BRAIN RESEARCH

E L S E V I E R Brain Research 690 (1995) 185-188

Research report

Increased neuropeptide Y secretion in the hypothalamic paraventricular nucleus of obese ( fa/fa) Zucker rats

Simon Dryden, Lucy Pickavance, Helen M. Frankish, Gareth Williams * Diabetes and Endocrinology Research Group, Department of Medicine, Unicersity of Liverpool, Lirerpool, UK

Accepted 2 May 1995

Abstract

NPY is synthesized in the hypothalamic arcuate nucleus (ARC), and NPY injected into the paraventricular nucleus (PVN), the main site of NPY release, induces hyperphagia and reduces energy expenditure. Hypothalamic NPY mRNA and NPY levels are increased in fatty Zucker rats, consistent with increased NPY release. This could explain the hyperphagia and reduced energy expenditure, which lead to obesity in the fatty Zucker rat. We have therefore compared NPY secretion in the PVN of conscious fatty and lean Zucker rats using push-pull sampling. The NPY secretory profile was consistently higher in fatty Zucker rats than in lean rats throughout the 3-h study period (P < 0.01), and mean NPY secretion over the whole 3 h was increased 2-fold in the fatty rats (P < 0.001). We conclude that fatty Zucker rats have increased NPY release in the PVN. This observation further supports the hypothesis that increased activity of the NPYergic ARC-PVN pathway may contribute to obesity in the fatty Zucker syndrome.

Keywords: Rat; Hypothalamus; Neuropeptide Y; Zucker; Obesity

I. Introduction

The fatty Zucker rat, homozygous for the fatty gene (fa/fa), is a well-characterized animal model of obesity and insulin resistance. Excessive weight gain is due mainly to impaired thermogenesis in brown adipose tissue (BAT), which is caused by inadequate central stimulation of the sympathetic nerves which supply it [4]. This is aggravated by hyperphagia, with a marked preference for carbohydrate and fat, and by increased triglyceride deposition in white adipose tissue under the influence of grossly raised circu- lating insulin levels [19].

The primary cause of the obese Zucker syndrome is unknown. Hypothalamic dysfunction could explain many of its key features [4], but the anatomical and neurochemi- cal basis of such a defect has not been identified. Neu- ropeptide Y (NPY) is a 36-amino acid peptide structurally related to pancreatic polypeptide [24]. It is one of the most abundant brain peptides and is highly concentrated in the

* Corresponding author. Department of Medicine, University of Liver- pool. PO Box 147, Liverpool. L69 3BX. UK. Fax: (44) (151) 706 5802.

0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00628-1

hypothalamus, notably the arcuate nucleus (ARC), where is synthesized [15], and the paraventricular nucleus (PVN) to which the ARC neurons project [5]. When injected into the PVN, NPY is one of the most powerful central appetite stimulants known, inducing striking carbohydrate-and fat-selective hyperphagia [7,17,23]. NPY also reduces en- ergy expenditure by inhibiting the sympathetic nerves that innervate and stimulate BAT [3]. Repeated NPY injection leads ultimately to obesity with features that resemble those of the fatty Zucker rat [23,27].

The ARC-PVN pathway is apparently stimulated by food restriction, diabetes and other conditions of negative energy balance; NPY and NPY mRNA levels are increased [7,8,26], and hypothalamic NPY receptor numbers reduced [9], consistent with down-regulation following enhanced release and increased NPY release in the PVN has been demonstrated in diabetic and food-deprived rats [10,11,20]. Through increased NPY secretion in the hypothalamus, the ARC-PVN projection may serve a homeostatic function in defending body weight by stimulating feeding and restrain- ing energy expenditure, especially under conditions of energy deficit [7,8].

In the fatty Zucker rat, obesity could be explained by

186 S. D~den et al. /Bra in Research 690 (1995) 185-188

increased NPY secretion in the PVN. NPY levels are increased in several hypothalamic regions including the ARC, PVN and DMH [2,14] and NPY mRNA levels are increased in the ARC [21]. Furthermore, NPY receptors are down-regulated in fatty Zucker rats [13].

This study aimed to determine whether NPY secretion in the PVN of fatty Zucker rats was greater than in lean Zucker rats.

2. Methods

Table 1

Plasma concentrations of glucose, insulin and corticosterone in lean and obese Zucker rats

Lean Fatty

n = 8 8

Mean daily food intake (g) 20.9 + 0.4 35.7 ± 1.2

Body weight (g) 287 ± 6 439 ± 10 * Glucose ( m m o l / l ) 8.4 + 0.6 7.9 ± 0.4

Insulin ( m U / I ) 18.1 ± 2.1 581 ± 68 * * Corticosterone ( n g / m l ) 15.6 ± 3.4 104 ± 352 * *

Statistical significance of differences vs. saline-treated rats: * P < 0.01,

• * P < 0 . 0 0 1 .

2.1. Animals

Male fatty (fa/fa) and lean (Fa/Fa) Zucker rats, obtained from Harlan Olac Ltd (Bicester, Oxon, UK) were housed in wire-bottomed cages and kept at 22 ___ 2°C in a room with a 12-h light-dark cycle (09.00-21.00 h). Water was freely available and standard laboratory chow (CRM, Biosure, Cambridge, UK) was given in excess to normal daily intake (approx. 100 g). Food intake and general condition were checked daily.

2.2. Surgery

Rats were anesthetized with using a mixture of di- azepam and Hypnorm (2.5 mg/kg i.p., and 0.5 ml /kg i.m. respectively), and with the incisor bar set 3 mm below the horizontal plane, a single 20 ga thin-walled stainless steel guide cannula was implanted into the right PVN at the coordinates 1.8 mm anterior to bregma, 0.4 mm lateral from the midline and 7.8 mm ventral from the dura, according to Paxinos and Watson, 1986 [18]. A stainless steel stylet was inserted into the guide cannula to prevent its blockage, and the animals were then allowed to recover for 3 days before any perfusions were carried out.

A standard concentric perfusion system was used, con- sisting of a 28-gauge stainless steel inner (push) cannula and a 23-gauge thin-walled outer (pull) cannula [16]. The inner and outer cannulae were connected by PE20 and PE50 tubing (Portex) to 1.0-ml gas-tight Hamilton glass syringes (Sigma, UK) mounted on a modified Biotech injection pump (Biotech Instruments, Herts, UK).

Perfusions of artificial cerebrospinal fluid (CSF), fil- tered through a Millipore filter before use, were carried out between 0900-1200 h with a flow rate of 20 /zl/min. Before each perfusion, a small air bubble was introduced into the pull tubing to allow flow to be monitored. The push-pull cannula was then inserted into the guide tube, and after a control perfusion of 15 min, a series of 15-min perfusions were carried out. The eluates were collected in plastic tubes containing 10 ~1 of 0.1 M HC1, frozen at -70°C and then lyophilized before assay for NPY.

The sites of the guide cannulae were verified by the injection of India ink before sacrifice and a frontal block

of brain, including the hypothalamus, was then sectioned on a vibrating microtome. Sections removed were then viewed under a microscope. Only those rats where staining occurred within the PVN (not in the 3rd ventricle), and where the tissue disruption due to the cannula insertion was dorsal to the PVN, were used for analysis of the collected eluates.

The rats were sacrificed by carbon dioxide inhalation (within 45-60 s) and immediately exsanguinated by car- diac puncture. Plasma was separated and stored at -40°C for subsequent measurement of glucose, insulin and corticosterone concentrations.

2.3. Assays

NPY concentrations in the samples (reconstituted in 100 /zl of acetate buffer) were measured by radioimmunoassay using NPY antiserum raised in a rabbit against porcine NPY (APT 144; Affiniti Research Products Ltd, Notting- ham, UK), ]251-labeled porcine NPY (Amersham Interna- tional, Aylesbury, Bucks, UK) and synthetic porcine NPY (Bachem Ltd, Saffron Walden, Essex, UK) as standard.

75

S 45

o) (n co --~ 3o

>- o. z 15

i i i i i t

30 60 90 120 150 180

Time after sta~ing I~rfuslon (mln)

Fig. 1. Hypothalamic neuropeptide Y secretion ( f m o l / m l ) in the paraven- tricular nucleus (PVN) of lean ( O ) and fatty (11) rats throughout a 3-h study period (n = 8 per group). Error bars are S.E.M. Statistical signifi- cance of differences vs lean animals: * P < 0.01, * " P < 0.001.

S. Dryden et al. / Brain Research 690 (1995) 185-188 187

Cross-reactivity with peptide YY and related peptides is < 1%. Assay sensitivity was < 0.5 fmo l /ml and the within-assay coefficient of variation was < 4%. All sam- ples from each study were measured in duplicate in a single assay, and NPY concentrations were expressed as fmol /ml .

Corticosterone and insulin concentrations were mea- sured using commercial radioimmunoassay kits, which each had a within-assay coefficient of variation of 4% (Shield Diagnostics, Dundee, UK), and plasma glucose by a glucose oxidase-based autoanalyzer.

2.4. Statistical analyses

Data are shown as mean + S.E.M. Differences between fatty and lean groups were analyzed using an unpaired Student's t-test, with a significance level of P < 0.05.

3. Results

3.1. Body weight, food intake and plasma analytes

These data are shown in Table 1. Fatty rats weighed 52% more than lean ones. Food intake in the fatty Zucker rats was 64% higher than in the lean rats ( P < 0.01). Fatty rats had significantly higher insulin and corticosterone levels than the lean animals (both P < 0.01), but plasma glucose levels were not significantly different between the groups.

3.2. NPY secretion in the PVN

Fig. 1 shows the NPY release in the PVN over a 3-h period. NPY release, measured in 15-min elutes, in the fatty Zucker rats was significantly increased compared with that of their lean counterparts at each interval shown

80

E so

E 4O

w

"¢ 20 >. a. z

0 Lean Fatty

Fig. 2. Mean NPY secretion during a 3-h period measured by push-pu l l

sampling in fatty and lean rats. Error bars are S.E.M.. Statistical signifi-

cance of differences between groups; * * P < 0.001.

( P < 0.01). The mean level of NPY release over the entire 3-h period was also increased 2-fold in these animals ( P < 0.01)(Fig. 2).

4. Discuss ion

The obesity associated with the fatty Zucker syndrome appears to be due to a combination of reduced energy expenditure and increased food intake, and increased in- sulin secretion [4,19]. NPY injected into the PVN or the nearby ventricular system mimics all of the above changes [27]. Overactivity of the NPY-ergic ARC-PVN projection in the obese rat could explain obesity and some other neuroendocrine dysfunctions in the syndrome. It has been already been shown that the fatty Zucker rat has increased NPY and NPY mRNA levels in these regions [2,14,21].

As NPY levels show circadian variation [1], the 3-h time period for measuring NPY secretion was at the same time for each animal, to minimise the effects of changes in NPY synthesis during the studies.

We therefore set out to test the hypothesis that the increased NPY levels in the PVN of the fatty Zucker rat are accompanied by increased NPY secretion in this im- portant hypothalamic region. This was confirmed: NPY concentrations in eluates from the PVN were consistently twice as high in the fatty animals as in lean rats. This suggests an increase in NPY release in this region, and implies an increase in transport of NPY along the ARC- PVN projection from its sites of synthesis in the ARC [21].

The cause of the increased NPY activity in the A R C - PVN projection in this model of obesity is unknown, but there are two main possibilities. Firstly, under normal conditions, insulin may inhibit NPY neurons in the ARC. Some studies have suggested that insulin inhibits hypotha- lamic NPY synthesis and decreases regional NPY concen- trations in normal and starved animals [7,8,22], although a recent report does not support this [6]. It has been proposed that the hypothalamus of the fatty rat is insulin-resistant, so that insulin's inhibitory effect may be lost, despite the fact that these animals have increased circulating insulin levels [22]. The second possible regulator of hypothalamic NPY is glucocorticoids. These have been found to increase NPY synthesis in some systems [12], and plasma levels cortico- sterone are increased in fatty rats [4]. However, the situa- tion is complex, as NPY injected into the PVN increases corticosterone levels [25] and the increases in fatty rats may be a consequence of the increased NPY release rather than the cause.

The relationship of the ARC-PVN projection to the fa mutation is unclear. It is possible that the gene product regulates NPY neurons more or less directly. For example, a protein secreted by adipose tissue, analogous to the recently sequenced and cloned ob gene in mice [28], could exert its effects on the CNS by modulating NPY synthesis, transport or release.

188 S. Dryden et al. / Brain Research 690 (199_5) 185-188

In summary, this study showed that NPY secretion in the PVN was significantly higher in fatty than in lean Zucker rats. Increased NPY secretion in the PVN, which is highly sensitive to NPY's central metabolic actions, could therefore be involved in development of obesity in the fatty Zucker rat. This is further evidence for a physio- logical role of NPY in the control of appetite and energy homeostasis in the rat, and in mediating the genetic obesity of the fatty Zucker rat.

Acknowledgements

The authors wish to thank Lilly Industries, the BBSRC and the Tobacco Products Research Trust for financial support of S.D., L.P. and H.M.F. respectively; Professor R.D. Myers of East Carolina University, USA, for his invaluable help, advice and discussion; Margaret Wooten for technical assistance; and Andrew Jones for care of the animals. Additional funding was provided by the Research & Development fund of the University of Liverpool, SmithKline-Beecham, and the Liverpool Diabetes Re- search Action fund.

References

[1] Akabayashi, A., Levin, N., Paez, X., Alexander, J.T. and Leibowitz, S.F., Hypothalamic neuropeptide Y and its gene expression: relation to light/dark cycle and circulating corticosterone, Mol. Cell Neu- rosci., 5 (1994) 210-218.

[2] Beck, B., Burlet, A., Nicolas, J-P. and Burlet, C., Hypothalamic neuropeptide Y in obese Zucker rats: implications in feeding and sexual behaviors, Physiol Behav., 47 (1990) 449-453.

[3] Billington, C.J., Briggs, J.E., Grace, M. and Levine, A.S., Effects of intracerebro-ventricular injections of neuropeptide Y on energy metabolism, Am. J. Physiol., 260 (1991) R321-327.

[4] Bray, G.A., Genetic and hypothalamic mechanisms for obesity - - finding the needle in the haystack, Am. J. Clin. Nutr., 50 (1989) 891-802.

[5] Chronwall, B.M., Dimaggio, D.A., Massari, V.J., Pickel, V.M., Ruggiero, D.A. and O'Donohue, T.L., The anatomy of neuropeptide Y containing neurons in the rat brain, Neuroscience, 15 (1985) 1159-1181.

[6] Cusin, I., Dryden, S., Wang, Q., Rohner-Jeanrenaud, F., Jeanrenaud, B. and Williams, G., Effects of sustained physiological hyperinsu- linemia on hypothalamic neuropeptide Y and NPY mRNA levels in the rat, Neuroendocrinology, 7 (1995) 193-197.

[7] Dryden, S., Frankish, H.M., Wang, Q. and Williams, G., Neuropep- tide Y and energy balance: one way ahead for the treatment of obesity, Eur. J. Clin. Invest., 24 (1994) 293-308.

[8] Frankish, H.M., Dryden, S., Hopkins, D., Wang, Q. and Williams, G., Neuropeptide Y the hypotbalamus and diabetes: insights into the central control of metabolism, Peptides, 16 (1995) 757-771.

[9] Frankish, H.M., McCarthy, H.D., Dryden, S., Kilpatrick, A. and Williams, G., Reduced neuropeptide Y receptor numbers in the hypothalamus of the streptozocin-diabetic rat: further evidence of increased NPY activity, Peptides, 14 (1993) 941-948.

[10] Kalra, S.P., Dube, M.G., Sahu, A., Phelps, C. and Kalra, P.S., Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food, Proc. Natl. Acad. Sci., 38 (1991) 10931-10935.

[11] Lambert, P.D., Wilding, J.P.H., Turton, M.D., Ghatei, M.A. and Bloom. S.R., Effect of food-deprivation and streptozocin-induced diabetes on hypothalamic neuropeptide Y release as measured by a radioimmunoassay-linked microdialysis procedure, Brain Res., 656 (1994) 135-140.

[12] Larsen, P.J., Jessop, D.S., Chowdrey, H.S., Lightman, S.L. and Mikkelsen, J.D., Chronic administration of glucocorticoids directly upregulates prepro-neuropeptide Y and Yl-receptor mRNA levels in the arcuate nucleus of the rat, J. Neuroendocrinol., 6 (1994) 153- 159.

[13] McCarthy, H.D., McKibbin, P.E., Holloway, B., Mayers, R. and Williams, G., Hypothalamic neuropeptide Y receptor characteriza- tion and induced feeding behavior in lean and obese Zucker rats, Life Sci., 49 (1991) 1491-1497.

[14] McKibbin, P.E., Cotton, S.J., McMillan, S., Holloway, B., Mayers, R., McCarthy, H.D. and Williams, G., Altered neuropeptide Y concentrations in specific hypothalamic regions of obese ( f a / f a ) Zucker rats, Diabetes, 40 (1991) 1423-1429.

[15] Morris, B.J., Neuronal localisation of neuropeptide Y gene expres- sion in rat brain, .L Comp. NeuroL, 290 (1989) 358-368.

[16] Myers, R.D., Chronic methods: intraventricular infusion, cere- brospinal fluid sampling and push-pull perfusion. In R.D. Myers (Ed), Methods in Psychobiology, Vol III, Academic Press, New York, 1977, pp. 281-315.

[17] Paez, X. and Myers, R.D., Insatiable feeding evoked in rats by recurrent perfusion of neuropeptide Y in the hypothalamus, Pep- tides, 12 (1991) 609-616.

[18] Paxinos, G. and Watson, C., The Rat Brain In Stereotaxic Coordi- nates, 2nd edn., Academic Press, Sydney, 1986.

[19] P~nicaud, L., Ferre, P., Terretaz, J., Kinebanyan, M.F., Leturque, A., Dore, E., Girard, J., Jeanrenaud, B. and Picon, L., Development of obesity in Zucker rats: early insulin resistance in muscles but normal sensitivity in white adipose tissue, Diabetes, 36 (1987) 626-631.

[20] Sahu, A., Sninsky, C.A., Phelps, C.P., Dube, M.G., Kalra, P.S. and Kalra, S.P., Neuropeptide Y release from the paraventricular nucleus in association with hyperphagia in streptozocin-induced diabetic rats, Endocrinology, 131 (1992) 2979-2985.

[21] Sanacora, G., Kershaw, M., Finklestein, J.A. and White, J.D., Increase in hypothalamic content of proneuropeptide Y messenger RNA in genetically obese Zucker rats and its regulation by food deprivation, Endocrinology, 127 (1990) 730-737.

[22] Schwartz, M.W., Figlewicz, D.P., Baskin, D.G., Woods, S.C. and Porte, D., Insulin in the brain. A hormonal regulator of energy balance, Endocr. ReL,., 13 (1992) 387-414.

[23] Stanley, B.G., Kyrkouli, S.E., Lampert, S. and Leibowitz, S.F., Neuropeptide Y chronically injected into the hypothalamus: a pow- erful neurochemical inducer of hyperphagia and obesity, Peptides, 7 (1986) 1189-1192.

[24] Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y, a novel brain peptide with structural similarities to peptide YY and pancre- atic polypeptide, Nature, 296 (1982) 659-660.

[25] Wahlestedt, C., Skakerberg, G., Ekman, R., Heilig, M., Sundler, F. and H~ikanson, R., Neuropeptide Y in the area of the hypothalamus paraventricular nucleus activates the pituitary-adrenocortical axis in the rat, Brain Res., 417 (1987) 33-48.

[26] Williams, G., Gill, J.S., Lee, Y.C., Cardoso, H.M., Okpere, B.E. and Bloom, S.R., Increased neuropeptide Y concentrations in specific hypothalamic regions of streptozocin-induced diabetic rats, Dia- betes, 38 (1989) 321-327.

[27] Zarjevski, N., Cusin, I., Vettor, R., Rohner-Jeanrenaud, F. and Jeanrenaud, B., Chronic intracerebroventricular neuropeptide Y ad- ministration to normal rats mimics hormonal and metabolic changes of obesity, Endocrinology, 133 (1993) 1753-1758.

[28] Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L. and Friedman, J.M., Positional cloning of the mouse obese gene and its human homologue, Nature, 372 (1994) 425-432.