unchanged hypothalamic neuropeptide y concentrations in hyperphagic, hypoglycemic rats: evidence for...

Peptides, Vol. 12, pp. 425--430. © Pergamon Press plc, 1991. Printed in the U.S.A. 0196-9781/91 $3.00 + .00

Unchanged Hypothalamic Neuropeptide Y Concentrations in Hyperphagic, Hypoglycemic

Rats: Evidence for Specific Metabolic Regulation of Hypothalamic NPY

S U S A N E. C O R R I N , 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 W I L L I A M S 1

Department of Medicine, The University of Liverpool, PO Box 147, Liverpool L69 3BX, United Kingdom

Rece ived 21 D e c e m b e r 1990

CORRIN, S. E., H. D. McCARTHY, P. E. McKIBBIN AND G. WILLIAMS. Unchanged hypothalamic neuropeptide Y concentrations in hyperphagic, hypoglycemic rats: Evidence for specific metabolic regulation of hypothalamic NPY. PEPTIDES 12(3) 425--430, 1991.--Hypothalamic concentrations of neuropeptide Y (NPY), a potent central appetite stimulant, increase dramatically in food-restricted and insulin-deficient diabetic rats. This suggests that NPY may drive hyperphagia in these conditions, which are characterized by weight loss and insulin deficiency. To test the hypothesis that insulin deficiency and weight loss are specific stimuli to hypothalamic NPY, we measured NPY concentrations in individual hypothalamic regions in rats with hyperphagia caused by insulin-induced hypoglycemia. Groups of 8 male Wistar rats were injected with ultralente insulin (20-60 U/kg) to induce either acute hypoglycemia (7 h after a single injection) or chronic hypoglycemia (8 days with daily injections). In hypoglycemic rats, plasma insulin concentrations were increased 6- to 7-fold compared with saline-injected controls; food intake was significantly increased with acute and chronic hypoglycemia and weight gain was significantly increased in the chronically hypoglycemic group. NPY concentrations were measured by radioimmunoassay in 8 hypothalamic regions microdissected from fresh brain slices. NPY concentrations were not increased in any region in either acute or chronic hypoglycemia. NPY therefore seems unlikely to mediate hyperphagia in hyperinsulinemia-induced hypoglycemia, supporting the hypothesis that weight loss is a specific stimulus to hypothalamic NPY and that insulin deficiency may be the metabolic signal responsible.

Hypoglycemia Hypothalamus Neuropeptide Y Food intake Rats

NEUROPEPTIDE Y, a 36 amino acid member of the pancreatic polypeptide family (31), is one of the most abundant peptides in the brain and is highly concentrated in major appetite-regulating areas of the hypothalamus (6, 36, 40). NPY injected into these areas in rodents causes dramatic increases in feeding; it is one of the most potent appetite stimulants known and can cause obesity with repeated administration (20, 21, 24, 28, 29). Central NPY injection also stimulates insulin and glucagon secretion (19). Hypothalamic levels of NPY and NPY mRNA are increased, particularly in appetite-regulating areas, in insulin-deficient diabetes (14, 26, 35, 37-39) and in starvation (5, 25, 34). This indicates that hypothalamic NPYergic activity is increased and suggests that NPY may drive the hyperphagia which is characteristic of both these conditions and which may represent a homeostatic response to weight loss.

These findings suggest that NPY in the hypothalamus may have a crucial role in integrating feeding behavior and energy balance and especially in defending body weight. The metabolic signals which regulate hypothalamic NPYergic activity, and particularly those which activate hypothalamic NPY in response to weight loss, are unknown. We have suggested that insulin deft-

~Requests for reprints should be addressed to Dr. Gareth Williams.

ciency, which is common to both diabetes and starvation, may be responsible (36--40). This possibility is consistent with the accumulating evidence that insulin is both an indicator of body weight and a satiety signal which acts on the brain to inhibit feeding (2).

The purpose of this study was to test the hypothesis that hypothalamic NPYergic activity is activated specifically by insulin deficiency, by measuring hypothalamic NPY levels in rats during acute and chronic insulin-induced hypoglycemia. Hypogly- cemia causes marked hyperphagia and ultimately weight gain (10, 11, 16, 17, 22), but, if our hypothesis is correct, hypothalamic NPY levels should not be increased because there is insulin excess rather than deficiency.

METHOD

Animals

Thirty-five male Wistar rats (Harlan Olac, Bicester, UK) with initial weights of 310---5 g were housed in individual wire-bot- tomed cages. They were maintained at a constant temperature of

425

426 CORRIN, McCARTHY, McKIBBIN AND WILLIAMS

22°C with a 12:12 h light:dark cycle, initiated at 0730 h. Food (CRM: Biosure, Cambridge, UK) and water were available freely throughout the study. The rats were habituated to being handled and to undergoing tail-prick blood sampling and daily subcutaneous injections of saline for 4 days before study.

Experimental Design

Acute hypoglycemia. Preliminary experiments were conducted giving subcutaneous injections of heat-treated bovine ultralente insulin (UltratardR: Novo Industri, Copenhagen, Denmark) at doses in the range of 10--60 U/kg. A dose of 60 U/kg was found to give a reproducible trough in blood glucose concentration occurring 6-8 h after injection, and caused a 500% increase in food intake between 0 and 8 h.

Sixteen rats, weighing 355 _+ 5 g, were selected for this study and were divided into two groups (n = 8), carefully matched for age and weight. Starting at 0800 h, rats from the first group were given a single subcutaneous injection of insulin (60 U/kg) while control rats received an equal volume of isotonic saline by the same route. Rats from each group were injected alternately at 15-minute intervals. Food and water intakes and blood glucose concentrations (using Ames Glucostix and Glucometer II: Miles Ltd., Ames Division, Slough, UK) were measured every 2 h and at sacrifice for each rat. Each animal was killed 7 h after injection; they were rapidly sacrificed by carbon dioxide inhalation, which produced unconsciousness within 45-60 sec- onds, and immediately exsanguinated by cardiac puncture.

Chronic hypoglycemia. Eleven rats received once-daily subcutaneous injections of Ultratard insulin at 1000 h, while 8 weight-matched controls received a subcutaneous injection of saline at the same time. Rats were maintained under the same conditions as above. Blood glucose concentrations were measured at 1000 h and 1900 h each day, and body weight and 24-h food and water intakes were also monitored daily throughout the study. The dose of insulin given was adjusted each day to keep the treated rats consistently hypoglycemic (1-3 mM) on each reading; the range of dosages given was 20-60 U/kg. Three rats died, presumably of hypoglycemia, during the study. After 8 days, the rats were sacrificed as above, insulin- and saline- treated rats being killed alternately between 1330 h and 1730 h. Blood was taken into fluoride-oxalate tubes for glucose measurement and serum was saved for insulin radioimmunoassay.

Microdissection Methods

The microdissection method has been described in detail (37). Briefly, slices of 350-500 p.m were cut from the hypothalamic area of fresh brain using a vibrating microtome. Selected areas of the hypothalamus were microdissected, either by using a fine scalpel blade or punching out with a blunt 18-gauge nee- dle (internal diameter, approximately 700 Ixm). The areas studied were: the medial preoptic area (MPO), lateral preoptic area (LPO), paraventricular nucleus (PVN), anterior hypothalamic area (AHA), ventromedial nucleus (VMH), dorsomedial nucleus (DMH), lateral hypothalamic area (LHA) and the arcuate nucleus together with the median eminence (ARC). The dissection was performed under x 16 magnification. All tissue from each region (sampled bilaterally from the appropriate slices) from a single rat was boiled in 400 Ixl of 0.5 M acetic acid for 10 minutes to extract NPY. The extracts were then frozen at -20°C until measurement of NPY and protein concentrations.

Assays

Blood glucose concentrations were measured using a glucose oxidase-based autoanalyzer. The insulin assay used a commer-

cial radioimmunoassay (RIA) kit (Novo Industri, Copenhagen, Denmark), using a rat insulin standard; the antiserum showed 98% cross-reactivity with bovine insulin. All samples were measured in duplicate in a single assay. The within-assay coefficient of variation was 4.9%.

The NPY assay employed 125I-labeled porcine NPY (Amer- sham International, Amersham, UK) and synthetic porcine NPY (Bachem Inc., Saffron Walden, UK) as standards, and an N-ter- minal-directed antiserum (Amersham), raised in rabbits. After incubation for 5 days, free and antibody-bound label were sepa- rated using dextran-coated charcoal (1). All samples from both the acute and chronic studies were measured in duplicate in a single assay. The sensitivity of the assay was 47 fmol/tube and the within-assay coefficient of variation was 6%. NPY concentrations were expressed as fmol/Ixg of protein, protein concentration in the extract being measured by the Coomassie Blue micromethod (Pierce and Warriner, Chester, UK).

Chromatographic Methods

To characterize NPY-like immunoreactivity in hypothalamic tissue, extracts from several hypothalamic areas were pooled from 4 rats in each group and subjected to high performance liquid chromatography (HPLC) using a LiChrospher-300 RP-8 10-1~m column (250×4 ram) (Merk: BDH, Poole, UK). The column was eluted at a flow rate of 1 ml per min and equili- brated at 20% acetonitrile in water containing 5% trifluoroacetic acid (1% TFA in water). UV absorbance was monitored at 280 nm at a sensitivity of 0.01 absorbance units for full-scale deflec- tion. Following the addition of the sample, a linear gradient was established from 20% rising to 48% acetonitrile in water containing 5% TFA over 20 minutes. Porcine NPY (Bachem Inc., Saffron Walden, UK) was used as a standard. Fractions collected at 1-min intervals were lyophilized, reconstituted in assay buffer and assayed for NPY as described above.

Statistical Analyses

All data are presented as mean---SEM. Differences in metabolic parameters, body weight and food intakes between insulin- and saline-treated rats were examined by the unpaired Student's t-test. The effects of insulin and saline on hypothalamic NPY concentrations were compared using two-way analysis of vari- ance (ANOVA). A significance level of p<0.05 was selected.

RESULTS

Metabolic Data

Acute hypoglycemia. At sacrifice, serum insulin levels were 7-fold higher on average in the insulin-treated rats (1552- 140 vs. 189- 15 pM; p<0.001). Final blood glucose concentrations of the insulin-treated group were significantly lower than in the saline-treated controls (3.4_+0.4 vs. 6.1_+0.2 mM; p<0.001) (Fig. la). Insulin-treated rats were markedly hyperphagic (Fig. lb) and polydipsic from 4 h after injection, consuming on average 480% more food (6.1 _+ 0.5 vs. 1.3 _+ 0.4 g/rat/7 h; p<0.001) and 330% more water (4.9 _+0.7 vs. 1.5 _+ 0.4 ml/rat/7 h; p<0.001) than the saline-injected controls during the 7 h of the study.

Chronic hypoglycemia. Dally blood glucose concentrations at 1000 h and 1900 h were significantly lower in the insulin-treated group of rats than in the saline-treated controls (p<0.001) throughout the study (Fig. 2a). Daily food intake increased by 25% (36.2---0.6 vs. 27.5-+0.7 g/rat/day; p<0.001) and dally water intake by 24% (36.9-+3 vs. 28.1+--1 ml/rat/day; p<O.05) in the

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FIG. 1. (a) Blood glucose concentrations in rats at 2, 4, 6, and 7 h after a single subcutaneous injection of ultralente insulin (60 U/kg) or saline. Values are given as mean---SEM (n = 8 rats per group). Insulin-treated vs. saline-treated groups: **p<0.01, ***p<0.001. (b) Cumulative food intake of rats after a single subcutaneous injection of insulin (60 U/kg) or saline. Values are given as mean---SEM (n= 8 rats per group). ***p<O.O01.

NPY AND HYPOGLYCEMIA 427

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FIG. 2. (a) Daily blood glucose concentrations at 1000 h and 1900 h in rats given a subcutaneous injection of insulin (20-60 U/kg) or saline at 1000 h each day for 8 days. Values are mean_. + SEM (n = 8 rats per group). ***p<0.001. (b) Weight gain of rats given subcutaneous injections of insulin (20--60 U/kg) or saline each day for 8 days. Values are mean---SEM (n = 8 rats per group). **p<0.01, ***p<0.001.

hypoglycemic rats. Weight gain was significantly increased in insulin-treated rats compared with control rats during the study (20.2--- 1.1% of initial weight vs. 11.8--- 1.2%; p<0.01), as shown in Fig. 2b. Final serum insulin levels in the insulin- treated group were increased 8-fold compared with saline-treated controls (1594---84 vs. 213 +-18 pM; p<0.001).

Hypothalamic NPY Concentrations

Protein concentrations in tissue extracts from various hypothalamic areas did not differ significantly between insulin-treated and control rats in either study.

Using HPLC, the porcine NPY standards eluted after 23 minutes at 48% acetonitrile. The dominant peak of NPY-like immunoreactivity in the hypothalamic extracts from both experimental groups eluted in the position of the NPY standard, con- finning the specificity of the NPY antiserum.

NPY concentrations in the eight hypothalamic regions in the acute and chronic hypoglycemia studies are shown in Fig. 3a and b. NPY concentrations showed regional variation in both control and insulin-treated rats, the highest being in the PVN. The pattern of distribution was comparable in both acute and

chronic experiments and was in general agreement with previous studies using radioimmunoassay of microdissected areas (26,37).

In the acutely hypoglycemic group, NPY levels did not differ significantly from those in the saline-treated control group [ANOVA: F(1,7)=0.43] and were closely similar in all 8 areas in the two groups (p>0.05).

In the chronic hypoglycemia study, NPY concentrations were again closely comparable in all 8 areas in the two groups, with no significant differences between them [ANOVA: F(1,7)= 0.39, p>0.05].

DISCUSSION

This study confirmed the findings of several previous reports that acute and chronic insulin-induced hypoglycemia stimulates eating and that, during chronic hypoglycemia, hyperphagia is sustained and leads to excessive weight gain (10, 11, 16, 17, 22). The mechanism of hypoglycemia-induced hyperphagia is unknown, although changes in various hypothalamic neurotrans- mitters and peptides with experimental actions on feeding behavior have been identified in hypoglycemic and neuroglycopenic animals. These findings include evidence of altered neuronal ac-


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FIG. 3. (a) NPY concentrations in the 8 hypothalamic regions examined, in rats given a single subcutaneous injection of insulin (60 U/kg) (filled bars) and in saline-injected controls (empty bars). Values are mean _ SEM (n = 8 rats per group). 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. (b) NPY concentrations in the 8 hypothalamic regions examined, in rats given subcutaneous injections of insulin (20-60 U/kg) (filled bars) or saline (empty bars) each day for 8 days. Values are mean- SEM (n = 8 rats per group). Key to regions as in (a).

tivities of monoamine transmitters (norepinephrine and serotonin) in the rat hypothalamus (9) and increased glucagon-like immunoreactivity in dogs (12).

The role of NPY in regulating feeding has recently attracted much interest. It is found in high concentrations in the hypothalamus, notably in the areas associated with the regulation of eating and drinking. When injected into the PVN, VMH, DMH and LHA, NPY powerfully stimulates feeding, with a preference for carbohydrate-rich food (28). NPY-induced hyperphagia does not diminish with repeated NPY administration and, indeed, leads to weight gain and obesity (29); NPY is the only one of the few known appetite-stimulating peptides with this action. Drinking is also stimulated by intrahypothalamic NPY administration (20). NPY injected into the PVN also activates the hypothalamo-pituitary-adrenocortical axis and stimulates corticosterone release (33). As the neuroendocrine and behavioral responses to hypoglycemia and neuroglycopenia include carbohydrate-specific hyperphagia, polydipsia and increased corticosterone secretion (9), hypothalamic NPY would seem a likely candidate for mediating these changes.

However, the present study found no changes in tissue NPY

levels in any of the 8 hypothalamic regions studied, during either acute or chronic insulin-induced hypoglycemia. Both hypoglycemic regimes induced hyperphagia, and chronic insulin treatment also caused excessive weight gain. The regions studied included those which are sensitive to the appetite-stimulating effects of NPY (i.e., PVN, VMH, DMH and LHA) and those which show increased levels of NPY and/or NPY mRNA in other hyperphagic states, namely starvation and insulin-deficient diabetes (i.e., PVN, DMH, VMH and ARC) (5, 25, 26, 34, 35, 37). It is possible that hypothalamic NPYergic activity in dis- crete areas might be altered so subtly that tissue levels of the peptide in individual nuclei were unaffected. However, we con- sider this unlikely, as other hypothalamic transmitters, such as the monoamines, show very rapid responses (within 10 min) to acute hypoglycemia (9) and as levels of NPY itself, in specific nuclei, change within a few hours of appetite-modulating stimuli such as the onset of darkness (15), the administration of fenfluramine (23), or food restriction and refeeding (25).

The current findings suggest that hypothalamic NPY is not simply activated as a general response to any condition which causes hyperphagia, but that it is stimulated selectively only under certain circumstances and presumably by specific stimuli. As mentioned above, NPY concentrations and NPY mRNA levels are increased in specific hypothalamic regions in food-restricted rats (5, 25, 34) and those with diabetes either induced by streptozotocin (14, 26, 37) or occurring spontaneously in the Bio- Breeding (BB) strain (26,38). Both starvation and uncontrolled insulin-deficient diabetes induce hyperphagia, which in the case of diabetes is strikingly carbohydrate selective (39). As circulating insulin levels are low in both starvation and in these diabetic models, we have suggested that insulin deficiency is the specific metabolic signal which activates hypothalamic NPY and that this in turn drives the hyperphagia of these conditions. This possibility is supported by the observations that refeeding after food restriction, or insulin replacement in diabetic animals, both factors which restore circulating insulin levels and abolish hyperphagia, normalize NPY levels in the PVN and other hypothalamic nuclei (14, 25, 26). Taken together, the previous work and the present study suggest that insulin deficiency specifically stimulates hypothalamic NPYergic activity, whereas insulin excess apparently has no effect.

These findings are relevant to the growing body of evidence suggesting that insulin is an important metabolic satiety signal which may regulate eating behavior and energy balance. Circu- lating insulin levels are generally proportional to body fat mass and insulin may cross the blood-brain barrier and enter the hypothalamus and other brain regions implicated in appetite control, in which insulin receptors have been identified (2, 7, 41). Several studies have shown that insulin injected into the hypothalamus or third ventricle inhibits feeding, whereas anti-insulin antibodies injected into the VMH stimulate feeding (2, 30, 32). Porte and colleagues have therefore hypothesized that circulating insulin levels signal body fat content to the brain and, through the "satiety" action of insulin, may modulate food intake so as to maintain a constant body weight (2,41). The mechanism through which insulin might act on the hypothalamus to affect feeding is unknown. One possibility is the NPYergic system which, according to our hypothesis, would be activated by insulin deficiency and stimulate feeding, which would help to coun- teract weight loss in insulinopenic conditions such as starvation and diabetes. NPY may therefore be an essential link in a circuit which regulates energy intake and acts to maintain body weight against loss. Recent studies have suggested that the links in this putative circuit which are regulated by insulin and NPY may function abnormally in obese rodents. Fatty Zucker rats have lower brain insulin content and insulin receptor density in

NPY AND HYPOGLYCEMIA 429

several brain regions compared with lean controls, despite mas- sive hyperinsulinemia; failure of insulin to exert its central hypophagic action may contribute to over-eating and obesity in this model (3, 8, 18). NPY concentrations and NPY mRNA levels are elevated in appetite-regulating hypothalamic nuclei in fatty Zucker rats (4, 13, 27), pointing to increased hypothalamic NPYergic activity as a possible cause of obesity and supporting the suggestion that central insulin levels and hypothalamic NPY may be inversely related.

A final point of interest is that hypoglycemia and neuroglycopenia may have divergent effects on norepinephrine and NPY- ergic activities in the hypothalamus: norepinephrine activity is markedly increased (9), whereas NPY is apparently unaffected. This difference is striking in view of the intimate anatomic and functional relationships between the two transmitters in the hypothalamus; both stimulate carbohydrate-specific hyperphagia when

injected into the PVN, and both are implicated in the activation of the pituitary-adrenocortical axis (9, 21, 33). This divergence may reflect the functional specificity of the NPYergic system and the selectivity of the metabolic signals which activate it.

In conclusion, we have found no evidence that acute or chronic insulin-induced hypoglycemia activates hypothalamic NPY. This is consistent with the hypothesis that insulin deficiency is a specific stimulus to NPYergic activity.

ACKNOWLEDGEMENTS

We are grateful to the British Diabetic Association, the Medical Re- search Council, the Mersey Regional Health Authority, the Nuffield Foundation, the Peel Medical Research Trust and Ames Division, Miles Ltd. for financial support; to Mr. Peter Hynes and Mrs. Linda Walmsley for care of the animals; and to Howard Corrin and Paul Shaw for their invaluable assistance.

REFERENCES

1. Adrian, T. E. Radioimmunoassay. In: Bloom, S. R.; Long, R. G., eds. Radioimmunoassay of gut regulatory peptides. Eastboume, UK, Saunders; 1982:3-10.

2. Baskin, D. G.; Figlewicz, D. P.; Woods, S. C.; Porte, D.; Dorsa, D. M. Insulin in the brain. Annu. Rev. Physiol. 49:335-347; 1987.

3. Baskin, D. G.; Stein, L. J.; Ikeda, H.; Woods, S. C.; Figlewicz, D. P.; Porte, D., Jr.; Greenwood, M. R. C.; Dorsa, D. M. Geneti- cally obese Zucker rats have abnormally low brain insulin content. Life Sci. 36:627-633; 1985.

4. Beck, B.; Burlet, A.; Nicolas, J.; Burlet, C. Hypothalamic neuropeptide Y in obese Zucker rats: Implications in feeding and sex- ual behaviours. Physiol. Behav. 47:449-453; 1990.

5. Calza, L.; Giardino, L.; Battistini, N.; Zanni, M.; Galletti, S.; Pro- topapa, F.; Velardo, A. Increases of neuropeptide Y-like immunoreactivity in the paraventricular nucleus of fasting rats. Neurosci. Lett. 104:99-104; 1989.

6. Chronwall, B. M.; DiMaggio, D. A.; Massari, V. H.; Pickel, V. M.; Ruggiero, D. A.; O'Donohue, T. L. The anatomy of neuropeptide Y-containing neurons in the rat brain. Neuroscience 15:1158- 1181; 1985.

7. Corp, E. S.; Woods, S. C.; Porte, D.; Dorsa, D. M.; Figlewicz, D. P.; Baskin, D. G. Localization of insulin binding sites in the rat hypothalamus by quantitative autoradiography. Neurosci. Lett. 70: 17-22; 1986.

8. Figlewicz, D. P.; Dorsa, D. M.; Stein, L. J.; Baskin, D. G.; Pa- quene, T.; Greenwood, M. R. C.; Woods, S. C.; Porte, D., Jr. Brain and liver insulin binding is decreased in Zucker rats carrying the 'fa' gene. Endocrinology 117:1537-1543; 1985.

9. Grunstein, H. S.; Smythe, G. A.; Bradshaw, J. E.; Compton, P. J. Tolbutamide increases hypothalamic serotonin activity in the rat. Diabetes 35:475-480; 1986.

10. Hausberger, F. X.; Hausberger, B. C. Effect of insulin and corti- sone on weight gain, protein and fat content of rats. Am. J. Phys- iol. 193:455-460; 1958.

11. Hoebel, B. G.; Teitelbaum, P. Weight regulation in normal and hypothalamic hyperphagic rats. J. Comp. Physiol. Psychol. 61:189- 193; 1965.

12. Kaneda, H.; Tominaga, H.; Marubashi, S.; Kamimura, T.; Kata- girl, T.; Sasaki, H. Effect of insulin-induced hypoglycemia and glucose-insulin infusion on brain glucagon-like materials in dog. Biomed. Res. 5:61-66; 1984.

13. McKibbin, P. E.; Cotton, S. J.; McMillan, S.; Holloway, B.; May- ers, R.; McCarthy, H. D.; Williams, G. Altered neuropeptide Y concentrations in specific hypothalamic regions of obese (fa/fa) Zucker rats: Possible relationship to obesity and neuroendocrine dis- turbances. Diabetes, in press; 1991.

14. McKibbin, P. E.; McMillan, S.; Cotton, S. J.; Williams, G. Does increased hypothalamic NPY drive hyperphagia and polydipsia in streptozotocin-diabetic rats? Diabetic Med. 7(Suppl. 1):4A; 1990.

15. McKibbin, P. E.; Rogers, P.; Williams, G. Increased neuropeptide Y in the lateral hypothalamic area; the trigger for dark phase eating? Life Sci., in press; 1991.

16. Mackay, E. M.; Callaway, J. W.; Barnes, R. H. Hyperalimentation in normal animals produced by protamine insulin. J. Nutr. 20:59- 66; 1940.

17. May, K. K.; Beaton, J. R. Hyperphagia in the insulin treated rat. Proc. Soc. Exp. Biol. Med. 127:1021-1024; 1968.

18. Melnyk, R. B. Decreased binding to hypothalamic insulin receptors in young genetically obese rats. Physiol. Behav. 40:237-241; 1987.

19. Mitsushi, A.; Masayuki, S.; Takashi, S. Neuropeptide Y and norepinephrine injected into the paraventricular nucleus of the hypothalamus activate endocrine pancreas. Biomed. Res. 10:431-436; 1989.

20. Morley, J. E.; Levine, S. E.; Gosnell, B.; Kneip, J.; Grace, M. Effect of neuropeptide Y on ingestive behaviors in the rat. Am. J. Physiol. 252:R599-609; 1987.

21. Morley, J. E.; Mitchell, J. E.; Levine, A. S. Appetite and satiety regulation: Pharmacological strategies. Psychopharmacol. Bull. 22: 745-749; 1986.

22. Panksepp, J. A.; Pollack, A.; Krost, K.; Meeker, R.; Ritter, M. Feeding in response to repeated protamine zinc insulin injections. Physiol. Behav. 14:487-494; 1975.

23. Rogers, P.; McKibbin, P. E.; Williams, G. Acute fenfluramine administration reduces neuropeptide Y concentrations in specific hypothalamic regions of the rat: Possible implications for the anorectic effect of fenfluramine. Peptides 12:251-255; 1991.

24. Rowland, N. E. Peripheral and central satiety factors in NPY-induced feeding. Peptides 9:989-994; 1988.

25. Sahu, A.; Kalra, P. S.; Kalra, S. P. Food deprivation and ingestion induce reciprocal changes in NPY concentrations in the paraventricular nucleus. Peptides 9:83-86; 1987.

26. Sahu, A.; Sninsky, C A.; Kalra, P. S.; Kalra, S. P. Neuropeptide Y concentration in microdissected hypothalamic regions and in vitro release from the medial basal hypothalamus-preoptic area of streptozotocin-diabetic rats with and without insulin substitution therapy. Endocrinology 126:192-198; 1990.

27. Sanacora, G.; Kershaw, M.; Finkelstein, J. A.; White, J. D. In- creased hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation. Endocrinology 127:730-737; 1990.

28. Stanley, B. G.; Daniel, D.; Chin, A. S.; Leibowitz, S. F. Paraven- tricular nucleus injections of peptide YY and neuropeptide Y prefer- entially enhance carbohydrate ingestion. Peptides 6:1205-1211; 1985.

29. Stanley, B. G.; Kyrkouli, S. E.; Lampert, S.; Liebowitz, S. F. Neuropeptide Y chronically injected into the hypothalamus: A pow- erful neurochemical inducer of hyperphagia and obesity. Peptides 7:1189-1192; 1986.

30. Strubbe, J. H.; Mein, C. G. Increased feeding in response to bilat- eral injection of insulin antibodies in the VMH. Physiol. Behav. 19: 309-313; 1977.

31. Tatemoto, K.; Carlquist, D. M.; Mutt, V. NPY--a novel brain peptide with structural similarities to peptide-YY and pancreatic polypeptide. Nature 296:659-660; 1982.

32. Vanderweele, D. A.; Pi-Sunyer, F. X.; Novin, D.; Bush, M. J. Chronic insulin infusion suppresses food ingestion and body weight


gain in rats. Brain Res. Bull. 5:7-12; 1980. 33. Wahlestedt, C.; Skagerberg, G.; Ekman, R.; Heilig, M.; Sundler,

F.; H~kanson, R. Neuropeptide Y in the area of the hypothalamic paraventricular nucleus activates the pituitary-adrenocortical axis in the rat. Brain Res. 417:33-38; 1986.

34. White, J. D.; Kershaw, M. Increased hypothalamic preproneuropeptide Y mRNA content following food deprivation. Mol. Cell. Neu- rosci., in press; 1990.

35. White, J. D.; Olchovsky, D.; Kershaw, M.; Berelowitz, M. In- creased hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in streptozotocin-diabetic rats. Endocrinology 126: 765-772; 1990.

36. Williams, G.; Bloom, S. R. Regulatory peptides, the hypothalamus and diabetes. Diabetic Med. 6:472--485; 1989.

37. Williams, G.; Gill, J. S.; Lee, Y. C.; Cardoso, H. M.; Okpere, B. E.; Bloom, S. R. Increased NPY concentrations in specific hypothalamic regions of streptozocin-induced diabetic rats. Diabetes 38:

321-327; 1989. 38. Williams, G.; Lee, Y. C.; Ghatei, M. A.; Cardoso, H. M.; Ball, J.

A.; Bone, A. J.; Baird, J. D.; Bloom, S. R. Elevated neuropeptide Y concentration in the hypothalamus of the spontaneously diabetic BB/E Wistar rat. Diabetic Med. 6:601~:~07; 1989.

39. Williams, G.; Steel, J. H.; Cardoso, H.; Ghatei, M. A.; Lee, Y. C.; Gill, J. S.; Burrin, J. M.; Polak, J. M.; Bloom, S. R. Increased hypothalamic neuropeptide Y concentrations in diabetic rat. Diabe- tes 37:763-772; 1988.

40. Williams, G.; Steel, J. H.; Polak, J. H.; Bloom, S. R. Neuropep- tide Y in the hypothalamus. In: Mutt, V.; Fuxe, K.; H6kfelt, T.; Lundberg, J. M., eds. Neuropeptide Y. New York: Raven Press; 1989:243-251.

41. Woods, S. C.; Porte, D.; Bobbioni, E.; lonescu, E.; Sauter, J.; Jeanrenaud, F. Insulin: Its relationship to the central nervous system, and to the control of food intake and body weight. Am. J. Clin. Nutr. 42:1063-1067; 1985.

unchanged hypothalamic neuropeptide y concentrations in hyperphagic, hypoglycemic rats: evidence for...

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