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Effect of fasting on regional levels of neuropeptide Y mRNA and insulin receptors in the rat hypothalamus: An autoradiographic study
MOLECULAR
AND CELLULAR
3,199-205 (19%)
NEUROSCIENCES
Effect of Fasting on Regional Levels of Neuropeptide Y mRNA and Insulin Receptors in the Rat Hypothalamus: An Autoradiographic Study’ JONATHAN L. MARKS,* Mu LI,* MICHAEL SCHWARTZ,~$ DANIEL PORTE, JR.,t’$ AND DENIS G. BASKIN?@ *Department of Clinical Endocrinology, Metabolism, Medical Research *Medicine and
Westmead Hospital, Westmead, New South Wales, Australia 2145; tDivision of Endocrinologyf Service, Veterans Affairs Medical Center, Seattle, Washington 98108; and Departments of SBiological Structure, University of Washington, Seattle, Washington 98105
Receivedfor publication January 23, 1992
Neuropeptide Y (NPY) and NPY mRNA are increased in the hypothalamic arcuate nucleus of rats after prolonged fasting but it is not known if NPY mRNA is also affected by more moderate fasting. NPY mRNA was quantified by in situ hybridization at five levels through the rostral/caudal axis of the arcuate nucleus and was found to be expressed in a markedly heterogenous fashion. NPY mRNA increased significantly in the mid portion of the arcuate nucleus after 24 h of fasting and further increased with fasting for 48 and 96 h. At the most caudal level of the arcuate nucleus, as well as in the dorsomedial nucleus of the hypothalamus and the reticular nucleus of the thalamus, NPY mRNA was unaffected by fasting. Serum insulin decreased after 24 h of fasting and decreased further with more prolonged fasting. Insulin receptor binding, which was highest in the same region of the arcuate nucleus that expressed NPY mRNA, was unaffected by fasting. These data show that NPY mRNA levels in the arcuate nucleus are sensitive to a relatively minor degree of food deprivation and are consistent with the hypothesis that circulating insulin modulates NPY mRNA expression in the arcuate nucleus o is92 Academic PMS, 1~. during mild to prolonged fasting.
INTRODUCTION Neuropeptide Y (NPY) is a 36-amino acid neuropeptide (1) that is found abundantly in the mammalian brain (2). NPY is one of the most potent appetite-stimulating agents known when administered into the third ventricle (3) or directly into various hypothalamic sites (4). That NPY affects food intake in uiuo is suggested by the relatively high levels of NPY peptide found in the hypothalamus (5,6). Nerve terminals containing NPY originate in neuronal cell bodies in the arcuate nucleus (7) and the brain stem (8) and are widely distributed in the hypothalamus. 1Presentedat the 34th Annual Meeting of the Endocrine Societyof Australia.
The paraventricular nucleus (PVN) receives a particularly dense innervation (7). Following 48 h of fasting in one study (9) but not until 72 h of fasting in another (5), NPY levels were increased in the PVN and arcuate nucleus but not in other hypothalamic nuclei. Release of NPY in the PVN was also increased after 72 h of fasting and in rats allowed to eat for only 4 h each day (10). Recently it has also been shown that NPY mRNA is increased markedly in the arcuate nucleus of rats fasted for 72 h (10). It is thus probable that, during moderate to more prolonged fasts and in trained rats, NPY synthesis in and release from nerve terminals of arcuate neurons are increased and that the hyperphagia of fasted rats is at least partially due to increased NPY activity. However, if hypothalamic NPY affects food intake in the rat under more normal feeding conditions, then NPY levels or release should increase with short periods of fasting and NPY mRNA in the arcuate nucleus should be affected similarly. To test this possibility, we measured NPY mRNA in the arcuate and other nuclei after 24 h as well as after more prolonged fasting. In streptozotocin-diabetic rats (6, 12), NPY immunoactivity is increased in several hypothalamic nuclei and NPY mRNA is increased in the arcuate nucleus (13). Because diabetes and fasting are hypoinsulinemic states and because locally administered insulin can decrease the level of NPY mRNA in the arcuate nucleus of fasted rats (14), it has been proposed that circulating insulin can decrease the synthesis of NPY in the hypothalamus (6, 11, 14). Considerable evidence suggests that circulating insulin acts as a satiety signal by entering the brain and then causing reduced food intake and body weight (15-18). One mechanism for this action could be the ability of insulin to decrease the synthesis and release of NPY at hypothalamic sites. In this study, we also wished to determine if hypothalamic NPY mRNA might be modulated by circulating insulin during fasting. Consequently, serum insulin levels following short-term as well as more prolonged fasting were compared to changes in hypothalamic NPY mRNA. 199
1044-7431/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Furthermore, we have compared the location of hypothalamic insulin receptor binding with the location of expression of hypothalamic NPY mRNA. MATERIALS
AND
METHODS
Adult female Sprague-Dawley rats, ages 12-16 weeks, were individually housed in perspex cages and maintained on a 12-h light and dark cycle beginning at 0600 h. Fasting animals had their food but not their water removed at 1400 h. Ad libitum fed and fasted animals were sacrificed at 1400 h using ip equithisin followed by perfusion of the brain via the left cardiac ventricle with 30 ml of ice-cold saline. Brains and livers were rapidly removed, frozen on dry ice, and stored at -70°C. Coronal sections (15 pm) were cut through the arcuate nucleus or liver on a cryostat. Five different levels throughout the rostrocaudal arcuate nucleus were identified on the basis of their similarity to figures in a stereotaxic atlas (19). Levels 1 (most caudal) through 5 (most rostral) were equivalent to bregma -4.16 mm, bregma -3.6 mm, bregma -3.3 mm, bregma -2.8 mm, and bregma -2.3 mm, respectively. Twelve adjacent slices were cut at each of these levels and used for either in situ hybridization or receptor binding. Representative slices from each region of each animal were stained with cresyl violet to confirm the correct localization of slides to the expected level. Northern blot hybridization for NPY mRNA was performed using a 36-base oligonucleotide probe as previously described (20, 21). In situ hybridization was performed using the same probe (20-22). The sections were autoradiographed on Hyperfilm ,&max film (Amersham, Arlington Heights, IL) for 4 days. Film images from the 4% and 96-h fasting experiments were analyzed using the MCID image analysis system (Imaging Research, St. Catherines, Ontario). Film densities were determined with 14C plastic sections of known radioactivity (American Radiolabeled Chemicals, St. Louis, MO). Film images from the 24-h fasting experiments were analyzed using the JAVA image analysis system (Jandel Scientific, Corte Madera, CA) and also standardized with 14C plastic sections (Amersham). The total NPY mRNA signal in bilateral arcuate nuclei was quantified in each brain slice by the following method. The area of the arcuate nucleus was measured and multiplied by the averaged signal density within this area. The area of the arcuate nucleus multiplied by the background density in the same slice was subtracted from this value. The resultant values for total NPY mRNA signal are presented as a percentage of fed controls. The background density was determined as the average density in a O.l-mm2 area of lateral hypothalamus for each section and was the same if the in situ hybridization was performed in the presence of 100-fold excess unlabeled probe. Using this method, the values for total NPY mRNA signal were unaffected by the subjective selection of arcuate area,
Tt!,l
AL. *-
provided all obvious signal clustered in the ventromedial part of the arcuate nucleus was included. Four brain slices from each arcuate level from each animal were analyzed. Total NPY mRNA signal in the hypothalamic dorsomedial nucleus was analyzed in the same way. NPY mRNA signal in the reticular nucleus of the thalamus was analyzed as a density only because of the large and irregular size of this region. Three density measurements were made in each of the bilateral nuclei and background density as described above was subtracted. Insulin receptor binding in the arcuate nucleus and liver was performed with 1251-insulin as previously described (23) and the resulting contact film autoradiographs were measured with the MCID system. Specific insulin binding was determined in the portion of the arcuate nucleus adjacent to the third ventricle and dorsal to the lateral extensions of the third ventricle. Three density recordings were taken on each side of and within a O.&mm distance from the third ventricle and averaged to give the insulin receptor density for each brain slice. Specific binding was defined as total insulin binding minus binding in the presence of 3.0 PLMunlabeled insulin. In liver, three density measurements were made on each tissue slice. Serum insulin and serum glucose were measured as previously described (24). The sensitivity of the insulin assay was 2 pU/ml. Statistical comparisons were made with the Mann-Whitney U test. RESULTS
Table 1 shows the effect of increasing duration of fasting on body weight, serum glucose, and insulin concentrations. A reduction in serum insulin was found after 24 h, with a further decrease with 48 h of fasting. Glucose values decreased after 48 h of fasting. Northern blot hybridization of newborn rat brain total RNA and adult rat hypothalamus total RNA showed a single band of approximately 800 bases (results not shown).
TABLE
1
The Effect of Fasting on Body Weight, Serum Insulin, and Serum Glucose in Adult Female Rats Fed, 96 h (n = 6) Body weight (% change) Serum insulin (&J/ml) Serum glucose (mm
+3.0
+ 0.5
Fasted, 24 h (n=4)
Fasted, 48 h (n = 3)
Fasted, (n=
-8.5
-11.1
-18.7
+ 0.4*
It 1.1’
7.6 + 0.8
4.6 f 0.5*
2.4 f 0.4*
12.6 f 0.6
10.7 ?I 1.3
7.8 f 0.9*
96 h 4)
+ 0.6*
5.4 k 0.5 *
Note. Animals, the numbers in parentheses were ad libitum fed or fasted for the times specified prior to sacrifice. Serum insulin was not assayed on animals fasted for 96 h. * P < 0.05 compared to the number in the column immediately to the left.
NPY
mRNA
AND
INSULIN
RECEPTORS
In Fig. 1, the distribution of NPY mRNA is shown in coronal sections of the brain at different levels of the arcuate nucleus. Figures lA-1C show the distribution of NPY mRNA at Levels 2,3, and 4 of the arcuate nucleus, respectively. Although the highest expression was at Levels 2 and 3, NPY mRNA was also expressed by the most caudal and rostra1 levels (not shown). In all cases, the NPY mRNA signal was located in the ventral part of the arcuate nucleus, adjacent to the third ventricle. In sections through Level 3, NPY mRNA was also present in the
FIG. 1. In situ hybridization autoradiographs fed animals; D, E, and F are from animals fasted in C and F. Abbreviations: an, arcuate nucleus; thalamus.
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dorsomedial nucleus of the hypothalamus. As previously reported (11, 25) NPY mRNA was expressed in the reticular nucleus of the thalamus, the hippocampus, and the cerebral cortex. Figures lD-1F show the effect of fasting for 96 h on NPY mRNA. There was an apparent increase in NPY mRNA signal from the arcuate nucleus, but not in other brain regions. When hybridizations were performed with loo-fold excess unlabeled probe, autoradiographs were uniformly faint and featureless (data not shown).
of NPY mRNA in coronal brain sections through the arcuate nucleus. A, B, and C are from for 96 h. Level 2 of the arcuate nucleus is shown in A and D, Level 3 in B and E, and Level 4 d, dentate gyrus; dm, dorsomedial nucleus of the hypothalamus; rn, reticular nucleus of the
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The quantification of NPY mRNA at different levels in the arcuate nucleus and the effect of fasting for 24 to 96 h are shown in Fig. 2. The highest expression of NPY mRNA was found at Levels 2 and 3 in both control and fasted animals. After 24 h of fasting, NPY mRNA signal had increased significantly at Levels l-4. By 48 h, this was significant and progressive only in Levels 2-4. Although an increase in NPY mRNA was present at Level 5 after 96 h of fasting, a very low value for the control
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tzIFed
r -
-
Reticular
Fasted
q
Fasted
2
1
3
4
Nut.
Dorsomedial
Nut.
FIG. 3. The effect of fasting for 96 h on NPY mRNA in the reticular nucleus of the thalamus and the dorsomedial nucleus of the hypothalamus. Measurements are the mean f SEM for the same slices used in Fig. 2 and came from four fasted and four fed rats. Slices used for assessment of reticular nucleus NPY mRNA were from Levels 3 and 4 and for dorsomedial nucleus NPY mRNA from Level 3. The uncorrected value for NPY mRNA concentration in the reticular nucleus of fed animals was 0.215 rC!i/g and the content in the dorsomedial nucleus of fed animals was 0.139 mm* &i/g.
100
0
$ 3 ap
120
0 24 Hours
E
~ 2 cy
AL.
200
2 I
ET
5
200 48 Hours
Fasted
100
I 02 E
P 0
1
2
4
3
5
300 r
98 Hours
1
2
3 Arcuate
4
Fasted
5
Level
FIG. 2. The effect of fasting on NPY mRNA at five levels of the arcuate nucleus. Results are the mean f SEM for total NPY mRNA signal in bilateral arcuate nuclei in an individual slice and were determined from in situ hybridization autoradiographs as described under Materials and Methods. Values for NPY mRNA have been standardixed as a percentage of the value at Level 2 from fed animals. (A) Six adult female rats were fed or fasted for 24 h. Values at Level 5 are from three rats. The uncorrected value for Level 2 fed animals was 0.065 mm2 &i/ g. (B) Six adult females were fed or fasted for 48 h. The uncorrected value for Level 2 fed animals was 0.112 mm2 #X/g. (C) Four adult females were fed or fasted for 96 h. Values from Level 5 were from three rats. The uncorrected value for Level 2 fed animals was 0.114 mm* rCi/ g. *P < 0.05 compared to the fed control.
animals makes this uncertain. The increases of NPY mRNA in the arcuate nucleus at Levels 2-4 were quite similar at each time point between 24 and 96 h of fasting, but the proportional increases were different. For example, at Level 4 there was a 30% increase in NPY mRNA expression after 24 h, a 100% increase after 48 h, and a 350% increase after 96 h of fasting, while at Level 2, there were 39, 50, and 100% increases, respectively. NPY mRNA expression was not altered by 96 h of fasting in the dorsomedial nucleus of the hypothalamus or in the reticular nucleus of the thalamus (Fig. 3). The distribution of insulin binding in the arcuate nucleus at Levels 2-4 is shown in Fig. 4. Relatively dense insulin binding was found in the arcuate nucleus, particularly in the ventromedial portion. The distribution of insulin binding in the arcuate nucleus can be compared to the distribution of NPY mRNA in the arcuate nucleus in Fig. 1. It is clear that the area of relatively dense insulin binding includes the area of NPY mRNA expression. When the density of insulin binding in the arcuate nucleus was measured (Table 2), similar values were found at Levels l-4, but a lower value was found at Level 5. After 96 h of fasting, there was no change in insulin binding at any of the levels of the arcuate nucleus or in Layer 2 of the cerebral cortex (data not shown). However in the liver, fasting for 72 h resulted in a 41% increase in insulin binding. DISCUSSION
The specificity of our oligonucleotide probe for NPY mRNA in the rat brain has been previously suggested by
NPY
mRNA
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INSULIN
RECEPTORS
DURING
203
FASTING
amus, we divided the mid to caudal extent of the hypothalamus into five levels and tested whether the expression and the response to fasting of NPY mRNA are heterogenous. At all five levels, NPY mRNA was found in the ventromedial portion of the arcuate nucleus, immediately adjacent to the third ventricle. Approximately 0.1 mm rostra1 to Level 5 and caudal to Level 1, NPY mRNA expression was no longer evident in the arcuate nucleus (data not shown). NPY mRNA was also found in the dorsomedial nucleus at Level 3. The expression of NPY mRNA varied considerably at different levels along the arcuate nucleus, with the highest expression consistently found in the mid to caudal region of the nucleus (Levels 2 and 3) in the fed or fasted state. Because of this marked variation, it is apparent that when using our method of quantitative autoradiography, very close anatomical matching of brain slices between control and experimental animals is mandatory. In response to fasting for 24 h and longer, NPY mRNA increased progressively in the mid arcuate nucleus (Levels 2, 3, and 4), but was not consistently altered in the most caudal or most rostra1 levels (Levels 1 and 5) of the arcuate nucleus. The greatest percentage increase with fasting for 48 h or more was found at Level 4. White and Kershaw (11) have reported that NPY mRNA is increased 2- to 3-fold in the arcuate nucleus of coronal rat brain slices after 3 days of fasting using quantitative in situ hybridization and in total RNA prepared from hypothalamic tissue blocks using solution hybridization. In overnight-fasted animals, NPY mRNA was not increased in RNA prepared from hypothalamic tissue blocks. The negative result in the latter experiment versus a positive result in our study after 24 h of fasting could be due to an increased sensitivity of the quantitative in situ hybridization method for detecting small changes in a mRNA species at discrete anatomical locations. How-
TABLE
2
The Effect of Fasting on Specific Insulin Binding at Different Levels through the Arcuate Nucleus and in the Liver, Measured by Quantitative Autoradiography FIG. through
4. Autoradiographs of insulin binding in coronal sections the rat brain at (A) Level 2, (B) Level 3, and (C) Level 4.
the similarity of signal distribution that was found to previous reports of the localization NPY mRNA (11, 20, 25) and immunoactivity (2, 7) in cell bodies. In this report, we have provided more evidence for the probe’s specificity by showing that excess unlabeled probe abolished the specific signal and that, when used for Northern blot analysis of rat brain RNA, the probe labeled a single band of a size similar to that reported for rat NPY mRNA (26). To investigate the regional effect of mild to severe food deprivation on NPY mRNA expression in the hypothal-
Fed animals (dpm/mm’)
Tissue Arcuate Arcuate Arcuate Arcuate Arcuate Liver
Level Level Level Level Level
1 2 3 4 5
167 182f 156 132 119f 254
+ 21 6 + 10 zk 7 5 + 29
Fasted animals (dpm/mm*) 172 166 165 151 115f 358
+ k + -c
16 13 11 10 6 k 31*
Note. Brain slices were taken from four adult female rats, fed or fasted for 96 h. Liver slices were taken from four adult females, fed or fasted for 72 h. Level 1 is the most caudal and Level 5 is the most rostra1 level in the arcuate nucleus. Values from Level 5 were significantly less than values from Level 1. * P < 0.05 compared to value to the left.
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ever, in the overnight-fasting experiment of White and Kershaw (ll), fewer animals were used and a shorter period of food deprivation and a smaller reduction in body weight were reported than in our experiment,. NPY mRNA levels were also increased in RNA prepared from mouse hypothalamic blocks after 16 h of fasting (27). In that report, a weight loss of 12% was similar to that following a 48-h fast in the rat, suggesting that a short period of fasting in the mouse may produce a greater metabolic disturbance. We found that fasting for 96 h had no effect on the NPY mRNA levels in the dorsomedial nucleus of the hypothalamus, despite its proximity to the arcuate nucleus, nor in the reticular nucleus of the thalamus. White and Kershaw have also found no effect of fasting on NPY mRNA in the reticular nucleus (11). The increase of NPY mRNA within the arcuate nucleus of the rat after 24 h of fasting is consistent with increased NPY levels in the PVN and arcuate nucleus of rats fasted for 48 h (9) and increased release of NPY in the PVN of rats trained to eat for short periods (10). It suggests that NPY synthesis is responsive to relatively minor changes in the nutritional state and that NPY release may affect food intake under conditions of relative abundance, including the diurnal variation. The cause for the increased NPY mRNA after 24 h or more prolonged fasting remains to be established. Increased release of NPY from nerve terminals may send intracellular signals to increase NPY expression. As previous reports have suggested that insulin can reduce NPY expression in the arcuate nucleus (6, 12-14) and as circulating insulin was reduced after 24 h of fasting, it is possible that the decrease in insulin could have contributed to the increased NPY mRNA. A further reduction of insulin levels with more prolonged fasting may have contributed to the progressive increase in NPY mRNA. Certainly, other metabolic and hormonal changes that occur with particularly prolonged fasting were potential contributors to the increase in NPY mRNA in the arcuate nucleus (28, 29). Further support for an effect of insulin on NPY expression in the arcuate nucleus comes from the presence of insulin binding (23,30) and insulin receptor mRNA (20) in this nucleus. However, the anatomical relationship between NPY mRNA and insulin binding in the arcuate nucleus has not been previously evaluated. We found relatively high amounts of insulin binding in all caudal to rostra1 levels of the arcuate nucleus. In particular, the region of highest insulin binding was adjacent to the ventral portion of the third ventricle and included the area of NPY mRNA expression. Therefore, an effect of insulin on NPY expression could be directly on NPY-ergic or adjacent neurons. It is interesting to note that in this area of NPY mRNA expression, insulin binding was approximately 60% of that in the liver, a classically insulin-sensitive organ.
ET
AL
The mechanism by which circulating insulin would gain access to the arcuate nucleus is not established, although it is clear that insulin crosses the blood-brain barrier (31) and can diffuse from the adjacent median eminence which lacks a blood-brain barrier (32). Despite marked changes in serum insulin levels with fasting, 4 days of fasting had no effect on insulin binding in the arcuate nucleus. This result was not due to insensitivity of our methods, because fasting led to an increase in liver insulin binding. We have previously found no effect of fasting on insulin binding in membranes prepared from medial hypothalamus (24), although it has been reported that reduced insulin binding occurs in membranes from the medial but not the lateral hypothalamus of fasted rats (33). Previous reports have shown or suggested that brain insulin binding is not regulated by changes in circulating insulin levels (24,34,35), but in this report more specific results were obtained because we were able to quantify binding in a specific anatomical layer. To conclude, we have found that NPY mRNA is increased in the arcuate nucleus following 24 h of fasting and that it increases further with more prolonged fasting. Because circulating insulin decreases after 24 h of fasting and because the arcuate nucleus contains a relatively high concentration of insulin receptors, it is possible that circulating insulin modulates the expression of NPY mRNA in the arcuate nucleus. ACKNOWLEDGMENTS This research was supported by the Merit Review Research Program, Medical Research Service, Department of Veterans Affairs; NIH Grant DK17047; a research grant from Diabetes Australia; and a Fellowship from the American Diabetes Association (J.L.M.). We thank John Breininger, Martha Du Ruz, and Kay Waite for expert technical assistance; Agnes Koltai for secretarial assistance; and Professor G. Johnston for the use of the JAVA system.
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AND CELLULAR
3,199-205 (19%)
NEUROSCIENCES
Effect of Fasting on Regional Levels of Neuropeptide Y mRNA and Insulin Receptors in the Rat Hypothalamus: An Autoradiographic Study’ JONATHAN L. MARKS,* Mu LI,* MICHAEL SCHWARTZ,~$ DANIEL PORTE, JR.,t’$ AND DENIS G. BASKIN?@ *Department of Clinical Endocrinology, Metabolism, Medical Research *Medicine and
Westmead Hospital, Westmead, New South Wales, Australia 2145; tDivision of Endocrinologyf Service, Veterans Affairs Medical Center, Seattle, Washington 98108; and Departments of SBiological Structure, University of Washington, Seattle, Washington 98105
Receivedfor publication January 23, 1992
Neuropeptide Y (NPY) and NPY mRNA are increased in the hypothalamic arcuate nucleus of rats after prolonged fasting but it is not known if NPY mRNA is also affected by more moderate fasting. NPY mRNA was quantified by in situ hybridization at five levels through the rostral/caudal axis of the arcuate nucleus and was found to be expressed in a markedly heterogenous fashion. NPY mRNA increased significantly in the mid portion of the arcuate nucleus after 24 h of fasting and further increased with fasting for 48 and 96 h. At the most caudal level of the arcuate nucleus, as well as in the dorsomedial nucleus of the hypothalamus and the reticular nucleus of the thalamus, NPY mRNA was unaffected by fasting. Serum insulin decreased after 24 h of fasting and decreased further with more prolonged fasting. Insulin receptor binding, which was highest in the same region of the arcuate nucleus that expressed NPY mRNA, was unaffected by fasting. These data show that NPY mRNA levels in the arcuate nucleus are sensitive to a relatively minor degree of food deprivation and are consistent with the hypothesis that circulating insulin modulates NPY mRNA expression in the arcuate nucleus o is92 Academic PMS, 1~. during mild to prolonged fasting.
INTRODUCTION Neuropeptide Y (NPY) is a 36-amino acid neuropeptide (1) that is found abundantly in the mammalian brain (2). NPY is one of the most potent appetite-stimulating agents known when administered into the third ventricle (3) or directly into various hypothalamic sites (4). That NPY affects food intake in uiuo is suggested by the relatively high levels of NPY peptide found in the hypothalamus (5,6). Nerve terminals containing NPY originate in neuronal cell bodies in the arcuate nucleus (7) and the brain stem (8) and are widely distributed in the hypothalamus. 1Presentedat the 34th Annual Meeting of the Endocrine Societyof Australia.
The paraventricular nucleus (PVN) receives a particularly dense innervation (7). Following 48 h of fasting in one study (9) but not until 72 h of fasting in another (5), NPY levels were increased in the PVN and arcuate nucleus but not in other hypothalamic nuclei. Release of NPY in the PVN was also increased after 72 h of fasting and in rats allowed to eat for only 4 h each day (10). Recently it has also been shown that NPY mRNA is increased markedly in the arcuate nucleus of rats fasted for 72 h (10). It is thus probable that, during moderate to more prolonged fasts and in trained rats, NPY synthesis in and release from nerve terminals of arcuate neurons are increased and that the hyperphagia of fasted rats is at least partially due to increased NPY activity. However, if hypothalamic NPY affects food intake in the rat under more normal feeding conditions, then NPY levels or release should increase with short periods of fasting and NPY mRNA in the arcuate nucleus should be affected similarly. To test this possibility, we measured NPY mRNA in the arcuate and other nuclei after 24 h as well as after more prolonged fasting. In streptozotocin-diabetic rats (6, 12), NPY immunoactivity is increased in several hypothalamic nuclei and NPY mRNA is increased in the arcuate nucleus (13). Because diabetes and fasting are hypoinsulinemic states and because locally administered insulin can decrease the level of NPY mRNA in the arcuate nucleus of fasted rats (14), it has been proposed that circulating insulin can decrease the synthesis of NPY in the hypothalamus (6, 11, 14). Considerable evidence suggests that circulating insulin acts as a satiety signal by entering the brain and then causing reduced food intake and body weight (15-18). One mechanism for this action could be the ability of insulin to decrease the synthesis and release of NPY at hypothalamic sites. In this study, we also wished to determine if hypothalamic NPY mRNA might be modulated by circulating insulin during fasting. Consequently, serum insulin levels following short-term as well as more prolonged fasting were compared to changes in hypothalamic NPY mRNA. 199
1044-7431/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Furthermore, we have compared the location of hypothalamic insulin receptor binding with the location of expression of hypothalamic NPY mRNA. MATERIALS
AND
METHODS
Adult female Sprague-Dawley rats, ages 12-16 weeks, were individually housed in perspex cages and maintained on a 12-h light and dark cycle beginning at 0600 h. Fasting animals had their food but not their water removed at 1400 h. Ad libitum fed and fasted animals were sacrificed at 1400 h using ip equithisin followed by perfusion of the brain via the left cardiac ventricle with 30 ml of ice-cold saline. Brains and livers were rapidly removed, frozen on dry ice, and stored at -70°C. Coronal sections (15 pm) were cut through the arcuate nucleus or liver on a cryostat. Five different levels throughout the rostrocaudal arcuate nucleus were identified on the basis of their similarity to figures in a stereotaxic atlas (19). Levels 1 (most caudal) through 5 (most rostral) were equivalent to bregma -4.16 mm, bregma -3.6 mm, bregma -3.3 mm, bregma -2.8 mm, and bregma -2.3 mm, respectively. Twelve adjacent slices were cut at each of these levels and used for either in situ hybridization or receptor binding. Representative slices from each region of each animal were stained with cresyl violet to confirm the correct localization of slides to the expected level. Northern blot hybridization for NPY mRNA was performed using a 36-base oligonucleotide probe as previously described (20, 21). In situ hybridization was performed using the same probe (20-22). The sections were autoradiographed on Hyperfilm ,&max film (Amersham, Arlington Heights, IL) for 4 days. Film images from the 4% and 96-h fasting experiments were analyzed using the MCID image analysis system (Imaging Research, St. Catherines, Ontario). Film densities were determined with 14C plastic sections of known radioactivity (American Radiolabeled Chemicals, St. Louis, MO). Film images from the 24-h fasting experiments were analyzed using the JAVA image analysis system (Jandel Scientific, Corte Madera, CA) and also standardized with 14C plastic sections (Amersham). The total NPY mRNA signal in bilateral arcuate nuclei was quantified in each brain slice by the following method. The area of the arcuate nucleus was measured and multiplied by the averaged signal density within this area. The area of the arcuate nucleus multiplied by the background density in the same slice was subtracted from this value. The resultant values for total NPY mRNA signal are presented as a percentage of fed controls. The background density was determined as the average density in a O.l-mm2 area of lateral hypothalamus for each section and was the same if the in situ hybridization was performed in the presence of 100-fold excess unlabeled probe. Using this method, the values for total NPY mRNA signal were unaffected by the subjective selection of arcuate area,
Tt!,l
AL. *-
provided all obvious signal clustered in the ventromedial part of the arcuate nucleus was included. Four brain slices from each arcuate level from each animal were analyzed. Total NPY mRNA signal in the hypothalamic dorsomedial nucleus was analyzed in the same way. NPY mRNA signal in the reticular nucleus of the thalamus was analyzed as a density only because of the large and irregular size of this region. Three density measurements were made in each of the bilateral nuclei and background density as described above was subtracted. Insulin receptor binding in the arcuate nucleus and liver was performed with 1251-insulin as previously described (23) and the resulting contact film autoradiographs were measured with the MCID system. Specific insulin binding was determined in the portion of the arcuate nucleus adjacent to the third ventricle and dorsal to the lateral extensions of the third ventricle. Three density recordings were taken on each side of and within a O.&mm distance from the third ventricle and averaged to give the insulin receptor density for each brain slice. Specific binding was defined as total insulin binding minus binding in the presence of 3.0 PLMunlabeled insulin. In liver, three density measurements were made on each tissue slice. Serum insulin and serum glucose were measured as previously described (24). The sensitivity of the insulin assay was 2 pU/ml. Statistical comparisons were made with the Mann-Whitney U test. RESULTS
Table 1 shows the effect of increasing duration of fasting on body weight, serum glucose, and insulin concentrations. A reduction in serum insulin was found after 24 h, with a further decrease with 48 h of fasting. Glucose values decreased after 48 h of fasting. Northern blot hybridization of newborn rat brain total RNA and adult rat hypothalamus total RNA showed a single band of approximately 800 bases (results not shown).
TABLE
1
The Effect of Fasting on Body Weight, Serum Insulin, and Serum Glucose in Adult Female Rats Fed, 96 h (n = 6) Body weight (% change) Serum insulin (&J/ml) Serum glucose (mm
+3.0
+ 0.5
Fasted, 24 h (n=4)
Fasted, 48 h (n = 3)
Fasted, (n=
-8.5
-11.1
-18.7
+ 0.4*
It 1.1’
7.6 + 0.8
4.6 f 0.5*
2.4 f 0.4*
12.6 f 0.6
10.7 ?I 1.3
7.8 f 0.9*
96 h 4)
+ 0.6*
5.4 k 0.5 *
Note. Animals, the numbers in parentheses were ad libitum fed or fasted for the times specified prior to sacrifice. Serum insulin was not assayed on animals fasted for 96 h. * P < 0.05 compared to the number in the column immediately to the left.
NPY
mRNA
AND
INSULIN
RECEPTORS
In Fig. 1, the distribution of NPY mRNA is shown in coronal sections of the brain at different levels of the arcuate nucleus. Figures lA-1C show the distribution of NPY mRNA at Levels 2,3, and 4 of the arcuate nucleus, respectively. Although the highest expression was at Levels 2 and 3, NPY mRNA was also expressed by the most caudal and rostra1 levels (not shown). In all cases, the NPY mRNA signal was located in the ventral part of the arcuate nucleus, adjacent to the third ventricle. In sections through Level 3, NPY mRNA was also present in the
FIG. 1. In situ hybridization autoradiographs fed animals; D, E, and F are from animals fasted in C and F. Abbreviations: an, arcuate nucleus; thalamus.
DURING
FASTING
201
dorsomedial nucleus of the hypothalamus. As previously reported (11, 25) NPY mRNA was expressed in the reticular nucleus of the thalamus, the hippocampus, and the cerebral cortex. Figures lD-1F show the effect of fasting for 96 h on NPY mRNA. There was an apparent increase in NPY mRNA signal from the arcuate nucleus, but not in other brain regions. When hybridizations were performed with loo-fold excess unlabeled probe, autoradiographs were uniformly faint and featureless (data not shown).
of NPY mRNA in coronal brain sections through the arcuate nucleus. A, B, and C are from for 96 h. Level 2 of the arcuate nucleus is shown in A and D, Level 3 in B and E, and Level 4 d, dentate gyrus; dm, dorsomedial nucleus of the hypothalamus; rn, reticular nucleus of the
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The quantification of NPY mRNA at different levels in the arcuate nucleus and the effect of fasting for 24 to 96 h are shown in Fig. 2. The highest expression of NPY mRNA was found at Levels 2 and 3 in both control and fasted animals. After 24 h of fasting, NPY mRNA signal had increased significantly at Levels l-4. By 48 h, this was significant and progressive only in Levels 2-4. Although an increase in NPY mRNA was present at Level 5 after 96 h of fasting, a very low value for the control
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tzIFed
r -
-
Reticular
Fasted
q
Fasted
2
1
3
4
Nut.
Dorsomedial
Nut.
FIG. 3. The effect of fasting for 96 h on NPY mRNA in the reticular nucleus of the thalamus and the dorsomedial nucleus of the hypothalamus. Measurements are the mean f SEM for the same slices used in Fig. 2 and came from four fasted and four fed rats. Slices used for assessment of reticular nucleus NPY mRNA were from Levels 3 and 4 and for dorsomedial nucleus NPY mRNA from Level 3. The uncorrected value for NPY mRNA concentration in the reticular nucleus of fed animals was 0.215 rC!i/g and the content in the dorsomedial nucleus of fed animals was 0.139 mm* &i/g.
100
0
$ 3 ap
120
0 24 Hours
E
~ 2 cy
AL.
200
2 I
ET
5
200 48 Hours
Fasted
100
I 02 E
P 0
1
2
4
3
5
300 r
98 Hours
1
2
3 Arcuate
4
Fasted
5
Level
FIG. 2. The effect of fasting on NPY mRNA at five levels of the arcuate nucleus. Results are the mean f SEM for total NPY mRNA signal in bilateral arcuate nuclei in an individual slice and were determined from in situ hybridization autoradiographs as described under Materials and Methods. Values for NPY mRNA have been standardixed as a percentage of the value at Level 2 from fed animals. (A) Six adult female rats were fed or fasted for 24 h. Values at Level 5 are from three rats. The uncorrected value for Level 2 fed animals was 0.065 mm2 &i/ g. (B) Six adult females were fed or fasted for 48 h. The uncorrected value for Level 2 fed animals was 0.112 mm2 #X/g. (C) Four adult females were fed or fasted for 96 h. Values from Level 5 were from three rats. The uncorrected value for Level 2 fed animals was 0.114 mm* rCi/ g. *P < 0.05 compared to the fed control.
animals makes this uncertain. The increases of NPY mRNA in the arcuate nucleus at Levels 2-4 were quite similar at each time point between 24 and 96 h of fasting, but the proportional increases were different. For example, at Level 4 there was a 30% increase in NPY mRNA expression after 24 h, a 100% increase after 48 h, and a 350% increase after 96 h of fasting, while at Level 2, there were 39, 50, and 100% increases, respectively. NPY mRNA expression was not altered by 96 h of fasting in the dorsomedial nucleus of the hypothalamus or in the reticular nucleus of the thalamus (Fig. 3). The distribution of insulin binding in the arcuate nucleus at Levels 2-4 is shown in Fig. 4. Relatively dense insulin binding was found in the arcuate nucleus, particularly in the ventromedial portion. The distribution of insulin binding in the arcuate nucleus can be compared to the distribution of NPY mRNA in the arcuate nucleus in Fig. 1. It is clear that the area of relatively dense insulin binding includes the area of NPY mRNA expression. When the density of insulin binding in the arcuate nucleus was measured (Table 2), similar values were found at Levels l-4, but a lower value was found at Level 5. After 96 h of fasting, there was no change in insulin binding at any of the levels of the arcuate nucleus or in Layer 2 of the cerebral cortex (data not shown). However in the liver, fasting for 72 h resulted in a 41% increase in insulin binding. DISCUSSION
The specificity of our oligonucleotide probe for NPY mRNA in the rat brain has been previously suggested by
NPY
mRNA
AND
INSULIN
RECEPTORS
DURING
203
FASTING
amus, we divided the mid to caudal extent of the hypothalamus into five levels and tested whether the expression and the response to fasting of NPY mRNA are heterogenous. At all five levels, NPY mRNA was found in the ventromedial portion of the arcuate nucleus, immediately adjacent to the third ventricle. Approximately 0.1 mm rostra1 to Level 5 and caudal to Level 1, NPY mRNA expression was no longer evident in the arcuate nucleus (data not shown). NPY mRNA was also found in the dorsomedial nucleus at Level 3. The expression of NPY mRNA varied considerably at different levels along the arcuate nucleus, with the highest expression consistently found in the mid to caudal region of the nucleus (Levels 2 and 3) in the fed or fasted state. Because of this marked variation, it is apparent that when using our method of quantitative autoradiography, very close anatomical matching of brain slices between control and experimental animals is mandatory. In response to fasting for 24 h and longer, NPY mRNA increased progressively in the mid arcuate nucleus (Levels 2, 3, and 4), but was not consistently altered in the most caudal or most rostra1 levels (Levels 1 and 5) of the arcuate nucleus. The greatest percentage increase with fasting for 48 h or more was found at Level 4. White and Kershaw (11) have reported that NPY mRNA is increased 2- to 3-fold in the arcuate nucleus of coronal rat brain slices after 3 days of fasting using quantitative in situ hybridization and in total RNA prepared from hypothalamic tissue blocks using solution hybridization. In overnight-fasted animals, NPY mRNA was not increased in RNA prepared from hypothalamic tissue blocks. The negative result in the latter experiment versus a positive result in our study after 24 h of fasting could be due to an increased sensitivity of the quantitative in situ hybridization method for detecting small changes in a mRNA species at discrete anatomical locations. How-
TABLE
2
The Effect of Fasting on Specific Insulin Binding at Different Levels through the Arcuate Nucleus and in the Liver, Measured by Quantitative Autoradiography FIG. through
4. Autoradiographs of insulin binding in coronal sections the rat brain at (A) Level 2, (B) Level 3, and (C) Level 4.
the similarity of signal distribution that was found to previous reports of the localization NPY mRNA (11, 20, 25) and immunoactivity (2, 7) in cell bodies. In this report, we have provided more evidence for the probe’s specificity by showing that excess unlabeled probe abolished the specific signal and that, when used for Northern blot analysis of rat brain RNA, the probe labeled a single band of a size similar to that reported for rat NPY mRNA (26). To investigate the regional effect of mild to severe food deprivation on NPY mRNA expression in the hypothal-
Fed animals (dpm/mm’)
Tissue Arcuate Arcuate Arcuate Arcuate Arcuate Liver
Level Level Level Level Level
1 2 3 4 5
167 182f 156 132 119f 254
+ 21 6 + 10 zk 7 5 + 29
Fasted animals (dpm/mm*) 172 166 165 151 115f 358
+ k + -c
16 13 11 10 6 k 31*
Note. Brain slices were taken from four adult female rats, fed or fasted for 96 h. Liver slices were taken from four adult females, fed or fasted for 72 h. Level 1 is the most caudal and Level 5 is the most rostra1 level in the arcuate nucleus. Values from Level 5 were significantly less than values from Level 1. * P < 0.05 compared to value to the left.
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MARKS
ever, in the overnight-fasting experiment of White and Kershaw (ll), fewer animals were used and a shorter period of food deprivation and a smaller reduction in body weight were reported than in our experiment,. NPY mRNA levels were also increased in RNA prepared from mouse hypothalamic blocks after 16 h of fasting (27). In that report, a weight loss of 12% was similar to that following a 48-h fast in the rat, suggesting that a short period of fasting in the mouse may produce a greater metabolic disturbance. We found that fasting for 96 h had no effect on the NPY mRNA levels in the dorsomedial nucleus of the hypothalamus, despite its proximity to the arcuate nucleus, nor in the reticular nucleus of the thalamus. White and Kershaw have also found no effect of fasting on NPY mRNA in the reticular nucleus (11). The increase of NPY mRNA within the arcuate nucleus of the rat after 24 h of fasting is consistent with increased NPY levels in the PVN and arcuate nucleus of rats fasted for 48 h (9) and increased release of NPY in the PVN of rats trained to eat for short periods (10). It suggests that NPY synthesis is responsive to relatively minor changes in the nutritional state and that NPY release may affect food intake under conditions of relative abundance, including the diurnal variation. The cause for the increased NPY mRNA after 24 h or more prolonged fasting remains to be established. Increased release of NPY from nerve terminals may send intracellular signals to increase NPY expression. As previous reports have suggested that insulin can reduce NPY expression in the arcuate nucleus (6, 12-14) and as circulating insulin was reduced after 24 h of fasting, it is possible that the decrease in insulin could have contributed to the increased NPY mRNA. A further reduction of insulin levels with more prolonged fasting may have contributed to the progressive increase in NPY mRNA. Certainly, other metabolic and hormonal changes that occur with particularly prolonged fasting were potential contributors to the increase in NPY mRNA in the arcuate nucleus (28, 29). Further support for an effect of insulin on NPY expression in the arcuate nucleus comes from the presence of insulin binding (23,30) and insulin receptor mRNA (20) in this nucleus. However, the anatomical relationship between NPY mRNA and insulin binding in the arcuate nucleus has not been previously evaluated. We found relatively high amounts of insulin binding in all caudal to rostra1 levels of the arcuate nucleus. In particular, the region of highest insulin binding was adjacent to the ventral portion of the third ventricle and included the area of NPY mRNA expression. Therefore, an effect of insulin on NPY expression could be directly on NPY-ergic or adjacent neurons. It is interesting to note that in this area of NPY mRNA expression, insulin binding was approximately 60% of that in the liver, a classically insulin-sensitive organ.
ET
AL
The mechanism by which circulating insulin would gain access to the arcuate nucleus is not established, although it is clear that insulin crosses the blood-brain barrier (31) and can diffuse from the adjacent median eminence which lacks a blood-brain barrier (32). Despite marked changes in serum insulin levels with fasting, 4 days of fasting had no effect on insulin binding in the arcuate nucleus. This result was not due to insensitivity of our methods, because fasting led to an increase in liver insulin binding. We have previously found no effect of fasting on insulin binding in membranes prepared from medial hypothalamus (24), although it has been reported that reduced insulin binding occurs in membranes from the medial but not the lateral hypothalamus of fasted rats (33). Previous reports have shown or suggested that brain insulin binding is not regulated by changes in circulating insulin levels (24,34,35), but in this report more specific results were obtained because we were able to quantify binding in a specific anatomical layer. To conclude, we have found that NPY mRNA is increased in the arcuate nucleus following 24 h of fasting and that it increases further with more prolonged fasting. Because circulating insulin decreases after 24 h of fasting and because the arcuate nucleus contains a relatively high concentration of insulin receptors, it is possible that circulating insulin modulates the expression of NPY mRNA in the arcuate nucleus. ACKNOWLEDGMENTS This research was supported by the Merit Review Research Program, Medical Research Service, Department of Veterans Affairs; NIH Grant DK17047; a research grant from Diabetes Australia; and a Fellowship from the American Diabetes Association (J.L.M.). We thank John Breininger, Martha Du Ruz, and Kay Waite for expert technical assistance; Agnes Koltai for secretarial assistance; and Professor G. Johnston for the use of the JAVA system.
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