Veterans Affairs San Diego Healthcare System and Department of Medicine, University of California, San Diego, La Jolla, California 92093
Submitted 31 October 2003 ; accepted in final form 6 April 2003
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ABSTRACT |
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fatty acid transporters; fatty acid translocase; free fatty acid utilization; troglitazone; rosiglitazone; pioglitazone
One possible mediator of FFA transport is fatty acid translocase (FAT). FAT has been shown to be identical to CD36, the scavenger receptor for oxidized LDL (48). FAT/CD36 is abundantly expressed in tissues with high metabolic capacity for fatty acids (2, 39). FAT/CD36 expression correlates well with FFA transport into rat heart as well as red and white muscle vesicles (2, 39). Transgenic mice overexpressing FAT/CD36 in muscle show an enhanced ability of muscle to oxidize FFA in response to stimulation and contraction, together with decreased triglyceride and FFA levels (31). FAT/CD36 deficiency has been proposed as a possible mechanism responsible for insulin resistance, defective fatty acid metabolism, and hypertriglyceridemia in the spontaneously hypertensive rat (56). Another proposed mediator of FFA uptake is plasma membrane fatty acid-binding protein (FABPpm). FABPpm expression correlates with FFA uptake or utilization in a variety of different circumstances (6, 68). Expression of FABPpm in 3T3 fibroblasts is associated with an increase in FFA uptake rates, and this increase reflects the addition of a saturable, high-affinity component inhibited by antibodies against FABPpm (70). A third possible mediator of FFA uptake is fatty acid transport protein (FATP-1). FATP-1 is expressed in tissues active in fatty acid utilization like skeletal muscle, heart, and fat (7). Overexpression of FATP-1 in cells is associated with an increase in uptake of FFA (61).
Because plasma FFA levels are increased (36, 64) and fatty acid utilization is impaired in type 2 diabetic patients (36), regulation of FFA uptake may also be impaired in this situation. Insulin decreases FFA uptake in muscle in vivo (69), possibly by an indirect effect to decrease FFA plasma levels, through inhibition of lipolysis in adipocytes (35). There is some evidence that insulin can activate FFA uptake into several cell types (20, 48), possibly by stimulating FAT/CD36 translocation to the plasma membrane (37). It is unclear whether insulin effects on FFA uptake into muscle represent another process in which insulin action is impaired in type 2 diabetic patients.
Troglitazone (Tgz), rosiglitazone (Rgz), and pioglitazone (Pio) are members
of the thiazolidinedione class of insulin-sensitizing drugs
(47). They have been shown to
lower plasma FFA levels in type 2 diabetic patients
(41,
53). Thiazolidinediones have
been identified as peroxisome profliferator-activated receptor-
(PPAR
) activators (62).
Both FAT and FATP have been shown to be regulatable by PPAR
(42,
46) and may participate in a
mechanism by which Tgz and Rgz increase FFA uptake in vitro into cultured
adipocytes (43).
The aim of the present study was to study the regulation of FFA transport by acute insulin and chronic thiazolidinedione treatment and compare these effects in cultured skeletal muscle cells of type 2 diabetic and nondiabetic subjects. This system has proved useful for muscle cells cultured from type 2 diabetic subjects, which have been shown to retain defects in insulin action and glucose metabolism reflective of those seen in vivo (18, 30). To gain insight into underlying mechanisms for regulation of FFA uptake, the effects of thiazolidinediones on FAT/CD36, FATP, and FABPpm mRNA and/or protein expression were also studied, along with the ultimate endpoint for FFA uptake, FFA oxidation.
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MATERIALS AND METHODS |
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Materials. Cell culture materials were purchased from Irvine Scientific (Irvine, CA) except for skeletal muscle basal medium, which was obtained from Clonetics (San Diego, CA). Human biosynthetic insulin was kindly supplied by Eli Lilly (Indianapolis, IN). Bovine serum albumin (BSA; Cohn fraction V) was purchased from Boehringer Mannheim (Indianapolis, IN) and phloretin from ICN Biomedicals (Costa Mesa, CA). [U-14C]palmitate, [9,10-3H]palmitate, L-[U-14C]glucose, and 2-[3H]deoxyglucose were obtained from New England Nuclear Life Science Products (Boston, MA). Unlabeled palmitate, FFA-free BSA, deoxyglucose, and L-glucose were purchased from Sigma (St. Louis, MO). TRIzol reagent was purchased from Gibco-BRL (Gaithersburg, MD), Nytran plus membrane from Schleicher & Schuell (Keene, NH), DECAprime II Random Priming Kit and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA from Ambion (Austin, TX), Chroma spin columns from Clontech (Palo Alto, CA), QuickHyb from Stratagene (San Diego, CA), and BioMax film from Eastman Kodak (Rochester, NY). FAT/CD36, FATP-1, and FABPpm cDNAs were kindly provided by Drs. Azeddine Ibrahimi of State University of New York (Stony Brook, NY), Andreas Stahl of the Whitehead Institute (Cambridge, MA), and Joseph Mattingly of the University of Missouri (Kansas City, MO), respectively. Reagents for electrophoresis and the AG-1X8 resin were obtained from Bio-Rad (Richmond, CA). Monoclonal CD36 and actin antibodies raised in mice were purchased from Serotec (Raleigh, NC) and Sigma, respectively. Anti-mouse IgG complexed to horseradish peroxidase and Hyperfilm were from Amersham (Arlington Heights, IL). SuperBlock and SuperSignal Chemiluminescent Substrate Kit were obtained from Pierce (Rockford, IL). Tgz was a kind gift of Dr. Alan Saltiel (Pfizer Parke-Davis Pharmaceuticals, Ann Arbor, MI); Rgz was provided by Dr. Stephen Smith (GlaxoSmithKline, Harlow, UK); and Pio was supplied by Dr. A. Kozai (Takeda Pharmaceuticals America, Lincolnshire, IL).
Human skeletal muscle cell culture. The methods for muscle biopsy
of the vastus lateralis and cell isolation and growth have been described in
detail previously (30). At
8090% confluence, cells were fused for 4 days in -MEM containing
2% FBS, 1% fungibact, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Fusion medium was changed every other day. Approximately 90% of the cells take
on the multinucleated morphology characteristic of mature, differentiated
myotubes (30); fibroblasts are
not present in the cultures. When indicated, 11.5 µM Tgz, 10 µM Rgz, or
10 µM Pio were added for 4 days at the initiation of
fusion/differentiation. The doses of the thiazolidinediones were determined in
preliminary studies to provide maximal stimulation of palmitate oxidation and
FFA uptake. Agents were dissolved in DMSO to a final concentration of 0.05%.
Previous work has shown that this treatment protocol does not alter the extent
of differentiation of myocytes into mature myotubes
(52).
FFA uptake assay. FFA transport was measured on cells grown on
12-well plates. After completion of differentiation and treatment, the cells
were rinsed twice with serum-free -MEM containing 0.1% BSA, pH 7.4.
Cells were then incubated in this medium at 37°C with 0 or 30 nM insulin
and 0 or 300 mM phloretin. After 70 min, the medium was removed by rinsing the
cells four times with room temperature reaction buffer (in mM: 150 NaCl, 5
KCl, 1.2 MgSO4, 1.2 CaCl2, 2.5
NaH2PO4, and 10 HEPES and 0.1% BSA, pH 7.4). Uptake was
initiated by replacing the reaction buffer with 1 ml of reaction buffer
premixed with substrate (0.3 µCi [14C]palmitate, final
concentration 20 µM). The substrate was prepared under N2 by
mixing with FFA-free BSA (12);
concentration of the stock solution was confirmed by colormetric assay for FFA
(Wako Chemicals, Richmond, VA). Transport was stopped after 1 min by
aspirating the substrate and washing five times with 4°C PBS. The cells
were solubilized by incubating them in 0.5 ml of 0.1 N NaOH with shaking.
Protein content was determined on an aliquot of the cell suspension by the
Bradford method (11). The
remaining volume was transferred to a scintillation vial, and scintillation
fluid was added and radioactivity measured. Total counts were determined by
placing 0.5 ml of substrate containing reaction buffer directly into the
scintillation vial. To compensate for FFA adhesion to the well and cells,
zero-time counts were subtracted. Transport was normalized to the amount of
cell protein. All measurements were performed in triplicate. The intra-assay
coefficient of variation was 7%. Whereas phloretin has been shown to inhibit a
number of carrier-mediated processes
(4), for this report
protein-mediated uptake is operationally defined as the difference between
total uptake and that measured in the presence of phloretin for each
individual set of cells. This correction would account for the radioactivity
that entered cells by diffusion and FFA absorbed to the cell surface that was
not removed during the washing procedure.
Glucose uptake assay. Glucose uptake measurements were carried out as described previously (30). Medium was added to the cells together with insulin (0 or 33 nM), and the cells were incubated for 6090 min in a 95% O2-5% CO2 incubator before washing and transport assay. An aliquot of the suspension was removed for protein analysis using the Bradford method. Uptake of L-glucose was used to correct each sample for the contribution of diffusion.
Measurement of palmitate oxidation. The procedure for assaying palmitate oxidation is a modification (10) of a method established for adherent cells (58). Cells were incubated in serum-free medium containing 10 µl of substrate (0.2 µCi [9,10-3H]palmitic acid, final concentration 2 µM) in a 95% O2-5% CO2 incubator at 37°C for 3 h. After incubation, an aliquot (100 µl) of the culture medium was placed over an ion exchange resin, and the column was washed twice with 0.75 ml of water. Intact palmitate (charged state) was retained by the resin, whereas the oxidized portion of palmitate passed freely through the resin column in the form of water (HO3H).
Northern analysis of FAT/CD36, FATP, and FABPpm. Total cellular
RNA was isolated from muscle cell cultures with TRIzol according to the
manufacturer's instructions. Ten micrograms of total RNA were separated
according to size by electrophoresis through a denaturing formaldehyde
11.5% agarose gel and transferred to a Nytran plus membrane. DNA probes
for Northern analysis were labeled by the decamer-priming method using the
DECAprime kit (Ambion) and purified with Chromaspin columns. Hybridization
with FAT/CD36, FATP, FABPpm, and GAPDH cDNA probes labeled with
[-32P]deoxycytidine triphosphate was carried out at 68°C
in 5 ml of QuickHyb, according to the manufacturer's instructions. To remove
nonspecific binding, membranes were washed twice at room temperature with
2x standard sodium citrate (SSC), 0.1% SDS buffer, and then once with
0.2x SSC and 0.1% SDS buffer, followed by one 30-min wash at 60°C
with 0.1x SSC and 0.1% SDS buffer. After being washed, membranes were
exposed to BioMax film at -70°C. The relative intensities of transcript
signals were compared quantitatively with a computer imaging program (NIH
Image) by using the GAPDH signal to normalize for loading differences.
Analysis of FAT/CD36 protein expression. Cell protein lysates were prepared as described previously (32). Western blot analysis was done as detailed previously (30). In summary, cell extracts were solubilized in Laemmli buffer, and 1020 µg of protein were separated according to size on 10% SDS-PAGE gels and electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked for 12h at room temperature with TBS, 0.05% Tween, and 5% nonfat milk, pH 7.4. The monoclonal CD36 antibody was diluted 1:250 in SuperBlock, pH 7.4, and incubated overnight at 4°C. For actin, the membrane was blocked with TBS, 0.05% Tween, and 5% nonfat milk, pH 7.4, overnight at 4°C. The monoclonal actin antibody was diluted 1:2,000 in TBS, 0.05% Tween, and 5% BSA, pH 7.4, and incubated at room temperature for 1 h. The secondary antibody for both CD36 and actin was anti-mouse IgG conjugated to horseradish peroxidase. Proteins were visualized with the SuperSignal Chemiluminescent Substrate Kit and exposed to Hyperfilm.
Statistical analysis. Statistical significance was evaluated using a two-tailed Student's t-test for paired or unpaired comparisons, where appropriate. Significance was accepted at the P < 0.05 level. Because of limitations in tissue availability, not all experiments in cell cultures were performed in cells from all individuals. There were no differences in the clinical characteristics of the subsets of subjects whose cells were used in each study.
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RESULTS |
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Acute regulation of FFA uptake. To determine whether insulin has a direct acute effect on FFA uptake, differentiated cells were treated with 30 nM insulin for 70 min before assay. Acute insulin treatment caused a modest (+16 ± 5%, n = 5) but significant (P < 0.025) stimulation of the carrier-mediated palmitate uptake (the phloretin-inhibitable portion) into nondiabetic muscle (Fig. 1B). There was no significant insulin effect on FFA uptake in diabetic muscle cells (Fig. 1B); this was true for either absolute values or relative (+19 ± 19%, n = 8, P = not significant) changes. In contrast, muscle cells from these same diabetic subjects retained significant insulin stimulation of glucose transport (+23 ± 10%, n = 5, P < 0.05), although this value was also reduced compared with the insulin effect in nondiabetic muscle (+44 ± 8%, n = 7, P < 0.005).
Chronic regulation of FFA uptake: effects of thiazolidinedione
treatment. Because treatment with antidiabetic thiazolidinediones
decreases FFA levels in vivo
(26,
35), their chronic effect on
FFA uptake into muscle cells was studied. Cells were treated with Tgz (11
µM), Rgz (10 µM), or Pio (10 µM) for 4 days during differentiation.
None of the agents influenced the differentiation of muscle cells, as
indicated by the extent of multinucleation or expression of sarcomere-specific
-actin (not shown), similar to previous results
(52). From the example
presented in Fig. 2A,
it can be seen that Rgz treatment increased total palmitate uptake in
nondiabetic muscle cells, whereas uptake in the presence of phloretin was
unaltered, indicating that thiazolidinedione treatment increased only the
protein-mediated component of uptake. Treatment effects on this
carrier-mediated component of FFA uptake are presented in
Fig. 2B. For
nondiabetic muscle cells, both Tgz (+32 ± 4%) and Rgz (+68 ±
26%) caused significant increases; Pio effects were not measured in
nondiabetic cells. In diabetic muscle cells, thiazolidinedione treatment had
the result of normalizing the impaired uptake activity
(Fig. 2B). The
thiazolidinediones were equally potent in elevating uptake in diabetic muscle:
Tgz, +87 ± 32%; Rgz, +70 ± 15%; and Pio, +98 ± 9%.
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One fate of FFA taken up into the muscle cell is oxidation. Under the same
circumstances where FFA uptake was upregulated by thiazolidinedione treatment,
-oxidation of palmitate was also increased by all three agents
(Fig. 2C) in both
nondiabetic and diabetic muscle cells, in confirmation of previous results
(15).
Effects of thiazolidinediones on expression of putative mediators of FFA uptake. To identify which gene products might be responsible for the protein-mediated component of FFA uptake and to gain initial insight into possible mechanisms by which thiazolidinediones could increase FFA uptake, the effects of thiazolidinedione treatment on FAT/CD36, FATP, and FABPpm mRNA expression and FAT/CD36 protein expression were determined.
RNA was isolated from cultures treated with 11 µM Tgz for 4 days during differentiation. FAT/CD36, FATP-1, and FABPpm mRNA expression were determined with Northern blotting. To normalize for loading differences, a GAPDH probe was used. A representative blot is shown in Fig. 3A. mRNAs for FAT/CD36, FATP, and FABPpm were all present in skeletal muscle cell cultures. Quantification of the signals, normalized for loading differences, is shown in Fig. 3B. Tgz treatment upregulated FAT/CD36 mRNA by 193 ± 65% (n = 6, P < 0.05). There was no significant change in either FABPpm or FATP mRNA expression.
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Protein was isolated from cultures treated with Tgz (11 µM), Rgz (10 µM), or Pio (10 µM) for 4 days, and Western blotting with a FAT/CD36 antibody was performed; results are shown in Fig. 4. All three thiazolidinediones increased FAT/CD36 protein expression: +90 ± 22% over control (P < 0.005) for Tgz, +146 ± 42% (P < 0.01) for Rgz, and +111 ± 37% (P < 0.025) for Pio.
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DISCUSSION |
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In vivo, insulin decreases FFA disposal in muscle (14, 21). Insulin could do this indirectly by decreasing FFA plasma levels and substrate availability to muscle. Conversely, insulin has also been shown to increase FFA uptake into skeletal muscle (21), possibly by stimulating translocation of FAT/CD36 to the plasma membrane (37). To distinguish between these processes, the effect of acute insulin treatment on FFA uptake into cultured human skeletal muscle cells was tested under conditions of fixed FFA availability. Human skeletal muscle cells grown in culture have been used to study muscle development and metabolism (9, 60). Although this experimental system lacks the neuronal, vascular, endocrine, and paracrine inputs of intact skeletal muscle, the cells maintain many of the morphological, biochemical, and metabolic properties of mature muscle (30).
Several investigators, including us (18, 25, 32), have found that muscle cells cultured from insulin-resistant individuals display impairments in insulin action on glucose uptake and glycogen synthesis. Thus at least a portion of insulin resistance for glucose metabolism in type 2 diabetes is intrinsic to skeletal muscle and not solely the result of the hyperglycemic, hyperinsulinemic, and hyperlipidemic environment characteristic of the condition. In the present work, we found that insulin stimulated the protein-mediated component of FFA uptake into muscle cells of nondiabetic subjects, a behavior similar to that seen in vivo (5). Differences were also seen in FFA uptake between nondiabetic and diabetic muscle cells and in diabetes-related impairments in both basal activity and insulin responsiveness. These differences suggest that at least a portion of defective FFA metabolism in type 2 diabetes is not acquired from the metabolic environment. These in vitro results are in agreement with several reports of impaired in vivo skeletal muscle FFA uptake and disposal measured in type 2 diabetic individuals (8, 29). Defects in FFA uptake and disposal have also been reported in subjects with impaired glucose tolerance in the presence of normal glucose, insulin, and lipid levels (44). Thus there is in vivo and in vitro (this report) evidence that impaired FFA uptake into skeletal muscle in type 2 diabetes is independent of the hormonal and metabolic environment. It should be noted that the type 2 diabetic subjects who supplied tissue for these studies were older and more obese than the nondiabetic subjects; the contribution of these differences to the impairment in FFA uptake is uncertain.
The insulin-sensitizing thiazolidinediones decrease plasma FFA levels in vivo (41, 53). Possible mechanisms for this response could include reduced lipolysis and FFA release by adipose tissue, reduced hepatic lipogenesis (reviewed in Ref. 5), or increased in FFA uptake into muscle. Indeed, the present results indicate that Tgz, Rgz, and Pio can all increase FFA uptake into muscle cells. Tgz and Rgz have been shown to increase FFA transport into 3T3-L1 adipocytes (24, 42) as well, so it is possible that similar mechanisms are functioning in adipocytes and muscle cells.
Reconciling findings of an in vitro effect of thiazolidinediones to stimulate FFA uptake and oxidation with in vivo results is complicated by the ability of thiazolidinediones to reduce circulating FFA levels. Thus a reduction in whole body FFA disposal or oxidation following thiazolidinedione treatment (50) could be due to a mass action effect of lowered substrate delivery to tissues. Conversely, when palmitate is infused to maintain a fixed level, FFA utilization is augmented after thiazolidinedione treatment (12, 26). This latter situation is closer to the in vitro condition, where the palmitate concentration is also fixed.
A reciprocal relationship between glucose and fatty acid metabolism has been established in many circumstances (57), although there are also situations where this relationship does not appear to hold (19). In light of this relationship, how is it possible to reconcile thiazolidinedione-induced increases in both glucose uptake and FFA uptake? One explanation would be if, at least for the limited time represented by these assays, the glucose were being directed toward storage in glycogen while palmitate was being oxidized to meet the immediate energy needs of the cell. If thiazolidinedione treatment were to shift the balance between oxidation of fatty acids and glucose oxidation predominantly to fatty acids, the net effect would be to direct glucose to nonoxidative pathways of utilization. That would be consistent with our finding that troglitazone treatment of human muscle cells, besides increasing glucose uptake, increased glycogen synthase activity and glucose incorporation into glycogen (52). This would also be consistent with the results of Miyazaki et al. (45), who reported that the increase in whole body glucose disposal following pioglitazone treatment of diabetic subjects could be accounted for by the increase in nonoxidative glucose metabolism, usually taken to represent glycogen synthesis.
Thiazolidinedione treatment increased only the protein-mediated component
of FFA uptake (Fig. 2),
suggesting that thiazolidinediones upregulate transporter levels or increase
transporter activity, the latter possibly through translocation to the cell
surface. Indeed, we did find that thiazolidinediones upregulated FAT/CD36 mRNA
and protein expression, whereas troglitazone did not alter FATP-1 and FABPpm
mRNA levels. These results would be in agreement with other reports where
PPAR ligands, including thiazolidinediones, have been shown to increase
FAT/CD36 expression in multiple cell types, including adipose tissue and
macrophages (23). Other
evidence suggests that changes in FATP-1 expression may not play a major role
in impairments in FFA uptake in obesity and type 2 diabetes
(10). However, there are data
that thiazolidinedione treatment increases both FAT and FATP mRNA levels in
adipose tissue (42,
46). Thus upregulation of FFA
uptake by thiazolidinediones and the specific proteins involved may show
tissue and/or species specificity.
From the current results we conclude that thiazolidinediones increase FFA
uptake into muscle cells, possibly through upregulation of FAT/CD36
expression. Although FFA uptake into muscle would have a positive impact on
circulating FFA levels, lipid accumulation in muscle has repeatedly been shown
to be associated with insulin resistance
(34,
51,
54,
55) in sedentary individuals.
How is this fact reconcilable with the demonstrated ability of
thiazolidinediones to improve insulin action? A similar dichotomy is observed
for FFA flux in macrophages. In those cells, thiazolidinediones upregulate
FAT/CD36 expression and oxidized LDL entry, generating additional ligands for
PPAR, further increasing FAT/CD36 expression and lipid uptake
(17,
49). Such a cycle could be
considered proatherogenic. However, in macrophages, thiazolidinediones also
increase expression of ATP Binding Cassette-A1, the transporter responsible
for reverse cholesterol efflux
(17); thus cholesterol esters
do not accumulate within the cell. A similar balancing act may occur in
skeletal muscle cells, as we have shown that thiazolidinediones increase both
palmitate uptake and oxidation (Fig.
2), resulting in an increase in overall FFA disposal. Studies
indicate that Tgz acts to increase the mitochondrial component of palmitate
oxidation (15). We therefore
hypothesize that thiazolidinediones are involved in directing FFA to the
mitochondria by upregulating FFA uptake mediators. Directing FFA to the
mitochondria for oxidation may prevent them (either in their free form or as
triglycerides) from causing insulin resistance. Consistent with this
postulate, thiazolidinediones have been found to increase uptake and oxidation
of FFA by muscle in several animal models of insulin resistance
(16,
33). Other investigators have
found a contrasting result, Tgz inhibition of palmitate oxidation in isolated
soleus muscle (13). The reason
for this discrepancy is not apparent but may reflect differences in the
experimental systems employed. A similar situation of augmented FFA disposal
in skeletal muscle occurs with endurance training, which increases
triglyceride localization around the mitochondria
(65). Interestingly, trained
individuals have elevated intramyocellular lipid levels yet do not display
insulin resistance (27); an
increased oxidative capacity of skeletal muscle in these individuals may
provide a protective effect lacking in insulin-resistant subjects. Because FFA
uptake into muscle is impaired in type 2 diabetes patients even when removed
from the in vivo environment, thiazolidenediones may improve insulin
sensitivity by modulating FFA uptake, intracellular transport, and
-oxidation, thereby mimicking the effects of training
(26).
In summary, we have shown that muscle FFA uptake is, in part, protein mediated and acutely affected by insulin and that, in muscle from type 2 diabetic subjects, the protein-mediated component was decreased and acute insulin regulation of FFA uptake was impaired. We also showed that thiazolidinedione treatment increases FFA metabolism in concert with increased expression of the lipid scavenger and transport protein FAT/CD36. The ability of thiazolidinediones to stimulate FFA metabolism in muscle may contribute to enhanced sensitivity of insulin-stimulated glucose metabolism in this tissue in type 2 diabetes observed after treatment.
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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