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J. Biol. Chem., Vol. 278, Issue 36, 34268-34276, September 5, 2003
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Contributes to Hepatic Steatosis, Triglyceride Clearance, and Regulation of Body Fat Mass*
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From the
Diabetes Branch, NIDDK and
¶Laboratory of Metabolism, NCI, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, January 2, 2003 , and in revised form, April 15, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(PPAR) is a
nuclear receptor that mediates the antidiabetic effects of thiazolidinediones.
PPAR is present in adipose tissue and becomes elevated in fatty livers,
but the roles of specific tissues in thiazolidinedione actions are unclear. We
studied the function of liver PPAR in both lipoatrophic A-ZIP/F-1
(AZIP) and wild type mice. In AZIP mice, ablation of liver PPAR reduced
the hepatic steatosis but worsened the hyperlipidemia, triglyceride clearance,
and muscle insulin resistance. Inactivation of AZIP liver PPAR also
abolished the hypoglycemic and hypolipidemic effects of rosiglitazone,
demonstrating that, in the absence of adipose tissue, the liver is a primary
and major site of thiazolidinedione action. In contrast, rosiglitazone
remained effective in non-lipoatrophic mice lacking liver PPAR,
suggesting that adipose tissue is the major site of thiazolidinedione action
in typical mice with adipose tissue. Interestingly, mice without liver
PPAR, but with adipose tissue, developed relative fat intolerance,
increased adiposity, hyperlipidemia, and insulin resistance. Thus, liver
PPAR regulates triglyceride homeostasis, contributing to hepatic
steatosis, but protecting other tissues from triglyceride accumulation and
insulin resistance. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(PPAR)1 is a
member of the nuclear hormone receptor superfamily. It can be activated by a
variety of ligands, including fatty acids, eicosanoids
(1), and
15-deoxy-
12,14-prostaglandin J2
(2). PPAR is the primary
molecular target of thiazolidinediones (TZDs), antidiabetic agents that act by
increasing insulin sensitivity (reviewed in Ref.
3). However, the target tissues
and mechanisms by which TZDs increase insulin sensitivity are not well
understood
(4–6).
TZDs increase glucose utilization in muscle and, at higher doses, inhibit
endogenous glucose production (largely a liver function)
(7–9).
However, muscle and liver have low PPAR levels. Several lines of
evidence suggest that TZDs act directly on adipose tissue, with secondary
effects in skeletal muscle and liver. 1) Adipose tissue is the only
insulin-responsive tissue expressing high levels of PPAR
(10–12).
2) TZDs stimulate insulin action in cultured adipose tissue
(13) but not in isolated
muscle (14) or primary
hepatocytes (15). 3)
PPAR is essential for adipocyte differentiation
(16,
17). 4) Overexpression of
PPAR and its activation by TZDs stimulate adipocyte differentiation
(18), leading to accumulation
of small adipocytes, which are more insulin-sensitive than large adipocytes
(19). 5) Finally, in
vivo, PPAR agonists cause massive changes in gene expression in
adipose tissue but affect a much smaller number of genes in liver and muscle
(20). Thus, adipose tissue
appears to be a direct target and the major site of TZD action.
Interestingly, in the near absence of adipose tissue, TZDs still have
beneficial effects. Troglitazone improved diabetes and hyperlipidemia in
lipoatrophic patients with various degrees of fat loss
(21) and in a mouse model of
late onset lipoatrophy (22).
In the A-ZIP/F-1 (hereafter AZIP) mouse, a model of severe congenital
lipoatrophic diabetes (23),
the effect of therapy depends on genetic background. In FVB/N AZIP mice, TZDs
lower circulating triglyceride levels but do not affect the diabetes
(24). The latter apparently
results from the opposite effects on muscle and liver, with increased insulin
sensitivity in skeletal muscle and decreased insulin sensitivity in liver
(25). In contrast, in C57BL/6J
AZIP mice, rosiglitazone improves both hyperlipidemia and
diabetes.2 Taken
together, these data suggest that PPAR agonists may act directly on
non-adipose tissues.
We focused on liver PPAR as a potential direct target of TZDs,
because PPAR mRNA levels are markedly elevated in steatotic livers of
the AZIP mice (24,
26,
27). Other mouse models of
hepatic steatosis also exhibit increased liver PPAR expression
(12,
22,
28–32),
indicating that increased PPAR mRNA levels are a general property of
steatotic liver. However, it is unclear whether the up-regulation of
PPAR causes the steatosis or whether the steatosis causes the elevated
PPAR levels.
The aim of this study is to understand the role of liver PPAR in
both wild type and adipose-deficient mice. We used the Cre-lox system to
inactivate hepatocyte PPAR and demonstrate that liver PPAR is an
important regulator of lipid homeostasis in wild type and lipoatrophic AZIP
mice, contributes to development of hepatic steatosis in lipoatrophy and in
diet-induced obesity, and mediates most of the effects of rosiglitazone in
lipoatrophic mice.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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fl/fl
(33), and Alb-Cre
(34) mice have been reported.
Hemizygous AZIP males (FVB/N background) were bred with
PPARfl/fl Alb-Cre females (mixed FVB/N, 129, and C57BL/6J
background). AZIP males from the PPARfl/+
offspring were then bred to Alb-Cre PPARfl/+
female littermates to generate mice: PPARfl/fl (named WT),
PPARfl/fl hemizygous for Alb-Cre (WT LKO),
PPARfl/fl hemizygous for the AZIP transgene (AZIP), and
PPARfl/fl hemizygous for both AZIP and Alb-Cre transgenes
(AZIP LKO). All mice studied were homozygous for the PPAR–floxed
allele, which had no affect on the phenotype of the wild type or AZIP mice.
Sex-matched littermates were used as controls. Mice were typically reared
three per cage on a 12-h light/dark cycle (lights on 0600–1800) and fed
water and NIH-07 diet (Zeigler Brothers, Inc., Gardners, PA) ad
libitum. When indicated, powder AIN-93G diet
(35) (Dyets, Bethlehem, PA) or
high fat diet (D12451
[GenBank]
; Research Diets, Inc., New Brunswick, NJ) was used.
Rosiglitazone (3 mg/kg/day; Smith Kline Beecham, West Sussex, United Kingdom)
was blended with food and administered for 5 weeks. Mice were euthanized in
the non-fasted state between 0900 and 1200. Tissues were fixed in neutralized
10% formalin and processed by American Histolabs (Gaithersburg, MD). Southern Blot Analysis—Genomic DNA was isolated from total liver of 11-week-old female mice, digested with BamHI, and analyzed as described (33).
RNA Analysis—Total liver RNA was prepared and analyzed by Northern blot by phosphorimaging as described previously (24, 26). RNase protection assay was perform as described (36).
Western Blot Analysis—Nuclear extracts from total liver were
isolated using an NE-PER nuclear and cytoplasmic extraction reagents kit
(Pierce, Rockford, IL). Hepa-1 cells were transfected with an expression
vector for PPAR1, pSG5-PPAR1 (provided by Dr. Wahli, Universite
de Lausanne, Lausanne, Switzerland), using LipofecAMINE reagent (Invitrogen).
10 µg of total protein from Hepa-1 cells and 10 µg of protein from
nuclear extracts were subjected to electrophoresis ona4to 15% Tris-HCl
gradient gel (Bio-Rad), transferred to Immobilon-P membranes (Millipore,
Bedford, MA), and probed according to the manufacturer's recommendations with
anti-PPAR antibody raised against a recombinant protein corresponding
to amino acids 6 to 105 (H-100; Santa Cruz Biotechnology, Inc., Santa Cruz,
CA). Immunoreactive proteins were detected using an enhanced chemiluminescence
blot detection system (Amersham Biosciences).
Biochemical Assays—Serum was obtained from tail or retro-orbital veins in the non-fasted state. Serum glucose, triglyceride, free fatty acid, insulin, and leptin were assayed as described (37). Serum adiponectin and serum estradiol were measure by radioimmunoassay (MADP-60HK; Linco Research, Inc., St. Charles, MO and DSL-39100; Diagnostic System Laboratories, Inc., Webster, TX, respectively). Tissue triglyceride was measured as described (26).
Euglycemic-hyperinsulinemic Clamps—Mice were fasted for 12 h. Clamps were performed are described (38, 39) with a target plasma insulin of 4 ng/ml and plasma glucose of 110–120 mg/dl.
Indirect Calorimetry—Oxygen consumption and carbon dioxide production were measured by indirect calorimetry in 40-week-old female mice as described (40).
In Vivo Triglyceride Secretion—Triglyceride secretion was measured as the increase in circulating triglyceride after inhibiting triglyceride clearance with WR1339 (41, 42). Mice were fed a fat-free diet (Frosted Flakes; Kellogg Company, Battle Creek, MI) for 4 h and then anesthetized with avertin (43). WR1339 (T-8761; Sigma; 100 µl of a 1:10 dilution in phosphate-buffered saline) was injected via tail vein, and blood samples were withdrawn at 0, 30, 60, and 120 min. Plasma triglycerides were measured colorimetrically. Data are expressed as mg triglyceride/kg body weight/h, assuming plasma volume is 3.5% of body weight.
Triglyceride Clearance—Triglyceride clearance was measured
in mice fasted for 4 h and then gavaged (at 
1200) with 400 µl of
peanut oil. Blood was taken hourly for 6 h from the tail vein, and plasma
triglycerides were measured colorimetrically.
Statistical Analysis—Values are reported as mean ± S.E. Statistical significance was determined using two-tailed t test or analysis of variance, as appropriate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Gene—We
generated liver-specific PPAR knockout mice by crossing
PPAR-floxed mice (33)
with Alb-Cre mice (34). The
wild type Alb-Cre PPARfl/fl (hereafter WT LKO)
offspring were then crossed with lipoatrophic AZIP
PPARfl/fl mice to produce AZIP mice lacking
PPAR in liver (AZIP LKO). Alb-Cre is expressed exclusively in
hepatocytes of the postpartum liver
(34), providing maximum
recombination by six weeks of age
(44). Therefore, we analyzed
mice at 11 weeks of age and older. Southern blot analysis demonstrated that
exon 2 of the PPAR gene, encoding the DNA binding domain, was indeed
deleted in both WT LKO and AZIP LKO livers (90 and 70%, respectively; see
Fig. 1a). The
remaining unrecombined floxed allele is likely to be from non-parenchymal
cells, which do not express the Alb-Cre
(45). To confirm the
efficiency of targeting, we measured liver PPAR mRNA levels. As
reported (24,
26), the AZIP livers expressed
six times more PPAR mRNA than the WT livers
(Fig. 1b). Northern
blot analysis revealed a 2- and 3-fold reduction of PPAR mRNA levels in
the WT LKO and AZIP LKO mice, respectively. RNase protection assay
demonstrated that in the AZIP LKO liver most of the residual PPAR mRNA
is a truncated transcript from the recombined null allele
(Fig. 1c). Similarly,
no detectible wild type PPAR mRNA was found in the WT LKO liver
(36). Truncated PPAR
transcripts carry a new stop codon, which is expected to produce 10- and
13-kDa translation products (PPAR1 or PPAR2, respectively),
which appear to be unstable
(33). Western blot analysis
revealed that the AZIP liver expressed predominantly PPAR2 isoform
(Fig. 1d), similarly
to the steatotic liver of the ob/ob mouse
(32). In the AZIP LKO mice,
PPAR2 protein levels were dramatically reduced; PPAR1 levels
were also affected; however, the amount of the residual PPAR1 protein
was comparable with that in the WT mice. In contrast to lipoatrophic mice, WT
liver contained predominantly PPAR1 band, which was not effected by the
knockout. Taken together, these data suggest that in the wild type liver most
of the detectable PPAR protein is produced by non-parenchymal cells,
whereas in the steatotic liver of the AZIP mice hepatocytes is the major
source of PPAR protein.
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Loss of Liver PPAR Improves Hepatic Steatosis in the
AZIP Mice—Inactivation of liver PPAR did not cause a major
phenotype in 11-week-old female non-lipoatrophic mice, with no effect on body
weight, liver weight, liver triglyceride, in vivo triglyceride
secretion, or serum glucose, insulin, triglyceride, or free fatty acid levels
(Fig. 2 and not shown). In
contrast, inactivation of liver PPAR significantly altered the AZIP
phenotype. The increased body and liver weights of the AZIP mice were reduced
by liver PPAR ablation (Fig. 2,
a and b) without effect on other organs (not
shown). The reduction in liver weight correlated with a decrease in liver
triglyceride content (30%), in vivo triglyceride secretion rate
(60%), and mRNA levels of several genes involved in fatty acid synthesis (up
to 40%), suggesting a multifaceted role for liver PPAR in triglyceride
homeostasis (see Fig. 2, c and
d and Table
I). Male AZIP LKO mice also had smaller, less steatotic livers
compared with the AZIP control mice, but this difference became significant
only at the age of 20 weeks (not shown). The more profound phenotype in
females was not because of the alteration of estradiol levels, because they
were comparable in WT, WT LKO, AZIP, and AZIP LKO mice (24.6 ± 1.6,
22.7 ± 2.7, 22.9 ± 1.3, and 26.7 ± 3.4 pg/ml,
respectively; seven-nine mice/group).
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Inactivation of Liver PPAR Impairs Triglyceride
Clearance—The reduced triglyceride secretion in the AZIP LKO mice
might be expected to lower circulating triglyceride levels. However,
disruption of liver PPAR actually increased the already elevated serum
triglyceride levels in the AZIP mice (Fig.
2e), suggesting a defect in triglyceride clearance. To
directly test this hypothesis, we measured serum triglyceride levels after an
oral lipid load (Fig.
2f). In WT mice, serum triglycerides peaked at 140 mg/dl
at 2 h and rapidly returned to baseline levels. In AZIP mice, serum
triglycerides rose to 874 mg/dl at 3 h, demonstrating impairment of
triglyceride clearance in the absence of adipose tissue. The AZIP LKO mice
were even more fat intolerant, with serum triglycerides reaching 1099 mg/dl at
4 h and remaining elevated 6 h after the lipid dose. Thus, disruption of liver
PPAR exacerbates the hyperlipidemia of AZIP mice by impairment of
triglyceride clearance.
Interestingly, whereas 11-week-old WT LKO mice did not have elevated serum
triglyceride levels, they did show a small delay in triglyceride clearance
(Fig. 2f). The area
under the curve above the baseline was 256% higher in the WT LKO than in the
WT mice (p = 0.008). These data suggest that liver PPAR
contributes to triglyceride clearance even in wild type, non-lipoatrophic
mice.
Loss of Liver PPAR Exacerbates Muscle Insulin
Resistance in the AZIP Mice—Because high circulating triglyceride
levels often correlate with increased muscle triglyceride content and insulin
resistance, we measured triglyceride levels in skeletal muscle. Loss of liver
PPAR caused a 33% increase in muscle triglyceride levels in the AZIP
mice (from 2.7 ± 0.3 to 3.6 ± 0.4 µmol/g; p = 0.09).
We did not detect significant differences in non-fasting serum glucose or
insulin levels in the AZIP and AZIP LKO mice; in both strains, serum glucose
levels were four times higher, and insulin levels were 100 times higher than
the WT controls (Fig. 2, g and
h). However, fasting blood glucose was significantly
higher in the AZIP LKO compared with the AZIP mice (257 ± 30 and 182
± 28 mg/dl, respectively; p = 0.04), suggesting that loss of
liver PPAR makes lipoatrophic mice even more diabetic.
To evaluate glucose homeostasis, we performed euglycemichyperinsulinemic
clamps (Fig. 2, i and
j). In WT mice, loss of PPAR in liver had no
measurable effect on any of the clamp parameters. In AZIP mice, endogenous
glucose production was less suppressed by insulin than in the WT controls (58
versus 93%), indicating hepatic insulin resistance. Loss of liver
PPAR did not cause significant changes in endogenous glucose production
or its suppression by insulin, suggesting that under the conditions used liver
insulin resistance was comparable in the AZIP and AZIP LKO mice
(Fig. 2j). In
contrast, inactivation of PPAR in liver did affect muscle insulin
sensitivity. In AZIP mice, muscle glucose uptake rate was 49% of the WT
controls but only 24% in the AZIP LKO mice (p = 0.04), indicating
that loss of liver PPAR worsens muscle insulin resistance in
lipoatrophic mice (Fig.
2i).
Increased Body Bat Mass in Mice Lacking Liver
PPAR—At the age of 11 weeks WT LKO did not have a
major phenotype, showing only a small impairment of triglyceride clearance
(Fig. 2). To study metabolic
consequences of this defect, we analyzed 40-week-old WT and WT LKO mice
(Fig. 3). Loss of liver
PPAR had no significant effect on body weight, liver weight, food
intake, or metabolic rate (Fig. 3,
a and b, and not shown). However, the WT LKO
mice had more adipose tissue (Fig.
3c). The small delay in triglyceride clearance observed
in 11-week-old WT LKO mice became more apparent with age, coinciding with
persistent hyperlipidemia (Fig. 3,
d and e). At the age of 40 weeks, the WT LKO
mice also had elevated serum glucose and insulin levels, indicating insulin
resistance (Fig. 3, f and
g). Increased adiposity in the WT LKO mice correlated
with a 2.3-fold increase in serum leptin and a 1.9-fold decrease in serum
adiponectin levels (not shown). Thus, in non-lipoatrophic mice, loss of liver
PPAR caused fat intolerance and with aging, hyperlipidemia, obesity,
and insulin resistance.
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To see whether an obesity phenotype can be also induced in younger mice, we
compared the effects of the AIN-93G (lipogenic, simple carbohydrate diet) and
NIH-07 (standard chow) diets on adipose tissue mass in WT and WT LKO mice
(Fig. 3h). WT and WT
LKO mice fed the NIH-07 diet and WT mice on the AIN-93G diet had a similar
amount of WAT and BAT. In contrast, after only five weeks on the AIN-93G diet
WT LKO mice had WAT depots two times larger than the controls. No difference
in body weight was detected because of the range of body weights in each
experimental group. However, within the same body mass range, the WT LKO mice
on the AIN-93G diet had the largest amount of WAT (not shown). Serum leptin
levels correlated with the WAT mass (not shown). Thus, the AIN-93G diet
unmasked a predisposition to obesity in liver-specific PPAR knockout
mice. Taken together, these data demonstrate that hepatic PPAR plays a
role in regulation of triglyceride partitioning between liver and other
tissues, contributing to regulation of body fat mass and glucose homeostasis
in non-lipoatrophic mice.
Liver PPAR Regulates Genes Involved in Lipid
Metabolism—To assess the direct transcriptional effects of liver
PPAR ablation, we measured target mRNA levels
(Table I). On a chow diet AZIP
mice have increased levels of lipogenic mRNA, including PPAR, sterol
response element-binding protein 1 (SREBP-1), fatty acid synthase (FAS),
acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD1)
(24). Liver PPAR
ablation reduced the FAS, ACC, and SCD1 mRNA levels (26, 33, and 41%,
respectively). Expression of SREBP-1, a master regulator of lipogenesis, was
not affected, suggesting that PPAR acts either downstream of or
parallel to SREBP-1.
AZIP mice have 10-fold elevated levels of liver CD36 mRNA, which encodes a
lipoprotein receptor/fatty acid transporter. Ablation of liver PPAR
reduced CD36 mRNA levels by only 16 and 39% in the AZIP and WT mice,
respectively. Low density lipoprotein receptor (LDLR) mRNA levels were reduced
by 45%, but only in the AZIP mice. In contrast, LDLR-related protein, hepatic
lipase, and endothelial lipase mRNA levels were increased in both WT LKO and
the AZIP LKO mice. Lipoprotein lipase mRNA and very low density lipoprotein
receptor mRNA were barely detectable in all strains and did not appear to be
affected by either AZIP transgene or PPAR null allele (not shown).
Levels of mRNAs encoding other lipid metabolism genes (acyl-CoA oxidase,
carnitine palmitoyl transferase, apolipoprotein B, microsomal triglyceride
transfer protein, fatty acid-binding protein) were not significantly affected
by ablation of liver PPAR.
Steatotic AZIP livers also have elevated levels of adipose
differentiation-related protein (ADRP) and adipose fatty acid-binding protein
(aP2) mRNA (3.6 and 2.4, respectively). ADRP is a protein covering lipid
droplets in non-adipose cells
(47), whereas aP2 is a
cytosolic fatty acid-binding protein, normally expressed in adipocytes
(48). The levels of both ADRP
and aP2 mRNA were reduced in the AZIP LKO mice, suggesting that liver
PPAR also contributes to regulation of lipid transport and storage
within the hepatocytes.
Liver PPAR Is Essential for Rosiglitazone Effects in
the AZIP Mice—To assess the contribution of liver PPAR to
the antidiabetic effects of rosiglitazone, a PPAR agonist, we first
analyzed rosiglitazone effects in the lipoatrophic AZIP mice
(Fig. 4). As noted previously
(24), rosiglitazone increased
liver weight and hepatic triglycerides and lowered serum triglycerides in the
AZIP mice (Fig. 4,
a–d). In addition, it elevated serum
alanine aminotransferase and aspartate aminotransferase levels, indicating
liver damage (not shown). All these effects were abolished in the AZIP LKO
mice, demonstrating that they are mediated by liver PPAR
expression.
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Previously, rosiglitazone treatment did not improve serum glucose and
insulin levels in AZIP mice with a pure FVB/N genetic background
(24). Here, using mice of
mixed genetic background, we observed partial improvement of the diabetes,
with a 2-fold decrease in serum glucose and 45% increase in serum insulin
(Fig. 4, e and
f; p = 0.001 and p = 0.09,
respectively). Loss of liver PPAR abolished the beneficial effect of
rosiglitazone in AZIP mice.
Taken together, these data demonstrate that, in the absence of adipose
tissue, liver PPAR mediates triglyceride lowering and contributes to
the hypoglycemic effects of rosiglitazone. Interestingly, rosiglitazone caused
a significant decrease in serum-free fatty acid levels in both AZIP and AZIP
LKO mice, suggesting that tissues other that liver and fat contribute to this
effect (Fig. 4g).
Rosiglitazone treatment had no effect on liver mRNA levels in WT mice (not
shown). In contrast, in the AZIP mice, rosiglitazone treatment increased mRNA
levels of ACC, FAS, SCD1, acyl-CoA oxidase, CD36, and aP2
(Fig. 4h and not
shown). All effects of rosiglitazone were abolished in the AZIP LKO mice,
suggesting a role for liver PPAR in regulation of genes involved in
lipogenesis, fatty acid transport, storage, and oxidation.
Liver PPAR Is Not Required for Rosiglitazone Effects in
Non-lipoatrophic Mice—We next asked whether liver PPAR is
essential for the antidiabetic effects of rosiglitazone in a typical mouse,
with adipose tissue. The WT and WT LKO mice were made insulin-resistant with a
high fat diet (Fig. 5). After
three months of high fat feeding, the WT and WT LKO mice had comparably
elevated serum glucose and insulin levels, suggesting a similar degree of
insulin resistance (Fig. 5, a and
b). Serum triglycerides tended to be slightly higher in
mice without liver PPAR (p = 0.2), whereas free fatty acids
were not different (Fig. 5, c and
d). Both WT and WT LKO mice responded to rosiglitazone by
lowering serum glucose, insulin, triglyceride, and free fatty acid levels and
by improving glucose and fat clearance
(Fig. 5,
a–d, and not shown). Thus, in
non-lipoatrophic mice liver PPAR is not required for the antidiabetic
and hypolipidemic effects of rosiglitazone.
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In both WT and WT LKO mice, rosiglitazone increased adipose tissue mass and reduced liver size and steatosis (Fig. 5, e and f, and not shown). These observations are consistent with the hypothesis that the primary effect was increased triglyceride storage in adipose tissue, with a secondary reduction in liver triglyceride levels. Rosiglitazone treatment increased serum adiponectin levels in both WT and WT LKO mice (2.9- and 3.6-fold, respectively), whereas the increase in serum leptin was significant only in the WT LKO mice.
Thus, rosiglitazone had very similar effects in the WT and WT LKO mice,
demonstrating that in non-lipoatrophic mice liver PPAR contributes
relatively little to the beneficial effects of rosiglitazone. These data
suggest that adipose tissue is the major site of thiazolidinedione action in
typical mice with adipose tissue.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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contributes to triglyceride
homeostasis, regulating both triglyceride clearance and the lipogenic program.
A role for hepatic PPAR had not been appreciated previously, possibly
because of its low abundance in non-steatotic liver. Inactivation of liver
PPAR reduced hepatic steatosis in both lipoatrophic AZIP mice and mice
with diet-induced obesity. In the AZIP mouse, this correlated with reduced
triglyceride output from the liver and lower expression of FAS, ACC, and SCD1,
suggesting that in lipoatrophic mice liver PPAR modulates hepatic
steatosis by regulating lipogenic genes.
Interestingly, reduction of hepatic steatosis did not improve the metabolic
phenotype of the AZIP mice, actually worsening the triglyceride clearance,
hyperlipidemia, and muscle insulin resistance. Inactivation of PPAR in
liver of the ob/ob mice caused similar effects
(36). These data suggest that
hepatic steatosis develops not only as a result of increased triglyceride
production but is also because of enhanced lipid uptake by liver. The degree
of hepatic steatosis and efficiency of triglyceride clearance in the AZIP
mouse depends on genetic background
(27). FVB/N AZIP mice have
high circulating triglyceride and fatty liver, whereas C57BL/6J AZIP mice have
low circulating triglyceride but much more steatotic liver. Thus, it is
possible that some the effects of liver PPAR deficiency might be
influenced by background genotype. Further understanding of the mechanisms
contributing to hepatic steatosis will come from identifying the relevant
modifier genes.
Improvement in hepatic steatosis has also been achieved in the
ob/ob mouse by inactivation of SCD1
(49) or of SREBP-1
(50). SREBP-1 proteins are
transcription factors that regulate multiple lipogenic genes, including FAS,
ACC, and SCD1 (51). Similar to
PPAR, SREBP-1 mRNA levels are elevated in steatotic livers
(24,
52). Transgenic mice
overexpressing the constitutively active form of SREBP-1 proteins have fatty
liver, supporting a role for SREBP-1 in development of the steatosis
(53,
54). Because liver-specific
disruption of PPAR did not affect SPEBP-1 levels, we conclude that
PPAR directly contributes to hepatic steatosis, acting either
downstream of or in parallel with SREBP-1.
Interestingly, whereas liver-specific disruption of PPAR and SREBP-1
both reduced lipogenesis and triglyceride content in liver
(50), elevation of circulating
triglyceride occurred only when PPAR was missing. These data suggest
that the role of hepatic PPAR in regulation of lipid uptake by liver is
not shared with SREBP-1. The target genes and molecular mechanisms by which
liver PPAR mediates lipid clearance remain largely unknown. Lack of
liver PPAR caused a 2-fold decreased in LDLR mRNA levels in the mice.
This is in agreement with a recently published observation
(55) that adenoviral
overexpression of PPAR1 increased LDLR mRNA levels in livers of the
PPAR
null mice. Thus, LDL receptor might be one of the mediators of
PPAR-regulated lipid clearance by liver. CD36, a lipoprotein
receptor/fatty acid transporter, is another likely candidate. PPAR
agonists increase CD36 mRNA levels in multiple tissues, including adipose,
muscle, liver, and macrophages
(30,
33,
56–59).
Loss of function mutation of CD36 gene significantly blunted hypolipidemic and
insulin sensitizing effects of pioglitazone in spontaneously hypertensive rats
(60). However, inactivation of
CD36 gene in mice reduced fatty acid uptake into adipose tissue, skeletal
muscle, and heart but had no effect on liver
(61). Thus, the role of CD36
in liver remains unclear.
Perhaps the most surprising observation is that liver PPAR ablation
caused increased adipose tissue mass and insulin resistance in
non-lipoatrophic mice. This is in marked contrast to the requirement for
PPAR for adipose tissue development
(16,
17,
62) and the observation that
some patients with lipodystrophy have PPAR mutations
(63–65).
Moreover, mice heterozygous for a PPAR null allele are protected from
high fat diet-induced obesity and have increased insulin sensitivity
(17,
66). A mechanistic explanation
for the obesity caused by liver-specific PPAR deficiency follows from
our observation that PPAR ablation reduces the ability of the liver to
take up triglyceride. This was the sole phenotype detected in young
non-lipoatrophic mice, supporting the idea that it has an etiologic role in
the later effects. Because of reduced hepatic uptake, more triglycerides are
stored in other sites such as adipose tissue and possibly muscle, thereby
causing insulin resistance. The obesity of the liver PPAR ablated mice
is dependent on dietary exposure and animal age, features that it shares with
the common forms of human obesity.
It has been well established that PPAR agonists, including TZDs, act
as insulin sensitizers in human and animal models, but the identity and
relative importance of target tissues are still debated
(5). The prevailing hypothesis
proposes that TZDs act directly on adipose tissue, with secondary effects in
skeletal muscle and liver. Improvement of insulin sensitivity in muscle,
liver, and adipose tissue
(7–9)
is accompanied by increase of peripheral adiposity
(21,
67,
68). Although insulin
signaling in muscle and adipose tissue could be stimulated by PPAR
agonists within a day, improvement of insulin action in liver requires longer
treatment (15) and higher
doses (8), consistent with an
indirect effect. When adipose tissue was present, ablation of liver
PPAR did not influence the effectiveness of rosiglitazone treatment.
Similarly, muscle-specific PPAR knockout mice also showed intact
response to rosiglitazone treatment
(69). Taken together, these
data demonstrate that in a typical mouse neither liver PPAR nor muscle
PPAR are essential for the beneficial effects of rosiglitazone,
suggesting that TZDs normally act via adipose tissue.
In contrast, in the absence of adipose tissue, liver becomes the major site
of TZD action. Loss of liver PPAR abolished most of the effects of
rosiglitazone in the AZIP mouse, including a reduction in circulating glucose
and triglyceride levels, and an increase in hepatic triglyceride content.
Thus, possible mechanisms by which TZDs improve metabolic abnormalities in
lipoatrophic mice may include redistribution of triglyceride away from the
circulating compartment into the liver and increased fat oxidation. TZDs
decrease respiratory quotient in lipoatrophic mice
(24) and humans
(21), but which tissue is
responsible for lipid oxidation is unclear. Liver is likely to contribute,
because rosiglitazone induced hepatic acyl-CoA oxidase mRNA in a
PPAR-dependent way. Our observation that liver PPAR is not
required for reduction of free fatty acids by rosiglitazone suggests that
tissues other that liver and adipose may also contribute to fat oxidation.
It is important to note that efficacy of TZD therapy in lipoatrophic mice
depends on background genotype. In FVB/N AZIP mice, rosiglitazone lowered
circulating triglyceride but had no effect on diabetes
(24), presumably because of
the opposite effects of muscle and liver
(25). In contrast, C57BL/6J
AZIP mice improved both hyperlipidemia and diabetes,2 similar to a
mouse model of late onset lipoatrophy
(22). In this study, in the
AZIP mice of mixed genetic background, rosiglitazone lowered hyperglycemia but
increased insulin levels, probably because of improvement of 
-cell
function. Which modifier genes determine the way lipoatrophic mice respond to
TZD therapy is not known. Given the range of responses even within the same
transgenic mouse model, it might be hard to predict the outcome of TZD therapy
in human lipoatrophy, a heterogeneous group of syndromes
(70,
71).
An important unanswered question concerns the similarity between mouse and
human hepatic steatosis. We are unaware of any published studies quantitating
PPAR levels in steatotic liver from humans. In typical type 2 diabetic
patients rosiglitazone reduced hepatic triglyceride
(9). Troglitazone treatment was
also efficacious in reducing liver size in a heterogeneous group of
lipodystrophic patients, correlating with an increase in adipose mass
(21). It is possible that
worsening of hepatic steatosis with TZD treatment is a species-specific
manifestation, similar to the hepatomegaly caused by PPAR agonists in
rodents (46,
72). However, more studies are
needed to evaluate the role of TZD therapy in patients with complete
lipoatrophy.
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FOOTNOTES |
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|| Present address: Merck Research Laboratories, Rahway, New Jersey 07065.
To whom correspondence should be addressed: Diabetes Branch, NIDDK, NIH, Bldg.
10, Rm. 8N-250, 10 Center Dr., Bethesda, MD 20892-1770. Tel.: 301-435-5370;
Fax: 301-402-5788; E-mail:
oksanag{at}bdg10.niddk.nih.gov.
1 The abbreviations used are: PPAR, peroxisome proliferator-activated
receptor ; TZD, thiazolidinediones; ACC, acetyl-CoA carboxylase; FAS,
fatty acid synthase; SCD1, stearoyl-CoA desaturase; SREBP-1, sterol response
element-binding protein; LDLR, low density lipoprotein receptor; WAT, white
adipose tissue; BAT, brown adipose tissue.
2 O. Gavrilova and M. L. Reitman, unpublished observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, WT LKO; 
, AZIP; 
, AZIP
LKO; *, significant effect of liver-specific PPAR
