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Food deprivation and hypothalamic neuropeptide gene expression: effects of strain background and the diabetes mutation
Molecular Brain Research, 11 (1991) 291-299 © Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$03.50 ADONIS 0169328X9270340Q
BRESM 70340
291
.
Food deprivation and hypotha!amic neuropeptide gene expression: effects of strain background and the diabetes mutation Streamson
C. Chua Jr., Andrew
W. Brown*, Jeehee Kim*, Karen L. Hennessey*, L. Leibel and Jules Hirsch
Rudolph
Laboratory of Human Behavior and Metabolism, Rockefeller University, New York, NY 10021 (U.S.A.) (Accepted 4 June 1991)
Key words: Hypothalamus; Food deprivation; Diabetes; Neuropeptide gene expression; Inbred mouse
We have used a novel method to identify genes expressed in the hypothalamus which may be ~te'+.~tially involved in controlling food intake and energy metabolism. We ~sumed that f~-,oddeprivation~ a powerful stimulus of food intake, would stimulate the activity of neural pathways involved in feeding behavior which should be reflected in en increase in the synthesis of any relevant neuropeptid¢ and its messenger RNA. A study of 5 neuropeptides in 5 strains of mice has identified neuropeptide Y (NPY) as a gene whose expression in the hypothalamus is controlled by nutritional status, suggesting that hypothalamic NPY neurons are a link in *.he neural network regulating feeding behavior and energy metabolism. In addition, we have studied the effect of the diabetes mutation on neuropeptide gene expression during fasting and refeeding. Our findings suggest that abnormal NPY and enkephalin gene expression in the hypothalamus may be two important determinants of the expression of the diabetes mutation.
INTRODUCTION Many approaches have been used to analyse the role of the hypothalamus in the integration of energy homeostasis through effects on ingestive behavior, energy utilization, and the endocrine system. (1) Lesions of the medial hypothalamus produce a characteristic syndrome of hyperphagia with hyperinsulinemia, increased metabolic efficiency, suppression of sexual behavior, and sham rage 29. These behavioral and metabolic derangements are due to the destruction of intrinsic neurons as well as fibers of passage. (2) The application of neurotransmitters and neuropeptides to specific areas of the brain have allowed investigations on the neural pathways regulating feeding 3t. However, the multiplicity of neurotransmitters which can stimulate or inhibit feeding makes it difficult to assess their relevance to any specific physiological state. (3) Direct intracellular recording from neurons in the conscious animal can give insight into the relationship between neuronal activity and behavior 32. The heterogeneity of neurons within the brain makes the biochemical characterization of neurons identified electrophysiologically extremely difficult. (4) Direct measurement of endogenous catecholamines in conscious animals
is a refinement on the direct neuronal recording strategy2°. This technique shows great promise and can be extended to any substance which can readily cross a dialysis membrane. We have utilized another approach to the identification of hypothalamic neuropeptides which are candidates for endogenous modulators of feeding behavior. Since the most powerful stimulus to feeding is food deprivation 9, we reasoned that food deprivation would increase the activity of neural pathways which are involved in feeding behavior while suppressing the activity of neurons which signal satiety. A prolonged increase of neural activity should be reflected in an increase in the rate of peptide release coupled to an increase in the synthesis of any relevant neuropeptide and its messenger RNA t°. As mRNA is synthesized in the nucleus and remains localized to the pefikaryon rather than being transported through the axon, measures of mRNA levels are specific to neurons in the area studied. In contrast, pepfides are transported and stored in synaptic vesicles. As a consequence, peptide levels could represent the contribution of many brain regions which project to hypothalamus. Therefore, we have studied the effects of fasting and refeeding on the levels of neuropeptide
Fellow of the Pew Center for Nutrition Excellence at the Rockefeller University. Correspondence: S.C. Chua Jr., Laboratory of Human Behavior and Metabolism, Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A. *
292 mRNAs in the hypothalamus to identify endogenous modulators of feeding behavior. Central injection of neuropeptide Y (NPY) has been found to have a strong stimulatory effect on food intake by decreasing the latency to feeding as well as increasing cumulative food intake 6"37. Further studies in rats have shown that food deprivation and refeeding produced reciprocal changes in NPY-immunoreactivity (NPY-ir) in the paraventricular nucleus 33. Moreover, diabetic rats have elevated levels of hypothalamic NPYir 43. It has been postulated that NPY neurosecretion is affected by food deprivation and diabetes 33"43 and may be responsible for the hyperphagia observed in these two models of abnormal metabolism. NPY can also potentially decrease energy expenditure by decreasing heart rate, respiratory rate, and inducing sleep is. Apart from such effects on energy balance, NPY has widespread endocrinologic effects which may secondarily affect food intake and energy utilization. For example, NPY has modulatory effects on the release of luteinizing hormone-releasing hormone (LHRH) tl, and corticotropin releasing factor (CRF) 17, and indirectly stimulates insulin release when injected centrally 2s. We have found that food deprivation increases messenger RNA (mRNA.) levels of NPY in the hypothalamus of CF1 and CD1 mice 4. NPY is a 36 amino acid peptide discovered due to the amidated nature of its amino- and carboxy-terminal tyrosines 3s. NPY is abundant in the central nervous system, with the highest concentrations found in the hypothalamus ~9. The peptide qualifies as a neurotransmitter since it is localized to synaptic vesicles 36 and affects electrical activity of certain neurons ~. NPY exerts some of its actions through a receptor which is coupled to a pertussis toxin-sensitive G protein 22.
creased metabolic efficiency. The db mouse displays a number of endocrinologic abnormalities, such as diabetes and infertility, which appear to be mediated at the level of the hypothalamus. In the C57BL/6J strain, db/db mice develop fl cell hyperplasia and hyperinsulinemia, with an associated insulin resistance 21. However, in the C57BL/Ks.I genetic background, db/db mice develop fl cell atrophy and hypoinsulinemia, with severe hyperglycemia. Despite extreme hyperphagia, the severely diabetic C57BL/Ks.I mutant mice experience weight loss due to glycosuria. Experimental ablation of the medio= basal hypothalamus prevents fl cell atrophy, suggesting that the hypothalamus plays a role in the atrophy of pancreatic fl cells 7. The db/db mouse exhibits infertility which is due in part to a decrease in sexual behavior. Some db/db males are fertile prior to the development of massive obesity. Female db/db rarely become pregnant. However, the ovaries of db/db females function normally when transplanted to histocompatible + / + hosts (unpublished observations). These observations indicate that the reproductive defect of db/db mice lies in the neuroendocrine system rather than the reproductive organs. Many of the phenotypic abnormalities of db/db mice can be ameliorated by dietary manipulations. Food restriction can improve the reproductive performance of db/db males. The feeding of a carbohydrate-free diet can prevent the /~ cell atrophy seen in db/db C57BL/KsJ mice 2s. We have detected subtle abnormalities in hypothalamic NPY and ENK gene expression which may contribute to the development and maintenance of obesity and diabetes in the db/db mouse.
In this paper, we report the effects of fasting and refeeding on hypothalamic expression of NPY and four other neuropeptide genes: dynorphin (DYN), enkephalin (ENK), somatostatin (SRIF), and thyrotropin releasing hormone (TRH). These responses were examined in 5 strains of mice. D Y N 16'3° and E N K 27'39 have been shown to increase food intake. T R H has inhibitory actions on feeding and drinking 41. SRIF was included since it does not have clearly demonstrable effects on food intake 3~. Any neuropeptide gene whose response to nutritional manipulation is conserved between different mouse strains would pies~mably be of particular physiological signifit:ance. In conjunction with these studies, we also examined the nutritional regulation of neuropeptide gene expression in the hypothalamus of the diabetes (db) mouse, a model of genetic obesity. The autosomal recessive db mutation s causes obesity due to hyperphagia and in-
Animals CD1 mice were purchased from Charles River. NZO mice were kindly provided by Dr. Jeffrey Friedman. FVB/N mice were obtained from Taconic. C57BL/6J and C57BL/KsJ mice were obtained from Jackson Laboratories. Mutant dbldb nfice of both C57BLi6J and C57BL/KsI strains were obtained from Jackson Labs. CD1 and NZO mice were 9 months of age. All other mice were 2-3 months old. All mice were housed in plastic cages with corn cob bedding. The animal room was maintained on a 12/12 h light-dark cycle with lights on at 06.30 EST. Pelleted Purina Chow 5001 was available freely except during food deprivation experiments. Water was always available.
MATERIALS AND METHODS
Food intake measurements For purposes of food intake measurements, mice were housed two to three per cage. The weight of the cage top with chow was determined daily. Weights of the mice and the chow were measured to the nearest 0.1 g on an Ohaus GT4000 balance. Food intake per mouse was the difference in the weights of the chow on two successive days~. We have previously found that daily handling of naive male and female mice caused a significant loss of body weight (unpublished observations). Therefore, food deprivation studies were not initiated until food intake and body weights were stable for at least 2 days. Food deprivation was initiated at 10.00 EST.
293 After 24 h, all mice were weighed and refeeding began.
Quantitative Northern blot analysis Groups of mice (4-6 mice per group) were fasted and refed as described above. Individual hypothalami were harvested at the appropriate times. The methods of brain dissection, total RNA purification, denaturing agarose gel electrophoresis, and capillary blotting have been previously described 4. Briefly, individual mouse hypothalami were homogenized in 7 M guanidine HCi, extracted once with a half volume of phenol--chloroform (1:1), and precipitated with 1/20 volume of 3 M sodium acetate (pH 4.5) and 112 volume of ethanol. The yield was typically 10 ug of total RNA per mouse hypothalamus. All of the RNA from one mouse hypothalamus was loaded on one lane of a formaldehyde agarose gel, electrophoresed until the Bromphenol blue had migrated 9 cm, and blotted to nylon supported nitrocellulose in 20× SSC. Northern blot analysis was used to determine, for each mouse hypothalamus, the levels for 5 neuropeptide mRNAs: DYN s, ENK 44, NPY 19, SRIF iS, and TRH 24. Simultaneous hybridizations for up to three different sized mRNAs were performed using cDNA inserts labelled with [a-32p]dCTP to a specific activity of >10 9 cpm//~g by random primer extension 12. Variation in loading was accounted for by normalizing to the actin mRNA content of each lane. The fluorographic signals were quantified by laser densitometry of appropriate exposures determined to be within the linear range c~f preflashed Kodak XAR film.
A
Statistical analyses All statistical tests were non-parametric in nature. The Wilcoxon rank sum test was used to compare the effect of fasting in food deprivation experiments and the influence of the db/db genotype in the same strain background. The Friedman test was used to as-
N
F
R1
R2
,, t
t
O
FoOd intake
N
R1
e
R2
i B
Body weight
ditll
Actin N
F
R1
R2
Fig. 1. Changes in food intake and body weight during fasting and refeeding in two strains of mice. A: food intake changes after food deprivation. B: body weight changes after food deprivation and refeeding. The food intakes and body weights are expressed as telative by setting median food intakes and body weights in the ad libitum fed state to 100%. The mice were assigned to one of 4 groups: (N) ad libitum fed; (F) 24 h food deprived; (R1) refed for 24 h after food deprivation; (R2) refed for 48 h after food deprivation. A symbol (+) denotes significant differences from the fed state (P < 0.025, Wilcoxon rank sum test).
Fig. 2. Northern blot of hypothalamic neuropeptide mRNAs during fasting and refeeding. CD1 female mice were assigned to one of four groups, as described in Fig. 1. Hypothalamic RNA was isolated and analyzed for neuropeptide mRNA content by Northern blot analysis. Three sequential hybridizations were performed and the same lanes from each hybridization are shown: first, TRH and NPY; second, DYN, ENK, and SRIF; and third, actin.
294 TABLE ! Hypothalamic neuropeptide mRNA content in five strains of mice during fasting and refeeding
Relative mRNA levels were determined for groups of mice (3-5 mice per group) in the ad libitum fed state (N), after a 24 h fast (F), and refed for up to 48 h after a 24 h fast (R1 = 24 h of refeeding, R2 = 48 h of refeeding). All values are medians with the median value of ad libitum fed mice set to 1.00 for each strain and for each neuropeptide mRNA. biD = not determined. Strain
Dynorphin
Strain
N
F
R1
R2
C57BI./6J C57BL/KsJ CD1 .~rB/N NZO
1.00 1.00 1.00 1.00 1.00
0.86 0.78* 0.91 1.40 0.95
1.10 0.81 0.99 1.97" 0.89
2.08" ND 1.16 ND 1.03
Strain
Enkephalin N
F
RI
17,2
C57BL/6J C57BL/KsJ CDI FVB/N NZO
1.00 1.00 1.00 1.00 1.00
0.64* 1.03 1.14" 2.59* 0.54*
0.42* 0.86 0.96 2.49 0.69
0.68* ND 0.92 ND 1.01
Strain
NPY t
C57BL/6J C57BUKsJ CD1 FVB/N NZO
N
F
RI
R2
1.00 1.00 1.00 1.00 1.00
3.42* 1.40* 2.11" 3.11" 1.96"
1.86" 0.97 1.26 1.98 1.23"
1.78 ND 1.13 ND 1.96"
SR I F N
F
R1
R2
C57BL/6J C57BL/KsJ CD1 FVB/N NZO
1.00 1.00 1.00 1.00 1.00
0.64 0.83 1.67" 2.84* 0.50
0.72 0.62 1.63" 3.18" 0.55
0.51 ND 1.30" ND 1.02
Strain
TRH N
F
RI
17,2
C57BI./6J C57BL/KsJ CD1 FVB/N NZO
1.00 1.00 1.00 1.00 1.00
1.55" 1.08 0.58* 3.74* 0.86
1.06 1.02 0.70* 4.34* 0.97
1.44 ND 0.85 ND 1.00
Strain
Animals per group
C57BL/6J C57BL/KsJ CD1 FVB/N NZO
N
F
RI
R2
4 4 4 5 4
5 5 5 5 4
4 5 4 4 3
5 ND 5 ND 4
*P < 0.05 (Wilcoxon test) for differences between nutritional states within one strain. *P < 0.025 (Friedman test) for differences between nutritional states across all strains of mice.
sess the effects of fasting and refeeding for one day on neuropeptide mRNA levels for the 5 strains of mice. Differences were considered to be significant when P values were less than 0.05.
5.8 g and FVB/N mice ate 5.5 g (P < 0.025, Wilcoxon rank sum test). The weight which had been lost during the fast was regained during the 24 h of refeeding.
RESULTS
The hypothalamic response to fasting and refeeding in f o u r strains o f mice Female mice of 4 strains (CD1, C57BL/6J, C57BL/ KsJ, and FVB/N) were subjected to a 24 h fast and refeeding for up to 48 h, while ad libitum fed mice served as controls. There were 3--6 mice in each group. The hypothalamic levels of the 5 neuropeptide m R N A s (Table I) were determined by Northern blot analysis. The indicated values in Table I represent the medians for each group of mice. All mice were studied individually, i.e. no samples were ever pooled. A representative blot for CD1 mice is shown in Fig. 2. Only NPY m R N A is significantly changed by fasting and refeeding for 24 h (P < 0.05, Friedman test), when 4 strains of mice are considered (Table I and Fig. 3). The inclusion of a fifth mouse strain, the N Z O strain (see below), in the analysis only changed the significance level ( P < 0.025, Friedman test). Fasting produced a significant increase
Food intake increases after a 24 h fast for female C57BLI6J and F V B / N mice The food intake of 12 female mice (10-12 weeks old) of two inbred strains, C57BL/6J and FVB/N, was measured in the ad libitum fed state and during refeeding after a 24 h of fast. In the free feeding state, C57BL/6J females (median body weight = 22.5 g with a range of 21.1-23.1 g) consumed 3.7 g of chow per mouse per day (range = 3.1-3.9 g) and FVB/N females (median body weight = 24.1 g with a range of 23.3-24.2 g) consumed 2.9 g of chow per mouse per day (range = 2.8-3.2 g). Fasting for 24 h produced a weight loss (Fig. 1B) of 6.6% of the original body weight for C57BL/6J mice and 9.9% for FVB/N mice ( P < 0.05, Wilcoxon rank sum test). In the first 24 h of refeeding, mice of both strains exhibited an increase in food intake (Fig. 1A): C57BL/B7 mice ate
295 TABLE !I Effect of the db gene on hypothalamic content of five neuropeptide mRNAs during fasting and refeeding Female mice (3-5 per group) were subjected to a 24 h fast (F) and refed for 24 h (R1) or 48 h (R2). Relative mRNA levels are expressed by setting the medians of ad libitum fed +/+ mice for each strain to 1.00. The ranges are in parentheses. ND - not determined. F
R2
Peptide mRNA
Strain
Genotype
N
RI
Dynorphin
C57BL/6J C57BL/6J C57BL/KsJ C57BL/KsJ
+/+ dbldb +1+ db/db
1.00 0.64* 1.00 0.50*
(0.71-2.28) (0.48-0.68) (0.95-1.43) (0.39-0.76)
0.86 0.86* 0.78* 0.80
(0.21-1.03) (0.70-1.14) (0.41-0.95) (0.51-0.93)
1.10 0.96 0.81 0.95
(0.92-1.70) (0.54-1.22) (0.49-1.46) (0.73-1.00)
2.08* (1.57-2.75) 0.79* (0.70-1.17) ND 1.05" (0.96--1.12)
Enkephalin
C57BL/6J C57BI.J6J C57BL/KsJ C57BL/KsJ
+/+ dbldb +/+ dbldb
1.00 0.18' 1.00 0.78*
(0.95-1.32) (0.15-0.29) (0.90-1.10) (0.70-0.78)
0.64* 0.26 1.03 0.28*
(0.51-0.66) (0.16-0.40) (0.77-1.32) (0.15-0.48)
0.42* 0.12 0.86 0.22*
(0.22-0.80) (0.08-0.18) (0.64-2.08) (0.18-0.40)
0.68* (0.51-0.74) 0.41" (0.38-0.53) ND 0.77 (0.70-0.91)
NPY
C57BL/6J C57BI.,t6J C57BL/KsJ C57BL/KsJ
+/+ db/db +/+ db/db
1.00 1.17 1.00 1.51'
(0.46-1.97) (1.04-1.22) (0.65-1.25) (1.24-1.58)
3.42* 1.59" 1.40" 1.42
(2.52-3.82) (1.25-1.92) (1.05-1.61) (1.12-1.61)
1.86" 1.07 0.97 1.19
(1.13-2.85) (1.00-1.30) (0.68-1.66) (1.03-1.78)
1.78 (1.37-2.65) 1.42 (0.97-2.21) ND 2.09 (1.43-2.39)
SRIF
C57BL/6J C57BI.J6J C57BL/KsJ C57BL/IC~J
+/+ dbldb +1+ db/db
1.00 0.94 1.00 2.02*
(0.42-1.24) (0.71-1.11) (0.64-1.14) (1.38-3.18)
0.64 1.19" 0.83 0.85*
(0.49-0.98) (1.09-1.50) (0.67-1.17) (0.62-1.58)
0.72 0.54* 0.62 1.08"
(0.41-0.93) (0.32-0.93) (0.52-0.92) (0.83-1.17)
0.51 (0.41-0.71) 0.59 (0.28-0.87) ND 1.53 (1.02-1.57)
TRH
C57BL/6J C57BI.,/6J C57BL/KsJ C57BIJKsJ
+/+ db/db +1+ db/db
1.00 0.40* 1.00 0.69*
(0.53--1.09) (0.32--0.49) (0.84-1.42) (0.69-0.74)
1.55" 0.84* 1.08 0.80
(1.51-2.09) (0.71-0.97) (0.98--1.40) (0.66-0.90)
1.06 0.77* 1.02 0.89
(0.39-1.25) (0.55-1.08) (0.80-1.72) (0.69-1.06)
1.44 (0.80-1.80) 0.81" (0.73-0.98) ND 1.18" (0.88-1.51)
*P < 0.05 (Wilcoxon test) for differences in the ad libitum fed state between genotypes. *P < 0.05 (Wilcoxon test) for differences due to nutritional state within one genotype.
of NPY m R N A in all strains. Refeeding caused a reduction of NPY m R N A to pre-fasting levels for 4 strains of mice. For the other neuropeptide m R N A s , there were inconsistent changes due to fasting and refeeding between strains. For example, fasting caused enkephalin m R N A to increase in two strains (FVB/N and CD1) but to decrease in another strain (C57BL/6J). These strain-specific alteratioas are consistent since all strains have been studied at least twice with the same qualitative results (data not shown). Hypothalamic N P Y m R N A is not reduced by refeeding in the N Z O mouse The N Z O mouse is regarded as a polygenic model of genetic obesity. Studies with these mice are difficult to interpret due to the lack of an appropriate group of mice to serve as controls. We have compared the hypothaiamic response of the N Z O mouse to four other strains of mice. One remarkable result is that the NPY m R N A levels of the N Z O mouse are not reduced by refeeding after food deprivation, even after 48 hours (Table I and Fig. 3). In the ad libitum fed state, hypothalamic NPY m R N A levels of N Z O mice are not significantly differ-
ent from those measured in CD1 mice, which were the same age (data not shown). Fasting produces a nearly two-fold increase in the female N Z O mouse as well as all other strains tested. However, refeeding of female N Z O mice for up to 48 h did not reduce the NPY m R N A to pre-fasting levels while refeeding for 48 h is sufficient to reduce NPY m R N A in the 4 other strains (see Table I and Fig. 3). Hypothalamic neuropeptJde m R N A content in db/db mice on two genetic backgrounds We performed fasting and refeeding studies on hypothalamic neuropeptide gene expression in db/db mice on two strain backgrounds: C57BL/6J and C57BL/KsJ. Hypothalamic neuropeptide m R N A contents (Table II and Fig. 4) are expressed as relative values by setting to 1.00 the median values of ad libitum fed wild type mice for each m R N A . We will initially describe differences between + / + and db/db mice in the fed state. Subsequently, we will describe the effects of food-deprivation and refeeding on db/db mice. In the fed state, only db/db mice of the C57BL/KsJ strain exhibit significantly higher levels of NPY m R N A in comparison to lean + / + mice of the same strain (Ta-
296 B
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C 57BL/6J
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Fig. 3. Relative hypothalamic NPY mRNA levels in 5 strains of mice during fasting and refeeding. Female mice of 5 strains were assigned to one of 4 groups, as described in Fig. 1. The bars represent the medians of each group and are expressed relative to the ad libitum fed state (N) for each strain. The ranges for each group are indicated. The number of mice in each group are indicated by numbers within the bars. A symbol (+) denotes significant differences from the control group (N) (P < 0.05, Wilcoxon rank sum test).
ble II and Fig. 4). Three neuropeptides, DYN, ENK, and TRH, show a significant reduction in mRNA levels in db/db mice of both strains when compared to lean +/+ mice (Table II) in the fed state. ENK mRNA showed a 5-fold reduction in C57BL/6J db/db mice whereas C57BL/KsJ db/db mice showed only a 20% reduction when compared to lean + / + C57BL/KsJ mice (Table II and Fig. 4). The decreases in DYN and TRH mRNA levels were quantitatively similar (approximately
50%) in mutant mice of both strains. There is a qualitative difference in the SRIF levels between mutant mice of different genetic backgrounds. In the fed state, C57BL/KsJ db/db mice exhibit 2-fold higher levels of SRIF mRNA when compared to lean +/+ mice of the same strain (Table II). This is in contrast to C57BL/6J db/db mice which have SRIF mRNA levels similar to lean C57BL/6J + / + mice. Fasting appears to ameliorate some of the abnormal levels of neuropeptide mRNAs associated with the db mutation. The "levels of DYN, SRIF, and TRH of the mutant mice after a fast of both strains are not significantly different from the levels seen in lean + / + mice in the fed and fasted states (Table II). The NPY mRNA levels in db/db and +t.!- mice are similar after food deprivation due to the fasting-induced increases in + / + mice. The major abnormality in the hypothalamus of fasting db/db mice is the reduced level of ENK mRNA which is reduced 3- to 4-fold relative to fasting + / + mice. The modest reduction of ENK mRNA in fed C57BL/KsJ db mice is further reduced by food deprivation. Refeeding of db/db mice for up to 48 h did not totally return neuropeptide mRNA levels to the pre-fasting state. For example, DYN and TRH mRNA levels are still increased to levels similar to + / + mice after refeeding for 2 days. SRIF mRNA was reduced in db/db C57BL/KsJ mice by fasting and for 1 day of refeeding. By the second day of refeeding, SRIF had increased to pre-fasting levels. DISCUSSION We have shown that two strains of inbred mice, C57BL/6J and FVB/N, exhibit hyperphagia after a short period of food deprivation. These two strains exhibit an increase in hypothalamic NPY and TRH mRNA levels in the fasting state. Since TRH has been shown to inhibit feeding and drinking 41, it is improbable that TRH is responsible for the post-fasting hyperphagia in these two strains of mice. Our findings suggest that hypothalamic NPY ~,e,.'rons may be endogenous regulators of energy homeostasis during conditions of caloric need. Of 5 neuropeptide mRNAs studied (DYN, ENK, NPY, SRIF, and TRH), only NPY showed significant and consistent changes in mRNA content with acute changes in nutritional state in 5 strains of mice. This is consistent with a study of diabetic rats which showed only a significant increase in NPY-ir in a survey of 10 neuropeptides 43. Moreover, studies with rats have also shown that food deprivation caused increases in hypothalamic NPY-ir 33 and NPY mRNA 3"35. The central nervous system response to NPY
297 NPY
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057BL/KsJ
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Fig. 4. Relative hypothalamic NPY and ENK mRNA levels in dbidb mice on two strain backgrounds. A,B: relative hypothalamic NPY mRNA levels. C,D: relative hypothalamic ENK mRNA levels. The mice were assigned to one of 4 groups, as described in Fig. 1. The
median values of lean +/+ mice for both strains have been set to 1.00. A symbol (+) represents significant effects (P < 0.05, Wilcoxon test) of the db/db genotype on mRNA levels relative to the +/+ genotype. Asterisks denote significant effects (P < 0.05, Wilcoxon test) of nutritional state on mRNA levels relative to the db/db fed (N) group.
may be conditioned by the availability of food: NPY may stimulate food intake or decrease metabolic rate. This type of response may of great survival value. During periods of low food availability, metabolic rate is decreased to conserve energy stores. When food becomes available, ingestive behavior is stimulated. Tids implies that there is an integration of external stimuli (food availability) and internal cues (caloric need). This view is consistent with hypothalamic NPY neurons as modulators of energy homeostasis rather than simple stimulators of the motor program controlling food ingestion. Our observation that all 5 of the neuropeptide mRNAs studied can be influenced by nutritional manipulation, depending on the strain used, suggests that many changes may occur in gene expression which are not directly involved in feeding behavior. For example, food deprivation caused marked increases in liver tyrosine aminotransferase activity in C57BL/6J mice but not in DBA/2J mice 2. These studies indicate that great caution must be exercised in interpreting experiments performed with only one strain, either inbred or outbred, of one species. Multiple strains of mice or rats, selected to be
as genetically divergent as possible, should be studied with the same experimental paradigm. In our study, the NZO females exhibited a persistent elevation in NPY mRNA during refeeding. However, this was observed only in I~,male NZO mice and not in male NZO mice (unpublished observations). Thus, the significance of elevated NPY mRNA levels during refeeding to the development of obesity remains unclear. The overexpression of hypothaiamic NPY mRNA in the db/db C57BL/Ks3 mouse may explain its characteristic hyperphagia and hyperinsulinemia, since NPY is known to stimulate feeding behavior 37 and insulin secretion 2s. Alternatively, it is possible that the high NPY mRNA levels in db/db C57BL/KsJ mice are due to glycosuria and weight loss. In fact, rats with streptozotocininduced diabetes have high levels of hypothalamic NPYir34 and N-PYmRNA42 Since NPY mRNA levels are not elevated in db/db C57BL/6J mice, which have a well compensated diabetes and are normoglycemic 21, it is likely that the high hypothalamic NPY mRNA levels are reflecting the state of negative energy balance of diabetic animals, as in streptozotocin-induced diabetes 34"43. Thus,
298 it is unlikely that increased NPY gene expression is responsible for the hyperphagia and obesity seen in db/db C57BL/6J mice. High levels of NPY m R N A may lead to high rates of secretion of NPY. This, in turn, may lead to increased stimulation of pancreatic fl cells. Moreover, hypothalamic NPY neurons from diabetic rats are more sensitive to ambient glucose levels than NPY neurons from normal rats 34. However, there have been no studies on the NPY receptor(s) or the target neurons for NPY which are responsible for NPY's effects of food intake and insulin secretion. These studies are required to understand the role of hypothalamic NPY neurons in metabolism. The high levels of NPY m R N A are surprising since db/db C57BL/KsJ mice have been found to lose up to 50% of the neurons in the arcuate nucleus 14, the area of the hypothalamus where NPY neurons are found. If there is a preferential preservation of NPY neurons, there must still be an increase in the amount of NPY mRNA synthesized. If there is a reduction in tl~e number of NPY neurons, there must be an even greater increase in the expression of the NPY gene in the surviving neurons. Destruction of NPY neurons in the rnediobasal hypothalamus 7 may remove the central stimulation to pancreatic fl cells, thus preventing the eventual fl cell atrophy seen in db/dbC57BL/KsJ mice. The underexpression of hypothalamic ENK m R N A in db/db mice is seen on both genetic back,r~unds. Moreover, fasting did not increase ENK m R N A ~e'Jels as was seen for DYN and TRH mRNA. However, this finding is not consistent with a previous study which found normal levels of Met- and Leu-enkephalin in the medioREFERENCES 1 Aibers, H.E., Ottenweller, J.E., Liou, S.Y., Lumpkin, M.D. and Anderson, E.R., Neuropeptide Y in the hypothalamus: effect on corticosterone and single-unit activity, Am. J. Physiol., 258 (1990) R376-R382. 2 Blake, R.L., Regulation of liver tyrosine aminotransferase activity in inbred strains and mutant mice. II. Studies on the starvation-induced enzyme adaptation, Can. I. Biochem., 48 (1970) 1043-1049. 3 Brady, L.S., Smith, M.A., Gold, P.W. and Herkenham, M., Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats, Neuroendocrinology, 52 (1990) 441-447. 4 Chua, S.C., Leibel, R.L. and Hirsch, J., Food deprivation and age modulate neuropeptide gene expression in the murine hypothalamus and adrenal gland, Moi. Brain Res., 9 (1991) 95101. 5 Civelli, O., Douglass, J., Goldstein, A. and Herbert, E., Sequence and expression of the rat prodynorphin gene, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 4291-4295. 6 Clark, J.T., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Neuropeptide Y and human pancreatic polypoptide stimulate feeding behavior in rats, Endocrinology, 115 (1984) 427-429. 7 Coleman, D.L. and Hummel, K.P., The effects of hypothalamic
basal hypothalamus of 3- and 6-week-old db/db mice 4°. The discrepancy may be explained by an age-dependent decrease of ENK m R N A in db/db mice since we used 10- to 12-week-old mice. This suggests that the underexpression of E N K is a secondary defect. ENK m R N A levels in neurons of the ventromedial nucleus (VMN) have been shown to be estrogen dependent. Diabetes mice have lower levels of circulating estrogens and lower brain uptake of estrogen ~3. The decrease of ENK m R N A in neurons of the ventromedial nucleus (VMN) may account for the functional sterility of db/db mice. The db/db mouse may have a decreased activity of the ENK neurons in the VMN which project to the midbrain central gray and have been suggested to be involved in sexual behavior 23. Fasting ameliorates some of the abnormalities in neuropeptide m R N A levels observed in db/db mice and refeeding for up to 48 h did not totally return neuropeptide m R N A levels to tbe abnormal pre-fasting state. This suggests that many of the abnormalities in neuropeptide expression can be ameliorated by fasting with effects lasting beyond the immediate period of food deprivation. The observation that NPY and E N K expression is not ameliorated by food deprivation suggests that abnormal NPY and E N K expression may be two important determinants of the expression of the diabetes mutation.
Acknowledgements. This work was supported by a grant from the NIH Obesity Core Center Grant 5P30AM26687. We wish to thank Drs. Douglas L. Coleman and Jeffrey Friedman for the use of the db/db mice and the NZO mice, respectively; and Dr. Neile Edens for careful reading of the m/muscript.
lesions in genetically diabetic mice, Diabetologia, 6 (1970) 263267. 8 Coleman, D.L., Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice, Diabetologia, 14 (1978) 141-148. 9 Collier, G.~ Body weight loss as a measure of motivation in hunger and thirst, Ann. N.Y. Acad. Sci., 157 (1969) 594-609. 10 Comb, M., Hyman, S.E. and Goodman, H.M., Mechanisms of trans-synaptic regulation of gene expression, Trends Neurosci., 10 (1987) 473-478. 11 Crowley, W.R. and Kalra, S.P., Neuropeptide Y stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamus in vitro: modulation by ovarian hormones, Neuroendocrinology, 46 (1987) 97-103. 12 Feinberg, A.P. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal Biochem., 132 (1983) 6-13. 13 Garris, D.R. and Coleman, D.L., Diabetes-associated changes in estradioi accumulation in the aging C57BL/KsJ mouse brain, Neurosci. Lett., 49 (1984) 285-290. 14 Garris, D.R., Morphometric analysis of obesity (ob/ob)-and diabetes (db/db)-associated hypothalamic neuronal degeneration in C57BL/KsJ mice, Brain Res., 501 (1989) 162-170. 15 Goodman, R.H, Jacobs, J.W., Dee, P.C. and Habener, J.F., Somatostatin-28 encoded in a cloned cDNA obtained from a rat
299 medullary thyroid carcinoma, 3. Biol. Chem., 257 (1982) 11561159. 16 Gosnell, B.A., Morley, J.E. and Levine, A.S., Opioid-induced feeding localization of sensitive brain sites, Brain Res., 369 (1986) 177-184. 17 Haas, D.A. and George, S.R., Neuropeptide Y-induced effects on hypothalamic corticotropin-releasing factor content and release are dependent on noradrenergic-adrenergic neurotransmission, Brain Res., 498 (1989) 333-338. 18 Haffstrand, A~ Intraventricular administration of neuropeptide Y (NPY) induces hypotension, bradycardia, and bradypnoea in the awake unrestrained male rat. Counteraction by NPY-induced feeding behavior, Acta Physiol. Scand., 128 (1986) 121123. 19 Higuchi, H., Yang, H.Y.T. and Sabol, S.L., Rat neuropeptide Y precursor gene expression, J. Biol. Chem., 263 (1988) 62886295. 20 Hoebel, B.G., Hernandez, L., Schwartz, D.H., Mark, G.P. and Hunter, G.A., Microdialysis studies on brain norepinephrine, serotonin, and dopamine release during ingestive behavior: theoretical and clinical implications, Ann. N.Y. Acad. Sci., 575 (1989) 171-193. 21 Hummel, K.P., Coleman, D.L. and Lane, P.W., The influence of genetic background on expression of mutations at the diabetes locus in the mouse. I. C57BIdKsJ and C57BId6J strains, Biochem. Genet., 7 (1972) 1-13. 22 llles, E and Regenold, J.T., Interaction between neuropeptide Y and noradrenaline on central catecholamine neurons, Nature, 344 (1990) 62-63. 23 Lauber, A.H., Romano, G.J. Mobbs, C.V., Howells, R.D. and Pfaff, D.W., Estradiol induction of proenkephalin messenger RNA in hypothalamus: dose-response and relation to reproductive behavior in the female rat, Mol. Brain Res., 8 (1990) 4754. 24 Lechan, R.M., Wu, P., Jackson, I.M.D., Wolf, H., Cooperman, S., Mandei, G. and Goodman, R.H., Thyrotropin-releasing hormone precursor: characterization in rat brain, Science, 231 (1986) 159-161. 25 Leiter, E.H., Coleman, D.L., Eisenstein, A.B. and Strack, I., Dietary control of pathogenesis in C57BIdKsJ db/db diabetes mice, Metabolism, 30 (1981) 554-562. 26 Les, E.P., Cage population density and efficiency of feed utilization in inbred mice, Lab. Anita. Care, 18 (1968) 305-313. 21 McLean, S. and Hoebel, B.G., Feeding induced by opiates injected into the paraventricular hypothalamus, Peptides, 4 (1983) 287-292. 28 Moltz, J.H. and McDonald, J.K., Neuropeptide Y: direct and indirect action on insulin secreuon in the rat, Pepti,',es, 6 (1985) 1155-1159. 29 Morgane, EJ., Historical and modem concepts of hypothalamic organization and function. In P.J. Morgane and J. Panksepp (Eds.), Handbook of the Hypothalamus, Vol. 1, Marcel Dekker, New York, 1979, pp. 1-64. 30 MtMey, J.E. and Levine, A.S., Involvement of dynorphin and
the kappa opioid receptor in feeding, Peptides, 4 (1983) 797800. 31 Morley, J.E., Bartness, T.J., Gosnell, B.A. and Levine, A.S., Peptidergic regulation of feeding. In J.R. Smythies and R.J. Bradley (Eds.), Int. Rev. Neurobiol., Vol. 27, Academic Press, Orlando, FL, 1985, pp. 207-298. 32 Rolls, E.T., Neurophysiology of feeding, Trends Neurosci., 1 (1978) 1-3. 33 Sahu, A., Kalra, P.S. and Kalra, S.E, Food deprivation and ingestion induce reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus, Peptides, 9 (1988) 83-86. 34 Sahu, A., Sninsky, C.A., Kaira, P.S. and Kalra, S.P., New ropeptide-Y concentration in microdissected hypothalamic regions and in vitro release from the medial ba~al hypothalamuspreoptic area of streptozotocin-diabetic rats with and without insulin substitution therapy, Endocrinology, 126 (1990) 192198. 35 Sanacora, G., Kershaw, M., Finkelstein, J.A. and White, J.D., Increased hypothalamic content of preproneuropeptide-Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation, Endocrinology, 127 (1990) 730737. 36 Sawchenko, EE. and Pfeiffer, S.W., Ultrastructural localization of neuropeptide Y and galanin immunoreactivity in the paraventricular nucleus of the hypothalamus in the rat, Brain Res., 474 (1988) 231-245. 37 Stanley, B.G. and Leibowitz, S.F., Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus, Life Sci., 35 (1984) 2635-2642. 38 Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y -a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature, 296 (1982) 659--660. 39 Tepperman, F.S. and Hirst, M., Effect of intrahypothalamic injection of (D-ala2-v-leus)enkephalin on feeding and temperature in the rat, Eur. J. Pharmacol., 96 (1983) 243-249. 40 Timmers, K.I., Palkovits, M. and Coleman, D.L., Unique alterations of neuropeptide content in median eminence, amygdala, and dorsal vagal complex of 3- and 6-week-old diabetes mutant mice, Metabolism, 39 (1990) 1158-1166. 41 Vijayan, E,. and McCann, S.M., Suppression of feeding and drinking activity in rats following intraventricular injection of thyrotropin releasing hormone (TRH), Endocrinology, 100 (1777) 1727. 42 White, J.D., Olchovsky, D., Kershaw, M. and Berelowitz, M., Increased hypothalamie content of preproneuropeptide-Y messenger ribonucleic acid in streptozotocin diabetic rats, Endocrinology, 126 (1990) 765-772. 43 Williams, G., Steel, J.H., Cardoso, H., Ghatei, M.A., Lee, Y.C., Gill, J.S., Burrin, J.M., Polak, J.M. and Bloom, S.R., Increased hypothalamic neuropeptide Y concentrations in diabetic rats, Diabetes, 37 (1988) 763-772. 44 Yoshikawa, K., Williams, C. and Sabol, S.L., Rat brain preproenkephalin mRNA, ~r. Biol. Chem., 259 (1984) 14301-14308.
BRESM 70340
291
.
Food deprivation and hypotha!amic neuropeptide gene expression: effects of strain background and the diabetes mutation Streamson
C. Chua Jr., Andrew
W. Brown*, Jeehee Kim*, Karen L. Hennessey*, L. Leibel and Jules Hirsch
Rudolph
Laboratory of Human Behavior and Metabolism, Rockefeller University, New York, NY 10021 (U.S.A.) (Accepted 4 June 1991)
Key words: Hypothalamus; Food deprivation; Diabetes; Neuropeptide gene expression; Inbred mouse
We have used a novel method to identify genes expressed in the hypothalamus which may be ~te'+.~tially involved in controlling food intake and energy metabolism. We ~sumed that f~-,oddeprivation~ a powerful stimulus of food intake, would stimulate the activity of neural pathways involved in feeding behavior which should be reflected in en increase in the synthesis of any relevant neuropeptid¢ and its messenger RNA. A study of 5 neuropeptides in 5 strains of mice has identified neuropeptide Y (NPY) as a gene whose expression in the hypothalamus is controlled by nutritional status, suggesting that hypothalamic NPY neurons are a link in *.he neural network regulating feeding behavior and energy metabolism. In addition, we have studied the effect of the diabetes mutation on neuropeptide gene expression during fasting and refeeding. Our findings suggest that abnormal NPY and enkephalin gene expression in the hypothalamus may be two important determinants of the expression of the diabetes mutation.
INTRODUCTION Many approaches have been used to analyse the role of the hypothalamus in the integration of energy homeostasis through effects on ingestive behavior, energy utilization, and the endocrine system. (1) Lesions of the medial hypothalamus produce a characteristic syndrome of hyperphagia with hyperinsulinemia, increased metabolic efficiency, suppression of sexual behavior, and sham rage 29. These behavioral and metabolic derangements are due to the destruction of intrinsic neurons as well as fibers of passage. (2) The application of neurotransmitters and neuropeptides to specific areas of the brain have allowed investigations on the neural pathways regulating feeding 3t. However, the multiplicity of neurotransmitters which can stimulate or inhibit feeding makes it difficult to assess their relevance to any specific physiological state. (3) Direct intracellular recording from neurons in the conscious animal can give insight into the relationship between neuronal activity and behavior 32. The heterogeneity of neurons within the brain makes the biochemical characterization of neurons identified electrophysiologically extremely difficult. (4) Direct measurement of endogenous catecholamines in conscious animals
is a refinement on the direct neuronal recording strategy2°. This technique shows great promise and can be extended to any substance which can readily cross a dialysis membrane. We have utilized another approach to the identification of hypothalamic neuropeptides which are candidates for endogenous modulators of feeding behavior. Since the most powerful stimulus to feeding is food deprivation 9, we reasoned that food deprivation would increase the activity of neural pathways which are involved in feeding behavior while suppressing the activity of neurons which signal satiety. A prolonged increase of neural activity should be reflected in an increase in the rate of peptide release coupled to an increase in the synthesis of any relevant neuropeptide and its messenger RNA t°. As mRNA is synthesized in the nucleus and remains localized to the pefikaryon rather than being transported through the axon, measures of mRNA levels are specific to neurons in the area studied. In contrast, pepfides are transported and stored in synaptic vesicles. As a consequence, peptide levels could represent the contribution of many brain regions which project to hypothalamus. Therefore, we have studied the effects of fasting and refeeding on the levels of neuropeptide
Fellow of the Pew Center for Nutrition Excellence at the Rockefeller University. Correspondence: S.C. Chua Jr., Laboratory of Human Behavior and Metabolism, Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A. *
292 mRNAs in the hypothalamus to identify endogenous modulators of feeding behavior. Central injection of neuropeptide Y (NPY) has been found to have a strong stimulatory effect on food intake by decreasing the latency to feeding as well as increasing cumulative food intake 6"37. Further studies in rats have shown that food deprivation and refeeding produced reciprocal changes in NPY-immunoreactivity (NPY-ir) in the paraventricular nucleus 33. Moreover, diabetic rats have elevated levels of hypothalamic NPYir 43. It has been postulated that NPY neurosecretion is affected by food deprivation and diabetes 33"43 and may be responsible for the hyperphagia observed in these two models of abnormal metabolism. NPY can also potentially decrease energy expenditure by decreasing heart rate, respiratory rate, and inducing sleep is. Apart from such effects on energy balance, NPY has widespread endocrinologic effects which may secondarily affect food intake and energy utilization. For example, NPY has modulatory effects on the release of luteinizing hormone-releasing hormone (LHRH) tl, and corticotropin releasing factor (CRF) 17, and indirectly stimulates insulin release when injected centrally 2s. We have found that food deprivation increases messenger RNA (mRNA.) levels of NPY in the hypothalamus of CF1 and CD1 mice 4. NPY is a 36 amino acid peptide discovered due to the amidated nature of its amino- and carboxy-terminal tyrosines 3s. NPY is abundant in the central nervous system, with the highest concentrations found in the hypothalamus ~9. The peptide qualifies as a neurotransmitter since it is localized to synaptic vesicles 36 and affects electrical activity of certain neurons ~. NPY exerts some of its actions through a receptor which is coupled to a pertussis toxin-sensitive G protein 22.
creased metabolic efficiency. The db mouse displays a number of endocrinologic abnormalities, such as diabetes and infertility, which appear to be mediated at the level of the hypothalamus. In the C57BL/6J strain, db/db mice develop fl cell hyperplasia and hyperinsulinemia, with an associated insulin resistance 21. However, in the C57BL/Ks.I genetic background, db/db mice develop fl cell atrophy and hypoinsulinemia, with severe hyperglycemia. Despite extreme hyperphagia, the severely diabetic C57BL/Ks.I mutant mice experience weight loss due to glycosuria. Experimental ablation of the medio= basal hypothalamus prevents fl cell atrophy, suggesting that the hypothalamus plays a role in the atrophy of pancreatic fl cells 7. The db/db mouse exhibits infertility which is due in part to a decrease in sexual behavior. Some db/db males are fertile prior to the development of massive obesity. Female db/db rarely become pregnant. However, the ovaries of db/db females function normally when transplanted to histocompatible + / + hosts (unpublished observations). These observations indicate that the reproductive defect of db/db mice lies in the neuroendocrine system rather than the reproductive organs. Many of the phenotypic abnormalities of db/db mice can be ameliorated by dietary manipulations. Food restriction can improve the reproductive performance of db/db males. The feeding of a carbohydrate-free diet can prevent the /~ cell atrophy seen in db/db C57BL/KsJ mice 2s. We have detected subtle abnormalities in hypothalamic NPY and ENK gene expression which may contribute to the development and maintenance of obesity and diabetes in the db/db mouse.
In this paper, we report the effects of fasting and refeeding on hypothalamic expression of NPY and four other neuropeptide genes: dynorphin (DYN), enkephalin (ENK), somatostatin (SRIF), and thyrotropin releasing hormone (TRH). These responses were examined in 5 strains of mice. D Y N 16'3° and E N K 27'39 have been shown to increase food intake. T R H has inhibitory actions on feeding and drinking 41. SRIF was included since it does not have clearly demonstrable effects on food intake 3~. Any neuropeptide gene whose response to nutritional manipulation is conserved between different mouse strains would pies~mably be of particular physiological signifit:ance. In conjunction with these studies, we also examined the nutritional regulation of neuropeptide gene expression in the hypothalamus of the diabetes (db) mouse, a model of genetic obesity. The autosomal recessive db mutation s causes obesity due to hyperphagia and in-
Animals CD1 mice were purchased from Charles River. NZO mice were kindly provided by Dr. Jeffrey Friedman. FVB/N mice were obtained from Taconic. C57BL/6J and C57BL/KsJ mice were obtained from Jackson Laboratories. Mutant dbldb nfice of both C57BLi6J and C57BL/KsI strains were obtained from Jackson Labs. CD1 and NZO mice were 9 months of age. All other mice were 2-3 months old. All mice were housed in plastic cages with corn cob bedding. The animal room was maintained on a 12/12 h light-dark cycle with lights on at 06.30 EST. Pelleted Purina Chow 5001 was available freely except during food deprivation experiments. Water was always available.
MATERIALS AND METHODS
Food intake measurements For purposes of food intake measurements, mice were housed two to three per cage. The weight of the cage top with chow was determined daily. Weights of the mice and the chow were measured to the nearest 0.1 g on an Ohaus GT4000 balance. Food intake per mouse was the difference in the weights of the chow on two successive days~. We have previously found that daily handling of naive male and female mice caused a significant loss of body weight (unpublished observations). Therefore, food deprivation studies were not initiated until food intake and body weights were stable for at least 2 days. Food deprivation was initiated at 10.00 EST.
293 After 24 h, all mice were weighed and refeeding began.
Quantitative Northern blot analysis Groups of mice (4-6 mice per group) were fasted and refed as described above. Individual hypothalami were harvested at the appropriate times. The methods of brain dissection, total RNA purification, denaturing agarose gel electrophoresis, and capillary blotting have been previously described 4. Briefly, individual mouse hypothalami were homogenized in 7 M guanidine HCi, extracted once with a half volume of phenol--chloroform (1:1), and precipitated with 1/20 volume of 3 M sodium acetate (pH 4.5) and 112 volume of ethanol. The yield was typically 10 ug of total RNA per mouse hypothalamus. All of the RNA from one mouse hypothalamus was loaded on one lane of a formaldehyde agarose gel, electrophoresed until the Bromphenol blue had migrated 9 cm, and blotted to nylon supported nitrocellulose in 20× SSC. Northern blot analysis was used to determine, for each mouse hypothalamus, the levels for 5 neuropeptide mRNAs: DYN s, ENK 44, NPY 19, SRIF iS, and TRH 24. Simultaneous hybridizations for up to three different sized mRNAs were performed using cDNA inserts labelled with [a-32p]dCTP to a specific activity of >10 9 cpm//~g by random primer extension 12. Variation in loading was accounted for by normalizing to the actin mRNA content of each lane. The fluorographic signals were quantified by laser densitometry of appropriate exposures determined to be within the linear range c~f preflashed Kodak XAR film.
A
Statistical analyses All statistical tests were non-parametric in nature. The Wilcoxon rank sum test was used to compare the effect of fasting in food deprivation experiments and the influence of the db/db genotype in the same strain background. The Friedman test was used to as-
N
F
R1
R2
,, t
t
O
FoOd intake
N
R1
e
R2
i B
Body weight
ditll
Actin N
F
R1
R2
Fig. 1. Changes in food intake and body weight during fasting and refeeding in two strains of mice. A: food intake changes after food deprivation. B: body weight changes after food deprivation and refeeding. The food intakes and body weights are expressed as telative by setting median food intakes and body weights in the ad libitum fed state to 100%. The mice were assigned to one of 4 groups: (N) ad libitum fed; (F) 24 h food deprived; (R1) refed for 24 h after food deprivation; (R2) refed for 48 h after food deprivation. A symbol (+) denotes significant differences from the fed state (P < 0.025, Wilcoxon rank sum test).
Fig. 2. Northern blot of hypothalamic neuropeptide mRNAs during fasting and refeeding. CD1 female mice were assigned to one of four groups, as described in Fig. 1. Hypothalamic RNA was isolated and analyzed for neuropeptide mRNA content by Northern blot analysis. Three sequential hybridizations were performed and the same lanes from each hybridization are shown: first, TRH and NPY; second, DYN, ENK, and SRIF; and third, actin.
294 TABLE ! Hypothalamic neuropeptide mRNA content in five strains of mice during fasting and refeeding
Relative mRNA levels were determined for groups of mice (3-5 mice per group) in the ad libitum fed state (N), after a 24 h fast (F), and refed for up to 48 h after a 24 h fast (R1 = 24 h of refeeding, R2 = 48 h of refeeding). All values are medians with the median value of ad libitum fed mice set to 1.00 for each strain and for each neuropeptide mRNA. biD = not determined. Strain
Dynorphin
Strain
N
F
R1
R2
C57BI./6J C57BL/KsJ CD1 .~rB/N NZO
1.00 1.00 1.00 1.00 1.00
0.86 0.78* 0.91 1.40 0.95
1.10 0.81 0.99 1.97" 0.89
2.08" ND 1.16 ND 1.03
Strain
Enkephalin N
F
RI
17,2
C57BL/6J C57BL/KsJ CDI FVB/N NZO
1.00 1.00 1.00 1.00 1.00
0.64* 1.03 1.14" 2.59* 0.54*
0.42* 0.86 0.96 2.49 0.69
0.68* ND 0.92 ND 1.01
Strain
NPY t
C57BL/6J C57BUKsJ CD1 FVB/N NZO
N
F
RI
R2
1.00 1.00 1.00 1.00 1.00
3.42* 1.40* 2.11" 3.11" 1.96"
1.86" 0.97 1.26 1.98 1.23"
1.78 ND 1.13 ND 1.96"
SR I F N
F
R1
R2
C57BL/6J C57BL/KsJ CD1 FVB/N NZO
1.00 1.00 1.00 1.00 1.00
0.64 0.83 1.67" 2.84* 0.50
0.72 0.62 1.63" 3.18" 0.55
0.51 ND 1.30" ND 1.02
Strain
TRH N
F
RI
17,2
C57BI./6J C57BL/KsJ CD1 FVB/N NZO
1.00 1.00 1.00 1.00 1.00
1.55" 1.08 0.58* 3.74* 0.86
1.06 1.02 0.70* 4.34* 0.97
1.44 ND 0.85 ND 1.00
Strain
Animals per group
C57BL/6J C57BL/KsJ CD1 FVB/N NZO
N
F
RI
R2
4 4 4 5 4
5 5 5 5 4
4 5 4 4 3
5 ND 5 ND 4
*P < 0.05 (Wilcoxon test) for differences between nutritional states within one strain. *P < 0.025 (Friedman test) for differences between nutritional states across all strains of mice.
sess the effects of fasting and refeeding for one day on neuropeptide mRNA levels for the 5 strains of mice. Differences were considered to be significant when P values were less than 0.05.
5.8 g and FVB/N mice ate 5.5 g (P < 0.025, Wilcoxon rank sum test). The weight which had been lost during the fast was regained during the 24 h of refeeding.
RESULTS
The hypothalamic response to fasting and refeeding in f o u r strains o f mice Female mice of 4 strains (CD1, C57BL/6J, C57BL/ KsJ, and FVB/N) were subjected to a 24 h fast and refeeding for up to 48 h, while ad libitum fed mice served as controls. There were 3--6 mice in each group. The hypothalamic levels of the 5 neuropeptide m R N A s (Table I) were determined by Northern blot analysis. The indicated values in Table I represent the medians for each group of mice. All mice were studied individually, i.e. no samples were ever pooled. A representative blot for CD1 mice is shown in Fig. 2. Only NPY m R N A is significantly changed by fasting and refeeding for 24 h (P < 0.05, Friedman test), when 4 strains of mice are considered (Table I and Fig. 3). The inclusion of a fifth mouse strain, the N Z O strain (see below), in the analysis only changed the significance level ( P < 0.025, Friedman test). Fasting produced a significant increase
Food intake increases after a 24 h fast for female C57BLI6J and F V B / N mice The food intake of 12 female mice (10-12 weeks old) of two inbred strains, C57BL/6J and FVB/N, was measured in the ad libitum fed state and during refeeding after a 24 h of fast. In the free feeding state, C57BL/6J females (median body weight = 22.5 g with a range of 21.1-23.1 g) consumed 3.7 g of chow per mouse per day (range = 3.1-3.9 g) and FVB/N females (median body weight = 24.1 g with a range of 23.3-24.2 g) consumed 2.9 g of chow per mouse per day (range = 2.8-3.2 g). Fasting for 24 h produced a weight loss (Fig. 1B) of 6.6% of the original body weight for C57BL/6J mice and 9.9% for FVB/N mice ( P < 0.05, Wilcoxon rank sum test). In the first 24 h of refeeding, mice of both strains exhibited an increase in food intake (Fig. 1A): C57BL/B7 mice ate
295 TABLE !I Effect of the db gene on hypothalamic content of five neuropeptide mRNAs during fasting and refeeding Female mice (3-5 per group) were subjected to a 24 h fast (F) and refed for 24 h (R1) or 48 h (R2). Relative mRNA levels are expressed by setting the medians of ad libitum fed +/+ mice for each strain to 1.00. The ranges are in parentheses. ND - not determined. F
R2
Peptide mRNA
Strain
Genotype
N
RI
Dynorphin
C57BL/6J C57BL/6J C57BL/KsJ C57BL/KsJ
+/+ dbldb +1+ db/db
1.00 0.64* 1.00 0.50*
(0.71-2.28) (0.48-0.68) (0.95-1.43) (0.39-0.76)
0.86 0.86* 0.78* 0.80
(0.21-1.03) (0.70-1.14) (0.41-0.95) (0.51-0.93)
1.10 0.96 0.81 0.95
(0.92-1.70) (0.54-1.22) (0.49-1.46) (0.73-1.00)
2.08* (1.57-2.75) 0.79* (0.70-1.17) ND 1.05" (0.96--1.12)
Enkephalin
C57BL/6J C57BI.J6J C57BL/KsJ C57BL/KsJ
+/+ dbldb +/+ dbldb
1.00 0.18' 1.00 0.78*
(0.95-1.32) (0.15-0.29) (0.90-1.10) (0.70-0.78)
0.64* 0.26 1.03 0.28*
(0.51-0.66) (0.16-0.40) (0.77-1.32) (0.15-0.48)
0.42* 0.12 0.86 0.22*
(0.22-0.80) (0.08-0.18) (0.64-2.08) (0.18-0.40)
0.68* (0.51-0.74) 0.41" (0.38-0.53) ND 0.77 (0.70-0.91)
NPY
C57BL/6J C57BI.,t6J C57BL/KsJ C57BL/KsJ
+/+ db/db +/+ db/db
1.00 1.17 1.00 1.51'
(0.46-1.97) (1.04-1.22) (0.65-1.25) (1.24-1.58)
3.42* 1.59" 1.40" 1.42
(2.52-3.82) (1.25-1.92) (1.05-1.61) (1.12-1.61)
1.86" 1.07 0.97 1.19
(1.13-2.85) (1.00-1.30) (0.68-1.66) (1.03-1.78)
1.78 (1.37-2.65) 1.42 (0.97-2.21) ND 2.09 (1.43-2.39)
SRIF
C57BL/6J C57BI.J6J C57BL/KsJ C57BL/IC~J
+/+ dbldb +1+ db/db
1.00 0.94 1.00 2.02*
(0.42-1.24) (0.71-1.11) (0.64-1.14) (1.38-3.18)
0.64 1.19" 0.83 0.85*
(0.49-0.98) (1.09-1.50) (0.67-1.17) (0.62-1.58)
0.72 0.54* 0.62 1.08"
(0.41-0.93) (0.32-0.93) (0.52-0.92) (0.83-1.17)
0.51 (0.41-0.71) 0.59 (0.28-0.87) ND 1.53 (1.02-1.57)
TRH
C57BL/6J C57BI.,/6J C57BL/KsJ C57BIJKsJ
+/+ db/db +1+ db/db
1.00 0.40* 1.00 0.69*
(0.53--1.09) (0.32--0.49) (0.84-1.42) (0.69-0.74)
1.55" 0.84* 1.08 0.80
(1.51-2.09) (0.71-0.97) (0.98--1.40) (0.66-0.90)
1.06 0.77* 1.02 0.89
(0.39-1.25) (0.55-1.08) (0.80-1.72) (0.69-1.06)
1.44 (0.80-1.80) 0.81" (0.73-0.98) ND 1.18" (0.88-1.51)
*P < 0.05 (Wilcoxon test) for differences in the ad libitum fed state between genotypes. *P < 0.05 (Wilcoxon test) for differences due to nutritional state within one genotype.
of NPY m R N A in all strains. Refeeding caused a reduction of NPY m R N A to pre-fasting levels for 4 strains of mice. For the other neuropeptide m R N A s , there were inconsistent changes due to fasting and refeeding between strains. For example, fasting caused enkephalin m R N A to increase in two strains (FVB/N and CD1) but to decrease in another strain (C57BL/6J). These strain-specific alteratioas are consistent since all strains have been studied at least twice with the same qualitative results (data not shown). Hypothalamic N P Y m R N A is not reduced by refeeding in the N Z O mouse The N Z O mouse is regarded as a polygenic model of genetic obesity. Studies with these mice are difficult to interpret due to the lack of an appropriate group of mice to serve as controls. We have compared the hypothaiamic response of the N Z O mouse to four other strains of mice. One remarkable result is that the NPY m R N A levels of the N Z O mouse are not reduced by refeeding after food deprivation, even after 48 hours (Table I and Fig. 3). In the ad libitum fed state, hypothalamic NPY m R N A levels of N Z O mice are not significantly differ-
ent from those measured in CD1 mice, which were the same age (data not shown). Fasting produces a nearly two-fold increase in the female N Z O mouse as well as all other strains tested. However, refeeding of female N Z O mice for up to 48 h did not reduce the NPY m R N A to pre-fasting levels while refeeding for 48 h is sufficient to reduce NPY m R N A in the 4 other strains (see Table I and Fig. 3). Hypothalamic neuropeptJde m R N A content in db/db mice on two genetic backgrounds We performed fasting and refeeding studies on hypothalamic neuropeptide gene expression in db/db mice on two strain backgrounds: C57BL/6J and C57BL/KsJ. Hypothalamic neuropeptide m R N A contents (Table II and Fig. 4) are expressed as relative values by setting to 1.00 the median values of ad libitum fed wild type mice for each m R N A . We will initially describe differences between + / + and db/db mice in the fed state. Subsequently, we will describe the effects of food-deprivation and refeeding on db/db mice. In the fed state, only db/db mice of the C57BL/KsJ strain exhibit significantly higher levels of NPY m R N A in comparison to lean + / + mice of the same strain (Ta-
296 B
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F
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C C57B1. / KsJ
FVB / N
4 < Z
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< Z n,
3
E 2
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1
N
4
F
R1
R2
N
F
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NzI
N
F
R1
R2
I
Fig. 3. Relative hypothalamic NPY mRNA levels in 5 strains of mice during fasting and refeeding. Female mice of 5 strains were assigned to one of 4 groups, as described in Fig. 1. The bars represent the medians of each group and are expressed relative to the ad libitum fed state (N) for each strain. The ranges for each group are indicated. The number of mice in each group are indicated by numbers within the bars. A symbol (+) denotes significant differences from the control group (N) (P < 0.05, Wilcoxon rank sum test).
ble II and Fig. 4). Three neuropeptides, DYN, ENK, and TRH, show a significant reduction in mRNA levels in db/db mice of both strains when compared to lean +/+ mice (Table II) in the fed state. ENK mRNA showed a 5-fold reduction in C57BL/6J db/db mice whereas C57BL/KsJ db/db mice showed only a 20% reduction when compared to lean + / + C57BL/KsJ mice (Table II and Fig. 4). The decreases in DYN and TRH mRNA levels were quantitatively similar (approximately
50%) in mutant mice of both strains. There is a qualitative difference in the SRIF levels between mutant mice of different genetic backgrounds. In the fed state, C57BL/KsJ db/db mice exhibit 2-fold higher levels of SRIF mRNA when compared to lean +/+ mice of the same strain (Table II). This is in contrast to C57BL/6J db/db mice which have SRIF mRNA levels similar to lean C57BL/6J + / + mice. Fasting appears to ameliorate some of the abnormal levels of neuropeptide mRNAs associated with the db mutation. The "levels of DYN, SRIF, and TRH of the mutant mice after a fast of both strains are not significantly different from the levels seen in lean + / + mice in the fed and fasted states (Table II). The NPY mRNA levels in db/db and +t.!- mice are similar after food deprivation due to the fasting-induced increases in + / + mice. The major abnormality in the hypothalamus of fasting db/db mice is the reduced level of ENK mRNA which is reduced 3- to 4-fold relative to fasting + / + mice. The modest reduction of ENK mRNA in fed C57BL/KsJ db mice is further reduced by food deprivation. Refeeding of db/db mice for up to 48 h did not totally return neuropeptide mRNA levels to the pre-fasting state. For example, DYN and TRH mRNA levels are still increased to levels similar to + / + mice after refeeding for 2 days. SRIF mRNA was reduced in db/db C57BL/KsJ mice by fasting and for 1 day of refeeding. By the second day of refeeding, SRIF had increased to pre-fasting levels. DISCUSSION We have shown that two strains of inbred mice, C57BL/6J and FVB/N, exhibit hyperphagia after a short period of food deprivation. These two strains exhibit an increase in hypothalamic NPY and TRH mRNA levels in the fasting state. Since TRH has been shown to inhibit feeding and drinking 41, it is improbable that TRH is responsible for the post-fasting hyperphagia in these two strains of mice. Our findings suggest that hypothalamic NPY ~,e,.'rons may be endogenous regulators of energy homeostasis during conditions of caloric need. Of 5 neuropeptide mRNAs studied (DYN, ENK, NPY, SRIF, and TRH), only NPY showed significant and consistent changes in mRNA content with acute changes in nutritional state in 5 strains of mice. This is consistent with a study of diabetic rats which showed only a significant increase in NPY-ir in a survey of 10 neuropeptides 43. Moreover, studies with rats have also shown that food deprivation caused increases in hypothalamic NPY-ir 33 and NPY mRNA 3"35. The central nervous system response to NPY
297 NPY
NPY
C57BL/6J
057BL/KsJ
250
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2.00
1.50
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1.50
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!
4
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R'2
~'~
C
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o.oo
......... +/+ N
I
I
ENK
..
D
, FI2
~,~b
,
I
ENK
C57BL/6J
C57BL/KsJ
1.40 1.20 1.00
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ee
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Fig. 4. Relative hypothalamic NPY and ENK mRNA levels in dbidb mice on two strain backgrounds. A,B: relative hypothalamic NPY mRNA levels. C,D: relative hypothalamic ENK mRNA levels. The mice were assigned to one of 4 groups, as described in Fig. 1. The
median values of lean +/+ mice for both strains have been set to 1.00. A symbol (+) represents significant effects (P < 0.05, Wilcoxon test) of the db/db genotype on mRNA levels relative to the +/+ genotype. Asterisks denote significant effects (P < 0.05, Wilcoxon test) of nutritional state on mRNA levels relative to the db/db fed (N) group.
may be conditioned by the availability of food: NPY may stimulate food intake or decrease metabolic rate. This type of response may of great survival value. During periods of low food availability, metabolic rate is decreased to conserve energy stores. When food becomes available, ingestive behavior is stimulated. Tids implies that there is an integration of external stimuli (food availability) and internal cues (caloric need). This view is consistent with hypothalamic NPY neurons as modulators of energy homeostasis rather than simple stimulators of the motor program controlling food ingestion. Our observation that all 5 of the neuropeptide mRNAs studied can be influenced by nutritional manipulation, depending on the strain used, suggests that many changes may occur in gene expression which are not directly involved in feeding behavior. For example, food deprivation caused marked increases in liver tyrosine aminotransferase activity in C57BL/6J mice but not in DBA/2J mice 2. These studies indicate that great caution must be exercised in interpreting experiments performed with only one strain, either inbred or outbred, of one species. Multiple strains of mice or rats, selected to be
as genetically divergent as possible, should be studied with the same experimental paradigm. In our study, the NZO females exhibited a persistent elevation in NPY mRNA during refeeding. However, this was observed only in I~,male NZO mice and not in male NZO mice (unpublished observations). Thus, the significance of elevated NPY mRNA levels during refeeding to the development of obesity remains unclear. The overexpression of hypothaiamic NPY mRNA in the db/db C57BL/Ks3 mouse may explain its characteristic hyperphagia and hyperinsulinemia, since NPY is known to stimulate feeding behavior 37 and insulin secretion 2s. Alternatively, it is possible that the high NPY mRNA levels in db/db C57BL/KsJ mice are due to glycosuria and weight loss. In fact, rats with streptozotocininduced diabetes have high levels of hypothalamic NPYir34 and N-PYmRNA42 Since NPY mRNA levels are not elevated in db/db C57BL/6J mice, which have a well compensated diabetes and are normoglycemic 21, it is likely that the high hypothalamic NPY mRNA levels are reflecting the state of negative energy balance of diabetic animals, as in streptozotocin-induced diabetes 34"43. Thus,
298 it is unlikely that increased NPY gene expression is responsible for the hyperphagia and obesity seen in db/db C57BL/6J mice. High levels of NPY m R N A may lead to high rates of secretion of NPY. This, in turn, may lead to increased stimulation of pancreatic fl cells. Moreover, hypothalamic NPY neurons from diabetic rats are more sensitive to ambient glucose levels than NPY neurons from normal rats 34. However, there have been no studies on the NPY receptor(s) or the target neurons for NPY which are responsible for NPY's effects of food intake and insulin secretion. These studies are required to understand the role of hypothalamic NPY neurons in metabolism. The high levels of NPY m R N A are surprising since db/db C57BL/KsJ mice have been found to lose up to 50% of the neurons in the arcuate nucleus 14, the area of the hypothalamus where NPY neurons are found. If there is a preferential preservation of NPY neurons, there must still be an increase in the amount of NPY mRNA synthesized. If there is a reduction in tl~e number of NPY neurons, there must be an even greater increase in the expression of the NPY gene in the surviving neurons. Destruction of NPY neurons in the rnediobasal hypothalamus 7 may remove the central stimulation to pancreatic fl cells, thus preventing the eventual fl cell atrophy seen in db/dbC57BL/KsJ mice. The underexpression of hypothalamic ENK m R N A in db/db mice is seen on both genetic back,r~unds. Moreover, fasting did not increase ENK m R N A ~e'Jels as was seen for DYN and TRH mRNA. However, this finding is not consistent with a previous study which found normal levels of Met- and Leu-enkephalin in the medioREFERENCES 1 Aibers, H.E., Ottenweller, J.E., Liou, S.Y., Lumpkin, M.D. and Anderson, E.R., Neuropeptide Y in the hypothalamus: effect on corticosterone and single-unit activity, Am. J. Physiol., 258 (1990) R376-R382. 2 Blake, R.L., Regulation of liver tyrosine aminotransferase activity in inbred strains and mutant mice. II. Studies on the starvation-induced enzyme adaptation, Can. I. Biochem., 48 (1970) 1043-1049. 3 Brady, L.S., Smith, M.A., Gold, P.W. and Herkenham, M., Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats, Neuroendocrinology, 52 (1990) 441-447. 4 Chua, S.C., Leibel, R.L. and Hirsch, J., Food deprivation and age modulate neuropeptide gene expression in the murine hypothalamus and adrenal gland, Moi. Brain Res., 9 (1991) 95101. 5 Civelli, O., Douglass, J., Goldstein, A. and Herbert, E., Sequence and expression of the rat prodynorphin gene, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 4291-4295. 6 Clark, J.T., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Neuropeptide Y and human pancreatic polypoptide stimulate feeding behavior in rats, Endocrinology, 115 (1984) 427-429. 7 Coleman, D.L. and Hummel, K.P., The effects of hypothalamic
basal hypothalamus of 3- and 6-week-old db/db mice 4°. The discrepancy may be explained by an age-dependent decrease of ENK m R N A in db/db mice since we used 10- to 12-week-old mice. This suggests that the underexpression of E N K is a secondary defect. ENK m R N A levels in neurons of the ventromedial nucleus (VMN) have been shown to be estrogen dependent. Diabetes mice have lower levels of circulating estrogens and lower brain uptake of estrogen ~3. The decrease of ENK m R N A in neurons of the ventromedial nucleus (VMN) may account for the functional sterility of db/db mice. The db/db mouse may have a decreased activity of the ENK neurons in the VMN which project to the midbrain central gray and have been suggested to be involved in sexual behavior 23. Fasting ameliorates some of the abnormalities in neuropeptide m R N A levels observed in db/db mice and refeeding for up to 48 h did not totally return neuropeptide m R N A levels to tbe abnormal pre-fasting state. This suggests that many of the abnormalities in neuropeptide expression can be ameliorated by fasting with effects lasting beyond the immediate period of food deprivation. The observation that NPY and E N K expression is not ameliorated by food deprivation suggests that abnormal NPY and E N K expression may be two important determinants of the expression of the diabetes mutation.
Acknowledgements. This work was supported by a grant from the NIH Obesity Core Center Grant 5P30AM26687. We wish to thank Drs. Douglas L. Coleman and Jeffrey Friedman for the use of the db/db mice and the NZO mice, respectively; and Dr. Neile Edens for careful reading of the m/muscript.
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the kappa opioid receptor in feeding, Peptides, 4 (1983) 797800. 31 Morley, J.E., Bartness, T.J., Gosnell, B.A. and Levine, A.S., Peptidergic regulation of feeding. In J.R. Smythies and R.J. Bradley (Eds.), Int. Rev. Neurobiol., Vol. 27, Academic Press, Orlando, FL, 1985, pp. 207-298. 32 Rolls, E.T., Neurophysiology of feeding, Trends Neurosci., 1 (1978) 1-3. 33 Sahu, A., Kalra, P.S. and Kalra, S.E, Food deprivation and ingestion induce reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus, Peptides, 9 (1988) 83-86. 34 Sahu, A., Sninsky, C.A., Kaira, P.S. and Kalra, S.P., New ropeptide-Y concentration in microdissected hypothalamic regions and in vitro release from the medial ba~al hypothalamuspreoptic area of streptozotocin-diabetic rats with and without insulin substitution therapy, Endocrinology, 126 (1990) 192198. 35 Sanacora, G., Kershaw, M., Finkelstein, J.A. and White, J.D., Increased hypothalamic content of preproneuropeptide-Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation, Endocrinology, 127 (1990) 730737. 36 Sawchenko, EE. and Pfeiffer, S.W., Ultrastructural localization of neuropeptide Y and galanin immunoreactivity in the paraventricular nucleus of the hypothalamus in the rat, Brain Res., 474 (1988) 231-245. 37 Stanley, B.G. and Leibowitz, S.F., Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus, Life Sci., 35 (1984) 2635-2642. 38 Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y -a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature, 296 (1982) 659--660. 39 Tepperman, F.S. and Hirst, M., Effect of intrahypothalamic injection of (D-ala2-v-leus)enkephalin on feeding and temperature in the rat, Eur. J. Pharmacol., 96 (1983) 243-249. 40 Timmers, K.I., Palkovits, M. and Coleman, D.L., Unique alterations of neuropeptide content in median eminence, amygdala, and dorsal vagal complex of 3- and 6-week-old diabetes mutant mice, Metabolism, 39 (1990) 1158-1166. 41 Vijayan, E,. and McCann, S.M., Suppression of feeding and drinking activity in rats following intraventricular injection of thyrotropin releasing hormone (TRH), Endocrinology, 100 (1777) 1727. 42 White, J.D., Olchovsky, D., Kershaw, M. and Berelowitz, M., Increased hypothalamie content of preproneuropeptide-Y messenger ribonucleic acid in streptozotocin diabetic rats, Endocrinology, 126 (1990) 765-772. 43 Williams, G., Steel, J.H., Cardoso, H., Ghatei, M.A., Lee, Y.C., Gill, J.S., Burrin, J.M., Polak, J.M. and Bloom, S.R., Increased hypothalamic neuropeptide Y concentrations in diabetic rats, Diabetes, 37 (1988) 763-772. 44 Yoshikawa, K., Williams, C. and Sabol, S.L., Rat brain preproenkephalin mRNA, ~r. Biol. Chem., 259 (1984) 14301-14308.