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Department of Physiology, University of California San Francisco, San Francisco, California 94143-0444
Address all correspondence and requests for reprints to: SuJean Choi, Department of Physiology, Box 0444, University of California San Francisco, San Francisco, California 94143-0444. E-mail: suchoi{at}itsa.ucsf.edu
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Abstract |
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Introduction |
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Antagonism by the agouti peptide or deletion of the melanocortin 4-receptor (MC4-R) results in obesity probably as a consequence of abnormal function of the MC4-R on neurons in the dorsomedial nuclei (DMN) and paraventricular nuclei (PVN), which are innervated by melanocortin-producing, POMC-expressing cells in the arcuate nuclei (ARC) (2). Neonatal treatment of rats with monosodium glutamate results in obese adults with reduced numbers of POMC neurons (3). Study of the ob/ob, db/db, and fa/fa phenotypes (the latter two lack leptin receptors) shows that abnormal leptin signaling results in elevated neuropeptide Y (NPY) synthesis in ARC (e.g. see Ref. 4). NPY, probably acting at receptors in the hypothalamus, causes increased food intake and insulin secretion (5). Although mice with a null mutation of the NPY gene exhibit few of the expected behavioral or metabolic effects known to be associated with acute provision or antagonism of NPY, the cross of NPY knockouts with ob/ob mice decreases the degree of obesity in the ob/ob mouse (6). In addition to the above signals that clearly regulate feeding and energy stores, it is increasingly apparent that other neuropeptide systems probably are involved in the circuits (e.g. Refs. 7, 8, 9, 10).
In rats, lesions of the medial hypothalamus cause obesities through mechanisms that are site dependent. Lesions that include the PVN produce obesity that depends on increased food intake, whereas those that include primarily the VMN result in a metabolic obesity that is independent of increased food intake (11, 12). However, electrolytic or neurotoxin lesions in the medial hypothalamus are seldom restricted to a single nucleus because of the close apposition of these cell groups (e.g. Refs. 11, 13, 14).
Because all four of the medial hypothalamic cell groups have been implicated in the regulation of energy balance (15), reported to contain leptin receptors (16, 17), and strongly interconnected (18, 19, 20, 21, 22), it is important to distinguish among them, if possible, to learn more about functions in each nucleus.
We used small injections of colchicine (23) to inhibit function in each of four medial hypothalamic cell groups so that we could determine the specific effects of inhibition in a given cell group on food intake, hormones, and fat stores. Previous studies using colchicine as a reversible neurotoxin have shown acute responses that are identical to those of irreversible excitotoxin lesions (23, 24). The results show that, although obesity occurs after disruption of function in 3 of the 4 medial hypothalamic nuclei, the response to inhibition in each cell group is unique using comparison of several endpoints.
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Materials and Methods |
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Surgery
All rats were anesthetized with a rodent cocktail consisting of
ketamine/xylazine/acepromazine (77/1.5/1.5 mg/kg; 1 ml/kg ip) and
placed in a stereotaxic apparatus. Injections of colchicine:
fluorescein-colchicine (1 µg/0.1 µl) or saline (0.1 µl) were made
using a Hamilton microsyringe and a 25-gauge needle. A 50:50 mixture of
colchicine and inactive fluorescein-colchicine (1 µg/0.1 µl;
Molecular Probes, Inc., Eugene, OR) was used to determine
the placement of injections and estimate the spread of the injectate.
Bilateral injections of either drug or vehicle were made in the ARC
(n = 27), VMN (n = 13), PVN (n = 12), and DMN (n =
20) using coordinates based on Paxinos and Watson (25). The upper
incisor bar was positioned -3.3 mm below horizontal zero and the
following stereotaxic coordinates from Bregma were used: VMN
[anterior-posterior (AP), -2.5 mm; medial-lateral (ML), ± 0.7 mm;
dorsal-ventral (DV), 9.2 mm]; DMN (]AP, -2.8 mm; ML, ± 0.7 mm; DV,
8.4 mm), ARC (AP, -2.4 mm; ML, ± 0.3 mm; DV, 10.0 mm), and PVN (AP,
-1.8 mm; ML, ± 0.5 mm; DV, 8.4 mm). To reduce pressure damage and
reflux, injections of either vehicle or drug were made over 1 min, and
the needle was removed 5 min later.
Sample collection
Five days after surgery, blood samples were taken in the morning
(0900–1030) by decapitation after 30-min restraint stress with a basal
sample collected from a tail nick at time 0. Trunk blood (5 ml) was
collected in tubes containing 0.3 M disodium EDTA (100
µl/tube). Brains were immediately postfixed in 10% formalin and
subsequently stored in a 30% sucrose solution. All brains were
sectioned and stained with cresyl violet to determine placement of
injections. Inactive fluorescein-colchicine from the injectate was
visualized in adjacent sections to locate its position and, by
implication, that of colchicine (23) in the site examined. All animals
with accurate bilateral placements were included. In addition to blood
samples, white adipose was collected, separated from brown adipose
tissue and weighed.
In a second set of experiments (Exp 2), rats were prepared as above with injections of colchicine into the same four medial hypothalamic nuclei but were also provided with permanent cannula guides placed over a lateral ventricle. The purpose of this experiment was to evaluate the effects of leptin on feeding, body weight, and metabolic indices in animals with colchicine inhibition of one of the four medial hypothalamic cell groups. Either leptin or saline was injected intracerebroventricularly (icv) 1 day after the lesions were made and, on day 5 were killed under basal, unstressed conditions. Most of the results from these experiments are reported elsewhere (25A ); however, we have combined the leptin and insulin data from both experiments and since icv leptin did not alter plasma leptin or insulin levels compared with saline injected animals 5 days after surgery.
RIAs
Blood samples were centrifuged at 3,000 rpm at 4 C to separate
plasma, which was subsequently stored at -20 C. Plasma B was measured
using an Immuchem double antibody corticosterone RIA kit (ICN Pharmaceuticals, Inc., Orangeburg, NY). Plasma insulin
and leptin were measured using rat insulin and leptin RIA kits
(Linco Research, Inc., St. Charles, MO).
Statistical analysis
Data were analyzed using ANOVA corrected for repeated measures
(when required). Scheffé analysis was used to test significance
of posthoc effects. Regression analysis with slope comparison was used
to test effects of colchicine disruption on the relationship of
circulating leptin to insulin. STATA (Stata Corp; College Station, TX)
and Statview (SAS Institute, Inc.; Carey, NC) were
commercial statistical packages used for the statistical analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), demonstrated discrete localization of
fluorescence within the cellular boundaries of each target nucleus.
Injections were scored as hits if the fluorescence marker was within
the cell group of interest. Occasionally, injections aimed for one
nucleus hit another medial hypothalamic cell group, in which case the
results from that rat were moved to the appropriate lesion group. If
the injections were out of the borders of any of the four target cell
groups, the rat was eliminated from the analyses. Approximately 30% of
the rats were eliminated from further study.
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) increased during both light and dark
periods during all 5 days (treatment P < 0.0001
(both); time P < 0.0001 (both)). By contrast, feeding
increased during the light but tended to decrease during the dark
periods in rats with VMN inhibition (time P < 0.0001;
time x treatment P < 0.003). There was no
increase during the light but again feeding tended to decrease during
the dark periods in rats after inhibition of the DMN (time
P < 0.001; time x treatment P <
0.003). Clearly two major patterns emerge from examination of food
intake rhythms: rats with ARC and PVN disruption eat more than normal
throughout the 24-h day, whereas rats with VMN disruption eat more
during the light period but tend to eat less during the dark, and rats
with DMN disruption eat normally during the light but tend to eat less
during the dark.
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) reflected food
intake patterns. Weight increased immediately and the increase was
sustained for 5 days after injections into the PVN and ARC [treatment:
PVN P < 0.02/ARC P < 0.01; time
P < 0.001 (both); time x treatment
P < 0.0001 (both)]. By contrast, there was only a
transient increase in body weight after VMN injections (time
P < 0.0001), and no increase after DMN injections
(time P < 0.0001; time x treatment
P < 0.0005). The cumulative increase in body weight
during the 5 days after injections showed that ARC- and PVN-disrupted
rats gained significantly more weight than their controls
(P < 0.001), whereas VMN- and DMN-disrupted rats did
not gain either more or less weight than their controls
(P > 0.2). Control injections of saline into any of
the four cell groups did not affect normal energy balance, and the
controls for each group were similar to each other (Fig. 2
, Table 1).
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).
Results collected from unstressed rats in Exp 2, 5 days
after colchicine and 4 days after icv leptin or saline injections were
pooled by lesion group with the current experiment. All data were
analyzed for the relationships between leptin and insulin. There is a
relationship between circulating leptin and insulin concentrations in
the pooled, sham-lesioned rats (Fig. 4).
A slope of approximately 0.4 and 25% of the variance in leptin
accounted for by the equation reflects the direct relationship that
exists between insulin and leptin (26). In colchicine-injected
rats, the relationships between insulin and leptin change, particularly
after PVN and DMN disruption (Fig. 5).
The best fit linear relationship describes 86% (PVN) and 51% (DMN) of
the variance between leptin and insulin in the lesioned rats, and the
slope became significantly steeper (P < 0.05 for both)
than that of the relationship in sham-lesioned rats. In ARC-lesioned
rats, there was no longer a significant relationship between leptin and
insulin. After VMN-lesions, although it was significant, the slope of
the relationship of leptin on insulin did not differ significantly from
that in sham-lesioned rats.
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. There
is a relatively constant caloric efficiency in sham-lesioned rats
during and across each experiment, with the exception of the ARC
sham-injection group, in which there was unusually high caloric
efficiency on the day before surgery (Fig. 6, insets).
Results from rats with colchicine injected into the various cell groups
are shown in the main panels of Fig. 6. In rats with both PVN or ARC
disruption, caloric efficiency on day 1 was very high compared with
sham-injected rats; thereafter, efficiency decreased steadily in the
two groups, and by day 4 caloric efficiency in both groups was
significantly below the value in concurrently examined sham-injected
rats. By contrast, rats with VMN- and DMN-colchicine injections did not
respond differently from their sham-injected controls during the 4 days
of study.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Disruption of both ARC and PVN causes
obesity, increased food intake during both light and dark periods,
elevated insulin and leptin levels and increased body weight. Although
the most difficult to distinguish from each other, the ARC and PVN can
be functionally dissociated by the altered relationships of leptin to
insulin after colchicine injections. Inhibition of the VMN results in
obesity, hyperinsulinemia, a distinct alteration of the pattern in food
intake, with increased feeding during light and a tendency to decrease
feeding during the dark period, in keeping with the proposed function
of the VMN as an amplifier of circadian rhythms (27). Inhibition of the
DMN results in a trend toward reduced feeding during the dark period,
slightly reduces body weight gain, and markedly increases the slope of
the relationship of leptin to insulin.
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The effects of ARC-PVN disruption on most of the variables measured are
entirely similar (Table 2). In both groups, food intake is elevated
both during the light and dark period, body and fat depot weights
increase, and there are increases in circulating insulin and leptin
levels. Moreover, after both ARC- and PVN-disruption, there was a
progressive decrease in caloric efficiency that occurred during the
days after colchicine injection. By day 4, caloric efficiency was
significantly below that in saline-injected controls, suggesting
strongly that other cell groups were compensating metabolically for the
increased stores induced by overeating. Data from Exp 2 (submitted)
show that the thermogenic uncoupling protein (UCP) in interscapular
brown adipose tissue is significantly increased in ARC- and tends to be
elevated in PVN-disrupted rats on day 5. No changes in UCP content were
observed in VMN- or DMN-lesioned rats (submitted). Increased
thermogenesis, driven by elevated UCP activity would contribute to the
decreased caloric efficiency observed in ARC- and PVN-colchicine
injected rats.
The only differences found between disruption of the ARC and PVN are the altered relationships between circulating leptin and insulin concentrations. The slope of the leptin:insulin relationship increased sharply after PVN-colchicine. By contrast, there was no significant linear relationship between leptin and insulin after ARC-colchicine. We interpret these findings to suggest that inhibition of PVN (and DMN, see below) activity results in augmentation of the effect of insulin on leptin secretion from white adipose tissue.
Disruption of the VMN results in characteristic effects that differ
strongly from those of ARC and PVN disruption (Table 2). Food intake
increases only in the light, not the dark, periods, total food intake
and body weight are usually not elevated by day 5, although the animals
become hyperinsulinemic, hyperleptinemic and obese. Clearly VMN
disruption induces a metabolic obesity as has been shown previously
after both colchicine injection (23, 27) and electrolytic lesions
(11, 12, 13). Recently we have suggested that a major role of the VMN is
simply to amplify signals generated by the circadian clock in the
suprachiasmatic nuclei (27), and the decreased amplitude of the daily
food intake and body weight rhythms exemplify this effect. The elevated
light phase and tendency to diminished dark phase food intake seen in
VMN-disrupted rats together with elevated trough corticosterone (B)
levels (23, 27) and insensitivity to B feedback (13) is not seen after
disruption of the other medial hypothalamic cell groups. It is
intriguing that chronic stress also alters food intake patterns and B
feedback in a similar fashion (28) and also significantly increases the
numbers of c-Fos positive cells in the VMN under basal conditions
(29).
Disruption of the DMN results in a syndrome that is readily
distinguishable from those resulting from disruption of the other three
medial nuclei (Table 2). Food intake is decreased at night; however,
body weight, insulin, leptin, and fat depot weights do not change.
These results agree well with those of Bellinger and Bernardis (14, 30, 31), who show that lesions of the DMN in weanling rats result in small
adults that eat less but strictly in proportion to body weight, and
thus have normal body composition. Our other studies (submitted) show
that rats with DMN disruption are more sensitive than sham-disrupted
rats to the effects of intracerebroventricularly administered leptin,
suggesting that the DMN normally abrogate, to some extent, the dominant
inhibitory effects of leptin, and possibly other signals of energy
stores, on food intake.
These studies also suggest that the distinguishable obesities
produced by discrete inhibition of the medial hypothalamus resemble the
distinct obesities that are mutation-induced, peptide-specific, and
present throughout development. Inhibition of activity in the ARC and
PVN causes a food-dependent obesity in which rats overeat during both
dark and light periods. This resembles effects on feeding observed in
Ay/a and MC4-R knockout mice, in
which POMC peptides synthesized in neurons of the ARC are prevented
from acting on their melanocortin 3 and 4-receptors found in high
concentrations in PVN and DMN. Because acute inhibition of the PVN, but
not the DMN, results in increased food intake and obesity and cuts
between the ARC and PVN proportionally decrease MSH (
-MSH)
immunostaining in the PVN and increase food intake (32), receptors
located in the PVN may, in part, mediate the obesity induced by
abnormalities in the melanocortin system.
Rats with VMN lesions have increased expression of NPY in the ARC and are insensitive to the action of leptin (33) similar to findings in ob/ob and db/db mice (4). Because the unique results of VMN inhibition resemble strongly the characteristics of obesity in leptin-deficient mutants, leptin receptors in the VMN may, in part, mediate the obesity induced by abnormalities in the leptin system. This suggests specific hypothalamic sites which should be examined more closely in the study of mechanisms of obesity; however, the results point toward but do not demonstrate mechanism. Moreover, the cell groups are massively interconnected, and it is clear that interfering with function in one affects function in the others (21, 34). Site-specific use of receptor agonists and antagonists must be performed to unravel further the various roads to obesity induced by alterations in medial hypothalamic cell groups by leptin, NPY, and melanocortin peptides.
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Acknowledgments |
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Footnotes |
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Received January 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-melanocyte stimulating hormone in the rat brain: an
immunohistochemical analysis. Brain Res 297:21–32[CrossRef][Medline]
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