NPY ablation in C57BL/6 mice leads to mild obesity and to an impaired refeeding response to fasting

Gabriella Segal-Lieberman, Daniel J. Trombly, Viral Juthani, Xiaomei Wang, and Eleftheria Maratos-Flier

Research Division, Joslin Diabetes Center and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neuropeptide Y (NPY) is an orexigenic (appetite-stimulating) peptide that plays an important role in regulating energy balance. When administered directly into the central nervous system, animals exhibit an immediate increase in feeding behavior, and repetitive injections or chronic infusions lead to obesity. Surprisingly, initial studies of Npy-/- mice on a mixed genetic background did not reveal deficits in energy balance, with the exception of an attenuation in obesity seen in ob/ob mice in which the NPY gene was also deleted. Here, we show that, on a C57BL/6 background, NPY ablation is associated with an increase in body weight and adiposity and a significant defect in refeeding after a fast. This impaired refeeding response in Npy-/- mice resulted in a deficit in weight gain in these animals after 24 h of refeeding. These data indicate that genetic background must be taken into account when the biological role of NPY is evaluated. When examined on a C57BL/6 background, NPY is important for the normal refeeding response after starvation, and its absence promotes mild obesity.

neuropeptide Y; C57BL/6 background


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FEEDING IS REGULATED by a complex interaction between peripheral and central signals. Peripheral signals including leptin (2, 17), ghrelin (41), and peptide YY (3-36) (4) are processed by key neurons in the hypothalamus. In the central nervous system (CNS), several peptidergic systems that are involved in feeding pathways have been identified. Orexigenic signals such as neuropeptide Y (NPY) (13, 38) or agouti-related peptide (AgRP) (25, 34) in the arcuate nucleus and melanin-concentrating hormone (MCH) (23, 31, 33) in the lateral hypothalamus all induce feeding when administered intracerebroventricularly at pharmacological doses. Anorectic signals, on the other hand, are primarily mediated via the melanocortin system. For example, alpha -melanocyte-stimulating hormone (alpha -MSH) and the melanocortin receptor agonist MTII lead to decreased feeding (16, 28, 39). In addition, 5-hydroxytryptamine (serotonin) is known to suppress appetite (7, 22, 40) via potential effects on the melanocortin system (18).

Additional information on the role of CNS peptides has been derived from genetic studies in which the signaling systems are disrupted. Genetic disruption of the melanocortin system results in obesity in both rodents and humans (20, 43). This disruption was reported to occur at the level of the gene encoding the proopiomelanocortin from which alpha -MSH is processed. In addition, slightly different obesity syndromes are seen when either of the central melanocortin receptors MC3-R (8, 10) or MC4-R (9) is disrupted. Results from studies on genetic disruption of orexigenic peptides have been less consistent. Mice lacking the gene for MCH are lean as a result of a decrease in feeding and an increase in energy expenditure (35). Mice lacking the MCH receptor MCHR-1 are also lean, although the primary cause of their leanness is increased motor activity (12, 26). In contrast, disruption of either the NPY gene or the AgRP gene does not affect body weight or feeding behavior (14, 30), whereas disruption of two NPY receptors, Y1 and Y5, leads to mild obesity (21, 24, 29). Further studies indicated that NPY is essential for the development of the full obesity syndrome in leptin-deficient ob/ob mice (15). NPY ablation in these animals attenuated their weight gain, which supports a role for this neuropeptide in mediating the increased adiposity in ob/ob mice. Interestingly, a subsequent study showed that Npy-/- mice backcrossed for two generations onto a C57BL/6 background had an anxiogenic-like phenotype and appeared to be hypoalgesic in a hot-plate paradigm in addition to demonstrating a modest deficit in refeeding after starvation (3).

A potential problem with the studies reported thus far is that many of the mice analyzed were of a mixed genetic background, 129/J and C57BL/6. These two backgrounds have different sensitivities to diet-induced obesity. Hence, due to lack of genetic homogeneity in the subject mice, mixed genetic background may confound feeding studies.

To further define the role of NPY in feeding behavior, in the present study we have backcrossed subject mice at least seven generations onto a C57BL/6 background. After being backcrossed, mice were evaluated in terms of body weight, adiposity, metabolic parameters, feeding patterns, and responses to fasting.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All studies were conducted with the approval of the Joslin Diabetes Center Animal Care and Use Committee. The creation of Npy-/- mice has been described previously (14), and these mice were the generous gift of Dr. Richard Palmiter, University of Washington, Seattle, WA. Npy+/- mice were bred and backcrossed for seven generations onto a C57BL/6 background at Taconic Farms (Germantown, NY) and were then bred to each other to generate homozygous Npy-/- mice and littermate wild-type (WT) controls. Genotypes were confirmed by PCR. Mice were weaned at 3 wk of age, genotyped, and housed in groups of 2-4 mice per cage. Animals were given access to a diet comprised of 9% fat by weight (LabDiet 5021) and water ad libitum and were maintained in a temperature-controlled room (24°C) under an alternating 12:12-h light-dark cycle.

Fasting/refeeding groups of studies. Groups of 20-wk-old mice were fasted for a period of 24 h starting at the beginning of the light cycle. At the end of the fast, preweighed food was reintroduced, and food intake was monitored for 1, 3, 6, 24, and 48 h. Body weight was measured before and immediately after the fast as well as 6, 24, and 48 h after refeeding.

Restricted-feeding paradigm. Mice were placed on a restricted-feeding paradigm as previously described (1). Six 4-mo-old male Npy-/- mice and seven age-matched WT controls were allowed limited access to food for only 4 h during the light cycle, between 1100 and 1500, for a period of 18 days. Body weights were recorded on a daily basis just before the animals received food. Mice were given a preweighed amount of food, and food intake was measured daily at the end of the 4-h feeding period.

Indirect calorimetry. Metabolic rate was measured for four 28-wk-old female Npy-/- mice and four WT control mice. These animals were those previously used at 20 wk of age for the manual fasting/refeeding study described earlier. Metabolic rate was measured by indirect calorimetry using an eight-chamber open-circuit Oxymax system component of the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH). Mice were maintained at ~24°C under a 12:12-h light-dark cycle (light period 0800-2000). Food and water were available ad libitum except where noted below. Animals were individually housed in specially built Plexiglas cages (5 × 4.5 × 8.5 in.), through which room air was passed at a flow rate of ~0.54 l/min. Exhaust air from each chamber was sampled at 1-h intervals for a period of 1 min. Sample air was sequentially passed through O2 and CO2 sensors (Columbus Instruments) for determination of O2 and CO2 content. All mice were acclimatized to the cage for 24 h before hourly recordings of physiological parameters commenced. Mice then underwent a 3-day course in the CLAMS: 24 h fed, 24 h fasted, and 24 h refed. Mice were weighed before each trial.

Food intake and diet composition. During acclimation and testing periods, female Npy-/- mice (n = 4; 28.6 g mean body wt) and WT littermates (n = 4; 26.5 g mean body wt) were individually housed in microisolation cages and maintained on milled Labdiet 5021 (9% fat by weight) under a light cycle from 0800 to 2000. During CLAMS monitoring, feeding was assessed using ground food placed in eight acrylic side feeders, each attached to a microisolation cage. Side feeders rested on balances directly linked to a computer for measuring continuous food intake. During each one of the feeding periods, cumulative food intake was automatically recorded every hour for 24 h.

Motor activity. Ambulatory activity of individually housed mice was evaluated within the metabolic chambers on a relative basis using an eight-cage rack OPTO-M3 sensor system (Columbus Instruments). Consecutive photobeam breaks occurring in adjacent photobeams were scored as an ambulatory movement. Cumulative ambulatory activity counts were recorded every hour throughout the light and dark cycles.

Body fat measurement. Eight-mo-old male Npy-/- and age-matched WT littermate controls were analyzed using dual-energy X-ray absorptiometry (DEXA; Lunar PIXImus2 mouse densitometer; GE Medical systems, Madison WI) as described by the manufacturer. Briefly, the machine was calibrated daily using a phantom provided by the manufacturer. The system has an image acquisition time of <5 min. The interassay coefficient of variation was 0.22%. Before being scanned, mice were intraperitoneally injected with a (1:1) mixture of tribromoethanol and tert-amyl alcohol (15 µl/g body wt), and an in vivo prediction of fat content was subsequently performed.

Statistics. Values are reported as group means ± SE. Interactions between genotype and physiological parameters across light and dark cycles were analyzed by one-way ANOVA (factorial) when appropriate. The level of significance was taken as P < 0.05. Statistical comparisons were made using Statview (Abacus Concepts, Berkley, CA) software.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body weights. Weights of both male and female Npy-/- mice vs. their respective WT counterparts were monitored on a weekly basis beginning from 3 wk of age until the animals were 17 wk old. As shown in Fig. 1A, both male Npy-/- mice and male WT animals exhibited similar body weights up to 11 wk of age. However, from week 12 onward, male Npy-/- mice exhibited a significantly higher body weight than their WT counterparts. At 12 wk, male Npy-/- mice weighed 29.14 ± 0.7 vs. 27.07 ± 0.5 g for WT males (P = 0.016), and this difference in body weight was maintained through week 17. A similar trend was observed for female Npy-/- mice compared with their WT counterparts (Fig. 1B). Both Npy-/- and WT females showed similar weights up to 14 wk of age. However, by week 15, female Npy-/- mice weighed 24.76 ± 1.5 vs. 22.06 ± 0.7 g for female WT animals (P = 0.03), and this increase in body weight was also maintained through week 17. Npy-/- females were further followed up to 37 wk of age and at this later time point still weighed significantly more than their WT counterparts (39.83 ± 1.52 vs. 30.38 ± 1.11 g, respectively, P = 0.0005; Fig. 1B). Thus, on a C57BL/6 background, both male and female Npy-/- mice exhibited increased body weights compared with WT mice.


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Fig. 1.   Body weights of male wild-type (WT) and neuropeptide Y-deficient [NPY knockout (KO), or Npy-/-] (A) and female WT and Npy-/- mice (B). Weights were measured weekly, beginning from 3 wk of age until the animals were 17 wk old (n = 13-14/group). Weights for female WT and Npy-/- mice were further monitored up to 37 wk of age. Results are means ± SE. *P < 0.05 vs. WT mice.

Body fat composition. To further define the role of NPY in body weight composition, the percentage of total body fat was measured in four 32-wk-old male Npy-/- and WT controls by DEXA scanning. As depicted in Fig. 2, Npy-/- mice had significantly higher total percent body fat compared with WT animals (40.87 ± 0.7 vs. 37.22 ± 0.7% respectively). In parallel, we also determined the percentage of lean body mass for these animals. The percent lean body mass for Npy-/- mice was 59.12 ± 0.7%, whereas that for WT animals was 62.77 ± 0.7% (P = 0.012).


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Fig. 2.   Percent body fat and percent lean body mass as determined by dual-energy X-ray absorptiometry scanning in 32-wk-old male WT and Npy-/- mice. *P = 0.012 vs. WT mice.

Fasting/refeeding. Under normal conditions, Npy-/- and WT mice have similar food intakes, with 20-wk-old animals from both groups consuming ~3.4 g of chow per day. To determine the effects, if any, of Npy ablation on refeeding after a prolonged fast, Npy-/- male and female mice and their WT counterparts were fasted for 24 h (from the beginning of one light cycle to the beginning of the light cycle on the successive day). After being fasted, animals were subsequently allowed to refeed ad libitum, and short-term food intake was measured for <= 6 h. In addition, food intake was determined over more prolonged intervals of 24 and 48 h. After being fasted for 24 h, all animals increased their food intake compared with their baseline intake; however, female Npy-/- mice ate significantly less than their WT counterparts (Fig. 3, A and B). This reduced refeeding behavior manifested itself as early as 1 h after the animals were allowed to refeed [0.9 ± 0.05 (WT) vs. 0.4 ± 0.07 g (Npy-/-), P < 0.0001] and persisted up to the 48-h time point [11.6 ± 0.06 (WT) vs. 9.4 ± 0.08 g (Npy-/-), P < 0.0001]. The greatest disparity in food intake was observed during the first 6 h of refeeding, during which WT mice ate more than twice as much as Npy-/- mice. A similar pattern was observed for male Npy-/- mice vs. their WT counterparts. However, the reduced food intake in male Npy-/- mice did not reach statistical significance (data not shown).


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Fig. 3.   Effect of fasting on food intake in female WT and Npy-/- mice. Animals were fasted for 24 h and subsequently allowed to refeed ad libitum. Short-term food intake was measured for <= 6 h (A), and food intake was also determined over more prolonged intervals of 24 and 48 h (B). *P < 0.0001 vs. WT mice. C: in parallel, food intake was measured by the Comprehensive Lab Animal Monitoring System (CLAMS) in both WT and Npy-/- mice fed ad libitum as well as in animals refed after a 24-h fast.

Food consumption was also monitored hourly using metabolic chambers. As shown in Fig. 3C, an analysis of food consumption in ad libitum-fed WT and Npy-/- mice revealed that, as expected, the majority of the feeding occurred during the dark cycle. Both groups of animals ate ~1 g of chow during the light cycle and about three times as much during the dark cycle. Mice were then fasted for 24 h, and food was reintroduced at the beginning of the light cycle. As shown in Fig. 3C, when WT mice were refed after a fast, they significantly increased their feeding rate compared with their basal feeding pattern. After a fast, these animals no longer exhibited the typical minimal eating seen during the light cycle, and the feeding throughout the 24-h interval became linear, with animals consuming equal amounts of chow (2.5 g) during the light and dark cycles. In contrast, after the fast, Npy-/- mice retained their feeding pattern seen before the fast and did not show the accelerated feeding observed in the WT animals after a fast.

Body weights of both female and male Npy-/- mice and their WT counterparts during the fasting and refeeding phases are shown in Fig. 4, A and B, respectively. During the 24-h fasting period, both WT and Npy-/- mice lost ~10% of their initial body weights. However, once allowed to refeed ad libitum, both male and female WT mice regained weight much more rapidly than Npy-/- mice. WT females returned to their initial weights within 24 h, whereas Npy-/- females regained weight at a much slower rate and failed to return to their initial body weights even after 48 h of refeeding (Fig. 4A). WT males lagged slightly behind WT females but had regained 96.4% of their body weights after 24 h of refeeding. At the same time, Npy-/- males, like the Npy-/- females, regained weight at a significantly slower rate compared with their WT counterparts (Fig. 4B).


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Fig. 4.   Body weights of 20-wk-old WT and Npy-/- mice during fasting and refeeding phases. Weights of female (A) and male (B) WT and Npy-/- mice were measured before and at the end of a 24-h fasting period. Body weights were subsequently measured during a 48-h refeeding period at the indicated intervals. Results are expressed as %basal body weight for each animal. *P < 0.004 vs. WT mice.

Because Npy-/- mice are slightly heavier than their WT counterparts, the food restriction experiment was also performed in a female cohort of Npy-/- mice in which the WT controls had been matched for weight. The same refeeding deficit was seen in the weight-matched animals (data not shown). We also examined refeeding in animals that had food withdrawn at the onset of the dark cycle and reintroduced 24 h later at the onset of the next dark cycle. Under this regimen, during the first 4 h of refeeding, Npy-/- and WT mice ate 2.36 ± 0.014 and 2.69 ± 0.085 g, respectively (P = 0.024). By 24 h of refeeding, Npy-/- and WT mice had eaten 5.05 ± 0.168 and 6.04 ± 0.0224 g, respectively (P = 0.01), and both groups had returned to their prefast body weights. These data further confirm that, after a fast, Npy-/- mice eat less than their WT counterparts. However, when animals are refed at the onset of the dark cycle, cumulative food intake during the first 4 h in both Npy-/- and WT mice is greater than when refeeding is initiated at the onset of the light cycle. Furthermore, the refeeding deficit of Npy-/- mice compared with their WT counterparts is attenuated when refeeding is initiated at the onset of the dark cycle.

Restricted feeding. To further examine the effect of NPY ablation on the response to altered food availability, we utilized a restricted-feeding paradigm (1). Four-month-old Npy-/- and WT mice were allowed access to food only during the light cycle, between 1100 and 1500. Neither normal nor NPY-ablated mice were able to fully adapt to the timed feeding (Fig. 5). Food intake during the first 24 h fell below 1 g in both groups (Fig. 5A). Food intake began to increase on the 2nd day, but Npy-/- animals lagged behind WT animals until day 11, when feeding curves overlapped. Subsequently, both sets of animals continued to slowly increase food intake toward normal (Fig. 5A). Over the first 4 days, both control and Npy-/- animals lost 15% of their initial body weight (Fig. 5B). The Npy-/- mice lost correspondingly more weight, and their weights finally stabilized at a lower level compared with control mice (Fig. 5B).


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Fig. 5.   Effect of NPY ablation on adaptation to altered food availability. Twenty-week-old WT and Npy-/- males were allowed restricted access to food for only 4 h during the light cycle (1100-1500). Food intake (A) and body weights (B) were monitored daily for 17 days under this restricted-feeding paradigm (*P < 0.04 vs. WT mice). SE for food intake in WT mice ranged from 0.001 to 0.11 g and for Npy-/- from 0.007 to 0.16 g. SE for body weight in WT mice ranged from 0.29 to 1.18% and for Npy-/- from 0.38 to 0.64%.

Metabolic analysis of Npy-/- mice. To further characterize potential defects in energy balance resulting from NPY ablation, the metabolic rates for both WT and Npy-/- animals were determined by indirect calorimetry using a CLAMS apparatus. Acclimated animals underwent a 3-day regimen in the CLAMS consisting of 24-h feeding, 24-h fasting, and 24-h refeeding. Oxygen consumption (VO2), when corrected for body weight, was the same in both Npy-/- and WT mice during fed, fasted, and refed phases; in both sets of animals, VO2 dropped significantly during the fasted interval and rose with refeeding (Fig. 6). Simultaneously, the respiratory exchange ratio (RER) was calculated as an index of carbohydrate and lipid metabolism. As shown in Fig. 7, A and B, the RER for both WT and Npy-/- mice was the same during both fed and fasted phases. However, during the first 9 h of refeeding, Npy-/- mice exhibited a marked reduction in RER compared with WT mice (Fig. 7C). This indicates that, during the first 9 h of refeeding, compared with WT mice, Npy-/- mice utilized lipids to a greater extent than carbohydrates as a source of oxidizable substrates. To rule out differences in locomotor activity as an underlying cause for the late-onset obesity observed in Npy-/- mice compared with their WT counterparts, their activity was evaluated. As shown in Fig. 8, locomotor activity was reduced for both animal groups in the fasted state compared with either the fed or refed states. However, no difference in activity was observed between Npy-/- mice and WT animals.


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Fig. 6.   Effect of NPY deficiency on oxygen consumption (VO2). VO2 for both female WT and Npy-/- mice was measured by indirect calorimetry with a CLAMS apparatus. VO2 was measured hourly for 3 consecutive days comprised of 24 h of feeding (A), 24 h of fasting (B), and 24 h of refeeding (C). SE for the fed state in WT mice ranged from 532 to 1,453 ml · kg-1 · h-1 and for Npy-/- from 181 to 685 ml · kg-1 · h-1. SE for fasted state in WT mice ranged from 399 to 1,154 ml · kg-1 · h-1 and for Npy-/- from 39 to 344 ml · kg-1 · h-1. SE for refed state in WT mice ranged from 613 to 1,824 ml · kg-1 · h-1 and for Npy-/- from 90 to 878 ml · kg-1 · h-1.



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Fig. 7.   Effect of NPY deficiency on respiratory exchange ratio (RER). In conjunction with VO2 measurements, RER was also determined for both female WT and Npy-/- mice. Conditions were those described in the legend to Fig. 6. SE for the fed state in WT mice ranged from 0.044 to 0.09 and for Npy-/- from 0.021 to 0.082. SE for fasted state in WT mice ranged from 0.023 to 0.07 and for Npy-/- from 0.001 to 0.037. SE for refed state in WT mice ranged from 0.034 to 0.085 and for Npy-/- from 0.01 to 0.07.



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Fig. 8.   Locomotor activity is unaffected by NPY ablation. In parallel with measurements for VO2 and RER described in Figs. 6 and 7, locomotor activity was also assessed for both WT and Npy-/- mice with a CLAMS apparatus during 3 days of feeding (A), fasting (B), and refeeding (C). SE for the fed state in WT mice ranged from 484 to 2,520 and for Npy-/- from 293 to 1,864. SE for fasted state in WT mice ranged from 2 to 2,071 and for Npy-/- from 94 to 1,686. SE for refed state in WT mice ranged from 14.5 to 1,936 and for Npy-/- from 82 to 958.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NPY appears to be an important regulator of energy balance. When administered intracerebroventricularly to rats, it leads to a robust induction of food intake. Furthermore, chronic infusion of NPY leads to increased adiposity and insulin resistance as well as to a decrease in basal energy expenditure (6, 27, 44). Interestingly, reports to date indicate that mice lacking NPY show no abnormality in terms of weight, food intake, or energy expenditure (5, 14, 27). Similarly, a recent study showed that mice lacking both NPY and AgRP have no obvious feeding or body weight deficits and have a normal response to starvation (30). However, NPY ablation in leptin-deficient ob/ob mice attenuated their obesity (15), indicating that increased NPY production induced by leptin deficiency in the ob/ob mouse contributes to the increased adiposity seen in this model.

In addition, a previous study reported that, compared with WT animals, Npy-/- mice backcrossed for two generations onto a C57BL/6 background had a diminished response to refeeding after a prolonged fast (3). In that study, the authors reported a 25% reduction in food intake after a fast by Npy-/- mice compared with their WT counterparts. Our study confirms these findings and shows an even greater reduction in postfast food intake of up to 50% for the Npy-/- mice compared with their WT counterparts. The differences between the study performed by Bannon et al. (3) and our study, in terms of the magnitude of the refeeding deficit in Npy-/- mice, may stem from the different degree of backcrossing of the animals in the two studies. Furthermore, the timing of the refed interval in the former study is unclear. If refeeding took place at the beginning of the dark cycle, this could potentially explain the lower magnitude of the refeeding deficit obseved in Npy-/- mice by Bannon et al. Similarly, other data suggest that NPY deficiency attenuates the hyperphagia seen with streptozotocin-induced diabetes (36). In aggregate, these data suggest that the absence of NPY may attenuate certain obese phenotypes and also cause subtle changes in feeding under conditions where hyperphagia is expected.

Genetic background plays an important role in energy balance and may affect the susceptibility of an animal to diet-induced obesity. For example, mice on a C57BL/6 background are more prone to becoming obese when placed on a high-fat diet than mice on a 129 background (32, 37). Most knockout mouse strains, including Npy-/- mice, are on a mixed 129 × C57BL/6 background. When Npy-/- mice were analyzed on this background, no differences were detected compared with WT mice in terms of body weight, basal food intake, and food intake in response to fasting (14), and it is possible that the mixed background may have obscured differences in energy balance.

In the present study, we reevaluated the phenotype of Npy-/- mice by utilizing animals backcrossed for seven generations onto a C57BL/6 background. We wanted to further explore the possibility that Npy-/- mice have an energy homeostasis deficit that may be masked on a mixed genetic background. Interestingly, we found that these mice exhibited mild obesity beginning at 12-13 wk of age. Under normal conditions, we did not observe differences in food intake between Npy-/- and WT mice. However, our methodology is inadequate for measuring small changes in food intake, and it is possible that a small increase in feeding over time in Npy-/- mice is responsible for their mild obesity. The obesity in our Npy-/- mice was associated with a small but statistically significant increase in total body fat as well as a decrease in lean body mass, leading to an alteration in the ratio of lean body mass to fat body mass. It should be noted that a similar increase in weight with no apparent change in food intake was also reported for Y1r- and Y5r-null mice (21, 29, 42). Thus the absence of either NPY ligand or its cognate receptors may lead to mild obesity. The mechanisms underlying this effect remain unclear and will be the subject of future studies.

We also found a significant deficit in refeeding after a 24-h fast when the fast was timed to begin and end at the onset of the light cycle. This effect was initially measured manually but was more dramatically demonstrated in mice studied in metabolic chambers. When food was returned to mice fasted for 24 h at the onset of the light cycle, WT mice immediately began eating at an accelerated rate, whereas Npy-/- mice ate at the rate of nonfasted animals. This failure to refeed adequately was associated with a persistent decrease in the RER, indicating that the mice were still in negative caloric balance and were preferentially utilizing fat for fuel. When the fasting and subsequent refeeding phases were initiated at the beginning of the dark cycle, the deficit in refeeding was not as dramatic and was consistent with the refeeding deficit previously reported (3). These data indicate that the refeeding response to a fast is mediated, at least in part, by upregulation and increased secretion of NPY in the hypothalamus (19). However, the data also indicate that environmental cues such as ambient light are important in initiating feeding and are likely to be reinforced by circadian changes in other hormones and neuropeptides that signal "feeding time." In the absence of environmental and circadian cues, the lack of NPY leads to a failure in the refeeding response. To confirm that the reduced refeeding in Npy-/- mice was not related to the slight increase in their adiposity, this experiment was repeated in weight-matched animals, and the same impairment was noted for both male and female Npy-/- mice. To evaluate the chronic response of Npy-/- mice to altered food availability, a restricted-feeding paradigm was utilized. Under this regimen, mice chronically fasted for 20 h and subsequently allowed to refeed only during a 4-h window in the light phase also exhibited a postfast refeeding impairment over a period of several days. It is somewhat paradoxical that impairment of NPY signaling, either at the level of the ligand or at the level of the receptor (Y1R and Y5R), leads to obesity despite a postfast refeeding deficit. Currently, the basis for this observation remains unclear. However, these findings suggest that refeeding after a fast and the maintenance of body weight under normal conditions may be regulated by different central pathways. Interestingly, unlike Npy-/- animals, mice lacking another orexigenic peptide, MCH, on either a mixed (35) or a pure C57BL/6 genetic background (E. Maratos-Flier, unpublished observations), do not show a postfast refeeding deficit, suggesting that the role of NPY in mediating food-seeking behavior after a fast is relatively specific.

VO2, carbon dioxide production, and activity in Npy-/- mice were the same as those of controls in the fed, fasted, and refed states. Both gas exchange and activity decreased significantly in both groups of mice when animals were fasted, declining to levels that were 50% of those seen in the fed state. It should be noted that the gas measurements are adjusted for body weight (Vgas · kg-1 · h-1). There is some debate as to the best method for reporting VO2; however, it should be noted that, without the weight adjustment, Npy-/- mice would have shown a 10% increase in VO2. This slight increase in metabolic rate is expected, given the known pharmacological effect of NPY to reduce metabolic rate (27). Similarly, RER was the same in both WT and Npy-/- animals except for the first 12 h of the refed state. The lower RER observed in Npy-/- mice during the first 9 h of refeeding after a 24-h fast reflects a continued reliance on fat as a fuel, given their inability to ingest sufficient carbohydrates as a source of fuel during the refeeding interval.

In both the fed and fasted states, a distinct diurnal variation in activity was seen. In contrast, in WT animals, the diurnal cycle was disrupted, as refed WT animals showed an increase in activity during the light cycle related to compensatory feeding. This increase in activity observed in WT but not in Npy-/- mice as food was reintroduced may reflect a difference in motivation to seek food after a fast. Although WT mice seemed to be aroused by the reintroduction of food, Npy-/- mice appeared to be much less stimulated by its presence.

In summary, these results indicate that NPY ablation on a C57BL/6 background leads to increased body weight despite apparently normal food intake as well as to a diminished refeeding response to starvation. Taken together, these findings further underscore the importance of NPY in regulating energy balance, in particular in mediating refeeding responses after a prolonged period of food deprivation


    ACKNOWLEDGEMENTS

We thank Dr. Richard Palmiter for generously providing the Npy-/- mice and for many helpful suggestions. We also thank Dr. Richard L. Bradley for helpful discussions, and Elena Nikiforova for valuable assistance on the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1 DK-56116 and RO1 DK-53978 to E. Maratos-Flier. E. Maratos-Flier is also a Principal Investigator on Program Project DK-56113.

Address for reprint requests and other correspondence: E. Maratos-Flier, Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215 (E-mail: emf1{at}joslin.harvard.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 11, 2003;10.1152/ajpendo.00491.2002

Received 11 November 2002; accepted in final form 9 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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