From the Diabetes Branch, NIDDK, National Institutes
of Health, Bethesda, Maryland 20892 and the ¶ Metabolism
Branch, NCI, National Institutes of Health,
Bethesda, Maryland 20892
Received for publication, July 30, 2002, and in revised form, November 6, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The metabolic phenotype of the A-ZIP/F-1 (AZIP)
lipoatrophic mouse is different depending on its genetic background. On
both the FVB/N (FVB) and C57BL/6J (B6) backgrounds, AZIP mice have a
similarly severe lack of white adipose tissue and comparably increased
insulin levels and triglyceride secretion rates. However, on the B6
background, the AZIP mice have less hyperglycemia, lower circulating
triglyceride and fatty acid levels, and lower mortality. AZIP
characteristics that are more severe on the B6 background include
increased liver size and liver triglyceride content. A unifying
hypothesis is that the B6 strain has higher triglyceride clearance into
the liver, with lower triglyceride levels elsewhere. This may account
for the observation that the B6 AZIP mice have less insulin-resistant
muscles and more insulin-resistant livers, than do the FVB AZIP mice.
B6 wild type, as well as B6 AZIP, mice have increased triglyceride
clearance relative to FVB, which may be explained in part by higher
serum lipase levels and liver CD36/fatty acid translocase mRNA
levels. Thus, it is likely that increased triglyceride clearance in B6,
as compared with FVB, mice contributes to the strain differences in
insulin resistance and lipid metabolism.
Diabetes, obesity, and atherosclerosis are examples of complex
genetic diseases (1, 2). They are caused by interactions among many
genes and environmental factors. Each susceptibility gene is thought to
make a relatively small contribution to the phenotype and it is likely
that different populations carry overlapping, but distinct sets of
susceptibility genes. In humans, a number of studies have identified
chromosomal regions that contain diabetes or obesity loci, but the
specific gene and responsible mutations/polymorphisms have been
definitively identified in only a few cases (3).
A number of major diabetes and obesity genes have been identified in
mice. Indeed, our current knowledge of the physiology of body
weight regulation and obesity is the direct result of the study of
mutant mice. Whereas most of the genes discovered cause monogenic
disease, they identify the pathways that are likely to be affected in
polygenic disease. Additionally, the background genotype has a profound
effect on the phenotype of monogenic mutants. For example, homozygous
ob or db (now officially
Lepob and Leprdb,
affecting leptin and leptin receptor, respectively) mutations on the B6
background cause obesity, mild hyperglycemia, and profoundly elevated
insulin levels. The same mutations on the C57BLKS/J background produce
severe diabetes and early mortality, with less obesity and
hyperinsulinemia (4, 5).
We have been studying the transgenic A-ZIP/F-1 (hereafter,
AZIP)1 mouse, which was
produced by adipose-selective expression of a dominant-negative protein
(6). These mice lack virtually all of their white adipose tissue (WAT).
The lack of WAT causes severe insulin resistance, hyperglycemia,
elevated serum triglycerides and fatty acids, and massively increased
liver triglyceride levels (6-9). The syndrome is remarkably similar to
that of patients with severe lipoatrophic diabetes (10, 11).
To date, studies of the AZIP mice have used a pure FVB genetic
background. Here we report the characterization of the AZIP phenotype
on the B6 background and compare it to the FVB phenotype. The FVB and
B6 backgrounds were chosen for practical reasons. The original AZIP
transgenic mouse was generated on the FVB background, which, for
technical ease, is commonly used to make transgenic mice (12). The
other strain, B6, is arguably the best characterized mouse strain,
particularly for metabolic phenotypes, and is among the first to have
its genome sequenced. The FVB and B6 strains are genetically quite
distant (13).
Here we report that genetic background has a profound effect on many
aspects of the AZIP phenotype, including opposite effects on muscle and
liver insulin resistance. Furthermore, we have observed differences in
triglyceride clearance between wild type FVB and B6 mice that may
explain some of the AZIP differences.
Mice--
All AZIP mice were studied as hemizygotes, produced by
breeding hemizygous males with wild type females. B6 AZIP mice were obtained by at least 13 generations of backcrossing AZIP males with B6
(Jackson Laboratory, Bar Harbor, ME) females. Although referred to as
B6, all of the B6 AZIP and control males have the FVB Y chromosome. The
B6 AZIP mice may carry advantageous FVB alleles, selectively retained
during the backcrossing, because some AZIP mice die before reaching
breeding age (see "Results"). FVB mice were originally obtained
from the Veterinary Resources Program, the National Institutes of
Health, and Harlan Sprague-Dawley. Mice were typically housed 3-5 per
cage, on a 12-h light/dark cycle (0600-1800), and fed NIH-07 rodent
chow (12.9 kcal % fat, Zeigler Brothers Inc., Gardners, PA) and water
ad libitum. Wild-type controls were generally littermates.
Animal experiments were approved by the NIDDK Animal Care and Use Committee.
Biochemical Assays--
Blood was obtained by tail bleed from
unaesthetized mice and was immediately analyzed for glucose
concentration. Glucose was measured using a Glucometer Elite (Bayer
Corp., Elkhart, IN). Triglycerides (number 339-11; Sigma),
nonesterified fatty acids (number 1383175; Roche Molecular
Biochemicals), cholesterol (number 401; Sigma), and insulin (number
SRI-13K; Linco Research Inc., St. Charles, MO) were assayed according
to the assay suppliers' procedures.
Tissue triglycerides were measured as follows (14). Tissue (~100 mg)
was homogenized in 3 ml of 2:1 chloroform:methanol and shaken at room
temperature for 4 h. Next, 1.5 ml of 0.1 M NaCl was
added, mixed, centrifuged, and the lower organic phase containing
triglycerides was transferred to a new glass tube and air-dried. The
residue was hydrolyzed (70 °C, 1 h) in 200 µl of 3 M KOH in 65% ethanol and the released glycerol was
quantitated using a radiometric glycerol kinase assay (15, 16). The
radiometric assay is more sensitive and accurate than the colorimetric assay.
Euglycemic-Hyperinsulinemic Clamps--
The clamp protocols are
based on those of Kim and Shulman (17-20) and have been described in
detail. The target plasma insulin level was 4 ng/ml and the target
plasma glucose was 110-120 mg/dl.
In Vivo Triglyceride Studies--
Triglyceride secretion was
studied by measuring the increase in circulating triglycerides after
lipase inhibition by WR1339 (21, 22). Briefly, mice were given ad
libitum access to a fat-free diet (Frosted Flakes, Kellogg, Battle
Creek, MI) for 4 h then anesthetized with ketamine (100 mg/kg;
Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (10 mg/kg;
Phoenix Scientific, St. Joseph, MO). WR1339 (Sigma, number T-8761, 100 µl of a 1:10 dilution in phosphate-buffered saline) was
injected via warmed tail vein and blood samples were withdrawn at 0, 1, and 2 h. Plasma triglycerides were measured colorimetrically. Data
are expressed as milligrams of triglyceride/kg of body weight/h,
assuming plasma volume is 3.5% of body weight.
Clearance of triglyceride (400 µl of peanut oil, delivered by gavage)
from the circulation was measured in mice previously fasted for 4 h. Blood was taken hourly via tail vein for 6 h and plasma
triglycerides were measured as described above.
Northern Blot Hybridization of RNA--
Total RNA was prepared,
hybridized, and quantitated by PhosphorImager as described previously
(9). Additional cDNA probes were prepared by reverse
transcriptase-PCR using the following primers (5' primer listed
first in each case). Acetyl-CoA carboxylase, 5'-GGGACTTCATGAATTTGCTGATTCTCAGTT and 5'-GTCATTACCATCTTCATTACCTCAATCTC; ApoB, 5'-TGGGATTCCATCTGCCATCTCGAG and 5'-GTAGAGATCCATCACAGGACAATG; CD36, 5'-CCTCTGACATTTGCAGGTCTATCTACGC and
5'-TAAGGCCATCTCTACCATGCCAAGGAGC; fructose-1,6-bisphosphatase, 5'-GATGAGCCTTCTGAGAAGGATGC and
5'-TCAGCCACCATGGAG/CCCCACATAC; low density lipoprotein receptor,
5'-CGCAGTGCTCCTCATCTGACTTGT and 5'-ACCCCAAGACGTGCTCCCAGGATGA; liver X
receptor Statistical Analysis--
Results are reported as mean ± S.E. Data were analyzed using SigmaStat (SPSS Inc., Chicago, IL) by
t test or two-way ANOVA followed by the Tukey test for
pairwise multiple comparisons.
B6 AZIP Mice Have Virtually No White Adipose Tissue--
To follow
up on preliminary observations suggesting an effect of genetic
background on the AZIP phenotype, we quantitated multiple
characteristics of the AZIP mice. No significant white adipose depots
were found in AZIP mice on either the B6 or FVB genetic background. The
brown adipose tissue phenotype of the B6 AZIP and FVB AZIP mice was
also similar, with relatively normal amounts and appearance at birth
followed by reduction in size (Table
I) and an inactive gross and
histological morphology in adulthood (data not shown).
Changes in Body Weight and Blood Chemistries--
FVB AZIP mice
are the same size at birth as wild type littermates, but rapidly fall
behind in growth, later catching up (males) or growing larger (females)
than their littermates (6). B6 AZIP males have a different growth
pattern, remaining smaller than their littermates (Fig.
1). The growth patterns of B6 AZIP and
FVB AZIP females are comparable (data not shown).
FVB AZIP mice are severely hyperglycemic, averaging 542 mg/dl in males,
compared with 164 mg/dl in controls (Fig. 1). In marked contrast, male
B6 AZIP mice have a transient hyperglycemia, peaking at ~300 mg/dl at
8-12 weeks of age, followed by near normal glucose levels thereafter.
The blood glucose levels in female B6 AZIP mice averaged 60 mg/dl
higher than the wild type controls and did not show the multiphasic
pattern of the male AZIP mice (data not shown). Serum insulin levels
were greatly and similarly elevated in B6 and FVB AZIP mice (Fig. 1).
These data suggest that the lower, more normal glucose levels in B6
AZIP mice are not because of more insulin secretion, but rather to less
insulin resistance.
Serum triglyceride levels are also affected by background genotype
(Fig. 1). Wild-type male FVB mice had higher triglyceride levels (172 mg/dl) than B6 mice (91 mg/dl). In the FVB mice the AZIP transgene
caused a remarkable increase in the triglyceride levels (to ~750
mg/dl) in young mice (the decrease in triglycerides with aging was not
seen in other mice at up to 17 weeks of age). In contrast, in male B6
AZIP mice, serum triglyceride levels averaged only 121 mg/dl. Female B6
wild type and AZIP mice had triglyceride levels of 59 and 51 mg/dl,
respectively. Thus, the AZIP transgene barely affects the B6
triglyceride levels, but causes a remarkable increase in FVB
triglyceride levels.
Serum fatty acid levels were similar in B6 wild type and AZIP mice.
Fatty acid levels in FVB wild type and AZIP mice were higher than in B6
mice, with the younger FVB AZIP mice tending to be higher than the wild
type FVB mice (Fig. 1).
Taken together, these data demonstrate no background genotype effect on
serum insulin levels, but appreciable background genotype effects on
body weight, blood glucose, serum triglyceride, and fatty acid levels.
In each case the B6 background gives a milder phenotype.
To see if the biochemically milder phenotype affects clinical outcome,
survival was scored, and indeed was markedly greater in the B6 AZIP
mice. At weaning (n = 292) B6 AZIP male and female mice
were present at 60 and 96% of the expected levels, respectively. For
comparison, FVB AZIP male and female mice were present at only 36 and
47% of the expected levels, respectively (6). Mortality between
weaning and 6 months of age was also less in the B6 than the FVB AZIP
mice, although this was not studied quantitatively.
Tissue Insulin Sensitivity in B6 and FVB AZIP Mice--
When
AZIP mice were studied anatomically, the B6 AZIP mice showed
remarkably greater hepatomegaly (4.4-fold enlarged in males and
5.2-fold in females) than did the FVB AZIP mice (2.2-fold in males)
(Table I, Fig. 2). (The weights of other
organs, brown adipose tissue, heart, spleen, and kidney, also showed
significant effects of background genotype, with the AZIP changes
generally greater on the FVB background, Table I.) The increased liver weight reflected massively increased liver triglyceride content, giving
the livers a lighter color and characteristic histology. Interestingly,
in the wild type mice liver triglyceride content was also greater
(~3-fold) in the B6 background.
Because organ triglyceride content correlates with insulin resistance,
euglycemic-hyperinsulinemic clamps were performed to evaluate the
individual tissue insulin responsiveness of the AZIP mice. Wild-type B6
and FVB mice were similar in all clamp parameters whereas the B6 AZIP
and FVB AZIP mice were quite different
(Table II, Fig.
3). The endogenous glucose production of
the FVB AZIP mice was suppressed almost as well as in the wild type FVB
mice (53 versus 69%, p = nonsignificant; in other studies the difference reached
significance (24)). However, the endogenous glucose production in B6
AZIP mice did not suppress at all during the clamp (Table II, Fig.
3A). Because endogenous glucose production is chiefly a
measure of liver glucose production, these data demonstrate that the B6
AZIP mice have more severe liver insulin resistance than do the FVB
AZIP mice.
During the clamp, whole body glucose uptake in FVB AZIP mice was only
32% of wild type controls, indicating severe muscle insulin resistance
(Fig. 3B). In contrast, B6 AZIP whole body glucose uptake in
mice was 66% of control levels. Direct measurement of muscle glucose
uptake with [14C]2-deoxyglucose gave similar results: in
FVB AZIP mice the rate was 22% of FVB wild type and in B6 AZIP mice it
was 63% of B6 wild type (Fig. 3C). This pattern was also
observed for whole body and muscle glycolysis and for glycogen
synthesis (Table II). Thus, the muscles of the FVB AZIP mice are more
insulin resistant than those of the B6 AZIP mice, the opposite of the
pattern observed for liver.
Lipid Metabolism--
Liver triglyceride levels were determined by
triglyceride synthesis, uptake, and secretion rates. To quantitate
triglyceride secretion, the rate of increase in circulating
triglycerides was measured after inhibiting clearance with Triton
WR1339 (21). Wild-type B6 and FVB mice had similar secretion rates,
which were increased significantly by the AZIP transgene (increased
3.1- and 3.6-fold in the B6 and FVB backgrounds, respectively) (Fig. 4A).
Triglyceride clearance was studied by measuring serum triglyceride
levels after an oral lipid load. In wild type FVB mice, serum
triglycerides peaked at 2 h (382 mg/dl) and then decreased gradually (Fig. 4B). The increase was accentuated (980 mg/dl) and occurred later (4 h) in FVB AZIP mice, demonstrating that triglyceride clearance on FVB background is greatly impaired in the
absence of WAT. Surprisingly, a totally different result was found for
the B6 mice. Serum triglycerides rose only minimally in wild type and
transgenic mice, peaking at 82 and 128 mg/dl in B6 wild type and B6
AZIP mice, respectively. This was not because of poor triglyceride
uptake from the intestine in B6 mice because, in the absence of
triglyceride clearance, serum triglyceride levels increased equally in
B6 and FVB mice after an oral lipid load (Fig. 4C). Thus, B6
mice show much more efficient clearance of circulating triglyceride
than do FVB mice.
Increased lipase activity should increase triglyceride clearance. Serum
lipase activity (without heparin), which in mice is largely a measure
of hepatic lipase (25), was higher in B6 than FVB mice (Table
III). Post-heparin plasma showed modest
increases in both hepatic and lipoprotein lipase activity in wild type
B6 as compared with FVB mice, but no striking effect of the AZIP transgene (Table III). Hepatic lipase mRNA levels were not
different between the groups, whereas endothelial lipase mRNA
levels were lower in the wild type FVB livers than in the other groups
(Fig. 5). Liver lipoprotein lipase
mRNA levels were <1/20 the level of adipose tissue levels, too low
to determine whether differences exist between the groups (data not
shown). These data suggest that increased lipase activity may
contribute to the increased triglyceride clearance.
Gene Expression--
Liver mRNA levels were measured in the B6
and FVB strains (Fig. 5). There was a significant background genotype
effect on mRNA levels of lipogenic genes (LXR
Unexpectedly, a huge, 20-fold elevation in the hepatic CD36 mRNA
level was observed in B6 wild type as compared with FVB wild type mice
(Fig. 6, top). The AZIP mice
on both genetic backgrounds had high CD36 mRNA levels, similar to
wild type B6, but 15-25-fold greater than wild type FVB, mice. In
contrast, CD36 mRNA levels in muscle were not different between
wild type and AZIP mice on either the FVB or B6 genetic backgrounds.
Taken together, these results demonstrate that there is a
liver-specific difference in the regulation of the
CD36 gene between the wild type FVB and B6 lines.
As an additional test of the contribution of CD36 to circulating
triglyceride levels, liver CD36 mRNA and serum triglyceride levels
were measured in four more strains (SWR/J, C3H/HeJ, BALB/cJ, and
129/SvJ) and repeated in the B6 and FVB mice (Fig. 6,
bottom). There is an approximate inverse correlation between
serum triglycerides and liver CD36 mRNA levels, but the CD36 levels
cannot completely account for the variation in serum triglycerides
among the six strains.
The AZIP Phenotype Depends on the Genetic Background--
The AZIP
phenotype on the B6 background is generally less severe than that on
the FVB background. Specifically, the B6 AZIP mice have less
hyperglycemia, lower circulating triglyceride and fatty acid levels,
faster triglyceride clearance, lower mortality, and less muscle insulin
resistance. Features of the AZIP phenotype that are similar on both
backgrounds include the severe lack of WAT, hugely increased insulin
levels, and increased triglyceride secretion. Characteristics of the
AZIP phenotype that are more profound on the B6 background include
hepatic insulin resistance and steatosis.
Previous studies of the effect of genetic background on diabetes using
ob/ob and db/db mice found
How Does Lipoatrophy Cause Diabetes?--
The lack of adipose
tissue is the primary cause of the metabolic symptoms known as
lipoatrophic diabetes as shown by the correlation between the degree of
fat loss and syndrome severity and by the reversal of all features by
adipose tissue transplantation (8). Adipose tissue ablation removes
triglyceride storage capacity and adipose hormones such as leptin and
adiponectin/Acrp30. The lack of fat is signaled to the liver, causing
an increase in hepatic fatty acid synthesis, triglyceride storage, and
secretion. The fatty liver can be thought of as an appropriate
compensatory response by the body to the deficit of adipose tissue
triglyceride. The changes in liver physiology are accomplished through
increased levels of the transcription factors sterol element-binding
protein 1c (SREBP-1c) (31) and peroxisome proliferator-activated
receptor-
The nature of the signals from WAT to the rest of the body, and the
route they take are not known. Leptin deficiency clearly contributes to
the insulin resistance and lipid abnormalities (36-39). Leptin may be
acting via the hypothalamus, elsewhere in the brain, and/or directly on
tissues such as liver and muscle (40, 41). Adiponectin/Acrp30 (42-44)
deficiency does not appear to be the major cause of the diabetes of the
A-ZIP/F-1 mice (39). Other possible contributors are other metabolic
and endocrine signals produced by WAT or indirect consequences of the
lack of WAT, such as increased availability of diet-derived substances.
Role of Genetic Background in the Lipoatrophic Phenotype--
Our
working hypothesis to explain the effect of the background genotype on
the AZIP metabolic phenotype focuses on hepatic triglyceride levels and
lipid clearance. The B6 mice have higher serum lipase levels and better
clearance of an oral lipid load. Presumably, more of the lipid ends up
in the liver in the B6 mice, whereas slightly less ends up in the
muscle. The increased liver triglyceride causes increased hepatic
insulin resistance, whereas the reduced muscle triglyceride levels lead
to less muscle insulin resistance. Increased hepatic lipogenesis may
also contribute. Previous reports have correlated muscle triglyceride
levels with muscle insulin resistance (45, 46), liver insulin
resistance with liver triglyceride content (47), and improvements in
one at the expense of the other (48, 49). However, the tissue triglycerides themselves are probably not the ultimate cause of decreased insulin signaling, but are rather a surrogate for molecules as yet unidentified (50).
The mechanistic details of the increased clearance are not yet known.
Increased circulating lipase activity, as in B6 mice, can theoretically
provide fatty acids for uptake into many organs. The reduced
triglyceride clearance by FVB AZIP as compared with wild type FVB mice
demonstrates that WAT can be a significant contributor. However, in the
B6 AZIP mice, despite the absence of WAT, rapid triglyceride uptake
still occurs, presumably some of which is into the liver. The
difference between the B6 and FVB genetic backgrounds in triglyceride
clearance may well be at the level of receptor-mediated lipoprotein
uptake, affecting ligand, receptor, or both. The different liver CD36
mRNA levels may contribute.
How do the insights derived from lipoatrophic mice apply to
lipoatrophic humans? The physiology is remarkably similar and we
believe that the insights are generally applicable. For example, treatment with leptin improves the diabetes and lipid abnormalities of
both mice (36, 39) and humans with lipoatrophy (51). The differences
between the FVB and B6 backgrounds is also relevant, suggesting that
human genetic heterogeneity greatly influences the observed
lipoatrophic phenotype.
Lipoprotein physiology is somewhat different between mice and humans.
Mice have higher high density lipoprotein, lower low density
lipoprotein, and are resistant to atherosclerosis. Thus to study
atherosclerosis in the mouse, a high cholesterol/high fat diet and/or
Ldlr
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 5'-TGCTGAGCTGCGAGGGCTGCAAGGG and
5'-CGCCTGTTACACTGTTGCTGGGC; SREBP2, 5'-CAGAGCCATGTGCACATCTGAACAGG and
5'-CTGTCACTGGAGTCAGGTGCTGGG. A cDNA probe for
glucose-6-phosphatase (23) was excised from plasmid DNA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Anatomical characterization of male AZIP mice at 28 weeks of age
0.05 and double symbols for p
0.005;
refers to AZIP versus wild type; * refers to FVB
versus B6; and + refers to interaction between
transgene status and background genotype. Muscle triglyceride (TG) in
wild type (but not AZIP) mice is unreliable due to the presence of
adipose tissue (Ref. 14 and O. Gavrilova, unpublished observations).
View larger version (16K):
[in a new window]
Fig. 1.
Growth and circulating glucose, insulin,
triglyceride, and fatty acid levels in male AZIP mice. Symbols
are: B6 wild type ( ), B6 AZIP (
), FVB wild type (
), and FVB
AZIP (
). Samples are from longitudinal follow up of single cohorts
of mice, and were obtained from unanesthetized mice in the nonfasting
state, between 0900 and 1200. Note the logarithmic scale for the
insulin data. Data are mean ± S.E., n = 4-9/group. Averages of all glucose values in mg/dl are (mean ± one S.D.): B6 wild type, 154 ± 18, n = 123; FVB
wild type, 164 ± 23, n = 81; FVB AZIP, 542 ± 104, n = 50. Averages of all triglyceride values in
mg/dl are: B6 wild type, 91 ± 26, n = 47; B6
AZIP, 120 ± 60, n = 50; FVB wild type, 172 ± 87, n = 72.
View larger version (79K):
[in a new window]
Fig. 2.
Liver appearance, histology, weight, and
triglyceride content and muscle triglyceride levels at 28 weeks of
age. In each pair of the indicated background genotype and
sex, the wild type control is in the left bar and the AZIP
in the right bar. Data are mean ± S.E.,
n = 5-9/group; n = 4/group for muscle
triglyceride.
Euglycemic-hyperinsulinemic clamp measurements
0.05 and double symbols for p
0.005;
refers to AZIP versus wild type; * refers to FVB
versus B6; and + refers to interaction between
transgene status and background genotype.
View larger version (24K):
[in a new window]
Fig. 3.
Liver and muscle insulin sensitivity in
female mice, 10 weeks of age. A, basal (left
bars) and clamp (right bars) endogenous glucose
production in mice of the indicated genotypes. The percent suppression
values for these data are in Table II. Statistical significance
(p < 0.02 by t test) between basal and
clamp endogenous glucose production (EGP) in each genotype
is indicated by *. B, whole body glucose uptake.
C, muscle glucose uptake. Statistical significance between
the FVB AZIP and B6 AZIP mice is indicated by the single
asterisk (*) (p = 0.06) and double
asterisks (**) (p < 0.05). Data are mean ± S.E., n = 5-6/group.
View larger version (20K):
[in a new window]
Fig. 4.
A, triglyceride secretion in mice after
WR1339 injection. Male mice 17 weeks old were used. Statistically
significant differences between the WT and AZIP mice are indicated by
an asterisk (*) (p < 0.05). No significant
difference between B6 AZIP and FVB AZIP mice was found. Data are
mean ± S.E., n = 3-5/group. B,
triglyceride clearance after peanut oil gavage in male mice 10 weeks
old. Data are mean ± S.E., n = 6-7/group. The
area under the curves (mean ± S.E.) are 1315 ± 82 for FVB
wild type, 3585 ± 598 for FVB AZIP, 361 ± 35 for B6 wild
type, and 395 ± 46 for B6 AZIP mice. C, normal uptake
of intestinal lipid in wild type B6 mice. Triglyceride clearance after
peanut oil gavage was inhibited by WR1339 injection (closed
symbols) as compared with controls (open symbols) in
both B6 and FVB mice. Data are mean ± S.E., n = 3-4/group.
Serum chemistries of male mice
0.05 and double symbols p
0.005;
refers to AZIP versus wild type; * refers to FVB
versus B6; and + refers to interaction between
transgene status and background genotype.
View larger version (44K):
[in a new window]
Fig. 5.
Liver mRNA levels of genes involved in
metabolism of glucose, triglycerides, and fatty acids. In each
group, the order of the bars is FVB wild type, FVB AZIP, B6
wild type, and B6 AZIP. mRNA levels were measured by Northern
blotting in livers from nonfasting mice euthanized between 0900 and
1200. Data are normalized with FVB wild type levels = 100. Data
are from male mice, age 28 weeks (mean ± S.E., n = 4-6/group). Statistical significance via two-way ANOVA: single
symbols are used for p 0.05 and double
symbols for p
0.005; ^ refers to AZIP
versus wild type and the single asterisk (*)
refers to FVB versus B6. No significant interaction between
transgene status and background genotype was detected. ACC,
acetyl-CoA carboxylase; G6P, glucose-6-phosphatase;
FAS, fatty acid synthase; CPT1, carnitine
palmitoyltransferase 1; PEPCK, phosphoenolpyruvate
carboxykinase; IRS-2, insulin receptor substrate 2;
AOX, acyl-CoA oxidase; LXR
, liver X receptor
; SCD-1, stearoyl-CoA desaturase 1;
F1-6BP, fructose-1,6-bisphosphatase.
, SREBP1,
PPAR
, acetyl-CoA carboxylase, fatty acid synthase, and
stearoyl-CoA desaturase 1), fatty acid oxidation genes (acyl-CoA
oxidase and carnitine palmitoyltransferase I), and gluconeogenic genes
(glucose-6-phosphatase and fructose-1,6-bisphosphatase), with
the B6 levels typically being 2-3-fold higher than the FVB levels.
Comparing AZIP to wild type mice, the lipogenic gene (SREBP1, PPAR
,
acyl-CoA carboxylase, fatty acid synthase, and SCD-1)
mRNA levels were higher in the AZIP mice. Except for a decrease in
acyl-CoA oxidase, no other mRNA levels were significantly different
between wild type and AZIP mice. Of these 13 mRNAs, none showed
significant transgene status x background genotype interaction. It is
remarkable that the background genotype has more effect on the mRNA
levels than the AZIP transgene, with increased lipogenesis in the B6 mice.
View larger version (17K):
[in a new window]
Fig. 6.
CD36 expression in liver and muscle.
mRNA levels were measured by Northern blot, quantitated by
PhosphorImager, and normalized to the FVB wild type levels.
Top, data are from male mice, aged 28 weeks (mean ± S.E., n = 4-6/group). Bottom, data are from
male mice at 6 weeks of age (mean ± S.E., n = 6/group). These six groups of mice were purchased from the Jackson
Laboratory 2 weeks before being studied.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell failure causing premature death on the C57BLKS/J (26) and
CBA/LtJ (27) backgrounds. In contrast, on the B6 (28), 129/J (29), and
FVB (30) backgrounds, the same mutations caused more obesity but milder
hyperglycemia, because of increased insulin production, with
-cell
hypertrophy and hyperplasia. Thus the studies of
ob/ob, db/db, and AZIP mice agree, suggesting that the B6 and FVB backgrounds have robust islets,
carrying alleles that promote
-cell production, longevity, and/or
insulin secretion. Because the FVB and B6 strains differ in their
insulin resistance phenotypes and because the confounding effects of
-cell failure do not complicate the studies, these strains can be
used to identify insulin resistance modifier genes, for example, by the
quantitative trait locus approach.
(PPAR
) (9). SREBP-1c increases lipid accumulation in
cultured hepatocytes (32) and livers of mice (33, 34) and may act by
increasing PPAR
expression (35). Recent experiments demonstrate that
liver-specific ablation of PPAR
greatly reduces hepatic triglyceride
levels in the AZIP mice, suggesting a role for PPAR
in driving
hepatic steatosis.2
/
or Apoe
/
mutations are
usually used to accelerate the process (52). The B6 background is more
susceptible to atherosclerosis than other strains and B6 mice have the
lowest circulating triglyceride levels of 17 mouse strains tested (53). Indeed, a direct comparison of ApoE-deficient mice on the B6 and FVB
backgrounds found the B6 mice to have lower total cholesterol, high
density lipoprotein cholesterol, ApoA-I, and ApoA-II levels and
larger atherosclerotic lesions (54). It is possible that mechanistic
understanding of the increased triglyceride clearance will shed light
on the B6 predisposition to atherogenesis. Possibly lipid uptake into
other tissues is also facilitated in the B6 strain. The FVB to B6
comparison points to triglyceride clearance as a modifier of the
diabetes and lipid phenotypes.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. I. J. Goldberg for the post-heparin lipase activity measurements. We thank Dr. S. Chua, Jr. for communicating results prior to publication, Drs. A. Attie, R. Eckel, I. J. Goldberg, R. Knapp, and D. J. Rader for helpful discussions, and Drs. C. J. Chou, P. Gorden, G. Waters, and L. Weinstein for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
§ Present address: 3 Dept. of Internal Medicine, 1 Faculty of Medicine, Charles University, Prague, Czech Republic.
To whom correspondence should be addressed: Merck Research
Laboratories, P.O. Box 2000, RY80M-213, Rahway, NJ 07065. Tel.: 732-594-4609; Fax: 732-594-3337; E-mail: marc_reitman@merck.com.
** Current address: Via Adda 17, 20010 Villastanza, Milano, Italy.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M207665200
2 O. Gavrilova, M. Haluzik, J. J. Cutson, L. Johnson, K. R. Dietz, C. Nicol, C. Vinson, F. Gonzalez, and M. L. Reitman, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: AZIP, A-ZIP/F-1; B6, C57BL/6J; FVB, FVB/N; WAT, white adipose tissue; PPAR, peroxisome proliferator-activated receptor; SREBP-1, sterol response element-binding protein 1; ANOVA, analysis of variance.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Comuzzie, A. G.,
and Allison, D. B.
(1998)
Science
280,
1374-1377 |
2. | Saltiel, A. R. (2001) Cell 104, 517-529[Medline] [Order article via Infotrieve] |
3. | Horikawa, Y., Oda, N., Cox, N. J., Li, X., Orho-Melander, M., Hara, M., Hinokio, Y., Lindner, T. H., Mashima, H., Schwarz, P. E., del Bosque-Plata, L., Oda, Y., Yoshiuchi, I., Colilla, S., Polonsky, K. S., Wei, S., Concannon, P., Iwasaki, N., Schulze, J., Baier, L. J., Bogardus, C., Groop, L., Boerwinkle, E., Hanis, C. L., and Bell, G. I. (2000) Nat. Genet. 26, 163-175[CrossRef][Medline] [Order article via Infotrieve] |
4. | Hummel, K. P., Coleman, D. L., and Lane, P. W. (1972) Biochem. Genet. 7, 1-13[Medline] [Order article via Infotrieve] |
5. | Coleman, D. L., and Hummel, K. P. (1973) Diabetologia 9, 287-293[Medline] [Order article via Infotrieve] |
6. |
Moitra, J.,
Mason, M. M.,
Olive, M.,
Krylov, D.,
Gavrilova, O.,
Marcus- Samuels, B.,
Feigenbaum, L.,
Lee, E.,
Aoyama, T.,
Eckhaus, M.,
Reitman, M. L.,
and Vinson, C.
(1998)
Genes Dev.
12,
3168-3181 |
7. |
Gavrilova, O.,
Leon, L. R.,
Marcus-Samuels, B.,
Mason, M. M.,
Castle, A. L.,
Refetoff, S.,
Vinson, C.,
and Reitman, M. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14623-14628 |
8. |
Gavrilova, O.,
Marcus-Samuels, B.,
Graham, D.,
Kim, J. K.,
Shulman, G. I.,
Castle, A. L.,
Vinson, C.,
Eckhaus, M.,
and Reitman, M. L.
(2000)
J. Clin. Invest.
105,
271-278 |
9. |
Chao, L.,
Marcus-Samuels, B.,
Mason, M. M.,
Moitra, J.,
Vinson, C.,
Arioglu, E.,
Gavrilova, O.,
and Reitman, M. L.
(2000)
J. Clin. Invest.
106,
1221-1228 |
10. | Foster, D. W. (1998) in Harrison's Principles of Internal Medicine (Fauci, A. S. , Braunwald, E. , Isselbacher, K. J. , Wilson, J. D. , Martin, J. B. , Kasper, D. L. , Hauser, S. L. , and Longo, D. L., eds), 14th Ed. , pp. 2209-2214, McGraw-Hill, New York |
11. | Reitman, M. L., Arioglu, E., Gavrilova, O., and Taylor, S. I. (2000) Trends Endocrinol. Metab. 11, 410-416[CrossRef][Medline] [Order article via Infotrieve] |
12. | Taketo, M., Schroeder, A. C., Mobraaten, L. E., Gunning, K. B., Hanten, G., Fox, R. R., Roderick, T. H., Stewart, C. L., Lilly, F., Hansen, C. T., and Overbeek, P. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2065-2069[Abstract] |
13. | Beck, J. A., Lloyd, S., Hafezparast, M., Lennon-Pierce, M., Eppig, J. T., Festing, M. F., and Fisher, E. M. (2000) Nat. Genet. 24, 23-25[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Burant, C. F.,
Sreenan, S.,
Hirano, K.,
Tai, T. A.,
Lohmiller, J.,
Lukens, J.,
Davidson, N. O.,
Ross, S.,
and Graves, R. A.
(1997)
J. Clin. Invest.
100,
2900-2908 |
15. | Bradley, D. C., and Kaslow, H. R. (1989) Anal. Biochem. 180, 11-16[Medline] [Order article via Infotrieve] |
16. | Brasaemle, D. L., Levin, D. M., Adler-Wailes, D. C., and Londos, C. (2000) Biochim. Biophys. Acta 1483, 251-262[Medline] [Order article via Infotrieve] |
17. | Rossetti, L., and Giaccari, A. (1990) J. Clin. Invest. 85, 1785-1792[Medline] [Order article via Infotrieve] |
18. | Youn, J. H., Kim, J. K., and Buchanan, T. A. (1994) Diabetes 43, 564-571[Abstract] |
19. |
Kim, J. K.,
Michael, M. D.,
Previs, S. F.,
Peroni, O. D.,
Mauvais-Jarvis, F.,
Neschen, S.,
Kahn, B. B.,
Kahn, C. R.,
and Shulman, G. I.
(2000)
J. Clin. Invest.
105,
1791-1797 |
20. |
Haluzik, M.,
Dietz, K. R.,
Kim, J. K.,
Marcus-Samuels, B.,
Shulman, G. I.,
Gavrilova, O.,
and Reitman, M. L.
(2002)
Diabetes
51,
2113-2118 |
21. | Borensztajn, J., Rone, M. S., and Kotlar, T. J. (1976) Biochem. J. 156, 539-543[Medline] [Order article via Infotrieve] |
22. |
Tietge, U. J.,
Bakillah, A.,
Maugeais, C.,
Tsukamoto, K.,
Hussain, M.,
and Rader, D. J.
(1999)
J. Lipid Res.
40,
2134-2139 |
23. |
Shelly, L. L.,
Lei, K. J.,
Pan, C. J.,
Sakata, S. F.,
Ruppert, S.,
Schutz, G.,
and Chou, J. Y.
(1993)
J. Biol. Chem.
268,
21482-21485 |
24. |
Kim, J. K.,
Gavrilova, O.,
Chen, Y.,
Reitman, M. L.,
and Shulman, G. I.
(2000)
J. Biol. Chem.
275,
8456-8460 |
25. | Peterson, J., Bengtsson-Olivecrona, G., and Olivecrona, T. (1986) Biochim. Biophys. Acta 878, 65-70[Medline] [Order article via Infotrieve] |
26. | Hummel, K. P., Dickie, M. M., and Coleman, D. L. (1966) Science 153, 1127-1128[Medline] [Order article via Infotrieve] |
27. | Leiter, E. H. (1981) Diabetes 30, 1035-1044[Abstract] |
28. | Ingalls, A. M., Dickie, M. M., and Snell, G. D. (1950) J. Hered. 41, 317-318 |
29. | Leiter, E. H., Coleman, D. L., Eisenstein, A. B., and Strack, I. (1980) Diabetologia 19, 58-65[Medline] [Order article via Infotrieve] |
30. | Chua, S. J., Mei Liu, S., Li, Q., Yang, L., Thassanapaff, V. T., and Fisher, P. (2002) Diabetologia 45, 976-990[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Shimomura, I.,
Bashmakov, Y.,
and Horton, J. D.
(1999)
J. Biol. Chem.
274,
30028-30032 |
32. |
Foretz, M.,
Guichard, C.,
Ferre, P.,
and Foufelle, F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12737-12742 |
33. | Shimomura, I., Matsuda, M., Hammer, R. E., Bashmakov, Y., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 77-86[Medline] [Order article via Infotrieve] |
34. |
Shimano, H.,
Horton, J. D.,
Shimomura, I.,
Hammer, R. E.,
Brown, M. S.,
and Goldstein, J. L.
(1997)
J. Clin. Invest.
99,
846-854 |
35. |
Fajas, L.,
Schoonjans, K.,
Gelman, L.,
Kim, J. B.,
Najib, J.,
Martin, G.,
Fruchart, J. C.,
Briggs, M.,
Spiegelman, B. M.,
and Auwerx, J.
(1999)
Mol. Cell. Biol.
19,
5495-5503 |
36. | Shimomura, I., Hammer, R. E., Ikemoto, S., Brown, M. S., and Goldstein, J. L. (1999) Nature 401, 73-76[CrossRef][Medline] [Order article via Infotrieve] |
37. | Gavrilova, O., Marcus-Samuels, B., Leon, L. R., Vinson, C., and Reitman, M. L. (2000) Nature 403, 850[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Ebihara, K.,
Ogawa, Y.,
Masuzaki, H.,
Shintani, M.,
Miyanaga, F.,
Aizawa-Abe, M.,
Hayashi, T.,
Hosoda, K.,
Inoue, G.,
Yoshimasa, Y.,
Gavrilova, O.,
Reitman, M. L.,
and Nakao, K.
(2001)
Diabetes
50,
1440-1448 |
39. |
Colombo, C.,
Cutson, J. J.,
Yamauchi, T.,
Vinson, C.,
Kadowaki, T.,
Gavrilova, O.,
and Reitman, M. L.
(2002)
Diabetes
51,
2727-2733 |
40. | Ahima, R. S., and Flier, J. S. (2000) Annu. Rev. Physiol. 62, 413-437[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Masuzaki, H.,
Paterson, J.,
Shinyama, H.,
Morton, N. M.,
Mullins, J. J.,
Seckl, J. R.,
and Flier, J. S.
(2001)
Science
294,
2166-2170 |
42. |
Fruebis, J.,
Tsao, T. S.,
Javorschi, S.,
Ebbets-Reed, D.,
Erickson, M. R.,
Yen, F. T.,
Bihain, B. E.,
and Lodish, H. F.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
2005-2010 |
43. | Berg, A. H., Combs, T. P., Du, X., Brownlee, M., and Scherer, P. E. (2001) Nat. Med. 7, 947-953[CrossRef][Medline] [Order article via Infotrieve] |
44. | Yamauchi, T., Oike, Y., Kamon, J., Waki, H., Komeda, K., Tsuchida, A., Date, Y., Li, M. X., Miki, H., Akanuma, Y., Nagai, R., Kimura, S., Saheki, T., Nakazato, M., Naitoh, T., Yamamura, K., and Kadowaki, T. (2002) Nat. Genet. 30, 221-226[CrossRef][Medline] [Order article via Infotrieve] |
45. | Krssak, M., Falk Petersen, K., Dresner, A., DiPietro, L., Vogel, S. M., Rothman, D. L., Roden, M., and Shulman, G. I. (1999) Diabetologia 42, 113-116[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Virkamaki, A.,
Korsheninnikova, E.,
Seppala-Lindroos, A.,
Vehkavaara, S.,
Goto, T.,
Halavaara, J.,
Hakkinen, A. M.,
and Yki-Jarvinen, H.
(2001)
Diabetes
50,
2337-2343 |
47. |
Seppala-Lindroos, A.,
Vehkavaara, S.,
Hakkinen, A. M.,
Goto, T.,
Westerbacka, J.,
Sovijarvi, A.,
Halavaara, J.,
and Yki-Jarvinen, H.
(2002)
J. Clin. Endocrinol. Metab.
87,
3023-3028 |
48. |
Klaman, L. D.,
Boss, O.,
Peroni, O. D.,
Kim, J. K.,
Martino, J. L.,
Zabolotny, J. M.,
Moghal, N.,
Lubkin, M.,
Kim, Y. B.,
Sharpe, A. H.,
Stricker-Krongrad, A.,
Shulman, G. I.,
Neel, B. G.,
and Kahn, B. B.
(2000)
Mol. Cell. Biol.
20,
5479-5489 |
49. |
Kim, J. K.,
Fillmore, J. J.,
Chen, Y., Yu, C.,
Moore, I. K.,
Pypaert, M.,
Lutz, E. P.,
Kako, Y.,
Velez-Carrasco, W.,
Goldberg, I. J.,
Breslow, J. L.,
and Shulman, G. I.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
7522-7527 |
50. |
Shulman, G. I.
(2000)
J. Clin. Invest.
106,
171-176 |
51. |
Oral, E. A.,
Simha, V.,
Ruiz, E.,
Andewelt, A.,
Premkumar, A.,
Snell, P.,
Wagner, A. J.,
DePaoli, A. M.,
Reitman, M. L.,
Taylor, S. I.,
Gorden, P.,
and Garg, A.
(2002)
N. Engl. J. Med.
346,
570-578 |
52. |
Knowles, J. W.,
and Maeda, N.
(2000)
Arterioscler. Thromb. Vasc. Biol.
20,
2336-2345 |
53. | Peters, L. L., and Paigen, B. (2002) MPD accession number 6224, Mouse Phenome Database Web Site, The Jackson Laboratory, Bar Harbor, ME (www.jax.org/phenome) |
54. |
Dansky, H. M.,
Charlton, S. A.,
Sikes, J. L.,
Heath, S. C.,
Simantov, R.,
Levin, L. F.,
Shu, P.,
Moore, K. J.,
Breslow, J. L.,
and Smith, J. D.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
1960-1968 |
55. |
Lutz, E. P.,
Merkel, M.,
Kako, Y.,
Melford, K.,
Radner, H.,
Breslow, J. L.,
Bensadoun, A.,
and Goldberg, I. J.
(2001)
J. Clin. Invest.
107,
1183-1192 |