Hypothalamic energy balance genes have been examined in the context of seasonal body weight regulation in the Siberian hamster. Most of these long photoperiod (LD)/short photoperiod (SD) comparisons have been of tissues collected at a single point in the light-dark cycle. We examined the diurnal expression profile of hypothalamic genes in hamsters killed at 3-h intervals throughout the light-dark cycle after housing in LD or SD for 12 wk. Gene expression of neuropeptide Y, agouti-related peptide, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, long-form leptin receptor, suppressor of cytokine signaling-3, melanocortin-3 receptor, melanocortin-4 receptor, and the clock gene Per1 as control were measured by in situ hybridization in hypothalamic nuclei. Effects of photoperiod on gene expression and leptin levels were generally consistent with previous reports. A clear diurnal variation was observed for Per1 in the suprachiasmatic nucleus in both photoperiods. Temporal effects on expression of energy balance genes were restricted to long-form leptin receptor in the arcuate nucleus and ventromedial nucleus, where similar diurnal expression profiles were observed, and melanocortin-4 receptor in the paraventricular nucleus; these effects were only observed in LD hamsters. There was no variation in serum leptin concentration. The 24-h profiles of hypothalamic energy balance gene expression broadly confirm photoperiodic differences that were observed previously, based on single time point comparisons, support the growing consensus that these genes have a limited role in seasonal body weight regulation, and further suggest limited involvement in daily rhythms of food intake.
- feeding behavior
the siberian hamster regulates its body weight with remarkable precision (17, 24), thereby providing a novel experimental paradigm for the study of the physiological control of energy balance. Transfer of adult male hamsters from long [long days (LDs)] to short photoperiod [short days (SDs)] induces body weight loss of 30–40% over a 12- to 15-wk period, while, conversely, individual SD-acclimated hamsters switched back to LDs can increase their body weight by up to 50% during a 2-wk period (27, 33). A key feature of body weight regulation in this and other seasonal species is the ability not only to defend an appropriate body weight against negative energy balance (e.g., food restriction or food deprivation), a capability common to mammalian species, but also to defend body weight according to the photoperiodic history of the animal (20, 31). Orexigenic and anorexigenic homeostatic genes in the hypothalamus, such as neuropeptide Y (NPY), agouti-related peptide (AGRP), proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART), clearly play an important role in the former process (4, 20), but the involvement of these “candidate” genes in seasonally programmed adjustments to body weight is less certain (24). Consequently, research has focused on the discovery of the components of this regulatory process (24, 28).
One of the limitations of studies of the known appetite regulatory genes has been that gene expression in a specific hypothalamic structure, such as the arcuate nucleus (ARC), has generally been compared for LD and SD groups at a single time point in the 24-h light-dark cycle. The time points selected for study are usually either early in, or in the middle of, the light phase. However, these timings do not take account of the possibility of 24-h profiles of gene expression that may be different in a 16:8-h light-dark cycle to a 8:16-h cycle. Furthermore, the timing of tissue sampling in the light phase of the cycle means that these energy balance genes are being quantified during a period when the behavior of the animal is characterized by low-energy intake, low levels of spontaneous activity, and sleep. The issue of temporal dynamics of mRNA expression across the light-dark cycle has been addressed for some energy balance genes in nonseasonal rodents (e.g., Refs. 15, 32, 36), and, although the results are relatively inconsistent, there is stronger evidence for diurnal variation in expression of orexigenic neuropeptides, such as NPY and AGRP, than for anorexigenic counterparts, such as POMC and CART. Twenty-four-hour expression profiles have also been established for a small number of “novel” genes in the Siberian hamster model (6, 28), but not for the “classical” energy balance genes in seasonal species. Thus, to further explore their role as possible regulators of seasonal body weight regulation, it is important to assess whether expression of individual genes varies across the 24-h day-night cycle in either photoperiod in addition to, or in the absence of, any apparent seasonal changes in gene expression. Quantitatively relevant diurnal variation in expression of genes that was different between the two photoperiods could have serious consequences for data interpretation.
To address this issue, hamsters that had been acclimated to LDs or SDs for 12 wk were killed at 3-h intervals across the respective light-dark cycles, and serum leptin concentrations and expression of a panel of eight energy balance genes in critical hypothalamic nuclei were analyzed. We also quantified expression of the mammalian clock gene, Per1, in the suprachiasmatic nucleus (SCN), since previous studies have demonstrated robust variation in expression across the light-dark cycle, according to photoperiod (23).
MATERIALS AND METHODS
All procedures were licensed under the United Kingdom Animals (Scientific Procedures) Act of 1986 and received approval from the Rowett Research Institute's Ethical Review Committee. Adult male hamsters (4–6 mo of age) were obtained from a breeding colony maintained at the Rowett Research Institute and held in LDs (16 h light, 8 h dark; lights on 0100–1700 GMT) at 21–22°C. All animals were fed a standard laboratory pellet diet (Rat and Mouse Breeder and Grower; Labsure; Special Diet Services, Witham, Essex, UK) ad libitum, and water was freely available. Experimental animals either continued in LDs or were transferred into SDs (8 h light, 16 h dark; lights on 0900–1700 GMT) for 12 wk. After this, groups of four LD or SD adult male hamsters were killed by cervical dislocation at 3-h intervals throughout a 24-h period, starting at lights on [Zeitgeber time (ZT) 0 (ZT0)] in each photoperiod. Safe lights were employed during the dark phase. Serum was prepared from trunk blood, and brains were rapidly dissected and frozen on dry ice.
Serum leptin was assayed using the Linco Multispecies kit (Biogenesis, Poole, UK), according to the manufacturer's instructions, and as validated previously for use with serum from the Siberian hamster (20).
Messenger RNA levels were measured using in situ hybridization. The riboprobes employed were generated from cloned cDNAs for NPY (19), AGRP (21), POMC (21), CART (1), suppressor of cytokine signaling (SOCS)-3 (34), melanocortin-3 receptor (MC3-R) (1), melanocortin-4 receptor (MC4-R) (1), leptin receptor, long form (OB-Rb) (21), and Per1 (23). Briefly, 20-μm coronal hypothalamic sections were collected onto sets of eight slides, with adjacent sections on consecutively numbered slides, permitting a number of mRNAs to be localized and quantified in each brain. Slides were fixed, acetylated, and hybridized overnight at 58°C using 35S-labeled cRNA probes (1–2 × 107 counts·min−1·ml−1). After hybridization, slides were treated with RNase A, desalted with a final high-stringency wash (30 min) in 0.1× saline sodium-citrate at 60°C, dried, and apposed to BioMax MR film (Kodak, Rochester, NY). Gene expression was quantified using the Image-Pro Plus system (Media Cybernetics, Silver Spring, MD), which determines the intensity and area of the hybridization signal on the basis of set parameters. Autoradiographic data were manipulated using a standard curve generated from 14C autoradiographic microscales (Amersham), and the integrated intensity of the hybridization signal was computed. Gene expression of NPY, AGRP, POMC, and CART in the ARC; OB-Rb and MC3-R in the ARC and ventromedial hypothalamus (VMH); SOCS3 in the ARC; and MC4-R in the paraventricular nucleus (PVN) were analyzed.
Diurnal variation in gene expression in either photoperiod was analyzed by one-way analysis of variance. Two-way analysis of variance was employed to examine the overall effect of photoperiod. Pairwise comparisons were made using the Student-Newman-Keuls method. Data that were not normally distributed were analyzed by ANOVA on ranks. Results are presented as means ± SE, and differences are considered significant where P < 0.05.
Serum leptin concentration.
There was no effect of time of day on serum leptin concentrations (Fig. 1 and Table 1) in either photoperiod [LD: F(7,24) = 0.2, P = 0.982; SD: F(7,22) = 0.9, P = 0.524]. However, leptin concentrations were higher overall in LD hamsters [F(1,46) = 37.2, P < 0.0001].
Neuropeptide gene expression.
There was no effect of time of day on NPY mRNA levels (Fig. 2 and Table 1) in either photoperiod [LD: F(7,21) = 1.2, P = 0.345; SD: F(7,26) = 0.8, P = 0.595], although gene expression was lower overall in SDs [F(1,43) = 5.3, P = 0.026]. There was no effect of time of day on AGRP mRNA levels in LDs [F(7,21) = 0.77, P = 0.619] or SDs (H = 12, P = 0.1). Gene expression was lower overall in SDs [F(1,43) = 7.6, P = 0.009]. There was no effect of time of day on POMC mRNA levels in either photoperiod [LD: F(7,24) = 0.4, P = 0.893; SD: F(7,22) = 0.8, P = 0.596], although gene expression was substantially lower overall in SDs [F(1,46) = 90.5, P < 0.0001]. There was no effect of time of day on CART mRNA levels in either photoperiod [LD: F(7,24) = 0.9, P = 0.522; SD: F(7,23) = 1.14, P = 0.373] and no overall effect of photoperiod [F(1,47) = 0.07, P = 0.792].
Receptor gene expression.
Time of day did affect OB-Rb mRNA levels (Fig. 2 and Table 1) in the ARC of LD animals [F(7,23) = 3.3, P = 0.014]. mRNA levels at ZT3 were lower than those at ZT18 and ZT21 (P < 0.05), suggesting that mRNA levels of this receptor were elevated during the dark phase in LD animals. This relationship was not seen in SD animals [F(7,23) = 0.6, P = 0.750]. The levels of OB-Rb mRNA in the ARC were affected by photoperiod [F(1,46) = 86.3, P < 0.0001]; levels in SD animals were reduced compared with those in LD animals. A similar diurnal variation in expression was observed in the VMH in LD hamsters [F(7,24) = 10, P < 0.0001], with expression levels being elevated at both ZT18 and ZT21 compared with all other time points (P < 0.05). Once again, this pattern was not observed in SD animals, and again there was a significant overall effect of photoperiod on OB-Rb mRNA levels [F(1,46) = 39, P < 0.0001], with levels being lower in SD animals.
MC3-R mRNA was also quantified in both the ARC and VMH. There was no effect of time of day on gene expression in either photoperiod for either neuroanatomical structure [LD/ARC: F(7,23) = 0.8, P = 0.595; SD/ARC: F(7,21) = 1.01, P = 0.452; LD/VMH: H(7) = 10.1, P > 0.05; SD/VMH: F(7,22) = 0.95, P = 0.490]. However, there were significant overall effects of photoperiod on MC3-R expression in both the ARC, with elevated levels in LD animals [F(1,44) = 8, P = 0.007], and the VMH, where expression was higher in SDs [F(1,45) = 24.4, P < 0.0001].
There was an effect of time of day on MC4-R gene expression in the PVN of LD hamsters [F(7,24) = 3.5, P = 0.010], with levels at ZT9 and ZT15 being significantly lower than levels at ZT3 (P < 0.05), but not in SD animals [F(7,21) = 1.37, P = 0.269]. There was a significant overall effect of photoperiod on MC4-R mRNA levels [F(1,45) = 24.3, P < 0.001], with expression in LD hamsters being lower than that in SD animals.
SOCS-3 gene expression.
There was no effect of time of day on levels of SOCS-3 mRNA (Fig. 2 and Table 1) in the ARC of either LD [F(7,24) = 1.26, P = 0.311] or SD hamsters [F(7,22) = 1.57, P = 0.197]. There was, however, a significant effect of photoperiod [F(1,46) = 75, P < 0.0001], with gene expression being consistently lower in SD hamsters.
Clock gene expression.
Per1 mRNA levels in the SCN (Fig. 3 and Table 1) exhibited diurnal variation in both photoperiods [LD: F(7,24) = 25.7, P < 0.001; SD: F(7,22) = 40.2, P < 0.0001], with a peak in gene expression at the ZT3 time point in both photoperiods. There was an effect of photoperiod on overall gene expression [F(1,46) = 30.4, P < 0.0001], driven by the low mRNA levels in the early dark phase in SDs.
Overall, the effects of time of day on the expression of hypothalamic energy balance genes were very limited (Table 1). By contrast, Per1 gene expression in the SCN demonstrated regulated profiles consistent with those reported previously (13, 22); pattern of expression varied, depending on photoperiodic background. Of the 10 nucleus/energy balance gene combinations examined, only 3 exhibited evidence of diurnal regulation. These putative rhythms were observed only in LDs and were all for receptor genes (OB-Rb in both ARC and VMH, and MC4-R in PVN). Recent studies in our laboratories characterizing the role of novel candidate energy balance genes in seasonal body weight regulation have also addressed the issue of 24-h expression profile, but without producing evidence of diurnal regulation. Components of the retinoid signaling pathway, namely cellular retinol binding protein 1, retinoic acid receptor, and retinoid X receptor in the dorsal medial posterior ARC (28), and the histamine H3 receptor in the dorsal medial posterior ARC (6), exhibited seasonal, but not diurnal, differences in gene expression.
It was particularly striking that there was no apparent rhythm in expression in either photoperiod for any of the classic ARC homeostatic neuropeptide genes, NPY, AGRP, POMC, or CART, and association with nocturnal food intake in hamsters is, therefore, limited. This contrasts with similar data obtained from freely feeding rats held in a 14:10-h light-dark cycle, where gene expression was assessed in hypothalamic blocks by ribonuclease protection assay (36). NPY mRNA levels were elevated during the early-mid-light phase, whereas POMC mRNA was maximal during the mid-late-light phase. Another study of rats, this time held in a 12:12-h light-dark cycle and with gene expression assessed by in situ hybridization, indicated that AGRP gene expression was maximal in the dark phase, whereas there were no differences across a 24-h period for POMC gene expression (15). Thus, in the latter study (15), AGRP mRNA exhibited an expression pattern that broadly paralleled feeding behavior. However, the most comprehensive investigation of diurnal expression patterns in relation to food intake is a recent study of C57BL/6J mice housed in a 12:12-h light-dark cycle, where a panel of genes, including five of those examined in the present study, was studied by quantitative real-time PCR of dissected hypothalami (32). Differences in gene expression between time points across the 24-h cycle were observed for AGRP and NPY, but not for POMC and CART. For AGRP, expression levels were highest around lights on and lowest around lights off, whereas, for NPY, the lowest level of gene expression was observed at lights on, but neither neuropeptide was clearly associated with food intake. There were few similarities with patterns reported from the similar studies of rats (15, 36).
Whereas there were no differences between individual time points across the 24-h cycle for MC3-R in either the ARC or the VMH in either photoperiod, MC4-R gene expression in the PVN of LD hamsters did show differences between time points. However, there was no consistent trend in the data relative to the light-dark cycle, and its biological relevance is presently obscure. There are no data for either melanocortin receptor from nonseasonal rodents with which to compare the hamster data reported here.
We observed pronounced variation in OB-Rb gene expression that was consistent in the two nuclei in LD hamsters, with a peak during the dark phase. This variation in receptor gene expression occurred in the absence of a marked profile in serum leptin concentration, itself an outcome consistent with a previous study of leptin in the Siberian hamster (12). It is interesting to note that leptin receptor expression in the hamster hypothalamus (both ARC and VMH) peaks during the middle of the dark phase, a pattern with some similarity to that reported from the mouse (32). However, there was no evidence of any accompanying changes in serum leptin concentration during this period, or of the decompression of a gene expression peak during the extended dark phase in SD hamsters. Nocturnal rises in blood leptin and leptin mRNA in adipose tissue have been widely reported in nonseasonal rodents (2, 3, 9, 16, 30, 32, 36), with discussion of the likely relationship between leptin and the temporal organization of food intake. These studies of the leptin signal have been accompanied by investigations of diurnal patterning in energy balance gene expression in attempts to establish a causative link to diurnal food intake. The results of these latter investigations are inconsistent (see discussion above and in Refs. 10, 32) and emphasize the complexity of the signaling involved in this process, although, of the expression profiles documented, those of leptin in adipose tissue and leptin receptor in hypothalamus most closely resemble the pattern of spontaneous food intake, where 20–25% of food was consumed during the light phase (32). An expression profile study of leptin receptor gene expression in the rat revealed a peak around lights off (36). Our study of the Siberian hamster extended these earlier investigations of the leptin signaling system to include SOCS-3, a leptin-responsive target gene that suppresses signaling downstream of the leptin receptor by inhibiting signal transducer and activator of transcription-3 phosphorylation. There was no variation in SOCS-3 in either photoperiod. Our laboratory has shown previously that SOCS-3 is expressed at high levels in LDs and that gene expression in this photoperiod, unlike in SDs, is not sensitive to peripheral leptin injection (33, 34). This suggests that the LD hamster is leptin resistant (7, 8), a conclusion supported by a body of in vivo data (5, 14, 29). It appears, therefore, that a diurnal variation in leptin receptor gene expression is revealed under conditions of high leptin levels and leptin insensitivity, but concealed when leptin levels are low and the animal is sensitive to leptin.
The 24-h profiles of ARC neuropeptide gene expression provide no evidence for the involvement of the products of these genes in daily patterns of food intake. If it is correct that leptin and its receptor are elevated in the hierarchy of genes regulating ad libitum feeding in mice (32), then the present data could indicate that Siberian hamsters are less robustly nocturnal in their food intake, as suggested elsewhere (12). The Siberian hamster has the ability to store food in cheek pouches or in a hoard. This may enable animals to ingest a more steady flow of energy throughout the light-dark cycle. Our own measures of food intake across the light and dark phases in each photoperiod suggest that hamsters from our colony are predominantly nocturnal in their food consumption. LD hamsters did not hoard appreciable amounts of food and consumed ∼80% of their daily intake during the dark phase (i.e., in 8 h). After correcting for hoarded food, SD hamsters (8 wk in SDs) consumed ∼75% of their daily intake during the dark phase (i.e., in 16 h). Daily intake in SDs was 71% of LD levels (J. G. Mercer, unpublished observations). However, it is important to note that this analysis does not take account of any food held in cheek pouches at the end of the dark phase, which could then be swallowed in the light phase. Food hoarding by Siberian hamsters has been the subject of extensive study by Wood and Bartness (35), including the observation that SD hamsters hoard food at a greater rate than LD hamsters. Thus the temporal pattern of food actually entering the gastrointestinal tract in the Siberian hamster may not be as rigidly diurnal as in rats and mice, although the animals are likely to be in negative energy balance toward the end of the light phase. This energy deficit may be more pronounced in LD hamsters, but does not induce any patterning in serum leptin concentration.
The overall effects of photoperiod on hypothalamic energy balance regulatory gene expression (Table 1) were generally consistent with previous reports, and the ability to detect quantitatively small changes in gene expression may have been enhanced by the relatively large sample size (n = 32) in the pooled data. The limited overall reduction in ARC NPY gene expression in SDs supports some published observations (4, 25), but not others (1, 20, 21, 29). The overall depressive effect of SDs on ARC AGRP gene expression was similar to the effect on the coexpressed (21) NPY mRNA. However, there is no clear consensus in the literature for the effect of photoperiod on expression of AGRP, whereas POMC gene expression is consistently downregulated in SDs in all published studies (1, 4, 20, 21, 25, 29). POMC was one of only two genes whose expression profiles in LDs and SDs were clearly separated at each of the eight time points. Whereas previous single time point studies in our laboratories have consistently reported increased CART gene expression in the ARC of SD animals (1, 4, 18, 20), this was not the case in the present study, and other groups have reported no effect of photoperiod on mRNA levels (26, 29). Here, CART mRNA levels were the least consistent in terms of LD-to-SD ratio across the eight time points. The overall effect of SDs was to reduce OB-Rb gene expression in both the ARC and the VMH, and SOCS3 gene expression in the ARC, in line with published studies (1, 20, 21, 33, 34). Similarly, the differential effect of SDs on MC3-R gene expression in the ARC and VMN confirmed published findings (20), whereas no clear effects of photoperiod on MC4-R gene expression in the PVN have been detected previously (1, 20). Blood leptin levels in the Siberian hamster are positively correlated with body adiposity (5, 11, 14, 20, 34), with lowest levels in SD hamsters with established seasonal weight loss.
The general absence of variation in circulating leptin and hypothalamic gene expression in Siberian hamsters suggests that, for many genes, the observed effect of photoperiod, measured at a single point in the light-dark cycle, is likely to be representative of overall change, or its absence. However, detailed assessment of the expression profiles of other genes in the different photoperiods suggests the possibility that comparisons may have been made that could have been influenced by temporal dynamics. Such sampling effects could explain some of the inconsistencies in the literature, for example for CART gene expression, where a small difference in time of sampling within the light phase might have a disproportionate influence on the outcome of LD/SD comparisons. Consequently, the potential importance of diurnal effects on gene expression should not be overlooked as new candidate molecules emerge and, particularly, as those molecules are evaluated in seasonal models. Despite these cautionary comments, the current studies support the growing consensus that the classical homeostatic energy balance genes play a role in compensatory mechanisms involved in responding to energy deficits, but have a more limited role in seasonal regulation of food intake and body weight (24). Furthermore, the lack of apparent association between leptin or gene expression profiles and feeding behavior questions their role in hour-to-hour food intake in the Siberian hamster.
Perspectives and significance.
Comparison of the data reported here from the Siberian hamster with those obtained from nonseasonal rodents emphasizes the importance that should be attached to the timing of sampling in studies assessing hypothalamic gene expression, and whether the tissues so sampled are representative of overall physiological state. Ideally, significant diurnal variation in gene expression should be either ruled out or characterized as new candidate molecules emerge. The generally weak association of expression profiles of individual genes with feeding behavior in any species highlights the limitations of our knowledge of the regulation of ad libitum feeding (32), as opposed to feeding under conditions of negative energy balance. The accumulating evidence from both seasonal and nonseasonal mammals indicates that ad libitum feeding, body weight defense against negative (or positive) energy balance, and the determination of the level of body weight that is to be defended are separate, albeit related, processes, probably involving different molecules and neuroanatomical structures. Despite substantial advance over the last two decades, the journey toward adequate understanding of these processes and the harnessing of that knowledge in the fight against obesity are still in their early stages.
This work was supported by the Scottish Government Rural and Environment Research and Analysis Directorate, by a Biotechnology and Biological Sciences Research Council Collaborative Awards in Science and Engineering studentship to C. Ellis, and by the European Commission FP6 Diabesity Project (LSHM-CT-2003-503041).
We thank Dr. G. Horgan, Biomathematics and Statistics Scotland, for advice on statistical analysis.
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.
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