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Agonist) Action
Department of Clinical Biochemistry (J.R.Z., J.W.R.), Karolinska Hospital, Karolinska Institute, S-171 76 Stockholm, Sweden; and Department of Molecular Endocrinology (T.D., J.W., M.W., J.V., Z.L., C.M., J.B., B.Z., D.E.M.) Merck Research Laboratories, Rahway, New Jersey 07065
Address all correspondence and requests for reprints to: David E. Moller, M.D., Director, Molecular Endocrinology, Merck Research Laboratories, RY80T-100, 126 East Lincoln Avenue, Rahway, New Jersey 07065. E-mail: david_moller{at}merck.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. However, their
mechanism of action and the in vivo target tissue(s)
that mediate insulin sensitization remain poorly defined. Although
PPAR messenger RNA expression has been reported in skeletal muscle,
the expression of PPAR within myocytes in intact muscle tissue has
not been examined. An antipeptide PPAR antibody was generated;
immunohistochemistry was then used to demonstrate that PPAR is
present within nuclei of myocytes [in both skeletal (white and red
fibers) and cardiac tissue (rodent and human)]. The effect of insulin
sensitizer treatment on muscle insulin action was studied using
ob/ob mice after 4 days dosing with a potent (6
nM PPAR Kd) TZD (10 mg/kg·day).
2-deoxyglucose (2-DOG) uptake was then assessed in freshly isolated
soleus muscles from lean vs. ob/ob vs. TZD-treated
ob/ob mice. In lean mouse muscles, 2-DOG uptake was
stimulated by 82%, 95%, 165% (with 25, 100, 2000 µU/ml insulin);
muscles from ob/ob were severely insulin resistant
(<80% stimulation with 2000 µU/ml insulin). Muscles from
TZD-treated ob/ob displayed a normal insulin response
with 100 (71%) or 2000 (158%) µU/ml insulin. Additional studies
were performed using ZDF rats treated with/without TZD for 7 days.
In vivo 2-DOG glucose uptake into soleus, gastrocnemius,
and diaphragm muscles was measured during euglycemic-hyperinsulinemic
clamp. Compared with lean rats, muscle 2-DOG uptake in ZDF was reduced
by 52% (soleus) or 71% (diaphragm). Partial (40–60%) normalization
of the reduced 2-DOG uptake was evident in TZD-treated ZDF rats. In
contrast to the effect of in vivo treatment on muscle
insulin action, preincubation of isolated soleus muscles from naive
lean or ob/ob mice for 5 h with 100 nM TZD did not
affect insulin-stimulated 2-DOG uptake. We conclude: 1) PPAR is
expressed in myocytes within skeletal and cardiac muscle. 2) In
vivo activation of PPAR by treatment of insulin-resistant
mice/rats with a potent TZD corrects impaired muscle insulin action. 3)
The lack of a direct effect on muscle after 5 h in
vitro TZD incubation suggests that changes in insulin action
may require a longer duration of PPAR activation or that improved
muscle insulin sensitivity may result from an indirect in
vivo effect of PPAR activation (e.g. changes
in systemic lipid metabolism). |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using cultured cell systems, thiazolidinediones have been shown to
promote adipocyte differentiation and accelerate the induction of
messenger RNAs (mRNAs) encoding adipocyte-specific genes such as fatty
acid binding protein (aP2) (9, 10). These findings led to the
hypothesis that these compounds might function as agonists for a
specific nu-clear receptor-peroxisome proliferator-activated
receptor (PPAR) that has been implicated in adipogenesis (11).
Two related isoforms (PPAR1 and PPAR2), which differ by the
addition of 30 N-terminal amino acids in PPAR2, occur as a result of
alternative promoter usage and mRNA splicing (12).
Three related PPAR family members—PPAR
, PPAR, and PPAR
(hNUC1)—exist and are subject to regulation by fatty acids and lipid
metabolites. Individual PPARs heterodimerize with the retinoid x
receptor (RXR); the PPAR-RXR complex binds to specific DNA response
elements (PPREs) in gene promoters and functions as a transcription
factor that can be activated by either RXR- or PPAR-specific ligands.
PPAR is expressed at high levels in adipose tissue; it mediates
transcriptional activation of the promoters for aP2 and other adipocyte
genes and is sufficient to induce in vitro adipocyte
differentiation (11, 13).
Several lines of evidence implicate PPAR activation as the
predominant mechanism for thiazolidinedione action: 1) It is now clear
that thiazolidinediones are high affinity PPAR-specific ligands that
can serve to transactivate PPAR-responsive gene promoters (14). 2)
In vivo efficacy in rodents generally correlates with
in vitro PPAR activity (3, 15). 3) Nonthiazolidinedione
PPAR agonists also exert antihyperglycemic effects in rodent NIDDM
models (16, 17). 4) Structurally distinct compounds that function as
selective RXR ligands activate PPAR/RXR heterodimers and cause
in vivo insulin sensitization in rodent NIDDM models (18).
5) Transcriptional activation of at least one gene (lipoprotein lipase)
by PPAR has been linked to one of the classic in vivo
effects of thiazolidinedione: triglyceride lowering (19).
Skeletal muscle is commonly viewed as the predominant tissue responsible for insulin mediated (in the fed state or during hyperinsulinemic clamp conditions) glucose disposal in both rat (20) and man (21). Because treatment of insulin-resistant humans and animals with thiazolidinediones has been shown to result in improved whole body glucose disposal under euglycemic hyperinsulinemic clamp conditions, substantial effects of insulin sensitizer therapy on insulin mediated glucose disposal in skeletal muscle can be implicated.
The potential effects of thiazolidinediones on muscle insulin
sensitivity are not well characterized. Thus, it is unclear whether
in vivo insulin sensitization is largely a secondary
consequence of improvement in the abnormal metabolic milieu
(e.g. FFA lowering) or whether there may be direct effects
on insulin-responsive target tissues. We and others have reported the
detection of PPAR mRNA in skeletal muscles derived from mouse (22),
rat (23), and man (23, 24, 25, 26, 27). However, to date the presence of PPAR
protein within myocytes (in either cardiac or skeletal muscle) has not
been determined. In addition, the hypothesis that activation of PPAR
can directly affect insulin action in intact skeletal muscles has not
been tested. Here, we used immunohistochemistry with a PPAR-specific
antiserum to characterize the expression of PPAR protein in muscle.
In addition, we used a potent, PPAR-selective thiazolidinedione to
assess the effects of in vivo PPAR activation on insulin
action in skeletal muscle (both during euglycemic hyperinsulimic clamp
and ex vivo in isolated soleus muscles). Finally, we
assessed the potential for direct effects of PPAR activation on
muscle insulin action.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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agonist compound
(5-[4-[2-(5
methyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl]-2,4-thiazolidinedione)
was kindly provided by Gerard Kieczykowski, Philip Eskola, Conrad
Santini, Joseph Leone, and Peter Cicala (Merck Research Laboratories,
Rahway, NJ). Male ob/ob and lean control ob/+ mice (10–12 weeks old from Jackson Laboratories, Bar Harbor, ME) and male Zucker diabetic fatty (ZDF) or control ZDF/+ lean rats (17 weeks old from Genetic Models, Inc., Indianapolis, IN) were allowed ad libitum access to water and Purina rodent chow (Ralston Purina, St. Louis, MO). The animals were untreated or dosed daily by oral gavage with vehicle (0.5% carboxymethylcellulose) or the TZD compound suspended in 0.5% carboxymethylcellulose at a specified mg of compound per kg animal weight. Normal Sprague-Dawley rats (6 weeks old from Jackson Laboratories) were were used for immunohistochemistry experiments.
Localization of PPAR protein in muscle by
immunohistochemistry
Polyclonal antibodies specific for peptide SEKTQLYNRPHEEPSNS,
which corresponds to amino acids 117–133 of human and mouse PPAR2,
were generated in rabbits (Cocalico Biologicals, Inc., Reamstown,
PA).
The antiserum recognized recombinant PPAR by Western blotting and
was tested for specificity using transiently transfected COS cells that
express PPAR or PPAR, or PPAR (3) as follows. Cells grown on
glass cover slides were fixed by first washing in PBS followed by
incubation in 2% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.3, for 30 min. Fixed cells were washed 2 times in PBS
containing 1% BSA (PBS/BSA) and nonspecific binding sites were blocked
by incubation in Pierce (Rockford, IL) Superblock
solution for 30 min followed by 2 x washing with PBS/BSA. The
blocked cells were permeabilized by incubation with 0.1% Triton X-100
for 10 min followed by 2 washes with PBS/BSA. Specific labeling was
then carried out by incubation with 5 µg/ml anti PPAR IgG. To
demonstrate antibody specificity, separate wells were incubated with
immune IgG in the presence of 20 µg/ml of immunizing peptide, an
irrelevant peptide of similar length but unrelated sequence, or in the
presence of nonimmune IgG. Incubation in primary antibody was carried
out for 2 h at room temperature followed by 7 washes in PBS/BSA.
Bound antibodies were visualized by incubation with a 1/200 dilution of
Cappel FITC-goat antirabbit IgG for 60 min at room temperature followed
by 7 washes with PBS/BSA. Following the final wash, cover slides were
mounted using p-phenylenediamine mounting media (28). Digital
micrographs were taken using a Kodak Megaplex camera mounted on a Zeiss
Axiophot microscope using a 63x phase contrast objective.
Cardiac muscle samples were collected from 200 g male Sprague-Dawley rats under deep Nembutal anesthesia in which the abdominal aorta was cannulated in a retrograde direction. The vasculature and heart was cleared of blood by perfusion with saline and the heart muscle was fixed by perfusion with 50 ml of Nakane’s (periodate/lysine/paraformaldehyde) fixative (29). Following perfusion fixation, tissue samples (left ventricle) were obtained. In separate experiments samples of soleus and gastrocnemius muscle were collected from rats which had been similarly perfused via a cannula inserted into the right ventricle. Perfused tissues were then fixed an additional 10 h in Nakane’s fixative. Muscle samples from a total of ten individual rats were examined by immunohistochemistry. Samples of human muscle were collected from the vastus lateralis portion of the quadriceps femorus under local anesthesia (mepivacain chloride 5 mg/ml). Upon excision, the muscle specimens were cleaned of connective tissue and placed in Nakane’s PLP fixative (within 15 min) for 6 h. The study protocol was reviewed and approved by the Institutional Ethical Committee of the Karolinska Institute, and informed consent was obtained from all subjects before participation. Skeletal muscle samples from three normal men (mean BMI 27.4 ± 1.8 kg/m2; mean age 55 ± 3 yr) were examined.
Following fixation, all samples were cryoprotected by sequential incubation in: 10% sucrose in 0.05 M phosphate buffer, pH 7.3, (1 h), 15% sucrose in 0.05 M phosphate buffer, pH 7.3, (4–12 h), 20% sucrose in 0.05 M phosphate buffer pH 7.3, (4 h), 20% sucrose plus 5% glycerol in 0.05 M phosphate buffer, pH 7.3, (1 h). Cryoprotected specimens were embedded in OCT medium (Miles) and rapidly frozen in liquid nitrogen and stored at -70 C until use. Cryosections (6 µ) were cut on a Reichert-Jung Cryocut 1800 equipped with a cryostat sectioning aid (Instumedics, Inc., Teaneck, NJ). All sections were pretreated with 3% H2O2 in methanol for 20 min to inactivate endogenous peroxidases, followed by 0.1% Triton-X 100 in PBS to increase permeability. Putative endogenous biotin binding was blocked with avidin solution (Vector Laboratories, Inc., Burlington, CA), followed by extensive washing in rinse buffer (0.1 M phosphate buffer, pH 7.8, containing 0.5% BSA and 0.01% Tween-20). Endogenous avidin binding sites were then blocked with biotin solution (Vector Laboratories, Inc., Burlingame, CA). Possible nonspecific staining was inhibited by preincubating the slides for 30 min in 1% normal donkey serum (Jackson Labs), followed by extensive washing in rinse buffer. To further reduce nonspecific labeling, sections were incubated for 30 min in a solution of 5% Carnation nonfat dry milk containing 0.1% BSA and 0.04% Na azide in 0.1 M phosphate buffer, pH 7.8. This solution was centrifuged for 5 min at 13000 rpm just before use.
Sections were immunolabeled by incubation with the primary
PPAR-specific antibody described above. Primary antibody incubations
were carried out at a concentration of 1 µg/ml IgG in rinse buffer
for 60 min at room temperature. Incubation was terminated by extensive
washing in rinse buffer. Bound antibodies were detected via
immunoperoxidase microscopy using the ABC technique (Elite kit;
Vector Laboratories, Inc.) with the following
modifications. Primary antibody was visualized using affinity purified
biotinylated F(ab')2 fragments of donkey antirabbit IgG from Jackson
Labs. Peroxidase reaction product was developed with a glucose
oxidase/DAB/nickel method to provide maximum sensitivity. In some cases
eosin was used as a counterstain. Labeled sections were then
dehydrated, cleared, and mounted with Permount. Digital
micrographs were taken using a Kodak Megaplex camera mounted on a Zeiss
Axiophot microscope using a 63x phase contrast objective.
Measurement of in vivo muscle 2-deoxyglucose uptake during
hyperinsulinemic euglycemic clamp
Hyperinsulinemic euglycemic clamps were performed in 17-week-old
Zucker diabetic fatty (ZDF) rats and Zucker/lean rats. The TZD compound
was administered to ZDF rats by oral gavage at a daily dose of 10 mg/kg
for 7–11 days. Sodium Nembutal-anesthetized rats were cannulated in
the left jugular vein for constant rate infusion of insulin (25
mU/kg·min) and 3H-glucose (New England Nuclear, Boston,
MA). The right jugular vein was cannulated for variable rate infusion
of glucose to maintain euglycemia; the left femoral vein was cannulated
for bolus infusions of 3H-glucose and
14C-2-deoxyglucose (NEN). Blood samples were obtained from
the cannulated left femoral artery for determination of plasma glucose
levels and 3H and 14C radioactivity. The rate
of 14C-2-deoxyglucose uptake into selected skeletal muscles
was determined as previously described (30).
2-Deoxyglucose uptake in isolated in vitro incubated soleus
muscles
All media were prepared from a stock solution of Krebs-Henseleit
buffer (KHB) containing 5 mM HEPES and 0.1% BSA (RIA
grade). The gas phase in the vial was maintained at 95%
O2/5% CO2 throughout all incubations. To
assess the effects of prior in vivo TZD treatment on
in vitro 2-deoxyglucose uptake, isolated soleus muscles from
lean ob/+, ob/ob, or TZD-treated ob/ob
mice were incubated for 20 min in KHB containing 20 mM
mannitol, and the specific additions as described for each experiment
in the figure legends. Thereafter, glucose uptake was assessed for 20
min as previously described (31). Muscles were incubated in KHB
containing 1 mM 2-deoxy-[1,2,3H]glucose (2.5
µCi/ml) and 19 mM [14C]mannitol (0.35
µCi/ml). 2-deoxyglucose uptake has been reported to directly reflect
glucose transport and not metabolism in mouse skeletal muscle when
performed under the present conditions (31). Following the incubations,
the muscles were processed as described by Wallberg-Henriksson et
al. (32) for the rat epitrochlearis muscle. Values are expressed
as micromoles of 2-deoxyglucose per milliliter of intracellular water
per hour.
Additional experiments were performed to assess whether TZD had a direct effect on insulin-stimulated 2-deoxyglucose uptake in skeletal muscle. Isolated soleus muscles from lean ob/+ or ob/ob mice were preincubated for 5 h in the presence or absence of 100 nM TZD. TZD concentration was maintained throughout all incubations. A stock solution of TZD was prepared in dimethylsulfoxide (DMSO) before addition to KHB. The final concentration of DMSO was adjusted to 0.002% in each incubation medium. Preincubation media was supplemented with 8 mM glucose and 12 mM mannitol. The media was changed after 2.5 h. Muscles were transferred to glucose-free KHB, which contained 25 or 2000 µU/ml insulin and incubated for 20 min. Thereafter, muscles were incubated to assess 2-deoxyglucose uptake as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein in skeletal and cardiac muscle peptide antibody,
COS-1 cells were plated on slides and transfected with pSG5,
pSG5-hPPAR2, pSG5-hPPAR, or pSG5-hPPAR. After 48 h,
the cells were permeabilized, exposed to the antibody and examined by
immunofluorescent microscopy. Predominant nuclear staining of cells was
observed only in the nuclei of cells transfected with hPPAR2 (not
shown). Additional experiments demonstrated that the signal observed by
immunofluorescence was completely blocked by immunizing peptide. As
predicted by the epitope used for immunization, the antibody recognized
both PPAR1 and PPAR2 by Western blot in differentiated 3T3L1
cells (not shown).
To visualize the distribution of PPAR in skeletal muscle, cryostat
sections of perfusion fixed rat soleus muscles (considered to be
predominantly red muscle fibers) were incubated with anti-PPAR
antibody followed by immunoperoxidase labeling. The results (Fig. 1
) demonstrate that immunoperoxidase
reaction product was easily seen within the nuclei of individual
myocytes. Reaction product was also often seen within the nuclei of
adjacent vascular endothelial cells. Reaction product was not detected
in the cytoplasmic areas of either the myocytes or the capillary
endothelial cells. The labeling of myocyte nuclei was very specific in
that simultaneous incubation with immunizing peptide completely blocked
labeling whereas incubation with a peptide of similar size but
unrelated sequence had no effect on the intensity of the label. In
addition, incubation with nonimmune IgG did not label this tissue.
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was differentially distributed between red and
white skeletal muscle fibers, samples were also collected from the
lateral head of rat gastrocnemius muscles (considered to be composed
primarily of white muscle fibers). The results (Fig. 2A) were similar to those obtained with
soleus muscles, demonstrating that, within the limits of this method,
both skeletal muscle fiber types contain roughly equal amounts of
immunodetectible PPAR. In addition, we determined that there was a
similar level of PPAR expression in the nuclei of cardiac (left
ventricle) myocytes (Fig. 2B). In all three sets of muscles,
immunolabeling was restricted to the nuclei and was not found
associated with any other cytoplasmic structures or with the
contractile apparatus.
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) demonstrated that
PPAR could be visualized within the nuclei of the myofiber syncytium
of the quadriceps muscle. Because these samples were examined and
recorded by direct microscopic observation, it is clear that the label
is actually within the nuclei of the myocytes themselves and could not
be confused with nuclei of adipose tissue, which is sometimes closely
associated with skeletal muscle.
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Effect of TZD treatment on in vivo muscle 2-deoxyglucose uptake in
ZDF rats
Based upon results from hyperinsulinemic euglycemic clamp studies,
ZDF rats displayed profound hyperglycemia and insulin resistance that
was substantially corrected by 7–11 day treatment with the TZD
compound (10 mg/kg-day). Elevated levels of free fatty acids were
suppressed following 1–2 days of treatment and elevated plasma
triglycerides were suppressed after 4 days in TZD treated ZDF rats
(data not shown). Mean nonfasting and fasting plasma glucose levels in
ZDF rats were 481 and 346 mg/dl, respectively, compared with 116 mg/dl
for both nonfasting and fasting levels in nondiabetic Zucker/lean rats.
The TZD dosing regimen resulted in 60% correction of both nonfasting
(266 mg/dl) and fasting (199 mg/dl) hyperglycemia. The glucose infusion
rate necessary to maintain euglycemia in the presence of extreme
hyperinsulinemia induced in all rats was 22.2 mg/kg·min in
Zucker/lean rats compared with only 4.1 mg/kg-min in ZDF rats. TZD
dosing for 7–11 days elicited a 100% correction of this parameter.
Skeletal muscle uptake of 2-deoxyglucose during the terminal euglycemic
phase was determined in each rat; a moderate to severe reduction in
insulin-stimulated glucose uptake was evident in several muscles from
ZDF vs. Zucker/lean rats (see Fig. 4).
Uptake of 2-deoxyglucose in soleus was decreased 50% in ZDF rats
compared with Zucker/lean rats, and TZD treatment resulted in a mean
value that was corrected by 60%. This value did not reach statistical
significance, possibly due to the small sample size. Uptake of
2-deoxyglucose in diaphragm from ZDF rats was reduced by 71% relative
to Zucker/lean rats; this parameter was corrected by 62% in
TZD-treated ZDF rats. In contrast to the reduced 2-deoxyglucose uptake
in soleus and diaphragm from ZDF rats, glucose uptake in gastrocnemius
was not significant different between ZDF and Zucker/lean rats.
However, TZD administration resulted in a significant (47%) increase
compared with untreated ZDF rats.
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). Basal,
submaximal, and maximal insulin-stimulated 2-deoxyglucose uptake
was markedly and significantly reduced in soleus muscles from
ob/ob compared with lean mice. Maximal insulin-stimulated
2-deoxyglucose uptake was reduced by 73% in soleus muscles from
ob/ob vs. lean mice. Basal glucose transport activity
in soleus muscle was fully restored to control levels in
ob/ob mice following TZD treatment. Furthermore,
insulin-stimulated rates of glucose transport (100 and 2,000 µU/ml)
were completely normalized in ob/ob mice following TZD
treatment. Submaximal insulin-stimulated (25 µU/ml) glucose uptake
rates were also improved, but not completely normalized to control
levels in soleus from TZD-treated ob/ob mice.
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).
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It should be noted that the concentration (100 nM) of the
specific TZD compound employed in the above noted experiments was
empirically determined to be 10-fold higher than the concentration
required for maximal activation of PPAR-mediated transcription under
two experimental conditions: 1) functional activation of a Gal4-PPAR
ligand-binding-domain chimeric receptor in transfected COS cells
(J. Berger, unpublished); 2) stimulation of adipocyte fatty
acid binding protein (aP2) mRNA expression in cultured 3T3-L1
preadipocytes (B. Zhang, unpublished).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is highly expressed in adipose tissue, but lower levels of
PPAR mRNA expression have been detected in skeletal muscle of
rodents and man (22, 23, 24, 25, 26, 27). In addition, there may be higher levels of
PPAR mRNA expression in skeletal muscle derived from nondiabetic
obese or obese NIDDM subjects (23, 24, 27). One shortcoming of previous
studies is that the contribution of contaminating adipocyte-derived
PPAR mRNA to the net amount of PPAR mRNA detected in samples of
muscle tissue cannot be excluded. To date, there are no published
reports of PPAR protein expression in muscle. We used antipeptide
antibodies that were specific for PPAR and immunoperoxidase labeling
to clearly demonstrate the presence of PPAR protein within the
nuclei of individual myocytes from both red and white skeletal muscle
fiber types in rat and human vastus lateralis. Although the signal
appeared strong relative to what one might expect from previously
reported low levels of PPAR mRNA, the immunoperoxidase methods we
used included an amplification step. For technical reasons, we were
unable to use immunoperoxidase staining to compare PPAR protein
abundance in fat vs. muscle. Nevertheless, this finding
provides a potential means by which TZD PPAR agonists could exert
direct insulin sensitizing effects in muscle. We also established that
PPAR protein was present in nuclei of left ventricular rat
cardiomyocytes. This result provides an important insight into a
potential PPAR-mediated toxicity because TZD insulin sensitizers
were shown to cause cardiac hypertrophy in preclinical animal studies
(35).
In addition to the well described effect of TZD PPAR agonists in
improving whole-body insulin sensitivity in insulin-resistant animals
and humans, in vivo treatment with these compounds can
reverse discrete defects in tissue insulin action. Thus, several
studies showed that in vivo treatment of insulin-resistant
mice or rats was associated with improved insulin-stimulated glucose
uptake in adipocytes derived from treated animals (4, 5, 36, 37). In
some cases, this was associated with up-regulation of previously
suppressed GLUT4 levels (5, 36). The effects of TZD treatment on
insulin action in muscle have not been well characterized.
In the present study, we used a potent and highly selective TZD PPAR
agonist (3) to demonstrate that in vivo treatment of
insulin-resistant rats or mice could affect skeletal muscle insulin
sensitivity. In ZDF rats with extreme hyperglycemia and insulin
resistance, in vivo treatment with this TZD fully normalized
whole-body glucose uptake under hyperinsulinemic clamp conditions. This
positive effect coincided with significant improvement in in
vivo 2-deoxyglucose glucose uptake into both diaphragm and
gastrocnemius muscles and a trend toward improved glucose uptake in
soleus muscle. Our experiments with ob/ob mice showed that 4
days of TZD treatment was sufficient to normalize hyperglycemia and
that this improvement was associated with normalization of severely
insulin-resistant glucose uptake measured ex vivo in soleus
muscles derived from treated and untreated mice. These results indicate
that improved insulin action in skeletal muscle is a clear consequence
of in vivo TZD treatment that is likely to substantially
contribute to net effects on insulin-stimulated glucose metabolism. To
our knowledge, there are only two prior reports where changes in muscle
insulin sensitivity per se were shown after in
vivo TZD treatment: 1) Chronic treatment of high fat-fed rats with
a potent TZD (BRL 49653) resulted in improved in
vivo glucose uptake into skeletal muscles under clamp conditions
(38). 2) Chronic in vivo TZD administration enhanced insulin
responsiveness of soleus muscles derived from dexamethasone-treated
rats (39).
A key unresolved question concerns the potential for direct effects of
TZDs (PPAR activation) on insulin-responsive target tissues.
Specifically, the hypothesis that TZDs might have direct effects on
intact muscle tissue has not been previously assessed. In the present
study, the potent TZD PPAR agonist was used to assess the potential
for direct effects on isolated soleus muscles. After a 5-h incubation
period (similar results, not shown, were obtained up to 9 h),
there was no TZD effect on maximal or submaximal insulin-mediated
glucose uptake in muscles derived from either lean or ob/ob
mice. In addition, insulin-stimulated glucose incorporation into
glycogen was unaffected by exposure to the TZD. Thus, despite the
presence of PPAR protein within this tissue, there was no apparent
direct effect of PPAR activation during this 5-h interval to
potentiate insulin-stimulated glucose uptake and storage as glycogen.
In contrast, we previously observed that in vitro incubation
of isolated rat adipose tissue with precisely the same TZD at the same
(or lower) concentration could clearly potentiate insulin-stimulated
glucose incorporation into glycogen and activation of glycogen synthase
within a 2–3 h incubation time period (40). Obviously, longer term
exposure to TZDs may be required for possible direct insulin
sensitizing effects in this tissue because PPAR mediated effects are
presumed to be based on changes in gene expression. Two prior studies
have reported that chronic incubation (20–24 h) of cultured muscle
cells (cardiomyocytes or BC3H-1 myocytes) with TZDs resulted in an
increase in glucose transport (41, 42). However, both studies noted
that there was a substantial increase in GLUT-1 expression, and in
neither case was insulin responsiveness definitively affected. We have
also observed that incubation of L6 myotubes, an insulin-responsive
muscle cell line, with various TZDs did not potentiate
insulin-stimulated glucose uptake in a significant manner (J. Berger,
unpublished).
In summary, we have definitively established that PPAR is expressed
in skeletal and cardiac myocytes and we have used two approaches to
demonstrate that in vivo administration of a TZD PPAR
agonist results in improved insulin-stimulated skeletal muscle glucose
uptake. Because there was no apparent direct effect of a 5-h exposure
of soleus muscles to a potent PPAR agonist upon insulin-stimulated
muscle glucose uptake, we suggest that a meaningful response of
skeletal muscle to PPAR activation may require longer exposure to
PPAR agonist. In recently completed additional studies conducted by
Wu et al. using ZDF rats, high doses of potent PPAR
agonists resulted in substantial improvement of in vivo
insulin-mediated skeletal muscle glucose uptake after 7–11 days but
not after 1–2 days of daily oral treatment (43). Alternatively, the
in vivo effect of TZD treatment on muscle insulin action may
predominantly occur as a secondary consequence of the improved
metabolic milieu. Thus, lowering of elevated FFA and triglyceride
levels, as well as partial improvements in elevated glucose and insulin
levels that may be mediated by effects of PPAR activation in fat
(and direct or indirect effects on the liver), could act in concert to
restore muscle insulin sensitivity.
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Acknowledgments |
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Received June 18, 1998.
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 K. B. Sotiropoulos, A. Clermont, Y. Yasuda, C. Rask-Madsen, M. Mastumoto, J. Takahashi, K. Della Vecchia, T. Kondo, L. P. Aiello, and G. L. King Adipose-specific effect of rosiglitazone on vascular permeability and protein kinase C activation: novel mechanism for PPAR{gamma} agonist's effects on edema and weight gain FASEB J, June 1, 2006; 20(8): 1203 - 1205. [Abstract] [Full Text] [PDF]
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F. Blaschke, Y. Takata, E. Caglayan, R. E. Law, and W. A. Hsueh Obesity, Peroxisome Proliferator-Activated Receptor, and Atherosclerosis in Type 2 Diabetes Arterioscler Thromb Vasc Biol, January 1, 2006; 26(1): 28 - 40. [Abstract] [Full Text] [PDF]
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 M. M. Masternak, K. A. Al-Regaiey, M. M. Del Rosario Lim, V. Jimenez-Ortega, J. A. Panici, M. S. Bonkowski, J. J. Kopchick, and A. Bartke Effects of Caloric Restriction and Growth Hormone Resistance on the Expression Level of Peroxisome Proliferator-Activated Receptors Superfamily in Liver of Normal and Long-Lived Growth Hormone Receptor/Binding Protein Knockout Mice J. Gerontol. A Biol. Sci. Med. Sci., November 1, 2005; 60(11): 1394 - 1398. [Abstract] [Full Text] [PDF]
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M. M. Masternak, K. A. Al-Regaiey, M. M. Del Rosario Lim, M. S. Bonkowski, J. A. Panici, G. K. Przybylski, and A. Bartke Caloric Restriction Results in Decreased Expression of Peroxisome Proliferator-Activated Receptor Superfamily in Muscle of Normal and Long-Lived Growth Hormone Receptor/Binding Protein Knockout Mice J. Gerontol. A Biol. Sci. Med. Sci., October 1, 2005; 60(10): 1238 - 1245. [Abstract] [Full Text] [PDF]
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 H. K.R. Karlsson, K. Hallsten, M. Bjornholm, H. Tsuchida, A. V. Chibalin, K. A. Virtanen, O. J. Heinonen, F. Lonnqvist, P. Nuutila, and J. R. Zierath Effects of Metformin and Rosiglitazone Treatment on Insulin Signaling and Glucose Uptake in Patients With Newly Diagnosed Type 2 Diabetes: A Randomized Controlled Study Diabetes, May 1, 2005; 54(5): 1459 - 1467. [Abstract] [Full Text] [PDF]
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M. Z. Strowski, Z. Li, D. Szalkowski, X. Shen, X.-M. Guan, S. Juttner, D. E. Moller, and B. B. Zhang Small-Molecule Insulin Mimetic Reduces Hyperglycemia and Obesity in a Nongenetic Mouse Model of Type 2 Diabetes Endocrinology, November 1, 2004; 145(11): 5259 - 5268. [Abstract] [Full Text] [PDF]
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 J. R. Colca, W. G. McDonald, D. J. Waldon, J. W. Leone, J. M. Lull, C. A. Bannow, E. T. Lund, and W. R. Mathews Identification of a novel mitochondrial protein ("mitoNEET") cross-linked specifically by a thiazolidinedione photoprobe Am J Physiol Endocrinol Metab, February 1, 2004; 286(2): E252 - E260. [Abstract] [Full Text] [PDF]
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Y. Xu, L. Lu, C. Greyson, J. Lee, M. Gen, K. Kinugawa, C. S. Long, and G. G. Schwartz Deleterious Effects of Acute Treatment With a Peroxisome Proliferator-Activated Receptor-{gamma} Activator in Myocardial Ischemia and Reperfusion in Pigs Diabetes, May 1, 2003; 52(5): 1187 - 1194. [Abstract] [Full Text] [PDF]
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C. Dello Russo, V. Gavrilyuk, G. Weinberg, A. Almeida, J. P. Bolanos, J. Palmer, D. Pelligrino, E. Galea, and D. L. Feinstein Peroxisome Proliferator-activated Receptor gamma Thiazolidinedione Agonists Increase Glucose Metabolism in Astrocytes J. Biol. Chem., February 14, 2003; 278(8): 5828 - 5836. [Abstract] [Full Text] [PDF]
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 J. A. Wagner Early Clinical Development of Pharmaceuticals for Type 2 Diabetes Mellitus: From Preclinical Models to Human Investigation J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5362 - 5366. [Full Text] [PDF]
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M. M. Meyer, K. Levin, T. Grimmsmann, N. Perwitz, A. Eirich, H. Beck-Nielsen, and H. H. Klein Troglitazone Treatment Increases Protein Kinase B Phosphorylation in Skeletal Muscle of Normoglycemic Subjects at Risk for the Development of Type 2 Diabetes Diabetes, September 1, 2002; 51(9): 2691 - 2697. [Abstract] [Full Text] [PDF]
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G. Jiang, Q. Dallas-Yang, Z. Li, D. Szalkowski, F. Liu, X. Shen, M. Wu, G. Zhou, T. Doebber, J. Berger, et al. Potentiation of Insulin Signaling in Tissues of Zucker Obese Rats After Acute and Long-Term Treatment With PPAR{gamma} Agonists Diabetes, August 1, 2002; 51(8): 2412 - 2419. [Abstract] [Full Text] [PDF]
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B. M. Jucker, T. R. Schaeffer, R. E. Haimbach, T. S. McIntosh, D. Chun, M. Mayer, D. H. Ohlstein, H. M. Davis, S. A. Smith, A. R. Cobitz, et al. Normalization of Skeletal Muscle Glycogen Synthesis and Glycolysis in Rosiglitazone-Treated Zucker Fatty Rats: An In Vivo Nuclear Magnetic Resonance Study Diabetes, July 1, 2002; 51(7): 2066 - 2073. [Abstract] [Full Text] [PDF]
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T. P. Combs, J. A. Wagner, J. Berger, T. Doebber, W.-J. Wang, B. B. Zhang, M. Tanen, A. H Berg, S. O'Rahilly, D. B. Savage, et al. Induction of Adipocyte Complement-Related Protein of 30 Kilodaltons by PPAR{gamma} Agonists: A Potential Mechanism of Insulin Sensitization Endocrinology, March 1, 2002; 143(3): 998 - 1007. [Abstract] [Full Text] [PDF]
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A. L. Hevener, D. Reichart, A. Janez, and J. Olefsky Thiazolidinedione Treatment Prevents Free Fatty Acid-Induced Insulin Resistance in Male Wistar Rats Diabetes, October 1, 2001; 50(10): 2316 - 2322. [Abstract] [Full Text]
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N. D. Oakes, P. G. Thalén, S. M. Jacinto, and B. Ljung Thiazolidinediones Increase Plasma-Adipose Tissue FFA Exchange Capacity and Enhance Insulin-Mediated Control of Systemic FFA Availability Diabetes, May 1, 2001; 50(5): 1158 - 1165. [Abstract] [Full Text]
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J. M. Way, W. W. Harrington, K. K. Brown, W. K. Gottschalk, S. S. Sundseth, T. A. Mansfield, R. K. Ramachandran, T. M. Willson, and S. A. Kliewer Comprehensive Messenger Ribonucleic Acid Profiling Reveals That Peroxisome Proliferator-Activated Receptor {{gamma}} Activation Has Coordinate Effects on Gene Expression in Multiple Insulin-Sensitive Tissues Endocrinology, March 1, 2001; 142(3): 1269 - 1277. [Abstract] [Full Text] [PDF]
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 R. Weindruch, T. Kayo, C.-K. Lee, and T. A. Prolla Microarray Profiling of Gene Expression in Aging and Its Alteration by Caloric Restriction in Mice J. Nutr., March 1, 2001; 131(3): 918S - 923. [Abstract] [Full Text]
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J.-M. Ye, P. J. Doyle, M. A. Iglesias, D. G. Watson, G. J. Cooney, and E. W. Kraegen Peroxisome Proliferator--Activated Receptor (PPAR)-{alpha} Activation Lowers Muscle Lipids and Improves Insulin Sensitivity in High Fat--Fed Rats: Comparison With PPAR-{gamma} Activation Diabetes, February 1, 2001; 50(2): 411 - 417. [Abstract] [Full Text]
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U. SMITH, S. GOGG, A. JOHANSSON, T. OLAUSSON, V. ROTTER, and B. SVALSTEDT Thiazolidinediones (PPAR{gamma} agonists) but not PPAR{alpha} agonists increase IRS-2 gene expression in 3T3-L1 and human adipocytes FASEB J, January 1, 2001; 15(1): 215 - 220. [Abstract] [Full Text] [PDF]
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M. Nawano, A. Oku, K. Ueta, I. Umebayashi, T. Ishirahara, K. Arakawa, A. Saito, M. Anai, M. Kikuchi, and T. Asano Hyperglycemia contributes insulin resistance in hepatic and adipose tissue but not skeletal muscle of ZDF rats Am J Physiol Endocrinol Metab, March 1, 2000; 278(3): E535 - E543. [Abstract] [Full Text] [PDF]
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L. Poretsky, N. A. Cataldo, Z. Rosenwaks, and L. C. Giudice The Insulin-Related Ovarian Regulatory System in Health and Disease Endocr. Rev., August 1, 1999; 20(4): 535 - 582. [Abstract] [Full Text] [PDF]
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J. G. Hunter, M. F. van Delft, R. A. Rachubinski, and J. P. Capone Peroxisome Proliferator-activated Receptor gamma Ligands Differentially Modulate Muscle Cell Differentiation and MyoD Gene Expression via Peroxisome Proliferator-activated Receptor gamma -dependent and -independent Pathways J. Biol. Chem., October 5, 2001; 276(41): 38297 - 38306. [Abstract] [Full Text] [PDF]
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