Physical
Training Increases P-Adrenoceptor Activity in High-Oxidative Skeletal
Density and Adenylate Cyclase Muscle of Diabetic Rats
Gilles Plourde, Suzanne Rousseau-Migneron,
and Andre Nadeau
The effects of physical training on p-adrenergic-receptor density (Bmax) and adenylate cyclase (AC) activity in soleus muscles (type I) and the deep red portion (type Ila) and superficial white portion (type Ilb) of vastus lateralis muscles in diabetic rats were investigated. Rats were rendered diabetic with streptozotocin ([STZ] 45 mg/ kg intravenously [IV]) and were either kept sedentary ([SD] n = 12) or submitted to a progressive IO-week treadmill running program ([TD] n = 13). A group of normal sedentary rats served as controls ([SC] n = 13). Plasma glucose levels were increased in SD rats in comparison with SC rats (21.3 + 1.4 mmol/L v 7.7 f 0.2; mean & SE, P c .OOl), but levels were partially reversed to normal by training (10.7 2 1.7; P c .Ol Y SD). The gastrocnemius nicotinamide adenine dinucleotide (NAD)-isocitrate dehydrogenase (ICDH) activity was significantly increased in TD rats in comparison to SC or SD rats (P < .OOl). The Bmax and antagonist affinity (I&) determined with ?odocyanopindolol (ICYP) were not affected by diabetes in any of the three types of muscle. In type I muscle, TD rats showed a significant 67% increase in Bmax compared with that of SD rats (TD 26.7 f 2.0 Y SD 16.0 + 1.0; P < .OOl). In type Ila muscle, Bmax was significantly higher by 68% in TD rats as compared with SD rats (TD 16.5 f 1.7 Y SD 9.8 -t 0.9 fmol/mg protein; P < .Ol). However, in type Ilb muscle, Bmax was not significantly modified by physical training (TD 8.3 2 0.6 Y SD 7.2 2 0.6). Kd was unaffected by training in each muscle type. The basal and sodium fluoride-stimulated AC activities were not modified by diabetes or training in each muscle tested. However, in type I muscle of TD rats, a significant 26% (P < .Ol) increase in the AC response to 10m5 mol/ L isoproterenol was observed in comparison to that of SD rats. Such an effect of training was not observed in the two other muscle types. These results indicate that physical training induced changes in Bmax and in AC response to 10e5 mol/L isoproterenol in skeletal muscles with a high oxidative capacity. How such changes may be implicated in the training-induced improvement of experimental diabetes mellitus remains to be established. Copyright 0 i992 by W.B. Saunders Comp&y
T
HE BENEFICIAL EFFECT OF physical training on the glucose homeostasis of rats with mild experimental diabetes mellitus is well documented.l-3 Although this may be explained in part by some enhancement in insulin sensitivity,“ the exact mechanism underlying this adaptive change remains to be elucidated. Skeletal muscle has been shown to be the site of the enhanced insulin sensitivity observed in trained nondiabetic rats.” Glucose utilization by skeletal muscle depends on the activation of its transport into the cell6 and the capacity of the cell to phosphorylate and oxidize glucose.’ Previous studies in nondiabetic rats suggested that P-adrenergic receptors play an important role in these phenomena. First, the increased hindiimb muscle glucose uptake present after acute exerciseX was prevented by P-adrenergic blockade with propranoloL9 Second, the oxidative capacity of skeletal muscle appeared to be positively correlated with P-adrenergic-receptor density in trained rats.“’ Third, and more importantly, the training-induced adaptations in oxidative enzyme activities was abolished by using P-blockers during the course of the conditioning program.” Previous studies in nondiabetic rats suggested that skeletal muscle P-adrenoceptor density is enhanced by training,“’ which contrasts with the decrease in p-adrenoceptor density observed at the heart level.*2 However, until now no study has examined the impact of training at the skeletal muscle level in diabetic rats. At the heart level, it has been reported that training did not produce a diminution of P-adrenoceptor density in diabetic rats,13 with these receptors being already diminished in number by diabetes itseIf.14 These observations in rat heart suggest that the changes in P-adrenoceptor status produced by training in nondiabetic rats cannot be automatically extrapolated to diabetic animals. Such an extrapolation would be particularly hazardous in a situation where an increased density is anticipated, Me?&o/ism,
Vol41, No 12 (December), 1992: pp 1331.1335
due to the role played by diabetes in protein catabolism and muscle-wasting and the importance of insulin in protein synthcsis.lz The present study was thus designed to examine the influence of physical training on P-adrenergic-receptor density and affinity in skeletal muscle of diabetic rats. Since it has been shown in nondiabetic rats that the effect of training on P-adrenoceptors may depend on fiber types,lO three muscles that differ in their relative content of fiber typesI were studied and compared: the soleus, mainly composed of slow-twitch, high-oxidative, and low-glycogenolytic fibers (type I); the deep red portion of the vastus lateralis, mainly composed of fast-twitch, intermediateto high-oxidative, and high-glycogenolytic fibers (type Ha); and the superficial white portion of the vastus lateralis, which consists almost entirely of fast-twitch, low-oxidative, and high-glycogenolytic fibers (type IIb). Moreover, the activity of adenylate cyclase was also measured in each muscle preparation, either in the basal state or after stimulation with isoproterenol or sodium fluoride. MATERIALS
AND
METHODS
Male Wistar rats were randomly assigned to three groups: sedentary control ([SC] n = 13), sedentary diabetic ([SD] n = 12), and trained diabetic ([TD] n = 13) rats. The animals were individually housed at 23°C under standard lighting (S:lIO AM to 7:00 PM)
From the Diabetes Research Unit. CHUL Research Center, Lava1 University Medical Center, Ste-Foy, QuCbec, Canada. Supported by the Medical Research Council of Canada and by Le Fonds de la Recherche en Sam& du Qut%ec. G.P. is the holder of a studentship from Le Fonds de la Recherche en Santk du Quebec. Address reptint requests to And+ Nadeau, MD, Diabetes Research Unit, Lava1 University Medical Center, 2705 Laker Blvd. Ste-Foy, QuCbec, Canada Cl V4G2. Copyright 0 1992 by W.B. Saunders Company 0026049519214112-OOlO$O3.OOlO 1331
1332
and fed with Purina Rat Chow (Agway, Country Foods Division, St Louis, MO) and tap water ad libitum. Experimental diabetes was induced with an intravenous (IV) injection of 45 mg/kg body weight streptozotocin (STZ) freshly dissolved in citrate buffer (200 FL, pH 4.5) in the nonfasted state. SC rats received an equivalent amount of citrate buffer. Rats treated with STZ were kept in the protocol only if, 1 week later, they had a urinary volume greater than or equal to 15 mL/d and a urinary glucose concentration greater than or equal to l%, as estimated by Diastix strips. Exercise training began 10 days after STZ injection and was achieved by having the rats run on a motor-driven treadmill (Quinton Instruments, Model 42-15, Seattle, WA) set at an 8 incline, according to a program adapted from Pattengale and Hol10szy.~~The rats were trained twice a day with 4 hours between each session, 5 days a week for 10 weeks; they initially ran for 10 minutes at 22 m/min for 3 weeks, then 40 minutes at 28 mimin for 3 weeks, and finally 60 minutes at 31 mimin for 4 weeks. The rats were killed by decapitation at the end of the lo-week training period and 64 hours after the last session of exercise, in order to examine, as much as possible, a training effect rather than a postexercise event. All three types of muscle were rapidly excised, washed in saline solution, weighed, frozen in liquid nitrogen, and stored at -80”C.t” Preparation of Skeletal Muscle Membranes After thawing the muscles, membranes were prepared by first mincing the tissue with scissors in a 0.32-mol/L sucrose solution at 4°C followed by two lo-second bursts with a Polytron homogenizer (Tekmar, Cincinnati, OH) set at high speed. The homogenates were filtered through a triple layer of cheesecloth and centrifuged at 40,000 x g for 20 minutes. Finally, the pellets were suspended and diluted in incubation buffer (50 mol/L Tris hydrochloride, pH 7.4 at 4°C) containing 1.1 mmol/L ascorbic acid and 3 mmol/L EGTA,is and homogenized with a motor-driven, glass-Teflon, Potter-Elvehjem mixer (Dyna-Mix, Model 143, Fisher Scientific, Pittsburgh, PA) set at high speed to give a protein concentration approximately corresponding to 0.30 mg/mL. Binding Assay For the binding assay, 300 p,L of muscle membrane preparations were incubated with increasing concentrations (5 to 300 pmol/L) of ( +-)-[lzsI]-iodocyanopindolol (ICYP) in a total volume of 500 pL incubation buffer. Preliminary experiments showed that binding of ICYP to skeletal muscle membranes reached equilibrium within 40 minutes and remained stable thereafter for at least 120 minutes at 37°C. Therefore, the membranes were incubated for 60 minutes at 37°C during the course of this study. Incubation was discontinued by the addition of 100 uL sheep y-globulin (0.5% wt/vol in Tris buffer) and 1.4 mL polyethylene glycol(l7% wt/vol in Tris buffer). Tubes were then vortexed and centrifuged for 30 minutes at 2,000 x g; the pellets were washed once with 1 mL polyethylene glycol (12% wtivol in Tris buffer) and centrifuged; the supernatant was discarded, and the radioactivity in the pellets was counted in a gamma counter. Specific binding was calculated as the difference between total and nonspecific binding measured in the presence of 10e5 mol/L (-)propranolol. lo The specific binding generally represented approximately 90% of total binding at the maximal concentration tested. A similar binding-assay procedure has recently been validated for the characterization of P-adrenoceptors in rat heart ventricular membranes.19 Adenylate Cyclase Activity For adenylate cyclase (AC) activity measurements, aliquots of 60 ug protein of skeletal membrane preparations were added to assay
PLOURDE, ROUSSEAU-MIGNERON,
AND NADEAU
tubes and incubated at 37°C for 5 minutes in a final volume of 150 uL 50 mmol/L Tris hydrochloride buffer (pH 7.5) containing 1 mmol/L isobutylmethylxanthine, 2.5 mmol/L MgClz, 10 mmol/L KCl, 4 mmol/L dithiothreitol, 0.25 mmol/L adenosine triphosphate (ATP), 10 p,mol/L guanosine triphosphate, an ATPregenerating system consisting of 1.5 mmol/L creatine phosphate and 0.1 mg/mL creatine phosphokinase, and [32P]ATP containing approximately 500,000 cpm. The reaction was discontinued by the addition of 100 FL 1% sodium lauryl sulfate, 40 mmol/L ATP, 1.4 mmol/L cyclic adenosine monophosphate (CAMP), and [3H]cAMP containing approximately 30,000 cpm (pH 7.5) followed by 3 minutes of boiling. AC activity was measured in the absence or presence of 10m5mol/L (-)isoproterenol or lo-* mol/L sodium fluoride. [32P]cAMP generation was measured by the method of Salomon et al.*O Other Laboratory Measurements Plasma glucose level was determined by an enzymatic method.*i Plasma insulin was measured by radioimmunoassay, using rat insulin as the standard and polyethylene glycol for separationz2 Glucagon was assayed with the 30K antibody of Unger and Eisentraut,23 with porcine glucagon as the standard and polyethylene glycol separation.** Protein concentration was measured according to the method of Lowry et a1,24using bovine serum albumin as the standard. Nicotinamide adenine dinucleotide-linked isocitrate dehydrogenase (ICDH) activity was measured according to the method of Vaughan and Newsholme25 in homogenates of gastrocnemius muscle. Drugs and Chemicals 1251(?)-labeled cyanopindolol (2,200 Ciimmol), [8-3H]cAMP, and [32P]ATP (30 Ciimmol) were purchased from New England Nuclear (Boston, MA). STZ was a gift of Upjohn of Canada (Don Mills, Ontario). (-)Propranolol hydrochloride, (-)isoproterenol hydrochloride, ascorbic acid, isobutylmethylxanthine, bovine serum albumin, and sheep y-globulin were obtained from Sigma Chemical (St Louis, MO). Polyethylene glycol8000 was purchased from Biotech Scientifique (Lachine, Quebec, Canada). Creatine phosphate, creatine phosphokinase, dithiothreitol, and guanosine triphosphate were obtained from Boehringer Mannheim (Dorval, Quebec, Canada). Diastix strips were purchased from Ames Division, Miles Laboratories (Rexdale, Ontario, Canada). Insulin and glucagon standards were obtained from Novo Research Institute (Copenhagen, Denmark). StatisticalAnalysis Results are presented as means f SE. Statistical comparisons between groups were performed by ANOVA, and comparison of means was performed by Duncan’s multiple-range test. Betaadrenergic-receptor density (Bmax) and antagonist affinity (&) were determined according to the method of Scatchard, using computerized iterative programs.*’ RESULTS
Some of the basic characteristics of the three groups of rats are shown in Table 1. The initial body weights were similar in each group, but a significant reduction in the rate of growth was observed in both SD and TD rats compared with SC rats. In comparison to SC rats, the weight of each of the muscles studied was lower in both SD and TD rats. Plasma glucose levels were significantly higher in SD and TD rats than in SC rats, but this was partly reversed to normal in TD rats. Plasma insulin levels were significantly
@ADRENOCEPTORS
1333
IN TRAINED DIABETIC RATS
Table 1. Body Weight, Muscle Weights, Basal Plasma Glucose, insulin, and Glucagon Levels, and Gastrocnemius
NAD-Linked ICDH
Activity in SC, SD, and TD Rats SC In = 13)
SD(n
=
12)
TD In = 13)
Body weight (9) Initial
206 + 2
209 + 1
208 + 2
Final
348 2 8
280 r ll*
276 + lO*
Muscle weights (g)
Tvpe 1
0.34 r 0.01
0.30 f 0.01 t
‘ype Ila
1.73 f 0.07
1.17 t O.lO*
1.34 k 0.08*
‘ype Ilb
1.45 + 0.09
1.14 k 0.12’
1.02 * 0.07*
Gastrocnemius
2.74 k 0.07
2.00 + 0.15*
2.20 + O.OE$ 10.7 + 1.7*11
0.28 + 0.01 t
Plasma levels 7.7 rT.0.2
21.3 + 1.4-J
insulin (pmol/LJ
303 + 16
161 + 18$
Glucagon (ng/L)
130 2 9
147 -t 5
Glucose (mmol/L)
1.01 + 0.07
NAD-ICDH (~mollminig)
0.82 2 0.09
156 t 14$ 121 + 69
with SD rats. However, in the muscle preparation mainly composed of type IIb fibers, Bmax was not significantly different between TD rats and SD rats. & was not modified by training in diabetic rats in each muscle preparation studied. AC activity in the three skeletal muscles studied, either in the basal state or during stimulation with 10es mol/L isoproterenol or lo-* mol/L sodium fluoride, is shown in Table 3. There was no difference between groups in the basal or sodium fluoride-stimulated AC activity for each type of muscle. However, a significant (P < .Ol) 26% increase in the 1O-5 mol/L isoproterenol-stimulated AC activity was observed in type I muscle of TD rats in comparison to SD rats. Such an effect of training was not observed with the two other muscle preparations.
1.40 t- 0.07*1/
DISCUSSION NOTE. Values are means + SE. lp < .Ol v SC. tP < .05 Y SC. SP < ,001 “SC. BP < .05vSD. l/I’ < .OOl vSD.
and similarly reduced in SD and TD rats. Although the increase in plasma glucagon levels observed in SD rats did not reach statistical significance, the values observed in TD rats were significantly lower than those in SD rats. The gastrocnemius NAD-linked ICDH activity was not significantly reduced in SD rats in comparison to SC rats. However, the activity of this enzyme was considerably greater in TD rats than in the two other groups. Bmax and Kd in type I, type IIa, and type IIb muscles are shown in Table 2. Bmax and Kd in each of these three skeletal muscles were similar between SD and SC rats. However, in TD rats there was a significant (P < .OOl) 67% increase in Bmax in type I muscle. In the muscle preparation rich in type IIa fibers, a significant (P < .Ol) 68% increase in Bmax was also observed in TD rats compared Table 2.
This study is the first to report on the impact of diabetes mellitus on the P-adrenergic-receptor AC system in skeletal muscle. Our results did not show any significant changes in Bmax or Kd in the three types of skeletal muscle studied. The basal and stimulated AC activities also were not modified by diabetes in any of the muscles tested, suggesting that chronic experimental diabetes mellitus as present in this study did not affect the /3-adrenergic-receptor AC system in skeletal muscle. This departs from the previously reported decrease in Bmax observed in heart muscle of chronically diabetic rats.13J4 Although the effect of physical training on skeletal muscle P-adrenoceptors of normal rats has already been investigated,‘O no such studies have yet been performed at the level of the three types of skeletal muscle in diabetic rats. The results of the present study demonstrated that physical training induced a significant 67% increase in Bmax of type I muscle in TD rats in comparison to that of SD rats. In type IIa muscle, physical training also induced a 68% increase in Bmax. However, in type IIb muscle, Bmax was not significantly modified in TD rats. This may be due to the fact that type IIb fibers were not recruited sufficiently
Bmax and Kd in Type I. Type Ila, and Type Ilb Muscles From SC, SD, and TD Rats
Table 3. Basal, Isoproterenol-,
and Sodium Fluoride-Stimulated TD Rats SodiumFluoride
Type 1
(lo-*
mol/L)
n
SC
18.5 2 1.3
97.4 +- 8.3
13
SD
16.0 2 1.0
97.0 + 12.1
12
Type 1
TD
26.7 -t 2.0*t
101.4 + 10.9
13
SC
6.6 k 0.8
11.2 + 1.3
26.3 r 2.4
13
SD
7.3 + 0.6
13.0 ? 0.9
24.7 k 1.0
12
8.1 + 0.8
16.4 t 1.2*
30.1 k 1.6
13
Type Ila SC SD TD
10.9 * 0.9 9.8 k
0.9
16.5 + 1.7%§
64.5 + 7.0
13
TD
56.2 i 8.1
12
Type Ila
67.0 t 5.7
13
Type Ilb SC
6.6 ? 0.8
74.6 k 7.2
13
SD
7.2 zk 0.6
61.9 2 4.4
12
TD
8.3 2 0.6
78.6 k 7.2
13
*P < .Ol II SC. ‘P < ,001 vSD.
Training Increases P-Adrenoceptor Activity in High-Oxidative Skeletal
Density and Adenylate Cyclase Muscle of Diabetic Rats
Gilles Plourde, Suzanne Rousseau-Migneron,
and Andre Nadeau
The effects of physical training on p-adrenergic-receptor density (Bmax) and adenylate cyclase (AC) activity in soleus muscles (type I) and the deep red portion (type Ila) and superficial white portion (type Ilb) of vastus lateralis muscles in diabetic rats were investigated. Rats were rendered diabetic with streptozotocin ([STZ] 45 mg/ kg intravenously [IV]) and were either kept sedentary ([SD] n = 12) or submitted to a progressive IO-week treadmill running program ([TD] n = 13). A group of normal sedentary rats served as controls ([SC] n = 13). Plasma glucose levels were increased in SD rats in comparison with SC rats (21.3 + 1.4 mmol/L v 7.7 f 0.2; mean & SE, P c .OOl), but levels were partially reversed to normal by training (10.7 2 1.7; P c .Ol Y SD). The gastrocnemius nicotinamide adenine dinucleotide (NAD)-isocitrate dehydrogenase (ICDH) activity was significantly increased in TD rats in comparison to SC or SD rats (P < .OOl). The Bmax and antagonist affinity (I&) determined with ?odocyanopindolol (ICYP) were not affected by diabetes in any of the three types of muscle. In type I muscle, TD rats showed a significant 67% increase in Bmax compared with that of SD rats (TD 26.7 f 2.0 Y SD 16.0 + 1.0; P < .OOl). In type Ila muscle, Bmax was significantly higher by 68% in TD rats as compared with SD rats (TD 16.5 f 1.7 Y SD 9.8 -t 0.9 fmol/mg protein; P < .Ol). However, in type Ilb muscle, Bmax was not significantly modified by physical training (TD 8.3 2 0.6 Y SD 7.2 2 0.6). Kd was unaffected by training in each muscle type. The basal and sodium fluoride-stimulated AC activities were not modified by diabetes or training in each muscle tested. However, in type I muscle of TD rats, a significant 26% (P < .Ol) increase in the AC response to 10m5 mol/ L isoproterenol was observed in comparison to that of SD rats. Such an effect of training was not observed in the two other muscle types. These results indicate that physical training induced changes in Bmax and in AC response to 10e5 mol/L isoproterenol in skeletal muscles with a high oxidative capacity. How such changes may be implicated in the training-induced improvement of experimental diabetes mellitus remains to be established. Copyright 0 i992 by W.B. Saunders Comp&y
T
HE BENEFICIAL EFFECT OF physical training on the glucose homeostasis of rats with mild experimental diabetes mellitus is well documented.l-3 Although this may be explained in part by some enhancement in insulin sensitivity,“ the exact mechanism underlying this adaptive change remains to be elucidated. Skeletal muscle has been shown to be the site of the enhanced insulin sensitivity observed in trained nondiabetic rats.” Glucose utilization by skeletal muscle depends on the activation of its transport into the cell6 and the capacity of the cell to phosphorylate and oxidize glucose.’ Previous studies in nondiabetic rats suggested that P-adrenergic receptors play an important role in these phenomena. First, the increased hindiimb muscle glucose uptake present after acute exerciseX was prevented by P-adrenergic blockade with propranoloL9 Second, the oxidative capacity of skeletal muscle appeared to be positively correlated with P-adrenergic-receptor density in trained rats.“’ Third, and more importantly, the training-induced adaptations in oxidative enzyme activities was abolished by using P-blockers during the course of the conditioning program.” Previous studies in nondiabetic rats suggested that skeletal muscle P-adrenoceptor density is enhanced by training,“’ which contrasts with the decrease in p-adrenoceptor density observed at the heart level.*2 However, until now no study has examined the impact of training at the skeletal muscle level in diabetic rats. At the heart level, it has been reported that training did not produce a diminution of P-adrenoceptor density in diabetic rats,13 with these receptors being already diminished in number by diabetes itseIf.14 These observations in rat heart suggest that the changes in P-adrenoceptor status produced by training in nondiabetic rats cannot be automatically extrapolated to diabetic animals. Such an extrapolation would be particularly hazardous in a situation where an increased density is anticipated, Me?&o/ism,
Vol41, No 12 (December), 1992: pp 1331.1335
due to the role played by diabetes in protein catabolism and muscle-wasting and the importance of insulin in protein synthcsis.lz The present study was thus designed to examine the influence of physical training on P-adrenergic-receptor density and affinity in skeletal muscle of diabetic rats. Since it has been shown in nondiabetic rats that the effect of training on P-adrenoceptors may depend on fiber types,lO three muscles that differ in their relative content of fiber typesI were studied and compared: the soleus, mainly composed of slow-twitch, high-oxidative, and low-glycogenolytic fibers (type I); the deep red portion of the vastus lateralis, mainly composed of fast-twitch, intermediateto high-oxidative, and high-glycogenolytic fibers (type Ha); and the superficial white portion of the vastus lateralis, which consists almost entirely of fast-twitch, low-oxidative, and high-glycogenolytic fibers (type IIb). Moreover, the activity of adenylate cyclase was also measured in each muscle preparation, either in the basal state or after stimulation with isoproterenol or sodium fluoride. MATERIALS
AND
METHODS
Male Wistar rats were randomly assigned to three groups: sedentary control ([SC] n = 13), sedentary diabetic ([SD] n = 12), and trained diabetic ([TD] n = 13) rats. The animals were individually housed at 23°C under standard lighting (S:lIO AM to 7:00 PM)
From the Diabetes Research Unit. CHUL Research Center, Lava1 University Medical Center, Ste-Foy, QuCbec, Canada. Supported by the Medical Research Council of Canada and by Le Fonds de la Recherche en Sam& du Qut%ec. G.P. is the holder of a studentship from Le Fonds de la Recherche en Santk du Quebec. Address reptint requests to And+ Nadeau, MD, Diabetes Research Unit, Lava1 University Medical Center, 2705 Laker Blvd. Ste-Foy, QuCbec, Canada Cl V4G2. Copyright 0 1992 by W.B. Saunders Company 0026049519214112-OOlO$O3.OOlO 1331
1332
and fed with Purina Rat Chow (Agway, Country Foods Division, St Louis, MO) and tap water ad libitum. Experimental diabetes was induced with an intravenous (IV) injection of 45 mg/kg body weight streptozotocin (STZ) freshly dissolved in citrate buffer (200 FL, pH 4.5) in the nonfasted state. SC rats received an equivalent amount of citrate buffer. Rats treated with STZ were kept in the protocol only if, 1 week later, they had a urinary volume greater than or equal to 15 mL/d and a urinary glucose concentration greater than or equal to l%, as estimated by Diastix strips. Exercise training began 10 days after STZ injection and was achieved by having the rats run on a motor-driven treadmill (Quinton Instruments, Model 42-15, Seattle, WA) set at an 8 incline, according to a program adapted from Pattengale and Hol10szy.~~The rats were trained twice a day with 4 hours between each session, 5 days a week for 10 weeks; they initially ran for 10 minutes at 22 m/min for 3 weeks, then 40 minutes at 28 mimin for 3 weeks, and finally 60 minutes at 31 mimin for 4 weeks. The rats were killed by decapitation at the end of the lo-week training period and 64 hours after the last session of exercise, in order to examine, as much as possible, a training effect rather than a postexercise event. All three types of muscle were rapidly excised, washed in saline solution, weighed, frozen in liquid nitrogen, and stored at -80”C.t” Preparation of Skeletal Muscle Membranes After thawing the muscles, membranes were prepared by first mincing the tissue with scissors in a 0.32-mol/L sucrose solution at 4°C followed by two lo-second bursts with a Polytron homogenizer (Tekmar, Cincinnati, OH) set at high speed. The homogenates were filtered through a triple layer of cheesecloth and centrifuged at 40,000 x g for 20 minutes. Finally, the pellets were suspended and diluted in incubation buffer (50 mol/L Tris hydrochloride, pH 7.4 at 4°C) containing 1.1 mmol/L ascorbic acid and 3 mmol/L EGTA,is and homogenized with a motor-driven, glass-Teflon, Potter-Elvehjem mixer (Dyna-Mix, Model 143, Fisher Scientific, Pittsburgh, PA) set at high speed to give a protein concentration approximately corresponding to 0.30 mg/mL. Binding Assay For the binding assay, 300 p,L of muscle membrane preparations were incubated with increasing concentrations (5 to 300 pmol/L) of ( +-)-[lzsI]-iodocyanopindolol (ICYP) in a total volume of 500 pL incubation buffer. Preliminary experiments showed that binding of ICYP to skeletal muscle membranes reached equilibrium within 40 minutes and remained stable thereafter for at least 120 minutes at 37°C. Therefore, the membranes were incubated for 60 minutes at 37°C during the course of this study. Incubation was discontinued by the addition of 100 uL sheep y-globulin (0.5% wt/vol in Tris buffer) and 1.4 mL polyethylene glycol(l7% wt/vol in Tris buffer). Tubes were then vortexed and centrifuged for 30 minutes at 2,000 x g; the pellets were washed once with 1 mL polyethylene glycol (12% wtivol in Tris buffer) and centrifuged; the supernatant was discarded, and the radioactivity in the pellets was counted in a gamma counter. Specific binding was calculated as the difference between total and nonspecific binding measured in the presence of 10e5 mol/L (-)propranolol. lo The specific binding generally represented approximately 90% of total binding at the maximal concentration tested. A similar binding-assay procedure has recently been validated for the characterization of P-adrenoceptors in rat heart ventricular membranes.19 Adenylate Cyclase Activity For adenylate cyclase (AC) activity measurements, aliquots of 60 ug protein of skeletal membrane preparations were added to assay
PLOURDE, ROUSSEAU-MIGNERON,
AND NADEAU
tubes and incubated at 37°C for 5 minutes in a final volume of 150 uL 50 mmol/L Tris hydrochloride buffer (pH 7.5) containing 1 mmol/L isobutylmethylxanthine, 2.5 mmol/L MgClz, 10 mmol/L KCl, 4 mmol/L dithiothreitol, 0.25 mmol/L adenosine triphosphate (ATP), 10 p,mol/L guanosine triphosphate, an ATPregenerating system consisting of 1.5 mmol/L creatine phosphate and 0.1 mg/mL creatine phosphokinase, and [32P]ATP containing approximately 500,000 cpm. The reaction was discontinued by the addition of 100 FL 1% sodium lauryl sulfate, 40 mmol/L ATP, 1.4 mmol/L cyclic adenosine monophosphate (CAMP), and [3H]cAMP containing approximately 30,000 cpm (pH 7.5) followed by 3 minutes of boiling. AC activity was measured in the absence or presence of 10m5mol/L (-)isoproterenol or lo-* mol/L sodium fluoride. [32P]cAMP generation was measured by the method of Salomon et al.*O Other Laboratory Measurements Plasma glucose level was determined by an enzymatic method.*i Plasma insulin was measured by radioimmunoassay, using rat insulin as the standard and polyethylene glycol for separationz2 Glucagon was assayed with the 30K antibody of Unger and Eisentraut,23 with porcine glucagon as the standard and polyethylene glycol separation.** Protein concentration was measured according to the method of Lowry et a1,24using bovine serum albumin as the standard. Nicotinamide adenine dinucleotide-linked isocitrate dehydrogenase (ICDH) activity was measured according to the method of Vaughan and Newsholme25 in homogenates of gastrocnemius muscle. Drugs and Chemicals 1251(?)-labeled cyanopindolol (2,200 Ciimmol), [8-3H]cAMP, and [32P]ATP (30 Ciimmol) were purchased from New England Nuclear (Boston, MA). STZ was a gift of Upjohn of Canada (Don Mills, Ontario). (-)Propranolol hydrochloride, (-)isoproterenol hydrochloride, ascorbic acid, isobutylmethylxanthine, bovine serum albumin, and sheep y-globulin were obtained from Sigma Chemical (St Louis, MO). Polyethylene glycol8000 was purchased from Biotech Scientifique (Lachine, Quebec, Canada). Creatine phosphate, creatine phosphokinase, dithiothreitol, and guanosine triphosphate were obtained from Boehringer Mannheim (Dorval, Quebec, Canada). Diastix strips were purchased from Ames Division, Miles Laboratories (Rexdale, Ontario, Canada). Insulin and glucagon standards were obtained from Novo Research Institute (Copenhagen, Denmark). StatisticalAnalysis Results are presented as means f SE. Statistical comparisons between groups were performed by ANOVA, and comparison of means was performed by Duncan’s multiple-range test. Betaadrenergic-receptor density (Bmax) and antagonist affinity (&) were determined according to the method of Scatchard, using computerized iterative programs.*’ RESULTS
Some of the basic characteristics of the three groups of rats are shown in Table 1. The initial body weights were similar in each group, but a significant reduction in the rate of growth was observed in both SD and TD rats compared with SC rats. In comparison to SC rats, the weight of each of the muscles studied was lower in both SD and TD rats. Plasma glucose levels were significantly higher in SD and TD rats than in SC rats, but this was partly reversed to normal in TD rats. Plasma insulin levels were significantly
@ADRENOCEPTORS
1333
IN TRAINED DIABETIC RATS
Table 1. Body Weight, Muscle Weights, Basal Plasma Glucose, insulin, and Glucagon Levels, and Gastrocnemius
NAD-Linked ICDH
Activity in SC, SD, and TD Rats SC In = 13)
SD(n
=
12)
TD In = 13)
Body weight (9) Initial
206 + 2
209 + 1
208 + 2
Final
348 2 8
280 r ll*
276 + lO*
Muscle weights (g)
Tvpe 1
0.34 r 0.01
0.30 f 0.01 t
‘ype Ila
1.73 f 0.07
1.17 t O.lO*
1.34 k 0.08*
‘ype Ilb
1.45 + 0.09
1.14 k 0.12’
1.02 * 0.07*
Gastrocnemius
2.74 k 0.07
2.00 + 0.15*
2.20 + O.OE$ 10.7 + 1.7*11
0.28 + 0.01 t
Plasma levels 7.7 rT.0.2
21.3 + 1.4-J
insulin (pmol/LJ
303 + 16
161 + 18$
Glucagon (ng/L)
130 2 9
147 -t 5
Glucose (mmol/L)
1.01 + 0.07
NAD-ICDH (~mollminig)
0.82 2 0.09
156 t 14$ 121 + 69
with SD rats. However, in the muscle preparation mainly composed of type IIb fibers, Bmax was not significantly different between TD rats and SD rats. & was not modified by training in diabetic rats in each muscle preparation studied. AC activity in the three skeletal muscles studied, either in the basal state or during stimulation with 10es mol/L isoproterenol or lo-* mol/L sodium fluoride, is shown in Table 3. There was no difference between groups in the basal or sodium fluoride-stimulated AC activity for each type of muscle. However, a significant (P < .Ol) 26% increase in the 1O-5 mol/L isoproterenol-stimulated AC activity was observed in type I muscle of TD rats in comparison to SD rats. Such an effect of training was not observed with the two other muscle preparations.
1.40 t- 0.07*1/
DISCUSSION NOTE. Values are means + SE. lp < .Ol v SC. tP < .05 Y SC. SP < ,001 “SC. BP < .05vSD. l/I’ < .OOl vSD.
and similarly reduced in SD and TD rats. Although the increase in plasma glucagon levels observed in SD rats did not reach statistical significance, the values observed in TD rats were significantly lower than those in SD rats. The gastrocnemius NAD-linked ICDH activity was not significantly reduced in SD rats in comparison to SC rats. However, the activity of this enzyme was considerably greater in TD rats than in the two other groups. Bmax and Kd in type I, type IIa, and type IIb muscles are shown in Table 2. Bmax and Kd in each of these three skeletal muscles were similar between SD and SC rats. However, in TD rats there was a significant (P < .OOl) 67% increase in Bmax in type I muscle. In the muscle preparation rich in type IIa fibers, a significant (P < .Ol) 68% increase in Bmax was also observed in TD rats compared Table 2.
This study is the first to report on the impact of diabetes mellitus on the P-adrenergic-receptor AC system in skeletal muscle. Our results did not show any significant changes in Bmax or Kd in the three types of skeletal muscle studied. The basal and stimulated AC activities also were not modified by diabetes in any of the muscles tested, suggesting that chronic experimental diabetes mellitus as present in this study did not affect the /3-adrenergic-receptor AC system in skeletal muscle. This departs from the previously reported decrease in Bmax observed in heart muscle of chronically diabetic rats.13J4 Although the effect of physical training on skeletal muscle P-adrenoceptors of normal rats has already been investigated,‘O no such studies have yet been performed at the level of the three types of skeletal muscle in diabetic rats. The results of the present study demonstrated that physical training induced a significant 67% increase in Bmax of type I muscle in TD rats in comparison to that of SD rats. In type IIa muscle, physical training also induced a 68% increase in Bmax. However, in type IIb muscle, Bmax was not significantly modified in TD rats. This may be due to the fact that type IIb fibers were not recruited sufficiently
Bmax and Kd in Type I. Type Ila, and Type Ilb Muscles From SC, SD, and TD Rats
Table 3. Basal, Isoproterenol-,
and Sodium Fluoride-Stimulated TD Rats SodiumFluoride
Type 1
(lo-*
mol/L)
n
SC
18.5 2 1.3
97.4 +- 8.3
13
SD
16.0 2 1.0
97.0 + 12.1
12
Type 1
TD
26.7 -t 2.0*t
101.4 + 10.9
13
SC
6.6 k 0.8
11.2 + 1.3
26.3 r 2.4
13
SD
7.3 + 0.6
13.0 ? 0.9
24.7 k 1.0
12
8.1 + 0.8
16.4 t 1.2*
30.1 k 1.6
13
Type Ila SC SD TD
10.9 * 0.9 9.8 k
0.9
16.5 + 1.7%§
64.5 + 7.0
13
TD
56.2 i 8.1
12
Type Ila
67.0 t 5.7
13
Type Ilb SC
6.6 ? 0.8
74.6 k 7.2
13
SD
7.2 zk 0.6
61.9 2 4.4
12
TD
8.3 2 0.6
78.6 k 7.2
13
*P < .Ol II SC. ‘P < ,001 vSD.
