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Long-Term Effects of CCK-Agonist and -Antagonist on Food Intake and Body Weight in Zucker Lean and Obese Rats
Peptides, Vol. 19, No. 2, pp. 291–299, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(97)00261-1
Long-Term Effects of CCK-Agonist and -Antagonist on Food Intake and Body Weight in Zucker Lean and Obese Rats KERSTIN MEEREIS-SCHWANKE,* HANNE KLONOWSKI-STUMPE,* LIESELOTTE HERBERG† AND CLAUS NIEDERAU*1 *Department of Medicine, Division of Gastroenterology, Hepatology and Infectious Diseases, and †Diabetes-Research Institute, Heinrich-Heine-University, Du¨sseldorf, Germany Received 2 April 1997; Accepted 18 August 1997 MEEREIS-SCHWANKE, K., H. KLONOWSKI-STUMPE, L. HERBERG AND C. NIEDERAU. Long-term effects of CCK-agonist and -antagonist on food intake and body weight in Zucker lean and obese rats. PEPTIDES 19(2) 291–299, 1998.—The present study evaluates long-term effects of the CCK-agonist caerulein and the CCK-A antagonist loxiglumide in obese and lean Zucker rats. Caerulein and loxiglumide altered food intake neither in obese nor in lean rats. By as yet unknown mechanisms, however, weight increase was accelerated by loxiglumide and reduced by caerulein in obese and lean rats. Caerulein increased pancreatic weight only in lean but not in obese rats. Thus, obese rats show a resistance of pancreatic CCK-A receptors. The failure of CCK-agonist and -antagonist to alter food intake suggests that this CCK-resistance is not responsible for obesity in the genetically altered rats. © 1998 Elsevier Science Inc. CCK CCK-receptor CCK-antagonists Caerulein Food intake Pancreatic growth Zucker rat Satiety
Loxiglumide
Gastrin
Leptin
intake in several species including humans (22,25,53). Nevertheless, CCK has been suggested to be involved in mediating satiety after a meal. The mechanisms involved in regulation of satiety by CCK, however, remain unsettled. Recent studies have suggested that effects of peripheral CCK on satiety—like other effects of CCK (17,18,45,54)—are mediated via CCK receptors on vagal afferent neurons (9,12,54,55,57). Genetically obese Zucker rats have a higher food intake when compared to their lean litter mates (10,27,29); this increase in food intake contributes to massive accumulation of adipose tissue (10,27,29). Little is known as yet about the mechanisms by which the obesity develops in the genetically altered Zucker rats. Recent studies have indicated that a defect in the leptin receptor may be responsible for the phenotype of the genetically obese Zucker rat (13,14,60). It has also been suggested that obese Zucker rats are less
THE peptide cholecystokinin (CCK) acts both as a peripheral hormone via CCK-A receptors and as a neurotransmitter within the brain and the enteric nervous system via CCK-B and CCK-A receptors (64). The CCK-A and -B receptors have been sequenced and are distinctly different whereas the gastrin receptor is identical with the CCK-B receptor (for review see [61]). In short-term experiments peripheral administration of CCK-A agonists inhibits food intake in various species including humans (for review see [22,47,50]); CCK in general refers to its sulfated form in the following text). Since food intake releases CCK from intestinal endocrine cells into the circulation (64), CCK may mediate postprandial satiety. Some studies have shown that peripheral administration of CCK-A antagonists increased food intake in several species (3,6,7,62,65). Other studies, however, failed to show an effect of CCK antagonists on food 1
Requests for reprints should be addressed to Prof. Dr. Claus Niederau, Department of Medicine, Division of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University Du¨sseldorf, Moorenstr. 5, D-40225 Du¨sseldorf, Germany. E-mail: [email protected]
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sensitive to the satiety effect of cholecystokinin (CCK) on meal size (26,28). Thus, CCK resistance might be a pathogenic factor for the development of obesity in these animals. We have recently corroborated that genetically obese Zucker rats are resistant to the satiety effect of CCK even at near physiological doses which result in plasma concentrations of CCK slightly above those seen after a meal (43). However, in the same studies the blockade of endogenous CCK released after a meal due to the administration of a CCK-A-receptor antagonist failed to alter the food intake in both lean and obese rats (43). Thus it was still unclear from the short-term studies whether CCK is involved in the long-term increase in body weight of growing rats and in particular in the development of obesity in genetically obese Zucker rats. The present studies evaluate whether a longterm administration of a CCK-agonist and CCK-antagonist alters food intake and body weight in obese and lean Zucker rats. METHOD Animals Genetically obese (fa/fa) male or female Zucker rats and their age-matched lean controls (FA/FA) were bred in the animal laboratories of the Diabetes-Research Institute at the Heinrich-Heine-University of Du¨sseldorf. Each animal was housed in a separate cage (Ebeco, Becker, Castrop-Rauxel, FRG) under the following standardized conditions: 12 h light/dark cycles with lights on at 0600 h; 25 6 2°C (mean 6 SD) room temperature; 55 6 5% air humidity. Animals were fed a standard rat diet (ssniff R, Soest, FRG). The daily food intake was recorded every morning at 0800 h. Manipulations and injections were done thereafter (0800 – 0900 h). Prior to the experiments, in which pancreatic weight, protein content, and DNA content, and fat cell volumes were measured, animals were fasted for 12 h with free access to drinking water. Experimental Design At the age of four weeks a mini-pump (Alzet model 2002, flow rate 0.5 ml/h; Savo, Kissleg, FRG) was surgically implanted in the subcutaneous fat of the neck region of obese and lean Zucker rats. The animals were allowed to recover for one week. Thus, the experiments were started at the age of five weeks and stopped at the age of 10 weeks; i.e. agents were given for a total period of five weeks. Caerulein (Takus®, Pharmacia, Erlangen, FRG) was given through the mini-pump at a dose of 4 mg/kg and the CCKantagonist loxiglumide (CR1505; D,L,-4-[3,4-dichlorobenzoylamino]-5-[N-3-methoxy-propyl-pentylamino]-5oxo-pentanoic acid, Rotta Research Laboratorium, Milano, Italy) was given at a dose of 5 mg/kg. In addition to the continuous administration of the CCK-agonist and antagonist the substances were also applied subcutaneously at a
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dose of 4 mg/kg and 5 mg/kg as a bolus injection at 0900 h. The subcutaneous bolus injection of caerulein may resemble peaks in plasma CCK bioactivity as seen after a large food intake. As shown previously the administration of 4 mg/kg caerulein as a single subcutaneous injection increases CCK bioactivity levels to values similar to what is seen after a meal (results not shown in detail here). Food intake was quantitatively assessed on a daily basis. Body weight was assessed at weekly intervals. Studies on blood samples, pancreatic tissue and adipose tissue were done at the end of the experiments (at week 10). Measurements of Plasma CCK by Bioassay Plasma CCK was measured by a specific and sensitive bioassay, as described previously in detail (20,21). The assay is based on the ability of CCK to stimulate amylase release from isolated rat pancreatic acini. Plasma was extracted and concentrated by adsorption onto octa-decylsilylsilica (C-18-Sep-Pak, Waters Associates, Milford, MA) cartridges. CCK was then eluted with 1 ml 80% ethanol– 0.2% trifluoroacetic acid, and the extracts were dried under nitrogen. Plasma extracts were incubated with isolated rat pancreatic acini for 30 min at 37°C, and amylase released into the incubation medium was assayed using the Phadebas amylase assay. Amylase release was compared with a doseresponse curve for CCK-8 (BioTrend, Ko¨ln, FRG). Plasma concentrations of CCK were not measurable in experiments in which loxiglumide was infused because even residual amounts of the CCK-receptor antagonist in the plasma samples interfere with the CCK-bioassay which is based on the ability of CCK to stimulate amylase release from isolated acini (20,21). The use of a radioimmunoassay of CCK, however, does also cause methodological problems in this situation. CCK-immunoreactivity is markedly elevated after infusion of a CCK-antagonist when compared with the control experiment in which saline is given instead of a CCK-antagonist (literature in [35]). The reason for this increase in CCK-immunoreactivity after administration of CCK-antagonists is unknown. Fat Cell Preparation Subcutaneous fat from the peritoneum was prepared after a mean laparotomy. Thereafter, the gonadal fat tissue was prepared from uterus and epididymis. Fat tissue was cleaned from connective tissue, weighed, and cut in small pieces with scissors. Fat cells were isolated by previously published methods (48,49). In brief, 100 mg fat tissue was put in a 3 ml KBR buffer solution containing 5 mg collagenase and incubated in a shaking water bath at 37°C for 50 min. Methylene blue was used for further identification and isolation of fat cells. An aliquot of the final supernatant was given on siliconiced glass, and photographs were taken to document the number of fat cells per mm3.
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Measurement of Pancreatic Weight, Protein Content and DNA Content The pancreas was prepared and cleaned from surrounding fat and connective tissue as previously reported (34,37). The whole pancreas was weighed and a 50 mg piece was used to measure the concentration of protein and DNA. Protein concentration was measured by the Lowry method (16) and DNA was measured using fluorescent Hoechst reagent 33258 (23). Statistical Analysis Differences between the experiments with infusion of NaCl versus loxiglumide were analyzed for statistical significance by analysis of variance using Tukey’s method (1). Analyses of multiple comparisons were done using Bonferroni– Holm’s correction (11). In the case of time-dependent changes in calorie intake and increase in body weight values for different experimental groups were compared by calculating the areas under the curve with the initial value subtracted from the following values which were assessed at weekly intervals. RESULTS The caerulein infusion via the mini-pump at 4 mg/kg resulted in plasma CCK bioactivity which markedly exceeded fasted values (p , 0.01 by analysis of variance) and still slightly exceeded fed values (p 5 0.05) (Fig. 1). The subcutaneous bolus injection of 4 mg/kg caerulein in additional to the mini-pump infusion resulted in plasma CCK values (30 min after the injection) which more clearly exceeded fed values (p , 0.01) and also slightly exceeded the values seen after CCK was only infused via the mini-pump (p , 0.05) (Fig. 1). CCK values were similar for lean and obese rats for all the experimental conditions indicated (p . 0.1) (Fig. 1). Administration of the CCK-agonist caerulein and the CCK-antagonist loxiglumide altered the food intake neither in obese nor in lean Zucker rats when compared to the saline control (Fig. 2). Data were virtually identical when analyzed separately for male vs. female animals (p . 0.2, data not shown in detail). As expected, the food intake was markedly increased over the whole time period in the genetically obese Zucker rats when compared to their lean littermates (Fig. 2). Correspondingly there was a marked accumulation of adipose tissue in the obese Zucker rats both at the gonadal and subcutaneous site (Fig. 3). The amount of adipose tissue was affected neither by the CCK-agonist nor by the CCK-antagonist when compared to the saline control both in females and in males as well as in obese and in lean rats (Fig. 3). The effects of the CCK agonist and antagonist on intake of calories and on body weight were similar for both gender; therefore the results are shown for the total number of both male and female animals. In contrast to the lack of an effect of CCK-agonist and antagonist on food intake, the increase in body weight was
FIG. 1. Plasma CCK concentrations as measured by the bioassay are given in pM from 10 animals for each separate condition as indicated on the X axis. Results are shown as mean 6 SEM for lean rats (open bars) and for obese rats (filled bars). When compared with the fed conditions the caerulein infusion via the minipump (at 4 mg/kg) only slightly increased plasma CCK bioactivity above fed values (p 5 0.05 by analysis of variance) whereas the additional subcutaneous bolus caerulein injection (at 4 mg/kg) resulted in plasma CCK values (30 min after the injection) which more clearly exceeded fed values (p , 0.01) and also slightly exceeded the values seen after CCK was only infused via the mini-pump (p , 0.05). CCK values were similar for lean and obese rats for all the experimental conditions indicated (p . 0.1).
slightly but significantly increased by the CCK-antagonist both in obese and lean rats (Fig. 4). In the obese rats the CCK agonist caerulein likewise significantly slowed the increase in body weight. In lean rats caerulein only slightly reduced the increase in body weight (p 5 0.05 when compared with the saline control) (Fig. 4). As expected the increase in body weight was markedly faster in obese vs. lean Zucker rats (p , 0.01) (Fig. 4). The long-term administration of the CCK-agonist caerulein significantly increased pancreatic weight, protein and DNA content of lean Zucker rats of both gender (p , 0.01) (pancreatic weights shown in Fig. 5). Thus caerulein induced both hypertrophy and hyperplasia under these circumstances. Since changes in pancreatic weights under all circumstances were closely related to changes in protein and DNA content, only data on weights are shown in detail. In contrast the same dose of caerulein failed to alter the pancreatic weight in obese rats (p . 0.2). In both obese and lean rats the CCK antagonist loxiglumide failed to alter the pancreatic weight (Fig. 5) and contents of protein and DNA after long-term administration (p . 0.2, respectively). DISCUSSION The present results corroborate that obese Zucker rats are resistant to the effects of CCK at the peripheral (e.g. pancreatic) CCK-A type receptor also after a long-term treatment over a period of several weeks. A near physiological
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FIG. 2. Food intake assessed as daily uptake of kJ (Y-axis) in obese animals left part and in lean litter mates right part during the study period as indicated on the X-axis. Animals which received NaCl are indicated by the black rectangles and closed lines, animals which received caerulein are indicated by the open triangles and broken lines, and animals which received the antagonist loxiglumide are indicated by the open rectangles and dotted lines. Agents were given between weeks five and ten (for further details see Methods). The obese animals showed markedly increased food intake from week five to ten when compared to the lean littermates (areas under the curve significantly different in saline, caerulein and loxiglumide groups with p , 0.01, respectively). However, in both lean and obese rats neither caerulein nor loxiglumide altered the food intake when compared to the saline control (p . 0.1 for comparisons of areas under the curve).
intake in obese and lean Zucker rats are due to differences in binding and action at the CCK-A receptor (26,28,43). Such a resistance of the satiety effect of CCK in obese rats might contribute to the larger meal size and thus the development of obesity in these genetically altered animals. The present results, however, show that this is not true on a long-term basis. Neither the CCK agonist nor the CCK antagonist altered the food intake in lean and obese rats over the five week period. The lack of the effect of CCK on food intake in the long-term experiments might be explained by a loss of the CCK resistance during the growth of the animals and/or by other factors which contribute to the increase in food intake to a greater part than CCK. The current experiments show that there was still a CCK-resistance at the pancreatic CCK-A type receptor in the longterm experiments. The recently characterized mutations in the leptin receptor of genetically obese Zucker rats most likely cause or contribute to the alterations of food intake and satiety in these animals (13,14,60). In any case, on a long-term basis CCK is not an essential and probably not even an important factor of controlling satiety in the rat. A previous study had shown that the CCK-A antagonist devazepide (also termed MK-329) increased total food intake over a period of 23 h—and in particular increased the average meal size and duration—in male obese Zucker rats whereas no effect was seen in male lean rats (58). The latter results were interpreted as evidence that the physiological
dose of the CCK-agonist caerulein increased pancreatic weight as well as DNA and protein contents after a longterm administration in lean rats whereas the same dose (with the same circulating concentrations of the peptide) failed to show any effect on pancreatic growth in obese animals. Thus there is still a CCK-resistance at the peripheral receptor after long-term treatment with a CCK agonist. Very similar to the short-term studies (43), however, the administration of a CCK-antagonist, which completely blocks all the actions of endogenous CCK released after a meal, failed to decrease pancreatic weight. Thus although near physiological increases in plasma CCK due to exogenous CCK increased pancreatic growth, the blockade of endogenous CCK did not have any effects on pancreatic weight. These results corroborate our own previous long-term studies in the mouse which also suggested that CCK is not an essential growth factor for the pancreas in this species (37). Recent short-term studies showed that obese Zucker rats are resistant to the satiety effect of near physiological concentrations of the CCK agonist caerulein (i.e. doses which result in circulating CCK bioactivity slightly above postprandial values) (43). A subcutaneous injection of 4 mg/kg caerulein inhibited the meal size over a 30 min period only in lean but not in obese Zucker rats (43). Further studies corroborate that the differential effects of CCK on food
FIG. 3. Fat cell volume in obese and lean Zucker rats are presented on the left and right sides of each set of columns as indicated in the figure. Values for gonadal tissue are shown on the left part of the figure and values for the subcutaneous tissue on the right part. Open bars represent rats which received NaCl, hatched bar rats which received caerulein, and black bars rats which received loxiglumide. Values represent means 6 SD of at least ten animals per group. There was a marked increase in fat cell volume in the genetically obese animals when compared to the lean littermates at both gonadal and subcutaneous sites (p , 0.01 by analysis of variance). Treatment with the CCK-agonist caerulein or the CCKantagonist loxiglumide did not alter the fat cell values in any of the different groups when compared with the NaCl control (p . 0.1).
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FIG. 4. The increase in body weight is presented as percent of the initial body weight at week five of the lean control animals which received NaCl injections. Animals which received NaCl are indicated by the black rectangles and closed lines, animals which received caerulein are indicated by the open triangles and broken lines, and animals which received the antagonist loxiglumide are indicated by the open rectangles and dotted lines. Agents were given between weeks five and ten (for further details see Methods). Experiments represent values of at least 10 animals, standard deviations were less than 10% and omitted for illustrative reason. The long-term administration of the CCK-antagonist loxiglumide significantly accelerated the increase in body weight both in obese and in lean rats (p , 0.01 when area under the curve was compared with the NaCl control). The CCK antagonist significantly reduced the increase in body weight in the obese rats (p , 0.05 when area under the curve was compared with the NaCl control) and also tended to reduce it in the lean littermates (p 5 0.05).
satiety effect is lost in the (male) obese Zucker rat. These conclusions are in line with our recent short-term studies (43). However, more recently Strohmayer and Greenberg (59) reported that devazepide increased food intake over a period of 60 min in male lean and obese Zucker rats, but not in females. In any case, both previous studies evaluated rather short-term periods when compared to the period of five weeks looked at in the present study. In contrast to the previous short-term studies the present experiments were not designed to study short-term meal patterns such a meal size and duration but intended to analyze daily food intake and body weight over a long time period. The present results did not provide any clue that the long-term effects of CCKagonists and antagonists on food intake differ between male and female rats. Considering the partial differences between the present results and those obtained by Strohmayer and Greenberg (58,59) one also needs to keep in mind that the latter authors had used MK-329 (devazepide) which differs from loxiglumide, the antagonist used in the present study. However, we do not see any evidence that these antagonists should differ in their effects on the action of endogenous or exogenous CCK in the type of experiments used.
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It remains unclear why the satiety effect of CCK is lost when the peptide is administered over a longer time period via a mini-pump and SC injections. Previously it has been shown that administration of CCK via a mini-pump does not exert a satiety effect and may even inhibit the effect of a bolus administration of CCK in a normal strain of rats (4). A similar attenuation of the CCK’s behavioral effects was also seen, however, with repetitive injections (4). In terms of food intake in lean rats the present results are in line with the previous results which looked only at a period of 2 weeks (4). However, the previous study and the present experiments differ in several other aspects: the present long-term administration of CCK over a period of five weeks significantly decreases body weight in lean (and obese) rats which was not seen in the two week-study which only evaluated a normal strain of (lean) rats (4). When one looks at the present results on body weight, the effect of chronic CCK administration indeed was seen only after the first two weeks in the lean animals (see Fig. 4). The present results also show further evidence that only some effects of chronic CCK administration show a tolerance: the action on pancreatic growth is still operative after a period of five weeks of CCK administration while CCK’s effects on satiety are lost. To our knowledge, there is as yet no experimental or theoretical evidence that chronic administration of CCKantagonist via a mini-pump or via repetitive injections may alter effects or binding of the agonist. The lack of the effects
FIG. 5. Pancreatic weights in obese rats are presented on the left side and corresponding values for lean rats on the right side. Experiment with saline are shown as open bars, experiments with caerulein are shown in hatched bars and experiments with loxiglumide are shown as black bars. Values represent means 6 SD of at least ten animals per group. Caerulein treatment significantly increased pancreatic weight in the lean Zucker rats (p , 0.01 when compared to the NaCl control) but not in the corresponding obese littermates (p . 0.2). Loxiglumide did not affect pancreatic weight in any of the groups (p . 0.2 when compared with the NaCl control).
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of the antagonist therefore supports the conclusion that endogenous CCK is not an essential satiety factor. Although the recent short-term experiments (43) and some other previous studies (13,14,26,28,60) at first sight suggest that CCK may play a physiological role in mediating satiety and may at least in part explain the increase in food intake and thus the development of obesity in genetically obese Zucker rats, the analysis of further recent shortterm experiments with a CCK-receptor antagonist already casted doubts on this hypothesis. The CCK antagonist loxiglumide—when given without a CCK agonist—failed to increase food intake in obese and lean Zucker rats (43). The lack of an effect of CCK-receptor blockade on food intake was similar in the recent short-term and the present longterm experiments. The doses of the CCK-A antagonist loxiglumide used are able to abolish the action of both near physiological and pharmacological doses of caerulein on food intake in lean and obese Zucker rats (43). Loxiglumide injections at doses ranging from 5–20 mg/kg are sufficient to completely inhibit the action of CCK at various gastrointestinal organs such as gallbladder, pancreas, stomach, small intestine and large intestine (4,33,35,36,42,56), even when CCK agonists are given at high doses resulting in circulating CCK levels markedly exceeding postprandial values. In the recent short-term experiments near physiological doses of exogenous CCK (given as subcutaneous injections without using a mini-pump) reduced food intake in lean Zucker rats whereas the blockade of endogenous CCK by the antagonist loxiglumide did not increase food intake in the same set of experiments. These two findings seem contradictory at first sight. However, one has to keep in mind that studies using exogenous CCK are associated with many pitfalls and do not prove that effects of exogenous peptide have physiological importance. In most previous studies pharmacological doses of CCK or CCK-agonists were administered which exceeded the physiological postprandial rise in plasma CCK. Only in recent years the post-prandial CCK rise has been defined more exactly (2,15,20,21,63). More recent studies—like the present one—tried to administer CCK at doses which mimic the rise in plasma CCK seen after a meal (2,15,63). However, such studies are still associated with potential pitfalls. There continues to be some controversy about the comparability and quality of radioimmunoassays and bioassays for CCK, and there is a controversy about which molecular forms of CCK are physiologically most important (for review see [37]). One also has to consider that systemic administration of a CCK preparation, even considering the dose which best mimics the postprandial rise in plasma CCK, might not reflect the true concentrations of the peptide at its place of action and the kinetics of its release in vivo. The development of specific CCK-receptor antagonists offers a new approach to evaluate the physiological role of CCK avoid-
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ing many pitfalls associated with previous studies. CCK has previously been thought to act as a physiological hormone or at least as a neurotransmitter for regulating gastric emptying, intestinal motility and insulin release because several studies had shown that intravenous infusion of CCK-A agonists at the doses which mimicked its postprandial rise in plasma altered these peripheral functions. Later studies with CCK-A antagonists however in many fields did not corroborate that CCK plays a physiological role for regulating insulin release, pancreatic growth or gastric emptying (34,37– 41). Thus, this discrepancy is just one more example of how important it is to study the physiology of a peptide with several different types of studies and techniques. In addition other recent studies also suggest that exogenous CCK inhibits food intake by vagal afferent pathways (9,12,43,54,55,57) whereas endogenously released CCK did not inhibit food intake (27). Thus from studies which administered physiological doses of CCK one cannot necessarily conclude that the actions seen are physiologically important since the blockade of endogenous CCK by a specific antagonist may show contradictory results. In the present experiments, both subcutaneous injections of CCK and administration of substances via a mini-pump were performed in order to mimic the rapid postprandial increase of this peptide and to have an increased basal CCK activity. Studies in which circulating levels of drugs are maintained at a relatively constant level (without additional peak increases) are associated with potential pitfalls because they may not mimic physiological conditions. We cannot definitely exclude that the results might have been different with either subcutaneous injections alone or with administration via a mini-pump alone. However, there is little evidence to support this hypothesis. Indeed the development of a behavioral tolerance has been documented both for the long-term administration of CCK via a mini-pump and for repetitive injections of CCK (4). Recent studies suggest that peripheral CCK-B (gastrin)receptor mediated effects are not involved in CCK’s effect on satiety because a CCK-B agonist did not inhibit food intake (43). The present long-term studies therefore did not use a CCK-B agonist or -antagonist. Neither the previous short-term nor the present long-term studies however exclude that central CCK might regulate food intake because the CCK-antagonist loxiglumide is an antagonist which shows a high specificity for the peripheral CCK-A receptor (24,46). The dose used is unlikely to alter binding of CCK at the B type of receptor (24,46). In addition peripheral CCK is believed to be unable to cross the blood-brain barrier although CCK-B and -A receptors occur in the central nervous system. However, feeding does not only increase plasma CCK but also releases CCK from the hypothalamus (8,30,32,51, for review see [52]). Indeed, recent studies suggested that hypothalamic CCK might regulate peripheral functions such as colonic motility and gastric
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emptying (5,19). It is as yet unknown whether CCK-A or -B receptors mediate the central satiety effects of CCK (literature in [47,50,52]). Some investigators showed that central application of the CCK-B receptor antagonists L-365,260 increased food intake (6) whereas other studies suggested that the central A-type receptor mediated satiety (for review see [52]). Central CCK might also elicit peripheral effects either via the vagal nerve or via release of other peptides such as met-enkephalin (31) and monoamines (44). In any case, the present experiments were not designed to study the role of central CCK for mediating food intake and satiety. It needs to be mentioned, however, that depolarization-evoked release of CCK from the hypothalamus is altered in obese when compared to lean Zucker rats (30). The mechanism for these central differences between fat and lean Zucker rats yet have to be elucidated. Although in the present long-term experiments CCKagonist and antagonist failed to alter food intake, the CCKagonist reduced the increase in body weight and the CCKantagonist accelerated the increase in body weight in particular in obese rats but to a lesser degree also in the lean rats. Since this difference cannot be explained by differences in food intake, the effects have to be explained either by an alteration in the energy expenditure or by an increased intestinal loss of calories. The latter explanation might be more intriguing because it is well known that CCK also exerts various actions on the intestinal tract (for review see [42]). For example, CCK markedly increases intestinal motility both in the small and large intestine and may thereby contribute to a loss of calories. The exact mechanism by which CCK-agonists and antagonists alter the body weight
during the growth of animals can only be clarified by further studies which quantitiate energy expenditure and loss of calories via the intestinal tract. In conclusion, genetically obese rats exhibit a CCKresistance at the peripheral CCK-A receptor site. Although this CCK-resistance can also be demonstrated in terms of the effect of CCK on satiety in a short-term fashion, longterm administration of CCK-agonist and antagonist failed to alter food intake both in obese and lean animals. Thus probably other factors than CCK are more important for the control of food intake and the loss of the satiety effect in the obese animals. The recently characterized mutations in the leptin receptor of genetically obese Zucker rats most likely cause the alterations of food intake and satiety in these animals (13,14,60). Surprisingly, despite of the failure of the CCK-agonist and antagonist to alter food intake in a long-term manner, the CCK-agonist reduced the increase in body weight and the CCK-antagonist accelerated the increase in body weight in particular in obese but also in lean rats. These effects might be due to either alterations in energy expenditure or in an increase loss of calories via the intestinal tract where CCK has various sites of action. In any case the long-term studies suggest that CCK does not play a major physiological role for the increase in body weight of growing animals and that CCK is not essential for the development of obesity in genetically obese Zucker rats. ACKNOWLEDGEMENTS The authors were supported by the Deutsche Forschungsgemeinschaft (SFB 351). The authors thank Ms. Schraven for her expert technical assistance and Ms. Herbrich for preparing the manuscript.
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tying and food intake by exogenous and endogenous CCK: role of capsaicin-sensitive vagal afferent pathway. Gastroenterology. 110:A672; 1996. Herberg, L.; Coleman, D. L. Laboratory animals exhibiting obesity and diabetes syndrome. Metabolism 26:59 –99; 1977. Holm, S. A simple sequentially rejective multiple test procedure. Scand. J. Statist. 6:65–70; 1979. Ho¨lzer, H. H.; Turkelson, C. M.; Solomon, T. E.; Raybould, H. E. Intestinal lipids inhibits gastric emptying via CCK and a vagal capsaicin afferent pathway in rats. Am. J. Physiol. 267:G625–G629; 1994. Idia, M.; Murakami, T.; Ishida, K.; Mizuno, A.; Kuwajima, M.; Shima, K. Substitution at codon 269 (glutamine 3 proline) of the leptin receptor (OB-R) cDNA is the only mutation found in the Zucker fatty (fa/fa) rat. Biochem. Biophys. Res. Commun. 224:597– 604; 1996. Idia, M.; Murakami, T.; Ishida, K.; Mizuno, A.; Kuwajima, M.; Shima, K. Phenotype-linked amino acid alteration in leptin receptor cDNA from Zucker fatty (fa/fa) rat. Biochem. Biophys. Res. Commun. 222:19 –26; 1996. Jansen, J. B. M. J.; Lamers, B. H. H. W. Radioimmunoassay of cholecystokinin in human tissue and plasma. Clin. Chim. Acta. 33:2197–2205; 1983. Labarca, C.; Paigen, K. A simple, rapid, and sensitive DNA assay procedure. Analyt. Biochem. 102:344 –352; 1990.
298 17. Li, Y.; Owyang, C. Vagal afferent pathway mediates physiological action of cholecystokinin on pancreatic enzyme secretion. J. Clin. Invest. 92:418 – 424; 1993. 18. Li, Y.; Hao, Y. B.; Owyang, C. High affinity CCK-A receptors on the vagus mediate cholecystokinin-stimulated pancreatic secretion in the rat. Gastroenterology. 110:A1094; 1996. 19. Liberge, M.; Arruebo, M. P.; Bueno, L. Role of hypothalamic cholecystokinin octapeptide in the colonic motor response to a meal in rats. Gastroenterology. 100:441– 449; 1991. 20. Liddle, R. A.; Goldfine, I. D.; Williams, J. A. Bioassay of plasma cholecystokinin in rats: effects of food, trypsin inhibitor, and alcohol. Gastroenterology. 87:542–549; 1984. 21. Liddle, R. A.; Goldfine, I. D.; Rosen, M. S.; Taplitz, R. A.; Williams, J. A. Cholecystokinin bioactivity in human plasma: molecular forms, responses to feeding, and relationship to gallbladder contraction. J. Clin. Invest. 75:1140 –1142; 1985. 22. Lieverse, R. J.; Jansen, J. B. M. J.; Masclee, A. A. M.; Lamers, C. B. H. W. Role of cholecystokinin in the regulation of satiation and satiety in humans. Ann. NY Acad. Sci. 713:268 – 272; 1994. 23. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurements with folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. 24. Makovec, F.; Chiste, R.; Bani, M.; Pacini, M. A.; Setnikar, I.; Rovati, L. A. New glutaramic acid derivatives with potent competitive and specific cholecystokinin-antagonistic activity. Arzneim. Forsch. 35:1048 –1051; 1985. 25. Makovec, F.; Bani, M.; Chiste`, R.; Revel, L.; Rovati, L. C.; Setnikar, I. Different peripheral and central antagonistic activity of new glutaramic acid derivatives on satiety induced by cholecystokinin in rats. Reg. Pept. 16:281–290; 1986. 26. McLaughlin, C. L.; Baile, C. A. Decreased sensitivity of Zucker obese rats to the putative satiety agent cholecystokinin. Physiol. Behav. 25:543–548; 1980. 27. McLaughlin, C. L.; Baile, C. A. Serum insulin, glucose and triglyceride responses of Zucker obese and lean rats to cholecystokinin. Physiol. Behav. 26:995–999; 1981. 28. McLaughlin, C. L.; Peikin, S. R.; Baile, C. A. Food intake response to modulation of secretion of cholecystokinin in Zucker rats. Am. J. Physiol. 244:R676 –R685; 1983. 29. McLaughlin, C. L.; Baile, C. A.; Buonomo, F. C. Effect of CCK antibodies on food intake and weight gain in Zucker rats. Physiol. Behav. 34:277–282; 1985. 30. McLaughlin, C. L.; Baile, C. A.; Della-Fera, M. A. Changes in brain CCK concentration with peripheral CCK injections in Zucker rats. Physiol. Behav. 36:477– 482; 1986. 31. McLaughlin, C. L.; Baile, C. A.; Della-Fera, M. A. Changes in brain met-enkephalin concentrations with peripheral CCK injections in Zucker rats. Physiol. Behav. 36:681– 686; 1986. 32. Micevych, P. E.; Go, V. L. W.; Yaksh, T. L. In vitro release of cholecystokinin from hypothalamus and frontal cortex of Sprague–Dawley, Zucker lean (Fa/2) and obese (fa/fa) rats. Peptides. 5:73– 80; 1984. 33. Niederau, C.; Niederau, M.; Williams, J. A.; Grendell, J. H. New proglumide analogue CCK-receptor antagonists: very potent and selective for peripheral tissues. Am. J. Physiol. 251:G856 –G860; 1986. 34. Niederau, C.; Liddle, R. A.; Williams, J. A.; Grendell, J. H. Pancreatic growth: Interaction of exogenous cholecystokinin, a protease inhibitor, and a cholecystokinin receptor antagonist in mice. Gut. 28:63– 69; 1987. 35. Niederau, C.; Heintges, T.; Rovati, L.; Strohmeyer, G. Effects of loxiglumide on gallbladder emptying in healthy volunteers. Gastroenterology. 97:1331–1336; 1989.
MEEREIS-SCHWANKE ET AL. 36. Niederau, M.; Niederau, C.; Strohmeyer, G.; Grendell, J. H. Comparative effects of CCK receptor antagonists on rat pancreatic secretion in vivo. Am. J. Physiol. 56:G150 –G157; 1989. 37. Niederau, C.; Lu¨then, R.; Niederau, M.; Strohmeyer, G.; Ferrell, L. D.; Grendell, J. H. Long-term effects of stimulation and inhibition at the CCK-receptor on morphology, function and growth of pancreas and intestine. Digestion. 46:217–225; 1990. 38. Niederau, C.; Karaus, M. Effects of agonists and antagonists of cholecystokinin on contractile and myoelectric activity of isolated muscle of colon and ileum in the dog and guinea pig. J. Gastrointest. Motility 2:160 –175; 1990. 39. Niederau, C.; Karaus, M. Effects of CCK-receptor blockade on intestinal motor activity in conscious dogs. Am. J. Physiol. 260:G315–G324; 1991. 40. Niederau, C.; Schwarzendrube, J.; Lu¨then, R.; Strohmeyer, G. The effects of a CCK-receptor blockade on circulating concentrations of glucose, insulin, C-peptide, and pancreatic polypeptide after various meals in healthy human volunteers. Pancreas. 7:1–10; 1992. 41. Niederau, C.; Mecklenbeck, W.; Heintges, T.; Lu¨then, R. CCK does not delay gastric emptying of regular meals in humans. Hepatogastroenterol. 40:380 –383; 1993. 42. Niederau, C.; Heintges, T.; Lu¨then, R. The effects of CCK on pancreatic function and morphology. Ann. NY Acad. Sci. 713:180 –196; 1994. 43. Niederau, C.; Meereis-Schwanke, K.; Klonowski-Stumpe, H.; Herberg, L. CCK-resistance in Zucker obese vs. lean rats. Reg. Pept. 1997; 70:97–104. 44. Orosco, M.; Duche, J. C.; Rybarczyk, M. C.; Rouch, C.; Cohen, Y.; Jacquot, C. Variability of changes in obese rat brain monoamines in response to cholecystokinin. Gen. Pharma. 17:665– 669; 1986. 45. Raybould, H. E.; Tache, Y. Cholecystokinin inhibits gastric motility and emptying via a capsaicin sensitive vagal pathway in rats. Am. J. Physiol. 255:G242–G246; 1988. 46. Reidelberger, R. D.; Solomon, T. E. Comparative effects of CCK-8 on feeding, sham feeding and exocrine pancreatic secretion in rats. Am. J. Physiol. 251:R1512–R1518; 1986. 47. Ritter, R. C.; Brenner, L. A.; Tamura, C. S. Endogenous CCK and the peripheral neutral substrates of intestinal satiety. Ann. NY Acad. Sci. 713:255–267; 1994. 48. Rodbell, M. Localization of lipoprotein lipase in fat cells of rat adipose tissue. J. Biol. Chem. 239:385–391; 1964. 49. Smith, E. B.; Slater, R. S. The microdissection of large atherosclerotic plaque to give morphologically and topographically defined fraction for analysis. Atherosclerosis. 15:57– 69; 1972. 50. Smith, G. P.; Gibbs, J. Satiating effect of cholecystokinin. Ann. NY Acad. Sci. 713:236 –241; 1994. 51. Schick, R. R.; Reilly, W. M.; Roddy, D. R.; Yaksh, T. L.; Go, V. W. L. Neuronal cholecystokinin-like immunoreactivity is postprandially released from primate hypothalamus. Brain. Res. 418:20 –26; 1987. 52. Schick, R. R.; Schusdziarra, V.; Yaksh, T. L.; Go, V. L. W. Brain regions where cholecystokinin exerts its effect on satiety. Ann. NY Acad. Sci. 713:242–254; 1994. 53. Schneider, L. H.; Murphy, R. B.; Gibbs, J.; Smith, G. P. Comparative potencies of CCK antagonists for the reversal of the satiating effects of cholecystokinin. In: Wang, R. A.; Schoenfeld, R., Eds. Cholecystokinin receptors antagonists. New York: Alan R. Liss Inc.; 1994; 263–284. 54. Schwartz, G. J.; McHugh, P. R.; Moran, T. H. Pharmacolog-
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ical dissociation of responses to CCK and gastric loads in rat mechanosensitive vagal afferents. Am. J. Physiol. 267:R303– R308; 1994. Schwartz, G. J.; McHugh, P. R.; Moran, T. H. Integration of vagal afferent responses to gastric loads and cholecystokinin in rats. Am. J. Physiol. 261:R64 –R69; 1991. Setnikar, I.; Bani, M.; Cereda, R.; Chiste, R.; Makovec, F.; Pacini, A.; Revel, L.; Rovati, L. C.; Rovati, L. A. Pharmacological characterization of a new potent and specific nonpolypeptidic cholecystokinin antagonist. Arzneim. Forsch. 6:703–707; 1987. South, E.; Ritter, R. C. Capsaicin application to central or peripheral vagal fibers attenuates CCK satiety. Peptides. 9:601– 612; 1988. Strohmayer, A. J.; Greenberg, D. Devazepide alters meal patterns in lean, but not obese, male Zucker rats. Physiol. Behav. 56:1037–1039; 1994. Strohmayer, A. J.; Greenberg, D. Devazepide increases food intake in male but not female Zucker rats. Physiol. Behav. 60:273–275; 1996. Takaya, K.; Ogawa, Y.; Isse, N.; Okazaki, T.; Satoh, N.; Masuzaki, H.; Mori, K.; Tamura, N.; Hosoda, K.; Nakao, K.
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Molecular cloning of rat leptin receptor isoform complementary DNAs—identification of a missense mutation in Zucker fatty (fa/fa) rats. Biochem. Biophys. Comm. 225:75– 83; 1996. Wank, A.; Pisegna R.; de Weerth, A. Cholecystokinin receptor family. Molecular cloning, structure, and functional expression in rat, guinea pig, and human. Ann. NY Acad. Sci. 713:49 – 66; 1994. Weatherford, S. C.; Laughton, W. B.; Salabarria, J.; Danho, W. D.; Tilley, J. W.; Netrerville, L. A.; Schwarzt, G. J.; Moran, T. H. CCK satiety is differentially mediated by high and low-affinity CCK receptors in mice and rats. Am. J. Physiol. 264:R244 –R249; 1993. Wiener, I.; Inoue, K.; Fagan, C. J.; Lilja, P.; Watson, L. C.; Thompson, J. C. Release of cholecystokinin in man: correlation of blood levels with gallbladder contraction. Ann. Surg. 194:321–327; 1981. Williams, J. A. Cholecystokinin: a hormone and a neurotransmitter. Biomed. Res. 3:107–115; 1982. Yok, D. P.; Brenner, L.; Ritter, R. C. CCK-receptors antagonists attenuate suppression of sham feeding by intestinal nutrients. Am. J. Physiol. 262:R554 –R561; 1992.
PII S0196-9781(97)00261-1
Long-Term Effects of CCK-Agonist and -Antagonist on Food Intake and Body Weight in Zucker Lean and Obese Rats KERSTIN MEEREIS-SCHWANKE,* HANNE KLONOWSKI-STUMPE,* LIESELOTTE HERBERG† AND CLAUS NIEDERAU*1 *Department of Medicine, Division of Gastroenterology, Hepatology and Infectious Diseases, and †Diabetes-Research Institute, Heinrich-Heine-University, Du¨sseldorf, Germany Received 2 April 1997; Accepted 18 August 1997 MEEREIS-SCHWANKE, K., H. KLONOWSKI-STUMPE, L. HERBERG AND C. NIEDERAU. Long-term effects of CCK-agonist and -antagonist on food intake and body weight in Zucker lean and obese rats. PEPTIDES 19(2) 291–299, 1998.—The present study evaluates long-term effects of the CCK-agonist caerulein and the CCK-A antagonist loxiglumide in obese and lean Zucker rats. Caerulein and loxiglumide altered food intake neither in obese nor in lean rats. By as yet unknown mechanisms, however, weight increase was accelerated by loxiglumide and reduced by caerulein in obese and lean rats. Caerulein increased pancreatic weight only in lean but not in obese rats. Thus, obese rats show a resistance of pancreatic CCK-A receptors. The failure of CCK-agonist and -antagonist to alter food intake suggests that this CCK-resistance is not responsible for obesity in the genetically altered rats. © 1998 Elsevier Science Inc. CCK CCK-receptor CCK-antagonists Caerulein Food intake Pancreatic growth Zucker rat Satiety
Loxiglumide
Gastrin
Leptin
intake in several species including humans (22,25,53). Nevertheless, CCK has been suggested to be involved in mediating satiety after a meal. The mechanisms involved in regulation of satiety by CCK, however, remain unsettled. Recent studies have suggested that effects of peripheral CCK on satiety—like other effects of CCK (17,18,45,54)—are mediated via CCK receptors on vagal afferent neurons (9,12,54,55,57). Genetically obese Zucker rats have a higher food intake when compared to their lean litter mates (10,27,29); this increase in food intake contributes to massive accumulation of adipose tissue (10,27,29). Little is known as yet about the mechanisms by which the obesity develops in the genetically altered Zucker rats. Recent studies have indicated that a defect in the leptin receptor may be responsible for the phenotype of the genetically obese Zucker rat (13,14,60). It has also been suggested that obese Zucker rats are less
THE peptide cholecystokinin (CCK) acts both as a peripheral hormone via CCK-A receptors and as a neurotransmitter within the brain and the enteric nervous system via CCK-B and CCK-A receptors (64). The CCK-A and -B receptors have been sequenced and are distinctly different whereas the gastrin receptor is identical with the CCK-B receptor (for review see [61]). In short-term experiments peripheral administration of CCK-A agonists inhibits food intake in various species including humans (for review see [22,47,50]); CCK in general refers to its sulfated form in the following text). Since food intake releases CCK from intestinal endocrine cells into the circulation (64), CCK may mediate postprandial satiety. Some studies have shown that peripheral administration of CCK-A antagonists increased food intake in several species (3,6,7,62,65). Other studies, however, failed to show an effect of CCK antagonists on food 1
Requests for reprints should be addressed to Prof. Dr. Claus Niederau, Department of Medicine, Division of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University Du¨sseldorf, Moorenstr. 5, D-40225 Du¨sseldorf, Germany. E-mail: [email protected]
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sensitive to the satiety effect of cholecystokinin (CCK) on meal size (26,28). Thus, CCK resistance might be a pathogenic factor for the development of obesity in these animals. We have recently corroborated that genetically obese Zucker rats are resistant to the satiety effect of CCK even at near physiological doses which result in plasma concentrations of CCK slightly above those seen after a meal (43). However, in the same studies the blockade of endogenous CCK released after a meal due to the administration of a CCK-A-receptor antagonist failed to alter the food intake in both lean and obese rats (43). Thus it was still unclear from the short-term studies whether CCK is involved in the long-term increase in body weight of growing rats and in particular in the development of obesity in genetically obese Zucker rats. The present studies evaluate whether a longterm administration of a CCK-agonist and CCK-antagonist alters food intake and body weight in obese and lean Zucker rats. METHOD Animals Genetically obese (fa/fa) male or female Zucker rats and their age-matched lean controls (FA/FA) were bred in the animal laboratories of the Diabetes-Research Institute at the Heinrich-Heine-University of Du¨sseldorf. Each animal was housed in a separate cage (Ebeco, Becker, Castrop-Rauxel, FRG) under the following standardized conditions: 12 h light/dark cycles with lights on at 0600 h; 25 6 2°C (mean 6 SD) room temperature; 55 6 5% air humidity. Animals were fed a standard rat diet (ssniff R, Soest, FRG). The daily food intake was recorded every morning at 0800 h. Manipulations and injections were done thereafter (0800 – 0900 h). Prior to the experiments, in which pancreatic weight, protein content, and DNA content, and fat cell volumes were measured, animals were fasted for 12 h with free access to drinking water. Experimental Design At the age of four weeks a mini-pump (Alzet model 2002, flow rate 0.5 ml/h; Savo, Kissleg, FRG) was surgically implanted in the subcutaneous fat of the neck region of obese and lean Zucker rats. The animals were allowed to recover for one week. Thus, the experiments were started at the age of five weeks and stopped at the age of 10 weeks; i.e. agents were given for a total period of five weeks. Caerulein (Takus®, Pharmacia, Erlangen, FRG) was given through the mini-pump at a dose of 4 mg/kg and the CCKantagonist loxiglumide (CR1505; D,L,-4-[3,4-dichlorobenzoylamino]-5-[N-3-methoxy-propyl-pentylamino]-5oxo-pentanoic acid, Rotta Research Laboratorium, Milano, Italy) was given at a dose of 5 mg/kg. In addition to the continuous administration of the CCK-agonist and antagonist the substances were also applied subcutaneously at a
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dose of 4 mg/kg and 5 mg/kg as a bolus injection at 0900 h. The subcutaneous bolus injection of caerulein may resemble peaks in plasma CCK bioactivity as seen after a large food intake. As shown previously the administration of 4 mg/kg caerulein as a single subcutaneous injection increases CCK bioactivity levels to values similar to what is seen after a meal (results not shown in detail here). Food intake was quantitatively assessed on a daily basis. Body weight was assessed at weekly intervals. Studies on blood samples, pancreatic tissue and adipose tissue were done at the end of the experiments (at week 10). Measurements of Plasma CCK by Bioassay Plasma CCK was measured by a specific and sensitive bioassay, as described previously in detail (20,21). The assay is based on the ability of CCK to stimulate amylase release from isolated rat pancreatic acini. Plasma was extracted and concentrated by adsorption onto octa-decylsilylsilica (C-18-Sep-Pak, Waters Associates, Milford, MA) cartridges. CCK was then eluted with 1 ml 80% ethanol– 0.2% trifluoroacetic acid, and the extracts were dried under nitrogen. Plasma extracts were incubated with isolated rat pancreatic acini for 30 min at 37°C, and amylase released into the incubation medium was assayed using the Phadebas amylase assay. Amylase release was compared with a doseresponse curve for CCK-8 (BioTrend, Ko¨ln, FRG). Plasma concentrations of CCK were not measurable in experiments in which loxiglumide was infused because even residual amounts of the CCK-receptor antagonist in the plasma samples interfere with the CCK-bioassay which is based on the ability of CCK to stimulate amylase release from isolated acini (20,21). The use of a radioimmunoassay of CCK, however, does also cause methodological problems in this situation. CCK-immunoreactivity is markedly elevated after infusion of a CCK-antagonist when compared with the control experiment in which saline is given instead of a CCK-antagonist (literature in [35]). The reason for this increase in CCK-immunoreactivity after administration of CCK-antagonists is unknown. Fat Cell Preparation Subcutaneous fat from the peritoneum was prepared after a mean laparotomy. Thereafter, the gonadal fat tissue was prepared from uterus and epididymis. Fat tissue was cleaned from connective tissue, weighed, and cut in small pieces with scissors. Fat cells were isolated by previously published methods (48,49). In brief, 100 mg fat tissue was put in a 3 ml KBR buffer solution containing 5 mg collagenase and incubated in a shaking water bath at 37°C for 50 min. Methylene blue was used for further identification and isolation of fat cells. An aliquot of the final supernatant was given on siliconiced glass, and photographs were taken to document the number of fat cells per mm3.
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Measurement of Pancreatic Weight, Protein Content and DNA Content The pancreas was prepared and cleaned from surrounding fat and connective tissue as previously reported (34,37). The whole pancreas was weighed and a 50 mg piece was used to measure the concentration of protein and DNA. Protein concentration was measured by the Lowry method (16) and DNA was measured using fluorescent Hoechst reagent 33258 (23). Statistical Analysis Differences between the experiments with infusion of NaCl versus loxiglumide were analyzed for statistical significance by analysis of variance using Tukey’s method (1). Analyses of multiple comparisons were done using Bonferroni– Holm’s correction (11). In the case of time-dependent changes in calorie intake and increase in body weight values for different experimental groups were compared by calculating the areas under the curve with the initial value subtracted from the following values which were assessed at weekly intervals. RESULTS The caerulein infusion via the mini-pump at 4 mg/kg resulted in plasma CCK bioactivity which markedly exceeded fasted values (p , 0.01 by analysis of variance) and still slightly exceeded fed values (p 5 0.05) (Fig. 1). The subcutaneous bolus injection of 4 mg/kg caerulein in additional to the mini-pump infusion resulted in plasma CCK values (30 min after the injection) which more clearly exceeded fed values (p , 0.01) and also slightly exceeded the values seen after CCK was only infused via the mini-pump (p , 0.05) (Fig. 1). CCK values were similar for lean and obese rats for all the experimental conditions indicated (p . 0.1) (Fig. 1). Administration of the CCK-agonist caerulein and the CCK-antagonist loxiglumide altered the food intake neither in obese nor in lean Zucker rats when compared to the saline control (Fig. 2). Data were virtually identical when analyzed separately for male vs. female animals (p . 0.2, data not shown in detail). As expected, the food intake was markedly increased over the whole time period in the genetically obese Zucker rats when compared to their lean littermates (Fig. 2). Correspondingly there was a marked accumulation of adipose tissue in the obese Zucker rats both at the gonadal and subcutaneous site (Fig. 3). The amount of adipose tissue was affected neither by the CCK-agonist nor by the CCK-antagonist when compared to the saline control both in females and in males as well as in obese and in lean rats (Fig. 3). The effects of the CCK agonist and antagonist on intake of calories and on body weight were similar for both gender; therefore the results are shown for the total number of both male and female animals. In contrast to the lack of an effect of CCK-agonist and antagonist on food intake, the increase in body weight was
FIG. 1. Plasma CCK concentrations as measured by the bioassay are given in pM from 10 animals for each separate condition as indicated on the X axis. Results are shown as mean 6 SEM for lean rats (open bars) and for obese rats (filled bars). When compared with the fed conditions the caerulein infusion via the minipump (at 4 mg/kg) only slightly increased plasma CCK bioactivity above fed values (p 5 0.05 by analysis of variance) whereas the additional subcutaneous bolus caerulein injection (at 4 mg/kg) resulted in plasma CCK values (30 min after the injection) which more clearly exceeded fed values (p , 0.01) and also slightly exceeded the values seen after CCK was only infused via the mini-pump (p , 0.05). CCK values were similar for lean and obese rats for all the experimental conditions indicated (p . 0.1).
slightly but significantly increased by the CCK-antagonist both in obese and lean rats (Fig. 4). In the obese rats the CCK agonist caerulein likewise significantly slowed the increase in body weight. In lean rats caerulein only slightly reduced the increase in body weight (p 5 0.05 when compared with the saline control) (Fig. 4). As expected the increase in body weight was markedly faster in obese vs. lean Zucker rats (p , 0.01) (Fig. 4). The long-term administration of the CCK-agonist caerulein significantly increased pancreatic weight, protein and DNA content of lean Zucker rats of both gender (p , 0.01) (pancreatic weights shown in Fig. 5). Thus caerulein induced both hypertrophy and hyperplasia under these circumstances. Since changes in pancreatic weights under all circumstances were closely related to changes in protein and DNA content, only data on weights are shown in detail. In contrast the same dose of caerulein failed to alter the pancreatic weight in obese rats (p . 0.2). In both obese and lean rats the CCK antagonist loxiglumide failed to alter the pancreatic weight (Fig. 5) and contents of protein and DNA after long-term administration (p . 0.2, respectively). DISCUSSION The present results corroborate that obese Zucker rats are resistant to the effects of CCK at the peripheral (e.g. pancreatic) CCK-A type receptor also after a long-term treatment over a period of several weeks. A near physiological
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FIG. 2. Food intake assessed as daily uptake of kJ (Y-axis) in obese animals left part and in lean litter mates right part during the study period as indicated on the X-axis. Animals which received NaCl are indicated by the black rectangles and closed lines, animals which received caerulein are indicated by the open triangles and broken lines, and animals which received the antagonist loxiglumide are indicated by the open rectangles and dotted lines. Agents were given between weeks five and ten (for further details see Methods). The obese animals showed markedly increased food intake from week five to ten when compared to the lean littermates (areas under the curve significantly different in saline, caerulein and loxiglumide groups with p , 0.01, respectively). However, in both lean and obese rats neither caerulein nor loxiglumide altered the food intake when compared to the saline control (p . 0.1 for comparisons of areas under the curve).
intake in obese and lean Zucker rats are due to differences in binding and action at the CCK-A receptor (26,28,43). Such a resistance of the satiety effect of CCK in obese rats might contribute to the larger meal size and thus the development of obesity in these genetically altered animals. The present results, however, show that this is not true on a long-term basis. Neither the CCK agonist nor the CCK antagonist altered the food intake in lean and obese rats over the five week period. The lack of the effect of CCK on food intake in the long-term experiments might be explained by a loss of the CCK resistance during the growth of the animals and/or by other factors which contribute to the increase in food intake to a greater part than CCK. The current experiments show that there was still a CCK-resistance at the pancreatic CCK-A type receptor in the longterm experiments. The recently characterized mutations in the leptin receptor of genetically obese Zucker rats most likely cause or contribute to the alterations of food intake and satiety in these animals (13,14,60). In any case, on a long-term basis CCK is not an essential and probably not even an important factor of controlling satiety in the rat. A previous study had shown that the CCK-A antagonist devazepide (also termed MK-329) increased total food intake over a period of 23 h—and in particular increased the average meal size and duration—in male obese Zucker rats whereas no effect was seen in male lean rats (58). The latter results were interpreted as evidence that the physiological
dose of the CCK-agonist caerulein increased pancreatic weight as well as DNA and protein contents after a longterm administration in lean rats whereas the same dose (with the same circulating concentrations of the peptide) failed to show any effect on pancreatic growth in obese animals. Thus there is still a CCK-resistance at the peripheral receptor after long-term treatment with a CCK agonist. Very similar to the short-term studies (43), however, the administration of a CCK-antagonist, which completely blocks all the actions of endogenous CCK released after a meal, failed to decrease pancreatic weight. Thus although near physiological increases in plasma CCK due to exogenous CCK increased pancreatic growth, the blockade of endogenous CCK did not have any effects on pancreatic weight. These results corroborate our own previous long-term studies in the mouse which also suggested that CCK is not an essential growth factor for the pancreas in this species (37). Recent short-term studies showed that obese Zucker rats are resistant to the satiety effect of near physiological concentrations of the CCK agonist caerulein (i.e. doses which result in circulating CCK bioactivity slightly above postprandial values) (43). A subcutaneous injection of 4 mg/kg caerulein inhibited the meal size over a 30 min period only in lean but not in obese Zucker rats (43). Further studies corroborate that the differential effects of CCK on food
FIG. 3. Fat cell volume in obese and lean Zucker rats are presented on the left and right sides of each set of columns as indicated in the figure. Values for gonadal tissue are shown on the left part of the figure and values for the subcutaneous tissue on the right part. Open bars represent rats which received NaCl, hatched bar rats which received caerulein, and black bars rats which received loxiglumide. Values represent means 6 SD of at least ten animals per group. There was a marked increase in fat cell volume in the genetically obese animals when compared to the lean littermates at both gonadal and subcutaneous sites (p , 0.01 by analysis of variance). Treatment with the CCK-agonist caerulein or the CCKantagonist loxiglumide did not alter the fat cell values in any of the different groups when compared with the NaCl control (p . 0.1).
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FIG. 4. The increase in body weight is presented as percent of the initial body weight at week five of the lean control animals which received NaCl injections. Animals which received NaCl are indicated by the black rectangles and closed lines, animals which received caerulein are indicated by the open triangles and broken lines, and animals which received the antagonist loxiglumide are indicated by the open rectangles and dotted lines. Agents were given between weeks five and ten (for further details see Methods). Experiments represent values of at least 10 animals, standard deviations were less than 10% and omitted for illustrative reason. The long-term administration of the CCK-antagonist loxiglumide significantly accelerated the increase in body weight both in obese and in lean rats (p , 0.01 when area under the curve was compared with the NaCl control). The CCK antagonist significantly reduced the increase in body weight in the obese rats (p , 0.05 when area under the curve was compared with the NaCl control) and also tended to reduce it in the lean littermates (p 5 0.05).
satiety effect is lost in the (male) obese Zucker rat. These conclusions are in line with our recent short-term studies (43). However, more recently Strohmayer and Greenberg (59) reported that devazepide increased food intake over a period of 60 min in male lean and obese Zucker rats, but not in females. In any case, both previous studies evaluated rather short-term periods when compared to the period of five weeks looked at in the present study. In contrast to the previous short-term studies the present experiments were not designed to study short-term meal patterns such a meal size and duration but intended to analyze daily food intake and body weight over a long time period. The present results did not provide any clue that the long-term effects of CCKagonists and antagonists on food intake differ between male and female rats. Considering the partial differences between the present results and those obtained by Strohmayer and Greenberg (58,59) one also needs to keep in mind that the latter authors had used MK-329 (devazepide) which differs from loxiglumide, the antagonist used in the present study. However, we do not see any evidence that these antagonists should differ in their effects on the action of endogenous or exogenous CCK in the type of experiments used.
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It remains unclear why the satiety effect of CCK is lost when the peptide is administered over a longer time period via a mini-pump and SC injections. Previously it has been shown that administration of CCK via a mini-pump does not exert a satiety effect and may even inhibit the effect of a bolus administration of CCK in a normal strain of rats (4). A similar attenuation of the CCK’s behavioral effects was also seen, however, with repetitive injections (4). In terms of food intake in lean rats the present results are in line with the previous results which looked only at a period of 2 weeks (4). However, the previous study and the present experiments differ in several other aspects: the present long-term administration of CCK over a period of five weeks significantly decreases body weight in lean (and obese) rats which was not seen in the two week-study which only evaluated a normal strain of (lean) rats (4). When one looks at the present results on body weight, the effect of chronic CCK administration indeed was seen only after the first two weeks in the lean animals (see Fig. 4). The present results also show further evidence that only some effects of chronic CCK administration show a tolerance: the action on pancreatic growth is still operative after a period of five weeks of CCK administration while CCK’s effects on satiety are lost. To our knowledge, there is as yet no experimental or theoretical evidence that chronic administration of CCKantagonist via a mini-pump or via repetitive injections may alter effects or binding of the agonist. The lack of the effects
FIG. 5. Pancreatic weights in obese rats are presented on the left side and corresponding values for lean rats on the right side. Experiment with saline are shown as open bars, experiments with caerulein are shown in hatched bars and experiments with loxiglumide are shown as black bars. Values represent means 6 SD of at least ten animals per group. Caerulein treatment significantly increased pancreatic weight in the lean Zucker rats (p , 0.01 when compared to the NaCl control) but not in the corresponding obese littermates (p . 0.2). Loxiglumide did not affect pancreatic weight in any of the groups (p . 0.2 when compared with the NaCl control).
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of the antagonist therefore supports the conclusion that endogenous CCK is not an essential satiety factor. Although the recent short-term experiments (43) and some other previous studies (13,14,26,28,60) at first sight suggest that CCK may play a physiological role in mediating satiety and may at least in part explain the increase in food intake and thus the development of obesity in genetically obese Zucker rats, the analysis of further recent shortterm experiments with a CCK-receptor antagonist already casted doubts on this hypothesis. The CCK antagonist loxiglumide—when given without a CCK agonist—failed to increase food intake in obese and lean Zucker rats (43). The lack of an effect of CCK-receptor blockade on food intake was similar in the recent short-term and the present longterm experiments. The doses of the CCK-A antagonist loxiglumide used are able to abolish the action of both near physiological and pharmacological doses of caerulein on food intake in lean and obese Zucker rats (43). Loxiglumide injections at doses ranging from 5–20 mg/kg are sufficient to completely inhibit the action of CCK at various gastrointestinal organs such as gallbladder, pancreas, stomach, small intestine and large intestine (4,33,35,36,42,56), even when CCK agonists are given at high doses resulting in circulating CCK levels markedly exceeding postprandial values. In the recent short-term experiments near physiological doses of exogenous CCK (given as subcutaneous injections without using a mini-pump) reduced food intake in lean Zucker rats whereas the blockade of endogenous CCK by the antagonist loxiglumide did not increase food intake in the same set of experiments. These two findings seem contradictory at first sight. However, one has to keep in mind that studies using exogenous CCK are associated with many pitfalls and do not prove that effects of exogenous peptide have physiological importance. In most previous studies pharmacological doses of CCK or CCK-agonists were administered which exceeded the physiological postprandial rise in plasma CCK. Only in recent years the post-prandial CCK rise has been defined more exactly (2,15,20,21,63). More recent studies—like the present one—tried to administer CCK at doses which mimic the rise in plasma CCK seen after a meal (2,15,63). However, such studies are still associated with potential pitfalls. There continues to be some controversy about the comparability and quality of radioimmunoassays and bioassays for CCK, and there is a controversy about which molecular forms of CCK are physiologically most important (for review see [37]). One also has to consider that systemic administration of a CCK preparation, even considering the dose which best mimics the postprandial rise in plasma CCK, might not reflect the true concentrations of the peptide at its place of action and the kinetics of its release in vivo. The development of specific CCK-receptor antagonists offers a new approach to evaluate the physiological role of CCK avoid-
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ing many pitfalls associated with previous studies. CCK has previously been thought to act as a physiological hormone or at least as a neurotransmitter for regulating gastric emptying, intestinal motility and insulin release because several studies had shown that intravenous infusion of CCK-A agonists at the doses which mimicked its postprandial rise in plasma altered these peripheral functions. Later studies with CCK-A antagonists however in many fields did not corroborate that CCK plays a physiological role for regulating insulin release, pancreatic growth or gastric emptying (34,37– 41). Thus, this discrepancy is just one more example of how important it is to study the physiology of a peptide with several different types of studies and techniques. In addition other recent studies also suggest that exogenous CCK inhibits food intake by vagal afferent pathways (9,12,43,54,55,57) whereas endogenously released CCK did not inhibit food intake (27). Thus from studies which administered physiological doses of CCK one cannot necessarily conclude that the actions seen are physiologically important since the blockade of endogenous CCK by a specific antagonist may show contradictory results. In the present experiments, both subcutaneous injections of CCK and administration of substances via a mini-pump were performed in order to mimic the rapid postprandial increase of this peptide and to have an increased basal CCK activity. Studies in which circulating levels of drugs are maintained at a relatively constant level (without additional peak increases) are associated with potential pitfalls because they may not mimic physiological conditions. We cannot definitely exclude that the results might have been different with either subcutaneous injections alone or with administration via a mini-pump alone. However, there is little evidence to support this hypothesis. Indeed the development of a behavioral tolerance has been documented both for the long-term administration of CCK via a mini-pump and for repetitive injections of CCK (4). Recent studies suggest that peripheral CCK-B (gastrin)receptor mediated effects are not involved in CCK’s effect on satiety because a CCK-B agonist did not inhibit food intake (43). The present long-term studies therefore did not use a CCK-B agonist or -antagonist. Neither the previous short-term nor the present long-term studies however exclude that central CCK might regulate food intake because the CCK-antagonist loxiglumide is an antagonist which shows a high specificity for the peripheral CCK-A receptor (24,46). The dose used is unlikely to alter binding of CCK at the B type of receptor (24,46). In addition peripheral CCK is believed to be unable to cross the blood-brain barrier although CCK-B and -A receptors occur in the central nervous system. However, feeding does not only increase plasma CCK but also releases CCK from the hypothalamus (8,30,32,51, for review see [52]). Indeed, recent studies suggested that hypothalamic CCK might regulate peripheral functions such as colonic motility and gastric
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emptying (5,19). It is as yet unknown whether CCK-A or -B receptors mediate the central satiety effects of CCK (literature in [47,50,52]). Some investigators showed that central application of the CCK-B receptor antagonists L-365,260 increased food intake (6) whereas other studies suggested that the central A-type receptor mediated satiety (for review see [52]). Central CCK might also elicit peripheral effects either via the vagal nerve or via release of other peptides such as met-enkephalin (31) and monoamines (44). In any case, the present experiments were not designed to study the role of central CCK for mediating food intake and satiety. It needs to be mentioned, however, that depolarization-evoked release of CCK from the hypothalamus is altered in obese when compared to lean Zucker rats (30). The mechanism for these central differences between fat and lean Zucker rats yet have to be elucidated. Although in the present long-term experiments CCKagonist and antagonist failed to alter food intake, the CCKagonist reduced the increase in body weight and the CCKantagonist accelerated the increase in body weight in particular in obese rats but to a lesser degree also in the lean rats. Since this difference cannot be explained by differences in food intake, the effects have to be explained either by an alteration in the energy expenditure or by an increased intestinal loss of calories. The latter explanation might be more intriguing because it is well known that CCK also exerts various actions on the intestinal tract (for review see [42]). For example, CCK markedly increases intestinal motility both in the small and large intestine and may thereby contribute to a loss of calories. The exact mechanism by which CCK-agonists and antagonists alter the body weight
during the growth of animals can only be clarified by further studies which quantitiate energy expenditure and loss of calories via the intestinal tract. In conclusion, genetically obese rats exhibit a CCKresistance at the peripheral CCK-A receptor site. Although this CCK-resistance can also be demonstrated in terms of the effect of CCK on satiety in a short-term fashion, longterm administration of CCK-agonist and antagonist failed to alter food intake both in obese and lean animals. Thus probably other factors than CCK are more important for the control of food intake and the loss of the satiety effect in the obese animals. The recently characterized mutations in the leptin receptor of genetically obese Zucker rats most likely cause the alterations of food intake and satiety in these animals (13,14,60). Surprisingly, despite of the failure of the CCK-agonist and antagonist to alter food intake in a long-term manner, the CCK-agonist reduced the increase in body weight and the CCK-antagonist accelerated the increase in body weight in particular in obese but also in lean rats. These effects might be due to either alterations in energy expenditure or in an increase loss of calories via the intestinal tract where CCK has various sites of action. In any case the long-term studies suggest that CCK does not play a major physiological role for the increase in body weight of growing animals and that CCK is not essential for the development of obesity in genetically obese Zucker rats. ACKNOWLEDGEMENTS The authors were supported by the Deutsche Forschungsgemeinschaft (SFB 351). The authors thank Ms. Schraven for her expert technical assistance and Ms. Herbrich for preparing the manuscript.
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