1 Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia; 2 Rheoscience, DK-2100 Copenhagen; and 3 Novo Nordisk, DK-2760 Måløv, Denmark
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ABSTRACT |
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Peroxisome proliferator-activated receptor
(PPAR) and PPAR
agonists lower lipid accumulation in muscle and
liver by different mechanisms. We investigated whether benefits could
be achieved on insulin sensitivity and lipid metabolism by the dual
PPAR
/
agonist ragaglitazar in high fat-fed rats. Ragaglitazar
completely eliminated high-fat feeding-induced liver triglyceride
accumulation and visceral adiposity, like the PPAR
agonist
Wy-14643 but without causing hepatomegaly. In contrast, the
PPAR
agonist rosiglitazone only slightly lessened liver triglyceride
without affecting visceral adiposity. Compared with rosiglitazone or
Wy-14643, ragaglitazar showed a much greater effect (79%, P
< 0.05) to enhance insulin's suppression of hepatic glucose
output. Whereas all three PPAR agonists lowered plasma triglyceride
levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and
rosiglitazone had greater insulin-sensitizing action in muscle than
Wy-14643, associated with a threefold increase in plasma adiponectin
levels. There was a significant correlation of lipid content and
insulin action in liver and particularly muscle with adiponectin levels
(P < 0.01). We conclude that the PPAR
/
agonist
ragaglitazar has a therapeutic potential for insulin-resistant states
as a PPAR
ligand, with possible involvement of adiponectin.
Additionally, it can counteract fatty liver, hepatic insulin
resistance, and visceral adiposity generally associated with PPAR
activation, but without hepatomegaly.
peroxisome proliferator-activated receptor subtypes; adipokines; tissue lipids; insulin resistance
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INTRODUCTION |
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INSULIN RESISTANCE IS A FUNDAMENTAL DEFECT of type 2 diabetes. It is central to the insulin resistance syndrome characterized by hyperglycemia, hyperinsulinemia, dyslipidemia, obesity, and hypertension. There is increasing evidence to suggest that central adiposity (8) and fatty liver (30) are also important features of this syndrome. It is clear that a lipid accumulation in muscle and liver can cause the development of insulin resistance (4, 26, 41). Realization of the role of excess lipids in the pathogenesis of insulin resistance has led to various strategies to improve insulin sensitivity by lowering excess lipid accumulation in liver and muscle (32).
Peroxisome proliferator-activated receptors (PPAR) are nuclear
transcription factors that include three subtypes: ,
(
), and
. PPAR
agonists, such as thiazolidinediones (TZDs), improve insulin action in peripheral tissues, attenuate hyperinsulinemia, and
lower circulating levels of lipids. PPAR
agonists are highly expressed in adipocytes and mediate their differentiation. A major mechanism of the insulin-sensitizing action of PPAR
agonists results
from the lowering of lipid supply to muscle and liver through a
"lipid-stealing" by PPAR
-mediated effects in adipose tissue
(22, 40). However, some concerns also arise over increases in fat mass and body weight associated with adipocyte proliferation (10, 14, 48).
Unlike PPAR, PPAR
mediates expression of genes regulating lipid
oxidation (22). PPAR
agonists, such as fibrates, have been used to treat hypertriglyceridemia and reduce cardiovascular risk
(24). A number of studies in insulin-resistant animal
models have shown marked decreases in liver triglyceride content and adiposity by PPAR
agonists (6, 10, 16, 37). Using a euglycemic hyperinsulemic clamp technique, our recent studies have
clearly shown that, while exerting these effects, the PPAR
agonist
Wy-14643 can also lessen insulin resistance and muscle lipid
accumulation in high-fat-fed rats. However, compared with a PPAR
agonist, the improvement of muscle insulin sensitivity by Wy-14643 was
much smaller for similar reductions in muscle lipids (49).
There are also opposite reports that PPAR
deficiency may even
protect insulin sensitivity (43). These data suggest that
other factors, such as PPAR
-responsive adipokines, may be involved
in the insulin-sensitizing action of PPAR
agonists. Currently, there
is enormous interest in the potential of combined PPAR
/
agonists
for enhancement of insulin action together with reductions in tissue
lipid accumulation and central adiposity (22, 32).
Although possible benefits of combined PPAR/
agonists have been
suggested in earlier reports in genetically obese insulin-resistant models (13, 29, 34), their effects on insulin action in liver and muscle in relation to adipokines in a nutritional model of
insulin resistance have not been demonstrated. Thus the aim of
the present study was to investigate whether a dual PPAR
/
compound would exert additional beneficial effects on liver steatosis, adiposity, and insulin sensitivity compared with selective activation of PPAR
or PPAR
. Lipid metabolism was examined in parallel, with
particular focus on liver and muscle lipid content and central adiposity, and their relationship with leptin and adiponectin was investigated.
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RESEARCH DESIGN AND METHODS |
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Study design.
All experimental procedures were approved by the Animal Experimentation
Ethics Committee (Garvan Institute/St Vincent's Hospital, Sydney) and
were in accordance with the National Health and Medical Research
Council of Australia Guidelines on Animal Experimentation. Ragaglitazar
[also known as NNC 61-0029 or DRF()2725] is a
2-ethoxy-3-[4-(2-phenoxazin-10-yl-ethoxy)-phenyl]-propionic acid,
structurally different from other PPAR
and PPAR
ligands including
TZDs, N-(2-benzoylphenyl)-L-tyrosine
derivatives,
-alkoxy-
-phenylpropanoic acids, or fibrates
(38). It is a full agonist of both PPAR
and PPAR
. In
vitro transactivation assay showed that the maximal activation of
ragaglitazar on PPAR
was 117% (EC50: 0.57 µM)
compared with rosiglitazone (100% with EC50 of 0.16 µM).
For the stimulation of PPAR
, ragaglitazar produces 97% (EC: 3.2 µM) of the maximal response induced by Wy-14643 (100% and
EC50: 12.6 µM) (38).
Assessment of PPAR and PPAR
activation in vivo.
The mRNA expression levels of liver peroxisome bifunctional enzyme
(PBE) and acyl-CoA oxidase (ACO) were determined to assess activation
of PPAR
. Total RNA was extracted from ~100 mg of liver using
Tri-Reagent (Sigma, St. Louis, MO). Real-time LightCycler RT-PCR was
employed to quantify ACO and PBE mRNA levels by use of the LightCycler
FastStart DNA Master SYBR Green 1 kit (Roche Molecular Biochemicals,
Mannheim, Germany) in a similar way as recently described
(44). The primer combinations were: 5'-GAT TCA AGA CAA AGC
CGT CCA AG-3' and 5'-TCC ACC AGA GCA ACA GCA TTG-3' for ACO, 5'-CGC ACT
TGA CAC ATT CCA GCT-3' and 5'- GGG CTA CTC ATC TAT GTT GTC CAC-3' for
PBE. Expression levels were normalized to the expression levels of
cyclophylin. Expression levels of phosphoenolpyruvate
carboxykinase (PEPCK) mRNA in retroperitoneal white adipose tissue, an
indicator of PPAR
activation in adipocytes (42, 46),
were determined by semiquantitative RT-PCR, as described previously
(20). Two primer sets (5 pmol of each oligo) were used
simultaneously, one specific for the gene in question and the other
specific for an internal standard (elongation factor 1a). For PEPCK,
forward primer 5'-ACAGGATGAGGAACCGTGC-3' and reverse primer
5'-CCTTGCCCTTATGCTCTGC-3' were used. For adipocyte protein-2 (aP2), forward primer 5'-AAGACAGCTCCTCCTCGAAGGTT-3' and reverse primer 5'-TGACCAAATCCCCATTTACGC-3' were employed. PCR products were
separated on a 6% polyacrylamide-7 M urea gel and analyzed using a
phosphorimager and ImageQuant (Molecular Dynamics). Results are
expressed as gene per internal standard. Activation of PPAR
in vivo
was also assessed by plasma concentration of adiponectin as a marker
(11) with a commercial radioimmunoassay kit (Linco, St.
Louis, MO).
Experimental protocol.
A week before the study, the left carotid artery and right jugular vein
of rats were cannulated, and cannulas were exteriorized in the back of
the neck under ketamine-xylazine (90:10 mg/kg ip) anesthesia. Rats were
handled daily to minimize stress. On the study day between 0900 and
1000, after animals had been fasted for 5 h, the cannulas were
connected to infusion apparatus (via the carotid line) and a
blood-sampling syringe (via the jugular line). The sampling line was
filled with sodium citrate (20.6 mM) to prevent clotting. After a
period (50-60 min) of settling, two basal blood samples were
collected for measurement of plasma parameters, as described previously
(49). Rats were allowed to settle for ~1-2 h and
then euthanized by pentobarbital sodium (~180 mg/kg). Muscles (red
and white quadriceps) and heart were immediately freeze-clamped with
aluminum tongs precooled in liquid nitrogen. Visceral (epididymal and
retroperitoneal) fat and liver were weighed before being frozen in
liquid nitrogen. Tissues were stored at 80°C for subsequent
analyses for basal metabolites. The hyperinsulinemic euglycemic clamp
was performed at an insulin infusion rate of 0.25 U · kg
1 · h
1
to elevate circulating insulin levels to approximately a half-maximal physiological concentration (25), with glucose infused at
variable rates (GIR) to maintain euglycemia. After plasma glucose
levels reached the steady state, a bolus of
2-deoxy-D-[2,6-3H]glucose and
D-[U-14C]glucose was injected (iv) to
determine glucose disappearance rate (Rd) and hepatic
glucose output rate (HGO). After the clamp, tissues of interest were
freeze-clamped to determine glucose uptake (Rg'), glycogen,
and lipid synthesis. Details of these measurements have been described
previously (25, 41, 49).
Metabolite measurements. Glucose concentrations were determined using a glucose analyzer (YSI 2300, Yellow Springs, OH). Plasma free fatty acid (FFA) and triglyceride levels were determined using enzymatic colorimetric methods by commercial kits (Sigma). Plasma insulin and leptin levels were determined with radioimmunoassay kits (Linco). Tissue triglycerides were extracted by the method of Bligh and Dyer (7), and content was determined by a Peridochrom Triglyceride GPO-PAP kit (Boehringer Mannheim, Mannheim, Germany). Tissue content of glycogen (9) and long-chain acyl (LCA)-CoAs (2) was determined as previously described.
Statistical analyses. All results are presented as means ± SE. A one-way analysis of variance (ANOVA) was used to assess the statistical significance across all groups. When tested as significant, a post hoc (Fisher paired least significant difference) test was used to establish differences between groups. All data were processed in Excel 5.0, and statistical analyses were performed using the Statview SE+Graphic Program (Abacus Concepts-Brain Power).
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RESULTS |
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Activation of PPAR and PPAR
by ragaglitazar.
Activation of PPAR
is known to upregulate the expression of liver
PBE, as shown in the HF-WY group (Fig.
1A). Although to a lesser
extent, ragaglitazar treatment also led to a dramatic increase
(3.1-fold) in PBE mRNA expression level in liver. Similarly, liver ACO
mRNA levels were significantly increased (P < 0.01) in both
HF-WY (35.8 ± 4.2) and HF-Raga (21.5 ± 5.9) groups but not
in the HF-Rosi group (8.7 ± 1.4) compared with the CH-Con (8.9 ± 0.8) or HF-Con (7.3 ± 1.4) group. The expression
levels of PEPCK mRNA in white adipose tissue were increased more than twofold in the HF-Raga group (Fig. 1B) in a pattern similar
to the HF-Rosi group. These data indicated that both PPAR
and
PPAR
were activated by ragaglitazar, an observation that was also
supported by a substantial (60%) reduction in plasma leptin levels in
all treated groups (Fig. 1C). There was a positive
correlation between plasma leptin concentrations and visceral fat
weight among all five groups (r = 0.75, P < 0.001, data not shown). Recently, elevated plasma
adiponectin concentration has been suggested as a specific indicator of
PPAR
but not PPAR
stimulation (11). Figure
1D shows that plasma adiponectin levels were raised
approximately threefold in both the HF-Raga group and the HF-Rosi
group. In addition, aP2 mRNA expression levels in retroperitoneal white adipose tissue were significantly higher in the HF-Raga group compared
with the HF-Con or CH-Con group [2.9 ± 0.16 vs. 1.5 ± 0.14 or 1.2 ± 0.04 arbitrary units (AU), respectively,
P < 0.01].
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Effects on basal lipid and glucose metabolism.
Table 1 shows basal metabolic parameters
in all groups. Except for a small decrease in body weight gain in the
HF-WY group (15% vs. HF-Con), there were no differences in weight gain
among the groups. Compared with CH-Con rats, visceral fat mass was
increased by 55% in the HF-Con group. This increase was prevented in
HF-WY and HF-Raga groups but not in HF-Rosi rats. Thus the visceral adiposity of both the HF-Raga and the HF-WY groups was significantly (P < 0.01) less (32 and 21%, respectively) than that
of the HF-Rosi group. There was severe hepatomegaly in HF-WY (58%
increase in liver weight vs. HF-Con), whereas neither ragaglitazar nor
rosiglitazone altered liver weight. Associated with a small reduction
in plasma glucose levels, elevated plasma insulin levels (34% vs.
CH-Con) in HF-Con were abolished by all drugs. A similar pattern of
decreases in plasma triglyceride levels by the three agonists
(16-28%) was found despite their relatively lower levels (vs.
CH-Con) in HF-Con, an adaptive response due to increased lipid
clearance (18). There was no significant effect on FFA
levels in any of the treated groups.
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Whole body insulin sensitivity and lipids during hyperinsulinemic
euglycemic clamp.
High-fat feeding induced insulin resistance, as evidenced by 50% of
the clamp GIR (Fig. 3A). The
underlying cause of insulin resistance was a combination of inhibited
insulin-mediated glucose disposal rate (Rd) in peripheral
tissues and impaired suppression of HGO. Compared with HF-Con, all
three PPAR agonists increased the GIR (HF-Rosi: 51%, HF-WY: 39%, and
HF-Raga: 66%) and Rd (15-22%). Increases in GIR and
Rd values were not significantly different among the
treated groups. Ragaglitazar substantially enhanced insulin's
suppressibility of HGO by 79%, whereas the apparent reduction of HGO
induced by rosiglitazone or Wy-14643 did not reach statistical
significance.
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Insulin action in liver and associated changes in lipids.
As shown in Fig. 4A, the
inhibition of insulin-stimulated glycogen synthesis by high-fat feeding
was completely overcome by rosiglitazone and ragaglitazar, but not by
Wy-14643. Although insulin-mediated de novo triglyceride synthesis from
glucose remained largely inhibited in all high-fat-fed groups compared
with CH-Con, there was a 41% increase in HF-Raga that was also
significantly higher than in HF-Rosi or HF-WY (P < 0.05, Fig. 4B). As in the basal state, triglyceride content
was reduced by all three drugs after the clamp (Table 2). However, its
reduction in rosiglitazone-treated rats was relatively small (24%)
and significantly less (P < 0.01) than the
reduction in liver triglyceride content in groups treated with Wy-14643
(45%) and ragaglitazar (88%). Indeed, the effect of ragaglitazar was
even greater than that of Wy-14643 (P < 0.05) and
reverted liver triglyceride content to the normal level of chow-fed
rats. When expressed as total hepatic triglyceride content per liver,
the levels in the high-fat-fed groups (354 ± 32, 245 ± 25, 239 ± 37, and 75 ± 13 µmol per liver in HF-Con, HF-Rosi, HF-WY, and HF-Raga, respectively) were positively correlated with HGO (Fig. 5A). Both liver
total triglyceride content and HGO were negatively correlated with
plasma adiponectin levels (Fig. 5, B and C).
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Insulin action in peripheral tissues and associated changes in lipids. As illustrated in Table 2, rosiglitazone and ragaglitazar enhanced insulin-mediated Rg' in both red (65 and 150%) and white (168 and 113%) muscles, whereas improvement of Rg' in HF-WY occurred mainly in white muscle (61%). All three agonists increased the Rg' in white adipose tissue (WAT), with the greatest improvement observed in HF-Raga (HF-Rosi: 79%, HF-WY: 74%, and HF-Raga: 147% above HF-Con). Compared with the HF-Rosi group, the Rg' values in the HF-Raga group were 30 and 38% higher in red muscle and WAT, respectively (P < 0.05), whereas white muscle showed similarly enhanced Rg' in these two groups. Compared with Wy-14643, ragaglitazar was clearly much more effective in enhancing insulin-mediated Rg' in muscle (79% vs. red quadriceps, P < 0.01, and 32% vs. white quadriceps, P < 0.08) and WAT (42%, P < 0.05). Because red muscle is the major muscle type for insulin-mediated Rg', we further investigated changes in glycogen and triglyceride synthesis in the red quadriceps. Glucose incorporation into glycogen (Fig. 4C) showed similar improvements to those shown for Rg', suggesting that the glycogen synthesis pathway has an important role in muscle insulin sensitivity enhanced by rosiglitazone and ragaglitazar. In comparison, there was only a small improvement of glucose incorporation into triglyceride by the three agonists (Fig. 4D), suggesting that de novo lipogenesis from glucose contributes very little to the enhanced insulin action in muscle. After the clamp, muscle LCA-CoA content was markedly decreased in all groups (P < 0.01 vs. basal values), and their levels remained significantly lower than the level of HF-Con (Table 2). There was a negative correlation between muscle LCA-CoA content and insulin-stimulated muscle Rg' (Fig. 5D) and plasma adiponectin levels (Fig. 5E). In contrast, insulin-stimulated muscle Rg' was positively correlated with plasma adiponectin levels (Fig. 5F).
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DISCUSSION |
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The present study demonstrates that combined activation of
PPAR/
markedly counteracts high-fat feeding-induced hepatic
steatosis and visceral adiposity in combination with a marked
improvement of insulin action. With use of a hyperinsulinemic
euglycemic clamp technique and double glucose tracers, in parallel with
determinants of lipid metabolism, we have clearly demonstrated
additional benefits on liver insulin sensitivity of dual activation of
PPAR
/
compared with selective activation of PPAR
.
Interestingly, although stimulating PPAR
, the PPAR
/
agonist
ragaglitazar did not cause hepatomegaly. As seen for selective PPAR
agonists, dual PPAR
/
activation substantially increased
circulating levels of adiponectin.
Liver steatosis is a characteristic in patients with type 2 diabetes
and obesity (39) and is closely associated with the insulin resistance syndrome (30). Our first important
finding was the complete prevention of high-fat feeding-induced liver steatosis by the dual PPAR/
activator ragaglitazar and associated substantial enhancement of insulin's suppressibility of HGO. It appears that the PPAR
agonist ragaglitazar decreased liver
triglyceride content primarily by activating PPAR
, as indicated by
increased expression levels of liver PBE and ACO mRNA, in a similar way to Wy-14643. These results are consistent with the finding in obese
Zucker rats that the PPAR
/
coligand KRP-297 reduces liver triglyceride content more effectively than rosiglitazone because it
stimulates FFA oxidation and inhibits lipogenesis (34).
There is evidence that rats treated with PPAR
activators have a
sustained increase in uncoupling protein (UCP)2 mRNA expression in the
liver and particularly the small intestine, where UCP2 is abundantly expressed (35). Further studies are required to
investigate whether this may provide a plausible mechanism to
facilitate energy depletion of the increased fatty acid oxidation
mediated by PPAR
or combined PPAR
/
activation. In addition to
PPAR
, PPAR
-mediated responses also appear to contribute to the
effects of ragaglitazar on liver. Like rosiglitazone, ragaglitazar
improved insulin-mediated hepatic glycogen synthesis. More importantly,
both ragaglitazar and rosiglitazone dramatically increased plasma
levels of adiponectin, a specific PPAR
-induced adipokine
(11) that has recently been shown to enhance insulin
action in liver (5). Although adiponectin has been shown
to promote muscle lipid oxidation (15), this effect has
not been described in liver. Our results clearly demonstrated a close
association of plasma adiponectin levels with triglyceride content and
insulin action in liver. PPAR
agonists have been reported to
downregulate gluconeogenic enzymes (3, 46, 48). However,
their relationship with adiponectin and lipids needs to be further investigated.
The second important finding of this study was that ragaglitazar almost
completely counteracted a high-fat feeding-induced increase in visceral
fat mass, an effect clearly different from that of rosiglitazone.
Although subcutaneous fat depots have been shown to be more responsive
to PPAR activation than visceral depots in humans (1)
and to potentially influence fat distribution, a recent study showed
that ragaglitazar does not alter subcutaneous fat weight
(27). This finding is of potential significance because of
body weight gain induced by PPAR
agonists alone (10, 14, 48). Additionally, insulin resistance is closely correlated with
visceral fat mass in rats (23) and humans
(8), and reduction of visceral fat can ameliorate insulin
resistance, particularly in the liver (4, 16, 17). PPAR
activators, such as fibrates and GW-9578, have been shown to reduce
adiposity in obese rodents (10, 16). In keeping with these
previous findings, the present study also found a reduction in visceral
adiposity by the selective PPAR
agonist Wy-14643. Because dual
PPAR
/
agonists (such as KRP-297) can increase FFA oxidation
(34), like selective PPAR
agonists, it is highly likely
that ragaglitazar reduces visceral adiposity development via
stimulating PPAR
-mediated FFA oxidation. It may be argued that the
lack of overall increased visceral fat mass upon combined PPAR
/
activation is associated with a lack of de novo adipogenesis. However,
like other dual PPAR
/
agonists (34), ragaglitazar
stimulated, rather than inhibited, adipogenesis, as indicated by an
increase in aP2 expression in adipose tissue compared with untreated
high-fat-fed rats. This suggests that the less visceral adiposity in
the ragaglitazar-treated group was not associated with reduced
adipogenesis. Although PPAR
-responsive adipokines, such as leptin
and adiponectin, may potentially influence body adiposity
(15), they do not seem to contribute to the reduced visceral adiposity by ragaglitazar, because rosiglitazone exerted similar effects on these adipokine plasma concentrations but without altering visceral adiposity.
Despite apparent responses mediated by PPAR, ragaglitazar did not
cause hepatomegaly. Most pure PPAR
agonists cause hepatomegaly in
rodents in a species-specific manner due to peroxisome proliferation (19). However, it is not clear how important
hepatomegaly is for PPAR
-mediated response in rodents. Our
results from ragaglitazar indicate that PPAR
-mediated effects in the
liver do not rely on enlargement of liver size (or peroxisome
proliferation). This interpretation is also supported by the effects of
fish oil in rats, where PPAR
is activated in the absence of
hepatomegaly (Cooney GJ and Kraegen EW, unpublished observations).
There are also reports that coactivation of PPAR
/
(33) or PPAR
/
(34) does not induce
hepatomegaly. Consistent with these observations, PPAR
-mediated
lipid oxidation in mitochondria has been shown to be independent of
peroxisome proliferation (28). It is possible that
activation of PPAR
interacts to prevent PPAR
-induced hepatomegaly or, alternatively, the stimulation of PPAR
by ragaglitazar was not
strong enough to produce hepatomegaly. Regardless of the mechanism, the
observed effects of ragaglitazar may have significant implications, because PPAR
agonists such as fibrates also lower lipids in the absence of hepatomegaly in humans.
Like rosiglitazone, ragaglitazar strongly enhanced insulin action in
muscle, a result similar to that previously reported in other
insulin-resistant models by dual PPAR/
agonists such as JTT-501
(29) and LY-465608 (13). These data suggest
that PPAR
-mediated effects may be mainly responsible for the
insulin-sensitizing action in muscle induced by ragaglitazar, because
the effects of PPAR
agonist Wy-14643 upon insulin-mediated
Rg' were relatively small and mainly seen in white muscle.
We have previously postulated that PPAR
activation may also improve
muscle insulin action by mechanisms independent of lipid
stealing (49). PPAR
agonists can alter
adipokines, which may affect insulin sensitivity (21).
Because leptin and adiponectin have been shown to improve insulin
action and increase lipid oxidation in muscle (15, 31, 45), we examined the effects of their circulating levels. The suppressed plasma leptin levels in all treated groups indicate that
leptin is unlikely to be involved in PPAR
-mediated insulin sensitization in muscle. Whereas PPAR
activation is known to suppress leptin expression (12), the decreased plasma
leptin levels in the HF-WY group may result from reduced visceral fat mass in a way similar to surgical removal (4). In
contrast, plasma adiponectin concentrations were threefold higher in
HF-Rosi and HF-Raga rats, and there was a strong correlation of plasma adiponectin levels with muscle LCA-CoA content, and even more so with
insulin-mediated Rg'. These results strongly suggest an
important role of adiponectin in PPAR
-mediated lipid metabolism and
insulin sensitization, as recently proposed (15). However, the distinct effects of ragaglitazar and rosiglitazone on liver triglyceride content, visceral adiposity, and HGO during the
hyperinsulinemic clamp suggest that these improvements could not be
entirely explainable by the elevated systemic adiponectin
concentrations. Furthermore, as these effects were substantially
similar to those caused by Wy-14643 without a significant increase in
plasma adiponectin concentrations, PPAR
-mediating changes
may be more likely to play an important role in reducing liver
triglyceride content and visceral adiposity.
We recognized that limitations might occur for comparisons based on a
single dose and that it could be argued that greater responses might be
obtained at higher doses of rosiglitazone and Wy-14643. However,
rosiglitazone-induced responses in this study were almost the same as
those obtained by our laboratory under similar conditions at a 3.8-fold
high dose administered by oral gavage (36). In terms of
Wy-14643, our pilot study showed that higher doses (e.g., 10 mg · kg1 · day
1)
caused appetite-averting effects with no further improvement of insulin
sensitivity (clamp GIR: 19.6 ± 4.2 mg · kg
1 · min
1,
n = 3). These data led us to believe that the responses
produced by both rosiglitazone and Wy-14643 had reached their plateau. Similar degrees of activation of PPAR
by rosiglitazone and
ragaglitazar in the present study were suggested by similar plasma
adiponectin levels. It was also noticed that there was no reduction in
liver LCA-CoAs in any of the drug-treated rats, a response
contradictory to that in muscle. The reason for this finding is not
known, but PPAR ligands are known to increase liver fatty acid-binding
protein, which has a strong affinity to bind LCA-CoAs and interacts
directly with PPAR
compounds (47).
In summary, the present study compared three types of PPAR agonists at
the levels of whole body and tissues. The results demonstrated that
combined activation of PPAR/
in the insulin-resistant
high-fat-fed rat exerts additional benefits to counteract fatty liver,
hepatic insulin resistance, and visceral adiposity while maintaining at least equivalent effectiveness to enhance muscle insulin action and
elevate circulating adiponectin concentrations to selective PPAR
stimulation. There is a close association between elevated plasma
adiponectin levels and improved insulin action in liver and muscle
induced by PPAR
/
(and PPAR
) activation. Our results suggest
that the PPAR
/
agonist ragaglitazar may have significant therapeutic potential in insulin-resistant states, with additional benefits of ameliorating fatty liver, hepatic insulin resistance, and
adiposity without any involvement of increased liver mass compared with
PPAR
activation alone.
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ACKNOWLEDGEMENTS |
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We thank J. Edema, Mercedes Ballesteros, and Nanna Hansen for excellent technical assistance, and the Biological Testing Facility staff for supportive animal care and maintenance.
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FOOTNOTES |
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This study was supported by the Australian National Health and Medical Research Council.
Address for reprint requests and other correspondence: J.-M. Ye, Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney, NSW 2010, Australia (E-mail: j.ye{at}garvan.org.au).
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.
10.1152/ajpendo.00299.2002
Received 8 July 2002; accepted in final form 23 October 2002.
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