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Elliot P. Joslin Laboratory (N.A.T., J.W.M., E.M.-F.), Joslin Diabetes Center, Division of Endocrinology (J.K.E., J.S.F.) and Department of Neurology (J.K.E.), Beth Israel Deaconess Medical Center, Program in Neuroscience (J.K.E.), Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Eleftheria Maratos-Flier, M.D., Joslin Diabetes Center, Room 620, One Joslin Place, Boston, Massachusetts 02215. E-mail: emarat{at}joslab.harvard.edu
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Abstract |
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Introduction |
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A number of hypothalamic factors are known to be important regulators
of feeding behavior in rodents. These include the orexigenic peptides
neuropeptide Y (NPY) (8, 9, 10, 11, 12, 13, 14) and agouti-related protein (AGRP) (15),
which are expressed in the arcuate nucleus (Arc) (11, 15);
melanin-concentrating hormone (MCH) (16, 17, 18), which is expressed in the
lateral hypothalamic area (LHA) and the zona incerta (ZI) (16, 17); and
the appetite-inhibiting peptide, 
MSH (19, 20), which is also
expressed in the arcuate (19). In the ob/ob mouse, levels of
messenger RNAs (mRNAs) of transcripts encoding the orexigenic peptides
are elevated (11, 15, 18), and the level of POMC transcript (which is
the precursor of MSH) is reduced (21). These changes are presumably
the result of the absence of leptin signaling.
In the present study, we sought to examine the expression of these peptides, by in situ hybridization histochemistry, in UCP-DTA mice that are hyperleptinemic and are resistant to exogenous leptin, with respect to both body weight and food intake (6). We expected that the leptin resistance might map to the hypothalamus and that levels of transcripts of orexigenic neuropeptides normally lowered by leptin would be elevated and the level of POMC would be decreased, as seen in ob/ob mice. Unexpectedly, we found that in the Arc, NPY and AGRP mRNA levels were appropriately reduced and that POMC mRNA was not low. In the lateral hypothalamus, MCH levels were also reduced. Interestingly, the UCP-DTA mice showed expression of NPY in the dorsal medial hypothalamic nucleus (DMH), an area in which NPY is not typically expressed in normal-weight animals.
These results indicate that these leptin targets in the Arc and lateral hypothalamus do not behave as though they are resistant to leptin and, indeed, the changes seen are consistent with the possibility that leptin is exerting the appropriate actions on these neurons. Hence, hyperphagia in the UCP-DTA animals is likely to be mediated by factors in the hypothalamus other than those studied here or by actions at distal sites. The finding of de novo expression of NPY in the DMH is of interest, because such induction has been reported in two other hyperleptinemic models of mouse obesity (agouti and melanocortin receptor 4 knockout (MC4-R KO) mice) (22, 23). This finding may therefore be a general indicator of hypothalamic resistance to leptin action.
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Materials and Methods |
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In situ hybridization histochemistry
Animals were deeply anesthetized with sodium pentobarbital (90
mg/kg wt ip) 1 h into the onset of the light cycle and perfused
transcardially with 20 ml saline and 50 ml 10% neutral buffered
formalin (Accustain, Sigma Chemical Co., St. Louis,
MO). Brains were removed and postfixed for 4 h and were
subsequently stored in 20% sucrose in diethylpyrocarbonate-treated PBS
for 24 h. Brains were frozen with powdered dry ice and sectioned
in the coronal plane (30 µm) by use of a sliding microtome (AO
Instrument Co., Buffalo, NY). The in situ hybridization
protocol was performed by a modification of a published protocol (24).
Briefly, brain slices (each series containing one-fourth of all
sections) were mounted on slides (Superfrost Plus, Fisher
Scientific International, Inc., Agawam, MA), postfixed in
4% formaldehyde, acetylated with acetic anhydride 0.25%, dehydrated
in ethanol, and stored at -20 C until hybridization. Antisense
riboprobes were generated with the use of a commercially available
in vitro transcription kit (Promega Corp.,
Madison, WI), employing 1 µg of the linearized DNA template,
35S uridine 5'-triphosphate (NEN, Boston, MA) and the
appropriate RNA polymerase (SP6 for NPY, MCH, and POMC, and T3 for AGRP
probes). Sections were hybridized with the appropriate riboprobe in
hybridization buffer (50% formamide, 0.1% SDS, 0.01% thiosulfate,
0.1 M dithiothreitol (DTT), 0.6 M NaCl, 10
mM Tris (pH 7.5), 1 mM EDTA, 5% dextran
sulfate, 0.01% sheared salmon sperm DNA, 0.05% total yeast transfer
RNA, 0.01% yeast transfer RNA and 1 x Denhardt’s) at 57 C in an
air oven for 18 h. Subsequently, tissue sections were treated with
ribonuclease A (20 µg/ml) for 30 min, rinsed in 300 mM
NaCl, 30 mM citrate [2 x saline-sodium citrate
(SSC)], washed in 300 mM NaCl, 30 mM citrate
(2 x SSC) and 1 mM DTT at 50 C for 1 h, and in
30 mM NaCl, 3 mM citrate (0.2 x SSC) and
1 mM DTT at 55 C and 60 C for 1 h each, dehydrated in
ethanol, 0.3 M ammonium acetate and 1 mM DTT,
air dried and exposed to Biomax MR film (Kodak, Rochester, NY) for
24–48 h, together with commercially available 14C
standards (Amersham Life Science, Buckinghamshire, UK) to verify
linearity of the film response and the consistency of signal detection
across films.
The absorbance of the autoradiographic images was measured with a computing densitometer (Molecular Dynamics, Inc., Sunnyvale, CA) and the ImageQuant software (Molecular Dynamics, Inc.). Briefly, a rectangle was drawn enclosing each nucleus of interest separately and was subsequently reproduced in the same dimensions over all the images containing hybridization signal from the hypothalamic nucleus being studied. The absorbances in the rectangular areas encompassing the nucleus of interest were integrated over each set of brain sections (one-fourth of all the sections from each brain). In addition, an adjacent area that did not contain specific hybridization signal was chosen to compute the background density and was subtracted from the absorbance measurements of signals over each hypothalamic nucleus being studied. Statistical comparisons of absorbance differences between animal groups were performed by the Mann-Whitney test (Statview 4.5, Abacus Concepts, Berkeley, CA), and P values less than 0.05 were considered significant.
For the purpose of display, scanned autoradiographic images were imported into the Adobe PhotoShop 4.0 package (Adobe Systems, Inc, San Jose, CA) as grayscale images, converted into duotone images (without any adjustment of the brightness, contrast, or texture of the individual panels), and printed on a Fujix printer (Fuji-Xerox Ltd. Pictography 3000, Tokyo, Japan). Slides were also dipped in NTB-2 emulsion (Kodak), exposed at 4 C for 2–3 weeks, developed, counterstained with thionin, dehydrated in graded ethanol series, and photographed with the BX60 microscope and the PM20 photomicrograph system (Olympus Corp., Japan). Additionally, densitometry data were plotted in per cent relative density units, with the highest absorbance in each figure set arbitrarily at 100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Using AGRP (Fig. 1) or
POMC (Fig. 2) antisense probes,
hybridization signal was present only in the Arc, with the expression
pattern for AGRP more closely resembling that of NPY, as suggested by
the hybridization signals in adjacent brain sections hybridized with
either NPY or AGRP probes. In addition, using an MCH antisense probe,
hybridization signal was detected only in the LHA and the ZI, starting
at the level of the anterior hypothalamic nucleus and caudally to the
mammillary nucleus complex (Fig. 1). Overall, the hybridization signal
in the Arc for NPY, AGRP, and POMC mRNA was present in approximately
6–7 slices from each series of brain sections, and hybridization
signal in the LHA and the ZI for MCH mRNA was detected in approximately
4–5 slices from each series of brain sections.
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and 3) and AGRP (Figs. 1 and 4) antisense probes, demonstrate that
expression of NPY and AGRP is decreased in the Arc of the UCP-DTA mice,
compared with their expression in control animals. In contrast,
qualitative examination suggests that the expression of NPY in the
cortex is similar in both UCP-DTA and control mice (Fig. 1). In
addition to expression of NPY in the Arc, there is prominent expression
of NPY in the caudal DMH (cDMH) in the UCP-DTA mice but not in the
FVB/NJ (control) mice (Figs. 1 and 3). Furthermore, representative
autoradiographic and bright-field images of emulsion-dipped slides of
coronal brain sections at the level of the Arc (caudal to the level
shown on Figs. 3 and 4), obtained after in situ
hybridization with a POMC antisense probe, are depicted in Figs. 2 and 5, and they show no difference in POMC
expression between UCP-DTA and control mice.
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and 6, and they demonstrate relatively
decreased MCH expression in UCP-DTA mice, compared with MCH expression
in the hypothalamus of control animals.
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). Expression of MCH in the LHA and the
ZI is decreased by 24% in UCP-DTA mice (n = 4), compared with MCH
expression in control animals (n = 4, P < 0.02)
(Fig. 7). In contrast, there is prominent expression of NPY in the DMH
of the UCP-DTA animals, although such expression is absent in control
mice (P < 0.02) (Fig. 8). Furthermore, expression of POMC in
the Arc does not differ between UCP-DTA and control animals (n =
5, P = 0.60) (Fig. 9).
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Discussion |
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Of the peptides examined in this study, NPY is the best characterized to date. Central administration of NPY to rodents promotes obesity, both by increased food intake (8, 9) and decreased BAT thermogenesis (10); and chronic intracerebroventricular (ICV) administration leads to obesity (9). Furthermore, NPY expression in the Arc of the hypothalamus is increased in several rodent models of obesity, including ob/ob mice (11), gold-thioglucose-treated mice (12), and Zucker fatty rats (13). NPY seems to be under negative control by leptin, because leptin administration reverses the increase in NPY mRNA observed in ob/ob mice (14, 25), and it reduces the increase in NPY observed with starvation (26). Although NPY has an important role in mediating leptin action in the central nervous system (14), mice with targeted disruption of NPY have a normal feeding phenotype and respond normally to exogenous leptin (27), suggesting that NPY is not essential for leptin action. However, breeding NPY-deficient animals into the ob/ob background produces mice that have an attenuated obese phenotype, confirming that NPY plays some role in the development of obesity in the ob/ob model (28).
AGRP is also expressed in the Arc and is implicated in the regulation of feeding behavior (15). This neuropeptide was recently characterized (15) on the basis of its homology with the agouti signaling protein (ASP) (29), a competitive antagonist of melanocortins at the melanocortin receptors -1 and -4 (MC1-R and MC4-R) (30). De novo hypothalamic expression of ASP as a consequence of a promoter rearrangement (31), with subsequent inhibition of MC4-R by the ASP, is the cause of the agouti obesity syndrome (31, 32, 33, 34). In contrast to ASP, AGRP is normally expressed in the hypothalamus, where it may play an important role in the regulation of energy balance and body adiposity (35), because AGRP is known to be overexpressed in the Arc of ob/ob mice (15), and transgenic animals overexpressing AGRP become obese (35).
POMC is also expressed in the arcuate hypothalamic nucleus (19), where
it is processed to yield several peptides (including melanocortins,
such as the MSH, and opiate peptides) (19). ICV administration of
MSH to rats results in decreased feeding (20), and rats given
monosodium-L- glutamate in the neonatal period become
POMC and MSH-deficient and subsequently become obese (19, 36).
Furthermore, POMC expression in the arcuate is decreased in both the
ob/ob and the leptin-resistant db/db mice (21, 37). The POMC mRNA increases with leptin treatment in the former group
(21, 37), suggesting that POMC may constitute one of the leptin targets
in the central nervous system. It has been recently demonstrated that
AGRP is a competitive antagonist at the MC3 and MC4-R (38, 39),
suggesting that endogenous melanocortins derived from POMC may act as
antagonists to AGRP at these receptors in the hypothalamus (39).
MCH is another important orexigenic peptide, and it was originally isolated from salmon pituitaries (40). MCH is present in the mammalian hypothalamus, specifically the ZI and the LHA (16, 17), where it is overexpressed in ob/ob mice; and its expression increases in both ob/ob and lean mice, in response to fasting (18). ICV administration of MCH to rodents increases food intake (18), although chronic ICV administration does not lead to obesity (41), perhaps as a result of compensatory changes in other neuropeptide systems involved in the regulation of food intake.
In this report, we substantially extend our previous observations, regarding the basis for hyperphagia in these mice (6), in a number of ways. First, we demonstrate that the decrease in hypothalamic NPY mRNA observed on Northern blots is caused by decreased arcuate expression of NPY, as determined by in situ hybridization. Second, we observe that two other orexigenic neuropeptides that are increased in states of decreased leptin levels or action, e.g. MCH (18) and AGRP (15), are also decreased in hypothalami of UCP-DTA mice. Third, POMC, which has been previously shown to be positively regulated by exogenous leptin (in ob/ob mice) and reduced in expression in either leptin-deficient (ob/ob mice) or leptin-resistant (db/db mice) states (21, 37, 42), is normal in UCP-DTA mice. Decreased expression of NPY, AGRP, and MCH in the UCP-DTA mice indicates that these peptides have responded appropriately to the high leptin levels. In the case of POMC, total lack of response to leptin would be associated with a 50–60% decrease in POMC expression (42). Our data indicate that POMC expression in UCP-DTA mice is normal, rather than decreased. Thus, leptin resistance seems not to exist at the level of these neurons, and changes in one or more additional neuropeptides that are capable of influencing food intake must underlie hyperphagia of these mice.
In contrast to the decreased NPY mRNA in the Arc, NPY expression is increased in the cDMH of the obese UCP-DTA mice. The DMH has been proposed to be an important site for integration of leptin action in the hypothalamus (43), and it has an abundant expression of the long form of the leptin receptor (14, 44, 45). Cells in the DMH are activated, as suggested by induction of c fos immunoreactivity, in response to systemic leptin administration (43, 46). Finally, leptin-activated cells, in the DMH, project and provide major input to the paraventricular nucleus (47).
The mechanisms leading to increased NPY expression in the DMH of the UCP-DTA mice are unclear. It is interesting that induction of NPY expression in the DMH has been previously demonstrated in three other paradigms: the obese agouti (Ay) mice (22), the obese MC4-R KO mice (22, 23), and lactating rats (48). In the case of the obese agouti and MC4-R KO mice, it has been suggested that NPY expression in the DMH results from loss of melanocortin input to the DMH (22). It is unlikely that there is reduced melanocortin input to the DMH in the UCP-DTA mice because, at the mRNA level, endogenous AGRP levels are decreased and POMC levels are normal, indicating that anorectic inputs to melanocortin receptors should be normal, or even enhanced, in UCP-DTA mice. This indicates that increased NPY expression in the DMH of UCP-DTA mice may reflect either impaired output from neurons expressing melanocortin receptors or may be the result of impaired signaling from leptin or from another, as yet unidentified, input. Notably, induction of the suppressor of cytokine signaling-3 (SOCS-3) protein has been recently demonstrated in response to leptin administration in the leptin-deficient ob/ob mice, as well as at baseline in the obese, hyperleptinemic, and leptin-resistant, yellow agouti (Ay) mice (49). The SOCS-3 may act as an inhibitor of leptin action and thereby explain leptin resistance (49). The induction of NPY expression in the DMH may contribute to the development and the perpetuation of the obese phenotype of the UCP-DTA mice or may be simply an indicator of leptin resistance.
In summary, our data indicate that expression of mRNAs encoding NPY and AGRP in the Arc are decreased in UCP-DTA mice, and expression of POMC mRNA in the Arc of UCP-DTA animals is appropriate to high leptin levels in these animals. Expression of MCH in the LHA and the ZI is also decreased in UCP-DTA mice. Our findings suggest that the pathways regulating the expression of these neuropeptides, according to the degree of body adiposity, remain intact in these animals. Further studies will address the mechanism for hyperphagia in these mice, in the face of appropriate leptin action on key regulatory hypothalamic neuropeptides, and the mechanisms whereby BAT deficiency produces these effects.
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Acknowledgments |
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Footnotes |
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Received April 15, 1998.
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