Chronic cerebroventricular galanin does not induce sustained hyperphagia or obesity

Peptides, Vol. 15, No. 7, pp. 1267-1272, 1994 Copyright© 1994ElsevierScienceLtd Printedin the USA.All rightsreserved 0196-9781/94$6.00 + .00

Pergamon 0196-9781(94)00105-7

Chronic Cerebroventricular Galanin Does Not Induce Sustained Hyperphagia or Obesity B. K. S M I T H , D. A. Y O R K ~ A N D G. A. B R A Y

Louisiana State University, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808-4124 Received 14 March 1994 SMITH, B. K., D. A. YORK AND G. A. BRAY. Chronic cerebroventriculargalanin does not induce sustained hyperphagia or obesity. PEPTIDES 15(7) 1267-1272, 1994.-- Acute centraladministrationofgalanin has been reported to increasefat consumption. These experiments were designed to test the hypothesis that repeated injections of galanin would elicit hyperphagia and weight gain and that this response would depend on the available diet. Male Sprague-Dawley rats were fed high (56% energy) or low (10% energy) fat diets. Galanin (300 pmol) or saline vehicle was injected into the third ventricle twice daily for 7 days and three times daily for another 6 days. On both the high-carbohydrate and high-fat diets, twice daily galanin increased daytime food intake, but there was a compensatory decrease in nighttime intake. The addition of a third, nighttime injection of galanin was ineffectivein producing an increase in total 24-h intake. There was no significantincrease in body weightduring galanin treatment in rats eating either diet although animals eating the high-fat diet gained more weight as reflected by a significant increase in epididymal fat pad weight. Galanin treatment had no effectson serum insulin, glucoseor corticosteroneconcentrations, measured at the end of the experiment. However, animals fed the high-fat diet had significantlyhigher insulin concentrations at the time of sacrifice. Although repeated central infusions of galanin reliably stimulated daytime intake of both diets, they failed to increase total daily energy intake or body weight. Galanin

Food intake

Body weight

Rat

lntracerebroventricular infusion

INTRODUCTION GALANIN is a 29 amino acid peptide that stimulates feeding when injected into the brain of rats (5,8) and ground squirrels (3). This behavioral effect of centrally administered galanin has been demonstrated for both the hypothalamus and the amygdala (4,9) and is not associated with alterations in water intake, general activity, or sleeping (9). There is some evidence that galanin induces the preferential eating of fat (20) particularly at the end of the dark period (12,21). However, galanin stimulated both fat and carbohydrate intake in rats maintained on pure macronutrient diets or on diets with a choice (low or high fat) (17,20). Furthermore, recent evidence suggests that galanin may stimulate existing macronutrient preferences, for example, the dominant preference for fat observed in Sprague-Dawley rats (17). The role of galanin in the physiological and endocrine changes associated with obesity is unclear at this time. Galanin injected into the paraventricular nucleus (PVN) of the hypothalamus inhibits ACTH release (6) and insulin release (22), effects that would oppose the development of obesity. Centrally administered galanin also elicits a reduction in energy expenditure that corresponds to the duration and dose range ofgalanin's feeding effect (14). Furthermore it has been shown that increased hypothalamic levels ofgalanin mRNA are associated with increased

Fat intake

body weight in obese Zucker rats, compared to lean rats, at 40 weeks-of-age (7). Repeated injections of NPY, another peptide that enhances food intake, produced an increase in fat deposition and in body weight ( 1,18,19,24). The consequences of repeated administration ofgalanin have not been examined. Because galanin appears to have a physiological role in the regulation of food intake, we decided to investigate the effects of chronic exposure to this peptide in nonobese animals fed either a high carbohydrate or high fat diet. METHOD

Animals Male Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed individually in hanging wire-mesh cages in a temperature-controlled room (22 ° 23°C) with lights on from 0700 to 1900 h. The animals were maintained on either a high-fat (HF, 56% energy value as fat) or a high-carbohydrate (HC, 10% fat) diet (see Table 1). Food and tap water were available ad lib and after 14 days exposure to the single choice diet, only weight-gaining animals were selected for surgery.

Requests for reprints should be addressed to David A. York, Ph.D.

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SMITH, YORK AND BRAY TABLE 1 COMPOSITION OF EXPERIMENTALDIETS High-CarbohydrateDiet*

Vitamin-free casein Corn starch Powdered sugar Corn oil Vegetable shortening Salt mix Vitamin mix Choline Dt-Methionine Alphacel nonnutritive bulk*

High-FatDietf

g/100kcal

%kcal

g/100 kcal

%kcal

6.00 11.55 4.95 0.67 0.44 0.88 0.22 0.05 0.03 2.50

24 46 20 6 4

6.00 5.00 0.00 0.67 5.56 0.88 0.22 0.05 0.03 2.50

24 20 0 6 50

Diet materialswere purchased from ICN Biomedicals,Inc. (Aurora, OH) except vegetableshortening was obtained from Proctor & Gamble (Cincinnati, OH) and powdered sugar from Albertsons, Inc. (Boise, ID). * 3.66 kcal/g. I" 4.78 kcal/g.

Stereotaxic Surgery

Data Analysis

Animals (275-325 g) were anesthetized with ketamine (80 mg]kg) and xylazine (12 mg/kg) and placed in a stereotaxic frame where a 25 gauge stainless steel guide cannula was positioned in the third ventricle at the midline (AP -2.8 mm from bregma; DV -8.1 mm from dura). The cannula was fixed to the skull with stainless steel screws and dental cement, and then occluded with a 31 gauge styler. The intraventricular location of each cannula was visually confirmed by injecting Coomasie Brilliant Blue dye through the cannula at the end of the experiment.

Data are presented as the mean + SEM. Analysis of variance with repeated measures was performed with diet and peptide as between-subject effects. The time factor was analyzed as a withinsubject effect. All main effects and all interactions among diet, peptide, and time were included. The Students' t-test was used to evaluate comparisons between groups for overall change in body weight and for measures of food intake at selected time points. RESULTS

EXPERIMENTAL Rat galanin 1-29 (Peninsula Laboratories, Belmont, CA) (300 pmol/0.5 ~1) or the same volume of 0.9% saline vehicle was infused through a 31 gauge injector that extended 0.5 mm beyond the guide cannula. Infusions were administered over 60 s with a Harvard syringe pump (model #22). One-half the number of animals in each diet group was given repeated intraventricular infusions ofgalanin twice daily (1000, 1400 h) on days 1-7, and then 3 times per day (1000, 1400, and 2300 h) for days 8-13. The control groups received saline vehicle alone at the same times. Baseline food intake was measured daily for 3 days before the experimental period, after which food intake was recorded three times daily, approximately 4 h after each daytime injection and at the end of the dark period. Body weights were obtained daily. At the end of the experiment (day 14), rats were killed by decapitation between 1000 and 1100 h. Trunk blood was collected in chilled tubes and the serum frozen for measurements of insulin, glucose, and corticosterone. Epididymal fat pads and the liver were dissected free and weighed.

Assays Insulin was analyzed with a radioimmunoassay kit (Linco Research, St. Louis, MO) using a rat insulin standard. Glucose was analyzed on a Beckman Synchron CX5 multichannel analyzer and corticosterone with a radioimmunoassay kit (ICN Biomedicals, Costa Mesa, CA).

Food Intake The repeated measures ANOVA for between subjects effects on daytime food intake showed a significant main effect of peptide [F(1, 13) = 10.97, p < 0.01] and diet IF(I, 13) = 10.91,p < 0.01 ], that is, that galanin treatment stimulated daytime food intake overall and that intake of the HC groups was different than that of the HF groups. However there were no main effects of peptide, diet, or peptide diet interaction for measures of nighttime or total daily food intake. In addition, within subjects analyses of days 1-13 revealed a significant day X peptide X diet interaction for daytime food intake [F(12, 156) = 3.31, p < 0.001] that was not seen for measures of nighttime [F(12, 156) = 0.63, p = 0.76] or total daily [F(12, 156) = 1.18, p = 0.32] food intake. This significant interaction is due to a significant day X diet effect [F(12, 156) = 4.40, p < 0.005] rather than a day x peptide effect [F( 12, 156) = 0.46, p = 0.91 ]. These effects can be explained by the different patterns of daytime feeding observed for the HC and the HF diets over time (Fig. 1A, 2A). Specifically, consumption of the HC diet was greater in galanin-treated rats than in saline-treated controls, but then declined. Conversely, intake of rats fed the HF diet was similar to that of controls for the first 3 days and then began to steadily increase. Thus, any day X peptide effect on daytime food intake was dependent on diet. Specific comparisons of daytime intake of the HC diet using Student's t-test showed that average food intake for days 1-5 was significantly greater in the galanin-treated rats compared to

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FIG. 1. Effect of galanin on (A) daytime, (B) nighttime, and (C) total food intake (mean ± SEM) of the high-carbohydrate (He) diet in Sprague-Dawley rats repeatedly injected with either galanin (n = 5) or saline (n = 3) into the third ventricle for 13 days.

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controls (GAL 8.7 + 0.6 g vs. SAL 4.8 + 1.8 g; p < 0.05) and then began to decline (Fig. 1). However, galanin-treated animals eventually compensated for the increased daytime intake by reducing their nighttime intake of the H C diet (see days 1--4) (GAL 7.8 _ 0.7 g vs. SAL 10.3 + 1.6 g; p = 0.14), thereby bringing total daily intake close to control levels (Fig. 1). The addition of a third daily injection of galanin in the early dark period on day 8 did not elicit any further increase in either nighttime or total daily food intake.

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FIG. 2. Effect of galanin on (A) daytime, (B) nighttime, and (C) total food intake (mean _+SEM) of the high-fat (HF) diet in Sprague-Dawley rats repeatedly injected with either galanin (n = 5) or saline (n = 5) into the third ventricle for 13 days.

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SMITH, Y O R K A N D B R A Y

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FIG. 3. Daily body weights (mean + SEM) of Sprague-Dawley rats before and during repeated injections of either galanin or saline into the third ventricle for 13 days. Animals were maintained on either the (A) highcarbohydrate or (B) high-fat diet.

+ 0.5 g vs. SAL 3.1 + 0.4 g; p < 0.005, days 8-13) infusion periods. Again, a compensatory decrease in nighttime food consumption was seen in galanin-treated rats (GAL 7.2 + 1.0 g vs. 10.0 + 0.9g; p = 0.08, days 4-7), resulting in 24-h intakes that were no different from controls (Fig. 2).

At baseline (preinjection days - 2 to 0), all experimental groups demonstrated a positive weight gain as seen in Fig. 3. In addition, the mean body weight of the animals was not different among groups at the beginning of the experiment (see Table 2). There was no significant main effect of peptide [F(1, 14) = 0.06, p = 0.82] or diet [F(1, 14) = 0.02, p = 0.90] on body weight when analyzed by repeated measures A N O V A for between subjects effects. Furthermore, there were no significant interactions involving peptide, that is, peptide × diet [F(1, 14) = 1.83, p = 0.20], peptide X day [F(12, 168) = 0.29, p = 0.77], and peptide X diet X day [F(12, 168) = 1.19, p = 0.32]. However there was a significant diet × day interaction [F(12, 168) = 18.45, p < 0.005]. Following an initial drop in weight at the start of the infusion period, all animals fed the H F diet slowly increased their body weights by the end of the experiment; animals fed the HC diet gradually lost weight whether they received galanin or vehicle injections (Fig. 3). This effect is further illustrated by the magnitude of mean weight change that occurred over the course of the experiment as a function of diet [F(1, 14) = 15.06, p < 0.005] as seen in Table 2. Table 2 shows the effect of galanin and diet on serum insulin, corticosterone, and glucose levels as well as body weight indices. A two-way A N O V A showed that there was no significant main effect of peptide on any of these variables compared to vehicle controls. However, there was a significant main effect of diet on insulin concentrations [F(1, 14) = 20.61, p < 0.001]; animals fed the H F diet had higher insulin concentrations than rats fed the HC diet at the time of sacrifice. In addition, the increased body weight of animals fed the H F diet was associated with significantly higher fat pad [F(1, 14) = 16.77, p < 0.005] and liver weights [F(1, 14) = 6.60, p < 0.05] compared to animals fed the HC diet. DISCUSSION

These studies have shown that repeated administration ofgalanin into the third ventricle did not significantly increase the 24-h food intake or body weight of nonobese rats. Although unilateral ICV injections of galanin twice daily for the first 7 days reliably increased food intake during the daylight hours, there was a compensatory decrease during the nighttime resulting in a 24-h energy intake that was not different from controls. This effect was seen whether the animals were eating the H F or the HC diet. When the frequency

TABLE 2 EFFECTS OF REPEATED ADMINISTRATIONOF INTRACEREBROVENTRICULARGALANIN AND DIET Saline HC Serum insulin (ng/ml)* Serum glucose (mg/dl) Serum corticosterone (ng/ml) Fat pad weight (g)t Liver weight:~ (g/100 g body weight) Initial body weight (g) Body weight change (g)f

1.0 + 0.0 133.0 _ 5.3 64.7 ___22.8 0.6 + 0.2 3.1 _+ 0.1 312.0 + 12.5 -42.3 ___ 6.8

Galanin HF

HC

HF

1.1 3.1 4.0 0.2 0.1

1.4 _+ 0.4 138.6 _+ 2.6 54.4 ___ 10.6 1.0 _+ 0.3 2.8 _+ 0.1

5.2 + 1.1 138.6 + 4.2 62.0 _+ 22.9 1.8 + 0.1 3.3 + 0.2

298.6 + 11.4 17.0 _+ 9.8

301.8 _ 8.4 -21.4 + 14.4

293.8 + 12.3 7.4 _+ 8.3

6.7 + 134.8 _+ 16.7 _+ 1.8 _+ 3.3 +

HC, High-carbohydrate diet; HF, high-fat diet. Values represent mean _ SEM for 3-5 rats per group. * p < 0.001 HC vs. HF diet. t P < 0.005 HC vs. HF diet. p < 0.05 HC vs. HF diet.

CHRONIC GALANIN INJECTIONS AND OBESITY

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of the injections was increased to three times daily, food intake was again stimulated during the daylight hours in animals eating the HF diet, but not in animals eating the HC diet. In the present experiment, we chose to administer galanin during the daylight hours because this is the time when minimal feeding occurs. The rat is a nocturnal animal and normally eats as much as 85% of 24-h energy intake at night (23). When animals are maintained on a 12:12 light:dark cycle there is an increase in food intake during the first 3 h of the dark period. When a third daily galanin injection was added on day 8, it was administered at 2300 h, several hours after the early dark feeding phase. From the data it appears that this third injection did not increase total nighttime intake of either diet. Galanin treatment alone did not produce a significant increase in body weight or fat pad weight in animals fed either a HC or HF diet. However, all animals fed the HF diet showed an increase in epididymal fat pad weight. In contrast, there was a small but persistent weight loss in both treatment and control groups fed the HC diet, and weight gain was attenuated in control animals fed the HF diet. We also observed that over the course of the experiment, rats injected with saline vehicle actually lost weight on the HC diet. The weight loss associated with central vehicle injections has been reported previously with both saline and artificial cerebrospinal fluid and appears to be reversed when the infusions are stopped (1,18). In our study, there appears to be an interaction between the HC diet and the presumed stress of these control injections. Finally, there remains the possibility that leakage of galanin from the cerebrospinal fluid could induce weight loss through some peripheral effect(s), as has been reported for peripheral administration of peptide YY (16). This event seems unlikely given that the doses of galanin used in these central injections, that is, 0.9 #g (300 pmol) per rat per injection, are similar to those used in other studies of the peripheral effects of galanin. For example, 1-4 #g/kg/h ofgalanin administered intravenously produced hyperglycemia and suppressed insulin levels in dogs (13). Also, intraperitoneal injections of galanin in doses of 0.11.0 ug increased serum CCK levels in rats (2). Although the central dose ofgalanin used in the present experiments was similar to that used for studies of peripheral responses (2,13), galanin has a short half life in the central nervous system (10) and would have to be transferred rapidly and completely for a peripheral response to occur. Regarding the possible effects of peripherally administered galanin on appetite or feeding, we were unable to find any report of this in the literature. Galanin injections did not induce any change in serum insulin, glucose, or corticosterone when measured at the time of sacrifice. However the HF diet significantly increased insulin levels independent ofgalanin treatment. Previous studies of galanin injected into the parventricular nucleus (PVN) have shown that this peptide inhibits both corticosterone and insulin release but has no effect on glucose levels when it is administered during the predark period (22). It is unlikely that the periodic injections of galanin given in

the present study would produce a sustained inhibition of these hormones. We are unable to explain the extreme variability in serum corticosterone levels among the experimental groups. The use of two and three ventricular injections per day in this experiment may have provided insufficient stimulation of feeding to induce weight gain and to overcome the compensatory reduction in nighttime food intake. The feeding behavior elicited by galanin has both a short latency of approximately 5 rain and a short duration ( 15-30 min) (8), producing its maximum effect within 60 min after injection. A small peptide, galanin is probably short lived in vivo due to rapid proteolytic degradation. For studies of chronic effects of cerebroventricular administration of a peptide, the use of osmotic minipumps may be necessary. Also, a higher dose of galanin may have been more effective in increasing total food intake and body weight. In studies of chronic neuropeptide Y (NPY) administration, the total daily dose employed was threefold (18) or sixfold higher (19) than the dose that produces the maximum acute feeding response. Moreover, the high doses of NPY used in those studies were injected directly into the PVN. The present study suggests the possibility of the development of tolerance to the feeding effects of galanin. From the data it appears that animals fed the HC diet were no longer responsive to galanin stimulation by day 6, when their daytime intake began to level off and then to diminish. A loss of stimulation was not apparent in animals fed the H F diet although the magnitude of galanin's acute effect on intake of HF diet was less than its effect on HC intake. Despite the fact that galanin clearly stimulates feeding when it is injected into the central nervous system, repeated administration of the peptide was ineffective in producing sustained hyperphagia and significant weight gain in nonobese rats. This is in contrast to chronic infusion of NPY that produces remarkable hyperphagia and obesity using a variety of diets and protocols (1,18,19,24). It may be worth noting that most studies of chronic NPY administration have used female rats, which have higher baseline fat intakes (18) and perhaps a greater propensity toward fat deposition. Other differences in the metabolic and endocrine effects of galanin and NPY have also been reported. NPY produces a long-lasting increase in respiratory quotient when injected into the PVN (15) indicating an increase in carbohydrate utilization. Although galanin has no effect on resting quotient, it elicits a short-lasting reduction in energy expenditure (14). NPY also stimulates the release of several hormones including corticosterone and insulin (11), two hormones that will promote fat deposition. In summary, the data reported in this paper do not support a role for galanin in promoting fat intake, chronic hyperphagia, or obesity. ACKNOWLEDGEMENT This research was supported by NIDDK Grant #32089.

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