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Increasing neuropeptide Y (NPY) signaling in the paraventricular nucleus (PVN) by recombinant adeno-associated virus (rAAV)-mediated overexpression of NPY in rats, results in hyperphagia and obesity in rats. To determine the importance of hyperphagia in the observed obesity phenotype, we pair-fed a group of AAV-NPY-injected rats to AAV-control-injected rats and compared parameters of energy balance to ad libitum fed AAV-NPY-injected rats. For 3 weeks, AAV-NPY-injected rats, received the same amount of food as ad libitum-fed rats injected with control rAAV They did not gain more body weight than these controls. When allowed access to food ad libitum, these AAV-NPY-injected rats increased food intake, which subsequently decreased when rats reached the same body weight as AAV-NPY-injected rats that were fed ad libitum for the entire study. These data indicate that overexpression of NPY in the PVN results in obesity by increasing food intake until a certain body weight is achieved.
Long-term central administration of neuropeptide Y (NPY) results in an obese phenotype, characterized by hyperphagia, increased lipogenesis in liver and adipose tissue, and elevated plasma concentrations of leptin, insulin, and corticosterone (1,2,3,4). Obesity is also induced when NPY is overexpressed in the adult hypothalamus of mice after hypothalamic injections of recombinant adeno-associated viral (rAAV) particles (5). Recently, we have shown (with the use of rAAV-NPY injected at the site of NPY receptors in the paraventricular nucleus (PVN)) that increased NPY signaling in the rat PVN is sufficient to result in obesity (6,7).
We here determine whether the obesity, induced by long-term NPY overexpression in the PVN in the adult rat, is dependent on increased food intake.
Rats were injected with rAAV-NPY (n = 18) or control (AAV-contr; n = 6) in the PVN of male Wistar rats (220–250 g; Crl-Wu, Charles River, Sulzfeld, Germany) and body weight gain examined for 50 days as described previously (7). Some of the rats (n = 9) were pair-fed to controls (AAV-NPY-pf) for 3 weeks. These rats obtained the same amount of food that was ingested by the AAV-contr rats the day before. Transmitters were placed under anesthesia (7) in the abdominal cavity (TA10TA-F40; Data Sciences International, St Paul, MN) to monitor locomotor activity and body temperature. Differences in body weight, food intake, body temperature, and locomotor activity were assessed using repeated measure analysis (SPSS for Windows, version 15.0; SPSS, Chicago, IL). When significant overall interactions were found, post hoc analyses were performed with t-tests or one-way ANOVA. All experimental procedures were approved by the Committee for Animal Experimentation of the University Medical Center Utrecht, the Netherlands.
Viral-induced NPY expression in the PVN at 50 days after injection was confirmed by in situ hybridization on coronal sections (16 µm) of the hypothalamus as described previously (6). AAV-infected areas were considered correctly targeted when bilateral staining was detected in the PVN, with the extension to the immediate surrounding area of the PVN. Animals with incorrectly targeted injection were excluded from analysis (in total, one rat from AAV-contr, two rats from AAV-NPY-al, and three rats from AAV-NPY-pf were excluded).
Food intake was analyzed over periods of 5 days. Rats that were injected with AAV-NPY and fed ad libitum (AAV-NPY-al) increased food intake during the first 3 weeks, after which it slowly decreased again. At the end of the study, food intake was no longer significantly different from controls (Figure 1a,b). Body weight gain was significantly increased in AAV-NPY-al rats compared to controls in the first part of the study; however, between day 35 and 50 the body weight gain was no longer significantly different (Figure 1c,d).
AAV-NPY rats that were pair-fed to the AAV-contr rats (AAV-NPY-pf) showed a decreased body weight gain when compared to controls (Figure 1c,d). This resulted in a cumulative body weight gain that was significantly lower than that of AAV-contr rats 20 days after injection (12.28 g ± 5.22 vs. 33.9 g ± 2.12 g for AAV-NPY-pf vs. AAV-contr rats, P < 0.01). The rats that were pair-fed consumed all their food in the first half of the dark period (data not shown).
The reduction in food intake seen in ad libitum-fed AAV-NPY-injected rats from day 40 tended toward those seen in controls. To determine whether this reduction was caused by adaptations downstream of viral-induced NPY signaling, we allowed the pair-fed animals to eat ad libitum from day 21 until the end of the experiment. Food intake of AAV-NPY-pf animals immediately increased when they were fed ad libitum, and food intake remained elevated until around day 40, after which food intake was not significantly increased compared to the control group (Figure 1a,b). Cumulative food intake from day 21–34 of AAV-NPY-pf rats was increased when compared to AAV-NPY-al rats (475.2 ± 39.0 g vs. 388.5 ± 16.5 g, P < 0.05).
Directly after being fed ad libitum, AAV-NPY-pf rats started to increase body weight gain. Twenty hours after their first day with ad libitum food, cumulative body weight gain of AAV-NPY-pf rats was already similar to that of control rats (day 21: 39.6 ± 3.38 vs. 37.7 ± 1.77 g for AAV-NPY-pf vs. AAV-contr rats). As of 4 days after ad libitum food, their cumulative body weight gain was significantly higher than that of controls (day 24: 60.6 ± 5.12 vs. 45 ± 2.33 g for AAV-NPY-pf vs. AAV-contr rats, P < 0.05). Between day 21 and 34, body weight gain per day was also increased when compared to AAV-NPY-al animals (P < 0.05) (Figure 1d). From day 34 after injection, body weight gain of the AAV-NPY-pf rats had normalized to the extent that average body weight gain per day was no longer significantly from controls and AAV-NPY-al rats.
In the light phase, body temperature was similar between groups. In the dark phase, body temperature was significantly reduced in AAV-NPY-injected animals, starting 2 weeks after injection, regardless whether the rats were fed ad libitum or pair-fed (Figure 1f). Dark phase body temperature remained significantly reduced during the entire experiment. As shown in Figure 1e–h, on day 20, the day before the pair-fed rats received ad libitum food intake, there was no difference in core body temperature and physical activity compared to AAV-NPY rats fed ad libitum. The AAV-NPY-pf and AAV-NPY-al rats were both different from the AAV-contr rats (Figure 1e–h).
Dark phase activity was reduced in both AAV-NPY-al and AAV-NPY-pf animals on day 6, and remained significantly reduced throughout the experiment (Figure 1h). There was no difference in light phase activity between the groups.
Fifty days after injection of the viral particles, body weight gain and fat percentage of AAV-NPY-pf animals (135.3 ± 10.91 g and 5.18 ± 0.57% of BW) was similar to that of AAV-NPY-al animals (150.8 ± 16.45 g and 5.40 ± 0.53% of BW), and significantly increased when compared to control animals (87.2 ± 1.38 g (P < 0.05) and 2.18 ± 0.21% of BW (P < 0.01)).
These results confirm that long-term overexpression of NPY in the PVN results in obesity by transiently increasing food intake and body weight gain (7). AAV-NPY-injected rats that were pair-fed to controls did not show this increased weight gain, supporting the hypothesis that increased food intake plays a major role in development of obesity by AAV-NPY overexpression in the PVN. When permitted to eat ad libitum, food-restricted AAV-NPY-injected animals directly consumed similar amounts of food as AAV-NPY ad libitum fed rats. Interestingly, food intake in these rats declined in a similar fashion as nonrestricted AAV-NPY-injected rats, resulting in normal food intake at the end of the study. Furthermore, the NPY-induced decline in body temperature and locomotor activity was observed in both pair-fed and ad libitum-fed rats, indicating that the effects of NPY on these parameters of energy expenditure are independent from food intake, and that NPY-induced decreases in body temperature and in locomotor activity play a minor role in body weight gain.
AAV-NPY-injected rats that were pair-fed to controls showed a lower body weight gain when compared to AAV-contr rats. AAV-NPY-pf rats ingested all their food in the first half of the dark period. As NPY overexpression specifically stimulates light phase food intake (6), the absence of food in the light phase might be perceived as starvation. Although central body temperature in the dark phase was decreased in the pair-fed rats, as assessed using telemetry probes, it is possible that the stressful condition of the pair-fed rats increases heat loss via the extremities, which would not be detected by the telemetry probes. The perceived starvation in AAV-NPY-injected rats during pair feeding may also explain that when given ad libitum access to food, they ate more than AAV-NPY-injected rats that had ad libitum access to food throughout the entire experiment.
Viral-mediated overexpression is a powerful tool to investigate the effect of long-term increased neuropeptide signaling for periods beyond those feasible with minipumps. It has been demonstrated before that gene expression from AAV vectors in the central nervous system remains stable for months (8). We have confirmed that levels of NPY mRNA by AAV-NPY in the PVN are similar in rats 23 and 50 days (6,7).
In summary, we show that NPY overexpression in the PVN of adult rats results in obesity mainly by increasing food intake until a certain body weight is achieved. Although a transient increase in food intake and body weight gain was observed, rats injected with AAV-NPY show an unremitting (food independent) reduction of body temperature and physical activity.
This study was supported by NWO ZonMW 903-039-193.