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Troglitazone Ameliorates Insulin Resistance in Patients with Werner’s Syndrome

First Department of Internal Medicine, Nagasaki University School of Medicine (K.I., H.S., M.I., H.T., H.Y., Y.Y., N.C., K.M., S.A., S.N.), Nagasaki; and the Laboratory of Biochemistry of Exercise and Nutrition, Institute of Health and Sports Sciences, University of Tsukuba (K.T.), Tsukuba, Ibaraki, Japan

Address all correspondence and requests for reprints to: Shigenobu Nagataki, M.D., First Department of Internal Medicine, Nagasaki University School of Medicine, 1–7-1 Sakamoto, Nagasaki 852, Japan.

	Abstract

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Insulin resistance in Werner’s syndrome (WS) is probably due to defective signaling distal to the insulin receptor. To analyze the metabolic effects of troglitazone (TRO) in these patients, we performed frequently sampled iv glucose tolerance tests. Glucose kinetics were analyzed by the minimal model. Five patients with WS (mean age, 41.2 yr; body mass index, 17.0 kg/m²) were treated with TRO (400 mg/day) for 4 weeks. Each subject underwent a 75-g OGTT and frequently sampled iv glucose tolerance tests. Treatment reduced the area under the curve of glucose and insulin in the OGTT by 26% and 43%, respectively. Glucose tolerance, as manifested by the glucose disappearance rate improved significantly (1.36 ± 0.16 to 1.94 ± 0.30%/min; P < 0.05). Although the first phase insulin secretion was unchanged, insulin sensitivity and glucose effectiveness increased significantly [0.47 ± 0.11 to 1.38 ± 0.37 x 10^-4 min/pmol·L (P < 0.05) and 1.72 ± 0.17 to 2.52 ± 0.24 x 10^-2 min^-1 (P < 0.05), respectively]. However, treatment did not change glucose effectiveness at zero insulin. In patients with WS, TRO ameliorates glucose intolerance mediated by increased insulin sensitivity as well as glucose effectiveness, as assessed by minimal model analysis. TRO may modulate the postreceptor signaling component and be a clinically useful regimen for the treatment of patients with the intracellular insulin signaling defect.

	Introduction

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WERNER’S SYNDROME (WS) is one of the progeria conditions characterized by various phenotypical abnormalities, including gray hair, bird-like face, slender extremities, cataract, and endocrine dysfunction (1, 2, 3, 4, 5). The condition is associated with glucose intolerance in approximately 70% of patients, a complication predominantly resulting from insulin resistance (6). Several studies have suggested that postreceptor abnormality is a critical site for insulin resistance, as insulin binding capacity, receptor affinity and autophosphorylation are normal, using skin fibroblasts and Epstein-Barr virus-transformed lymphocytes from patients with WS (6, 7, 8). These findings are in agreement with hyperinsulinemic euglycemic glucose clamp studies that showed reduced insulin sensitivity in WS patients (6, 7, 8, 9). Although a reduced number of insulin receptors in the fibroblasts was shown in one WS patient (8), no structural changes in the insulin receptor molecule were detected in five WS patients in our laboratory (5). Considered together, it is likely that insulin resistance found in WS patients is due to a deterioration in postreceptor insulin signaling.

Thiazolidinedione derivatives, including troglitazone (TRO), have been recently developed as new antidiabetic drugs for sensitizing insulin action (10, 11, 12, 13, 14, 15, 16). However, the exact biochemical mechanism of the insulin-sensitizing action remains to be elucidated. In vitro and animal studies have demonstrated that the agent sensitizes insulin action at target tissues without any effect on ß-cell insulin secretion, and that this sensitizing biological action involves both the receptor and postreceptor levels in peripheral and hepatic tissues (10, 11). The clinical usefulness of these new drugs was shown recently in patients with noninsulin-dependent diabetes mellitus (12, 13, 14, 15, 16). In addition to insulin sensitization, the use of [³H]glucose produces a marked reduction in hepatic glucose output in response to TRO treatment (14). We used TRO in a group of WS patients and assessed its glucose-metabolic effect using the hyperinsulinemic-euglycemic clamp (17). Our results indicated that TRO can potentially ameliorate a postreceptor defect, thus lowering plasma glucose levels.

In the present study, the glucose-lowering effect of TRO was investigated in WS patients using Bergman’s minimal model analysis to further understand the insulin-sensitizing mechanism of the drug.

	Subjects and Methods

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Subjects and protocol

Five patients (4 women and 1 man) with WS and 10 normal control subjects were invited to participate in the present study, and informed consent was obtained. The clinical characteristics of WS patients are summarized in Table 1. Four patients (cases 1–4) had diabetes, and 1 subject (case 5) had impaired glucose tolerance according to the criteria of the National Diabetes Data Group. Three patients were treated with diet therapy only; the other 2 patients required insulin to maintain appropriate plasma glucose levels (fasting plasma glucose, <7.6 mmol/L; 2 h plasma glucose, <12 mmol/L). All patients with WS were hospitalized and treated with a weight-maintaining diet (30 Cal/kg ideal BW) and/or insulin administration. To stabilize the metabolic state of the patients, they were observed for 4 weeks before TRO administration, and plasma glucose was measured 7 times/day. A steady state of plasma glucose was maintained for at least 2 weeks, then TRO was administered at 400 mg/day for the next 4 weeks. We also performed a modified frequently sampled iv glucose tolerance test (FSIGT) and a 75-g oral glucose tolerance test (OGTT) on separate days after an overnight fast, before and after treatment.

FSIGT

After 12 h of fasting, FSIGT was performed as we described previously (18, 19, 20, 21). In brief, baseline samples for glucose and insulin were obtained, and then glucose was administered in the contralateral antecubital vein (300 mg/kg BW) within 1 min. An additional infusion was administered (30 mU/kg BW) to the antecubital vein from 20–25 min after the administration of glucose. Blood samples were frequently obtained up until 180 min.

Data analysis

The glucose disappearance rate (Kg) was calculated as the slope of the least square regression line relating the natural logarithm of glucose concentration to time, from at least four blood samples withdrawn between 10–19 min. The endogenous plasma insulin response was expressed as the area under the insulin curve 0–19 min after the administration of glucose. The integrated area of plasma insulin above the basal level was calculated using the trapezoidal method (22). Insulin sensitivity (Si) and glucose effectiveness (S_G) were estimated by Bergman’s minimal model analysis (23, 24, 25). In this analysis, fluctuations in circulating glucose levels over time are described by the following differential equations: dG(t)/dt = -p1 [G(t) - Gb] - X(t) G(t), and dX(t)/dt = -p2 X(t) + p3 [I(t) - Ib], where G(t) is the plasma glucose concentration, I(t) represents the serum insulin concentration, and Gb and Ib represent baseline concentrations. X(t) represents the time course of peripheral insulin effects. Parameter p1 represents the effect of glucose per se at basal insulin levels to normalize its own concentration in plasma independent of increased insulin. This parameter is known as S_G and has been verified through comparison with studies in which the insulin secretory response was suppressed (26). The ratio of p3 to p2 defines Si, which represents the insulin-dependent increase in the net glucose disappearance rate.

Because S_G includes the contribution of basal insulin to insulin-dependent tissues and the contribution of hyperglycemia per se to both insulin-independent and insulin-dependent tissues, we calculated glucose effectiveness at zero insulin (GEZI) (29). The basal insulin component is known as the basal insulin effect (BIE) and basal insulin (Ib). GEZI is the difference between the total S_G and the BIE, i.e. BIE = Ib x Si and GEZI = S_G - (Ib x Si) (29).

Measurements

Plasma glucose was measured by a glucose autoanalyzer (Clinalyzer RX-40, JEOL, Tokyo, Japan), and plasma insulin was assayed by RIA kits (Insulin Riabead II, Dainabot, Tokyo, Japan). Statistical analysis was performed using Wilcoxon signed rank test. Statistical differences were considered significant at P < 0.05.

	Results

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Weight and blood pressure remained stable in all subjects throughout the treatment period. During the same period, no significant changes were observed in total cholesterol, triglyceride, high density lipoprotein, free fatty acid and other routine clinical laboratory tests. In patients with WS treated with diet only (cases 3 and 4), plasma glucose levels began to fall 10 days after treatment with TRO, especially postprandial glucose. Although the plasma glucose level did not change in cases 1 and 2 treated with diet and insulin, the daily insulin requirement was reduced from 38 to 26 U/day and from 22 to 0 U/day, respectively, after 4 weeks of TRO therapy.

75-g OGTT

The mean levels of plasma glucose and insulin at each indicated time point of the OGTT before and after treatment are depicted in Fig. 1. Although the basal levels of plasma glucose and insulin before and after treatment were not significantly different, significant reductions in plasma glucose and insulin levels were detected at 90 min and at 90 and 120 min, respectively. The values calculated from the area under the glucose and insulin curves were reduced by 26% (from 156.6 ± 21.4 to 116.6 ± 10.3 mmol/L·min; P < 0.05) and 43% (from 93,040 ± 13,057 to 53,003 ± 889 pmol/L·min; P < 0.05), respectively.

View larger version (15K): [in this window] [in a new window]   Figure 1. Results of OGTT in patients with WS. Data represent the mean ± SEM of plasma glucose (top) and insulin (bottom). Closed circles, Before treatment; open circles, after treatment. *, P < 0.01.

The results of FSIGT and the minimal model analysis in control subjects and WS patients before and after treatment are shown in Table 2 and Fig. 2, A–C. Basal glucose and insulin levels did not change significantly. Glucose tolerance, assessed by the glucose disposal rate (Kg), improved significantly after treatment in every WS subject (Table 2 and Fig. 2A). Treatment did not change the first phase insulin secretion (1825 ± 315 to 1834 ± 315 pmol/L·min). The results of FSIGT were subjected to the minimal model analysis. Treatment resulted in a significant increase in insulin sensitivity (Si; Table 2 and Fig. 2B) and glucose effectiveness (S_G; Table 2 and Fig. 2C). S_G indicates the total effect of fractional glucose disappearance independent of insulin increase and includes the effect of basal insulin. Therefore, we attempted to separate glucose effectiveness at zero insulin (GEZI) from S_G. No significant changes in GEZI were observed after treatment (Table 2 and Fig. 3).

View larger version (13K): [in this window] [in a new window]   Figure 2. A, Glucose tolerance (Kg) in control subjects and patients with WS before and after TRO treatment. Data points represent the values in different patients, with the mean ± SEM represented by the horizontal line and vertical bars, respectively. Open circles, Before treatment; closed circles, after treatment. B, Insulin sensitivity (Si) in control subjects and patients with WS before and after TRO treatment. Data points represent the values in different patients, with the mean ± SEM represented by the horizontal line and vertical bars, respectively. Open circles, Before treatment; closed circles, after treatment. C, Glucose effectiveness (SG) in control subjects and patients with WS before and after TRO treatment. Data points represent the values in different patients, with the mean ± SEM represented by the horizontal line and vertical bars, respectively. Open circles, Before treatment; closed circles, after treatment.

View larger version (14K): [in this window] [in a new window]   Figure 3. Glucose effectiveness (SG) in control subjects and patients with WS before and after TRO treatment. Open bars, Glucose effectiveness at basal insullin (BEI); closed bars, glucose effectiveness at zero insulin (GEZI).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Although the glucose clamp technique is an established method for assessment of insulin sensitivity and secretion, it is technically difficult and only reflects insulin sensitivity at a static glucose concentration. In contrast, the minimal model analysis offers a simple interpretation of the action of insulin and glucose to normalize plasma glucose after a glucose bolus injection. Another advantage provided by this method is the ability to assess insulin sensitivity (Si) and glucose effectiveness (S_G). Assessment of both parameters is important because these two effects on glucose tolerance are synergistic. The Si obtained using the minimal model method is a valid alternative to the clamp study (27, 28, 32, 33). The minimal model can be used to simultaneously determine the relationship among insulin sensitivity, glucose effectiveness, and pancreatic function during a single FSIGT.

We demonstrated here that the glucose intolerance in WS was associated predominantly with diminished Si, and after treatment with TRO, both Si and S_G significantly increased without any change in the first phase insulin secretion. To make sure that the insulin-sensitizing effect (the increase in Si) would be dependent on TRO treatment, we discontinued the TRO treatment in some of the patients, but diet or insulin therapy was continued. Plasma glucose rose again to levels similar to those observed just before TRO treatment. This reverse effect suggests that the amelioration of the drug seen in this study is strongly associated with the TRO treatment.

In diabetic patients with WS, the insulin response to glucose is exaggerated, and euglycemic glucose clamp studies have shown reduced insulin sensitivity and responsiveness (6, 7, 8, 9). In vitro studies have shown that insulin binding and tyrosine kinase activity are normal using Epstein-Barr virus-transformed lymphocytes from patients with WS (7). We have also demonstrated that the number of insulin receptors in abdominal fibroblasts derived from WS patients was not reduced compared with that in control fibroblasts (8). These results indicate that insulin resistance associated with WS arises probably from a postreceptor defect.

The present results suggest that the reduced Si in WS results from a postreceptor defect. We showed that administration of TRO to patients with WS improved their glucose tolerance through an increase in Si. Our results indicate that TRO targets mainly postreceptor signaling in WS.

TRO did improve Si in WS, but not completely. The magnitude of insulin sensitivity recovery was approximately double and similar to the doubling of insulin sensitivity in obese men and women with normal glucose tolerance or impaired glucose tolerance (15). This comparison suggest that TRO-regulatable insulin resistance may contribute to a similar extent to total insulin resistance in WS as well as noninsulin-dependent diabetes mellitus.

In addition to Si, amelioration of S_G by TRO was observed in WS patients in this study. The S_G value is also an important indicator of glucose tolerance produced by the minimal model analysis (34). In general, S_G reflects the total effect of glucose on fractional glucose disappearance independent of insulin increase, including the effect of the basal insulin level. Therefore, to exclude the effect of basal insulin, calculation was performed to generate S_G at zero insulin (GEZI), representing the peripheral tissue glucose uptake occurring independent of circulating insulin. In this way, the increase in total S_G seen in this study was not due to alteration of GEZI. That is, the BIE (BIE = Ib x Si) appeared to be a major contributor to the TRO-induced increase in total S_G.

Interestingly, even though TRO induces transcriptional activation of PPAR-2, leading to differentiation of adipocytes, any significant changes in weight in WS patients were not observed before and after TRO treatment. During this study a weight-maintaining diet was given to the patients, so that no changes in their weight would be expected. The current study demonstrated that microscopic examination of adipose tissue in Zucker fatty rats treated with TRO revealed two significant histological changes: an increase in the number of small adipocytes, probably due to differentiation from preadipocytes, and a decrease in the number of triglyceride-rich large adipocytes. However, the weight of total adipose tissue was not significantly changed (Kadowaki, T., Tokyo University, Tokyo, Japan, unpublished observations).

In our study, treatment of five diabetic WS patients with TRO ameliorated hyperglycemia through improvement of the stimulated as well as the basal insulin effect without changing insulin secretion. TRO may be a potent drug to ameliorate diabetes-associated WS. The analysis in the present study is the first to quantitate each parameter of the metabolic effect of TRO during treatment of WS.

	Acknowledgments

 
We thank T. Kadowaki for useful comments. Troglitazone was provided in 200-mg capsules by Sankyo (Tokyo, Japan).

Received November 14, 1996.

Revised February 26, 1997.

Accepted May 2, 1997.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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