- Email: [email protected]
Actions of GIP
Peptides, Vol. 2, Suppl. 2, pp. 241-245, 1981. Printed in the U.S.A.
Actions of GIP J. C. B R O W N , M. D A H L , S. K W A U K , C. H . S. M c l N T O S H , A N D R. A . P E D E R S O N
S. C. O T T E
Department of Physiology, University of British Columbia Vancouver, British Columbia, Canada V6T 1W5
BROWN, J. C., M. DAHL, S. KWAUK, C. H. S. MclNTOSH, S. C. OTI'E AND R. A. PEDERSON. Actions of G1P. PEPTIDES 2: Suppl. 2,241-245, 1981.--Two structurally similar peptides were isolated from a preparation of GIP using an HPLC system. The major peptide corresponds to GIP~_42 and the minor has the sequence GIP a-~2. GIP1_42 has both insulinotropic and somatostatinotropic activities, whereas GIP~..42has only insignificant activity. GIP was also shown to potentiate insulin release initiated by D-glyceraldehyde, L-leucine/L-glutamine and 2-keto-isocaproic acid. No potentiation was observed with 2-ketocaproate. The 4 substrates studied are all metabolized but via different mechanisms. HPLC
GIP
Insulinotropic effect
Somatostatinotropic effect
AN amino acid sequence for GIP, based on sequences determined for trypsin and cyanogen bromide cleaved fragments was reported in 1971 [3,5]. Recently the purest preparations of GIP have been shown by isotachophoresis, high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC) to contain a second minor peptide component [4]. The existence of the second peptide component'in what were considered to be homogeneous preparations of the hormone warranted a re-investigation of the primary structure of natural GIP preparations. The results of a new sequence determination for GIP have been reported [10]. The corrected sequence showed that the previously published primary structure was one glutamine residue too long, at position 30. In the same study it was confirmed that the GIP preparation was heterogeneous. Simultaneous sequence analyses of this minor component suggested that it was identical in structure to the major component in the preparation except that it lacked residues 1 and 2. The second peptide was therefore considered to be composed of residues 3-42 of the major peptide component in the GIP preparation. The insulinotropic action of GIP1-42 and the peptide GIP3-42 have been studied in the isolated perfused rat pancreas and stomach preparations. The interaction of GIP with some initiators of insulin release was also investigated to ascertain if the potentiation of insulin release was associated with a particular aspect of glucose metabolism. METHOD
Isolated Perfused Pancreas The isolated perfused rat pancreas was used to assay for insulinotropic activity [8,16]. Male Wistar rats (250-300 g) were anaesthetized with pentobarbital (60 mg/kg). The pancreas and upper duodenum were perfused at a flow rate of 4.0 ml/min, with a non-recirculating Krebs-Ringer bicarbonate medium containing 1% bovine serum albumin (Sigma RIA grade) and 3% dextran (Sigma clinical grade). The perfusate was gassed with 95% 02-5% COz to pH 7.4 and
C o p y r i g h t © 1981 A N K H O
G|P1-42
GIP3-42
warmed to 37°C. The portal venous effluent was collected at 60 sec intervals and stored at -20°C until assayed, for IRI (immunoreactive insulin).
Isolated Perfused Stomach Male Wistar rats (250-300 g) were used in the studies to investigate the release of SLI (somatostatin-like immunoreactivity) from the isolated perfused stomach. The animals were anaesthetized with 60 mg/kg pentobarbital (intraperitoneally), following an overnight fast (15-18 hr). Arterial perfusion was accomplished via a cannula placed in the aorta, adjacent to the coeliac artery. A second cannula, to collect total venous effluent, was inserted into the portal vein. The perfusate was a Krebs-Ringer bicarbonate buffer containing 0.2% bovine serum albumin (Sigma RIA grade), 3% dextran T70 (Pharmacia, Sweden) and 80 mg% glucose. The solution was gassed continuously with 95% 02-5% CO2 to a pH of 7.4. The perfusion rate was maintained at 3 ml/min and portal vein effluent was collected at 60 sec intervals into ice-cold tubes containing 1000 K I U aprotinin/ml (Trasylol, Bayer, Germany). A 20 min equilibration period was allowed, prior to collection of samples. All samples were stored at -20°C until assayed for SLI.
Radioimmunoassay for Insulin Insulin RIA was performed on all pancreatic effluent samples. The assay used purified rat insulin standards and iodinated bovine insulin (Novo, Copenhagen). Antisera were raised in guinea pigs to purified human insulin and stored lyophilized. The assays were controlled by a pooled sample (40/xU/ml) at the beginning and end of each assay and a variance of -+5 txU/ml resulted in invalidation of the assay. SLI was determined by a specific RIA as previously described [11,12] with minor modifications. Perfusate samples were diluted 1:5 or 1:10 with assay buffer, prior to measurement and all perfusion media and test substances were checked to ensure there was no interference in the RIA.
I n t e r n a t i o n a l Inc.--0196-9781/81/060241-05501.00/0
BROWN ET AL.
242 Inter- and intra-assay variation and sensitivity of the assay were similar to those described in the earlier reports [ 11,12].
~-IOng/ml H PLC-GIP Grodienf] IO000-
Purification of GIP GIP was purified as described earlier (7, 6). This preparation (GIP III) was further purified by H P L C on a/zBondapak C18 column (3.9×300 mm) using a Waters pump and variable wave length detector. The solvent system used for the preparative runs was 68% water, 32% acetonitrile and 0.1% trifluoroacetic acid (TFA) to a final concentration of 0.1%. The flow rate was 3.0 ml/min at a pressure of 2,600 psi. Two components were separated and collected and are referred to as HPLC I and HPLC III. The HPLC purified peptides were subjected to analyses on a Dionex amino acid analyzer, following hydrolysis for 20 hr at 110°C in vacuo. The peptides were introduced into the perfusion systems employing a gradient maker. The gradient maker consisted of two identical chambers connected at the base by polyethylene tubing. Perfusate was pumped from the chamber closest to the perfused organ which was then constantly fed by perfusate from the second chamber. Gradual mixing of the contents of both chambers occurred so that a gradient was established, the slope of which could be regulated by changing the concentration of the peptide in the second vessel. In the experiments in which metabolic compounds were being infused the preparations were preperfused for 15 min with a low concentration (without effect) of the substrate under investigation. A rapid switch to the required concentration of the metabolic compound at the end of the equilibration period was possible through the use of two parallel recirculating perfusate systems. In these experiments GIP was introduced through a side arm by means of a Harvard infusion pump, at a flow rate of 200 ~l/min, to achieve a final concentration of 5 rig/rain.
Substrates a-Ketocaproic acid and a-ketoisocaproic acid (purchased from Sigma) were infused at a concentration of 5 mM. D-glyceraldehyde (Sigma) and L-glutamine (Sigma) were used at a concentration of 10 mM and L-leucine (Fisher) at a concentration of 20 raM. All results are expressed as mean___S.E.M. and statistical significance was determined using the unpaired Student's t-test. RESULTS
8000-
c-
E
6000-
I-.-t
r-r-
o 8.9 mM Glucose {n- 5)
4000-
* 8.9mM Glucose÷HPLC-GIP_f(n-6) A 8.9 mM Glucose+HPLC-GIP~(n-6)
2000-
0 I0
20
50
Time (min) FIG. 1. Mean IRI release from the isolated perfused pancreas preparation, in response to a graded increase in infusion of GIP-HPLC I (GIPa-42) and GIP-HPLC III (GIPH2). A square wave glucose infusion (8.9 raM) was given throughout.
fused pancreas in the presence of 8.9mM glucose, The peptides were infused using a gradient system so that a gradual increase in concentration was produced. The " r a m p " effect produced was from 0 to 10 ng/ml. GIP (HPLC-III) was a powerful stimulator of insulin release whereas only an insignificant effect was observed with the N-terminal shortened peptide.
High Pressure Liquid Chromatography
Somatostatinotropic Effect of GIP-HPLC I and I11
GIP III when subjected to H P L C in the solvent system described revealed the heterogeneity described earlier [4,10]. The T F A system gave reasonable recoveries, approximately 66%, after the acetonitrile was evaporated in a stream of Nz and the resulting aqueous solution lyophilized. Aliquots of two of the components I and III were subjected to hydrolysis and amino acid analysis. Peak III had the composition of the corrected GIP1-42 and peak I had the composition of GIPa-42 as reported (10). Peak I differed in that it had one tyrosine and one alanine less than GIP.
SLI release has been measured in the isolated perfused rat stomach, in response to a gradient of GIP3-42 and G I P H z ' from 0-50 ng/ml. Figure 2 shows that GIP3-4z has no SLI releasing capabilities whereas GIP1-4z had the full biological effect. The broken line is only an approximation of the peptide concentration change.
Insulinotropic Activity of GIP-HPLC I and III Figure 1 shows the effect of the infusion of GIP (HPLC-I) and GIP (HPLC-III) on insulin release from the isolated per-
lnsulinotropic Activity of GIP in the Presence of Substrates Figure 3 shows that 10ram D-glyceraldehyde produced a biphasic release of insulin in the absence o f glucose. The preparations were pre-perfused with buffer containing 5.0 mM. A square wave G I P infusion at a final concentration of 5.0 ng/ml produced a further significant increase in IRI release. This increase was also seen to be biphasic and re-
A C T I O N O F GIP
243
I0 mM D-Glyceraldehyde 15ng/ml GIPI
2000 1600
I
c, 1200" o.
±r~3
-J 8 0 0 " 0o
o D-Glyceraldehyde alone(n-4)
400-
D-Glyceroldehyde+GtP (n=5)
•
0 0
I0
20 Time
30
40
in M i n u t e s
2400"
FIG. 2. SLI (somatostatin-like immunoreactivity) release from the isolated perfused stomach of the rat. GIP3-42 was without effect as a somatostatinotropic agent.
c;
turned to normal values within 3 min of the end of the infusion. Both a-ketocaproic and t~-ketoisocaproic acid (5.0 mM) induced biphasic increases in insulin secretion. The preparations were pre-perfused with buffer containing 2.5 mM of the appropriate buffer. A 20-min square wave infusion of GIP (5.0 ng/ml) produced a significant increase in IRI release in the studies with 5.0 mM a-ketoisocaproic acid (Fig. 5) but did not have a significant effect when 5.0 mM c~-ketoisocaproic acid was used (Fig. 4). Neither 20 mM L-leucine nor 10mM L-glutamine when added to the perfusate separately, evoked a measurable insulin response in the presence or absence of GIP. However, a mixture of the two amino acids at these concentrations elicited a small increase. A 5.0 ng/ml square wave GIP infusion evoked a rapid and sustained monophasic increase in IR1 release (Fig. 6). The insulin secretory rate remained elevated and did not return to control levels following termination of the GIP infusion. DISCUSSION The heterogeneity of GIP preparations [4,10] has been confirmed and a solvent system for preparative H P L C producing acceptable yields has been developed. GIP1-4~ (HPLC III) has been shown to have both IRI and SLI releasing activities. Surprisingly the shortened peptide GIPa_42 had little or no biological activity. A technique to produce gradually increasing concentrations of the peptides in perfusion systems has been used in this study as opposed to earlier studies, where a square wave stimulus was employed. This " r a m p " increase which is more akin to a physiological release has shown that the biphasic nature of IRI release to GIP is an artifact of the square wave stimulus (Fig. l). The substrates used in this study were chosen because of the demonstrated ability to initiate insulin release in the absence of glucose. Although they enter intermediary metabolic pathways at different areas and cannot be considered to have a common oxidative pathway, they do share with glucose the ability to evoke responses in the B-cell. The most notable of these changes is the increase in islet content of pyridine nucleotides, an effect which has been repeatedly suggested as a link between substrate oxidation and stimulus for insulin release [l, 9, 14]. The different metabolic paths involved in the oxidation of the substrates provides an investigative tool whereby the
1800,
E
? ii'i
~ 12oocr p
, o
t,, o" b /' ~ l , ~ \ '~o.O
o t,,
o / o\ o. o g,,
d'o
600-
o
%%
I
,
,'5 Time (min)
FIG. 3. Biphasic IRI release to I0 mM D-glyceraldehyde from the isolated perfused rat pancreas in the absence of glucose. A square wave of GIP (5 ng/ml) produced a further significant increase in IRI.
I 25 ]
5 0 m M a:-
mM
[
Kelocaproic
5nt]/ml
GIP
j
Acid I
3000o
~ ',
o
"-" 2000.c_ E "x
n,"
",
,'°'o,
;' ' '~
'
~8 °''°
,o,o" ,
"°R"o/'
V
I000" +
io
)'5
(n=6)
,)'5
T i m e (rain.)
FIG. 4. The insulinotropic effect in response to infusion of 5.0 mM a-ketocaproic acid and the absence of potentiation by 5 ng/ml GIP in the isolated perfused rat pancreas.
244
BROWN E T A L .
Lz51 mM
5 0 m M e:- Keloisocoproic
I
5~g~, G1P
3000"
Acid
[
I o ~ K I C alone(n=S} • ¢~ KIC~GIP (n~6)
/!
2000-
I000-
Time (rain)
FIG. 5. The insulinotropic effect in response to the infusion of 5.0 mM c~-ketoisocaproic acid. A potentiation of insulin release can be seen following GIP infusion (5.0 ng/ml).
ItOn',MI L-GIn
IOmM I
L-Glutamine÷2OmM 5 ng/ml GIP
J
L-Leucine I
3000' • L-Leu~-L-GIn÷GIP (n=6] o L-Leu+L-GIn
(n=7)
.E 2000 E
~
Iooo.
0 , 15
,
t
30
45
Time (rain)
FIG. 6. Absence of a stimulatory effect of 20 mM L-leucine and 10 mM L-glutamine on insulin release from the isolated perfused rat pancreas. Addition of a square wave of GIP stimulation (5.0 ng/ml) produced a significant increase in IRI release.
specificity of GIP's interaction with the products produced by the metabolic degradation of glucose can be examined. D-glyceraldehyde is phosphorylated by the islet [2] and enters glycolysis at the triosephosphate level. It mimics the effects of glucose on pro-insulin biosynthesis and 45Ca net uptake and, like glucose, metabolism of it yields both lactate and CO2 [13]. Since both glucose and glyceraldehyde are catabolized through a common metabolic pathway it is not surprising that GIP potentiates the insulin release which is initiated by either of these compounds. A similar result has previously been reported using a 1 /zg/ml dose of GIP in an isolated islet preparation [ 17,18]. The mixture of the amino acids L-leucine and L-glutamine did not prove to be a very potent initiator of insulin release from the isolated perfused pancreas, but a relatively large potentation was observed in the presence of GIP. The similarities between t~-keto acid- and glucose-induced insulin release have recently been reported in detail [9]. Both the keto acids have been shown to evoke similar changes to glucose in B-cell electrical activity and in levels of islet ATP, ADP, AMP and pyridine nucleotides. The metabolism of a-ketoisocaproate and a-ketocaproate in islet tissue is not completely delineated [9]. It has been proposed that mitochondrial degradation to CO2, NADH and acyl CoA derivatives or transamination to L-alpha-hydroxycarboxylic acids represents the means by which the 2 keto acids are metabolized [15]. GIP potentiated the insulin release evoked by 2 ketoisocaproate but not by 2 ketocaproate, indicating that mechanisms by which the two substrates elicit insulin release must differ. The four substrates studied are all metabolized, but through different mechanisms. GIP potentiated the insulin release initiated by three of these; D-glyceraldehyde, L-leucine/L-glutamine and c~-ketoisocaproic acid, which indicated that this action was not specific for any of the metabolic pathways involved. ACKNOWLEDGEMENTS These studies have been supported by research grants from the Medical Research Council of Canada, The British Columbia Health Care Research Foundation and the Vancouver Foundation.
REFERENCES
1. Ashcroft, S. J. H. Glucoreceptor mechanisms and the control of insulin release and biosynthesis. Diabetologia 18: 5--15, 1980. 2. Ashcroft, S. J. H., L. Chatra, C. Weerasinghe and P. J. Randle. Interrelationship of islet metabolism, adenosine triphosphate content and insulin release. Biochem. J. 132: 223-231, 1973. 3. Brown, J. C. A gastric inhibitory polypeptide. I. The aminoacid composition and tryptic peptides. Can. J. Biochem. 49: 255261, 1971. 4. Brown, J. C., M. Dahl, S. Kwauk, C. H. S. Mclntosh, M. Mueller, S. C. Otte and R. A. Pederson. Properties and actions of GIP. In: Gut Hormones, 2nd ed., edited by S. R. Bloom and J. M. Polak. Edinburgh: Churchill Livingstone, 1981, pp. 248255. 5. Brown, J. C. and J. R. Dryburgh. A gastric inhibitory polypeptide. II. The complete amino acid sequence. Can. J. Biochem. 49: 867-872, 1971.
6. Brown, J. C., V. Mutt and R. A. Pederson. Further purification of a polypeptide demonstrating enterogastrone activity. J. Physiol. 209: 57-64, 1970. 7. Brown, J. C., R. A. Pederson, E. Jorpes and V. Mutt. Preparation of highly active enterogastrone. Can. J. Physiol. Pharmac. 47" 113--114, 1969. 8. Grodsky, G. M., R. Fanska and I. Lundquist. Interrelationships between a- and/3-anomers of glucose affecting both insulin and glucagon secretion in the perfused rat pancreas. Endocrinology 97: 573--580, 1975. 9. Hutton, J. C., A. Sener, A. Herchuelz, I. Atwater, S. Kawazu, A. C. Boschero, G. Somers, G. Davis and W. J. Malaisse. Similarities in the stimulus-secretion coupling mechanisms of glucose- and a-keto acid-induced insulinrelease. Endocrinology 106: 203-219, 1980.
ACTION OF GIP 10. J6rnvall, H., M. Carlquist, S. Kwauk, S. C. Otte, C. H. S. McIntosh, J. C. Brown and V. Mutt. Amino acid sequence and heterogeneity of gastric inhibitory polypeptide (GIP). FEBS Left. 123: 205-210, 1981. 11. McIntosh, C., R. Arnold, E. Bothe, H. Becket, J. K6bbefling and W. Creutzfeldt.. Gastrointestinal somatostatin in man and dog. Metabolism 27: Suppl. 1, 1317-1320, 1978. 12. Mclntosh, C., R. Arnold, E. Bothe, H. Becker, J. K6bberling and W. Creutzfeidt. Gastrointestinal somatostatin: extraction and radioimmunoassay in different species. Gut 19: 655-663, 1978. 13. Malaisse, W. J., A. Herchuelz, J. Levy, A. Sener, D. G. Pipeleers, G. Davis, G. Somers, E. Van Obberghen. The stimulus-secretion coupling of glucose-induced insulin release. XIX. The insulinotropic effect of glyceraldehyde. Molec. cell. Endocr. 4: 1-12, 1976. 14. Malaisse, W. J., A. Sener, A. Herchuelz and J. C. Hutton. Insulin release: the fuel hypothesis. Metabolism 28: 373-386, 1979.
245 15. Panten, U. Effects of alpha-ketomonocarboxylic acids upon insulin secretion and metabolism of isolated pancreatic islets. Naunyn-Schmiedeberg's Arch. Pharmac. 291: 405--420, 1975. 16. Pederson, R. A. and J. C. Brown. The insulinotropic action of gastric inhibitory polypeptide in the perfused isolated rat pancreas. Endocrinology 99: 780-785, 1976. 17. Schauder, P., J. C. Brown, H. Frerichs and W. Creutzfeldt. Gastric inhibitory polypeptide: effect on glucose-induced insulin release from isolated rat pancreatic islets in vitro. Diabetologia 11: 483-484, 1965. 18. Schauder, P., B. Schindler, U. Panten, J. C. Brown, H. Frerichs and W. Creutzfeldt. Insulin release from isolated rat pancreatic islets induced by a-ketoisocaproic acid, L-leucine, D-glucose or D-glyceraldehyde: effect of gastric inhibitory polypeptide or glucagon. Molec. cell. Endocr. 7:115-- 123, 1977.
Actions of GIP J. C. B R O W N , M. D A H L , S. K W A U K , C. H . S. M c l N T O S H , A N D R. A . P E D E R S O N
S. C. O T T E
Department of Physiology, University of British Columbia Vancouver, British Columbia, Canada V6T 1W5
BROWN, J. C., M. DAHL, S. KWAUK, C. H. S. MclNTOSH, S. C. OTI'E AND R. A. PEDERSON. Actions of G1P. PEPTIDES 2: Suppl. 2,241-245, 1981.--Two structurally similar peptides were isolated from a preparation of GIP using an HPLC system. The major peptide corresponds to GIP~_42 and the minor has the sequence GIP a-~2. GIP1_42 has both insulinotropic and somatostatinotropic activities, whereas GIP~..42has only insignificant activity. GIP was also shown to potentiate insulin release initiated by D-glyceraldehyde, L-leucine/L-glutamine and 2-keto-isocaproic acid. No potentiation was observed with 2-ketocaproate. The 4 substrates studied are all metabolized but via different mechanisms. HPLC
GIP
Insulinotropic effect
Somatostatinotropic effect
AN amino acid sequence for GIP, based on sequences determined for trypsin and cyanogen bromide cleaved fragments was reported in 1971 [3,5]. Recently the purest preparations of GIP have been shown by isotachophoresis, high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC) to contain a second minor peptide component [4]. The existence of the second peptide component'in what were considered to be homogeneous preparations of the hormone warranted a re-investigation of the primary structure of natural GIP preparations. The results of a new sequence determination for GIP have been reported [10]. The corrected sequence showed that the previously published primary structure was one glutamine residue too long, at position 30. In the same study it was confirmed that the GIP preparation was heterogeneous. Simultaneous sequence analyses of this minor component suggested that it was identical in structure to the major component in the preparation except that it lacked residues 1 and 2. The second peptide was therefore considered to be composed of residues 3-42 of the major peptide component in the GIP preparation. The insulinotropic action of GIP1-42 and the peptide GIP3-42 have been studied in the isolated perfused rat pancreas and stomach preparations. The interaction of GIP with some initiators of insulin release was also investigated to ascertain if the potentiation of insulin release was associated with a particular aspect of glucose metabolism. METHOD
Isolated Perfused Pancreas The isolated perfused rat pancreas was used to assay for insulinotropic activity [8,16]. Male Wistar rats (250-300 g) were anaesthetized with pentobarbital (60 mg/kg). The pancreas and upper duodenum were perfused at a flow rate of 4.0 ml/min, with a non-recirculating Krebs-Ringer bicarbonate medium containing 1% bovine serum albumin (Sigma RIA grade) and 3% dextran (Sigma clinical grade). The perfusate was gassed with 95% 02-5% COz to pH 7.4 and
C o p y r i g h t © 1981 A N K H O
G|P1-42
GIP3-42
warmed to 37°C. The portal venous effluent was collected at 60 sec intervals and stored at -20°C until assayed, for IRI (immunoreactive insulin).
Isolated Perfused Stomach Male Wistar rats (250-300 g) were used in the studies to investigate the release of SLI (somatostatin-like immunoreactivity) from the isolated perfused stomach. The animals were anaesthetized with 60 mg/kg pentobarbital (intraperitoneally), following an overnight fast (15-18 hr). Arterial perfusion was accomplished via a cannula placed in the aorta, adjacent to the coeliac artery. A second cannula, to collect total venous effluent, was inserted into the portal vein. The perfusate was a Krebs-Ringer bicarbonate buffer containing 0.2% bovine serum albumin (Sigma RIA grade), 3% dextran T70 (Pharmacia, Sweden) and 80 mg% glucose. The solution was gassed continuously with 95% 02-5% CO2 to a pH of 7.4. The perfusion rate was maintained at 3 ml/min and portal vein effluent was collected at 60 sec intervals into ice-cold tubes containing 1000 K I U aprotinin/ml (Trasylol, Bayer, Germany). A 20 min equilibration period was allowed, prior to collection of samples. All samples were stored at -20°C until assayed for SLI.
Radioimmunoassay for Insulin Insulin RIA was performed on all pancreatic effluent samples. The assay used purified rat insulin standards and iodinated bovine insulin (Novo, Copenhagen). Antisera were raised in guinea pigs to purified human insulin and stored lyophilized. The assays were controlled by a pooled sample (40/xU/ml) at the beginning and end of each assay and a variance of -+5 txU/ml resulted in invalidation of the assay. SLI was determined by a specific RIA as previously described [11,12] with minor modifications. Perfusate samples were diluted 1:5 or 1:10 with assay buffer, prior to measurement and all perfusion media and test substances were checked to ensure there was no interference in the RIA.
I n t e r n a t i o n a l Inc.--0196-9781/81/060241-05501.00/0
BROWN ET AL.
242 Inter- and intra-assay variation and sensitivity of the assay were similar to those described in the earlier reports [ 11,12].
~-IOng/ml H PLC-GIP Grodienf] IO000-
Purification of GIP GIP was purified as described earlier (7, 6). This preparation (GIP III) was further purified by H P L C on a/zBondapak C18 column (3.9×300 mm) using a Waters pump and variable wave length detector. The solvent system used for the preparative runs was 68% water, 32% acetonitrile and 0.1% trifluoroacetic acid (TFA) to a final concentration of 0.1%. The flow rate was 3.0 ml/min at a pressure of 2,600 psi. Two components were separated and collected and are referred to as HPLC I and HPLC III. The HPLC purified peptides were subjected to analyses on a Dionex amino acid analyzer, following hydrolysis for 20 hr at 110°C in vacuo. The peptides were introduced into the perfusion systems employing a gradient maker. The gradient maker consisted of two identical chambers connected at the base by polyethylene tubing. Perfusate was pumped from the chamber closest to the perfused organ which was then constantly fed by perfusate from the second chamber. Gradual mixing of the contents of both chambers occurred so that a gradient was established, the slope of which could be regulated by changing the concentration of the peptide in the second vessel. In the experiments in which metabolic compounds were being infused the preparations were preperfused for 15 min with a low concentration (without effect) of the substrate under investigation. A rapid switch to the required concentration of the metabolic compound at the end of the equilibration period was possible through the use of two parallel recirculating perfusate systems. In these experiments GIP was introduced through a side arm by means of a Harvard infusion pump, at a flow rate of 200 ~l/min, to achieve a final concentration of 5 rig/rain.
Substrates a-Ketocaproic acid and a-ketoisocaproic acid (purchased from Sigma) were infused at a concentration of 5 mM. D-glyceraldehyde (Sigma) and L-glutamine (Sigma) were used at a concentration of 10 mM and L-leucine (Fisher) at a concentration of 20 raM. All results are expressed as mean___S.E.M. and statistical significance was determined using the unpaired Student's t-test. RESULTS
8000-
c-
E
6000-
I-.-t
r-r-
o 8.9 mM Glucose {n- 5)
4000-
* 8.9mM Glucose÷HPLC-GIP_f(n-6) A 8.9 mM Glucose+HPLC-GIP~(n-6)
2000-
0 I0
20
50
Time (min) FIG. 1. Mean IRI release from the isolated perfused pancreas preparation, in response to a graded increase in infusion of GIP-HPLC I (GIPa-42) and GIP-HPLC III (GIPH2). A square wave glucose infusion (8.9 raM) was given throughout.
fused pancreas in the presence of 8.9mM glucose, The peptides were infused using a gradient system so that a gradual increase in concentration was produced. The " r a m p " effect produced was from 0 to 10 ng/ml. GIP (HPLC-III) was a powerful stimulator of insulin release whereas only an insignificant effect was observed with the N-terminal shortened peptide.
High Pressure Liquid Chromatography
Somatostatinotropic Effect of GIP-HPLC I and I11
GIP III when subjected to H P L C in the solvent system described revealed the heterogeneity described earlier [4,10]. The T F A system gave reasonable recoveries, approximately 66%, after the acetonitrile was evaporated in a stream of Nz and the resulting aqueous solution lyophilized. Aliquots of two of the components I and III were subjected to hydrolysis and amino acid analysis. Peak III had the composition of the corrected GIP1-42 and peak I had the composition of GIPa-42 as reported (10). Peak I differed in that it had one tyrosine and one alanine less than GIP.
SLI release has been measured in the isolated perfused rat stomach, in response to a gradient of GIP3-42 and G I P H z ' from 0-50 ng/ml. Figure 2 shows that GIP3-4z has no SLI releasing capabilities whereas GIP1-4z had the full biological effect. The broken line is only an approximation of the peptide concentration change.
Insulinotropic Activity of GIP-HPLC I and III Figure 1 shows the effect of the infusion of GIP (HPLC-I) and GIP (HPLC-III) on insulin release from the isolated per-
lnsulinotropic Activity of GIP in the Presence of Substrates Figure 3 shows that 10ram D-glyceraldehyde produced a biphasic release of insulin in the absence o f glucose. The preparations were pre-perfused with buffer containing 5.0 mM. A square wave G I P infusion at a final concentration of 5.0 ng/ml produced a further significant increase in IRI release. This increase was also seen to be biphasic and re-
A C T I O N O F GIP
243
I0 mM D-Glyceraldehyde 15ng/ml GIPI
2000 1600
I
c, 1200" o.
±r~3
-J 8 0 0 " 0o
o D-Glyceraldehyde alone(n-4)
400-
D-Glyceroldehyde+GtP (n=5)
•
0 0
I0
20 Time
30
40
in M i n u t e s
2400"
FIG. 2. SLI (somatostatin-like immunoreactivity) release from the isolated perfused stomach of the rat. GIP3-42 was without effect as a somatostatinotropic agent.
c;
turned to normal values within 3 min of the end of the infusion. Both a-ketocaproic and t~-ketoisocaproic acid (5.0 mM) induced biphasic increases in insulin secretion. The preparations were pre-perfused with buffer containing 2.5 mM of the appropriate buffer. A 20-min square wave infusion of GIP (5.0 ng/ml) produced a significant increase in IRI release in the studies with 5.0 mM a-ketoisocaproic acid (Fig. 5) but did not have a significant effect when 5.0 mM c~-ketoisocaproic acid was used (Fig. 4). Neither 20 mM L-leucine nor 10mM L-glutamine when added to the perfusate separately, evoked a measurable insulin response in the presence or absence of GIP. However, a mixture of the two amino acids at these concentrations elicited a small increase. A 5.0 ng/ml square wave GIP infusion evoked a rapid and sustained monophasic increase in IR1 release (Fig. 6). The insulin secretory rate remained elevated and did not return to control levels following termination of the GIP infusion. DISCUSSION The heterogeneity of GIP preparations [4,10] has been confirmed and a solvent system for preparative H P L C producing acceptable yields has been developed. GIP1-4~ (HPLC III) has been shown to have both IRI and SLI releasing activities. Surprisingly the shortened peptide GIPa_42 had little or no biological activity. A technique to produce gradually increasing concentrations of the peptides in perfusion systems has been used in this study as opposed to earlier studies, where a square wave stimulus was employed. This " r a m p " increase which is more akin to a physiological release has shown that the biphasic nature of IRI release to GIP is an artifact of the square wave stimulus (Fig. l). The substrates used in this study were chosen because of the demonstrated ability to initiate insulin release in the absence of glucose. Although they enter intermediary metabolic pathways at different areas and cannot be considered to have a common oxidative pathway, they do share with glucose the ability to evoke responses in the B-cell. The most notable of these changes is the increase in islet content of pyridine nucleotides, an effect which has been repeatedly suggested as a link between substrate oxidation and stimulus for insulin release [l, 9, 14]. The different metabolic paths involved in the oxidation of the substrates provides an investigative tool whereby the
1800,
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? ii'i
~ 12oocr p
, o
t,, o" b /' ~ l , ~ \ '~o.O
o t,,
o / o\ o. o g,,
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600-
o
%%
I
,
,'5 Time (min)
FIG. 3. Biphasic IRI release to I0 mM D-glyceraldehyde from the isolated perfused rat pancreas in the absence of glucose. A square wave of GIP (5 ng/ml) produced a further significant increase in IRI.
I 25 ]
5 0 m M a:-
mM
[
Kelocaproic
5nt]/ml
GIP
j
Acid I
3000o
~ ',
o
"-" 2000.c_ E "x
n,"
",
,'°'o,
;' ' '~
'
~8 °''°
,o,o" ,
"°R"o/'
V
I000" +
io
)'5
(n=6)
,)'5
T i m e (rain.)
FIG. 4. The insulinotropic effect in response to infusion of 5.0 mM a-ketocaproic acid and the absence of potentiation by 5 ng/ml GIP in the isolated perfused rat pancreas.
244
BROWN E T A L .
Lz51 mM
5 0 m M e:- Keloisocoproic
I
5~g~, G1P
3000"
Acid
[
I o ~ K I C alone(n=S} • ¢~ KIC~GIP (n~6)
/!
2000-
I000-
Time (rain)
FIG. 5. The insulinotropic effect in response to the infusion of 5.0 mM c~-ketoisocaproic acid. A potentiation of insulin release can be seen following GIP infusion (5.0 ng/ml).
ItOn',MI L-GIn
IOmM I
L-Glutamine÷2OmM 5 ng/ml GIP
J
L-Leucine I
3000' • L-Leu~-L-GIn÷GIP (n=6] o L-Leu+L-GIn
(n=7)
.E 2000 E
~
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0 , 15
,
t
30
45
Time (rain)
FIG. 6. Absence of a stimulatory effect of 20 mM L-leucine and 10 mM L-glutamine on insulin release from the isolated perfused rat pancreas. Addition of a square wave of GIP stimulation (5.0 ng/ml) produced a significant increase in IRI release.
specificity of GIP's interaction with the products produced by the metabolic degradation of glucose can be examined. D-glyceraldehyde is phosphorylated by the islet [2] and enters glycolysis at the triosephosphate level. It mimics the effects of glucose on pro-insulin biosynthesis and 45Ca net uptake and, like glucose, metabolism of it yields both lactate and CO2 [13]. Since both glucose and glyceraldehyde are catabolized through a common metabolic pathway it is not surprising that GIP potentiates the insulin release which is initiated by either of these compounds. A similar result has previously been reported using a 1 /zg/ml dose of GIP in an isolated islet preparation [ 17,18]. The mixture of the amino acids L-leucine and L-glutamine did not prove to be a very potent initiator of insulin release from the isolated perfused pancreas, but a relatively large potentation was observed in the presence of GIP. The similarities between t~-keto acid- and glucose-induced insulin release have recently been reported in detail [9]. Both the keto acids have been shown to evoke similar changes to glucose in B-cell electrical activity and in levels of islet ATP, ADP, AMP and pyridine nucleotides. The metabolism of a-ketoisocaproate and a-ketocaproate in islet tissue is not completely delineated [9]. It has been proposed that mitochondrial degradation to CO2, NADH and acyl CoA derivatives or transamination to L-alpha-hydroxycarboxylic acids represents the means by which the 2 keto acids are metabolized [15]. GIP potentiated the insulin release evoked by 2 ketoisocaproate but not by 2 ketocaproate, indicating that mechanisms by which the two substrates elicit insulin release must differ. The four substrates studied are all metabolized, but through different mechanisms. GIP potentiated the insulin release initiated by three of these; D-glyceraldehyde, L-leucine/L-glutamine and c~-ketoisocaproic acid, which indicated that this action was not specific for any of the metabolic pathways involved. ACKNOWLEDGEMENTS These studies have been supported by research grants from the Medical Research Council of Canada, The British Columbia Health Care Research Foundation and the Vancouver Foundation.
REFERENCES
1. Ashcroft, S. J. H. Glucoreceptor mechanisms and the control of insulin release and biosynthesis. Diabetologia 18: 5--15, 1980. 2. Ashcroft, S. J. H., L. Chatra, C. Weerasinghe and P. J. Randle. Interrelationship of islet metabolism, adenosine triphosphate content and insulin release. Biochem. J. 132: 223-231, 1973. 3. Brown, J. C. A gastric inhibitory polypeptide. I. The aminoacid composition and tryptic peptides. Can. J. Biochem. 49: 255261, 1971. 4. Brown, J. C., M. Dahl, S. Kwauk, C. H. S. Mclntosh, M. Mueller, S. C. Otte and R. A. Pederson. Properties and actions of GIP. In: Gut Hormones, 2nd ed., edited by S. R. Bloom and J. M. Polak. Edinburgh: Churchill Livingstone, 1981, pp. 248255. 5. Brown, J. C. and J. R. Dryburgh. A gastric inhibitory polypeptide. II. The complete amino acid sequence. Can. J. Biochem. 49: 867-872, 1971.
6. Brown, J. C., V. Mutt and R. A. Pederson. Further purification of a polypeptide demonstrating enterogastrone activity. J. Physiol. 209: 57-64, 1970. 7. Brown, J. C., R. A. Pederson, E. Jorpes and V. Mutt. Preparation of highly active enterogastrone. Can. J. Physiol. Pharmac. 47" 113--114, 1969. 8. Grodsky, G. M., R. Fanska and I. Lundquist. Interrelationships between a- and/3-anomers of glucose affecting both insulin and glucagon secretion in the perfused rat pancreas. Endocrinology 97: 573--580, 1975. 9. Hutton, J. C., A. Sener, A. Herchuelz, I. Atwater, S. Kawazu, A. C. Boschero, G. Somers, G. Davis and W. J. Malaisse. Similarities in the stimulus-secretion coupling mechanisms of glucose- and a-keto acid-induced insulinrelease. Endocrinology 106: 203-219, 1980.
ACTION OF GIP 10. J6rnvall, H., M. Carlquist, S. Kwauk, S. C. Otte, C. H. S. McIntosh, J. C. Brown and V. Mutt. Amino acid sequence and heterogeneity of gastric inhibitory polypeptide (GIP). FEBS Left. 123: 205-210, 1981. 11. McIntosh, C., R. Arnold, E. Bothe, H. Becket, J. K6bbefling and W. Creutzfeldt.. Gastrointestinal somatostatin in man and dog. Metabolism 27: Suppl. 1, 1317-1320, 1978. 12. Mclntosh, C., R. Arnold, E. Bothe, H. Becker, J. K6bberling and W. Creutzfeidt. Gastrointestinal somatostatin: extraction and radioimmunoassay in different species. Gut 19: 655-663, 1978. 13. Malaisse, W. J., A. Herchuelz, J. Levy, A. Sener, D. G. Pipeleers, G. Davis, G. Somers, E. Van Obberghen. The stimulus-secretion coupling of glucose-induced insulin release. XIX. The insulinotropic effect of glyceraldehyde. Molec. cell. Endocr. 4: 1-12, 1976. 14. Malaisse, W. J., A. Sener, A. Herchuelz and J. C. Hutton. Insulin release: the fuel hypothesis. Metabolism 28: 373-386, 1979.
245 15. Panten, U. Effects of alpha-ketomonocarboxylic acids upon insulin secretion and metabolism of isolated pancreatic islets. Naunyn-Schmiedeberg's Arch. Pharmac. 291: 405--420, 1975. 16. Pederson, R. A. and J. C. Brown. The insulinotropic action of gastric inhibitory polypeptide in the perfused isolated rat pancreas. Endocrinology 99: 780-785, 1976. 17. Schauder, P., J. C. Brown, H. Frerichs and W. Creutzfeldt. Gastric inhibitory polypeptide: effect on glucose-induced insulin release from isolated rat pancreatic islets in vitro. Diabetologia 11: 483-484, 1965. 18. Schauder, P., B. Schindler, U. Panten, J. C. Brown, H. Frerichs and W. Creutzfeldt. Insulin release from isolated rat pancreatic islets induced by a-ketoisocaproic acid, L-leucine, D-glucose or D-glyceraldehyde: effect of gastric inhibitory polypeptide or glucagon. Molec. cell. Endocr. 7:115-- 123, 1977.