TY - JOUR
AU - Mojsov,, Svetlana
AB - Abstract 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 β-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. 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 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, 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. 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 β-cells. The unique property of the mammalian β-cell is its ability to secrete insulin only in response to elevated levels of glucose in circulation. This secretory response of the β-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 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 GLP-1 (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 secretion from the pancreatic α-cells (Kreymann et al., 1987; Orskov et al., 1988), and stimulates somatostatin release from the δ-cells (Orskov et al., 1988). The ability of GLP-1 to inhibit the release of glucagon from pancreatic α-cells is especially important, because this effect contributes to the overall decrease of glucose levels in circulation. 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 β-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). 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 complications, 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 β 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 β-cells are destroyed as a result of an autoimmune disorder. However, the pancreatic α-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 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 α-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. 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 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 GLP-1 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 Ca2+ flux, and was unable to activate phosphorylase A. In contrast to these findings, several recent reports have described GLP-1 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 GLP-1 in regulating glucose uptake from the liver, skeletal muscle and adipose tissues can not be resolved unless the physiological conditions leading to the proposed insulin-like effects of GLP-1 in glucose sensitive peripheral tissues are properly defined and examined. Otherwise, this issue will remain controversial. Ligand specificity of the mammalian GLP-1 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 Ca2+ 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 μM 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 GLP-1 (7–36) amide, with an inhibition concentration 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), GLP-1c (1–30) are about 3 to 10 fold higher than the values obtained for mammalian GLP-1s. 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 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 DLSKQMEEEAVRLFIAWLVKGR-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 GLP-1 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 GLP-1 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 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 (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 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 GLP-1 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, 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 G-protein 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 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 I-125-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 μM range) of GLP-1 (7–36) amide, zebrafish GLP-1 and exendin-4. As shown in Figure 6, zebrafish GLP-1 receptor 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 GLP-1, 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 structural specificity as the zebrafish GLP-1 receptor. On the basis of these findings we propose that the structural differences that exist in the ligand binding specificity of the GLP-1/GLP-1 receptor system in mammals and teleost fish may reflect in part the differences in the mechanism by which GLP-1 regulates glucose metabolism in these evolutionary distant species. 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: mojsov@rockvax.rockefeller.edu Open in new tabDownload slide 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 β-, α- and δ-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. Open in new tabDownload slide 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 β-, α- and δ-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. Open in new tabDownload slide 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. Open in new tabDownload slide 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. Open in new tabDownload slide 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. Open in new tabDownload slide 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. Open in new tabDownload slide 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 μM) 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. Open in new tabDownload slide 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 μM) 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. Open in new tabDownload slide 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. Open in new tabDownload slide 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. Open in new tabDownload slide Fig. 6. Zebrafish GLP-1 receptor has a high binding specificity towards the structures of zebrafish GLP-1, human GLP-1 (7–36) amide and exendin-4. Competitive binding experiments with the recombinant zebrafish 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 zebrafish GLP-1 receptor was displaced with increasing concentrations (pM to μM) of human GLP-1 (7–36) amide (open triangles), exendin-4 (crossed lines) and zebrafish GLP-1 (closed triangles). Experimental conditions were the same as the ones described in Legend to Figure 4. Open in new tabDownload slide Fig. 6. Zebrafish GLP-1 receptor has a high binding specificity towards the structures of zebrafish GLP-1, human GLP-1 (7–36) amide and exendin-4. Competitive binding experiments with the recombinant zebrafish 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 zebrafish GLP-1 receptor was displaced with increasing concentrations (pM to μM) of human GLP-1 (7–36) amide (open triangles), exendin-4 (crossed lines) and zebrafish GLP-1 (closed triangles). Experimental conditions were the same as the ones described in Legend to Figure 4. Table 1. Summary of the competitive binding experiments with recombinant human GLP-1R and zebrafish GLP-1R. Open in new tab Table 1. Summary of the competitive binding experiments with recombinant human GLP-1R and zebrafish GLP-1R. Open in new tab 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. References Andrews , P.C. , and P. Ronner. 1985 . Isolation and structures of glucagon and glucagon-like peptide from catfish pancreas. J. Biol. Chem. , 260 3910 -3914. PubMed Bell , G.T. , R.F. Santere, and G.T. Mullenbach. 1983 . Hamster preproglucagon contains the sequences of glucagon and two related peptides. 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TI - Glucagon-like Peptide-1 (GLP-1) and the Control of Glucose Metabolism in Mammals and Teleost Fish
JF - Integrative and Comparative Biology
DO - 10.1093/icb/40.2.246
DA - 2000-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/glucagon-like-peptide-1-glp-1-and-the-control-of-glucose-metabolism-in-R4b0vUnAtV
SP - 246
VL - 40
IS - 2
DP - DeepDyve
ER -