INTRODUCTION

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AN INCREASE IN DIETARY calories and fat has been shown to induce weight gain and increase adipose tissue mass in both rodents and humans. Several studies have suggested that weight gain may depend not only on caloric intake but also on the macronutrient composition of the diet, with adipose tissue mass increasing in proportion to the amount of fat in the diet (4, 8, 20). Leptin, the protein product of the ob gene, regulates food intake and energy expenditure and is involved in a signaling pathway from adipose tissue that acts to regulate body fat stores (27). Leptin circulates in proportion to total body fat mass (19), and its levels rise with increasing adiposity (7, 10, 18). The reason for the failure of elevated leptin levels, associated with weight gain, to restrain further increase in weight is not known; this has led to the suggestion that leptin resistance occurs.

The mechanisms for leptin resistance are uncertain. It has been suggested that leptin may have a limited ability to restrain intake of highly palatable, high-fat diets that are normally associated with an increase in adiposity (26). Previous studies in mice suggest that defects downstream of the leptin receptor in the hypothalamus may account for leptin resistance (11). The observation that in obese humans elevated plasma leptin is not associated with an increase in cerebrospinal fluid levels of leptin has led to the hypothesis that the leptin transport system may be defective or saturated in obese humans (6, 24). In addition, the finding that C57BL/6 and AKR diet-induced obese mice have preserved sensitivity to centrally administered leptin, despite resistance to peripherally administered leptin, is consistent with this concept (25).

Although there is substantial information regarding the actions of both high-fat diets and leptin in rodent models, little is known about the effect of diet on weight gain in marsupials. In addition, there is no information on the effects of leptin on body weight and adipose tissue mass in marsupials. This is of interest because marsupials have evolved separately from eutherian mammals since the early Cretaceous period, ~150 million years ago. Sminthopsis crassicaudata (the fat-tailed dunnart) is an Australian nocturnal marsupial (10-20 g), with a lifespan of ~2 yr in captivity. One of the unique features of this marsupial is that it stores ~25% of total adipose tissue in the tail (15). Removal of tail fat results in reaccumulation of the fat in subcutaneous adipose tissue depots, demonstrating that, like other mammals, S. crassicaudata regulates the total amount of fat stored (15). However, it is unknown whether an increase in dietary calories and fat will induce an increase in adiposity in S. crassicaudata and whether leptin regulates food intake and body weight. We have shown that S. crassicaudata prefer a diet of mealworms to their usual laboratory diet (13). Mealworms have a higher caloric density and fat and protein content (2.99 kcal/g, 30% fat, 60% protein) than laboratory diet (1.01 kcal/g, 20% fat, 25% protein), but the effect of these diets on body weight and fat mass have not been studied.

The aims of this study were to determine in S. crassicaudata 1) the effect of a high-calorie, higher-fat diet on body weight and fat stores and 2) the effect of peripheral leptin administration on body weight, tail width, and food intake when animals were fed laboratory diet alone (low calorie), compared with when they were given a choice between laboratory diet and the higher-calorie and -fat diet of mealworms.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

All experimental protocols were approved by the Animal Ethics Committee of the University of Adelaide.

Animals, Diets, and Drugs

A laboratory diet (1.01 kcal/g, containing ~70% water, and 20% fat, 25% protein, 55% carbohydrate by dry weight), consisting of a mixture of Woofs dog food (Ridley Agri Products) and Whiskas cat food (Uncle Ben's), as described by Bennett et al. (2), and water were available ad libitum, except where stated. Daily intake of laboratory diet was determined by weighing plastic bowls containing food immediately before and 24 h after feeding. To correct for weight loss through evaporation, bowls of food were placed in empty cages and weighed as above. Live mealworms (Tenebrio spp. larvae) (2.99 kcal/g, containing ~55% water, and 30% fat, 60% protein, 10% carbohydrate by dry weight) were provided ad libitum where stated. The mealworms were presented in a small jar, and intake was recorded by weighing the jar at each time point. Data for food intake are expressed as kilocalories per gram body weight or total kilocalories eaten.

Purified r-Met human leptin, 5 mg/ml, (Amgen) was stored at 70°C and diluted in sterile PBS, pH 7.2, to the required concentration before use. PBS or leptin (2.5 mg/kg) was administered intraperitoneally twice daily at 0830 and at 1600. Sterile 29-gauge needles were used, and injection volume did not exceed 350 µl.

Measurements

Body temperature. Body temperature (°C) was measured by inserting a thermocouple probe (Fluke 52K/J Electronic thermometer) 5 mm into the rectum.

Blood glucose. Blood was sampled by puncturing the orbital sinus using a 29-gauge needle and withdrawing blood into a 1-ml syringe. Approximately 50 µl of blood was collected, and the blood glucose concentration was measured immediately using a glucometer (Medisense, Waltham, MA).

Oxygen consumption and respiratory quotient. Oxygen consumption (O₂) and respiratory quotient (RQ) were determined at 24°C for each animal using previously established methods (14). Measurements were made over a 15-min period, and animals were studied in random order. Individual S. crassicaudata were placed in a clear Perspex cylindrical chamber (22 cm long × 8 cm in diameter) that was located in a large, photoperiod-controlled, constant-temperature cabinet (Quantech). The air outflow from the chamber was connected to an infrared gas (O₂ and CO₂) analyzer (Dept. of Physiology, University of Adelaide). The expired gas was initially passed through a column containing drierite crystals, CaSO₄, to remove water vapor. This dry air was then pumped through the gas analyzer at a known rate by a pump connected to a flow meter (calibrated to 575.09 ml/min). Fresh air entered the chamber from small inlet ports at the opposite end to the outlet port. Steady-state O₂ and CO₂ production (CO₂) were calculated using the Haldane equation modified for an open-flow system (9).

Briefly

(1)

and

(2)

substituting back and replacing I

(3)

where I is inspired, E is expired, F is fraction of total gas, and E is the flow rate through the analyzers.

CO₂ was determined by I_CO2 was taken to be zero. Both O₂ and CO₂ were expressed as milliliters per gram per hour after first converting the measured values to STPD by multiplying by (P₁ × T_s)/(P_s × T₁), where P₁ is atmospheric pressure, P_s is standard pressure (760 mmHg), T₁ is experimental temperature, and T_s is standard temperature (0°C).

RQ was calculated as

Statistical Analyses

Experimental Protocols

Experiment 2: Determination of effect of leptin on body weight, tail width, body temperature, food intake, O₂, and RQ and whether these responses are modified by diet. S. crassicaudata were divided into four groups of similar initial body weight (n = 9-10). Body weight, tail width, body temperature, and food intake were measured daily for 5 days. In each animal, PBS was injected (ip) at 1600 on day 5, at 0830 and 1600 on day 6, and at 0830 on day 7. On day 7 at 1600, one-half of the animals continued to receive PBS, and one-half were injected (ip) with purified r-Met human leptin in a dose of 2.5 mg/kg. Animals received twice-daily (ip) injections of PBS or leptin and their allocated diets for a further 12 days. In each of the PBS- and leptin-treated groups, one-half of the animals were fed with laboratory diet only and one-half with a combination of laboratory diet and mealworms. Baseline O₂ and RQ were determined for each animal on day 5, day 13 (i.e., 7 days after randomization to leptin treatment or PBS), and day 19 (i.e., 12 days of leptin treatment). Body weight, tail width, body temperature, and food intake were measured daily at 1530.

To determine whether there were any acute effects of leptin on food consumption, food intake was recorded in each animal at 0.5, 1, 2, 4, and 24 h after the 1600 injection on days 7 and 15.

Experiment 3: Determination of effect of leptin on blood glucose concentration and whether the responses are modified by diet. The protocol was similar to that used in expt 2. Baseline blood glucose was determined on day 5 at 0830 in 24 animals. Animals were then divided into three groups (n = 8) matched for initial body weight and blood glucose concentration. Injections of PBS were given as in expt 2. On day 7 at 1600, one group of animals received (ip) leptin at 2.5 mg/kg and was then refed with laboratory diet and mealworms. The other two groups received PBS; one of these then received laboratory diet and the other both laboratory diet and mealworms. Leptin or PBS injections were continued twice daily for a further 12 days. The blood glucose concentration was measured at 0830 on days 8 and 18.

	RESULTS

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Experiment 1: Effect of a Preferred Food

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Mean ± SE cumulative food intake in kilocalories (A) and in kilocalories per gram body weight (B), body weight (g; C), and tail width (mm; D) of long-day-housed (16 h light, 8 h dark) Sminthopsis crassicaudata given either standard laboratory diet () or mealworms () ad libitum for 22 days. Before commencement of the study, animals had constant access to standard laboratory diet only.

Body weight. At baseline, body weight was 16.7 ± 0.43 and 16.8 ± 0.42 g in the laboratory diet- and mealworm-fed groups, respectively (P = 0.89). Body weight increased by ~13% in response to a diet of mealworms, and by day 21 body weights were 15.5 ± 0.40 and 19.3 ± 0.44 g in the laboratory diet- and mealworm-fed groups, respectively (diet F_1,263 = 17.84, P = 0.002; diet × day F_21,263 = 10.78, P < 0.0001) (Fig. 1C).

Tail width. At baseline, tail widths were 3.78 ± 0.14 and 4.01 ± 0.36 mm in the laboratory diet- and mealworm-fed groups, respectively (P = 0.61). Tail width increased by ~17.7% in response to a diet of mealworms, and by day 21 tail widths were 3.52 ± 0.25 and 4.87 ± 0.32 mm in the laboratory diet- and mealworm-fed groups, respectively (diet F_1,143 = 5.42, P = 0.04; diet × day F_11,143 = 16.56, P < 0.0001) (Fig. 1D).

Body temperature. Baseline body temperatures were 34.0 ± 0.24 and 35.0 ± 0.29°C in the laboratory diet- and mealworm-fed groups, respectively (P = 0.03). Body temperature did not change significantly in response to diet, and on day 21 body temperature was 33.2 ± 0.38 and 33.5 ± 0.24°C in the laboratory diet- and mealworm-fed groups, respectively.

Experiment 2: Effect of Leptin on Body Weight and Tail Width in Relation to Diet

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Mean ± SE cumulative food intake (kcal) of long-day-housed (16 h light, 8 h dark) S. crassicaudata (from days 7 to 19) given intraperitoneal injections of PBS vehicle or leptin (2.5 mg/kg) twice daily and refed with either laboratory diet alone (A) or with a choice between laboratory diet and mealworms (B). , PBS + laboratory diet; , leptin + laboratory diet; , PBS + laboratory diet and mealworms; , leptin + laboratory diet and mealworms.

ANIMALS FED BOTH LABORATORY DIET AND MEALWORMS. Cumulative food intake did not differ significantly over the baseline period (days 1-7) between the PBS (94.91 ± 5.35 kcal)- and leptin (80.65 ± 5.25 kcal)-allocated groups (drug F_1,132 = 2.14, P = 0.16). There was, however, a significant drug × day interaction (drug × day F_6,132 = 4.48, P = 0.0005). Leptin had no significant effect on total food intake, and between days 7 and 19 cumulative food intake was 217.5 ± 16.5 and 190.0 ± 14.9 kcal in the PBS- and leptin-treated groups, respectively (drug F_1,246 = 1.08, P = 0.31; drug × day F_12,246 = 1.68, P = 0.07) (Fig. 2B).

FOOD CHOICE IN ANIMALS FED BOTH LABORATORY DIET AND MEALWORMS. In animals fed both laboratory diet and mealworms there was a significant reduction in the intake of laboratory diet in leptin-treated animals compared with controls. Cumulative intakes of laboratory diet from days 7-19 were 135.77 ± 26.05 and 75.87 ± 23.18 kcal in the PBS- and leptin-treated groups, respectively (drug F_1,246 = 3.66, P = 0.07; drug × day F_12,246 = 2.75, P = 0.002) (Fig. 3A). There was a nonsignificant increase in mealworm intake between the two groups. Cumulative mealworm intake from days 7-19 was 81.71 ± 24.65 and 111.25 ± 30.74 kcal in the PBS- and leptin-treated groups, respectively (Fig. 3B).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Mean ± SE cumulative food intake (kcal/g) of laboratory diet (A) and mealworms (B) of long-day-housed (16 h light, 8 h dark) S. crassicaudata (from days 7 to 19) given intraperitoneal injections of PBS vehicle or leptin (2.5 mg/kg) twice daily and refed with a choice between laboratory diet and mealworms. , PBS; , leptin.

Body weight. ANIMALS FED LABORATORY DIET ONLY. Body weight did not change over the baseline period (between days 1 and 7) in either the PBS (15.13 ± 0.36 vs. 15.51 ± 0.44 g)- or leptin (14.87 ± 0.38 vs. 14.62 ± 0.44 g)-allocated groups and was not significantly different between the two groups. Administration of leptin was associated with a fall in body weight of 8.34 ± 0.42%, and by day 19, body weight was 15.25 ± 0.26 and 13.4 ± 0.41 g in the PBS- and leptin-treated groups, respectively (drug F_1,341 = 7.41, P = 0.02; drug × day F_18,341 = 5.44, P < 0.0001) (Fig. 4A).

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Mean ± SE body weights (g; A and B) and tail widths (mm; C and D) of long-day-housed (16 h light, 8 h dark) S. crassicaudata given intraperitoneal injections of PBS vehicle or leptin (2.5 mg/kg) twice daily and refed from day 7 with either laboratory diet alone (A and C) or with a choice between laboratory diet and mealworms (B and D). Baseline body weights and tail widths were measured from days 1 to 6, and the first leptin injection was given on day 7. Placebo injections of PBS were given twice daily for 2 days before the first leptin injection. , PBS + laboratory diet; , leptin + laboratory diet; , PBS + laboratory diet and mealworms; , leptin + laboratory diet and mealworms. b.i.d., Twice daily

ANIMALS FED BOTH LABORATORY DIET AND MEALWORMS. Body weight did not change over the baseline period (between days 1 and 7) in either the PBS (14.63 ± 0.44 vs. 14.62 ± 0.54 g)- or leptin (14.51 ± 0.43 vs. 15.04 ± 0.50 g)-allocated groups, and body weight was not significantly different between the two groups. After commencement of the diets, both PBS- and leptin-treated animals showed a significant increase in body weight from baseline (P < 0.05), and on day 19 body weight was 15.84 ± 0.51 and 15.41 ± 0.53 g in the PBS- and leptin-treated animals, respectively (Fig. 4B).

Tail width. ANIMALS FED LABORATORY DIET ONLY. Tail width did not change over the baseline period (between days 1 and 7) in either the PBS (4.20 ± 0.14 vs. 4.19 ± 0.12 mm)- or leptin (4.33 ± 0.38 vs. 4.32 ± 0.22 mm)-allocated groups, and there was no significant difference between these groups. Leptin administration was associated with a decrease in tail width of 17.1 ± 0.1%, and by day 19, tail width was 4.00 ± 0.14 and 3.58 ± 0.20 mm in the PBS- and leptin-treated groups, respectively (drug F_1,341 = 0.25, P = 0.62; drug × day F_18,341 = 6.80, P < 0.0001) (Fig. 4C).

ANIMALS FED BOTH LABORATORY DIET AND MEALWORMS. Tail width did not change over the baseline period (between days 1 and 7) in either the PBS (3.98 ± 0.14 vs. 3.95 ± 0.15 mm)- or leptin (3.96 ± 0.19 vs. 3.97 ± 0.13 mm)-allocated groups, and there was no significant difference between the groups. Leptin administration had no significant effect on tail width, and on day 19 tail width was 4.13 ± 0.18 and 3.96 ± 0.16 mm in the PBS- and leptin-treated groups, respectively (Fig. 4D).

Body temperature. ANIMALS FED LABORATORY DIET ONLY. Body temperature did not change over the baseline period (between days 1 and 7) in either the PBS (34.52 ± 0.29 vs. 34.67 ± 0.21°C)- or leptin (35.10 ± 0.24 vs. 34.91 ± 0.24°C)-allocated groups, and there was no significant difference between the groups. On day 19 body temperature was 32.41 ± 0.68 and 32.78 ± 0.72°C in the PBS- and leptin-treated groups, respectively, and did not differ significantly from baseline.

ANIMALS FED BOTH LABORATORY DIET AND MEALWORMS. Body temperature did not change over the baseline period (between days 1 and 7) in either the PBS (34.71 ± 0.34 vs. 34.67 ± 0.23°C)- or leptin (34.28 ± 0.27 vs. 34.29 ± 0.18°C)-allocated groups, and body temperature did not differ significantly between these groups. On day 19 body temperature was 32.84 ± 0.48 and 33.57 ± 0.52°C in the PBS- and leptin-treated groups, respectively, and did not differ significantly from baseline.

Metabolic parameters. ANIMALS FED LABORATORY DIET ONLY. At baseline (day 5), O₂ and RQ did not differ between the PBS- and leptin-allocated groups. Administration of leptin had no significant effect on O₂ compared with PBS-treated controls on either days 13 or 19. On day 13, RQs were not significantly different from baseline; however, on day 19, leptin-treated animals had a significantly higher RQ than the PBS-treated controls (P = 0.05) (Table 1).

ANIMALS FED BOTH LABORATORY DIET AND MEALWORMS. At baseline (day 5), O₂ and RQ did not differ between the PBS- and leptin-allocated groups. On day 19, O₂ of the leptin-treated animals was less than that of the PBS-treated controls (P = 0.04). RQ was similar in the PBS- and leptin-treated groups on days 13 and 19 (Table 1).

Effect of leptin on short-term food intake and food choice. ANIMALS FED LABORATORY DIET ONLY. Leptin administration had no significant effect on subsequent 24-h food intake (laboratory diet) at any of the time points studied, on either day 7, after the first leptin injection, or on day 15, after 9 days of leptin treatment (Table 2).

ANIMALS FED BOTH LABORATORY DIET AND MEALWORMS. Leptin administration had no significant effect on subsequent 24-h food intake (both laboratory diet and mealworms) at any of the time points studied, on either day 7, after the first leptin injection, or on day 15, after 9 days of leptin treatment (Table 2).

FOOD CHOICE IN ANIMALS FED BOTH LABORATORY DIET AND MEALWORMS. On day 7, after the first leptin injection, leptin reduced intake of laboratory diet over the next 24 h compared with the control group (drug F_1,94 = 3.5, P = 0.08; drug × time F_4,94 = 5.10, P = 0.001) (Fig. 5A). On day 15, after 8 days of leptin treatment, leptin had no significant effect on the intake of laboratory diet at any of the time points studied (drug F_1,94 = 2.75, P = 0.12; drug × time F_4,94 = 1.92, P = 0.12) (Fig. 5B). Compared with the PBS-treated animals, leptin had no significant effect on the subsequent 24-h intake of mealworms on either day 7, after the first leptin injection (drug F_1,94 = 2.07, P = 0.1686; drug × time F_4,94 = 2.24, P = 0.07) (Fig. 5C), or on day 15, after 8 days of leptin treatment (drug F_1,94 = 1.02, P = 0.33; drug × time F_4,94 = 0.90, P = 0.47) (Fig. 5D).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Mean ± SE food choice in long-day-housed (16 h light, 8 h dark) S. crassicaudata given an intraperitoneal injection of PBS vehicle or leptin (2.5 mg/kg) and refed with a choice between laboratory diet and mealworms. Cumulative intake of laboratory diet (kcal) on day 7, after the first leptin injection (A), and on day 15, after 8 days of leptin treatment (B), and cumulative intake of mealworms (kcal) on day 7, after the first leptin injection (C), and on day 15, after 8 days of leptin treatment (D), are shown. Injections were given at lights out, and food intake was measured 0.5, 1, 2, 4, 6, and 24 h after injections. Open bars, PBS; filled bars, leptin.

Experiment 3: Effect of Leptin on Blood Glucose Concentration in Relation to Diet

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

We have demonstrated that in the marsupial S. crassicaudata, body weight and fat mass (tail width) increased when animals were fed a highly palatable, preferred diet of mealworms, which are calorie dense and have a greater fat and protein content than standard laboratory diet. Furthermore, although leptin administration induces a decrease in body weight and fat mass in animals fed standard laboratory diet, leptin fails to effect either body weight or overall energy intake when animals are fed both laboratory diet and mealworms. These data suggest that an increased intake of dietary fat, protein, and calories results in increased adiposity, associated with resistance to the effect of exogenously administered leptin.

In the current study, the diet of mealworms resulted in a 13% increase in body weight and a 17.7% increase in tail width, indicating an increase in body fat stores in S. crassicaudata. These findings are consistent with studies in rats showing that an increase in dietary fat and caloric density results in increased body weight under ad libitum feeding conditions (23). Animals that received a mealworm diet consumed 10% more calories and 39% more calories as fat than animals receiving standard laboratory diet. Small daily increases in energy intake may have been sufficient to predispose S. crassicaudata to an increase in adiposity. However, an increased intake of fat alone may predispose to increased adiposity. We have shown previously that, as in rodents (4, 23) and humans (8), body fat stores in S. crassicaudata increase in response to increased dietary fat, and this is independent of total calories (20).

The effects of leptin on body weight in both lean and genetically obese rodent models have been studied extensively. In the ob/ob leptin-deficient mouse, leptin administration caused a reduction in food intake of up to 40% within 4 days, and this was associated with a dramatic fall in body weight of ~40% within 33 days (12). Leptin has also been shown to decrease body weight in the monkey and dog (21). In wild-type mice, leptin administration induces a fall in body fat from 12.2 to 0.67% (12). In S. crassicaudata fed standard laboratory diet, leptin administration induced a decrease in body weight and food intake. The observation that tail width, a reflection of fat stores (15), decreased suggests that fat mass was reduced. To our knowledge, this is the first demonstration that leptin can induce a decrease in food intake and cause weight loss in marsupials.

Leptin has been shown to have both short-term (5) and relatively sustained (25) effects on energy intake. In S. crassicaudata, as well as inducing an overall decrease in cumulative energy intake, leptin administration had acute effects on food consumption. When animals were fed laboratory diet alone, leptin induced a 17 and 22% reduction in 24-h food intake on days 7 and 15, respectively. Although this did not reach statistical significance, our findings compare with a 10% decrease in 24-h food intake in lean mice following leptin administration (5).

We cannot necessarily conclude that either the increase in calories or the effect of any specific macronutrient in the mealworm diet was responsible for the observed effects, because the diets were markedly different not only in fat content but also in protein content. In addition, as mealworms are highly palatable to S. crassicaudata, there remains the possibility that food intake was prejudiced by dietary preference (13). Nevertheless, our data are consistent with previous findings in rodents (10, 25). Studies in mice have shown that high-fat diets induce an increase in leptin levels associated with the development of obesity. The mice, however, became obese without decreasing caloric intake (10). These studies further suggest that a high content of dietary fat changes the set point for body weight, partly by limiting the actions of leptin. It has also been demonstrated that diet-induced obese mice exhibit resistance to peripheral leptin but retain sensitivity to centrally administered leptin (25). This finding is consistent with the observation that leptin transport into the cerebrospinal fluid is saturable (6). However, in contrast to the above findings, it has been shown in diet-induced obese rats that the response to intracerebroventricular leptin is attenuated compared with control animals (26). The mechanisms responsible for diet-induced leptin resistance remain unknown. It has been suggested that dietary weight gain might impair the satiety signal due to either reduced receptor sensitivity, occupation of receptors by endogenous leptin molecules (26), or interference with the leptin postreceptor signaling pathways (10). In addition, an increase in fat may stimulate hypothalamic centers involved in appetite regulation that are downstream of the initial leptin target (10). Further studies are required in this area.

Overall calorie intake was slightly decreased by leptin treatment in animals offered a choice of foods, but this decrease did not reach statistical significance. Furthermore, the proportion of laboratory diet and mealworms consumed differed significantly between the leptin- and PBS-treated animals. After the first injection of leptin on day 7, animals offered a choice of foods reduced their 24-h intake of the laboratory diet compared with PBS-injected controls. This decrease was counteracted by a nonsignificant increase in the intake of the preferred high-calorie diet of mealworms. Consequently, an increased proportion of the higher-fat, calorie-dense mealworms was consumed relative to the PBS-injected controls. As a result, the total amount of fat consumed was comparative between the two groups. The failure of leptin treatment to decrease the intake of mealworms is consistent with the hypothesis that leptin has a limited ability to restrain intake of highly palatable diets (26).

The effect of leptin on body weight is not due solely to a reduction in food intake, but also to an overall increase in energy expenditure. For example, in the ob/ob mouse, single intraperitoneal doses of leptin have been shown to increase O₂ (12, 16, 22), indicating an increase in energy expenditure. In normal mice, leptin prevents the decrease in energy expenditure during the nadir of the diurnal cycle and also prevents the decrease in energy expenditure that would normally occur with weight loss (16). In S. crassicaudata fed laboratory diet alone, body temperature and O₂ were similar in both PBS- and leptin-treated animals. This was despite the significant fall in body weight in the leptin-treated animals, and our results are therefore consistent with previous observations (12, 16, 22). Among the animals offered a choice of foods, O₂ was observed to be lower in the leptin-treated animals compared with PBS-treated controls at day 15. This finding can be attributed to an increase in O₂ in PBS-treated animals as opposed to a decline in leptin-treated animals. The absolute O₂ in the leptin-treated animals did not change from baseline to day 15. The increased weight in the PBS-treated animals was presumably associated with an increased energy expenditure. Why this same effect was not observed in the leptin-treated animals is unknown.

In addition to effects on energy expenditure, leptin administration has also been shown to affect nutrient partitioning. In ob/ob mice, leptin causes a decrease in RQ, indicating an increase in fat oxidation (16). In S. crassicaudata fed laboratory diet only, RQ increased in the leptin-treated animals, indicating a change from fat to carbohydrate utilization. RQ, however, was only measured after 6 and 9 days of leptin treatment. An initial decrease in RQ may have been observed if measurements were taken closer to the onset of leptin treatment, as the animals oxidized fat in response to leptin. Substrate utilization switches from fat to carbohydrate as fat stores become depleted, and RQ increases as a consequence. Despite the consumption of a higher-fat diet by the leptin-treated animals that received both laboratory diet and mealworms, there was no change in RQ. This suggests that fat was stored rather than oxidized.

In S. crassicaudata, plasma glucose levels failed to change significantly in response to leptin. This finding is consistent with previous studies. Although in the hyperinsulinemic and hyperglycemic ob/ob mouse leptin administration decreases serum glucose and insulin, lean controls show no change in glucose or insulin with leptin administration (22). In addition, Kamohara et al. (17) found that neither intracerebroventricular nor intravenous leptin infusion affected plasma glucose or insulin levels in wild-type lean mice. The above study did, however, show acute effects of leptin infusion on glucose metabolism, with a 5-h infusion increasing glucose turnover and glucose uptake but decreasing hepatic glycogen content.

Whether a higher dose of leptin may have overcome the effect of the mealworm diet used in this study was not addressed. The dose used, and the frequency of leptin administration, was based on published studies in other species and the results of several preliminary studies. The dose chosen was the minimum dose that reproducibly produced a decrease in body weight and food intake in this animal. The object of the study was to determine whether this dose of leptin would also prevent and increase in adiposity when the animals were fed a preferred diet that contained more calories and increased fat and protein compared with carbohydrate. The issue of whether a suprapharmacological dose may have had an effect was not addressed. Additional studies are also needed to determine the effect of specific macronutrients and how these interact with caloric content. A further limitation of this study is the absence of a pair-fed control group of animals to account for changes in weight on the measured parameters, and this should be included in future study designs. Nevertheless, our observations are consistent with those in other species.

We conclude that leptin has effects on food intake, energy expenditure, and fat mass in S. crassicaudata. Furthermore, S. crassicaudata, like rodents, exhibits dietary-induced resistance to the actions of leptin, which fails to decrease the intake of a highly palatable, calorie-dense, high-fat diet or to induce either weight loss or prevent weight gain when this diet is offered. These findings provide further evidence that the actions of leptin are highly conserved and, by implication, may function similarly in humans.

Perspectives

Although we are unable to separate the effects of total calories from that of specific nutrients in this study, our data, together with similar data in rodents, suggest that from a therapeutic standpoint leptin is most likely to be effective for treating human obesity when combined with a low-fat, low-calorie diet (or modified protein diet).