Departments of 1 Biochemistry and 2 Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461; and 3 Department of Medicine and Liver Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
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ABSTRACT |
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Human obesity is
associated with elevated plasma leptin levels. Obesity is also an
important risk factor for cholesterol gallstones, which form as a
result of cholesterol hypersecretion into bile. Because leptin levels
are correlated with gallstone prevalence, we explored the effects of
acute leptin administration on biliary cholesterol secretion using lean
(FA/) and obese (fa/fa) Zucker rats. Zucker
(fa/fa) rats become obese and hyperleptinemic due to
homozygosity for a missense mutation in the leptin receptor, which
diminishes but does not completely eliminate responsiveness to leptin.
Rats were infused intravenously for 12 h with saline or
pharmacological doses of recombinant murine leptin (5 µg · kg
1 · min
1)
sufficient to elevate plasma leptin concentrations to 500 ng/ml compared with basal levels of 3 and 70 ng/ml in lean and obese rats,
respectively. Obesity was associated with a marked impairment in
biliary cholesterol secretion. In biles of obese compared with lean
rats, bile salt hydrophobicity was decreased whereas
phosphatidylcholine hydrophobicity was increased. High-dose leptin
partially normalized cholesterol secretion in obese rats without
altering lipid compositions, implying that both chronic effects of
obesity and relative resistance to leptin contributed to impaired
biliary cholesterol elimination. In lean rats, acute leptin
administration increased biliary cholesterol secretion rates. Without
affecting hepatic cholesterol contents, leptin downregulated
hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) reductase, upregulated activities of both sterol
27-hydroxylase and cholesterol 7
-hydroxylase, and lowered plasma
very low-density lipoprotein cholesterol concentrations. Increased
biliary cholesterol secretion in the setting of decreased cholesterol
biosynthesis and increased catabolism to bile salts suggests that
leptin promotes elimination of plasma cholesterol.
obesity; liver; bile salts; phospholipids; lipoproteins
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INTRODUCTION |
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HUMAN OBESITY IS ASSOCIATED with altered cholesterol homeostasis including increased production and turnover (33, 37), as well as secretion of excess cholesterol from the liver into bile (43). Among the clinical consequences are cholesterol gallstones, which occur with high frequency in individuals who are obese, lose weight rapidly (29), or experience frequent fluctuations in weight (59). Leptin is a 16-kDa circulating hormone that is secreted by adipocytes and plays a critical role in regulation of body weight. Plasma leptin levels are elevated in most obese individuals (10) and are highly correlated with the frequency of gallstone disease in Mexican Americans (14).
Although rarely the cause of human obesity, spontaneous mutations in
leptin or its receptor constitute the genetic basis for obesity in
several well-established rodent models (17). Zucker rats
become obese and hyperleptinemic due to a Q269P missense mutation that
is present in all isoforms of the leptin receptor (42).
However, leptin receptors harboring the fa mutation appear to retain residual function, as evidenced by blunted physiological responses of obese (fa/fa) Zucker rats to pharmacological
doses of leptin (13, 16, 67). Unlike human obesity in
which whole body cholesterol production is increased (33,
37), endogenous rates of cholesterol synthesis do not differ in
lean and obese Zucker rats (32). Nevertheless, when
compared with other monogenic animal models of obesity due to mutations
in leptin or the leptin receptor (17), Zucker rats more
closely parallel humans, in whom obesity is associated with leptin
resistance (10). The aim of this study was to examine
systematically the influence of obesity and leptin on biliary lipid
secretion using lean (FA/) and obese (fa/fa)
Zucker rats. Our findings demonstrate that obesity in this animal model
is associated with a marked impairment in biliary cholesterol
elimination. Consistent with a residual capacity of obese Zucker rats
to respond to leptin, this defect in biliary lipid secretion was
partially reversed by high-dose intravenous leptin. In lean rats, acute
leptin administration promoted excess biliary cholesterol secretion. We
further show that leptin downregulated cholesterol biosynthesis,
upregulated cholesterol catabolism, and decreased plasma very
low-density lipoprotein (VLDL) cholesterol concentrations. Taken
together, these findings suggest that an important biological function
of leptin is to promote biliary clearance of plasma cholesterol.
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MATERIALS AND METHODS |
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Materials
Recombinant murine leptin was a gift from Amgen (Thousand Oaks, CA). All chemicals were obtained from Sigma (St. Louis, MO) unless otherwise noted. [4-14C]cholesterol (60 mCi/mmol, DuPont) was obtained from New England Nuclear (Boston, MA) and was purified by silicic acid chromatography (49). 3-Hydroxy-3-methyl[3-14C]glutaryl-coenzyme A ([14C]HMG-CoA, 57 mCi/mmol) and [5-3H]mevalonolactone (24 Ci/mmol) were purchased from Amersham (Arlington Heights, IL).Animals
Lean and obese male Zucker rats (Charles River Laboratories, Wilmington, MA) 11-13 wk of age were maintained on chow diet with water ad libitum while being subjected to cycles of 12 h light (6 AM to 6 PM) alternating with 12 h dark. After an overnight fast, rats were anesthetized by an intraperitoneal injection of 50 mg/kg body wt of pentobarbital sodium. With the use of sterile technique, a catheter was placed surgically in the left jugular vein and advanced into the right atrium (44). Catheters were exteriorized through a 1-cm incision on the back of the neck, which was then closed with surgical clips. Blood (1 ml) was sampled for baseline lipoprotein analysis (see Experimental Design), and then catheters were flushed with 1 ml of heparin (100 U/ml) to maintain patency. Rats were allowed to recover for 3 to 4 days before experiments.Experimental Design
At 6 AM, conscious unrestrained rats were administered either a bolus injection (2 min) of 150 µg of leptin (100 µg/ml in 150 mM NaCl) or the same volume of saline. This was followed by a 6-h period of continuous infusion (Harvard Infusion Pump, Harvard Apparatus, South Natick, MA) of leptin (100 µg/ml in 150 mM NaCl) at 5 µg · kgAnalytical Techniques
Biliary lipids.
Biliary cholesterol concentrations were quantified by HPLC
(66). Phospholipid concentrations in bile were determined
by an inorganic phosphorous procedure (4), and bile salt
concentrations were measured enzymatically (4). Biliary
bile salt species were analyzed by HPLC (45) using a
Beckman Ultrasphere ODS column (4.6 mm × 250 mm, 5 µm; mobile
phase methanol:0.01 M KH2PO4 75:25 vol:vol, pH
5.35). Bile salt hydrophobic index was determined according to Heuman
(23). Bile flow rates were calculated assuming hepatic
bile density of one (9), so that bile volumes were equivalent to weights. Cholesterol, phospholipid, and bile salt secretion rates
(µmol · kg1 · h
1) were
calculated as products of lipid concentrations and bile flow rates.
Tissue lipids. Hepatic contents of free cholesterol, total cholesterol, and triglyceride were determined enzymatically according to Carr et al. (5). Briefly, frozen samples of liver tissue were extracted in chloroform:methanol (2:1 vol:vol), and phases were separated by addition of 20 vol% of 0.05% H2SO4. Appropriate volumes of the lower organic phase were mixed together in glass test tubes with 1 ml of Triton X-100 dissolved in chloroform (1% wt/vol), dried under a stream of nitrogen, and resuspended in 0.5 ml of H2O. Enzymatic assays were performed by adding 50-µl aliquots of sample to individual wells of a 96-well microtiter plate. Free or total cholesterol was measured by addition of 150 µl of free cholesterol C enzymatic reagent (Wako Chemical, Richmond, VA) or cholesterol high-performance reagent (Boehringer Mannheim/Roche Diagnostics, Indianapolis, IN), respectively. Triglycerides were measured by sequential addition of 75 µl each of the two reagents of the triglycerides-GB reagent kit (Boehringer Mannheim/Roche Diagnostics) as described (5). Plates were incubated at room temperature for 1 h and then analyzed using a Titertek Multiskan Plus microplate reader (Eflab, Finland) set to 492 nm. Hepatic phospholipid concentrations were determined from phosphorus content (4), which was measured after organic extraction.
Plasma lipids. Total plasma cholesterol concentrations were determined by HPLC (66), and triglyceride concentrations were determined enzymatically (Boehringer Mannheim/Roche). Plasma lipoproteins were fractionated by fast-performance liquid chromatography (FPLC) into VLDL, low-density lipoproteins (LDL), and high-density lipoproteins (HDL) using two prepacked Pharmacia Biotech (Piscataway, NJ) Superose 6 HR10/30 columns connected in series (26). Samples (0.2 ml) were applied to columns equilibrated with phosphate-buffered saline (0.15 M NaCl, 2.6 mM KCl, 5 mM Na2EDTA, 3 mM NaN3, and 10 mM phosphate buffer, pH 7.4) and eluted at a flow rate of 18 ml/h. Cholesterol concentrations in fractions (0.3 ml) were determined by mixing 150 µl of each fraction plus 200 µl of cholesterol 50 reagent (Sigma) in individual wells of a 96-well microtiter plate. Color was developed for 10 min at 37°C and then analyzed using a microplate reader set at 492 nm, as described in Tissue lipids. Plasma concentrations of VLDL-, LDL-, and HDL cholesterol were calculated as products of total plasma cholesterol concentrations and relative FPLC peak areas of respective lipoprotein fractions.
Phosphatidylcholine molecular species. Molecular species of phosphatidylcholines in liver and bile were quantified as previously described (8, 41). Briefly, phosphatidylcholines were purified using a Hibar 5-µm silica 4.6 × 250-mm LiChrospher Si-100 HPLC column and a mobile phase consisting of isopropanol-hexane-ethanol-25 mM phosphate buffer (pH 7.0)-glacial acetic acid (495:367:100:57:0.3 by vol). Phosphatidylcholines were hydrolyzed to form diglycerides using phospholipase C from Bacillus cereus (Boehringer Mannheim/Roche). Benzoate derivatives were prepared and then fractionated by reverse phase HPLC (Beckman Ultrasphere 5 µm ODS 2.5 × 250-mm column; mobile phase methanol-water-acetonitrile 942:39:19 by vol). Benzoate derivatives were detected by absorbance at 230 nm so that relative peak areas directly represented mole fractions of individual phosphatidylcholine molecular species. Peak identities were established according to Patton and Robins (41). For indeterminant peaks, molecular species were identified by matrix-assisted time-of-flight mass spectrometry (47) using a Voyager Biospectrometry workstation (Perseptive Biosystems, Framingham, MA).
Hepatic enzyme activities.
Hepatic microsomes and mitochondria were prepared by differential
ultracentrifugation (50), and protein concentrations were determined according to Lowry et al. (30). Cholesterol
concentrations in microsomes and mitochondria determined by gas liquid
chromatography (72) were incorporated into the
calculations of the specific radioactivity of the
[4-14C]cholesterol substrate used in determining the
catalytic activities of the microsomal cholesterol 7-hydroxylase and
mitochodrial sterol 27-hydroxylase.
Analysis of Biliary Lipid Secretion Rates
Over the range of bile salt secretion rates in the current study, secretion rates of cholesterol and phospholipids varied linearly as functions of bile salt secretion rates (Fig. 1, A-D). Physiologically, the slopes of these lines, (i.e.,
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Statistical Analysis
Analysis of variance was employed to detect differences among mean values, whereas analysis of covariance was performed to compare slopes of linear regressions (74). Pair-wise differences among means and slopes were assessed according to the Fischer's protected least squares differences method (15). ![]() |
RESULTS |
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There were no differences between weights of rats infused with leptin [321 ± 15 g (mean ± SE), lean; 511 ± 64 g, obese] or saline (313 ± 12 g, lean; 512 ± 34 g, obese). As indicated by identical liver-to-body weight ratios for lean (0.034 ± 0.001) and obese rats (0.035 ± 0.001), liver masses scaled in proportion to body weight. Therefore, normalization to either liver or body weight provided an equivalent basis for comparing biliary lipid secretion rates in lean and obese rats.
Figure 1 presents biliary lipid secretion rates normalized to body
weight for lean and obese rats infused with saline or leptin. Bile salt
concentrations and secretion rates were similar in all groups (47 ± 3 mM and 123 ± 6 µmoles · kg1 · h
1 , respectively) and decreased threefold to 16 ± 1 mM and 41 ± 2 µmoles · kg
1 · h
1,
respectively, during the 5-h bile collection period. Results of
regression analyses for data in Fig. 1 are presented in Table 1. For lean rats infused with saline
(Fig. 1), there were statistically significant positive correlations
between biliary bile salt secretion rates and secretion rates of
cholesterol (Fig. 1A) and phospholipid (Fig. 1C),
as well as between secretion rates of cholesterol and phospholipid
(Fig. 1E). The slope values in Table 1 represent the
magnitudes of coupling between secretion of bile salts, cholesterol, and phospholipids in lean rats. In contrast, there was no correlation between bile salt secretion rates and secretion rates of cholesterol (Fig. 1B) and phospholipid (Fig. 1D) for obese
rats during saline infusion. This indicated that obesity in Zucker rats
is associated with uncoupling of cholesterol and phospholipid secretion
from bile salt secretion. As shown in Fig. 1F, cholesterol,
and phospholipid secretion for obese rats were highly correlated, with
a similar
Ch/
PL value as observed in saline-infused lean rats.
Therefore, coupling of biliary cholesterol secretion to phospholipid
secretion remained intact in obese rats, despite uncoupling of
secretion of these lipids from bile salt secretion.
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Leptin administration (Fig. 1) in lean rats increased Ch/
BS
without influencing
PL/
BS (Fig. 1, A and C,
Table 1). This was associated with a twofold increase in
Ch/
PL in
lean rats (Fig. 1E, Table 1). Leptin infusion in obese rats
resulted in statistically significant correlations between bile salt
secretion rates and secretion rates of cholesterol (
Ch/
BS, Fig.
1B) and phospholipid (
PL/
BS, Fig. 1D).
There was appreciable data scatter in Fig. 1, B and
D, which was reflected in the low r2
values of 0.151 and 0.053, respectively (Table 1). To insure that the
apparent influence of leptin was not an artifact of pooling biliary
lipid secretion rates, the same effect was demonstrated by determining
slope values for each individual rat and then comparing the means of
slope values for leptin- vs. saline-infused rats. Due to the data
scatter, however, we could not exclude the possibility that only a
subset of obese rats were responsive to leptin. As shown in Fig.
1F and Table 1, leptin administration also resulted in a
slight (10%), but not significant, increase in the value of
Ch/
PL compared with saline infusion in obese rats.
Five major bile salt species were identified in lean and obese rats
(Fig. 2), and these comprised 85% of
biliary bile salts. Because the HPLC technique utilized in this study
did not resolve tauro-
-muricholate and tauro-
-muricholate, these
isomers were integrated as a single peak. Figure 2,
A-C, compares biliary bile salt species at three time
periods during bile collection (i.e., 0.5-1 h, 1.5-2 h and
4.5-5 h). Early during the bile collection period (Fig.
2A), the distribution of biliary bile salts species was
similar in lean and obese rats. With increasing time of biliary drainage, the proportion of tauromuricholates increased in obese rats,
whereas the proportion of taurocholate decreased (Figs. 2, B
and C) so that during the final bile collection period (Fig. 2C), tauromuricholates were significantly higher and
taurocholate significantly lower. The hydrophobic index is a calculated
concentration-weighted average of hydrophobicities of individual bile
salts present in a mixture (23) that allows the overall
hydrophobicity of biliary bile salts to be represented by a single
value. Figure 2D plots bile salt hydrophobicities as
functions of bile salt secretion rates. At early points after bile duct
cannulation (i.e., at high bile salt secretion rates), hydrophobic
indexes of lean and obese rats were similar. As bile salt secretion
rates decreased due to drainage of the endogenous bile salt pool, the
mixture of bile salts in bile from obese rats became more hydrophilic
as evidenced by a decrease in the hydrophobic index of bile salts. The
opposite trend was observed in lean rats in which the hydrophobic index increased at low secretion rates, indicating a more hydrophobic mixture
of bile salts in bile. Although leptin infusion did not affect the
hydrophobic index of obese rats, it prevented an increase in
hydrophobic index for lean rats at low bile salt secretion rates.
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Table 2 presents hepatic lipid contents
of lean and obese Zucker rats. Contents of cholesterol (total,
free, and esterified) as well as of phospholipid were similar
in saline- and leptin-infused lean and obese rats. Hepatic triglyceride
contents were sevenfold higher in obese compared with lean rats but
were not changed by leptin administration. Analysis by
thin-layer chromatography (71) demonstrated no changes in
the proportions of phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols,
phosphatidylserines, or sphingomyelins (data not shown). Biliary
phospholipids were >95% phosphatidylcholines in all groups of rats.
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Figure 3 demonstrates the effects of
obesity as well as leptin infusion on molecular species of
phosphatidylcholines in bile and in liver. HPLC resolved seven major
peaks corresponding to eight phosphatidylcholine molecular species,
which accounted for >87% of phosphatidylcholines. Unlike bile salts,
the molecular species of phosphatidylcholines in biles did not vary
during the bile drainage period. The peak representing 16:0-18:1
was not separated from 18:0-22:6 phosphatidylcholine. However,
Patton and Robins (41) have shown that the 16:0-18:1
molecular species constitutes ~85 and ~60% of this peak in rat
bile and liver, respectively. Compared with biliary
phosphatidylcholines in saline-infused lean rats, Fig. 3A
demonstrates in saline-infused obese rats a 30% reduction in the major
biliary phosphatidylcholine, 16:0-18:2. In these obese rats, there
was a reciprocal twofold increase in 16:0-18:1 (18:0-22:6).
Figure 3B shows that the same qualitative differences
between lean and obese rats were also present in liver. In addition,
there was in liver a higher percentage of 18:0-18:1 in obese
compared with lean rats. Figure 3C presents the ratios of
molecular species in bile normalized to the same molecular species in
liver. Data plotted in this format reveal relative enrichment of each
molecular species in bile compared with liver. Values that exceed 1 (e.g., 16:0-18:2) are relatively enriched in bile compared with
liver, and molecular species with ratios <1 are relatively enriched in
liver (e.g., 18:0-20:4). Whereas in lean rats, leptin infusion
decreased the proportion of 18:0-18:1 phosphatidylcholine in bile,
it did not significantly alter the enrichment of this molecular species
in bile. In obese rats, leptin did not significantly change the molar
percentages of 16:0-18:2 phosphatidylcholine in liver or bile.
However, leptin slightly increased the enrichment of this species in
bile compared with liver.
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To examine whether cholesterol recruited by leptin for secretion into
bile might be derived from newly synthesized cholesterol or cholesterol
destined for catabolism into bile salts, we measured hepatic activities
of HMG-CoA reductase, cholesterol 7-hydroxylase, and sterol
27-hydroxylase. Figure 4 demonstrates
that leptin infusion into lean or obese rats resulted in >50%
reductions in HMG-CoA reductase activity. In contrast, leptin treatment
doubled the hepatic activities of cholesterol 7
-hydroxylase and
sterol 27-hydroxylase in obese rats. In lean rats, the activities of
both enzymes also tended to increase in response to leptin, but the
differences from saline-infused controls did not reach statistical
significance.
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Finally, we examined the influence of leptin on plasma cholesterol and
its distribution among lipoproteins. For each rat, cholesterol
concentrations in plasma and its distribution among lipoproteins were
determined at baseline (time 0) as well as 6 and 12 h
after the start of a saline or leptin infusion. Figure 5 displays representative elution
profiles for plasma lipoproteins from lean and obese rats before
infusion with leptin or saline. Peak designations as VLDL, LDL, and HDL
are in accordance with Liao et al. (28). Because columns
were loaded with identical volumes of plasma (200 µl), quantitative
as well as qualitative differences were apparent between lean and obese
rats. Consistent with earlier findings (28), each
lipoprotein fraction contained more cholesterol in obese compared with
lean rats, and this difference was most pronounced for VLDL. In
addition, the peak fractions of LDL and HDL for obese rats eluted
slightly earlier, consistent with larger particle sizes. Because VLDL
eluted at the void volume of the Superose 6 gel filtration columns,
potential differences in VLDL sizes could not be resolved.
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Figure 6 summarizes results for plasma
cholesterol and individual lipoprotein fractions. At each point in
time, total plasma cholesterol concentrations (Fig. 6A) were
higher in obese compared with lean rats. This was generally the case
for VLDL, LDL, and HDL (Fig. 6, B-D), with the
exception that all differences did not achieve statistical
significance. In lean rats, there was a 60% increase in total plasma
cholesterol over the 12-h period of the experiment for both leptin- and
saline-infused rats, and this was principally due to an increase in the
LDL fraction (Fig. 6C). Leptin infusion had no effect on
total plasma cholesterol concentrations in lean or obese rats. However,
as shown in Fig. 6B, leptin administration decreased VLDL
cholesterol in both lean and obese rats compared with saline infusion.
Significant differences were observed in both lean and obese rats at
6 h. This difference was maintained at 12 h for obese but not
lean rats. As shown in Fig. 6, C and D, leptin
administration did not affect the plasma concentrations of LDL or HDL.
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DISCUSSION |
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Even before cloning and characterization of leptin and its receptor, abnormalities in biliary lipid metabolism were reported in genetically obese rats. In the Koletsky corpulent (cp/cp) rat, obesity is due to a null mutation in the leptin receptor (60). Using the SHR/N-corpulent strain of the Koletsky (cp/cp) rat, Turley (61) demonstrated reductions in the capacity of bile salts to promote the biliary secretion of cholesterol and phospholipids. This was attributed in part to depressed hepatic cholesterol synthesis in the SHR/N-corpulent rat strain (62). St. George et al. (56) reported quite different obesity-related changes in the JCR:LA-corpulent rats, which are also derived from the Koletsky rat: biles of obese rats were enriched with cholesterol relative to bile salts and phospholipids. These observations notwithstanding, a primary pathophysiological role for leptin in hepatic cholesterol elimination has remained unexplored.
This study was designed to examine the role of leptin in biliary
cholesterol elimination using the Zucker rat, in which homozygosity for
the fa mutation gives rise to obesity but does not
completely eliminate leptin responsiveness. We observed that obesity in
Zucker rats was associated with uncoupling of cholesterol and
phospholipid secretion from biliary secretion of bile salts. At a dose
of leptin that we have shown previously to elevate plasma leptin
concentrations to 500 ng/ml compared with basal levels of 3 and 70 ng/ml in lean and obese rats, respectively (44), biliary
lipid secretion was partially normalized in obese
(fa/fa) rats. This treatment induced hypersecretion of biliary cholesterol in lean (FA/) rats.
In a previous study on the influence of acute intravenous leptin
administration in rats (44), a 6-h infusion period was sufficient to observe changes in hepatic glucose production that were
correlated with alterations in gene expression of hepatic glucokinase
and phosphoenolpyruvate carboxykinase. To begin to explore the
influence of leptin on biliary lipid metabolism, the current study was
designed to test for an effect of leptin under similar experimental
conditions, in which bile fistulae were placed immediately after a 6-h
infusion period. An important limitation of this experimental design is
that steady-state conditions with respect to bile acid and cholesterol
metabolism (27, 53) may not have been achieved under these
conditions. In addition, changes in enzyme activities of cholesterol
7-hydroxylase, sterol 27-hydoxylase, and HMG-CoA reductase (Fig. 4)
may have been indicative of posttranslational regulation. Despite these
possibilities, our data are consistent with a key role for leptin in
regulating biliary lipid secretion.
Although the hepatocellular mechanisms by which bile salts promote
biliary secretion of cholesterol and phospholipid molecules remain
incompletely understood, accumulating evidence suggests that
physical-chemical events that occur at the canalicular (apical) plasma
membrane play a critical role in coupling of biliary lipid secretion.
As illustrated in Fig. 7, bile salts are
actively secreted into bile by ABCB11 (trivially referred to as the
bile salt export pump, Bsep), an ATP-dependent transporter localized to
the canalicular membrane (19). Translocation of
phosphatidylcholines from inner to outer membrane hemi-leaflet is
accomplished by ABCB4 (trivially known as Mdr2), a canalicular
ATP-dependent P-glycoprotein (46, 52). Detergent-like bile
salt molecules interact with the exoplasmic leaflet of the canalicular
membrane and promote the formation of biliary vesicles composed of
phosphatidylcholines and cholesterol (11).
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Bile salt-membrane interactions that promote biliary lipid secretion (Fig. 7) are influenced by the molecular species of bile salts as well as the membrane composition. Accordingly, secretion rates of vesicles into bile vary in proportion to the hydrophobicity of biliary bile salts (9, 12). Variations in bile salt hydrophobicities as functions of bile salt secretion rates (Fig. 2C) might have been expected to produce nonlinear relationships between bile salt secretion rates and secretion rates of cholesterol and phospholipid. Presumably due to data scatter, however, these relationships were best modeled as linear relationships (Fig. 1). Over the range of bile salt concentrations in the current study, a diminished capacity of more hydrophilic bile salts to interact with the canalicular membrane may have contributed to uncoupling of biliary lipid secretion in obese rats (Fig. 1, A and C, Table 1).
Studies using both native (18, 73) and model membranes (7, 34, 65) have suggested that the phospholipid composition of the canalicular membrane may influence bile salt-membrane interactions and biliary lipid secretion. In particular, we have demonstrated that substitution of the more hydrophilic 16:0-18:2 phosphatidylcholine with the more hydrophobic 16:0-18:1 species significantly diminishes interactions between bile salts and model membranes (7). Therefore, depletion of 16:0-18:2 phosphatidylcholine in membranes of obese rat livers and enrichment with 16:0-18:1 phosphatidylcholine (Fig. 3) may also have led to uncoupling of lipid secretion.
Partial restoration of lipid coupling (Fig. 1, B and D, Table 1) in the absence of changes in bile salt hydrophobicity (Fig. 2D) or phosphatidylcholine composition (Fig. 3) suggest that leptin's acute regulatory effects on biliary lipid secretion are not due to changes in physical-chemical interactions among lipids. Stimulation of ABCB4 activity represents a potential canalicular mechanism by which leptin administration promoted biliary phosphatidylcholine secretion in obese rats. In transgenic mice, the level of ABCB4 expression regulates coupling of biliary phosphatidylcholine secretion to bile salt secretion (55). In these mice, however, cholesterol secretion is not tightly coupled to phospholipid secretion (55). In Zucker rats, there was tight coupling of cholesterol to phospholipid secretion in the absence or presence of leptin infusion (Fig. 1F). Moreover in lean rats, the increase in coupling of cholesterol to bile salt secretion occurred without a concomitant increase in phosphatidylcholine secretion rates. Taken together, these findings argue against regulation of biliary lipid secretion by leptin via ABCB4.
The influence of acute leptin administration on hepatic cholesterol
metabolism is depicted in Fig. 7. Free and esterified cholesterol
contents of livers from lean or obese rats remained unchanged (Table
2), indicating that leptin did not simply mobilize preformed hepatic
stores of cholesterol for secretion into bile. To determine whether
leptin might have increased synthesis of cholesterol for biliary
secretion, we measured activities of HMG-CoA reductase in liver (Fig.
4). Consistent with an earlier study by McNamara (32), we
observed similar specific activities in lean and obese Zucker rats.
However, 50% reductions in both lean and obese rats revealed that
leptin suppressed rather than increased cholesterol biosynthesis. In
the liver, cholesterol is also catabolized by conversion to bile acids.
This occurs via a classic pathway for which the microsomal enzyme
cholesterol 7-hydroxylase is rate limiting, and an alternative
pathway that is initiated by mitochondrial sterol 27-hydroxylase.
Although a leptin-induced decrease in cholesterol catabolism could have
led to increased biliary cholesterol secretion, acute leptin
administration instead promoted twofold increases in the specific
activities of cholesterol 7
-hydroxylase and sterol 27-hydroxylase in
obese rats and nonsignificant increases in activities of each enzyme in
lean rats (Fig. 4). Therefore, leptin increased cholesterol catabolism
in addition to suppressing its biosynthesis.
Decreases in plasma VLDL cholesterol concentrations (Fig. 6B) in response to leptin suggests a probable source of excess biliary cholesterol, as is illustrated in Fig. 7. Elevated levels of VLDL in obese Zucker rats are the result of overproduction of VLDL lipoprotein particles (48, 68) rather than defects in their catabolism (68). Therefore, leptin most likely decreased plasma VLDL cholesterol concentrations by suppressing production. Because VLDL and biliary cholesterol appear to derive from a common hepatic pool (36, 54, 58), the decrease in VLDL production may have, in turn, increased the availability of cholesterol for biliary secretion.
In summary, our findings suggest that leptin plays an integral role in biliary elimination of plasma cholesterol and that uncoupling of cholesterol and phospholipid secretion from bile salt secretion in Zucker (fa/fa) rats represents a combined metabolic defect. Chronic obesity is associated with decreases in bile salt hydrophobicity and increases in phosphatidylcholine hydrophobicity, which favor uncoupling. Although acute administration did not reverse these changes, leptin partially restored coupling in obese animals and promoted hypersecretion of biliary cholesterol in lean rats. Collectively, these leptin-induced changes in lipid metabolism imply an integrated regulatory response to promote cholesterol elimination. Because leptin has been shown to inhibit hepatic triglyceride synthesis (64), we postulate that a primary effect of leptin in livers of Zucker rats may have been to inhibit VLDL formation by limiting triglyceride supply. To prevent accumulation of cholesterol that was no longer packaged and secreted in VLDL, the liver responded by downregulating synthesis, upregulating catabolism to bile acids, and increasing biliary cholesterol secretion.
It is important to note that our data do not exclude the possibility
that leptin directly regulates hepatic activities of cholesterol
7-hydroxylase, sterol 27-hydoxylase, and HMG-CoA reductase.
Hepatocytes express leptin receptors (6, 75), which
mediate insulin-like signaling (75). In primary hepatocyte cultures (1), in the isolated perfused rat liver
(35) and in vivo (44), leptin enhances the
inhibitory effects of insulin on glycogenolysis and hepatic glucose
production. In hepatocytes, insulin downregulates cholesterol
7
-hydroxylase (63, 69) and sterol 27-hydoxylase
(63) while upregulating HMG-CoA reductase (20,
57). Therefore, potentiation of insulin action would not likely
explain the influence of leptin on enzyme activities as observed in our
experiments (Fig. 4). Similarly, evidence has been presented that
activation of peroxisome proliferator-activated receptor-
downregulates cholesterol 7
-hydroxylase (31, 40) and
may alter bile acid composition via sterol 12
-hydroxylase (24). Leptin increases peroxisome proliferator-activated
receptor-
expression in adipocytes (70), and its
influence in hepatocytes remains unknown. Further investigation will be
required to elucidate cellular and molecular mechanisms whereby leptin
regulates hepatic sterol metabolism.
The proposed role for leptin in biliary elimination of plasma cholesterol is supported by recent observations in leptin-deficient ob/ob mice, which are hypercholesterolemic principally due to elevations in plasma HDL concentrations (39). Silver et al. (51) have demonstrated that chronic treatment of ob/ob mice with low-dose leptin (i.e., amounts insufficient to reduce body weight) reduced plasma HDL cholesterol concentrations by promoting hepatic clearance. In separate studies, Bouchard et al. (2) showed that the ob mutation confers resistance to cholesterol gallstone formation in otherwise gallstone-susceptible C57BL/6 mice. These observations, taken in context with our current findings and epidemiological data (14), suggest an integral role for leptin in cholesterol homeostasis as well as in the pathogenesis of cholesterol gallstones.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael McCaleb (Amgen, Thousand Oaks, CA) for providing the leptin used in this study.
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FOOTNOTES |
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This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48873 (D. E. Cohen) and DK-26756 (S. Shefer). S. VanPatten was supported by National Institutes of Health Training Grant GM-07491.
Address for reprint requests and other correspondence: D. E. Cohen, Liver Research Center, Ullmann 625, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: dcohen{at}aecom.yu.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 3 February 2001; accepted in final form 13 April 2001.
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