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Departments of Neuroscience and Biostatistics, Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Obese, diabetic C57BL/Ks db/db mice that lack the long-form leptin receptor exhibit no decrease in body weight or food intake when treated with leptin. Here we compared responses to leptin in two strains of db/db mice: C57BL/6J mice that are hyperglycemic and hyperinsulinemic and C57BL/Ks that are hyperglycemic and normo- or hypoinsulinemic. Chronic intraperitoneal infusion of 10 µg leptin/day partially reversed hyperglycemia in C57BL/6J male mice but exaggerated the diabetic state of female mice. Bolus intraperitoneal injections of 40 µg leptin/day did not effect glucose in either strain of male db/db mice, whereas chronic intraperitoneal infusion of 20 µg leptin/day significantly reduced fasting blood glucose in male mice from both strains, especially C57BL/6J mice. Food intake, body weight, rectal temperature, and body fat did not change. Chronic intraperitoneal infusion of 10 µg leptin/day significantly reduced body fat in lean db/+ C57BL/6J but not in C57BL/Ks mice. Thus peripherally administered leptin is active in mice that have only short-form leptin receptors, and the response is dependent on the method of leptin administration and the background strain.
fasting glucose; glucose tolerance; body fat
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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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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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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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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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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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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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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.| |
ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-53903-01A1.
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FOOTNOTES |
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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.
Received 11 October 2000; accepted in final form 23 January 2001.
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METHODS
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DISCUSSION
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