From the Garvan Institute of Medical Research (J.-M.Y., M.A.I., D.G.W., G.J.C., E.W.K.), Sydney, Australia; and Novo Nordisk A/S (P.J.D.), Bagsverd, Denmark.
Address correspondence and reprint requests to Ji-Ming Ye, PhD, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010, Australia. E-mail: j.ye{at}garvan.unsw.edu.au . P.J.D. is currently affiliated with F.Hoffman La-Roche, PRBM, Basel, Switzerland.
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ABSTRACT |
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INTRODUCTION |
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In comparison, PPAR- is widely expressed in liver, muscle, kidney,
and intestine (8) and mediates
expression of genes promoting fatty acid ß-oxidation
(7,9,10).
Activation of the PPAR-
also lowers circulating lipids. For example,
the fibrate class PPAR-
agonists are effective drugs in clinical use to
treat hypertriglyceridemia
(7,11).
Given that lipid over-supply/accumulation leads to insulin resistance, one
would expect that lowering lipids with a PPAR-
agonist should also
attenuate insulin resistance, as does a PPAR-
agonist in
insulin-resistant states caused by lipid oversupply/accumulation
(7). However, studies in humans
have been inconclusive, with reports showing both improved
(12,13)
and unimproved insulin sensitivity
(14,15).
It might be that the PPAR-
agonists, though lowering circulating
lipids, have little effect on muscle lipids in those reports in which insulin
sensitivity is not improved. Thus far, we are aware of only one study that
examined the effects on muscle lipids of a PPAR-
agonist
(16). Although this study
showed some lowering of muscle triglyceride, no proper assessment of muscle
insulin sensitivity was made. In addition, the PPAR-
agonist
bezafibrate used in this study
(16) may also activate the
PPAR-
receptor and PPAR-ß
(10), a third subtype of PPARs
with unknown function. A more recent study in diabetic db/db mice
appears to add more uncertainty by showing that the PPAR-
agonist
WY14643 was almost ineffective in overcoming insulin resistance in spite of a
marked reduction in circulating triglyceride levels
(17). However, this conclusion
was based only on the fasting plasma glucose levels and neither muscle insulin
sensitivity nor muscle lipid content was examined.
Thus, we believe that the consequences of PPAR- stimulation on
muscle insulin sensitivity and lipid metabolism should be further
investigated, and apparent contradictions between effects of PPAR-
and
PPAR-
agonists on insulin sensitivity resolved. The aim of our study
was to compare the consequences of PPAR-
activation on muscle lipid
accumulation and insulin action in insulin-resistant high fatfed rats
using its specific agonist WY14643 and to compare the responses with specific
PPAR-
activation using pioglitazone. Regarding muscle lipid content,
recent evidence suggests that levels of the long-chain acyl-CoAs (LCACoAs) may
be more important mediators of muscle insulin resistance than triglyceride
accumulation (18), and we have
therefore also compared effects of the PPAR-
and PPAR-
agonists
on these lipid metabolites in muscle.
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RESEARCH DESIGN AND METHODS |
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After the acclimatization period, rats (300 g) were fed a high-fat
diet isocalorically (350 kJ/day given at 4:00 P.M.). The nutrient composition
of the fat diet expressed as a percentage of energy was as follows: 59% fat,
21% protein, and 20% carbohydrate with quantities of fibers, vitamin, and
minerals equal to those in the standard diet. Starting from the second week of
the high-fat feeding, rats were administered WY14643 or pioglitazone (Eli
Lilly, Indianapolis, IN) as an additive in the high-fat diet (each at 3 mg
· kg-1 · day-1) for 2 weeks. Body weight
was recorded daily. No appetite-averting effect was observed for either of the
compounds at the dose used.
Experimental protocol. Rats were divided into the following three groups: high fatfed controls, high fatfed treated with WY14643, and high fatfed treated with pioglitazone. Subgroups were studied in both basal and insulin-stimulated states. A week prior to study, the left carotid artery and right jugular vein were cannulated under ketamine/xylazine (90 mg/10 mg per kg i.p.) anesthesia. The cannulae were exteriorized in the back of the neck. Rats were handled daily to minimize stress. On the study day, after a 5-h fast, the cannulae were connected to infusion apparatus (via the carotid line) and a blood-sampling syringe (via the jugular line) between 9:00 and 10:00 A.M. The sampling line was filled with sodium citrate (20.6 mmol/l) to prevent blood from clotting. During the experiment, rats were allowed free access to water. After a 50- to 60-min period of settling, two basal blood samples (0.4 ml each) were collected at -30 and 0 min in tubes containing EDTA-K (5 µl) to act as an anticoagulant. After a rapid centrifugation, erythrocytes were suspended in 0.25 ml sterile normal saline (0.9% NaCl) and returned to the rat. Blood and plasma glucose concentrations were measured immediately and an aliquot of plasma was frozen in liquid nitrogen and stored at -80°C for subsequent measurement of triglycerides, nonesterified free fatty acids (NEFAs), glycerol, insulin, and leptin concentrations.
For the basal subgroups, the rats were usually allowed to settle for 2
h before being killed by an injection of overdose pentobarbital (
180
mg/kg). Muscles (including red and white quadriceps) were freeze-clamped with
aluminum tongs precooled in liquid nitrogen. Visceral (epididymal and
retroperitoneal) fat, liver, heart, and intrascapular brown adipose tissue
(BAT) were weighed and frozen in liquid nitrogen. The collected tissues were
kept at -80°C until assay.
Hyperinsulinemic-euglycemic clamp. Insulin was infused at a rate of 0.25 U · kg-1 · min-1 while euglycemia was maintained by infusing 30% glucose. During the clamp, 0.05 ml blood was taken every 10 min to measure blood glucose concentration for adjustment of the glucose infusion rate (GIR). After blood glucose levels reached steady state (4.5 ± 0.1 mmol/l blood glucose), a bolus of 2-deoxy-D-[2,6-3H]glucose (150 x 106 dpm) and D-[U-14C]glucose (100 x 106 dpm) in 0.1 ml normal saline was quickly injected via the jugular vein. Blood samples (0.2 ml) were taken at 2, 5, 10, 15, 20, 30, and 45 min after the injection to determine the tracer disappearance curve. After 45 min, the rat was killed and tissues collected as described above. Glucose disappearance rate (Rd) and hepatic glucose output rate were calculated from the disappearance of [14C]glucose. The area under the tracer disappearance curve of 2-deoxy-D-[2,6,-3H]glucose together with the counts of phosphorylated [3H]glucose from individual tissues was used to calculate insulin-stimulated glucose metabolic index (Rg'), an estimate of tissue glucose uptake (19). Insulin-mediated glycogen synthesis rate during the clamp was assessed by measuring [14C]glucose incorporation into glycogen (20). [14C]Glucose incorporation into lipids was determined by counting [14C] in the extracted triglycerides (21). Plasma levels of lipids, insulin, and leptin during the clamp were obtained from averaged values of blood samples taken before the tracer injection and at the end of experiment.
Metabolite measurements. Plasma glucose was determined using a glucose analyzer (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH). Plasma NEFA was determined spectrophotometrically using an acyl-CoA oxidase-based colorimetric kit (NEFA-C; Wako Pure Chemical Industries, Osaka, Japan). Both plasma triglycerides and glycerol were measured using enzymatic colorimetric methods (Triglyceride INT, procedure 336 and GPO Trinder; Sigma, St. Louis, MO). Plasma insulin and leptin were determined by radioimmunoassay using commercial kits (Linco, St. Louis, MO). Tissue triglycerides were extracted and measured by a Peridochrom Triglyceride GPO-PAP kit (Boehringer Mannheim, Germany) as previously described (22). LCACoAs were extracted from muscle and measured by high-performance liquid chromatography using a symmetry C18-reversed phase column (5 µm, 3.9 x 150 mm, Waters Corporation) as described previously (20,22,23). The sum of four major species consisting of palmitoyl (16:0), stearoyl (18:0), oleoyl (18:1), and linoleoyl (18:2) is presented to reflect LCACoAs.
Statistical analyses. All results are presented as means ± SE. A one-way analysis of variance followed by post-hoc (Fisher projected least squares difference [PLSD]) tests was used to assess the statistical significance between groups. All data were processed in Excel 5.0 and statistical analyses were performed using Macintosh Statview SE + Graphic program (Abacus Concepts-Brain Power).
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RESULTS |
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The effects of WY14643 and pioglitazone on tissue triglyceride content are shown in Fig. 1. Both agonists substantially lowered content of muscle triglyceride (-34 and -28%, respectively, Fig. 1A). WY14643 had an additional action to lower liver triglycerides by -54% when expressed as micromoles per gram of liver weight (Fig. 1B). When adjusted for the increased total liver weight, the total triglyceride content in the organ was still 26% lower in WY14643-treated rats than that of high fatfed control rats (231 ± 20 vs. 314 ± 17 µmol/liver, P < 0.01). In comparison, pioglitazone had no effect on total triglyceride content in the liver (308 ± 33 µmol/liver, P > 0.05 vs. high fatfed controls).
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Consistent with the changes in muscle triglyceride content, total LCACoAs were substantially lowered in muscle of rats treated with WY14643 and pioglitazone (-41 and -42%, respectively) compared with high fatfed control rats (Fig. 2A). In WY14643-treated rats, most of the measured major species (stearoyl 18:0, oleoyl 18:1, and linoleoyl 18:2) were significantly reduced, whereas in pioglitazone-treated rats, linoleoyl-CoA was the major species reduced (-69%, 15.2 ± 2.3 vs. 4.7 ± 0.7 nmol/g high fatfed controls, P < 0.001).
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Figure 3 shows insulin-stimulated glucose metabolic index (Rg') in individual tissues. WY14643 enhanced Rg' in red (47%) and white (63%) muscles as well as in white adipose tissue (90%) (P < 0.05 vs. high fatfed control values). Compared with WY14643, the improvement of Rg' induced by pioglitazone was greater (P < 0.01 vs. WY14643) in both the red and white muscles with increases of 125 and 169% above the high fatfed control values, respectively. Pioglitazone also improved Rg' in white adipose tissue (110%). Neither agonist had any significant effect on Rg' in the heart or BAT.
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Consistent with the increases in insulin-mediated Rg' in muscle and fat tissue, pioglitazone enhanced [14C]glucose incorporation into glycogen (57%) in muscle and into lipids (250%) in adipose tissue (Table 3). In the WY14643 group, increased [14C] glucose incorporation into lipids was found only in fat (106%). Although their values were similar among all groups when expressed as micromoles per gram of weight, the total [14C]glucose incorporations into glycogen and lipids in the liver were increased by 80 and 49%, respectively, above the high fatfed control values in the WY14643 group (Table 3).
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Muscle triglyceride contents were similar to respective basal levels in all postclamp groups (data not shown). However, compared with the individual basal values (Fig. 2A), muscle total LCACoAs were decreased by 59, 45, and 68% in the high fatfed control, WY14643, and pioglitazone groups, respectively, in response to insulin stimulation (Fig. 2B, P < 0.01). Among the clamp subgroups, muscle total LCACoAs in pioglitazone-treated rats were significantly lower than those in high fatfed controls and the most apparent reduction occurred in linoleoyl- and oleoyl-CoAs. Both circulating triglyceride levels and muscle LCACoA content were inversely correlated with muscle insulin sensitivity (Rg') (Fig. 4).
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DISCUSSION |
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The question as to whether accumulation of muscle lipids is causally
related to the development of muscle insulin resistance, and conversely
whether lowering cytosolic muscle lipid accumulation leads to improved insulin
action, is of importance for our study. One reason for our interest in
performing the present study was that, although not directly studied, indirect
evidence tended to indicate that PPAR- agonists might reduce muscle
lipid supply without significantly improving insulin action in
insulin-resistant states (17).
This would appear contradictory to muscle lipid availability as an important
causative factor modulating muscle insulin resistance, and also against a
reduction of lipid supply to muscle being a principal factor in the
insulin-sensitizing action of PPAR-
activators. However, the findings
from both PPAR-
and PPAR-
activation in the present study are
consistent with a lipid supply hypothesis of muscle insulin resistance.
Although PPAR-
and PPAR-
agonists lower lipids by entirely
different mechanisms, with PPAR-
mediating lipid oxidation mainly in
the liver
(7,9)
and PPAR-
sequestering lipids in adipose tissue
(6), both enhance muscle
insulin action accompanied by a lowering of muscle lipid accumulation.
Although resolving an apparent contradiction of the lipid supply hypothesis of modulation of muscle insulin sensitivity does not prove the causality implicit in the hypothesis, our group has recently demonstrated that incubation of isolated soleus muscle with physiological concentrations of various fatty acids can significantly inhibit insulin-mediated glucose uptake and glycogen synthesis and impair insulin-mediated Akt/protein kinase B phosphorylation (24). Similar findings have also recently been reported in cultured murine C2C12 myotubes (25). Furthermore, in the isolated soleus preparation, fatty acidinduced insulin resistance was accompanied by muscle LCACoA accumulation (24). These in vitro studies support a causal link between muscle lipid accumulation and insulin resistance.
The present study provides evidence that improved muscle insulin action in
vivo by a PPAR- or PPAR-
agonist is associated with a reduction
of LCACoAs. LCACoAs are the activated state of fatty acids within cells, and
their intramuscular levels are closely associated with various
insulin-resistant states, such as high-fat feeding
(18), chronic glucose infusion
(26), triglyceride
emulsion/heparin infusion (2),
or an acute human growth hormone infusion
(23). Recently, we have found
an inverse association of muscle LCACoA levels with insulin sensitivity in
humans (22). Although precise
mechanisms in which increased LCACoAs cause insulin resistance are still
unclear, LCACoAs have been reported to inhibit the activity of hexokinase and
pyruvate dehydrogenase (27)
and glycogen synthesis in the liver
(28), and it is possible that
similar inhibitory effects of LCACoAs may also occur in muscle. In addition,
they are precursors for the synthesis of diacylglycerol, an endogenous
activator of protein kinase C (PKC)
(29). Lipid-induced activation
of some PKC subtypes may impair the insulin signaling pathway
(3). Therefore, it is likely
that lowered muscle LCACoA content by WY14643 and pioglitazone may improve
muscle response to insulin action by alleviating these factors that cause
insulin resistance.
In spite of a number of similarities, several apparent differences were
found between effects of PPAR- and PPAR-
activation. At a dosage
that produced similarly increased levels of whole-body GIR and
Rd, pioglitazone had a greater effect than WY14643 on
enhancing muscle Rg' as well as [14C]glucose incorporation into
glycogen. Although dose-response relations and muscle drug bioavailability
might elucidate reasons for this, it was noteworthy that the greater muscle
insulin sensitization with pioglitazone was in parallel with a greater
lowering of muscle LCACoAs and circulating triglyceride levels during the
clamp. Compared with WY14643, pioglitazone reduced muscle linoleoyl CoA (18:2)
and oleoyl-CoA (18:1) more effectively. The most abundant LCACoA
specieslinoleoyl CoAis the predominant fatty acid component in
safflower oil used to make the high-fat diet. In cultured murine C2C12
myotubes (25) and the
incubated rat soleus preparation
(24), linoleoate significantly
inhibited insulin-mediated glucose uptake/phosphorylation at physiological
concentrations. Therefore, a greater reduction in linoleoyl-CoA levels may be
involved in the greater improvement of muscle insulin sensitivity with
pioglitazone treatment.
However, we cannot exclude possible local effects of both PPAR- and
PPAR-
in muscle. There is evidence that PPAR-
receptor levels in
muscle may be higher
(8,30)
than previously thought (31).
The possibility of local intramuscular action of TZDs is also suggested in
some (32) but not other
(31) in vitro studies. It
remains to be further investigated as to whether muscle PPAR-
also
contributes to pioglitazone-induced improvement of insulin sensitivity or
whether some TZDs have additional insulin-sensitizing action independent of
PPAR-
-mediated responses. In comparison, PPAR-
is abundantly
expressed in muscle
(8,30).
Although it is not clear whether PPAR-
also mediates lipid
ß-oxidation in muscle, WY14643 can increase the expression and activity
of muscle pyruvate dehydrogenase kinase similarly to starvation
(33). Activation of this
kinase could phosphorylate and thus inactivate the pyruvate dehydrogenase
complex. Therefore, it may well be that the local effects mediated by
PPAR-
have partially offset, by substrate competition
(34), an otherwise much
greater improvement of muscle insulin action in response to WY14643-reduced
lipid accumulation.
The apparent lesser effectiveness of WY14643 than pioglitazone in enhancing
Rg' in muscle on the basis of similar GIR and Rd suggests
that enhanced glucose uptake in tissues other than muscle and fat may also
contribute to the glucose disposal. Because PPAR- is highly expressed
in liver
(7,9),
we assessed a possible hepatic contribution by determining
[14C]glucose incorporation into glycogen and lipids in the liver.
As expected, there were substantial increases in total [14C]glucose
incorporation into both glycogen (80%) and lipids (49%) in WY14643-treated
rats, suggesting that the liver may be another important site of glucose
disposal induced by PPAR-
stimulation.
Insulin resistance is closely correlated with an excess accumulation of visceral fat (35), and lowering central fat has been a target to improve insulin action. In animals such as rats, insulin resistance is ameliorated when visceral fat is reduced by surgical removal (36), pharmacological interventions (4), or food restriction (37). WY14643 treatment substantially reduced total liver triglyceride content and visceral fat (estimated as epididymal and retroperitoneal fat stores). This may be also involved in its enhancement of whole-body insulin sensitivity.
The human PPAR- shares functional characteristics with the rodent
PPAR-
, in that the activation of PPAR-
leads to reduction of
circulating lipids (38).
However, PPAR-
induced hepatomegalypresumably by promoting
peroxisome proliferationis thought to be species-specific for rodents
(39). In fact, the fibrate
class of PPAR-
agonists was safely used in humans to treat
hypertriglyceridemia, and no hepatomegaly was reported
(11). In the present study, we
used WY14643 because of its high specificity as a PPAR-
agonist with
virtually no affinity for PPAR-
and PPAR-ß
(10,17,40).
Our results are consistent with recent reports showing that other PPAR-
activators also improve insulin sensitivity and lower lipid availability in
insulin-resistant rodent models, such as obese Zucker rats, high fatfed
mice (41), and
sucrose-lardfed rats
(17). These data taken
together suggest that lowering muscle lipid accumulation might be a key factor
for an improvement of insulin sensitivity in humans with PPAR-
activators. Although still controversial, some recent clinical studies have
shown improved insulin sensitivity by fibrates
(12,13).
The controversy over the consequence of PPAR-
induced lipid
lowering in humans may be resolved by using a hyperinsulinemic-euglycemic
clamp technique together with muscle lipid measurement.
In summary, using the hyperinsulinemic-euglycemic clamp technique, we have
found that the PPAR- activation with WY14643 ameliorates insulin
resistance in the high fatfed rat along with its lipid-lowering action.
The association of reduced lipids, particularly muscle LCACoAs, with the
improvement of insulin sensitivity was similar for both PPAR-
and
PPAR-
agonists. These results are consistent with reduced muscle lipid
accumulation being central to PPAR-mediated improvement in insulin action.
Because the finding of PPAR-
activation-reduced visceral fat mass was
consistent with another recent report
(41), we suggest that
compounds with combined PPAR-
and PPAR-
stimulating action may
have significant therapeutic potential in insulin-resistant states, with less
tendency toward increased adiposity compared with PPAR-
activation
alone.
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ACKNOWLEDGMENTS |
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We wish to thank J. Edema, L. Croft, and D. Wilks for their excellent technical assistance and the Biological Testing Facility staff for their support of animal care and maintenance.
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FOOTNOTES |
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Received for publication June 1, 2000 and accepted in revised form October 11, 2000
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REFERENCES |
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