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Prince Henry’s Institute of Medical Research (B.A.H., A.R., I.J.C.) and Department of Physiology (A.J.T.), Monash University, Clayton, Victoria 3168, Australia; Agriculture Victoria (F.R.D.), Victorian Institute of Animal Science, Werribee, Victoria 3030, Australia; and Animal Science (D.B., G.B.M.), Faculty of Agriculture, University of Western Australia, Nedlands, Western Australia 6009, Australia
Address all correspondence and requests for reprints to: Iain J. Clarke, Ph.D., Prince Henry’s Institute, Monash Medical Centre, Level 4, Block E, 246 Clayton Road, P.O. Box 5152, Victoria 3168, Australia. E-mail: iain.clarke{at}med.monash.edu.au
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Introduction |
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Other peptides that may play a role in appetite regulation include the
opioids and various ligands for the melanocortin receptors. The
neuropeptides ß-endorphin (ß-end), ACTH, and 
-MSH are encoded by
the POMC gene, and all are thought to be involved in the regulation of
appetite (7, 8, 9) and in neuroendocrine function (5, 10). For example,
central administration of ß-end has been shown to stimulate food
intake (11) through the µ- and 
-opioid receptor subtypes (12, 13).
Long-term food restriction has been shown to decrease the expression of
POMC in the ARC (3) and to reduce ß-end secretion in the
posterior-lateral median eminence of ewe lambs (4). Enkephalins (ENKs)
are also opioid peptides that bind predominantly to the -subtype of
the opioid receptor and stimulate feeding in sheep (11) and rodents
(7).
Melanin-concentrating hormone (MCH) is a newly identified peptide that
appears to be an important regulator of energy homeostasis. Studies
investigating the effect of MCH have produced conflicting results, with
initial indications of an inhibitory effect on food intake (14),
whereas more recent studies suggest a stimulatory role (15, 16). MCH
gene knock-out mice are hypophagic and lean, supporting the notion that
the neuropeptide stimulates food intake (17). The POMC-derived
peptides, ACTH and -MSH, bind to the melanocortin 3-receptor (MC3-R)
and melanocortin 4-receptor (MC4-R) and have also been implicated in
the regulation of feeding behavior (18, 19). MCH and -MSH have both
been shown to elicit mutually antagonistic effects on food intake and
the stress axis, although MCH is thought not to act on either MC3-R or
MC4-R (20) but on a novel MCH receptor (21). The POMC-derived
neuropeptides, ENK, and MCH all interact with the neuroendocrine
system, having effects on the reproductive, GH, and stress axes (5, 22-
24).
We have developed a ruminant model to investigate the effects of long-term alterations in body weight (adiposity) on the expression of appetite-regulating peptides. This allows very accurate definition of endocrine and metabolic parameters (through serial blood sampling) as well as body composition. In the present study, we sought to relate all of these factors to alterations in the expression of hypothalamic peptides involved in opioid and MC signaling, viz. POMC, ENK, and MCH. The expression of NPY was also measured as an indicator of response to altered body weight, since increased expression in animals of low body weight is well documented (1, 2, 3).
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Materials and Methods |
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Experimental design
Adult Corriedale ewes of mean body weight 55 ± 1 kg were
ovariectomized (to remove possible confounding effects of stage of
estrous cycle) and maintained on natural photoperiod. Ten animals were
randomly divided into two groups (FAT and THIN) and fed, respectively,
either a supplemented diet (lucerne hay ad libitum + 1 kg
lupin grain/week) to increase body weight or a restricted diet (lucerne
hay, 
400 g per day). The animals were weighed at various time points
to monitor the effect of diet on body weight. Once both groups had
reached the desired weight (after 3 months), the lupin grain was
removed from the FAT animals to stabilize their body weight and to
standardize the dietary content. Sheep were subsequently kept on either
an ad libitum lucerne hay diet (FAT) or a low nutritional
plane (restricted lucerne hay) to maintain the established differences
in body weight for a further 5 months. This was achieved by regular
weighing of the animals and adjustment of the restricted diet to
maintain the body weight difference of the two groups.
At the time of experimentation the two groups had average body weights of 60 ± 1 kg (FAT) and 37 ± 3 kg (THIN) (P < 0.001). One external jugular vein was cannulated, and serial blood samples (6 ml) were taken at 10-min intervals for 8 h (0800 h–1600 h). One hundred microliters of plasma were pooled to determine the plasma concentrations of leptin, cortisol, glucose, lactate, insulin, urea, and nonesterified fatty acids. The animals were injected (iv) with an overdose of pentobarbitone (Lethobarb, May and Baker Pty. Ltd., Australia) and decapitated. Using a peristaltic pump, the heads were perfused via both carotid arteries with 2 liters of heparinized (12, 500 U/liter) normal saline, followed with 2 liters of 4% paraformaldehyde/0.1 M sodium phosphate buffer (pH 7.4), and finally with 4% paraformaldehyde in buffer plus 20% sucrose. The brains were then removed and the hypothalamus was dissected out and placed into 4% paraformaldehyde in buffer plus 30% sucrose for 1 month until frozen and then sectioned.
Morphometric analysis
After decapitation, each animal was subjected to body
composition analysis. The liver, kidney, spleen, abdominal fat, and
emptied stomach were removed and weighed. Body composition on the
eviscerated carcass was determined using dual energy x-ray
absorptiometry (DXA) (QDR4500, Hologic, Inc., Waltham,
MA). The DXA utilizes an x-ray source located under a flat bed
and relies on differential attenuation of low [38 kiloelectronvolts
(keV)] and high (70 keV) x-rays by bone and soft tissue to
determine body composition in sheep (25).
RIAs
LH. Plasma samples (100 µl) were assayed in duplicate
using the method of Lee et al. (26) using NIH-oLH-S18 as a
standard. For five assays the average sensitivity was 0.2 ng/ml, the
intraassay coefficient of variation (CV) was less than 10% over the
range 1.2–18.5 ng/ml, and the interassay CV was less than 20%.
GH. Plasma samples (200 µl) were assayed in duplicate using the method of Thomas et al. (27) and NIDDK-oGH-I-4 as a standard. For three assays the average sensitivity was 0.5 ng/ml, the intraassay CV was less than 10% over the range 1.4–17.2 ng/ml, and the interassay CV was 13.6%.
TSH. The TSH RIA was performed using ovine thyroid-stimulating antiserum (rabbit) NIDDK-anti-oTSH-1 (AFP-C33815). Bovine TSH, NIDDK-bTSH-I-2 (AFP-7196A), was used for iodination and as the assay standard. For five assays the average sensitivity was 108 pg/ml, the intraassay CV was less than 10% between the range 621-1841 pg/ml, and the interassay CV was 5.1%.
Plasma leptin was assayed in duplicate (100 µl) using a double-antibody RIA developed by Blache et al. (28). The limit of detection was 100 pg/ml, and the intraassay CV was 4.3% at 0.74 ng/ml and 7.7% at 1.41 ng/ml. In the linear part of the standard curve the amount of bovine leptin recovered was within 2.5% accuracy, and serially diluted samples were also measured to within 2.5% accuracy. Plasma cortisol was assayed in duplicate (100 µl), using the RIA outlined by Bocking et al. (29). The sensitivity of the assay was 0.2 ng/ml and the intraassay CV was 5.3%. Plasma insulin was assayed using a kit (Linco Research, Inc., St. Charles, MO) with human insulin as a standard and validated for ovine insulin in our laboratory (30). Plasma nonesterified fatty acid (NEFA) levels were measured with an enzymatic kit assay outlined by Sechen et al. (31). Plasma glucose and lactate concentrations were measured in 25-µl samples using a YSI2300 STAT glucose/L-lactate analyzer (YSI, Inc., Yellow Springs, OH). The measurable range was between 0–30 mM for glucose and 0–16 mM for lactate. Plasma urea levels were measured using a modified enzymatic kit assay (Sigma, St. Louis, MO; procedure number 640). All samples were analyzed in a single assay with an intraassay variation of 2.3%. Quantitative analysis of free T4 (FT4) was determined by the AxSym system (Abbott Laboratories, Diagnostic Division, Abbott Park, IL), which utilized a microparticle enzyme immunoassay. Analysis of free T3 (FT3) was carried out using the RIA-gnost FT3 kit (CIS-Bio International, Sorgues, France).
In situ hybridization
Frozen 20-µm sections were cut using a cryostat and saved into
2% paraformaldehyde/cryoprotectant solution at -20 C. At least two
sections per animal were anatomically matched and mounted onto Super
Frost Plus slides (Menzel-Glaser, Braunschweig, Germany) and
dried at room temperature overnight.
In situ hybridization was performed using 35S-dUTP-labeled (NEN Life Science Products, Boston, MA) riboprobes following the method of Simmons et al. (32). The cDNA and plasmid inserts used were 1) a 400-bp ovine POMC insert in pBSSK (33), 2) a 639-bp rat preproenkephalin insert in pGem, 3) a 400-bp rat MCH insert in PCRII, and 4) a 511-bp rat NPY insert in pBSM13. The amplification, purification, and linerarization of plasmid DNA were performed using standard techniques (34). All cRNA probes were synthesized using a Gemini System II kit (Promega Corp., Annandale, New South Wales, Australia). Hybridization was carried out at 53 C in a humid chamber for a minimum of 16 h. After posthybridization, treatment slides were taped to an x-ray cassette and exposed to BioMax film (Eastman Kodak Co., Rochester, NY) at room temperature. ENK and MCH in situ slides were exposed to film for 1 week, whereas NPY and POMC were exposed for 5 days. Slides were dipped in Ilford K5 photographic emulsion (Ilford Australia, Mount Waverly, Australia) and exposed at 4 C for 3–7 days (depending on the probe), and then developed using Ilford Phenisol x-ray developer, stop bath and Hypam fixer. For ENK and MCH, the slides were exposed to emulsion for 1 week, NPY for 5 days, and POMC for 3 days. Sections were counterstained with 1% cresyl violet, dehydrated, and coverslipped using DPX. Emulsion-dipped slides were analyzed at a cellular level, counting the number of labeled cells and silver grains per cell. At least 20 cells per section were randomly selected and analyzed by counting silver grains under 400x magnification using the microcomputer imaging device (MCID) M1 system from the Imaging Research, Inc. (Brock University, St. Catharines, Ontario, Canada). Labeled cells were counted under 20x magnification.
Data analysis
Pulse analysis was used to study the secretory profile of LH and
GH, and mean daily levels of TSH were analyzed without pulse analysis.
LH pulses were calculated as previously outlined by Clarke (35). Mean
GH concentration, interpulse interval, pulse amplitude, and baseline
concentration were calculated using the TURBOPULSAR program and
parameters outlined by Henry et al. (30).
Statistical analysis
Body composition and organ weight data were corrected for body
weight and are presented in grams/kg. All data were checked for
homogeneity of variance; glucose, NEFA, and GH interpulse interval were
subjected to log transformation. Nontransformed data are presented as
means ± SEM. Body weight measurements were analyzed
by repeated measures ANOVA, and least significant differences were used
to test for significant differences between the mean values. All other
data including in situ hybridization, hormone, and metabolic
data were analyzed by single-factor ANOVA.
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Results |
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and Table 1)
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and
Table 1
)
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Expression of NPY, POMC, ENK, and MCH
The expression of NPY was influenced by body weight with higher
expression in THIN animals (Fig. 3); this
was not examined further due to the lack of expression in the FAT
group. The expression of ENK was measured in the periventricular
(PERI), paraventricular (PVN), and ventromedial (VMH) nuclei of the
hypothalamus (Fig. 4). Body weight
influenced the expression of ENK in all three regions (Figs. 4 and 5). The number of ENK-labeled cells was
higher (P < 0.05) in the PERI of the THIN animals, but
there was no difference between the groups in the number of silver
grains per cell in this region. In both the PVN and VMH the number of
ENK-labeled cells was significantly (P < 0.05 and
P < 0.01, respectively) lower in the THIN animals, as
was the number of silver grains per cell (P < 0.05 and
P < 0.01, respectively). The expression of POMC was
localized to the ARC; the number of POMC-labeled cells
(P = 0.118) and the number of silver grains per cell
(P = 0.468) were similar between FAT and THIN animals
(Fig. 6). MCH was found in the lateral
hypothalamus. There was no effect of body weight on the number of
MCH-labeled cells (Fig. 7), but a
significantly (P < 0.05) higher number of silver
grains per cell was seen in the THIN animals (Fig. 7).
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Discussion |
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Many species, including humans, have been shown to hold a set-point body weight, with only small fluctuations throughout adult life. Alterations in body weight lead to changes in both energy expenditure and appetite regulation (36, 37), to maintain the set-point. NPY is a good example for the neural regulation of this adaptation response to changes in nutritional status. Consistent with previous studies (see Introduction), the expression of NPY was greater in the ARC of THIN animals. This would stimulate appetite and lower the metabolic rate, in an effort to promote weight gain. Furthermore, NPY also regulates neuroendocrine systems that control the reproductive axis, and this may be causally linked to alterations in body weight. NPY can elicit differential effects on the secretion of LH. High doses inhibit the reproductive axis in sheep (38), as in other species, but intracerebral infusion of NPY antiserum blocks the preovulatory LH surge in ewes (6), suggesting a fundamental involvement of this neurotransmitter in the positive feedback action of estrogen. The ability of NPY to inhibit LH secretion (38) may indicate a link between the lowered nutritional status and lower LH secretion in THIN animals. With regard to GH secretion, high doses of NPY are inhibitory in the rat (39), and plasma GH levels are lowered during undernutrition in this species. In cows (40), however, high doses of NPY had no effect on GH secretion, suggesting that in higher mammals, such as humans or sheep, NPY may not mediate alterations in the secretion of GH.
Studies in rodents implicate both the PVN and VMH in the satiety response (41), and the expression of ENK was lower in both of these areas in the THIN animals. In sheep, the VMH is important in the central satiety response (42), but the role of the PVN is less well defined. The present data suggest that ENK may be one of the appetite-regulating peptides that responds to chronically altered nutritional status and adiposity. Since this is a ubiquitous peptide and is likely to serve a number of functions, it is perhaps not surprising that there were differential effects of adiposity in different regions of the hypothalamus. Since the VMH is important in the regulation of energy expenditure, alterations in ENK expression at this level may be directly involved in appetite and energy homeostasis.
In the PERI, the expression of ENK was higher in THIN animals, an effect that may be relevant to the secretion of LH via the GnRH system. Expression is greater in this nucleus during the luteal phase (43) (when LH levels are low) than during the follicular phase, consistent with our result in THIN animals. ENK expression in this region may also be relevant to changes in GH secretion due to altered adiposity. ENK stimulates GH secretion (44) by modulating somatotropin release-inhibiting factor (SRIF), and one could invoke a mechanism whereby ENK inhibits SRIF in the PERI to enhance GH secretion in THIN animals.
MCH is a peptide that stimulates appetite (see Introduction) and is found in neurons of the lateral hypothalamus (45). This region has been deemed the feeding center and lesions result in hypophagia and weight loss (7, 42). MCH expression was higher throughout this area in THIN animals and could indicate a move toward a lower metabolic rate and increased appetite to promote weight gain, as in the case of NPY. MCH has been shown to stimulate the GnRH/LH system (22), but connections between the lateral hypothalamus and other neuroendocrine systems remain to be identified for this species. The elevated expression of MCH in THIN animals, however, is not consistent with the reported (22) stimulatory effect on the reproductive axis.
Previous reports in sheep have shown that undernutrition decreases the
expression (3) of the POMC gene and reduces the levels of ß-end
(measured by push-pull perfusion) (4) in ewe lambs. The results of the
current study conflict with this data, although there was a trend
toward a reduction in the expression of mRNA for POMC in thin animals,
which was not found to be significant. One possible explanation for
this may be the greater degree of undernutrition imposed on the animals
in the previous two studies (20–30% of maintenance diet). On the
other hand, our results are consistent with a recent study in adult
castrated rams showing that POMC expression was not altered with
changes in nutrition (46). In addition, diet-induced obesity had no
effect on the hypothalamic concentrations of -MSH or POMC in male
rats (47). We therefore conclude that, with alterations in adiposity
under metabolically stable conditions, there is no alteration in the
expression of the gene for POMC-derived peptides.
There was no effect of adiposity on plasma concentrations of cortisol, a result that is consistent with previous studies in the ruminant (48). This is also consistent with our observation (30) that leptin does not affect the HPA axis in ovariectomized ewes, suggesting that under metabolically stable conditions cortisol levels are unchanged. In the human (49) and the rodent (50), reduced food intake causes an increase in cortisol levels, but this may be due to fluctuations in caloric intake and may not be related to body weight or adiposity.
It is extremely difficult to discriminate between the effects of body weight and caloric intake with respect to thyroid status, and many studies produce equivocal results. Consistent with a previous study (51), the plasma concentrations of FT4 and FT3 were lower in THIN sheep. We found that the FT3:FT4 ratio was higher in THIN animals. Changes in the hypothalamo-pituitary-thyroid axis due to altered body weight appear to manifest within the thyroid gland with lowered T4 and altered conversion to T3; there does not seem to be any effect at the hypothalamic level as plasma TSH levels were similar in FAT and THIN animals. The secretion of thyroid hormones in THIN sheep (lower FT3) indicates a move toward lower metabolic rate, which is reflected in the data on metabolic parameters (see below).
Neuroendocrine function was altered with adiposity, but plasma glucose and urea levels were similar in the FAT and THIN animals. This demonstrates that the animals of both groups were metabolically stable and suggests that neither of these metabolites is involved in conveying information regarding adiposity to the appetite-regulating and neuroendocrine systems of the brain. The plasma NEFA levels were lower in THIN animals, reflecting a reduction in fat stores, either as adipose tissue or circulating triglycerides. Whether this metabolite signals to the brain is not known, but it does not seem likely, since NEFA levels are increased with obesity as well as during negative energy balance (42). Sustained hyperinsulinemia can, however, reduce NPY mRNA levels in the ARC and reduce NPY secretion in the PVN of the rat (52). This may explain, in part, the lowered NPY expression in FAT animals. Our model will allow the study of the effects of altered insulin status in the face of normoglycemia.
The newly identified hormone leptin, which is produced by adipocytes, is thought to signal to the brain as a satiety factor (10). Plasma leptin levels were 5-fold higher in the FAT animals, and this may be a primary cause of alterations in gene expression of appetite-regulating peptides. It must be noted, however, that obesity may be associated with the development of leptin resistance (53). Whether the altered leptin status of the animals is related to the alteration in GH secretion is a matter that needs to be addressed. In rodents, it has been found that leptin stimulates GH secretion (54), and a single intracerebral injection of leptin stimulated GH secretion in pigs (55). On the other hand, we found no effect of intracerebral leptin infusion on GH secretion in normally fed sheep (30), and leptin inhibited GHRH-stimulated GH secretion from cultured ovine pituitary cells (56). The latter would be more consistent with the lowered levels of GH in the FAT animals. Alternatively, leptin resistance in these animals may be preventing the proposed stimulatory effects of leptin.
We employed DXA analysis to determine body composition and found that the FAT animals had higher estimated fat mass and percent body fat with reduced lean body mass, characteristic of the development of obesity (51). The combination of body composition analysis and measurements of metabolic parameters in plasma (see above) suggests that these THIN animals were metabolically stable and there was no deterioration of lean tissue or bone mineral content. Organ weights can be considered an indication of lean body mass, and our results for kidney, liver, and heart were consistent with the DXA analysis. The emptied stomach was enlarged in the FAT animals, indicating greater stomach capacity in this group. These data indicate that our animals not only had different body weights, but that the body composition was altered in line with the differences in adiposity.
In conclusion, we have shown that body weight influences the expression of appetite- regulating peptides in the hypothalamus and alters neuroendocrine status in the absence of ovarian hormones. The appetite stimulators NPY and MCH appear to be involved in the metabolic response to altered body weight, whereas ENK in the PERI may be linked to the secretion of GH and possibly LH. Our results suggest that altered expression of appetite-regulating peptides can be linked with the endocrine and metabolic adaptations that occur with long-term changes in adiposity.
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Acknowledgments |
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Footnotes |
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2 Supported by a stipend from Prince Henry’s Institute of Medical
Research.
Received September 7, 1999.
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References |
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-MSH antagonize feeding behaviour stimulated by
kappa opiate agonists. Peptides 7:843–848[CrossRef][Medline]
-MSH or
pro-opiomelanocortin concentrations in dietary-obese and
food-restricted rats. Biochem Biophys Res Commun 258:574–577[CrossRef][Medline]
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