increased neuropeptide y concentrations in specific hypothalamic regions of lactating rats: possible...

Pergamon Peptides, Vol. 15, No. 1, pp. 83-87, 1994

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Increased Neuropeptide Y Concentrations in Specific Hypothalamic Regions of Lactating Rats: Possible Relationship to Hyperphagia and Adaptive Changes in Energy Balance

U S M A N H. MALABU,* A N N E KILPATRICK,* M A R K WARE,* R I C H A R D G. V E R N O N t A N D G A R E T H WILLIAMS*

*Department of Medicine, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England and ~-Hannah Research Institute, Ayr KA6 5HL, Scotland

Received 12 May 1993

MALABU, U. H., A. KILPATRICK, M. WARE, R. G. VERNON AND G. WILLIAMS. Increasedneuropeptide Yconcentrations in specific hypothalamic regions of lactating rats: Possible relationship to hyperphagia and adaptive changes in energy balance. PEPTIDES 15(1) 83-87, 1994.--Lactation is accompanied by hyperphagia and a reduction in brown adipose tissue (BAT) thermogenesis, which are unexplained. Neuropeptide Y (NPY) powerfully stimulates feeding and inhibits BAT thermogenesis when injected into the paraventricular nucleus and other specific regions of the rat hypothalamus. We have tested the hypothesis that hypothalamic NPY activity is increased in lactating rats. Lactating rats consumed over four times as much food as nonlactating controls (n = 10; p < 0.001). Final plasma insulin concentrations in lactating rats were lower than in controls (6.8 + 0.8 vs. 11.7 _+ 2. l pmol/l; p < 0.05) although plasma glucose and corticosterone concentrations were comparable (p > 0.05). Lactating rats showed significantly higher NPY levels than controls in specific hypothalamic regions, namely the arcuate nucleus-median eminence complex (a 41% rise; p < 0.001), paraventricular nucleus (35%; p < 0.001), ventromedial nucleus (66%; p = 0.003), and dorsomedial nucleus (78%; p < 0.001). Other hypothalamic regions showed no significant differences between groups. Increased NPY concentrations in specific hypothalamic regions, particularly the arcuate nucleus where NPY is synthesized, suggest increased activity of the hypothalamic NPYergic system in lactation. Neuropeptide Y may mediate hyperphagia and reduced BAT thermogenesis in lactation. Hypoinsulinemia may be a stimulus to hypothalamic NPY in lactation, as has been postulated in other conditions of negative energy balance.

Neuropeptide Y Lactation Hyperphagia Hypothalamus Rat

LACTATION has profound effects on energy balance, which can be viewed as attempts to meet the energy demands of milk production itself and of hypertrophy of the mammary glands. Food intake increases several fold, while thermogenesis in brown adipose tissue (BAT) is markedly reduced (11,32-34,39). The mechanisms that drive hyperphagia and suppress BAT activity in lactation are not known, but may reside in the hypothalamus, which is important in the regulation of both food intake and energy expenditure. Recently, much interest has focused on neuropeptide Y (NPY), a major hypothalamic peptide that is now firmly implicated in the normal control of nutritional state and is also thought to mediate hyperphagia under various conditions (14,27,44). Neuropeptide Y is a 36-amino acid peptide structurally related to pancreatic polypeptide. It is synthesized in arcuate nucleus (ARC) neurons that project upwards through the lateral hypothalamic area (LHA), to end in the paraventric-

ular nucleus (PVN) and dorsomedial nucleus (DMH), which are both regions that influence feeding (3,15). When injected into the PVN, DMH, LHA, or the ventromedial nucleus (VMH), NPY is an extremely potent stimulator of feeding (22,30,31) and its administration into the third ventricle or PVN reduces energy expenditure, apparently by decreasing sympathetic stimulation of BAT (5,9). Repeated intrahypothalamic injection causes sustained hyperphagia and ultimately obesity (31).

The activity of the hypothalamic NPYergic system is apparently increased in conditions of negative energy balance, such as fasting, food restriction, insulin-deficient diabetes, and intense exercise. In all these conditions, hypothalamic NPY levels are increased, especially in the NPY-sensitive regions that regulate energy metabolism (4,16,21,25,43). Rises in hypothalamic NPY mRNA levels have been identified in underfeeding and diabetes (41,42), and NPY binding site numbers in hypothalamic tissue

Requests for reprints should be addressed to Dr. Usman Malabu.

83

84 MALABU ET AL.

are reduced in diabetic rats, consistent with downregulation of NPY receptors following enhanced endogenous release (12). Recently, push-pull sampling has demonstrated increased NPY release within the PVN in food-deprived and diabetic rats (15,26). Increased NPY synthesis, transport, and release could explain the hyperphagia and reduced BAT activity in these conditions. Because its actions tend to increase body weight, NPY may act homeostatically to counteract the effects of excessive energy def- icits (27,44).

The aim of this study was to determine whether the activity of the hypothalamic NPYergic system is increased in lactation, as appears to be the case in other hyperphagic states. We have therefore compared regional hypothalamic NPY concentrations between lactating and nonlactating rats.

METHOD

A group of 20 age-matched, adult female Wistar rats, origi- nally obtained from A. Tuck & Sons (Rayleigh, Essex, UK), was studied. They were habituated to a 12:12 h light:dark cycle (lights on at 0700 h) and frequent handling, and were kept singly in identical cages. Ambient temperature was maintained at 22°C, and standard rat chow (CRM; Labsure, Poole, Dorset, UK) and water were freely available throughout the study. Food intake and body weight were monitored daily for 3 days before sacrifice. Ten rats were lactating (range, 13-17 days after delivery; mean, 16 days) and were each nursing 10 pups, which were constantly kept in the same cage; litter size was adjusted at birth. The other 10 rats were randomly cycling, nonlactating controls.

Rats were killed from each group alternately between 0900 and 1600 h, using carbon dioxide inhalation followed imme- diately by cardiac puncture and exsanguination. Plasma was separated and stored at -40°C for subsequent measurement of glucose, insulin and corticosterone concentrations.

Hypothalamic Microdissection

The brain was quickly removed and a frontal slice including the hypothalamus was excised by vertical cuts 1 mm anterior to the body of the optic chiasm and 1 mm posterior to the mam- millary bodies. The caudal surface was mounted on a prechilled metal stage using contact adhesive (Superglue®; Loctite, Welwyn Garden City, Hertfordshire, UK). The brain slice was then transferred to ice-cold isotonic saline and cut into 330- and 500- um sections using a vibrating microtome, as previously described (8,43). Individual hypothalamic regions were then punched out with a blunt-ended 19-ga needle and boiled in 0.1 M hydrochloric acid for 10 min to extract NPY. The areas studied were the medial preoptic area (MPO), lateral preoptic area (LPO), PVN, anterior hypothalamic area (AHA), LHA, VMH, DMH, and the area at the base of the third ventricle that includes the arcuate nucleus and the median eminence (ARC/ME). The extracts were frozen and kept at -40°C until measurement of NPY and protein concentrations.

Assays

Neuropeptide Y concentrations were measured in 35- or 60- ~1 aliquots of hypothalamic extract using a radioimmunoassay (RIA) that employed [~2~I]labeled porcine NPY (Amersham In- ternational, Amersham, Buckinghamshire, UK) and porcine NPY as standard (Bachem Inc., Saffron Walden, Oxfordshire, UK). Neuropeptide Y antiserum (APT 140; Affiniti Research Products Ltd., likeston, Derby, UK), raised in our laboratory in a rabbit against porcine NPY, was used at a final concentration of 1:65,000. The assay sensitivity was <1 fmol/tube and the

intra-assay coefficient of variation was 4.2%. Cross-reactivity with pancreatic polypeptide and other related peptides is < 1%. Neu- ropeptide Y-like immunoreactivity determined using this ex- traction and assay methodology has been shown by high per- formance liquid chromatography to be identical to that of syn- thetic porcine NPY (24). All samples were measured in duplicate in a single assay. Protein concentrations in hypothalamic extracts were analyzed using a modified Lowry method (17).

Final plasma glucose levels were measured using a glucose- oxidase-based autoanalyzer. Plasma insulin concentration was determined by RIA using a kit that employed rat insulin as standard; the intra-assay coefficient of variation of the R1A was 5%. Corticosterone was analyzed using a commercial RIA kit (IDS, Tyne & Wear, UK), with an assay sensitivity of 0.5 ng/ml and an intra-assay coefficient of variation of <4%.

Statistical A nalyses

Two-way analysis of variance (ANOVA) was used to examine the effects of lactation on hypothalamic NPY concentrations, using group and hypothalamic region as independent variables. As significant effects were found, regional hypothalamic NPY concentrations were then compared between the two groups using the unpaired Student's t-test. Because of the large number of com- parisons tested, a significance level of p < 0.01 was selected for the t-test. Differences in metabolic data between the groups were examined using the unpaired Student's t-test with a significance level ofp < 0.05. Data are quoted throughout as mean _+ SEM.

RESULTS

Body Weight and Metabolic Data

The food intake, body weight, and metabolic date are shown in Table 1. During the 3-day study period, lactating rats ate over four times as much as the nonlactating group (p < 0.001). The lactating rats weighed 22% more than the nonlactating rats (p < 0.001). Final plasma insulin levels were 40% lower in the lactating rats than the controls (p < 0.05), although plasma glucose levels were similar in both groups (p > 0.05). Plasma corticosterone concentrations were higher in the lactating group, but the difference fell short of statistical significance.

Regional Hypothalamic NPY Concentrations

The NPY levels in the eight individual hypothalamic nuclei are shown in Fig. 1. Two-way ANOVA demonstrated a significant effect of region, F(7, 139) = 70, p < 0.001, and of group, F(I, 139) = 54, p < 0.001. As in previous studies (t4,I6,23), highest overall concentrations were found in the PVN and ARC/

TABLE 1 CHARACTERISTICS OF CONTROL AND LACTATING RATS

Controls Lactating (n = 10) (n = 10)

Mean daily food intake (g) Final body weight (g) Final plasma insulin (pmol/l) Final plasma glucose (retool/I) Plasma cortieosterone (pmol/l)

13.9+ 0.3 60.1 + 0.9* 222.9_+ 3.1 272.3 _+ 4.2*

11.7+ 2.1 6.8+_ 0.8t 7.1+ 0.5 6.6_+ 0.3

187.2 _+ 60.6 232.0 + 41.6

Data are mean _+ SE. *t Significance of differences between control and lactating groups:

*p < 0.001, tP < 0.05.

HYPOTHALAMIC NPY IN LACTATION 85

12

10 ¢,=.

0

.-I 6

0

E " - 4 ) . - Q. z 2

MPO LPO PVN AHA LHA VMH DMHARC/ME

FIG. 1. Neuropeptide Y concentrations (fmol/pg of protein) in eight hypothalamic regions in controls (open columns) and lactating rats (black columns), at an average of 16 days after onset of lactation. MPO, medial preoptic area; LPO, lateral preoptic area; PVN, paraventricular nucleus; AHA, anterior hypothalamic area; LHA, lateral hypothalamic area; VMH, ventromedial nucleus; DMH, dorsomedial nucleus; ARC/ME, arcuate nucleus plus median eminence. Data are mean _ SEM. Signif- icance of differences between groups indicated: **p < 0.01, ***p < 0.001.

ME. Striking differences within several nuclei were observed between controls and lactating rats. The lactating rats showed marked increases in NPY levels in four nuclei: ARC/ME (a 41% increase; p < 0.001), PVN (a 35% increase; p < 0.001), VMH (66% increase; p = 0.003), and DMH (78% increase; p < 0.001).

DISCUSSION

Neuropeptide Y injected centrally is an extremely potent appetite stimulant, and also suppresses sympathetic stimulation of BAT and reduces energy expenditure (5,9,22,30,31). Its apparently adaptive changes in the hypothalamus in various hyperphagic states suggest that it plays a role in the regulation of food intake and energy balance (27,44). Increased activity of the hypothalamic NPYergic system could therefore mediate the hyperphagia and reduced BAT thermogenesis of lactation. Neu- ropeptide Y levels throughout the hypothalamus, and especially the regions implicated in the control of energy metabolism, have not previously been reported in lactation, although Ciofi et al. (6) have recently found NPY immunostaining to be increased in the external layer of the median eminence in lactating rats.

We found that lactating rats had significantly higher NPY concentrations in the ARC/ME, PVN, VMH, and DMH compared with controls. As the arcuate nucleus is the major source of NPY mRNA within the hypothalamus (28,29,41,42), increased NPY concentrations in this region suggest increased NPY synthesis. Indeed, this has recently been confirmed by Pelletier and Tong (23), who found a 50% increase in preproNPY mRNA in the arcuate nucleus of rats that had been lactating for 4 days. Neuropeptide Y neurons in the arcuate nucleus project pre- dominantly to the PVN and DMH (3); increased NPY levels in all these sites therefore point to enhanced activity of the hypothalamic NPYergic system. The 35-80% increases in NPY concentrations in the PVN and other NPY-sensitive sites are broadly comparable with those previously reported in fasting, exercise, and diabetes (4,16,25,43); increased NPY release in the PVN has recently been demonstrated in fasting and diabetes, using push-pull sampling (15,26). Neuropeptide Y injected into the PVN, DMH, and VMH is the most potent appetite stimulant

yet identified (22,31 ), and when injected into the third ventricle or PVN, it also reduces energy expenditure, possibly by inhibiting the firing of the sympathetic nerves supplying BAT (5,9). En- hanced activity of the NPY-containing arcuato-paraventricular projection could therefore cause hyperphagia and reduce BAT thermogenesis, and so mediate the major changes in energy balance known to occur in lactation. Increased regional NPY concentrations cannot be interpreted with certainty and could even result from decreased release, causing local accumulation, rather than increased activity of these neurons. However, the finding of increased NPY mRNA levels consistent with enhanced synthesis (23), together with the striking parallels with the other hyperphagic states in which increased NPY release has now been documented (14,25,26,43), support our contention that NPYergic activity is stimulated.

The signals that might activate hypothalamic NPY during lactation have not been identified. The mechanical stimulation of suckling may account for an 80-90% increase in feeding but the central pathways and neurotransmitters involved have not been defined. Hyperprolactinaemia does not appear to cause increase in NPY gene expression in lactation, as neither central prolactin injection nor hyperprolactinemia induced by peripheral pituitary implants had this effect; rather, NPY may evoke prolactin release (23).

Other possible endocrine signals include gonadal and adrenocortical steroids, both of which are known to modulate NPY gene expression and peptide concentrations in the hypothalamus. In lactation, circulating progesterone concentrations are raised, whereas estradiol levels are reduced. The effects on NPY of ma- nipulating these steroids within the physiological range have not yet been fully characterized. Experimental changes in estrogen status appear to affect hypothalamic NPY and to modulate the effects of NPY itself on gonadotropin release (14), although in- terestingly, NPY immunostaining in the mediobasal hypothalamus was not affected by the stages of the estrous cycle, or even by gonadectomy (23).

Circulating corticosterone levels rise in lactation (37,38), although not significantly so in our study; variable terminal stress responses may have obscured differences between the groups. Glucocorticoids may stimulate hypothalamic NPY synthesis, as dexamethasone increases NPY mRNA levels in hypothalamic neurones in vitro (7). Interactions between NPY and corticosterone may be complex, as NPY injected into the PVN stimulates corticosterone secretion, possibly by inducing release of corticotropin releasing factor (CRF) in the median eminence (2,40). Corticosterone suppresses BAT activity in obese rodents (35,46) and may specifically inhibit the activation of the sympathetic system by dietary signals (46). Raised corticosterone levels could therefore suppress sympathetic activity in brown fat in lactating rats, possibly acting through increased NPY activity in the hypothalamus.

An alternative stimulus to hypothalamic NPY may be low circulating insulin levels, which are a consistent feature of lactation and may be due to both reduced secretion and enhanced destruction by the mammary gland (13,18,37,38,45). Several conditions of negative energy balance, such as starvation, insulin- deficient diabetes, and strenuous physical exercise, which all show evidence of increased hypothalamic NPY activity, are also characterized by hypoinsulinemia. Raising circulating insulin levels, by insulin replacement therapy in diabetic rats (21,25) or by carefully monitored insulin treatment in fasting (19), lowers hypothalamic NPY concentrations to normal. Despite its high molecular weight, insulin readily enters specific brain regions including the hypothalamus, in which the ARC contains plentiful insulin receptors (27,36). Insulin may directly inhibit NPY ac-

86 MALABU ET AL.

tivity, as its injection into the third ventricle of lean rats prevents the rises in NPY m R N A and NPY levels that normally accom- pany fasting (28,29). Peripheral insulin administration, which achieved circulating insulin levels within the high physiological range, can also reduce NPY levels in the ARC of fasted rats (19). This further suggests that circulating insulin may inhibit various aspects of the activity of the hypothalamic NPYergic pathways. As with corticosterone, injection of NPY into the PVN may stimulate insulin secretion acutely (l). Any tendency of increased NPY release in the PVN to raise circulating insulin levels is presumably overcome in lactation by the changes in insulin turnover mentioned above, and by the regulatory effect of prevailing blood glucose levels.

In conclusion, regional hypothalamic NPY concentrations are markedly raised in lactating rats. Together with the dem-

onstration of increased preproNPY m R N A levels in the ARC (23), this strongly suggests hyperactivity of the NPYergic system in the hypothalamus. This may contribute to the hyperphagia and reduced BAT thermogenesis, which are crucial adaptive changes in lactation.

ACKNOWLEDGEMENTS

U.H.M. was supported financially by the Association of Common- wealth Universities, We also acknowledge the generosity of the Nutfield Foundation, Mason Medical Research Trust, the Peel Medical Trust, and the Scottish Ot~ce's Agriculture and Fisheries Department for ad- ditional support. We are grateful to the staff of the Animal Unit at the Hannah Research Institute for their conscientious care of the rats, and to Dr. H. D. McCarthy for his useful comments.

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