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Department of Physiology, University of California, San Francisco, California 94143-0444
Address all correspondence and requests for reprints to: Dr. SuJean Choi, Department of Physiology, Box 0444, 513 Parnassus Avenue, University of California, 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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies of rodent obesity models resulting from genetic
mutations have clearly demonstrated that both leptin (4) and
melanocortins (5, 6) are important signaling systems for regulating
energy homeostasis. Melanocortins are peptide products cleaved from the
POMC protein that bind to melanocortin-4 receptors (7). Leptin appears
to act as a feedback signal and alters the synthesis and secretion of
critical regulators of energy balance such as neuropeptide Y (NPY) and
POMC-derived peptides (Fig. 1
). In
leptin-deficient obese rodents, NPY is increased, and POMC is decreased
in the arcuate (8). However, POMC in the arcuate may have a greater
regulatory role in energy balance than NPY, as destruction of the ARC
by neonatal administration of the excitotoxin, monosodium glutamate,
which reduces the number of both arcuate NPY (orexigenic signal) and
POMC (anorexigenic signal) neurons, results in obesity (9).
Intra-cerebroventricular injections of leptin have been shown to
decrease food intake, body weight, and NPY content in the ARC, PVN, and
DMN nuclei and increase uncoupling protein (UCP) messenger RNA in brown
adipose tissue (BAT) (10, 11). Moreover, the effects of leptin on
energy balance are enhanced when leptin is directly injected into the
VMN (12) and to an even greater extent when it is injected into the ARC
(13). These studies combined with the fact that leptin receptors are
found in various hypothalamic cell groups (3) demonstrate that leptin
may have different roles in the regulation of energy balance depending
on the site of action.
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The close physical proximity of the medial hypothalamic cell groups
makes it difficult to lesion each site selectively to determine its
functional role(s) in the regulation of energy balance. Electrolytic or
neurochemical lesions (14) are most often not confined to the nucleus
of interest. By contrast, small volumes of colchicine can be injected
selectively within the boundaries of specific cell groups (Fig. 2) where the agent is taken up by neurons
(15) and subsequently disrupts function (16).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgery
Animals 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. Each midline hypothalamic site was
bilaterally injected using a Hamilton microsyringe (25 gauge) under
anesthesia on day 0 with either a solution of colchicine (1 µg/0.1
µl) mixed with fluorescein-colchicine (Molecular Probes, Inc., Eugene, OR) or saline (0.1 µl). Fluorescein-colchicine
is inactive, but was used to determine the placement of injection sites
and estimate the spread of injectate. Bilateral injections of either
drug or vehicle were made in the ARC (n = 22), VMN (n = 25),
PVN (n = 17), and DMN (n = 23) using coordinates based on the
atlas of Paxinos and Watson (17). The upper incisor bar was positioned
at -3.3 mm below horizontal zero, and the following stereotaxic
coordinates were used for the four hypothalamic cell groups: from
bregma: 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. Pressure damage and reflux was
reduced by injecting vehicle or drug over a 1-min period and waiting 5
min after injections before removing the microsyringe.
At the time of hypothalamic injections, a 22-gauge guide cannula (Plastics One, Roanoke, VA) was implanted into a lateral ventricle for subsequent icv injection of 3 µg leptin or vehicle (3 µl) on postsurgical day 1.
Sample collection
On postsurgical day 5, blood samples were taken in the morning
(0900–1030 h) by decapitation. Trunk blood (5 ml) was collected in
tubes containing 0.3 M disodium EDTA (100 µl/tube).
Brains were immediately postfixed in 10% formalin for 24 h and
then stored in a 30% sucrose solution. Brains were sliced at 30 µm,
and the tip of the injection needle was localized in cresyl
violet-stained sections; fluorescence from injected
fluorescein-colchicine was localized in adjacent sections (Fig. 2
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Rats were included in the study only if the needle tip was clearly
directed at the intended nucleus, and fluorescence was within the
borders of the cell group and did not extend to neighboring groups of
interest. In addition to blood samples and brains, we collected
interscapular BAT (iBAT) and white adipose tissue (WAT) from perirenal,
epididymal, sc (inguinal), and mesenteric sites. BAT was collected and
cleaned of WAT for measurement of UCP content.
RIAs
Blood samples were centrifuged at 3000 rpm at 4 C to separate
plasma, which was subsequently stored at -20 C. Plasma insulin and
leptin were measured using rat insulin and leptin RIA kits (Linco Research, Inc., St. Charles, MO). UCP was measured as previously
(18), using reagents provided by Dr. Jean Himms-Hagen, University of
Ottawa (Ottawa, Canada).
Statistical analysis
Data were analyzed using ANOVA corrected for repeated measures
(when required). Scheffe analysis was used to test the significance of
post-hoc effects. Regression analysis with slope comparisons
was used to test the effects of colchicine disruption on the
relationship of circulating leptin to fat mass. StatView (SAS Institute, Inc., Carey, NC) was the commercial statistical
package used for all analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Compared with icv saline injections,
icv leptin caused a decrease in both body weight gain (by ANOVA,
P < 0.001 for time and time x injection
interaction) and food intake (by ANOVA, P < 0.01 for
icv injection, time, and injection x time interaction). Leptin
injected icv on day 1 reduced dark period food intake on days 2 and 3
and decreased body weight on days 1.5–4 compared with saline-injected
(icv) control values (by post-hoc test, P <
0.05). Leptin did not significantly decrease sc fat depot weight on day
5, but did reduce caloric efficiency only on day 1, the day of
injection (P < 0.05). Leptin had no effect on the
thermogenic UCP content of iBAT on day 5. UCP increases thermogenesis,
and thus decreases caloric efficiency (20).
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, left half) and fat
depots, as previously observed (19). Leptin injected on day 1 did not
affect any of these colchicine-injected groups (Fig. 4, left half, columns 1 and
2). Because food intake was increased only during the light
period in rats with VMN lesions and tended to decrease during the dark
period, there was no significant cumulative increase in food intake in
these rats (by repeated measures ANOVA, P = 0.66),
although cumulative food intake was significantly increased in rats
with ARC and PVN lesions (by ANOVA: lesion, P <
0.0003; time, P < 0.0001; lesion x time
interaction, P < 0.0001 for both). Rats with DMN
lesions exhibited marked decreases in food intake after leptin
injection (by ANOVA: leptin, P < 0.05; time,
P < 0.0001; leptin x time interaction,
P < 0.0001) and body weight (by ANOVA: time,
P < 0.0001; leptin x time interaction,
P < 0.0001) after leptin injections (Fig. 4, bottom left half). Subcutaneous fat depot weights (Fig. 4, third column) were increased in rats with ARC, PVN, and VMN
lesions (P < 0.05 all three) and were not affected by
leptin injections (not shown in Fig. 4). Although rats with DMN lesions
did not have increased fat weight compared with sham-lesioned controls,
they did respond to leptin injection with a significant decrease in sc
fat compared with saline-lesioned controls (P < 0.03;
not shown). All lesioned rats, regardless of icv saline or leptin
injections, had significantly increased plasma insulin and leptin
levels (Fig. 5; P <
0.05) compared with sham-lesioned animals.
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, columns 1–3) differed in their
responses to the signals of obesity. Caloric efficiency progressively
decreased over days 1–4 in rats with ARC and PVN lesions regardless of
whether they had received icv leptin (by ANOVA: time, P
< 0.0001). By contrast, caloric efficiency did not change in rats with
VMN or DMN lesions with or without leptin (Fig. 4, fourth
column). This unresponsiveness in caloric efficiency, after VMN
and DMN lesions, was not a function of exogenous leptin, as results
very similar to these have been reported in a separate experiment on
different animals in which the same four lesions were made without icv
cannulation and leptin injections (19). UCP content in iBAT (Fig. 4, right column) increased in rats with ARC and PVN lesions,
probably accounting in part for the decreased caloric efficiency in
these rats [the increase in UCP was significant (P <
0.01) in ARC-lesioned and borderline (P = 0.1)
in PVN-lesioned rats). Outflow of the sympathetic nervous system
is primarily responsible for changes in iBAT UCP content (20),
suggesting that the ARC- and PVN-lesioned groups, which are capable of
responding to increased energy stores with increased UCP content, both
still receive signals of energy stores and can act appropriately in
response to those signals. Clearly, ARC and PVN lesions, which blocked
decreases in food intake and body weight gain after icv leptin
injections, did not block responses to signals of excess energy,
although they appeared to be unresponsive to leptin per se.
By contrast, obese rats with VMN lesions did not respond with
significant changes in caloric efficiency or UCP content in iBAT to
either saline or leptin, suggesting that this group either does not
detect or does not respond to signals of elevated energy stores.
Disruption of the medial hypothalamic cell groups produced distinct
changes not only in caloric efficiency but also in the relationship
between fat mass and leptin. The relationship between mesenteric fat
mass and circulating leptin in all sham-lesioned rats is shown in Fig. 6. A slope of approximately 0.9 and 34%
of the variance in leptin accounted for by the equation reflect the
well described direct relationship that exists between fat mass and the
hormone it secretes (21). In colchicine-injected rats, compared with
saline-injected sham animals, the relationship between mesenteric fat
mass and leptin changes, particularly after ARC and VMN disruption
(Fig. 7). After ARC disruption, leptin
concentrations are elevated and unrelated to fat mass; there is a
calculated slope of 2.26, but only 10% of the variance in leptin is
accounted for by the equation (P = 0.396). After VMN
disruption, there is a much steeper slope of the relation of leptin to
fat mass than after sham lesion or any other nuclear disruption; in
this group, the slope of the relationship increased to 5.3, and
approximately 72% of the variance in leptin is accounted for by the
equation (P < 0.001). After both PVN and DMN
disruption, the relation of leptin to fat mass tended to increase, but
not as dramatically as after VMN disruption; the slopes were
approximately 1.9, and 39–48% of the variance in leptin was accounted
for (P < 0.01 for both). These altered slopes after
nuclear disruption also held for the relationships of leptin to
perirenal and epididymal fat masses (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lesions of the ARC, PVN, and VMN produced obese rats, which, compared
with sham-lesioned controls, had increased signals of excess energy
(8), as shown by elevated plasma leptin and insulin concentrations on
day 5. Although lesions of all three cell groups produced obesity, ARC
and PVN lesions produced different changes in feeding patterns compared
with VMN lesions. ARC and PVN lesions increased food intake during both
dark and light periods, whereas lesions of the VMN resulted in
increased food intake only during the light period and a tendency for
reduced feeding during the dark period, confirming our previous
experiments (19). Increases in body weight and food intake after
lesions of the ARC, PVN, or VMN were not altered by the acute icv
injection of leptin. Although the action of leptin on food intake was
blocked by both ARC and VMN lesions, lesions of the ARC may mediate a
greater role in the regulation of food intake by leptin. In
sham-lesioned animals, icv leptin selectively decreased feeding during
the dark, and lesions of the ARC, but the not the VMN, produced changes
in dark period feeding. Previous studies have also shown that bilateral
injections of leptin into the ARC are more effective in reducing food
intake and body weight gain than injections into the VMN (13).
Furthermore, it seems likely that POMC-derived MSH peptides mediate
much of the feeding, body weight, and UCP responses to leptin, as
injections of a melanocortin receptor antagonist inhibits the effects
of leptin (10). It is likely that ARC pathways to both VMN (24) and PVN
(25) are activated by an action of leptin at the ARC, thus causing both
sympathetic and behavioral effects (see Fig. 1). By contrast, DMN
lesions resulted in increased sensitivity to icv leptin. This probably
results from blockade of signals from cell groups in the lateral
hypothalamus that contain orexigenic peptides (26), which innervate the
dorsomedial nuclei either directly (27) or indirectly through ARC and
PVN pathways (26, 28) to modulate medial hypothalamic control of energy
balance.
Arcuate lesions result in the loss of NPY and POMC neuronal activity,
and paraventricular lesions result in the loss in action of NPY and
POMC products. Thus, both lesions would be expected to produce similar
effects on food intake. In support of this hypothesis, we have reported
that small knife cuts placed between the ARC and PVN, which deprive the
PVN of some innervation from ARC, also result in elevated food intake,
during both light and dark periods, similar to the effects of either
ARC or PVN lesions (29). Moreover, a significant percentage of the
variance in food intake in those rats is explained by the inverse
relationship between food intake and the amount of 
MSH (POMC
product) detected in the PVN by immunocytochemical staining (Bell,
M. E., unpublished). The current results combined with the effects
of a PVN-ARC knife cut suggest that it is the removal of a tonic
inhibitory input from ARC to PVN nuclei that results in increased food
intake, in accord with the obesity-causing effects of melanocortin 4
receptor knockouts (6).
However, ARC lesions can be distinguished from PVN lesions. Differences between ARC and PVN lesions can be found in altered relationships among circulating leptin concentrations, fat mass, and insulin. After ARC disruption, there is no relationship between fat mass and circulating leptin or between insulin and leptin (19), suggesting either that those leptin receptors that respond acutely to peripheral leptin input are blocked or that ARC efferents have major autonomic effects. Systemically injected labeled leptin localizes only in the ARC, median eminence, and choroid plexus (30). Based on the results of ARC lesions, it appears that systemic leptin signals are not heeded, given the lack of relationship of leptin to either fat mass or insulin. Alternatively, it is possible that direct or, more likely, indirect projections from the ARC normally participate in the autonomic regulation of leptin secretion from fat as well as the regulation of insulin secretion. In vitro studies have demonstrated that norepinephrine inhibits leptin secretion from adipose cells (31). ARC lesions may abolish the normal noradrenergic regulation of leptin secretion from fat, thereby eliminating the relationship between leptin and fat mass. Dysregulation of insulin secretion resulting from ARC lesions (possibly through lack of signaling to the VMN) could abolish the relationship between leptin and insulin. PVN disruption only slightly increased the slope of the leptin-fat relationship, suggesting that after inactivating the PVN, the leptin signal is still recognized (perhaps by ARC), but that a small change in the efferent control of leptin secretion occurs.
Disruption of the PVN and ARC can also be differentiated by the subsequent changes in the leptin-insulin relationship. Our previous studies demonstrated that ARC disruption abolished the normal relationship between leptin and insulin, whereas the slope of the relationship increased after PVN and DMN disruption (19). It seems reasonable to conclude that occupied leptin receptors in ARC regulate energy balance in PVN and elsewhere, and that when the axonal output of cells bearing these receptors is blocked by colchicine, the peripheral leptin-signaling system on NPY (11, 32)- and POMC (10, 33)-synthesizing cells is disabled.
Although three of the four hypothalamic nuclei, when inhibited, cause obesity and insensitivity to icv leptin, there are clear differences in the capacity of the energy expenditure systems to respond to this inhibition. The response to obesity resulting from overeating in rats with ARC and PVN lesions was a progressive decrease in caloric efficiency and either an increase or a tendency for an increase in iBAT UCP. This suggests strongly that the activity in neither ARC nor PVN is required for the energetic response to increased signals of energy stores. On the other hand, VMN-inhibited rats that were also obese did not respond with changes in either caloric efficiency or UCP, suggesting that the VMN and/or DMN are required for this energetic response. Because DMN inhibition did not result in obesity, it is unclear how to interpret the lack of change in caloric efficiency or UCP in these essentially normal rats. Lesions of the VMN inhibit sympathetic outflow (1). The changes in caloric efficiency and UCP observed after ARC and PVN lesions, but not after VMN lesions, suggest strongly that activity in VMN is responsible for this adjustment to overeating. It is clear that the decreased food intake and body weight gain after icv leptin does not require DMN activity, as these were exaggerated by DMN inhibition. Thus, leptin reception at the DMN is not essential for these responses, and the major effect of DMN inhibition was to augment the responsivity of the rats to icv leptin.
Our studies, using small injection volumes of colchicine, distinguish between feeding and metabolic mechanisms that produce obesity induced by the inhibition of specific hypothalamic nuclei. Our previous studies have shown that both the concentration of colchicine and the volume of injectate must be small to confine the effects to a single nucleus (15). Others, using higher concentrations and larger injection volumes of colchicine into the VMN, did not find the marked selectivity on food intake that we observed (34). We suspect that the larger volume and concentration of colchicine used in those studies may have spilled over from the target VMN and blocked function in other cell groups, such as the ARC.
Obesity induced by alterations in function in or communication with PVN can be blocked by limiting caloric intake to that of normal rats (35). By contrast, obesity caused by inhibiting the VMN is metabolic and is not reversed by food restriction (35). Our data not only demonstrate that PVN and VMN lesions result in feeding and metabolically-dependent obesity, respectively, but that responsivity to signals of excess energy, such as leptin, is also dependent on functional activity of specific medial hypothalamic cell groups. There is strong conservation of hypothalamic neuro- and chemical anatomy between rodents and man (26). Thus, if human obesity is derived from altered activity in structures functionally analogous to the rat ARC and PVN, one would predict that with a good deal of will power and reduced food intake, normal energy stores could be regained. However, this study suggests strongly that after VMN disruption, rats are no longer capable of responding to increased signals of energy stores with decreased caloric efficiency and increased thermogenesis to the signals of obesity. Because the VMN amplify circadian rhythms induced by the circadian clock in the suprachiasmatic nuclei (36), this raises the possibility that aging in some individuals, accompanied by both damped circadian rhythms (37) and progressive obesity (38), may in part be ascribed to uncoupling and relative insensitivity of VMN neurons to signals of metabolism and the circadian cycle. In this case, nutritional control would help, but not overcome, the tendency toward obesity.
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Acknowledgments |
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Footnotes |
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Received April 6, 1999.
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References |
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