| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Departments of Medicine (M.W.S., D.G.B.), Biochemistry (J.C.E., R.D.P.), and Biological Structure (D.G.B.), University of Washington, and Puget Sound Veterans Affairs Health Care System (M.W.S., D.G.B.), Seattle, Washington 98108
Address all correspondence and requests for reprints to: Michael W. Schwartz, M.D., Puget Sound Veterans Affairs Health Care System (151), 1660 South Columbian Way, Seattle, Washington 98108. E-mail: mschwart{at}u.washington.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In ob/ob mice, the effect of leptin to lower NPY messenger RNA (mRNA) levels has been detected only in the arcuate nucleus (6). Arcuate nucleus neurons, therefore, appear to be uniquely sensitive to leptin’s effects on NPY gene expression. Similarly, fasting increases NPY mRNA expression in the arcuate nucleus of normal rats without affecting NPY mRNA levels in other brain areas (9). The molecular mechanisms that regulate NPY gene expression in arcuate nucleus neurons are unknown, but could involve changes in NPY gene transcription, stability of the NPY mRNA transcript, or both. Efforts to study the control of NPY gene expression at the molecular level, however, are complicated by the highly localized nature of the response of NPY neurons to hormonal and metabolic stimuli. Conventional assays of gene transcription and mRNA stability that use cell culture systems are limited by the fact that neurons maintained in cell culture are unlikely to possess the functional characteristics that distinguish the arcuate nucleus from other neuronal populations. We, therefore, developed an approach that provides a measure of NPY gene transcription in brain tissue in vivo. This was accomplished using mice with a targeted mutation of the NPY gene in which the lacZ reporter gene is inserted into the first exon, where it is under control of the NPY gene promoter (10). The product of the lacZ gene, ß-galactosidase (ßgal), is, therefore, expressed in NPY neurons after activation of the NPY promoter.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Exp 1
To investigate the gene dosage effect of the mutant NPY allele
on the number of cells staining positively for ßgal
(ßgal+) in the brain, NPY+/- mice
(n = 6) and NPY-/- littermates (n = 9) weighing
29.0 ± 2.0 and 28.1 ± 2.0 g, respectively
(P = 0.76), were killed by CO2 inhalation
in the early light phase (between 0900–1200 h). All animals had
ad libitum access to food before death. Brains were rapidly
removed, frozen on crushed dry ice, and stored at -70 C until
assay.
Exp 2
To determine the effect of fasting on the number of
ßgal+ cells in the arcuate nucleus,
NPY+/- mice, weighing 25.3 ± 1.0 g, were either
fed ad libitum (n = 10) or food deprived for 48 h
(n = 10), then killed, and brains were removed as described for
Exp 1. Drinking water was available to both groups at all times.
Exp 3
To determine the effect of genetic leptin deficiency on the
number of ßgal+ cells in the arcuate nucleus,
obese ob/ob mice heterozygous for targeted disruption of the
NPY gene (ob/ob, NPY+/-) received single daily
ip injections of either murine leptin (70 µg; n = 8; a gift from
Zymogenetics) or saline vehicle (n = 8) for 4 days. Food intake
and body weight were measured daily, and mice were killed and brains
removed as described above between 0800–1200 h on the morning of the
fifth day. Untreated littermates heterozygous for both the
ob mutation and targeted disruption of the NPY gene
(ob/+, NPY+/-; n = 5; weight, 25.9 ±
0.9 g) were also killed for comparison to data obtained in
leptin-deficient mice. Blood from each animal was obtained by cardiac
puncture, separated into serum, and stored at -20 C until assay of
glucose and insulin concentrations.
ßGal staining and quantitation
Coronal cryostat sections (14 µm) were thaw-mounted on slides,
washed for 2 min in iced PBS, and fixed for 5 min in ice-cold 0.02%
glutaraldehyde-3.8% formaldehyde. Fixed tissue sections were incubated
at 37 C in a solution containing PBS, potassium ferricyanide, potassium
ferrocyanide, MgCl, and galactopyranoside dissolved in
dimethylsulfoxide (11). ßgal+ cells were
identified by their blue-staining nuclei. To determine the time course
of ßgal staining in the arcuate nucleus, coronal sections from
NPY-/- (n = 2) and NPY+/- mice (n
= 3) were incubated as described above for a total duration of 72
h. At selected time points, the slides were removed, and the number of
ßgal+ cells in the arcuate nucleus was
determined from three sections from each animal. The slides were then
returned to the incubation solution. Based on the results of this
experiment, slides were incubated for 24 h in all subsequent
experiments. Quantitation of ßgal+ cell number
in the arcuate nucleus and thalamic reticular nucleus was performed by
a technician blinded to the study conditions. Brain areas were imaged
in 24-bit color using a Sony color CCD camera (Sony Corp., Park Ridge,
NJ). Thresholding of color images was based on color intensity, hue,
and saturation, using an automated algorithm in the image analysis
software (MCID, St. Catherines, Canada). ßGal+
cells were defined on the basis of a suprathreshold density detected in
a contiguous area of sufficient size to exclude background staining
artifact. In each coronal brain slice, the number of
ßgal+ cells was determined in both the
hypothalamic arcuate nucleus and the thalamic reticular nucleus by
computing the total number of suprathreshold pixels and dividing by the
mean pixel number per cell. This estimate was employed to eliminate
quantitation artifact due to overlap of adjacent cells that stain
positively for ßgal. A total of 10 sections were selected at
equidistant intervals throughout the entire rostro-caudal extent of the
arcuate nucleus of each animal (-2.0 to -3.5 mm caudal to bregma),
and the mean number of ßgal+ cells from each
anatomical region was determined for each study group.
Plasma assays
For Exp 2, plasma glucose concentrations were measured by the
glucose oxidase method (Glucose Analyzer II, Beckman Instruments, La
Brea, CA). Plasma immunoreactive insulin levels were measured by RIA
using a rat insulin standard (13).
Statistical analysis
Unless otherwise noted, data are presented as the mean ±
SEM. Comparisons between data sets in Exp 1 and 2 used
two-tailed Student’s t test. One-way ANOVA with Fisher’s
test for multiple comparisons was used to compare data obtained in the
three groups of animals studied in Exp 3. The null hypotheses of no
difference between groups was rejected at P <
0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). To determine the optimal
incubation time for detecting differences in lacZ gene
expression, we measured the time course of the appearance of
ßgal+ cells in the arcuate nucleus of
NPY-/- and NPY+/- mice (Fig. 2A). The maximum number of
ßgal+ cells was detected at 24 h.
ßGal+ cells were detectable after 1-h
incubation in NPY-/-, but not NPY+/-, mice.
The time required for the appearance of the half-maximal
ßgal+ cell number was reduced by 60% (3.5
vs. 9.0 h) in mice with two, compared with only one,
mutant allele, and the maximal number of ßgal+
cells detected in NPY-/- mice was 203%
(P < 0.05) of that detected in NPY+/-
mice. This leftward shift in the time course of appearance of
ßgal+ cells in mice bearing two copies,
compared with a single copy, of the lacZ allele suggests
that ßgal+ cell number increases in parallel
with increases in lacZ gene transcription. We selected a
24-h incubation time to detect differences in
ßgal+ cell number in subsequent experiments,
because this duration yields the maximum number of
ßgal+ cells and is associated with an
approximate doubling of the number of ßgal+
cells in mice with two copies of the lacZ allele compared
with that in mice that have one copy.
|
|
). Throughout the
rostro-caudal extent of both brain regions, however,
NPY-/- mice had more ßgal+ cells
than did NPY+/- mice, with a mean increase of 260%
detected in both the thalamus and the hypothalamus.
Exp 2
To determine whether fasting increases transcription of the NPY
gene in arcuate nucleus neurons, the number of
ßgal+ cells in brain sections from
NPY+/- mice deprived of food for 48 h were compared
with those obtained from controls fed ad libitum. In fasted
mice, the number of arcuate nucleus neurons staining positively for
ßgal was visibly increased (Fig. 3).
The mean number of ßgal+ cells per section of
arcuate nucleus varied considerably (from 2–12) throughout its
rostro-caudal extent in NPY+/- mice that were fed ad
libitum, with the maximal number of ßgal+
cells located in the midregion of the arcuate nucleus (Fig. 4). Although the distribution pattern of
ßgal+ cells throughout the length of the
arcuate nucleus was similar in food-deprived NPY+/- mice,
the mean ßgal+ cell number was consistently
higher than that in fed controls (Fig. 4). In contrast, no effect of
food deprivation was observed on the number of
ßgal+ cells in the thalamic reticular nucleus
(Fig. 4). Thus, fasting increased mean ßgal+
cell number in the arcuate nucleus by 260%, but had no effect in the
reticular nucleus (Fig. 4). The effect of fasting to increase
expression of the lacZ reporter gene, therefore, was
localized to the arcuate nucleus.
|
|
). This decline in food intake was
associated with a significant decrease in body weight (-12.6%; Table 1). Leptin also significantly reduced
serum glucose (-37%) and insulin concentrations (-95%) in
ob/ob, NPY+/- mice to values similar to those
measured in lean ob/+, NPY+/- mice (Table 1).
The distribution of ßgal+ cells throughout the
arcuate nucleus in both leptin-treated and saline-treated
ob/ob, NPY+/- mice was similar to that in
leptin-replete ob/+, NPY+/- mice (data not
shown). However, the mean number of arcuate nucleus
ßgal+ cells was 67.6% higher in saline-treated
ob/ob, NPY+/- mice (P < 0.05)
than in untreated lean littermates (ob/+,
NPY+/- mice), as shown in Fig. 5B. This effect of genetic
leptin deficiency was attenuated by 36.2% (P < 0.05)
in leptin-treated ob/ob, NPY+/- mice, although
the values remained significantly above those observed in nonobese,
leptin-replete ob/+, NPY+/- mice (Fig. 5B).
Therefore, leptin administration for 4 days to leptin-deficient mice
significantly attenuated the increase in the number of
ßgal+ neurons in the arcuate nucleus detected
in leptin-deficient mice. Compared with leptin-replete ob/+,
NPY+/- mice, the saline-treated ob/ob,
NPY+/- mice also showed elevations of reticular nucleus
ßgal+ cell number (Fig. 5B), but leptin
administration did not reduce ßgal+ cell number
in the reticular nucleus.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We quantified ßgal expression in neurons by counting the number of
ßgal+ cell nuclei, which provides an indirect
measurement of lacZ expression. The rationale for this
approach was based on the time course of ßgal+
staining in brain sections from mice bearing either one
(NPY+/-) or two lacZ alleles
(NPY-/-; Fig. 2A). This analysis revealed an effect of
lacZ allele number on both the rate of appearance and the
maximal number of ßgal+ cells in the arcuate
nucleus compared with those in NPY+/- mice; the incubation
time required to yield the half-maximal ßgal+
cell number was reduced by 60%, and the maximal
ßgal+ cell number was doubled in
NPY-/- mice. One explanation for this result is that
differences in the rate of reporter gene transcription determine
whether the amount of ßgal that accumulates intracellularly will be
sufficient to permit detection in our assay. Based on the assumption
that the amount of ßgal synthesized per neuron varies in direct
proportion to the number of lacZ alleles, these observations
suggest that differences in lacZ gene transcription are
reflected by differences in ßgal+ cell number
that vary with the duration of the assay. As incubation for 24 h
yielded an approximate doubling of ßgal+ cells
in NPY-/- compared with NPY+/- mice, we
selected this duration to estimate lacZ gene transcription
in subsequent experiments.
Because the lacZ gene was inserted into the first exon at the initiation codon of NPY, the 5'-flanking sequences are unaffected. Thus, the promoter and enhancer elements that regulate NPY should now regulate the lacZ gene, although it is possible that regulatory elements may lie downstream of the initiation codon that have been deleted, disrupted, or inappropriately displaced. It is unlikely that cell-specific enhancers in the NPY gene were affected, however, because ßgal cells were previously detected only in areas known to express NPY in both the brain and the periphery (10). This method, therefore, should be generally applicable for measuring the in vivo regulation of gene transcription in animals in which a lacZ gene is targeted appropriately to a gene of interest. In the nervous system, this method relies on the targeting of ßgal to the cell nucleus to prevent its transport to axon terminals, although in other tissues, this step may not be required.
The well documented effect of fasting to increase NPY mRNA levels within arcuate nucleus neurons (8, 17, 18, 19) could reflect an increase in NPY gene transcription, increased stability of its mRNA transcript (20), or both. Our results provide direct evidence that increased NPY gene expression during fasting involves an increase in transcription. Although our findings do not exclude the possibility that fasting also affects NPY mRNA stability, such an effect is unlikely to explain the differences in ßgal staining that we observed. This is because the lacZ mRNA produced from the targeted allele bears only a small portion of the 5'-untranslated region of NPY fused to lacZ mRNA, with a 3'-untranslated region derived from the mouse protamine 1 gene. Thus, proteins that might stabilize NPY mRNA are unlikely to recognize the lacZ reporter gene mRNA.
The effect of fasting to increase NPY gene transcription in NPY+/- mice was detected throughout the rostro-caudal length of the arcuate nucleus and was most prominent in its midregion and rostral portions. This finding is similar to the regional pattern of increase in the NPY mRNA content of the arcuate nucleus of fasted rats (17). In contrast, fasting did not influence NPY gene transcription in the thalamus, suggesting that arcuate nucleus neurons possess unique characteristics that permit physiological changes in the hormonal or metabolic milieu to be transduced into changes in NPY gene transcription. A wide variety of metabolic and hormonal responses to fasting are implicated as signals to arcuate nucleus neurons that may influence expression of the NPY gene (1, 8, 18, 19, 20, 21, 22, 23, 24, 25). To investigate the role of leptin deficiency as a stimulus to arcuate nucleus NPY gene transcription, we studied obese, leptin-deficient ob/ob mice heterozygous for the mutant NPY allele. We found that genetic leptin deficiency increased NPY gene transcription in the arcuate nucleus, although the effect appeared to be somewhat smaller than that elicited by fasting. The increase in ßgal+ cell number was also smaller than the 4-fold increase in NPY mRNA previously reported in the hypothalamus of ob/ob mice (5). Although our findings suggest that hypothalamic NPY gene transcription is increased in ob/ob mice, an increase in mRNA stability may also contribute to increased NPY mRNA expression in the hypothalamus of these animals.
Leptin-deficient ob/ob mice suffer from a variety of neuroendocrine and metabolic disturbances, many of which have the potential to influence hypothalamic NPY gene expression. To investigate whether leptin deficiency per se increases hypothalamic NPY gene transcription, we administered leptin to ob/ob, NPY+/- mice for 4 days. We found that ßgal+ cell number in leptin-treated ob/ob, NPY+/- mice was significantly reduced compared with that in vehicle-treated ob/ob, NPY+/- controls. This finding strengthens the conclusion that leptin inhibits NPY gene transcription in the arcuate nucleus in vivo. Leptin treatment, however, did not completely normalize the number of ßgal+ neurons detected in the arcuate nucleus of ob/ob mice despite marked suppression of food intake. Thus, factors other than leptin deficiency may stimulate NPY gene transcription in the hypothalamus of ob/ob mice. One caveat to this conclusion is that the time course over which a decrease in lacZ gene transcription results in reduced ßgal staining is unknown. As ßgal+ cells may persist transiently after lacZ transcription ceases, measures of ßgal staining may underestimate the extent to which NPY gene transcription is inhibited by leptin.
Pathways other than those containing NPY appear to be capable of mediating at least some of leptin’s effects in the brain. For example, leptin receptors in the arcuate nucleus are abundantly expressed by neurons containing POMC (26). Therefore, it is possible that leptin alters the function of a population of neurons that make synaptic contacts with arcuate nucleus NPY neurons and that changes in the firing rate of these neurons in response to leptin produce changes in NPY gene transcription. This possibility suggests that both direct (27) and indirect mechanisms may contribute to leptin’s effects on NPY gene transcription.
As overeating, obesity, and numerous neuroendocrine and metabolic defects in ob/ob mice are attenuated by genetic NPY deficiency (12), NPY activation appears to be a key component of the response to a sustained reduction in leptin signaling. The ability of leptin to constrain activation of the NPY pathway, therefore, is likely to be an integral component of its role in energy homeostasis. This observation supports our conclusion that inhibition of hypothalamic NPY gene transcription is an important mechanism by which leptin regulates body weight.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received October 7, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  Z. Hawke, T. R. Ivanov, D. A. Bechtold, H. Dhillon, B. B. Lowell, and S. M. Luckman PACAP Neurons in the Hypothalamic Ventromedial Nucleus Are Targets of Central Leptin Signaling J. Neurosci., November 25, 2009; 29(47): 14828 - 14835. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Stengel, M. Goebel, M. Million, M. P. Stenzel-Poore, P. Kobelt, H. Monnikes, Y. Tache, and L. Wang Corticotropin-Releasing Factor-Overexpressing Mice Exhibit Reduced Neuronal Activation in the Arcuate Nucleus and Food Intake in Response to Fasting Endocrinology, January 1, 2009; 150(1): 153 - 160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. X. Huang, X. G. Luo, L. Lu, and B. Liu Effects of Age and Strain on Yolk Sac Utilization and Leptin Levels in Newly Hatched Broilers Poult. Sci., December 1, 2008; 87(12): 2647 - 2652. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Barnes, S. D. Primeaux, and G. A. Bray Food deprivation increases the mRNA expression of {micro}-opioid receptors in the ventral medial hypothalamus and arcuate nucleus Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2008; 295(5): R1385 - R1390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Scarlett, X. Zhu, P. J. Enriori, D. D. Bowe, A. K. Batra, P. R. Levasseur, W. F. Grant, M. M. Meguid, M. A. Cowley, and D. L. Marks Regulation of Agouti-Related Protein Messenger Ribonucleic Acid Transcription and Peptide Secretion by Acute and Chronic Inflammation Endocrinology, October 1, 2008; 149(10): 4837 - 4845. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. T. Phillips and R. D. Palmiter Role of Agouti-Related Protein-Expressing Neurons in Lactation Endocrinology, February 1, 2008; 149(2): 544 - 550. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Dridi, C. Ververken, F. B. Hillgartner, A. Lutgarde, E. Van der Gucht, L. Cnops, E. Decuypere, and J. Buyse FAS inhibitor cerulenin reduces food intake and melanocortin receptor gene expression without modulating the other (an)orexigenic neuropeptides in chickens Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2006; 291(1): R138 - R147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Cho, M. J. Blaser, F. Francois, J. P. Mathew, X. Y. Ye, J. D. Goldberg, and E. J. Bini Helicobacter pylori and Overweight Status in the United States: Data from the Third National Health and Nutrition Examination Survey Am. J. Epidemiol., September 15, 2005; 162(6): 579 - 584. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nagamori, M. Ishibashi, T. Shiraishi, Y. Oomura, and K. Sasaki Effects of Leptin on Hypothalamic Arcuate Neurons in Wistar and Zucker Rats: An In Vitro Study Exp Biol Med, November 1, 2003; 228(10): 1162 - 1167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Hill and J. E. Levine Abnormal Response of the Neuropeptide Y-Deficient Mouse Reproductive Axis to Food Deprivation But Not Lactation Endocrinology, May 1, 2003; 144(5): 1780 - 1786. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Watanobe Leptin directly acts within the hypothalamus to stimulate gonadotropin-releasing hormone secretion in vivo in rats J. Physiol., November 15, 2002; 545(1): 255 - 268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Watanobe and S. Habu Leptin Regulates Growth Hormone-Releasing Factor, Somatostatin, and alpha -Melanocyte-Stimulating Hormone But Not Neuropeptide Y Release in Rat Hypothalamus In Vivo: Relation with Growth Hormone Secretion J. Neurosci., July 15, 2002; 22(14): 6265 - 6271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Abu-Elheiga, M. M. Matzuk, K. A. H. Abo-Hashema, and S. J. Wakil Continuous Fatty Acid Oxidation and Reduced Fat Storage in Mice Lacking Acetyl-CoA Carboxylase 2 Science, March 30, 2001; 291(5513): 2613 - 2616. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. F. Breininger and D. G. Baskin Fluorescence In Situ Hybridization of Scarce Leptin Receptor mRNA using the Enzyme-Labeled Fluorescent Substrate Method and Tyramide Signal Amplification J. Histochem. Cytochem., December 1, 2000; 48(12): 1593 - 1600. [Abstract] [Full Text]
|
|
|
|
|
|
T. M. Loftus, D. E. Jaworsky, G. L. Frehywot, C. A. Townsend, G. V. Ronnett, M. D. Lane, and F. P. Kuhajda Reduced Food Intake and Body Weight in Mice Treated with Fatty Acid Synthase Inhibitors Science, June 30, 2000; 288(5475): 2379 - 2381. [Abstract] [Full Text]
|
|
|
|
|
|
M. C. Lebrethon, E. Vandersmissen, A. Gerard, A. S. Parent, J. L. Junien, and J. P. Bourguignon In Vitro Stimulation of the Prepubertal Rat Gonadotropin-Releasing Hormone Pulse Generator by Leptin and Neuropeptide Y through Distinct Mechanisms Endocrinology, April 1, 2000; 141(4): 1464 - 1469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Baskin, M. W. Schwartz, R. J. Seeley, S. C. Woods, D. Porte , Jr., J. F. Breininger, Z. Jonak, J. Schaefer, M. Krouse, C. Burghardt, et al. Leptin Receptor Long-form Splice-variant Protein Expression in Neuron Cell Bodies of the Brain and Co-localization with Neuropeptide Y mRNA in the Arcuate Nucleus J. Histochem. Cytochem., March 1, 1999; 47(3): 353 - 362. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
