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Troglitazone Regulation of Glucose Metabolism in Human Skeletal Muscle Cultures from Obese Type II Diabetic Subjects¹

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.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
To determine the effects of troglitazone on abnormal skeletal muscle glucose metabolism, muscle cultures from type II diabetic patients were grown for 4–6 weeks and then fused for 4 days either without or with troglitazone (1–5 µg/mL; chronic studies) or had troglitazone added for 90 min (1–5 µg/mL) at completion of fusion (acute studies). Acute troglitazone treatment stimulated glucose uptake, but not glycogen synthase (GS) activity 2-fold (P < 0.05) in a dose-dependent fashion and to the same extent as the addition of maximal (33 nmol/L) insulin. Maximal chronic troglitazone (5 µg/mL for 4 days) increased both glucose uptake (from 9.0 ± 1.5 to 40.9 ± 8.1 pmol/mg protein·min; P < 0.05) and GS fractional velocity (from 5.4 ± 0.7% to 20.6 ± 6.3%; P < 0.05) by approximately 4-fold. At each concentration of chronic troglitazone, glucose uptake rates were similar in the absence and presence of maximal (33 nmol/L) insulin concentrations. In contrast, insulin-stimulated GS activity was greater (P < 0.05) when maximal chronic troglitazone and acute insulin were combined than when chronic troglitazone alone was used. After 4 days of troglitazone, GLUT1 messenger ribonucleic acid and protein increased about 2-fold (P < 0.05) without a change in GLUT4 or GS messenger ribonucleic acid and protein. We conclude that troglitazone has both acute and chronic effects to improve skeletal muscle glucose metabolism of obese type II diabetic subjects. These effects involve direct insulin mimetic stimulatory actions as well as indirect insulin-sensitizing properties.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
INSULIN resistance is a major pathophysiological abnormality in obese patients with type II diabetes (1). Skeletal muscle is the principal tissue of insulin-mediated glucose disposal and the major site of peripheral insulin resistance in such subjects (2). New therapeutic agents have recently been developed to help reduce the severity of insulin resistance in this disorder. One of these is troglitazone, a member of the thiazolidinedione class of compounds that has been shown to improve glucose tolerance in insulin-resistant states. Troglitazone treatment reduces hyperglycemia, hyperinsulinemia, and hypertriglyceridemia in animal models of diabetes and in type II diabetic subjects (3, 4, 5, 6, 7). In addition to these beneficial effects, troglitazone therapy has been shown to enhance insulin-stimulated glucose uptake into peripheral tissues, including skeletal muscle (6, 7). At the cellular level, troglitazone has been shown to improve insulin action in skeletal muscle, liver, and adipose tissue (8, 9, 10, 11). However, to date, there has been no direct assessment of how this drug works in insulin-resistant human skeletal muscle from type II diabetic subjects.

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.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Experimental subjects

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.

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.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Glucose uptake activity

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.

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of acute troglitazone treatment on glucose uptake in muscle cultures from obese type II diabetic subjects. Fused multinucleated myotubes from type II diabetic subjects were washed and incubated in serum-free 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.

View larger version (21K): [in this window] [in a new window]   Figure 2. Effect of chronic troglitazone treatment on glucose uptake in muscle cultures from obese type II diabetic subjects. Muscle cultures from type II diabetic subjects were fused in the absence or presence of varying concentrations of troglitazone (1, 2, and 5 µg/mL) for 4 days. Cultures were washed and incubated in serum-free MEM for 90 min in the absence (open bars) or presence (solid bars) of 33 nmol/L insulin 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 culture. , P < 0.05 vs. corresponding value in untreated muscle cultures of type II diabetic subjects.

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.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of acute troglitazone treatment on GS activity in muscle cultures from obese type II diabetic subjects. Fused multinucleated myotubes from type II diabetic subjects were washed and incubated in serum-free MEM for 60 min in the absence (open bars) or presence (solid bars) of 33 nmol/L insulin and/or 5 µg/mL troglitazone and assayed for GS activity 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 culture. , P < 0.05 vs. corresponding value in untreated muscle cultures of type II diabetic subjects.

View larger version (19K): [in this window] [in a new window]   Figure 4. Effect of chronic troglitazone treatment on GS activity in muscle cultures from obese type II diabetic subjects. Muscle cultures from type II diabetic subjects were fused in the absence or presence of varying concentrations of troglitazone (1, 2, and 5 µg/mL) for 4 days. Cultures were washed and incubated in serum-free MEM for 90 min in the absence (open bars) or presence (solid bars) of 33 nmol/L insulin and assayed for GS activity 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 culture. , P < 0.05 vs. corresponding value in untreated muscle cultures of type II diabetic subjects.

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).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Compounds of the thiazolidinedione class, including troglitazone, have been shown to have potent glucose-lowering effects in diabetic animals and humans (5, 6, 7, 28, 29, 30). These compounds act primarily to improve impaired insulin action in human skeletal muscle, but by unknown mechanisms. The current work was therefore undertaken to determine how troglitazone influences insulin action and glucose metabolism in human skeletal muscle cell cultures obtained from insulin-resistant obese type II diabetic subjects, focusing on changes in glucose metabolism and gene expression.

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.

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of troglitazone on GLUT1 mRNA and protein expression in muscle cultures from obese type II diabetic subjects. Muscle cultures from six type II diabetic subjects were fused in the absence (-) or presence (+) of 5 µg/mL troglitazone for 4 days. RNA and total membranes were prepared, and assays were performed as described in Materials and Methods. A, Quantitation of GLUT1 mRNA. B, Representative autoradiogram of Western blot for GLUT1 protein in cultures from two diabetic subjects. C, Total membrane immunoreactive GLUT1 protein levels. Data from seven nondiabetic (ND) control subjects are shown for comparison. Results are the mean ± SEM. , P < 0.05 vs. value in nondiabetic muscle culture. , P < 0.05 vs. value in untreated muscle cultures of type II diabetic subjects.

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.

	Footnotes

 
¹ This work was supported by funds from the Medical Research Service, Department of Veteran Affairs and Veteran Affairs Medical Center (San Diego, CA), Parke Davis Pharmaceutical Research Division, Warner Lambert Co., the Whittier Institute for Diabetes Research, and Grant MO1-RR-00827 from the General Clinical Research Branch, Division of Research Resources, NIH.

Received October 28, 1997.

Revised December 31, 1997.

Accepted January 14, 1998.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. DeFronzo RA, Bonadonna RC, Ferrannini E. 1992 Pathogenesis of NIDDM. A balanced overview. Diabetes Care. 15:318–368.[Abstract]
2. Baron AD, Laasko M, Brechtel G, Edelman SV. 1991 Reduced capacity and affinity of skeletal muscle for insulin-mediated glucose uptake in non-insulin dependent diabetic subjects. J Clin Invest. 87:1186–1194.
3. Fujiwara T, Yoshioka S, Yoshioka T, Ushiyama I, Horikoshi H. 1988 Characterization of new oral antidiabetic agent CS-045. Studies in KK and ob/ob mice and Zucker fatty rats. Diabetes. 37:1549–1558.[Abstract]
4. Fujiwara T, Wada M, Fukuda K, et al. 1991 Characterization of CS-045, a new oral antidiabetic agent. II. Effects on glycemic control and pancreatic islet structure in C57L/Ksj db/db mice. Metabolism. 40:1213–1218.[CrossRef][Medline]
5. Iwamoto Y, Kuzuya T, Matsuda A, et al. 1991 Effect of new oral antidiabetic agent CS-045 on glucose tolerance and insulin secretion in patients with NIDDM. Diabetes Care. 14:1083–1086.[Abstract]
6. Suter SL, Nolan JJ, Wallace P, Gumbiner B, Olefsky J. 1992 Metabolic effects of new oral hypoglycemic agents CS-045 in NIDDM subjects. Diabetes Care. 15:193–203.[Abstract]
7. Nolan JJ, Ludvik B, Beerdsen P, Joyce M, Olefsky J. 1994 Improvement in glucose tolerance and insulin resistance in obese subjects treated with troglitazone. N Engl J Med. 331:1188–1193.[Abstract/Free Full Text]
8. Ciaraldi TP, Gilmore A, Olefsky JM, Golderg M, Heidenreich KA. 1996 In vitro studies on the action of CS-045, a new antidiabetic agent. Metabolism. 39:1056–1062.
9. Tafuri SR. 1996 Troglitazone enhances differentiation, basal glucose uptake, and GLUT1 protein levels in 3T3–L1 adipocytes. Endocrinology. 137:4706–4712.[Abstract]
10. Ciaraldi TP, Huber-Knudsen K, Hickman M, Olefsky JM. 1995 Regulation of glucose transport in cultured muscle cells by novel hypoglycemic agents. Metabolism. 44:976–982.[CrossRef][Medline]
11. Bahr M, Spelleken M, Bock M, von Holtey M, Eckel J. 1996 Acute and chronic effects of troglitazone (CS-045) on isolated rat ventricular cardiomyocytes. Diabetologia. 39:766–774.[CrossRef][Medline]
12. Henry RR, Abrams L, Nikoulina S, Ciaraldi TP. 1995 Insulin action and glucose metabolism in non-diabetic control and NIDDM subjects: comparison using human skeletal muscle cell cultures. Diabetes. 44:936–946.[Abstract]
13. Henry RR, Ciaraldi TP, Abrams-Carter L, Mudaliar S, Park KS, Nikoulina SE. 1996 Glycogen synthase activity is reduced in cultured skeletal muscle cells of NIDDM subjects: biochemical and molecular mechanisms. J Clin Invest. 98:1231–1236.[Medline]
14. Ciaraldi T, Abrams L, Nikoulina S, Mudaliar S, Henry RR. 1995 Glucose transport in cultured human skeletal muscle cells. Regulation by insulin and glucose in nondiabetic and non-insulin-dependent diabetes mellitus subjects. J Clin Invest. 96:2820–2827.
15. Henry RR, Ciaraldi TP, Mudaliar S, Abrams L, Nikoulina S. 1996 Acquired defects of glycogen synthase activity in cultured human skeletal muscle cells: influence of high glucose and insulin levels. Diabetes. 45:400–407.[Abstract]
16. National Diabetes Data Group. 1979 Classification and diagnosis of diabetes mellitus and other categories of glucose tolerance. Diabetes. 28:1039–1057.[Medline]
17. Blau HM, Webster C. 1981 Isolation and characterization of human muscle cells. Proc Natl Acad Sci USA. 78:5623–5627.[Abstract/Free Full Text]
18. Sarabia V, Lam L, Burdett E, Leiter LA, Klip A. 1990 Glucose uptake in human and animal muscle cells in culture. Biochem Cell Biol. 68:536–542.[Medline]
19. Ailhaud G. 1982 Adipose cell differentiation in culture. Mol Cell Biochem. 49:17–31.[Medline]
20. Klip A, Li G, Logan WJ. 1984 Induction of sugar uptake response to insulin by serum depletion in fusing L6 myoblast. Am J Physiol. 247:E291–E296.
21. Bradford MM. 1976 A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal Biochem. 71:248–254.[CrossRef]
22. Walker PS, Ramlal T, Sarabia V, et al. 1990 Glucose transport activity in L6 muscle cells is regulated by the cordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription. J Biol Chem. 265:1516–1523.[Abstract/Free Full Text]
23. Burnette WN. 1989 "Western blotting:" electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 112:195–203.
24. Wang AM, Doyle MV, Mark DF. 1989 Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA. 86:9717–9721.[Abstract/Free Full Text]
25. Browner MF, Nakano K, Bang AG, Fletterick RF. 1989 Human muscle glycogen synthase cDNA sequence: a negatively charged protein with an asymmetric charge distribution. Proc Natl Acad Sci USA. 86:1443–1447.[Abstract/Free Full Text]
26. Fukumoto H, Seino S, Imura H, Seino Y, Bell GI. 1988 Characterization and expression of human hepG2/erythrocyte glucose-transporter gene. Diabetes. 37:657–661.[Abstract]
27. Birnbaum MJ. 1989 Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell. 57:305–315.[CrossRef][Medline]
28. Hofmann C, Lerenz K, Colca JR. 1991 Glucose transport deficiency in diabetic animals is corrected by treatment with the oral antihyperglycemic agent pioglitazone. Endocrinology. 129:1915–1925.[Abstract]
29. Stevenson RW, Hutson NJ, Krupp MN, et al. 1990 Actions of novel antidiabetic agent englitazone in hyperglycemic hyperinsulinemic ob/ob mice. Diabetes. 39:1218–1227.[Abstract]
30. Shargill NS, Tatoyan A, Fukishima M. 1986 Effect of ciglitazone on glucose uptake and insulin sensitivity in skeletal muscle of the obese (ob/ob) mouse: distinct insulin and glucocorticoid effects. Metabolism. 35:64–70.[CrossRef][Medline]
31. Thorburn AW, Gumbiner B, Bulacan F, Brechtel G, Henry RR. 1991 Multiple defects in muscle glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin dependent diabetes mellitus. J Clin Invest. 87:489–495.
32. Horikoshi H, Okuno A, Fujiwara T, Shiota M, Sugano T. 1993 Peripheral effects of a new antidiabetic agent, CS-045: acute stimulation of insulin-induced glucose uptake in perfused rat hindlimb [Abstract 188]. Diabetes 42(Suppl 1):59A.
33. Lee M-K, Olefsky JM. 1995 Acute effects of troglitazone on in vivo insulin action in normal rats. Metabolism. 44:1166–1169.[CrossRef][Medline]
34. Shibata H, Nii S, Kobayashi M, et al. 1993 Phase I study of a new hypoglycemic agent CS-045 in healthy volunteers. Safety and pharmacokinetics in repeated administration. Rinsho Iyaku. 9:1519–1537.
35. Weinstein SP, Holand A, O’Boyle E. 1993 Effects of thiazolidinediones on glucocorticoid-induced insulin resistance and GLUT4 glucose transporter expression in rat skeletal muscle. Metabolism. 42:1365–1369.[CrossRef][Medline]
36. Sandouk T, Rada D, Hofmann C. 1993 The antidiabetic agent pioglitazone increases expression of glucose transporters in 3T3–F442A cells by increasing messenger ribonucleic acid transcript stability. Endocrinology. 133:352–359.[Abstract]
37. El-Kebbi I, Roser S, Pollet RJ. 1994 Regulation of glucose transport by pioglitazone in cultured muscle cells. Metabolism. 43:953–958.[CrossRef][Medline]
38. Mueckler M. 1994 Facilitative glucose transporters. Eur J Biochem. 219:713–725.[Medline]
39. Ciaraldi TP, Mudaliar S, Henry RR. 1995 Relationship between skeletal muscle GLUT1/GLUT4 and glucose disposal [Abstract 567]. Diabetes 44(Suppl 1):165A.
40. Bak JK, Moller N, Schmitz O, Saaek A, Pedersen O. 1992 In vivo insulin action and muscle glycogen synthase activity in type II (non-insulin-dependent) diabetes mellitus: effects of diet treatment. Diabetologia. 35:777–784.[Medline]
41. Lawrence Jr JC, Roach PJ. 1997 New insights into the role and mechanism of glycogen synthase activation by insulin. Diabetes. 46:541–547.[Abstract]
42. Kellerer M, Kroder G, Tippmer S, et al. 1994 Troglitazone prevents glucose-induced insulin resistance of insulin receptor in Rat-1 fibroblasts. Diabetes. 43:447–453.[Abstract]
43. Maegawa H, Ide R, Hasegawa M, et al. 1995 Thiazolidine derivatives ameliorate high glucose-induced insulin resistance via the normalization of protein-tyrosine phosphatase activities. J Biol Chem. 270:7724–7730.[Abstract/Free Full Text]
44. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Wilson TM, Kliewer SA. 1995 An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor (PPAR). J Biol Chem. 270:12953–12956.[Abstract/Free Full Text]
45. Park KS, Ciaraldi TP, Abrams-Carter L, Mudaliar S, Nikoulina SE, Henry RR. 1997 PPAR gene expression is elevated in skeletal muscle of obese and type II diabetic subjects. Diabetes. 46:1230–1234.[Abstract]
46. Park KS, Abrams-Carter L, Mudaliar S, Nikoulina S, Henry RR. 1997 Troglitazone effects on gene expression in human skeletal muscle involve regulation of PPAR [Abstract 85]. Diabetes. 46(Suppl 1):22A.
47. Khoursheed M, Miles PD, Gao KM, Lee MK, Moossa AR, Olefsky JM. 1995 Metabolic effects of troglitazone on fat-induced insulin resistance in the rat. Metabolism. 44:1489–1494.[CrossRef][Medline]
48. Lee M-K, Miles PDG, Khoursheed M, Gao K-M, Moossa AR, Olefsky JM. 1994 Metabolic effects of troglitazone on fructose-induced insulin resistance in the rat. Diabetes. 43:1435–1439.[Abstract]
49. Yagi N, Takasu N, Higa S, Ishikawa K, Murakami K, Mimura G. 1995 Effect of troglitazone, a new oral antidiabetic agent, on fructose-induced insulin resistance. Horm Metab Res. 27:439–441.[Medline]
50. Stevenson RW, McPherson RK, Persson LM, et al. 1996 The antihyperglycemic agent englitazone prevents the defect in glucose transport in rats fed a high-fat diet. Diabetes. 45:60–66.[Abstract]

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