widespread increases in regional hypothalamic neuropeptide y levels in acute cold-exposed rats

Neuroscience Vol. 54, No. I, pp. 127-132, 1993 Printed in Great Britain

0306-4522/93 $6.00 + 0.00 Pergamon press Ltd

0 1993 IBRO

WIDESPREAD INCREASES IN REGIONAL HYPOTHALAMIC NEUROPEPTIDE Y LEVELS IN

ACUTE COLD-EXPOSED RATS

H. D. MCCARTHY,*? A. P. KILPATRICK,* P. TRAYHURN$ and G. WILLIAMS*

*Department of Medicine, University of Liverpool, Liverpool L69 3BX, U.K. IDivision of Biochemical Sciences, Rowett Research Institute, Bucksbum, Aberdeen AB2 9SB, U.K.

Ahstrati-Neuropeptide Y injected into the hypothalamus or third ventricle stimulates feeding and inhibits the sympathetic activation of brown adipose tissue. To clarify the involvement of hypothalamic neuropeptide Y in cold-induced thermogenesis, groups of rats exposed to 4°C for 2.5 or 18 h were compared with warm-adapted rats (22°C). Neuropeptide Y was measured in eight selected hypothalamic regions, including those known to be involved in the regulation of energy expenditure. Activation of brown adipose tissue was confirmed by significant six- to nine-fold increases in brown adipose tissue uncoupling protein messenger RNA. Compared with warm-adapted controls, neuropeptide Y levels were significantly raised by 80-170% in several hypothalamic regions of rats exposed to cold for 2.5 h, namely the medial preoptic area, paraventricular nucleus, ventromedial nucleus, dorsomedial nucleus and lateral hypothalamic area. Neuropeptide Y levels in 18-h cold-exposed rats were similarly elevated in these regions and were also significantly increased in the anterior hypothalamic area (75%). By contrast, neuropeptide Y levels in the arcuate nucleus, the main hypothalamic site of synthesis, were not increased by cold exposure, being significantly reduced by 21% after 2.5 h exposure and comparable with controls after 18 h.

As neuropeptide Y injection inhibits brown adipose tissue activation, we suggest that the rapid and dramatic increases in neuropeptide Y levels in specific hypothalamic regions occur because cold exposure might inhibit the release of neuropeptide Y and so cause accumulation of neuropeptide Y in these sites. By removing a central neuropeptide Y-ergic inhibitory influence on the sympathetic outflow to brown adipose tissue, such changes may lead to increased energy expenditure. At normal environmental temperatures, neuropeptide Y may therefore tonically inhibit brown adipose tissue activity to maintain body temperature and regulate energy homeostasis.

Exposure to cold stimulates metabolic rate and heat production, which serves to maintain homeo- thermy.i3 In small mammals such as rats, cold stimulates metabolic rate primarily through non-shivering thennogenesis (NST), i.e. sympathetically mediated activation of thermogenesis in brown adipose tissue (BAT). 16~‘9 The central mechanisms which regulate the increased sympathetic output to BAT apparently involve several hypothalamic regions. These include the preoptic area which is crucial in thermoregulation and NST,) and other areas which have neural connec- tions with BAT, notably the paraventricular nucleus, ventromedial nucleus and the anterior hypothalamic area.“.*’

The neuropharmacology of the hypothalamic control of BAT has recently received much attention.

tTo whom correspondence should be addressed at: Depart- ment of Human Nutrition, University of Southampton, Bassett Crescent East, Southampton SO9 3TU, U.K.

Abbreviations: AHA, anterior hypothalamic area; ARC, arcuate nucleus; BAT, brown adipose tissue; CRF, corticotrophin-releasing factor; DMN, dorsomedial nucleus; LHA, lateral hypothalamic area; LPO, lateral preoptic area; MPO, medial preoptic area; NPY, neuropeptide Y; NST, non-shivering thermogenesis: pNPY, porcine NPY; PVN, paraventricular nucleus; VMN, ventromedial nucleus.

Certain neuropeptides with established roles in the hypothalamic regulation of appetite may also be important in the central modulation of sympathetic outflow to BAT.” For example, corticotrophin- releasing factor (CRF), an important endogenous inhibitor of food intake,** powerfully stimulates the sympathetic outflow to BAT, oxygen consumption and body temperature when injected intracerebroventricularly.‘*‘O Recently, neuropeptide Y (NPY) has also been implicated in the hypothalamic control of energy balance and thermoregulation.

NPY is a 36-amino acid peptide which is distributed widely throughout the central and peripheral nervous systems and is concentrated particularly within the hypothalamus, where its principal site of synthesis is the arcuate nucleus (ARC).3o The ARC produces NPY-containing axons which pass through the lateral hypothalamic area (LHA) to end in the medial preoptic area (MPO), paraventricular nucleus (PVN) and dorsomedial nucleus (DMN).*s4* NPY potently stimulates feeding when injected into several hypothalamic areas of the rat, including the PVN, ventromedial nucleus (VMN), LHA, DMN and MP0.29,38 Its repeated administration leads to obesity;39 not all the excess weight gain can be attributed to increased energy intake, suggesting that NPY also reduced energy expenditure. This possibility is

127

supported,by the finding that hypothermia resulted from NPY injected intracerebroventricularly in mice and dogs,“~” or into the preoptic area of rats.3” Furthermore, NPY has been shown directly to decrease BAT thermogenesis when injected into the third ventricle (measured as a reduction in mitochondrial GDP binding) and its injection directly into the PVN suppresses the firing rate of the sympathetic nerves supplying BAT.‘.“’

In view of NPY’s experimental effects in inhibiting BAT function, it would be predicted that rats exposed to a cold environment would show reduced hypothaiamic NPY release, to permit activation of BAT.

We have therefore compared NPY levels in microdissected hypothalamic regions in rats acclimatized to a normal environmenta temperature (22°C) and in rats exposed to cold (4’C) for either 2.5 or 18 h. These periods of cold exposure stimulate synthesis of mitochondrial uncoupling protein in BAT and leads to an unmasking of its GDP-binding sites which play a central role in the activation of thermogenesis.~’ BAT levels of mRNA coding for uncoupling protein rise rapidly on cold exposure,‘,“,” whereas prolonged exposure to cold results in a gradual increase in the level of the protein itself, following an initial delay.40

EXPERIMENTAL PROCEDURES

Animuls

Male hooded rats (Rowett strain, Rowett Institute), aged seven weeks, were studied. They had previously been acclimatized to an ambient temperature of 22’C and a 12: I2 h light: dark cycIe (lights on at 07.00 h). Standard laboratory chow and tap water were available ad fibitum.

At 19.00 h on the evening before the study, nine rats were caged individually and placed in a room at a constant temperature of 4”C, with the same light: dark cycle as previously. Because of the time taken to perform the dissection procedure detailed below, these rats were exposed for 18. 20 h. The following day, nine rats were individually caged and placed at 4°C at staggered intervals, such that each rat was exposed to the cold for 2.5 h. The remaining nine rats were continuously m~n~ned at room temperature. Ail animals remained alert and we11 throughout.

Rats were killed within 60 s by carbon dioxide inhalation and immediately exsanguinated. Plasma was stored at - 70°C and later assayed for glucose, insulin and corticosterone levels. BAT was rapidly removed from the interscapular site, frozen in liquid nitrogen, and stored at -80% until measurement of the uncoupling protein mRNA (see below).

Hypothalamic dissection

The brain was quickly removed and a coronal block of tissue containing the hy~thalamus was cut on a vibrating microtome into slices 350-500 pm thick. Eight selected hypothalamic areas were microdissected as previously describedZh and the tissue boiled in 400 ~1 of 0.1 M HCl and sonicated for 30 s to extract NPY, Samples were frozen at - 70°C until assayed for NPY and protein content. The areas studied were: MPO, lateral preoptic area (LPO), anterior hypothalamic area (AHA), PVN, VMN, LHA, DMN and the arcuate nucleus together with median eminence (ARC).

Assal’s

Plasma glucose was measured by a glucose oxidase-based autoanalyser. Insulin was measured by radioimmunoassay

using a commercial kit which employed rat rnsuiiu ;i’, standard (Novo Biolabs Ltd, Cambridge, 1i.K.) The within-assay coefficient of variation was 4.0’%,. C‘ortico- sterone was measured using a commercial kit (IDS. Tync and Wear, U.K.), with an assay sensitivity of0.5 ngml and an intra-assay coefficient of variation of t4”.,,$

NPY was measured in 35- lOO-~1 samples using an m house assay employing [‘251]labelled porcine NPY (pNPY b (Amersham International, Amersham. U.K.), pNPY as standard (Bachem. Saffron Walden, U.K.) and rabbit anti- pNPY antiserum used at a finai dilution of I : 30,000. The assay sensitivity was 3 fmol/tube and the intra-assay coefficient of variation was ~4%. We have previously shown that NPY-like immunoreactivity in hypothalamic extracts as detected by this assay coelutes on high-perfor~~ce Iiquid chromatography with that for synthetic pNPY” and that there is no significant cross-reactivity ( < I %) with the related peptides, pancreatic polypeptide and peptide YY.

Protein content of the hypothaiamic extracts was measured by the Lowry method2’ and NPY concentrations in each region were expressed as fmoliiig protein.

Northern blotting .for uncoupling protein messenger RNA

Levels of mitochondrial uncoupling protein mRNA in BAT was assessed as an index of the acute activation of thermogenesis foliowing cold exposure. Although mitochondrial GDP binding measurements have traditionally been used for this purp0se.j’ the logistics of the present study made it impractical to perform binding studies on sufficient numbers of the samples generated. Mitochondr~ need to be isolated from fresh tissue and CiDP binding measurements undertaken immediately, in contrast to mRNA studies which can be processed from material that has been stored frozen (~40 ‘C).

Uncoupling protein mRNA was measured by northern blotting using a 27-mer oligonucleotide corresponding to a region of the gene coding for the protein that IS highly conserved across mammalian species.‘” The method has previously been described.’

Briefly, total RNA was extracted from frozen tissue by a guanidinium isothiocyanate-phenol method,” and 20 or 25 pg of RNA per lane was then applied to a 1.4% agarose gel and subjected to horizontal-gel electrophoresis (BRL, Life Technologies Inc., U.S.A.). The RNA was transferred overnight to a nitrocellulose membrane (Hybond-C Extra: Amersham International, U.K.) by capillary blotting. After prehybridization, hybridization wis conducted at 4?C for 3 h with 5 x sodium chloride-sodium citrate buffer. 0.5% sodium dodecyl sulphate, 5 x Denhardt’s and a 20 pM concentration of the oljgonucleotide. This was modified for enhanced chemiluminescence by addition of an amino group to the S-end, to which alkaline phosphatase was attached using the “E-link PlusTM” system kit (Cambridge Research Biochemicals, U.K.). After washing and removal of excess buffer, the nitrocellulose membrane sprayed with Lumi- PhosT” (Cambridge Research Biochemicals), and sealed in a polythene bag. The membrane was then exposed to film (Hyperfilm’r”-ECL), Amersham International, U.K.) for two days. After development of the film, the 1.5 kb band corresponding to the mRNA for uncoupling protein was quantitated by densitometry.

Statistical analvses

Values are expressed as mean _+ S.E.M. For NPY levels, differences between groups were tested by two-way (ANOVA) coupled to a posf hoc Scheffe test. Where group effects were observed, differences between groups within individual regions were analysed using a Student’s r-test for unpaired data. A P value of 0.05 or less was taken as significant. For uncoupling protein mRNA band densities were normalized (controls = 1.0 arbitrary units) and differences between groups were tested by an unpaired Student’s l-test.

Hypothalamic neuropeptide Y and cold exposure

Table 1. Effect of cold exposure on uncoupling protein messenger RNA levels in brown adipose tissue and on plasma hormone and glucose concentrations

129

Control Cold exposure (22°C) 2.5 h 18h

Uncoupling protein mRNA (arbitrary units) l.OkO.2 6.6 f 2.5* 9.0 f 2.7+ Corticosterone (ng/ml) 37.2 k 2.4 64.1 f 3.6*** 56.6 +4.1*** Insulin (ng/ml) 7.1 + 1.7 6.3 f 0.4 5.3 If: 0.4 Glucose (mmol/l) 7.0 f 0.3 8.3 + 0.4** 8.0 f 0.2;

*P < 0.05 vs control; l *P < 0.01 vs control; ***P i 0.001 vs control.

RESULTS

Changes in uncoupling protein messenger RNA and plasma analytes

These data are shown in Table 1. Levels of uncoupling protein increased substantially (P < 0.05) following cold exposure, indicating that thermogenesis had been activated. After 2.5 and 18 h, levels increased 6.6-fold and 9-fold, respectively, compared with controls maintained at room temperature.

Cold exposure significantly raised corticosterone levels (72 and 52% for 2.5 and 18 h, respectively, P < 0.01). Plasma glucose levels were slightly but significantly raised in the cold-exposed groups (P < 0.01 and P < 0.05 for 2.5 and 18 h, respectively). Insulin levels were unaltered by cold exposure.

Hypothalamic neuropeptide Y levels

NPY levels in the eight hypothalamic regions of all three groups are shown in Fig. 1. Two-way ANOVA revealed a significant effect attributable to group

(Fw, = 36.7, P < O.OOOl), and region (F,,,** = 35.1, P c 0.0001) and a significant interaction between group and region (F17,,88 = 3.68, P = 0.027).

Striking differences in NPY levels within several regions were observed between warm-adapted and cold-exposed rats. Cold exposure for 2.5 h resulted in dramatic increases in NPY concentrations in five regions, namely, the MPO (by 96%, P < O.OOOl), PVN (109%, P < O.Ol), VMN (126%, P < O.OOOl), LHA (SO%, P c 0.0001) and DMN (170%, P <

MPD LPO AHA PVN VMN LHA DMN ARC

Hypothalamic area

Fig. 1. NPY levels in eight hypothalamic regions in rats exposed either to a warm environment (22”C, solid bars) or to 4°C for 2.5 (single-hatched bars) and 18 h (cross-hatched bars). Values = mean + S.E.M., n = 9 per group. *P c 0.05;

**p < 0.01; ***p < 0.005; tP < 0.001.

0.005). After 18 h cold-exposure, NPY levels were similarly elevated in these nuclei and did not differ significantly from those in the 2.5-h cold-exposed group. NPY levels in the AHA were also increased in cold-exposed rats but only reached significance at 18 h (75% increase over warm-adapted values, P < 0.02). Levels in the LPO also showed a tendency to rise, but failed to reach significance in either cold-exposed group.

By contrast, NPY levels in the ARC showed a completely different pattern, with no tendency to rise. Indeed, levels fell significantly by 21%. (P c 0.05) after 2.5 h cold exposure and a level virtually identical to controls after 18 h (P = not significant).

DISCUSSION

Exposure to cold is the most potent stimulus to thermogenesis in BAT. The sequence of events which result in BAT activation involve afferent thermal sig- nals from the skin and temperature-sensitive neurons within the hypothalamus, co-ordination within the hypothalamus, and activation of sympathetic outflow to the periphery.rg The precise neural pathways involved, and the neurotransmitters which mediate these processes, are largely unknown. Recent studies have yielded mounting evidence to suggest that NPY may function in the central regulation of BAT activity.

At the start of this study, we hypothesized that rats exposed to a cold environment would show reduced NPY-ergic activity. This assumption was based on the observations that intrahypothalamic injection of NPY induced hypothermia and reduced sympathetic outflow to BAT.5*‘4*33 However, the possible physiological role of NPY in thermogenesis has not been systematically investigated.

This study showed that cold exposure which stimulated thermogenesis in BAT also markedly increased the concentrations of NPY in several hypothalamic regions which are involved in thermoregulation. These rapid changes indicate that cold exposure and hypothalamic NPY turnover are inextricably linked, although this experiment does not allow us to define precisely the changes which occurred in NPY systems, and the relationship to increased sympathetic outflow to BAT.

The level of a peptide within a particular region reflects the dynamics of its synthesis, transport, stor- age and release. The elevated regional levels observed in this study could therefore be a consequence of

130 H. LT. MCCARTHY et trl

increased synthesis and transport to these sites, or decreased release, or a combination of these processes. The majority of NPY in the hypothalamus arises from synthesis within the ARC,? although there is also an input from NPY-synthesizing neurons origin- ating in the brainstem which projects to the PVN.” In this study, the absence of any rise in NPY in the ARC strongly suggests that the synthesis of the peptide was not increased, and the significant fall in NPY concentrations after 2.5 h of cold exposure may even indicate that synthesis was inhibited. NPY concentrations in the sites of projection of the ascending input from the brainstem could rise through increased synthesis and transport in this pathway, although this seems unlikely in view of the relative length of this projection and the very rapid time-course of the changes. We were unable to examine NPY concentrations in the brainstem nuclei because of the time needed to dissect the hypothalamic nuclei.

An alternative explanation for the increase in NPY

concentrations in the regions supplied by the ARC is inhibition of its release from the nerve endings, leading to accumulation of the peptide in the presynaptic nerve terminals. As NPY injected into the hypothalamus is known to reduce BAT activity, presumably by reducing the intensity of the sympathetic outflow to the tissue,5.‘4 reduced endogenous release of NPY could lead to disinhibition of BAT activity. Some regions which showed prominent increases in NPY levels are implicated in energy expenditure regulation (PVN, MPO, VMN) and the PVN is a specific site where NPY injection reduces BAT thermogenesis. Inhibition by cold exposure of NPY release in areas

of the hypothalamus known to be involved in temperature regulation would therefore be physiologically appropriate, since it may reflect release of a central inhibitory influence on sympathetic outflow to the periphery, so allowing the necessary increase in thermogenesis. From these changes in NPY levels following acute cold exposure, possibly due to an inhibition of release, it is tempting to speculate that perhaps hypothalamic NPY may have a physiological role in the central control of thermogenesis and body temperature regulation. At normal environmental temperatures, NPY-containing systems terminating in the hypothalamus may provide a tonic inhibitory influence on sympathetic outflow to BAT. As discussed above, this hypothesis will need to be further tested by clarifying other aspects of NPY turnover in the hypothalamus, through studies of NPY synthesis, receptor changes and release in circumscribed hypothalamic regions which can be measured using push-pull sampling.22

These findings (of increased NPY concentrations in the MPO, PVN, VMN, DMN and LHA without any associated rise in the ARC) contrast fundamentally with those seen in the fatty Zucker rat where we have demonstrated similar increases in hypothalamic NPY content, notably in the MPO, PVN and DMN.‘” However, in this situation, a significant increase in

NPY is also found in the ARC- the hypothalamic site of synthesis and this increase in NPY content IS coupled to increased NPY mRNA expression in the ARC.36 Increased synthesis together with increased regional levels suggest that the activity of the NPY- ergic projections is increased. This is supported by the findings that in the fatty Zucker rat hypothalamic NPY receptors are downregulated and the feeding response to exogenous NPY is blunted.2’ The events leading to NPY release may also be stimulated in these animals, and this could contribute to their decreased thermogenesis which is a major factor in their obesity.’ Similarly, in food deprivation, where the drive to eat is increased, NPY concentrations are elevated in

the ARC as well as in the PVN and other sites of projection.4 In addition, release of NPY within the PVN is increased22 and increased NPY synthesis in the ARC has been confirmed by demonstrating rises in NPY mRNA levels.’ Food deprivation differs fundamentally from cold exposure, in that energy expenditure is decreased in the former and greatly stimulated in the latter. We suggest that changes in BAT activity may be mediated by increased and decreased NPY release, respectively.

Exactly how hypothalamic NPY release is regulated is unknown, but presumably it is affected by local neurotransmitter concentrations, or perhaps by changes in circulating hormones stimulated by cold exposure. In this experiment, we observed that cold exposure resulted in a significant rise in circulating corticosterone levels. Glucocorticoid receptors are distributed throughout the hypothalamus and are present on many NPY-containing neurons.‘” Cortico- sterone may act here to block NPY release, as Corder ef al. have shown that chronic treatment of rats with glucocorticoids resulted in an accumulation of NPY in hypothalamic neurons.” These changes were attributed both to increased synthesis and to reduced release; as discussed above, the unchanged levels in the ARC in these acute experiments argue against any substantial increase in intrahypothalamic NPY synthesis.

A second, but less powerful stimulus for BAT thermogenesis in rats is overfeeding, especially if stimulated by a varied and palatable high-energy diet. It has been shown that such a “cafeteria diet” can activate thermogenesis.3s The central mechanisms regulating sympathetic outflow to BAT under these conditions are not clear, but may overlap with those which control cold-induced non-shivering thermogenesis. Cafeteria feeding for seven weeks which induced hyperphagia and obesity in rats, resulted in increased NPY concentrations in several hypothalamic nuclei, notably the MPO, PVN and ARC.” There were no increases in NPY mRNA, arguing against increased synthesis and again suggesting that NPY may have accumulated in these sites through reduced release. Chronic cafeteria feeding and acute cold exposure both induce BAT activation; should NPY act physiologically to inhibit BAT activity. then

Hypothalamic neuropeptide Y and cold exposure 131

reduced NPY release would be anticipated in both NIT’s experimental effects in inhibiting BAT activity. these situations. As these changes in NPY in the hypothalamus are

the most acute and widespread yet reported for any

CONCLUSION manipulation, this may indicate that NPY’s primary role is in regulating energy expenditure.

In summary, we have shown that acute cold exposure in rats results in a marked increase in NPY levels in several hypothalamic nuclei implicated in Acknowledgements-We are grateful to the Cancer Re-

thermoregulation and feeding. The cause of these rapid search Campaign for the support of HDM, and to Jackie Keith for skilled assistance. We would also like to thank Dr

increases are as Yet unclear, but they may represent Thue Schwartz, (Rigshospitallt, Copenhagen, Denmark), inhibition of release which would be consistent with for kindly providing the NPY antiserum.

1.

2.

I.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19. 20.

21.

22.

23.

24.

25.

REFERENCES

Arase K., York D. A., Shimizu H., Shargill N. and Bray G. A. (1988) Effects of corticotropin-releasing factor on food intake and brown adipose tissue thennogenesis in rats. Am. J.‘Phyiiol. 255, E255-E259.- Bai F. L.. Yamano M.. Shiotani Y.. Emson P. C.. Smith A. D.. Powell J. F. and Tohvama M. (1985) An arcuato- paravent&lar and dorsomedial hypothalamic neuropeptide Y-containing system which iacks noradrenahne in the rat. Brain Res. 331, 172-175. Banet M., Hensel H. and Lieberman H. (1978) The central control of shivering and non-shivering thermogenesis in the rat. 1. Physiol. 283, 569-584. Beck B., Jhanwar-Uniyal M., Burlet A., Chapleur-chateau M., Leibowitz S. F. and Burlet C. (1990) Rapid and localized alterations of neuropeptide Y in discrete hypothalamic nuclei with feeding status. Brain Rex 528, 245-249. Billington C. J., Briggs J. E., Grace M. and Levine A. S. (1991) Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism. Am. J. Physiol. 260, R321-R327. Bouillaud F., Riquier D., Mory G. and Thibault J. (1984) Increased level of mRNA for the uncoupling protein in brown adipose tissue of rats during thermogenesis induced by cold exposure or norepinephrine infusion. J. biol. Chem. 259, 11,583-l 1,586. Brady L. S., Smith M. A., Gold P. W. and Herkenham M. (1990) Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-derpived rats. Neuroendocrinology 52, 441-447. Brander F., Keith J. S. and Trayhum P. (1992) A 27-mer oligonucleotide probe for the detection and measurement of the mRNA for uncoupling protein in brown adipose tissue of different species. Comp. Biochem. Physiol. 104B, 125-131. Bray G. A., York D. A. and Finkler J. S. (1989) Experimental obesity: a homeostatic failure due to defective nutrient stimulation of the sympathetic nervous system. Vitamins Hormones 45, l-125. Brown M. R., Fischer L. A., Rivier J., Speiss J., Rivier C. and Vale W. (1982) Corticotropin-releasing factor: effects on the sympathetic nervous system and oxygen consumption. Life Sci. 30, 207-210. Chomcxynski P. and Sacci N. (1987) Single-step method of RNA isolation by guanidinium isothiocyanate-phenol- chloroform extraction. Analyt. Biochem. 162, 156-159. Corder R., Pralong F., Turnill D., Saudan P., Muller A. F. and Gaillard R. C. (1988) Dexamethasone treatment increases neuropeptide Y levels in rat hypothalamic neurones. Life Sci. 43, 1879-1886. Davis T. R. A. and Mayer J. (1955) Demonstration and quantitative determination of the contributions of physical and chemical thermogenesis on acute exposure to cold. Am. J. Physiol. 181, 675-678. Egawa M., Yoshimatsu H. and Bray G. A. (1991) Neuroceptide Y suppresses sympathetic activity to interscapular brown adipose tissue in rats. Am. J. Physiol.. 260,. R328-R3j4. _ _ _ _ E&ban J.. Chover A. J.. Sanchez P. A.. Mica J. A. and Gibert-Rahola J. (1989) Central administration of neuropeptide Y induces hypothermia in mice. Possible interaction with central noradrenergic systems. Life Sci. 45, 2395-2400. Foster D. 0. and Frydman M. L. (1978) Non-shivering thermogenesis in the rat 2: Measurement of blood flow with microspheres point to brown adipose tissue as the dominant site of the calorigenesis induced by noradrenaline. Can. J. Physiol. 56, 110-l 12. Freeman P. H. and Wellman P. (1987) Brown adipose tissue thermogenesis induced by low level electrical stimulation of hypothalamus in rats. Brain Res. Bull. 18, 7-l 1. Gustafsson J. A., Carlstedt-Duke J., Poellinger L., Okret S., Wilkstrom A. C., Bronnegard M., Gillner M., Dong Y., Fuxe K., Cintra A., Harfstrand A. and Agnati L. (1987) Biochemistry, molecular biology and physiology of the gluwcorticoid receptor. Endocr. Rev. 8, 185-234. Himms-Hagen J. (1984) Non-shivering thermogenesis. Brain Res. Bull. 12, 151-160. Holt S. J., Wheal H. V. and York D. A. (1987) Hypothalamic control of brown adipose tissue thermogenesis in Zucker lean and obese rats. Effects of electrical stimulation of the ventromedial nucleus and other hypothalamic centres. Brain Res. 405, 221-233. Jacobsson A., Stadler U., Glotzer M. A. and Koxak L. P. (1985) Mitochondrial uncoupling protein from mouse brown fat. Molecular cloning, genetic mapping and mRNA expression. J. biol. Chem. 260, 16,250-16,254. Kalra S. P., Dube M. G., Sahu A., Phelps C. P. and Kalra P. S. (1991) Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food. Proc. natn. Acad. Sci. 88, 10,931-10,935. Lowry O., Rosebrough, N., Farr A. and Randall R. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-270. Macfarlane F. and Trayhum P. (1990) Oligonucleotide probe for the cross-species measurement of the mRNA for uncoupling protein in brown adipose tissue. Biochem. Sot. Trans. 18, 1261. McCarthy H. D., McKibbin P. E., Holloway B., Mayers R. and Williams G. (1991) Hypothalamic neuropeptide Y characteristics and NPY-induced feeding responses in lean and obese Zucker rats. Life Sci. 49, 1491-1497.

132 H. D MCCAKTHY et (I/.

26

27

28

29

30 31

32.

33

34. 35. 36.

31

38.

39.

40.

41.

42.

43.

McKibbin P. E., Cotton S. J., Macmillan S., Holloway B., Mayers R., McCarthy H. D. and Williams G. (1Wi ) Altercd neuropeptide Y concentrations in specific hypothalamic regions of obese ( faifa) Zucker rats: possible relationship To obesity and neuroendocrine disturbances. Diabetes 40, 1423 1429. Morioka H., Inui, A.. Inoue T., Sakatani N.. Oya M.. Mizuno N. and Baba S. (1986) Neuropeptide Y dccrcases recral temperature after intracerebroventricular administration in conscious dogs. Kobe J. Med. Sci. 32, 45 47. Morley J. E. and Levine A. S. (1982) Corticotropin-releasing factor, grooming and ingestive behaviors. /.ifr .%i 31, 1459 1464. Morley J. E., Levine A. S. Gosnell B. A., Kneip J. and Grace M. (1987) Effect of neuropeptide Y on ingestive behaviors in the rat. Am. J. Physsiol. 252, R599%R609. Morris B. J. (1989) Neuronal localisation of neuropeptide Y gene expression in rat brain. J. camp. Neural. 290,358- 368. Reichling S., Pate1 H. V., Freeman K. B.. Kates A. L. and Himms-Hagen J. (1988) Attenuated cold-induced increase in mRNA for uncoupling protein in brown adipose tissue of obese (objob) mice. Biochem. ceN. Biol. 66, 193- 198. Rogers P., McKibbin P. E. and Williams G. (1991) Acute fenfluramine administration reduces neuropeptide Y concentrations in specific hypothalamic regions of the rat: possible implication for the anorectic effect of fenfluramine. Peptides 12, 251L2.55. Roscoe A. K. and Myers R. D. (1991) Hypothemia and feeding induced simultaneously m rats by perfuslon of neuropeptide Y in preoptic area. Pharmac. Biochem. Behac. 39, 1003-1009. Rothwell N. J. (1989) Central control of brown adipose tissue. Proc. Nutr. Sot. 48, 197~~206. Rothwell N. J. and Stock M. J. (1979) A role for brown adipose tissue in diet-induced thermogenesis. Nufure 281,31&35. Sanacora G.. Kershaw M., Finkelstein J. A. and White J. D. (1990) Increased hypothalamic content of prepro- neuropeptide Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation. Endocrinology 127, 730-731. Sawchenko P. E., Swanson L. W., Grzanna R., Howe P. R. C., Bloom S. R. and Polak J. M. (1985) Colocalization of neuropeptide Y immunoreactivity in brainstem catecholaminergic neurones that project to the paraventricular nucleus of the hypothalamus. J. camp. Neural. 241, 138- 153. Stanley B. G. and Leibowitz S. F. (1985) Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulator of feeding behavior. Proc. natn. Acad. Sci. U.S.A. 82, 3940-3943. Stanley B. G., Kyrkouli S. E., Lampert S. and Leibowitz S. E. (1986) Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity. Peprides 7, 1189-I 192. Trayhurn P., Ashwell M.. Jennings G.. Richard D. and Stirling D. M. (1987) Effect of warm or cold exposure on GDP binding and uncoupling protein in rat brown fat. Am. J. Physiol. 252, E237-E243. Trayhurn P. and Milner R. E. (1989) A commentary on the interpretation of in t&-o biochemical measures of brown adipose tissue thermogenesis. Can. J. Physiol. Pharmac. 67, 81 I-819. Ueda S., Nishmura M., Takahashi H., Yoshimura M. and Kawata M. (1991) lmmunohistochemical study of the neuropeptide Y immunoreactivity in the paraventricular nucleus and the preoptic area of the rat following experimental lesions. Biomed. Res. 12, 249.-258. Wilding J. P. H., Gilbey S. G., Mannan M., Aslam N., Ghatei M. A. and Bloom S. R. (1992) Increased neuropeptide Y content in individual hypothalamic nuclei, but not neuropeptide Y mRNA, in diet-induced obesity in rats. J. Endocr. 132, 299.-304.

(Accepted 16 December 1992)

widespread increases in regional hypothalamic neuropeptide y levels in acute cold-exposed rats

Documents