INTRODUCTION

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IN 1973, KOLETSKY REPORTED the appearance of a spontaneous mutation that produced obesity in a hypertensive strain of rats (14). The autosomal recessive mutation was, in some cases, called corpulent. After introgression into the LA and spontaneously hypertensive rat (SHR)/N strains, rats carrying the corpulent mutation became useful models for carbohydrate-sensitive hyperlipoproteinemia (19) and for the relationships among hyperlipidemia, obesity, and coronary heart disease (22).

Corpulent rats were the second genetically obese rat to be reported. The first was the spontaneous mutation called fatty reported in 1961 (31). The phenotypes of both mutations are similar and include hyperphagia and obesity.

Recent work has shown that fatty and corpulent are allelic mutations of the leptin receptor (Lepr) on rat chromosome 5 (15). The rat fatty mutation (Lepr^fa) is a point mutation of codon 269 (CAGCCG, Q269P) near the ligand-binding domain of the receptor (4, 12, 20, 25). The substitution does not affect mRNA levels of Lepr, but may affect intracellular trafficking of the various isoforms of the receptor. Although transient transfection studies of a mouse Lepr cDNA containing the Q269P mutation showed normal affinity for leptin, but decreased binding (5, 21), binding of leptin by the choroid plexus was not different in frozen sections of brains from lean and fa/fa rats (9). In addition to the reports of decreased binding in transfection systems, there is now considerable evidence for deficits in the intracellular signaling function of Lepr^fa (6, 8, 28, 30).

The Koletsky mutation (Lepr^fak) is a point mutation that causes a premature stop at codon 763 (TATTAG, Tyrstop) before the transmembrane domain. This truncates all known isoforms of the receptor (26, 29). Thus, unlike the Lepr^fa mutation, the Lepr^fak mutation is null.

The decreased binding and deficits in intracellular signaling function of the Lepr^fa suggest that the effects of exogenous leptin on food intake and body weight should be less in fa/fa rats than in their lean littermates. This possibility has been investigated, and the results are inconsistent.

Single injections of leptin into the lateral (LV) or third ventricle (3V) decreased food intake in nondeprived lean and fa/fa rats in two experiments (16, 27), but not in a third (23).

Cusin et al. (7) reported that male fa/fa rats were 2-10× less responsive than male +/fa rats to the inhibitory effects of leptin on intake and body weight when a single injection of leptin was administered into the LV 3-4 h before refeeding after 24 h of food deprivation. The magnitude of the difference of potency of leptin in fa/fa and +/fa rats is uncertain, however, because doses <3 µg were tested in +/fa rats, but not in fa/fa rats.

In the only multiple-injection study that has been reported, Wang et al. (27) injected 2.5 µg leptin into the LV for 5 consecutive days in three fa/fa and three +/?fa rats and observed equivalent body weight loss, decreases of food and water intakes, and decreases of respiratory quotient.

One chronic infusion study has been reported (1). Murine leptin, infused systemically by a minipump for 7 days, significantly decreased food intake in +/?fa, but not fa/fa, rats. Leptin also produced a larger decrease in body weight in +/?fa rats than in fa/fa rats.

There is also evidence of decreased sensitivity to endogenous leptin in fa/fa rats. This conclusion is on the basis of experiments in which LV injection of an antibody, apparently specific for mouse and rat leptin, increased food intake in lean Sprague-Dawley rats, but not in fa/fa rats (3).

Given the inconsistent effects of leptin in fa/fa rats, we investigated the fa^k/fa^k rat because the fa^k/fa^k rat does not express any functional isoforms of the leptin receptor, and thus exogenous leptin should have no effect on body weight or food intake in fa^k/fa^k rats. The primary purpose of the experiments reported here was to test that prediction by administering exogenous leptin into the 3V for 5 consecutive days in +/+, +/fa^k, and fa^k/fa^k rats. Administration of leptin into the 3V gives leptin relatively direct access to its receptors in the ventromedial tuberal region of the hypothalamus, which does not depend on the receptor-mediated transport of leptin across the choroid plexus into the cerebrospinal fluid or from the blood into the hypothalamus. Thus if leptin had no effect in fa^k/fa^k rats, this could not be due to a failure in the transport of leptin to its receptors in the hypothalamus (16).

A second purpose of the experiments was to investigate further the synergistic effects of leptin and CCK-8 on food intake and body weight that have been reported in mice and rats. Intraperitoneal administration of leptin and CCK-8 decreased food intake in mice more in the first 3 and 24 h after injection than leptin or CCK-8 alone (2, 18). No synergistic effect, however, was observed in a 30-min intake test given 3 h after leptin (intraperitoneally) and immediately after CCK-8 (intraperitoneally) (18). The only study of this phenomenon in rats (Sprague-Dawley) found a significant synergism between leptin in the LV and CCK-8 (intraperitoneally) on food intake and body weight 24 h later, but not 3 h after the leptin injection (17). Because of its potential importance, we looked for this synergistic effect under our experimental conditions by administering CCK-8 (intraperitoneally) on the second and fifth days of central leptin administration just before a 30-min test of 10% sucrose intake.

	METHODS

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Eight obese (fa^k/fa^k) and eight lean (2 +/+, 2 +/fa^k and 4 +/?) LA/N rats were obtained from the breeding colony at Vassar College. Rats were housed individually in wire mesh-bottomed plastic cages and maintained on a 12:12-h light-dark cycle (lights off at 1400) in a room with a mean temperature of 23°C. Rats had ad libitum access to water and ground rat chow (Rodent Laboratory Chow 5001, Ralston Purina, St. Louis, MO), except as described below.

Surgery. Rats were surgically implanted with 22-gauge, stainless steel, guide cannulas (Plastics One, Roanoke, VA) into the 3V under surgical anesthesia (Chloropent; a mixture of chloral hydrate and pentobarbital, 0.8 ml/300 g for lean, 0.7 ml/300 g for obese rats). With the skull flat from bregma to lambda, stereotaxic coordinates were 2.8 mm posterior to bregma on the sagittal suture and 8.5 mm below the dura. The cannula was secured to the skull using stainless steel screws and cranioplastic cement before being occluded with an obturator (Plastics One). Rats were allowed at least 1 wk to recover from surgery. For 3V injections, rats were restrained in a towel and the obturator was removed. Injections were made with a Hamilton syringe connected to a 28-gauge cannula injector that protruded 1 mm beyond the end of the cannula. Cannula placement was confirmed by 3V administration of angiotensin II (0.15 µg, Sigma, St. Louis, MO) dissolved in 5 µl artificial cerebrospinal fluid. If a rat did not drink 5 ml of water within 30 min after angiotensin, it was not used. During the course of the experiment, angiotensin tests were repeated every 2 wk to ensure that the cannulas were patent. All of the results reported here were obtained from rats that met this criterion of patency.

Protocol. Experiments began 2-3 wk after surgery. The experimental protocol was run Mondays through Fridays (Table 1). Rats had continuous access to chow and water on Saturday and Sunday. Vehicle, 3 µl of sterile buffer of Trizma base (Sigma) dissolved in deionized water (0.1 M, pH 8), was injected into the 3V on each day at 0930 for 5 days. During the next week, one dose of leptin (0.125, 0.625, 1.25, 2.5, or 5 µg in 3 µl of vehicle for lean rats; 20 or 80 µg for obese rats; Prepro Tech, Rocky Hill, NJ) was injected into the 3V on each day at 0930 for 5 days. There were 5 days of vehicle treatment before each dose of leptin treatment. Lean rats received the doses of leptin in the order of decreasing magnitude; obese rats received 20 µg and then 80 µg.

Statistical analysis. The effects of leptin and vehicle treatments on body weight were analyzed by computing the body weight change from Monday at 0900 to Thursday at 0900. (The body weight on Thursday was used because some of the measurements of body weight on Friday at 0900 were missing.) The differences in body weight from the initial body weight on Monday were calculated in grams and as percentages.

	RESULTS

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Lean rats. After 3 consecutive days of 3V administration, all doses of leptin larger than 0.125 µg/day produced a significant weight loss compared with vehicle treatment in lean rats (Fig. 1). This response was dose-related for grams lost [F(5,35) = 16.33; P < 0.001] and percent body weight lost [F(5,35) = 17.46, P = 0.0001]. The body weight loss in response to leptin was not dependent on the rat's body weight just before the first injection of leptin (P values >0.07). The body weight loss after the first day of leptin treatment was maintained, but did not change significantly, despite two more injections of leptin [F(2,8) = 0.04, P = 0.96].

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Lean rats (+/+ and +/?fak on LA/N) exhibited a dose-dependent loss of body weight (g) and percent body weight during 3 days of leptin treatment at dose indicated. Loss of body weight was calculated by subtracting the body weight on Thursday at 0900 from initial body weights on Monday at 0900. Data are means ± SE of 7 or 8 animals/group. * P < 0.05 vs. vehicle injection; aP < 0.05 vs. 0.125 µg; b P < 0.05 vs. 0.625 µg.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Data are means ± SE of 8 lean rats during first 3 days of leptin (2.5 µg/day) or first 3 days of vehicle treatment during preceding week. Leptin decreased body weight (t values  4.71, P values < 0.01; A) and food intake (t values  3.23, P values < 0.05; B) on all 3 days compared with vehicle. There were no significant differences across the 3 days for weight loss. Decrease of intake on day 1, however, was significantly less than on days 2 and 3 [F(2) = 7.28, P < 0.01]. * P < 0.05 compared with vehicle.

Obese rats. As predicted by the nature of the Lepr^fak mutation, 3 days of treatment with 20 or 80 µg of leptin had no effect on body weight in fa^k/fa^k rats compared with vehicle treatment as measured by grams [F(2,11) = 0.06, P = 0.94] or percent body weight [F(2,11) = 0.06, P = 0.94; Fig. 3]. These relatively large doses of leptin also did not significantly decrease intake of chow [F(2,14) = 2.86, P = 0.09] over 3 days [F(2,14) = 0.77, P = 0.48; Table 3].

View larger version (17K): [in this window] [in a new window]   Fig. 3.   In fak/fak rats, leptin (20 or 80 µg/day) did not decrease body weight expressed either as grams or percent body weight. Data are means ± SE of 5-8 rats/group.

Effect of CCK-8. No dose of 3V leptin changed the magnitude of the inhibitory effect of CCK-8 on 30-min intake of 10% sucrose compared with its corresponding week of 3V vehicle in lean rats (Table 4). Furthermore, no dose of 3V leptin changed the 5-min interval intakes in the 30-min tests after CCK-8 or saline administration compared with 3V vehicle; the results of the experiments using 2.5 µg/day of leptin are presented in Fig. 4. There was also no significant difference in the potency of CCK-8 in lean and obese rats during a week of 3V vehicle injections (Table 5).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Intracerebroventricular leptin (2.5 µg) and CCK-8 (4 µg/kg ip) administration did not alter pattern of interval intake compared with that produced by Tris buffer vehicle (intracerebroventricularly) and CCK-8 [F(1,280) = 0.86, P = 0.61]. Data are means ± SE of intake of a 10% sucrose solution in 8 lean (+/+ and +/?fak) rats/group on days 1 and 2. Results are similar for days 4 and 5 (see Table 4).

	DISCUSSION

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The major result of these experiments is that 3V administration for 5 days of a dose of leptin that was 120 times larger than the smallest dose that decreased body weight in lean rats and 600 times larger than the smallest dose that decreased food intake in lean rats had no significant effect on food intake or body weight in fa^k/fa^k rats. This is consistent with the null mutation of Lepr in the fa^k/fa^k rat. The lack of response to leptin in the fa^k/fa^k rat is quite different from the variable response to LV or 3V administration of leptin in the fa/fa rat (1, 7, 16, 23, 27). Although differences in rat strain, preparation of leptin, and experimental protocol may account for the variability of results obtained in fa/fa rats, the fact that leptin produces some effect on food intake or body weight in the fa/fa rat indicates that the missense mutation does not result in complete loss of function of the receptor. In this context, the null mutation in fa^k/fa^k provides an advantage for the experimental analysis of the effects of leptin on the brain for the control of energy intake, expenditure, and storage.

Although 3V administration of leptin for 5 days decreased body weight and food intake in lean rats, there were differences in the sensitivity and temporal patterns of these effects. Although 0.125 µg/day decreased food intake, 0.625 µg/day was the smallest dose that produced significant loss of body weight. The pattern was also different. All efficacious doses of leptin decreased body weight as much on the first day of treatment as on the second or third. The effect of leptin on intake of chow was also equal across the first 3 days of treatment with 0.125, 0.625, and 1.25 µg/day (Table 2), but 2.5 µg/day decreased intake more on days 2 and 3 than on day 1, and 5 µg/day decreased intake more on day 2 than on day 1 or 3 (Table 2). It is possible that the increased effect of 2.5 and 5.0 µg/day on days 2 and 3 was due to the injection of CCK-8 (intraperitoneally) on day 2, because injections of CCK-8 (intraperitoneally) have been reported to increase the inhibitory effect of leptin on daily food intake in mice (18) and rats (17), but further experiments are necessary to evaluate that possibility.

We failed to detect any synergism among five doses of exogenous leptin (3V) and 4 µg/kg of CCK-8 (intraperitoneally) on intake of 10% sucrose in 30-min tests (Table 4 and Fig. 4). It is possible, of course, that such synergism might be observed in lean rats with other doses of leptin or CCK-8.

It is important to note, however, that endogenous leptin also did not increase the inhibitory potency of CCK-8 under these same test conditions. There was no significant difference between the decrease of intake in lean rats that have normal circulating leptin and the decrease of intake in fa^k/fa^k rats that cannot respond to their high circulating levels of leptin (29) because of the null mutation of Lepr (Table 5).

Our results are consistent with the failure to detect synergism between exogenous leptin and CCK-8 in 30-min intake tests in BALB/c mice (18), ob/ob mice (24) , and Sprague-Dawley rats (17). All of these results do not support the suggestion (10, 11, 13) that leptin decreases meal size during spontaneous eating by increasing the satiating potency of endogenous CCK released from the small intestine during a meal. That suggestion requires further testing.

In summary, very large doses of murine leptin administered into the 3V for 5 consecutive days had no effect on food intake or body weight in fa^k/fa^k rats. This is consistent with their mutation of Lepr that truncates all known isoforms of the receptor. Because leptin (3V) produced dose-related effects on food intake and body weight in +/+ and +/fa^k rats of the LA/N, the fa^k/fa^k rat is a good experimental model in which to analyze the controls of food intake, energy expenditure, and energy storage in the complete absence of the effects of leptin.

Perspectives

In the mouse, three null Lepr mutations are available (db^Pas, db³¹, db^NCSU) and in the rat, only one (fa^k). The null mice can be compared with Lep^db, fa^k can be compared with fa (Zucker) in which the receptor is expressed, but a Gln269Pro transversion affects intracellular trafficking or possibly signal transduction of Lepr.

To be most useful for studies of comparative physiology, the mutations should be examined on the same strain background. Such lines can be created once potentially interesting effects have been seen in extant lines. The Lepr^fak mutation represents a valuable research tool for understanding the molecular physiology of the leptin axis.