AMER. ZOOL., 40:246–258 (2000)
Glucagon-like Peptide-1 (GLP-1) and the Control of Glucose Metabolism in
Mammals and Teleost Fish1
SVETLANA MOJSOV2
Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York
PART I
ROLE
OF INSULIN, GLUCAGON AND GLP-1
IN THE CONTROL OF GLUCOSE HOMEOSTASIS
IN MAMMALS
The two pancreatic hormones, insulin
and glucagon, are the key hormones that
control glucose homeostasis in mammals.
Insulin actions, leading to a decrease in circulating levels of glucose, are exerted at the
level of the liver, skeletal muscle and adipocytes, while glucagon actions in the liver
lead to increased levels of circulating glucose.
The experimental work with GLP-1 in
the last 15 years provided conclusive experimental evidence in support of the long
standing hypothesis that an additional
1 From the symposium A Tribute to Erika M. Plisetskaya: New Insights on the Function and Evolution
of Gastroenteropancreatic Hormones presented at the
Annual Meeting of the Society for Integrative and
Comparative Biology, 6–10 January 1999, at Denver,
Colorado.
2 E-mail: [email protected]
mechanism of the regulation of glucose homeostasis in mammals is provided by the
gastrointestinal tract (Zunz and LaBarre,
1929; Unger and Eisentraut, 1969; Creutzfeldt, 1979). The initial experiments with
GLP-1 (7–37) in the rat perfused pancreas
(Mojsov et al., 1987) and with GLP-1 (7–
36) amide in the pig perfused pancreas
(Holst et al., 1987), which demonstrated the
potent stimulatory effect of GLP-1 on insulin secretion, revived the interest in the
incretin concept (Creutzfield, 1979; Creutzfeldt and Ebert, 1985) and the proposed
function of the entero-insular axis (Unger
and Eisentraut, 1969). The concept of the
entero-insular axis postulated that some
gastrointestinal substances, also known as
incretins (Creutzfeldt, 1979), which are secreted in the gut in response to glucose uptake and ingested nutrients, will regulate
glucose metabolism by influencing the release of different hormones from the pancreatic islet cells. The function of the entero-insular axis is to coordinate the actions
of the intestinal and pancreatic hormones,
246
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SYNOPSIS. Glucose metabolism in mammalian species and teleost fish is controlled
by different metabolic pathways. These include differences in the function of several major hormones, especially insulin and GLP-1. The major physiological role
of GLP-1 in mammals is to connect the consumption of nutrients with glucose
metabolism. The glucose lowering effects of GLP-1 in the postprandial state of
mammals are regulated predominantly through metabolic pathways that integrate
different physiological processes. These are: (i) stimulation of insulin release from
the pancreatic b-cell during hyperglycemia and (ii) inhibition of nutrient absorption in the gastrointestinal tract. These effects are mediated by a same type of a
highly selective GLP-1 receptor, often referred to as the ‘‘pancreatic GLP-1 receptor.’’ In teleost fish GLP-1 increases glucose levels through the activation of
glycogenolysis and gluconeogenesis from liver. Functional characterization of the
recombinant GLP-1 receptor from zebrafish, which is the first example of a recombinant fish GLP-1 receptor, demonstrated that zebrafish GLP-1 receptor has
a binding specificity towards a wider range of GLP-1 structures than the mammalian GLP-1 receptor. This property of the zebrafish GLP-1 receptor, and most
likely other fish GLP-1 receptors, sets apart the structure of the zebrafish GLP-1
receptor from the structures of mammalian GLP-1 receptors. These differences in
the binding specificity between the zebrafish and mammalian GLP-1 receptors
might reflect in part the differences in the mechanism by which GLP-1 regulates
glucose metabolism in mammals and teleost fish.
GLP-1 RECEPTORS
IN
MAMMALS
and, thus, accelerate the secretions of the
islet hormones in response to uptake of nutrients. The finding that in human subjects
GLP-1 is released in circulation after consumption of a meal (Kreymann et al., 1987)
reinforced the notion that GLP-1 might be
one of the postulated incretins.
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incretin functions of GLP-1. Early experiments with GLP-1 demonstrated that the
potent stimulatory effects of GLP-1 on insulin secretion were manifested in a glucose-dependent manner. Thus, in the rat
perfused pancreas GLP-1 stimulated insulin
release only under conditions of elevated
glucose, a state typically induced by nutrient uptake. GLP-1 had no effect on insulin
secretions from the rat perfused pancreas at
basal glucose concentrations of 3 mM (Weir
et al., 1989). Similarly, the effect of GLP1 (7–36) amide administration on insulin
release in healthy human subjects was more
pronounced at higher glucose concentrations (Kreymann et al., 1987). These early
observations were later confirmed in animal
studies, which demonstrated that the effect
of GLP-1 (7–37) on insulin release was significant only under conditions of hyperglycemia (Hargrove et al., 1996).
In addition to its stimulatory effects on
insulin secretion, GLP-1 inhibits glucagon
FIG. 1. Schematic representation of the known GLP-1 effects in mammals that regulate glucose metabolism
through the interaction of GLP-1 with specific high affinity receptors (designated with red triangles) expressed
in the pancreatic b-, a- and d-cells, respectively, and in the gastrointestinal tract. The labeling of the GLP-1
receptors on different cell types in the pancreatic islets is arbitrary and illustrates the known GLP-1 effects on
secretions of insulin, glucagon and somatostatin. The identity of the intestinal cells expressing GLP-1 receptors
is not known yet. Representation of GLP-1 receptor expression in intestinal cells illustrates only the known
GLP-1 effects in the gastrointestinal tract.
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GLP-1 and the incretin concept
In mammals, the major physiological
role of GLP-1 is to connect the consumption of nutrients with glucose metabolism
through a network of regulatory pathways
that are integrated predominantly at the level of the pancreatic b-cells. The unique
property of the mammalian b-cell is its ability to secrete insulin only in response to elevated levels of glucose in circulation. This
secretory response of the b-cells ensures
that glucose levels are maintained at constant levels at all the times regardless of the
nutritional input.
Several lines of evidence established the
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SVETLANA MOJSOV
secretion from the pancreatic a-cells (Kreymann et al., 1987; Orskov et al., 1988), and
stimulates somatostatin release from the dcells (Orskov et al., 1988). The ability of
GLP-1 to inhibit the release of glucagon
from pancreatic a-cells is especially important, because this effect contributes to the
overall decrease of glucose levels in circulation.
GLP-1 and Type 2 (non-insulin
dependent) and Type 1 (insulin dependent)
diabetes mellitus
The importance of the accurate functions
of the regulatory mechanisms that maintain
circulating insulin and glucose levels constant is demonstrated in the disorders of
glucose metabolism referred to as insulin
resistance and manifested in patients with
Type 2 (non-insulin dependent) diabetes
mellitus (NIDDM). In these individuals the
blood glucose levels are elevated despite elevated levels of insulin in circulation. The
persistence of elevated glucose and insulin
levels leads to number of secondary com-
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Gastrointestinal effects of GLP-1
GLP-1 contributes to the maintenance of
circulating glucose levels also through its
actions in the gastrointestinal tract, where
GLP-1 inhibits gastric emptying (Wettergren et al., 1993) and small bowel motility
in a fed, but not fasting state (Tolessa et al.,
1998). The former effect is especially beneficial after an ingestion of a meal when
there is a large and rapid increase in glucose
concentrations in circulation, and provides
the pancreatic b-cell with a certain lag period during which it can adapt its secretory
response with a proper release of insulin.
Recent evidence suggests that the gastrointestinal effects of GLP-1 may outweigh
the insulinotropic effects of GLP-1 on glucose metabolism (Nauck et al., 1997).
Thus, all the available evidence indicates
that the glucose lowering effects of GLP-1
in the postprandial state of mammals are
achieved primarily through its stimulatory
effects on insulin secretion during hyperglycemia, and through the inhibition of gastric emptying which slows down nutrient
absorption in the gastrointestinal tract (Fig.
1).
plications, the most prominent of which are
manifested as cardiovascular disease and
hypertension.
The origins of the metabolic disorders in
the regulation of insulin-glucose homeostasis are complex and include a genetic predisposition to the disease. The most common disorder in individuals with Type 2 diabetes mellitus is manifested at the level of
the peripheral tissues, that are the sites of
glucose turnover, such as skeletal muscle,
liver and adipose tissue. These tissues, and
especially skeletal muscle, are unable to utilize glucose from circulation despite elevated levels of insulin. This condition is often referred to as insulin resistance. Disorders in individuals with Type 2 diabetes
mellitus are also found at the level of the
pancreatic b cells. These cells have lost the
ability to rapidly secrete insulin in response
to elevated levels of glucose in circulation.
This condition is most prominently displayed after a consumption of a meal when
there is a rapid increase of glucose in circulation.
Clinical studies with patients with Type
2 diabetes mellitus demonstrated that the
incretin effects of GLP-1 are preserved in
this group of patients (Nauck et al., 1993;
Nathan et al., 1992; Gutniak et al., 1992).
Administration of GLP-1 to individuals
with Type 2 diabetes together with a meal
eliminated the postprandial rise of glucose
in circulation (Nathan et al., 1992). The
rapid and effective decrease of circulating
glucose levels detected in these early clinical studies with patients with Type 2 diabetes mellitus was most likely accomplished through the combined effects of
GLP-1 on insulin secretion and inhibitory
effects on gastric emptying (Willms et al.,
1996) (Figure 1).
In individuals with Type 1 (insulin dependent) diabetes mellitus (IDDM) the pancreatic b-cells are destroyed as a result of
an autoimmune disorder. However, the pancreatic a-cells are intact and are able to secrete glucagon. Administration of GLP-1 to
this group of patients lowered their circulating glucose levels (Gutniak et al., 1992;
Creutzfeldt et al., 1996). These observations provide additional evidence in support
of the conclusions that the glucose lowering
GLP-1 RECEPTORS
IN
MAMMALS
Tissue-specific distribution of GLP-1
receptors, incretin concept and the
mechanism of regulation of glucose
metabolism by GLP-1
The existence of specific GLP-1 receptors was established in competitive binding
experiments performed in insulin secreting
cell lines derived from rat insulinomas
(Goke and Conlon, 1988; Mojsov, 1992).
However, only after the cloning of the rat
and human GLP-1 receptors (Thorens,
1992; Thorens et al., 1993), it became apparent that the incretin effects of GLP-1 in
the pancreas, and GLP-1 effects in intestine,
are mediated by a single type of GLP-1 receptors, commonly referred to as the ‘‘pancreatic type’’ (Wei and Mojsov, 1995; Dunphy et al., 1998).
The availability of recombinant GLP-1
receptors provided new experimental tools
which were used to support and extend the
conclusions obtained from the biochemical,
physiological and clinical experiments. As
a result of the application of molecular biology approaches several other tissues, in
addition to the pancreas and intestine, were
identified as targets for GLP-1 action (Lankat-Buttgereit et al., 1994; Wei and Mojsov,
1995; Bullock et al., 1996; Dunphy et al.,
1998). These include lung, kidney, heart
and brain. The molecular biology experiments taken together with the functional experiments demonstrated that GLP-1 also
has a function in the regulation of physiological processes that are not connected to
the control of glucose metabolism, and that
these GLP-1 effects are mediated by the
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249
pancreatic type of the GLP-1 receptor. Currently, the role of GLP-1 in the central nervous system is a subject of intensive research. The available information indicates
that GLP-1 regulates appetite and nutrient
consumption (Dijk et al., 1997; Turton et
al., 1996; Thiele et al., 1997), and, thus,
contributes to the regulatory pathways that
control the feeding behavior of mammals.
By RNase protection experiments GLP1 receptor transcripts were not detected in
peripheral tissues which are the sites of glucose turnover, such as liver, skeletal muscle
and adipocytes (Wei and Mojsov, 1995;
Bullock et al., 1996; Dunphy et al., 1998).
The absence of GLP-1 receptor transcripts
in the liver is consistent with the initial observations that GLP-1 was unable to stimulate gluconeogenesis from rat hepatocytes
(Blackmore et al., 1991). In this set of experiments GLP-1 also did not have any effects on several intracellular messengers involved in mediating glucose metabolism in
the liver, such as, for example, cAMP and
Ca21 flux, and was unable to activate phosphorylase A. In contrast to these findings,
several recent reports have described GLP1 mediated effects in the liver (Valverde et
al., 1994). In these experiments GLP-1 exhibited insulin-like effects on glucose metabolism in healthy (Valverde et al., 1994)
and diabetic (Morales et al., 1997) rats.
Similarly, GLP-1 effects in skeletal muscle
and adipocytes have been described by
some research groups (Valverde et al.,
1993; Villanueva-Penacarillop et al., 1994)
and disputed by others (Furnsinn et al.,
1995; Hansen et al., 1998). There is a general agreement that, if indeed GLP-1 modulates glucose metabolism in glucose sensitive peripheral tissues of mammals, then
these effects will be mediated by a GLP-1
receptor that is structurally distinct from the
pancreatic form of the receptor (Dunphy et
al., 1998).
The question of the possible role of GLP1 in regulating glucose uptake from the liver, skeletal muscle and adipose tissues can
not be resolved unless the physiological
conditions leading to the proposed insulinlike effects of GLP-1 in glucose sensitive
peripheral tissues are properly defined and
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effects of GLP-1 are achieved through a
mechanism outlined in Figure 1. In the case
of individuals with Type 1 diabetes, the incretin effects of GLP-1 are manifested
through the inhibition of glucagon secretion
from the a-cells (Kreymann et al., 1987).
The findings that GLP-1 is very effective
in stimulating insulin secretion in patients
with Type 2 diabetes mellitus during hyperglycemia demonstrated the therapeutic
potential of GLP-1, and initiated efforts to
develop a new type of ‘‘GLP-1 based’’ therapeutic agents for treatment of disorders of
glucose metabolism in patients with Type
2, and possibly Type 1 diabetes mellitus.
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SVETLANA MOJSOV
examined. Otherwise, this issue will remain
controversial.
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Ligand specificity of the mammalian GLP1 receptor
The experiments in the rat hepatocytes
that demonstrated that GLP-1 did not antagonize the stimulatory effects of glucagon
on intracellular cAMP levels and Ca21 flux
provided the first preliminary evidence that
physiological effects of GLP-1 are mediated through a GLP-1 specific receptor and
not through the glucagon receptor (Blackmore et al., 1991). These conclusions were
further confirmed in binding experiments
utilizing the insulin secreting RIN 1046-38
and RIN-5mF cell lines (Mojsov, 1992;
Goke and Conlon, 1988), and when the recombinant GLP-1 receptor was characterized (Thorens, 1992; Thorens et al., 1993).
Collectively, these experiments established
that glucagon is only a weak agonist of the
mammalian GLP-1 receptor with the inhibition concentration IC50 in the 10 mM concentration range.
As already mentioned earlier, there is
only a single known type of GLP-1 receptors in mammals. The GLP-1 receptor has
the structural characteristics found in all the
G-protein coupled receptors (GPCRs) consisting of seven membrane spanning domains connected to each other by three extracellular and three intracellular loops, as
is the case with the other known members
of this class of GPCRs for metabolic hormones, like glucagon, secretin and glucose
dependent insulinotropic hormone (GIP)
(Segre and Goldring, 1993). All these receptors contain a large extracellular domain
where several cysteine residues are located.
Their cytoplasmic domains contain several
potential phosphorylation sites.
The GLP-1 receptor has an exquisite
specificity towards GLP-1 (7–36) amide
and GLP-1 (7–37), which are the two biologically active forms of GLP-1 (Mojsov et
al., 1987; Weir et al., 1989; Orskov et al.,
1993). In the competitive binding experiments performed in the RIN 1046-38 cell
line the specific binding of I-125-GLP-1
(7–37) was displaced in the same concentration range with GLP-1 (7–37) and GLP1 (7–36) amide, with an inhibition concen-
tration IC50 of 0.6 nM (Mojsov, 1992). In
contrast, GLP-1 receptor has only a weak
affinity towards the biologically inactive
forms of the mammalian GLP-1s (GLP-1
(1–37), and GLP-1 (1–36) amide) (Mojsov,
1992; Goke and Conlon, 1988). Thus, the
amino terminal hexapeptide extension
found in the sequences of GLP-1 (1–37)
and GLP-1 (1–36) amide changes dramatically the high affinity of the GLP-1 receptor
towards GLP-1 (7–37) and GLP-1 (7–36)
amide structures. The entire sequence of
these two peptides is necessary for the recognition by the GLP-1 receptor. Thus, the
lack of the amino terminal histidine (position 7 in the sequence) leads to a 1,000 fold
lower affinity of the GLP-1 receptor towards this peptide structure (Mojsov, 1992;
Gefel et al., 1990). The sequential deletion
of the 2 carboxyl terminal amino acids of
GLP-1 (7–36) amide leads to a more gradual loss of binding affinity (Mojsov, 1992),
while the deletion of the third carboxyl terminal residue leads to a GLP-1 (7–33)
structure completely devoid of insulinotropic activity (Gefel et al., 1990).
The mammalian GLP-1 receptor has also
high affinity towards the sequences of four
GLP-1 peptides encoded by the Xenopus
laevis proglucagon mRNA (Irwin et al.,
1997). IC50s measured in competitive binding assays for the Xenopus GLP-1b (1–30)
and GLP-1b (1–32) are similar to the ones
determined for mammalian GLP-1 (7–37)/
GLP-1 (7–36) amide, while IC50s for Xenopus GLP-1a (1–37), GLP-1a (1–32), GLP1c (1–30) are about 3 to 10 fold higher than
the values obtained for mammalian GLP1s. Each of these four Xenopus GLP-1s
shows high structural homology to the sequence of the mammalian GLP-1 (7–37)/
GLP-1 (7–36) amide (Fig. 2) consisting of
54% for Xenopus GLP-1a and 66% for
Xenopus GLP-1b and GLP-1c.
Despite the stringent structural requirements needed for the recognition of GLP-1
by its receptor, it was found that GLP-1 receptor has also a high affinity towards the
structure of the exendin-4 peptide (Eng et
al., 1992), which shares only 52% amino
acid sequence identity with the sequences
of GLP-1 (7–37) and GLP-1 (7–36) amide.
This level of structural homology is similar
GLP-1 RECEPTORS
IN
MAMMALS
AND
TELEOST FISH
251
to the one that exists between the amino
acid sequences of GLP-1 and glucagon
(Fig. 2), and yet glucagon is only a weak
agonist of the mammalian GLP-1 receptor,
as already mentioned earlier.
Exendin-4 is a 39 residue long peptide
isolated from the venom of the Heloderma
suspectum (Eng et al., 1992), and is found
exclusively in the lizard species (Pohl and
Wank, 1998; Chen and Drucker, 1997). The
effects of GLP-1 and exendin-4 are indistinguishable from each other in both functional experiments with the recombinant
GLP-1 receptor (Thorens et al., 1993; Goke
et al., 1993) and in the ability to stimulate
insulin secretion (Eng and Eng, 1992).
Deletion of the first 9 amino acids from
the amino terminus of exendin leads to an
exendin- (9–39) peptide that is still recognized by the GLP-1 receptor, but is unable
to stimulate the cAMP-mediated signal
transduction pathway (Thorens et al.,
1993). Thus, exendin-4 is a full agonist of
the GLP-1 receptor, while exendin- (9–39)
is the only known GLP-1 receptor antagonist (Schirra et al., 1998). This property of
exendin (9–39) was especially useful in delineating the incretin effects of GLP-1 (Kolligs et al., 1995; Wang et al., 1995; Mark
et al., 1999).
Chimeric peptide structures containing
domains from GLP-1 and exendin sequences were unable to mimic fully the interactions of the GLP-1 receptor with
GLP-1 and exendin-4 (Parker et al., 1998).
For example, the chimeric peptide that
contained residues 9–23 of exendin-4 and
residues 29–36 of GLP-1 (i.e., structure
having the sequence DLSKQMEEEAVR
LFIAWLVKGR-NH2) (Fig. 2) was recognized by the GLP-1 receptor with a decreased binding affinity, but was without
any functional activity. This observation is
in agreement with the earlier findings that
exendin (9–39) is an antagonist of the GLP1 receptor, and indicates that different domains in the GLP-1 (and exendin-4) sequences are responsible for the binding to
the GLP-1 receptor and for the activation
of a cAMP-mediated signal transduction
pathway.
Ligand specificity of the mammalian GLP1 receptor towards zebrafish GLP-1
The remarkable specificity of the mammalian GLP-1 receptors towards the structures of GLP-1 (7–37), GLP-1 (7–36) amide, Xenopus GLP-1s and exendin-4 suggests that the binding pocket of mammalian
GLP-1/GLP-1 receptor system can accommodate only a highly constrained peptide
conformation specified by the amino acid
sequences of these peptides.
The sequences of GLP-1s from a large
number of teleost fish are known (Fig. 3),
(reviewed in Plisetskaya and Mommsen,
1996). Comparison with the sequences of
biologically active forms of mammalian
GLP-1s shows that there is about 60–70%
sequence identity between the mammalian
and fish GLP-1s (Fig. 3). The main differences are found in discrete amino acid domains located in the middle part of the sequence and in the carboxyl terminal domains. The contribution of these different
amino acid domains to the formation of the
binding pocket of mammalian GLP-1 receptor can be determined in competitive
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FIG. 2. Comparison of the amino acid sequence of mammalian GLP-1 (Bell et al., 1983; Lopez et al., 1983;
Heinrich et al., 1984) with the sequences of GLP-1s from Xenopus (Irwin et al., 1997), exendin-4 (Eng et al.,
1992) and human glucagon (Bromer, 1983). Shaded areas represent identical amino acid residues.
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SVETLANA MOJSOV
binding experiments. The affinity of the
mammalian GLP-1 receptor towards the sequence of the zebrafish GLP-1 was examined first, because the sequence of zebrafish
GLP-1 shows a high degree of divergence
FIG. 4. Human GLP-1 receptor has a high binding
specificity towards the structures of GLP-1 (7–36) amide, and exendin-4, but not zebrafish GLP-1. Competitive binding experiments with the recombinant human
GLP-1 receptor expressed transiently into COS-7 cells
in which the binding of the human I-125-GLP-1 (7–
36) amide to the recombinant human GLP-1 receptor
was displaced with increasing concentrations (pM to
mM) of human GLP-1 (7–36) amide (open triangles),
exendin-4 (crossed lines) and zebrafish GLP-1 (closed
triangles). Competitive binding experiments were performed 48 hr after transfection of GLP-1 receptor
cDNA into COS-7 cells as described previously (Wei
and Mojsov, 1996). Each data point represents an average of three independent measurements.
(32%) with the mammalian GLP-1 sequence (Fig. 3).
The results from the competitive binding
experiments showed that amino acid substitutions in the zebrafish GLP-1 sequence
dramatically decrease the binding affinity of
the mammalian GLP-1 receptor towards the
zebrafish GLP-1 (Fig. 4). The inhibition
concentration IC50 is about 1,000 fold higher for zebrafish GLP-1 than for mammalian
GLP-1 (7–36) amide and exendin-4 (Table
1). Thus, the degree of divergence that exists between the mammalian and zebrafish
GLP-1s, although smaller than that between
the sequences of mammalian GLP-1 and
exendin-4 (32% vs. 48%, respectively) (Fig.
2), is sufficient to change dramatically the
affinity of the mammalian GLP-1 receptor
towards zebrafish GLP-1 sequence.
The results shown in Figure 4 confirm
and extend previous conclusions that only
a highly specific conformation of GLP-1
structure is recognized by the mammalian
GLP-1 receptor. This suggests that such a
conformation is formed by the amino acid
sequences of GLP-1 peptides found in vertebrates that evolved after zebrafish.
PART II
ROLE OF INSULIN, GLUCAGON AND GLP-1
IN THE CONTROL OF GLUCOSE METABOLISM
IN TELEOST FISH
Increasing evidence suggests that glucose
metabolism in teleost fish and mammals is
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FIG. 3. Comparison of the amino acid sequences of the biologically active forms of mammalian GLP-1 with the
sequences of GLP-1 from selected number of teleost fish. The sequence of anglerfish GLP-1s is from Lund et
al. (1982), Lund et al. (1983), the sequence of tilapia from Nguyen et al. (1995), the sequence of salmon from
Plisetskaya et al. (1986), the sequence of daddy sculpin from Conlon et al. (1987), the sequence of catfish from
Andrews and Ronner (1985), the sequence of trout I from Irwin and Wong (1995), the sequence of goldfish
from Yuen et al. (1997) and the sequence of zebrafish from Mommsen and Mojsov (1998). Shaded areas
represent identical amino acids.
GLP-1 RECEPTORS
IN
MAMMALS
TELEOST FISH
253
1991; Wright et al., 1998) reinforces the
conclusions that the precise balance of circulating insulin and glucose levels is not
needed for the maintenance of metabolic
processes in teleost fish.
Characterization of a GLP-1 receptor
from zebrafish
It is clear that the general mechanism of
regulation of glucose metabolism in teleost
fish needs to be defined independently from
the one that exist in mammals. Consequently, the role of GLP-1 in the control of glucose metabolism in teleost fish needs to be
considered in the context of the metabolic
pathways that regulate their glucose metabolism in the absence of a feedback loop that
maintains a steady levels of insulin and glucose in circulation.
The cloning, functional characterization
and determination of the tissue distribution
of GLP-1 receptors in teleost fish represent
initial steps in understanding the mechanism by which GLP-1 regulates glucose
metabolism in teleost fish, especially in
view of the findings that GLP-1 and glucagon have similar effects on glucose metabolism in fish hepatocytes.
Ideally, the cloning and characterization
of GLP-1 receptors in teleost fish should be
accomplished in species of fish where the
glucagon-like effects of GLP-1 in fish hepatocytes have been well characterized, as
is the case with the catfish and rockfish (Plisetskaya and Mommsen, 1996). But, because the reagents for the molecular biology approaches in these teleosts have not
been developed yet, the zebrafish represents
a better starting point for these studies.
A combination of a RT-PCR and homology based cloning strategy was used to isolate a G-protein coupled receptor from a
cDNA library prepared from a whole 6-day
old fish. The deduced amino acid sequence
of the putative receptor contained all the
structural features found in this class of Gprotein coupled receptors (GPCRs) and
showed about 50% structural homology
with the sequence of the human GLP-1 receptor (Fig. 5). The main differences between the sequences of the zebrafish receptor and mammalian GLP-1 receptor are
found in their cytoplasmic domain, which
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controlled by different metabolic pathways.
These differences are manifested in the
functions of the two major hormones, insulin and GLP-1 (reviewed in Plisetskaya
and Mommsen, 1996), as well as glucose
transporters (Wright et al., 1998). The
available information indicates that fish can
tolerate and experience large fluctuations in
circulating insulin and glucose levels
(Mommsen and Plisetskaya, 1991). They
can exist in the state of elevated insulin and
elevated glucose levels without the metabolic consequences that develop in humans
characterized by insulin resistance and Type
2 diabetes mellitus (NIDDM).
The early experiments with GLP-1 demonstrated that GLP-1 exerted powerful effects on glycogenolysis and gluconeogenesis in hepatocytes of different species of teleost fish (Mommsen et al., 1987). These
findings, that were reproduced in multiple
experiments with both mammalian and fish
GLP-1 peptides, and in different species of
teleosts (Mommsen and Moon, 1989;
Mommsen and Moon, 1990) established an
important paradigm for comparative and
evolutionary physiology. They demonstrated that an evolutionarily conserved peptide
could regulate glucose metabolism in mammals and teleost fish through different regulatory pathways.
In contrast to the glucose lowering effects of GLP-1 in mammals, the effects of
GLP-1 on glucose metabolism in fish hepatocytes are similar to those of glucagon,
the only known difference being that GLP1 is more potent than glucagon in stimulating gluconeogenesis and glycogenolysis in
most, if not all species of teleost fish tested
(reviewed in Plisetskaya and Mommsen,
1996). At present, GLP-1 seems to be one
of the most potent direct metabolic hormones in fish species.
GLP-1 did not have a significant effect
on insulin secretion from the dispersed pancreatic cells of coho salmon (Plisetskaya
and Mommsen, 1996), another finding that
sets apart the physiological functions of
GLP-1 in fish from those in mammalian
species. This observation taken together
with the findings that glucose does not regulate insulin secretion from the fish endocrine pancreas (Mommsen and Plisetskaya,
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is considerably longer in the zebrafish receptor. Some of the key structural features
are completely conserved. For example, all
the cysteine residues are located at identical
positions in the amino terminal extracellular
domain and the first intracellular loop. In
addition, the mammalian ‘‘RLAK’’ structural motif found in the sequence preceding
the sixth membrane spanning domain is located in the corresponding position of the
zebrafish receptor. This motif is essential
for interaction of all known GPCRs with
the intracellular G-proteins (Okamoto et al.,
1991). The presence in the zebrafish receptor sequence suggested that it could be
characterized in functional experiments by
transfecting its cDNA into cell lines of
mammalian origin.
Ligand specificity of the zebrafish GLP-1
receptor
The ligand specificity of the putative zebrafish GLP-1 receptor was determined in
competitive binding experiments in which
the binding of the radioiodinated human I125-GLP-1 (7–36) amide to the recombinant zebrafish GLP-1 receptor, transiently
expressed in mammalian COS-7 cells, was
displaced with increasing concentrations
(pM to mM range) of GLP-1 (7–36) amide,
zebrafish GLP-1 and exendin-4. As shown
in Figure 6, zebrafish GLP-1 receptor
Downloaded from http://icb.oxfordjournals.org/ at Pennsylvania State University on February 23, 2013
FIG. 5. Comparison of the deduced amino acid sequences of zebrafish (top lines) and human (bottom lines)
GLP-1 receptors. Shaded areas represent identical amino acids. The horizontal lines above the sequences represent the position of the membrane spanning domains.
GLP-1 RECEPTORS
IN
MAMMALS
AND
TELEOST FISH
255
TABLE 1. Summary of the competitive binding experiments with recombinant human GLP-1R and zebrafish
GLP-1R.
IC50*
hGLP-1R
zfGLP-1R
hGLP-1
(7–36)amide
zf GLP-1
Exendin-4
3 nM
0.9 nM
mM range
2 nM
1 nM
0.9 nM
* IC50’s were calculated from displacement curves
obtained in competitive binding experiments shown in
Figure 4 and Figure 6, and represent concentration of
peptides that inhibited the specific binding by 50%.
showed similar binding affinities towards
human GLP-1, exendin-4 and zebrafish
GLP-1 with similar IC50s for all these peptides (Table 1). Thus, in contrast to the
mammalian GLP-1 receptor, the binding
pocket of zebrafish GLP-1 receptor can accommodate a wider range of structural conformations which are specified not only by
the amino acid sequence of zebrafish GLP1, but also by the sequences of mammalian
GLP-1 (7–37)/GLP-1 (7–36) amide and exendin-4. The broad ligand specificity of the
zebrafish GLP-1 receptor sets apart its
structure from the structures of the mammalian GLP-1 receptors.
GLP-1 receptors from other species of
teleost fish need to be cloned and characterized before these conclusions can be generalized. However, the functional experiments demonstrating that mammalian and
fish GLP-1s, including zebrafish GLP-1,
stimulate gluconeogenesis and glycogenolysis in catfish and rockfish hepatocytes in a
similar concentration range (Plisetskaya
and Mommsen, 1996; Mommsen and Mojsov, 1998) indicate that GLP-1 receptors in
all teleosts would probably contain similar
ACKNOWLEDGMENTS
I am deeply grateful to Dr. Erika Plisetskaya who provided the impetus for the
studies on the molecular characterization of
GLP-1 receptors in fish. I want to thank Dr.
Thomas P. Mommsen for many discussions
about fish physiology and comparative evolution, Yang Wei for his contribution to the
cloning and characterization of the zebrafish GLP-1 receptor, and Drs. Stella C. Martin and Gerhard Heinrich for the zebrafish
cDNA library. I want to especially thank
Drs. Stacia Sower and Mark Sherdan for
organizing the Symposium in honor of Dr.
Plisetskaya and providing a forum to review the current status of our knowledge of
the evolution and function of gastroenteropancreatic hormones.
The research described in this paper was
supported by grants from National Science
Foundation IBN 9513989 and Diabetes Action Research and Educational Foundation.
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