Skeletal muscle glycogen synthase activity in subjects with non-insulin-dependent diabetes mellitus after glyburide therapy

Skeletal Muscle Glycogen Synthase Activity in Subjects Non-Insulin-Dependent Diabetes Mellitus After Glyburide Bulangu L. Nyomba,

Daniel Freymond,

ltamar Raz, Karen Stone,

David M. Mott,

With Therapy

and Clifton Bogardus

Sulfonylureas are used in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) largely because of their ability to enhance insulin secretion and possibly to potentiate insulin action. In this study, we investigated the effects of chronic glyburide treatment on glycogen synthase activity determined in skeletal muscle biopsies taken during euglycemic hyperinsulinemic clamps in nine Pima Indians with NIDDM. Insulin was infused at the rate of 40 mU/m’/min (low dose) followed by 460 mU/m*/min (high dose). Compared with the fasting value, the mean glycogen synthase activity assayed at low glucose-6-phosphate (G6Pj concentration (active glycogen synthase) showed no significant changes during insulin infusion before glyburide treatment. After glyburide treatment, the mean active glycogen synthase increased by 39% (P < .05) above the fasting value during the high-dose insulin infusion. Total glycogen synthase activity assayed at high G6P concentration did not change after glyburide treatment. Changes of insulin-stimulated active glycogen synthase associated with glyburide treatment correlated with changes in total body glucose disposal rates (r = .70, P < .05) during euglycemic clamps. We conclude that glyburide treatment of subjects with NIDDM is associated with an increase in insulin action in vivo and concomitantly with improved insulin action on skeletal muscle glycogen synthase. 0 1590 by W.B. Saunders Company.

ULFONYLUREAS are thought to lower blood glucose concentration and promote glucose tolerance by both pancreatic and extrapancreatic mechanisms.‘.2 They exert a stimulatory effect on insulin secretion when given acutely, whereas this effect may be present, absent, or transient after chronic administration. At the extrapancreatic level, studies suggest that sulfonylureas potentiate insulin action.2 The assessment of extrapancreatic mechanisms of action of sulfonylureas in man was made possible by the glucose clamp technique. 3-7 Therapy with these agents increases insulin-mediated total body glucose disposa13-’ and suppresses endogenous glucose production4,6,7 in both normal subjects and in subjects with non-insulin-dependent diabetes mellitus (NIDDM). In vitro, sulfonylureas enhance insulinstimulated glucose uptake and transport in a variety of tissues.‘.2,s,9 They also increase insulin-stimulated glycogen synthase activity in cultured liver’0-‘2 and fat cells,13 but they are not known to exert any effect on skeletal muscle glycogen synthase, and it is not known whether these agents improve glucose metabolism by an effect on glycogen synthase in human subjects. Since skeletal muscle is a major site of insulin action,‘43’5 we evaluated the effects of glyburide therapy on insulin stimulation of skeletal muscle glycogen synthase in subjects with NIDDM. We report that in the same subjects that improve glucose tolerance, glyburide potentiates insulin stimulation of skeletal muscle glycogen synthase.

S

From the Clinical Diabetes and Nutrition Section, National Institutes of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Phoenix, AZ. Supported by a grant from the Upjohn Company, Kalamazoo, MI. Address reprint requests to Bulangu L. Nyomba, MD, Clinical Diabetes and Nutrition Section, National Institutes of Health, 4212 N 16th St, Room 541. Phoenix, AZ 85016. @ 1990 by W.B. Saunders Company. 00260495/90/391 I-001 7$03.00/O

1204

METHODS Subjects

and Study

Protocol

Nine Southwestern American Indians, six males and three females, entered the study. They gave informed consent and the studies were approved by the ethical committees of the National Institutes of Health and the Indian Health Service, and by the Gila River Indian Community where the subjects were living. All of the subjects had NIDDM according to the National Diabetes Data Group Criteria,16 but were otherwise in good health as assessed by physical examinations, electrocardiography, and routine hematological, biochemical, and urine tests. None of the subjects had taken any medicine for the preceding month. Subjects were admitted at the Phoenix Clinical Research Ward of the National Institutes of Health and fed a weight-maintenance diet (50% carbohydrate, 30% fat, and 20% protein). After at least 2 days of this diet, an oral glucose tolerance test (OGTT) was performed.16 Body composition was determined by underwater weighing with simultaneous determination of residual lung volume. Percent fat and fat-free mass (FFM) were calculated.” After at least 7 days on the ward, a hyperinsulinemic euglycemic clamp was performed, with indirect calorimetry and muscle biopsies (see below). These studies were performed before and after approximately 12 weeks of treatment with glyburide. The characteristics of the subjects are shown in Table 1. Glyburide

Treatment

Treatment with glyburide (Micronase, Upjohn, Kalamazoo, MI) was started 2 days after the baseline euglycemic hyperinsulinemic clamp. The dose of glyburide was progressively increased while subjects were on the research ward up to a maximum dose of 20 mg/d or until fasting plasma glucose declined to the normal range. Subjects were then observed as outpatients at weekly intervals for a mean duration of 11 weeks. They were then readmitted for a week, maintained on their usual glyburide dose, and studied as outlined above. Euglycemic

Clamp,

Indirect

Calorimetry,

and Muscle Biopsy

A euglycemic hyperinsulinemic clamp was performed as previously reported. I8 After a IO-hour overnight fast, an indwelling catheter was placed in an antecubital vein for infusion of insulin, 20% glucose, and [3-3H]-glucose. Another catheter was placed

Merabolism, Vol39,

No 11 (November), 1990: pp 1204-1210

1205

EFFECT OF GLYBURIDE ON GLYCOGEN SYNTHASE

Table 1. Subject Characteristics Sex (M/F) Age W Body weight (kg) Body fat (%) Duration of diabetes (yr) Glyburida therapy (mg) Duration of treatment (wk)

(mean f SEMI 513 29 & 2 121.5

+ 9.1

36.5

? 2.8

4.6 + 1.4 14 * 2 11.6 + 1.6

performed under local anesthesia. The samples were collected with a Bergstram” muscle biopsy needle (Depuy, Phoenix, AZ), and frozen in liquid nitrogen within 15 seconds. The second muscle biopsy was performed on the contralateral thigh between 340 and 360 minutes, while insulin continued to be infused. The third muscle biopsy was performed between 440 and 460 minutes at a site that was 10 cm proximal to the first biopsy. The samples were stored at - 70°C until assayed.

Calculations retrograde in a dorsal vein of the contralateral hand for blood sampling. To arterialize the blood, the hand was kept in a preheated warming box at 70°C for the duration of the test. Two to three blood samples were drawn during the 30 minutes (-25, - 18, and - 1 minute) preceding insulin infusion for the measurement of glucose, insulin, and catecholamines. The clamp was initiated by a primed, continuous low-dose infusion of short-acting insulin (Velosulin, Nordisk, Bethesda, MD) at a rate of 40 mU/m*/min for 360 minutes. A primed (30 &i), continuous (0.30 pCi/min) infusion of [3-‘HI-glucose was begun at 220 minutes of the low-dose insulin infusion and continued until 340 minutes. The mean blood [3-3H]glucose-specific activity was 2,807 + 285 dpm/mg before glyburide treatment and 2,016 r 199 dpm/mg after glyburide treatment. The respective mean coefficients of variation (CVs) were 3.8% + 0.6% and 2.7% + 0.4%, indicating that [3-‘HI-glucose distribution had reached steady-state by the time endogenous glucose production was calculated (see below). A primed, continuous high-dose insulin infusion (400 mU/m*/min) was started after 360 minutes and continued for 100 minutes. Blood samples were obtained every 5 minutes for the determination of plasma glucose concentrations, while samples for insulin and catecholamine concentrations were obtained at time 315 and 330 minutes of low-dose insulin infusion and at time 4 15 and 430 minutes of high-dose insulin infusion. Blood for the determination of [3-3H]-glucose-specific activity was drawn every 10 minutes from 300 to 340 minutes. The plasma glucose concentration was kept at approximately 100 mg/dL for all subjects by varying the rate of the glucose infusion, which was started when the plasma glucose concentration decreased to 100 mg/dL. The rate of decline of the plasma glucose concentration was never more than 1.0 mg/dL/min, in an attempt to avoid rapid changes in glucose concentrations that might precipitate secretion of hormones counterregulatory to insulin.‘4 Indirect calorimetry measurements were performed, starting at 280 minutes of the low-dose insulin infusion and continuing for the rest of the clamp.” A clear plastic, ventilated hood was placed over the subject’s head. Room air was drawn through the hood and its flow rate measured by a pneumotachograph (Gould, Recording Systems Division, Cleveland, OH). A constant amount of expired air mixed with room air within the hood was aspirated and analyzed for its carbon dioxide and oxygen fractions. The oxygen analyzer was a Zirconium cell analyzer and the carbon dioxide analyzer was an infrared analyzer (both by Applied Electrochemistry, Sunnyvale, CA). The gas analyzers and the flowmeter were connected to a desk-top computer (Hewlett-Packard, Palo Alto, CA). The computer recorded continuous, integrated, Sminute calorimetric measurements used for calculations between 300 and 340 minutes, and 400 and 440 minutes according to the start of the insulin infusion. The protein oxidation rate during the test was estimated from urea nitrogen production rate measured in the urine collected during the clamp. The nonprotein respiratory quotient was then calculated and the substrate oxidation rates were determined from the equations of Lusk.” During the 18 minutes before the start of the insulin infusion, the first of three percutaneous biopsies of the vastus lateralis muscle was

Rates of glucose infusion (exogenous glucose disposal rates), expressed per kg FFM per minute, were averaged from 300 to 340 minutes during the low-dose, and from 400 to 440 minutes during the high-dose insulin infusions. The appearance rate of glucose in the plasma was calculated from the blood [3-3H]-glucose-specific activities using Steele’s non-steady-state equations.*’ The total glucose disposal rate during the low-dose insulin infusion is equal to the glucose appearance rate assessed at the same time. The endogenous glucose production was estimated as the difference between the isotopically derived glucose appearance rate and the exogenous glucose disposal rate. When the isotopically determined appearance rate was equal to or smaller than the exogenous glucose infusion rate, the endogenous glucose production rate was assumed to be fully suppressed and the total glucose disposal rate equaled exogenous glucose disposal rate. Insulin-stimulated carbohydrate oxidation rates were calculated from indirect calorimetry data by using the mean values obtained during the 40-minute calculation periods of both insulin infusions. The carbohydrate nonoxidative disposal rate (storage rate), expressed in mg per kg FFM per minute was considered as the difference between the total glucose disposal rate and the carbohydrate oxidation rate.

Biochemical Assays Muscle glycogen synthase and glycogen synthase phosphatase activities were determined as previously described.‘8,22 Physiologically active glycogen synthase was assayed at low glucose-6phosphate (G6P) concentration (0.17 mmol/L) and is expressed as units per gram wet weight. Total glycogen synthase activity (U/g wet wt) was assayed at a high G6P concentration (10.8 mmol/L). Glycogen synthase phosphatase activity of muscle homogenate was measured only in the fasting state, as the change in percent active form of rabbit glycogen synthase D during a 20-minute incubation. One unit of glycogen synthase equals the incorporation of 1 rmol UDP-glucose into glycogen per minute. The interassay CV for synthase and synthase phosphatase assays were 6% and 15%. respectively. Plasma insulin concentration was determined by radioimmunoassay (RIA) using AUTOPAK INSULIN system on a Concept 4 radioassay analyzer (ICN, Horsham, PA). Plasma concentrations of epinephrine, norepinephrine and dopamine were determined by radioenzymatic assay using CAT-A-KIT assay system (Amersham, Arlington Heights, IL). Plasma glucose concentrations were measured by the glucose oxidase method using a Beckman glucose analyzer (Fullerton, CA), and tritiated glucose-specific activity in blood samples was determined after precipitating protein with perchloric acid.‘j

Statistics Repeated measures ANOVA and Friedman test were used for comparison of glycogen synthase and other clamp results before and after glyburide treatment. Other data were compared using paired t test and Wilcoxon’s test for paired observations. Correlations were Pearson’s product moments (r) and Kendall’s rank correlation coefficients (TAU). The parametric and nonparameter tests were

1206

NYOMBA ET AL

performed using Statistical Analysis System (SAS Institute, Cary, NC) and Statistical Package for Social Sciences (SPSS Inc, Chicago, IL) computer softwares, respectively. Data are shown as the mean * SEM, unless otherwise

Table 2. Euglycemic Clamp Data (mean + SEM) Before and After Glyburide Therapy BeforeGlyburide

indicated. Eody Weight (kg)

RESULTS

Glyburide therapy was associated with an increase in fasting and glucose induced insulin secretion, and an improvement of glucose tolerance (Fig 1). The area under the 3-hour plasma insulin concentration was increased from 6,227 + 2,868 pU/mL to 11,678 + 3,390 &J/mL (P < .OOl), while the area under the glucose curve was reduced from 54,282 * 3,765 mg/dL to 40,318 f 3,112 mg/dL (P < .05). Fasting plasma glucose was significantly lower after glyburide therapy than before (P < .Ol). Subjects also gained weight. The increase in body weight was due to an increase in fat-free body mass (Table 2). Glyburide therapy was also associated with enhanced total body and hepatic sensitivity to insulin as shown by increased glucose disposal rate and decreased endogenous glucose production rate during euglycemic hyperinsulinemic clamps (Table 2). The profile of muscle glycogen synthase activity during the euglycemic clamps is shown in Fig 2. Compared with the fasting values, the glycogen synthase activity assayed at low G6P concentration (active glycogen synthase) decreased by 19% during the low-dose insulin infusion, and increased by 13% during the high-dose insulin, before glyburide therapy, but these changes were not statistically significant. After glyburide therapy, active glycogen synthase increased by 16% and 39% (P < .05) during the low-dose and high-dose insulin infusions, respectively. The fractional activity of glycogen synthase showed similar changes. A two-factor repeated measures ANOVA showed a barely significant effect of glyburide (F = 4.60, P = .06), but no effect of insulin (F = .27, p = NS) on active glycogen synthase. There was a significant interaction (F = 7.40, P -c.05) between glyburide and insulin, indicating a potentiation of insulin’s effect on active glycogen synthase activity by glyburide. This analysis disclosed a similar interaction (F = 8.60, P < .05) between glyburide and insulin on the synthase fractional activity, but none of them had a significant effect when taken separately. The glycogen synthase activity measured at high G6P concentration (total glycogen synthase) did not change with insulin infusion either before

IOOJI

0

60

120

160 Time

(min)

Fig 1. Plasma glucose and insulin concentrations (mean + SEM) during a 75-g OGTT before (0-j and after W-0) glyburide therapy in nine Pima Indians with NIDDM.

FFM (kg) Fat mass (kg)

After Glyburide

121.5

* 9.1

125.1

+ 9.1’

75.5

f 3.7

78.7

+ 3.4t

46.1

+ 6.2

46.4

r 6.5

Plasma glucose (mg/dL) Before insulin infusion

217 + 20

157 f 13t

Low-dose insulin

103 + 1

103 * 1

High-dose insulin

104 f 1

102 f 1

Before insulin infusion

17 f 5

28 ? 8t

Low-dose insulin

71 *9

Plasma insulin (plJ/mL)

High-dose insulin

1,099

i 97

91 + ll$ 1,183

+ 107

Glucosedisposalrate (mg/kg FFM . min) Low-dose insulin

3.23

+ 0.47

4.11

+ 0.57s

High-dose insulin

8.42

2 0.85

11.27

+ 1.21t

Carbohydrate oxidation (mg/kg FFM *min) Low-dose insulin

1.48 + 0.31

1.94 f 0.24

High-dose insulin

2.64

3.11 f 0.25

? 0.33

Carbohydrate storage (mg/kg FFM . min) Low-dose insulin

1.75

k 0.29

2.18 + 0.38

High-dose insulin

5.78

t 0.66

8.16 + 1.031

1.48 * 0.35

0.35 + 0.14*

Endogenous glucose productions (mg/kg FFM - min)

lf < .05: tP -z .Ol: $P < ,001 “before glyburide treatment. SDetermined during low-dose insulin infusion.

or after glyburide treatment. Paired comparisons of before and after glyburide data showed a statistically significant (P-c.Ol) increase of active glycogen synthase measured at low-dose insulin infusion (Fig 2). Active or total glycogen synthase determined in the fasting state or during the high-dose insulin infusion did not change significantly after glyburide treatment. Glyburide treatment was associated with an increase of fasting glycogen synthase phosphatase activity in seven of nine subjects. In the remaining two subjects, the phosphatase activity decreased following gly-

c 4

0.8

Fig 2. Glycogen synthase activity determined in the fasting state and during insulin infusion at the rates of 40 mU/m2/min (low dose) and 400 mUlm’lmin (high dose). Glycogen synthase activity (U/g muscle) was determined at (A) 0.17 mmol/L (active glycogen synthasej and iCj 10.8 mmol/L (total glycogen synthasel of GBP. (Bj The ratio between the two values (fractional activity) is also shown. Data on total glycogen synthase during the high-dose insulin infusion were available for eight subjects. By repeated messures ANOVA there was a significant interaction between glyburide and insulin on active glycogen synthase (F = 7.40. P < .05) and the synthase fractional activity (F = 8.60, P < .05). 0. Before glyburide therapy: I%,after glyburide therapy.

1207

EFFECT OF GLYBURIDE ON GLYCOGEN SYNTHASE

buride treatment. The average phosphatase activity increased by 29%, although this increase was not statistically significant (P = .lO, Fig 3). There was a positive correlation between the changes in active glycogen synthase associated with glyburide treatment and the corresponding changes in glucose disposal rate during the low-dose (r = .70, TAU = 0.44, both P < 0.05) (Fig 4), but not during the high-dose (r = .37, TAU = 0.25, both P = NS) insulin infusion. However, changes in glycogen synthase fractional activity were correlated with changes in glucose disposal rates at both low-dose (r = .68, TAU = 0.43, both P < 0.05) (Fig 5) and high-dose(r = .70, TAU = 0.42, both P < .05) (data not shown) insulin infusion. Furthermore, the changes in fractional activity measured during the high-dose insulin infusion were correlated (r = .75, P < .05, TAU = 0.39, P = .07) with the corresponding changes in nonoxidative glucose disposal rate (data not shown). Plasma concentrations of epinephrine, norepinephrine, and dopamine during euglycemic clamps were obtained in three subjects. Because of the small number of subjects, no statistical analysis was performed on these data. However, it is clear that epinephrine concentration increased considerably above basal during insulin infusion (Table 3). This increase was greater during the low-dose than the high-dose insulin. This increase of epinephrine levels during insulin infusion was substantially lower after glyburide treatment. A smaller increase of plasma norepinephrine levels was observed during insulin infusion, but this increase was not affected by glyburide therapy. Dopamine levels remained

:

0.30-

z co”

0.25 -

s-0) 8r; $r o E +eQ ‘5 2

l

0.20 l

0.15-

l l

O.lO-

l l

0.05-

.E

l

l

O-

g g f

l

-0.05-O.IOd

I

-0.6

-0.2

0.2

0.6

1

1

1

1

1

1.0

1.4

I.6

2.2

2.6

Chanqe in Glucose Disposal Rate (mq/kqFFM/min) ot Low Dose Insulin Infusion Fig 4. Relationship between changes in insulin-stimulated glycogen synthase activity associated with glyburide therapy and the corresponding changes in glucose disposal rate at low-dose (r = .70, TAU = 0.44, both P< .051 of insulin infusion. Insulin doses are defined in the legend to Fig 2.

essentially constant during insulin infusion both before and after glyburide treatment (Table 3). DISCUSSION

The purpose of the present study was to investigate the effects of glyburide therapy on skeletal muscle glycogen synthase in subjects with NIDDM. Each subject was compared with himself before and after glyburide treatment. We observed an increase of insulin stimulation of skeletal muscle glycogen synthase activity in parallel with an augmentation of insulin-mediated total body glucose disposal rates, following chronic glyburide therapy. This treatment significantly improved glucose tolerance in all subjects and was also associated with an increase of fasting and glucose-induced insulin secretion, as well as insulin suppression of endogenous glucose production. 0.5-

ii + E Q s g

l

1.25-

l.OO-

% ;

sz o”FE (3 .;”

0.2-

0.3l

0.50

0. I l

0 a -0.

I -

-0.6

0.25

l

o-

-0.2-

0

I -0.2

I 0.2

l

1

I

0.6

I .o

Chanqe in Glucose Disposal at Low Dose Insulin

13 -

Before

After

Fig 3. Effect of glyburide therapy on skeletal muscle glycogen synthase phosphatase activity. The mean and individual phosphatase values are given before and after glyburide therapy.

l

0

E 5

zh

s f

0.4-

cu .gt

0.75-

co” c

$ 0 f ‘,E ln .E

1 1.4

I.8

2.2

Rate (mq/kqFFM/min)

Fig 5. Relationship between changes in insulin-stimulated glycogen synthase fractional activity induced by glyburide therapy and the corresponding changes of glucose disposal rate at lowdose (r = .68. TAU = 0.43, both P < .051 insulin infusion. Insulin doses defined in the legend to Fig 2.

1208

NYOMBA ET AL

Table 3. Mean (k SEM. pg/mL)

Plasma Dopamine.

Epinephrina,

Treatment

and Norepinephrine

During Insulin Infusion Before and After Glyburide

of Three Diabetic Subjects

Befwe Glyburide Time (min)

DOP

After Glyburide

EPI

NOR

DOP

EPI

NOR

-25

9.7 * 3.4

10.7 * 9.5

137.3

f 26.1

9.7 f 4.1

21.7

r 2.3

164.0

+ 17.8

-18

5.7 + 3.2

25.0

_t 13.6

113.7

_+ 17.9

7.3 _t 3.7

19.0 _t 2.1

157.7

_t 21.9

315

17.0 * 7.4

388.3

k 186.3

253.3

f 33.6

14.3 + 3.7

129.3

+ 91.8

236.3

it 43.2

330

13.7 f 3.7

316.0

+ 117.1

205.7

+ 40.0

14.0 * 7.4

125.7

t 81.3

244.3

k 36.4

410

16.0 + 6.6

245.3

k 103.2

222.3

f 39.7

17.3 f 8.7

108.3

+ 59.4

214.7

+ 49.4

425

18.3 e 6.1

291.7

+ 150.6

23 1 .O + 49.0

89.7

f 43.2

216.0

+ 65.9

25.0

+ 13.6

Abbreviations: DOP, dopamine; EPI, epinephrine; NOR, norepinephrine.

Sulfonylureas are known to increase insulin secretion24 and this effect was thought to explain the reduction of hyperglycemia by these agents in NIDDM. This view is supported by studies that show a little25 or noz6 effect of sulfonylureas on metabolic control in subjects with insulin dependent diabetes. Even though others27,28have found an improvement of glucose control in NIDDM by sulfonylureas while plasma insulin levels were unchanged, such effects could be explained by increased chronic exposure to insulin, and account should be taken of the insulin:glucose relationship in these subjects. In balance, these studies suggest that at the extrapancreatic level sulfonylureas act to enhance the effects of insulin, whose secretion is increased by sulfonylurea treatment. As previously shown,‘4 skeletal muscle is a major site of postprandial, insulin-mediated glucose metabolism, and this is accounted for mostly by glucose storage as glycogen. Insulin-mediated glucose storage in vivo is closely correlated with insulin activation of glycogen synthase in skeletal muscle,22,29 and this tissue is a major site of the insulin resistance characteristic of NIDDM.14 Our findings of enhanced insulin stimulation of glycogen synthase activity in skeletal muscle after glyburide therapy, and the presence of correlations between changes in glycogen synthase activity following this treatment and the corresponding changes in insulin-mediated glucose disposal rates are in line with those previous studies. Sulfonylureas have been reported to increase glycogen content of fat and liver30-32by decreasing glycogenolysis3’ or increasing glycogen synthesis.‘0-‘3 Sulfonylureas also decreased glycogenolysis in rat skeletal muscle,30 but no stimulatory effect of these drugs on muscle glycogen synthase was known. The present study therefore extends our knowledge of the mode of action of sulfonylureas on glucose metabolism in human skeletal muscle. How glyburide affects glycogen synthase activity is presently unknown. A few investigators”*” using isolated fat or liver cells have reported that sulfonylureas exert a direct effect on glycogen synthase. However, others’*21”1’2found only a potentiating effect on insulin action. Whether one or both of these mechanisms are involved cannot be adequately assessed in the current, in vivo study, since both insulin secretion and insulin action improved during glyburide therapy. Nevertheless, statistical analysis showed the presence of a significant interaction between insulin and glyburide on active glycogen synthase and the synthase frac-

tional activity, suggesting that glyburide potentiates insulin action on glycogen synthase. During the low-dose insulin infusion, glycogen synthase activity decreased (unsignificantly) below the fasting level in subjects with NIDDM before glyburide therapy. This phenomenon was attributed to the release of epinephrine,‘4v33 which inhibits glycogen synthase activity via the activation of synthase kinase.34 The marked increase of plasma epinephrine levels (Table 3) could explain the lack of synthase activation by insulin before glyburide therapy. Conversely, glyburide might have uncovered insulin action on glycogen synthase by preventing epinephrine release (Table 3). It is also possible that this sulfonylurea activated glycogen synthase by activating the low-K, CAMP-phosphodiesterase, an enzyme also activated by insulin,35 thereby decreasing tissue concentration of cAMP’~ and inhibiting CAMP-dependent protein kinase.37 Glycogen synthase may also be activated via synthase phosphatase activation. We have recently reported an association between fasting glycogen synthase phosphatase activity and insulin stimulation of glycogen synthase in human skeletal muscle.‘* In the present study, insulin-stimulated glycogen synthase activity increased after glyburide therapy, but the mean change of synthase phosphatase activity was not statistically significant (Fig 3). Margolis3* recently reported an increased activation of glycogen synthase by glyburide in obese Zucker rat liver without any effect of the drug on synthase phosphatase. It has been shown that sulfonylureas enhance insulinstimulated glucose uptake in various tissues,‘B2 and these compounds increase the number of glucose transporters in cell membranes.8*9 Altan et alI3 showed that glyburideinduced activation of glycogen synthase in rat adipose tissue is dependent on extracellular glucose concentrations. However, the role of glucose in the regulation of glycogen synthase is questionable in human skeletal muscle.39 At the plasma membrane level, sulfonylureas have been found by someJo- but not a1143*44 investigators, to increase insulin receptor number. It was recently proposed that sulfonylureas may facilitate insulin receptor translocation by their effects on membrane fluidity.28 However, Jacobs et aY4 found no effect of chlorpropamide on insulin receptor tyrosine kinase, which is believed to transmit the insulin signal to cellular effector systems,45 and may be necessary for the receptor internalization.46

1209

EFFECT OF GLYBURIDE ON GLYCOGEN SYNTHASE

The increase in insulin-stimulated glycogen synthase activity after glyburide therapy was associated with an increased insulin-mediated whole body capacity for carbohydrate storage, an important mechanism of insulin action4’ With the observed decrease in hepatic glucose production, this phenomenon reflects an increased anabolic state induced by glyburide therapy, as also evidenced by the increase in lean (fat-free) body mass. Bogardus et a?’ have previously shown that tolazamide therapy reduced the resting metabolic rate in subjects with NIDDM, and proposed that this, together with a reduced protein turnover, may contribute to prevent weight loss in diabetic subjects. The present study is in keeping with this previous report and demonstrates that in contrast to what was previously thought49 sulfonylurea treatment induces an increase of lean body mass in association

with an improvement of insulin sensitivity and metabolic control. In summary, glyburide therapy was associated with an increase of insulin stimulation of glycogen synthase in human skeletal muscle. This effect was associated with enhanced total body and liver sensitivity to insulin and an increase in lean body mass. ACKNOWLEDGMENT

We thank Carol Lamkin and her nursing staff, Vicky Boyce and her dietary staff, and the technical staff, including Vera Rodriguez, Tom Anderson, Debbie Wolfe-Lopez, Jane11 Ramsey, Harlan Osife, John Brown, and Victoria Ossowski, for their professional assistance. We are also indebted to Charlesetta Lincoln for her secretarial work. Most of all, we thank the volunteers.

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