Peripheral insulin administration attenuates the increase in neuropeptide Y concentrations in the hypothalamic arcuate nucleus of fasted rats

Peptides.Vol. 13, pp. 1097-1102, 1992

0196-9781/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.

Printed in the USA.

Peripheral Insulin Administration Attenuates the Increase in Neuropeptide Y Concentrations in the Hypothalamic Arcuate Nucleus of Fasted Rats U S M A N H. M A L A B U , 1 H. D A V I D M c C A R T H Y ,

P A U L I N E E. M C K I B B I N A N D G A R E T H

WILLIAMS

Department o f Medicine, University o f Liverpool, P.O. Box 147, Liverpool L69 3BX, UK R e c e i v e d 25 M a r c h 1992 MALABU, U. H., H. D. McCARTHY, P. E. McK1BBIN AND G. WILLIAMS. Peripheral insulin administration attenuates the increase in neuropeptide Y concentrations in the hypothalamic arcuate nucleus offasted rats. PEPTIDES 13(6) 1097-1102, 1992.--Fasting increases neuropeptide Y (NPY) concentrations in the arcuate nucleus (ARC), its site of synthesis, and in other regions of the rat hypothalamus. Neuropeptide Y is a potent central orexigenic agent and may therefore stimulate appetite during fasting. We tested the hypothesis that low plasma insulin levels stimulate ARC levels of NPY in fasted rats. Compared with freely fed controls (n = 8), rats fasted for 72 h (n = 8) showed significantly lower plasma insulin levels (28.9 + 1.6 vs. 52.6 _ 5.7 pmol/ 1;p < 0.001) and higher ARC NPY concentrations ( 14.2 _+ 1.8 vs. 8.4 + 2.2 fmol/#g protein; p < 0.001). Fasted rats treated with subcutaneous insulin (5 U/kg/day; n = 10), which nearly normalized plasma insulin (46.6 _+ 2.8 pmol/l), showed intermediate ARC NPY levels ( 11.2 ___1.4 fmol/#g protein; p < 0.01 vs. controls and untreated fasted rats). Insulin administered peripherally, therefore, attenuates fasting-induced NPY increases in the ARC, supporting the hypothesis that hypoinsulinemia stimulates hypothalamic NPY. Neuropeptide Y

Fasting

Hypothalamus

Insulin

FASTED or food-restricted animals show intensified food-seeking behavior and increased feeding when food again becomes freely available. The neurotransmitter(s) mediating these lifesaving behaviors, and the metabolic or other signals that trigger them, are not known. Various neurotransmitter changes have been described in the hypothalamus and other appetite-regulating brain regions of fasted animals, including striking alterations in hypothalamic neuropeptide Y (NPY) (4-6,9,1 l, 18,21,25-27). Neuropeptide Y is a 36 amino acid peptide, structurally related to pancreatic polypeptide. It is very abundant in the brain and is found at particularly high concentrations in the hypothalamus. It is synthesized in arcuate nucleus (ARC) neurons, which project upwards through the lateral hypothalamic area (LHA) to end in the paraventricular nucleus (PVN) and dorsomedial nucleus (DMH), both regions involved in regulating feeding behavior. When injected into the PVN, DMH, LHA, or the ventromedial nucleus (VMH), NPY is an extremely potent stimulator of both food-seeking behavior and eating (18,26,27). In food-deprived or food-restricted animals, NPY concentrations rise in the ARC and PVN (22); increases in the ARC are due, at least in part, to increased synthesis of the peptide, as NPY mRNA levels here are also elevated (24). Some reports suggest Requests for reprints should be addressed to Dr. Usman H. Malabu.

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an extremely rapid and dramatic NPY rise in the ARC, with concentrations increasing tenfold within 48 h of food deprivation (4). Increased NPY synthesis, together with elevated NPY levels in hypothalamic regions supplied by the ARC neurons, suggests that NPYergic activity is stimulated, and increased NPY release within the PVN (which is exquisitely sensitive to NPY) has recently been demonstrated by push-pull sampling in fasted rats (9). Neuropeptide Y could therefore drive food seeking and compensatory hyperphagia in starvation. The increased NPY levels and release in the PVN are reversed by a few h of refeeding (4,9), further suggesting that this apparently adaptive change is physiologically relevant. Many metabolic and endocrine disturbances occur in starvation. The specific signal that might activate hypothalamic NPY has not yet been identified, but there is increasing evidence that a fall in circulating insulin levels could be responsible. This possibility is supported by the observations that regional hypothaiamic NPY levels and/or NPY mRNA levels are increased in other hypoinsulinemic conditions, namely diabetes due to Bcell destruction (either by streptozotocin administration, or occurring spontaneously in the BB rat) and intensive exercise (12,22,29-33). Insulin can apparently cross the blood-brain

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barrier, and many brain regions, including the ARC and other hypothalamic nuclei, contain insulin receptors (6,10,35). Insulin injected intracerebroventricularly (ICV) suppresses feeding (2,28), suggesting that it could function as a satiety factor. As circulating insulin levels are generally proportional to body fat content, it may be involved in the long-term regulation of body weight and composition (34). Direct evidence that insulin may influence NPYergic activity in the hypothalamus has come from the elegant studies of Schwartz et al., who found that insulin injected into the third ventricle reduced the rise in NPY mRNA levels in the ARC, which normally occurs in food-deprived lean Zucker rats (24). However, this route of administration undoubtedly produced very high insulin concentrations in the ARC and other hypothalamic regions immediately adjacent to the third ventricle, which may be far in excess of those achieved by the physiological entry of insulin to the brain from the circulation. In this study, we therefore examined the relationship between circulating insulin levels and hypothalamic NPY in food deprivation. We measured regional hypothalamic NPY concentrations in freely fed rats and in rats that were food deprived for 72 h. We tested the hypothesis that low circulating insulin levels were responsible for stimulating hypothalamic NPY in fasting by treating a group of food-deprived rats with subcutaneous insulin. METHOD Male Wistar rats were obtained from Harlan Olac, Bicester, UK. They were weaned at 3 weeks of age and were housed individually in wire-bottomed cages at a room temperature of 22°C with a 12:12 h light:dark cycle. The study began when the rats weighed 210-260 g. They were habituated to frequent handling. Water was provided freely throughout the study.

Experimental Design A control group (n = 8) was freely fed, while two other groups housed in a separate room were starved for 72 h, starting at 0800 h. Body weight, blood glucose concentration (measured in tailprick samples using a reflectance meter), and general condition were checked twice daily, at 0800 h and 1900 h, during this period. One group of food-deprived rats (n = 10) was given heattreated bovine ultralente insulin (Ultratard®: Novo Industri, Copenhagen, Denmark), injected subcutaneously twice daily after blood glucose monitoring, at a dose of 5 U/kg/day. The first injection was given at 0800 h at the start of the fast, and the last at 0800 h on day 3. Insulin was to be withheld if a rat's blood glucose level fell below 2.0 mmol/1, but this did not occur during the period of food deprivation. The dosage and timing of injections were decided following a series of pilot studies in which the time course and hypoglycemic effect of different insulin dosages (2.5-7.5 U/kg) were studied in rats deprived of food for 24-72 h. The dosage used is an order of magnitude smaller than that which we previously used to study the effects of severe hypoglycemia (7). An equal volume of isotonic saline was injected subcutaneously into the remaining food-deprived rats (n = 9) and control rats, at the same times. All animals remained in good clinical condition throughout the study, except for one of the food-deprived, saline-treated group that died on day 2. Animals from all three groups were killed in rotation between 0900 and 1600 h on day 3, by carbon dioxide inhalation followed by immediate exsanguination by cardiac puncture. Plasma was separated and stored at - 4 0 ° C for subsequent measurement of glucose and insulin concentrations.

Hypothalamic Microdissection The brain was rapidly removed and the hypothalamic area cut into 330- and 500-#m slices using a vibrating microtome, as previously described (30). Tissue from each hypothalamic region was punched out and boiled for 10 min in 400 ~1 of 0.1 M hydrochloric acid and then sonicated for 30 s to disperse the tissue and extract NPY. The extracts were stored at - 4 0 ° C until assayed for NPY and protein concentrations. The hypothalamic areas investigated were: the medial preoptic area (MPO), the lateral preoptic area (LPO), the paraventricular nucleus (PVN), the ventromedial nucleus (VMH), the dorsomedial nucleus (DMH), the lateral hypothalamic area (LHA), and a wedge of tissue at the base of the third ventricle that includes the arcuate nucleus and the median eminence (ARC/ME).

Assays Final plasma glucose concentration was measured using a glucose-oxidase based autoanalyzer. Plasma insulin concentration was measured by radioimmunoassay (RIA) using a kit that employed rat insulin as standard (Novo Biolabs Ltd., Cambridge, U K). The within-assay coefficient of variation was <5% and the cross-reactivity with bovine insulin was 98%. Neuropeptide Y concentrations were measured by RIA using NPY antiserum (kindly provided by Dr. Thue Schwartz, Rigshospitalet, Copenhagen), |25I-labeled porcine NPY (Amersham International, Amersham, UK), and synthetic porcine NPY (Bachem Ltd., Saffron Walden, UK) as standard (1). The sensitivity of the assay was 4.9 fmol/tube and the within-assay coefficient of variation was <5%. Neuropeptide Y-like immunoreactivity identified by this assay coelutes with synthetic porcine NPY on reverse-phase high performance liquid chromatography (20), and the cross-reactivity with related peptides (pancreatic polypeptide and peptide YY) is < 1%. Protein concentration was assayed using the Lowry method (l 3). All samples were measured in duplicate in a single assay and NPY concentration was expressed as fmol/#g protein.

Statistical Analyses Two-way analysis of variance (ANOVA) was used to examine the effects of food deprivation, with or without insulin treatment, on hypothalamic NPY concentrations, using group and hypothalamic region as independent variables. As significant effects were found, the unpaired Student's t-test was then used to examine regional differences in NPY concentrations. The significance level was set at p < 0.01. A correlation between plasma insulin or glucose concentrations and ARC NPY levels was tested. A p value < 0.05 was taken as significant. Data are quoted throughout as mean + SEM. Differences between the groups in metabolic data were examined using one-way ANOVA. RESULTS

Body Weight and Metabolic Data Changes in body weight are shown in Fig. I. Body weight continued to increase in the freely fed group but fell at closely similar rates in both food-deprived groups, and after 24 h, was significantly lower than controls (p < 0.001) in both groups. After 72 h of food deprivation, the saline-injected, food-deprived group had lost 19.2%, and the insulin-treated, food-deprived group 20.7% of their initial body weight (both p < 0.001 vs. controls); there were no significant differences between the two food-deprived groups. Plasma glucose concentrations are shown in Fig. 2. In the control group, these were maintained at 5.9 + 0.1 mmol/1, but

INSULIN A N D NPY IN F A S T I N G

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FIG. 1. Percentage changes in body weight during the study, in freely fed control rats (I-q;n = 8), saline-injected, food-deprived rats (m; n = 8), and insulin-treated, food-deprived rats ( l ; n = 10). Error bars are SEM. Significance of differences from controls: ***p < 0.001.

levels were significantly lower in both food-deprived groups by 24 h and remained consistently so after this time. Overall blood glucose concentrations during the study were 4.6 _+ 0.3 m m o l / l in saline-treated, food-deprived rats and 4.4 _+ 0.3 mmol/1 in the insulin-treated, food-deprived group (NS between these groups; both p < 0.001 vs. controls). Throughout the study, none of the insulin-treated rats had preinjection blood glucose values of <2.0 mmol/l. Mean terminal plasma glucose concentrations, taken 1-7 h after the last injection, were significantly lower in the insulin-treated than in the saline-treated, food-deprived group (2.5 _ 0.3 vs. 4.9 _ 0.6 mmol/1; p < 0.001), and four insulin-treated rats had values of < 2.0 mmol/l (range, 0.81.9 mmol/l). Final plasma insulin concentrations in the saline-treated, food-deprived group were 55% of those in the controls (28.9 _+ 1.6 vs. 52.6 +_ 5.7 pmol/1; p < 0.001), but levels were close to

FIG. 3. Neuropeptide Y concentrations in the ARC plotted against final plasma insulin concentrations, in the controls (O), untreated food-deprived rats (m), and insulin-treated, food-deprived rats (O).

controls in the insulin-treated group (46.6 + 2.8 pmol/l; NS vs. controls, p < 0.001 vs. saline-injected, food-deprived group). Figures 3 and 4 show NPY concentrations in the ARC plotted against final plasma insulin and glucose concentrations, respectively, for all the rats studied. There were significant negative correlations between ARC NPY concentrations and both insulin (r = -0.39, p < 0.05; Fig. 3) and glucose concentrations (r = -0.41, p < 0.05; Fig. 4).

Regional Hypothalamic NPY Concentrations Neuropeptide Y levels in the individual hypothalamic nuclei are shown in Fig. 5. ANOVA demonstrated a significant effect of region, F(7, 209) = 172, p < 0.001, and of group, F(2, 209) = 3.49, p = 0.032). As in previous studies (7,12,29), NPY concentrations in control animals were highest in the ARC, PVN, and MPO. Seventy-two h of food deprivation in the saline-injected group resulted in a 69% increase in NPY levels in the ARC compared with freely fed control rats (14.2 + 1.8 vs. 8.4 ___ 2.2 fmol/t~g protein; p < 0.001). Starvation did not affect

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FIG. 5. Neuropeptide Y levels (fmol//~g protein) in individual microdissected hypothalamic regions, in controls (open columns), salinetreated, food-deprived (hatched columns), and insulin-treated, food-deprived rats (black columns). Values are mean _ SEM. Key to hypothalamic regions: MPO, medial preoptic area; LPO, lateral preoptic area; PVN, paraventricular nucleus; AHA, anterior hypothalamic area; VMH, ventromedial nucleus; DMH, dorsomedial nucleus; LHA, lateral hypothalamic area; ARC, arcuate nucleus together with the median eminence. *p < 0.01 vs. controls, **p < 0.001 vs. controls, +p < 0.001 vs. fasted insulin-treated rats.

NPY levels in any other region studied. Insulin treatment of food-deprived rats resulted in a 33% lowering of NPY levels in the ARC compared with saline-injected, food-deprived rats (p < 0.001), the value remaining significantly higher than in the controls (11.2 ___ 1.4 fmol//zg protein; p < 0.01). In addition, insulin treatment lowered NPY levels in the LHA to 40% of those in freely fed control rats (p < 0.001). DISCUSSION

We have confirmed that prolonged food deprivation (72 h) produced a significant rise in NPY concentrations in the ARC. As this is the hypothalamic region where most NPY is synthesized (19), this strongly suggests increased activity of the hypothalamic NPYergic system that projects through the LHA to the PVN and DMH. This is in agreement with previous studies, and supports the hypothesis that enhanced NPYergic activity might drive the food-seeking behavior and hyperphagia of food-deprived animals (4,20). We examined the effects of insulin treatment, using a regimen designed to try to maintain circulating insulin levels within a broadly physiological range above the low levels associated with food deprivation. Insulin treatment significantly reduced the rise in NPY levels in the ARC produced by food deprivation, which suggests that hypoinsulinemia might be a stimulus that activates hypothalamic NPY in starvation. This possibility is supported by the weak but significant correlation between ARC NPY levels and final plasma insulin concentrations (Fig. 3). This is consistent with evidence of increased hypothalamic NPYergic activity in other insulin-deficient states, such as diabetes and intense exercise (12,30,32). There is accumulating evidence of an inverse relationship between insulin availability at the hypothalamic level and the activity of the hypothalamic NPY system (32). In addition to the NPY increases in the hypoinsulinemic states mentioned above, insulin has recently been shown to have a direct inhibitory effect on NPY, as its ICV injection in lean Zucker rats suppressed the increase in NPY gene expression that normally follows starvation (24). Our study

extends the latter findings by demonstrating that an increase in peripheral insulin levels within the physiological range, rather than supraphysiological local hypothalamic insulin levels, may inhibit NPYergic activity in the hypothalamus. Under normal circumstances, the rise in circulating insulin levels induced by refeeding after starvation might act to reduce the activity of the hypothalamic NPYergic system to its resting level. The possible time course of this action is not known, although Beck et al. have suggested that the rise in ARC NPY levels may be reversed within 6 h of refeeding (4). In addition, NPY injected into the PVN induces insulin secretion (17,19); another effect of stimulating the NPYergic system may therefore be an attempt to correct insulin deficiency. From our study, it is not clear whether hypoinsulinemia itself or some other metabolic effect of insulin deficiency might be responsible for increasing hypothalamic NPYergic activity. However, the evidence that insulin can enter the brain, that the ARC and other regions contain plentiful insulin receptors (2,6,34), and that ICV-injected insulin reduces NPY mRNA levels in the ARC (24) all argue in favor of a direct effect of the hormone. Interestingly, the ambient plasma glucose concentration may also influence hypothalamic NPY, independently of insulin, as there was a weak but significant negative correlation between ARC NPY levels and final plasma glucose concentrations (Fig. 4). An inhibitory action of glucose on NPY has previously been suggested by our observation that diabetic rats deprived of food show further rises in NPY concentrations in the ARC and other regions; in these animals, blood glucose falls to near-normal concentrations, but insulin levels remain low (16). Neuropeptide Y inhibiting actions of both insulin and glucose could cooperate to suppress hypothalamic NPY activity after feeding (4,21), as the circulating levels of both increase postprandially. The insulin regimen that we used requires comment. We chose twice-daily injections of long-acting insulin in preference to implanted minipumps, as they allowed greater flexibility, and particularly the option of withholding insulin should potentially dangerous hypoglycemia occur after prolonged fasting. In any event, hypoglycemia (blood glucose < 2.0 mmol/l) did not occur in any animal until after the last insulin injection, given after 72 h without food. The regimen of 5 U/kg/day divided into two doses was determined by pilot studies. The final plasma insulin levels measured spanned the period between 1-8 h after the last injection and so broadly reflect the overall levels achieved during the study, although insulinemia would have declined during the few hours before the 0800 h injection. Insulin levels in the treated group were close to control values; higher insulin dosages might have matched freely fed levels more precisely but would undoubtedly have increased the risks of severe hypoglycemia after shorter periods of food deprivation. We have previously reported that hypoglycemia, either acute or chronic, induced with much larger insulin dosages (60 U/kg/day) in rats with free access to food did not cause any NPY alterations in the same regions that we studied here (7). Neuropeptide Y concentrations in other regions in this study raise additional questions. We found no change in the LHA in saline-injected, food-deprived rats, but a significant fall in the insulin-treated, food-deprived group. The LHA is a region through which the axons of the ARC NPY neurons pass en route to the PVN; the fall in NPY concentrations could possibly arise if transport to the PVN for release were increased without a proportionate increase in NPY synthesis. An important divergence from other studies of food deprivation (4,5,20) is that we did not find any significant NPY increase in the PVN of starved rats. We have previously found increased NPY concen-

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trations in the PVN of streptozotocin-diabetic rats and in fatty Zucker rats (15,29), as have other groups (3,21), but we have not detected any significant alterations in the PVN of rats subjected to 40 days' moderate food restriction that caused 25% weight loss (11), or in a preliminary form of the present study in which rats were also food deprived for up to 72 h (unpublished results). As the PVN is a major site of release of the peptide, local concentrations here will be highly dependent on the balance between the input of NPY from the ARC and its release; unchanged tissue levels may therefore be due to an increase in release that parallels a rise in synthesis and transport, and therefore do not exclude an overall increase in the activity of the system. Insulin deficiency at the hypothalamic level and its possible role in activating the NPYergic system may have repercussions in other disorders of nutrition and energy balance. Rats with spontaneous obesity (thefa/fa (fatty) Zucker and cp/cp JCR:LA corpulent rats) are obese because of a combination of hyperphagia and reduced energy expenditure. Neuropeptide Y levels in the ARC and other regions are increased in the obese mutants (3,15), and increased NPY m R N A (24) and reduced NPY receptor numbers (14), consistent with downregulation of receptors due to enhanced endogenous N P Y release, have been demonstrated in the fatty Zucker rat. As in the insulin-deficient models, increased NPYergic activity could contribute to obesity, as NPY's central actions include both hyperphagia and reduced energy expenditure. However, the obese rats differ fundamentally from

other models in that their circulating insulin levels are grossly elevated, in parallel with the insulin insensitivity of the liver and other peripheral tissues. This apparent paradox may be explained by the fact that the brain of the fatty Zucker rat is also apparently insensitive to insulin, possibly because insulin receptor numbers in the brain are reduced (33,34): insulin injected ICV neither suppresses feeding nor suppresses NPY m R N A levels in the ARC, whereas it exerts both these effects in lean Zucker rats (8,24). The interaction between circulating insulin and NPY in the hypothalamus may therefore have a fundamental significance in the control of energy balance (32). In conclusion, peripheral administration of relatively low insulin dosages to food-deprived rats significantly reduced the rise in NPY levels normally seen in the ARC of such animals. This supports the hypothesis that a fall in circulating insulin levels is an important stimulus that activates the hypothalamic NPYergic system, and further suggests that the interaction of insulin with this system may be important in controlling nutritional state. ACKNOWLEDGEMENTS We are indebted to the Commonwealth Fund who support Dr. Malabu as a Commonwealth Scholar, and to the Cancer Research Campaign and the British Diabetic Association for their support of Drs. McCarthy and McKibbin, respectively. We are very grateful to the Nuffield Foundation, the Mason Medical Research Fund, and the Peel Medical Trust for additional funding; to Mr. Peter Hynes and his staff for their meticulous care of the animals; and to Mrs. Anne Kilpatrick for her skilled technical assistance.

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