Am J Physiol Gastrointest Liver Physiol 295: G776–G783, 2008.
First published August 21, 2008; doi:10.1152/ajpgi.90275.2008.
Reduced absorption of saturated fatty acids and resistance to diet-induced
obesity and diabetes by ezetimibe-treated and Npc1l1⫺/⫺ mice
Eric D. Labonté,1 Lisa M. Camarota,1 Juan C. Rojas,1 Ronald J. Jandacek,1 Dean E. Gilham,1
Joanna P. Davies,2 Yiannis A. Ioannou,2 Patrick Tso,1 David Y. Hui,1 and Philip N. Howles1
1
Department of Pathology, Genome Research Institute, University of Cincinnati, Cincinnati, Ohio, and the 2Department
of Human Genetics, The Mount Sinai School of Medicine, New York, New York
Submitted 2 April 2008; accepted in final form 13 August 2008
THE COMPARATIVELY NEW DRUG ezetimibe has been shown to
dramatically reduce absorption of dietary and biliary cholesterol and is now used alone or as an adjunct therapy for
reducing plasma cholesterol (3, 29, 43). Results from initial
studies with gene knockout mice are consistent with the drug
having a direct effect on the cholesterol absorption pathway
mediated by the Niemann-Pick type C-1 like protein 1
(NPC1L1) (2, 11). Both ezetimibe treatment and NPC1L1 gene
inactivation effectively reduce hypercholesterolemia in humans and atherosclerosis in animal models (3, 9, 10, 42, 45,
46). Although the physiological impact of ezetimibe and
NPC1L1 on cholesterol absorption is unquestioned, the mechanisms of action of both the drug and the protein are not clear.
NPC1L1 has been reported to be located on the apical surface
of enterocytes as expected for a transport protein (2, 24).
However, other reports have localized this protein to intracellular compartments (8, 51). Similarly, ezetimibe has been
reported to inhibit NPC1L1 function by direct interaction with
the protein (16), but reports from several groups indicate that
ezetimibe also interacts with SR-BI, the class B type I scavenger receptor (1, 13, 28, 30, 44). These reports suggest that
SR-BI serves as a plasma membrane cholesterol transporter
that acts in concert with NPC1L1 to facilitate cholesterol
absorption (13, 30).
Although current reports indicate that ezetimibe treatment
and NPC1L1 gene inactivation do not affect dietary fat (triglyceride) absorption (45– 47), a different cholesterol absorption inhibitor, disodium ascorbyl phytostanyl phosphate (36),
has been shown to reduce weight gain in rodent models fed
diets high in fat and cholesterol (14, 48, 49). These results
suggest that inhibition of cholesterol absorption may impact
dietary fat absorption and/or metabolism and protect against
diet-induced obesity. Interestingly, ezetimibe treatment does
reduce plasma triglyceride levels in some animal models fed
diets high in fat and cholesterol (45), suggesting that
ezetimibe-sensitive pathways may modulate fat metabolism.
The possibility that inhibiting cholesterol absorption may decrease fat absorption, or that ezetimibe directly inhibits some
step in the fat absorption process has substantial clinical
significance since surgical (31, 50) and pharmaceutical (6, 40)
inhibition of fat absorption has become a therapeutic approach
for treating obesity and its comorbidity, type II diabetes.
Hydrolysis and absorption of dietary fat (mainly triglycerides) are extremely efficient (⬎90%). However, because of the
caloric density of lipids, relatively small decreases in absorption can significantly impact energy balance. The end products
of fat digestion, i.e., monoglycerides and free fatty acids,
partition into bile salt-mixed micelles and are delivered, along
with dietary and biliary cholesterol, to the intestinal brush
border for absorption. Whether fatty acid absorption is solely
by passive diffusion or also by facilitated transport remains a
matter of debate. Some studies suggest that FAT/CD36 (fatty
acid transporter/cluster determinant 36) plays a role in fatty
acid absorption by enterocytes (12, 35) although other mechanisms are clearly indicated (18). However, another school of
thought purports that fatty acids cross the apical membrane of
enterocytes by passive diffusion and that apparent transport
against a concentration gradient is mediated by rapid intracellular modification of the fatty acids that prevent them from
diffusing back across the cell membrane (21, 27, 34). Thus
“protein-facilitated” absorption of fatty acids need not result
from transporter activity. The family of fatty acid transport
Address for reprint requests and other correspondence: P. N. Howles, Dept.
of Pathology, Genome Research Institute, Univ. of Cincinnati, 2120 E. Galbraith Rd., Cincinnati, OH 45237 (e-mail: [email protected]).
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.
diabetogenic diet; glucose metabolism; insulin sensitivity; Western
diet; sucrose polybehenate
G776
0193-1857/08 $8.00 Copyright © 2008 the American Physiological Society
http://www.ajpgi.org
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Labonté ED, Camarota LM, Rojas JC, Jandacek RJ, Gilham
DE, Davies JP, Ioannou YA, Tso P, Hui DY, Howles PN. Reduced
absorption of saturated fatty acids and resistance to diet-induced
obesity and diabetes by ezetimibe-treated and Npc1l1⫺/⫺ mice. Am J
Physiol Gastrointest Liver Physiol 295: G776 –G783, 2008. First
published August 21, 2008; doi:10.1152/ajpgi.90275.2008.—The impact of NPC1L1 and ezetimibe on cholesterol absorption are well
documented. However, their potential consequences relative to absorption and metabolism of other nutrients have been only minimally
investigated. Thus studies were undertaken to investigate the possible
effects of this protein and drug on fat absorption, weight gain, and
glucose metabolism by using Npc1l1⫺/⫺ and ezetimibe-treated mice
fed control and high-fat, high-sucrose diets. Results show that lack of
NPC1L1 or treatment with ezetimibe reduces weight gain when
animals are fed a diabetogenic diet. This resistance to diet-induced
obesity results, at least in part, from significantly reduced absorption
of dietary saturated fatty acids, particularly stearate and palmitate,
since food intake did not differ between groups. Expression analysis
showed less fatty acid transport protein 4 (FATP4) in intestinal
scrapings of Npc1l1⫺/⫺ and ezetimibe-treated mice, suggesting an
important role for FATP4 in intestinal absorption of long-chain fatty
acids. Concomitant with resistance to weight gain, lack of NPC1L1 or
treatment with ezetimibe also conferred protection against diet-induced hyperglycemia and insulin resistance. These unexpected beneficial results may be clinically important, given the focus on NPC1L1
as a target for the treatment of hypercholesterolemia.
NPC1L1 AND EZETIMIBE AFFECT SATURATED FAT ABSORPTION AND FATP4 EXPRESSION
MATERIALS AND METHODS
Animals and diets. Npc1l1⫺/⫺ mice were generated by homologous
recombination in embryonic stem cells and backcrossed 10 times into
the C57BL/6J background as previously described (8). Wild-type
C57BL/6J littermate mice were used in the control groups. Animals
were maintained in a temperature- and humidity-controlled room with
a 12-h light-dark cycle and free access to water and rodent chow
(Teklad LM485). Experimental mice were fed either a basal diet
(D12328, Research Diets, New Brunswick, NJ) or one of two diabetogenic diets (high in fat and sucrose). The caloric composition of
diabetogenic diets were 58.5% fat (coconut oil), 25% sucrose, 16.5%
protein (D12331, Research Diets) or 58.5% fat (lard), 27% sucrose,
14.5% protein (F3282, Bio-Serv, Frenchtown, NJ). The Western diet
(TD88137, Harlan Teklad, Madison, WI) was 42% fat (milk fat), 43%
sucrose, and 15% protein. For diets that included ezetimibe, Zetia
tablets (Schering, Kenilworth, NJ) were ground and thoroughly mixed
into portions of the diet at 10 mg/100 g food. Weight gain was
followed by weekly measurements taken at the same time of day
throughout a study. Body composition (fat and lean mass) was
determined with a 1H-magnetic resonance spectroscopy (EchoMRI100; EchoMedical Systems, Houston, TX) before animals were fed
high-fat diets and periodically thereafter, usually near the end of the
light cycle. All animal protocols were approved by the institutional
animal care and use committee at the University of Cincinnati.
Lipid absorption. Fat absorption in mice was determined by the
sucrose polybehenate method essentially as previously described (25).
Individually housed mice were fed the test diet for 3 days, after which
their cages were changed and fecal samples were collected on each of
3 subsequent days. On the 4th and 5th days, fecal pellets were
recovered from each cage and the fatty acid content and composition
of excreted lipids were determined by gas chromatography. Since
behenic acid is essentially absent from the fat sources used in the test
diet and is entirely excreted when given as sucrose polybehenate,
absorption is calculated from the difference between diet and feces in
the ratio behenate/total fatty acid. The calorie composition of the test
diet was 58% fat (1:1 mix of lard and coconut oil so that long- and
medium-chain fatty acids would be similarly represented and readily
detected), 27% sucrose, and 15% protein (nonfat powdered milk).
Cholesterol absorption was determined by the dual-isotope method as
previously described (7, 19).
Blood chemistries. Plasma lipids were measured in tail blood
collected after an overnight fast (8 –10 h) by use of standard colorimetric kits (Thermo Electron, Waltham, MA). Blood glucose was
measured at the same time with AccuCheck glucometers (Roche
Applied Science, Penzberg, Germany). Plasma insulin was measured
by radioimmunoassay (Wako Chemicals, Richmond, VA). Glucose
tolerance tests were also performed after an 8- to 10-h overnight fast.
Baseline glucose levels were determined and the test was initiated by
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oral gavage or intraperitoneal injection of 2 g glucose per kilogram
body weight. Blood was obtained from the tail vein 5, 15, 30, 60, and
120 min after glucose administration.
Western blots. Mucosal scrapings from intestines of overnightfasted mice were homogenized in 5 volumes of 10 mM Tris 䡠 HCl, 0.25
M sucrose, 5 mM benzamidine, 1 mM EDTA, pH 7.4, plus a protease
inhibitor cocktail, with 10 strokes of a Dounce homogenizer. Debris
was pelleted at 1,000 g for 10 min and cell membranes were subsequently pelleted at 100,000 g for 1 h, followed by resuspension in the
above buffer. All steps were carried out on ice or at 4°C and all
fractions were stored at ⫺80°C until analysis. Cytosolic and membrane proteins (50 –100 ␮g) were resolved by SDS-PAGE, transferred
to polyvinylidene difluoride or nylon membranes, and detected with
antibodies against liver fatty acid binding protein (L-FABP) (SC50380, Santa Cruz Biotechnology), FATP4 (a kind gift from Dr.
Nicholas Davidson, Washington University School of Medicine),
NPC1L1 (a kind gift of Dr. Helen Hobbs, University of Texas
Southwestern Medical School), SR-BI (NB400-101, Novus Biologicals), CD36 (mybiosource.com), and ␣-tubulin (MS-581-P0, Lab
Vision). Immunoreactive proteins were detected by goat anti-rabbit
IgG conjugated to horseradish peroxidase (Dako) and visualized by
enhanced chemiluminescence (Pierce) or by FITC-conjugated donkey
anti-rabbit IgG, followed by goat anti-FITC IgG conjugated to alkaline phosphatase and visualized by enhanced chemifluorescence (Amersham). Protein concentrations were determined by a bicinchoninic
acid kit (Pierce) or the Lowry procedure.
Statistical analysis. Results are presented as means ⫾ SD. Statistical significance (P ⬍ 0.05) between groups was assessed by one-way
ANOVA with Tukey’s post hoc test for all pairwise comparisons, or
by Student’s unpaired t-test, by use of Prism 4 (GraphPad Software)
or SigmaPlot 9.01 (Systat Software).
RESULTS
Ezetimibe-treated and NPC1L1-null mice are resistant to
diet-induced obesity. As an initial test to determine whether
ezetimibe treatment or lack of NPC1L1 protein affects nutrient
absorption or metabolism in a physiologically relevant way,
weight gain and other metabolic parameters were monitored
while mice were fed diets enriched with fat and sucrose. In the
first study, weight gain was monitored for several weeks while
mice were fed either a basal diet, similar in composition to
standard rodent chow, or a high-fat, high-calorie, “diabetogenic” diet with 58% of calories from fat and 26% of calories
from sucrose, with or without ezetimibe at 100 ␮g per gram of
food. As shown in Fig. 1, both ezetimibe-treated and Npc1l1⫺/⫺ mice
were resistant to diet-induced weight gain when fed the diabetogenic diet. Control animals (C57BL/6J) gained 23.5 ⫾ 2.2 g
whereas the other groups gained an average of only 7.8 ⫾ 1.3 g
after 14 wk on the diabetogenic diet (P ⬍ 0.001). These
differences were significant after only 3 wk on the diet (P ⬍
0.015). Data shown are from male mice (4 – 6 per group) but
female mice exhibited the same phenotype. Equivalent results
were obtained when similar groups of mice were fed the
standard “Western” diet, which has a less extreme fat content
(42% of calories compared with 58% of calories in diabetogenic diets) and may serve as a better model for diet-induced
human disease (data not shown).
To determine whether the lack of NPC1L1 function affected
diet-induced obesity or overall body growth, body composition
of control and Npc1l1⫺/⫺ mice was measured by NMR after 3
wk on the Western diet. The data in Table 1 show that accrual
of fat mass accounted for essentially all of the difference
between control and Npc1l1⫺/⫺ animals in weight gained.
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proteins (FATPs), which are fatty acyl:CoA synthetase enzymes, appear to be important in this regard in several cell
types (22, 38), and studies indicate that FATP4, specifically,
may be important for intestinal absorption of long-chain fatty
acids (17, 20).
We have initiated experiments to investigate the relationship
between inhibition of cholesterol absorption and efficiency of
dietary fat absorption, with specific focus on susceptibility to
diet-induced obesity and diabetes. Our present results show
that wild-type mice treated with ezetimibe and Npc1l1⫺/⫺ mice
are resistant to diet-induced weight gain and hyperglycemia
compared with control mice, which may result, in part, from
decreased fat absorption. Interestingly, our data show that
ezetimibe treatment or lack of NPC1L1 protein significantly
reduces absorption of saturated fatty acids by a mechanism that
may involve reduced expression of FATP4.
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NPC1L1 AND EZETIMIBE AFFECT SATURATED FAT ABSORPTION AND FATP4 EXPRESSION
Fig. 1. Ezetimibe treatment or Niemann-Pick type C-1 like protein 1
(NPC1L1) gene inactivation prevents diet-induced weight gain. A: body weight
of control mice fed diabetogenic diet with or without added ezetimibe and of
Npc1l1⫺/⫺ mice fed diabetogenic diet was measured for several weeks (n ⫽
4 – 6 per group). B: data expressed as change from starting weight over time.
Differences between control mice fed diabetogenic diet and all other groups
are significant at 3 wk and all times later.
Initially, lean mass and fat mass were the same for the two
groups. Lean mass was not changed in either group after 3 wk
of Western diet. However, fat mass increased more than
twofold in control mice (2.15 ⫾ 0.65 to 5.06 ⫾ 1.22 g) but did
not change significantly in Npc1l1⫺/⫺ mice (1.72 ⫾ 0.60 vs.
2.25 ⫾ 0.96 g). The relative change in fat mass is further
illustrated by the twofold change in fat-to-lean ratio for control
mice (0.10 ⫾ 0.03 to 0.22 ⫾ 0.05). This ratio did not change
for Npc1l1⫺/⫺ mice (0.08 ⫾ 0.05 vs. 0.10 ⫾ 0.04). Similar
results were obtained with the diabetogenic diets and with
ezetimibe-treated mice (data not shown).
Lipid absorption is decreased in ezetimibe-treated and
NPCL1-null mice. These striking differences caused us to
reexamine the effect of ezetimibe and the impact of NPC1L1
on dietary triglyceride as well as cholesterol absorption. Cholesterol absorption was measured by the standard dual-isotope
method (7, 19) and the results are presented in Fig. 2. Similar
Table 1. Western diet adds fat mass to control but not
Npc1l1⫺/⫺ mice
to previous reports (2, 8), cholesterol absorption was inhibited
90% by the absence of NPC1L1 and 80% by ezetimibe treatment. To measure dietary triglyceride absorption, a sensitive
and physiologically accurate method was used. Instead of
monitoring the appearance in serum of digestion products from
an acutely delivered bolus of oil, fecal excretion of dietary fat
was measured and normalized to the excretion of a nonabsorbable fat, sucrose polybehenate, incorporated into the diet.
Briefly, a trace amount of the marker was mixed with a test diet
of desired composition (see MATERIALS AND METHODS) and fecal
samples were collected after the animals had consumed this
diet for 3 to 5 days. Absorption was calculated from the
difference in fat content and composition between the diet and
fecal samples as described (25). The data in Fig. 2 show that
both ezetimibe treatment and lack of NPC1L1 inhibited fat
absorption to a similar degree under normal eating conditions.
Control animals absorbed 94.9% of dietary fatty acids whereas
drug-treated and knockout mice absorbed only 86.9 and 89.7%,
respectively, of the fat from a high-fat, high-sucrose diet (P ⫽
0.021 for ezetimibe and P ⫽ 0.033 for Npc1l1⫺/⫺ vs. controls).
Absorption of specific fatty acids was also determined by
this method for control, ezetimibe-treated, and Npc1l1⫺/⫺
mice, and the results are presented in Table 2. Only a small
effect was observed for the unsaturated fatty acids oleate (18:1)
and linoleate (18:2). Absorption of these fatty acids was ⱖ95%
in controls and 2–5% less in the experimental groups. Longchain saturated fatty acids were absorbed less efficiently than
unsaturated fatty acids by all groups, as expected. However,
Table 2. Reduced fatty acid absorption by Npc1l1⫺/⫺
and ezetimibe-treated mice
% Absorbed
Start
Body weight, g
Fat mass, g
Lean mass, g
Fat/lean ratio
Weight gained, g
Western Diet, 3 wk
Control
Npc1l1⫺/⫺
Control
Npc1l1⫺/⫺
25.77⫾1.16
2.16⫾0.65
21.68⫾0.78
0.10⫾0.03
26.38⫾1.08
1.72⫾0.60
22.51⫾0.90
0.08⫾0.03
29.67⫾1.16
5.06⫾1.22
22.67⫾0.46
0.22⫾0.05
3.90⫾1.10
27.13⫾1.31*
2.25⫾0.96*
22.52⫾0.63
0.10⫾0.04*
0.75⫾0.52*
Values are means ⫾ SD. *P ⬍ 0.01 vs. control on diet.
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Laurate
Myristate
Palmitate
Stearate
Oleate
Linoleate
Control
Ezetimibe
Npc1l1⫺/⫺
97.9⫾1.6
93.5⫾2.6
89.0⫾5.3
68.8⫾10.4
95.9⫾0.6
97.1⫾0.3
94.4⫾2.8*
87.7⫾5.2†
79.2⫾7.3*
49.4⫾13.7*
91.2⫾3.7*
94.9⫾2.0†
94.9⫾2.1*
84.5⫾4.8*
80.4⫾5.2*
49.9⫾10.3*
92.1⫾3.0*
95.1⫾1.2*
Values are means ⫾ SD. *P ⬍ 0.045 vs. control; †P ⬍ 0.06 vs. control.
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Fig. 2. Dietary fat and cholesterol absorption are decreased in ezetimibetreated and Npc1l1⫺/⫺ mice. Cholesterol absorption (left, n ⫽ 3– 4) was
measured by the dual-isotope method and fat absorption (right, n ⫽ 4 –5) was
measured by the sucrose polybehenate method as described in MATERIALS AND
METHODS. Mice were fed basal chow. *P ⬍ 0.001 compared with controls;
**P ⫽ 0.033, ***P ⫽ 0.025 compared with controls.
NPC1L1 AND EZETIMIBE AFFECT SATURATED FAT ABSORPTION AND FATP4 EXPRESSION
Fig. 3. Western blot analysis of proteins potentially important for fatty acid
absorption. The protein from mucosal scrapings of the small intestine from
control, ezetimibe-fed, and Npc1l1⫺/⫺ mice were subjected to Western blot for
analysis of expression of fatty acid transport protein 4 (FATP4), liver fatty acid
binding protein (L-FABP), CD36, and NPC1L1. L-FABP was assayed in
cytosolic fractions; all others were assayed in total cell membrane fractions.
Mice were fasted 12 h before sample collection. Each blot is representative of
at least 3 independent sample collections.
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contribute to decreased fat absorption in drug-treated mice.
However, recent studies (14) show that CD36 does not contribute to absorption of palmitate and stearate, which are the
fatty acids most affected in our experiments. L-FABP is
another protein that has been suggested to play a role in fatty
acid transport and chylomicron production (4, 38). However,
Western blots of cytosol from intestinal homogenates revealed
no difference in the amount of L-FABP present in the different
groups of mice (Fig. 3), indicating that this protein does not
directly contribute to decreased fat absorption by Npc1l1⫺/⫺
and ezetimibe-treated mice. Figure 3 also confirms the absence
of NPC1L1 from knockout mice.
Lack of NPC1L1 function prevents diet-induced hyperglycemia. Since obesity often correlates with hyperglycemia as well
as higher plasma lipids, the effects of ezetimibe and NPC1L1
on glucose metabolism were also investigated. Line 1 of Table 3
summarizes the results from a cohort of C57BL/6J mice from
Jackson Laboratories that were fed diabetogenic diet (lard
based, BioServ) with or without ezetimibe. After 13 wk,
fasting plasma glucose was significantly elevated in mice fed
the diabetogenic diet (179 ⫾ 27 mg/dl) but not in mice fed
diabetogenic diet with ezetimibe (129 ⫾ 19 mg/dl). As expected, plasma cholesterol was also elevated by the diabetogenic diet but remained normal when ezetimibe was included
(data not shown). Lines 2 and 3 of Table 3 summarize results
from a cohort of control and Npc1l1⫺/⫺ mice bred in house
(from the same colony) and fed either basal or diabetogenic
diet (coconut oil-based, Research Diets) for 8 wk. As in the
previous study, plasma glucose of Npc1l1⫺/⫺ mice was not
changed by the diabetogenic diet compared with basal diet,
whereas plasma glucose of control mice was elevated. Maintenance of normal glycemia by ezetimibe-treated and Npc1l1⫺/⫺
mice, despite consuming these calorie-dense diets, suggests
that blocking function of the NPC1L1 pathway protects against
diet-induced diabetes as well as hypercholesterolemia.
To further investigate this difference in diet-induced hyperglycemia, glucose tolerance and insulin sensitivity were measured. Glucose tolerance tests were performed on the three
groups of mice fed either a basal diet or high-fat diet for 10 wk.
After an overnight fast (8 –10 h), baseline glucose was measured and the glucose tolerance test was initiated with an
intraperitoneal injection of glucose (2 g/kg body wt). Blood
glucose was monitored at 5, 15, 30, 60, and 120 min thereafter.
As shown in Fig. 4, there were no differences in glucose
tolerance between the groups fed basal diet (dotted lines),
suggesting that ezetimibe treatment and NPC1L1 inactivation
do not directly affect glucose metabolism in mice. However,
when the animals were fed the diabetogenic diet for 10 wk, a
significant difference in glucose tolerance was observed between groups. Control mice exhibited a loss of glucose tolerance, as depicted by the delayed glucose clearance curve in
Fig. 4 (solid line, solid circles). However, glucose tolerance
was maintained in ezetimibe-treated and Npc1l1⫺/⫺ mice despite the diabetogenic diet (Fig. 4, solid lines, solid squares and
triangles). Insulin secretion during the glucose tolerance test
was also measured but did not differ between groups. In
addition to glucose tolerance, insulin sensitivity was also
assessed. Insulin tolerance tests were performed on Npc1l1⫺/⫺
and control mice after 6 wk of high-fat diet. As shown in Fig. 5A,
control mice exhibited a diet-induced loss of insulin sensitivity
whereas Npc1l1⫺/⫺ mice retained a robust response. Insulin
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drug treatment and lack of NPC1L1 significantly impaired
absorption of saturated fatty acids in a graded manner that
correlated with chain length. Thus stearate (18:0) absorption
was decreased from ⬃69% in controls to ⬃50% in experimental mice, and palmitate (16:0) absorption was decreased from
⬃91% in controls to ⬃80% in experimental mice. Absorption
of medium-chain saturated fatty acids was more moderately
affected. Myristate (14:0) absorption was decreased 7–10%
compared with controls, and laurate (12:0) absorption was
reduced ⬃4%. Data shown are from mice that were naive to
diabetogenic diet except for the test period. Virtually identical
results were obtained from a different cohort of mice that were
fed diabetogenic diet for 6 wk (data not shown).
FATP4 expression is suppressed by ezetimibe and by the
lack of NPC1L1. To begin to understand the mechanism(s) by
which ezetimibe and NPC1L1 might affect absorption of longchain saturated fatty acids, intestinal expression of proteins
thought to contribute to fatty acid absorption was investigated.
Membrane and cytosol fractions were prepared from mucosal
scrapings of the duodena and jejuna from fasted ezetimibetreated, Npc1l1⫺/⫺, and control mice that had been maintained
on basal diet. Western blots were used to determine the relative
amounts of FATP4, CD36, and L-FABP (liver fatty acid
binding protein) protein present in these preparation. FATP4 is
the only member of the fatty acyl:CoA synthetase family
present at significant levels in small intestine (41). It has a
distinct preference for very long-chain fatty acids and for
long-chain saturated fatty acids, and some studies suggest that
it plays a role in dietary fat absorption (17, 20). As shown in
Fig. 3, Western blot analysis revealed that FATP4 was present
at much lower levels in membrane fractions from Npc1l1⫺/⫺
and ezetimibe-treated mice than from control animals. Quantitation of the bands in Fig. 3 (normalized to tubulin) indicates
that FATP4 is reduced ⬃50% by ezetimibe and ⬃60% by the
complete absence of NPC1L1. These data are consistent with
the chain length- and saturation-dependent decrease in dietary
fatty acid absorption by these mice.
CD36 has also been reported to be important for fat absorption, especially for very long-chain fatty acids (12, 35). Western blots revealed a ⬃35% decrease of CD36 (normalized to
␣-tubulin) in intestinal preparations from ezetimibe-treated
mice but normal levels in preparations from Npc1l1⫺/⫺ mice
(Fig. 3). Thus it is possible that lower levels of CD36 may
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NPC1L1 AND EZETIMIBE AFFECT SATURATED FAT ABSORPTION AND FATP4 EXPRESSION
Table 3. Diet effects on blood glucose of control
and Npc1l1⫺/⫺ mice
C57BL/6J
Control
Npc1l1⫺/⫺
Chow/Basal
Diabetogenic
Diabetogenic With Ezetimibe
146⫾17
113⫾13
102⫾18
179⫾27*
144⫾34†
102⫾28†
129⫾19
n.d.
n.d.
Values are means ⫾ SD, in mg/dl. C57BL/6J cohort of mice were from
Jackson Laboratories, 13 wk on diet, n ⫽ 4 – 6, *P ⬍ 0.03 vs. chow and
diabetogenic diet ⫹ ezetimibe; control and Npc1l1⫺/⫺ cohorts of mice were
bred in-house, 10 wk on diet, n ⫽ 6 each, †P ⫽ 0.06. n.d., Not determined.
DISCUSSION
It is well recognized that NPC1L1 plays a key role in
cholesterol absorption and that the drug ezetimibe potently
inhibits this process (2, 8, 11, 16), although the exact
mechanisms involved are poorly understood (1, 28, 30).
Studies of Npc1l1⫺/⫺ mice have focused primarily on diminished intestinal cholesterol absorption (2, 8, 11) and
other functions for NPC1L1 have not been thoroughly
investigated. However, the NPC1L1 protein is found in liver
and other tissues as well (2, 8). Similarly, the notion that
ezetimibe’s overall effect on plasma cholesterol derives
solely from its action in the intestine has not been rigorously
proven even though the drug is known to be present at low
levels in circulation (15, 37). The present report shows that
the NPC1L1 pathway also affects dietary fat absorption,
primarily that of long-chain saturated fatty acids, possibly
by influencing the amount of FATP4 present in enterocyte
membranes. Furthermore, blocking NPC1L1 function with
ezetimibe treatment or eliminating the protein by gene
ablation confers resistance to diet-induced obesity and diabetes.
Fig. 4. Ezetimibe-treated and Npc1l1⫺/⫺ mice are protected from diet-induced
glucose intolerance. Mice were subjected to a glucose tolerance test while fed
basal chow (dotted lines, open symbols) and again after 10 wk of diabetogenic
diet (solid lines, solid symbols). After baseline glucose was measured, tests
were initiated by intraperitoneal injection of 2 g glucose/kg body wt. Blood
glucose was monitored from the tail vein for 2 h thereafter (n ⫽ 6 – 8 per
group). *P ⬍ 0.05 control basal vs. control diabetogenic, **P ⬍ 0.05 control
diabetogenic vs. ezetimibe diabetogenic and Npc1l1⫺/⫺ diabetogenic.
AJP-Gastrointest Liver Physiol • VOL
Fig. 5. Npc1l1⫺/⫺ and ezetimibe-treated mice are protected from diet-induced
loss of insulin sensitivity. A: control and Npc1l1⫺/⫺ mice were subjected to an
insulin tolerance test after 6 wk of Western diet. After baseline glucose was
measured, animals were given insulin (0.75 U/kg) by intraperitoneal injection
and glucose was monitored for 2 h thereafter (n ⫽ 8 –10 per group). Groups
were significantly different, *P ⬍ 0.01, at all times after time 0. B: insulin
sensitivity was similarly measured in different groups of mice fed Western diet
with or without ezetimibe for 3 wk (n ⫽ 8 per group). Groups were significantly different, *P ⬍ 0.02, at 15 and 30 min.
The differences in weight gain between control vs. drugtreated and gene knockout mice were not due to differences in
food intake. The latter was carefully monitored by two different methods in independent experiments and no differences
were detected. However, decreased fat absorption by Npc1l1⫺/⫺
and ezetimibe-treated mice results in decreased calorie intake
from the same amount of food. Although the decrease in total
fat absorbed by Npc1l1⫺/⫺ and ezetimibe-treated mice compared with control mice does not appear dramatic, this decrease
may account for some of the difference in weight gain between
groups over the period of time they were fed the high-fat,
high-calorie diets. The amount of weight not gained because of
reduced fat absorption by the Npc1l1⫺/⫺ and ezetimibe-treated
mice can be estimated from the fat composition of the diet
(36% by weight), the amount eaten per day (2.2 g), and the
difference in overall fat absorption (⬃7%). This calculation
predicts that Npc1l1⫺/⫺ and ezetimibe-treated mice would gain
⬃0.06 g/day less than control mice, which could account for
⬃50% of the observed difference in weight between groups
after several weeks. Thus decreased fat absorption may not
account for all of the difference in weight gained by these
groups. Another possibility, not addressed by the experiments
reported here, is that systemic effects of ezetimibe and
NPC1L1 function in other tissues may modulate overall metabolic rates in mice. Feeding diets enriched in unsaturated fatty
acids, such as in safflower oil, might distinguish between these
possibilities since absorption should be nearly equivalent between the groups.
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tolerance tests were also performed on a separate group of mice
fed high-fat diet with or without ezetimibe for 3 wk. Consistent
with previous data, ezetimibe-treated mice were also more
insulin sensitive than control mice after the dietary challenge
(Fig. 5B).
NPC1L1 AND EZETIMIBE AFFECT SATURATED FAT ABSORPTION AND FATP4 EXPRESSION
AJP-Gastrointest Liver Physiol • VOL
location of transporters and other proteins important to the
absorption process.
Hyperglycemia, insulin resistance, is a comorbidity of
obesity. Thus maintenance of euglycemia and insulin sensitivity by Npc1l1⫺/⫺ and ezetimibe-treated mice fed highfat diets may derive, in part, from their total resistance to the
diet-induced weight gain experienced by control mice. However, it is also possible that absence of NPC1L1 function
systemically may affect glucose and/or fatty acid metabolism by tissues other than intestine, such as heart and
muscle. Results of initial experiments with mice on basal
diet are consistent with this hypothesis (not shown). Such
systemic effects, if present, might be direct or indirect. For
example, location of GLUT4 (glucose transporter 4) is sensitive to
cholesterol levels in cells, with more being localized in the cell
membrane when cholesterol is low (5, 32), a condition that might
exist in the absence of NCP1L1 function (8, 39).
The experiments reported in this study used high-fat,
high-calorie diets as a central component to explore the
effects of NPC1L1 and ezetimibe on aspects of the metabolic syndrome other than hypercholesterolemia, specifically weight gain and hyperglycemia. Three novel and
intriguing effects were revealed when mice were fed these
diets: 1) diet-induced obesity was prevented, 2) saturated fat
absorption was reduced, and 3) diet-induced hyperglycemia
was prevented in ezetimibe-treated and Npc1l1⫺/⫺ mice.
Taken together, these results suggest that a clinically relevant, ezetimibe-sensitive and/or NPC1L1-mediated pathway
has important physiological functions that extend well beyond facilitation of cholesterol absorption in the intestinal
tract.
ACKNOWLEDGMENTS
Fat absorption was measured by the Cincinnati Mouse Metabolic Phenotyping Center NIH U24DK59630.
GRANTS
This work was supported by National Institutes of Health (NIH) Grants
R01DK76907 and R01HL78900, by U. S. Department of Agriculture Grant
CSREES 2002-01135, and by American Heart Association Grant 0525340B
(Great Rivers Affiliate).
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