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Different Influence of CGRP(8-37), an Amylin and CGRP Antagonist, on the Anorectic Effects of Cholecystokinin and Bombesin in Diabetic and Normal Rats
Peptides, Vol. 18, No. 5, pp. 643–649, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(97)00124-1
Different Influence of CGRP(8–37), an Amylin and CGRP Antagonist, on the Anorectic Effects of Cholecystokinin and Bombesin in Diabetic and Normal Rats T. A. LUTZ,* 1 T. R. PIEBER,† B. WALZER,* E. DEL PRETE* AND E. SCHARRER* *Institute of Veterinary Physiology, University of Zuerich, Winterthurerstrasse 260, CH 8057 Zuerich, Switzerland †Department of Internal Medicine, Karl-Franzens-University Graz, Auenbruggerplatz 15, A 8036 Graz, Austria Received 18 September 1996; Accepted 2 January 1997 LUTZ, T. A., T. R. PIEBER, B. WALZER, E. DEL PRETE AND E. SCHARRER. Different influence of CGRP(8–37), an amylin and CGRP antagonist, on the anorectic effects of cholecystokinin and bombesin in diabetic and normal rats. PEPTIDES 18(5) 643–649, 1997.—Because previous studies had suggested that the anorectic effects of cholecystokinin (CCK) and bombesin (BBS) depend partly on the release of amylin or calcitonin gene-related peptide (CGRP), we investigated the influence of the amylin and CGRP receptor antagonist CGRP(8–37) on the anorectic effects of CCK and BBS in streptozotocin (STZ)-diabetic and nondiabetic rats. STZ-diabetic rats had significantly lower plasma amylin and insulin concentrations than nondiabetic control rats. Amylin (5 mg/kg or 2.5 mg/rat) injected IP at dark onset after 24-h food deprivation elicited an anorectic effect of similar extent in STZ-diabetic and control rats. Under similar conditions, CCK (0.25 and 2 mg/kg) and BBS (5 mg/kg) reduced food intake in both STZ-diabetic and nondiabetic rats. These effects were markedly attenuated by CGRP(8–37) (10 mg/kg) in nondiabetics but not in STZ-diabetic rats. It is concluded that part of the anorectic effects of CCK and BBS depend on the release of amylin from pancreatic B-cells. q 1997 Elsevier Science Inc. Amylin Cholecystokinin Bombesin Diabetic rat Plasma amylin concentration
CGRP CGRP(8–37) Radioimmunoassay
AMYLIN is released from pancreatic beta-cells in response to meal ingestion (9). Under most experimental conditions, amylin seems to be coreleased with insulin in a constant ratio [ for review, see (12)]. Amylin has a potent anorectic effect in rats and mice when administered centrally or peripherally (3,10,26–28,33–35). Abdominal vagotomy did not attenuate the anorectic effect of peripherally administered amylin (27,35). The latter may hence be mediated by amylin action at receptor sites within the central nervous system (1,4,6). Amylin has been regarded as a peripheral satiety peptide as it reduces food intake in rats at low, near physiological doses without inducing a conditioned taste aversion (26,28). Its exact role in the control of feeding behavior, however, remains to be established. We have presented evidence in a recent study (29) that amylin or CGRP may mediate part of the anorectic effects of cholecystokinin (CCK) [ for review, see (25)], bombesin (BBS) (30), and glucagon (18). Because each of the latter peptides increases insulin secretion (21,25,32), and because insulin secretion is 1
Streptozotocin
Food intake
usually associated with amylin secretion [ for review, see (12)], the attenuation of the anorectic effects of these peptides by the amylin and CGRP receptor antagonist calcitonin gene-related peptide [CGRP](8–37), which also reduces amylin’s anorectic effect (29), may be interpreted by CCK, BBS, and glucagon reducing food intake in part due to a stimulation of amylin release from pancreatic B-cells (29). An involvement of CGRP, however, could not be excluded. Amylin expression and secretion is greatly reduced in betacell deficiency, such as insulin-dependent diabetes mellitus [ for review, see (12)] or after treatment of rats with streptozotocin (STZ) (8,37), which selectively destroys pancreatic beta cells (2). STZ treatment, however, also depletes sensory neurons of CGRP (15,41). If our hypothesis, that one component of CCK’s and BBS’s anorectic effects depends on the release of amylin were true, coadministration of CGRP(8–37) with CCK or BBS would not be expected to affect the anorectic effects of the latter peptides in STZ-diabetic rats where CCK- and BBS-induced release of amylin is supposed to be low.
Requests for reprints should be addressed to T. A. Lutz.
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It was therefore the aim of the present study to compare the influence of the amylin and CGRP receptor antagonist CGRP(8– 37), which has previously been shown to block the anorectic effect of exogenous amylin (29), on the anorectic effects of CCK or BBS in STZ-diabetic rats with nondiabetic control rats. In addition, amylin’s anorectic efficacy was studied in STZ-diabetic and intact control rats. METHOD
Adult male rats (ZUR:SD; Institut fu¨r Labortierkunde, University of Zuerich, Switzerland) were used for the experiments and were adapted to the housing conditions for at least 2 weeks before treatment with STZ. Mean body weight was approximately 200 g before STZ treatment and 350 g (STZ-diabetic rats) and 600 g (nondiabetic rats), respectively, after finishing all experiments when a blood sample was taken from the aorta (see below). A separate group of 33 rats without pretreatment was used to investigate the influence of CGRP(8–37) on the anorectic effect of CGRP. Mean body weight was approximately 325 g. Rats were housed in individual wire cages in a temperaturecontrolled room (21 { 17C) and on a 12 h/12 h light/dark cycle (lights on 0800 h). Rats were fed a medium fat diet containing 18% fat, 46% carbohydrates, and 13% protein (26), because diabetes-induced hyperphagia is less pronounced when fed a fatenriched diet (16). The same diet was used in the experiment investigating the influence of CGRP(8–37) on CGRP’s anorectic effect. Rat amylin, rat CGRP, the amylin and CGRP receptor antagonist human CGRP(8–37), CCK, and BBS were obtained from Peninsula Laboratories (Belmont, CA). All peptides were dissolved in an appropriate volume of 0.9% NaCl to yield injection volumes of 1 ml/kg. Injection of 0.9% NaCl served as control. Amylin was injected at a dose of 5 mg/kg or 2.5 mg/rat, CCK was injected at a dose of 0.25 or 2 mg/kg, BBS at a dose of 5 mg/kg (see Results for details), CGRP at a dose of 5 mg/kg, and CGRP(8–37) at a dose of 10 mg/kg. In all experiments, rats were injected IP at dark onset (2000 h) after 24-h food deprivation. Water was available ad lib throughout the study. Food intake was measured by weighing the feeding cups ( {0.1 g) after correcting for spillage. Depending on the respective experiment, STZ-diabetic and nondiabetic rats were divided into two or three groups based on their body weight and basal food intake measured on the day before the experiment. Experiments were performed in a counterbalanced design with each animal tested under all conditions. The interval between single trials of an experiment was 5 days. Similarly, three groups were formed for the experiment investigating the influence of CGRP(8–37) on CGRP’s anorectic effect. This experiment was performed in a single trial. STZ Treatment In 18 rats, diabetes was induced by an IP injection of STZ (50 mg/kg; Sigma Chemical, Co., St. Louis, MO) after an overnight fast (17). Fasting was continued for about 3 h after the injection. STZ was dissolved in ice-cold citrate buffer (Na3-citrate, 75 mmol/l, pH 4.5; injection volume 2 ml/kg) that was kept on ice during the injection procedure, and all rats were injected within 5 min after STZ preparation (7). Sixteen rats were treated with citrate buffer vehicle only to serve as nondiabetic controls. Experiments began approximately 4 weeks after STZ treatment when STZ-diabetic rats had attained a mean body weight
of approximately 280 g and nondiabetic rats of approximately 400 g. During this period, rats were closely monitored daily but left untreated otherwise. Verification of Diabetic State Ten days after STZ treatment, a blood sample was taken from the retrobulbar ophthalmic venous plexus and blood glucose concentration determined by the glucose oxidase method on a COBAS MIRA automatic analyser (Hoffmann–La Roche, Basle, Switzerland). Only undeprived rats with a blood glucose concentration above 18 mmol/l were considered diabetic and hence used in the experiments. Blood glucose concentration was also determined in nondiabetic citrate-treated control rats. About 5 months after STZ treatment and after finishing all experiments, the diabetic state of STZ-treated rats was again assessed to test if rats had to be excluded retrospectively from analysis. Only rats confirmed to be diabetic in this second assessment were included in the analyses. Rats were food deprived for 24 h prior to the test. They were then offered their usual diet at dark onset for 30 min. Then rats were etherized and, after laparotomy, a blood sample was taken from the aorta. Blood was immediately transferred into chilled fluoride oxalate (determination of plasma glucose concentration) or EDTA blood tubes (determination of amylin, and insulin by radioimmunoassay [RIA; see below]), free fatty acids, triglycerides, and b-OH-butyrate). Plasma was separated by centrifugation and stored at 0207C until analysis. Aprotinin (500 kallikrein inactivator units/ml; Bayer, Leverkusen, Germany) was added to plasma for RIA and stored at 0707C until analysis. Plasma concentrations of glucose, free fatty acids (NEFA C; Wako Chemicals, Neuss, Germany), triglycerides (Triglyceride PAP; Hoffmann–La Roche), and b-OH-butyrate ( b-HBA; Sigma) were determined on an automatic COBAS MIRA analyzer. Radioimmunoassay for Amylin and Insulin Plasma concentrations of amylin and insulin were determined by radioimmunoassay (RIA). Plasma amylin concentration was determined as described earlier (39,40). Briefly, primary antibody T-486–6 (rabbit anti-amylin) was diluted with assay buffer [Na2HPO4 50 mmol/l (pH 7.2), aprotinin 1%, EDTA 0.25%, Triton X-100 0.2%, fish gelatin 0.1%, Na azide 0.02%; all reagents from Sigma] to a final dilution of 1:50,000. Rat amylin (Peninsula Laboratories) dissolved in assay buffer was used as standard. Sample or standards were incubated with antibody at 47C for 72 h. Then amylin tracer ([ 125I]amylin, 9,000–10,000 cpm; Amersham, Little Chalfont, UK) was added and samples were again incubated at 47C for an additional 96 h. Bound amylin was separated from free by a polyethylene-glycol supported second antibody (goat anti-rabbit IgG; Sigma) precipitation method. The detection limit of the assay was below 2 pmol/l, intra- and interassay coefficients of variation were below 5%. Plasma insulin concentration was determined with a commercial RIA kit (rat insulin RIA kit, Linco Research Inc., St. Charles, MO). Statistics Results are presented as mean { SEM. The treatment groups of STZ-diabetic and nondiabetic control rats were compared using paired Student’s t-test or repeated-measures ANOVA with the Student Newman–Keuls post hoc test where appropriate. The treatment groups of rats without pretreatment were compared using one-way ANOVA with the Student Newman–Keuls post hoc
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ANORECTIC EFFECT OF AMYLIN, CCK, AND BBS IN DIABETIC RATS
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TABLE 1 POSTPRANDIAL PLASMA CONCENTRATION OF GLUCOSE, AMYLIN, INSULIN, FREE FATTY ACIDS, TRIGLYCERIDES, AND b-OHBUTYRATE IN STREPTOZOTOCIN (STZ)-DIABETIC AND NONDIABETIC RATS FED FOR 30 min AFTER 24-h FOOD DEPRIVATION Plasma Concentration Nondiabetic Rats (n Å 15)
Glucose (mmol/l) Amylin (pmol/l) Insulin (pmol/l) Amylin/insulin ratio Free fatty acids (mmol/l) Triglycerides (mmol/l) b-OH-butyrate (mmol/l)
8.5 { 0.2 27.1 { 2.5 605.3 { 73.7 (n Å 14) 0.049 { 0.007 (n Å 14) 0.50 { 0.03 0.8 { 0.1 193 { 9
STZ-Diabetic Rats (n Å 10)
30.8 { 2.1† 14.3 { 2.2* 161.8 { 26.4† 0.103 { 0.018* 1.01 { 0.16† 4.1 { 0.7† 2055 { 649*
Plasma taken in anesthetized rats from the aorta. Fluoride oxalate plasma for glucose; EDTA plasma for amylin, insulin, free fatty acids, triglycerides, and b-OH-butyrate. *† Significantly different from nondiabetic rats (unpaired Student’s t-test; * p õ 0.01, † p õ 0.001, respectively).
test. Differences in plasma hormone concentrations and concentrations of plasma metabolites between STZ-diabetic and nondiabetic rats were statistically evaluated using the unpaired Student’s t-test. A value of p õ 0.05 was considered significant. RESULTS
Verification of Diabetic State; Characterization of Diabetic Rats Based on the blood glucose concentration determined 10 days after STZ treatment, induction of the diabetic state was successful in 11 rats [mean blood glucose 30.2 { 2.2 (18.5–40.2) mmol/ l]. These 11 rats were used in the experiments as diabetic rats. Nondiabetic control rats had a mean blood glucose concentration of 6.3 { 0.3 (4.4–8.9) mmol/l. Ten of the 11 diabetic rats exhibited marked polyuria and polydipsia, and body weight gain was markedly lower in STZdiabetic rats than in nondiabetic controls [body weight on the day of STZ treatment: 202 { 3 g in STZ-diabetic rats vs. 200 { 3 in nondiabetic controls; 4 weeks after STZ treatment: 276 { 12 g vs. 396 { 9 ( p õ 0.001); 5 months after STZ treatment: 315 { 27 g vs. 598 { 13 (p õ 0.001)]. Food intake was not measured daily, but whenever it was measured, STZ-diabetics consumed more food than nondiabetic rats [e.g., 4 weeks after STZ treatment: 12-h dark phase intake 23.3 { 1.4 g in STZdiabetics vs. 20.2 { 0.5 in nondiabetics ( p õ 0.05); 12-h light phase intake 5.0 { 0.7 g vs. 2.8 { 0.5 ( p õ 0.05)]. The diabetic state of the STZ-diabetic rats was reexamined after the termination of experiments 5 months after STZ injection (see Table 1). Based on the postprandial plasma glucose level, one rat had to be excluded from the diabetic group as it no longer fulfilled the criteria for diabetic hyperglycemia (plasma glucose level 9.4 mmol/l). This rat was excluded from the study, although its blood glucose concentration at first assessment was 20.1 mmol/l. Compared to other diabetic rats, polydipsia was not striking, and body weight in this rat was approximately 660 g 5 months after STZ treatment.
FIG. 1. Influence of amylin (5 mg/kg) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic (n Å 10) or nondiabetic (n Å 16) rats. *,**Significant difference compared with respective saline control (paired Student’s t-test; *p õ 0.05; **p õ 0.01).
Postprandial plasma glucose concentration was markedly elevated in the remaining 10 STZ-diabetic rats compared to nondiabetic controls (Table 1) and only these 10 rats were included in all analyses. STZ-diabetic rats had significantly lower postprandial plasma amylin and insulin concentrations than nondiabetic rats. However, insulin concentration was relatively more depressed than amylin concentrations, which resulted in an increased amylin/insulin ratio in STZ-diabetic rats (Table 1). Levels of free fatty acids, triglycerides, and b-OH-butyrate were markedly elevated in STZ-diabetic rats. Anorectic Effect of Amylin in STZ-Diabetic and Nondiabetic Rats Amylin (5 mg/kg) injected at dark onset after 24-h food deprivation significantly reduced cumulative food intake compared to saline controls for 30 min in STZ-diabetic and for 2 h in nondiabetic rats (Fig. 1). The relative reduction in food intake 30 min after injection was similar in STZ-diabetic (18%) and nondiabetic rats (19%). Similar results were obtained when amylin was injected at a dose of 2.5 mg/rat (Fig. 2).
FIG. 2. Influence of amylin (2.5 mg/rat) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic (n Å 10) or nondiabetic (n Å 15) rats. *,** Significant difference compared with respective saline control (paired Student’s t-test; *p õ 0.05; **p õ 0.01).
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FIG. 3. Influence of CCK (0.25 mg/kg) or CCK (0.25 mg/kg) / CGRP(8–37) (10 mg/kg) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic ( n Å 10) or nondiabetic (n Å 16) rats compared with respective saline control. *,**Significant difference between groups (repeated-measures ANOVA with Student Newman–Keuls post hoc test; *p õ 0.05; ** p õ 0.01).
FIG. 5. Influence of BBS (5 mg/kg) or BBS (5 mg/kg) / CGRP(8– 37) (10 mg/kg) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic (n Å 10) or nondiabetic (n Å 16) rats compared with respective saline control. **,***Significant difference between groups (repeated-measures ANOVA with Student Newman–Keuls post hoc test; **p õ 0.01; *** p õ 0.001; a,b,c values with different letters differ significantly, p õ 0.05).
Following 24-h food deprivation, STZ-diabetic rats did not eat more than nondiabetic controls during the first 2 h after injection (Figs. 1–5).
attenuated at 1 h. In STZ-diabetic rats, however, CGRP(8–37) did not influence CCK’s anorectic effect (Fig. 4).
Influence of CGRP(8–37) on CCK’s Anorectic Effect in STZDiabetic and Nondiabetic Rats
Influence of CGRP(8–37) on BBS’s Anorectic Effect in STZDiabetic and Nondiabetic Rats
CCK (0.25 mg/kg) significantly reduced cumulative food intake in 24-h food deprived nondiabetic rats for 1 h after injection compared to saline-treated controls (Fig. 3). This effect was totally antagonized by the simultaneous administration of the amylin receptor antagonist CGRP(8–37) (10 mg/kg). CCK only nonsignificantly reduced cumulative food intake in STZ-diabetic rats (Fig. 3). At a higher dose, CCK (2 mg/kg) significantly reduced cumulative food intake in both STZ-diabetic and nondiabetic rats compared to their respective controls (Fig. 4). The relative reduction in food intake was similar in both groups. The anorectic effect of CCK was almost completely reversed by CGRP(8–37) 30 min after injection in nondiabetic rats and
After the injection of BBS (5 mg/kg), cumulative food intake was significantly reduced for 2 h in both 24-h food-deprived STZ-diabetic and nondiabetic rats (Fig. 5). BBS’s anorectic effect was significantly reduced by about 50% by CGRP(8–37) in nondiabetic rats, whereas CGRP(8–37) had a minor, nonsignificant impact on BBS’s anorectic effect in STZ-diabetic rats (Fig. 5).
FIG. 4. Influence of CCK (2 mg/kg) or CCK (2 mg/kg) / CGRP(8– 37) (10 mg/kg) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic (n Å 10) or nondiabetic (n Å 16) rats compared with respective saline control. *,**,***Significant difference between groups (repeated-measures ANOVA with Student Newman–Keuls post hoc test; *p õ 0.05; ** p õ 0.01; ***p õ 0.001; a,b values with different letters differ significantly, p õ 0.05).
Influence of CGRP(8–37) on CGRP’s Anorectic Effect in Rats IP injection of CGRP(5 mg/kg) significantly reduced cumulative food intake for 1 h in 24-h food-deprived normal rats (Fig. 6).
FIG. 6. Influence of CGRP(5 mg/kg; n Å 11) or CGRP(5 mg/kg) / CGRP(8–37) (10 mg/kg; n Å 11) injected IP at dark onset on cumulative food intake in 24-h food-deprived rats compared with saline control (n Å 11). *,**Significant difference between groups (ANOVA with Student Newman–Keuls post hoc test; *p õ 0.05; ** p õ 0.01; a,b values with different letters differ significantly, p õ 0.05).
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ANORECTIC EFFECT OF AMYLIN, CCK, AND BBS IN DIABETIC RATS Coadministration of CGRP(8–37) (10 mg/kg) completely abolished CGRP’s anorectic effect. DISCUSSION
The main finding of this study, that the amylin and CGRP receptor antagonist CGRP(8–37) did not influence the anorectic effect of CCK or BBS in STZ-diabetic rats whereas it markedly attenuated this effect in nondiabetic controls, is consistent with the hypothesis that part of the anorectic effects of CCK and BBS in nondiabetic rats are mediated by amylin release. The finding that CGRP(8–37) attenuated the anorectic effects of CCK and BBS in nondiabetic rats confirms our previous study (29). In the first two experiments, we have shown that exogenous amylin reduces food intake in both STZ-diabetic and nondiabetic rats to a similar degree. However, a comparative complete dose– response study was not performed. That STZ-diabetic rats in our study were not completely depleted of amylin may explain why upregulation of amylin receptors, which presumably parallels upregulation of insulin receptors in STZ-diabetic rats (42), was probably not sufficient to cause hyperresponsiveness of STZ-diabetic rats to amylin. Morley and Flood also reported that amylin reduces food intake similarly in STZ-diabetic and nondiabetic mice food deprived overnight (33). The anorectic effect observed in nondiabetic rats confirms previous studies in that IP injections of amylin effectively reduce food intake in food-deprived rats (26–29). The second experiment with amylin (2.5 mg/rat) was performed because the difference in body weight between STZ-diabetic and nondiabetic rats is probably mainly due to a difference in adipose tissue mass rather than lean body mass. Dosage of amylin per rat rather than relative to rats’ body weight may therefore be more appropriate because amylin is probably mainly distributed in the extracellular water space, as has recently been shown for an amylin peptide analogue (11). Greatly reduced amylin expression and secretion in beta-cell deficiency after STZ treatment (20,37) are reflected in a decreased plasma amylin concentration as observed in the present study. The component of CCK’s and BBS’s anorectic effect that could be attenuated by simultaneous injection of the amylin and CGRP receptor antagonist CGRP(8–37) in a previous (29) and the present study in nondiabetic rats is supposed to be caused by the stimulatory effect of CCK and BBS on beta-cell secretion. Both CCK and BBS increase insulin secretion (21,25), and because amylin and insulin are usually cosecreted (12), we presume that CCK and BBS have a similar stimulating effect on amylin as on insulin secretion. Amylin may therefore be responsible for part of CCK’s and BBS’s effects in nondiabetic rats. The role of endogenous amylin in the control of food intake, however, remains to be clarified in further studies. It has to be kept in mind that CGRP(8–37) has antagonistic effects on receptors for both amylin and CGRP, which is structurally closely related to amylin (12). Plasma concentration of CGRP, which is an important neurotransmitter in the central and peripheral nervous system, also increases in response to food intake (44,45), and injection of CGRP has been shown to reduce food intake in rats and mice (24,36). We have confirmed CGRP’s anorectic action after peripheral administration in rats in the present study even at a much lower dose than reported previously (36). Similar to amylin’s anorectic effect (29), CGRP’s anorectic effect was blocked by coadministration of CGRP(8–37). Because recent studies have shown that STZ-treatment depletes certain sensory neurons of CGRP (15,41), and because the anorectic effects of both CCK and BBS seem to be at least
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in part nervally transmitted to the central nervous system (25,30), the lacking effect of CGRP(8–37) to attenuate CCK’s and BBS’s anorectic effects in STZ-diabetic rats could be explained by CGRP depletion in sensory neurons rather than amylin depletion in pancreatic B-cells. This alternative interpretation, however, appears to be rather unlikely considering our preliminary findings where we have shown that IP injected CCK (2 mg/ kg) had no effect on plasma CGRP levels compared with vehicle in 24-h food-deprived rats 30 min (20.9 { 4.6 pmol/l vs. 19.7 { 3.5 pmol/l) or 60 min (25.6 { 5.2 pmol/l vs. 19.6 { 3.5 pmol/l; n Å 8 in each group) after injection and refeeding. CGRP concentrations were similar to postprandial concentrations observed previously in humans (44). The amylin-dependent components of CCK’s and BBS’s anorectic effects presumably are of minor importance in STZ-diabetic rats because the secretory capacity of beta-cells for amylin is greatly reduced after STZ pretreatment (8,37). Our study confirms the expectation that CGRP(8–37) does not influence CCK’s and BBS’s anorectic effects under these conditions. Because CGRP(8–37) attenuated the anorectic effects of amylin, CCK, BBS, glucagon (29), and CGRP(present study), it could be argued that CGRP(8–37) acts as an unspecific antagonist at peptide receptors. Because CGRP(8–37), however, failed to reduce vasopressin’s anorectic effect in rats (29), we suggested in our previous study (29) that CGRP(8–37) is unlikely to attenuate CCK’s and BBS’s anorectic effects by directly blocking their respective receptors. This was confirmed in the present study because if the former were true, CGRP(8–37) would have been expected to reduce CCK’s and BBS’s effects in STZ-diabetic rats. The anorectic effect of BBS appeared to be somewhat stronger in nondiabetic than in STZ-diabetic rats, although this difference did not reach the level of significance [multifactor analysis of variance p Ç 0.29 for interaction between BBS injection and diabetic state (30 min after injection), p Ç 0.20 (1 h after injection)]. This may point, however, to the reduced amylin-depending component of BBS’s effect in the STZ-diabetic rats. The similar potency of the higher dose of CCK (2 mg/kg) to reduce food intake in both groups was somewhat surprising because of the diminished contribution of amylin to CCK’s effect in STZ-diabetic rats. The reason for this is unknown at present but the lacking component of CCK’s effect may have been compensated by other as yet unidentified factors in the present study. In another study (23), however, the anorectic effect of CCK (10 mg/kg IP) appeared to be moderately stronger in nondiabetic (54% reduction of food intake) than in STZ-diabetic rats (38% reduction). The authors, however, did not state if this difference was significant, and baseline food intake was significantly higher in STZ-diabetic rats, which impedes comparison of the data. Compared to previous studies (26–29), a higher amylin dose (5 mg/kg) was necessary to elicit a similar degree of anorexia as observed previously with a lower dose (1 mg/kg). Use of a different strain of rats (ZUR:SD in this study; ZUR:SIV in previous studies), although both being Sprague–Dawley rats, may explain this difference. Preliminary experiments have shown that amylin at a dose of 1 or 3 mg/kg inconsistently reduced food intake in ZUR:SD rats. Similar observations were made with CGRP because CGRP at a dose of 1 mg/kg significantly reduced food intake in ZUR:SIV rats while leaving food intake unaffected in ZUR:SD rats (unpublished). This strain of rats (ZUR:SD) may have a lower sensitivity to anorectic peptides in general, because similar to amylin and CGRP, a higher dose of CCK (2 mg/kg) was necessary to elicit an anorectic effect of similar strength compared to previous studies (0.25 mg/kg) (29). The lower sensitivity to some satiating
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peptides may also explain the higher growth rates in ZUR:SD compared to ZUR:SIV rats kept under similar conditions (e.g., body weight in ZUR:SIV rats at 10 weeks of age: about 280 g; ZUR:SD rats: about 340 g). Verification of Diabetic State Successful induction of the diabetic state with STZ was verified before and after the experiments and only those rats that fulfilled the criteria of hyperglycemia on both occasions were included in further analyses. STZ-diabetic rats exhibited a higher food intake than nondiabetic controls under free fed conditions. That this was not very pronounced under the present experimental conditions may have been due to the use of a fat-enriched diet, which attenuates diabetic hyperphagia compared with high-carbohydrate diets (16). It has to be kept in mind, however, that hyperphagia (g per kg body weight) was more marked if the lower body weight of STZdiabetic rats is taken into account. Clinical findings and parameters of carbohydrate and lipid metabolism confirmed the classification into diabetic or nondiabetic. The finding of an increased amylin/insulin ratio in STZ-diabetic rats despite a significant reduction in both plasma amylin and insulin agrees with numerous previous studies that showed a dissociation of amylin and insulin expression and release in spontaneously diabetic and STZ-diabetic rats (8,19,20,22,37). This seems to be directly correlated to the development of hyperglycemia
(39). The smaller reduction in plasma amylin than insulin concentration may also be due to production of amylin in pancreatic delta cells (14) or in extrapancreatic production sites, such as in the gastrointestinal mucosa (13,31,38,43). Expression of insulin, however, has also been described in the duodenal wall (5). Because CGRP leakage from sensory neurons is the main source of circulating CGRP, plasma CGRP concentrations may be lower in STZ-diabetic than in nondiabetic rats due to CGRP depletion in sensory neurons after STZ treatment (15,41,44). However, these have not been measured in diabetic rats in the present study. In conclusion, the present study has shown that the amylin and CGRP receptor antagonist CGRP(8–37), which antagonizes anorexia induced by amylin and CGRP, attenuated the anorectic effects to IP injected CCK and BBS in nondiabetic rats whereas it was ineffective in STZ-diabetic rats. Part of the anorectic effects of CCK and BBS in nondiabetic rats may therefore be mediated by the release of amylin from the pancreas. Due to amylin depletion in the pancreas of STZ-diabetic rats, the amylindependent component of CCK’s and BBS’s anorectic effect appears to be absent after STZ treatment. An involvement of blockade of CGRP receptors by CGRP(8–37), however, cannot be ruled out at present. ACKNOWLEDGEMENT
We gratefully acknowledge Barbara Schneider for her help with laboratory analyses. This work was supported by the Swiss National Foundation (Grant #3100-045583.95/1).
REFERENCES 1. Aiyar, N.; Baker, E.; Martin, J.; Patel, A.; Stadel, J. M.; Willette, R. N.; Barone, F. C. Differential calcitonin gene-related peptide (CGRP) and amylin binding sites in nucleus accumbens and lung: Potential models for studying CGRP/amylin receptor subtypes. J. Neurochem. 65:1131–1138; 1995. 2. Ar’Rajab, A.; Ahren, B. Long-term diabetogenic effect of streptozotocin in rats. Pancreas 8:50–57; 1993. 3. Balasubramaniam, A.; Renugopalakrishnan, V.; Stein, M.; Fischer, J. E.; Chance, W. T. Syntheses, structures and anorectic effects of human and rat amylin. Peptides 12:919–924; 1991. 4. Beaumont, K.; Kenney, M. A.; Young, A. A.; Rink, T. J. High affinity amylin binding sites in rat brain. Molecul. Pharmacol. 44:493– 497; 1993. 5. Bendayan, M.; Park, I.-S. Presence of extrapancreatic islets of Langerhans in the duodenal wall of the rat. Diabetologia 34:604–606; 1991. 6. Bhogal, R.; Smith, D. M.; Bloom, S. R. Investigation and characterization of binding sites for islet amyloid polypeptide in rat membranes. Endocrinology 130:906–913; 1992. 7. Brand, C. L.; Rolin, B.; Jorgensen, P. N.; Svendsen, I.; Kristensen, J. S.; Holst, J. J. Immunoneutralization of endogenous glucagon with monoclonal glucagon antibody normalizes hyperglycemia in moderately streptozotocin-diabetic rats. Diabetologia 37:985–993; 1994. 8. Bretherton–Watt, D.; Ghatei, M. A.; Bloom, S. R.; Jamal, H.; Ferrier, G. J. M.; Girgis, S. I.; Legon, S. Altered islet amyloid polypeptide (amylin) gene expression in rat models of diabetes. Diabetologia 32:881–883; 1989. 9. Butler, P. C.; Chou, J.; Carter, W. B.; Wang, Y.-N.; Bu, B.-H.; Chang, D.; Chang, J.-K.; Rizza, R. A. Effects of meal ingestion on plasma amylin concentration in NIDDM and nondiabetic humans. Diabetes 39:752–756; 1990. 10. Chance, W. T.; Balasubramaniam, A.; Zhang, F. S.; Wimalawansa, S. J.; Fischer, J. E. Anorexia following the intrahypothalamic administration of amylin. Brain Res. 539:352–354; 1991. 11. Colburn, W. A.; Gottlieb, A. B.; Koda, J.; Kolterman, O. G. Pharmacokinetics and pharmacodynamics of AC137 (25,28,29 tripro-amylin, human) after intravenous bolus and infusion doses in patients with insulin-dependent diabetes. J. Clin. Pharmacol. 36:13–24; 1996.
12. Cooper, G. J. S. Amylin compared with calcitonin gene-related peptide: Structure, biology, and relevance to metabolic disease. Endocr. Rev. 15:163–201; 1994. 13. D’Este, L.; Wimalawansa, S. J.; Renda, T. G. Amylin-immunoreactivity is co-stored in a serotonin cell subpopulation of the vertebrate stomach and duodenum. Arch. Histol. Cytol. 58:537 – 547; 1995. 14. DeVroede, M.; Foriers, A.; VanDeWinkel, M.; Madsen, O.; Pipeleers, D. Presence of islet amyloid polypeptide in rat islet B and D cells determines parallelism and dissociation between rat pancreatic islet amyloid polypeptide and insulin content. Biochem. Biophys. Res. Commun. 182:886–893; 1992. 15. Fernyhough, P.; Diemel, L. T.; Brewster, W. J.; Tomlinson, D. R. Deficits in sciatic nerve neuropeptide content coincide with a reduction in target tissue nerve growth factor messenger RNA in streptozotocin-diabetic rats: Effects of insulin treatment. Neuroscience 62:337–344; 1994. 16. Friedman, M. I. Hyperphagia in rats with experimental diabetes mellitus: A response to a decreased supply of utilizable fuels. J. Comp. Physiol. Psychol. 92:109–117; 1978. 17. Friedman, M. I.; Ramirez, I. Food intake in diabetic rats: Relationship to metabolic effects of insulin treatment. Physiol. Behav. 56:373–378; 1994. 18. Geary, N.; Le Sauter, J.; Noh, N. Glucagon acts in the liver to control spontaneous meal size in rats. Am. J. Physiol. 264:R116–R122; 1993. 19. Gedulin, B.; Cooper, G. J. S.; Young, A. A. Amylin secretion from the perfused pancreas: Dissociation from insulin and abnormal elevation in insulin-resistant diabetic rats. Biochem. Biophys. Res. Commun. 180:782–789; 1991. 20. Inoue, K.; Hisatomi, A.; Umeda, F.; Nawata, H. Relative hypersecretion of amylin to insulin from rat pancreas after neonatal STZ treatment. Diabetes 41:723–727; 1992. 21. Ipp, E.; Unger, R. H. Bombesin stimulates the release of insulin and glucagon, but not pancreatic somatostatin, from the isolated perfused dog pancreas. Endocr. Res. Commun. 6:37–42; 1979. 22. Jamal, H.; Bretherton–Watt, D.; Suda, K.; Ghatei, M. A.; Bloom, S. R. Islet amyloid polypeptide-like immunoreactivity (amylin) in
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rats treated with dexamethasone and streptozotocin. J. Endocrinol. 126:425–429; 1990. Kapas, L.; Obal, F., Jr.; Farkas, I.; Payne, L. C.; Sary, G.; Rubicsek, G.; Krueger, J. M. Cholecystokinin promotes sleep and reduces food intake in diabetic rats. Physiol. Behav. 50:417–420; 1991. Krahn, D. D.; Gosnell, B. A.; Levine, A. S.; Morley, J. E. Effects of calcitonin gene-related peptide on food intake. Peptides 5:861– 864; 1984. Liddle, R. A. Cholecystokinin. In: Walsh, J. H.; Dockray, G. J., Eds. Gut peptides: Biochemistry and physiology. New York: Raven Press; 1994:175–216. Lutz, T. A.; Del Prete, E.; Scharrer, E. Reduction of food intake in rats by intraperitoneal injection of low doses of amylin. Physiol. Behav. 55:891–895; 1994. Lutz, T. A.; Del Prete, E.; Scharrer, E. Subdiaphragmatic vagotomy does not influence the anorectic effect of amylin. Peptides 16:457– 462; 1995. Lutz, T. A.; Geary, N.; Szabady, M. M.; Del Prete, E.; Scharrer, E. Amylin decreases meal size in rats. Physiol. Behav. 58:1197–1202; 1995. Lutz, T. A.; Del Prete, E.; Szabady, M. M.; Scharrer, E. Attenuation of the anorectic effects of glucagon, cholecystokinin, and bombesin by the amylin receptor antagonist CGRP(8–37). Peptides 17:119– 124; 1996. McCoy, J. G.; Avery, D. D. Bombesin: Potential integrative peptide for feeding and satiety. Peptides 11:595–607; 1990. Miyazato, M.; Nakazato, M.; Shiomi, K.; Aburaya, J.; Toshimori, H.; Kangawa, K.; Matsuo, H.; Matsukura, S. Identification and characterization of islet amyloid polypeptide in mammalian gastrointestinal tract. Biochem. Biophys. Res. Commun. 181:293 – 300; 1991. Moore, C. X.; Cooper, G. J. S. Co-secretion of amylin and insulin from cultured islet beta-cells: Modulation by nutrient secretagogues, islet hormones and hypoglycemic agents. Biochem. Biophys. Res. Commun. 179:1–9; 1991. Morley, J. E.; Flood, J. F. Amylin decreases food intake in mice. Peptides 12:865–869; 1991. Morley, J. E.; Morley, P. M. K.; Flood, J. F. Anorectic effects of amylin in rats over the life span. Pharmacol. Biochem. Behav. 44:577–580; 1993.
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35. Morley, J. E.; Flood, J. F.; Horowitz, M.; Morley, P. M. K.; Walter, M. J. Modulation of food intake by peripherally administered amylin. Am. J. Physiol. 267:R178–R184; 1994. 36. Morley, J. E.; Farr, S. A.; Flood, J. F. Peripherally administered calcitonin gene-related peptide decreases food intake in mice. Peptides 17:511–516; 1996. 37. Mulder, H.; Ahren, B.; Sundler, F. Differential expression of islet amyloid polypeptide (amylin) and insulin in experimental diabetes in rodents. Mol. Cell. Endocrinol. 114:101–109; 1995. 38. Nicholl, C. G.; Bhatavdekar, J. M.; Mak, J.; Girgis, S. I.; Legon, S. Extra-pancreatic expression of the rat islet amyloid polypeptide (amylin) gene. J. Mol. Endocrinol. 9:157–163; 1992. 39. Pieber, T. R.; Stein, D. T.; Ogawa, A.; Alam, T.; Ohneda, M.; McCorkle, K.; Chen, L.; McGarry, J. D.; Unger, R. H. Amylininsulin relationships in insulin resistance with and without diabetic hyperglycemia. Am. J. Physiol. 265:E446–E453; 1993. 40. Pieber, T. R.; Roitelman, Lee, Y.; Luskey, K. L.; Stein, D. T. Direct plasma radioimmunoassay for rat amylin-(1–37): concentrations with acquired and genetic obesity. Am. J. Physiol. 267:E156–E164; 1994. 41. Sango, K.; Verdes, J. M.; Hikawa, N.; Horie, H.; Tanaka, S.; Inoue, S.; Sotelo, J. R.; Takenaka, T. Nerve growth factor (NGF) restores depletions of calcitonin gene-related peptide and substance P in sensory neurons from diabetic mice in vitro. J. Neurol. Sci. 126:1–5; 1994. 42. Thompson, C. S.; Sykes, R. M.; Muddle, J.; Dashwood, M. R. Distribution of 125I-labelled insulin-binding sites in peripheral tissues of the normal and diabetic rat: an autoradiographic study. J. Endocrinol. 128:85–89; 1991. 43. Toshimori, H.; Narita, R.; Nakazato, M.; Asai, J.; Mitsukawa, T.; Kangawa, K.; Matsuo, H.; Matsukura, S. Islet amyloid polypeptide (IAPP) in the gastrointestinal tract and pancreas of man and rat. Cell Tissue Res. 262:401–406; 1990. 44. Wimalawansa, S. J. Effects of in vivo stimulation on molecular forms of circulatory calcitonin and calcitonin gene-related peptide in man. Mol. Cell. Endocrinol. 71:13–19; 1990. 45. Zelissen, P. M. J.; Koppeschaar, H. P. F.; Lips, C. J. M.; Hackeng, W. H. L. Calcitonin gene-related peptide in human obesity. Peptides 12:861–863; 1991.
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Different Influence of CGRP(8–37), an Amylin and CGRP Antagonist, on the Anorectic Effects of Cholecystokinin and Bombesin in Diabetic and Normal Rats T. A. LUTZ,* 1 T. R. PIEBER,† B. WALZER,* E. DEL PRETE* AND E. SCHARRER* *Institute of Veterinary Physiology, University of Zuerich, Winterthurerstrasse 260, CH 8057 Zuerich, Switzerland †Department of Internal Medicine, Karl-Franzens-University Graz, Auenbruggerplatz 15, A 8036 Graz, Austria Received 18 September 1996; Accepted 2 January 1997 LUTZ, T. A., T. R. PIEBER, B. WALZER, E. DEL PRETE AND E. SCHARRER. Different influence of CGRP(8–37), an amylin and CGRP antagonist, on the anorectic effects of cholecystokinin and bombesin in diabetic and normal rats. PEPTIDES 18(5) 643–649, 1997.—Because previous studies had suggested that the anorectic effects of cholecystokinin (CCK) and bombesin (BBS) depend partly on the release of amylin or calcitonin gene-related peptide (CGRP), we investigated the influence of the amylin and CGRP receptor antagonist CGRP(8–37) on the anorectic effects of CCK and BBS in streptozotocin (STZ)-diabetic and nondiabetic rats. STZ-diabetic rats had significantly lower plasma amylin and insulin concentrations than nondiabetic control rats. Amylin (5 mg/kg or 2.5 mg/rat) injected IP at dark onset after 24-h food deprivation elicited an anorectic effect of similar extent in STZ-diabetic and control rats. Under similar conditions, CCK (0.25 and 2 mg/kg) and BBS (5 mg/kg) reduced food intake in both STZ-diabetic and nondiabetic rats. These effects were markedly attenuated by CGRP(8–37) (10 mg/kg) in nondiabetics but not in STZ-diabetic rats. It is concluded that part of the anorectic effects of CCK and BBS depend on the release of amylin from pancreatic B-cells. q 1997 Elsevier Science Inc. Amylin Cholecystokinin Bombesin Diabetic rat Plasma amylin concentration
CGRP CGRP(8–37) Radioimmunoassay
AMYLIN is released from pancreatic beta-cells in response to meal ingestion (9). Under most experimental conditions, amylin seems to be coreleased with insulin in a constant ratio [ for review, see (12)]. Amylin has a potent anorectic effect in rats and mice when administered centrally or peripherally (3,10,26–28,33–35). Abdominal vagotomy did not attenuate the anorectic effect of peripherally administered amylin (27,35). The latter may hence be mediated by amylin action at receptor sites within the central nervous system (1,4,6). Amylin has been regarded as a peripheral satiety peptide as it reduces food intake in rats at low, near physiological doses without inducing a conditioned taste aversion (26,28). Its exact role in the control of feeding behavior, however, remains to be established. We have presented evidence in a recent study (29) that amylin or CGRP may mediate part of the anorectic effects of cholecystokinin (CCK) [ for review, see (25)], bombesin (BBS) (30), and glucagon (18). Because each of the latter peptides increases insulin secretion (21,25,32), and because insulin secretion is 1
Streptozotocin
Food intake
usually associated with amylin secretion [ for review, see (12)], the attenuation of the anorectic effects of these peptides by the amylin and CGRP receptor antagonist calcitonin gene-related peptide [CGRP](8–37), which also reduces amylin’s anorectic effect (29), may be interpreted by CCK, BBS, and glucagon reducing food intake in part due to a stimulation of amylin release from pancreatic B-cells (29). An involvement of CGRP, however, could not be excluded. Amylin expression and secretion is greatly reduced in betacell deficiency, such as insulin-dependent diabetes mellitus [ for review, see (12)] or after treatment of rats with streptozotocin (STZ) (8,37), which selectively destroys pancreatic beta cells (2). STZ treatment, however, also depletes sensory neurons of CGRP (15,41). If our hypothesis, that one component of CCK’s and BBS’s anorectic effects depends on the release of amylin were true, coadministration of CGRP(8–37) with CCK or BBS would not be expected to affect the anorectic effects of the latter peptides in STZ-diabetic rats where CCK- and BBS-induced release of amylin is supposed to be low.
Requests for reprints should be addressed to T. A. Lutz.
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It was therefore the aim of the present study to compare the influence of the amylin and CGRP receptor antagonist CGRP(8– 37), which has previously been shown to block the anorectic effect of exogenous amylin (29), on the anorectic effects of CCK or BBS in STZ-diabetic rats with nondiabetic control rats. In addition, amylin’s anorectic efficacy was studied in STZ-diabetic and intact control rats. METHOD
Adult male rats (ZUR:SD; Institut fu¨r Labortierkunde, University of Zuerich, Switzerland) were used for the experiments and were adapted to the housing conditions for at least 2 weeks before treatment with STZ. Mean body weight was approximately 200 g before STZ treatment and 350 g (STZ-diabetic rats) and 600 g (nondiabetic rats), respectively, after finishing all experiments when a blood sample was taken from the aorta (see below). A separate group of 33 rats without pretreatment was used to investigate the influence of CGRP(8–37) on the anorectic effect of CGRP. Mean body weight was approximately 325 g. Rats were housed in individual wire cages in a temperaturecontrolled room (21 { 17C) and on a 12 h/12 h light/dark cycle (lights on 0800 h). Rats were fed a medium fat diet containing 18% fat, 46% carbohydrates, and 13% protein (26), because diabetes-induced hyperphagia is less pronounced when fed a fatenriched diet (16). The same diet was used in the experiment investigating the influence of CGRP(8–37) on CGRP’s anorectic effect. Rat amylin, rat CGRP, the amylin and CGRP receptor antagonist human CGRP(8–37), CCK, and BBS were obtained from Peninsula Laboratories (Belmont, CA). All peptides were dissolved in an appropriate volume of 0.9% NaCl to yield injection volumes of 1 ml/kg. Injection of 0.9% NaCl served as control. Amylin was injected at a dose of 5 mg/kg or 2.5 mg/rat, CCK was injected at a dose of 0.25 or 2 mg/kg, BBS at a dose of 5 mg/kg (see Results for details), CGRP at a dose of 5 mg/kg, and CGRP(8–37) at a dose of 10 mg/kg. In all experiments, rats were injected IP at dark onset (2000 h) after 24-h food deprivation. Water was available ad lib throughout the study. Food intake was measured by weighing the feeding cups ( {0.1 g) after correcting for spillage. Depending on the respective experiment, STZ-diabetic and nondiabetic rats were divided into two or three groups based on their body weight and basal food intake measured on the day before the experiment. Experiments were performed in a counterbalanced design with each animal tested under all conditions. The interval between single trials of an experiment was 5 days. Similarly, three groups were formed for the experiment investigating the influence of CGRP(8–37) on CGRP’s anorectic effect. This experiment was performed in a single trial. STZ Treatment In 18 rats, diabetes was induced by an IP injection of STZ (50 mg/kg; Sigma Chemical, Co., St. Louis, MO) after an overnight fast (17). Fasting was continued for about 3 h after the injection. STZ was dissolved in ice-cold citrate buffer (Na3-citrate, 75 mmol/l, pH 4.5; injection volume 2 ml/kg) that was kept on ice during the injection procedure, and all rats were injected within 5 min after STZ preparation (7). Sixteen rats were treated with citrate buffer vehicle only to serve as nondiabetic controls. Experiments began approximately 4 weeks after STZ treatment when STZ-diabetic rats had attained a mean body weight
of approximately 280 g and nondiabetic rats of approximately 400 g. During this period, rats were closely monitored daily but left untreated otherwise. Verification of Diabetic State Ten days after STZ treatment, a blood sample was taken from the retrobulbar ophthalmic venous plexus and blood glucose concentration determined by the glucose oxidase method on a COBAS MIRA automatic analyser (Hoffmann–La Roche, Basle, Switzerland). Only undeprived rats with a blood glucose concentration above 18 mmol/l were considered diabetic and hence used in the experiments. Blood glucose concentration was also determined in nondiabetic citrate-treated control rats. About 5 months after STZ treatment and after finishing all experiments, the diabetic state of STZ-treated rats was again assessed to test if rats had to be excluded retrospectively from analysis. Only rats confirmed to be diabetic in this second assessment were included in the analyses. Rats were food deprived for 24 h prior to the test. They were then offered their usual diet at dark onset for 30 min. Then rats were etherized and, after laparotomy, a blood sample was taken from the aorta. Blood was immediately transferred into chilled fluoride oxalate (determination of plasma glucose concentration) or EDTA blood tubes (determination of amylin, and insulin by radioimmunoassay [RIA; see below]), free fatty acids, triglycerides, and b-OH-butyrate). Plasma was separated by centrifugation and stored at 0207C until analysis. Aprotinin (500 kallikrein inactivator units/ml; Bayer, Leverkusen, Germany) was added to plasma for RIA and stored at 0707C until analysis. Plasma concentrations of glucose, free fatty acids (NEFA C; Wako Chemicals, Neuss, Germany), triglycerides (Triglyceride PAP; Hoffmann–La Roche), and b-OH-butyrate ( b-HBA; Sigma) were determined on an automatic COBAS MIRA analyzer. Radioimmunoassay for Amylin and Insulin Plasma concentrations of amylin and insulin were determined by radioimmunoassay (RIA). Plasma amylin concentration was determined as described earlier (39,40). Briefly, primary antibody T-486–6 (rabbit anti-amylin) was diluted with assay buffer [Na2HPO4 50 mmol/l (pH 7.2), aprotinin 1%, EDTA 0.25%, Triton X-100 0.2%, fish gelatin 0.1%, Na azide 0.02%; all reagents from Sigma] to a final dilution of 1:50,000. Rat amylin (Peninsula Laboratories) dissolved in assay buffer was used as standard. Sample or standards were incubated with antibody at 47C for 72 h. Then amylin tracer ([ 125I]amylin, 9,000–10,000 cpm; Amersham, Little Chalfont, UK) was added and samples were again incubated at 47C for an additional 96 h. Bound amylin was separated from free by a polyethylene-glycol supported second antibody (goat anti-rabbit IgG; Sigma) precipitation method. The detection limit of the assay was below 2 pmol/l, intra- and interassay coefficients of variation were below 5%. Plasma insulin concentration was determined with a commercial RIA kit (rat insulin RIA kit, Linco Research Inc., St. Charles, MO). Statistics Results are presented as mean { SEM. The treatment groups of STZ-diabetic and nondiabetic control rats were compared using paired Student’s t-test or repeated-measures ANOVA with the Student Newman–Keuls post hoc test where appropriate. The treatment groups of rats without pretreatment were compared using one-way ANOVA with the Student Newman–Keuls post hoc
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TABLE 1 POSTPRANDIAL PLASMA CONCENTRATION OF GLUCOSE, AMYLIN, INSULIN, FREE FATTY ACIDS, TRIGLYCERIDES, AND b-OHBUTYRATE IN STREPTOZOTOCIN (STZ)-DIABETIC AND NONDIABETIC RATS FED FOR 30 min AFTER 24-h FOOD DEPRIVATION Plasma Concentration Nondiabetic Rats (n Å 15)
Glucose (mmol/l) Amylin (pmol/l) Insulin (pmol/l) Amylin/insulin ratio Free fatty acids (mmol/l) Triglycerides (mmol/l) b-OH-butyrate (mmol/l)
8.5 { 0.2 27.1 { 2.5 605.3 { 73.7 (n Å 14) 0.049 { 0.007 (n Å 14) 0.50 { 0.03 0.8 { 0.1 193 { 9
STZ-Diabetic Rats (n Å 10)
30.8 { 2.1† 14.3 { 2.2* 161.8 { 26.4† 0.103 { 0.018* 1.01 { 0.16† 4.1 { 0.7† 2055 { 649*
Plasma taken in anesthetized rats from the aorta. Fluoride oxalate plasma for glucose; EDTA plasma for amylin, insulin, free fatty acids, triglycerides, and b-OH-butyrate. *† Significantly different from nondiabetic rats (unpaired Student’s t-test; * p õ 0.01, † p õ 0.001, respectively).
test. Differences in plasma hormone concentrations and concentrations of plasma metabolites between STZ-diabetic and nondiabetic rats were statistically evaluated using the unpaired Student’s t-test. A value of p õ 0.05 was considered significant. RESULTS
Verification of Diabetic State; Characterization of Diabetic Rats Based on the blood glucose concentration determined 10 days after STZ treatment, induction of the diabetic state was successful in 11 rats [mean blood glucose 30.2 { 2.2 (18.5–40.2) mmol/ l]. These 11 rats were used in the experiments as diabetic rats. Nondiabetic control rats had a mean blood glucose concentration of 6.3 { 0.3 (4.4–8.9) mmol/l. Ten of the 11 diabetic rats exhibited marked polyuria and polydipsia, and body weight gain was markedly lower in STZdiabetic rats than in nondiabetic controls [body weight on the day of STZ treatment: 202 { 3 g in STZ-diabetic rats vs. 200 { 3 in nondiabetic controls; 4 weeks after STZ treatment: 276 { 12 g vs. 396 { 9 ( p õ 0.001); 5 months after STZ treatment: 315 { 27 g vs. 598 { 13 (p õ 0.001)]. Food intake was not measured daily, but whenever it was measured, STZ-diabetics consumed more food than nondiabetic rats [e.g., 4 weeks after STZ treatment: 12-h dark phase intake 23.3 { 1.4 g in STZdiabetics vs. 20.2 { 0.5 in nondiabetics ( p õ 0.05); 12-h light phase intake 5.0 { 0.7 g vs. 2.8 { 0.5 ( p õ 0.05)]. The diabetic state of the STZ-diabetic rats was reexamined after the termination of experiments 5 months after STZ injection (see Table 1). Based on the postprandial plasma glucose level, one rat had to be excluded from the diabetic group as it no longer fulfilled the criteria for diabetic hyperglycemia (plasma glucose level 9.4 mmol/l). This rat was excluded from the study, although its blood glucose concentration at first assessment was 20.1 mmol/l. Compared to other diabetic rats, polydipsia was not striking, and body weight in this rat was approximately 660 g 5 months after STZ treatment.
FIG. 1. Influence of amylin (5 mg/kg) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic (n Å 10) or nondiabetic (n Å 16) rats. *,**Significant difference compared with respective saline control (paired Student’s t-test; *p õ 0.05; **p õ 0.01).
Postprandial plasma glucose concentration was markedly elevated in the remaining 10 STZ-diabetic rats compared to nondiabetic controls (Table 1) and only these 10 rats were included in all analyses. STZ-diabetic rats had significantly lower postprandial plasma amylin and insulin concentrations than nondiabetic rats. However, insulin concentration was relatively more depressed than amylin concentrations, which resulted in an increased amylin/insulin ratio in STZ-diabetic rats (Table 1). Levels of free fatty acids, triglycerides, and b-OH-butyrate were markedly elevated in STZ-diabetic rats. Anorectic Effect of Amylin in STZ-Diabetic and Nondiabetic Rats Amylin (5 mg/kg) injected at dark onset after 24-h food deprivation significantly reduced cumulative food intake compared to saline controls for 30 min in STZ-diabetic and for 2 h in nondiabetic rats (Fig. 1). The relative reduction in food intake 30 min after injection was similar in STZ-diabetic (18%) and nondiabetic rats (19%). Similar results were obtained when amylin was injected at a dose of 2.5 mg/rat (Fig. 2).
FIG. 2. Influence of amylin (2.5 mg/rat) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic (n Å 10) or nondiabetic (n Å 15) rats. *,** Significant difference compared with respective saline control (paired Student’s t-test; *p õ 0.05; **p õ 0.01).
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FIG. 3. Influence of CCK (0.25 mg/kg) or CCK (0.25 mg/kg) / CGRP(8–37) (10 mg/kg) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic ( n Å 10) or nondiabetic (n Å 16) rats compared with respective saline control. *,**Significant difference between groups (repeated-measures ANOVA with Student Newman–Keuls post hoc test; *p õ 0.05; ** p õ 0.01).
FIG. 5. Influence of BBS (5 mg/kg) or BBS (5 mg/kg) / CGRP(8– 37) (10 mg/kg) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic (n Å 10) or nondiabetic (n Å 16) rats compared with respective saline control. **,***Significant difference between groups (repeated-measures ANOVA with Student Newman–Keuls post hoc test; **p õ 0.01; *** p õ 0.001; a,b,c values with different letters differ significantly, p õ 0.05).
Following 24-h food deprivation, STZ-diabetic rats did not eat more than nondiabetic controls during the first 2 h after injection (Figs. 1–5).
attenuated at 1 h. In STZ-diabetic rats, however, CGRP(8–37) did not influence CCK’s anorectic effect (Fig. 4).
Influence of CGRP(8–37) on CCK’s Anorectic Effect in STZDiabetic and Nondiabetic Rats
Influence of CGRP(8–37) on BBS’s Anorectic Effect in STZDiabetic and Nondiabetic Rats
CCK (0.25 mg/kg) significantly reduced cumulative food intake in 24-h food deprived nondiabetic rats for 1 h after injection compared to saline-treated controls (Fig. 3). This effect was totally antagonized by the simultaneous administration of the amylin receptor antagonist CGRP(8–37) (10 mg/kg). CCK only nonsignificantly reduced cumulative food intake in STZ-diabetic rats (Fig. 3). At a higher dose, CCK (2 mg/kg) significantly reduced cumulative food intake in both STZ-diabetic and nondiabetic rats compared to their respective controls (Fig. 4). The relative reduction in food intake was similar in both groups. The anorectic effect of CCK was almost completely reversed by CGRP(8–37) 30 min after injection in nondiabetic rats and
After the injection of BBS (5 mg/kg), cumulative food intake was significantly reduced for 2 h in both 24-h food-deprived STZ-diabetic and nondiabetic rats (Fig. 5). BBS’s anorectic effect was significantly reduced by about 50% by CGRP(8–37) in nondiabetic rats, whereas CGRP(8–37) had a minor, nonsignificant impact on BBS’s anorectic effect in STZ-diabetic rats (Fig. 5).
FIG. 4. Influence of CCK (2 mg/kg) or CCK (2 mg/kg) / CGRP(8– 37) (10 mg/kg) injected IP at dark onset on cumulative food intake in 24-h food-deprived streptozotocin-diabetic (n Å 10) or nondiabetic (n Å 16) rats compared with respective saline control. *,**,***Significant difference between groups (repeated-measures ANOVA with Student Newman–Keuls post hoc test; *p õ 0.05; ** p õ 0.01; ***p õ 0.001; a,b values with different letters differ significantly, p õ 0.05).
Influence of CGRP(8–37) on CGRP’s Anorectic Effect in Rats IP injection of CGRP(5 mg/kg) significantly reduced cumulative food intake for 1 h in 24-h food-deprived normal rats (Fig. 6).
FIG. 6. Influence of CGRP(5 mg/kg; n Å 11) or CGRP(5 mg/kg) / CGRP(8–37) (10 mg/kg; n Å 11) injected IP at dark onset on cumulative food intake in 24-h food-deprived rats compared with saline control (n Å 11). *,**Significant difference between groups (ANOVA with Student Newman–Keuls post hoc test; *p õ 0.05; ** p õ 0.01; a,b values with different letters differ significantly, p õ 0.05).
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ANORECTIC EFFECT OF AMYLIN, CCK, AND BBS IN DIABETIC RATS Coadministration of CGRP(8–37) (10 mg/kg) completely abolished CGRP’s anorectic effect. DISCUSSION
The main finding of this study, that the amylin and CGRP receptor antagonist CGRP(8–37) did not influence the anorectic effect of CCK or BBS in STZ-diabetic rats whereas it markedly attenuated this effect in nondiabetic controls, is consistent with the hypothesis that part of the anorectic effects of CCK and BBS in nondiabetic rats are mediated by amylin release. The finding that CGRP(8–37) attenuated the anorectic effects of CCK and BBS in nondiabetic rats confirms our previous study (29). In the first two experiments, we have shown that exogenous amylin reduces food intake in both STZ-diabetic and nondiabetic rats to a similar degree. However, a comparative complete dose– response study was not performed. That STZ-diabetic rats in our study were not completely depleted of amylin may explain why upregulation of amylin receptors, which presumably parallels upregulation of insulin receptors in STZ-diabetic rats (42), was probably not sufficient to cause hyperresponsiveness of STZ-diabetic rats to amylin. Morley and Flood also reported that amylin reduces food intake similarly in STZ-diabetic and nondiabetic mice food deprived overnight (33). The anorectic effect observed in nondiabetic rats confirms previous studies in that IP injections of amylin effectively reduce food intake in food-deprived rats (26–29). The second experiment with amylin (2.5 mg/rat) was performed because the difference in body weight between STZ-diabetic and nondiabetic rats is probably mainly due to a difference in adipose tissue mass rather than lean body mass. Dosage of amylin per rat rather than relative to rats’ body weight may therefore be more appropriate because amylin is probably mainly distributed in the extracellular water space, as has recently been shown for an amylin peptide analogue (11). Greatly reduced amylin expression and secretion in beta-cell deficiency after STZ treatment (20,37) are reflected in a decreased plasma amylin concentration as observed in the present study. The component of CCK’s and BBS’s anorectic effect that could be attenuated by simultaneous injection of the amylin and CGRP receptor antagonist CGRP(8–37) in a previous (29) and the present study in nondiabetic rats is supposed to be caused by the stimulatory effect of CCK and BBS on beta-cell secretion. Both CCK and BBS increase insulin secretion (21,25), and because amylin and insulin are usually cosecreted (12), we presume that CCK and BBS have a similar stimulating effect on amylin as on insulin secretion. Amylin may therefore be responsible for part of CCK’s and BBS’s effects in nondiabetic rats. The role of endogenous amylin in the control of food intake, however, remains to be clarified in further studies. It has to be kept in mind that CGRP(8–37) has antagonistic effects on receptors for both amylin and CGRP, which is structurally closely related to amylin (12). Plasma concentration of CGRP, which is an important neurotransmitter in the central and peripheral nervous system, also increases in response to food intake (44,45), and injection of CGRP has been shown to reduce food intake in rats and mice (24,36). We have confirmed CGRP’s anorectic action after peripheral administration in rats in the present study even at a much lower dose than reported previously (36). Similar to amylin’s anorectic effect (29), CGRP’s anorectic effect was blocked by coadministration of CGRP(8–37). Because recent studies have shown that STZ-treatment depletes certain sensory neurons of CGRP (15,41), and because the anorectic effects of both CCK and BBS seem to be at least
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in part nervally transmitted to the central nervous system (25,30), the lacking effect of CGRP(8–37) to attenuate CCK’s and BBS’s anorectic effects in STZ-diabetic rats could be explained by CGRP depletion in sensory neurons rather than amylin depletion in pancreatic B-cells. This alternative interpretation, however, appears to be rather unlikely considering our preliminary findings where we have shown that IP injected CCK (2 mg/ kg) had no effect on plasma CGRP levels compared with vehicle in 24-h food-deprived rats 30 min (20.9 { 4.6 pmol/l vs. 19.7 { 3.5 pmol/l) or 60 min (25.6 { 5.2 pmol/l vs. 19.6 { 3.5 pmol/l; n Å 8 in each group) after injection and refeeding. CGRP concentrations were similar to postprandial concentrations observed previously in humans (44). The amylin-dependent components of CCK’s and BBS’s anorectic effects presumably are of minor importance in STZ-diabetic rats because the secretory capacity of beta-cells for amylin is greatly reduced after STZ pretreatment (8,37). Our study confirms the expectation that CGRP(8–37) does not influence CCK’s and BBS’s anorectic effects under these conditions. Because CGRP(8–37) attenuated the anorectic effects of amylin, CCK, BBS, glucagon (29), and CGRP(present study), it could be argued that CGRP(8–37) acts as an unspecific antagonist at peptide receptors. Because CGRP(8–37), however, failed to reduce vasopressin’s anorectic effect in rats (29), we suggested in our previous study (29) that CGRP(8–37) is unlikely to attenuate CCK’s and BBS’s anorectic effects by directly blocking their respective receptors. This was confirmed in the present study because if the former were true, CGRP(8–37) would have been expected to reduce CCK’s and BBS’s effects in STZ-diabetic rats. The anorectic effect of BBS appeared to be somewhat stronger in nondiabetic than in STZ-diabetic rats, although this difference did not reach the level of significance [multifactor analysis of variance p Ç 0.29 for interaction between BBS injection and diabetic state (30 min after injection), p Ç 0.20 (1 h after injection)]. This may point, however, to the reduced amylin-depending component of BBS’s effect in the STZ-diabetic rats. The similar potency of the higher dose of CCK (2 mg/kg) to reduce food intake in both groups was somewhat surprising because of the diminished contribution of amylin to CCK’s effect in STZ-diabetic rats. The reason for this is unknown at present but the lacking component of CCK’s effect may have been compensated by other as yet unidentified factors in the present study. In another study (23), however, the anorectic effect of CCK (10 mg/kg IP) appeared to be moderately stronger in nondiabetic (54% reduction of food intake) than in STZ-diabetic rats (38% reduction). The authors, however, did not state if this difference was significant, and baseline food intake was significantly higher in STZ-diabetic rats, which impedes comparison of the data. Compared to previous studies (26–29), a higher amylin dose (5 mg/kg) was necessary to elicit a similar degree of anorexia as observed previously with a lower dose (1 mg/kg). Use of a different strain of rats (ZUR:SD in this study; ZUR:SIV in previous studies), although both being Sprague–Dawley rats, may explain this difference. Preliminary experiments have shown that amylin at a dose of 1 or 3 mg/kg inconsistently reduced food intake in ZUR:SD rats. Similar observations were made with CGRP because CGRP at a dose of 1 mg/kg significantly reduced food intake in ZUR:SIV rats while leaving food intake unaffected in ZUR:SD rats (unpublished). This strain of rats (ZUR:SD) may have a lower sensitivity to anorectic peptides in general, because similar to amylin and CGRP, a higher dose of CCK (2 mg/kg) was necessary to elicit an anorectic effect of similar strength compared to previous studies (0.25 mg/kg) (29). The lower sensitivity to some satiating
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peptides may also explain the higher growth rates in ZUR:SD compared to ZUR:SIV rats kept under similar conditions (e.g., body weight in ZUR:SIV rats at 10 weeks of age: about 280 g; ZUR:SD rats: about 340 g). Verification of Diabetic State Successful induction of the diabetic state with STZ was verified before and after the experiments and only those rats that fulfilled the criteria of hyperglycemia on both occasions were included in further analyses. STZ-diabetic rats exhibited a higher food intake than nondiabetic controls under free fed conditions. That this was not very pronounced under the present experimental conditions may have been due to the use of a fat-enriched diet, which attenuates diabetic hyperphagia compared with high-carbohydrate diets (16). It has to be kept in mind, however, that hyperphagia (g per kg body weight) was more marked if the lower body weight of STZdiabetic rats is taken into account. Clinical findings and parameters of carbohydrate and lipid metabolism confirmed the classification into diabetic or nondiabetic. The finding of an increased amylin/insulin ratio in STZ-diabetic rats despite a significant reduction in both plasma amylin and insulin agrees with numerous previous studies that showed a dissociation of amylin and insulin expression and release in spontaneously diabetic and STZ-diabetic rats (8,19,20,22,37). This seems to be directly correlated to the development of hyperglycemia
(39). The smaller reduction in plasma amylin than insulin concentration may also be due to production of amylin in pancreatic delta cells (14) or in extrapancreatic production sites, such as in the gastrointestinal mucosa (13,31,38,43). Expression of insulin, however, has also been described in the duodenal wall (5). Because CGRP leakage from sensory neurons is the main source of circulating CGRP, plasma CGRP concentrations may be lower in STZ-diabetic than in nondiabetic rats due to CGRP depletion in sensory neurons after STZ treatment (15,41,44). However, these have not been measured in diabetic rats in the present study. In conclusion, the present study has shown that the amylin and CGRP receptor antagonist CGRP(8–37), which antagonizes anorexia induced by amylin and CGRP, attenuated the anorectic effects to IP injected CCK and BBS in nondiabetic rats whereas it was ineffective in STZ-diabetic rats. Part of the anorectic effects of CCK and BBS in nondiabetic rats may therefore be mediated by the release of amylin from the pancreas. Due to amylin depletion in the pancreas of STZ-diabetic rats, the amylindependent component of CCK’s and BBS’s anorectic effect appears to be absent after STZ treatment. An involvement of blockade of CGRP receptors by CGRP(8–37), however, cannot be ruled out at present. ACKNOWLEDGEMENT
We gratefully acknowledge Barbara Schneider for her help with laboratory analyses. This work was supported by the Swiss National Foundation (Grant #3100-045583.95/1).
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