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Amphibian glucagon family peptides: potent metabolic regulators in fish hepatocytes
Regulatory Peptides 99 Ž2001. 111–118 www.elsevier.comrlocaterregrep
Amphibian glucagon family peptides: potent metabolic regulators in fish hepatocytes Thomas P. Mommsen a,) , J. Michael Conlon b, David M. Irwin c a
Department of Biochemistry and Microbiology, UniÕersity of Victoria, P.O. Box 3055, Victoria, BC, Canada V8W 3P6 b Department of Biomedical Sciences, Creighton UniÕersity Medical School, Omaha, NE, USA c Department of Laboratory Medicine and Pathobiology, UniÕersity of Toronto, Toronto, ON, Canada Received 20 August 2000; received in revised form 30 January 2001; accepted 6 February 2001
Abstract Peptides analogous to glucagon-like peptide-1 ŽGLP-1. have been isolated from amphibian pancreas and intestine, and their amino acid sequences and cDNA structures elucidated. Just like their mammalian counterpart, these peptides are potent insulinotropins in mammalian pancreatic cells. We show here that these peptides also exert strong glycogenolytic actions when applied to dispersed fish hepatocytes. We compared the potencies of three synthetic GLP-1s from Xenopus laeÕis and two native GLP-1s from Bufo marinus in the activation of glycogenolysis in the hepatocytes of a marine rockfish Ž Sebastes caurinus . and two freshwater catfish Ž Ameiurus nebulosus and A. melas ., and demonstrated their effectiveness in increasing the degree of phosphorylation of glycogen phosphorylase. We also compared the glycogenolytic potency of the peptides with those of human GLP-1 and glucagons from human and B. marinus. Sensitivity to these peptides is species-specific, with the rockfish responding at lower concentrations to GLP-1s and the two catfish reacting better to glucagons. However, the relative potency of the amphibian GLP-1s and glucagons is similar in the three species. Xenopus GLP-1C Ž xGLP-1C. is consistently more potent than xGLP-1B, while xGLP-1A displays the smallest activation of glycogenolysis. Similarly, Bufo GLP-1Ž32. —the peptide with the highest amino acid sequence identity to xGLP-1C—always shows a higher potency than Bufo GLP-1Ž37., which is closely related to xGLP-1B. The relative hierarchy of these glycogenolytic GLP-1s differs from their ranking as insulinotropins in mammalian b-cells. In the rockfish system, Bufo glucagon-36, a C-terminally extended glucagon, is more potent than the shorter bovine glucagon and Bufo glucagon-29 in the activation of glycogenolysis; when tested in A. nebulosus hepatocytes, bovine and amphibian glucagons are equipotent. Amphibian GLP-1s and glucagons activate glycogenolysis in fish hepatocytes through increased phosphorylation of glycogen phosphorylase, implying involvement of the adenylyl cyclaserprotein kinase A system in signal transduction. We conclude that the broad physiological effectiveness of GLP-1 has been retained throughout vertebrate evolution, and that both insulinotropic activity and glycogenolytic actions belong to the repertoire of GLP-1. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Insulinotropin; Glycogenolysis, Fish hepatocytes; Glucagon; Glycogen phosphorylase
1. Introduction Glucagon-like peptides in vertebrates are expressed in pancreatic endocrine tissue, intestine and brain, and display a wide variety of biological activities. The proglucagon gene coencodes glucagon, glucagon-like peptides-1 ŽGLP1. and GLP-2. While the functions of glucagon as key regulator of hepatic metabolic functions are fairly well
) Corresponding author. Tel.: q1-250-721-6508; fax: q1-250-7218855. E-mail address: [email protected] ŽT.P. Mommsen..
defined, the field of functions for the GLPs is expanding rapidly. The GLPs regulate insulin release ŽGLP-1, mammalian pancreas w1,2x., acid secretion ŽGLP-1, mammals., lung function ŽGLP-1, mammals., glycogenolysis, gluconeogenesis and lipolysis ŽGLP-1, fish liver w3x., feeding ŽGLP-1, brain w4,5x., gut glucose uptake and oxidation ŽGLP-1, fish w6x. and intestinal growth ŽGLP-2, mammalian intestine w7x.. While significant differences seem to exist in the expression and processing of glucagon-like peptides in different vertebrates and different tissues w3,8– 10x, very strong common themes exist in the structure of GLP-1 in all vertebrate groups. Nevertheless, as we have pointed out before w3x, the peptides are functionally sepa-
0167-0115r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 1 1 5 Ž 0 1 . 0 0 2 4 1 - 5
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rated, with a strong glycogenolytic action in all fish species examined to date as opposed to a powerful insulinotropic action in mammals. From our previous observation of a lack of glycogenolytic action of fish and mammalian GLP1s in amphibians and reptiles w11x, we postulated the recently confirmed strong insulinotropic action of amphibian GLPs in mammalian B cells w12,13x. Unfortunately, proof of direct insulinotropic action for the peptides in frogs is still outstanding, largely due to the lack of simple and reproducible assays for anuran insulins, although the peptides have recently been purified and characterized w13–16x. The presence of different proglucagon transcripts, encompassing multiple in-series GLP-1s in Xenopus laeÕis w12x and Rana pipiens w17x, and the confirmation of the presence of similar peptides in the pancreas of the cane toad Bufo marinus w13x have led to the availability of the peptides Žsynthetic for Xenopus and native for Bufo .. Therefore, we now have the ability to test potential physiological roles in nonhomologous systems and assess structure–function relationships between glucagons and GLP-1s. Because of our long-standing interest in the structure and function of GLP-1s in the vertebrate lineage, we decided to test amphibian GLPs and glucagons in established fish hepatocyte assay systems.
2. Materials and methods 2.1. Animals Marine copper rockfish Ž Sebastes caurinus, 150–450 g. were captured in Trincomali Channel, BC, while brown bullhead catfish Ž Ameiurus nebulosus, 80–250 g. were captured in Beaver Lake, Victoria, BC. Fish were returned to the University of Victoria, where they were maintained either in flowing seawater Žrockfish. at 28‰ and 12–148C or flowing freshwater Žbullheads. at 17–198C. Studies were undertaken on feeding animals of both sexes in spring and early summer after the fish had been laboratory acclimated for at least a year. Experiments with black bullheads Ž A. melas, 80–150 g. were carried out on fish raised at the University of Ottawa. 2.2. Hepatocyte isolation Dispersed hepatocytes were prepared as previously reported for these three species w18x. Cell viability, estimated by Trypan blue exclusion, always exceeded 90%. The time between isolation and use of hepatocytes for experimentation did not exceed 6 h, although some glycogen phosphorylase experiments were done on cells that had been kept
Table 1 Glucagon family peptides used
References: Human GLP-1Ž7 – 37. —synthetic w32x; X. laeÕis—synthetic w12x; B. marinus—natural isolates w13x; rainbow trout Ž Oncorhynchus mykiss . — synthetic ŽrtGLP-2. w9x. Bovine glucagon is sequence-identical to human glucagon. Xenopus glucagon-like peptides may also occur in C-terminally extended forms w12x; only the unextended forms were tested here. The dash indicates that residues are identical to the line directly above.
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overnight at 48C. Hepatocytes were suspended to 100–300 mg mly1 in complete fish Hanks’ medium Žcontaining 1.5 mM CaCl 2 and 2% bovine serum albumin, at pH 7.63. w18x.
man GLP-1Ž7 – 37. was kindly donated by Dr. Svetlana Mojsov ŽRockefeller University.. Peptide concentrations were confirmed by amino acid analyses of hydrolysates.
2.3. Glucose production
3. Results
Cells Ž2.5–5 mg. were transferred to 1.5-ml microcentrifuge tubes and the volume brought to 50 ml with complete fish Hanks’ medium. Cells were allowed to rest at RT for at least 90 min. Incubations were initiated by adding equal amounts of hormone solution or Hanks’ medium. Perchloric acid Žfinal concentration 0.3 M. was added to terminate the incubations at timed intervals; the tubes were placed on ice until centrifuged Ž16,000 = g for 2 min., and the supernatants were assayed enzymatically for glucose w19x.
3.1. Rockfish
2.4. Glycogen phosphorylase (GPase, EC 2.4.1.1) The enzyme was measured after a timed incubation as outlined w20x. Briefly, cells were arrested in their phosphorylation status by addition of equal amounts of ice-cold stopping buffer, yielding final concentrations of 25 mM b-mercaptoethanol, 5 mM EDTA, 5 mM EGTA, 100 mM KF, 0.5 mg mly1 bovine glycogen Žnot dialyzed. and 20 mg mly1 aprotinin adjusted to pH 7.4. Cells were sonicated and processed immediately. GPase activities were assayed using a two-step process. Assay medium consisted of 150 mM NaF, 50 mM glucose-1-P, 1.0% bovine glycogen, 2.5 mM EDTA and 50 mM imidazole, adjusted to pH 6.5. To 20 ml of this medium, we added an equal volume of 1 mM Žfor rockfish. or 2 mM Žfor catfish. caffeine, or 2 mM AMP in imidazole buffer, providing estimates of GPase a and total GPase, respectively. Reactions were started with the addition of 10 ml homogenate and terminated after 30 min at RT using 50 ml cold 12% trichloroacetic acid. The supernatants obtained after centrifugation were assayed for inorganic phosphate w21x.
The amphibian GLP-1 peptides potently activate glycogenolysis in rockfish hepatocytes, as assessed by glucose production from endogenous glycogen. While the maximum glycogenolytic activity is similar, the potency between six GLP-1s tested differs by three orders of magnitude ŽFig. 1, Table 2.. Bufo GLP-1Ž32., the most potent, achieves half-maximal stimulation of glucose production below 200 pM, slightly below the concentration of endogenous GLP-1 found in the plasma of salmonid fishes w3x, but somewhat above normal plasma concentration in humans w22x. Bufo GLP-1Ž37. and Xenopus GLP-1C were slightly poorer activators of glycogenolysis. Bufo GLP-1Ž37. is characterized by six amino acid substitutions and a Cterminal extension of five residues compared with Bufo GLP-1Ž32.. Bufo GLP-1 and Xenopus GLP-1C show high sequence similarity and can be considered as homologs of the same peptide, but the dose–response curve for the Xenopus peptide is right-shifted by more than a factor of five. GLP-1C is the most potent Xenopus GLP-1. Least active is xGLP-1A, containing nine substitutions com-
2.5. Chemicals Chemicals were purchased from Sigma ŽSt. Louis, MO, USA. or Roche ŽLachine, PQ, Canada.. 2.6. Peptides The peptides used are listed in Table 1. X. laeÕis GLPs were identified through expression analysis and synthesized using a Symphony peptide synthesizer ŽRainin, Woburn, MA. w12x. The final products were purified by HPLC. B. marinus peptides Žtwo GLP-1s and two glucagons. were isolated and purified from pancreatic extracts w13x. Independent sequencing analysis confirmed the presence of single peptides in all preparations. Bovine glucagon was purchased from Calbiochem. Synthetic hu-
Fig. 1. Activation of glycogenolysis in isolated copper rockfish Ž S. caurinus . hepatocytes by different GLP-1s. Values are means of 6–12 independent observations"SEM and normalized to the maximum response attained in the presence of human GLP-1Ž7 – 37. , usually at 10y6 M.
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Table 2 Concentrations of glucagon family peptides eliciting half-maximal activation of glycogenolysis in rockfish hepatocytes Peptide
EC 50 "SEM ŽnM.
n
Copper rockfish Ž S. caurinus . Human GLP-1Ž7 – 37. Bufo GLP-1Ž32. Bufo GLP-1Ž37. Xenopus GLP-1A Xenopus GLP-1B Xenopus GLP-1C Bovine glucagon Bufo glucagon-29 Bufo glucagon-36
8.54"1.59 0.068"0.013 0.615"0.069 89.9"15.1 8.49"1.04 1.40"0.26 5.06"2.19 4.93"0.77 1.66"0.829
12 11 10 12 6 6 12 10 10
Dispersed rockfish hepatocytes were incubated with peptide hormones for 30 min, as described in Section 2. Glucose produced from endogenous glycogen was measured enzymatically. Concentration response curves were established Žsee Figs. 1 and 2. for a minimum of eight hormone concentrations, spread over at least four orders of magnitude, and concentrations eliciting the half-maximal response are presented above; ns number of independent observations.
pared with xGLP-1C. The potency between these two peptides differs by almost two orders of magnitude. The short Bufo GLP-1Ž32. is about 40 times more potent than human GLP-1Ž7 – 37. . Similar forms of the human truncated GLP-1 ŽGLP-1Ž7 – 37. used here, GLP1Ž7 – 36amide. , GLP-1Ž7 – 36. . possess indistinguishable potencies in the rockfish system Ždata not shown.. We also
Fig. 2. Activation of glycogenolysis in isolated copper rockfish Ž S. caurinus . hepatocytes by different glucagons. For comparison with Fig. 1, mammalian GLP-1 and Bufo GLP-1Ž32. are included. Values are means of 6–12 independent observations"SEM and normalized to the maximum response attained in the presence of human GLP-1Ž7 – 37. , usually at 10y6 M. Rates at the three highest concentrations for B. marinus glucagon-36 Žindicated by the dashed line. are significantly Ž p- 0.05, paired t-test. below the maximum rates for glucagon-36.
tested three glucagons. Bovine glucagon and Bufo glucagon-29, differing only at the C-terminal residue, are equipotent in the rockfish system, and slightly more effective in activating glycogenolysis at lower concentrations than human GLP-1Ž7 – 37. ŽFig. 2.. No differences are observed in the maximum activation of glycogenolysis between glucagons and GLPs. Although identical to Bufo glucagon-29 in its N-terminal region, the C-terminally extended Bufo glucagon-36 activates glycogenolysis in an entirely different pattern. Firstly, the dose–response curve is left-shifted compared with the other two glucagons by almost an order of magnitude. Secondly, while the same maximum activation is reached, concentrations of the peptide exceeding 0.1 mM activate glycogenolysis less than those at lower concentrations, resulting in a bell-shaped curve ŽFig. 2.. The difference in potency between Bufo glucagon and GLP-1 is also reflected in the specific activation of hepatocyte glycogen phosphorylase ŽFig. 3.. Again, in rockfish liver cells, GLP-1 is much more potent than glucagon. Although glycogen phosphorylase is only one of the components necessary in the cascade leading from receptor binding of the hormone to glucose release into the hepatocyte medium, the activation of glycogen phosphorylase gives a fairly good representation of overall glucose re-
Fig. 3. Activation of glycogen phosphorylase ŽGPase. in isolated rockfish Ž S. caurinus . hepatocytes by GLP-1Ž32. and glucagon-29 from B. marinus. Values from one representative experiment in duplicates. GPase activity is expressed as the ratio of GPase caffeine rGPaseAMP =100 Ž%GPase in the phosphorylated a-form.. Cells were exposed to the peptides for 8 min at room temperature before processing and assaying for GPase activity ratio. Arrowheads on the x-axis indicate the graphically determined value for the peptide concentration eliciting a half-maximal response Žcf. Figs. 1 and 2..
T.P. Mommsen et al.r Regulatory Peptides 99 (2001) 111–118
Fig. 4. Activation of glycogenolysis in isolated brown bullhead Ž A. nebulosus . hepatocytes by different GLP-1s. Values are means of four Žhuman and xGLP-1A. or three Žall others. independent observations" SEM and normalized to the maximum response attained in the presence of bovine glucagon, usually at 10y6 M Žcf. Fig. 5..
lease. Half-maximal activation points are reached at around 200 pM for Bufo GLP-1Ž32. and 3–4 nM for Bufo glucagon-29 ŽFigs. 2 and 3, respectively., resulting in a 15-fold potency difference in favour of GLP-1. The data also confirm that the phosphorylation status of GPase can serve as a highly sensitive indicator of GLP-1 action and
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Fig. 5. Activation of glycogenolysis in isolated brown bullhead Ž A. nebulosus . hepatocytes by different glucagons. Values are means of four Žbovine glucagon. or three Žothers. independent observations in duplicates"SEM. Only one observation in duplicates was available for Bufo glucagon-36. Values are normalized to the maximum response attained in the presence of bovine glucagon, usually at 10y6 M. The most active amphibian GLP-1 Ž Bufo GLP-1Ž32.. is included for comparison with Fig. 4.
imply that protein kinase A and the adenylyl cyclase systems are important components in the cascade.
Table 3 Concentrations of glucagon family peptides eliciting half-maximal activation of glycogenolysis in catfish hepatocytes Peptide
EC 50 "SEM ŽnM.
n
Brown bullhead catfish (A. nebulosus) Human GLP-1Ž7 – 37. 13.0"3.1 Bufo GLP-1Ž32. 40.4"30.6 Xenopus GLP-1A 574"178 Xenopus GLP-1B 770"215 Xenopus GLP-1C 480"75.7 Bovine glucagon 4.36"1.99 Bufo glucagon-29 0.53"0.28
4 3 4 4 3 4 3
Black bullhead catfish (A. melas) Human GLP-1Ž7 – 37. 12.3"2.9 Xenopus GLP-1B 456"38 Xenopus GLP-1C 60.3"11.6 Bovine glucagon 3.36"1.52
7 7 7 6
Dispersed catfish hepatocytes were incubated with peptide hormones for 30 min, as described in Section 2. Glucose produced from endogenous glycogen was measured enzymatically. Concentration response curves were established Žsee Figs. 4, 5 and 7. for a minimum of eight hormone concentrations, spread over at least four orders of magnitude, and concentrations eliciting the half-maximal response are presented above; ns number of independent observations.
Fig. 6. Activation of glycogen phosphorylase ŽGPase. in isolated hepatocytes by GLP-1Ž32. and B. marinus glucagon-29. Values from two experiments in duplicates"SD. Activity ratio and experimental procedure as in Fig. 3. Arrowheads on the x-axis indicate the graphically determined value for the peptide concentration eliciting a half-maximal response Žcf. Figs. 4, 5 and 7..
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3.2. Catfish Just as in the rockfish hepatocytes, the amphibian peptides activate glycogenolysis in hepatocytes from two species of catfish, but important species differences in specific responses are apparent. Firstly, catfish cells respond better to human GLP-1Ž7 – 37. than to any of the amphibian GLPs; however, even for GLP-1Ž7 – 37. , the concentration eliciting the half-maximal response lies in the 10–20 nM range. Secondly, the relative potency within the amphibian GLP-1s resembles that found for the rockfish, albeit at much higher concentrations: Bufo GLP-1Ž32. is the most potent, with the analogous xGLP-1C a distant second ŽFig. 4, Table 3., while Bufo GLP-1Ž37. and xGLP-1A are equally ineffective in the brown bullhead. In liver cells of black bullhead Žcf. Fig. 7., where we tested only two Xenopus peptides, xGLP-1C is more potent than xGLP-1B. As expected, rainbow trout GLP-2 failed to alter the rate of glycogen breakdown. Even at the highest concentration used Ž6.6 = 10y6 M., the maximum is not reached for any xGLP-1 or the C-terminally extended Bufo GLP-1Ž37.. Thirdly, liver cells isolated from two congeneric catfish species Ž A. nebulosus and A. melas . respond much better to glucagon than to any GLP-1 ŽFigs. 4–7, Table 3.. Half-maximal activation of glycogenolysis is achieved at low nanomolar concentrations for glucagons, but requires more than 10 times higher concentrations for GLP-1Ž7 – 37. . The same pattern is noted when the degree of phosphoryla-
Fig. 7. Activation of glycogenolysis in isolated black bullhead Ž A. melas . hepatocytes by glucagon family peptides. Values are means of seven Žhuman GLP-1. or six Žall others. independent observations in duplicates "SEM. Values are normalized to the maximum response attained in the presence of human GLP-1Ž7 – 37. , usually at 10y6 M. For synthetic rainbow trout GLP-2, the SEMs are often smaller than the symbol.
tion of glycogen phosphorylase is assessed directly ŽFig. 6.. Finally, in contrast to the rockfish cells, where the C-terminally extended Bufo glucagon-36 was more potent and where it showed a bell-shaped dose–response relationship, all glucagons seem to be equipotent in the brown bullhead assay system. However, the existence of a bellshaped curve cannot be ruled out based on our limited data for Bufo glucagon-36 ŽFig. 5..
4. Discussion Just like their piscine or mammalian counterparts, amphibian GLPs are powerful activators of glycogenolysis and glycogen phosphorylase in fish hepatocytes, but substantial differences exist in species-specific responses. While rockfish hepatocytes respond to GLP peptides at much lower concentrations than to glucagons, the reverse is true for cells isolated from the two catfish species. Therefore, glycogenolysis in the catfish can be considered ‘glucagon-driven’ as opposed to ‘GLP-driven’. It would be worthwhile to analyze the relative potencies in relation to actual plasma concentrations, or even better, with concentrations of the endogenous peptides reaching the liver in these species. If the observation that similar concentrations of both peptides Ž0.4–0.5 nM. reach the liver in the rainbow trout w23x can be extrapolated to other species, then, indeed, glucagon- and GLP-1-driven metabolic regulation should be distinguished at least for the liver. The EC 50 for the most potent peptides when tested in the piscine systems falls into the low to mid-nanomolar range, and thus, it might be argued that they are somewhat above the physiological concentrations of these peptides. However, a few counterarguments should be mentioned. First, physiological concentrations of these peptides are considerably higher in the fish Žlow nanomolar range w3x. than in mammals. Second, the dispersed hepatocytes tend to be less sensitive to hormonal stimulation than cells maintained in perifusion columns. Using prostaglandin E 2 to activate glycogenolysis in rockfish liver cells, we found that the dose–response curve was displaced by more than an order of magnitude towards higher sensitivity when cells were immobilized in perifusion columns as opposed to maintained in suspension ŽBusby and Mommsen, in preparation.. Finally, accumulation of glucose from glycogenolysis is a relatively crude indicator, among other things requiring relatively lengthy incubations Ž30 min. for detection of the signal, while peak activation of glycogen phosphorylase is reached within about 8 min of hormone application Žcf. Figs. 3 and 6.. After peak stimulation, the enzyme activity falls off rapidly at low hormone concentrations, but remains high at supraphysiological hormone concentrations w20x ŽMommsen and Busby, unpublished.. Eventhough we do not know the physiological concentrations of any of the amphibian glucagon family peptides,
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we feel we can make a fairly strong case that we are indeed analyzing physiologically important phenomena. Independent of these species-dependent responses, the potency ranking for amphibian GLPs is similar in the three species and thus internally consistent. In every case, the Bufo GLP-1Ž32. is the most potent, followed by the corresponding peptide from Xenopus, xGLP-1C. A similar relationship exists between Bufo GLP-1Ž37. and the corresponding xGLP-1B. xGLP-1A is the least active of all GLP-1s tested. A corresponding peptide has not been found in Bufo Žpeptides. or Rana ŽcDNA., raising again the question whether this specific Xenopus peptide may have a function distinct from the other GLP-1 peptides. However, it should be pointed out that the Xenopus peptides were synthesized based on cDNA analysis and presumed gene products w12x, while the Bufo peptides were natural isolates from pancreatic tissue w13x, and hence, the next step should be to show that, indeed, a peptide corresponding to xGLP-1A is synthesized and secreted in X. laeÕis. Interestingly, the relative potency for amphibian GLP-1 peptides to human GLP-1Ž7 – 37. is species-dependent—in the rockfish, three amphibian GLP1s are more potent than human GLP-1Ž7 – 37. , while in both catfish species, GLP1Ž7 – 37. is more powerful than any amphibian GLP-1 peptide. Furthermore, the ranking of the amphibian peptides in their insulinotropic potency in isolated rat pancreas differs fundamentally from the fish liver test system w12x. Just like the fish test system, similar maximum rates are reached, but differences are apparent in receptor-binding constants and in the ability to increase cAMP in CHO cells transfected with the human GLP-1 receptor. The potency ranking in terms of receptor binding and cAMP production is xGLP-1BŽ30. ) human GLP-1Ž7 – 36amide. ) xGLP-1A ) xGLP-1C w12x. Functional relationships between the different GLP-1 peptides indicate that, ultimately, the potency depends somewhat on the test system. The rockfish are GLP-1driven, while the two catfish are glucagon-driven. We have no data on GLP-1 versus glucagon receptors in the rockfish, but it appears that, although catfish respond to GLP-1, the effects of glucagon may prevail in vivo, and the physiological raison d’etre ˆ for the GLP-1 is as yet unknown. Conversely, in the rockfish, GLP-1 is certainly a powerful hormone, partially occupying the functional position of glucagon. The terminally extended glucagon from B. marinus behaves differently in the two fish test systems. The peptide is more powerful than the shorter version in the rockfish hepatocytes; however, there is no difference in the catfish. In both systems Žbut note that n s 1 for the brown bullhead., the longer peptide is inhibitory at concentrations exceeding 3–6 = 10y8 M. It is possible that peptide processing in the hepatocytes is at the root of the species difference, and that the C-terminal extension may serve to protect against degradation in the rockfish, but not in the
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catfish liver cells. In fishes, liver has been identified as the main site for glucagon degradation w23x, although degradation pattern and dynamics have not yet been addressed in any detail, or with attention to potential species differences. In mammalian models for GLP-1, Ala2 may be a key site in determining peptide degradation, and substitution with Ser 2 substantially increases the half-life of the peptide in circulation w24x. It is interesting to note that throughout the fish w3x and amphibians w15,17,25x, Ala2 is conserved in all GLP-1 peptides, while glucagons contain Ser in this position. The metabolic functions of GLP-1 peptides in fish are by no means restricted to glycogenolysis, but also extend to other functions, such as hepatic gluconeogenesis and lipolysis w3x, and intestinal glucose transport w6x, with most functions mediated by adenylyl cyclase, cAMP and protein kinase A w26x and the secondary involvement of intracellular calcium w27x. Overall, these roles seem to be fish-specific, accentuate those of glucagon, and generally oppose the actions of insulin. Also, they are yet to be found in other vertebrate groups. Some hepatic actions of GLP-1 have been described for mammals, in alleged absence of GLP-1 receptors w28x, but these encompass actions opposing those noted in fish, including decreases in glucose output and increases in glycogen synthesis w29x. With the role in glycogenolysis confirmed and the function in insulin release still under debate for the fish, our data support the notion on comparable structure–function relationships in glycogenolytic potential in fish and insulinotropic action in mammals. Using directed alanine substitutions along the entire GLP-1 molecule, studies with mammals have identified residues His1 , Gly 4 , Thr 7, Asp 9 , Phe 22 and Ile 23 to be primarily important for binding to the rat pancreatic GLP-1 receptor. Additionally, Phe 6 , Tyr 13 , Glu15 and Leu26 play important roles. It is interesting to note that the many different fish GLP-1s, covering a much longer evolutionary range than the ‘highly conserved’ mammalian GLP-1, also have conserved the residues mentioned above. As we have shown here, the different frog GLP-1s follow a similar pattern with similarly conserved residues, although a few comments seem warranted. The chemical nature of residue 23 ŽIle in mammals and Ile or Val in fish or other amphibians. may be less important than what the previous studies imply. Bufo GLP-1Ž32. containing an Ile 23 and Bufo GLP-1Ž37. with its Tyr 23 show similar effectiveness in increasing insulin release in a rodent test system w12x, and the two peptides are also indistinguishable in their ability to activate glycogenolysis in our rockfish liver test system. While specific actions for GLP-1 in amphibians remain to be determined, we can anticipate that insulinotropic functions prevail and that direct hepatic actions, similar to those in fishes, are unlikely, since we could not find any glycogenolytic actions of GLP-1 in dispersed frog hepatocytes w11x. Apart from pancreas and intestine, other actions
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of GLP-1 appear to be similar and widespread in the vertebrate lineage—actions in brain, depression of feeding Žmediated by cAMP. w4,26,30x and alterations in intestinal transport and metabolism w6,26,31x. Acknowledgements This study was supported by research grants from the Natural Sciences and Engineering Research Council of Canada ŽTPM and DMI.. We thank Dr. Thomas W. Moon ŽUniversity of Ottawa. for access to black bullhead catfish and laboratory facilities. References w1x Mojsov S. Glucagon-like peptide-1 ŽGLP-1. and the control of glucose metabolism in mammals and teleost fish. Am Zool 2000;28:246–58. w2x Nauck MA. Is glucagon-like peptide 1 an incretin hormone? Diabetologia 1999;42:373–9. w3x Plisetskaya EM, Mommsen TP. Glucagon and glucagon-like peptides in fishes. Int Rev Cytol 1996;168:187–257. w4x Turton MD, O’Shea D, Gunn I, Beak SA, Edwards CMB, Meeran K, et al. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 1996;379:69–72. w5x Silverstein JT, Bondareva VM, Leonard JBK, Plisetskaya EM. Neuropeptide regulation of feeding in catfish Ictalurus punctatus: a role for glucagon-like peptide-1 ŽGLP-1.? Comp Biochem Physiol Part B 2001;29:623–31. w6x Soengas JL, Moon TW. Transport and metabolism of glucose in isolated enterocytes of the black bullhead, Ictalurus melas: effects of diet and hormones. J Exp Biol 1998;201:3263–73. w7x Munroe DG, Gupta AK, Kooshesh F, Vyas TB, Rizkalla G, Wang H, et al. Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2. Proc Natl Acad Sci U S A 1999;96:1569–73. w8x Hasegawa S, Terazono K, Nata K, Takada T, Yamamoto H, Okamoto H. Nucleotide sequence determination of chicken glucagon precursor cDNA. Chicken preproglucagon does not contain glucagon-like peptide II. FEBS Lett 1990;264:117–20. w9x Irwin DM, Wong J. Trout and chicken proglucagon: alternative splicing generates mRNA transcripts encoding glucagon-like peptide 2. Mol Endocrinol 1995;9:267–77. w10x Chen YE, Drucker DJ. Tissue-specific expression of unique mRNAs that encode proglucagon-derived peptides or exendin 4 in the lizard. J Biol Chem 1997;272:4108–15. w11x Mommsen TP, Moon TW. Glucagon-like peptides: structure–function relationship and evolution. In: Davey KG, Peter RE, Tobe SS, editors. Perspectives in Comparative Endocrinology. Ottawa: National Research Council of Canada, 1994:493–8. w12x Irwin DM, Satkunarajah M, Wen Y, Brubaker PL, Pederson RA, Wheeler MB. The Xenopus proglucagon gene encodes novel GLP1-like peptides with insulinotropic properties. Proc Natl Acad Sci U S A 1997;94:7915–20. w13x Conlon JM, Abdel-Wahab YHA, O’Harte FPM, Nielsen PF, Whittaker J. Purification and characterization of insulin, glucagon, and two glucagon-like peptides with insulin-releasing activity from the pancreas of the toad, Bufo marinus. Endocrinology1998;139:3442–8.
w14x Shuldiner AR, Bennett C, Robinson EA, Roth J. Isolation and characterization of two different insulins from an amphibian, Xenopus laeÕis. Endocrinology 1989;125:469–77. w15x White AM, Secor SM, Conlon JM. Insulin and proglucagon-derived peptides from the horned frog, Ceratophrys ornata ŽAnura:Leptodactylidae.. Gen Comp Endocrinol 1999;115:143–54. w16x Conlon JM, Yano K, Chartrel N, Vaudry H, Storey KB. Freeze tolerance in the wood frog Rana sylÕatica is associated with unusual structural features in insulin but not in glucagon. J Mol Endocrinol 1998;21:153–9. w17x Irwin DM, Sivarajah P. Proglucagon cDNAs from the leopard frog, Rana pipiens, encode two GLP-1-like peptides. Mol Cell Endocrinol 2000;162:17–24. w18x Mommsen TP, Moon TW, Walsh PJ. Hepatocytes: isolation, maintenance and utilization. In: Hochachka PW, Mommsen TP, editors. Biochemistry and Molecular Biology of Fishes. Amsterdam: Elsevier, 1994:355–73. w19x Passonneau JV, Lowry OH. Methods of Enzymatic Analysis. Totowa, NJ, USA: Humana Press, 1993:1–403. w20x Moon TW, Busby ER, Cooper GA, Mommsen TP. Fish hepatocyte glycogen phosphorylase—a sensitive indicator of hormonal activation. Fish Physiol Biochem 1999;21:15–24. w21x Fiske CH, Subbarow Y. The colorimetric determination of phosphorous. J Biol Chem 1925;66:375–8. w22x Hvidberg A, Toft Nielsen M, Hilsted J, Orskov C, Holst JJ. Effect of glucagon-like peptide-1 Žproglucagon 78–107amide. on hepatic glucose production in healthy man. Metabolism 1994;43:104–8. w23x Plisetskaya EM, Sullivan CV. Pancreatic and thyroid hormones in rainbow trout Ž Salmo gairdneri .: what concentration does the liver see? Gen Comp Endocrinol 1989;75:310–5. w24x Deacon CF, Knudsen LB, Madsen K, Wiberg FC, Jacobsen O, Holst JJ. Dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1 which have extended metabolic stability and improved biological activity. Diabetologia 1998;41:271–8. w25x Cavanaugh ES, Nielsen PF, Conlon JM. Isolation and structural characterization of proglucagon-derived peptides, pancreatic polypeptide and somatostatin from the urodele Amphiuma tridactylum. Gen Comp Endocrinol 1996;101:12–20. w26x Mommsen TP, Mojsov S. Glucagon-like peptide-1 activates the adenylyl cyclase system in rockfish enterocytes and brain membranes. Comp Biochem Physiol 1998;121B:49–56. w27x Moon TW, Gambarotta A, Capuzzo A, Fabbri E. Glucagon and glucagon-like peptide signaling pathways in the liver of two fish species, the American eel and the black bullhead. J Exp Zool 1997;279:62–70. w28x Wei Y, Mojsov S. Tissue-specific expression of the human receptor for glucagon-like peptide-1: brain, heart and pancreatic forms have the same deduced amino acid sequences. FEBS Lett 1995;358:219– 24. w29x Lopez-Delgado MI, Morales M, Villanueva-Penacarrillo ML, ´ ˜ Malaisse WJ, Valverde I. Effects of glucagon-like peptide 1 on the kinetics of glycogen synthase a in hepatocytes from normal and diabetic rats. Endocrinology 1998;139:2811–7. w30x Mommsen TP. Glucagon-like peptides in fishes: the liver and beyond. Am Zool 2000;40:259–68. w31x Wojdemann M, Wettergren A, Sternby B, Holst JJ, Larsen S, Rehfeld JF, et al. Inhibition of human gastric lipase secretion by glucagon-like peptide-1. Dig Dis Sci 1998;43:799–805. w32x Mojsov S, Weir GC, Habener JF. Insulinotropin: glucagon-like peptide I Ž7–37. co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J Clin Invest 1987;79:616–9.
Amphibian glucagon family peptides: potent metabolic regulators in fish hepatocytes Thomas P. Mommsen a,) , J. Michael Conlon b, David M. Irwin c a
Department of Biochemistry and Microbiology, UniÕersity of Victoria, P.O. Box 3055, Victoria, BC, Canada V8W 3P6 b Department of Biomedical Sciences, Creighton UniÕersity Medical School, Omaha, NE, USA c Department of Laboratory Medicine and Pathobiology, UniÕersity of Toronto, Toronto, ON, Canada Received 20 August 2000; received in revised form 30 January 2001; accepted 6 February 2001
Abstract Peptides analogous to glucagon-like peptide-1 ŽGLP-1. have been isolated from amphibian pancreas and intestine, and their amino acid sequences and cDNA structures elucidated. Just like their mammalian counterpart, these peptides are potent insulinotropins in mammalian pancreatic cells. We show here that these peptides also exert strong glycogenolytic actions when applied to dispersed fish hepatocytes. We compared the potencies of three synthetic GLP-1s from Xenopus laeÕis and two native GLP-1s from Bufo marinus in the activation of glycogenolysis in the hepatocytes of a marine rockfish Ž Sebastes caurinus . and two freshwater catfish Ž Ameiurus nebulosus and A. melas ., and demonstrated their effectiveness in increasing the degree of phosphorylation of glycogen phosphorylase. We also compared the glycogenolytic potency of the peptides with those of human GLP-1 and glucagons from human and B. marinus. Sensitivity to these peptides is species-specific, with the rockfish responding at lower concentrations to GLP-1s and the two catfish reacting better to glucagons. However, the relative potency of the amphibian GLP-1s and glucagons is similar in the three species. Xenopus GLP-1C Ž xGLP-1C. is consistently more potent than xGLP-1B, while xGLP-1A displays the smallest activation of glycogenolysis. Similarly, Bufo GLP-1Ž32. —the peptide with the highest amino acid sequence identity to xGLP-1C—always shows a higher potency than Bufo GLP-1Ž37., which is closely related to xGLP-1B. The relative hierarchy of these glycogenolytic GLP-1s differs from their ranking as insulinotropins in mammalian b-cells. In the rockfish system, Bufo glucagon-36, a C-terminally extended glucagon, is more potent than the shorter bovine glucagon and Bufo glucagon-29 in the activation of glycogenolysis; when tested in A. nebulosus hepatocytes, bovine and amphibian glucagons are equipotent. Amphibian GLP-1s and glucagons activate glycogenolysis in fish hepatocytes through increased phosphorylation of glycogen phosphorylase, implying involvement of the adenylyl cyclaserprotein kinase A system in signal transduction. We conclude that the broad physiological effectiveness of GLP-1 has been retained throughout vertebrate evolution, and that both insulinotropic activity and glycogenolytic actions belong to the repertoire of GLP-1. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Insulinotropin; Glycogenolysis, Fish hepatocytes; Glucagon; Glycogen phosphorylase
1. Introduction Glucagon-like peptides in vertebrates are expressed in pancreatic endocrine tissue, intestine and brain, and display a wide variety of biological activities. The proglucagon gene coencodes glucagon, glucagon-like peptides-1 ŽGLP1. and GLP-2. While the functions of glucagon as key regulator of hepatic metabolic functions are fairly well
) Corresponding author. Tel.: q1-250-721-6508; fax: q1-250-7218855. E-mail address: [email protected] ŽT.P. Mommsen..
defined, the field of functions for the GLPs is expanding rapidly. The GLPs regulate insulin release ŽGLP-1, mammalian pancreas w1,2x., acid secretion ŽGLP-1, mammals., lung function ŽGLP-1, mammals., glycogenolysis, gluconeogenesis and lipolysis ŽGLP-1, fish liver w3x., feeding ŽGLP-1, brain w4,5x., gut glucose uptake and oxidation ŽGLP-1, fish w6x. and intestinal growth ŽGLP-2, mammalian intestine w7x.. While significant differences seem to exist in the expression and processing of glucagon-like peptides in different vertebrates and different tissues w3,8– 10x, very strong common themes exist in the structure of GLP-1 in all vertebrate groups. Nevertheless, as we have pointed out before w3x, the peptides are functionally sepa-
0167-0115r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 1 1 5 Ž 0 1 . 0 0 2 4 1 - 5
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rated, with a strong glycogenolytic action in all fish species examined to date as opposed to a powerful insulinotropic action in mammals. From our previous observation of a lack of glycogenolytic action of fish and mammalian GLP1s in amphibians and reptiles w11x, we postulated the recently confirmed strong insulinotropic action of amphibian GLPs in mammalian B cells w12,13x. Unfortunately, proof of direct insulinotropic action for the peptides in frogs is still outstanding, largely due to the lack of simple and reproducible assays for anuran insulins, although the peptides have recently been purified and characterized w13–16x. The presence of different proglucagon transcripts, encompassing multiple in-series GLP-1s in Xenopus laeÕis w12x and Rana pipiens w17x, and the confirmation of the presence of similar peptides in the pancreas of the cane toad Bufo marinus w13x have led to the availability of the peptides Žsynthetic for Xenopus and native for Bufo .. Therefore, we now have the ability to test potential physiological roles in nonhomologous systems and assess structure–function relationships between glucagons and GLP-1s. Because of our long-standing interest in the structure and function of GLP-1s in the vertebrate lineage, we decided to test amphibian GLPs and glucagons in established fish hepatocyte assay systems.
2. Materials and methods 2.1. Animals Marine copper rockfish Ž Sebastes caurinus, 150–450 g. were captured in Trincomali Channel, BC, while brown bullhead catfish Ž Ameiurus nebulosus, 80–250 g. were captured in Beaver Lake, Victoria, BC. Fish were returned to the University of Victoria, where they were maintained either in flowing seawater Žrockfish. at 28‰ and 12–148C or flowing freshwater Žbullheads. at 17–198C. Studies were undertaken on feeding animals of both sexes in spring and early summer after the fish had been laboratory acclimated for at least a year. Experiments with black bullheads Ž A. melas, 80–150 g. were carried out on fish raised at the University of Ottawa. 2.2. Hepatocyte isolation Dispersed hepatocytes were prepared as previously reported for these three species w18x. Cell viability, estimated by Trypan blue exclusion, always exceeded 90%. The time between isolation and use of hepatocytes for experimentation did not exceed 6 h, although some glycogen phosphorylase experiments were done on cells that had been kept
Table 1 Glucagon family peptides used
References: Human GLP-1Ž7 – 37. —synthetic w32x; X. laeÕis—synthetic w12x; B. marinus—natural isolates w13x; rainbow trout Ž Oncorhynchus mykiss . — synthetic ŽrtGLP-2. w9x. Bovine glucagon is sequence-identical to human glucagon. Xenopus glucagon-like peptides may also occur in C-terminally extended forms w12x; only the unextended forms were tested here. The dash indicates that residues are identical to the line directly above.
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overnight at 48C. Hepatocytes were suspended to 100–300 mg mly1 in complete fish Hanks’ medium Žcontaining 1.5 mM CaCl 2 and 2% bovine serum albumin, at pH 7.63. w18x.
man GLP-1Ž7 – 37. was kindly donated by Dr. Svetlana Mojsov ŽRockefeller University.. Peptide concentrations were confirmed by amino acid analyses of hydrolysates.
2.3. Glucose production
3. Results
Cells Ž2.5–5 mg. were transferred to 1.5-ml microcentrifuge tubes and the volume brought to 50 ml with complete fish Hanks’ medium. Cells were allowed to rest at RT for at least 90 min. Incubations were initiated by adding equal amounts of hormone solution or Hanks’ medium. Perchloric acid Žfinal concentration 0.3 M. was added to terminate the incubations at timed intervals; the tubes were placed on ice until centrifuged Ž16,000 = g for 2 min., and the supernatants were assayed enzymatically for glucose w19x.
3.1. Rockfish
2.4. Glycogen phosphorylase (GPase, EC 2.4.1.1) The enzyme was measured after a timed incubation as outlined w20x. Briefly, cells were arrested in their phosphorylation status by addition of equal amounts of ice-cold stopping buffer, yielding final concentrations of 25 mM b-mercaptoethanol, 5 mM EDTA, 5 mM EGTA, 100 mM KF, 0.5 mg mly1 bovine glycogen Žnot dialyzed. and 20 mg mly1 aprotinin adjusted to pH 7.4. Cells were sonicated and processed immediately. GPase activities were assayed using a two-step process. Assay medium consisted of 150 mM NaF, 50 mM glucose-1-P, 1.0% bovine glycogen, 2.5 mM EDTA and 50 mM imidazole, adjusted to pH 6.5. To 20 ml of this medium, we added an equal volume of 1 mM Žfor rockfish. or 2 mM Žfor catfish. caffeine, or 2 mM AMP in imidazole buffer, providing estimates of GPase a and total GPase, respectively. Reactions were started with the addition of 10 ml homogenate and terminated after 30 min at RT using 50 ml cold 12% trichloroacetic acid. The supernatants obtained after centrifugation were assayed for inorganic phosphate w21x.
The amphibian GLP-1 peptides potently activate glycogenolysis in rockfish hepatocytes, as assessed by glucose production from endogenous glycogen. While the maximum glycogenolytic activity is similar, the potency between six GLP-1s tested differs by three orders of magnitude ŽFig. 1, Table 2.. Bufo GLP-1Ž32., the most potent, achieves half-maximal stimulation of glucose production below 200 pM, slightly below the concentration of endogenous GLP-1 found in the plasma of salmonid fishes w3x, but somewhat above normal plasma concentration in humans w22x. Bufo GLP-1Ž37. and Xenopus GLP-1C were slightly poorer activators of glycogenolysis. Bufo GLP-1Ž37. is characterized by six amino acid substitutions and a Cterminal extension of five residues compared with Bufo GLP-1Ž32.. Bufo GLP-1 and Xenopus GLP-1C show high sequence similarity and can be considered as homologs of the same peptide, but the dose–response curve for the Xenopus peptide is right-shifted by more than a factor of five. GLP-1C is the most potent Xenopus GLP-1. Least active is xGLP-1A, containing nine substitutions com-
2.5. Chemicals Chemicals were purchased from Sigma ŽSt. Louis, MO, USA. or Roche ŽLachine, PQ, Canada.. 2.6. Peptides The peptides used are listed in Table 1. X. laeÕis GLPs were identified through expression analysis and synthesized using a Symphony peptide synthesizer ŽRainin, Woburn, MA. w12x. The final products were purified by HPLC. B. marinus peptides Žtwo GLP-1s and two glucagons. were isolated and purified from pancreatic extracts w13x. Independent sequencing analysis confirmed the presence of single peptides in all preparations. Bovine glucagon was purchased from Calbiochem. Synthetic hu-
Fig. 1. Activation of glycogenolysis in isolated copper rockfish Ž S. caurinus . hepatocytes by different GLP-1s. Values are means of 6–12 independent observations"SEM and normalized to the maximum response attained in the presence of human GLP-1Ž7 – 37. , usually at 10y6 M.
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114
Table 2 Concentrations of glucagon family peptides eliciting half-maximal activation of glycogenolysis in rockfish hepatocytes Peptide
EC 50 "SEM ŽnM.
n
Copper rockfish Ž S. caurinus . Human GLP-1Ž7 – 37. Bufo GLP-1Ž32. Bufo GLP-1Ž37. Xenopus GLP-1A Xenopus GLP-1B Xenopus GLP-1C Bovine glucagon Bufo glucagon-29 Bufo glucagon-36
8.54"1.59 0.068"0.013 0.615"0.069 89.9"15.1 8.49"1.04 1.40"0.26 5.06"2.19 4.93"0.77 1.66"0.829
12 11 10 12 6 6 12 10 10
Dispersed rockfish hepatocytes were incubated with peptide hormones for 30 min, as described in Section 2. Glucose produced from endogenous glycogen was measured enzymatically. Concentration response curves were established Žsee Figs. 1 and 2. for a minimum of eight hormone concentrations, spread over at least four orders of magnitude, and concentrations eliciting the half-maximal response are presented above; ns number of independent observations.
pared with xGLP-1C. The potency between these two peptides differs by almost two orders of magnitude. The short Bufo GLP-1Ž32. is about 40 times more potent than human GLP-1Ž7 – 37. . Similar forms of the human truncated GLP-1 ŽGLP-1Ž7 – 37. used here, GLP1Ž7 – 36amide. , GLP-1Ž7 – 36. . possess indistinguishable potencies in the rockfish system Ždata not shown.. We also
Fig. 2. Activation of glycogenolysis in isolated copper rockfish Ž S. caurinus . hepatocytes by different glucagons. For comparison with Fig. 1, mammalian GLP-1 and Bufo GLP-1Ž32. are included. Values are means of 6–12 independent observations"SEM and normalized to the maximum response attained in the presence of human GLP-1Ž7 – 37. , usually at 10y6 M. Rates at the three highest concentrations for B. marinus glucagon-36 Žindicated by the dashed line. are significantly Ž p- 0.05, paired t-test. below the maximum rates for glucagon-36.
tested three glucagons. Bovine glucagon and Bufo glucagon-29, differing only at the C-terminal residue, are equipotent in the rockfish system, and slightly more effective in activating glycogenolysis at lower concentrations than human GLP-1Ž7 – 37. ŽFig. 2.. No differences are observed in the maximum activation of glycogenolysis between glucagons and GLPs. Although identical to Bufo glucagon-29 in its N-terminal region, the C-terminally extended Bufo glucagon-36 activates glycogenolysis in an entirely different pattern. Firstly, the dose–response curve is left-shifted compared with the other two glucagons by almost an order of magnitude. Secondly, while the same maximum activation is reached, concentrations of the peptide exceeding 0.1 mM activate glycogenolysis less than those at lower concentrations, resulting in a bell-shaped curve ŽFig. 2.. The difference in potency between Bufo glucagon and GLP-1 is also reflected in the specific activation of hepatocyte glycogen phosphorylase ŽFig. 3.. Again, in rockfish liver cells, GLP-1 is much more potent than glucagon. Although glycogen phosphorylase is only one of the components necessary in the cascade leading from receptor binding of the hormone to glucose release into the hepatocyte medium, the activation of glycogen phosphorylase gives a fairly good representation of overall glucose re-
Fig. 3. Activation of glycogen phosphorylase ŽGPase. in isolated rockfish Ž S. caurinus . hepatocytes by GLP-1Ž32. and glucagon-29 from B. marinus. Values from one representative experiment in duplicates. GPase activity is expressed as the ratio of GPase caffeine rGPaseAMP =100 Ž%GPase in the phosphorylated a-form.. Cells were exposed to the peptides for 8 min at room temperature before processing and assaying for GPase activity ratio. Arrowheads on the x-axis indicate the graphically determined value for the peptide concentration eliciting a half-maximal response Žcf. Figs. 1 and 2..
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Fig. 4. Activation of glycogenolysis in isolated brown bullhead Ž A. nebulosus . hepatocytes by different GLP-1s. Values are means of four Žhuman and xGLP-1A. or three Žall others. independent observations" SEM and normalized to the maximum response attained in the presence of bovine glucagon, usually at 10y6 M Žcf. Fig. 5..
lease. Half-maximal activation points are reached at around 200 pM for Bufo GLP-1Ž32. and 3–4 nM for Bufo glucagon-29 ŽFigs. 2 and 3, respectively., resulting in a 15-fold potency difference in favour of GLP-1. The data also confirm that the phosphorylation status of GPase can serve as a highly sensitive indicator of GLP-1 action and
115
Fig. 5. Activation of glycogenolysis in isolated brown bullhead Ž A. nebulosus . hepatocytes by different glucagons. Values are means of four Žbovine glucagon. or three Žothers. independent observations in duplicates"SEM. Only one observation in duplicates was available for Bufo glucagon-36. Values are normalized to the maximum response attained in the presence of bovine glucagon, usually at 10y6 M. The most active amphibian GLP-1 Ž Bufo GLP-1Ž32.. is included for comparison with Fig. 4.
imply that protein kinase A and the adenylyl cyclase systems are important components in the cascade.
Table 3 Concentrations of glucagon family peptides eliciting half-maximal activation of glycogenolysis in catfish hepatocytes Peptide
EC 50 "SEM ŽnM.
n
Brown bullhead catfish (A. nebulosus) Human GLP-1Ž7 – 37. 13.0"3.1 Bufo GLP-1Ž32. 40.4"30.6 Xenopus GLP-1A 574"178 Xenopus GLP-1B 770"215 Xenopus GLP-1C 480"75.7 Bovine glucagon 4.36"1.99 Bufo glucagon-29 0.53"0.28
4 3 4 4 3 4 3
Black bullhead catfish (A. melas) Human GLP-1Ž7 – 37. 12.3"2.9 Xenopus GLP-1B 456"38 Xenopus GLP-1C 60.3"11.6 Bovine glucagon 3.36"1.52
7 7 7 6
Dispersed catfish hepatocytes were incubated with peptide hormones for 30 min, as described in Section 2. Glucose produced from endogenous glycogen was measured enzymatically. Concentration response curves were established Žsee Figs. 4, 5 and 7. for a minimum of eight hormone concentrations, spread over at least four orders of magnitude, and concentrations eliciting the half-maximal response are presented above; ns number of independent observations.
Fig. 6. Activation of glycogen phosphorylase ŽGPase. in isolated hepatocytes by GLP-1Ž32. and B. marinus glucagon-29. Values from two experiments in duplicates"SD. Activity ratio and experimental procedure as in Fig. 3. Arrowheads on the x-axis indicate the graphically determined value for the peptide concentration eliciting a half-maximal response Žcf. Figs. 4, 5 and 7..
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3.2. Catfish Just as in the rockfish hepatocytes, the amphibian peptides activate glycogenolysis in hepatocytes from two species of catfish, but important species differences in specific responses are apparent. Firstly, catfish cells respond better to human GLP-1Ž7 – 37. than to any of the amphibian GLPs; however, even for GLP-1Ž7 – 37. , the concentration eliciting the half-maximal response lies in the 10–20 nM range. Secondly, the relative potency within the amphibian GLP-1s resembles that found for the rockfish, albeit at much higher concentrations: Bufo GLP-1Ž32. is the most potent, with the analogous xGLP-1C a distant second ŽFig. 4, Table 3., while Bufo GLP-1Ž37. and xGLP-1A are equally ineffective in the brown bullhead. In liver cells of black bullhead Žcf. Fig. 7., where we tested only two Xenopus peptides, xGLP-1C is more potent than xGLP-1B. As expected, rainbow trout GLP-2 failed to alter the rate of glycogen breakdown. Even at the highest concentration used Ž6.6 = 10y6 M., the maximum is not reached for any xGLP-1 or the C-terminally extended Bufo GLP-1Ž37.. Thirdly, liver cells isolated from two congeneric catfish species Ž A. nebulosus and A. melas . respond much better to glucagon than to any GLP-1 ŽFigs. 4–7, Table 3.. Half-maximal activation of glycogenolysis is achieved at low nanomolar concentrations for glucagons, but requires more than 10 times higher concentrations for GLP-1Ž7 – 37. . The same pattern is noted when the degree of phosphoryla-
Fig. 7. Activation of glycogenolysis in isolated black bullhead Ž A. melas . hepatocytes by glucagon family peptides. Values are means of seven Žhuman GLP-1. or six Žall others. independent observations in duplicates "SEM. Values are normalized to the maximum response attained in the presence of human GLP-1Ž7 – 37. , usually at 10y6 M. For synthetic rainbow trout GLP-2, the SEMs are often smaller than the symbol.
tion of glycogen phosphorylase is assessed directly ŽFig. 6.. Finally, in contrast to the rockfish cells, where the C-terminally extended Bufo glucagon-36 was more potent and where it showed a bell-shaped dose–response relationship, all glucagons seem to be equipotent in the brown bullhead assay system. However, the existence of a bellshaped curve cannot be ruled out based on our limited data for Bufo glucagon-36 ŽFig. 5..
4. Discussion Just like their piscine or mammalian counterparts, amphibian GLPs are powerful activators of glycogenolysis and glycogen phosphorylase in fish hepatocytes, but substantial differences exist in species-specific responses. While rockfish hepatocytes respond to GLP peptides at much lower concentrations than to glucagons, the reverse is true for cells isolated from the two catfish species. Therefore, glycogenolysis in the catfish can be considered ‘glucagon-driven’ as opposed to ‘GLP-driven’. It would be worthwhile to analyze the relative potencies in relation to actual plasma concentrations, or even better, with concentrations of the endogenous peptides reaching the liver in these species. If the observation that similar concentrations of both peptides Ž0.4–0.5 nM. reach the liver in the rainbow trout w23x can be extrapolated to other species, then, indeed, glucagon- and GLP-1-driven metabolic regulation should be distinguished at least for the liver. The EC 50 for the most potent peptides when tested in the piscine systems falls into the low to mid-nanomolar range, and thus, it might be argued that they are somewhat above the physiological concentrations of these peptides. However, a few counterarguments should be mentioned. First, physiological concentrations of these peptides are considerably higher in the fish Žlow nanomolar range w3x. than in mammals. Second, the dispersed hepatocytes tend to be less sensitive to hormonal stimulation than cells maintained in perifusion columns. Using prostaglandin E 2 to activate glycogenolysis in rockfish liver cells, we found that the dose–response curve was displaced by more than an order of magnitude towards higher sensitivity when cells were immobilized in perifusion columns as opposed to maintained in suspension ŽBusby and Mommsen, in preparation.. Finally, accumulation of glucose from glycogenolysis is a relatively crude indicator, among other things requiring relatively lengthy incubations Ž30 min. for detection of the signal, while peak activation of glycogen phosphorylase is reached within about 8 min of hormone application Žcf. Figs. 3 and 6.. After peak stimulation, the enzyme activity falls off rapidly at low hormone concentrations, but remains high at supraphysiological hormone concentrations w20x ŽMommsen and Busby, unpublished.. Eventhough we do not know the physiological concentrations of any of the amphibian glucagon family peptides,
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we feel we can make a fairly strong case that we are indeed analyzing physiologically important phenomena. Independent of these species-dependent responses, the potency ranking for amphibian GLPs is similar in the three species and thus internally consistent. In every case, the Bufo GLP-1Ž32. is the most potent, followed by the corresponding peptide from Xenopus, xGLP-1C. A similar relationship exists between Bufo GLP-1Ž37. and the corresponding xGLP-1B. xGLP-1A is the least active of all GLP-1s tested. A corresponding peptide has not been found in Bufo Žpeptides. or Rana ŽcDNA., raising again the question whether this specific Xenopus peptide may have a function distinct from the other GLP-1 peptides. However, it should be pointed out that the Xenopus peptides were synthesized based on cDNA analysis and presumed gene products w12x, while the Bufo peptides were natural isolates from pancreatic tissue w13x, and hence, the next step should be to show that, indeed, a peptide corresponding to xGLP-1A is synthesized and secreted in X. laeÕis. Interestingly, the relative potency for amphibian GLP-1 peptides to human GLP-1Ž7 – 37. is species-dependent—in the rockfish, three amphibian GLP1s are more potent than human GLP-1Ž7 – 37. , while in both catfish species, GLP1Ž7 – 37. is more powerful than any amphibian GLP-1 peptide. Furthermore, the ranking of the amphibian peptides in their insulinotropic potency in isolated rat pancreas differs fundamentally from the fish liver test system w12x. Just like the fish test system, similar maximum rates are reached, but differences are apparent in receptor-binding constants and in the ability to increase cAMP in CHO cells transfected with the human GLP-1 receptor. The potency ranking in terms of receptor binding and cAMP production is xGLP-1BŽ30. ) human GLP-1Ž7 – 36amide. ) xGLP-1A ) xGLP-1C w12x. Functional relationships between the different GLP-1 peptides indicate that, ultimately, the potency depends somewhat on the test system. The rockfish are GLP-1driven, while the two catfish are glucagon-driven. We have no data on GLP-1 versus glucagon receptors in the rockfish, but it appears that, although catfish respond to GLP-1, the effects of glucagon may prevail in vivo, and the physiological raison d’etre ˆ for the GLP-1 is as yet unknown. Conversely, in the rockfish, GLP-1 is certainly a powerful hormone, partially occupying the functional position of glucagon. The terminally extended glucagon from B. marinus behaves differently in the two fish test systems. The peptide is more powerful than the shorter version in the rockfish hepatocytes; however, there is no difference in the catfish. In both systems Žbut note that n s 1 for the brown bullhead., the longer peptide is inhibitory at concentrations exceeding 3–6 = 10y8 M. It is possible that peptide processing in the hepatocytes is at the root of the species difference, and that the C-terminal extension may serve to protect against degradation in the rockfish, but not in the
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catfish liver cells. In fishes, liver has been identified as the main site for glucagon degradation w23x, although degradation pattern and dynamics have not yet been addressed in any detail, or with attention to potential species differences. In mammalian models for GLP-1, Ala2 may be a key site in determining peptide degradation, and substitution with Ser 2 substantially increases the half-life of the peptide in circulation w24x. It is interesting to note that throughout the fish w3x and amphibians w15,17,25x, Ala2 is conserved in all GLP-1 peptides, while glucagons contain Ser in this position. The metabolic functions of GLP-1 peptides in fish are by no means restricted to glycogenolysis, but also extend to other functions, such as hepatic gluconeogenesis and lipolysis w3x, and intestinal glucose transport w6x, with most functions mediated by adenylyl cyclase, cAMP and protein kinase A w26x and the secondary involvement of intracellular calcium w27x. Overall, these roles seem to be fish-specific, accentuate those of glucagon, and generally oppose the actions of insulin. Also, they are yet to be found in other vertebrate groups. Some hepatic actions of GLP-1 have been described for mammals, in alleged absence of GLP-1 receptors w28x, but these encompass actions opposing those noted in fish, including decreases in glucose output and increases in glycogen synthesis w29x. With the role in glycogenolysis confirmed and the function in insulin release still under debate for the fish, our data support the notion on comparable structure–function relationships in glycogenolytic potential in fish and insulinotropic action in mammals. Using directed alanine substitutions along the entire GLP-1 molecule, studies with mammals have identified residues His1 , Gly 4 , Thr 7, Asp 9 , Phe 22 and Ile 23 to be primarily important for binding to the rat pancreatic GLP-1 receptor. Additionally, Phe 6 , Tyr 13 , Glu15 and Leu26 play important roles. It is interesting to note that the many different fish GLP-1s, covering a much longer evolutionary range than the ‘highly conserved’ mammalian GLP-1, also have conserved the residues mentioned above. As we have shown here, the different frog GLP-1s follow a similar pattern with similarly conserved residues, although a few comments seem warranted. The chemical nature of residue 23 ŽIle in mammals and Ile or Val in fish or other amphibians. may be less important than what the previous studies imply. Bufo GLP-1Ž32. containing an Ile 23 and Bufo GLP-1Ž37. with its Tyr 23 show similar effectiveness in increasing insulin release in a rodent test system w12x, and the two peptides are also indistinguishable in their ability to activate glycogenolysis in our rockfish liver test system. While specific actions for GLP-1 in amphibians remain to be determined, we can anticipate that insulinotropic functions prevail and that direct hepatic actions, similar to those in fishes, are unlikely, since we could not find any glycogenolytic actions of GLP-1 in dispersed frog hepatocytes w11x. Apart from pancreas and intestine, other actions
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of GLP-1 appear to be similar and widespread in the vertebrate lineage—actions in brain, depression of feeding Žmediated by cAMP. w4,26,30x and alterations in intestinal transport and metabolism w6,26,31x. Acknowledgements This study was supported by research grants from the Natural Sciences and Engineering Research Council of Canada ŽTPM and DMI.. We thank Dr. Thomas W. Moon ŽUniversity of Ottawa. for access to black bullhead catfish and laboratory facilities. References w1x Mojsov S. Glucagon-like peptide-1 ŽGLP-1. and the control of glucose metabolism in mammals and teleost fish. Am Zool 2000;28:246–58. w2x Nauck MA. Is glucagon-like peptide 1 an incretin hormone? Diabetologia 1999;42:373–9. w3x Plisetskaya EM, Mommsen TP. Glucagon and glucagon-like peptides in fishes. Int Rev Cytol 1996;168:187–257. w4x Turton MD, O’Shea D, Gunn I, Beak SA, Edwards CMB, Meeran K, et al. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 1996;379:69–72. w5x Silverstein JT, Bondareva VM, Leonard JBK, Plisetskaya EM. Neuropeptide regulation of feeding in catfish Ictalurus punctatus: a role for glucagon-like peptide-1 ŽGLP-1.? Comp Biochem Physiol Part B 2001;29:623–31. w6x Soengas JL, Moon TW. Transport and metabolism of glucose in isolated enterocytes of the black bullhead, Ictalurus melas: effects of diet and hormones. J Exp Biol 1998;201:3263–73. w7x Munroe DG, Gupta AK, Kooshesh F, Vyas TB, Rizkalla G, Wang H, et al. Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2. Proc Natl Acad Sci U S A 1999;96:1569–73. w8x Hasegawa S, Terazono K, Nata K, Takada T, Yamamoto H, Okamoto H. Nucleotide sequence determination of chicken glucagon precursor cDNA. Chicken preproglucagon does not contain glucagon-like peptide II. FEBS Lett 1990;264:117–20. w9x Irwin DM, Wong J. Trout and chicken proglucagon: alternative splicing generates mRNA transcripts encoding glucagon-like peptide 2. Mol Endocrinol 1995;9:267–77. w10x Chen YE, Drucker DJ. Tissue-specific expression of unique mRNAs that encode proglucagon-derived peptides or exendin 4 in the lizard. J Biol Chem 1997;272:4108–15. w11x Mommsen TP, Moon TW. Glucagon-like peptides: structure–function relationship and evolution. In: Davey KG, Peter RE, Tobe SS, editors. Perspectives in Comparative Endocrinology. Ottawa: National Research Council of Canada, 1994:493–8. w12x Irwin DM, Satkunarajah M, Wen Y, Brubaker PL, Pederson RA, Wheeler MB. The Xenopus proglucagon gene encodes novel GLP1-like peptides with insulinotropic properties. Proc Natl Acad Sci U S A 1997;94:7915–20. w13x Conlon JM, Abdel-Wahab YHA, O’Harte FPM, Nielsen PF, Whittaker J. Purification and characterization of insulin, glucagon, and two glucagon-like peptides with insulin-releasing activity from the pancreas of the toad, Bufo marinus. Endocrinology1998;139:3442–8.
w14x Shuldiner AR, Bennett C, Robinson EA, Roth J. Isolation and characterization of two different insulins from an amphibian, Xenopus laeÕis. Endocrinology 1989;125:469–77. w15x White AM, Secor SM, Conlon JM. Insulin and proglucagon-derived peptides from the horned frog, Ceratophrys ornata ŽAnura:Leptodactylidae.. Gen Comp Endocrinol 1999;115:143–54. w16x Conlon JM, Yano K, Chartrel N, Vaudry H, Storey KB. Freeze tolerance in the wood frog Rana sylÕatica is associated with unusual structural features in insulin but not in glucagon. J Mol Endocrinol 1998;21:153–9. w17x Irwin DM, Sivarajah P. Proglucagon cDNAs from the leopard frog, Rana pipiens, encode two GLP-1-like peptides. Mol Cell Endocrinol 2000;162:17–24. w18x Mommsen TP, Moon TW, Walsh PJ. Hepatocytes: isolation, maintenance and utilization. In: Hochachka PW, Mommsen TP, editors. Biochemistry and Molecular Biology of Fishes. Amsterdam: Elsevier, 1994:355–73. w19x Passonneau JV, Lowry OH. Methods of Enzymatic Analysis. Totowa, NJ, USA: Humana Press, 1993:1–403. w20x Moon TW, Busby ER, Cooper GA, Mommsen TP. Fish hepatocyte glycogen phosphorylase—a sensitive indicator of hormonal activation. Fish Physiol Biochem 1999;21:15–24. w21x Fiske CH, Subbarow Y. The colorimetric determination of phosphorous. J Biol Chem 1925;66:375–8. w22x Hvidberg A, Toft Nielsen M, Hilsted J, Orskov C, Holst JJ. Effect of glucagon-like peptide-1 Žproglucagon 78–107amide. on hepatic glucose production in healthy man. Metabolism 1994;43:104–8. w23x Plisetskaya EM, Sullivan CV. Pancreatic and thyroid hormones in rainbow trout Ž Salmo gairdneri .: what concentration does the liver see? Gen Comp Endocrinol 1989;75:310–5. w24x Deacon CF, Knudsen LB, Madsen K, Wiberg FC, Jacobsen O, Holst JJ. Dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1 which have extended metabolic stability and improved biological activity. Diabetologia 1998;41:271–8. w25x Cavanaugh ES, Nielsen PF, Conlon JM. Isolation and structural characterization of proglucagon-derived peptides, pancreatic polypeptide and somatostatin from the urodele Amphiuma tridactylum. Gen Comp Endocrinol 1996;101:12–20. w26x Mommsen TP, Mojsov S. Glucagon-like peptide-1 activates the adenylyl cyclase system in rockfish enterocytes and brain membranes. Comp Biochem Physiol 1998;121B:49–56. w27x Moon TW, Gambarotta A, Capuzzo A, Fabbri E. Glucagon and glucagon-like peptide signaling pathways in the liver of two fish species, the American eel and the black bullhead. J Exp Zool 1997;279:62–70. w28x Wei Y, Mojsov S. Tissue-specific expression of the human receptor for glucagon-like peptide-1: brain, heart and pancreatic forms have the same deduced amino acid sequences. FEBS Lett 1995;358:219– 24. w29x Lopez-Delgado MI, Morales M, Villanueva-Penacarrillo ML, ´ ˜ Malaisse WJ, Valverde I. Effects of glucagon-like peptide 1 on the kinetics of glycogen synthase a in hepatocytes from normal and diabetic rats. Endocrinology 1998;139:2811–7. w30x Mommsen TP. Glucagon-like peptides in fishes: the liver and beyond. Am Zool 2000;40:259–68. w31x Wojdemann M, Wettergren A, Sternby B, Holst JJ, Larsen S, Rehfeld JF, et al. Inhibition of human gastric lipase secretion by glucagon-like peptide-1. Dig Dis Sci 1998;43:799–805. w32x Mojsov S, Weir GC, Habener JF. Insulinotropin: glucagon-like peptide I Ž7–37. co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J Clin Invest 1987;79:616–9.