
mutations in the GLP molecule may be an adaptive response to the low biological
potency of bowfin insulin (Conlon et al., 1991).
Trout GLP shows potent insulin-releasing activity both in vitro (Figs 1 & 2) and
in vivo (Fig. 7) but the magnitudes of the maximum responses are less than those of
paddlefish and lamprey GLP and, unlike these peptides, trout GLP appears to act only
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by activation of the GLP-1 receptor (Figs 5 & 6). As a result of a putative whole
genome duplication occurring in the ancestral fish lineage around 320-350 million
years ago (Vandepoele et al., 2004), many genes encoding neuroendocrine peptides that
exist in a single copy in tetrapods are found as two paralogs in teleost fish.
Consequently, as expected, the trout genome contains a second proglucagon gene that
encodes a GLP-1 containing the single amino substitution Arg30→ Pro compared with
the peptide used in this study (Irwin and Wong, 1995). The insulinotropic activity of
this paralog has yet to be determined.
The world is currently experiencing a global pandemic of obesity-related type 2
diabetes mellitus (T2DM) and while several therapeutic options are available for
treatment of patients, none is completely satisfactory (Bailey, 2018). None of the
existing treatments, alone or in combination, completely normalizes blood glucose
concentrations and prevents debilitating complications. Thus, there is a definite need
for new types of safe and more effective anti-diabetic agents. The present study has
demonstrated that GLP-1 peptides from lamprey and paddlefish, by interacting with
both the GLP-1 and glucagon receptors to stimulate insulin release and lower blood
glucose concentrations, have the potential to act as templates for development of new
therapeutic agents for treatment of patients with T2DM. Pre-clinical trials involving
administration of dual GLP-1/glucagon receptor agonists have shown positive results
including reduction of food intake, weight loss, inhibition of glucagon release, and β-
cell proliferation as well as normalization of blood glucose concentrations [reviewed in
(Brandt et al., 2018)]. Previous work has shown that peptides derived from dogfish
glucagon possess potent insulin releasing actions in vitro (O’Harte et al., 2016a) and
antihyperglycaemic activity in obese mice with insulin resistance and impaired glucose
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tolerance (O’Harte et al., 2016b). Future studies will investigate the activities of long-
acting peptidase-resistant analogues of lamprey and paddlefish GLP-1.
5. Conflict of interest
The authors declare that there is no conflict of interest that could be perceived as
prejudicing the impartiality of the research reported.
6. Contributions
P.R.F., J.M.C. and Y.H.A. conceived and designed the study. GVG performed the in
vitro and animal experiments. GVG and JMC purified the synthetic peptides and wrote
the manuscript. All authors analyzed and interpreted the data and have approved the
final submission.
Acknowledgements
The authors wish to thank Bernard Thorens (University of Lausanne, Switzerland) for
GLP-1 receptor-transfected CHL cells, Cecilia Unson (The Rockefeller University,
USA) for glucagon receptor-transfected HEK293 cells, and Jaqueline Naylor
(MedImmune, Cambridge, UK) for CRISPR/Cas9-engineered INS-1 cells. The study
was supported by the Northern Ireland Department of Education and Learning (DEL),
and Ulster University Strategic Funding.
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