Intracerebroventricular neuropeptide Y produces hyperinsulinemia in the presence and absence of food

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Physiology & Behavior, Vol. 60, No. 3, 685-692, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All tights reserved 0031-9384/96 $15.00 + .00

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PII S0031-9384(96)00033-9

Intracerebroventricular Neuropeptide Y Produces Hyperinsulinemia in the Presence and Absence of Food J O N A T H A N L. MARKS, 1 K A Y W A I T E A N D L I N D A DAVIES

Department of Clinical Endocrinology, Westmead Hospital, Westmead NSW, Australia Received 19 December 1994 MARKS, J. L., K. WAITE AND L. DAVIES. lntracerebroventricular neuropeptide Yproduces hyperinsulinemia in the presence and absence of food. PHYSIOL BEHAV 60(3) 685-692, 1996.--Acute administration of neuropeptide Y into the hypothalamus or cerebral ventricles produces hyperphagia and hyperinsulinemia. However, it is not known to what extent the hyperinsulinemia depends on the food intake. Consequently, serum insulin and glucose, as well as food and water consumption, were measured over 3 h, following injection of 1-20 p.g neuropeptide Y into the third ventricle of adult female rats. In the presence of food, 1-10 ~g neuropeptide Y produced a dose-dependent increase in food and water intake and serum insulin. Insulin levels were closely correlated with the quantity of food ingested. In the absence of food, 1-20 p.g neuropeptide Y produced a dose-dependent increase in water intake, whereas 1-5 o.g produced a dose-dependent increase in serum insulin. We concluded that ICV neuropeptide Y can stimulate insulin secretion even at low doses and this response does not completely depend on food intake. Neuropeptide Y

Hypothalamus

Insulin

Hyperphagia

NEUROPEPTIDE Y (NPY) is a 36-amino acid neuropeptide (27) that is widely distributed in the mammalian brain (2). High levels of NPY are found in the hypothalamus, particularly in the paraventricular nucleus (PVN) (3,31), and this results from a dense innervation of NPY terminals from neurons in the arcuate nucleus and brain stem (6). Hypothalamic administration of NPY has potent effects on body metabolism, including an increase in food intake (4,7,24,29,32), body weight (24,32), hyperinsulinemia (1,12,20,29), and glucocorticoid secretion (15). Consequently, endogenous hypothalamic NPY release may have an important role in controlling whole body metabolism (14). Chronic central administration of NPY leads to adiposity, marked hyperinsulinemia (24,32), and other features consistent with the obese state (5,32,33). As hypothalamic NPY expression is increased in several genetic rodent models of obesity and hyperinsulinemia (3,22,30), excess hypothalamic NPY could be involved in the pathogenesis of obesity (10). The ability of hypothalamic NPY to stimulate insulin release is of particular interest in this context. First, there is a close relationship between hyperinsulinemia and obesity (10), and second, circulating (17,18) and hypothalamic (23) insulin can inhibit hypothalamic NPY expression. From these observations, a negative feedback relationship between hypothalamic NPY and circu-

lating insulin has been proposed that may be involved in the regulation of food intake, body weight, and adiposity (23). Not only does injection of NPY into the third ventricle stimulate insulin secretion when food is available (12,20), but injection of NPY into the PVN (1), lateral ventricle (29), and brain stem (9) stimulates insulin secretion in its absence. Thus, NPY acting in the brain may have a more direct mechanism for producing hyperinsulinemia than from the response to ingested food. However, in the one study where this direct hyperinsulinemic response was reported following PVN injection, marked and possibly unphysiological hyperphagia was reported when the same NPY doses were injected into the PVN in the presence of food (1). In a recent study, injection of NPY into the PVN did not affect insulin secretion unless food was also present (28). Therefore, there are contradictory data about the direct effects of hypothalamic NPY on insulin secretion and the possibility that it only occurs at high NPY doses. Marked hyperinsulinemia has been reported following chronic NPY infusion into the lateral ventricle (32,33), even when NPYinduced hyperphagia has been prevented. However, chronic NPY infusion also produced increased adiposity, hyperlipidemia, and corticosteronemia (32), and these changes could have independently influenced insulin secretion. Consequently, chronic infu-

1 TO whom requests for reprints should be addressed.

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sion studies have only provided circumstantial evidence that endogenous hypothalamic NPY directly stimulates insulin secretion. Indeed, studies that used fasted (11,22) and underweight (8) rodents have not supported this hypothesis. In these conditions, there has been reported increased expression and release of hypothalamic NPY, associated with profound hypoinsulinemia. Therefore, the purpose of this study was to determine first, if the insulinemic response to hypothalamic NPY is dependent on food intake, and second, if it occurs at NPY doses that have small to moderate effects on ingestive behavior. As recent studies have shown that endogenous hypothalamic NPY can promote food intake (13,26), we reasoned that effects of exogenous NPY at doses that promote food intake from a small to moderate extent would be likely to duplicate the effects of endogenous hypothalamic NPY. Furthermore, by using an acute study, the confounding secondary effects of NPY on body metabolism would be avoided. Consequently, we have measured the effect of increasing NPY doses on ingestive behavior, serum insulin, and glucose in the presence and absence of food. Injection into the third ventricle (ICV) was used to allow consistent delivery of NPY to the same parts of the hypothalamus and thereby ensure consistent insulin secretory and ingestive responses. However, the advantages of ICV injection were offset by substantially reduced potency compared to injection into the hypothalamic substance. Furthermore, it is possible that some of the effects observed were due to movement of NPY outside the hypothalamus. METHOD

Animals Studies were performed on adult female Wistar rats, weighing 200-280 g. Animals were housed in individual hanging cages and maintained on a 1 2 / 1 2 h light/dark cycle beginning at 0600 h. Animals were fed pellets of Young Milling rat chow (Young, NSW), (carbohydrate 56%, protein 21%, and fat 4%) ad lib.

Surgical Procedures A permanent cannula consisting of an external 22-gauge stainless steel guide sleeve and obturator (Plastics One, Roanoke, VA) was inserted under stereotaxic control into the third ventricle. Surgery was performed under IP xylazine (4 m g / k g ) and ketamine (100 m g / k g ) anesthesia. The coordinates were 2.3-2.5 mm caudal to bregma and 7.5-8.0 mm ventral to the cortical surface on the midline sagittal sinus. The incisor bar was used to place lambda and bregma on a horizontal plane. The sagittal sinus was pulled out of the path of the cannula prior to insertion. The cannula was secured to the dorsal skull with screws and dental acrylic. The animals received 12.5 mg IM ampicillin postoperatively and if recovery was slow 10 ml of IP Ringer's solution (NaCI 147 m M , KC1 4 m M , and CaCI 2 m M ) . Subsequently, animals were fed ad lib and only used if they regained weight following the initial weight loss after ICV cannulation.

Experimental Procedures Seven to 14 days after surgery, at 1200 h, the food hopper was removed and weighed and the tail was snipped 1 - 2 mm from the tip. Sixty minutes later, the animal and water bottle were weighed. The food hopper was returned to the cage in the Fed group and not in the Fasted group. Immediately afterwards, at time 0, a sample of blood (60-100 p.l) was milked from the tail tip into a capillary tube. The obturator was removed and replaced with an internal 28-gauge cannula connected by PE60

tubing to a Hamilton syringe. Sterile isotonic saline (5 Izl) _ human NPY(Auspep, Melbourne, VIC.) was injected into the cannula over 20 s. The obturator was replaced and the animal was returned to its cage. Subsequent samples of blood were collected from the initial tail wound at 30, 60, 120, and 180 min and stored on ice until the end of the procedure. In the Fed animals receiving 1 ixg NPY and some of their saline controls, some blood samples could not be collected at 180 min and data are therefore reported up to 120 rain for this group. Serum was separated and stored at - 2 0 ° C until assayed. As some serum samples showed evidence of hemolysis, bacitracin (Sigma Chemical Co.), 0.25 m g / m l final concentration, was added to blood collecting tubes to prevent any insulin degradation from red cell contents. After 180 min, the remaining food, including spilled remains, and the water bottle were weighed to determine intake of food and water. Two or three days later the procedure was repeated in nearly all cases on the same animal except saline was replaced with NPY or vice versa.

Serum Assays Serum insulin was assayed using the reagents from the Phadeseph (Pharmacia, North Ryde, NSW) double antibody RIA. The reagents were used in one-fourth volume of that specified in the package insert and the serum sample of 25 Ixl and primary antibody incubated at 4°C overnight. Subsequently the labelled insulin was added and incubated for 2 h at room temperature. The subsequent steps followed the package insert. The sensitivity of the assay was 0.75 IxU/ml, the interassay CV was < 10%, and the intra-assay CV was < 8% using the standards supplied. Single samples were assayed using rat insulin kindly supplied by Eli Lilly Australia Pty Ltd. (West Ryde, NSW) to define the standard curve. The upper limit of the assay was 2 5 0 / ~ U / m l . An occasional serum sample that reached this value and that could

TABLE 1 EFFECT OF ICV NPY ON FOOD AND WATER INTAKE IN FED AND FASTED ANIMALS

1 ~g NPY Saline 1 ~.g NPY Saline 2 ~g NPY Saline 2 g.g NPY Saline 5 Izg NPY Saline 5 g.g NPY Saline 10 p.g NPY Saline 10 Ixg NPY Saline 20 ~g NPY Saline 20 g.g NPY Saline

FoodAccess

FoodIntake (g)

Water Intake

n 11 11 7 6 14 13 9 8 9 9 7 8 13 16 6 7 8 7 9 9

no no yes yes no no yes yes no no yes yes no no yes yes no no yes yes

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3.2 5- 0.6 0.75 5- 0.5 6.7 5- 2.5* 0.8 5- 0.3 3.1 5- 0.6* 1.0 5- 0.2 6.1 5. 1.9" 1.0 + 0.3 5.3 5. 1.6":~ 1.1 + 0.2 11.6 5. 1.4*t:~ 0.7 5. 0.2 7.3 5. 1.3*t~: 0.8 + 0.2 12.3 + 1.2"*~: 0.7 5. 0.2 7.9 5. 1.6*t:~ 0.4 ± 0.2 8.4 5. 1.4" 0.6 5- 0.2

All data are presented as the mean 5- SE. * p < 0.05 compared to saline. t P < 0.05 compared to the 1 ~g dose. p < 0.05 compared to the 2 Izg dose. § p < 0.05 compared to the 5 ~g dose. I p < 0.05 compared to the 10 ~g dose.

(g)

H Y P O T H A L A M I C NEUROPEPTIDE Y A N D INSULIN RELEASE

not be repeated as a dilution was called 250 floU/ml. Serum glucose was analysed using a Beckmann II glucose analyser.

Data Analysis Data are expressed as the mean + SE. Serum insulin and glucose data presented have been calculated as the difference from time 0 values. Insulin area under the curve (AUC) data in f l o U / m l / h units were calculated using the trapezoid method and values are reported as total A U C and net AUC. The latter is calculated as the difference from time 0 values. Statistical analysis was peformed using Fisher PLSD after A N O V A and signifi-

687

cant differences presented are between individuals. It should be noted that insulin A U C results for Fed animals receiving 1 flog NPY and their controls are taken up to 120 and not 180 rain and are therefore underestimates compared to higher NPY dose groups. RESULTS In Fed animals, ICV NPY produced a dose-dependent increase in food and water intake (Table 1). Food intake was significant with the 1 I.Lg dose, maximal with the 10 I~g dose, and submaximal with the 20 flog dose. Water intake was significant

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FIG. 1. The effect of ICV saline ( O ) or NPY ( 0 ) on serum insulin in Fed animals. The NPY dose was (A) 1, (B) 2, (C) 5, (D) 10, or (E) 20 v,g in 5 V,1 saline injected at time 0. The number of animals used is the same as in Table 1. Baseline values for serum insulin in v,U/ml were: (A) 25 + 2 and 21 + 2; (B) 28 + 3 and 27 + 3; (C) 28 + 4 and 23 + 4; (D) 58 + 22 and 50 d- 8; and (E) 32 + 2 and 23 + 2, for the NPY and saline groups, respectively. * p < 0.05 for the experimental group compared to the saline group; " p < 0.05 compared to the 1 /,~gdose; b p < 0.05 compared tothe 2 p g dose; c P < 0.05 compared to the 5/~g dose; d P < 0.05 compared to 10 p g dose.

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FIG. 2. Correlation between food intake and net insulin AUC from the values described in Tables 1 and 2. Each point represents the data from individual animals, saline (O), 1 (A), 2 (O), 5 ( I ) , 10 (O), and 20 p.g

AUC was compared to food intake in individual animals at all NPY doses (Fig. 2). The correlation coefficient was 0.8. Blood glucose was increased by ICV NPY in Fed animals, but the effect was small and reached significance only at some time points with the 10 and 20 wg doses (Fig. 3). In Fasted animals, 1 - 5 i~g ICV NPY produced a dose-dependent increase in serum insulin (Fig. 4). The effect was greatest at 30 min and was not significant by 60 rain. The increase in serum insulin produced by the 10 ~ g dose was submaximal and by the 20 wg dose it was not significant. Net A U C for insulin was increased with the 1 i~g dose, maximal with the 5 wg dose, and submaximal with the 10 wg dose (Table 2). The effect of food availability on the insulinemic response to ICV NPY was assessed by dividing the net insulin AUC in Fasted by the Fed groups (Table 2). The value was 0.15 at the 1 I~g dose and decreased to 0.01 at the 10 ~ g dose. In Fasted animals, ICV NPY had no effect on serum glucose (Fig. 5).

NPY (zx).

DISCUSSION

with the 1 Ixg dose and maximal with the 5 and 10 vLg doses. In Fasted animals, ICV NPY produced a dose-dependent increase in water intake, maximal at the 10 and 20 ~ g doses (Table 1). Figure 1 shows the effect o f ICV NPY on serum insulin in the Fed group. There was a dose-dependent increase in serum insulin that was present by 30 rain and was maintained for at least 3 h. Net A U C for insulin also showed a dose-dependent increase that was maximal at the 10 I~g dose (Table 2). However, the effect of the 10 ~ g NPY dose on insulin levels may be somewhat exaggerated because of high baseline values in this group. To determine the association of insulin levels with food intake, the net insulin

As previously reported, acute ICV injection of NPY (4,7,21) produced a dose-dependent increase in food intake and water consumption in satiated rats. A small but significant effect on food and water ingestion was found with the lowest dose tested, 1 I~g, whereas the largest effect was found with 10 wg. Previous studies using ICV injection found that 1 - 3 i~g NPY was required to increase food intake acutely in satiated rats and a maximal effect was found with 1 0 - 1 6 I~g (4,7). Similar to our study, higher doses produced a submaximal or absent feeding response (4,7). By contrast, studies using intrahypothalamic injection have shown lower doses of NPY-stimulate food and water ingestion. For example 0.1 Ixg NPY injected into the PVN (24), perifomical region (25), or ventromedial hypothalamus (21) stimulated food

TABLE 2 THE EFFECT OF ICV NPY ON TOTAL AND NET INSULIN AUC IN FED AND FASTED ANIMALS

1 I~g NPY Saline 1 wgNPY Saline 2 ~g NPY Saline 2 ILgNPY Saline 51~gNPY Saline 5 IxgNPY Saline 10 Ixg NPY Saline 10 I~g NPY Saline 20 I~g NPY Saline 201~gNPY Saline

Food Access

Total Insulin AUC (IzU/ml.hr)

Net Insulin AUC (p.U/ml.hr)

no no yes yes no no yes yes no no yes yes no no yes yes no no yes yes

78 + 10 61 + 7 133 5: 25* 44 5: 5 69 + 9 77 5:11 343 5: 67"* 78 + 16 114 4- 19"t 68 5: 5 418 5: 84"t 134 5 : 7 9 111 + 2 105 5:15 1361 5: 311't$§ 131 + 31 96 ± 8 73 5- 8 505 4- 86"t¶ 78 4- 7

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HYPOTHALAMIC NEUROPEPTIDE Y AND INSULIN RELEASE

and water intake, whereas 0.5-1 ~ g induced a similar or greater ingestive response to 10 Itg administered ICV in our study. Although not investigated, likely factors contributing to reduced sensitivity after ICV vs. intrahypothalamic NPY injection are a diffusion barrier between the third ventricle and NPY sensitive hypothalamic sites and dilution of NPY in the CSF. In Fed animals, we found a marked insulinemic response to ICV NPY. Serum insulin was increased 30 mix) after ICV NPY in a dose-dependent manner and remained elevated for at least 3 h. The change in insulin levels over 3 h as assessed by net insulin AUC also increased in a dose-dependent fashion in response to ICV NPY. Because there was a strong correlation between the amount of food eaten and the net insulin AUC, we concluded that

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the increase in insulin secretion was largely due to the amount of food ingested. A similar correlation has been noted between serum insulin and food intake following chronic NPY infusion in the lateral ventricle (5). In Fasted animals, we have confirmed that ICV NPY can increase serum insulin and demonstrated that this occurs by a dose-dependent mechanism. Similar to the orexigenic and some other metabolic effects of centrally administered NPY (4,7,19), this response diminished at supramaximal NPY doses. Although the effect was small, and short-lived compared to that in Fed animals, it still occurred at NPY doses that stimulated ingestive behavior to a moderate extent. Consequently, provided ICV NPY was acting in the adjacent hypothalamus, these data support the

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hypothesis that endogenous hypothalamic NPY directly stimulates insulin secretion. The mechanism by which ICV NPY increased serum insulin in the absence of food was not apparent from our study. ICV NPY did not increase blood glucose in the Fasted group and therefore it was unlikely that hyperglycemia contributed to the increased insulin levels. Furthermore, as the experiments were performed at least 1 h after food withdrawal and at a time that rats eat very little, it was unlikely that this NPY effect was due to increased absorption of gut contents. Leturque et al. (16) have shown that in the postabsorptive state the rat gut does not contribute glucose to the circulation and we have found similar

results following ICV NPY administration (unpublished data). Therefore, it is probable that ICV NPY has direct hormonal a n d / o r neural mechanisms for stimulating insulin release and these may include modulation of autonomic nervous system activity (28). The finding in this study that ICV NPY induced insulin release was large in the presence of and considerably smaller in the absence of food is consistent with the proposed negative feedback relationship between insulin and hypothalamic NPY (23) being relevent in the long-term control of food intake and body weight. In the presence of food, hypothalamic NPY probably increases food intake and, as suggested in some studies

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HYPOTHALAMIC NEUROPEPTIDE Y AND INSULIN RELEASE

(28,33), may potentiate the insulin release resulting from the ingested food. The resulting hyperinsulinemia might well stimulate lipogenesis and contribute to increased adiposity (10). At the same time, the hyperinsulinemia could increase satiety and limit excess weight gain by inhibiting hypothalamic NPY expression (18,23). By contrast, in the absence of food, if hypothalamic NPY stimulates insulin release to a much smaller extent, then the insulin might have little effect on hypothalamic NPY expression or on satiety. In addition, if the animal was starved or underfed, additional factors such as hypoglycemia may further decrease insulin secretion and thus permit the association of increased hypothalamic expression of NPY and profound hypoinsulinemia that has been observed in these states (8,11,17,22).

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In summary, our results have shown that ICV NPY not only substantially increases insulin secretion when food is available, but can modestly increase insulin secretion when food is unavailable. The ability of low doses of NPY to produce this effect suggests that endogenous hypothalamic NPY may have a similar action. ACKNOWLEDGEMENTS This study was supported by a research grant from Diabetes Australia, a priming project grant from the National Health and Medical Research Council of Australia, and a Postgraduate Fellowship from the Medical Foundation of Sydney University for J. L. M.

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FIG. 5. The effect of ICV saline (O) or NPY (Q) on serum glucose in Fasted animals from the animals in Fig. 4. Baseline values for serum glucose in mM were: (A) 6.8 + 0.3 and 6.6 + 0.2; (B) 7.7 ± 0.4 and 7.6 + 0.3; (C) 6.9 + 0.2 and 7.7 ± 0.3; (D) 8.2 ± 0.4 and 8.2 :i: 0.3; and (E) 7.2 ± 0.2 and 7.0 + 0.1, for the NPY and saline groups, respectively.

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