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Molecular Neuroendocrinology Group, Aberdeen Centre for Energy Regulation and Obesity, Rowett Research Institute, Bucksburn, Aberdeen, Scotland AB21 9SB, United Kingdom
Address all correspondence and requests for reprints to: Dr. J. G. Mercer, Molecular Neuroendocrinology Group, Aberdeen Center for Energy Regulation and Obesity, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, Scotland, United Kingdom AB21 9SB. E-mail: jgm{at}rri.sari.ac.uk
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Abstract |
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Introduction |
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In the absence of any specific knowledge of the systems that encode body weight cycles, a number of studies have examined hypothalamic neuropeptide gene expression during photoperiod and food-deprivation-induced weight change in the Siberian hamster (7, 8, 9), to begin to define the neuroendocrine changes that accompany body weight change under these contrasting circumstances. Candidate genes have been selected on the basis of their involvement in the regulation of energy balance. Although complete food deprivation will result in weight loss and the consequent engagement of compensatory systems, this is an inappropriate paradigm for comparison with short photoperiod-induced weight reduction. If the causes and consequences of photoperiodic weight change are to be defined, longer-term food restriction will be required, to allow more subtle manipulations. In particular, it will be important to differentiate between hypothalamic changes that induce weight change and those that are secondary, consequential events.
In the present study, we have employed two food-restriction paradigms of relevance to programmed weight change and the cycle of seasonal body weight regulation, and to issues such as how the brain integrates the leptin signal into the hypothalamic circuitry. Food was restricted in long photoperiod, with the intent of mimicking short photoperiod-induced body weight trajectory (10) and distinguishing between imposed and programmed weight change. Our restriction in short photoperiod replicated the characteristic features of the seasonal cycle of body weight regulation and allowed definition of hypothalamic systems that are activated or suppressed by inappropriately low body weight, even when this weight is lost coincident with seasonal weight change. In combination, these manipulations test the hypothesis that, at the level of known neuropeptide systems, the hypothalamus recognizes the difference between weight change brought about by imposed negative energy balance and seasonal, and thus (by definition) appropriate, body weight change.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental protocols
Exp 1: Mimicking SD body weight changes by food restriction in
LDs. Hamsters were divided into three groups matched for body
weight. One group remained in LDs and was fed ad libitum
(LD-ADLIB, n = 10) throughout the 12-wk experiment. A second group
was transferred to SDs and was also fed ad libitum
throughout (SD-ADLIB, n = 10). The final group remained in LDs but
received a restricted ration of food (LD-REST, n = 10) from wk 2
onwards, such that the group mean body weight tracked that of the SD
hamsters (10). Body weights and food intakes were measured
daily for all animals during the restriction period. Animals in the
LD-REST group received a measured amount of food each day in the second
half of the light phase. The degree of food restriction imposed on the
LD-REST group did not exceed 33% of LD-ADLIB intake at any point
during the study. One animal in the LD-REST group failed to eat its
daily food ration and was withdrawn from the study. At the end of the
12-wk study, all animals were killed, by cervical dislocation, in the
middle of the light phase. Trunk blood was collected in lithium-heparin
tubes, and brains were frozen on dry ice and stored at -70 C. The
following tissue weights were recorded: paired testes, liver, paired
kidney, interscapular brown adipose tissue (IBAT), pooled
retroperitoneal white adipose tissue (RWAT), inguinal white adipose
tissue (IWAT), and epididymal white adipose tissue (EWAT). The latter
depot was not dissected free of associated reproductive tissue.
Exp 2: Food restriction during SD-induced body weight change. In these studies, we set out to verify that adjustments in defended body weight (5) also occur under laboratory conditions of photoperiod switching (Exp 2a), before establishing the pattern of hypothalamic gene expression in SD-REST hamsters before refeeding (Exp 2b). Hamsters were divided into two groups matched for body weight. Both groups were transferred to SDs and were fed ad libitum for 28 d. Food restriction was then imposed upon one group (SD-REST, n = 5) with the intention of generating a body weight differential between SD-REST and SD-ADLIB animals (n = 5) of approximately 25% within 3 wk. To achieve this body weight reduction, food availability was initially set at 60% of the average ad libitum intake on d 28 (i.e. a 40% restriction). Animals in the SD-REST group received a measured amount of food each day in the second half of the light phase. Body weight and food intake were measured weekly during ad libitum feeding and daily for all animals during the restriction period. In Exp 2a, the SD-REST group was returned to ad libitum feeding after 18 d (d 46 of the study). Food intakes and body weights were measured daily until d 91; and body weight, weekly, until day 126 (wk 18). In Exp 2b, hamsters were divided into two weight-matched groups and transferred into SDs with food and water available ad libitum for the first 28 d. After 28 d, one of the groups was switched to a restricted feeding regime (SD-REST, n = 8) of approximately 60% of the d 28 ad libitum intake, while the other group continued to be fed ad libitum (SD-ADLIB, n = 8). On d 49, all animals were killed early in the light phase. Trunk blood, brain, and other tissues were collected as detailed for Exp 1.
RIA
Plasma concentrations of leptin were measured using the Linco
Multispecies kit (catalog no. XL-85K, lot XLR-1188;
Biogenesis, Poole, UK), according to the manufacturer’s
instructions and as detailed elsewhere for Siberian hamsters (11, 12). Serial dilutions of pooled plasma generated a curve that
was parallel to the standard curve provided as part of the kit. The
assay had a lower detection limit of 2 ng/ml leptin human equivalent
(HE).
Hypothalamic gene expression
Messenger RNA levels were quantified by in situ
hybridization, in 20-µm coronal hypothalamic sections, using
techniques described in detail elsewhere (13, 14).
Riboprobes complementary to the leptin receptor [long form; OB-Rb;
(8)], the melanocortin-3 and -4 receptors [MC3-R and
MC4-R; (9)], and a number of appetite/body weight-related
neuropeptides were generated from cDNA fragments. The panel of
neuropeptide genes included NPY, ACTH-releasing factor (CRF)
(13), agouti-related protein (AGRP), the precursor POMC
(8), and cocaine- and amphetamine-regulated transcript
[CART; (9)]. As previously described (8),
forebrain sections were collected from the very caudal extent of the
arcuate nucleus (ARC) through to the rostral extent of the
paraventricular nucleus (PVN) onto two sets of eight slides. Six or
seven sections were mounted on each slide. Accordingly, the first set
of slides spanned the hypothalamic region, approximating -2.7 mm to
-1.46 mm, relative to Bregma, according to the atlas of the mouse
brain (15). These slides therefore contained the full
extent of the ARC. The second set of slides continued through to -0.58
mm, relative to Bregma, and therefore encompassed the caudal and
rostral extent of the PVN. One slide from each animal was hybridized
with each probe. Briefly, slides were fixed, acetylated (optional), and
hybridized overnight at 58 C using
[35S]-labeled cRNA probes (1–2 x
107 cpm/ml). Slides were treated with
ribonuclease A, desalted with a final high-stringency wash (30 min) in
0.1x SSC at 60 C or 75 C, dried, and apposed to Hyperfilm ß-max
(Amersham Pharmacia Biotech UK Ltd., Little Chalfont,
Buckinghamshire, UK). Autoradiographic images were quantified using the
Image-Pro Plus system (Media Cybernetics, Silver
Spring, MD). Image analysis was typically performed on four or five
sections from the ARC, and two or three sections from the PVN,
according to the probe used in hybridization. Data were manipulated
using a standard curve generated from 14C
autoradiographic microscales (Amersham Pharmacia Biotech), and the integrated intensity of the hybridization
signal was computed.
Statistical analysis
Data were analyzed by t test or one-way ANOVA
followed by Student’s-Newman-Keuls multiple-comparison test, as
appropriate, using SigmaStat statistical software (Jandel Corp.,
Erkrath, Germany). Where data failed equal variance or normality tests
they were analyzed by Mann Whitney rank sum test or one-way ANOVA on
ranks followed by Dunn’s multiple-comparison test. Results are
presented as means ± SEM, and differences
are considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). SD-ADLIB and LD-REST groups
decreased in body weight by 23.8% and 22%, respectively, as a
consequence of either SD exposure or imposed food restriction. There
were significant differences in final body weight between the groups
(equal variance test failed, ANOVA on Ranks; P <
0.001; LD-ADLIB > SD-ADLIB = LD-REST). Twelve weeks in SDs
reduced testes weight by 90% and also reduced combined kidney weight
in the SD-ADLIB group compared with either LD-ADLIB or LD-REST (Table 1). Liver and adipose
tissue weights were higher in LD-ADLIB hamsters than in either SD-ADLIB
or LD-REST groups, but there were no differences between SD-ADLIB and
LD-REST groups.
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).
Within the experimental population (pooled data), plasma leptin
concentration was strongly correlated with body weight (r =
0.84, P < 0.001, n = 29) and the weight of
IWAT (r = 0.86, P < 0.001, n = 29), RWAT
(r = 0.95, P < 0.001, n = 29), and IBAT
(r = 0.92, P < 0.001, n = 29) depots.
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). An imposed 10-wk food intake
restriction in LDs, to mimic SD body weight trajectory (LD-REST), gave
rise to differences in expression of five of the six ARC genes
examined; NPY was the only gene for which differences in expression did
not attain statistical significance. AGRP gene expression was elevated
in LD-REST hamsters compared with the LD-ADLIB group. In the LD-REST
group, an intermediate concentration of POMC mRNA was observed that was
lower than the LD-ADLIB level but higher than that of the SD-ADLIB
group. CART gene expression was lower in the LD-REST group than in the
SD-ADLIB group of similar body weight. OB-Rb gene expression was
elevated in the LD-REST hamsters, relative to both LD-ADLIB and
SD-ADLIB groups. Food restriction in LDs (LD-REST) reduced MC3-R gene
expression compared with the LD-ADLIB group; this reduction was similar
to that seen in SD-ADLIB animals. Data for OB-Rb gene expression in the
VMN exhibited differences between the groups similar to those observed
in the ARC (Fig. 4), although mRNA levels
in the LD-REST group were not significantly elevated, relative to the
LD-ADLIB animals. These relationships were not observed in the DMN
(Fig. 4). The VMN contains the greatest abundance of hypothalamic MC3-R
mRNA. The relative levels of MC3-R gene expression in the VMN were the
opposite of those recorded in the ARC; both SD-ADLIB and LD-REST groups
had elevated mRNA levels compared with the LD-ADLIB controls (Fig. 5). In the hypothalamic PVN, SD-ADLIB
hamsters had reduced CRF gene expression, compared with LD-ADLIB
controls (Fig. 3), but there was no effect of food restriction on CRF
gene expression (LD-ADLIB vs. LD-REST). MC4-R gene
expression in the PVN did not differ significantly among the three
treatment groups.
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). During the 18-d restriction, the
SD-REST animals fell to 65% of their starting weight, whereas the body
weights of SD-ADLIB hamsters on d 46 had fallen to 84% of their
initial level. This represented a body weight differential of 24%
between SD-REST and SD-ADLIB hamsters (SD-ADLIB, 32.18 ±
1.22 g vs. SD-REST, 24.42 ± 0.64 g;
P < 0.001). Upon return to ad libitum
feeding, the body weight of SD-REST hamsters increased such that, by d
70, both groups were at 77% of their starting weight. Both groups then
continued to lose weight through to 126 d (18 wk) in SDs.
Differences between the groups in body, testes, and adipose tissue
weights on d 126 were not statistically significant (data not shown).
Food intake by SD-REST hamsters averaged 63% of that consumed by
the SD-ADLIB group during the 18-d restriction period (SD-ADLIB,
60.7 ± 2.4 g vs. SD-REST, 38.3 ± 0.7
g; normality test failed, Mann Whitney rank sum test, P
< 0.05; Fig. 6b). When released from this restriction on d 46, SD-REST
hamsters expressed a voluntary hyperphagia, relative to SD-ADLIB
animals. During the first 7 d after the restoration of excess food
(d 47–54), SD-REST hamsters consumed 20% more food than SD-ADLIB
hamsters (SD-ADLIB, 25.8 ± 1.7 g vs. SD-REST,
31.1 ± 1.08 g; P < 0.05). This relative
hyperphagia seemed to extend beyond the first week of refeeding,
given that food intake over 18 d was 72.0 ± 4.9 g and
81.3 ± 0.7 g for SD-ADLIB and SD-REST hamsters,
respectively, although this difference was not statistically
significant. Compensation for the imposed restriction was incomplete,
because by d 91, when body weight trajectories were similar in the two
groups, the cumulative intake of SD-ADLIB hamsters, over the period
28–91 d, still exceeded that of the SD-REST group, although this trend
was not statistically significant (SD-ADLIB, 216.5 ± 12.6 g
vs. SD-REST, 206.9 ± 3.0 g).
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). Food intake
by SD-REST hamsters averaged 59.4% of that consumed by the
SD-ADLIB group during the 21-d restriction period, and a body weight
differential of 23.5% was established between the hamster groups
(SD-ADLIB, 31.54 ± 0.73 g vs. SD-REST,
24.14 ± 0.85 g). All tissue weights except testes were
reduced in REST hamsters (Table
2).
Plasma leptin levels were reduced by over 70% in REST hamsters
(Fig. 7; SD-ADLIB, 7.33 ± 0.77ng/ml vs.
SD-REST, 2.15 ± 0.13 ng/ml; P < 0.001, equal
variance test failed, Mann-Whitney rank sum test), although
concentrations in the latter group were close to the lower sensitivity
limit of the assay. The positive correlation between plasma leptin
concentration and body weight in SD-ADLIB hamsters approached, but
did not achieve, statistical significance (r = 0.695;
P < 0.1; data not shown).). Reductions in POMC, CART, and MC3-R
mRNA in the ARC and MC4-R gene expression in the PVN in SD-REST
hamsters approached, but did not attain, statistical significance
(POMC, P = 0.07; CART, P = 0.09; MC3-R,
P = 0.11; MC4-R, P = 0.054). There was
a trend toward increased OB-Rb gene expression in the VMN, but not the
DMN, of REST hamsters, but this difference did not attain statistical
significance (VMN: SD-ADLIB, 100 ± 4.6% vs. SD-REST,
176.7 ± 34.6%, normality test failed, Mann Whitney rank sum
test, P = 0.08; DMN: SD-ADLIB, 100 ± 8.6%
vs. SD-REST, 110.0 ± 12.3, P = 0.51).
There was no effect of food restriction on MC3-R mRNA levels in the VMH
(SD-ADLIB, 100 ± 9.1% vs. SD-REST, 94.3 ±
7.9%; P = 0.64), or CRF gene expression in the PVN
(Fig. 8).
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Discussion |
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In the present study, we have used the effects of imposed seasonally-inappropriate body weight reduction to highlight those neuropeptides that are involved in body weight defense in the hamster, including the anabolic peptides, NPY and AGRP, and their catabolic counterparts, POMC and CART. In Exp 1, these changes are juxtaposed to the hypothalamic changes that accompany seasonally-appropriate weight change of similar magnitude. Involvement of anabolic or catabolic neuropeptides in the latter body weight trajectory could be at the level of gradual change in gene expression and protein synthesis, leading to regulated food intake and body weight. Alternatively, a more abrupt switch in the activity of one or more of the components of the signaling system could push the animal along an appropriate body weight trajectory. By contrast with the neuropeptides listed above, little is known of the effect of energetic status or physiological weight regulation on the expression of the melanocortin (MC3 and MC4) receptor genes.
By mimicking SD weight loss by food restriction in LDs, we demonstrated opposite effects of short photoperiod and food restriction on CART and OB-Rb gene expression in the hypothalamic ARC. Similar trends were observed for OB-Rb gene expression in the VMN. Three other neuropeptide genes (AGRP, POMC, CRF) exhibited changes in expression in the same direction, but of different magnitude, in response to the different stimuli. Only MC3-R gene expression fell into the category where changes in the ARC (or VMN) in SD-ADLIB and LD-REST groups, compared with LD-ADLIB controls, were effectively indistinguishable. There were no differences between the treatment groups for NPY or MC4-R gene expression, the former data supporting the conclusions of earlier studies of the Siberian hamster (7, 8). The second food restriction protocol investigated adapted a paradigm previously employed classically to demonstrate the concept of the seasonal cycle of defended body weight (5). Food restriction, superimposed on SD weight loss, established a profile of hypothalamic gene expression that was typical of an animal in negative energy balance, despite the simultaneous changes in mRNA levels induced by short photoperiod itself.
In Exp 1, a food restriction regime was imposed in LDs to mimic SD weight trajectory, i.e. to produce broadly equivalent long-term changes in body weight and adiposity within the same time scale as SD-induced change. However, assessment of the cumulative food intake of hamsters in the SD-ADLIB and LD-REST groups suggested that the latter group engaged an energy-conserving strategy as part of their body weight defense. Differences in tissue turnover and metabolism were also indicated by the relative reduction in the mass of the kidneys in SD hamsters, a change that was independent of weight loss per se. Plasma leptin was positively correlated with body weight in both ADLIB groups but not in the LD-REST group. In view of the comparatively limited body weight range in the latter group, the precise significance of this observation is uncertain. Nevertheless, plasma leptin concentrations generally reflected body and adipose tissue weight, but the respective leptin signals seemed to be perceived differently according to whether body weight was or was not appropriate to the seasonal cycle. Whereas SD-ADLIB and LD-REST hamsters had similar leptin concentrations, differential regulation of OB-Rb gene expression in the ARC and VMN was suggestive of differences in signal integration. In SDs (SD-ADLIB), and as described previously (8), OB-Rb gene expression was reduced, relative to expression levels in freely-feeding animals in the opposite photoperiod (LD-ADLIB). By contrast, low leptin levels up-regulated OB-Rb gene expression in LD-REST hamsters, an outcome that is consistent with other experimental paradigms of imposed negative energy balance, albeit more acute manipulations, such as food deprivation and cold exposure (16, 17, 18). In agreement with other studies, OB-Rb mRNA in both the ARC and the VMN was sensitive to low plasma leptin, whereas the DMN was not (16, 17, 18). One of the intriguing issues raised by seasonal mammals, such as the Siberian hamster, is how signals arising from peripheral energy stores, such as leptin secretion from adipose tissue, can be integrated into the hypothalamic regulatory circuitry without acting to oppose programmed weight change (19). This argument can be summarized as follows: the loss of body fat that forms a large part of weight loss in SDs (20) results in low blood leptin concentrations, yet this declining lipostatic feedback signal does not act orexigenically to oppose the SD weight trajectory. It may be, of course, that gradual changes in leptin signal do not instigate any hypothalamic response, provided they exceed some threshold concentration, although the data for chronically REST hamsters would suggest that this is not the case. Part of the difficulty in interpreting the leptin signal arises from the two distinct forms of feedback provided by this hormone. Leptin levels reflect the normal pattern of food intake, and the absence of leptin is a potent signal of starvation (21, 22). In addition, basal levels of leptin provide feedback on the levels of body fat storage (23), and it is likely that chronic changes in leptin that reflect body adiposity will be read quite differently from acute changes that result from energetic challenges. Regulated sensitivity to leptin feedback may be critical for the maintenance of seasonal body weight (24, 25); in this example, reduced sensitivity to a chronic decline in leptin may be appropriate to animals undergoing programmed body weight reduction.
The profiling of hypothalamic gene expression in Exp 1 demonstrated additional effects of SDs (i.e. LD-ADLIB vs. SD-ADLIB) that were generally consistent with those reported previously (8, 9). In a study of adult male Siberian hamsters kept in SDs for 18 wk (8), POMC gene expression was reduced in the ARC (see also 7), and AGRP mRNA was elevated. In the present study, differences in AGRP gene expression did not reach statistical significance. Furthermore, elevated CART gene expression and reduced ARC MC3-R mRNA in the SD-ADLIB group were consistent with differences observed in juvenile female hamsters 8–12 wk after transfer to SDs at weaning (9). The reduction in CRF gene expression in SDs followed similar trends in an earlier study of the closely related Djungarian hamster (13). Two of the hypothalamic neuropeptide genes investigated, POMC and CART, differed in expression level between SD-ADLIB and LD-REST groups despite their similar body weight trajectories and plasma leptin levels. In addition, AGRP gene expression was elevated in LD-REST hamsters compared with the LD-ADLIB group. In general, the changes induced by 10 wk of food restriction (LD-REST) reflected the ongoing state of negative energy balance, low plasma leptin, and inappropriate body weight, with elevated AGRP and OB-Rb gene expression and low POMC mRNA levels compared with LD-ADLIB controls. MC3-R gene expression in the ARC was also reduced in LD-REST hamsters.
One of the principal aims of Exp 1 was to begin to unravel the signals regulating hypothalamic gene expression in the SD hamster. By mimicking in LDs the magnitude and trajectory of SD body weight change, it was possible to assess whether changes in the activity of regulatory signals were likely to be secondary to weight loss or low leptin signal, or to have a more direct primary role in seasonal weight regulation and signal integration. CART was the only neuropeptide studied where change in gene expression in response to SDs was suggestive of a primary role in weight loss. The increase in gene expression of this catabolic peptide supported our earlier findings that CART mRNA was elevated in juvenile female hamsters after just 2 wk in SDs and before major divergence of growth trajectories (9). In our recent studies, we observed that, of the gene expression changes known to accompany SD weight loss, only those to POMC mRNA were consistent with direct regulation by plasma leptin concentrations for both male and female hamsters (9). In the present study, this relationship was weakened because the reduction in POMC gene expression in SDs (SD-ADLIB) exceeded that in the LD-REST group that had a similar/lower plasma leptin. The reduction in POMC gene expression in SD Syrian hamsters (26), which do not lose weight (27) and which show an upward trend in plasma leptin concentration (11), makes reduced gonadal steroid feedback in SDs a more plausible regulator of POMC gene expression.
This raises an important issue for our understanding of the role of candidate hypothalamic genes in seasonal body weight regulation. One component of the physiological response of the Siberian hamster to short photoperiod is the regression of the reproductive system and an accompanying fall in gonadal steroid synthesis. The reduction in gonadal steroid feedback is likely to be directly responsible for part of the weight loss observed on transfer to short photoperiods; surgical gonadectomy reduces body weight in LD hamsters independent of any photoperiod change (14, 20). However, the consequences for hypothalamic gene expression and localized peptide concentrations of reduced gonadal steroid feedback remain to be established. With the description, in the present study, of the effect on hypothalamic parameters of matched body weight in intact hamsters, the outcome of further studies to duplicate the strategy employed in Exp 1 in weight-stable gonadectomized hamsters is likely to provide additional critical insight into the role of these regulatory pathways. In the present study, there was little evidence to suggest that the male reproductive system was sensitive to sustained inappropriately low body weight or low plasma leptin concentration, whereas photoperiod effects were readily apparent.
The changes in hypothalamic gene expression in response to food restriction were more pronounced, at least in terms of the anabolic neuropeptides, NPY and AGRP, in Exp 2b, when restriction was applied coincident with photoperiod-induced weight loss. In Exp 1, 10-wk food restriction in LDs produced an average 22% reduction in body weight; but although AGRP mRNA was markedly elevated, differences in NPY gene expression were not statistically significant. In Exp 2b, a 40% restriction, for 3 wk, reduced body weight by 32% relative to the start of the restriction period but by only 23.5% compared with SD-ADLIB controls. A similar food restriction regime in Exp 2a induced a clear compensatory hyperphagia once an unlimited food supply was restored. This hyperphagia, which is not observed on refeeding after 48 h of food deprivation in LD male hamsters (e.g. 14), and the apparent hypersensitivity of the anabolic neuropeptide systems to this manipulation in SDs may be the result of the animals approaching their body weight nadir, where the further depletion of fat stores would constitute a challenge that might threaten survival in the wild. There were no indications, however, of increased sensitivity of catabolic neuropeptide gene expression to food restriction in SDs compared with LDs. POMC and CART mRNA levels were affected (reduced) to a similar extent in both photoperiods, although differences between groups were not always statistically significant. CRF gene expression in the PVN was not influenced by food restriction in either photoperiod. The failure of differences in POMC and CART gene expression to attain statistical significance in Exp 2b seems to be the result of interanimal variation in this experiment, although the fact that the effects of food restriction were being superimposed upon a level of gene expression that was already subject to the regulatory impact of photoperiod manipulation may also be important. In both Exp 1 and Exp 2b, AGRP gene expression was more sensitive to negative energy balance than NPY gene expression, the latter being relatively insensitive to negative energy balance, at least that induced by food deprivation in LDs (8, 13, 14). This is an interesting outcome, in view of the extensive coexpression of NPY and AGRP mRNAs in ARC neurones (8, 28, 29, 30). The opposing effects of SDs on POMC and CART mRNAs are also indicative of transcript-specific regulation at the level of individual neurones (31).
Until the recent transgenic knockout of the melanocortin-3 receptor gene (32, 33), the physiological function of this receptor was largely unknown, although its pattern of expression within the hypothalamus (34) was suggestive of a role in energy balance. By contrast, the melanocortin-4 receptor was known to mediate effects of melanocortins on energy balance (35), the transgenic knockout being associated with hyperphagia and obesity but normal lean body mass (36). Significantly, the phenotype of the MC3-R knockout mouse is characterized by hypophagia, a relatively normal body weight, but increased fat mass and reduced lean mass (32, 33). Analysis of mice lacking both MC3-R and MC4-R reinforced the likelihood of these receptors having separate, nonredundant roles in energy homeostasis (32). We recently observed lower levels of MC3-R gene expression in the ARC of juvenile female hamsters in SDs (9). This finding in growth-restricted female hamsters has now been extended to weight loss in adult males; and significantly, there was no difference between MC3-R mRNA levels in SD males and LD males food-restricted to the same body weight in Exp 1; both groups had lower MC3-R gene expression than LD-ADLIB controls. Thus, low POMC gene expression in the ARC is associated with low levels of MC3-R mRNA in the same nucleus. However, analysis of the VMN in the same animals revealed increases in receptor mRNA levels. The expression of MC3-R mRNA in AGRP and POMC neurones in the ARC has led to the suggestion that, in this location at least, the MC3-R may be an autoreceptor (37). In this event, the function of the MC3 receptor in the inhibition of energy storage would be performed by receptors in other anatomical locations. It is not clear whether the VMN would constitute one such candidate.
Food restriction superimposed on SD weight reduction in seasonal mammals provides some of the best evidence that mammals directly regulate their body weight. The characteristic features of the elegant data from the Siberian hamster, published nearly 20 yr ago (5), were replicated here using an abrupt, square-wave transformation between LDs and SDs, rather than a natural (ambient) shortening photoperiod. This model serves to emphasize the difference between compensatory and programmed body weight regulation, the former being essentially a defense mechanism, whereas the latter provides a means of effecting advantageous long-term changes in body weight and, moreover, in the level of body weight that will be defended. Knowledge of the hypothetical body weight comparator system is sparse, and considerable research effort will be required to establish its detailed mechanics. Here, we have provided evidence that broadly equivalent chronic diminutions of the leptin signal may be read differently, according to the manipulation employed in their generation. In the seasonal mammal, photoperiod and melatonin may interact with the leptin system at the level of the leptin receptor. Furthermore, CART expressed in the ARC seems to be the most likely candidate among the studied neuropeptides to be an effector of seasonally-appropriate weight loss or growth restriction.
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Acknowledgments |
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Footnotes |
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Abbreviations: ADLIB, Fed ad libitum; AGRP, agouti-related protein; ARC, arcuate nucleus; CART, cocaine- and amphetamine-regulated transcript; CRF, ACTH-releasing factor; DMN, dorsomedial nucleus; EWAT, epididymal white adipose tissue; HE, human equivalent; IBAT, interscapular brown adipose tissue; IWAT, inguinal white adipose tissue; LD, long day; MC, melanocortin; MC3-R and MC4-R, melanocortin-3 and -4 receptors ; PVN, paraventricular nucleus; REST, restricted ration of food; RWAT, retroperitoneal white adipose tissue; SD, short day; VMN, ventromedial nucleus.
Received January 31, 2001.
Accepted for publication June 27, 2001.
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 E. Krol and J. R Speakman Regulation of body mass and adiposity in the field vole, Microtus agrestis: a model of leptin resistance J. Endocrinol., February 1, 2007; 192(2): 271 - 278. [Abstract] [Full Text] [PDF]
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A. Tups, M. Helwig, S. Stohr, P. Barrett, J. G. Mercer, and M. Klingenspor Photoperiodic regulation of insulin receptor mRNA and intracellular insulin signaling in the arcuate nucleus of the Siberian hamster, Phodopus sungorus Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2006; 291(3): R643 - R650. [Abstract] [Full Text] [PDF]
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P. Barrett, A. W. Ross, A. Balik, P. A. Littlewood, J. G. Mercer, K. M. Moar, T. Sallmen, J. Kaslin, P. Panula, S. Schuhler, et al. Photoperiodic Regulation of Histamine H3 Receptor and VGF Messenger Ribonucleic Acid in the Arcuate Nucleus of the Siberian Hamster Endocrinology, April 1, 2005; 146(4): 1930 - 1939. [Abstract] [Full Text] [PDF]
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A. W. Ross, L. M. Bell, P. A. Littlewood, J. G. Mercer, P. Barrett, and P. J. Morgan Temporal Changes in Gene Expression in the Arcuate Nucleus Precede Seasonal Responses in Adiposity and Reproduction Endocrinology, April 1, 2005; 146(4): 1940 - 1947. [Abstract] [Full Text] [PDF]
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Z A Archer, S M Rhind, P A Findlay, C E Kyle, M C Barber, and C L Adam Hypothalamic responses to peripheral glucose infusion in food-restricted sheep are influenced by photoperiod J. Endocrinol., March 1, 2005; 184(3): 515 - 525. [Abstract] [Full Text] [PDF]
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 E. Krol, P. Redman, P. J. Thomson, R. Williams, C. Mayer, J. G. Mercer, and J. R. Speakman Effect of photoperiod on body mass, food intake and body composition in the field vole, Microtus agrestis J. Exp. Biol., February 1, 2005; 208(3): 571 - 584. [Abstract] [Full Text] [PDF]
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 J. R. Speakman Obesity: The Integrated Roles of Environment and Genetics J. Nutr., August 1, 2004; 134(8): 2090S - 2105S. [Abstract] [Full Text] [PDF]
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A. Tups, C. Ellis, K. M. Moar, T. J. Logie, C. L. Adam, J. G. Mercer, and M. Klingenspor Photoperiodic Regulation of Leptin Sensitivity in the Siberian Hamster, Phodopus sungorus, Is Reflected in Arcuate Nucleus SOCS-3 (Suppressor of Cytokine Signaling) Gene Expression Endocrinology, March 1, 2004; 145(3): 1185 - 1193. [Abstract] [Full Text] [PDF]
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S. D. Bilbo and R. J. Nelson Photoperiod Influences the Effects of Exercise and Food Restriction on an Antigen-Specific Immune Response in Siberian Hamsters Endocrinology, February 1, 2004; 145(2): 556 - 564. [Abstract] [Full Text] [PDF]
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W. L. Peacock, E. Krol, K. M. Moar, J. S. McLaren, J. G. Mercer, and J. R. Speakman Photoperiodic effects on body mass, energy balance and hypothalamic gene expression in the bank vole J. Exp. Biol., January 1, 2004; 207(1): 165 - 177. [Abstract] [Full Text] [PDF]
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A. W. Ross, C. A. Webster, J. G. Mercer, K. M. Moar, F. J. Ebling, S. Schuhler, P. Barrett, and P. J. Morgan Photoperiodic Regulation of Hypothalamic Retinoid Signaling: Association of Retinoid X Receptor {gamma} with Body Weight Endocrinology, January 1, 2004; 145(1): 13 - 20. |
