Melanocortinergic Activation by Melanotan II Inhibits Feeding and Increases Uncoupling Protein 1 Messenger Ribonucleic Acid in the Developing Rat
2007
[b]Abstract]The hypothalamic neurocircuitry that regulates energy homeostasis in adult rats is not fully developed until the third postnatal week. In particular, fibers from the hypothalamic arcuate nucleus, including both neuropeptide Y (NPY) and {alpha}-MSH fibers, do not begin to innervate downstream hypothalamic targets until the second postnatal week. However, {alpha}-MSH fibers from the brainstem and melanocortin receptors are present in the hypothalamus at birth. The present study investigated the melanocortin system in the early postnatal period by examining effects of the melanocortin receptor agonist melanotan II (MTII) on body weight, energy expenditure, and hypothalamic NPY expression. Rat pups were injected ip with MTII (3 mg/kg body weight) or saline on postnatal day (P) 5 to P6, P10–P11, or P15–P16 at 1700 and 0900 h and then killed at 1300 h. Stomach weight and brown adipose tissue uncoupling protein 1 mRNA were determined. In addition, we assessed central c-Fos activation 90 min after MTII administration and hypothalamic NPY mRNA after twice daily MTII administration from P5–P10 or P10–P15. MTII induced hypothalamic c-Fos activation as well as attenuating body weight gain in rat pups. Stomach weight was significantly decreased and uncoupling protein 1 mRNA was increased at all ages, indicating decreased food intake and increased energy expenditure, respectively. However, MTII had no effect on NPY mRNA levels in any hypothalamic region. These findings demonstrate that MTII can inhibit food intake and stimulate energy expenditure before the full development of hypothalamic feeding neurocircuitry. These effects do not appear to be mediated by changes in NPY expression.
[b]Introduction]IN THE ADULT rodent, the arcuate nucleus of the hypothalamus (ARH) plays a key role in body weight regulation, in part by responding to numerous peripheral signals of metabolic status, including leptin. These effects are mediated primarily by two populations of neurons, the orexigenic neuropeptide Y (NPY)/agouti-related protein (AgRP) neurons and the anorexigenic {alpha}-MSH neurons. These neuronal populations relay information to other hypothalamic sites that mediate food intake and energy expenditure, including the paraventricular nucleus of the hypothalamus (PVH), the dorsomedial nucleus of the hypothalamus (DMH), the perifornical region (PFR), and the lateral hypothalamic area (LHA), as well as the brainstem.
Although pathways mediating energy homeostasis during the early postnatal period are not well understood, the mechanisms appear to be less complex than in the adult. Importantly, the ARH neurocircuitry that regulates energy homeostasis in the adult rat is not fully developed at birth, such that ARH neurons do not begin to innervate downstream hypothalamic targets until postnatal day (P) 5 to P11. Before P6, ingestion appears to be mainly stimulated by dehydration, whereas the primary inhibitory signal is gastric distention. By P9–P12, rat pups begin responding to caloric signals; however, 2-deoxyglucose and insulin, which decrease available glucose, do not stimulate food intake until P25–P30. Furthermore, exogenous leptin has no effect on food intake during the first 3 wk of postnatal life, indicating functional immaturity of downstream hypothalamic pathways during the entire preweaning period. Peripheral metabolic and caloric signals thus appear to have a minimal role in food intake in the developing rat.
The early postnatal period is a time of rapid body growth and therefore high energy demands, suggesting a strong orexigenic drive or low anorexigenic signals. Although the major orexigenic neurocircuitry, i.e. the ARH NPY/AgRP neuronal projections, are not established in the early postnatal period, hypothalamic NPY content is abundant during this time. In fact, during development, NPY is expressed in a number of hypothalamic regions that typically do not show expression in adult rats. In the adult rat hypothalamus, NPY is expressed mainly in the ARH with an additional low level of expression in the central compact region of the DMH (DMHp). In addition to these regions, during development, there is a unique, transient expression of NPY in the noncompact zone of the DMH (DMHnc), the PFR, the LHA, and the PVH (8, 9). We hypothesize that these transient NPY populations drive food intake before the establishment of ARH feeding neurocircuitry and/or promotes the transition to independent ingestion. This transient NPY expression peaks at approximately P16 and subsequently declines to an adult-like expression by P30 (8, 9), suggesting the establishment of a tonic inhibitory signal that persists through adulthood. A likely candidate for this inhibitory signal is {alpha}-MSH. Evidence for this includes the induction of NPY expression in the DMHnc in specific models of reduced melanocortin signaling, including lactation, the melanocortin 4 receptor (MC4R) knockout mouse (11), and the agouti mouse (11). In addition, site-directed administration of the nonselective melanocortin receptor agonist melanotan II (MTII) greatly attenuates this NPY induction during lactation (12). The early postnatal period, before downstream innervation by arcuate melanocortinergic fibers, may similarly be considered a period of reduced melanocortin signaling, thereby providing a permissive environment for the novel NPY expression.
Orexigenic drive likely dominates under most conditions during development; however, anorexigenic mechanisms are not absent. Although the key anorexigenic pathway mediated by ARH {alpha}-MSH fibers is not fully established in the early postnatal period, {alpha}-MSH projections originating from the brainstem are widespread in the hypothalamus at birth, as are melanocortin receptors. Therefore, the components of a functional melanocortin system are present in rodent neonates. Because the projections of the endogenous melanocortin receptor antagonist AgRP, which originate solely from ARH NPY neurons, are not yet established during the early postnatal period, this would in fact suggest an enhanced capacity for {alpha}-MSH-mediated effects. To investigate the role of the melanocortin system in the developing rat, the present study used the melanocortin receptor agonist MTII to determine whether the melanocortin system can regulate food intake and energy expenditure during the early postnatal period. In addition, we investigated the ability of MTII to inhibit the transient hypothalamic NPY expression observed during the early postnatal period.
[b]Materials and Methods] Animals
All animals were maintained under a 12-h light, 12-h dark (lights on at 0600 h) cycle and constant temperature (23 ± 2 C). Pregnant female rats were housed individually and checked for birth of pups every morning. The day of birth was considered P0, and litters were adjusted to eight male pups on P2. Adult male rats were housed individually. All animal procedures were approved by the Oregon National Primate Research Center Institutional Animal Care and Use Committee.
Drug preparation and administration
MTII (NeoMPS, San Diego, CA) was diluted in sterile saline and injected ip. Vehicle animals were injected with sterile saline. MTII was injected ip instead of icv because of possible confounding effects that would result from intracranial cannulation in suckling pups. In addition, previous studies have shown that ip MTII administration decreases food intake in adult rats. MTII was made fresh before use, and approximate volume of injection was 100 µl for pups and 250 µl for adult rats.
MTII dose response
To determine an appropriate dose of MTII, we examined the effects of ip MTII at various doses on body weight and food intake. Offspring of pregnant Wistar females (Charles River Laboratories, Wilmington, MA) were tested at P10–P11. Pups within each litter (five litters total) were randomly assigned to receive 0 (saline), 0.1, 3.0, or 10.0 mg/kg MTII (final n = 7–8 per dose). Before drug administration, the dam was removed from the home cage, and pups were weighed and then injected ip with either MTII or saline at 1700 h. Immediately after injections, the dam was returned to the home cage (dam absent <10 min). The following day, injections were repeated at 0900 h. Pups were reweighed and killed by decapitation at 1300 h. Stomachs were removed and weighed as an index of food intake. Subsequently, a dose of 3.0 mg/kg MTII was used in all additional studies.
c-Fos activation in response to peripheral MTII administration
There is some controversy in the literature as to whether MTII administered peripherally effectively crosses the blood-brain barrier (BBB). Therefore, we first assessed whether ip MTII administration would activate c-Fos in the CNS under our conditions. In addition, because BBB function develops progressively through to the fourth postnatal week, we examined whether the extent of central c-Fos activation was affected by age. Wistar rats (Simonsen Laboratories, Gilroy, CA) were tested during the early postnatal period (P6 or P15) or in adulthood (P90). For both P6 and P15 animals, two litters were used at each age, with n = 4 at P6 and n = 8 at P15. For adults, three saline-injected and four MTII-injected animals were assessed. To minimize any nonspecific stress effects of the treatment, adult rats were acclimatized for 7 d before testing by daily saline injections at 1100 h. Rat pups were not acclimatized before testing because of possible confounding effects of repeated handling and maternal separation and because preliminary investigation demonstrated that saline-injected naive pups exhibited minimal c-Fos activation. On the day of testing, adult rats and pups were injected ip with 3.0 mg/kg MTII or saline at 1100 h. For adult rats, food and water were removed at this time, and, for pups, the mother was removed and the cage was placed on a heating pad. Animals were killed with pentobarbital 90 min after injection and then perfused transcardially with saline, followed by ice-cold, phosphate-buffered 4% paraformaldehyde (pH 7.4). The brains were removed, postfixed in paraformaldehyde overnight, and then saturated in 25% sucrose. Brains were frozen in –40 C isopentane and stored at –80 C before immunohistochemical analysis for c-Fos (as described below).
[b]Acute MTII administration]
Offspring of pregnant Wistar females (Charles River Laboratories) were tested on P5, P10, or P15. These ages were chosen to represent a spectrum of hypothalamic feeding neurocircuitry development. Pups within each litter (four to seven litters were tested per age) were randomly assigned to either the saline or MTII condition, with four pups per drug condition per litter (final n = 14–16). Before drug administration, the dam was removed from the home cage, and pups were weighed and then injected ip with either 3.0 mg/kg MTII or saline at 1700 h. Immediately after injections, the dam was returned to the home cage. The following day, injections were repeated at 0900 h. Pups were reweighed and killed by decapitation at 1300 h. Stomachs were removed and weighed as an index of food intake. Interscapular brown adipose tissue (BAT) was removed to a microcentrifuge tube containing a ribonuclease inactivator (RNAlater; Ambion, Austin, TX) and then stored at –20 C for analysis of uncoupling protein 1 (UCP1) (an index of BAT thermogenesis) mRNA by real-time PCR (as described below).
In a subset of the animals (two litters per age), pup behaviors were observed and quantified for 1 h after injection by an investigator blind to the treatment group. These behaviors included latency to feed, defined as the latency for an individual pup to attach to a nipple (measured in the morning only), number of yawns observed (measured in the evening only), and total time spent grooming (measured at P15, in the evening only).
[b]Chronic MTII administration]
To determine whether melanocortin receptor activation inhibits transient hypothalamic NPY expression, MTII was administered over 5 d at two different developmental stages. Offspring of pregnant Sprague Dawley females (Simonsen Laboratories) were randomly assigned to either the saline or MTII condition, with four pups per drug condition per litter. Two litters were tested per age group. Before drug administration, the dam was removed from the cage and returned on completion of injections. Pups were injected ip with MTII or saline twice daily (at 0900 and 1700 h) for 5 consecutive days, from P5 to P10 or P10 to P15, with the first injection at 1700 h and the last injection at 0900 h. Pups were weighed before each injection. On the final day (P10 or P15), pups were killed by decapitation at 1300 h. Brains were rapidly removed, frozen on powdered dry ice, and then stored at –80 C for NPY mRNA analysis by in situ hybridization (as described below), with six animals per group.
Immunohistochemistry for c-Fos
Immunohistochemistry was performed to detect c-Fos immunoreactivity as described previously. Briefly, perfused brains were sectioned (25 µm) on a microtome in a one-in-three coronal series through the extent of the hypothalamus and brainstem. Free-floating sections were rinsed in 0.05 M potassium PBS and then preincubated in blocking buffer, consisting of 0.05 M potassium PBS with 0.4% Triton X-100 and 2% donkey serum, for 30 min. Sections were then incubated with rabbit anti-c-Fos antibody (1] RNA isolation and real-time RT-PCR analysis
Real-time RT-PCR was performed on BAT as described previously. Briefly, BAT was homogenized in 800 µl Trizol reagent (Invitrogen, Carlsbad, CA), and total cellular RNA was isolated according to the specifications of the manufacturer. Total RNA was further purified using the RNeasy Mini kit (Qiagen, Valencia, CA). The quality and concentration of the RNA were determined by measuring the absorbance at 260 and 280 nm, and RNA integrity was confirmed by bioanalysis (Agilent 2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA). RNA samples (1 µg) were reverse transcribed using random hexamer primers (Promega, Madison, WI). The RT product was then diluted 1]
In situ hybridization
Brains were sectioned (20 µm) on a cryostat in a one-in-four coronal series through the entire extent of the hypothalamus. NPY mRNA levels were determined in one series by in situ hybridization as described previously (8, 9). Briefly, a cRNA probe for NPY was transcribed from a 511-bp cDNA (obtained from Dr. S. L. Sabol, National Institutes of Health, Bethesda, MD) in which 25% of the UTP was 35S labeled (PerkinElmer, Wellesley, MA). Brain sections were fixed in phosphate-buffered 4% paraformaldehyde (pH 7.4) and treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). Sections were then rinsed in 2x sodium saline citrate (SSC), dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air dried. The NPY probe (specific activity, 5–6 x 108 dpm/µg; saturating concentration, 0.3 µg/ml·kb) was diluted in hybridization buffer (50% formamide, 6.25% dextran sulfate, 0.7% Ficoll, and 0.7% polyvinylpyrolidone) and then the sections were exposed to the labeled probe overnight in a humidified chamber at 55 C. After incubation, the slides were washed in 4x SSC, ribonuclease A at 37 C and in 0.1x SSC at 60 C. Slides were then dehydrated through a graded series of alcohols and dried. For histological analysis of the distribution of NPY mRNA, slides were dipped in Kodak NTB emulsion (Eastman Kodak, Rochester, NY) diluted 1]
Quantification of NPY mRNA
Images of silver grain distribution were captured under dark-field illumination using a CoolSNAPHQ CCD camera (Photometrics, Tucson, AZ) and analyzed using the MetaMorph Imaging system (Universal Imaging Corp., West Chester, PA). Silver grains were analyzed using a sampling box that encompassed the entire region of interest (ROI) and measured as the area occupied by silver grains within the ROI multiplied by the OD. Background labeling, determined using the same sampling box over an adjacent region that contained no NPY gene expression, was subtracted from this measurement. For the DMH, the sampling box encompassed both the central compact zone (DMHp) and the surrounding scattered neurons of the noncompact zone (DMHnc). To distinguish between these two regions, a second ROI was drawn to outline only the DMHp, and this measurement (minus its corresponding background measurement) was subtracted from the entire DMH measurement to produce a measure of the DMHnc. Measurements were taken bilaterally through the complete rostrocaudal extent of the ARH, DMH, and PFR. For data analysis, the brain sections were anatomically matched across animals from all groups, with equal numbers of sections sampled per animal. The mean value per region per animal was determined and used for statistical analysis.
Statistical analyses
To account for litter effects (because two to four pups per litter received the same treatment), data were analyzed using nested-design ANOVA for the factors of drug treatment and litter, with litter nested under treatment. For all data, individual ANOVAs were conducted at each age. Because c-Fos immunohistochemistry and NPY in situ hybridizations were conducted as different assays at each age, separate statistical analyses were conducted at each age for each hypothalamic region. Body weights for chronic MTII administration were analyzed by repeated-measures nested-design ANOVAs, with day treated as a repeated measure and litter nested under treatment. Any significant main or interaction effects were further analyzed by Newman-Keuls post hoc analysis. Statistical analyses were conducted using Statistica software (StatSoft, Tulsa, OK). In all cases in which significant litter effects were observed, the effect was attributable to differences among saline-injected animals or in the magnitude of response to MTII and not a difference in the direction of the response. All values are represented as the mean ± SEM. Statistical significance was defined as P < 0.05.
Results]MTII dose response
In rat pups, stomach weight at the time of death was used as an index of food intake. Increasing doses of MTII resulted in significantly decreased stomach weight (Fig. 1AGo) at all doses (0.1, 3.0, and 10.0 mg/kg), as demonstrated by a significant main effect of dose (F(3,11) = 52.13, P < 0.0001), with saline > 0.1 mg/kg > 3.0 mg/kg = 10.0 mg/kg (P < 0.05). In addition, a significant litter effect was observed (F(16,11) = 2.88, P < 0.05). Body weight (Fig. 1BGo) showed a similar dose response effect (main effect of dose, F(3,11) = 76.65, P < 0.0001), with saline > 0.1 mg/kg > 3.0 mg/kg = 10.0 mg/kg (P < 0.005). A dose of 3.0 mg/kg MTII was used in all subsequent studies.
MTII dose response effects on body weight (A) and stomach weight (B). Rat pups (age P10–P11) were injected ip with MTII at 0.1, 3.0, or 10.0 mg/kg or saline vehicle at 1700 h and at 0900 h the following day and then were killed at 1300 h. *, P < 0.0001 vs. vehicle; **, P < 0.001 vs. vehicle and P < 0.01 vs. 0.1 mg/kg MTII dose. Values represent the mean ± SEM of seven to eight animals per group.
Effects of MTII on food intake and energy expenditure
Acute MTII administration resulted in significantly decreased stomach weight (Fig. 4Go) at all ages tested (P < 0.001), with significant main effects of treatment and litter at each age (P6 treatment effect, F(1,22) = 79.12, P < 0.0001; litter effect, F(6,22) = 12.97, P < 0.0001; P11 treatment effect, F(1,18) = 180.15, P < 0.0001; litter effect, F(12,18) = 5.39, P < 0.001; P16 treatment effect, F(1,24) = 126.143, P < 0.0001; litter effect, F(6,24) = 5.83, P < 0.001). In addition, MTII significantly attenuated body weight gain (Fig. 5Go) at P6 compared with saline, and P11 and P16 pups showed a loss in body weight (P < 0.001 compared with saline). Significant main effects of treatment were observed at all ages, and significant litter effects were observed at P11 and P16 (P6 treatment effect, F(1,22) = 81.63, P < 0.0001; P11 treatment effect, F(1,18) = 320.56, P < 0.0001; litter effect, F(12,18) = 3.53, P < 0.01; P16 treatment effect, F(1,24) = 313.38, P < 0.0001; litter effect, F(6,24) = 3.30, P < 0.05).
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Body weight in response to acute MTII administration. MTII (3.0 mg/kg) or saline was administered ip at 1700 h and then 0900 h, and then the animals were killed at 1300 h. Values represent the change in weight from the time of the first injection until death 20 h later. Values represent the mean ± SEM of 14–16 animals per group. **, P < 0.001 compared with saline.
Chronic MTII administration from P5–P10 (Fig. 6AGo) or from P10–P15 (Fig. 6BGo) resulted in an attenuation of body weight gain on all days (P < 0.01) during the entire administration period, with a more pronounced attenuation in the P10–P15 group. In both age groups, the effect was primarily attributable to a decreased weight gain during the first 2 d. This is consistent with previous studies showing that the effects of MTII on body weight diminish over time. This was demonstrated by significant treatment x day interaction effects for both P5–P10 (F(4,48) = 3.03, P < 0.05) and P10–P15 (F(4,44) = 20.35, P < 0.0001) pups. In addition, there was a significant litter x day interaction effect in P10–P15 (F(8,44) = 22.90, P < 0.0001) pups only.
Body weight in response to chronic MTII administration. MTII (3.0 mg/kg) or saline was administered ip daily at 0900 and 1700 h from P5 to P10 (A) or P10 to P15 (B), and values represent the cumulative weight gain across this period (mean ± SEM of seven to eight animals per group). *, P < 0.01 compared with saline; **, P < 0.001.
MTII significantly increased BAT UCP1 mRNA levels (Fig. 7Go) in rat pups at all ages examined (P6, P11, and P16; P < 0.05), indicative of increased energy expenditure. This was demonstrated by significant main effects of treatment (P6, F(1,8) = 10.64, P < 0.05; P11, F(1,8) = 11.54, P < 0.01; P16, F(1,8) = 11.44, P < 0.01). The increase in UCP1 mRNA was more pronounced in older pups.
BAT thermogenesis in response to MTII. UCP1 mRNA was quantified by real-time PCR in BAT from rat pups at P6, P11, or P16 in response to acute MTII administration. MTII (3.0 mg/kg) or saline was administered ip at 1700 h and then 0900 h, and the animals were killed at 1300 h. Values represent the mean ± SEM of four saline and eight MTII animals per group. *, P < 0.05 compared with saline; **, P < 0.01.
Behavioral effects of MTII administration
As shown in Table 1Go, MTII significantly increased the latency to feed in P11 pups (main treatment effect, F(1,6) = 37.92, P < 0.001; litter effect, F(8,6) = 58.23, P < 0.0001), with a marginal increase (P = 0.065) in P16 pups and no significant effect in P6 pups. MTII also significantly increased the number of yawns (P < 0.0005) in animals at all ages (main treatment effects at P6, F(1,10) = 76.75, P < 0.0001; P11, F(1,6) = 67.47, P < 0.0001; P16, F(1,12) = 173.75, P < 0.0001). In addition, time spent grooming was significantly increased by MTII at P16 (main treatment effect, F(1,12) = 63.43, P < 0.0001).
[b]Discussion]* The present studies demonstrate that, before the full maturation of central feeding neurocircuitry, melanocortin receptor activation via the agonist MTII can decrease food intake, increase sympathetic outflow, and subsequently attenuate body weight gain in rodent neonates as early as P5. Although both central and peripheral administration of MTII are known to reduce food intake in adult rodents, peripheral administration has been shown previously to produce limited or no central c-Fos activation in adult rats, whereas central administration shows abundant central activation. In contrast, we found significant central c-Fos activation in rat pups after peripheral MTII administration, with the greatest activation seen at P15. The same dose and route of MTII produced no central c-Fos activation in adult rats, confirming previous studies. These findings suggest that the BBB may be more permeable to MTII during development, allowing centrally mediated effects to be observed. In addition, the greater extent of c-Fos activation in P15 compared with P6 pups may reflect increased levels of melanocortin receptors and/or additional development of downstream pathways mediating melanocortin receptor activation with age. At P15, the most pronounced activation in the hypothalamus was observed in the PVH and VMH. Although PVH c-Fos activation has been shown previously in response to central MTII administration in adult rats, activation in the VMH has not been reported. Regions activated by MTII included numerous sites involved in energy homeostasis, namely the PVH, VMH, ARH, and solitary tract nucleus. All of these regions express MC4Rs, which have been shown to mediate MTII effects on food intake and metabolism. MTII also activates MC3 receptors, which are also expressed in the ARH and VMH; therefore, some of the c-Fos activation observed in these regions may have been mediated through this receptor subtype. It is also important to note that any c-Fos immunoreactivity observed could be the result of either direct MTII activation of a given region or an indirect activation via other central regions. It should be noted that, because melanocortin receptors are also expressed in peripheral tissue, it remains possible that some of the MTII effects observed may be mediated, in part, via these peripheral receptors.
Peripheral MTII administration (P5–P6, P10–P11, or P15–P16) significantly decreased stomach content weight, suggesting a decrease in milk intake. Rat pups also displayed an attenuated body weight gain that was most pronounced in P16 pups when there was in fact a loss in body weight. MTII administration resulted in a small but significant increased latency to feed, although only in P11 pups. In addition, MTII administration increased yawning (P5, P10, and P15) and time spent grooming (measured at P16 only) during the first hour after injection. Both yawning and grooming behaviors have been attributed previously to hypothalamic activation of MC4Rs, suggesting activation of central melanocortin pathways. As seen with acute administration, chronic MTII administration over 5 d (P5–P10 or P10–P15) also attenuated body weight gain in pups, with a greater effect in older pups. Although the effect on body weight was substantial after the first day of MTII administration, subsequent rate of body weight gain was similar between MTII and saline animals but remained at a lower level in the MTII group. A similar tachyphylactic response to chronic MTII administration has been observed in adult rodents and may be attributable in part to decreased circulating leptin levels or other secondary effects of reduced energy intake.
In the present studies, maternal milk provided the sole nutritional source for pups; consequently, the lower stomach content weight observed reflects an MTII-mediated inhibition of suckling and not necessarily adult-like feeding. Because suckling differs considerably from adult feeding behavior, a number of previous studies have used models of adult-like independent ingestion to study the ontogeny of food intake controls in pups. These studies have demonstrated that, before P6, food intake is primarily inhibited by gastric fill, and, by P9, independent ingestion can be inhibited by nutritive signals. In comparison, nutritive signals do not appear to inhibit suckling until at least P14. We, however, observed MTII-mediated inhibition of milk intake in suckling pups at all ages studied, from P6 to P16. This inhibition therefore does not appear to reflect the developmental progression of inhibitory ingestive controls but instead likely reflects activation of central melanocortin receptors that are already present at birth. Importantly, these studies demonstrate that, not only does MTII inhibit solid food intake in adult rats, but it can inhibit suckling-mediated milk intake as early as P6, a time when food intake is primarily mediated by gastric fill.
It is possible that, in the early postnatal period, vagal feedback can activate brainstem {alpha}-MSH neurons that project to the PVH even early in development. In addition to effects on food intake, MTII administration significantly increased BAT UCP1 mRNA levels in rat pups. Up-regulation of UCP1, which mediates BAT thermogenesis, is indicative of increased ß-adrenergic sympathetic outflow and subsequently increased energy expenditure. In adult rats, MTII has been shown to increase BAT UCP1 levels in response to central but not peripheral administration, indicating a centrally mediated mechanism. That we observed a significant increase in UCP1 mRNA in pups with peripheral MTII administration again suggests increased BBB permeability to MTII in rat pups and a central site of action. The neuroanatomical pathways mediating melanocortin effects on BAT thermogenesis are thought to involve PVH neurons that express melanocortin receptors. Intra-PVH MTII administration both increases oxygen consumption and inhibits food intake. We also demonstrated previously an increase in UCP1 mRNA levels in response to intra-DMH MTII administration in lactating rats. In addition, there appears to be an independent pathway in the caudal brainstem, as evidenced by elevated UCP1 mRNA in BAT after fourth ventricle MTII administration in chronic decerebrate rats. Because we observed MTII-induced c-Fos activation in both the hypothalamus and the brainstem in rat pups, the UCP1 activation and effects on food intake may have been mediated by either of these pathways.
In the adult, an increase in energy expenditure via BAT thermogenesis is predominantly used to maintain body weight homeostasis. Such a mechanism would at first glance appear to be detrimental to the developing rat, when appropriate energy utilization is critical to sustain rapid growth and development. However, under certain conditions, such as low ambient temperature, BAT thermogenesis may be critical for survival through defense of body temperature. Indeed, as early as 5 h after birth, rodent neonates can increase UCP1 levels in response to either cold or ß-adrenergic stimulation. Our findings suggest that sympathetic outflow to BAT, mediated through melanocortin receptor activation, is functional and responsive at birth. Increased energy expenditure through this mechanism, in addition to the decrease in food intake, likely both contributed to the effects that we observed of MTII on body weight.
Although our studies demonstrate that rat pups have the capacity for anorexigenic effects, orexigenic drive is expected to dominate during development to sustain rapid growth. We propose that the transient hypothalamic NPY expression (in the DMHnc, PFR, PVH, and LHA) observed during development may drive food intake in pups before the development of ARH projections. An orexigenic role for this population is suggested by adult rat models of reduced melanocortin signaling, including the lactating rat and the MC4R knockout mouse, which show a similar induction of NPY although limited to the DMHnc. We have shown previously that this DMH-NPY expression mediates hyperphagia in the lactating rat and is inhibited by MTII. We therefore hypothesized that the novel hypothalamic NPY induction during development similarly drives food intake and can be inhibited by MTII administration. However, we did not observe a significant MTII-induced reduction of NPY mRNA in any hypothalamic region. Although we have shown previously that MTII inhibits lactation-induced NPY expression in the DMHnc, these studies used MTII injection directly into the DMHnc, resulting in increased BAT UCP1 mRNA levels and decreased food intake. It is thus possible that the MTII effects we observed on food intake and energy expenditure in rat pups were also mediated in part through NPY neurons of the DMHnc. Although the lack of a decrease in DMHnc-NPY suggests that peripheral MTII administration may not have adequately penetrated the hypothalamus to down-regulate NPY expression, this seems unlikely because we saw robust c-Fos activation in the PVH. Another possibility is that competing mechanisms may have obscured any observable effects of MTII on NPY mRNA in the DMH. Alternately, a signal other than {alpha}-MSH may provide the primary inhibition of NPY expression in this region. Additional investigation is needed to determine whether {alpha}-MSH is the primary inhibitory signal, what the role of this transient NPY population is during development, and how the various regions involved (i.e. the DMHnc, PFR, LHA, and PVH) are related in function and regulation.
In summary, we demonstrated that, before the development of ARH projections, melanocortin receptor activation can inhibit food intake and increase energy expenditure. These effects were observed as early as P5, although the effectiveness of MTII was greater at P15, likely attributable to increased permeability of MTII and/or additional development of the melanocortin system.
Melanocortin MC4 receptor-mediated feeding and grooming in rodents 2013
* Abstract] Melanocortin receptors mediate melanocortin-induced pigmentation and grooming respectively. Grooming is a low priority behavior that is concerned with care of body surface. Activation of central melanocortin MC4 receptors is also associated with meal termination, and continued postprandial stimulation of melanocortin MC4 receptors may stimulate natural postprandial grooming behavior as part of the behavioral satiety sequence.
Indeed, melanocortins do not suppress food intake or induce grooming behavior in the melanocortin MC4 receptor-deficient/null. This clinical study has focus on how melanocortins affect grooming behavior through the MC4R, and how melanocortin MC4 receptors mediate feeding behavior. Peptide review illustrates how melanocortins were the most likely candidates to mediate grooming and feeding based on the natural behaviors they induced. PMID:23872405.
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