Metabolic Responses to Intracerebroventricular Leptin and Restricted Feeding

Metabolic Responses to Intracerebroventricular Leptin and Restricted Feeding

Physiology & Behavior, Vol. 65, Nos. 4/5, pp. 839–848, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99 $–see fr...

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Physiology & Behavior, Vol. 65, Nos. 4/5, pp. 839–848, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99 $–see front matter

PII S0031-9384(98)00243-1

Metabolic Responses to Intracerebroventricular Leptin and Restricted Feeding TIANLUN WANG,*1 DIANE L. HARTZELL,* BARBRA S. ROSE,* WILLIAM P. FLATT,* MARTIN G. HULSEY,* NANDA K. MENON,† RONALD A. MAKULA† AND CLIFTON A. BAILE* *Departments of Animal Science and Foods and Nutrition, †Department of Biochemistry and Molecular Biology, The University of Georgia, 712 Boyd Graduate Studies Research Center, Athens, GA 30602 Received 28 July 1997; Accepted 19 August 1998 WANG, T., D. L. HARTZELL, B. S. ROSE, W. P. FLATT, M. G. HULSEY, N. K. MENON, R. A. MAKULA AND C. A. BAILE. Metabolic responses to intracerebroventricular leptin and restricted feeding. PHYSIOL BEHAV 65(4/5) 839–848, 1999.—Leptin is a hormone secreted by adipocytes, which plays an important role in the control of food intake and metabolic processes. In the current study, a dose-dependent relationship was shown between a bolus intracerebroventricular rat recombinant leptin administration and reductions in food intake and body weight in Sprague–Dawley rats. During the 24 h postinjection period, food intake was decreased by 24, 26, and 52% with 0.625, 2.5, and 10 mg of leptin, respectively. Body weight was reduced by 2, 3, and 5% at 24 h after leptin administration at the doses of 0.156, 2.5, and 10 mg, respectively. Furthermore, indirect calorimetry demonstrated that five daily i.c.v. injections of leptin resulted in an increase in heat production per unit of metabolic body size and fat oxidation by approximately 10 and 48%, respectively. In contrast, food-restricted rats that consumed the equivalent amount of food as leptin-treated rats for 5 days decreased their energy expenditure by 10%. Food restriction was found to decrease respiratory quotient in a similar pattern as the leptin administration. When ad lib feeding was resumed, food-restricted rats quickly recovered their normal food intakes, body weights, and metabolism. Conversely, leptin treatment has prolonged effects on body weight resulting from different metabolic responses than food restriction. Leptin not only suppresses food intake, but also enhances energy expenditure to reduce fat depots. © 1999 Elsevier Science Inc. Heat production

Respiratory quotient

Body weight

Food intake

Lepob mice (2,6,7,16,24). Rodent obese models other than Lepob/Lepob mice, such as the agouti mouse, tub, fat, or the AKR/J mouse (22), BAT-deficient mouse (12), and NZO mice (15,19) all exhibit higher leptin levels and apparent resistance to peripheral leptin than their lean controls. In this study, rat recombinant leptin was used to determine the dose response relationship between intracerebroventricularly (i.c.v.) injected rat leptin and changes in body weight, food intake, and water intake in nonobese Sprague–Dawley rats. In addition, the effect of daily i.c.v. injection of rat leptin on energy metabolism was investigated and compared with that of food restriction.

COMPLEX interactions between genes and the environment are responsible for body weight and body composition. Epidemiological studies and clinical research indicate that body weight remains remarkably constant over long periods of time in adults, suggesting that there are physiological control mechanisms for the regulation of body fat stores (3). An excess of energy intake over expenditure results in increased fat depots. Leptin is a circulating peptide encoded by the obese gene and secreted by adipocytes. It is postulated that the rate of leptin production increases with adiposity, which is a signal for the brain to regulate body weight and adiposity through changes in food intake and metabolism. Plasma leptin concentrations have been shown to be correlated to body fat depot status in normal and obese animals and humans (10–13,17,22). Administration of recombinant mouse or human leptin normalizes food intake and energy expenditure as well as other metabolic and reproductive physiological aspects of Lepob/ 1To

Water intake


Animals and Housing Adult male Sprague–Dawley rats (Harlan, Indianapolis, IN) were housed individually in suspended stainless steel

whom requests for reprints should be addressed. E-mail: [email protected]




cages in Experiment 1. The room was temperature controlled at 258C and on a 12 L:12 D cycle where the lights were on at 0400 h. The rats of Experiments 2 and 3 were housed in identical cages before the acclimation period of the experiments and then moved into automated open-circuit respiration chambers in the Calorimetry Laboratory. The light/dark cycles for Experiments 2 and 3 were 0300/1500 and 1000/2200 h, respectively. The ambient room temperature was maintained at 258C. The animals had free access to ground PMI Rodent Chow #5012 (Richmond, IN) and tap water unless otherwise noted. Lateral Cerebroventricular Cannulation and Verification of Cannula Placement The rats were anesthetized with 1 mL/kg of a 3:2:1 v/v/v mixture of ketamine HCl (Ketaset®, Fort Dodge Laboratories, Inc., Fort Dodge, IA; 100 mg/mL), acepromazine maleate (PromAce, Fort Dodge Laboratories, Inc., Fort Dodge, IA; 10 mg/mL), and xylazine (Rompun, Miles Inc., Schawnee Mission, KS; 20 mg/mL), and, when necessary, methoxyflurane (Metafane®, Pittman-Moore, NJ). The dorsum of the head was shaved and vacuumed. The rat was placed in a stereotaxic instrument (Kopf Instruments, Tujunga, CA) and the skin was disinfected with chlorhexidine (Nolvasan®, Fort Dodge, IA). A 22-gauge guide cannula (313GC, Plastics One, Roanoke, VA) was implanted into the right lateral cerebroventricle of each rat. The stereotaxic coordinates used were: AP, 0.8 mm, ML, 1.2 mm with respect to bregma, and DV, 23.5 mm from the skull surface. The cannula was held in place with three stainless steel machine screws (Small Parts Inc., Miami Lakes, FL) and cranioplastic cement (Plastics One) attached to the skull. A 28-gauge stylet (313DC, Plastics One) was installed into the guide cannula when the rat was not receiving injections. A recovery period following surgery extended at least a week before rats were subjected to an angiotensin II (ANG II) drinking test. ANG II (Sigma, St. Louis, MO) was dissolved in sterile artificial cerebrospinal fluid (aCSF) at a concentration of 10 ng/mL. An increased drinking response of at least 3 mL of water following an i.c.v. injection of 100 ng ANG II confirmed correct cannula placement. Cannula placement was verified again by the same drinking response to ANG II after the completion of each experiment, and the rats were euthanized with CO2. A 0.14% methylene blue solution (10 mL) was injected into the ventricle, and the brain was extracted from the skull. Cannula placement was confirmed by the appearance of dye in the ventricular system. Rat Leptin Source Recombinant rat leptin (The University of Georgia) was produced by transformed Escherichia coli. The leptin gene was obtained from Dr. Tohru Funahashi (14). It has an N-terminal Ndel site for cloning into Pet systems. The synthesized gene was cloned into Pet21b. The Pet21b construct was transformed into BL21 (DE3). Cultures were grown to an optical density of 1.0, measured at a wavelength of 550 nm, and then induced with 1 mM IPTG. Cells were grown in a media containing yeast extract, tryptone, and sodium phosphate. The cells were induced by a temperature shift for 3 h and then were harvested and frozen overnight at 2208C. Cells were disrupted by sonication in Tris-HCl buffer (pH 8.0, 50 mM) containing 5 mM EDTA, then centrifuged at 15,000 3 g for 30 min, and the supernatant was discarded. The pellet was washed and solubilized in 50 mL Tris-HCl containing 8 M urea, 2 mM phenylmethylsulfonyl fluoride, and 5 mM EDTA

FIG. 1. Silver stained 12% SDS polyacrylamide gel of recombinant rat leptin run under reducing conditions. Lane 1, molecular weight marker; lanes 2, vehicle, Tris-HCl buffer (50 mM PH 7.5, 1 mM EDTA, 1 mM dithiothretol, and 10% glycerol); lanes 3 and 4, 0.05 and 0.025 mg recombinant rat leptin, respectively.

at room temperature for 15 min. This was then centrifuged at 15,000 3 g for 10 min at 158C. The pellet was discarded and the supernatant was dialyzed, and then centrifuged at 15,000 3 g for 30 min. Storage of the leptin supernatant was at 48C. The leptin was dissolved in vehicle, Tris-HCl buffer (Tris-HCl 50 mM pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol) and diluted to appropriate concentrations. The product displayed a molecular weight of 14 kDa, and a purity approximately 98%, as judged by silver staining of SDS-PAGE analysis (Fig. 1). The specificity of the bioactivity of the protein was examined in Leprdb/Leprdb mice that do not respond to leptin (16) due to a point nonsense mutation in the leptin receptor gene (9,20). Daily intraperitoneal administration of rat leptin resulted in significant body weight loss in lean mice, but not in Leprdb/Leprdb mice (Fig. 2). Intracerebroventricular Injections All solutions injected into the lateral ventricle were given in a volume of 10 mL. Twenty-three-centimeter lengths of PE-20 tubing were affixed to the injector cannulae (C313I, Plastics One) and stored in 70% v/v isopropanol. Before each injection, the PE-20 tubing with an injector cannula was attached to 10 mL Hamilton syringe, and flushed with sterile aCSF, followed by the vehicle. Ten microliters of the injection solution were drawn into the PE-20 tubing. The stylet was removed from the guide cannula and the injector cannula was inserted. The injection lasted over 20 s, after which the injector cannula was left in place for an additional 20 s before being replaced by the stylet. Indirect Calorimetry A computer-controlled open circuit calorimetry system for rats (Oxymax, Columbus Instrument Co., Columbus, OH) was used in Experiments 2 and 3. It consisted of 10 respiration chambers, each with a feed hopper, water bottle, and wire floor. Oxygen consumption, carbon dioxide production, respiratory quotient (RQ), and heat production were measured for each rat at 16.5-min intervals. Airflow was controlled and measured using a mass flowmeter for each chamber. Gas com-



FIG. 2. Effect of leptin in 12-week-old male C57BL/6J 1/1 mice and C57BL/6J Leprdb/Leprdb mice. Mice received two intraperitoneal injections (50 mg leptin or 0.1 mL vehicle each) at 2 h before the beginning of the dark phase and 1 h before the onset of the light phase on each treatment day for 3 days. Body weight is expressed as the cumulative difference from the body weight on the day before the initial injection (day 0). Baseline weights for each group of Leprdb/Leprdb mice were: leptin, 36.66 6 0.42 g (n 5 6); and vehicle, 37.54 6 1.09 g (n 5 6). **Significantly different from the vehicle-injected 1/1 mice (p , 0.01). Data expressed as least square mean 6 standard error of the least square mean.

position of incoming outdoor air and exhaust gas were measured using an infrared gas analyzer for carbon dioxide, and an electrochemical oxygen sensor battery system, based on limited diffusion metal air battery for oxygen. The gas analyz-

ers were calibrated daily using cylinders of primary gas standard mixtures with known concentrations of CO 2, O2, and N2. The calculation for the RQ was: CO2 production (liters)/O2 consumption (liters). The percentages heat production (HP)







F(4, 39)


DBW (g/day) 1–24 h 24–48 h 48–72 h FI (g/day) 1–24 h

0.91* 1.60*†‡ 5.06

26.53† 6.30‡ 6.81

24.22ab 20.10ab 6.73

210.35† 2.25†‡ 4.25

218.30‡ 23.13* 3.66

8.57 3.61 0.96

,0.01 0.01 0.44




12.11* (252%) 13.95* (247%) 21.34


48–72 h WC(ml/day) 1–24 h

18.69† (226%) 20.54† (221%) 25.54


26.16 †

19.36† (224%) 23.49† (23%) 27.74


24–48 h

21.05†‡ (217%) 25.81† (21%) 28.40



45.14 ‡

26.94*† (240%) 35.71 41.68 37.81

23.95* (247%) 44.10 52.30 37.83


55.04 38.91 37.84

36.08†‡ (220%) 44.76 46.56 37.75


24–48 h 48–72 h 24 hr RT (8C)

35.04†‡ (222%) 51.46 42.83 37.95

0.83 0.81 0.22

0.51 0.53 0.92

Data shown are least square means. Values within a row with a common superscript (*, †, or ‡) are not significantly different (p $ 0.05).



FIG. 3. (A) Daily food intake (g/day) of i.c.v. leptin (10 mg/day, n 5 5) and vehicle (n 5 4)treated rats. (B) Body weight change (g). Treatments were administered on days 0, 1, 2, 3, and 4. **Significantly different from the vehicle-injected rats (p , 0.01). Data expressed as least square mean 6 standard error of the least square mean.

resulting from fat and carbohydrates oxidized were based on the RQ (4). Heat production calculations were based on the Brouwer (5) equation, HP (kcal) 5 3.866 O2 consumption (liters) 1 1.200 CO2 production (liters) 2 0.518 CH4 production (liters) 2 1.431 urinary nitrogen excreted (g). For laboratory animals, where there is no detectable methane produced, and where the nitrogen content of the diet is not varied, the formula used for the Oxymax program was as follows: HP 5

3.820 O2 consumption (liters) 1 1.150 CO2 production (liters). The notation for metabolic body size (MBS) is body weight in kilograms raised to the 0.75 power (kg0.75). Experiment 1 Five treatments were randomly assigned to 10 360 g i.c.v. cannulated rats that responded positively to ANG II accord-



FIG. 4. (A) Daily respiratory quotient (RQ) of rats administered i,c.v. leptin (10 mg/day, n 5 5) or vehicle (n 5 4). (B) Daily heat production per metabolic body size (MBS; kcal/kg0.75). Rats received treatments on days 0, 1, 2, 3, and 4. **Significantly different from the vehicle-injected rats (p , 0.01). Data expressed as least square mean 6 standard error of the least square mean.

ing to replicated 5 3 5 Latin square design. The five treatments were: 0, 0.156, 0.625, 2.5, and 10 mg leptin injected into the lateral ventricle. Each rat received one of the five treatments on a given day followed by 2 days of recovery. All injections were performed at 1 h before the beginning of the dark cycle. Body weight, food intake, and water intake were measured immediately before injection. Rectal temperature was measured 24 h after each i.c.v. injection.

Experiment 2 Ten i.c.v. cannulated rats (375.6 6 5.9 g) received either 10 mg of leptin in 10 mL of vehicle (n 5 5) or 10 mL of vehicle (n 5 5) injected daily for 5 days. Prior to treatment, all rats had a positive drinking response to ANG II and were acclimated in the 10 open-circuit respiration chambers. On the last 2 days of the acclimation period, 10 mL of aCSF was injected into the



lateral ventricle to adapt the rats to the i.c.v. injection procedure. Each day at 1400 h, the chambers were opened, and the rats were weighed and injected. Food cups and water bottles were weighed and refilled. Food spillage was weighed and the papers lining the chamber floor were changed. The data from the Oxymax system was included in the analysis only after the chambers regained equilibrium. Experiment 3 Ten noncannulated rats (380.7 6 8.1 g) were moved into 10 open-circuit respiration chambers. The rats had 2 days of adaptation before they were randomly assigned to two feeding regimes: restricted fed (n 5 5) and ad lib fed (n 5 5). The restricted-fed rats were pair fed on a daily basis to the rats receiving leptin in Experiment 2. The following calculation was used to determine the amount of food offered to the restricted-fed rats: the average food intake divided by the average body weight 24 h after leptin injection multiplied by the individual body weight of rats assigned within this experiment. The restricted-fed rats received half of the daily allotment of food at 1000 h and the other half at 2200 h. Ad lib-fed rats were also fed daily at 1000 h, and at 2200 h their food was stirred in the cup to bring the fresh feed to the top surface. Body weight, food cups, and water bottles of both groups were measured daily at 1000 h. On Day 6, the restricted-fed rats were returned to ad lib feeding. The recovery period was monitored for 3 days. Data Analysis The data were subjected to PROC GLM (SAS for Windows 6.1, USA, 1995). A two-way analysis of variance (ANOVA) with repeated measurements was applied. Significant F-values were followed by the preplanned multiple comparisons using the least-square means. Probability values less than 0.05 were considered significant. RESULTS

Experiment 1 Data from two rats that died due to the loss of stylets during the experiment were not included in the statistical analysis. Table 1 summarizes the dose response of i.c.v. injected leptin on body weight change, food intake, water intake, and rectal temperature. During the first 24-h period after the i.c.v. injection, food intake was reduced by 24, 26, and 52% with 0.625, 2.5, and 10 mg of leptin, respectively. Body weight was decreased by 2, 3, and 5% at 24 h after leptin administration at the doses of 0.156, 2.5, and 10 mg, respectively. There were 40 and 47% reductions in the first 24-h water consumption after injection of 2.5 and 10 mg of leptin, respectively. The i.c.v. leptin injection treatments resulted in no rectal temperature changes [Table 1, F(4, 35) 5 0.22, p 5 0.923]. Experiment 2 One rat of the vehicle-injected group lost its cannula, reducing the sample size to four. Significant differences in daily food intake were observed between the leptin-treated group and the vehicle-treated group, F(1, 7) 5 235.68, p , 0.001. The reduction of food intake by leptin occurred after the first i.c.v. leptin administration and persisted throughout the entire 5-day treatment period (Fig. 3A, p , 0.01). The two groups did not differ in water intake during the pretreatment or treatment periods, F(1, 7) 5 0.33, p 5 0.58, and F(1, 7) 5

2.71, p 5 0.14, respectively. The rats that received i.c.v. leptin experienced progressive body weight loss [Fig. 3B, F(1, 7) 5 149.06, p , 0.001] during the five daily leptin administrations (p , 0.05). A two-way ANOVA, F(1, 7) 5 163.42, p , 0.001, revealed a difference in RQ between leptin and vehicle injections in rats. The reducing effect of leptin could be observed after the initial i.c.v. leptin injection (Fig. 4A, p , 0.01). The leptintreated rats maintained lower RQ (p , 0.01) without fluctuations during the treatment period. The RQ of the vehicle-treated group did not differ from pretreatment period. Average estimated percentages of heat production from carbohydrates and fat metabolism were 93.6 and 6.4% for vehicle-treated rats, and 45.9 and 54.1% for leptin-treated rats, respectively. Both groups of rats produced essentially the same total amount of heat during the 24-h period, F(1, 7) 5 0.80, p . 0.40. The 24-h heat production adjusted for metabolic body size of the first 3 days did not show any differences between the two groups (p . 0.20). After the fourth leptin treatment, 24-h heat production per metabolic body size of the leptintreated group increased over the vehicle-treated group (14% increase, Fig. 4B, p , 0.01). After the fifth leptin injection, a 12% increase of 24-h heat production was observed compared to the vehicle-injected group (p , 0.01). Experiment 3 During the 5-day restricted feeding regime, rats consumed all the feed offered. The rats were returned to ad lib feeding following the 5-day restricted feeding period where they ate greater amounts of food than the ad lib-fed rats during the 3-day recovery period [Fig. 5A, F(1, 8) 5 27.12, p , 0.001]. This compensatory food intake response was seen on the first and third days of the recovery period (p , 0.01). Body weight gain varied greatly between the restricted-fed group and the group with free access to food, which continued to gain weight both during the treatment as well as the recovery periods [Fig. 5B, F(1, 8) 5 251.24, p , 0.001, and F(1, 8) 5 115.19, p , 0.001, respectively]. Unlike the leptin-treated rats that experienced continual weight loss every day throughout the treatment period in Experiment 2, the restricted-fed rats did not lose significant weight during Day 3 to Day 4 of the treatment. In addition, immediately after ad lib feeding was resumed in the restricted-fed rats, body weight increased and was similar to control rats. Similar to i.c.v. leptin, restricted feeding decreased RQ [Fig. 6A, F(1, 8) 5 1.29.47, p , 0.001]. Once ad lib feeding recommenced, no difference in RQ was evident between the restricted-fed group and ad lib-fed group (p . 0.05). On the third day, restricted-fed rats had a higher RQ than the control rats (p , 0.01). Heat production was 93.6% from the oxidation of carbohydrates in both the ad lib-fed rats and the restricted-fed rats during the entire experiment, except during the period of food restriction. During the food-reduction period, the restricted-fed rats averaged 50.7 and 49.3% of heat production from carbohydrates and fat utilization, respectively. Restricted feeding resulted in contrasting responses with leptin by decreasing daily heat production per MBS [Fig. 6B, F(1, 8) 5 8.84, p , 0.05]. Reduced heat production in feedrestricted rats was not apparent until day two of food restriction (Fig. 6B, p , 0.05). The restricted-fed rats produced 89, 88, 88, and 84% of the daily heat per MBS that ad lib rats produced after 2, 3, 4, and 5 days, respectively, on the restricted feed regime (p , 0.05). The heat production on the basis of



FIG. 5. (A) Daily food intake (g/day) of ad lib-fed (n 5 5) and restricted-fed (n 5 5) rats. (B) Body weight change (g). Two feeding regimes were offered to rats on days 0, 1, 2, 3, and 4. **Significantly different from ad lib-fed rats (p , 0.01). Data expressed as least square mean 6 standard error of the least square mean.

MBS was normalized to the same level as the control group on the first day of cessation of food restriction (p . 0.20). DISCUSSION

Leptin has a high degree of homology among mice, rats, and humans. Recombinant mouse and human leptin have been shown to be biologically active not only in lean and

Lepob/Lepob mice but also in the lean rats. The present study used recombinant rat leptin to demonstrate the effects of leptin in nonobese Sprague–Dawley rats. It showed a dose-dependent relationship between a bolus i.c.v. leptin administration and the reduction in food intake and body weight. In the present study, we measured rectal temperature at 24 h after i.c.v. injection. We did not observe an increase in rectal temperature caused by i.c.v. leptin administration in Sprague–



FIG. 6. (A) Daily respiratory quotient (RQ) of ad lib-fed and restricted-fed rats. (B) Daily heat production per metabolic body size (MBS; kcal/kg0.75). Two feeding regimes were offered to rats on days 0, 1, 2, 3, and 4. **Significantly different from ad lib-fed rats (p , 0.01). Data expressed as least square mean 6 standard error of the least square mean.

Dawley rats even in the face of an increased metabolic rate, indicating that leptin’s effect of increasing heat production could not be attributed to increased body temperature; the rats had apparent normal thermoregulation. Pellymounter et al. (24) only found an increase in body temperature of hypothermic Lepob/Lepob mice and not of lean mice. The results of our study confirmed that leptin does not influence the body temperature of normothermic animals.

In addition to changes in food intake and body weight, we observed an elevation in heat production and a decline in RQ after leptin injection. Decreases in food intake, body weight, and RQ occurred within the first day of leptin treatment, whereas increases in heat production became apparent only after the fourth leptin injection. Scarpace et al. (25) suggested that synthesis of new uncoupling protein may account for the delayed response in energy expenditure.



In Experiment 3, we pair fed a group of rats with the leptintreated rats of Experiment 2 to dissociate the effects of leptin on food intake from its effect on energy metabolism. The pair feeding resulted in a significant decrease in heat production per metabolic body size, while leptin induced pronounced elevation in energy expenditure. These data confirmed that leptin has a dual effect on energy intake and expenditure. Leptin-treated rats increased their energy expenditure despite the reduced food intake. Therefore, leptin’s effect on energy expenditure is dissociated from its effect on food intake, suggesting that leptin disrupted the mechanisms that normally coordinate food intake and energy expenditure in the energy balance equation. Leptin and food restriction increased utilization of fat as their energy source, as indicated by the decreases in RQ. The above findings support previous observations made in Lepob/Lepob mice reported by Hwa et al. (18) that i.c.v. injection of leptin increased thermogenesis and mobilized fat in Lepob/Lepob mice. Recent in vitro study indicates that leptin may also directly alter lipid partitioning in skeletal muscle when the maximal responses were observed at a concentration of leptin that is 100- to 1,000-fold higher than that in serum (23). The pattern of weight loss in leptin-treated rats differed slightly from that of food-restricted rats. It is reported that leptin’s weight-reducing effect did not start to exceed that of pair-fed Lepob/Lepob mice until the seventh day of leptin treatment in Lepob/Lepob mice (21). In the current study, a greater divergence in weight loss might have been observed if leptin treatments were continued.

Body weight, RQ, and heat production in food-restricted rats returned to normal within 24 h after the food restriction regime stopped, with a compensatory food intake. In another study, the recovery response was monitored in rats injected i.c.v. with rat leptin at a dose of 2.5 mg/day for 4 days. Food intake normalized within 3 days of the last treatment, but body weight had not recovered even after 8 days posttreatment when the animals were sacrificed (1). The sustained effect of leptin on body weight after termination of i.c.v. leptin treatment was also observed in lean Long–Evans rats (26) as well as in obese (Leprfa/Leprfa) Zucker rats (unpublished observation). The prolonged or irreversible specific effect of leptin on adipose tissue may account for the sustained effect on body weight. Eight days after the withdrawal of treatment, there was no difference in liver, kidney, heart, gastrocnemius muscle, and soleus muscle weights between control and leptintreated rats (1). However, epididymal and retroperitoneal fat pad weights of leptin-treated rats were considerably reduced from those of the control rats. Pair-feeding studies demonstrated that leptin exerts adipose-reducing effects greater than those induced by reduction in food intake (8,21). Thus, leptin has a more dramatic adipose tissue depleting effect. In summary, exogenously i.c.v. administration of rat leptin resulted in different metabolic responses in Sprague–Dawley rats than those in a food restriction paradigm, which matched the food intake to that of leptin-treated rats. Leptin not only inhibits food intake but also increases energy expenditure and depletes fat storage. The basis for this response remains to be fully elucidated.

REFERENCES 1. Azain, M. J.; Wang, T.; Hulsey, M. G.; Hartzell, D. L.; Baile, C. A.: Adipose tissue specific effects of intracerebroventricular leptin in rats. J. Anim. Sci. 75:167; 1997. 2. Barash, I. A.; Cheung, C. C.; Weigle, D. S.; Ren, H.; Kabigting, E. B.; Kuijper, J. L.; Clifton, D. K.; Steiner, R. A.: Leptin is a metabolic signal to the reproductive system. Endocrinology 137:3144–3147; 1996. 3. Bray, G.A.: Treatment for obesity: A nutrient balance/nutrient partition approach. Nutr. Res. 49:33–45; 1991. 4. Brody, S.: Methods in animal calorimetry. In: Brody, S., ed. Bioenergetics and growth. New York: Reinhold Publishing Corporation; 1945:307–313. 5. Brouwer, E.: Report of subcommittee on constants and factors. In: Blaxter, K. L., ed. Energy metabolism. Proceedings of the 3rd symposium, European assoc. animal prod., publ. 11. London: Academic Press; 1965. 6. Campfield, L. A.; Smith, F. J.; Guisez, Y.; Devos, R.; Burn, P.: Recombinant mouse OB protein: Evidence for a peripheral signal linking adiposity and central neural networks. Science 269: 546–549; 1995. 7. Chehab, F. E.; Lim, M. E.; Lu, R. H.: Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat. Genet. 12:318–320; 1996. 8. Chen, G.; Koyama, K.; Yuan, X.; Lee, Y.; Zhou, Y.; O’Doherty, R.; Newgard, C. B.; Unger, R. H.: Disappearance of body fat in normal rats induced by adenovirus-mediated leptin gene therapy. Proc. Natl. Acad. Sci. USA 93:14795–14799; 1996. 9. Chua, S. C., Jr.; Chung, W. K.; Wu-Peng, X. S.; Zhang, Y.; Liu, S.-M.; Tartaglia, L.; Leibel, R. L.: Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science 271: 994–996; 1996. 10. Considine, R. V.; Sinha, M. K.; Heiman, M. L.; Kiaucinas, A.; Stephens, T. W.; Nyce, M. R.; Ohannesian, J. P.; Marco, C. C.; McKee, L. J.; Bauer, T. L.; Caro, J. F.: Serum immunoreactiveleptin concentrations in normal-weight and obese humans. N. Engl. J. Med. 334:292–295; 1996.

11. Dua, A.; Hennes, M. I.; Hoffmann, R. G.; Maas, D. L.; Krakower, G. R.; Sonnenberg, G. E.; Kissebah, A. H.: Leptin: A significant indicator of total body fat but not of visceral fat and insulin insensitivity in African-American Women. Diabetes 45:1635–1637; 1996. 12. Frederick, R. C.; Hamann, A.; Anderson, S.; Lollmann, B.; Lowell, B. B.; Flier, J. S.: Leptin levels reflect body lipid content in mice: Evidence for diet-induced resistance to leptin action. Nat. Med. 1:1311–1314; 1995. 13. Frederick, R. C.; Lollmann, B.; Hamann, A.; Napolitano-Rosen, A.; Kahn, B. B.; Lowell, B. B.; Flier, J. S.: Expression of OB mRNA and its encoded protein in rodents. J. Clin. Invest. 96:1658–1663; 1995. 14. Funahashi, T.; Shimomura, I.; Hiraoka, H.; Arai, T.; Takahashi, M.; Nakamura, T.; Nozaki, S.; Yamashita, S.; Takemura, K.; Tokunaga, K.; Matsuzawa, Y.: Enhanced expression of rat obese (ob) gene in adipose tissues of ventromedial hypothalamus (VMH)lesioned rats. Biochem. Biophys. Res. Commun. 211:469–475; 1995. 15. Halaas, J.; Boozer, C.; Denton, D.; Blairwes, J.; Friedman, J.; Fidahuse, N.: Physiological-response to long-term peripheral and central leptin infusion in lean and obese mice. Proc. Natl. Acad. Sci. USA 9416:8878–8883; 1997. 16. Halaas, J. L.; Gajiwala, K. S.; Maffei, M.; Cohen, S. L.; Chait, B. T.; Rabinowitz, D.; Lallone, R. L.; Burley, S. K.; Friedman, J. M.: Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269:543–546; 1995. 17. Hickey, M. S.; Considine, R. V.; Israel, R. G.; Mahar, T. L.; McCammon, M. R.; Tyndall, G. L.; Houmard, L. A.; Caro, J. F.: Leptin is related to body fat content in male distance runners. Am. J. Physiol. 271:E938–E940; 1996. 18. Hwa, J. J.; Ghibaudi, L.; Compton, D.; Fawzi, A. B.; Strader, C. D.: Intracerebroventricular injection of leptin increases thermogenesis and mobilizes fat metabolism in ob/ob mice. Horm. Metab. Res. 28:659–663; 1996. 19. Igel, M.; Becker, W.; Herberg, L.; Joost, H.-G.: Hyperleptinemia in the New Zealand Obese (NZO) mouse reflects disruption of

848 leptin signaling from its receptor. Endocrinology 138:4234–4239; 1997. 20. Lee, G.-H.; Proenca, R.; Montez, J. M.; Carroll, K. M.; Darvishzadeh, J. G.; Lee, J. I.; Friedman, J. M.: Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632-635; 1996. 21. Levin, N.; Nelson, C.; Gurney, A.; Vandlen, R.; de Sauvage, F.: Decreased food intake does not completely account for adiposity reduction after ob protein infusion. Proc. Natl. Acad. Sci. USA 93:1726–1730; 1996. 22. Maffei, M. J.; Halaas, J.; Rayussin, E.; Pratley, R. E.; Lee, G. M.; Zhang, Y.; Fei, H.; Kim, S.; Lallone, R.; Ranganathan, S.; Kern, P. A.; Friedman, J. M.: Leptin levels in human and rodent: Measurement of plasma leptin and ob RNA in obese and weightreduced subjects. Nat. Med. 1:1155–1161; 1995.

WANG ET AL. 23. Muoio, D. M.; Dohn, G. L.; Fiedorek, F. T., Jr.; Tapscott, E. B.; Coleman, R. A.: Leptin directly alters lipid partitioning in skeletal muscle. Diabetes 46:1360–1363; 1997. 24. Pelleymounter, M. A.; Cullen, M. J.; Baker, M. B.; Hecht, R.; Winters, D.; Boone, T.; Collins, F.: Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540–543; 1995. 25. Scarpace, P. J.; Matheny, M.; Pollock, B. H.; Tumer, N.: Leptin increases uncoupling protein expression and energy expenditure. Am. J. Physiol. 273:E226–E230; 1997. 26. Seeley, R. J.; van Dijk, G.; Campfield, L. A.; Smith, F. J.; Burn, P.; Neligan, J. A.; Bell, S.M.; Baskin, D. G.; Woods, S. C.; Schwartz, M. W.: Intracerebroventricular leptin reduces food intake and body weight of lean rats but not obese Zucker rats. Horm. Metab. Res. 28:664–668; 1996.