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Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808
Address all correspondence and requests for reprints to: Ruth Harris, Ph.D., Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808. E-mail: harrisrb{at}mhs.pbrc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that leptin does exchange between parabiosed mice, although the half-life of leptin and the rate of blood exchange between parabiotics are such that a concentration gradient is maintained when one animal in a pair has high circulating concentrations of the protein and the other partner has none (13). We confirmed previous observations (2) that ob/ob partners of lean mice reduce their food intake, serum glucose, and insulin, and lose weight. However, we also found that lean partners of ob/ob mice lost a significant amount of body fat. Loss of fat is usually associated with a reduction in leptin expression (14); but in the parabiosis preparation, serum leptin concentrations and adipose leptin expression in the lean partners of ob/ob mice were maintained despite a 30% reduction in body fat content. These observations led to the conclusion that ob/ob mice produced another circulating factor, or factors, responsible for the inappropriate stimulation of leptin expression (15).
The objective of experiments described here was to examine the effects of parabiosis between db/db and ob/ob or lean mice on body composition and serum hormones of the mice. In Coleman’s study, body weight, serum glucose, and insulin were measured; but body composition was not determined (2). Our results confirm that ob/ob partners of db/db mice experience a rapid weight loss (2), which is exclusively body fat in ob/ob mice, whereas lean partners of db/db mice lose a combination of fat and lean tissue. The results also suggest that leptin-infused ob/ob mice produce a circulating factor that promotes growth of lean body mass in the db/db parabiont.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exp 1: Parabiosis between db/db and ob/ob female mice
Seven-week-old female C57Bl/6J-m db/db mice were
obtained from a breeding colony maintained at the Pennington Biomedical
Research Center and derived from breeding pairs supplied by Dr. Gary
Truett. Seven-week-old female C57Bl/6J ob/ob mice were purchased from
Jackson Laboratories (Bar Harbor, ME). The mice were housed in shoe-box
cages with free access to chow (Purina mouse chow 5015: Purina Mills,
St. Louis, MO) and water, in a room maintained at 80 F with the lights
on 12 h/day from 1100 h.
Body weights were recorded for 1 week and then mice matched into pairs.
Members of pairs containing two obese mice of the same genotype were
matched for body weight. The mice were joined surgically by parabiosis,
as described previously (13). The total number of pairs made and the
fate of those pairs are described in Table 1
. Disharmony, which represents an
immunological intolerance between partners, occurred within 11 days of
surgery. Many of the obese mice were unable to maintain body
temperature and died of hypothermia during the study. Experimental
pairs were housed two per cage, body weights were recorded daily, and
blood exchange was determined 15 days post operation on all surviving
pairs, as described previously (13). The rate of blood exchange ranged
from 1.0–4.0% of each animal’s blood volume exchanging in every
minute, consistent with values reported previously for mice and rats
(15, 16). Rectal temperatures of the mice were recorded every 2 days
for the first 2 weeks after surgery and then once per week. The pairs
were maintained for 18 days after surgery. All animal procedures were
approved by the Pennington Biomedical Research Center Institutional
Animal Care and Use Committee.
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(TNF) (Mouse TNF Quantikine kit; R&D Systems, Minneapolis,
MN). The two carcasses were separated; organs were dissected, weighed,
and returned to the carcass. The gastrointestinal tract was weighed,
cleaned, reweighed, and returned to the carcass. Gut content was used
as an index of food intake. Carcass composition of each member of a
pair was determined as described previously (17). Statistically significant differences between body weights and rectal temperatures of pairs in different treatment groups were determined by repeated-measures ANOVA and post hoc Duncan’s multiple-range test (P < 0.05). Differences in body composition and serum hormones were determined by two-way ANOVA, with genotype and genotype of partner as independent variables and pair number as a covariate, and post hoc Duncan’s multiple-range test at P < 0.05 (Statistica; StatSoft, Tulsa, OK).
Exp 2: Parabiosis between db/db and db/+ male mice
Seven-week-old male C57Bl/6J-m db/db and db/+ mice
were obtained from a breeding colony maintained at the Pennington
Biomedical Research Center. The db/+ mice were identified by coat
color, given that wild-type mice had a misty coat. They were housed as
described above, and mice were joined in parabiotic pairs after 1 week.
Members of pairs containing two obese or two lean mice were matched for
body weight. The total number of pairs made and the fate of those pairs
are described in Table 1
. Experimental design was exactly as described
for Exp 1, except that the mice were maintained for 25 days after
surgery.
The initial statistical analysis was as described above; however, ANOVA failed to detect differences between db/+ partners of db/db mice and their controls, because of the large variance contributed by db/db mice. Therefore, statistically significant differences in body composition and serum hormone concentration between groups of male db/+ mice or groups of male db/db mice were determined by unpaired Student’s t test (P < 0.05).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Control pairs of ob/ob-ob/ob mice and
db/db-db/db mice maintained weight, whereas ob/ob-db/db pairs lost
weight continuously during the postoperative period. One-way ANOVA with
repeated measures (day) showed no effect of treatment group on body
weight but a significant effect of day (P < 0.0001)
and a significant interaction between treatment and day
(P < 0.0001). The ob/ob-db/db pairs weighed
significantly less than the ob/ob pairs by postoperative day 8, and
less than db/db pairs by postoperative day 11. Statistical analysis of
rectal temperature showed a significant effect of genotype
(P < 0.03), partner (P < 0.004), and
interactions of genotype x partner, partner x day, and partner x
day x genotype (P < 0.0001). All of the obese mice had
low rectal temperatures (approximately 35.5 C) during the week after
surgery; but by the end of the experiment, the temperatures of ob/ob
partners of db/db mice were significantly increased. Many of the pairs
that included obese mice died from hypothermia. The physiological basis
of impaired thermoregulation in the db/db mice is not clear but was
probably associated with the stressful aspects of surgery and
parabiosis. Organ weights of the mice are shown in Table 2. ob/ob partners of db/db mice were
significantly smaller than their controls. A large portion of the
weight difference was caused by a reduction in body fat, with all fat
pads, including intrascapular brown adipose tissue (IBAT), responding
equally on a relative basis. In addition to loss of fat, there were
significant reductions in the weights of liver, pancreas, and spleen.
There was little effect of parabiosis to ob/ob mice on organ weights of
db/db mice; the only statistically significant effect was a reduction
in IBAT. There were many significant genotype effects and interactions
with treatment (parabiotic partner) on body composition, as summarized
in Table 3. In ob/ob mice, parabiosis to
db/db mice caused a significant reduction in carcass weight, fat, ash,
and water but had no significant effect on protein. In contrast,
parabiosis of a db/db mouse to an ob/ob mouse caused a small, but
significant, reduction in carcass fat and a significant (30%) increase
in carcass protein. We have previously demonstrated that carcass gut
content is a good indicator of food intake during the previous 24
h in both ob/ob and lean mice (13). Based on this association,
parabiosis between an ob/ob and a db/db mouse caused a substantial
reduction in intake of the ob/ob mouse but had no effect on the intake
of the db/db parabiont.
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. The leptin RIA kit was not
specific, because it detected the same level of leptin in members of
ob/ob-ob/ob pairs as in db/+-db/+ pairs (see Table 7). However,
assuming the values to be relative rather than absolute, db/db mice had
excessive levels of serum leptin that exchanged inefficiently across
the parabiotic union, so that a concentration gradient remained between
the two parabionts. Female db/db mice had higher leptin concentrations
than males (see Table 7), and this resulted in ob/ob partners of db/db
mice having 3-fold higher leptin levels than db/+ partners of db/db
mice. ob/ob mice that received leptin from the db/db mice had
significantly reduced serum glucose and insulin but elevated
corticosterone concentrations. All of the obese mice had elevated serum
concentrations of TNF.
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. There
was a significant (P < 0.0001) effect of treatment
group, day, and interaction between treatment and day, on body weights
of the mice. All treatment groups were significantly different from one
another on every day of the study. There was a significant effect of
genotype (P < 0.0001) and partner (P
< 0.005) on rectal temperature but no effect of day and no significant
interactions. Parabiosis to a db/+ mouse moderated the hypothermia of
db/db mice. The weights of organs from db/db-db/+ male pairs after 25
days of parabiosis are shown in Table 5,
and body composition is shown in Table 6.
Although the data were initially analyzed by two-way ANOVA, the large
variance contributed by db/db mice prevented detection of differences
between db/+ partners of db/db mice and their controls. Therefore,
statistically significant differences between groups of male db/+ mice
or groups of male db/db mice were determined by unpaired Students
t test (P < 0.05) and are marked by
superscripts in the tables. Parabiosis of a db/+ mouse to a
db/db partner caused significant reductions in the weights of the
carcass, liver, spleen, kidneys, white fat depots, IBAT, and femur, and
reduced femur density. These changes were reflected as significant
reductions in carcass fat, lean tissue, and ash, although gut content,
and (by implication) food intake, were not significantly changed. In
contrast, parabiosis of a db/db mouse to a db/+ animal inhibited some
of the weight loss experienced by members of db/db-db/db pairs and
caused a significant increase in liver weight and femur length. The
only significant changes in carcass composition were small increases in
carcass water and a 30% increase in gut content, implying increased
food intake.
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. db/+ partners of db/db mice had
significantly reduced serum glucose and insulin but elevated
corticosterone concentrations, compared with their controls. The db/+
partners of db/db mice also had elevated serum concentrations of
TNF, compared with members of db/+-db/+ pairs. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The failure of leptin to suppress food intake of db/+ partners of db/db mice is consistent with the results of an experiment in which female mice were infused from Alzet pump (18). Lean mice are transiently sensitive to the hypophagic effects of leptin and recover food intake within a few days of initiation of leptin administration. This recovery of a normal food intake may represent development of resistance to leptin, similar to that reported for obese humans and dietary obese mice (19, 20). In lean mice, parabiosis to a db/db mouse resulted in a significant reduction in carcass protein, as well as carcass fat, compared with db/+-db/+ controls. This reduction in lean mass may have resulted from either protein breakdown or a failure to deposit protein, because the db/+-db/db parabionts were male and would have continued to grow throughout the experimental period, whereas ob/ob-db/db pairs were female and would have been on a slower growth curve than the male mice. However, other differences between the two sets of mice in this experiment may also account for the loss of protein from db/+ partners of db/db mice. It is possible that the loss of lean tissue occurred during the later stages of parabiosis in male mice, a period that would have been missed in the shorter experiment with female obese mice. Alternatively, the excessive amount of fat in ob/ob mice provided an abundant source of energy that was not available in the lean mice and may have protected lean tissue in ob/ob partners of db/db mice. In vitro studies have shown that adipocytes exposed to leptin for an extended period of time become insulin resistant (21), whereas leptin has an insulin-like effect on glucose uptake by cultured myotubes (22). If these tissue-specific responses are maintained in vivo they would also contribute the exclusive loss of fat from ob/ob partners of db/db mice. However, the loss of protein from lean mice suggests that in vivo, tissue-specific responses to leptin are modulated by the size of energy stores available in different tissues. The extreme nature of the weight loss in parabiosed ob/ob mice and the weight loss in db/+ mice that had maintained food intake implied that negative energy balance in these animals was exaggerated by a stimulation of energy expenditure. No measures of energy expenditure were made in this study, but others have shown an increase in metabolic rate after either peripheral (5) or central (23) leptin administration.
The most surprising result from this study was the small, but significant, decrease in carcass fat content and the 30% increase in carcass protein content of db/db partners of ob/ob mice. In vitro studies have shown that leptin promotes nutrient uptake by muscle tissue and cells, having an insulin-like effect on glucose uptake (24) and inhibiting insulin’s down-regulation of lipid utilization (22). There is no information available on the effect of leptin on muscle protein synthesis or turnover in vivo, but it has been reported that leptin inhibits protein breakdown in cultured C2C12 myoblasts (25). The results from this study imply that leptin infusion into ob/ob mice leads to the release of another circulating factor that is carried back to the db/db mouse and promotes protein deposition. Because the genetic abnormality in db/db mice causes a mutation of the intracellular portion of the long-form leptin receptor, these animals have substantial amounts of leptin and functional short-form leptin receptors (26). In contrast, ob/ob mice have functional long- and short-form receptors but do not have any leptin. Therefore, the results from this experiment suggest that release of the second factor is mediated by the long-form leptin receptor, because mutation of this receptor is the only difference that remains between db/db mice and ob/ob mice infused with leptin. There are various possible reasons as to why this response was not observed in db/db partners of db/+ mice. Male db/db mice had more lean tissue and less fat than the female mice, and this gender difference may have masked any changes that occurred in partners of db/+ mice. Alternatively, the release of the growth factor may be dependent on a reproductive hormone that is present only in the female mice. However, a more likely explanation is related to the enhanced responsiveness of ob/ob mice to leptin. We have demonstrated not only that ob/ob mice are much more responsive to leptin than lean mice but also that they do not adapt to leptin administration and will continue to respond for as long as leptin is administered (18). In this study, the ob/ob partners of female db/db mice received large quantities of leptin and would have produced relatively large amounts of the factor that influenced lean tissue in their db/db partners. In the lean partners of db/db mice, not only was less leptin crossing the parabiotic union, but the lean mice may have down-regulated peripheral aspects of their response to leptin in addition to becoming resistant to the satiating effects of leptin. Although db/db partners of db/+ did not gain lean tissue, compared with their controls, they did maintain a slightly higher body temperature, seemed to eat more, and lost less weight than members of db/db-db/db pairs, indicative of beneficial aspects of parabiosis to a lean mouse. We did not attempt to identify the factor in this study, but others have reported that GH secretion can be modulated by leptin (27, 28) and that there is a correlation between serum leptin and IGF-1 concentrations in very old men (29).
The measurements of serum hormones in the parabiosed mice demonstrate that, as with humans (30), leptin is present at higher concentrations in female than male db/db mice. The amount of leptin present in ob/ob partners of female db/db mice was substantially greater than that in db/+ partners of male db/db mice. It is impossible to determine from this study whether the differences in concentration were totally accounted for by the difference in serum leptin concentrations in male and female db/db mice, or whether ob/ob mice have a reduced ability to clear leptin from the circulation. The maintenance of a concentration gradient in serum leptin for both types of pairs confirms our previous observation that the 30-min half-life of leptin is too short to allow equilibrium across the parabiotic union, because clearance rates exceed the slow rate of blood exchange between partners (13). The infusion of leptin into ob/ob partners of db/db mice caused a substantial drop in serum insulin and glucose concentrations. Although it has been demonstrated in vitro that leptin has a direct inhibitory effect on pancreatic insulin secretion (31, 32), it is likely that reduced food intake and weight loss contributed to the drop in both insulin and glucose in the mice in this study. Leptin also caused a significant elevation of corticosterone in both ob/ob and db/+ partners of db/db mice, which may have been caused either by direct activation of the hypothalamic-pituitary-adrenal axis by leptin or by stress associated with parabiosis to db/db mice and the resulting changes in physiology and metabolism.
In this experiment, serum TNF was measured in the parabionts. This
cytokine is released both by adipose tissue and by macrophages and is
present at elevated concentrations in genetically obese mice (33). It
has been demonstrated that leptin promotes cytokine production by
murine peritoneal macrophages (10), and TNF has been shown to
suppress food intake, increase thermogenesis, promote lipolysis, and
induce peripheral insulin resistance (34). Because all of these
activities would promote weight loss, TNF was measured to determine
whether leptin stimulated release of this cytokine, which then
contributed to weight loss in parabiotic partners of db/db mice. The
results showed that TNF was elevated in all obese parabionts,
including members of ob/ob-ob/ob pairs, making it unlikely that it
contributed to the weight loss in partners of db/db mice. TNF
concentrations were greatly increased in db/+ partners of db/db mice,
compared with members of db/+-db/+ pairs, but it was not possible to
determine whether the TNF was transferred across the parabiotic
union from the db/db mouse or whether release was stimulated in the
db/+ parabiont by the leptin that was carried across the parabiotic
union.
In conclusion, these experiments confirm that the exchange of leptin between parabiosed db/db and ob/ob mice inhibits food intake of ob/ob mice and causes an extremely rapid loss of weight that is exclusively adipose tissue. In contrast, db/+ partners of db/db mice maintain a normal food intake but lose both fat and lean tissue, presumably because of an increase in energy expenditure. The increase in carcass protein content of db/db partners of ob/ob mice suggests that leptin infusion into ob/ob mice causes release of a second factor that is carried back into the db/db mouse, where it promotes muscle protein synthesis or inhibits protein breakdown.
Received May 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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