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Original Studies |
Department of Medicine, University of California-San Diego, La Jolla, California 92093; and Veterans Affairs Medical Center, San Diego, California 92161
Address all correspondence and requests for reprints to: Robert R. Henry, M.D., Veterans Affairs Medical Center, San Diego (V111G), 3350 La Jolla Village Drive, San Diego, California 92161.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In recent studies from our laboratory, we have shown that human skeletal muscle grown in culture from needle biopsy specimens retain the metabolic characteristics of intact skeletal muscle (12). Muscle cultures from type II diabetic subjects demonstrate decreased glucose transport and glycogen synthase (GS) activity even after 4–6 weeks of growth in normal medium conditions (12, 13, 14, 15).
In the current study, we sought to exploit the advantages of this muscle culture system to investigate the cellular mechanisms by which troglitazone improves defects of glucose metabolism in skeletal muscle from type II diabetic subjects.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Fourteen patients with type II diabetes provided muscle tissue
for troglitazone treatment studies. Acute troglitazone studies were
conducted in cultures from six type II diabetic patients, four of whom
were treated with oral antidiabetic agents (metformin-2, glipizide-1,
glipizide/metformin-1), one with insulin, and one with diet only.
Chronic troglitazone treatment studies were performed in cultures from
eight diabetic subjects. Six of these patients were treated with oral
antidiabetic agents (glyburide-1, glipizide-1, glipizide/metformin-4),
and two were treated with diet only. Data from seven healthy
nondiabetic controls subjects are provided for comparative purposes.
Glucose tolerance was determined in all subjects after a 75-g oral
glucose tolerance test (16). Characteristics of the study groups are
summarized in Table 1
. Diabetic patients
had their medication withheld on the morning of biopsy. None of the
nondiabetic control subjects had a family history of type II diabetes
or were taking any medications known to influence glucose metabolism.
The experimental protocol was approved by the committee on human
investigation of the University of California-San Diego. Informed
written consent was obtained from all subjects after explanation of the
protocol.
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Cell culture materials were purchased from Irvine Scientific (Irvine, CA), except for skeletal muscle growth medium and bovine insulin, which were obtained from Clonetics Corp. (San Diego, CA). FBS was purchased from Gemini (Calabasas, CA). All radioisotopes were obtained from DuPont-New England Nuclear (Boston, MA). Polyclonal antisera against GLUT1 (RaGLUTRANS) and GLUT4 (RaIRGT) were purchased from East Acres Biologicals (Cambridge, MA). Polyclonal antisera raised to the COOH-terminal sequence of GS (13) was a gift from Dr. L. Groop (Malmo, Sweden). An antirabbit IgG conjugated with horseradish peroxidase and the enhanced chemiluminescence kit were obtained from Amersham (Arlington Heights, IL). BSA (fraction V), peptastatin, leupeptin, phenylmethylsulfonylfluoride, 2-deoxyglucose, L-glucose, glycogen, glucose-6-phosphate (G-6-P), and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Troglitazone [(±)-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-ylmethoxy)benzyl]-2,4 thiazolidinedione] was provided by Dr. Alan Saltiel at Parke-Davis Pharmaceutical (Ann Arbor, MI).
Cell culture and treatment
The methods for muscle biopsy, cell isolation, and growth have been detailed previously (12). Percutaneous biopsies were obtained from vastus lateralis muscle using a 5-mm side-cutting needle. The method used for clonal growth of human skeletal muscle is a modification of those of Blau and Webster (17) and Sarabia et al. (18). Medium was changed every other day. Troglitazone was dissolved in dimethylsulfoxide and added to cells with medium. The final concentration of dimethylsulfoxide did not exceed 0.05%. For acute studies, troglitazone was added at a concentration of 1, 2, or 5 µg/mL for 90 min with or without 33 nmol/L insulin. In the chronic studies, troglitazone was added at concentrations of 1, 2, and 5 µg/mL for 4 days beginning at the initiation of fusion. At completion of fusion, cells were incubated in the absence or presence of 33 nmol/L insulin for 90 min.
Troglitazone treatment had no significant effect on cell number or protein. There were no obvious differences in cell morphology after treatment, including no visible accumulation of lipid, as determined by Oil Red O staining. Levels of messenger ribonucleic acid (mRNA) for glyceraldehyde-3-phosphate dehydrogenase, an enzyme highly induced during adipocyte differentiation (19) or by troglitazone in adipocyte precursors (9), was not altered by troglitazone treatment of human skeletal muscle cell (HSMC) (not shown).
Glucose uptake assay
The procedure for glucose uptake measurement was modified from that described by Klip et al. (20) and has been described in detail (12). This procedure measures the uptake (transport plus phosphorylation) of deoxyglucose. Under these study conditions phosphorylation was not rate limiting for deoxyglucose accumulation. Protein was measured by the Bradford dye-binding method (21).
Membrane preparation
Cells for membrane preparation were grown in 100-mm dishes, and total membranes were prepared by the method developed by Walker et al. for L6 cells (22), as described in an earlier publication (12). GLUT1 and GLUT4 protein expression was measured in total membranes prepared on parallel plates to those used for glucose transport assays.
Detection of glucose transporter protein
Membrane preparations were diluted 1:1 in 2 x Laemmli’s buffer without ß-mercaptoethanol and heated for 5 min at 90 C. Proteins were separated on 10% SDS-PAGE gels and then transferred to a nitrocellulose membrane. GLUT1 was identified using a rabbit polyclonal antisera (RaGLUTRANS) that recognizes human GLUT1. A polyclonal antisera specific for GLUT4 (RaIRGT) was also employed. The second antibody was antirabbit IgG conjugated with horseradish peroxidase. Immune complexes were detected using an enhanced chemiluminescence kit. Quantification was performed with a scanning laser densitometer (Scan Analysis, Biosoft, Cambridge, UK). All results were normalized for protein. Standards for GLUT1 (human erythrocyte membranes) and GLUT4 (human adipocyte low density microsomes) were included in all gels to permit comparison of results from different blots.
GS activity
The activity of GS was measured as described in detail previously (12). GS activity is expressed as fractional velocity (FV = % of the ratio of activity at 0.1 mmol/L G-6-P/10 mmol/L G-6-P).
Determination of GS protein
Western blot analysis was performed by the method of Burnette (23), as detailed previously (12). In brief, cell sonicates were solubilized in Laemmli’s buffer, and proteins were separated on 8% SDS-PAGE gels and electrophoretically transferred to a nitrocellulose membrane. GS protein was identified using affinity-purified polyclonal antibody raised in rabbits against an oligopeptide (12-mer) specific for the carboxyl-terminal sequence of the enzyme. The secondary antibody for GS was antirabbit IgG conjugated with horseradish peroxidase. Proteins were visualized with an ECL detection kit and exposed to autoradiograph film (XAR-5 Omat, Eastman Kodak, Rochester, NY).
Quantitation of GLUT1, GLUT4, and GS mRNA by reverse transcriptase-PCR
To measure muscle GLUT1, GLUT4, and GS mRNA expression, a quantitative competitive PCR assay was developed (24). The internal standards were designed to use the same primers as the target, but to yield different sized PCR products (308 vs. 340 bp for GLUT1, 268 vs 245 bp for GLUT4, and 262 vs. 310 bp for GS).
Two synthetic genes, which were used to produce complementary RNA (cRNA) for each internal control, were constructed by amplification of a heterologous DNA fragment with a pair of composite primers. The amplified sequences contain the sequences for GLUT1, GLUT4, and GS primers that are contiguous to the heterologous DNA fragment. These sequences also contain an EcoRI restriction site at the 5'-end and a poly(deoxythymidine) and BamHI restriction site at the 3'-end. After digestion with EcoRI and BamHI, the fragments for GLUT1 and GLUT4 were subcloned into a pGEM T vector, and the GS fragment was subcloned into a pGEM 3Z vector. The resulting pKSP-1 (GLUT1 and GLUT4) and pKSP-2 (GS) plasmids were used as a template for transcription by T7 RNA polymerase according to the transcription protocol of Promega (Madison, WI). The cRNAs were purified using phenol chloroform extraction and a Qiagen Oligotex mRNA kit (Chatsworth, CA), then quantitated by absorbance spectrophotometry at 260 nm and stored at -70 C.
PCR primers were synthesized on an automated solid phase DNA synthesizer, using standard phosphoramidite chemistry with purification through Sep-Pak columns (Applied Biotechnology, Milford, MA). The upstream primer for GS was ATC ATG CCA GCG CGG ACC AA, and the downstream primer was GCA CTG CTA TTG AAG AGG CC (25). The upstream primer for GLUT1 was ATC ATC GGT GTG TAC TGC GG, and the downstream primer was GGT CAT GGG TCA CGT CAG CT (26). The upstream primer for GLUT4 was CAG AGC TAC AAT GAG ACG TGG, and the downstream primer was CAT AGG AGG CAG CAG CGT TG (27).
For reverse transcriptase-PCR, total RNA was extracted from muscle cultures using Trizol reagent (Life Technologies, Grand Island, NY). Total RNA (0.25 µg) and increasing concentrations of each cRNA construct were reverse transcribed with 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies) with 200 ng random hexamers in a 20-µL reaction volume for 1 h at 37 C, then heated to 70 C for 15 min.
PCR amplification was performed using 2 µL of the reverse transcriptase reaction in PCR buffer (50 mmol/L KCl, 1.5 mmol/L MgCl2, and 10 mmol/L Tris-HCl, pH 8.3), 200 µmol/L deoxy-NTP, 25 pmol of forward and reverse primers, and 1 U AmpliTaq Gold (Perkin-Elmer/Cetus, Norwalk, CT) in a final volume of 50 µL in a DeltaSystemII thermocycler (Ericomp, San Diego, CA). Cycling parameters were 94 C for 1 min, 57 C for 1 min, and 72 C 1 min 30 s for 33 cycles for GLUT4; and 94 C for 1 min, 60 C 1 min, and 72 C 1 min 30 s for 30 cycles for GLUT1 and GS. Samples were incubated at 95 C for 12 min for initial denaturation and for 7 min at 72 C at the end of the last cycle. Ten microliters of the amplification products were separated on a 3% agarose gel, stained with ethidium bromide, photographed, and quantitated using NIH Image software (available from the NIH), or gels were photographed with a DC40 camera and quantitated using Kodak Digital Science 1D Image Analysis software. Both methods gave equivalent results. The ratio of each target product/cRNA standard was plotted against the number of copies of cRNA added to yield the equivalence point between cRNA and target mRNA. The r value of the standard curves was between 0.95–1.00, with an interassay variation of about 15%.
Statistical analysis
Statistical significance was evaluated using repeated measure ANOVA or Student’s t test for paired comparison. Significance was accepted at the P < 0.05 level.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Acute insulin administration (33 nmol/L for 90 min) increased
glucose uptake in diabetic muscle cultures from 9.5 ± 0.8 to
13.7 ± 1.2 pmol/mg protein·min (P < 0.01; Fig. 1). Acute administration of troglitazone
alone (5 µg/mL for 90 min) resulted in significant stimulation of
glucose uptake to levels (14.0 ± 1.5 pmol/mg protein·min;
P < 0.01) comparable to those achieved after acute
insulin treatment (P = NS) and to those present in
nondiabetic control muscle (15.2 ± 1.9 pmol/mg protein·min;
P = NS) in the absence of insulin. The ability of
troglitazone to stimulate glucose uptake was dose dependent and maximal
at 5 µg/mL (Fig. 1). The combination of 33 nmol/L insulin and 5
µg/mL troglitazone had no greater effect on muscle glucose uptake
than either insulin or troglitazone alone (16.1 ± 1.6
vs. 13.7 ± 1.2 and 14.0 ± 1.5 pmol/mg
protein·min, respectively; all P = NS). Acute
treatment with troglitazone did not alter GLUT1 or GLUT4 protein levels
in the total membrane fraction.
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). After maximal chronic troglitazone
treatment (5 µg/mL for 4 days), glucose uptake was increased in both
the absence (from 9.0 ± 1.5 to 40.9 ± 8.1;
P < 0.05) and presence of added insulin (from
13.1 ± 1.8 to 33.9 ± 4.5 pmol/mg protein·min;
P < 0.05) and to similar values (P =
NS). Although chronic troglitazone treatment increased
insulin-independent and insulin-dependent glucose uptake in diabetic
muscle in a dose-dependent manner, the effect on insulin-independent
uptake was disproportionately greater (4.5-fold vs.
2.5-fold; P < 0.05). As a result of the greater
increases in insulin-independent values, there was no significant
incremental glucose uptake response to acute insulin administration at
any troglitazone concentration. The absolute rates of both
insulin-independent and insulin-stimulated glucose uptake after maximum
troglitazone treatment were significantly greater than the
corresponding rates of glucose uptake present in nondiabetic control
muscle (40.9 ± 8.1 vs. 15.2 ± 1.9 and 33.9
± 4.5 vs. 20.4 ± 2.3 pmol/mg protein·min,
respectively; both P < 0.05). The absolute rates of
glucose uptake were 2- to 3-fold higher after chronic compared to acute
maximal troglitazone treatment. Chronic troglitazone treatment was also
able to up-regulate transport in cells from obese nondiabetic subjects
by 2- to 3-fold; transport rates after treatment were similar in
nondiabetic and diabetic cells.
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In untreated diabetic muscle, GS FV rose nearly 2-fold (from
6.9 ± 1.3% to 11.9 ± 2.2%; P < 0.05) in
response to acute insulin stimulation (Fig. 3). Acute troglitazone treatment (5
µg/mL for 90 min) had no significant effect on GS FV in either the
absence (9.0 ± 2.6% vs. 6.9 ± 1.3%;
P = NS) or presence (11.1 ± 3.2% vs.
11.9 ± 2.2%; P = NS) of maximal (33 nmol/L)
insulin.
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). However, unlike the glucose transport
response, maximal troglitazone treatment resulted in corresponding
increases in GS FV in the absence (from 5.4 ± 0.7% to 20.6
± 6.3%; P < 0.05) and presence (8.2 ± 1.0% to
27.8 ± 6.1%; P < 0.05) of added insulin, such
that a significant incremental response to insulin was maintained. As a
result of these marked increases after maximal chronic troglitazone,
both insulin-independent and insulin-stimulated GS FV increased to the
corresponding values present in nondiabetic control subjects (both
P = NS from nondiabetic control values). Troglitazone
treatment was also able to increase both basal and insulin-stimulated
GS FV by 3-fold in cells from obese nondiabetic subjects.
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GLUT1 protein (13.6 ± 2.3 vs. 26.2 ± 3.5 arbitrary units/10 µg protein; P < 0.05), but not GLUT1 mRNA (84 ± 13 vs. 105 ± 16 x 105 copies/µg total RNA; P = NS), was lower in diabetic than in nondiabetic control muscle. After 4 days of troglitazone treatment, significant increases occurred in GLUT1 mRNA (from 84 ± 13 to 122 ± 25 x 105 copies/µg total RNA; P < 0.05) and GLUT1 protein (from 13.6 ± 2.3 to 27.1 ± 6.9 arbitrary units/10 µg protein; P < 0.05). GLUT1 protein was normalized by troglitazone treatment (27.1 ± 6.9 vs. 26.2 ± 3.5 AU/10 µg protein; P = NS). Troglitzone treatment had no significant effect on GLUT1 protein in nondiabetic cells. GLUT4 and GS mRNA and protein were unchanged after 4 days of troglitazone treatment (data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Impaired glucose uptake/transport and GS activity in skeletal muscle, especially in response to insulin, are major defects in type II diabetes (2, 31) that continue to be manifest in the metabolic behavior of skeletal muscle cultures (12, 13, 14, 15). Using these insulin-resistant skeletal muscle cultures, we have confirmed that troglitazone has acute effects on glucose uptake that are virtually identical to maximal insulin concentrations. Interestingly, these acute insulinomimetic effects were selective, stimulating glucose uptake but not GS activity. As glucose transport and GS in muscle tend to be activated in parallel by insulin, such findings imply that troglitazone may not act acutely through the classic insulin receptor-signal transduction cascade, but through some undefined alternative pathway. Acute stimulation of glucose transport by troglitazone was not associated with a change in total cellular GLUT1 or GLUT4 protein content. Therefore, the effects of troglitazone on glucose transport possibly result from enhanced activity and/or translocation of either or both of these transporter isoforms and/or from stimulation of hexokinase. As insulin (20–22 pmol/L from serum) was present in the medium during cell differentiation, but not during acute stimulation unless noted, we cannot rule out an effect of acute troglitazone exposure to potentiate the residual effect of that insulin, leading to transport stimulation. However, if such potentiation were to occur, it would also have to be specific for signaling to transport, as basal GS activity was unaltered.
In addition to the acute insulinomimetic effect on glucose uptake, troglitazone has been shown to acutely potentiate insulin-stimulated glucose uptake when infused with insulin into normal rats (32, 33). In the current study, troglitazone was not shown to have acute insulin-potentiating properties on either glucose uptake or GS activity when evaluated at maximal combined insulin and troglitazone concentrations. However, to effectively rule out an acute insulin-potentiating effect of troglitazone, muscle cultures would need to be evaluated further at submaximal insulin and troglitazone concentrations.
To evaluate chronic effects on muscle glucose metabolism, troglitazone was added over a range of concentrations during the 4 days of fusion to multinucleated myotubes. Chronic treatment of diabetic muscle cultures with troglitazone increased both insulin-independent and insulin-stimulated glucose uptake activity in a dose-dependent manner. Studies in healthy volunteers showed that oral troglitazone administration (400 mg, twice daily, for 7 days) resulted in steady state plasma concentrations of 2 µg/mL (34). Thus, the concentrations employed in the current study are in the therapeutic range. At the highest dose of troglitazone (5 µg/mL) tested, glucose uptake in the absence of added insulin was more than 3-fold greater than when maximal acute insulin was added to diabetic muscle cells not treated with troglitazone. In addition, as occurred after acute troglitazone administration, more prolonged administration of troglitazone alone stimulated glucose uptake as much as adding maximal acute insulin to troglitazone-treated cells. Prolonged troglitazone treatment also increased insulin-independent and insulin-stimulated glucose uptake of insulin-resistant diabetic muscle to significantly higher levels than those achieved in nondiabetic control cells. Thus, these studies provide further evidence that chronic troglitazone has pronounced effects on muscle glucose transport, independent of and greater than maximal acute insulin administration alone. Such results are consistent with previous studies demonstrating effects of thiazolidinedione to increase insulin-independent and insulin-stimulated glucose transport into animal muscle (28, 30, 35), 3T3-L1 and 3T3-F442A adipocytes (9, 36), BC3H-1 myocytes (37), L6 myocytes and myotubes (10), as well as cardiac myocytes (11). Thus, our studies confirm that when administered chronically, troglitazone appears to have insulin-sensitizing properties.
Glucose transport into insulin target tissues such as adipose and
muscle is mediated by the transporter isoforms GLUT1 and GLUT4 (38).
GLUT1 protein is believed to be primarily responsible for
insulin-independent and GLUT4 to be primarily responsible for
insulin-stimulated glucose transport in muscle tissue (38). We have
again demonstrated that GLUT1 expression is reduced in diabetic muscle,
an observation made in both muscle cultures (12) (Fig. 5) and skeletal muscle biopsies (39).
After 4 days of troglitazone treatment, GLUT1 mRNA levels increased
significantly in concert with GLUT1 protein levels in these diabetic
muscle cultures (Fig. 5). These results are consistent with a
gene-enhancing effect of chronic troglitazone on GLUT1 expression,
which could contribute to the dramatic improvement in
insulin-independent transport activity that resulted.
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Impairments in GS activity are found in skeletal muscle biopsies and cultures from type II diabetic subjects (13, 31, 40) and involve multiple kinetic defects, with variable changes in total enzyme activity and protein expression (13, 31). The response of GS to chronic troglitazone treatment was similar to that of glucose transport, with dose-dependent increases in both insulin-independent and insulin-stimulated fractional velocities from diabetic muscle. Thus, troglitazone can also normalize the defective muscle GS activity from insulin-resistant obese diabetic subjects. However, at the highest troglitazone concentration, insulin-stimulated GS activity, unlike glucose transport, was significantly increased compared to its activity in the absence of insulin. This effect could not be explained by enhanced gene expression, as neither mRNA nor protein levels of GS were influenced by chronic troglitazone treatment. In agreement with the current results, troglitazone has been shown to increase GS activity in BC3H-1 myocytes and HepG2 cells by increasing the affinity for G-6-P while not altering total activity (8). The ability of troglitazone treatment to increase GS activity while not influencing total activity or protein expression suggests enhanced activation of the enzyme through dephosphorylation. The key enzymes regulating GS phosphorylation state, GS kinase-3 and protein phosphatase-1G (41), may represent additional sites of troglitazone action.
The mechanisms by which troglitazone acts to stimulate glucose
transport, GS, and gene expression remain to be defined, but may be
distinct for the two responses. Although acute effects of troglitazone
have been reported both in vivo on glucose disposal (33) and
in vitro to reduce hyperglycemia-induced inhibition of
insulin receptor kinase activity (42, 43), most actions require
prolonged exposure. The ability of troglitazone and other
thiazolidinediones to augment glucose transport appear to be at least
in part dependent on protein synthesis through increases in transporter
expression. Such effects may be mediated through peroxisome
proliferator activated receptors (PPAR), specifically the PPAR
isoform, a nuclear receptor for which troglitazone has recently been
identified as a ligand (44). PPAR is present in HSMC (45) and
appears to be up-regulated by troglitazone (46). However, as noted
earlier, effects on transporter localization and intrinsic activity as
well as activation of hexokinase or other proteins cannot be dismissed.
Similar to the response in human muscle cultures, stimulation of GS in
liver cells by troglitazone has been shown to be independent of protein
synthesis (8), but still slow in onset.
The ability of troglitazone to improve glucose tolerance while lowering insulin levels in hyperinsulinemic, insulin-resistant humans (5, 6, 7) and animals (3, 4, 35, 47, 48, 49) has been characterized as an insulin-sensitizing action. Yet the major result of troglitazone treatment in the current study was to increase glucose transport and GS activity independent of added insulin. Insulin stimulation was either maintained or overcome, certainly not augmented, compared to that by troglitazone alone. The argument can be made that human muscle cultures are only modestly insulin responsive, and insulin-independent glucose transport might be the major activity in these cells. However, increases in insulin-independent glucose transport in response to thiazolidinedione treatment have also been reported in L6 myocytes and myotubes (10), BC3H-1 myocytes (36), cardiomyocytes (11) and 3T3-L1 adipocytes (9), cell lines with a wide range of insulin responsiveness. Furthermore, adipocytes from thiazolidinedione-treated rodents display increases in insulin-independent glucose transport (50) and lipogenesis (29) that parallel increases in basal whole body glucose utilization (28, 30, 35). Insulin stimulation of GS in HSMC is of the same magnitude as that seen in muscle biopsies after insulin infusion (31), and troglitazone is able to stimulate FV to nearly the same extent as insulin. As insulin-independent glucose transport in muscle cultures occurs in the complete absence of insulin, these data, as well as the current results, suggest that troglitazone may serve, at least acutely, as an insulin mimicker to stimulate glucose metabolism, with decreases in circulating insulin levels being an adaptation to improvement in glucose utilization. Regardless of the exact mechanism, troglitazone is capable of inducing major increases in glucose metabolism in muscle from subjects with type II diabetes, reversing the defects in glucose transport and GS characteristic of this disease.
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Footnotes |
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Received October 28, 1997.
Revised December 31, 1997.
Accepted January 14, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). J Biol Chem. 270:12953–12956. gene expression is elevated
in skeletal muscle of obese and type II diabetic subjects. Diabetes. 46:1230–1234.[Abstract]
[Abstract 85]. Diabetes.
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 I. Gabriely, R. Wozniak, M. Hawkins, and H. Shamoon Troglitazone Amplifies Counterregulatory Responses to Hypoglycemia in Nondiabetic Subjects J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 521 - 528. [Abstract] [Full Text]
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 C. Fürnsinn, B. Brunmair, S. Neschen, M. Roden, and W. Waldhäusl Troglitazone Directly Inhibits CO2 Production from Glucose and Palmitate in Isolated Rat Skeletal Muscle J. Pharmacol. Exp. Ther., May 1, 2000; 293(2): 487 - 493. [Abstract] [Full Text]
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S. Schinner, C. Dellas, M. Schroder, C. A. Heinlein, C. Chang, J. Fischer, and W. Knepel Repression of Glucagon Gene Transcription by Peroxisome Proliferator-activated Receptor gamma through Inhibition of Pax6 Transcriptional Activity J. Biol. Chem., January 11, 2002; 277(3): 1941 - 1948. [Abstract] [Full Text] [PDF]
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MEM for 90 min in the absence
(open bars) or presence (solid bars) of
33 nmol/L insulin and/or 1–5 µg/mL troglitazone and assayed for
glucose uptake as described in Materials and Methods.
Data from seven nondiabetic (ND) control subjects are shown for
comparison. Results are the mean ± SEM. *,
P < 0.05 vs. corresponding basal
value. 
, P < 0.05 vs.
corresponding value in nondiabetic muscle cultures. 
,
P < 0.05 vs. corresponding value in
untreated muscle cultures of type II diabetic subjects.
