THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 40, Issue of October 5, pp. 37402–37408, 2001
Printed in U.S.A.

Severe Hypercholesterolemia, Hypertriglyceridemia, andAtherosclerosis in Mice Lacking Both Leptin and the Low Density
Lipoprotein Receptor*
Received for publication, November 8, 2000, and in revised form, May 29, 2001
Published, JBC Papers in Press, July 9, 2001, DOI 10.1074/jbc.M010176200

Alyssa H. Hasty‡, Hitoshi Shimano§¶, Jun-ichi Osuga, Ichiji Namatame, Akimitsu Takahashi§,Naoya Yahagi, Stéphane Perrey, Yoko Iizuka, Yoshiaki Tamura, Michiyo Amemiya-Kudo,
Tomohiro Yoshikawa, Hiroaki Okazaki, Ken Ohashi, Kenji Harada, Takashi Matsuzaka§,
Hirohito Sone§, Takanari Gotoda, Ryozo Nagai, Shun Ishibashi, and Nobuhiro Yamada§From the Department of Metabolic Diseases, University of Tokyo, Tokyo, 113-8655, Japan, and the §Division of
Endocrinology and Metabolism, Department of Internal Medicine, University of Tsukuba, Ibaraki 305-8575, Japan

Leptin-deficient mice (ob/ob) are an excellent murine
model for obesity, insulin resistance, and diabetes, all ofwhich are components of a multiple risk factor syndromethat, along with hypercholesterolemia, precipitates a potential high risk for atherosclerosis. In the current study,
we show an unexpectedly severe hyperlipidemia in ob/ob
mice on a background of low density lipoprotein receptor(LDLR) deficiency (ⴚ/ⴚ). Doubly mutant mice (LDLRⴚ/ⴚ;ob/ob) exhibited striking elevations in both total plasmacholesterol (TC) and triglyceride (TG) levels (1715 ⴞ 87
and 1016 ⴞ 172 mg/dl, respectively), at age 3– 4 months,
resulting in extensive atherosclerotic lesions throughout
the aorta by 6 months. Lipoprotein analyses revealed theelevated TC and TG levels to be due to a large increase inan apoB-containing broad-␤ remnant lipoprotein fraction. While fasting, diet restriction, and low level leptin
treatment significantly lowered TG levels, they caused
only slight changes in TC levels. Hepatic cholesterol andtriglyceride contents as well as mRNA levels of cholesterologenic and lipogenic enzymes suggest that leptin deficiency increased hepatic triglyceride production but did
not change cholesterol production in ob/ob mice regardless of their LDLR genotype. These data provide evidence
that the hypertriglyceridemia and hypercholesterolemia
in the doubly mutant mice are caused by distinct mechanisms and point to the possibility that leptin might havesome impact on plasma cholesterol metabolism, possibly
through an LDLR-independent pathway. This model will
be an excellent tool for future studies on the relationshipbetween impaired fuel metabolism, increased plasma
remnant lipoproteins, diabetes, and atherosclerosis.

Dyslipidemia, diabetes, and obesity are among the many risk
factors associated with coronary artery disease. Althoughmany human patients present with varying combinations ofthese risk factors, most animal models produced to date have

* This work was supported in part by Promotion of Fundamental
Studies in Health Science of the Organization for Pharmaceutical
Safety and Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ Recipient of a fellowship under the Japan Society for the Promotionof Science Postdoctoral Fellowship Program for Foreign Researchers.
¶ To whom correspondence should be addressed: Dept. of Internal
Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki, Japan 305-8575. Fax: 81-298-63-2170;
E-mail: shimano-tky@umin.ac.jp.

focused on only one or two and their effects on atherogenesis.
Leptin-deficient mice (ob/ob)1 are an excellent murine modelfor obesity, insulin resistance, and diabetes, all of which arecomponents of a multiple risk factor syndrome that, along withhypercholesterolemia, precipitates a potential high risk for atherosclerosis (1, 2). These mice also have fatty livers, presumably due to hepatic overproduction of triglycerides, reflecting
an imbalanced energy metabolism. However, they show only amodest increase in plasma triglyceride and HDL cholesterollevels (3) and thus do not develop atherosclerosis on a regularchow diet as shown in the current study. It has been reported
that ob/ob mice might have an impaired secretion of very low
density lipoproteins (VLDL) (4); however, extensive studies on
apoB-containing lipoprotein metabolism in these mice have notbeen undertaken.
Remnant lipoprotein particles are the apolipoprotein B(apoB)-containing lipoproteins that remain in the circulation
after hepatic VLDL and intestinal chylomicrons are lipolyzed.
They include lipoproteins in the size range of VLDL, intermediate density lipoproteins (IDL), and low density lipoproteins(LDL). Remnant particles can exist in individuals in a normolipidemic state, especially postprandially, but are generally
processed by lipoprotein lipase and quickly cleared fromplasma. However, the triglyceride-rich lipoproteins that are
increased in diabetic patients as well as cholesterol-rich remnants referred to as ␤-VLDL that are found in patients with
type III hyperlipidemia are also remnant lipoproteins and inthese cases are known to be atherogenic. Several mouse modelsof chronically increased remnant lipoproteins have recently
been created by targeted disruption of genes that are crucial inlipoprotein metabolism. These include apoE-deficient mice (5,6), LDL receptor-deficient mice on a high fat diet (7), andLDLR/LDLR-related protein (LRP) double knockout mice (8),confirming the importance of apoE as a ligand and LDLR and
LRP as receptors for plasma remnant clearance. Recently, it
was reported that disruption of the LDLR gene unmasked
severe hyperlipidemia with increased remnant particles in sterol regulatory element-binding protein (SREBP)-1a transgenic

1
The abbreviations used are: ob/ob, leptin-deficient mice; LDLR, lowdensity lipoprotein receptor; TC, total plasma cholesterol; TG, total
plasma triglycerides; LRP, LDLR-related protein; HDL, high density
lipoproteins; LDL, low density lipoproteins; VLDL, very low density
lipoprotein; IDL, intermediate density lipoproteins; SREBP, sterol regulatory element-binding protein; TgSREBP-1a;LDLR⫺/⫺, SREBP-1atransgenic mice crossed with LDLR(⫺/⫺) mice; apo, apolipoprotein;
HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline.

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This paper is available on line at http://www.jbc.org

Severe Hyperlipidemia and Atherosclerosis in LDLR⫺/⫺;ob/ob Micemice (TgSREBP-1a;LDLR⫺/⫺), providing another model forstudying the effects of excessive remnant lipoproteins inplasma (9). In this model, hepatic overexpression of nuclear
SREBP-1a, a potent transcription activator for both cholesterol
and triglyceride synthesis, caused overproduction of lipids andlarge VLDL in the liver. Normally, these large particles can betaken up by the LDLR very efficiently, which prevented thedevelopment of hyperlipidemia in the TgSREBP-1a mice; however, in the absence of the LDLR, remnant lipoproteins accumulated in plasma, providing support for the importance of the
LDLR in remnant clearance. The TgSREBP-1a;LDLR⫺/⫺
mouse model is different from other hyperlipidemic animalmodels because hepatic lipid overproduction, rather than simply a lack of lipoprotein clearance, contributes to the pathological mechanism. This observation prompted us to study themetabolic consequences of hepatic triglyceride overproduction
as seen in ob/ob mice, on the background of a state of impaired
remnant clearance.
We sought to analyze the effects of leptin deficiency on remnant lipoprotein metabolism by producing mice that were deficient in both leptin and the LDLR (LDLR⫺/⫺;ob/ob). These
doubly mutant mice showed extreme elevations in both plasmacholesterol and triglyceride levels and also had extensive atherosclerotic lesions throughout the aorta. While fasting, diet
restriction, and low level leptin treatment of LDLR⫺/⫺;ob/ob
mice significantly lowered TG levels, they caused only slightchanges in total plasma cholesterol (TC) levels. This model
supports the idea that a relationship exists between energy
imbalance/hepatic overproduction of triglycerides and plasma
remnant accumulation during impaired lipoprotein clearance.
EXPERIMENTAL PROCEDURES

Animals—LDLR(⫺/⫺) mice (10) were back-crossed onto a C57Bl/6
background to the 10th generation. ob/⫹ mice on a C57Bl/6 backgroundwere purchased from Jackson Laboratories and were also maintained inour colonies. To obtain leptin deficient (ob/ob) on a background of LDLR
deficiency, ob/⫹ mice were crossed with LDLR(⫺/⫺) mice, and the F1progeny of these matings (LDLR⫹/⫺;ob/⫹) were then crossed to obtainmice that had either zero, one, or both normal LDLR alleles and wereleptin-deficient (LDLR⫺/⫺;ob/ob, LDLR⫹/⫺;ob/ob, and LDLR⫹/⫹;ob/
ob, respectively) as well as control LDLR(⫺/⫺), LDLR(⫹/⫺), and wild
type mice. Male and female mice were caged separately, and data was
combined for all experiments, since no gender difference was noted.
Mice were maintained and cared for according to the regulations of theTokyo University Animal Care Committee. Mice were kept in microisolator cages with 12-h light/dark cycles and were fed ad libitum except
during food restriction procedures. All mice were fed a normal rodent
chow diet containing 0.075% cholesterol (MF; Oriental Yeast Co., Ltd.
(Osaka, Japan)). A cholesterol-free diet was also purchased from Oriental Yeast Co. and fed to mice ad libitum for the cholesterol-free dietstudies. For caloric restriction, mice were given 1.6 g of normal chowdiet per day, and control age-matched mice were fed ad libitum.
Plasma Analyses—Blood collections were performed via optical venous plexus puncture with EDTA-treated tubes. Collections were performed between 10 and 12 a.m. on animals fed ad libitum. Samples
were preserved with EDTA and NaN3. Plasma cholesterol, triglyceride,and HDL cholesterol levels were measured with colorimetric assays by
Determiner TC555, TG555, and HDL-C (Kyowa Medex, Co., Ltd. Tokyo,
Japan), respectively. Glucose was measured by a standard enzymatic
method. Insulin levels were tested with an enzyme-linked immunosorbent assay kit by LABIS (Japan) for mouse insulin. For analysis oflipoprotein distribution, pooled plasma samples from three mice pergroup were subjected to high performance liquid chromatography(HPLC) (SRL, Japan). Estimation of HDL levels in some samples wereobtained from HPLC plots by calculation of the area under the curve.
Agarose gel electrophoresis for lipoproteins was performed per standard protocol on gels purchased from Ciba/Corning.
Determination of Liver Cholesterol and Triglyceride Content—Livers
were immediately collected and snap frozen in liquid nitrogen. A 50-mg
piece of liver was homogenized in PBS. Folch’s reagent (CHCl3/MeOH,
2:1) (0.75 ml) was added to the homogenate. The nonaqueous phase wascollected, and 30 ␮l of 200 mg/ml Triton X-100 in CHCl3 was added.
Samples were dried and used for cholesterol and triglyceride analysis as

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TABLE I
Blood TC and TG levels in 12–16-week-old miceMice were maintained on a chow diet (0.075% cholesterol). TC and TG(mg/dl) of 3– 4-month-old mice were measured as described under “Experimental Procedures.” Values are given as the mean ⫾ S.E.
GenotypenCholesterol

Wild type
LDLR(⫹/⫺)LDLR(⫺/⫺)LDLR⫹/⫹;ob/ob
LDLR⫹/⫺;ob/ob
LDLR⫺/⫺;ob/ob

4
3
6
5
6
6

81 ⫾ 16149 ⫾ 11265 ⫾ 40119 ⫾ 25282 ⫾ 29
1715 ⫾ 87

Triglyceridemg/dl

60 ⫾ 12100 ⫾ 12161 ⫾ 25120 ⫾ 1278 ⫾ 81016 ⫾ 172

described above for plasma samples.
RNA Preparation and Northern Blotting—Total RNA was preparedfrom the snap frozen livers with TRIzol reagent (Life Technologies,Inc.). RNA (12 ␮g) was electrophoresed through formalin-denatured
agarose gels and transferred to Hybond-N membranes (Amersham
Pharmacia Biotech). cDNA probes were prepared as described (11, 12).
Probes were labeled with [␣-32P]dCTP using Megaprime DNA Labeling
System kit (Amersham Pharmacia Biotech). Membranes were hybridized with the radiolabeled probes in Rapid-hyb Buffer (Amersham
Pharmacia Biotech) and washed in 0.1⫻ SSC, 0.1% SDS at 65 °C.
Membranes were exposed to Eastman Kodak Co. XAR-5 film for 2–5 h
at ⫺80 °C.
Lipoprotein Lipase Measurements—Lipoprotein lipase measurements in postheparin plasma (10 min after intravenous injection of 0.1
mg/Kg heparin) were performed using a modification of the standard
technique by Muir (21).
Leptin Treatment—Leptin was purchased from Calbiochem and was
prepared according to the manufacturer’s instructions. Leptin was diluted in PBS for individual mice according to their weight, and 0.3 ␮g/g
body weight was injected in 100-␮l doses per day.
Atherosclerotic Lesion Analyses—Whole aortae were collected andstained with Sudan IV as described (7, 13). Cross-sections of proximal
aorta were prepared and stained with Oil-Red-O by SKK (Japan) per
standard techniques as described (14).
Statistical Analyses—All values are stated as mean ⫾ S.E., anddifferences between groups were evaluated with Student’s t test.
RESULTS

Plasma Analyses—Blood was collected from wild type,
LDLR(⫺/⫺), LDLR(⫹/⫺), LDLR⫹/⫹;ob/ob, LDLR⫹/⫺;ob/ob,
and LDLR⫺/⫺;ob/ob mice that were 3– 4 months of age. Plasma
was analyzed for TG and TC levels (Table I). LDLR⫹/⫹;ob/ob
mice had slightly elevated TC levels (119 ⫾ 25 mg/dl), as waspreviously reported (3). LDLR⫹/⫺;ob/ob mice showed a significant elevation in TC levels to 282 ⫾ 29 mg/dl with no increasein TG levels; however, the LDLR⫺/⫺;ob/ob mice showed adramatic increase in TC levels with an average of 1715 ⫾ 87
mg/dl with values ranging from 1430 to 2030 mg/dl. TG levelsin these mice were also highly elevated (1016 ⫾ 172 mg/dl).
Since well known phenotypes of ob/ob mice such as obesity,
increased plasma glucose, and insulin levels are age-dependent, a time course study of TC and TG levels from additional
animals was performed (Fig. 1). At 1 month (upon weaning),
the LDLR⫺/⫺;ob/ob mice already displayed modest elevations
in both TC and TG levels (369 ⫾ 20 mg/dl and 137 ⫾ 26 mg/dl,
respectively; n ⫽ 6). These values increased significantly to716 ⫾ 48 and 393 ⫾ 97 after mice were placed on a normal dietfor 2 weeks. The LDLR⫺/⫺;ob/ob mice showed an age-dependent increase in TC and TG levels between 1 and 4 months, withvalues becoming maximal at 3– 4 months, followed by a gradualdecrease to 585 ⫾ 44 and 306 ⫾ 36 mg/dl (n ⫽ 3) by 8 months.
LDLR⫹/⫺;ob/ob mice also showed a similar pattern of reaching
peak TC levels at 3– 4 months of age and maintained stable TC
levels of ⬃280 mg/dl thereafter. TG levels in LDLR⫹/⫺;ob/ob
mice were not different from wild type animals at any time
(Fig. 1). HDL levels in all groups of mice were estimated from
HPLC results and remained unchanged over the course of the37404

Severe Hyperlipidemia and Atherosclerosis in LDLR⫺/⫺;ob/ob Mice

FIG. 1. Time course study of TG and TC in LDLRⴚ/ⴚ;ob/ob and
LDLRⴙ/ⴚ;ob/ob mice. Blood was collected from LDLR⫺/⫺;ob/ob and
LDLR⫹/⫺;ob/ob mice monthly, from age 1 month (at weaning) to 8
months and analyzed for TG and TC levels as described under “Experimental Procedures.” LDLR⫺/⫺;ob/ob mouse values are shown in closed
squares, and LDLR⫹/⫺;ob/ob values are in open circles. The top panel
shows TC values, and the bottom panel shows TG levels. Error bars
represent S.E. n ⬎ 6 for 1– 6-month time points; n ⫽ 3 for 7- and8-month data.

study. Wild type mice had HDL levels of 54 mg/dl at 3 months
and 47 mg/dl at 8 months. LDLR(⫺/⫺) HDLs were slightly
elevated to 69 and 65 mg/dl at 3 and 8 months. LDLR⫹/⫹;ob/ob
mice were 89 and 56 mg/dl, LDLR⫹/⫺;ob/ob were 69 and 62
mg/dl, and LDLR⫺/⫺;ob/ob had levels of 90 and 94 mg/dl at 3
and 8 months, respectively. Glucose levels in LDLR⫺/⫺;ob/ob
mice were ⬃200 mg/dl at 2 months and rose to between 300 and
400 mg/dl from 2 to 7 months. There were no significant differences between LDLR⫺/⫺;ob/ob and LDLR⫹/⫺;ob/ob mice
during the study (data not shown). Insulin levels in LDLR⫺/⫺;ob/ob and LDLR⫹/⫺;ob/ob mice rose steadily from ⬃30 to⬃300 ng/ml blood between 1 and 7 months (data not shown).
Lipoprotein Profiles—Plasma samples from 12–16-week-old
mice were further analyzed for the distribution of lipoproteins(Fig. 2). As expected, wild type mouse plasma contained primarily HDL-sized lipoproteins. LDLR⫹/⫹;ob/ob mice showed aunique profile containing mostly HDL with a shoulder on the
HDL peak with particles sized between LDL and HDL, as has
been recently described (15). This peak most likely contains
HDL1 particles and will be referred to as such. LDLR(⫺/⫺)
mice displayed elevated levels of LDL, similar to previous reports (10). The distribution of lipoproteins in LDLR⫹/⫺;ob/ob
mice was similar to the LDLR⫹/⫹;ob/ob mice except that the
HDL1 peak was higher and broader. In contrast, plasma from
LDLR⫺/⫺;ob/ob mice contained a severely elevated and broadened lipoprotein peak ranging from VLDL/IDL-sized particles
to LDL-sized particles, presumably remnant lipoproteins, andretained HDL levels similar to wild type animals.
Further analysis of lipoprotein profiles was performed bylipoprotein-agarose gel electrophoresis (data not shown). As
expected, LDLR⫹/⫹;ob/ob mouse plasma had a pattern similar
to wild type mice, although the ␣-band was slightly broader,presumably due to the HDL1 particles. Interestingly, LDLR⫹/⫺;ob/ob mice had both ␣ and ␤ migrating particles, andLDLR⫺/⫺;ob/ob plasma contained a broad ␤-migrating band
similar to that seen from apoE(⫺/⫺) lipoproteins.

FIG. 2. HPLC analyses of serum samples from LDLRⴚ/ⴚ;ob/ob
mice. Blood was collected from 3– 4-month old mice, and three pooled
mouse serum samples for each group were separated by gel filtration
chromatography as described under “Experimental Procedures.” A, theentire protein profile with VLDL/IDL, LDL, HDL1, and HDL peaks
indicated. B, a magnification of the HDL1/HDL region of the graph forbetter clarity. The key for genotype of mice is shown in the inset of A.

Whole lipoprotein fractions (d ⬍ 1.21) were separated frompooled plasma samples of each group (n ⫽ 3) and were subjected to SDS-polyacrylamide gel electrophoresis to analyze
apoproteins (Fig. 3). LDLR(⫺/⫺) mice had elevated apoB-100
levels as compared with wild type mice (lanes 1 and 2). LDLR⫹/⫹;ob/ob mouse plasma contained barely detectable apoBs (lane3); however, LDLR⫹/⫺;ob/ob plasma did have apoB-100 andlow levels of B-48 (lane 4). The amounts of apoBs in thesegroups were consistent with their LDL peaks in the HPLC. In
striking contrast, the LDLR⫺/⫺;ob/ob lipoproteins consisted ofhighly elevated levels of both apoB-100 and apoB-48, with agreater ratio of B-100 to B-48 particles (lane 5), indicating that
their elevated TC and TG levels may be at least partially due toan increase in particle number. In addition, the LDLR⫺/⫺;ob/ob mice had an apparent increase in apoAIV and apoE. Thisis probably due to the increased remnant lipoproteins. In comparison, apoE(⫺/⫺) mouse lipoproteins are enriched in apoAIV,
and LDLR(⫺/⫺) mice on a high fat diet show increases in apoE
(7).
Lipoprotein lipase activities were measured in pooled samples from mice to determine if lipolytic activity was decreasedin the LDLR⫺/⫺;ob/ob mice. Postheparin lipolytic activity forwild type mice was 0.45 ␮mol of FFA/ml/min. LDLR(⫺/⫺),LDLR⫹/⫹;ob/ob, and LDLR⫺/⫺;ob/ob all had slightly elevatedlipolytic activity (0.61, 0.72, and 0.57 ␮mol of FFA/ml/min).
Liver Analyses—To determine if the increased plasma TC
and TG in the LDLR⫺/⫺;ob/ob mice was related to hepaticcholesterologenic and lipogenic enzymes, livers were collectedfrom 6-month-old animals and analyzed for both cholesterol
and triglyceride content as well as various enzyme RNA levels.
The livers of LDLR(⫺/⫺) mice as well as all three ob/ob groups
were found to contain slightly, but significantly, higher levels

Severe Hyperlipidemia and Atherosclerosis in LDLR⫺/⫺;ob/ob Mice

37405

TABLE II
Liver cholesterol and triglyceride content
Liver cholesterol and triglyceride content was measured as describedunder “Experimental Procedures.” For all groups, n ⫽ 3. Values are
given as mean ⫾ S.E. in mg/g of liver.
Genotype

Cholesterol contenta

Wild type
LDLR(⫺/⫺)LDLR⫹/⫹;ob/ob
LDLR⫹/⫺;ob/ob
LDLR⫺/⫺;ob/ob

1.77 ⫾ 0.32
2.91 ⫾ 0.22
4.13 ⫾ 0.9
3.45 ⫾ 0.1
5.01 ⫾ 0.5

Triglyceride contentb

mg/g of liver27.0 ⫾ 5.2
24.2 ⫾ 3.3
244 ⫾ 4.2
222 ⫾ 40278 ⫾ 40ap ⬍ 0.05 wild type versus all other groups; LDLR(⫺/⫺) versus
LDLR⫺/⫺;ob/ob.
bp ⬍ 0.005 wild type and LDLR(⫺/⫺) versus all ob/ob groups.

FIG. 3. SDS-polyacrylamide gel electrophoresis of lipoproteinsfrom LDLRⴚ/ⴚ;ob/ob mice. Lipoproteins were isolated from pooled
serum samples of 3– 4-month-old mice by ultracentrifugation to a density of 1.21 as described under “Experimental Procedures.” Lipoproteins
were loaded onto 3–20% SDS-polyacrylamide gels, electrophoresed, andstained with Coomassie Blue. Mouse genotypes are as follows: lane 1,
wild type; lane 2, LDLR(⫺/⫺); lane 3, LDLR⫹/⫹;ob/ob; lane 4, LDLR⫺/⫺;ob/ob; lane 5, LDLR⫺/⫺;ob/ob.

of cholesterol than wild type mice (Table II), which is consistent
with previous reports (9, 16). LDLR⫺/⫺;ob/ob mice containedslightly higher levels of cholesterol than the other two ob/obgroups and the LDLR(⫺/⫺) mice (p ⬍ 0.05) (Table II). Triglyceride levels were ⬃10 times higher in the three ob/ob groups
compared with the wild type and LDLR(⫺/⫺) mice, but there
was no association between LDLR genotype and liver triglyceride content in the ob/ob mice. These data indicate that leptin
deficiency causes a marked increase in liver triglyceride levels
but only a small increase in liver cholesterol content.
Northern blot analyses on liver RNAs revealed that cholesterologenic enzymes HMG-CoA synthase (Fig. 4), HMG-CoA
reductase and SREBP-2 (data not shown), were unchanged
among all five groups. In contrast, lipogenic enzyme RNAs
(SREBP-1, fatty acid synthase, ATP citrate lyase, and stearoylCoA desaturase-1) were increased in the ob/ob groups compared with wild type and LDLR(⫺/⫺); however, there was very
little difference in the ob/ob mice between the three LDLR
genotypes. ApoAI was slightly decreased in ob/ob mice; however, apoAII, and apoE RNA levels were unchanged in the
ob/ob mice compared with controls. A control probe for theribosomal enzyme 36B4 demonstrates equal loading (Fig. 4).
Special Diets—To determine the possible mechanism bywhich lipoproteins become elevated in the LDLR⫺/⫺;ob/ob
mice, initial studies were focused on the possibility that theincreased caloric intake of ob/ob mice was overwhelming andsurpassed backup lipoprotein clearance systems when the
LDLR was not present. To test this hypothesis, LDLR⫹/⫺;ob/ob and LDLR⫺/⫺;ob/ob mice were fasted for 2 days, after
which glucose, TC, and TG levels were analyzed (n ⫽ 3 in eachgroup). Glucose levels were reduced by 57 and 44% in LDLR⫹/⫺;ob/ob and LDLR⫺/⫺;ob/ob mice, respectively. Furthermore,
TG levels fell by 58 and 53%, respectively, in the fasted mice. In
contrast, TC levels increased slightly (although not significantly; p ⫽ 0.12) in the LDLR⫺/⫺;ob/ob mice from 585 ⫾ 44 to
712 ⫾ 47 mg/dl, while TC levels in the LDLR⫹/⫺;ob/ob mice
were unchanged (265 ⫾ 14 to 244 ⫾ 12 mg/dl). These data areconsistent with the faster turnover rate of fatty acids andtriglycerides than cholesterol.
Following this short term fasting experiment, a long term
caloric restriction diet, in which 4-week-old LDLR⫺/⫺;ob/ob

FIG. 4. Northern blot analysis of total liver RNA from LDLRⴚ/ⴚ;ob/ob mice. Livers were collected from 6-month-old mice, and total
RNA was prepared as described under “Experimental Procedures.”Total RNA was electrophoresed on formalin-denatured gels, transferred
to nylon membranes, and probed with 32P-labeled probes. Lanes 1–5
contain wild type (wt), LDLR(⫺/⫺), LDLR⫹/⫹;ob/ob, LDLR⫹/⫺;ob/ob,
and LDLR⫺/⫺;ob/ob RNA samples, respectively. Probes used (from top
to bottom) were SREBP-1, fatty acid synthase (FAS), stearoyl-CoA
desaturase (SCD-1), HMG-CoA synthase, ATP citrate lyase (ACL),
apoE, apoAI, apoAII, and a control probe, 36B4.

mice were given only 50% of what a wild type mouse would
consume, was initiated. Both diet-restricted mice (n ⫽ 2) andmice fed ad libitum (n ⫽ 3) showed a significant increase in TC
over the 2-week study, confirming the age-dependent increasein TC; however, this increase was more drastic in the mice fed
ad libitum, so that after 2 weeks there was a significant difference in TC levels between the two groups (Fig. 5). TG levels
increased significantly in the control group but remained unchanged in the diet-restricted group (Fig. 5), a pattern thatparalleled changes in glucose levels in the mice (data notshown). There was a slight reduction in HDL levels in bothcontrol and diet-restricted LDLR⫺/⫺;ob/ob mice (92 ⫾ 35 to
89 ⫾ 28 and 90 ⫾ 35 to 79 ⫾ 32, respectively); however, there
were no significant differences, reflecting the smaller contribu-

37406

Severe Hyperlipidemia and Atherosclerosis in LDLR⫺/⫺;ob/ob Mice

FIG. 5. TC and TG of LDLRⴚ/ⴚ;ob/ob mice placed on caloric
restriction. Four-week-old LDLR⫺/⫺;ob/ob mice were placed on a
restricted diet (50% of wt calories/day) for 2 weeks (n ⫽ 2). Age-matched
control mice were fed ad libitum (n ⫽ 3). Blood samples were collected
before starting the diet and at 2 weeks. Serum samples were analyzed
for TC and TG as described under “Experimental Procedures.” TC areshown in circles, and TG are in squares. Diet-restricted mice are represented by open shapes, and control mice are represented by filledshapes. Error bars represent S.E. *, p ⬍ 0.01 for base-line versus 2-weekvalues of all parameters excluding TG of diet-restricted mice. **, p ⬍0.005 for TC levels of diet-restricted and ad libitum mice at 2 weeks.

tion of HDL to the hyperlipidemia in these mice. Based onthese studies, while the elevated TG levels in the LDLR⫺/⫺;ob/ob mice were basically associated with the amount of shortterm food consumption, the increased caloric intake of thesemice, due to their leptin deficiency, cannot account for theelevations in TC levels.
To determine whether the increase in TC levels was derived
from the cholesterol that was present in the normal chow diet,these mice were placed on a cholesterol-free diet. After 2 weeks
on the cholesterol-free diet, there were no detectable changes inTC (542 ⫾ 20 to 560 ⫾ 102 mg/dl) or TG levels (272 ⫾ 19 to281 ⫾ 34 mg/dl) of LDLR⫺/⫺;ob/ob animals. HDL levels in
LDLR⫺/⫺ mice were unchanged by the cholesterol-free diet(83 ⫾ 12 to 86 ⫾ 13.5 mg/dl). HDL levels in LDLR⫺/⫺;ob/ob
mice were decreased from 75 ⫾ 5 to 65 ⫾ 13 mg/dl; however, it
was not statistically significant. Glucose levels were reduced by
29% in LDLR⫹/⫺;ob/ob and LDLR⫺/⫺;ob/ob mice.
Leptin Treatment—To determine whether the high plasma
TC and TG levels observed in the LDLR⫺/⫺;ob/ob were reversible with leptin treatment, 6 – 8-week-old LDLR(⫺/⫺), LDLR⫹/⫺;ob/ob, and LDLR⫺/⫺;ob/ob mice were injected with a low
dose of leptin (0.3 ␮g of leptin/g body weight) daily for 10 days.
This low dose of leptin has been shown to be suboptimal forweight reduction so that body weight in the treated mice was
not significantly changed by this treatment (15). Control mice
were injected daily with a similar volume of PBS. The
LDLR(⫺/⫺) mice did not show any significant changes in TC,
TG, or glucose levels (data not shown). Mice in all groups
gained weight within the 10-day treatment (predictable fromtheir age), but there were no significant differences due to theleptin treatment (Table III). Insulin levels were typical of ob/ob
mice of this age (20 –30 ng/ml) throughout the study. Insulinlevels in the PBS-treated mice increased during the treatment,
while leptin treatment tended to retard this increase in both
LDLR⫺/⫺;ob/ob and LDLR⫹/⫺;ob/ob groups, although there
was no significant difference due to a high variability (data notshown). Glucose levels were slightly decreased in the leptintreated LDLR⫺/⫺;ob/ob and LDLR⫹/⫺;ob/ob mice (Table III).
TG levels were substantially decreased in the leptin-treated
LDLR⫺/⫺;ob/ob mice, while they tended to be increased in the
PBS-treated mice during the course of the study (Table III). In
contrast, TC levels in the leptin-treated LDLR⫺/⫺;ob/ob mice
were only slightly reduced, whereas those of the PBS-treated
LDLR⫺/⫺;ob/ob increased significantly (p ⬍ 0.05) during thecourse of the study (Table III). HPLC analyses of pooled serum
samples from leptin and PBS-injected LDLR⫺/⫺;ob/ob mice
are shown are Fig. 6. Leptin treatment caused a substantial
decrease in the VLDL/IDL fraction compared with little change
seen in the PBS-injected mice (data not shown). This noticeable
shift from VLDL/IDL size particles to particles in the LDL size
range is consistent with the marked reduction in TG and minimal decrease in TC detected from total serum. Interestingly,
HDL levels appeared to increase upon leptin injection. The
leptin-injected LDLR⫹/⫺;ob/ob mice also displayed a loweringin TG levels, which was concomitant with a reduction in the
HDL1 peak compared with their base-line profile and with
PBS-injected animals (data not shown). These data indicatethat leptin treatment in LDLR⫺/⫺;ob/ob mice can revert their
hypertriglyceridemia but not their hypercholesterolemia, since
the remnant cholesterol was presumably converted to LDL
cholesterol.
Atherosclerosis—Animals were sacrificed at 6 months of age,
and whole aortae and hearts were collected for the analysis ofatherosclerotic development. As expected, wild type mice had
no detectable lesions in either their whole aortae (Fig. 7, panels
A) or the proximal aortic sinus (data not shown). LDLR(⫺/⫺)
mice had slight lesions in the arch region of the whole aorta andfatty streak lesions on the cusps in the aortic sinus (Fig. 7,
panels B, and Fig. 8B). Although the aortae of all three ob/obgroups were coated on the outside extensively with adipose
tissue, the LDLR⫹/⫹;ob/ob mice had no detectable lesions ineither the whole aorta (Fig. 7, panels C) or the aortic sinus (Fig.
8A). Interestingly, while the LDLR⫹/⫺;ob/ob had elevated TC
levels, comparable with LDLR(⫺/⫺) mice, they had no detectable atherosclerotic lesions in either the whole aorta (Fig. 7,
panels D) or the proximal aortic sinus (Fig. 8C). In contrast,
LDLR⫺/⫺;ob/ob had extensive lesions throughout the aorta(Fig. 7, panels E; Fig. 8D). Lesions were smaller in the abdominal and thoracic aortic regions but were most extensive in theaortic arch. These data indicate that LDL cholesterol is astronger risk factor for atherogenesis than are the combined
effects of obesity, diabetes, and insulin resistance in murine
models on a normal diet.
DISCUSSIONLDLR⫺/⫺;ob/ob Mice Are a Good Model for Hyperlipoproteinemia—There are several available animal models for elevated remnant lipoprotein particles: apoE(⫺/⫺) mice,
LDLR(⫺/⫺) mice on a high fat diet, TgSREBP-1a/LDLR⫺/⫺,
and LDLR and LRP double knockout mice (5–9). All of thesemodels were shown to have large increases in remnant particles, although the mechanisms, such as lack of receptors andligands and overproduction of VLDL, are different. Our current
animals provide another model of increased remnant lipoproteins with several unique characteristics. One feature of thismodel is the increase in apoB-100-containing lipoproteins inaddition to apoB-48. Mice produce both apoB-100 and apoB-48
in their livers; however, apoB-48 particles are more predominant in the previously mentioned models of hyperlipidemia.
Considering the noticeable elevations in apoB-100 in humans
with hyperlipidemia, this remnant model could be more relevant to the study of human disease. Currently, the mechanism
by which apoB-100 is preferentially accumulated in the plasmaof LDLR⫺/⫺;ob/ob is unknown. Another characteristic of thismodel is the sustained levels of HDL cholesterol. While othermouse models show an increase in VLDL/LDL lipoproteins,they are also characterized by low HDL levels. The LDLR⫺/⫺;ob/ob and LDLR⫹/⫺;ob/ob, in contrast, maintain normal, if notslightly elevated, HDL levels, making them a valuable tool forthe study of the distinct role of remnant lipoproteins in atherogenesis without the confounding variable of low HDL.

Severe Hyperlipidemia and Atherosclerosis in LDLR⫺/⫺;ob/ob Mice

37407

TABLE III
Effect of leptin treatment in LDLR⫺/⫺;ob/ob mice
There was no significant difference between base line and 10 days post-treatment in controls and leptin-treated mice unless indicated.
Control

Leptin-treatedGroup

LDLR⫹/⫺;ob/ob
Body weight (g)Glucose (mg/dl)
TG (mg/dl)
TC (mg/dl)
LDLR⫺/⫺;ob/ob
Body weight (g)Glucose (mg/dl)
TG (mg/dl)
TC (mg/dl)aBase line

10 daysBase line

10 days

21 ⫾ 3194 ⫾ 2684 ⫾ 17235 ⫾ 2130 ⫾ 2201 ⫾ 31193 ⫾ 27a
227 ⫾ 632.5 ⫾ 6
303 ⫾ 41
128 ⫾ 17249 ⫾ 1636 ⫾ 5215 ⫾ 67
114 ⫾ 42190 ⫾ 2121 ⫾ 5295 ⫾ 84364 ⫾ 182
803 ⫾ 3629 ⫾ 2303 ⫾ 35459 ⫾ 174951 ⫾ 28a23 ⫾ 7377 ⫾ 196
401 ⫾ 246
636 ⫾ 9028 ⫾ 4196 ⫾ 48270 ⫾ 102593 ⫾ 128p ⬍ 0.05 compared with base line.

FIG. 6. HPLC analysis of serum samples from LDLRⴚ/ⴚ;ob/ob
mice treated with leptin. Blood was collected from LDLR⫺/⫺;ob/ob
mice before and at 7 and 10 days after injections with 3 ␮g/g bodyweight leptin. Serum was separated and pooled for HPLC analyses as
described under “Experimental Procedures.” Solid lines, base-line values; large dashes, 7-day time point; small dashes, 10-day time point (asindicated on the figure).

FIG. 8. Cross-sections of the proximal aorta from LDLRⴚ/ⴚ;ob/ob mice. Hearts collected from 6-month-old mice (n ⫽ 3 for eachgroup) were fixed in formalin and embedded in OCT, and frozen crosssections of the proximal aorta were cut as described under “Experimental Procedures.” Sections with stained with Oil-Red-O and counterstained with hematoxylin. Sections shown are representative. A is froman LDLR⫹/⫹;ob/ob mouse; B is from an LDLR(⫺/⫺) mouse; C is froman LDLR⫹/⫺;ob/ob; and D is from an LDLR⫺/⫺;ob/ob mouse.

FIG. 7. En fac aortas from LDLRⴚ/ⴚ;ob/ob mice. Mice (n ⫽ 3 foreach group) were sacrificed at 6 months, and whole aortas were collected and stained for lipids as described under “Experimental Procedures.” Shown are representative aortas from wild type mice (panels A),
from LDLR(⫺/⫺) mice (panels B), from LDLR⫹/⫹;ob/ob mice (panels C),from LDLR⫹/⫺;ob/ob (panels D), and from LDLR⫺/⫺;ob/ob mice(panels E).

Hypertriglyceridemia and Hypercholesterolemia Are Caused
by Distinct Mechanisms—One important conclusion that canbe made from this study, is that the hypertriglyceridemia andhypercholesterolemia in the LDLR⫺/⫺;ob/ob mice are causedby distinct mechanisms. This distinction becomes apparent
when hepatic lipid production is contrasted with plasma lipid
levels. ob/ob mice show hepatic overproduction of triglycerides

and have fatty livers, presumably due to the activation of
nuclear SREBP-1 (see Fig. 4 and Ref. 16), a key transcription
factor for lipogenic enzymes (12). Fasting and caloric restriction in mice have both been shown to inhibit hepatic SREBP-1c
production (17, 18) and also reverted the hypertriglyceridemia
in LDLR⫺/⫺;ob/ob mice. These data suggest that overconsumption of dietary calories by ob/ob mice and the subsequent
increase in hepatic production of triglycerides through activation of SREBP-1 lead to hepatic overproduction and oversecretion of triglyceride-rich VLDL particles. These particles tend toaccumulate as remnant lipoproteins due to LDLR deficiency,
and this burden of triglycerides might be beyond the capacity ofthe plasma lipoprotein lipase activity, leading to hypertriglyceridemia. In contrast, hepatic production of cholesterol has
been reported to be decreased in ob/ob mice with a slightincrease in liver cholesterol content (16). In addition, cholesterol synthesis has been shown to be at normal levels in
LDLR(⫺/⫺) mice, because although there is a decreased returnof cholesterol through the LDLR, this is compensated for by anincrease in LDLR-independent influx (19). Therefore, it is unlikely that the hypercholesterolemia in the LDLR⫺/⫺;ob/ob37408Severe Hyperlipidemia and Atherosclerosis in LDLR⫺/⫺;ob/ob Micemice is caused by hepatic overproduction of cholesterol. The
current data on mRNA levels of cholesterologenic enzymes andliver cholesterol content from the doubly mutant mice also
support this interpretation. Taken together with the increased
apoB-100 in plasma, suggesting an increase in lipoprotein particle number, it can be speculated that impaired catabolism of
remnant particles is the main cause of hypercholesterolemia inthese mice. It is interesting to compare the LDLR⫺/⫺;ob/ob
mice with TgSREBP-1a/LDLR⫺/⫺ mice (9), which show a similar phenotype, with a dramatic increase in TC and TG levels(1050 mg/dl and 900 mg/dl, respectively). This severe hyperlipidemia in TgSREBP-1a/LDLR⫺/⫺ mice was primarily due tothe plasma appearance of very large VLDL particles that contained mainly apoB-48 and were enriched in both cholesterol
and triglycerides. However, in contrast to our LDLR⫺/⫺;ob/ob
mice, the TgSREBP-1a;LDLR⫺/⫺ mice had a marked elevationin cholesterol synthesis and liver cholesterol content in addition to the increase in triglyceride synthesis. Therefore, although LDLR deficiency may similarly serve to unmask hyperlipidemia in both animal models, the mechanism causing
hypercholesterolemia appears to be different.
An intriguing question brought up by these studies is thetime course of the hyperlipidemia. Peak TC and TG levels wereseen at between 3 and 4 months of age with a rapid drop in TG
levels at 5 months and a gradual reduction in TC between 5
and 8 months. The mechanism by which both hypertriglyceridemia and hypercholesterolemia were ameliorated during thelater stages of the study is currently unknown. It is probably
related to the natural course of energy metabolism in ob/ob
mice. To clarify this issue, detailed analysis on individual ob/ob
phenotypes such as obesity, glucose/insulin metabolism, andhepatic lipid synthesis in a time course study will be required.
Involvement of Leptin in Plasma Clearance of Non-HDL Cholesterol—It has previously been shown that leptin plays a rolein HDL metabolism (15, 20), and we now show that leptin also
has strong effects on remnant lipoprotein metabolism. The
unexpectedly dramatic hypercholesterolemia in LDLR⫺/⫺;ob/ob mice compared with LDLR⫺/⫺ mice, if not due to hepaticcholesterol overproduction (as discussed above), may possibly
be due to the impairment of LDLR-independent lipoprotein
clearance. This accumulation of cholesterol-rich remnant lipoproteins appears to take place chronically and cumulatively,
as supported by the observation that TC levels increased in anage-dependent manner and short term leptin treatment did not
produce a marked effect. While ob/ob mice have long been
known to have drastically increased blood glucose and insulinlevels because of energy imbalance and insulin resistance, thecurrent study demonstrates that ob/ob mice have the potential
for hypertriglyceridemia and hypercholesterolemia and moreover shows a novel potential link between leptin and plasmacholesterol metabolism. This is, to our knowledge, the firstobservation that leptin might play a role in plasma non-HDL
cholesterol metabolism. However, precise kinetic studies areneeded to confirm this speculation.
HDL1 Cholesterol in ob/ob Mice Is Not Proatherogenic—It
has been known for some time that ob/ob mice have slightly
elevated TC levels, which can be completely accounted for by anincrease in HDL (3). However, not much emphasis has beenplaced upon lipoprotein production and metabolism in theseanimals. Recently, extensive studies on HDL metabolism in
ob/ob mice have been reported. These studies have shown thatthe increase in HDL cholesterol is due to both regular HDLcholesterol and HDL1 cholesterol, a larger particle that shows
up in a peak between LDL and HDL, that HDL clearance in
ob/ob mice is impaired, and that low dose leptin treatment

significantly reduces HDL levels (15). They further demonstrated that the altered HDL catabolism is due to defective
HDL uptake, recycling, and degradation by hepatocytes (20).
We have confirmed their observations for increased normal
HDL and HDL1 cholesterol in LDLR⫹/⫹;ob/ob mice and show
a more dramatic increase in these particles in LDLR⫹/⫺;ob/ob
mice (Figs. 2 and 3). However, it is hard to conclude that HDL1
is cleared through LDL receptors from plasma, since LDLR⫺/⫺;ob/ob mice had a slightly decreased HDL1 fraction (Fig. 2
and 3). Of interest is the difference in atherosclerotic lesion
development between the LDLR(⫺/⫺) and the LDLR⫹/⫺;ob/ob
mice. While the TC and HDL cholesterol levels between the twogroups were nearly identical, there was a noticeable differencein their lipoprotein profiles, with LDLR⫹/⫺;ob/ob mice having
an HDL1 peak in place of the LDL peak seen in LDLR(⫺/⫺)
mice. This difference in lipoprotein profiles probably contributed to the differences in atherogenesis noted between the twogroups of mice; LDLR(⫺/⫺) mice showed signs of lesions in theaortic sinus and the aortic arch, whereas the LDLR⫹/⫺;ob/ob
mice were completely lesion-free. Therefore, it can be said that
this HDL1 is not proatherogenic, although whether it is antiatherogenic remains to be determined. Future studies comparing these two groups of mice more extensively will help todefine the protective effect of this HDL1 pool of lipoproteins in
the development of atherosclerosis.
The current studies with LDLR⫺/⫺;ob/ob mice demonstrate
important findings about remnant lipoprotein metabolism, discussed above, and provide a good model for the contributions ofmultiple risk factors, including hyperinsulinemia, diabetes,
and increased remnant particles, in the development of atherosclerosis. This model also raises the possibility of the therapeutic potential of leptin for treatment of dyslipidemia andatherosclerosis.
REFERENCES
1. Coleman, D. L. (1978) Diabetologia 14, 141–148
2. Zhang, Y., Proenca, R., Maffel, M., Barone, M., Leopold, L., and Friedman,
J. M. (1994) Nature 372, 425– 432
3. Nishina, P. M., Lowe, S., Wang, J., and Paigen, B. (1994) Metabolism 43,
549 –553
4. Li, X., Grundy, S. M., and Patel, S. B. (1997) J. Lipid Res. 38, 1277–1288
5. Plump, A. S., Smith, J. D., Hayek, T., Aalto-Setälä, K., Walsh, A., Verstuyft,
J. G., Rubin, E. M., and Breslow, J. L. (1992) Cell 71, 343–353
6. Zhang, S. H., Reddick, R. L., Piedrahita, J. A., and Maeda, N. (1992) Science
258, 468 – 471
7. Ishibashi, S., Goldstein, J. L., Brown, M. S., Herz, J., and Burns, D. K. (1994)
J. Clin. Invest. 93, 1885–1893
8. Rohlmann, A., Gotthardt, M., Hammer, R. E., and Herz, J. (1998) J. Clin.
Invest. 101, 689 – 695
9. Horton, J. D., Shimano, H., Hamilton, R. L., Brown, M. S., and Goldstein, J. L.
(1999) J. Clin. Invest. 103, 1067–1076
10. Ishibashi, S., Brown, M. S., Goldstein, J. L., Gerard, R. D., Hammer, R. E., andHerz, J. (1993) J. Clin. Invest. 92, 883– 893
11. Shimano, H., Horton, J. D., Hammer, R. E., Shimomura, I., Brown, M., andGoldstein, J. L. (1996) J. Clin. Invest. 7, 1575–1584
12. Shimano, H., Yahagi, N., Amemiya-Kudo, M., Hasty, A., Osuga, J., Tamura,Y., Shionoiri, F., Iizuka, Y., Ohashi, K., Harada, K., Gotoda, T., Ishibashi,S., and Yamada, N. (1999) J. Biol. Chem. 274, 35832–35839
13. Yagyu, H., Ishibashi, S., Chen, Z., Osuga, J., Okazaki, M., Perrey, S.,
Kitamine, T., Shimada, M., Ohashi, K., Harada, K., Shionoiri, F., Yahagi,N., Gotoda, T., Yazaki, Y., and Yamada, N. (1999) J. Lipid Res. 40,
1677–1685
14. Paigen, B., Morrow, A., Holmes, P. A., Mitchell, D., and Williams, R. A. (1987)
Atherosclerosis 68, 231–240
15. Silver, D. L., Jiang, X.-C., and Tall, A. R. (1999) J. Biol. Chem. 274, 4140 – 4146
16. Shimomura, I., Bashmakov, Y., and Horton, J. (1999) J. Biol. Chem. 274,
30028 –30032
17. Horton, J. D., Bashmakov, Y., Shimomura, I., and Shimano, H. (1998) Proc.
Natl. Acad. Sci. U. S. A. 95, 5987–5992
18. Yahagi, N., Shimano, H., Hasty, A., Amemiya-Kudo, M., Okazaki, H., Tamura,Y., Iizuka, Y., Shionoiri, F., Ohashi, K., Osuga, J., Harada, K., Gotoda, T.,
Nagai, R., Ishibashi, S., and Yamada, N. (1999) J. Biol. Chem. 274,
35840 –35844
19. Osono, Y., Woollett, L. A., Herz, J., and Dietschy, J. M. (1995) J. Clin. Invest.
95, 1124 –1132
20. Silver, D. L., Wang, N., and Tall, A. R. (2000) J. Clin. Invest. 105, 151–159
21. Muir, J. R. (1967) Clin. Chim. Acta 17, 312–314