Departments of Neuroscience and Biostatistics, Pennington
Biomedical Research Center, Baton Rouge, Louisiana 70808
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INTRODUCTION |
THE CYTOKINE LEPTIN IS
RELEASED from adipose tissue and has been hypothesized to act as
a feedback signal in regulation of energy balance (40).
Leptin production increases in proportion to the size of body fat
stores (11), and stimulation of a hypothalamic leptin
receptor suppresses food intake (16). Thus, in theory, food intake should be inhibited as body fat increases to maintain a
stable body weight. Parabiosis studies with obese C57BL/Ks-misty (m) (BL/Ks) mice, homozygous for the
autosomal recessive diabetes mutation (db/db mice), indicate
that these animals are unable to respond to a circulating feedback
signal in the regulation of energy balance (8). This
diabetes mutation is a single nucleotide substitution in an exon near
the COOH terminus of a leptin receptor that has a long intracellular
domain, the long-form leptin receptor (ObRb) (7). ObRb is
present at low concentrations in most tissue and at high concentrations
in the hypothalamus, where it is thought to mediate the effects of
leptin on food intake (37). In addition to ObRb, there are
other leptin receptors (ObRa, ObRc, and ObRd) with short intracellular
domains [short-form leptin receptors (ObRs)] and one splice
variant (ObRe) that has the properties of a secreted receptor
(37) thought to act as a circulating binding protein
(29). Results from in vitro studies with transfected cells
suggest that the short-form leptin receptors, which retain one Janus
kinase binding site, have signaling capability
(4). These receptors are present at high concentrations in
most peripheral tissues in addition to the brain (31). It
has been proposed that the short-form receptors function as transport
proteins (12); however, their true physiological relevance
has yet to be demonstrated.
If ObRb is the only leptin receptor capable of signal transduction,
db/db mice, which express the short- but not the long-form receptors, would be expected to be totally unresponsive to leptin. Central or peripheral administration of recombinant leptin has no
effect on food intake or body weight in BL/Ks db/db mice
(16). These observations support the hypothesis that their
obesity is secondary to defective leptin receptor function; therefore,
we were surprised to find decreases in blood glucose concentrations of
C57 BL/6J-m Leprdb
(BL/6J) db/db mice that received peripheral
infusions of leptin (R. B. S. Harris, unpublished observations).
It is well documented that background strain can influence phenotype,
such as the strain-dependent responsiveness of mice fed a high-fat diet
(39); similarly, it is established that the diabetic
phenotype of db/db mice is determined by the genetic background on which the mutation is expressed (23). Thus
BL/Ks db/db mice are severely diabetic with extreme
hyperglycemia and a temporary hyperinsulinemia followed by islet
degeneration. In contrast, expression of the diabetes gene on a BL/6J
background results in less severe diabetes and pancreatic hyperplasia,
rather than islet degeneration (23). The studies described
below provide evidence that leptin influences glycemic control in both
male and female adult db/db mice, and the response to acute
or chronic leptin administration is minimized by the BL/Ks background
in both lean heterozygous (db/+) and obese diabetic
(db/db) mice.
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METHODS |
All mice were housed individually with free access to water and
chow, except where specified (Purina 5001; Purina Mills, MO), in a room
maintained at 78-80°F with lights on 12 h a day from 6:00
AM. All procedures were approved by the Pennington Center Institutional
Animal Care and Use Committee. BL/6J mice were obtained from a
colony maintained at the Pennington Center. db and m
genes were expressed in repulsion of one another, so that
wild-type mice could be identified by their misty coat color and
heterozygotes by their dark coat. Misty mice are small with stunted
growth (38); therefore, heterozygote lean mice were used
as controls for obese db/db mice. C57BL/KS/J-m
+/+ Leprdb (BL/Ks) mice were
purchased from The Jackson Laboratory (Bar Harbor, ME).
Experiment 1: infusion of 20 µg leptin/day in male obese and
lean BL/6J mice.
Pilot data (not shown) suggested that fasting glucose decreased in male
db/db mice infused with leptin from an intraperitoneal Alzet osmotic minipump (Alza, CA). This experiment was
designed to attempt to replicate these observations and to
test whether the response was specific to leptin and not attributable
to a contaminant in a particular supply of protein.
Daily body weights and food intakes of male obese and lean BL/6J mice,
aged 11-13 wk, were recorded for 1 wk, and then each genotype was divided into three weight-matched groups of five or six
animals. Each mouse was fitted with an intraperitoneal Alzet pump that
delivered 0.25 µl/h for 2 wk. One group in each genotype was infused
with PBS, one with 20 µg PeproTech leptin/day (recombinant
murine leptin; PeproTech) and the third with 20 µg R&D leptin/day
(recombinant murine leptin, R&D Systems). On day 9 of
infusion, mice were fasted for 5 h before the start of an oral
glucose tolerance test (OGTT). They were gavaged with 50 mg glucose,
and blood samples were collected by tail bleeding at 0, 10, 30, and 60 min for determination of glucose (Sigma kit 510, Sigma Chemical) and
insulin (rat insulin RIA kit, Linco Research) concentrations.
Corticosterone (corticosterone RIA kit; ICN Biomedicals) was measured
in the time zero sample, and remaining samples were combined
for measurement of leptin (mouse leptin RIA kit; Linco). On day
13 of infusion, rectal temperatures of the mice were measured at
7:00 AM, and the animals were fasted for 6 h before decapitation. Serum glucose, insulin, free fatty acids (FFA; NEFA C kit; Wako Chemicals) and corticosterone were measured. Organs and fat pads were
weighed and returned to the carcass for analysis (18). A
small piece of liver was used to determine lipid and glycogen content
(20).
Experiment 2: chronic infusion of 20 µg leptin/day in female
obese and lean mice.
In the previous experiment, the hyperglycemia of male db/db
mice was improved by infusion of 20 µg leptin/day. This experiment tested whether hyperglycemia also was decreased in leptin-infused female db/db mice. Baseline food intakes and body weights of
14 obese and 14 lean 4-wk-old female mice were recorded for 1 wk. Blood
glucose concentration was measured after a 5-h fast, and the mice were
fitted with intraperitoneal Alzet pumps delivering PBS or 20 µg
leptin/day. On days 2, 5, and 8 of infusion,
blood glucose was measured after a 5-h fast. The mice were killed on day 13. Trunk blood was collected for measurement of
glucose, insulin, corticosterone, FFA, and leptin concentrations. Serum leptin receptor [ObRe; 120 kDa (28)] was determined by
Western blot using a polyclonal antimouse leptin receptor antibody
(Affinity BioReagents) under conditions described previously
(20). Actin was detected using a monoclonal antibody
(monoclonal AC-40) and a peroxidase conjugated anti-mouse IgG secondary
antibody. Spot density was determined using a ChemiImager 4000 system
(Alpha Innotech), and the concentration of ObRe was expressed as a
ratio to actin. The retroperitoneal, mesenteric, and inguinal fat pads and liver were weighed. Retroperitoneal fat was snap-frozen for measurement of leptin mRNA expression by Northern blot
(19). The remaining carcass, less gut content, was
analyzed for fat content.
Experiment 3: chronic peripheral infusion of 10 µg leptin/day
in male and female obese and lean mice.
The two previous experiments indicated that leptin decreased the
hyperglycemia in male but not female db/db mice. Therefore, this study directly tested the effect of chronic peripheral leptin infusion in lean and obese, male and female mice and tested whether a
lower dose of leptin was effective in triggering the response in obese mice.
Daily body weights and food intakes of male and female 7-wk-old obese
and lean mice were recorded for 1 wk before the mice from each gender
and genotype were divided into two weight-matched groups of six mice.
Each animal was fitted with an intraperitoneal Alzet pump that
delivered either PBS or 10 µg leptin/day. Because 50% of the
leptin-infused female obese mice became hyperglycemic (HG), additional
animals were added to this group and, retrospectively, all mice were
subdivided into those that became HG and those that maintained the same
levels of fasting glucose [normoglycemia (NG)] as the
PBS-infused group. The level of fasting glucose used to allocate mice
to the HG group was arbitrarily set at 300 mg/dl (16.7 mmol/l). Fasting
glucose was in the HG range for all male obese mice.
On day 9 of infusion, an OGTT was performed and samples were
analyzed for glucose, insulin, corticosterone, and leptin
concentrations, as described above. On day 13, rectal
temperatures of the mice were measured at 7:00 AM, and the mice were
fasted for 6 h before decapitation. Trunk blood was collected for
measurement of circulating glucose, insulin, FFA, and corticosterone
concentrations. Organs and fat pads were weighed and returned to the
carcass, which was analyzed for composition. A small piece of liver was
frozen for determination of lipid and glycogen content.
Experiment 4: acute central leptin administration in lean and
obese mice.
The previous experiments showed that peripheral infusion of low doses
of leptin had metabolic effects in db/db mice, yet these mice do not express the ObRb and thus should not show leptin-induced changes in food intake and body weight (5). In this study
we tested whether central injections of leptin decreased food intake of
BL/6J db/db mice.
Sixteen obese and twenty-four lean 4-wk-old, female mice were fitted
with lateral ventricle (intracerebroventricular) cannulas and allowed
to recover from surgery for 5 days. The obese mice were divided into
two weight-matched groups (29 ± 1.0 g), and the lean mice
were divided into three groups of eight (18 ± 0.6 g). On the
day of experiment, mice were food deprived from 8:00 AM to 2:00 PM, and
one group of obese and two groups of lean mice received a bilateral
injection of 1 µl PBS into the lateral ventricle. The remaining
groups received equivolume injections delivering a total of 5 µg
leptin. Most mice were fed ad libitum, but one group of PBS lean mice
was pair-fed the average intake of leptin-treated lean mice. This group
lagged 1 day behind the others to facilitate pair-feeding. Food intakes
and body weights were recorded 6 and 18 h after injection. Mice
were then food deprived for 6 h before measurement of fasting
serum glucose and FFA concentration. Food was returned to the cage, and
intake was recorded 48, 72, and 96 h after the injection.
Experiment 5: acute effects of peripheral leptin injection on
glucose tolerance in obese mice.
The previous study produced no body weight or food intake responses to
central leptin in db/db mice. In this study we tested whether a single peripheral injection of leptin influenced the hyperglycemia or the insulin response to a glucose challenge in db/db mice. Daily food intakes and body weights of eight
male and eight female obese mice, aged 6-8 wk, were recorded for 4 days. On day 5, food was removed from the cages at the start
of the light cycle and the mice were injected intraperitoneally with 0.2 ml PBS or 30 µg leptin in 0.2 ml PBS. Two hours later an OGTT was
performed, as described above. The experiment was repeated 1 wk later
with the treatment groups switched, in a crossover design.
Experiment 6: leptin injection and infusion in BL/Ks and BL/6J
obese mice.
Others have reported that repeated peripheral leptin injections have no
effect on the food intake or body weight of BL/Ks db/db mice
(5, 34), whereas in experiments 1 and
3 described above, chronic leptin infusion decreased fasting
blood glucose concentrations in male BL/6J db/db mice. In
this study we tested whether the effects of daily bolus injections of
40 µg leptin were the same as those of a chronic infusion of 20 µg
leptin/day in both BL/6J and BL/Ks obese mice.
Body weights and food intakes of 10 BL/6J and 12 BL/Ks obese, 8-wk-old
male mice were recorded for 5 days. On day 0, mice were food
deprived for 7 h, from the start of the light period, before
measurement of blood glucose concentration in a tail blood sample. On
each of the next 5 days, the mice were injected twice (7:00 AM, 6:00
PM) with either 0.2 ml PBS or 20 µg leptin in 0.2 ml PBS. Fasting
glucose was measured 7 h after the morning injection. On day
5, rectal temperature was measured and an OGTT was performed. Fasting glucose also was measured on days 6 and
7, when injections had stopped.
Daily measurement of food intake and body weight continued until
day 13, when mice were anesthetized with isofluorane for intraperitoneal placement of an Alzet pump delivering PBS or 20 µg
leptin/day for 7 days. Rectal temperatures were recorded daily, and
fasting blood glucose was measured for the first 4 days of infusion. On
day 5 of infusion an OGTT was performed, and on day 7 mice were killed in the fasting state.
Trunk blood was collected for measurements of serum glucose, insulin,
corticosterone, triglycerides (Sigma kit 350.2; Sigma Chemical) and FFA
concentrations. Organs were weighed, and carcasses of BL/Ks mice were
analyzed for composition. Retroperitoneal fat pads, liver, and skeletal
muscle from the BL/6J mice were snap-frozen for determination of
glucose transporters and leptin receptor by Western blot and liver
phosphoenolpyruvate carboxykinase (PEPCK) mRNA
expression by RNase protection assay. These assays are not described,
because no differences in glucose transporters or PEPCK expression were
found. Epididymal fat leptin mRNA expression was determined by Northern
blot (19).
Experiment 7: leptin infusion or injection in
BL/Ks obese mice.
In the previous experiment, leptin reduced fasting glucose in BL/6J but
not BL/Ks obese mice. To test whether stress-induced hepatic
gluconeogenesis was masking a leptin response in BL/Ks mice, we
minimized stress by housing the mice on bedding instead of grids and
limiting the number of blood samples collected.
Body weights of 28 8-wk-old male BL/Ks obese mice were recorded daily
for 5 days, and then blood glucose concentration was measured after a
7-h fast during the light period. Glucose was measured in tail blood
using a glucometer (Accu-Chek Instant; Boehringer
Mannheim). A reading was taken from the first drop of blood because
glucose concentration doubled within 30 s of handling the mice.
The mice were divided into four weight-matched groups the next day. Two
groups received twice daily intraperitoneal injections delivering a
total of 40 µg leptin/day or PBS, and two groups were infused from
intraperitoneal Alzet pumps delivering either 20 µg leptin/day or
PBS. Fasting blood glucose was measured after 5 days of treatment. On
day 7, injected mice received their normal morning
injection, and all mice were killed in the fasting state 5 h
later. Serum ObRe, glucose, insulin, leptin, corticosterone, and FFA
concentrations were measured. Retroperitoneal fat and liver were
weighed. Epididymal fat leptin expression and intrascapular brown fat
(IBAT) uncoupling protein 1 (UCP1) mRNA expression were determined by Northern blot (20).
Experiment 8: leptin infusion in lean BL/6J and
BL/Ks mice.
The two previous experiments indicated that hyperglycemia in BL/6J
obese mice was more responsive to leptin than that of BL/Ks obese mice.
In this experiment we tested whether the severity of diabetes or the
background strain of the obese mice accounted for the difference by
measuring the responses to chronic leptin infusion in lean
(db/+) mice from both strains.
After 5 days of baseline measurements, 18 BL/6J and 18 BL/Ks 8-wk-old
lean mice were divided into two weight-matched groups per strain. All
mice were fitted with an intraperitoneal Alzet pump that delivered PBS
or 10 µg leptin/day for 14 days. On day 9 of infusion an
OGTT was performed. On day 12, the mice were food deprived
for 5 h before decapitation. Serum glucose, insulin, corticosterone, and FFA concentrations were measured. Liver and five
fat depots were dissected and weighed. Most tissues were returned to
the carcass for carcass composition. Small samples of liver, epididymal
fat, and IBAT were collected for determination of liver glycogen and
lipid content, adipose leptin mRNA expression, and IBAT UCP1 mRNA
expression, respectively.
Statistical analysis.
Daily body weight, OGTT glucose or insulin concentrations, fasting
glucose concentrations, and food intake were modeled separately with
repeated-measures ANOVA independently for each strain (BL/Ks vs. BL/6J)
or genotype (obese vs. lean). Measurements made the day before leptin
administration were used as covariates in the analysis of body weight
or food intake, and time zero glucose or insulin
concentrations were used as covariates in analysis of the OGTT
responses. Post hoc comparisons of treatment means at different time
points were tested by t-test, and significance levels were
unadjusted for multiple comparisons (Statistical Analysis Software for
Windows, release 6.12: SAS Institute, Cary, NC). Differences
in single time point measures were determined by t-test or
by ANOVA and post hoc Duncan's multiple-range test (Statistica, StatSoft).
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RESULTS |
Experiment 1: infusion of 20 µg leptin/day in male obese and lean
BL/6J mice.
In this experiment, lean and obese male mice were chronically infused
with 20 µg leptin/day. There was no statistically significant effect
of leptin infusion on body weight or food intake of either genotype of
mouse, although leptin-treated lean mice weighed less than their
controls (see Fig. 1). Placement of the
minipump caused a significant, but temporary, drop in food intake of
all of the mice. Rectal temperature measured on day 9 of
infusion was higher in lean than obese mice, but there was no effect of
leptin in either genotype (obese: 36.2 ± 0.4, lean: 37.5 ± 0.2°C).
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Fig. 1.
Daily body weights (A and C) and food intakes
(B and D) of obese (A and
B) and lean (C and D) male mice
infused with 20 µg leptin/day for 13 days in experiment 1.
Food intake is expressed as a percentage of baseline intake, which was
the average of the last 4 days of the baseline period. Data are
means ± SE for groups of 5 or 6 mice.
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There was no effect of 20 µg leptin/day on fasting insulin or on the
insulin response to glucose in the OGTT in obese mice (see Fig.
2). Fasting glucose in all leptin-infused
obese mice was significantly lower (P < 0.002) than
that of PBS-infused mice, and the difference was maintained throughout
the OGTT (see Fig. 2). There were no differences in area under the
curve above baseline for either glucose or insulin, suggesting that the
effect of leptin was independent of glucose-stimulated insulin release
(data not shown). There was no effect of leptin on any aspect of the
OGTT in lean mice (data not shown). Leptin had no effect on the weights of soleus muscle, pancreas, kidneys, liver, liver lipid, or liver glycogen content of either genotype of mouse (data not shown) or on the
body composition of obese mice (see Table
1). There were significant reductions in
fat pad weights and carcass fat content of leptin-infused lean mice
(see Table 1). At the end of the experiment (day 13 of
infusion), fasting glucose was reduced in leptin-infused obese mice,
but the difference did not reach statistical significance
(P < 0.09). Serum insulin concentrations were
significantly reduced in leptin-treated lean mice (control: 1.3 ± 0.2 ng/ml, PeproTech leptin: 0.5 ± 0.1 ng/ml, R&D leptin: 0.9 ± 0.2 ng/ml). There were no effects of leptin on serum FFA or
corticosterone concentrations in lean or obese mice (data not shown).
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Fig. 2.
Insulin (A and C) and glucose (B
and D) during an oral glucose tolerance test (OGTT)
performed after 9 days of leptin infusion in obese (A and
B) and lean (C and D) mice in
experiment 1. E: glucose area under the curve
above baseline. Data are means ± SE for groups of 5 or 6 mice.
*Significant difference (P < 0.05) between control and
leptin-infused obese mice.
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Table 1.
Body composition and serum hormones of male BL/6J mice infused with 20 µg of leptin for 13 days in experiment 1
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Experiment 2: chronic infusion of 20 µg leptin/day in female
obese and lean mice.
In this experiment, infusion of 20 µg leptin/day in obese and lean
female mice had no effect on food intake or body weight of either
genotype (data not shown). Fasting glucose did not change in either
group of lean mice (Fig. 3A).
In leptin-treated obese mice, glucose was significantly higher on
day 5 than before leptin infusion (Fig. 3B) but
the increase was attributable to only three of seven leptin-treated
mice (Fig. 3C). At the end of the study, corticosterone was
elevated in leptin-treated obese mice, but the response did not reach
statistical significance (control: 84 ± 25 ng/ml, leptin:
150 ± 21 ng/ml, P < 0.07). In lean mice, leptin
significantly reduced the weights of fat depots and percentage of
carcass fat (control: 11.4 ± 0.6%, leptin: 7.1 ± 1.3%,
P < 0.02). White adipose leptin mRNA expression was
significantly higher in obese than lean mice, but was markedly reduced
in the three leptin-treated HG mice compared with control or leptin NG animals (Fig. 3D). There was no effect of leptin on leptin
expression in the lean mice or on serum leptin receptor concentration
in either obese or lean animals (data not shown).
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Fig. 3.
Fasting glucose for female mice infused with 20 µg leptin/day in
experiment 2. Data are means + SE for groups of 7 mice.
In obese mice (B), there was a significant difference in
glucose on day 5 of infusion compared with preinfusion
concentrations. A: lean mice. C: changes in
individual fasting glucose values from days 0 to
5 in obese mice, indicating that only 3 mice became
hyperglycemic. D: retroperitoneal fat leptin mRNA expression
measured after 13 days of infusion of 20 µg leptin/day. Values with
different superscripts were significantly different (P < 0.05). Leptin NG, leptin normoglycemic; leptin HG, leptin
hyperglycemic.
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Experiment 3: chronic peripheral infusion of 10 µg leptin/day in
male and female obese and lean mice.
Body weight was significantly reduced from day 4 of infusion
in lean male mice infused with 10 µg leptin/day [Fig.
4A; baseline: not significant
(NS), leptin: P < 0.04, day: P < 0.0001, interaction: NS], and food intake was inhibited from day
2 of infusion (Fig. 4B; baseline: NS, leptin:
P < 0.004, day: P < 0.0001, interaction: NS). The difference in weights of control and
leptin-treated mice was ~1.0 g at the end of the study. The small
increase in weight gain of leptin-treated obese males did not reach
significance (Fig. 4; baseline: P < 0.007, leptin: P < 0.08, day: P < 0.0001, interactions: NS), and there were no differences in food intake of
obese male mice (Fig. 4D).
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Fig. 4.
Daily body weights (A, C, E, and
G) and food intakes (B, D,
F, and H) of male (A-D) and female
(E-H) mice infused intraperitoneally with 10 µg
leptin/day for 13 days. A, B, E, and
F: lean mice; C, D, G, and
H: obese mice. Data are means ± SE for groups of 6 mice. *Significant (P < 0.05) differences between
treatment groups.
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Leptin significantly decreased body weight of lean females by ~1.0 g
(Fig. 4E; baseline: P < 0.001, leptin:
P < 0.02, day: P < 0.0001, interaction: NS), but had no effect on food intake (Fig.
4F). Exactly 50% of leptin-treated obese female mice became HG but fasting glucose did not reach 300 mg/dl in any of the
controls. There was no overall effect of leptin on food intake or body
weight when all leptin-treated mice (leptin NG + leptin HG) were
analyzed as one group. Post hoc analysis, treating leptin NG (fasting
glucose <300 mg/dl) and leptin HG (fasting glucose >300 mg/dl) as
separate groups, showed that HG mice had a significantly
(P < 0.05) lower body weight than controls from
day 8 of infusion (Fig. 4G) and a significantly
reduced food intake by day 6 of infusion (Fig. 4H). There were no differences between leptin NG and control mice.
In lean male mice, leptin inhibited insulin release during the OGTT
(Fig. 5A; baseline: NS,
leptin: P < 0.02, time: P < 0.0006, interaction: NS), with significant differences between control and
leptin-treated mice 10 and 30 min after glucose gavage, but had no
effect on serum glucose (Fig. 5B). Differences in area under
the curve above baseline did not reach statistical significance (control: 57 ± 17, leptin: 28 ± 6, P < 0.08). There was no effect of leptin treatment on fasting glucose or
insulin or on the response to the OGTT in obese male or lean female
mice (data not shown). In obese females, the OGTT results were analyzed
with all leptin-treated mice in one group and resulted in no
significant effect of leptin on either glucose or insulin. Post hoc
analysis, separating leptin NG and HG groups, showed that insulin was
significantly lower in leptin HG mice than control or leptin NG mice
(Fig. 5C). Conversely, leptin HG mice were HG, but there
were no differences between control and leptin NG mice (Fig.
5D).
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Fig. 5.
Serum glucose (B and D) and insulin
(A and C) during an OGTT in lean male
(A and B) and obese female (C and
D) mice infused with 10 µg leptin/day in experiment
3. Data are means + SE for groups of 6 mice. *Significant
difference (P < 0.05) between leptin-treated and
control animals. Leptin HG obese females had fasting glucose
concentrations that exceeded 16.7 mmol/l (300 mg/dl); leptin NG and
control obese females had fasting glucose concentrations <16.7
mmol/l.
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There was no effect of leptin on rectal temperatures of the mice,
although obese mice had lower temperatures than lean mice (Table
2; P < 0.01). Leptin
tended to increase fat depot size in obese male mice, and this change
was significant in epididymal fat (Table 2). In lean females there was
a trend for all fat depots to be reduced in size, and body fat content
was 30% lower in leptin-treated mice than controls, but this did not
reach statistical significance (Table 2; P = 0.09). In
obese females, leptin HG animals weighed less but had the same amount
of body fat as control and leptin NG mice. This response was depot
specific, as inguinal fat was significantly increased, whereas uterine
and mesenteric pads were significantly decreased (Table 2;
P < 0.05). Liver lipid content also was decreased,
although liver weight was not different among the three groups of obese
female mice. The difference in carcass weights of control and leptin HG
mice was accounted for by a combination of protein, water, and ash
(Table 2). Serum leptin was significantly increased in leptin-infused
lean mice (P < 0.05), but the proportional increase
was greater in female (4-fold) than male animals (2-fold), as shown in
Table 3. Fasting corticosterone and
glucose concentrations were substantially increased in leptin HG
females (Table 3; P < 0.001).
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Table 2.
Organ weights body composition of male and female mice infused with 10 µg leptin/day for 13 days in experiment 3
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Table 3.
Serum hormones and metabolites in obese and lean mice infused with 10 µg leptin/day for 13 days in experiment 3
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Experiment 4: acute central leptin administration in lean and obese
mice.
A central injection of leptin caused significant weight loss in lean
mice 18 and 48 h after injection (Fig.
6A; leptin: P < 0.006, time: P < 0.0001, leptin × time:
P < 0.05). Food intake was significantly inhibited
only at 18 h after leptin injection (Fig. 6C; leptin:
P < 0.06, time: P < 0.0001, leptin × time: P < 0.05). Statistical analysis
did not include the pair-fed mice, because their intake was under
external control. Although these mice were offered the amount of food
eaten by the leptin group, spillage reduced intake even further.
Despite this difference in intake, both the leptin-treated and pair-fed
groups lost similar amounts of weight, which was recovered 4 days after
the injection. Central injection of leptin had no effect in obese mice
(Fig. 6, B and D). There was no effect of leptin
on fasting glucose or FFA concentrations of either genotype, measured
24 h after the injection (data not shown).
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Fig. 6.
Weight change (A and B) and food intake
(C and D) of female mice that received a single
intracerebroventricular (icv) injection of 5 µg leptin at time
0. A and C, lean mice; B and
D, obese mice. Data are means ± SE for groups of 8 mice. *Significant differences between treatment groups
(P < 0.05).
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Experiment 5: acute effects of peripheral leptin injection on
glucose tolerance in obese mice.
Leptin injection caused a significantly greater insulin release in
db/db mice during an OGTT performed 2 h after an
intraperitoneal injection of 30 µg leptin. Leptin significantly
increased serum insulin concentrations in male mice (Fig.
7A; leptin: P < 0.003, time: P < 0.0001, interaction: NS) at all
time points (P < 0.02) except 60 min
(P < 0.06). Area under the curve above baseline also
was significantly increased by leptin (control: 268 ± 27 units,
leptin: 436 ± 42 units, P < 0.01), but there was
no effect on glucose during the OGTT (Fig. 7B). Females gave
a similar response to males, with leptin significantly increasing
insulin concentrations 15 and 30 min after glucose administration (Fig.
7C; leptin: P < 0.02, time:
P < 0.0004, interaction: P < 0.04),
but had no effect on glucose concentrations (Fig. 7D).
During the OGTT, insulin concentrations were higher in male than female
mice, but glucose did not decline, indicating a greater degree of
insulin resistance in males than females. Serum leptin was elevated
during the test in leptin-injected mice (male mice: control = 59 ± 4 ng/ml, leptin = 178 ± 25 ng/ml,
P < 0.02; female mice: control = 183 ± 8 ng/ml, leptin = 365 ± 31 ng/ml, P < 0.0004), but there was no effect of leptin on the body weight or the
amount of food consumed by male or female mice during the 24 h
after injection (data not shown).
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Fig. 7.
Glucose tolerance serum insulin (A and C) and
glucose (B and D) for obese mice injected with 30 µg leptin 2 h before the test in experiment 5. A and B, males; C and D,
females. Data are means + SE for groups of 8 mice. *Significant
differences (P < 0.05) between control and
leptin-injected groups.
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Experiment 6: leptin injection and infusion in BL/Ks
and BL/6J obese mice.
There was no effect of daily injections of 40 µg leptin or of
infusing 20 µg leptin/day on food intake or body weight of obese BL/6J or BL/Ks mice (data not shown). Fasting serum glucose was substantially higher in BL/Ks than BL/6J mice (35 vs. 18 mmol/l), but
there was no effect of leptin injection in either strain (data not
shown). Daily fasting glucose was significantly (P < 0.02) reduced in leptin-infused BL/6J mice, compared with controls, but
there was no effect in BL/Ks mice (Fig.
8) and there was no effect of leptin
injections on the response to an OGTT in either strain of mice (data
not shown). The data from the OGTT performed on the 5th day of leptin
infusion is not shown. Baseline insulin was nonsignificantly lower in
leptin-treated mice than controls, and, when this was included as a
covariate, there was no effect of leptin on glucose-stimulated insulin
release during the OGTT in either BL/6J (baseline: P < 0.01, leptin: NS, time: P < 0.03, leptin × time:
NS) or BL/Ks mice (baseline: P < 0.02, leptin: NS,
time: P < 0.001, leptin × time: NS). In BL/6J
mice, baseline glucose was lower in leptin-treated mice (15 ± 2 vs. 23 ± 4 mmol/l), but leptin had no effect on glucose measured
during the OGTT when baseline glucose was used as a covariate
(baseline: P < 0.0001, leptin: NS, time:
P < 0.05, leptin × time: NS). There was no
effect of leptin on glucose concentrations in BL/Ks mice. Rectal
temperatures were not changed by leptin injection or infusion in either
strain of obese mice (data not shown).
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Fig. 8.
Daily fasting glucose in C57BL/6J (BL/6J; A)
and C57BL/Ks (BL/Ks; B) mice infused with 20 µg leptin/day
from days 13 to 19 in experiment 6.
Data are means ± SE for groups of 5 or 6 mice. Leptin infusion
caused a significant (P < 0.02) reduction in fasting
glucose of BL/6J mice. C: OGTT area under the curve above
baseline.
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Serum leptin, glucose, corticosterone, and triglyceride concentrations
were all substantially higher in BL/Ks than BL/6J mice, measured after
5 days of injection or infusion of leptin (Table 4), whereas serum insulin concentration
was lower. The only significant effect of leptin was on glucose, which
was lower in leptin-treated mice (Table 4). There was a significant
interaction between strain and leptin treatment for FFA on days
5 and 7 of infusion. FFA increased in BL/Ks mice, but
decreased in BL/6J mice (Table 4). There was no effect of leptin on
carcass composition of BL/Ks mice (data not shown) or on fat pad or
liver weights or on liver lipid or glycogen content in either strain of
mouse, but carcass weights, fat depots, and liver weights were greater
for BL/6J than BL/Ks mice (data not shown).
There was a substantial (P < 0.02) downregulation of
leptin expression in adipose tissue of leptin-treated BL/6J, but not BL/Ks mice (Fig. 9A). There
was no effect of leptin on ObRs in either adipose or liver tissue (Fig.
9, C and D), but there was a trend
(P < 0.09) for a reduction in serum leptin receptor of BL/6J mice with no effect in BL/Ks mice (Fig. 9B).
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Fig. 9.
A: epididymal fat leptin mRNA expression (10 µg
mRNA/lane) in BL/6J obese mice infused with leptin in experiment
6. *Leptin treatment significantly downregulated leptin expression
in BL/6J mice. B: serum leptin receptor (ObRe) for both
BL/6J and BL/Ks db/db mice infused with leptin for 6 days in
experiment 6. C and D: liver and
adipose short-form leptin receptor (ObRs) in BL/6J db/db
mice. There was no effect of leptin on receptor protein in either
tissue. Data are means + SE for groups of 5 or 6 mice. C, control;
L, leptin.
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Experiment 7: leptin infusion or injection in BL/Ks
obese mice.
There was no effect of leptin on body weight in BL/Ks mice that were
either injected or infused with leptin for 7 days, although the
placement of pumps alone caused weight loss in all infused mice (data
not shown). There were no differences in pretreatment serum glucose
concentrations, but there were significant effects of leptin and method
of administration after 5 days (Fig.
10; baseline: NS, leptin:
P < 0.0001, pump: P < 0.03, pump × leptin: P < 0.001). Leptin infusion caused a
significant decrease in fasting glucose, compared with the three other
treatment groups.
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Fig. 10.
Fasting glucose in BL/Ks obese mice injected or infused
with leptin in experiment 7. Data are means + SE for
groups of 7 mice. Glucose was measured after a 7-h fast on the day
before placement of the Alzet pump (pretreatment) and after 5 days of
leptin treatment. Values for day 5 that do not share a
common superscript are significantly different (P < 0.05).
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Liver and fat pad weights and leptin mRNA expression reflected the
reduced body weight of infused mice (Table
5). Epididymal fat of leptin-infused mice
was significantly reduced compared with that in PBS-infused mice
(P < 0.05). Serum glucose was significantly lower in
leptin-infused mice than in either of the groups of injected animals
(P < 0.05). There were no differences in serum
insulin, serum ObRe concentration, or in IBAT UCP mRNA expression (data not shown). Corticosterone was lowest in leptin-infused mice and highest in leptin-injected animals.
Experiment 8: leptin infusion in lean BL/6J and
BL/Ks mice.
All of the data for the two strains of lean mice infused with leptin
were analyzed together to test for effects of both strain and leptin
treatment, and a summary of the results for repeated-measures analysis
of data from this experiment is shown in Table
6. There were significant effects of
strain and of leptin and a significant interaction between strain and
leptin for body weight. Weight loss caused by leptin treatment was
greater in BL/6J than BL/Ks mice (Fig.
11, A and B).
There was a significant effect of leptin and of strain on food intake
and an interaction between strain, day, and leptin (see Table 6).
Intakes of BL/Ks mice remained slightly below baseline after the pump
implantation, whereas intakes of BL/6J mice returned to baseline levels
within a few days of surgery (Fig. 11, C and D).
BL/6J mice infused with leptin had a significantly (P < 0.05) lower food intake than control mice for the first 3 days of
infusion, whereas the difference was significant (P < 0.05) only on day 2 for BL/Ks mice (Fig. 11, C
and D).
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Fig. 11.
Daily body weights (A and B) and food
intakes (C and D) of groups of BL/6J
(A and C) and BL/Ks (B and
D) lean mice infused with PBS or 10 µg leptin/day in
experiment 8. Data are means ± SE for groups of 9 mice. *Significant differences (P < 0.05) between
control and leptin-infused mice.
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There were no differences in fasting serum glucose between strains or
between treatment groups within each strain. There was a significant
effect of strain on fasting insulin, which was lower in BL/Ks than
BL/6J mice (0.75 ± 0.08 vs. 1.00 ± 0.10 ng/ml,
P < 0.03). There was no effect of leptin on insulin
concentration at any time point of the OGTT for either strain of mouse
(data not shown). IBAT, inguinal, retroperitoneal, epididymal
and mesenteric fat, carcass fat, carcass water. and corticosterone were
all significantly increased in BL/Ks, compared with BL/6J mice
(P < 0.01), but there was no effect of leptin and no
interaction (Table 7). Some of the strain
differences were large; therefore, the effect of leptin was determined
within each strain. In BL/6J mice, leptin caused a significant
(P < 0.05) reduction in the weights of IBAT,
perirenal, retroperitoneal, and inguinal fat and in carcass
fat content (Table 7). Serum leptin was increased three- to fourfold by
leptin infusion in both strains of mice, but there was no effect of
strain or leptin on UCP1 or leptin mRNA expression, on liver glycogen
or lipid content, or on hormone concentrations measured at the end of
the study (data not shown).
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DISCUSSION |
These experiments demonstrate that recombinant murine leptin
decreases fasting glucose in obese male db/db mice despite
the lack of a functional ObRb. Previous experiments have only examined food intake and body weight in db/db mice treated with
leptin, and our results confirm that neither peripheral nor central
injections of leptin have any significant effect on either of these two
parameters. This surprising, but repeatable, change in glucose
indicates that leptin has biological effects that are mediated by
receptors other than the hypothalamic ObRb receptor. The same response
was induced by two different sources of leptin, confirming that it was
specific and could not be attributed to a contaminant in the protein
preparation. There was no effect of leptin on rectal temperature of
either db/db or db/+ mice, which excludes the
possibility of a secondary response to a contaminating endotoxin in the
protein. This observation also demonstrates that leptin does not induce
hyperthermia in normal mice and that the reversal of hypothermia in
leptin-treated ob/ob mice (20, 34) is mediated
by the ObRb.
None of the experiments described here identified the site of action of
leptin in db/db mice, and further studies are in progress to
determine whether the response is induced by activation of ObRs or by
non-specific activation of other receptors. Injection of leptin into
the lateral ventricles did not change food intake, body weight, or
blood glucose of obese mice. In addition, circulating leptin
concentrations are extremely high in db/db mice, and leptin transport into the brain is close to saturation when leptin levels are
within the normal range for a lean animal (1). Thus it is
unlikely that the small change in leptin caused by the peripheral infusion would have changed central concentrations of leptin, supporting the notion that the response in db/db mice was
mediated in the periphery.
The first three experiments demonstrated gender-specific responses to
leptin in db/db mice. Infusion of 20 µg leptin/day
significantly improved the hyperglycemia in male mice, whereas both 10 and 20 µg leptin/day caused ~50% of obese female mice to develop
symptoms typical of insulin insufficiency, evidenced by hyperglycemia, hypoinsulinemia, reduced food intake, and loss of lean tissue. None of
these symptoms was apparent in obese females infused with PBS. All
obese males infused with 10 µg leptin/day were HG, but they were also
hyperinsulinemic, and insulin release was stimulated during the OGTT,
indicating a normal pancreatic response to a glucose stimulus. The
phenotype of leptin HG females in experiment 3 was identical
to that found in BL/Ks mice. It was somewhat surprising that females,
rather than males, developed insulin insufficiency as diabetes
progresses faster in male than female BL/Ks db/db mice
(27) and other strains of obese mice (14).
The gender differences in susceptibility have been attributed to
inactivation of sex steroids by hepatic steroid sulfotransferase
(27). Hepatic glucose production and insulin sensitivity
are determined by the balance between estrogens and androgens, with
estrogen inhibiting hyperglycemia (27). Both male and
female db/db mice, however, are sterile, and the uterus in
female mice is immature in appearance. Therefore, it is unlikely that
estrogen derived from the reproductive system is responsible for the
gender difference in response to leptin. Adipose tissue is also a site
of estrogen production, but this would be effective in both male and
female mice. Alternatively, it has been reported that leptin stimulates
UCP2 activity in white fat (35) and that overexpression of
UCP2 inhibits glucose-stimulated insulin release from pancreatic islets
(6). Therefore, it also is possible that leptin stimulated
pancreatic expression of UCP2, which, in turn, inhibited insulin
release. Further studies are needed to identify mechanisms responsible
for the severity of diabetes observed in 50% of the leptin-treated
obese female mice and whether they are reversible, as this would
provide some indication of whether leptin promotes islet degeneration
or simply inhibits some aspect of the signaling process that promotes
insulin release. Because lean female mice did not become diabetic, the
ObRb must protect against this response and, because only 50% of the
db/db mice became HG, leptin may be interacting with a
recessive gene on the X chromosome.
Lean male mice infused with 10 µg leptin/day in experiment
3 showed a greater inhibition of food intake, but a smaller change in body fat, than female mice. In addition, circulating concentrations of leptin were greater in female than male mice despite a similar rate
of leptin infusion in experiment 3 or the same peripheral injection in experiment 5. This difference in leptin
concentrations may represent a difference in leptin clearance between
the genders and contribute to the variable responses between male and
female lean mice. There may also be interactions between gonadal
steroids and leptin that determine the energetic response to
exogenously applied hormone. Because leptin had no effect on food
intake of the female mice, it is possible that estrogen modified the
central response to leptin, consistent with reports of variations in
hypothalamic expression of the long-form receptor during the estrous
cycle (2). Further studies are needed to clarify this
difference between genders in normal mice and whether steroid hormones
modulate the effects of leptin on energy expenditure and food intake.
In experiment 5, reduced insulin sensitivity was suggested,
because a single 30-µg injection of leptin exaggerated insulin release in response to glucose ingestion. We previously reported an
identical response to a single injection of leptin in lean mice
(17), and these results indicate that this effect is
independent of the ObRb. The increased insulin release is unlikely to
be associated with a direct effect of leptin on islet function because
in vitro studies have demonstrated that leptin inhibits insulin release (25). Because increased insulin release did not occur in
mice that had been injected with leptin for 5 days (experiment
6) or in lean or obese mice infused with leptin for 5 or more days
(experiments 1-6), the stimulation must be a transient
response to leptin administration and may also be dose dependent.
Differences in response to leptin injection and infusion were also
apparent in experiments 6 and 7. Daily injections
of leptin had no effect in male db/db mice, whereas constant
infusion of leptin had no effect on body weight, food intake, or body
composition of BL/6J obese mice but caused a significant reduction in
fasting glucose concentration. In BL/Ks db/db mice, the
effect of leptin on fasting glucose was detectable only when
experimental conditions were designed to minimize stress. The lack of
response to leptin injections implies that intermittent boluses of the
protein, which has a half-life of ~30 min (21), are
unable to activate the same mechanisms as those stimulated by constant
infusion of low concentrations of leptin. It is well established that
intraperitoneal injections (34) or infusion (20) of small quantities of leptin reverses the
hyperglycemia in male (34) and female (20)
ob/ob mice that are leptin deficient. This response occurs
with a dose of leptin that is lower than that required for a
significant change in food intake or body weight of the mice
(34), suggesting that it is mediated by leptin receptors
other than the hypothalamic long-form receptors, which are responsible
for the effect on food intake.
The mechanisms responsible for the reduction in fasting glucose of male
db/db mice were not identified in the experiments described
here, although others (24) reported that either
intravenous or central infusion of leptin increased glucose turnover in
lean mice, promoting hepatic glucose output, reducing hepatic glycogen content, but stimulating muscle glucose uptake and doubling whole body
glycolysis. Therefore, measures of glucose and lipid use are needed to
determine whether increased glucose use or decreased hepatic glucose
output accounts for the fall in fasting glucose in leptin-treated obese
mice. An increase in energy expenditure also could account for a
reduction in glucose without any change in food intake or body
composition. Leptin increases energy expenditure in ob/ob
mice (34) and in food-deprived, but not fed, mice
(13), and stimulates norepinephrine turnover
(9) and expression of UCP (10) in brown
adipose tissue. These are thought to be centrally mediated responses
(22) and may not be present in mice lacking ObRb.
Measurement of IBAT UCP1 mRNA expression and rectal temperatures of
obese and lean mice in these experiments did not provide any evidence
for stimulation of thermogenesis by leptin, but the loss of weight
without any change in food intake of leptin-treated lean female mice
(experiment 3) suggests that energy expenditure was
increased. All of the animals used in these experiments were housed at
80°F, and it is possible that this relatively high ambient temperature prevented any significant change in brown fat
thermogenesis, which is absent at thermoneutrality (32).
The fall in fasting glucose concentration of leptin-treated male BL/6J
obese mice was similar to that reported by Grasso et al.
(15) for female BL/Ks db/db mice treated with a
leptin-related synthetic peptide. Specifically, daily injections of 1 mg of peptide [LEP-(116-130)] caused
weight loss and reduced blood glucose by ~20%, independent of any
change in food intake. There also was no change in body temperature of
the mice, consistent with our results. One obvious difference between
the studies is the amount of protein used, because a constant infusion
of 20 µg leptin over 24 h produced a similar change in glucose
in BL/6J mice as was produced by 1-mg injections of the peptide in
BL/Ks mice. Smaller amounts of peptide may produce a response in BL/6J
mice, which are more responsive to leptin than BL/Ks obese mice.
It has previously been reported that genetic modifiers in the
background strain determine the phenotypic expression of the diabetes
gene. Obesity and diabetes are associated with 
-cell hyperactivity
in BL/6J db/db mice, whereas BL/Ks mice experience islet
atrophy (23). Experiments described here showed additional differences between the two strains. Fat pads were larger but serum
leptin concentrations were lower in BL/6J than BL/Ks db/db mice. Because there were no strain-related differences in adipose leptin expression of PBS-infused db/db mice, the increase in
circulating leptin was probably due to inhibition of renal clearance in
BL/Ks mice, which were uremic by the end of the study. The excessive levels of corticosterone in these mice indicated that they were in an
unstable condition, either because of the manipulations involved in the
study or because of the late stage of their diabetes. In less stressful
conditions (experiment 7), the BL/Ks mice had lower levels
of corticosterone, and a small effect of leptin on fasting glucose was
detectable as long as blood was sampled within seconds of handling the
mice. The rapid elevation of glucose once BL/Ks db/db mice
were disturbed suggests that the leptin response in these animals is
easily masked by sympathetic stimulation of hepatic glucose output.
There are a number of possible explanations for the differences between
the two strains of adult db/db mice. If leptin reduces serum
glucose by augmenting insulin action, then there would be a requirement
for elevated levels of both insulin and leptin. This combination of
factors was not present in the BL/Ks mice, which had normal
concentrations of insulin despite extreme hyperglycemia. Alternatively,
if an increase in free leptin is essential to the response, the Western
blots suggest that there was less effect of leptin on the soluble
receptor in BL/Ks mice, which would make less free leptin available for
activation of peripheral receptors. Finally, genetic modifiers that
determine the phenotype of BL/Ks db/db mice may interfere
with the metabolic pathway activated by leptin in BL/6J mice and this
possibility was explored in experiment 8. There was a
significant inhibitory effect of leptin on weight gain and body fat
content of BL/6J but not BL/Ks mice, confirming that background strain
determined leptin sensitivity. Both in vivo and in vitro studies have
demonstrated that leptin induces insulin resistance in adipose tissue
(17, 33); therefore, the increased adiposity of BL/Ks lean
mice, compared with BL/6J mice, may be explained by their insensitivity
to leptin.
One issue raised by these experiments is how a small increase in leptin
can produce a significant response in mice that already have
excessively high serum leptin concentrations. The Western blots for
ObRe suggest that the circulating receptor, or binding protein, is
regulated in synchrony with adipose leptin expression. Under normal
conditions, it would be essential for the binding protein to be
downregulated when leptin production decreased, to retain some free
protein for maintenance of leptin tone on reproductive and immune
systems (26, 30). If infusion of recombinant leptin causes
a positive feedback, downregulation of leptin mRNA expression, and an
accompanying decline in binding protein in BL/6J mice, then the final
outcome would be a significant increase in the amount of free leptin
present with only a small change in total leptin concentration. There
was no evidence for downregulation of ObRs in tissue from these mice,
consistent with our previous measurements in leptin-infused lean and
ob/ob mice (20). These observations, made on a
small number of animals, imply differential regulation of the receptor isoforms.
Peripheral infusion of leptin into lean mice caused a small, transient
inhibition of food intake, which contrasts with the substantial
inhibition of intake produced by central injection of leptin
(36). The amount of leptin used here caused only a three-
to fourfold increase in circulating leptin, which presumably resulted
in only small changes in the amount of leptin reaching the brain. The
transient nature of the response can be explained in several different
ways. The first is that the mice developed "leptin resistance" in
response to continuous exposure to increased concentrations of leptin.
Transport across the blood-brain barrier could have reached saturation
(1) or hypothalamic receptors may have become unresponsive
to leptin, due to either a downregulation of receptor expression or
postreceptor events (3). Alternatively, the reduction in
food intake may have reflected shifts in energy use of muscle, liver,
and adipose tissue of leptin-treated mice, and the return of intake to
normal levels may have been achieved once a new metabolic equilibrium
had been established.
In conclusion, these experiments demonstrate that leptin has
bioactivity in obese mice that have no functional long-form receptor. Our results do not distinguish between a direct effect of leptin on
ObRs and non-specific binding of leptin to other receptors, but
experiments are in progress to address this issue. The results comparing BL/6J and BL/Ks mice demonstrate a significant effect of
background strain on leptin activity in both lean and obese mice,
suggesting that the Ks background modifiers interfere with the site of
leptin action.
Perspectives
The effects of leptin on food intake, energy balance, and
substrate use (24) are assumed to be mediated through the
ObRb, which is present at high concentrations in the hypothalamus. The results from experiments described here demonstrate that leptin does
have metabolic activity that is independent of the ObRb and may be
mediated in the periphery. The majority of studies investigating leptin
function has focused on its effects in the brain, working through the
long-form receptor. Obese humans and other animals have high
circulating concentrations of leptin, but leptin transport into the
brain is saturated at concentrations that are only slightly above those
found in normal weight animals (1). Therefore, the
increase in peripheral leptin in obese animals has a minimal impact on
outcomes mediated by central receptors, but, if leptin has functions
that are independent of the central long-form receptor, then there may
be physiological consequences of increased peripheral leptin that are
relevant to both lean and obese individuals.
This work was supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grant RO1-DK-53903-01A1.
Address for reprint requests and other correspondence: R. Harris, Dept. of Foods and Nutrition, Dawson Hall, Univ. of Georgia, Athens, GA 30602-3622 (E-mail: harrisrb{at}arches.uga.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
TOP
ABSTRACT
INTRODUCTION
