Drt-fa).

Altered expression of muscle glucose transporter in diabetic fatty Zucker rats (ZDF/Drt-fa)

GLUT-4

JACOB E. FRIEDMAN, JAMES E. DE VENTE, RICHARD G. PETERSON, AND G. LYNIS DOHM Department of Biochemistry, School of Medicine, East Carolina University, Greenville, North Carolina 27858; and Department of Anatomy, Indiana University School of Medicine, Indianapolis, Indiana 46202

FRIEDMAN, JACOB E., JAMES E. DE VENT@, RICHARD G. PETERSON, AND G. LYNIS DOHM. ALtered expression of muscZe glucose transporter GLUT-4 in diabetic fatty Zucker rats (ZDF/ Drt-fa). Am. J. Physiol. 261 (Endocrinol. Metab. 24): E782E788, 1991.-W, examined GLUT-4 glucose transporter protein and mRNA in muscle tissue from a new rodent model of non-insulin-dependent diabetes mellitus (NIDDM), the male obese Zucker diabetic fatty (ZDF) rat [ZDF/Drt-fa(FlO)]. We also determined whether prevention of hyperglycemia might affect GLUT-4 expression by feeding the intestinal a-glucosidase inhibitor acarbose (40 mg/lOO g diet) in the diet of male ZDF rats for 19 wk, starting at least 1 wk before the onset of diabetes. Fasting glucose was four- to sixfold greater in diabetic ZDF rats (24.1 t 6.7 mM) compared with lean or obese nondiabetic rats. Fasting insulin in diabetic ZDF rats (0.5 t 0.1 rig/ml) was similar to lean rats (0.4 t 0.1) but greatly reduced compared with obese nondiabetic rats (18.7 t 4.0 rig/ml). Acarbose treatment significantly reduced fasting glucose levels to 13.4 of: 1.4 mM, while insulin levels increased to 1.6 t 0.3 rig/ml. GLUT-4 protein levels in diabetic ZDF rats were reduced -40% in red quadriceps and mixed gastrocnemius muscles but were unchanged in white quadriceps muscle. Acarbose treatment was associated with a twofold increase in GLUT-4 protein and mRNA in mixed gastrocnemius muscle. These data indicate that, in this obese model of NIDDM without hyperinsulinemia, there is reduced muscle GLUT-4 protein in red but not white muscle fiber types. The decrease in muscle GLUT-4 expression in this model of NIDDM can be prevented by acarbose treatment, which reduces hyperglycemia and increases P-cell responsiveness. skeletal muscle; non-insulin-dependent bose

diabetes mellitus;

acar-

STATES such as obesity and noninsulin-dependent diabetes mellitus (NIDDM) are characterized by reduced insulin-stimulated glucose transport in skeletal muscle, the major site of whole body glucose disposal (9, 35). The majority of the glucose transport response in insulin-sensitive tissues is mediated by the glucose transporter protein isoform GLUT-4, the expression of which is limited to muscle and adipose tissue (2, 16, 20). Previous experiments have shown reduced expression of muscle GLUT-4 protein and mRNA in streptozotocin (STZ)-induced diabetes and a return to normal with insulin injection (1,12,36). Although muscle GLUT-4 downregulation appears to coincide with hyperglycemia (1, 22), in several hyperinsulinemic-hypergly-

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cemic models of obesity and NIDDM, muscle GLUT-4 protein appears to be unchanged (23, 27, 37). These observations suggest that changes in muscle GLUT-4 protein in NIDDM may depend on the combination of impaired insulin secretion and the emergence of moderate-to-severe hyperglycemia. As in human NIDDM, animal models vary in their degree of obesity, glucose intolerance, insulin response to glucose, and susceptibility to diabetes. In the present study we examined levels of GLUT-4 glucose transporter protein and mRNA in muscles of a new rodent model of NIDDM, the male obese Zucker diabetic fatty (ZDF) rat [ZDF/Drt-fa(FlO)]. In the ZDF rat, obesity and insulin resistance preceed the development of hyperglycemia (32). As in human NIDDM patients, there is a progressive loss of glucose-stimulated insulin secretion and frank hyperglycemia, probably due to the loss of GLUT2 expression in the ,&cell recently described in this rodent model of NIDDM (30). Altered expression of muscle glucose transporters in any genetic model of NIDDM could be due to an abnormality in the GLUT-4 gene or susceptibility to metabolic factors such as hyperinsulinemia and/or hyperglycemia (1). To determine whether expression of GLUT-4 in the ZDF rat is altered by prevention of hyperglycemia, we added acarbose, an intestinal cr-glucosidase inhibitor, to the diet of male ZDF rats beginning at 7 wk of age, at least 1 wk before the onset of diabetes. Acarbose has affinity for sucrase, glucoamylase, and maltase and has been shown to delay the rise in postprandial glucose. Unlike other antihyperglycemic agents such as insulin and sulfonyl ureas, acarbose has no known direct effects on peripheral insulin sensitivity or ,&cell function (5). Our findings indicate that, in diabetic ZDF rats without hyperinsulinemia, there is a downregulation of muscle GLUT-4 expression in red but not white muscle fiber types and that early administration of acarbose to prediabetic rats reduces hyperglycemia, increases ,&cell responsiveness, and prevents the loss of GLUT-4 expression in skeletal muscle. METHODS

In this study we employed as a model of NIDDM a colony of partially inbred obese ZDF rats [(ZDF/Drtfa(FlO)] in which all male members become diabetic by 10 wk of age as described previously (32). Because sex-

1991 the American

Physiological

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Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 16, 2018. Copyright © 1991 American Physiological Society. All rights reserved.

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matched nondiabetic obese ZDF rats do not exist, we used as controls age-matched lean male heterozygotes (Fa/+). The partially inbred ZDF rats (ZDF/Drt-fa) are from the Diabetes Animal Support Facility, Department of Anatomy, Indiana University School of Medicine. Diabetic obese ZDF rats and lean ZDF rats were fed standard lab Chow (Purina 5008), which included 10% sucrose, ad libitum. A second group of diabetic ZDF rats were fed standard lab Chow plus 10% sucrose supplemented with acarbose (40 mg/lOO g diet) for 19 wk, beginning at 7 wk of age. Sucrose was added to the diet to increase the effectiveness of acarbose, since sucrose is one of the primary carbohydrates whose digestion is blocked by acarbose. Glucose levels in the fed state were obtained weekly on all animals between 800 and 9:00 A.M. by tail vein collections and were analyzed using a Beckman glucose II analyzer. Fed and fasting plasma insulin levels were measured by double-antibody radioimmunoassay by the method of Morgan and Lazarow (29) using a rat insulin standard (Eli Lilly). Insulin secretion was assessedby measuring glucose-stimulated insulin secretion after administration of 2 g/kg glucose by oral gavage. Animals were anesthetized at 26 wk of age by intraperitoneal injection of pentobarbital sodium, and the quadriceps and gastrocnemius muscles were removed and immediately frozen between two tongs chilled to the temperature of liquid nitrogen. Muscles were stored at -70°C until analysis. Muscle GLUT-4 protein was detected initially by Western blot analysis and was quantitated using dotblot analysis. The gastrocnemius sample was obtained by powdering the entire muscle at the temperature of liquid nitrogen and weighing 200 mg of a well-mixed sample. The red and white portions of the quadriceps were obtained by visual dissection of the muscle. These discrete portions of muscle were chosen to address the question of whether GLUT-4 was decreased in a fiber type specific manner. Muscle (ZOOmg) was homogenized using a Polytron homogenizer in buffer containing (in mM) 25 N-Z-hydroxyethylpiperazine-N’-Z-ethanesulfonic acid (HEPES), 4 EDTA, and 25 benzamidine and 1 PM each of leupeptin, pepstatin, and aprotinin. After homogenization, membranes were sedimented by centrifuging at 150,000 g in a Beckman 50 Ti rotor for 1 h. The pellet was resuspended in homogenization buffer containing 1% Triton X-100, incubated for 1.5 h at 4°C and was centrifuged again at 150,000 g for 1 h. For Western analysis, samples of supernatant containing 50 ,ug protein (BCA procedure; Pierce Chemical, Rockford, IL) were mixed with Laemmli sample buffer (25) containing 2.5% dithiothreitol and 1% sodium dodecyl sulfate (SDS). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on an 8% resolving gel and were transferred from the gel to an Immobilon membrane by electrotransfer. The membrane was blocked for 2 h with 5% Carnation low-fat instant milk in tris(hydroxymethyl)aminomethane (Tris)-buffered saline (TBS), followed by incubation at 25°C for 16 h in polyclonal antibody ECU4 specific for the last 12 amino acids of the GLUT-4 carboxy terminus (21). The membranes were then washed alternatively in TBS and TBS0.05% Tween and probed for 4 h with 1251-labeledgoat

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anti-rabbit immunoglobulin G. Membranes were washed, dried, and subjected to autoradiography for 48 h at -70°C and the resulting autoradiograms were analyzed by densitometry. For dot-blot analysis, 5 pugprotein from each sample were diluted to 0.01% Triton using the original homogenization buffer, and samples were applied in triplicate to an Immobilon membrane filter placed on a Minifold dot-blot apparatus (Schleicher and Schuell, Keene, NH). Membranes were allowed to dry, were rewet in TBS, and were analyzed as outlined above. The resulting autoradiogram was scanned by densitometry using a Titertek Multiskan MCC/340 plate reader at a wavelength of 540 nm. Muscle GLUT-4 mRNA was detected in muscle using Northern analysis. Total RNA was isolated from 200 mg of tissue using the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction described previously (3). Total RNA was size fractionated on a 1.8% agarose-formaldehyde gel and was transferred by capillary diffusion to 0.45 pm Hybond-N membranes (Amersham). The RNA was visualized and photographed by ultraviolet transillumination to ensure that the RNA was intact and to allow calculation of molecular mass based on migration of the 28s and 18s ribosomal subunits. The entire @DNA insert of GT-2 (20), which encodes for the murine insulin-sensitive glucose transporter protein GLUT-4, was used to probe the RNA after 32P random labeling using the multiprime labeling system (Amersham). Blots were prehybridized for 4 h at 45°C in a solution of 50% deionized formamide, ~5 standard sodium citrate (SSC), ~5 Denhardt’s solution, 0.1 mg/ml yeast tRNA, 0.5 mM NaP04, 0.5 mg/ml sodium pyrophosphate, 1% SDS, and 2.5% H20. Hybridization was carried out at 45°C for 16 h in 50% deionized formamide and ~5 SSC using the 32P-labeled cDNA probe. The membranes were washed at high stringency (65°C X0.1 SSC, 0.1% SDS, 15 min) and were autoradiographed at -70°C using Kodak XAR-5 film. The membranes were then stripped and reprobed with a 1.9kilobase (kb) cDNA clone for ,&actin (4). The resulting autoradiograms were quantitated by densitometry, and the levels of expression of GLUT-4 mRNA were normalized to that of P-actin. All differences between groups were analyzed using one-way analysis of variance with Neuman-Keuls post hoc analysis, with P < 0.05 as statistically significant. All values are presented as means t SE. RESULTS

Glucose levels in the diabetic ZDF rat were four- to sixfold higher compared with either lean or obese nondiabetic rats (Table 1). As shown in Fig. 1, acarbose treatment of diabetic ZDF rats dramatically lowered blood glucose throughout the study. Acarbose lowered both fasting and postchallenge glucose levels relative to untreated obese diabetic ZDF rats (P < 0.01; Table 1). Glucose levels were still significantly greater in acarbosetreated diabetic ZDF rats compared with either lean or nondiabetic obese rats (P < 0.01). Although both fasting and 30-min postchallenge insulin levels were similar in diabetic ZDF rats compared


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TABLE 1. Plasma glucose and insulin in lean, nondiabetic obese, lean ZDF, diabetic obeseZDF, and acarbose-treated diabetic obeseZDF rats

Group

Lean (Fa/-) Obese (fa/fa) Lean ZDF (fa/+) Diabetic ZDF (falfa) Acarbose-treated diabetic

Fasting Glucose, mM

6.320.4 7.8t0.4 4.6t0.5 24.lt6.7" 13.4+1.4*-f-

Fasting Insulin, w/ml

7.3tl.O 18.7t4.0* 0.4t0.1 0.5t0.1 1.6+0.3*?

30-Min Glucose, mM

Postchallenge Insulin, w/ml

ND ND

ND ND

9.1t0.5 58.5&2.2* 19.9*2.9*-t

0.8kO.2 0.9t0.3 12.4+3.4*"f

ZDF (falfa) Values are means t SE; n = 5-8 animals/group. Blood samples were obtained from animals after an 18-h fast and 30 min after a glucose load (2.0 g/kg) administered by oral gavage. Data from previously published results [lean (Fa/-) and obese (fa/fa) see Ref. lo] are presented to compare insulin resistance of obese nondiabetic rats (36 wk old). ND, not determined. * Significantly greater than lean rats, P < 0.01. t Significantly different from diabetic Zucker diabetic fatty (ZDF) rats, P < 0.01. 40

muscle GLUT-4 protein in diabetic obese ZDF rats (Fig. 2) . As in our previous studies (10,21) we detected a single n32 band of -45kDa protein using polyclonal antibody ECU4 by Western blot. To determine whether the changes we observed in GLUT-4 protein were fiber type specific, we performed quantitative dot blots using equivalent amounts of protein from red quadriceps, white quadriceps, and mixed gastrocnemius muscle. Rat heart muscle was used as a standard because heart contains higher levels of GLUT-4 protein than skeletal muscle. The linearity of the GLUT-4 dot-blot signal in skeletal 0”““““““““” 1 muscle was verified using 2.0, 5.0, and 10.0 pg muscle 0 1 2 3 4 5 6 7 8 9 1011 121314151617181920 protein, and we obtained a correlation coefficient of 0.957, 0.963, and 0.974 for red vastus, white vastus, and WEEKS OF TREATMENT mixed gastrocnemius, respectively. A correlation coeffiO0 LEAN l -0 DIABETIC A -A DIABETIC-ACARBOSE cient of 0.965 was obtained when identical samples were FIG. 1. Weekly blood glucose levels in lean, diabetic Zucker diabetic analyzed by Western and dot-blot analysis. fatty (ZDF), and diabetic ZDF acarbose-treated rats. Blood samples As shown in Fig. 3, GLUT-4 protein in diabetic ZDF were obtained from tail vein collections as outlined in METHODS. rats was significantly reduced by 40 and 45% in red Beginning of curve indicates rats at 7 wk of age. quadriceps and mixed gastrocnemius muscles, respectively (P < 0.05), whereas GLUT-4 protein was slightly with lean fa/+ rats, diabetic ZDF rats were insulin de& cient relative to obese rats (Table 1). Although acarbose although not statistically lower by 19% in the white treatment significantly increased fasting insulin levels quadriceps muscle. Acarbose treatment of diabetic ZDF rats was associated with a significant 1.5- and X-fold more than threefold (P < O.Ol), insulin levels were still increase in GLUT-4 protein in red quadriceps and mixed well below levels found in obese nondiabetic rats. Postgastrocnemius muscle, respectively (P < 0.01). GLUT-4 challenge plasma insulin was increased significantly by protein in white quadriceps muscle was not changed acarbose treatment in diabetic ZDF rats (P < 0.01). significantly as a result of acarbose treatment. Final body weight was significantly lower in diabetic Northern analysis was used to determine steady-state ZDF rats (P < 0.01) compared with obese nondiabetic or mRNA levels of GLUT-4 and @-actin using equivalent acarbose-treated diabetic ZDF rats (Table 2). As reported amounts of RNA obtained from mixed gastrocnemius previously by others (26, 34) gastrocnemius muscle muscle (Fig. 4). GLUT-4 mRNA was lower by -20% in weight in fa/fa obese rats was less than muscles in lean diabetic ZDF rats and was significantly increased by animals (P < 0.05). Although muscle membrane protein twofold in acarbose-treated diabetic ZDF rats (P < 0.01) per gram of tissue and RNA per gram of tissue were not compared with untreated diabetic ZDF animals (Fig. 4). statistically different, these parameters were slightly When GLUT-4 mRNA in diabetic ZDF rats was exlower in diabetic ZDF animals and tended to be higher pressed relative to total tissue RNA, the slight decrease in diabetic acarbose-treated rats (Table 2). in total RNA combined with a small decrease in GLUTWestern blot analysis was used to detect GLUT-4 4 mRNA resulted in a significant decrease of 30% in protein in muscles of lean obese diabetic and obese total GLUT-4 mRNA (P < 0.05). Likewise, when GLUTdiabetic ZDF acarbose-fed rats. We (10) and others (23, 4 mRNA in acarbose-treated diabetic ZDF rats was ex27, 37) have found that the hyperinsulinemia of obesity pressed relative to total RNA, total GLUT-4 mRNA is not associated with any change in muscle GLUT-4 levels were increased 2.6-fold over diabetic ZDF rats (P protein. By contrast, there was a significant decrease in c 0.01). 36


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2. Body weight and muscle analysis of lean, obesenondiabetic, lean ZDF, diabetic obeseZDF, and acarbose-fed diabetic obeseZDF rats

TABLE

Group

Final Body Wt, g

Lean @a/-) Obese (fa/fa) Lean ZDF vu/+) Diabetic ZDF (fa/fa) Acarbose-treated diabetic ZDF

398.1k4.4 560.2fll.l* 488.7zk9.9 479.1f29.8 622.0+17.8t

Gastrocnemius w g

Membrane Protein, mg/g muscle

2.203kO.055

f&54+0.34

1.911rtO.O66* 2.619kO.079 1.752+0.091* 1.903+0.061*

7.98kO.27 8.28zlzO.31

Total RNA, rg/g muscle

ND ND

8.02kO.45

467k86 355&90

8.57k0.99

694+140

Cfalfa)

Values are means + SE; n = 5-8 animals/group. Significantly different from lean, *P < 0.05, t P < 0.01. Data from previously published results [lean (Fa/-) and obese (fa/fa) see Ref. lo] are presented to compare muscle characteristics of obese nondiabetic rats (36 wk old). ND, not determined.

LEAN CONTROL DIABETIC DIABETIC - ACARBOSE

48.5K-

/

“

o-

2. Western blot of glucose transporter protein GLUT-4 in mixed gastrocnemius muscle from lean (fa/+), obese diabetic ZDF, and acarbose-fed obese diabetic ZDF rats. Total muscle membranes were prepared as outlined in METHODS, and 50 fig protein were subjected to SDS-polyacrylamide gel electrophoresis, transferred to Immobilon membrane, and immunoblotted with polyclonal antibody ECU4 to GLUT4 protein. Results are representative of 2 separate blots of muscles obtained from 57 different rats/group. Molecular mass markers are in kilodaltons. *Significantly less than lean control, P < 0.01. FIG.

‘i

-- - \ b %

m 8

and starch digestion and slows the rate of glucose absorption from the gut (5). In diabetic obese ZDF animals Skeletal muscle is the primary tissue responsible for given acarbose before development of hyperglycemia, insulin-stimulated glucose disposal (7). Glucose transplasma glucose levels were significantly lower, although port is normally the rate-limiting step in muscle glucose not completely normalized, relative to lean or obese metabolism (24) and is mediated by a unique tissueanimals. Surprisingly, acarbose treatment was associated specific glucose transporter protein GLUT-4 (11). Prewith a marked increase in glucose-stimulated insulin vious studies have established that expression of GLUTsecretion in diabetic ZDF rats. Previous studies in dia4 glucose transporter protein and mRNA are signifibetic populations have generally found little or no change cantly reduced in skeletal muscle from STZ-induced in insulin secretion with acarbose treatment (6). The diabetic animals and are restored to normal levels with profound effects in the present study could be due to the insulin therapy (1, 12, 36). However, in various animal fact that acarbose was administered before the onset of models of genetic obesity and NIDDM, investigators diabetes, thereby preventing some of the loss of P-cell have been unable to detect any significant change in GLUT-4 protein expression in skeletal muscle (23, 27, function caused by glucose toxicity (7). Direct evidence 37). Recently, a new animal model of NIDDM, the obese in support of this hypothesis has recently been provided Zucker diabetic fatty (ZDF) rat (ZDF/Drt-fa) has been by Johnson et al. (18) and Orci et al. (30) who found striking reductions in GLUT-2 protein and mRNA in developed in which obesity and insulin resistance preceed the development of hyperglycemia. Diabetes in this the P-cell of diabetic ZDF rats. With acarbose treatment, levels of GLUT-2 were still well below model resembles the onset, course, and symptoms of immunodetectable values of nondiabetic rats but were more than threefold NIDDM in humans (32). The purpose of the present above untreated diabetic ZDF rats (30). Thus early adstudy was to determine whether muscle GLUT-4 expresministration of acarbose prevented severe hyperglycemia sion is downregulated in this rodent model of NIDDM and may have preserved GLUT-2 levels enough to preand whether prevention of hyperglycemia using acarbose vent the loss of glucose responsiveness in the P-cell of could influence GLUT-4 expression in skeletal muscle. Acarbose is a potent orally administered intestinal CY- this model of NIDDM. We found the concentration of GLUT-4 protein was glucosidase inhibitor that delays the time course of sugar DISCUSSION


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I 80 3. Comparison of relative GLUT-4 protein levels in muscles from lean control (n = 8), diabetic ZDF (n = 7), and acarbosetreated diabetic ZDF (n = 6) rats. GLUT-4 was determined by dot-blot analysis as described in METHODS and was expressed as percent of lean control, means + SE. *Signifkantly less than lean control, P < 0.05. ttsignificantly greater than diabetic ZDF group, P < 0.01. FIG.

60 40 20 -

20

-o-

0

WHITE QUADRICEPS

RED QUADRICEPS 0

LEAN CONTROL

-

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86881 DIABETIC-ACARBOSE

decreased 40-45% in red muscle fibers of the quadriceps muscle and in mixed gastrocnemius muscle of diabetic ZDF rats. Our findings of decreases in both GLUT-4 protein and mRNA levels are in good agreement with previous studies (1, 12, 36), which indicate that chronic regulation of GLUT-4 in muscle occurs at a pretranslational level. Previous studies have suggested that hyperglycemia plays a major role in the downregulation of

GLUT4

)3-Actin

6

i x

i 1.9Kb

160 ,140 120 0

100

LEAN

CONTROL

80

I

DIABETIC

60

m

DIABETIC-ACARBOSE

40 20 0 FIG. 4. Northern blot analysis of GLUT-4 mRNA levels in gastrocnemius muscle from lean control, diabetic ZDF, and diabetic ZDF acarbose-treated rats. Total RNA extraction, gel electrophoresis, hybridization, and scanning densitometry were performed as outlined in METHODS. Results are percent of lean control, means f SE from 2 separate analyses of muscles obtained from 5 rats/group and are expressed relative to @-actin. *Significantly greater than diabetic ZDF at P < 0.01.

TREATED

muscle GLUT-4 mRNA (1) and glucose transporter protein (1,22,33). However, decreased GLUT-4 protein has not been observed in skeletal muscle of hyperinsulinemic-hyperglycemic models of NIDDM (23,27,37). Our observations were made on animals with relative hypoinsulinemia accompanying hyperglycemia and suggest that, while muscle GLUT-4 expression may be under control of ambient glucose, the response may depend on the presence of low levels of insulin. The hyperinsulinemia in the obese nondiabetic rats, although very significant, does not correlate with any change in muscle GLUT-4 protein (10). On the other hand, obese diabetic ZDF animals were hypoinsulinemic and severly hyperglycemic from an early age (Fig. 1 and Table 1). Our results suggest that decreased muscle GLUT-4 expression in this model of NIDDM may depend on the loss of insulin secretion as well as maintainence of a prolonged period of hyperglycemia. It is well known that insulin deficiency can inhibit protein synthesis in skeletal muscle (17) and may have contributed to the decreased muscle and body weight found in obese diabetic ZDF animals. The finding that insulin levels were increased with acarbose feeding suggests that restored pancreatic function may have contributed to increased transporter expression as well as to lowering hyperglycemia. These findings differ with respect to our previous study in which there was an -20% decrease in muscle GLUT4 protein in obese patients with or without NIDDM compared with nondiabetic nonobese controls (8). Thus, while obesity appears to influence muscle GLUT-4 expression in humans, this does not appear to be the case in rodent models of obesity. Conversely, with respect to NIDDM, no further change in muscle GLUT-4 is observed in human muscle (8, 31), whereas in rodents with hyperglycemia there is a marked decrease in muscle GLUT-4 expression. Whether these differences are due to the duration of diabetes, the degree of P-cell destruction, or to species differences between humans and rats is unknown. It should be emphasized that the genetically obese ZDF rat demonstrates a significantly higher level of diabetes than most other obese models of NIDDM (13), and this underscores the importance of different


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genetic backgrounds when considering the diabetic trait. The lack of change in white skeletal muscle suggests that GLUT-4 levels are regulated in a fiber type specific manner. Red (types I and IIA) skeletal muscles express much higher levels of GLUT-4 protein and mRNA than white (type IIB) muscle, and this correlates highly with insulin responsiveness in these muscles in vitro (14, 21). Lillioja et al. (28) have also reported a significant association between muscle fiber type and in vivo insulin action in humans. We recently observed 70% higher levels of muscle GLUT-4 protein in mixed gastrocnemius muscle of middle-aged endurance athletes compared with sedentary aged-matched controls, and this correlated with increased percentage of type I fibers (15). Because most human muscles are composed of a mixture of fiber types (1.9) and because fiber composition varies widely between . individ .uals (19, 28), the data sugge 1stthat muscle fiber type may be a very important factor to consider when examining the expression of GLUT-4 in both rat and human muscle. In summary, the present study demonstrates that, in this obese model of NIDDM without hyperinsulinemia, muscle GLUT-4 mRNA and protein are decreased in specific muscle fiber types. Our results indicate that reduction of chronic hyperglycem ia by acarbose treatment increases ,&cell responsiveness and prevents the loss of muscle GLUT-4 expression. We gratefully acknowledge Mary-Ann Neel and Leah A. Little for assistance in carrying out the animal protocols used in this study, and we thank the laboratory of Dr. D. Fletcher for performing the insulin assays. This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Research Service Award DK-08477-01 to J. E. Friedman, NIDDK Grant DK-381416 to G. L. Dohm, and a Grant-in-Aid from Miles Laboratory to R. G. Peterson. Address for reprint requests: J. E. Friedman, Dept. of Biochemistry, School of Medicine, Case Western Reserve Univ., 2119 Abington Rd., Cleveland, OH 44106. Received

8 March

1991; accepted

in final

form

24 July

1991.

REFERENCES 1. BOUREY, R., L. KORANYI, D. E. JAMES, M. MUECKLER, AND M. A. PERMUTT. Effects of altered glucose homeostasis on glucose transporter expression in skeletal muscle of the rat. J. CZin. Invest. 86: 542-547, 1990. 2. CHARRON, M. J, F. C. BROSIUS, S. L. ALPER, AND H. F. LODISH. A glucose transport protein expressed predominantly in insulinresponsive tissues. Proc. NutZ. Acad. Sci. USA 86: 2535-2539,1989. 3. CHOMCZYNSKI, P., AND N. SACCHI. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. 4. CLEVELAND, D. W., M. A. LOPATA, R. J. MACDONALD, N. J. COWAN, W. J. RUTTER, AND M. W. KIRSCHNER. Number and evolutionary conservation of cy- and P-tubulin and cytoplasmic ,& and cu-actin genes using specific cloned cDNA probes. Cell 20: 95105, 1980. 5. CLISSOLD, S. O., AND C. EDWARDS. Acarbose-a prelimiary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential. Drugs 35: 214-243, 1988. 6. CREUTZFELDT, W. (Editor). Proceedings of the First International Symposium on Acurbose. Amsterdam: Excerpta Medica, 1982. 7. DEFRONZO, R. A. The triumvirate: ,&cell, muscle, liver: a collusion responsible for NIDDM. Diabetes 37: 667-687, 1988. 8. DOHM, G. L., C. W. ELTON, J. E. FRIEDMAN, P. F. PILCH, W. J. PORIES, S. M. ATKINSON, JR., AND J. F. CARO. Decreased expres-

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10.

11.

12.

13

14.

15.

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17. 18.

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20.

21.

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23.

24.

25. 26.

27.

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RATS

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sion of glucose transporter in muscle from insulin-resistant patients. Am. J. Physiol. 260 (Endocrinol. Metub. 23): E459-E463, 1991. DOHM, G. L., E. B. TAPSCBTT, W. J. PORIES, D. J. DABBS, E. G. FLICKINGER, D. MEELHEIM, T. FUSHIKI, S. M. ATKINSON, C. W. ELTON, AND J. F. CARO. An in vitro human muscle preparation suitable for metabolic studies. Decreased insulin stimulation of glucose transport in muscle from morbidly obese and diabetic subjects. J. CZin. Invest. 82: 486-494, 1988. FRIEDMAN, J. E., W. M. SHERMAN, M. J. REED, C. W. ELTON, AND G. L. DOHM. Exercise training increases glucose transporter protein GLUT4 in skeletal muscle of obese Zucker (fulfa) rats. FEBS Lett. 268: 13-16, 1990. FUKOMOTO, H. T., T. KAYANO, J. B. BUSE, Y. EDWARDS, P. F. PILCH, G. I. BELL, AND S. SEINO. Cloning and characterization of the major insulin-responsive glucose transporter expressed in human skeletal muscle and other insulin-responsive tissues. J. BioZ. Chem. 264: 7776-7779,1989. GARVEY, W. T., T. P. HUECKSTEADT, AND M. J. BIRNBAUM. Pretranslational suppression of an insulin-responsive glucose transporter in rats with diabetes mellitus. Science Wash. DC 24: 60-63, 1989. GREENHOUSE, D. D., 0. E. MICHAELIS, AND R. G. PETERSON. The development of fatty and corpulent rat strains. In: New Models of Genetically Obese Ruts for Studies in Diabetes, Heart Disease, and Complications of Obesity, edited by C. T. Hansen. Bethesda, MD: National Institutes of Health, Division of Research Services, Veterinary Resources Branch, 1988, p. 3-6. HENRIKSEN, E. J., R. E. BOUREY, K. J. RODNICK, L. KORANYI, M. A. PERMUTT, AND J. 0. HOLLOSZY. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am. J. Physiol. 259 (Endocrinol. Metub. 22): E593-E598, 1990. HOUMARD, J. A., P. C. EGAN, P. D. NEUFER, J. E. FRIEDMAN, W. S. WHEELER, R. G. ISRAEL, AND G. L. DOHM. Elevated skeletal muscle glucose transporter levels in exercise-trained middle-aged men. Am. J. Physiol. 261 (Endocrinol. Metub. 24): E437-E443, 1991. JAMES, D. E., R. BROWN, J. NAVARRO, AND P. F. PILCH. Insulinregulatable tissues express a unique insulin-sensitive glucose transport protein. Nature Lond. 333: 183-185, 1990. JEFFERSON, L. S. Role of insulin in the regulation of protein synthesis. Diabetes 29: 487-496, 1980. JOHNSON, J. H., A. OGAWA, L. CHEN, L. ORCI, C. B. NEWGARD, T. ALAM, AND R. UNGER. Underexpression of P-cell high K, glucose transporters in noninsulin-dependent diabetes. Science Wash. DC 250: 546-549,199O. JOHNSON, M. A., J. POLGAR, D. WEIGHTMAN, AND D. APPLETON. Data on the distribution of fiber types in thirty-six human muscles. An autopsy study. J. Neural. Sci. 18: 11-129, 1973. KAESTNER, K. H., J. R. CHRISTY, J. C. MCLENITHAN, L. T. BRAITERMAN, P. CORNELIUS, P. H. PEKALA, AND M. D. LANE. Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3Ll adipocytes. Proc. NutZ. Acud. Sci. USA 86: 3150-3154, 1989. KERN, M., J. A. WELLS, M. STEPHENS, C. W. ELTON, J. E. FRIEDMAN, E. B. TAPSCOTT, P. H. PEKALA, AND G. L. DOHM. Insulin responsiveness in skeletal muscle is determined by glucose transporter (GLUT4) protein level. Biochem. J. 270: 397-400,199O. KLIP, A., T. RAMLAL, P. J. BILAN, G. D. CARTEE, E. A. GULVE, AND J. 0. HOLLOSZY. Recruitment of GLUT-4 glucose transporters by insulin in diabetic rat skeletal muscle. Biochem. Biophys. Res. Commun. 172: 728-736,199O. KORANYI, L., D. E. JAMES, M. MUECKLER, AND M. A. PERMUTT. Glucose transporter levels in spontaneously obese (db/db) insulinresistant mice. J. CZin. Invest. 85: 962-967, 1990. KUBO, K., AND J. E. FOLEY. Rate-limiting steps for insulin-mediated glucose uptake into perfused rat hindlimb. Am. J. Physiol. 250 (Endocrinol. Metub. 13): ElOO-E102, 1986. LAEMLLI, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Lond. 227: 680-685,197O. LANZA-JACOBY, S., AND M. L. KAPLAN. Alterations in skeletal muscle proteins in obese and nonobese rats. Int. J. Obesity 8: 451-456,1984. LE MARCHAND-BRUSTEL, Y., C. OLICHON-BERTHE, T. GREMEAUX, J. F. TANTI, N. ROCHET, AND E. VAN OBBERGHEN. Glu-


E788

28.

29. 30.

31.

32.

GLUT-4

IN

ZUCKER

case transporter in insulin sensitive tissues of lean and obese mice. Effect of the thermogenic agent BRL 26830A. Endocrinology 127: 2687-2695,199O. LILLIOJA, S., A. A. YOUNG, C. L. CULTER, J. L. IVY, W. G. H. ABBOTT, J. K. ZAWADZKI, H. YKI-JARVINEN, L. CHRISTIN, T. W. SECOMB, AND C. BOGARDUS. Skeletal muscle capillary density and fiber type are possible determinants of in vivo insulin resistance in man. J. CZin. Inuest. 80: 415-424, 1987. MORGAN, C. R., AND A. LAZAROW. Immunoassay of insulin: two antibody system. Diabetes 12: 115-126, 1963. ORCI, L., M. RAVAZZOLA, D. BAETENS, L. INMAN, M. AMHERDT, R. G. PETERSON, C. B. NEWGARD, J. H. JOHNSON, AND R. H. UNGER. Evidence that downregulation of ,&cell glucose transporters in noninsulin dependent diabetes may be the cause of diabetic hyperglycemia. Proc. N&L. Acad. Sci. USA 87: 9953-9957, 1990. PEDERSEN, O., J. F. BAK, P. H. ANDERSEN, S. LUND, D. E. MOLLER, J. S. FLIER, AND B. F. KAHN. Evidence against altered expresssion of GLUT1 or GLUT4 in skeletal muscle of patients with obesity or NIDDM. Diabetes 39: 865-870, 1990. PETERSON, R. G., W. N. SHAW, M. A. NEEL, L. A. LITTLE, AND J. EICHBERG. Zucker diabetic fatty rat as a model for non-insulin-

DIABETIC

33.

34.

35.

36.

37.

FATTY

RATS

dependent diabetes mellitus. Inst. Lab. Anim. Res. News 32: 16-19, 1990. RAMLAL, T., S. RASTOGI, M. VRANIC, AND A. KLIP. Decrease in glucose transporter number in skeletal muscle of mildly diabetic (streptozotocin-treated) rats. Endocrinology 125: 890-897, 1989. SHAPIRA, J. F., I. KIRCHER, AND R. J. MARTIN. Indices of skeletal muscle growth in lean and obese Zucker rats. J. Nutr. 110: 13131318,198O. SHERMAN, W. M., A. L. KATZ, C. L. CUTLER, J. VAN DYKE, AND J. L. IVY. Glucose transport: locus of insulin resistance in obese Zucker rats. Am. J. Physiol. 255 (Endocrinol. Metab. 18): E374E382,1988. STROUT, H. V., P. P VICARIO, C. BISWAS, R. SAPERSTEIN, E. J. BRADY, P. F. PILCH, AND J. BERGER. Vanadate treatment of streptozotocin diabetic rats restores expression of the insulinresponsive glucose transporter in skeletal muscle. Endocrinology 126: 2728-2732,199O. YAMAMOTO, T., H. FUKUMOTO, G. KOH, H. HIDEKI, K. YASUDA, M. KAZUHIRO, I. HITOSHI, H. IMURA, AND Y. SEINO. Liver and muscle-fat glucose transporter gene expression in obese and diabetic rats. Biochem. Biophys. Res. Commun. 175: 995-1002, 1991.


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