Diabetes
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ciaraldi, T. P.
Right arrow Articles by Henry, R. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ciaraldi, T. P.
Right arrow Articles by Henry, R. R.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?
Diabetes 51:30-36, 2002
© 2002 by the American Diabetes Association, Inc.

Regulation of Glucose Transport and Insulin Signaling by Troglitazone or Metformin in Adipose Tissue of Type 2 Diabetic Subjects

Theodore P. Ciaraldi1, Alice P.S. Kong1, Neelima V. Chu1, Dennis D. Kim1, Sunita Baxi1, Mattias Loviscach1, Ray Plodkowski1, Richard Reitz2, Michael Caulfield2, Sunder Mudaliar1, and Robert R. Henry1

1 VA San Diego Healthcare System and the Department of Medicine, University of California, San Diego, California
2 Quest Diagnostics, San Juan Capistrano, California


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Type 2 diabetic subjects failing glyburide therapy were randomized to receive additional therapy with either metformin (2,550 mg/day) or troglitazone (600 mg/day) for 3–4 months. Biopsies of subcutaneous abdominal adipose tissue were obtained before and after therapy. Glycemic control was similar with both treatments. Metformin treatment increased insulin-stimulated whole-body glucose disposal rates by 20% (P < 0.05); the response to troglitazone was greater (44% increase, P < 0.01 vs. baseline, P < 0.05 vs. metformin). Troglitazone-treated subjects displayed a tendency toward weight gain (5 ± 2 kg, P < 0.05), increased adipocyte size, and increased serum leptin levels. Metformin-treated subjects were weight-stable, with unchanged leptin levels and reduced adipocyte size (to 84 ± 4% of control, P < 0.005). Glucose transport in isolated adipocytes from metformin-treated subjects was unaltered from pretreatment. Glucose transport in both the absence (321 ± 134% of pre-Rx, P < 0.05) and presence of insulin (418 ± 161%, P < 0.05) was elevated after troglitazone treatment. Metformin treatment had no effect on adipocyte content of GLUT1 or GLUT4 proteins. After troglitazone treatment, GLUT4 protein expression was increased twofold (202 ± 42%, P < 0.05). Insulin-stimulated serine phosphorylation of Akt was augmented after troglitazone (170 ± 34% of pre-Rx response, P < 0.05) treatment and unchanged by metformin. We conclude that the ability of troglitazone to upregulate adipocyte glucose transport, GLUT4 expression, and insulin signaling can contribute to its greater effect on whole-body glucose disposal.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Major characteristics of type 2 diabetes include impaired utilization of glucose and resistance to the ability of insulin to stimulate glucose uptake and disposal in tissue (1). Insulin stimulation of glucose uptake is initiated by the hormone binding to cell surface receptors with activation of the intrinsic tyrosine kinase activity of the receptor (rev. in [2]). Critical events in the subsequent phosphorylation cascades include receptor-mediated tyrosine phosphorylation of insulin receptor substrates (IRSs), especially IRS-1, followed by association of IRS-1 with the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and activation of the kinase. The lipid products of PI3K contribute to activation of the serine kinase Akt (protein kinase B), followed by translocation of the insulin-regulated glucose transporter isoform, GLUT4, from intracellular pools to the plasma membrane (3,4). Numerous investigations have established PI3K as essential, if not sufficient, for glucose transport stimulation (3,4), whereas the role of Akt is still controversial (5,6). Each of these events and proteins represent a potential site or cause of insulin resistance.

Glucose intolerance in type 2 diabetes is manifested by defects in glucose transport into muscle (6,7) and adipose tissue (8). In adipose tissue from diabetic subjects, defective glucose transport has been shown to result from a reduced complement of insulin-stimulated GLUT4 in intracellular pools (9). Insulin receptor tyrosine kinase activity is also impaired in adipocytes of obese type 2 diabetic subjects (10). Both the expression and insulin-stimulated phosphorylation of IRS-1 are reduced in adipose tissue from diabetic subjects (11). Events downstream of IRS-1 are also impacted, as insulin stimulation of PI3K activity and phosphorylation of Akt are impaired (11). Thus, adipose tissue insulin resistance in type 2 diabetes involves defects in both insulin signaling pathways and final effector systems, i.e., glucose transporters.

A number of therapeutic approaches have proven useful in controlling hyperglycemia and improving insulin action in type 2 diabetic patients. These include weight loss (12), exercise (13), sulfonylureas (14), biguanides such as metformin (15), and thiazolidinediones (16,17). More recently, combination therapies have been used with increasing frequency and effectiveness (18). Although the general mechanisms by which these various therapies exert their hypoglycemic effects are known, tissue-specific effects in human type 2 diabetes are less well understood.

In the current study, type 2 diabetic individuals failing control with sulfonylurea therapy had either metformin or the thiazolidinedione troglitazone added to their treatment regimen. The goal was to match the extent of glycemic control and compare the impact of the therapies on previously reported defects in insulin signaling and glucose transport in isolated adipocytes as well as on whole-body glucose disposal and insulin action.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and treatment protocol.
A total of 21 male and female type 2 diabetic subjects (between the ages of 30 and 70 years) who were poorly controlled (HbA1c >8.5% and fasting plasma glucose >140 mg/dl) on maximal doses of any sulfonylurea agents were recruited. Except for diabetes, the subjects were healthy and on no other medications known to influence glucose metabolism. After being screened, their existing sulfonylurea medication was discontinued, and all subjects were uniformly started on glyburide 10 mg b.i.d. for 4 weeks. Baseline studies were performed, and then the subjects were randomized to either the troglitazone or metformin treatment group. Treatment involved troglitazone titration of up to 600 mg per day or metformin up to 2,550 mg per day for 4–6 weeks as required to achieve glycemic goals. After 3–4 months of troglitazone or metformin treatment, patients were readmitted for repeat studies. Subjects were counseled to consume a fixed calorie diet for the duration of the study. Insulin action was determined by a 3-h hyperinsulinemic (300 mU · m–2 · min–1)–euglycemic (5.0–5.5 mmol/l) clamp, and the glucose disposal rate was measured during the last 30 min of the clamp (19). Glucose and insulin (20) levels were determined with standard techniques. Serum leptin levels were measured by radioimmunoassay (21). 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 the protocol was explained.

Dual-energy X-ray absorptionometry scanning.
Whole-body composition was determined by dual-energy X-ray absorptionometry (DEXA) scanning (22). Anthropometric measurements of skinfold thickness and waist-to-hip ratio were performed by a single experienced individual.

Materials.
Human biosynthetic insulin was kindly supplied by Eli Lilly (Indianapolis, IN). Collagenase was purchased from Worthington (Freehold, NJ). Bovine serum albumin (BSA; Cohn fraction V) was obtained from Roche (Indianapolis, IN). [14C]3-O-methylglucose was purchased from New England Nuclear (Boston, MA). Antibodies were obtained from the following sources: GLUT1 and GLUT4 were from Biogenesis (Kingston, NH), pS473-Akt was from New England Biolabs (Beverly, MA), IRS-1 and p85 were from Upstate Biotechnology (Sarnac Lake, NY), Akt 1/2 and p110ß were from Santa Cruz Biotechnology (Santa Cruz, CA), and peroxisome proliferator–activated receptor-{gamma} (PPAR-{gamma}) was from Biomol (Plymouth Station, MA). Horseradish peroxidase–conjugated anti-mouse and anti-rabbit IgGs were from Amersham (Arlington Heights, IL), and SuperSignal-enchanced chemiluminesence substrate was from Pierce (Rockford, IL). Electrophoresis reagents were purchased from Bio-Rad (Richmond, CA). All other chemicals were reagent grade and purchased from Sigma (St. Louis, MO).

Adipose tissue biopsy and preparation of human adipocytes.
Adipose tissue was obtained by needle biopsy of the lower subcutaneous abdominal depot using a 5-mm side-cutting needle. Lidocaine (1%) was infiltrated in a square-field fashion, and the biopsy was taken from the center of the field. Isolated adipocytes were prepared by a modification (8) of the method of Rodbell (23). After digestion and filtration, the cells were washed twice in a buffer consisting of 150 mmol/l NaCl, 5 mmol/l KC1, 1.2 mmol/l MgSO4, 1.2 mmol/l CaC12, 2.5 mmol/l NaH2PO4, 10 mmol/l HEPES, and 2 mmol/l pyruvate (pH 7.4), supplemented with 4% BSA. The cells were then divided, and one portion was washed twice more in the same buffer and resuspended at ~2–5 x 105 cells/ml for glucose transport assay. The major portion of the cells were washed twice in a buffer (HEPES washing salts [HWS]) consisting of 116 mmol/l NaCl, 5 mmol/l KCl, 0.5 mmol/l MgSO4, 0.7 mmol/l CaCl2, 25 mmol/l HEPES, 5 mmol/l glucose, and 2% BSA (pH 7.4) and resuspended at ~1 x 105 cells/ml before cell extraction.

Cell counts were performed by a modification of method III of Hirsch and Gallian (24), in which cells were fixed in 2% osmium tetroxide and counted with a model ZB Coulter Counter (Coulter Electronics, Hialeah, FL) equipped with a 400-µm aperture tube. Osmium fixed adipocytes were sized using a light microscope with a calibrated ocular scale. Diameters of 150–200 cells were measured for calculation of cell volume.

3-0-methylglucose transport.
Adipocytes (~5 x 105 cells/ml) were incubated for 60 min at 37°C in the absence or presence of insulin (8.5 nmol/l). Transport activity was assessed by measuring the initial rates of uptake of 3-0-methylglucose (15–20 µmol/l), using a modification (8) of the method of Whitesell and Gliemann (25).

Adipocyte treatment and extraction.
Adipocytes in HWS were incubated for 50 min at 37°C with gentle shaking. Cells were treated with or without insulin for 15 min at 37°C and then rapidly washed twice in 17°C, insulin-free, BSA-free HWS buffer as previously described (26). A twice-concentrated solubilization buffer was then added: final concentrations were -20 mmol/l Tris-HCl, 145 mmol/l NaCl, 10% glycerol, 5 mmol/l EDTA, 1% Triton X-100, 0.5% NP-40, 200 µmol/l sodium orthovanadate, 200 µmol/l phenylmethylsulfonyl fluoride, 1 µmol/l leupeptin, 1 µmol/l pepstatin, and 10 mg/ml aprotinin (pH 7.5). After lysis/extraction for 30 min at 4°C with repeated vortexing, nonsolubulized material was removed by centrifugation at 14,000g (10 min, 4°C), and the total cell extracts were stored at -70°C before analysis.

Electrophoresis and Western blotting.
Procedures for the electrophoresis, transfer, and Western blotting of proteins are similar to standard methods (27). Detection was by enhanced chemiluminescence followed by densotometric analysis. Quantitation of the blots was performed using ScanAnalysis software (Biosoft, Cambridge, U.K.). An internal standard, prepared from adipocytes of a single subject, was included in each gel to permit for correction of variability between blots.

Statistical analysis.
Statistical analysis was performed using the StatView II program (Abacus Concepts, Berkeley, CA). Statistical significance was evaluated with Students’s t test and repeated measures analysis of variance. Paired analysis was used to determine the effect of treatment in the same subject. Data are presented as means ± SE. Significance was accepted at the P < 0.05 level. Because of limitations in tissue availability, not all analyses could be performed in all subjects. The number of subjects studied is given in the table and figure legends.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experimental subjects and in vivo effects of treatment.
After initial screening, type 2 diabetic subjects failing control on glyburide alone were randomized into two groups and fully characterized, including analysis of body composition by waist/height measurements, skinfold thickness, DEXA scanning, hyperinsulinemic-euglycemic clamps, and adipose tissue biopsies. At baseline, the subjects in the two groups were matched for age, obesity (BMI), and other clinical characteristics, including insulin action on whole-body glucose disposal (Table 1). Subjects then had either metformin or troglitazone added to their treatment regimen, and the studies were repeated 3–4 months later. Posttreatment HbA1c levels were comparable in the two groups: 7.0 ± 0.4 vs. 7.5 ± 0.3% for troglitazone and metformin, respectively (P = NS). In addition, relative changes in fasting glucose, HbA1c, and insulin levels were the same in both groups (Table 1), further indication that the extent of glycemic control was matched between groups. Insulin-stimulated whole-body glucose disposal improved with metformin treatment, by 20 ± 9% over the paired baseline value (P < 0.05). The improvement in insulin action after troglitazone treatment (44 ± 7%, P < 0.01) was significantly greater (P < 0.05) than the relative change in the metformin group (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1 Effects of treatment on clinical characteristics of type 2 diabetic subjects

 
The metformin subjects maintained their weight (Table 1). Their body composition, as determined by percent fat from DEXA analysis, was also stable (posttreatment value = 99 ± 5% of baseline, P = NS). Serum leptin levels were unaltered with metformin treatment (Table 1). Even with a preservation of overall adiposity, there was a significant reduction in the average size of subcutaneous adipocytes (16 ± 4% decrease, P < 0.005) in the metformin treatment group. Conversely, there was a modest but statistically significant weight gain in the troglitazone-treated group (Table 1), whereas percent body fat (104 ± 5% of baseline, P = NS) did not change. Serum leptin levels increased (24 ± 15%), but not significantly (P < 0.1). There was a trend for fat cell volume to also increase with troglitazone treatment (30 ± 25%, P < 0.1), although this difference also did not attain statistical significance. Initially, subcutaneous adipocyte volumes were similar in the two groups (635 ± 56 vs. 596 ± 25 pL for troglitazone and metformin groups, respectively, P = NS). After treatment, fat cells from the troglitazone-treated group were significantly larger (P < 0.05) than those in the metformin group.

Adipocyte glucose transport and transporter expression.
In the baseline studies, adipocytes from both groups were well matched for glucose transport activity in both the absence and presence of added insulin (Fig. 1). Acute insulin exposure caused a 2- to 2.5-fold increase in transport activity in both groups. Metformin treatment had no consistent effect on adipocyte glucose transport in either the absence (29 ± 32% increase over baseline) or presence (83 ± 81% increase, P = 0.35) of insulin. Troglitazone treatment resulted in large increases in both basal (221 ± 133% over baseline, P < 0.05) and insulin-stimulated (318 ± 161%, P < 0.05) glucose transport (Fig. 1). The relatively greater increase in insulin-stimulated transport activity translated into an elevation of insulin responsiveness after troglitazone treatment, from an average 2.3-fold stimulation at baseline to 3.2-fold at posttreatment.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. Effect of treatment on glucose transport into adipocytes. Absolute rates of 3-O-methylglucose transport were measured in adipocytes obtained before ({blacksquare}) and after ({square}) drug treatment with acute (60 min) incubation in the absence (-) or presence (+) of insulin (8.2 nmol/l). Results are average + SE, n = 10 for the troglitazone group and n = 11 for the metformin group. *P < 0.05 vs. absence of insulin for same treatment condition; {dagger}P < 0.05 vs. paired pre-Rx value.

 
The total cellular complements of adipocyte GLUT1 and GLUT4 were determined by Western blotting. Metformin had no effect on either GLUT1 (19 ± 30% over baseline, not shown) or GLUT4 (9 ± 15% below baseline) (Fig. 2) expression in adipocytes. With troglitazone, there was a tendency for GLUT1 protein to increase (64 ± 42%, P = 0.16, not shown) and a doubling in GLUT4 expression (102 ± 44% over baseline, P < 0.05) (Fig. 2). The changes, or lack thereof, in GLUT1 and GLUT4 expression mirror those seen in basal and insulin-stimulated glucose transport activities (Fig. 1).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 2. Effect of treatment on GLUT4 glucose transporter protein expression in adipocytes. Adipocytes were isolated from subcutaneous abdominal adipose tissue obtained before (baseline, {blacksquare}) and after ({square}) treatment. A: Representative Western blot. B: Quantitation of blots, normalized to total extract protein expression. Results are average + SE, n = 10 for the troglitazone group and n = 11 for the metformin group. *P < 0.05 vs. paired pre-Rx value.

 
Insulin signaling.
To determine what impact glycemic control might have on insulin signaling, the expression of key components in a pathway leading to glucose transport stimulation were measured by Western blotting. Baseline expression of the proteins of interest did not differ significantly between groups (Table 2). Neither metformin nor troglitazone treatment had any consistent or significant effect on the total cellular protein expression of IRS-1, p85{alpha}, p110ß, or Akt 1/2 (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2 Effects of treatment on signaling protein expression

 
Limitations of tissue availability prevented analysis of insulin receptor kinase and PI3K activities. However, Ser473 phosphorylation of Akt, a reliable downstream indicator of activation of the enzyme (28) that was also impaired in adipocytes from diabetic subjects (11), was determined. Because S473 phosphorylation of Akt is routinely undetectable in the absence of insulin (Fig. 3), the results are presented as the amount of pS473-Akt in the insulin-stimulated condition and normalized to the amount of Akt protein in the same sample. Insulin exposure resulted in the generation of a phosophorylated form of Akt to a comparable extent in both groups at baseline. Insulin stimulation of Akt phosphorylation in adipocytes was unaltered after metformin treatment (Fig. 3). However, the insulin effect was increased (70 ± 34% over baseline response, P < 0.05) with troglitazone treatment.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 3. Influence of in vivo treatment on insulin-stimulated Akt serine phosphorylation in adipocytes. Cell extracts were prepared from adipocytes obtained before ({blacksquare}) and after ({square}) drug treatment with acute (15 min) incubation in the absence (-) or presence (+) of insulin (8.2 nmol/l). A: Representative Western blot. B: Quantitation of pS473-Akt in insulin-treated cells normalized to total Akt 1/2 protein in the same sample. Results are average + SE, n = 7 for both the troglitazone and metformin groups. *P < 0.05 vs. paired pre-Rx value.

 
Modulation of PPAR-{gamma} expression.
The nuclear receptor PPAR-{gamma} has been identified as a target for troglitazone and other thiazolidindiones (29). PPAR-{gamma} protein expression was determined by Western blotting with an antibody that recognizes a region common to the {gamma}1 and {gamma}2 isoforms. Adipocyte content of PPAR-{gamma} was similar in the troglitazone and metformin groups before treatment (14.2 ± 5.8 vs. 14.5 ± 4.5 arbritrary units/10 µg protein for troglitazone and metformin groups, respectively). Neither treatment had any consistent effect on PPAR-{gamma} protein expression; there was a 32 ± 69% increase over control with troglitazone and a 50 ± 52% increase over control with metformin (P = NS for both).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Glucose intolerance in type 2 diabetes is the consequence of a number of defects, including impaired insulin secretion by the pancreatic ß-cell, resistance of peripheral tissues to the glucose-utilizing effects of insulin, and augmented hepatic glucose production (rev. in [1]). Pharmacological approaches have been developed to treat each of these problems. Combination therapy with these agents is an increasingly popular approach because multiple defects are often present in diabetic subjects, and many individuals respond only partially to single agents. Any understanding of the molecular mechanism(s) by which multiple therapies act to improve glycemic control is complicated by common therapeutic consequences. Questions include which responses might be specific for a certain drug on a particular tissue and which are the result of generalized changes in glucose and insulin levels. These issues were addressed in type 2 diabetic subjects who failed sulfonylurea therapy when the dose of troglitazone or metformin was titrated to attain a common therapeutic target. We were able to achieve this goal of matched glycemic control because changes in glucose, insulin, and HbA1c levels were similar in the troglitazone and metformin treatment groups (Table 1). Despite the comparable glycemic control, troglitazone treatment was twice as effective as metformin in improving whole-body insulin action and glucose disposal, suggesting that factors other than changes in circulating glucose and insulin levels are responsible for the difference. Other investigators have also found a greater efficacy of troglitazone when compared with metformin on glucose disposal in type 2 diabetic subjects (30).

A common consequence of tight glycemic control is weight gain, although this appears to occur less frequently with metformin (18). Our results are in agreement with this behavior, as troglitazone-treated subjects demonstrated modest weight gain, whereas metformin-treated subjects did not. One postulated mechanism by which troglitazone could increase insulin action even in the presence of weight gain is augmented apoptosis of large, insulin-resistant adipocytes coupled with proliferation and differentiation (31) of smaller, more insulin-sensitive adipocytes. Although evidence in support of this hypothesis has been obtained in rodents (32), our results in humans are the opposite, showing a trend toward an increase in cell size in subcutaneous adipose tissue with troglitazone treatment. A more likely explanation may be a redistribution of fat stores, as several investigators have found thiazolidinedione treatment to reduce visceral fat mass and increase subcutaneous fat (33,34). That supposition would be supported by the lack of any significant effect of troglitazone treatment on whole-body fat content; more of it may now be in the subcutaneous depots, a subtle distribution that could not be detected by changes in waist-to-hip ratio or abdominal skinfold thickness. Although we did not directly measure the mass of the different adipose depots, an increase in adipocyte size from subcutaneous fat would be consistent with an increase in the size of that depot.

Although a tight relationship between adiposity and serum leptin levels is a common observation (35), the effects of antidiabetic therapy on leptin in the absence of weight loss are more mixed. In agreement with the lack of effect seen in the current study, short-term metformin treatment did not influence leptin levels in type 2 diabetic women (36). The effects of treatment on leptin levels in the current report are in general agreement with the behavior of body weight: a tendency toward increases in both parameters with troglitazone and no change after metformin.

Even though the major portion of insulin-mediated glucose disposal occurs in skeletal muscle, a strong relationship between whole-body glucose disposal and glucose transport into adipocytes has been demonstrated (8). A similar tendency, although not statistically significant (P = 0.18), was observed in the current subjects. Adipocytes from individuals with type 2 diabetes display defects in glucose transport (8) and GLUT4 expression as well as insulin-stimulated IRS-1–associated PI3K activity and Akt phosphorylation (11)—behaviors that are also found, for the most part, in diabetic skeletal muscle (6,7,37). Thus, in many ways, insulin resistance in diabetic adipocytes is reflective of that in skeletal muscle. Troglitazone treatment was able to reverse many of the major defects identified in diabetic adipocytes as well as in impaired glucose transport, reduce GLUT4 expression, and reduce insulin stimulation of Akt phosphorylation. If S473 phosphorylation of Akt is taken as an indirect downstream marker of PI3K activity, a commonly accepted supposition (28), then the results suggest that troglitazone treatment also improved insulin-stimulated PI3K activity. Thus, troglitazone treatment ameliorates diabetes-related impairments in both insulin signaling and final effector systems. None of these defects were influenced by metformin treatment. There are reports of thiazolidinediones improving adipocyte glucose uptake and GLUT4 expression in various animal models of insulin resistance (38,39), results that mirror the current findings in human adipocytes and suggest that the effects are general to the class of thiazolidinediones and not specific for troglitazone.

The ability of metformin to reduce hyperglycemia through effects on hepatic glucose production has been well documented both in vivo (40,41) and in vitro (16). The mechanisms for this action include both reduced gluconeogenesis and increased glyogenesis (16). Information on metformin actions on glucose disposal in other tissues is more varied. Metformin has been reported to upregulate GLUT1 expression in human skin fibroblasts (39). Conversely, metformin had no effect on GLUT1 or GLUT4 expression in skeletal muscle of obese (fa/fa) Zucker rats (42). The question of direct effects of metformin on glucose disposal in nonhepatic tissues versus the consequences of relieving glucotoxicity remains to be resolved.

The ability of troglitazone to increase whole-body glucose uptake has been demonstrated in a number of insulin-resistant animal models (43,44) as well as in subjects with type 2 diabetes (17,45). This response is observed in the absence or presence of sulfonylureas, although, just as we found, thiazolidinedione treatment augments glycemic control over that seen with sulfonylurea monotherapy (46,47). The fact that both troglitazone (46) and rosiglitazone (47) gave a similar result indicates that this is a general effect of thiazolidinediones and not limited to troglitazone. Treatment of insulin-resistant animals with several different thiazolidinediones have been shown to increase glucose transport and/or GLUT4 expression in adipocytes (48). Treatment of fully differentiated 3T3-L1 adipocytes with troglitazone, which is more reflective of the therapeutic situation in adult diabetic subjects, resulted in an increase in basal glucose uptake and upregulation of GLUT1 expression that was similar to certain aspects of our findings in adipocytes. In addition, we have reported that troglitazone treatment of human skeletal muscle cells in culture also caused increases in GLUT1 expression (49), implicating control of glucose transporter expression as a key contributor to the antihyperglycemic actions of this family of drugs.

Thiazolidinediones are thought to exert their glucose and lipid fatty acid–lowering actions by activating PPAR-{gamma} (29), thereby altering the transcription of genes involved in glucose and lipid metabolism. Expression of PPAR-{gamma} is greatest in adipose tissue (29), and it has been proposed that adipose tissue is a major target for thiazolidinedione action. Increases in adipose tissue GLUT4 expression after troglitazone treatment can certainly explain the augmented glucose transport into adipocytes that those subjects displayed. As there were no changes in the expression of several key proteins involved in insulin activation of PI3K and Akt, the mechanism responsible for increased insulin-stimulated phosphorylation of Akt is uncertain but may involve control of an inactivating phosphatase or proteins involved in intracellular localization of signaling complexes. The fact that the changes in adipocytes reported here are specific for troglitazone and do not occur in response to metformin suggest that they involve mechanisms beyond correction of glucotoxicity and hyperinsulinemia, as those changes were common to both treatments. The troglitazone effect is most likely occurring through activation of PPAR-{gamma} (31) and not through upregulation of the expression of the receptor.

Several conclusions can be drawn from the current results. One is that although troglitazone and metformin may be equally effective in controlling hyperglycemia, they do so by different mechanisms and by acting on different tissues. The greater efficacy of troglitazone to increase glucose disposal, as opposed to reducing glucose production, is due, at least in part, to the ability of the thiazolidinedione to increase glucose uptake and insulin action in adipose tissue. The relationship between increased glucose uptake in subcutaneous adipocytes and further improvements in whole-body glucose disposal, occurring in response to troglitazone and not metformin, also suggests that adipose tissue can make a major contribution to whole-body glucose disposal.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Medical Research Service, Department of Veterans Affairs and VA San Diego Healthcare System, by Pfizer Parke-Davis, and by grant MO1 RR-00827 from the General Clinical Research Branch, Division of Research Resources, National Institutes of Health. Dr. Loviscach was supported by an American Diabetes Association Mentor-based Fellowship Award.

We thank Debra Armstrong and Leslie Carter at the VA San Diego Healthcare System for assistance with the clamp, biopsy, and assay procedures and Kay Griver for performing anthropometric measurements.


   FOOTNOTES
 
Address correspondence and reprint requests to Dr. Robert R. Henry, Department of Medicine (V111G), VA San Diego Healthcare System, 3350 La Jolla Village Dr., San Diego, CA 92161. E-mail: rrhenry{at}vapop.ucsd.edu.

Received for publication 6 August 2001 and accepted in revised form 17 October 2001.

T.P.C. and S.M. have received honoraria for speaking engagements from Pfizer Parke Davis. R.R.H. has received honoraria for speaking engagements and grant/research support from Pfizer.

T.P.C. and A.P.S.K. contributed equally to this study.

BSA, bovine serum albumin; DEXA, dual-energy X-ray absorptionometry; HWS, HEPES washing salts; IRS, insulin receptor substrate; PI3K, phosphatidylinositol 3-kinase; PPAR{gamma}, peroxisome proliferator–activated receptor {gamma}.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Shulman GI: Cellular mechanisms of insulin resistance. J Clin Invest 106:171–176, 2000[Medline]
  2. Viramaki A, Ueki K, Kahn CR: Protein-protein interactions in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest 103:931–943, 1999[Medline]
  3. Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M: Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. J Biol Chem 269:3568–3573, 1994[Abstract/Free Full Text]
  4. Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR: Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biochem 14:4902–4911, 1994
  5. Kitamura T, Ogawa W, Sakaue H, Hino Y, Kuroda S, Takata M, Matsumoto M, Maeda T, Konishi H, Kikkawa U, Kasuga M: Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport. Mol Cell Biol 18:3708–3717, 1997[Abstract/Free Full Text]
  6. Andréasson K, Galuska D, Thörne A, Sonnenfeld,T, Wallberg-Henriksson H: Decreased insulin-stimulated 3-O-methylglucose transport in in vitro incubated muscle strips from type II diabetic subjects. Acta Physiol Scand 142:255–260, 1991[Medline]
  7. Dohm, GL Tapscott EB, Pories WJ, Dabbs DJ, Flickinger EG, Meelheim D, Fushiki T, Atkinson SM, Elton CW, Caro JF: An in vitro human muscle preparation suitable for metabolic studies. J Clin Invest 82:486–494, 1988
  8. Ciaraldi TP, Kolterman OG, Scarlett JA, Kao M, Olefsky JM: Role of glucose transport in the post-receptor defect of non-insulin dependent diabetes mellitus. Diabetes 31:1016–1022, 1982[Medline]
  9. Garvey WT, Maianu L, Huecksteadt TP, Birnbaum MJ, Molina JM, Ciaraldi TP: Pretranslational suppression of GLUT4 glucose transporters causes insulin resistance in type II diabetes. J Clin Invest 87:1072–1081, 1991
  10. Freidenberg GR, Reichart D, Olefsky JM, Henry RR: Reversibility of defective adipocyte insulin receptor kinase activity in non-insulin-dependent diabetes mellitus. J Clin Invest 82:1398–1406, 1988
  11. Smith U, Axelsen M, Carvalho E, Eliasson B, Jansson PA: Insulin signaling and action in fat cells: associations with insulin resistance and type 2 diabetes. Ann N Y Acad Sci 892:119–126, 1999[Medline]
  12. Day C: Thiazolidinediones: a new class of antidiabetic drugs. Diabet Med 16:179–192, 1999[Medline]
  13. Williams KV, Kelley DE: Metabolic consequences of weight loss on glucose metanolism and insulin action in type 2 diabetes. Diabetes Obes Metab 2:121–129, 2000[Medline]
  14. Tudor-Locke CE, Bell RC, Meyers AM: Revisiting the role of physical activity and exercise in the treatment of type 2 diabetes. Can J Appl Physiol 25:466–492, 2000[Medline]
  15. Zimmerman BR: Sulfonylureas. Endocrinol Metab Clin N Am 26:511–522, 1997[Medline]
  16. Wiernsperger NF, Bailey CJ: The antihyperglycemic effect of metformin: therapeutic and cellular mechanisms. Drugs 58 (Suppl. 1):31–39, 1999
  17. Maggs DG, Buchana TA, Burant CF, Cline G, Gumbiner B, Hsueh WA, Inzucchi S, Kelley D, Nolan J, Olefsky JM, Polonsky KS, Silver D, Valiquett TR, Shulman GI: Metabolic effects of troglitazone monotherapy in type 2 diabetes mellitus: a randomized, double-blind, placebo-controlled trial. Ann Int Med 128:176–185, 1998[Abstract/Free Full Text]
  18. DeFronzo RA: Pharmacologic therapy for type 2 diabetes. Annals Int Med 131:281–303, 1999[Abstract/Free Full Text]
  19. Thorburn AW, Gumbiner B, Bulacan F, Wallace P, Henry RR: Intracellular glucose oxidation and glycogen synthase activity are reduced in non-insulin dependent (type II) diabetes independent of impaired glucose uptake. J Clin Invest 85:522–529, 1990
  20. Desbuquois B, Aurbach GD: Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radio-immunoassays. J Clin Endocrinol Metab 33:732–738, 1971[Medline]
  21. Ma Z, Gingerich RL, Santiago JV, Klein S, Smit CH, Landt M: Radioimmunoassay of leptin in human plasma. Clin Chem 42:942–946, 1966[Abstract/Free Full Text]
  22. Roubenoff R, Kehayias JJ, Dawson-Hughes B, Heymsfield SB: Use of dual-energy X-ray absorptiometry in body composition studies: not yet a "gold standard". Am J Clin Nutr 58:589–591, 1993[Free Full Text]
  23. Rodbell M: Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375–380, 1964[Free Full Text]
  24. Hirsch J, Gallian E: Methods for the determination of adipose cell size in man and animals. J Lip Res 9:110–119, 1968[Abstract]
  25. Whitesell RR, Gliemann J: Kinetic parameters of 3-O-methylglucose and glucose in adipocytes. J Biol Chem 254:5276–5283, 1979[Free Full Text]
  26. Thies RS, Molina JM, Ciaraldi TP, Freidenberg GR, Olefsky JM: Insulin-receptor autophosphorylation and endogenous substrate phosphorylation in human adipocytes from control, obese, and NIDDM subjects. Diabetes 39:250–259, 1990[Abstract]
  27. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 22:680–686, 1970
  28. Kohn AD, Takeuchi F, Roth RA: Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. J Biol Chem 271:21920–21926, 1996[Abstract/Free Full Text]
  29. Spiegelman BM: PPAR-{gamma}: adipogenic regulator and thiazolidinedione receptor. Diabetes 47:507–514, 1999[Abstract]
  30. Inzucchi SE, Maggs DG, Spollet GR, Page SL, Rife FS, Walton V, Shulman GI: Efficacy and metabolic effects of metformin and troglitazone in type II diabetes mellitus. N Engl J Med 338:867–872, 1998[Abstract/Free Full Text]
  31. Adams M, Montague CT, Prins JB, Holder JC, Smith SA, Sanders L, Digby JE, Sewter CP, Lazar MA, Chatterjee KK, O’Rahilly S: Activators of peroxisome proliferator-activated receptor gamma have depot specific effects on human preadipocyte differentiation. J Clin Invest 100:3149–3153, 1997[Medline]
  32. Okuno A, Tamemoto H, Tobe K, Ueki K, Mori Y, Iwamot K, Umesono K, Akanuma Y, Fujiwara T, Horikoshi H, Yazaki Y, Kadowaki T: Troglitazone increases the number of small adipocytes without change of white adipose tissue mass in obese Zucker rats. J Clin Invest 101:1354–1361, 1998[Medline]
  33. Kelly IE, Han TS, Walsh K, Lean ME: Effects of a thiazolidinedione compound on body fat and fat distribution of patients with type 2 diabetes. Diabetes Care 22:288–293, 1999[Abstract/Free Full Text]
  34. Banerji M, Lebovitz H, Dugbartey M: Rosiglitazone selectively increases subcutaneous but not visceral adipose tissue mass in type 2 diabetes mellitus (Abstract). Diabetes 50 (Suppl. 2):A90
  35. Fried SK, Ricci MR, Russell CD, Laferrere B: Regulation of leptin production in humans. J Nutr 130:3127S–3131S, 2000[Abstract/Free Full Text]
  36. Guler S, Cakir B, Demirbas B, Gursoy G, Serter R, Aral Y: Leptin concentrations are related to glycemic control, but do not change with short-term oral antidiabetic therapy in female patients with type 2 diabetes mellitus. Diabetes Obes Metab 2:313–316, 2000[Medline]
  37. Kim Y-B, Nikoulin SE, Ciaraldi TP, Henry RR, Kahn BB: Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes. J Clin Invest 104:733–741, 1999[Medline]
  38. Arakawa K, Ishihara T, Aoto M, Inamasu M, Saito A, Ikezawa K: Actions of novel antidiabetic thiazolidinedione, T-174, in animal models of non-insulin-dependent diabetes mellitus and in cultured muscle cells. Br J Pharmacol 125:429–436, 1998[Medline]
  39. Hamann A, Benecke H, Greten H, Matthaei S: Metformin increases glucose transporter protein and gene expression in human fibroblasts. Biochem Biophys Res Comm 196:382–387, 1993[Medline]
  40. Hundal RS, Krssak M, Dufour S, Laurent D, Lebon V, Chandramouli V, Inzucchi SE, Schumann WC, Petersen KF, Landu BR, Shulman GI: Mechanism by which metformin reduces glucose production in type 2 diabetes. Diabetes 49:2063–2069, 2000[Abstract/Free Full Text]
  41. Fery F, Plat L, Balasse EO: Effects of metformin on the pathways of glucose utilization after oral glucose in non-insulin-dependent diabetes mellitus patients. Metabolism 46:227–233, 1997[Medline]
  42. Rouru J, Koulu M, Peltonen J, Santti E, Hanninen V, Pesonen U, Huupponen R: Effects of metformin treatment on glucose transporter proteins in subcellular fractions of skeletal muscle in (fa/fa) Zucker rats. J Pharmacol 1182–1187, 1995
  43. Koursheed M, Miles PDG, Gao K-M, Lee M-K, Moos AR, Olefsky JM: Metabolic effects of troglitazone on fat-induced insulin resistance in the rat. Metabolism 44:1489–1494, 1995[Medline]
  44. Sugimoto M, Takeda N, Nakashima K, Okumura S, Takami K, Yoshino K, Hattori J, Ishimori M, Takami R, Sasaki,A, Yasuda K: Effects of troglitazone on hepatic and peripheral insulin resistance induced by growth hormone excess in rats. Metabolism 47:783–787, 1998[Medline]
  45. Petersen KF, Krssak M, Inzucchi S, Cline GW, Dufour S, Shulman GI: Mechanism of troglitazone action in type 2 diabetes. Diabetes 49:827–831, 2000[Abstract]
  46. Buysschaert M, Bobbioni E, Starlie M, Frith L: Troglitazone in combination with sulphonylurea improves glycemic control in type 2 diabetic patients inadequately controlled by sulphonylurea therapy alone. Diabet Med 16:147–153, 1999[Medline]
  47. Wolffebbuttel BH, Gomis R, Squatrito S, Jones NP, Patwardha RN: Addition of low dose rosiglitazone to sulphonylurea therapy improves glycemic control in type 2 diabetic patients. Diabet Med 17:40–47, 2000[Medline]
  48. Kanoh Y, Bandyopadhyay G, Sajan MP, Standaert ML, Farese RV: Thiazolidinedione treatment enhances insulin effects on protein kinase C-zeta/lambda activation and glucose transport in adipocytes of non-diabetic and Goto-Kakizaki type II diabetic rats. J Biol Chem 275:16690–16696, 2000[Abstract/Free Full Text]
  49. Park KS, Ciaraldi TP, Abrams-Carter L, Mudaliar S, Nikoulina SE, Henry RR: Troglitazone regulation of glucose metabolism in human skeletal muscle cultures from obese type II diabetic subjects. J Clin Endocrinol Metab 83:1636–1643, 1998[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 DiabetesHome page
R. Basu, P. Shah, A. Basu, B. Norby, B. Dicke, V. Chandramouli, O. Cohen, B. R. Landau, and R. A. Rizza
Comparison of the Effects of Pioglitazone and Metformin on Hepatic and Extra-Hepatic Insulin Action in People With Type 2 Diabetes
Diabetes, January 1, 2008; 57(1): 24 - 31.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
G. Ranganathan, R. Unal, I. Pokrovskaya, A. Yao-Borengasser, B. Phanavanh, B. Lecka-Czernik, N. Rasouli, and P. A. Kern
The lipogenic enzymes DGAT1, FAS, and LPL in adipose tissue: effects of obesity, insulin resistance, and TZD treatment
J. Lipid Res., November 1, 2006; 47(11): 2444 - 2450.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
T. P. Ciaraldi, D. K. Oh, L. Christiansen, S. E. Nikoulina, A. P. S. Kong, S. Baxi, S. Mudaliar, and R. R. Henry
Tissue-specific expression and regulation of GSK-3 in human skeletal muscle and adipose tissue
Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E891 - E898.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
C. A. Collier, C. R. Bruce, A. C. Smith, G. Lopaschuk, and D. J. Dyck
Metformin counters the insulin-induced suppression of fatty acid oxidation and stimulation of triacylglycerol storage in rodent skeletal muscle
Am J Physiol Endocrinol Metab, July 1, 2006; 291(1): E182 - E189.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
A. P. M. Viljanen, K. A. Virtanen, M. J. Jarvisalo, K. Hallsten, R. Parkkola, T. Ronnemaa, F. Lonnqvist, P. Iozzo, E. Ferrannini, and P. Nuutila
Rosiglitazone Treatment Increases Subcutaneous Adipose Tissue Glucose Uptake in Parallel with Perfusion in Patients with Type 2 Diabetes: A Double-Blind, Randomized Study with Metformin
J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6523 - 6528.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
C.-Y. Lin, T. Gurlo, L. Haataja, W. A. Hsueh, and P. C. Butler
Activation of Peroxisome Proliferator-Activated Receptor-{gamma} by Rosiglitazone Protects Human Islet Cells against Human Islet Amyloid Polypeptide Toxicity by a Phosphatidylinositol 3'-Kinase-Dependent Pathway
J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6678 - 6686.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
J. Klein, S. Westphal, D. Kraus, B. Meier, N. Perwitz, V. Ott, M. Fasshauer, and H H. Klein
Metformin inhibits leptin secretion via a mitogen-activated protein kinase signalling pathway in brown adipocytes
J. Endocrinol., November 1, 2004; 183(2): 299 - 307.
[Abstract] [Full Text] [PDF]

Home page
 Diabetes CareHome page
I. Bogacka, H. Xie, G. A. Bray, and S. R. Smith
The Effect of Pioglitazone on Peroxisome Proliferator-Activated Receptor-{gamma} Target Genes Related to Lipid Storage In Vivo
Diabetes Care, July 1, 2004; 27(7): 1660 - 1667.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
H. Bays, L. Mandarino, and R. A. DeFronzo
Role of the Adipocyte, Free Fatty Acids, and Ectopic Fat in Pathogenesis of Type 2 Diabetes Mellitus: Peroxisomal Proliferator-Activated Receptor Agonists Provide a Rational Therapeutic Approach
J. Clin. Endocrinol. Metab., February 1, 2004; 89(2): 463 - 478.
[Full Text] [PDF]

Home page
 Arch Intern MedHome page
S. R. Salpeter, E. Greyber, G. A. Pasternak, and E. E. Salpeter
Risk of Fatal and Nonfatal Lactic Acidosis With Metformin Use in Type 2 Diabetes Mellitus: Systematic Review and Meta-analysis
Arch Intern Med, November 24, 2003; 163(21): 2594 - 2602.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
I. Pavo, G. Jermendy, T. T. Varkonyi, Z. Kerenyi, A. Gyimesi, S. Shoustov, M. Shestakova, M. Herz, D. Johns, B. J. Schluchter, et al.
Effect of Pioglitazone Compared with Metformin on Glycemic Control and Indicators of Insulin Sensitivity in Recently Diagnosed Patients with Type 2 Diabetes
J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1637 - 1645.
[Abstract] [Full Text] [PDF]

Home page
 Diabetes Spectr.Home page
V. R. Aroda and R. R. Henry
Thiazolidinediones: Potential Link Between Insulin Resistance and Cardiovascular Disease
Diabetes Spectr, April 1, 2003; 16(2): 120 - 125.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
S. A. Phillips, T. P. Ciaraldi, A. P.S. Kong, R. Bandukwala, V. Aroda, L. Carter, S. Baxi, S. R. Mudaliar, and R. R. Henry
Modulation of Circulating and Adipose Tissue Adiponectin Levels by Antidiabetic Therapy
Diabetes, March 1, 2003; 52(3): 667 - 674.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
K. A. Virtanen, K. Hallsten, R. Parkkola, T. Janatuinen, F. Lonnqvist, T. Viljanen, T. Ronnemaa, J. Knuuti, R. Huupponen, P. Lonnroth, et al.
Differential Effects of Rosiglitazone and Metformin on Adipose Tissue Distribution and Glucose Uptake in Type 2 Diabetic Subjects
Diabetes, February 1, 2003; 52(2): 283 - 290.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
K. Hallsten, K. A. Virtanen, F. Lonnqvist, H. Sipila, A. Oksanen, T. Viljanen, T. Ronnemaa, J. Viikari, J. Knuuti, and P. Nuutila
Rosiglitazone but Not Metformin Enhances Insulin- and Exercise-Stimulated Skeletal Muscle Glucose Uptake in Patients With Newly Diagnosed Type 2 Diabetes
Diabetes, December 1, 2002; 51(12): 3479 - 3485.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
M. M. Meyer, K. Levin, T. Grimmsmann, N. Perwitz, A. Eirich, H. Beck-Nielsen, and H. H. Klein
Troglitazone Treatment Increases Protein Kinase B Phosphorylation in Skeletal Muscle of Normoglycemic Subjects at Risk for the Development of Type 2 Diabetes
Diabetes, September 1, 2002; 51(9): 2691 - 2697.
[Abstract] [Full Text] [PDF]