From the Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032
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ABSTRACT |
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Abnormalities of plasma high density lipoprotein
(HDL) levels commonly reflect altered metabolism of the major HDL
apolipoproteins, apoA-I and apoA-II, but the regulation of
apolipoprotein metabolism is poorly understood. Two mouse models of
obesity, ob/ob and db/db, have markedly
increased plasma HDL cholesterol levels. The purpose of this study was
to evaluate mechanisms responsible for increased HDL in
ob/ob mice and to assess potential reversibility by leptin administration. ob/ob mice were found to have increased HDL
cholesterol (2-fold), apoA-I (1.3-fold), and apoA-II (4-fold). ApoA-I
mRNA was markedly decreased (to 25% of wild-type) and apoA-II
mRNA was unchanged, suggesting a defect in HDL catabolism. HDL
apoprotein turnover studies using nondegradable radiolabels confirmed a
decrease in catabolism of apoA-I and apoA-II and a 4-fold decrease in
hepatic uptake in ob/ob mice compared with wild-type, but
similar renal uptake. Low dose leptin treatment markedly lowered HDL
cholesterol and apoA-II levels in both ob/ob mice and in
lean wild-type mice, and it restored apoA-I mRNA to normal levels
in ob/ob mice. These changes occurred without significant
alteration in body weight. Moreover, ob/ob neuropeptide
Y In general, HDL1 levels
are inversely related to atherosclerosis susceptibility in humans and
mice (1, 2). The protective properties of HDL may be due to its role in
reverse cholesterol transport and to antioxidant and anti-inflammatory
effects in the arterial wall (3). In humans, low HDL cholesterol levels may be associated with defects in synthesis or catabolism of the major
HDL apolipoprotein, apoA-I, with catabolic defects being more common
(4, 5). Low HDL is often accompanied by hypertriglyceridemia, obesity,
and insulin resistance, and obese subjects characteristically have
accelerated catabolism of HDL apolipoproteins (4). A common belief is
that low HDL cholesterol reflects increased core lipid exchange between
HDL and triglyceride-rich lipoproteins, leading to modifications of HDL
composition and size that result in increased catabolism of HDL
particles (6). HDL from hypertriglyceridemic subjects with low HDL is
smaller and more susceptible to renal filtration and degradation
(7).
However, the liver is one of the principal organs of HDL apolipoprotein
degradation, and it is unclear how the hepatic catabolism of the major
HDL apoproteins is regulated or mediated (8, 9). Recently, an authentic
HDL receptor, scavenger receptor B-I (SR-BI), was identified (10).
SR-BI mediates the selective uptake of HDL cholesteryl esters in the
liver and steroidogenic tissues; however, SR-BI mice do not have
defects in catabolism of HDL apoproteins (11, 12), indicating that this
receptor is unlikely to mediate hepatic uptake of apoA-I and
apoA-II.
Intriguingly, two monogenic mouse obesity models, ob/ob and
db/db, have greatly increased plasma HDL cholesterol levels
(13). This contrasts with obese humans with low HDL and other mouse models of obesity and diabetes, such as lethal yellow mice, tubby mice,
and the brown adipose-ablated transgenic mouse UCP-DTA, all of which
have normal HDL levels (13, 14). ob/ob mice were found to
have a nonsense mutation in the leptin gene (15), and db/db
mice were shown to have a splicing defect in the gene encoding the
leptin receptor (16), resulting in defective leptin production and
signaling, respectively. Because ob/ob and db/db
represent specific mutations in leptin and its receptor, these findings suggested to us that leptin might play a role in the regulation of HDL
apoprotein metabolism. As an initial step in investigating this
hypothesis, we have characterized HDL apolipoprotein metabolism in
ob/ob and db/db mice, and determined whether
leptin deficiency is responsible for the high HDL levels.
Animals--
All mice used in these studies were female
wild-type, ob/ob, and db/db mice of the pure
inbred strain C57BL/6J (purchased from The Jackson Laboratory, Bar
Harbor, ME). Mice were between the ages of 8 and 13 weeks old. All mice
were fed chow diet. All mice were fasted for 4 h before plasma collections.
Lipoprotein Analysis--
Native gel electrophoresis was carried
out on plasma from mice fasted for 4 h and separated on a 4-20%
polyacrylamide gel (Lipo-Gel, Zaxis) and stained with Sudan Black. HDL
size was determined using a high molecular weight calibration kit
(Amersham Pharmacia Biotech). HDL fractions (1.063 < Catabolic Assay--
HDL was radiolabeled with
125I-N-methyl tyramine cellobiose (500 cpm/ng),
and 10 µg was injected into the femoral vein. Asialoglycophorin (Sigma) was conventionally labeled with 125I (600 cpm/ng),
and 5 µg was injected in the femoral vein. Fractional catabolic rate
was calculated by fitting a least squares multiexponential curve to
each set of turnover data as previously described (17).
Leptin Treatment--
ob/ob, db/db, and
lean wild-type mice were injected once daily with with either 0.3 µg/g of body weight of leptin (R&D Systems) or 1.0 µg/g of body
weight. Control groups of ob/ob, db/db, and lean wild-type
mice were injected once daily with saline. All mice were 8 weeks of age
at the beginning of the experiments. Saline-injected mice were pair-fed
along with the leptin-injected mice of the same genotype.
Northern Blot Analysis--
10 µg of total liver RNA was
hybridized with radiolabeled cDNAs of mouse apoA-I, apoA-II, and a
28 S oligonucleotide. ApoA-I and ApoA-II signal levels are reported
under "Results" as ApoA-I or ApoA-II signal/28 S rRNA signal. 2.5 µg of liver poly(A+) RNA was hybridized with radiolabeled
cDNAs of mouse SR-BI or LDL receptor. Both signal levels were
standardized with a radiolabeled mouse B-actin cDNA. PhosphorImager
analysis was used for all quantitations. Data were reported for all
Northern blots as mean ± S.D. (arbitrary units).
Western Blot Analysis--
SR-BI protein levels in 50 µg of
mouse liver membranes were detected using polyclonal antibodies against
mouse SR-BI as described previously (18). SR-BI signals levels on x-ray
film were quantified by scanning laser densitometry.
Hepatic Lipase and Phospholipid Transfer Protein (PLTP)
Assays--
Both assays were performed as described previously (19,
20). Hepatic lipase assays and PLTP assays were performed on plasma from n = 5 and n = 10 (for each
genotype) mice, respectively.
Statistical Analysis--
Probability values were calculated for
each experiment using the Student t test.
Apolipoprotein Composition and apoA-I and apoA-II mRNA Levels
in ob/ob Mice--
Previous studies employed nonspecific precipitation
methods to show increased HDL cholesterol in ob/ob and
db/db mice (13). Fractionation of plasma lipoproteins from
ob/ob mice by fast protein liquid chromatography confirmed a
marked increase in the main peak of HDL relative to plasma from
wild-type mice, as well as a shoulder in the region where a large
subclass of HDL (HDL1) and LDL elute (Fig.
1A). Native gel
electrophoresis of plasma from ob/ob and wild-type mice
showed that ob/ob mice have an increased amount and size of
HDL (mean increase in diameter, 0.4 nm) and increased HDL1,
as well as decreased size, but an unchanged amount of LDL (Fig.
1B). Thus, the hypercholesterolemia of ob/ob mice is due to a marked increase in HDL and HDL1.
The apoprotein composition of HDL was next determined in
ob/ob and db/db mice. ApoA-II levels were 4-fold
(ob/ob) and 3-fold (db/db) increased relative to
wild-type mice (Fig. 1C, wt, 286 ± 26;
ob/ob, 1136 ± 156; db/db, 788 ± 88 (mean ± S.D); p < 0.005; n = 6 for wt and ob/ob, n = 3 for db/db
(arbitrary units)). A more modest elevation in apoA-I was also observed
(Fig. 1C, 1.3- and 1.2-fold, respectively; p < 0.005). The marked increase in apoA-II correlates well with
increased HDL cholesterol, as apoA-II levels are an important
determinant of HDL cholesterol levels in mice (21). The increased
plasma apoA-I and apoA-II protein levels in ob/ob mice were
not due to increased expression of their respective genes (Fig.
1D): hepatic apoA-II mRNA abundance was similar to that of wild-type mice, and surprisingly, apoA-I
mRNA levels were reduced 4-fold in ob/ob livers (Fig.
1D, wt, 0.047 ± 0.017; ob/ob, 0.01 ± 0.001 (mean ± S.D., arbitrary units); p < 0.05;
n = 6 for each genotype).
HDL Apolipoprotein Clearance in ob/ob Mice--
The greatly
increased plasma levels of apoA-II and, to a lesser extent, apoA-I with
no change or an opposite change in their mRNA levels suggested that
the increase in HDL is due to a catabolic defect of HDL apoproteins. To
test this idea, the proteins of intact HDL particles from
ob/ob mice were conjugated to a radioiodinated nondegradable
tracer, N-methyl-tyramine cellobiose (22), and injected into
groups of ob/ob and wild-type mice. Fig.
2A shows that the removal of
the plasma HDL tracer in ob/ob mice was slower than in
wild-type mice. The HDL protein fractional catabolic rate (17) was
significantly decreased relative to wild-type mice (0.138 ± 0.029 and .067 ± 0.011 pools/h, respectively; p < 0.01). Moreover, the initial (0-4 h) plasma decay of both apoA-I and apoA-II was markedly reduced, with apoA-I levels decaying at a slower
rate than apoA-II in ob/ob mice (Fig. 2B). The
level of the HDL protein tracer in livers of ob/ob mice at
the 24-h time point was profoundly reduced (4.3-fold) relative to
wild-type livers (Fig. 2C). No significant difference in the
HDL tracer levels were seen in kidneys of ob/ob and
wild-type mice, suggesting that ob/ob mice have a HDL
protein catabolic defect specific for the liver. The livers of
ob/ob mice are moderately enlarged as a result of increased
fat deposition (23). To eliminate the possibility that ob/ob
mice have a more general defect in liver function resulting from
increased liver weight, the activity of the well characterized hepatic
receptor for asialoglycophorin was examined (24). Radioiodinated
asialoglycophorin was injected into ob/ob and wild-type
mice. Fig. 2D shows that the plasma clearance of
asialoglycophorin was slightly faster in ob/ob mice relative to wild-type mice, with similar fractional catabolic rates (0.29 ± 0.04 and 0.21 ± 0.11, respectively). Taken together, these
data indicate that ob/ob mice have a liver-specific defect
in HDL protein catabolism.
Examination of Factors Known to Effect HDL Turnover--
Next, we
examined the possibility that factors known to alter HDL catabolism
might be altered in ob/ob mice. First, the level of the HDL
receptor SR-BI was evaluated by Western blot analysis and found to be
unchanged (Fig. 3A,
ob/ob, 174 ± 55; wt, 151 ± 8 densitometric
units; n = 6 for each genotype); SR-BI mRNA levels were also unchanged (Fig. 3B, wt, 1.9 ± 0.6;
ob/ob, 1.9 ± 0.3 arbitrary units; n = 6 for each genotype). Second, the LDL receptor is known to catabolize
large apolipoprotein E (apoE)-containing HDL particles (25). However,
LDL receptor mRNA levels were found to be unchanged (Fig.
3A, wt, 0.38 ± 0.043; ob/ob, 0.39 ± 0.046 arbitrary units; n = 6 for each genotype). Third,
the activity of hepatic lipase, an enzyme shown to promote the uptake
of HDL by cultured hepatocytes (25), was found to be normal (wt,
3695 ± 311 cpm transferred/µl of plasma/h; ob/ob,
4284 ± 339 cpm transferred/µl of plasma/h). Fourth, the
activities of PLTP (which circulates bound to HDL and mediates the
transfer and exchange of phospholipids between different lipoproteins
(19)) was found to be increased (wt, 203 ± 8 cpm transferred/µl
of plasma/h; ob/ob, 397 ± 65 cpm transferred/µl of
plasma/h; p < 0.001). However, PLTP mRNA levels in
the liver of ob/ob mice were unchanged (data not shown). The increased PLTP activity, with no increase in PLTP mRNA, suggests that the increased activity is secondary to increased concentration of
HDL particles. These data indicated that it is unlikely that these
factors account for the high HDL level in ob/ob mice.
Leptin Treatment of ob/ob and Lean Wild-type Mice--
These
experiments indicate major defects in HDL metabolism in
ob/ob mice: both decreased synthesis of apoA-I, as implied
by decreased apoA-I mRNA, and decreased catabolism of apoA-I and apoA-II. To determine whether abnormalities of HDL were reversible with
leptin treatment, low doses of leptin were injected into ob/ob and wild-type mice. Doses of leptin that are
suboptimal for weight reduction were used throughout these studies so
that no major effects of leptin on adiposity occurred, as measured by
body weight. Fig. 4A shows
that mice injected with leptin or saline (and pair-fed) had similar
body weights during the course of the experiment (Fig. 4A),
although leptin indeed reduced food intake (data not shown). Fasting
levels of plasma HDL cholesterol in leptin-injected ob/ob
mice were significantly decreased below levels in saline-injected
ob/ob mice (Fig. 4B). After cessation of
injections, HDL cholesterol rebounded to saline-injected levels. There
was no decrease in plasma HDL cholesterol levels in leptin and
saline-injected wild-type mice (Fig. 4B), nor in
db/db mice injected with leptin (day 0, 90 ± 17 mg/dl;
day 14, 86 ± 17 mg/dl). However, injection of 3-fold more leptin
into wild-type and ob/ob mice resulted in a 25% decrease in
HDL cholesterol in both groups (Fig. 4D), without a decrease
in body weight in wild-type mice (Fig. 4C). Once again,
db/db mice injected with leptin showed no decrease in plasma
HDL cholesterol levels (day 0, 89 ± 4.2 mg/dl; day 14, 90 ± 7.3 mg/dl). At both lower (Fig. 4E) and higher (Fig.
4G) doses of leptin, the decrease in plasma HDL cholesterol in ob/ob mice was associated with a 50-60% decrease in HDL
apoA-II levels, with no decrease in apoA-II in saline-injected
ob/ob mice. Similarly, apoA-II levels were decreased by
45-50% in leptin-injected wild-type mice (Fig. 4, F and
H). Leptin did not change steady state HDL apoA-I levels in
ob/ob or wild-type mice (Fig. 4, E-H). These
results indicate a substantial reversal of the increased HDL
cholesterol and apoA-II by low dose leptin in ob/ob mice. Moreover, a parallel process occurred in wild-type mice at a 3-fold higher leptin dosage. That more leptin was necessary to cause a
decrease in plasma HDL cholesterol in wild-type mice indicates that
wild-type mice are less sensitive to the effects of leptin than are
ob/ob mice, as previous reports suggested for body weight changes and as noted here (26-28). In addition, leptin treatment did
not alter levels of either SR-BI or LDL receptor mRNA in livers of
ob/ob and wild-type mice (Fig. 3B).
ApoA-I mRNA Levels after Leptin Treatment--
Although these
findings could result if leptin reverses the catabolic defect of HDL
proteins in ob/ob mice, apoA-I levels were unchanged by
leptin treatment (Fig. 4, E-H). This may be explained if
leptin also increases the low levels of apoA-I mRNA observed in
ob/ob livers (Fig. 1D), thereby increasing apoA-I synthesis. Indeed, apoA-I mRNA from ob/ob mice treated
with leptin was restored to levels of wild-type mice, whereas
apoA-I mRNA levels in ob/ob mice treated with
saline and db/db mice treated with leptin remained at a low
level, with no change in apoA-II mRNA levels (Fig.
5). Thus, low dose leptin essentially
normalized apoA-II levels (Fig. 4, E and G) and
apoA-I mRNA (Fig. 5) in ob/ob mice. However, HDL
cholesterol levels remained above wild-type levels (Fig. 4,
B and D).
HDL Cholesterol Levels in Neuropeptide Y
(NPY This study has revealed major alterations in the metabolism of
apoA-I and apoA-II in ob/ob and db/db mice. In
the case of apoA-I, a striking 4-fold decrease in apoA-I mRNA is
offset by a comparable decrease in hepatic catabolism, so that plasma
levels are only slightly elevated. Low dose leptin administration
reversed the decrease in apoA-I mRNA in ob/ob mice,
without changing plasma apoA-I levels. These findings, which imply that
leptin enhances the turnover of plasma apoA-I, are reminiscent of its
stimulation of glucose turnover without significant changes in plasma
level (30). In contrast to apoA-I, hepatic apoA-II mRNA was
unchanged; thus, a marked hepatic catabolic defect resulted in
increased HDL apoA-II levels. In mice, increased plasma apoA-II levels
are commonly associated with increased HDL cholesterol (21). Therefore, increased apoA-II may be the predominant factor responsible for increased HDL cholesterol in ob/ob mice. The parallel
defects in hepatic catabolism of apoA-I and apoA-II provide novel
evidence for a specific liver catabolic process that is down-regulated in ob/ob mice.
Although altered catabolism of HDL apoproteins commonly underlies
variation in HDL cholesterol levels in humans (4, 5, 31), the
regulation and molecular mechanisms of apolipoprotein catabolism are
obscure. Our findings indicate a catabolic pathway for apoA-I and
apoA-II that is liver-specific, down-regulated in ob/ob
mice, and possibly regulated by leptin. A variety of factors that could
potentially be responsible for these effects were excluded, including
SR-BI levels, LDL receptor mRNA, hepatic lipase activity, and PLTP
and hepatic PLTP mRNA levels. Other potential pathways include the
LDL receptor-related protein (32) and an apoE-proteoglycan pathway
(25). However, the phenotype of mice overexpressing the LDL
receptor-related protein inhibitor RAP suggests that LDL
receptor-related protein is unlikely to represent the major catabolic
factor for apoA-I and apoA-II (33). Moreover, apoE accumulation was not
particularly prominent in ob/ob or db/db mice
(Fig. 1C), suggesting that it may not be the ligand
primarily regulating HDL apoprotein metabolism in these animals.
ob/ob mice do have increased lipoprotein lipase (LPL) activity when measured relative to the entire adipose mass (34). LPL
acts by hydrolyzing triglycerides from apoB-containing particles in
plasma (35). However, mice overexpressing LPL do not show increases in
HDL apolipoproteins (36). In addition, leptin has recently been shown
to increase LPL expression in vivo (37); therefore, in our
experiments to decrease HDL cholesterol, it is unlikely that LPL is
responsible for the HDL phenotype in ob/ob mice. Thus, the
parallel severe defects in apoA-I and apoA-II catabolism in
ob/ob mice suggest down-regulation of a novel process, possibly a receptor, mediating HDL particle uptake in the liver.
Increased apoA-I gene expression in transgenic mice is associated with
a potent, consistent anti-atherogenic effect in different mouse models
(38, 39). The molecular mechanisms of regulation of apoA-I gene
expression have been intensively studied, at both the transcriptional
and posttranscriptional levels. The proximal 256 base pairs of the
human apoA-I gene contain sufficient information to direct high level
hepatic expression (40), and a number of transcription factors appear
to be active on the proximal promoter, such as apoA-I regulatory
protein-I (41), retinoid X receptor ob/ob mice have multiple metabolic defects, including
obesity, insulin-resistant diabetes, and hypercortisolism (47, 48). Potentially, any of these or other abnormalities in ob/ob
mice could be responsible for the metabolic derangement of HDL
apoprotein metabolism. Several lines of evidence indicate that the
increased HDL levels of ob/ob mice are not solely and simply
secondary to obesity or diabetes. First, several other obese diabetic
mouse models do not have increased HDL levels (13, 49). Second, in the
present study, compound ob/ob, NPY There are many differences in lipoprotein metabolism between mice and
humans. These include the presence of cholesteryl ester transfer
protein in humans, mediating core lipid exchange between HDL and
triglyceride-rich lipoproteins (51), and the absence of a correlation
between HDL cholesterol and apoA-II levels in human (4). However, it is
striking that the high HDL phenotype of ob/ob mice is
opposite that observed in humans. Because obese humans usually have
elevated plasma leptin levels (52), and some form of central resistance
to leptin has been postulated (53), this contrast raises the intriguing
possibility that in obese humans there could be direct peripheral
actions of elevated leptin on the liver (54, 55), leading to
accelerated hepatic degradation of HDL apoproteins. Alternatively,
there could be central effects of elevated leptin levels that influence
HDL metabolic pathways.
/
mice, despite marked attenuation of diabetes and
obesity phenotypes, showed no change in HDL cholesterol levels relative
to ob/ob mice. Thus, increased HDL levels in
ob/ob mice reflect a marked hepatic catabolic defect for
apoA-I and apoA-II. In the case of apoA-I, this is offset by decreased
apoA-I mRNA, resulting in apoA-II-rich HDL particles. The studies
reveal a specific HDL particle catabolic pathway that is down-regulated
in ob/ob mice and suggest that HDL apolipoprotein turnover
may be regulated by obesity and/or leptin signaling.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
< 1.21) were isolated from fasted mice by buoyant density
ultracentrifugation, and apolipoproteins were separated through a
4-20% SDS-polyacrylamide gel. Coomassie Blue-stained
SDS-polyacrylamide gel electrophoresis gels were destained, and
proteins were quantified using scanning densitometry. Isolation of HDL
by chemical precipitation (HDL reagent, Sigma) and enzymatic
measurements (Wako Pure Chemical Industries, Ltd.) was carried out
according to the manufacturers' instructions.
RESULTS
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Fig. 1.
Characterization of plasma lipoproteins and
their expression levels in ob/ob, db/db, and wild-type
mice. A, plasma lipoprotein cholesterol distribution as
determined by fast protein liquid chromatography (19). Each profile
represents pooled plasma from six wild-type ( ) and six
ob/ob (
) mice fasted for 4 h. B, a
representative native gel electrophoresis analysis of plasma from
individual ob/ob (n = 3) and wild-type mice
(n = 3). C, a representative
SDS-polyacrylamide gel electrophoresis analysis of HDL apoproteins from
two individual wt, ob/ob, and db/db mice.
D, a representative Northern blot analysis of hepatic RNA
from duplicate ob/ob and wild-type mice.
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Fig. 2.
Analysis of HDL apoprotein metabolism in
ob/ob and wild-type mice. A, kinetics
of 125I-N-methyl tyramine cellobiose HDL
(125I-NMTC HDL) plasma clearance. 125I-NMTC HDL
was injected intravenously into five mice of each genotype. The
percentage of radioactivity remaining in plasma (expressed as the
mean ± S.E.) was determined at the times indicated (*,
ob/ob versus wild-type, p < 0.05). Similar results were obtained in a separate experiment using
conventionally labeled 125I-HDL (data not shown).
B, kinetics of 125I-NMTC apoA-I and apoA-II
plasma clearance. The HDL was isolated by ultracentrifugation from
pooled plasma collected from samples shown in A and
analyzed by SDSpolyacrylamide gel electrophoresis, and apoA-I and
apoA-II bands were counted. Each data point represents the percentage
of radioactivity remaining relative to time 0. C, amount of
125I-NMTC HDL remaining in liver and kidney at 24 h
after the start of the experiment. Entire livers and kidneys were
homogenized and the amount of radioactivity was reported as the mean
radioactivity per g of tissue ± S.E. (*, ob/ob liver
versus wild-type liver, p < 0.0001).
D, plasma turnover of 125I-asialoglycophorin
(ASGP). 125I-Asialoglycophorin was injected
intravenously into three ob/ob and wild-type mice, and
plasma radioactivity was measured at the indicated times and expressed
as the mean percentage of radioactivity remaining ± S.E. relative
to time 0.
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Fig. 3.
SR-B1 expression levels and LDL mRNA
levels in ob/ob and wild-type mice. A,
representative Western blot analysis of SR-BI protein levels in liver
membranes from three ob/ob and three wild-type mice.
B, representative Northern blot analysis of SR-BI and LDL
receptor mRNA levels detected in poly(A+) RNA from
individual mice of each genotype injected with saline or leptin for 14 days (S, saline; L, leptin). Three mice treated
with leptin or saline (shown in Fig. 4D) were examined for
SR-BI and LDL receptor mRNA levels.
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Fig. 4.
Effects of leptin on HDL metabolism.
Mice were injected intraperitoneally once daily with either 0.3 µg of
human recombinant leptin per g of body weight (A, B, E, and
F) or 1.0 µg/g (C, D, G, and H).
Control mice were injected with saline and pair-fed along with
leptin-injected mice. A, body weight was measured at the
times indicated and expressed as mean ± S.E. (n = 5). B, plasma samples were isolated at the times indicated
following a 4-h fast, and HDL was isolated by chemical precipitation.
Total cholesterol content of HDL was measured and expressed as
mean ± S.E. (n = 5; * p < 0.01).
Arrows indicate the start and stop of all injections.
C, body weight as determined in A
(n = 4 for all groups of mice). D, HDL was
isolated by chemical precipitation and total cholesterol was measured
at time 0 and 7 and 14 days after the start of the experiment. Values
are reported as mean ± S.E. (n = 4 for all groups
of mice; *, leptin-injected versus saline-injected mice,
p < 0.01). E-H, apoA-I and apoA-II were
measured from HDL fractions (1.063 < < 1.21) isolated from
pooled plasma from each animal as described in Fig. 1B. Each
data point represents the percentage remaining relative to
time 0. E and F,
, A-II, saline;
, A-I, 0.3 µg/g leptin;
, A-I, saline;
, A-II, 0.3 µg/g leptin.
G and H,
, A-II, saline;
, A-I, 1.0 µg/g
leptin;
, A-I, saline;
, A-II, 1.0 µg/g leptin.
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Fig. 5.
Leptin restores apoA-I
mRNA levels in ob/ob mice. ApoA-I and
apoA-II mRNA levels were determined in hepatic RNA
isolated from mice at day 14 of the leptin injection experiment shown
in Fig. 3, C, D,G, and H. Significant differences
were measured for apoA-I mRNA levels between
leptin-treated and saline-treated ob/ob and db/db
mice (p < 0.003). There was no significant difference
between wild-type mice (wt) (both leptin- and
saline-treated) and ob/ob leptin-treated mice
(p > 0.18).
/
),ob/ob Mice--
The leptin administration
studies showed that HDL abnormalities were partly reversible without
changes in body weight. To further examine the relationship between HDL
and obesity, we measured HDL levels in ob/ob NPY-deficient
mice. Mice deficient in NPY and carrying the ob/ob mutation
show a 51% reduction in adiposity and a marked attenuation of
hyperphagy and diabetes-associated phenotypes caused by the
ob/ob mutation (29). Therefore, we asked whether decreases
in plasma insulin, glucose, and adiposity would have a proportional
effect on HDL cholesterol levels in ob/ob mice. Despite the
attenuation of obesity in npy
/
ob/ob mice (29), a lack of NPY on the ob/ob
background did not reduce plasma HDL levels relative to the
ob/ob mice carrying a functional npy gene (Fig.
6). Thus, significant decreases in obesity-related phenotypes do not appear to reduce the high HDL cholesterol levels in ob/ob mice.
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Fig. 6.
HDL cholesterol levels in ob/ob
npy /
mice. HDL was isolated by chemical
precipitation from pooled plasma from littermates of the designated
genotypes, and total cholesterol was measured enzymatically (wild-type
(wt), n = 7; ob/ob,
n = 23; ob/ob npy
/
,
n = 13) (*, ob/ob and ob/ob
npy
/
versus wild-type,
p < 0.01). Values are expressed as the mean ± S.E.
DISCUSSION
(42), thyroid hormone receptor
(43), and early growth response factor I (44). Although a number of
pharmacological and dietary factors may signal through these and other
transcription factors to affect apoA-I levels, apoA-I gene expression
has proven relatively insensitive to physiological change. Some of the
states in which there is altered apoA-I gene expression in rodents
include experimental hyperthyroidism (1.7-2.5-fold increased apoA-I
mRNA) (45), and nephrotic syndrome (2-fold increased apoA-I
mRNA) (44). High fat diets are also associated with a 40% increase
in apoA-I synthesis, reflecting increased translational efficiency of
apoA-I mRNA (46). The finding of a 4-fold decrease in apoA-I
mRNA in ob/ob mice that is completely reversed by low
dose leptin administration is notable and may indicate that leptin
signaling pathways play a major role in vivo in the
regulation of apoA-I synthesis.
/
mice had
elevated HDL levels similar to ob/ob littermates, despite marked attenuation of diabetes and obesity (29). Third, a developmental study in ob/ob mice indicated that increased HDL levels
precede the onset of severe obesity (13), and this has also been
confirmed for the decrease in hepatic apoA-I mRNA
levels.2 Fourth, a major part
of the HDL abnormalities of ob/ob mice was reversed by low
doses of leptin, beneath the threshold needed for weight reduction.
Fifth, significant lowering of HDL cholesterol and apoA-II levels was
produced in lean wild-type mice by leptin treatment, again without
change in body weight. As to hypercortisolism, we have found that
MCR4
/
mice, which are obese due to a defect in central
leptin signaling, have an elevation in HDL cholesterol and apoA-II
levels similar to that of ob/ob mice,2 and these
animals do not have elevated plama corticosteroid levels (50). This
ensemble of evidence suggests that there may be effects of leptin on
HDL metabolism that are independent of diabetes, obesity, and
hypercortisolism per se. However, because the reduction of
HDL cholesterol in response to leptin administration was only partial
in ob/ob mice (Fig. 4, B and D), there
may be an independent effect of obesity on HDL levels that is not
reversed without significant changes in body weight.
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ACKNOWLEDGEMENTS |
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We thank G. Hollopeter and R. D. Palmiter for plasma samples from npy/
ob/ob mice, F. Rinninger for providing us with NMTC, J. L. Breslow for many helpful discussions, and I. Tabas for comments on
the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL22682 (to A. R. T.) and National Institutes of Health Research Fellowship Award HL09928-01 (to D. L. S.).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.
To whom correspondence should be addressed: Div. of Molecular
Medicine, Department of Medicine, 622 W. 168th St., Columbia University, New York, NY 10032. Tel.: 212-305-5789; Fax: 212-305-5052; E-mail: dls51{at}columbia.edu.
The abbreviations used are: HDL, high density lipoprotein; LDL, low density lipoprotein; apo, apolipoprotein; SR-BI, scavenger receptor B-I; wt, wild-type; PLTP, phospholipid transfer protein; NPY, neuropeptide Y; LPL, lipoprotein lipase; NMTC, N-methyltyramine cellobiose.
2 D. Silver and A. Tall, unpublished results.
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REFERENCES |
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