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Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine (N.S., Y.O., H.M., J.H., T.O., Y.Y., S.N., K.H., K.N.), Kyoto; and Shionogi Research Laboratories, Shionogi Co. (G.K., T.T., M.T., M.H.), Osaka, Japan
Address all correspondence and requests for reprints to: Yoshihiro Ogawa, M.D., Ph.D. Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. E-mail: ogawa{at}kuhp.kyoto-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hypothalamus seems to be the main control center of satiety and energy expenditure (19). It has been demonstrated that recombinant leptin decreases the hypothalamic production of neuropeptide Y, a potent stimulator of food intake (17, 20). Peripheral administration of leptin induces expression of the immediate early gene c-fos, a marker of neuronal activation, in the hypothalamus (21). A leptin receptor has been isolated that is encoded by the db gene (22). The receptor has several alternatively spliced isoforms, one of which is expressed at a high level (23) in the hypothalamus and is missing in db/db mice due to a point mutation leading to its aberrant transcript (24, 25). These findings, taken together, suggest that leptin exerts its satiety effect via the hypothalamus.
Bilateral lesions of the ventromedial hypothalamus (VMH) result in the development of obesity (19), and thus VMH-lesioned animals have been widely used to study the mechanisms of body weight regulation. Previous studies have revealed that bilateral VMH lesions in one of a parabiotic pair lead to an increase in food intake and obesity in the lesioned rats, but result in a decrease in food intake and body weight leading to death by starvation in the unlesioned animals (26, 27). These findings strongly suggest that VMH-lesioned rats overproduce a circulating satiety factor to which the unlesioned animals can respond but VMH-lesioned rats cannot. However, the molecular basis for such a factor has been undefined. In the present study, to explore the pathophysiological significance of leptin in VMH-lesioned rats, we examined its synthesis and secretion in VMH-lesioned rats compared with those in sham-operated controls. We also studied the effects of iv or intracerebroventricular (icv) injection of recombinant leptin on food intake and body weight in these animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VMH lesions
Rats were anesthetized by ip injection of Nembutal (50 mg/kg;
Abbott Laboratories, North Chicago, IL). Bilateral VMH lesions were
made through stereotaxically positioned stainless steel electrodes (50
mA, 40 sec) using coordinates from the atlas of Paxinos and Watson
(2.56 mm posterior to the bregma, ±0.4 mm lateral, 9.5 mm from the
dural surface) (28). At the end of the experiments, areas of the
hypothalamic lesions in all animals were determined histologically by
hematoxylin and eosin staining. Only the animals that showed bilateral
VMH lesions were included in the present study. Control animals
received sham VMH lesions (no current passing through the
electrode).
Tissue and plasma samplings
Rats were used before and 1, 5, 10, and 30 days after the VMH
lesions. After rats were decapitated at 0900 h, blood was sampled,
and the white adipose tissue (WAT) was removed immediately from the
mesenteric, sc abdominal, and epididymal fat pads. Plasma glucose,
triglyceride, and insulin levels were measured as previously described
(3, 13). Plasma leptin levels were determined using the RIA for rat
leptin as described below. The adipose tissue was frozen in liquid
nitrogen and stored at -70 C until use.
RNA extraction and Northern blot analysis
Total RNA extraction was carried out, and Northern blot analysis
was performed with the 32P-labeled rat ob
complementary DNA (cDNA) probe as previously described (3). A human
ß-actin genomic probe (Wako Pure Chemical, Osaka, Japan) was used to
monitor the amount of total RNA in each sample. Rat ob
messenger RNA (mRNA) levels were normalized to the ß-actin mRNA
levels in the adipose tissue to correct for differences in the amount
of RNA applied. mRNA levels (arbitrary units) were expressed relative
to those of mesenteric WAT from 8-week-old Sprague-Dawley rats before
the VMH lesions (the ob mRNA level in 10 µg total RNA from
the mesenteric WAT in 8-week-old rats was defined as 1 U).
RIA for rat leptin
A full-length rat ob cDNA clone (3) was used as
template in the PCR with primers (sense,
5'-GTGCCTATCCACAAAGTCCAGGAT-3'; antisense,
5'-GCATTCAGGGCTAAGGTCCAACTG-3') selected to amplify sequences
corresponding to amino acids 22–167 (3). The PCR product was subcloned
into the pET-3c expression vector (Takara Shuzo Co., Shiga, Japan)
(29). Expression and purification of recombinant rat leptin-(22–167)
were performed as previously described (13, 29). Recombinant rat leptin
was radioiodinated by the chloramine-T method. The specific activity of
[125I]rat leptin ranged from 62.5–72.5 µCi/µg. An
antiserum for rat leptin was raised in Japanese white rabbits immunized
with recombinant rat leptin, which was used at a 1:5000 final dilution.
RIA for rat leptin was performed following the method of RIA for human
leptin (13). Rat plasma leptin levels were determined using the RIA for
rat leptin.
Preparation of recombinant human leptin
A full-length human ob cDNA clone (4) was used as
template in PCR with primers (sense, 5'-TACGTACCCATCCAAAAAGTCCAA-3';
antisense, 5'-AGGCCT-CAGCACCCAGGGCTGAG-3') selected to amplify
sequences corresponding to amino acids 22–166 (4). The PCR product was
subcloned into the NcoI site of the pTrc99A vector
(Pharmacia LKB Biotechnology, Piscataway, NJ), and the resulting pTacOb
was verified by sequencing. Escherichia coli DH5
was
transformed with pTacOb, and the recombinant human leptin protein was
expressed and purified essentially as previously described (30). In
brief, proteins were concentrated and suspended in a solution
containing 50 mM Tris-HCl (pH 8.5), 100 mM
NaCl, 1 mM dithiothreitol, and 8 M urea;
dialyzed against graded concentrations of urea containing 50
mM Tris-HCl (pH 8.5) and 100 mM NaCl; and
resuspended in 50 mM Tris-HCl (pH 8.5) and 100
mM NaCl. The protein solution was fractionated by gel
filtration on a HiLoad Superdex 75pg 16/60 column (Pharmacia LKB
Biotechnology), and fractions were analyzed by SDS-PAGE and staining
for protein with Coomassie blue. Protein concentrations were determined
by the Bradford method (31).
iv and icv injections of recombinant leptin
One day after the VMH lesions, rats were injected with
recombinant leptin or vehicle from the tail vein. For icv injection, a
stainless steel cannula (od, 1.09 mm; Becton Dickinson, Sparks, MD) was
implanted as previously described (32) in the skull of rats 5 days
before the VMH lesions, using coordinates (6.5 mm anterior to the
lambdoidal suture, ±1.4 mm lateral to the midline, 4.5 mm from the
dural surface) (28). The icv cannula placement was confirmed in all
animals by introducing Evans blue dye after the experiments. Only the
animals that showed bilateral VMH lesions and correct icv cannula
placement were included in the present study. The test solution was
injected with a microsyringe into sham-operated and VMH-lesioned rats.
Cumulative food intake was measured 1 day after the iv or icv
injection. Body weight change was measured after the VMH lesions. In
normal rats, a single iv or icv injection of recombinant leptin caused
dose-related reductions in food intake and body weight (iv, 0.2–1.0
mg/0.5 ml saline·rat; icv, 0.25–2.0 µg/10 µl saline·rat)
(Satoh, N., Y. Ogawa, G. Katsuura, and K. Nakao, unpublished data),
which were comparable to those into mice reported previously (14, 15, 16, 17, 18).
In the present study, therefore, leptin was injected at the maximal
doses (iv, 1.0 mg/rat; icv, 2.0 µg/rat).
Statistical analysis
All data were expressed as the mean ± SEM. The
statistical significance of differences in mean values was assessed by
Duncan’s multiple range test after one-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). VMH-lesioned rats gained weight more rapidly than
sham controls during the first postoperative day, resulting in
125.7 ± 4.8 and 260.5 ± 17.4 g gains in sham-operated
and VMH-lesioned rats, respectively, at the end of the experiments
(Fig. 1B). In VMH-lesioned rats, the plasma insulin level was elevated
throughout the experiments (Fig. 1E). Plasma glucose and triglyceride
levels were increased in VMH-lesioned rats 1 day after the surgery;
thereafter, these levels were roughly equivalent in sham-operated and
VMH-lesioned rats (Fig. 1, C and D).
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). The rank order
of the ob mRNA level was epididymal WAT > sc WAT
> mesenteric WAT (Fig. 2B). In the present study, no significant
changes in the ob mRNA level were noted in WAT examined in
sham-operated rats throughout the experiments (Fig. 2B).
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). Thereafter,
ob gene expression was markedly augmented in mesenteric and
sc WAT during the progression of obesity in VMH-lesioned rats. The
ob mRNA levels in mesenteric WAT from VMH-lesioned rats were
8.9- and 7.5-fold higher than those from sham-operated rats 10 and 30
days after the VMH lesions, respectively (P < 0.01).
Expression of the ob gene was also augmented in sc WAT in
VMH-lesioned rats 10 and 30 days after the VMH lesions (4.7- and
4.0-fold, respectively, vs. sham-operated rats;
P < 0.01). By contrast, ob gene expression
remained unchanged in epididymal WAT in VMH-lesioned rats throughout
the experiments (Fig. 2).
RIA for rat leptin
In the standard curve of the RIA for rat leptin (Fig. 3), the minimal detectable quantity was 0.1 ng/tube, and
the 50% binding intercept was 1.3 ng/tube. Intra- and interassay
coefficients of variation were 5.3% (n = 10) and 5.9% (n =
10), respectively.
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). Plasma leptin levels were significantly elevated in
VMH-lesioned rats compared with those in sham-operated controls 1 day
after the VMH lesions (2.1-fold vs. sham-operated rats;
P < 0.05). Plasma leptin levels continued to be
elevated in VMH-lesioned rats 5, 10, and 30 days after the VMH lesions
compared with those in sham-operated rats (3.3-, 3.9-, and 9.1-fold,
respectively, vs. sham-operated rats; P <
0.01; Fig. 4).
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), indicating
that the monomeric recombinant human leptin (22–166) was obtained.
Western blot analysis using an antiserum raised against the
NH2-terminal portion, 22–36, of human leptin revealed that
the Mr of the purified monomeric protein is
identical to that of the native protein in the adipose tissue from
obese patients (Ogawa, Y., G. Katsuura, N. Satoh, and K. Nakao,
unpublished data). Fraction 34, on the other hand, gave a single band
of approximately 30 kDa in size under nonreducing conditions, whereas a
band with a Mr of 16 kDa was detected under
reducing conditions (Fig. 5), suggesting dimer formation during the
purification process. The monomeric recombinant leptin protein was used
for further analysis.
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). By contrast, no significant changes in food
intake were observed in VMH-lesioned rats that received iv injections
of leptin. In sham-operated rats that received a 1.0-mg leptin
treatment, body weight gain was reduced significantly in leptin-treated
groups, but was increased in vehicle-treated groups (4.0 ±
2.9 g loss and 8.7 ± 1.6 g gain, respectively,
vs. the pretreatment values; Fig. 6B). In VMH-lesioned rats,
however, no significant differences in body weight gain were noted
between leptin- and vehicle-treated groups (31.2 ± 6.7 and
30.2 ± 4.7 g gains, respectively, vs. the
pretreatment values; Fig. 6B).
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). Body weight gain was also significantly reduced in
sham-operated rats after icv injection of leptin at the same dose
(0.3 ± 2.2 g gain in vehicle-treated groups and 27.7 ±
3.1 g loss in leptin-treated groups vs. the
pretreatment values; Fig. 7B). By contrast, icv injection of leptin did
not affect body weight gain in VMH-lesioned rats (24.0 ± 11.0 and
19.4 ± 9.8 g gains in vehicle- and leptin-treated groups,
respectively, vs. the pretreatment values).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, the VMH lesions resulted in significant hyperinsulinemia (38). It has been shown that insulin stimulates ob gene expression in the development of obesity (39). Therefore, hyperinsulinemia may be involved in the augmentation of ob gene expression and leptin secretion in VMH-lesioned rats. In this regard, we recently observed that ob gene expression remains increased in genetically obese KKAy mice after 72 h of fasting, when their obese phenotype remained but hyperinsulinemia was normalized (9). These observations suggest that augmented expression of the ob gene is due not only to hyperinsulinemia but also to the obese phenotype per se in VMH-lesioned rats. The relationship between hyperinsulinemia and responsiveness to leptin is not clear at present. However, it has been demonstrated that leptin can be effective in mice with diet-induced obesity (16), which must be hyperinsulinemic. It is, therefore, unlikely that hyperinsulinemia results in loss of the satiety effect of leptin in VMH-lesioned rats. It is conceivable that loss of the satiety effect of leptin in VMH-lesioned rats is due to the VMH lesions themselves. Direct microinjections of recombinant leptin into various hypothalamic regions are ongoing in our laboratory to determine whether the VMH is indeed the direct site of action of leptin or is located upstream and/or downstream to it.
The VMH has been implicated in the integration of the autonomic nervous system. In VMH-lesioned animals, sympathetic activity decreases, whereas vagal activity increases (19). Recent studies have shown that sympathetic stimulation decreases ob gene expression in adipose tissue (40). Dysregulation of the autonomic nervous system might be responsible for the augmentation of ob gene expression in VMH-lesioned rats. It has been demonstrated that recombinant leptin increases the otherwise decreased metabolic rate, body temperature, and locomotor activity in ob/ob mice (14). Furthermore, leptin causes an increase in noradrenaline turnover to brown adipose tissue (41). Collectively, these findings suggest that leptin increases sympathetic outflow. It is, therefore, tempting to speculate that the VMH lesions lead to a defect in the coupling of leptin-mediated afferent signals to the efferent autonomic neural pathways, which might contribute to the phenotypic changes in VMH-lesioned rats.
The present study represents the first report of plasma leptin levels in rats determined by RIA. The plasma levels in rats are comparable to those determined by RIA in humans (12, 13). Furthermore, plasma leptin levels were significantly elevated during the development of obesity in VMH-lesioned rats compared with those in sham-operated rats, which is consistent with previous reports, using immnoblotting method, that plasma leptin levels are increased in several models of rodent obesity (8, 10, 11). In the present study, ob mRNA levels were not statistically increased in mesenteric, sc, and epididymal WAT 1 and 5 days after the VMH lesions in VMH-lesioned rats compared with sham-operated rats. Differences in the time course between ob mRNA and plasma leptin levels may be due to differences in sensitivity between Northern blot analysis and RIA. It has been reported that normal weight animals made parabiotic with db/db mice, fa/fa rats, or VMH-lesioned rats reduce their food intake and body weight, and die of starvation (26, 27, 33, 34). The phenotypic changes in normal animals may be explained in part by an increase in circulating leptin supplied by the obese partners. Further studies are needed to determine the range of plasma leptin levels required to induce a satiety effect in normal animals.
We and others have demonstrated that ob gene expression is augmented in ob/ob and db/db mice and fa/fa rats, all of which have a defect in leptin or its receptor (2, 23, 24, 35, 36, 37). Furthermore, during fasting or food restriction, ob gene expression is down-regulated in normal animals, but not in these genetically obese animals (42, 43). These findings suggest that a normal action of leptin is required for proper regulation of ob gene expression. Indeed, treatment with recombinant leptin decreases adipose tissue expression of the ob gene in ob/ob mice (44). We have, therefore, postulated that augmented expression of the ob gene is due at least partly to the defective interactions between leptin and its receptor in ob/ob and db/db mice and fa/fa rats (3). Similarly, the augmentation of ob gene expression in VMH-lesioned rats is attributable to loss of the satiety effect of leptin.
The present study demonstrates that ob gene expression is markedly up-regulated in mesenteric and sc WAT, but remains unchanged in epididymal WAT during the progression of obesity in VMH-lesioned rats. These findings indicate that adipose tissue expression of the ob gene is augmented in a region-specific manner in VMH-lesioned rats. Funahashi et al. (6) previously showed that after the VMH lesions in rats, ob gene expression is augmented more rapidly in mesenteric WAT than in sc WAT. In the present study, however, no such difference was noted between these two fat pads. It has been shown that gold thioglucose-induced VMH lesions in mice induce obesity with differences in cellularity and weight among different fat pads (45). Differences in cellularity and weight among different fat pads might result in regional differences in ob gene expression in the WAT from VMH-lesioned animals.
In conclusion, we have demonstrated augmented expression of the ob gene and loss of the satiety effect of leptin in VMH-lesioned rats. The present study provides evidence that leptin represents one of the blood-borne satiety factors to which VMH-lesioned rats cannot respond and suggests that loss of the satiety effect of leptin contributes to the development of obesity and the obesity-related phenotypes in VMH-lesioned rats.
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Footnotes |
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Received August 20, 1996.
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