From the
The role of the enzyme lipoprotein lipase (LPL) in
atherosclerosis is uncertain. To generate an animal model of LPL
deficiency, we targeted the LPL gene in embryonic stem cells with a
vector designed to disrupt the COOH terminus of the protein and used
these cells to generate LPL-deficient mice. Germ line transmission of
the disrupted LPL allele was achieved with two chimeric males, and
offspring from each of these animals were phenotypically identical.
Pups homozygous (-/-) for LPL deficiency died within 48 h
of birth with extreme elevations of serum triglycerides (13,327 mg/dl)
associated with essentially absent LPL enzyme activity in heart and
carcass. Newborn heterozygous (+/-) LPL-deficient pups had
lower LPL enzyme activity and higher triglycerides (370 versus 121 mg/dl) than wild type (+/+) littermates. Adult
heterozygotes had higher triglycerides than wild type mice with ad
libitum feeding (236 mg/dl for +/- versus 88
mg/dl for +/+) and after fasting for 4 h (98 mg/dl for
+/- versus 51 for +/+) or 12 h (109 mg/dl
for +/- versus 56 mg/dl for +/+).
Triglycerides were present as very low density lipoprotein particles
and chylomicrons, but high density lipoprotein cholesterol levels were
not decreased in +/- animals. Plasma heparin-releasable LPL
activity was 43% lower in +/- versus +/+
adult animals. LPL activity, mRNA, and protein were lower in the
tissues of +/- versus +/+ mice.
Homozygous LPL deficiency caused by disruption of the COOH terminus
of the enzyme is lethal in mice. Heterozygous LPL deficiency caused by
this mutation is associated with mild to moderate hypertriglyceridemia
without affecting static HDL cholesterol levels. Heterozygous
LPL-deficient mice could be useful for determining if
hypertriglyceridemia, independently or in combination with other
discrete defects, influences atherosclerosis.
Hypertriglyceridemia is a risk factor for coronary heart disease
(1), but the biology underlying this epidemiology is obscure. Elevated
triglycerides may cause vascular damage through both direct and
indirect mechanisms. Remnants of triglyceride-rich lipoproteins may be
directly atherogenic
(2) , a hypothesis bolstered by the recent
demonstration of triglyceride-rich lipoproteins in human
atherosclerotic lesions
(3) . Elevated triglycerides might affect
vascular health indirectly by decreasing HDL
Defects in lipoprotein lipase
(LPL) can cause hypertriglyceridemia. LPL is increasingly recognized as
a multifunctional protein. In addition to hydrolyzing triglycerides in
VLDL and chylomicrons, the LPL protein may also function as an
apolipoprotein, associating with the surface of various lipoproteins to
promote binding to the low density lipoprotein (LDL) receptor-related
protein/
LPL
could also impact atherogenesis independent of effects on circulating
triglycerides. Macrophages from human and rabbit atherosclerotic
lesions express LPL
(13, 14, 15) , and higher
levels of macrophage LPL expression are found in inbred strains of mice
that are susceptible to atherosclerosis
(16) , suggesting that
local expression of LPL could promote uptake of lipoproteins by
vascular tissue
(17) . Thus genetic defects in LPL could have
opposing effects on atherosclerotic risk; systemically decreased
function could increase the concentration of atherogenic,
triglyceride-rich particles, but decreased macrophage expression could
decrease foam cell formation.
Human heterozygous LPL deficiency is
probably common. It has been postulated to form a subset of familial
combined hyperlipidemia
(18) , a very common genetic disorder
associated with vascular disease. However, heterozygous LPL deficiency
could have phenotypically different effects depending on genetic and
physiological backgrounds. This might explain why some families with
heterozygous LPL deficiency manifest familial
hypertriglyceridemia
(19) , while others have lipid abnormalities
associated with increased atherosclerotic risk
(20) . Further
complicating study of human heterozygous LPL deficiency is the fact
that detection of this disorder is difficult given the large number of
mutations in the LPL gene
(21) .
With a goal of generating an
animal model of LPL deficiency within a homogeneous genetic background
suitable for studying atherosclerosis, we have inactivated the LPL gene
in mice by homologous recombination using a targeting vector designed
to disrupt the COOH-terminal domain of the LPL protein.
E14
ES cell electroporation, positive (using G418) and negative (using
ganciclovir) selection, and injections into the blastocysts of C57BL/6J
embryos were carried out as described
(25) . Chimeric (on the
basis of coat color) males were mated with C57BL/6J females to generate
F1 animals, and these F1 animals were crossed with each other to
generate the mice characterized in this study.
The strategy for inactivating the mouse LPL gene is shown in
Fig. 1
. The mouse LPL gene has 10 exons, 9 of which are
translated. The targeting vector contains
Additional confirmation of authentic targeting is shown in panelC. Tail DNA from animals shown to be +/+ or
+/- by Southern blotting was subjected to PCR using an
upstream primer just 5` to the NEO insertion site in exon 8 and a
downstream primer complementary to NEO. The expected
Homozygotes were born viable, initially appeared healthy, but
uniformly died within 48 h. The 100% mortality for this genotype was
significantly higher than death rates for +/- and
+/+ animals (). Autopsy findings from four
homozygotes (data not shown) were nonspecific, although atelectasis and
congestion in the lungs and hepatic congestion were seen consistently.
There were no pancreatic abnormalities detected.
Also shown in
are serum lipids from mice
As
expected, LPL enzyme activity was decreased in +/- and
essentially absent in -/- pups (Fig. 3). For these
experiments, the heart was removed and assayed, then the carcass minus
the heart (and milk-filled stomach, as described under
``Experimental Procedures'') was assayed separately.
The fasting data of Fig. 4were obtained after a 12-h fast
(11:00 p.m. to 11:00 a.m.), an intervention that may represent a
considerable physiological stress in mice
(32) . However, a 4-h
fast (7:00 a.m. to 11:00 a.m.) resulted in essentially the same
difference in triglycerides between +/- and +/+
animals ().
As with neonates, HDL cholesterol levels
were not lower in adult +/- animals. Ad libitum fed
adults with heterozygous LPL deficiency tended to have higher HDL
cholesterols than their +/+ littermates (118 ± 14 for
+/- versus 100 ± 9 for +/+, p = 0.3339).
Lipoprotein analysis by gel filtration
chromatography showed that the increased triglycerides in heterozygous
LPL-deficient animals were due to an increase in VLDL/chylomicrons
(Fig. 5, toppanels, fractions1-10). Consistent with measurements of HDL
cholesterol by PEG precipitation, HDL cholesterol measured by gel
filtration tended to be higher in +/- animals (middlepanels, fractions 30-38). In males,
``shoulders'' to the right of the VLDL triglyceride peak
(fractions 10-15) and to the left of the HDL cholesterol
peak (fractions 20-30) were consistently more prominent
in heterozygotes.
LPL message and protein were decreased in heterozygotes
(Fig. 7). Both the 3.6- and 3.4-kb LPL mRNA species detected by
others in mouse tissues
(23) were decreased in +/-
(lane2) compared to +/+ (lane1) adipose tissue. Similar differences were seen in
comparisons of adipose tissue total RNA from five different pairs of
+/+ and +/- animals. A decrease in LPL mRNA for
heterozygotes was also seen in heart (not shown). LPL protein mass was
decreased in +/- (lane4) compared to
+/+ (lane3) adipose tissue. Similar
differences were seen in comparisons of adipose tissue protein from
three different pairs of +/+ and +/- animals.
We present evidence consistent with the inactivation of the
LPL gene in mice. Targeting of the LPL gene with a vector designed to
disrupt the COOH terminus of the LPL protein results in severe
hypertriglyceridemia and essentially absent enzyme activity in
homozygotes. These animals die within 2 days of birth. Heterozygotes
have decreased enzyme activity and mild to moderate
hypertriglyceridemia with both feeding and fasting. The homozygous and
heterozygous states appear to have different effects on HDL levels.
Homozygous LPL deficiency in humans causes a distinctive phenotype,
the chylomicronemia syndrome, that is not necessarily
lethal
(33) . Naturally occurring defects in LPL activity have
also been described in cats
(34) , mice lacking mast cells
(W/W
HDL cholesterol was absent from the serum of
LPL -/- mice, an expected finding since the major sources
of lipid for HDL generation are thought to be remnant cholesterol and
phospholipid from chylomicron/VLDL metabolism. Whether apolipoprotein
A-I, the major protein of HDL, is produced normally in LPL
-/- animals is unknown. These mice could be used to
determine if nascent HDL-like particles are present in the absence of
LPL activity.
In human populations, lower levels of LPL enzyme
activity are associated with lower levels of HDL
cholesterol
(39) . Given this association, one might predict that
the lower (but not absent) LPL activity of LPL +/- mice
would also be associated with lower HDL cholesterol. This was not
observed. There was no difference in HDL cholesterol between
+/- and +/+ adult mice, and levels were almost
significantly higher in heterozygous neonates (). At least
three groups have overexpressed human LPL in transgenic
mice
(40, 41, 42) , and all report decreases in
triglyceride-rich lipoproteins without significant effects on HDL
cholesterol. These results and our findings in LPL +/- mice
suggest that, with the exception of the LPL -/- state, LPL
activity in mice has a limited role in determining HDL cholesterol
levels.
That heterozygous LPL-deficient mice have increased
triglycerides seems sensible, but this result was not predictable. In
humans, 94% of young heterozygotes have normal triglycerides
(19) as do mice heterozygous for the cld mutation
(36) . The mechanism responsible for
hypertriglyceridemia in our mice, especially with fasting, is
uncertain. Mutations generally cause gain of function, loss of
function, or have dominant negative effects. The latter class of
mutation could be relevant to our mice since active LPL is probably a
dimer and one could envision an inactive but stable monomer having a
prolonged dominant negative effect on triglyceride metabolism. This
mechanism does not appear to be operative in our +/- mice.
LPL activity (Fig. 6), message, and protein (Fig. 7) are
lower in the tissues of heterozygotes, making it likely that our
mutation causes loss of function through a decrease in LPL protein.
Consistent with this hypothesis, COOH-terminal truncation of human LPL
at residue 381, essentially the same site disrupted in mouse LPL in the
current study, causes a loss of catalytic activity as well as a
decrease in protein mass in transient transfection studies
(43) .
Thus, the phenotype of mice generated by COOH-terminal disruption of
LPL may be similar to the phenotype generated by targeting regions of
the LPL gene 5` to the exon 8 site chosen in this study.
Why then do
LPL +/- mice have elevated triglycerides in the fasted state
on a low fat diet? At least four explanations are possible. First, the
disruption of the COOH terminus of the LPL molecule could somehow
impair the metabolism of triglyceride-rich lipoproteins as suggested by
cell culture studies
(12) . We do not find evidence of a protein
with altered size in +/- tissues to support this hypothesis,
but Western blots were done with a single antibody, raising the
possibility that an altered protein is produced but not recognized by
our antibody. Second, LPL deficiency could affect triglyceride-rich
particle composition and impair clearance. Third, LPL deficiency could
reduce hepatic reuptake of apo B-containing particles with a net effect
of lipoprotein overproduction by the liver
(44) . Our enzyme
assay was not sufficiently sensitive to detect LPL activity in mouse
liver, although we have been able to detect very low levels of LPL
protein in this tissue by Western blotting (not shown). Fourth, LPL
deficiency is likely to increase the concentration of remnant particles
in these animals, a possibility suggested by the
``shoulders'' of particles seen to the right of the
triglyceride peak and to the left of the cholesterol peak for males in
Fig. 5
. Remnants are noncompetitive inhibitors of LPL activity,
and even low concentrations of these particles could substantially
interfere with VLDL hydrolysis
(45) .
There are other mouse
models of elevated triglycerides. Overexpression of apo CIII in mice
results in hypertriglyceridemia
(46) . This condition is
corrected by overexpression of apo E
(47) , supporting the
hypothesis that excess apo CIII displaces apo E from triglyceride-rich
lipoproteins, thereby hindering clearance by apo E-dependent
mechanisms. Overexpression of apo CI probably operates through a
similar mechanism to increase triglycerides
(48) . Overexpression
of apo CII, a co-factor for LPL activity, surprisingly also increases
triglycerides, perhaps by interfering with the ability of VLDL to bind
glycosaminoglycans
(49) . Each of these models was generated
using human transgenes in mice. Heterozygous LPL-deficient mice have
the theoretical advantage of manifesting hypertriglyceridemia that is
not dependent on the expression of a protein from another species.
Another potential advantage of our model is the lack of an effect on
HDL cholesterol levels. In species with cholesteryl ester transfer
protein activity, triglyceride concentrations are inversely related to
HDL cholesterol levels, making it difficult to decide whether
beneficial effects on vascular disease are due to decreased
triglycerides or increased HDL-C. LPL +/- mice suitably
backcrossed into the C57BL/6J background could be useful for
determining how hypertriglyceridemia alone affects atherosclerosis. In
addition, assessing atherosclerotic disease in LPL +/- mice
in the setting of diabetes mellitus, ethanol intake, and
hypothyroidism, or after crossing these animals with cholesteryl ester
transfer protein transgenic mice, with heterozygotes for apo E
deficiency, or with heterozygotes for LDL receptor deficiency, could
provide insight into whether commonly observed human lipid phenotypes
confer atherosclerotic risk.
Data are presented as mean ±
S.E. in mg/dl.
We thank Marie La Regina, Division of Comparative
Medicine, Washington University School of Medicine, for performing
autopsies on mice, and Kimberly Kluckman for performing blastocyst
injections.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
cholesterol levels, by rendering LDL particles more
atherogenic, or by affecting clotting.
-macroglobulin receptor (4), LDL
receptor
(5, 6) , and extracellular
proteoglycans
(7, 8) . The LPL protein can be
functionally divided into two major domains: an NH
-terminal
domain containing the catalytically active site, and a COOH-terminal
domain
(9) . The latter is essential for catalytic
activity
(10) , binds lipoproteins
(11) , and is probably
responsible for the LPL-mediated catabolism of triglyceride-rich
lipoproteins by the LDL receptor-related
protein/
-macroglobulin receptor
(12) .
Vector Construction and Generation of LPL-deficient
Mice
An 8.3-kb SacI/Sau3AI genomic fragment
was isolated from a EMBL3 library constructed using Balb/c
DNA
(22) . Restriction mapping, Southern blotting (with detection
by oligonucleotides based on the mouse LPL cDNA sequence in Ref. 23),
and sequencing showed that this fragment contained exons 7-10 of
the mouse gene with essentially the same structure described by Zechner
et al.(24) . A 1.8-kb EcoRI/HindIII
fragment of the plasmid pKJ-1 containing the neomycin resistance
cassette driven by the PGK promoter was inserted by blunt-end ligation
at a HindIII site in exon 8. This manipulation was shown to
abolish the HindIII site and interrupt exon 8, resulting in a
predicted mouse LPL protein disrupted following the leucine residue at
position 380 as numbered in Ref. 23. This modified genomic clone was
then inserted at the XhoI site of a Bluescript plasmid (a gift
from Fred Fiedorek, Chapel Hill, NC) containing the 3.4-kb thymidine
kinase fragment from pHSV-106 to generate the targeting vector.
Animal Genotyping
DNA isolated from tail (for most
animals) or liver (from recently expired animals) was subjected to
Southern blotting or PCR. For Southern blotting, 5-15 µg of
DNA was electrophoresed followed by acid depurination, treatment with
NaOH, and transfer using standard techniques. Blots were probed with a
random-primed 0.8-kb BamHI/PvuII fragment of the
mouse LPL gene spanning the 3` end of intron 5 and the 5` end of exon
6. As expected, preliminary blots showed that this probe did not
hybridize with the targeting vector. For PCR genotyping of potential
heterozygotes, an upstream primer corresponding to mouse exon 8 (5`-TTT
ACA CGG AGG TGG ACA TCG GA) and a downstream primer corresponding to a
region near the 3` end of the neomycin resistance cassette (5`-TCG CCT
TCT ATC GCC TTC TTG AC) were used in reactions containing genomic DNA
and 2 mM MgCl subjected to the following cycling
parameters: 5 min at 94 °C for 1 cycle, and 1 min at 94 °C/2
min at 55 °C/3 min at 72 °C for 30 cycles.
Mouse Housing, Handling, and Diet
Animal rooms
were illuminated between 7:00 a.m. and 7:00 p.m. Mice were fed a 50/50
mixture of PicoLab rodent chow 20 and mouse chow 20 (product numbers
5053 and 5058) with a total fat content of 6.75%. Animals were
weaned at 21 days of age. For blood collection via the retro-orbital
plexus, animals were lightly anesthetized with methoxyflurane. Deep
anesthesia was used for those animals subjected to exsanguination via
inferior vena cava venipuncture.
Lipid and Lipoprotein Analysis
Triglycerides,
cholesterol, and phospholipids were assayed enzymatically using
commercially available kits (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan). For measurements of total lipids, serum was kept at 4
°C or on ice and assayed within 2 h for triglycerides and
phospholipids to decrease the chances of endogenous
hydrolysis
(26) . Cholesterol determinations were generally done
the following day after storing samples at -70 °C. HDL
cholesterol was measured after polyethylene glycol precipitation of apo
B-containing lipoproteins
(27) . For separation of lipoproteins
by gel filtration, serum samples from two animals with similar total
lipids were pooled and separated by FPLC using two Superose 6 columns
in series
(28) . The 50 fractions collected per condition were
analyzed the same day for triglycerides and the following day for
phospholipids and cholesterol.
Determination of LPL Enzyme Activity, mRNA, and
Protein
LPL activity was assayed as the salt-inhibitable ability
of triplicate samples to hydrolyze an emulsion containing radiolabeled
triolein
(29) . For postheparin plasma activity, animals were
injected intraperitoneally with 200 units of porcine heparin; 30 min
later blood was collected from the inferior vena cava. Previous studies
have shown this procedure to be suitable in mice
(30) . For other
assays, individual tissues were flash frozen in liquid nitrogen,
weighed, and made 4% (w/v) in assay buffer
(29) . For newborn
pups, the heart was removed and processed, the milk-filled stomach was
removed and discarded (to prevent dilution of radioactive emulsion by
milk lipids), and the remaining carcass (minus heart and stomach) was
frozen, weighed, and homogenized in assay buffer. For determination of
LPL message, total RNA was prepared from tissues by centrifugation in
cesium chloride and mRNA was detected by Northern blotting as described
previously
(31) . For determination of LPL protein, tissue
extracts were subjected to SDS-polyacrylamide gel electrophoresis and
Western blotted, and LPL protein was detected with chicken anti-bovine
milk LPL as described
(29) . Each gel included one lane loaded
with purified bovine LPL as a positive control.
Statistical Information
Differences were assessed
using unpaired, two-tailed t tests unless otherwise specified.
For the data shown in Fig. 4, ANOVA was specifically not used
because groups contained different numbers of animals.
Figure 4:
Serum lipids in adult mice. Blood was
obtained by retro-orbital bleeding from mice at age 7-9 weeks
between 10:00 a.m. and noon. Serum was assayed immediately or stored at
-70 °C for assay within 24 h. Data from the leftside of the figure represent mice fasted for 12 h, and
data from the rightside represent ad libitum fed mice. +/+ indicates wild type, and +/-
indicates heterozygotes as determined by PCR genotyping of tail DNA.
Data represent means ± S.E. for 3-8 animals per
group.
3 kb of mouse DNA
upstream and
5 kb downstream of the exon 8 HindIII site
where the neomycin resistance cassette (NEO) was inserted. Predicted
restriction fragments detected by the LPL probe spanning the intron
5/exon 6 junction are shown at the bottom of the figure for native and
targeted genes. With HindIII, the native fragment is 8.4 kb
but the targeted fragment is 14.4 kb since insertion of NEO abolishes
the natural HindIII site in exon 8. With Tth111I, the
native fragment is 10.3 kb but the targeted fragment is 7.7 kb since
NEO introduces a new Tth111I I site 5` to the natural
Tth111I site.
Figure 1:
Strategy for inactivation of the mouse
LPL gene. Schematic diagrams of the targeting vector, native (wild
type) mouse gene, a successfully targeted allele, and the predicted
restriction fragments resulting from digestion of genomic DNA with
HindIII and Tth111I are shown. S represents
SacI, TK represents thymidine kinase (for negative
selection in the presence of ganciclovir), and NEO represents
the neomycin resistance cassette (for positive selection with G418).
Also shown is the location of the LPL genomic probe (spanning the
intron 5/exon 6 junction) used for detection of fragments by Southern
blotting.
Evidence for correct targeting of the LPL
gene in ES cells and mice is shown in Fig. 2. Southern blots of
ES cell DNA are shown in panelA, blots of F1
F1 mouse DNA are in panelB, and results of a PCR
assay using mouse tail DNA are in panelC. For
panelA, lanes 1-4 contain
HindIII-digested DNA detected with the LPL probe shown in
Fig. 1
. Wild type (+/+) cells contain only the 8.4-kb
native fragment (lane1), but targeted clones also
contain the 14.4-kb targeted allele (lanes 2-4). When
this same DNA was probed with a NEO cDNA (lanes5-8), only the 14.4-kb targeted allele was detected
in targeted clones (lanes 6-8), consistent with the
presence of the NEO cassette in this fragment.
Tth111I-digested DNA from +/+ cells showed only the
10.3-kb native band (lane9), but the expected 7.7-kb
band is also present in targeted cells (lane10).
Figure 2:
Southern blots and PCR assays of correctly
targeted ES cells and mice. PanelA shows
representative Southern blots of ES cells. Fifteen µg of genomic
DNA from non-electroporated ES cells (lanes1,
5, and 9) and correctly targeted ES cell clones
(lanes2-4, 6-8, and
10) was digested with HindIII or Tth111I,
Southern blotted, and detected with the LPL probe indicated
schematically in Fig. 1 (lanes 1-4, 9, and
10) or a NEO probe (lanes 5-8), consisting of a
BamHI/PvuII fragment complementary to the
phosphotransferase coding region from the vector pKJ-1. Correctly
targeted HindIII-cut DNA contained the predicted 14.4-kb
mutant band, which hybridized with both LPL and NEO probes. Correctly
targeted Tth111I-cut DNA contained the predicted 7.7-kb mutant
band (lane10). PanelB shows DNA
from wild type (+/+), heterozygous (+/-), and
homozygous (-/-) mice after HindIII or
Tth111I digestion. As expected, heterozygotes have both mutant
and native alleles after digestion with both enzymes while homozygotes
have only the mutant allele (14.4 kb for HindIII, 7.7 kb for
Tth111I). PanelC shows a PCR assay using
the same DNA from the +/- mice in panelB.
Assays were performed as described under ``Experimental
Procedures'' using an upstream primer complementary to exon 8 of
the mouse LPL gene and a downstream primer complementary to NEO. As
expected, the predicted 600-bp band was seen in DNA from
+/- mice and with the targeting vector (labeled
Plasmid) but not in +/+ mice or in the negative
control lane (labeled NoDNA).
Five male chimeric mice were generated using these ES cells. Two
independent chimeras transmitted the mutant allele through the germ
line, and analyses of progeny from each of these animals were
identical. The same alleles detected in ES cells were detected in the
offspring of F1 F1 crosses (panelB). For
HindIII, heterozygotes (+/-) carried both the
8.4-kb native and 14.4-kb mutant alleles while homozygotes
(-/-) had only the 14.4-kb allele. For Tth111I,
+/- animals carried both the 10.3-kb native and 7.7-kb
mutant alleles while -/- mice had only the 7.7-kb fragment.
600-bp band
was amplified from heterozygotes (+/-, lanes2 and 3) but not wild type animals (+/+,
lanes1 and 4). This assay was subsequently
used for rapid genotyping. In other studies, confirmation of the
homozygous state was also performed by PCR using the primers of
panelC in conjunction with primers (separated by
127 bp) flanking the NEO insertion site in exon 8. With the latter
primers, wild type animals showed a 127-bp band, homozygotes had a
1.8-kb band (due to the presence of the NEO cassette between the
primers), and heterozygotes had both bands (data not shown).
12 h after birth.
Homozygotes had marked elevations of triglycerides, phospholipids, and
total cholesterol, but no detectable HDL cholesterol. This extreme
hyperlipidemia was feeding-dependent. One homozygote was kept from
nursing (verified by the absence of milk in the stomach), and in this
single animal at the time of sacrifice lipids were: triglycerides, 296
mg/dl; cholesterol, 95 mg/dl; phospholipids, 178 mg/dl. Triglycerides
(p = 0.0019), cholesterol (p = 0.0008),
and phospholipids (p = 0.0057) were significantly
higher in heterozygotes than in wild type animals at 12 h. HDL
cholesterol levels were not decreased in +/- animals; in
fact, HDL cholesterol was almost significantly higher in +/-
compared to +/+ pups (p = 0.0730).
Figure 3:
LPL enzyme activity in the heart and
carcass of neonatal mice. Pups were allowed to suckle and then
sacrificed 12 h after birth. Tissues (not including the milk-filled
stomach) were flash-frozen in liquid nitrogen and then thawed once for
determination of LPL enzyme activity as described under
``Experimental Procedures.'' Data represent means ±
S.E. for 4 animals for -/-, 8 animals for +/-,
and 6 animals for +/+, with assays for each animal done in
triplicate.
Heterozygotes developed normally. Over 100 animals were weighed at
weekly intervals for the first two months of life, and although
+/- animals tended to be slightly heavier than +/+
littermates, this difference was not significant (p =
0.5513). Heterozygotes had higher triglycerides than wild type animals
with fasting and with ad libitum feeding ( Fig. 4and
). With fasting, triglycerides were 2-fold elevated
in +/- versus +/+ animals regardless of
sex. With ad libitum feeding, the genotype-specific
differences in triglycerides were amplified with the highest elevations
seen in heterozygous LPL-deficient females. There were no significant
differences between groups for total cholesterol or phospholipids.
Figure 5:
Separation of serum lipoproteins in mice
by gel filtration chromatography. Blood was obtained from the inferior
vena cava under deep anesthesia as described under ``Experimental
Procedures.'' For each profile, serum from two ad libitum fed animals known to have similar lipid levels was pooled and
separated using two Superose 6 columns in series. Fifty fractions were
collected, and each was assayed for triglyceride, cholesterol, and
phospholipid content. VLDL (which refers to both VLDL and chylomicrons
since animals were studied in the fed state) and HDL peaks are
appropriately labeled. The expected elution position for LDL is
approximately fractions 20-25. Similar results were seen in at
least two independent profiles for each sex and genotype. , wild
type;
, heterozygote.
LPL enzyme activity was decreased in animals
carrying one copy of the mutant allele (Fig. 6). Activity in
plasma 30 min after the intraperitoneal injection of heparin (panelA) was 43% lower in heterozygotes (146 ± 25
µmol of FFA/ml/h for +/- versus 258 ± 19
for +/+, n = 4 animals per genotype, p = 0.0121). This decrease was also reflected in enzyme
activity assayed in individual tissues (panelB). For
heart, kidney, epididymal/parametrial and inguinal adipose tissue,
lung, psoas muscle, and brain, LPL activity was consistently decreased
in +/- versus +/+ animals. These
differences were statistically significant only for heart and kidney
(both p < 0.05) due to considerable animal-to-animal
variation, especially for adipose tissue. Since these experiments were
performed using the F2 generation representing a mixture of the the
129/Ola and C57BL/6J strains, the genetic heterogeneity of these
animals is probably a major source of the observed variation in LPL
enzyme activity.
Figure 6:
LPL enzyme activity in wild type
(+/+, solid bars) and heterozygous LPL-deficient
(+/-, open bars) mice. For panelA, ad libitum fed animals were injected
intraperitoneally with heparin and 30 min later plasma was obtained and
LPL enzyme activity assayed as described under ``Experimental
Procedures.'' In panelA, * indicates p = 0.0121 versus +/+; data represent the
mean ± S.E. for 4 animals (2 males and 2 females) for each
genotype with plasma values for each individual animal determined in
triplicate. For panelB, ad libitum fed
animals (different from the animals used in panelA)
were sacrificed and tissues flash-frozen in liquid nitrogen. fatE represents epididymal or parametrial adipose tissue,
while fatI represents inguinal adipose tissue. In
panelB, * indicates p < 0.05; data
represent the mean ± S.E. for 6 animals (4 males and 2 females)
for each genotype with each individual tissue value determined in
triplicate. Sex differences were minimal except in fat, where
epididymal/parametrial activities were 3-4-fold higher and
inguinal activities were 4-6-fold higher in females than
males.
There were tissue-specific differences in the
magnitude of the decrease in enzyme activity associated with the mutant
allele. For example, in multiple assays involving six different
side-by-side comparisons of age and weight-matched +/+
versus +/- animals, the decrease in heart activity
was consistently less than 50% (mean decrease 28.4%) while the decrease
in renal activity was consistently greater than 50% (mean decrease
62.4%).
Figure 7:
LPL
mRNA and protein mass in +/+ versus +/- mice.
Adipose tissue RNA was subjected to Northern blotting (lanes1 and 2), and adipose tissue protein was
subjected to Western blotting (lanes3 and
4). For lanes1 and 2, 20 µg of
total RNA was separated on agarose gels containing formaldehyde. Equal
loading and equal RNA integrity for each genotype was verified by
analysis of ethidium-stained 28 and 18 S ribosomal RNA intensities
before transfer. The LPL message (indicated by the 3.6-kb and 3.4-kb
markers) was detected using a random-primed mouse LPL cDNA. For
lanes3 and 4, 1 µg of protein was
separated by SDS-polyacrylamide gel electrophoresis with the
55-kDa LPL protein (visible in both lanes at the position
indicated by the arrowhead to the right of lane
4) detected using an iodinated chicken anti-bovine LPL antibody.
Numbers to the left of lane3 indicate the positions of protein size standards. Results shown
for LPL mRNA are representative of comparisons of 5 different pairs of
+/+ and +/- animals. Results for LPL protein are
representative of comparisons of 3 different pairs of +/+ and
+/- animals.
mice; Ref. 35), and mice homozygous for
combined lipase deficiency (cld/cld mice; Ref. 36). Our
homozygotes resemble cld/cld mice. Triglycerides are similar
at 12 h postpartum (
10,000 mg/dl for cld/cld,
13,000
mg/dl for LPL -/-), and animals uniformly die within 48 h
of birth. cld/cld mice presumably die because of microinfarcts
in critical organs caused by dense packing of chylomicrons in
capillaries (37), and a similar mechanism probably operates in LPL
-/- mice. The cld mutation affects the
posttranslational processing of both LPL and hepatic
lipase
(38) , but the similarities between LPL -/-
and cld/cld mice suggest that LPL is the lipase most critical
for neonatal survival.
Table:
Neonatal mortality rates and serum lipids for
LPL-deficient mice
Table:
Serum triglycerides in adult heterozygous
LPL-deficient and wild type mice
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.