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Rat tail suspension causes a decline in insulin receptors
Exp Toxic Patho11993; 45: 291-295 Gustav Fischer Verlag Jena
Departments of Internal Medicine", Pediatrics3! and Surgery'!; The University of Texas Medical Branch at Galveston, Galveston, Texas Department of Anthropology2l, Washington University, St. Louis
Rat tail suspension causes a decline in insulin receptors5) C. A. STUART!), L. S. KIDDER'!, R. A. PIETRZYK ", G. L. KLEIN", and D. 1. SIMMONS 4)
With 4 figures Received: January 28,1992; Revised: October 5,1992; Accepted: November 23,1992 Address for correspondence: Prof. CHARLES A. STUART, M. D. University of Texas Medical Branch at Galveston, MRB 3.142, Rt. J60 Galveston, Texas 77555-1060; Telephone: (409) 772-1923. Key words: Tail suspension; Insulin resistance; Insulin-like growth factor I (lGF-I); Microsomal membrane; IGF-I; Insulin binding; Insulin receptors; Hyperinsulinernia; Receptors, insulin; Skeletal muscle, IGF-I receptors; Liver, membrane insulin receptors.
Summary Decreased muscular activity results in weakness and muscular atrophy. Coincident with this protein catabolic state is glucose intolerance and hyperinsulinemia. Rats were tail suspended for 7 to 14 days to accomplish unloading of the hindlimbs. Insulin resistance was documented in these animals by a 14 day tail suspension-related 26 % increase in serum glucose in spite of a 253 % increase in serum insulin concentration. Microsomal membranes were prepared from hindlimb muscles and specific binding of insulin and insulin-like growth factor I (IGF-I) were determined in these membranes. Insulin binding was decreased by 27 % at 7 days and by 21 % at 14 days. In contrast, IGF-I binding was unchanged at 7 days and was increased by 24 % at 14 days. Liver membrane insulin receptors also had declined by 14 days of suspension, suggesting that the change in insulin receptors was a generalized, humorally-mediated phenomenon. These data suggest that tail suspension in rats results in insulin resistance, hyperinsulinemia, a decline in insulin receptors in liver and muscle, and a relative increase in muscle membrane IGF-I receptors. These data are consistent with the hypothesis that resi-
stance to insulin' s effects on protein metabolism in skeletal muscle may contribute to the protein catabolism associated with decreased muscular acti vity .
Introduction A prolonged decrease in muscular aCtIVIty results in muscle atrophy and weakness (1, 2). MOREY and coworkers described a rat model of decreased muscular work and decreased motor activity which involved suspension by the tail such that hindlimbs did not come in contact with the
Supported in part by grants from the National Aeronautics and Space Administration (NAG 9-172 and NTG-003)
5)
cage floor (12,25). This model as well as other rat hindlimb immobilization models have been used extensively to evaluate anatomic, functional, and biochemical consequences of muscular disuse (1, 5, 10,24). Disuse ofthe rat hindlimb results in decreased muscle weight, decreased muscle strength, decreased muscle oxygen utilization, and an increase in the relative number of fast twitch muscle fibers. The decreased muscular activity is associated with an early decrease in muscle protein synthesis and a later increase in protein breakdown (2, 10). The biochemical mechanisms which translate the change in contractile activity to these many alterations and particularly to changes in protein synthesis and breakdown are unknown. Since resistance to insulin's effects of glucose metabolism is induced by muscular inactivity in rats (17) and man (14, 22), we hypothesized that resistance to insulin's effects on protein metabolism in muscle might also be induced by muscular inactivity. Decreased insulin suppression of protein-breakdown or decreased insulin stimulation of protein synthesis would result in a net catabolic change in muscle protein balance. Direct quantitation of muscle membrane insulin binding was performed in order to determine if insulin resistance in muscle might be associated with a decrease in insulin receptors.
Material and methods
Materials Male Sprague-Dawley rats were from Harlan SpragueDawley (Houston, TX). Suspended rats and littermate controls were 6-8 weeks of age (180-200 g) at the beginning of each study. Littermate controls were housed in individual cages and ExpToxic Pathol45 (1993) 5-6
291
were pair fed according to the amount of food consumed the previous day by the suspended animals. Human insulin was the generous gift of Dr. RONALD CHANCE of Lilly Research Labs. Insulin was labeled with 1251 according to minor modifications of the dilute chloramine-T method of Freychet et al. (7). Insulin-like growth factor I (IGF-I) was purchased from Amgen (Thousand Oaks, CA). IGF-I was iodinated exactly as was insulin. '25I_IGF_I was further purified by high performance liquid chromatography using a C 18 reverse phase column. Other chemicals were of reagent grade.
Rat tail suspension The animals were prepared by fixing a thin strip of adhesive orthopedic moleskin along the length of the tail, leaving the sides open to permit radial growth. A small split ring was placed at the tip, and the array was secured with a gauze bandage wrap. The ring was then clipped to a swivel on an overhead "arm" which projected over the middle of the cage. The height of this arm was adjusted to maintain the animals in a 35-40 ° head-down tilt throughout the course of the study. This system allows the animal mobility and the capacity to groom and feed (25). Littermate controls were pair fed and allowed ad lib activity in individual cages.
Preparation of muscle and liver microsomal membranes At the end of 0, 7, or 14 days of suspension each animal was sacrificed by decapitation. The animals were not food restricted overnight, but were sacrificed in the morning at least two hours after removal of food. The lower hindlimbs were disarticulated from the thigh and foot and all muscles were dissected free and placed in saline on ice. Livers were also harvested and placed in ice cold saline. Muscle or liver was weighed and homogenized in a mixture of 0.25 M sucrose, 5 mM EDT A, 2 mM phenylmethylsulfonyl fluoride, and 100 mM imidazole, pH 7.4, using a Polytron (Brinkmann, Westbury, NY) for 60 seconds at a concentration of 1 g tissue per 2 ml homogenization buffer. The homogenate was centrifuged for 15 minutes at 1000 xg, the supernate was removed and recentrifuged at 100,000 xg for 60 minutes. The centrifuged pellet was then resuspended in 2 mM phenylmethylsulfonyl fluoride and I mM imidazole, pH 7.4, in a volume of 0.5 ml/g original tissue wet weight.
Radioimmunoassay for serum insulin Rat serum was assayed for immunoreactive insulin using a kit purchased from INCST AR (Stillwater, MN). The antiserum used in this assay was raised in rabbits against porcine insulin. The insulin standard was rat insulin extracted from rat pancreases and partially purified. Standards ranged 1 to 25 ng/ml and sample size was 200 ul of rat serum. Rat insulin was parallel to but 5 fold less potent than porcine insulin in displacing radiolabeled porcine insulin from the antibody. Since polyethylene glycol precipiation reduced the measured value consistently by 40 percent, the data used were those from non-precipitated serum. Glucose in rat serum was quantitated by the glucose oxidase method using a YSI Glucose Analyzer (Yellow Springs Instrument Company, Yellow Springs, Ohio). 292
Exp Toxic Pathol45 (1993) 5-6
Ligand binding Muscle membranes were then incubated for 12-15 hours at 4 °C at a concentration of 500 ug/ml in a binding buffer, consisting of 100 mM Hepes, 120 mM sodium chloride, 1.2 mM magnesium sulfate, 1 mM EDTA, 10 mM glucose, 15 mM sodium acetate, and I % BSA, pH 8.0. '25I-insulin or 125I-IGF-I was added to the binding buffer resulting in a final labeled ligand concentration of 0.05 nM; nonspecific binding was defined as the '25I counts bound in the presence of at least 200 fold molar excess of unlabeled insulin or IGF-I. SCATCHARD analysis (19) was calculated from binding studies performed using a constand amount of radiolabeled ligand and several dilutions of unlabeled ligand. Total specific binding is the percent of the labeled ligand bound in the absence of unlabeled ligand with the nonspecific ally bount count subtracted. SCATCHARD plot analysis used a two receptor mathematical model previously described (21). In cases where affinity does not change, total specific binding is directly proportional to the quantitation of high affinity binding sites determined by this model.
Statistics Data were analyzed using STUDENT'S t test or analysis ofvariance where appropriate. Data are expressed as means and standard errors of the mean except where noted.
Results The effect of 14 days of tail suspension on serum glucose and insulin: Six rats were suspended by the tail method described above. Littermate controls were pair fed for the same 14 days. Animals were allowed free access to food and water overnight and were sacrificed in the morning about 2 hr after food was removed. Each animal had approximately 2 ml blood for assay of glucose and insulin obtained by cardiac puncture just prior to sacrifice by decapitation. Figure 1 shows the results of those measurements. Both glucose and insulin concentrations were significantly higher in the suspended animals than in the controls. Glucose concentration was increased (8.28 ± 0.33 vs 6.56 ± 0.17 mmollL, n 6) by 26 % and insulin concentration was 253 % higher (44.1 ± 2.8 vs 12.5±2.7 pmollL, n 6) in the suspended rats (both p < 0.001). This increase in glucose in spite of a large increase in insulin documents insulin resistance of glucose metabolism has been induced by tail suspension.
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The effect of tail suspension on specific insulin binding to muscle membranes: Specific binding of insulin to membranes obtained from 24 control rats and 12 rats suspended for 7 days and 12 rats suspended for 14 days in shown in figure 2. These data represent the pooling of data from four separate experiments each with six suspended and six littermate controls. The data from controls at 7 and 14 days were not different and were combined. As can be seen, suspension for 7 days was associated with a decrease in insulin binding from 3.32 ± 0.19 to 2.38 ± 0.46 %/mg membrane protein (p < 0.02). At 14 days of suspension, the
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Fig. 3. The effects of tail suspension on the specific binding of insulin to hindlimb skeletal muscJe membranes and liver membranes: The data displayed represent the mean and SEM of total specific binding of 125I-insulin to muscle membranes and liver membranes from 6 suspended rats and 6 control animals. The decrease in binding in liver membranes was statistically significant, but the decline in muscle membrane binding did not achieve significance.
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Fig. I. Serum glucose and insulin concentrations after 14 days of tail suspension: Panel A displays the serum glucose concentration in six controls rats and six rats subjected to tail suspension for 14 days. Serum insulin concentrations from the same animals are displayed in panel B. The asterisk denotes significantly increased compared with the control data.
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Fig. 2. The effects of tail suspension on the specific binding of insulin and IGF-I to hindlimb skeletal muscle membranes: The data displayed are from 12 rats subjected to tail suspension for 7 days and 12 rats suspended for 14 days and from groups of an equal number of littermates who were not suspended but were pair fed. The binding data from the control groups were not different between 7 days and 14 days and were pooled for this figure. The asterisk denotes significant difference (p < 0.05) form the control data.
decreased insulin binding persisted but did not worsen (2.64 ± 0.25, p < 0.02 compared with controls).
The effect of tail suspension on specific binding of IGF-I to muscle membranes: IGF-I specific binding was performed on the same membrane preparations as used for quantitation of insulin binding described above. The results of these studies are also presented in figure 2. In contrast to the suspension related decline in insulin binding, IGF-I binding did not decline at 7 days (5.05 ± 0.33 vs control of 4.72 ± 0.40 %/mg membrane protein, p 0.32) and was increased (5.82 ± 0.50, p < 0.05 compared with controls) at 14 days.
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Fig. 4. Scatchard analysis of insulin binding to liver membranes: The data shwon represent the mean of Scatchard analyses of insulin binding data from membranes prepared from six 14 day suspended rats and six control rats. Affinity was not different (0.30 ± 0 .01 vs 0.35 ± 0.04 Llmole control, p = 0.46), but insulin receptor number declined (=.175 ± 0.005 vs 0.098 ± 0.002 pmollmg membrane protein, p < 0.(01) in proportion to the change in total specific binding shown in figure 3.
The dTect of 14 days of tail suspension on insulin binding to liver microsomal membranes: If the observed decline in insulin receptors in skeletal muscle was a primary inactivity-related event, then this decline might be limited to muscle. However, if insulin resistance were due to another effect, such as a post-receptor defect, then the resulting hyperinsulinemia could cause down regulation of insulin receptors in other insulin responsive tissues such as liver. We, therefore, quantitated liver insulin receptors in tail-suspended rats. Six Sprague Dawley rats were tail suspended for 14 days as described in Methods. Membranes from livers and hindlimb muscles were prepared and specific insulin binding was determined as described above. Figure ExpToxic Pathol45 (1993) 5-6
293
3 shows the results of these studies. Insulin binding was decreased by 45 % in liver membranes from suspended animals (2.11 ± 0.05 vs 3.83 ± 0.57 %/mg control, p < 0.001). The muscle membrane insulin binding tended to also be decreased by 34 %, but this difference did not achieve statistical significance (p = 0.15). Figure 4 shows Scatchard analysis of studies of insulin binding to liver membranes. The decrease in total specific binding was accompanied by a decline in number of receptors with no change in the receptor affinity. Similar SCATCHARD analysis of the muscle membrane insulin binding from the same rats also showed that the high affinity portion of the curves from control and suspended rats were parallel and the x-axis intercepts suggested (but not statistically significant) a 20 % decrease in muscle insulin receptor number in the suspended animals.
Discussion Tail suspension of young male Sprague-Dawley rats for seven to fourteen days was associated with a decrease in specific insulin binding to microsomal membranes from lower hindlimb fast and slow twitch muscles. IGF-I binding to these membranes was unchanged at seven days and was increased at fourteen days of tail suspension, indicating that the observed decline in insulin receptors was not due to a global decline in cell surface receptors. Tail suspension for 14 days resulted in a similar decline in hepatic microsomal membrane insulin receptors. Serum insulin increased more than 3 fold and glucose increased by 26 % during tail suspension, documenting insulin resistance in glucose metabolism was induced by the model. The biochemical mechanism by which muscular inactivity results in muscle catabolism is unknown. The observation that insulin receptor levels decline in unloaded muscle during tail suspension is consistent with the hypothesis that inactivity-induced insulin resistance is due to a decline in muscle insulin receptors. The decline in insulin receptors could then lead to a loss of insulin's anabolic effects on muscle protein net balance and could help explain the catabolic state. However, our data could alternatively be explained by a primary defect in intracellular muscle translocation of glucose transporters, with the apparent decrease in insulin receptors being a secondary event caused by compensatory hyperinsulinemia, which in tum downregulated cell surface insulin receptors. The liver membrane data showing a similar decline in insulin receptors support this latter explanation. Muscle inactivity induced by hindlimb suspension or immobilization in a shortened position is associated with a decrease in the synthesis of all proteins in the affected muscle (2). Hindlimb suspension does not cause atrophy of all of the muscles but exerts most of its effects on the slow extensors of the ankle (16). Booth has suggested that the concentrations of some rapidly turning over proteins (i.e., cytochrome C) may decline in the early phase of immobilization, but that as the synthesis of more slowly turning over structural proteins declines, the relative concentrations of rapidly turning over proteins may appear 294
Exp Toxic Patho145 (1993) 5-6
to increase back toward normal (2). The changes we have demonstrated in ligand binding were quantitated in relation to membrane protein. It is possible that the decline in insulin binding we observed was simply part of a decline in all skeletal muscle proteins and the decline in insulin receptors was not directly involved as a mechanism of the protein catabolism. These data could be explained if the rate of insulin receptor turnover were more rapid than that of the average of all membrane proteins. That the IGF-I binding did not decrease with the insulin binding and perhaps changed in the opposite direction at 14 days suggests either that the IGF-I receptor turnover rate was less than the average of membrane proteins or that these receptors are regulated independently from insulin receptors in relation to the muscular inactivity. Hyperinsulinemia and resistance to insulin's effects on glucose metabolism have been associated with muscular inactivity in animals (2, 17) and in man (13, 14, 22). In human studies, insulin binding to circulating mononuclear cells was not altered by muscular inactivity (22), but muscle insulin binding was not measured directly. Muscular inactivity resulting in a decline in muscle insulin receptors could account for insulin resistance and subsequent hyperinsulinemia. On the other hand, hyperinsulinemia due to an independent mechanism may induce a decline in insulin receptors through down regulation (3, 4, 15). In our previous studies of the effects of seven days of absolute bedrest in humans, we found no change in serum IGF-I concentrations (22). If IGF-I blood concentrations did not change in suspended rats, and the circulating hormone level was the major determinant of the tissue receptor concentration, then IGF-I binding would not change. The later increase in IGFI binding to muscle membranes might be explained by an inactivity-related decline in production of IGF-I within muscle tissue as a local mediator (6,9,23) and a subsequent up-regulation of the IGF-I receptors. Rats which are maintained with inactivity of specific muscle groups by hindlimb pinning or casting or by tail suspension undergo a stress response in catecholamines and glucocorticoid release in the early stages, but these changes return to baseline by 5-7 days (18). We did not quantitate serum glucocorticoids during these studies but based on similar studies by others (8, 11, 20), it is not likely that insulin-counterregulatory hormones are directly responsible for the changes in insulin and IGF-I receptor numbers. The studies reported here show that rat tail suspension induced insulin resistance with marked hyperinsulinemia. There was a decline in insulin binding to muscle membranes with hindlimb unloading for 7-14 days. Inactivity of muscle may result in tissue specific insulin resistance through a decrease in muscle cell surface insulin receptors. However, a humoral mechanism causing at least part of this decline in cell surface insulin receptors is suggested by the concomitant fall in liver insulin receptors. Acknowledgement: The authors wish to thank Ms. TRACY MILLER for her expert secretarial assistance in preparation of this manuscript.
References 1. BOOTH FW: Effect of limb immobilization of skeletal muscle. J Appl Physiol 1982; 52: 1113-1118. 2. BOOTH FW: Physiologic and biochemical effects of immobilization on muscle: Clin Orthop 1987; 219: 15-20. 3. CARO JF, AMATRUDA JM: Insulin receptors in hepatocytes: postreceptor events mediate down regulation. Science 1980;210: 1029-1031. 4. CLARK, RB: Desensitization of hormonal stimuli coupled to regulation of cyclic AMP levels. Adv Cyclic Nucleotide Protein Phosphorylation Res 1986; 20: 151-208. 5. DESPLANCHES D, MAYET MH, SEMPORE B, et al.: Structural and functional responses to prolonged hindlimb suspension in rat muscle. J Appl Physiol1987; 63: 558-563. 6. EDWALL D, SCHALLING M, JENNISCHE E, et al.: Induction of insulin-like growth factor I messenger ribonucleic acid during regeneration of rat skeletal muscle. Endocrinology 1989; 124: 820-825. 7. FREYCHET P, ROTH J, NEVILLE DM. Monoidoinsulin: Demonstration of its biologic activity and binding to fat cells and liver membranes. Biochem Biophys Res Commun 1971; 43: 400-408. 8. GANGONG WF, GOLD NI, HUME DM. Effect of hypothalamic lesions on plasma 17-hydroxycorticoid response to immobilization in the dog. Federation Proc 1955; 14: 54. 9. ISGAARD J, NILSSON A, VIKMAN K, et al.: Growth hormone regulates the level of insulin-like growth factor-I mRNA in rat skeletal muscle. J Endricol1989; 120: 107-112. 10. JASPERS SR, FAGAN JM, SATARUG S, et al.: Effects of immobilization on rat hind limb muscles under nonweight-bearing conditions. Muscle Nerve 1988; 11: 458-466. 11. JASPERS SR, TISCHLER ME: Role of glucocorticoids in the response of rat leg muscles to reduced activity. Muscle Nerve 1986; 9: 554-556. 12. KIDDER LS, KLEIN GL, STUART CA, et al.: Musculoskeletal effects of sodium fluoride during hypokinesia: Putative roles of insulin and IGF-l. Bone Mineral 1990; 11: 305-318. 13. LIPMAN RL, RASKIN P, LOVE T, et al.: Glucose intolerance
during decreased physical activity in man. Diabetes 1972; 21: 101-107. 14. LIPMAN RL, SCHNURE JJ, BRADLEY EM, et al.: Impairment of peripheral glucose utilization in normal subjects by prolonged bed rest. J Lab Clin Med 1970; 76: 221-230. 15. MARSHALL S, OLEFSKY JM: Effects of insulin incubation on insulin binding, glucose transport, and insulin degradation by isolated rat adipocytes. Evidence for hormoneinduced desensitization at the receptor and postreceptor level. J Clin Invest 1980; 66: 763-772. 16. MICHEL RN, GARDINER PF: To what extent is hind limb suspension a model of disuse. Muscle Nerve 1990; 13: 646-653. 17. NICHOLSON WF, WATSON P~, BOOTH FW: Glucose uptake and glycogen synthesis in muscles from immobilized limbs. J Appl Physiol1984; 56: 431-438. 18. POPOVIC V, POPOVIC P, HONEYCUT C: Hormonal changes in antiorthostatic rats. Physiologist 1982; 25, Suppl: S77-S78. 19. SCATCHARD G: The attractions of proteins for small molecules and ions. Ann N Y Acad Sci 1949; 51: 660-666. 20. SEIDER MJ, NICHOLSON WF, BOOTH FW: Insulin resistance for glucose metabolism in disused soleus muscle of mice. Am J Physiol1982; 242: E12-E18. 21. STUART CA: Characteristics of a novel insulin receptor from stingray liver. J BioI Chern 1988; 263: 7881-7886. 22. STUART CA, SHANGRAW RE, PRINCE MJ, et al.: Bedrestinduced insulin resistance occurs primarily in muscle. Metabolism 1988; 37: 802-806. 23. TURNER JD, ROTWEIN P, NOVAKOFSKI J, et al.: Induction of mRNA for IGF-I and -II during growth hormone-stimulated muscle hypertrophy. Am J Physiol 1988; 255: E513-E517. 24. WINIARSKI AM, Roy RR, ALFORD EK, et al.: Mechanical properties of rat skeletal muscle after hindlimb suspension. Exp Neuro11987; 96: 650-660. 25. WRONSKI TJ, MOREY ER: Skeletal abnormalities in rats induced by simulated weightlessness. Metab Bone Dis Relat Res 1982; 4: 69-75.
ExpToxic Pathol45 (1993) 5--6
295
Departments of Internal Medicine", Pediatrics3! and Surgery'!; The University of Texas Medical Branch at Galveston, Galveston, Texas Department of Anthropology2l, Washington University, St. Louis
Rat tail suspension causes a decline in insulin receptors5) C. A. STUART!), L. S. KIDDER'!, R. A. PIETRZYK ", G. L. KLEIN", and D. 1. SIMMONS 4)
With 4 figures Received: January 28,1992; Revised: October 5,1992; Accepted: November 23,1992 Address for correspondence: Prof. CHARLES A. STUART, M. D. University of Texas Medical Branch at Galveston, MRB 3.142, Rt. J60 Galveston, Texas 77555-1060; Telephone: (409) 772-1923. Key words: Tail suspension; Insulin resistance; Insulin-like growth factor I (lGF-I); Microsomal membrane; IGF-I; Insulin binding; Insulin receptors; Hyperinsulinernia; Receptors, insulin; Skeletal muscle, IGF-I receptors; Liver, membrane insulin receptors.
Summary Decreased muscular activity results in weakness and muscular atrophy. Coincident with this protein catabolic state is glucose intolerance and hyperinsulinemia. Rats were tail suspended for 7 to 14 days to accomplish unloading of the hindlimbs. Insulin resistance was documented in these animals by a 14 day tail suspension-related 26 % increase in serum glucose in spite of a 253 % increase in serum insulin concentration. Microsomal membranes were prepared from hindlimb muscles and specific binding of insulin and insulin-like growth factor I (IGF-I) were determined in these membranes. Insulin binding was decreased by 27 % at 7 days and by 21 % at 14 days. In contrast, IGF-I binding was unchanged at 7 days and was increased by 24 % at 14 days. Liver membrane insulin receptors also had declined by 14 days of suspension, suggesting that the change in insulin receptors was a generalized, humorally-mediated phenomenon. These data suggest that tail suspension in rats results in insulin resistance, hyperinsulinemia, a decline in insulin receptors in liver and muscle, and a relative increase in muscle membrane IGF-I receptors. These data are consistent with the hypothesis that resi-
stance to insulin' s effects on protein metabolism in skeletal muscle may contribute to the protein catabolism associated with decreased muscular acti vity .
Introduction A prolonged decrease in muscular aCtIVIty results in muscle atrophy and weakness (1, 2). MOREY and coworkers described a rat model of decreased muscular work and decreased motor activity which involved suspension by the tail such that hindlimbs did not come in contact with the
Supported in part by grants from the National Aeronautics and Space Administration (NAG 9-172 and NTG-003)
5)
cage floor (12,25). This model as well as other rat hindlimb immobilization models have been used extensively to evaluate anatomic, functional, and biochemical consequences of muscular disuse (1, 5, 10,24). Disuse ofthe rat hindlimb results in decreased muscle weight, decreased muscle strength, decreased muscle oxygen utilization, and an increase in the relative number of fast twitch muscle fibers. The decreased muscular activity is associated with an early decrease in muscle protein synthesis and a later increase in protein breakdown (2, 10). The biochemical mechanisms which translate the change in contractile activity to these many alterations and particularly to changes in protein synthesis and breakdown are unknown. Since resistance to insulin's effects of glucose metabolism is induced by muscular inactivity in rats (17) and man (14, 22), we hypothesized that resistance to insulin's effects on protein metabolism in muscle might also be induced by muscular inactivity. Decreased insulin suppression of protein-breakdown or decreased insulin stimulation of protein synthesis would result in a net catabolic change in muscle protein balance. Direct quantitation of muscle membrane insulin binding was performed in order to determine if insulin resistance in muscle might be associated with a decrease in insulin receptors.
Material and methods
Materials Male Sprague-Dawley rats were from Harlan SpragueDawley (Houston, TX). Suspended rats and littermate controls were 6-8 weeks of age (180-200 g) at the beginning of each study. Littermate controls were housed in individual cages and ExpToxic Pathol45 (1993) 5-6
291
were pair fed according to the amount of food consumed the previous day by the suspended animals. Human insulin was the generous gift of Dr. RONALD CHANCE of Lilly Research Labs. Insulin was labeled with 1251 according to minor modifications of the dilute chloramine-T method of Freychet et al. (7). Insulin-like growth factor I (IGF-I) was purchased from Amgen (Thousand Oaks, CA). IGF-I was iodinated exactly as was insulin. '25I_IGF_I was further purified by high performance liquid chromatography using a C 18 reverse phase column. Other chemicals were of reagent grade.
Rat tail suspension The animals were prepared by fixing a thin strip of adhesive orthopedic moleskin along the length of the tail, leaving the sides open to permit radial growth. A small split ring was placed at the tip, and the array was secured with a gauze bandage wrap. The ring was then clipped to a swivel on an overhead "arm" which projected over the middle of the cage. The height of this arm was adjusted to maintain the animals in a 35-40 ° head-down tilt throughout the course of the study. This system allows the animal mobility and the capacity to groom and feed (25). Littermate controls were pair fed and allowed ad lib activity in individual cages.
Preparation of muscle and liver microsomal membranes At the end of 0, 7, or 14 days of suspension each animal was sacrificed by decapitation. The animals were not food restricted overnight, but were sacrificed in the morning at least two hours after removal of food. The lower hindlimbs were disarticulated from the thigh and foot and all muscles were dissected free and placed in saline on ice. Livers were also harvested and placed in ice cold saline. Muscle or liver was weighed and homogenized in a mixture of 0.25 M sucrose, 5 mM EDT A, 2 mM phenylmethylsulfonyl fluoride, and 100 mM imidazole, pH 7.4, using a Polytron (Brinkmann, Westbury, NY) for 60 seconds at a concentration of 1 g tissue per 2 ml homogenization buffer. The homogenate was centrifuged for 15 minutes at 1000 xg, the supernate was removed and recentrifuged at 100,000 xg for 60 minutes. The centrifuged pellet was then resuspended in 2 mM phenylmethylsulfonyl fluoride and I mM imidazole, pH 7.4, in a volume of 0.5 ml/g original tissue wet weight.
Radioimmunoassay for serum insulin Rat serum was assayed for immunoreactive insulin using a kit purchased from INCST AR (Stillwater, MN). The antiserum used in this assay was raised in rabbits against porcine insulin. The insulin standard was rat insulin extracted from rat pancreases and partially purified. Standards ranged 1 to 25 ng/ml and sample size was 200 ul of rat serum. Rat insulin was parallel to but 5 fold less potent than porcine insulin in displacing radiolabeled porcine insulin from the antibody. Since polyethylene glycol precipiation reduced the measured value consistently by 40 percent, the data used were those from non-precipitated serum. Glucose in rat serum was quantitated by the glucose oxidase method using a YSI Glucose Analyzer (Yellow Springs Instrument Company, Yellow Springs, Ohio). 292
Exp Toxic Pathol45 (1993) 5-6
Ligand binding Muscle membranes were then incubated for 12-15 hours at 4 °C at a concentration of 500 ug/ml in a binding buffer, consisting of 100 mM Hepes, 120 mM sodium chloride, 1.2 mM magnesium sulfate, 1 mM EDTA, 10 mM glucose, 15 mM sodium acetate, and I % BSA, pH 8.0. '25I-insulin or 125I-IGF-I was added to the binding buffer resulting in a final labeled ligand concentration of 0.05 nM; nonspecific binding was defined as the '25I counts bound in the presence of at least 200 fold molar excess of unlabeled insulin or IGF-I. SCATCHARD analysis (19) was calculated from binding studies performed using a constand amount of radiolabeled ligand and several dilutions of unlabeled ligand. Total specific binding is the percent of the labeled ligand bound in the absence of unlabeled ligand with the nonspecific ally bount count subtracted. SCATCHARD plot analysis used a two receptor mathematical model previously described (21). In cases where affinity does not change, total specific binding is directly proportional to the quantitation of high affinity binding sites determined by this model.
Statistics Data were analyzed using STUDENT'S t test or analysis ofvariance where appropriate. Data are expressed as means and standard errors of the mean except where noted.
Results The effect of 14 days of tail suspension on serum glucose and insulin: Six rats were suspended by the tail method described above. Littermate controls were pair fed for the same 14 days. Animals were allowed free access to food and water overnight and were sacrificed in the morning about 2 hr after food was removed. Each animal had approximately 2 ml blood for assay of glucose and insulin obtained by cardiac puncture just prior to sacrifice by decapitation. Figure 1 shows the results of those measurements. Both glucose and insulin concentrations were significantly higher in the suspended animals than in the controls. Glucose concentration was increased (8.28 ± 0.33 vs 6.56 ± 0.17 mmollL, n 6) by 26 % and insulin concentration was 253 % higher (44.1 ± 2.8 vs 12.5±2.7 pmollL, n 6) in the suspended rats (both p < 0.001). This increase in glucose in spite of a large increase in insulin documents insulin resistance of glucose metabolism has been induced by tail suspension.
=
=
The effect of tail suspension on specific insulin binding to muscle membranes: Specific binding of insulin to membranes obtained from 24 control rats and 12 rats suspended for 7 days and 12 rats suspended for 14 days in shown in figure 2. These data represent the pooling of data from four separate experiments each with six suspended and six littermate controls. The data from controls at 7 and 14 days were not different and were combined. As can be seen, suspension for 7 days was associated with a decrease in insulin binding from 3.32 ± 0.19 to 2.38 ± 0.46 %/mg membrane protein (p < 0.02). At 14 days of suspension, the
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Fig. 2. The effects of tail suspension on the specific binding of insulin and IGF-I to hindlimb skeletal muscle membranes: The data displayed are from 12 rats subjected to tail suspension for 7 days and 12 rats suspended for 14 days and from groups of an equal number of littermates who were not suspended but were pair fed. The binding data from the control groups were not different between 7 days and 14 days and were pooled for this figure. The asterisk denotes significant difference (p < 0.05) form the control data.
decreased insulin binding persisted but did not worsen (2.64 ± 0.25, p < 0.02 compared with controls).
The effect of tail suspension on specific binding of IGF-I to muscle membranes: IGF-I specific binding was performed on the same membrane preparations as used for quantitation of insulin binding described above. The results of these studies are also presented in figure 2. In contrast to the suspension related decline in insulin binding, IGF-I binding did not decline at 7 days (5.05 ± 0.33 vs control of 4.72 ± 0.40 %/mg membrane protein, p 0.32) and was increased (5.82 ± 0.50, p < 0.05 compared with controls) at 14 days.
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Fig. 4. Scatchard analysis of insulin binding to liver membranes: The data shwon represent the mean of Scatchard analyses of insulin binding data from membranes prepared from six 14 day suspended rats and six control rats. Affinity was not different (0.30 ± 0 .01 vs 0.35 ± 0.04 Llmole control, p = 0.46), but insulin receptor number declined (=.175 ± 0.005 vs 0.098 ± 0.002 pmollmg membrane protein, p < 0.(01) in proportion to the change in total specific binding shown in figure 3.
The dTect of 14 days of tail suspension on insulin binding to liver microsomal membranes: If the observed decline in insulin receptors in skeletal muscle was a primary inactivity-related event, then this decline might be limited to muscle. However, if insulin resistance were due to another effect, such as a post-receptor defect, then the resulting hyperinsulinemia could cause down regulation of insulin receptors in other insulin responsive tissues such as liver. We, therefore, quantitated liver insulin receptors in tail-suspended rats. Six Sprague Dawley rats were tail suspended for 14 days as described in Methods. Membranes from livers and hindlimb muscles were prepared and specific insulin binding was determined as described above. Figure ExpToxic Pathol45 (1993) 5-6
293
3 shows the results of these studies. Insulin binding was decreased by 45 % in liver membranes from suspended animals (2.11 ± 0.05 vs 3.83 ± 0.57 %/mg control, p < 0.001). The muscle membrane insulin binding tended to also be decreased by 34 %, but this difference did not achieve statistical significance (p = 0.15). Figure 4 shows Scatchard analysis of studies of insulin binding to liver membranes. The decrease in total specific binding was accompanied by a decline in number of receptors with no change in the receptor affinity. Similar SCATCHARD analysis of the muscle membrane insulin binding from the same rats also showed that the high affinity portion of the curves from control and suspended rats were parallel and the x-axis intercepts suggested (but not statistically significant) a 20 % decrease in muscle insulin receptor number in the suspended animals.
Discussion Tail suspension of young male Sprague-Dawley rats for seven to fourteen days was associated with a decrease in specific insulin binding to microsomal membranes from lower hindlimb fast and slow twitch muscles. IGF-I binding to these membranes was unchanged at seven days and was increased at fourteen days of tail suspension, indicating that the observed decline in insulin receptors was not due to a global decline in cell surface receptors. Tail suspension for 14 days resulted in a similar decline in hepatic microsomal membrane insulin receptors. Serum insulin increased more than 3 fold and glucose increased by 26 % during tail suspension, documenting insulin resistance in glucose metabolism was induced by the model. The biochemical mechanism by which muscular inactivity results in muscle catabolism is unknown. The observation that insulin receptor levels decline in unloaded muscle during tail suspension is consistent with the hypothesis that inactivity-induced insulin resistance is due to a decline in muscle insulin receptors. The decline in insulin receptors could then lead to a loss of insulin's anabolic effects on muscle protein net balance and could help explain the catabolic state. However, our data could alternatively be explained by a primary defect in intracellular muscle translocation of glucose transporters, with the apparent decrease in insulin receptors being a secondary event caused by compensatory hyperinsulinemia, which in tum downregulated cell surface insulin receptors. The liver membrane data showing a similar decline in insulin receptors support this latter explanation. Muscle inactivity induced by hindlimb suspension or immobilization in a shortened position is associated with a decrease in the synthesis of all proteins in the affected muscle (2). Hindlimb suspension does not cause atrophy of all of the muscles but exerts most of its effects on the slow extensors of the ankle (16). Booth has suggested that the concentrations of some rapidly turning over proteins (i.e., cytochrome C) may decline in the early phase of immobilization, but that as the synthesis of more slowly turning over structural proteins declines, the relative concentrations of rapidly turning over proteins may appear 294
Exp Toxic Patho145 (1993) 5-6
to increase back toward normal (2). The changes we have demonstrated in ligand binding were quantitated in relation to membrane protein. It is possible that the decline in insulin binding we observed was simply part of a decline in all skeletal muscle proteins and the decline in insulin receptors was not directly involved as a mechanism of the protein catabolism. These data could be explained if the rate of insulin receptor turnover were more rapid than that of the average of all membrane proteins. That the IGF-I binding did not decrease with the insulin binding and perhaps changed in the opposite direction at 14 days suggests either that the IGF-I receptor turnover rate was less than the average of membrane proteins or that these receptors are regulated independently from insulin receptors in relation to the muscular inactivity. Hyperinsulinemia and resistance to insulin's effects on glucose metabolism have been associated with muscular inactivity in animals (2, 17) and in man (13, 14, 22). In human studies, insulin binding to circulating mononuclear cells was not altered by muscular inactivity (22), but muscle insulin binding was not measured directly. Muscular inactivity resulting in a decline in muscle insulin receptors could account for insulin resistance and subsequent hyperinsulinemia. On the other hand, hyperinsulinemia due to an independent mechanism may induce a decline in insulin receptors through down regulation (3, 4, 15). In our previous studies of the effects of seven days of absolute bedrest in humans, we found no change in serum IGF-I concentrations (22). If IGF-I blood concentrations did not change in suspended rats, and the circulating hormone level was the major determinant of the tissue receptor concentration, then IGF-I binding would not change. The later increase in IGFI binding to muscle membranes might be explained by an inactivity-related decline in production of IGF-I within muscle tissue as a local mediator (6,9,23) and a subsequent up-regulation of the IGF-I receptors. Rats which are maintained with inactivity of specific muscle groups by hindlimb pinning or casting or by tail suspension undergo a stress response in catecholamines and glucocorticoid release in the early stages, but these changes return to baseline by 5-7 days (18). We did not quantitate serum glucocorticoids during these studies but based on similar studies by others (8, 11, 20), it is not likely that insulin-counterregulatory hormones are directly responsible for the changes in insulin and IGF-I receptor numbers. The studies reported here show that rat tail suspension induced insulin resistance with marked hyperinsulinemia. There was a decline in insulin binding to muscle membranes with hindlimb unloading for 7-14 days. Inactivity of muscle may result in tissue specific insulin resistance through a decrease in muscle cell surface insulin receptors. However, a humoral mechanism causing at least part of this decline in cell surface insulin receptors is suggested by the concomitant fall in liver insulin receptors. Acknowledgement: The authors wish to thank Ms. TRACY MILLER for her expert secretarial assistance in preparation of this manuscript.
References 1. BOOTH FW: Effect of limb immobilization of skeletal muscle. J Appl Physiol 1982; 52: 1113-1118. 2. BOOTH FW: Physiologic and biochemical effects of immobilization on muscle: Clin Orthop 1987; 219: 15-20. 3. CARO JF, AMATRUDA JM: Insulin receptors in hepatocytes: postreceptor events mediate down regulation. Science 1980;210: 1029-1031. 4. CLARK, RB: Desensitization of hormonal stimuli coupled to regulation of cyclic AMP levels. Adv Cyclic Nucleotide Protein Phosphorylation Res 1986; 20: 151-208. 5. DESPLANCHES D, MAYET MH, SEMPORE B, et al.: Structural and functional responses to prolonged hindlimb suspension in rat muscle. J Appl Physiol1987; 63: 558-563. 6. EDWALL D, SCHALLING M, JENNISCHE E, et al.: Induction of insulin-like growth factor I messenger ribonucleic acid during regeneration of rat skeletal muscle. Endocrinology 1989; 124: 820-825. 7. FREYCHET P, ROTH J, NEVILLE DM. Monoidoinsulin: Demonstration of its biologic activity and binding to fat cells and liver membranes. Biochem Biophys Res Commun 1971; 43: 400-408. 8. GANGONG WF, GOLD NI, HUME DM. Effect of hypothalamic lesions on plasma 17-hydroxycorticoid response to immobilization in the dog. Federation Proc 1955; 14: 54. 9. ISGAARD J, NILSSON A, VIKMAN K, et al.: Growth hormone regulates the level of insulin-like growth factor-I mRNA in rat skeletal muscle. J Endricol1989; 120: 107-112. 10. JASPERS SR, FAGAN JM, SATARUG S, et al.: Effects of immobilization on rat hind limb muscles under nonweight-bearing conditions. Muscle Nerve 1988; 11: 458-466. 11. JASPERS SR, TISCHLER ME: Role of glucocorticoids in the response of rat leg muscles to reduced activity. Muscle Nerve 1986; 9: 554-556. 12. KIDDER LS, KLEIN GL, STUART CA, et al.: Musculoskeletal effects of sodium fluoride during hypokinesia: Putative roles of insulin and IGF-l. Bone Mineral 1990; 11: 305-318. 13. LIPMAN RL, RASKIN P, LOVE T, et al.: Glucose intolerance
during decreased physical activity in man. Diabetes 1972; 21: 101-107. 14. LIPMAN RL, SCHNURE JJ, BRADLEY EM, et al.: Impairment of peripheral glucose utilization in normal subjects by prolonged bed rest. J Lab Clin Med 1970; 76: 221-230. 15. MARSHALL S, OLEFSKY JM: Effects of insulin incubation on insulin binding, glucose transport, and insulin degradation by isolated rat adipocytes. Evidence for hormoneinduced desensitization at the receptor and postreceptor level. J Clin Invest 1980; 66: 763-772. 16. MICHEL RN, GARDINER PF: To what extent is hind limb suspension a model of disuse. Muscle Nerve 1990; 13: 646-653. 17. NICHOLSON WF, WATSON P~, BOOTH FW: Glucose uptake and glycogen synthesis in muscles from immobilized limbs. J Appl Physiol1984; 56: 431-438. 18. POPOVIC V, POPOVIC P, HONEYCUT C: Hormonal changes in antiorthostatic rats. Physiologist 1982; 25, Suppl: S77-S78. 19. SCATCHARD G: The attractions of proteins for small molecules and ions. Ann N Y Acad Sci 1949; 51: 660-666. 20. SEIDER MJ, NICHOLSON WF, BOOTH FW: Insulin resistance for glucose metabolism in disused soleus muscle of mice. Am J Physiol1982; 242: E12-E18. 21. STUART CA: Characteristics of a novel insulin receptor from stingray liver. J BioI Chern 1988; 263: 7881-7886. 22. STUART CA, SHANGRAW RE, PRINCE MJ, et al.: Bedrestinduced insulin resistance occurs primarily in muscle. Metabolism 1988; 37: 802-806. 23. TURNER JD, ROTWEIN P, NOVAKOFSKI J, et al.: Induction of mRNA for IGF-I and -II during growth hormone-stimulated muscle hypertrophy. Am J Physiol 1988; 255: E513-E517. 24. WINIARSKI AM, Roy RR, ALFORD EK, et al.: Mechanical properties of rat skeletal muscle after hindlimb suspension. Exp Neuro11987; 96: 650-660. 25. WRONSKI TJ, MOREY ER: Skeletal abnormalities in rats induced by simulated weightlessness. Metab Bone Dis Relat Res 1982; 4: 69-75.
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