Insulin stimulation of muscle protein synthesis in obese Zucker rats is not via a rapamycin-sensitive pathway

James D. Fluckey1,2, Steven C. Pohnert1, S. Greg Boyd1, Ronald N. Cortright1, Todd A. Trappe2, and G. Lynis Dohm1

1 Department of Biochemistry, East Carolina University School of Medicine, Greenville, North Carolina 27834; and 2 Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The obese Zucker rat is resistant to insulin for glucose disposal, but it is unknown whether this insulin resistance is accompanied by alterations of insulin-mediated muscle protein synthesis. We examined rates of muscle protein synthesis either with or without insulin in lean and obese Zucker rats with the use of a bilateral hindlimb preparation. Additional experiments examined insulin's effect on protein synthesis with or without rapamycin, an inhibitor of protein synthesis. Protein synthesis in red and white gastrocnemius was stimulated by insulin compared with control (no insulin) in obese (n = 10, P < 0.05) but not in lean (n = 10, P > 0.05) Zucker rats. In white gastrocnemius, rapamycin significantly reduced rates of protein synthesis compared with control in lean (n = 6) and obese (n = 6) rats; however, in red gastrocnemius, the attenuating effect of rapamycin occurred only in obese rats. The addition of insulin to rapamycin resulted in rates of synthesis that were similar to those for rapamycin alone for lean rats and to those for insulin alone (augmented) for obese rats in both tissues. Our results demonstrate that insulin enhances protein synthesis in muscle that is otherwise characterized as insulin resistant. Furthermore, rapamycin inhibits protein synthesis in muscle of obese Zucker rats; however, stimulation of protein synthesis by insulin is not via a rapamycin-sensitive pathway.

signal transduction; translation; p70S6k


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE PHENOTYPIC EXPRESSION of obesity in the Zucker rat is associated with both normoglycemia and hyperinsulinemia compared with phenotypically lean littermates (2). Despite normal glycemia, insulin-stimulated glucose transport in muscle is significantly lowered in obese vs. lean Zucker rats (6). The obese Zucker rat is, therefore, insulin resistant (3, 12, 24) and has been used to study mechanisms of type 2 diabetes (20). The etiology of insulin resistance in the obese Zucker rat is unclear; however, upstream regulators of the insulin signal transduction pathway may be limiting (1, 5, 27). Furthermore, it is not known whether impaired insulin signaling for glucose disposal affects other actions, such as protein synthesis, that are elicited by this important metabolic hormone.

Several reports suggest that insulin is a modulator of muscle protein synthesis (8, 9, 13, 16, 18, 19). Although mechanisms of insulin action are complex and not completely understood, there is mounting evidence that the anabolic effect of insulin is a result of signal transduction through a rapamycin-sensitive pathway (4, 11, 21, 26). Activation of the rapamycin-sensitive pathway by insulin in vitro leads to elevated protein synthesis. Conversely, incubation of cells with rapamycin, a potent antibiotic that inhibits signal transduction through this pathway, leads to attenuated protein synthesis, even in the presence of insulin (4). Therefore, functional insulin signal transduction through this pathway may be of paramount importance for the maintenance of muscle protein synthesis. It is unclear what effect insulin may have on this important metabolic process in animals where some of the key upstream activators of the rapamycin-sensitive pathway are impaired (1, 5, 27). In the first portion of this study, we examined the effect of insulin on muscle protein synthesis in lean and obese Zucker rats. In the second, we examined whether or not insulin activation of muscle protein synthesis in obese Zucker rats operates through a rapamycin-sensitive pathway.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The animal use and care committee of East Carolina University School of Medicine approved all methods used in the study. In the first portion of the study, lean (n = 10) and obese (n = 10) Zucker rats (~12 wk old) were perfused using a bilateral hindlimb preparation to determine the effect of insulin on muscle protein synthesis (8, 9). Briefly, after anesthesia with pentobarbital sodium (65 mg/kg ip), animals were laparotomized, and polythene catheters were placed into the left and right iliac arteries, where one limb received medium containing insulin but the other received medium without insulin. Using this technique, we were able to determine rates of fiber-specific muscle protein synthesis with and without insulin within each animal. Hindlimbs were perfused with a Krebs-Henseleit (KHB) medium (pH 7.4), containing time-expired washed bovine red blood cells (hematocrit ~30%), 4.5% bovine serum albumin, 11.25 mM glucose, 2 mM phenylalanine, and all other amino acids at physiological concentrations, as described by Jefferson et al. (14). Flow rate for each hindlimb was maintained at 5 ml/min (10 ml/min for both hindlimbs combined) by means of flow-matched peristaltic pumps. Before the introduction of red blood cells, the KHB was filtered through a nitrocellulose filter (Millipore, 0.8 µm pore size), and L-[2,3,4,5,6-3H]phenylalanine (Amersham Life Science, Arlington Heights, IL) was added to the medium to yield a final concentration of 0.5 µCi/ml. Porcine insulin (Lilly, Indianapolis, IN) was provided at 20,000 µU/ml to one limb when necessary. Hindlimbs receiving treatment were counterbalanced within groups, and the medium was not circulated (single-pass perfusion preparation). Lean and obese animals perfused on a given day were selected at random with equal numbers within each group. The medium used for animals on a given day was made with sufficient volume so that all animals were perfused with the same medium.

The medium and hindlimb preparation was maintained at 37°C during the perfusion protocol and gassed with humidified O2 (95%) and CO2 (5%). PO2 was maintained at >90% saturation. A 15-min washout period with KHB medium preceded the 35-min bilateral hindlimb perfusion. Insulin used in the first portion of the study and/or rapamycin used in the second portion of the study were present for the entire 35-min period. After the 35-min period, entire muscles were excised in the order of soleus, extensor digitorum longus, and gastrocnemius, with gastrocnemius being further partitioned into red and white portions. Soleus and extensor digitorum longus muscles were obtained to examine differences of hindlimb muscle mass in lean and obese rats. Gastrocnemius muscles were obtained to examine rates of muscle protein synthesis in tissue predominantly composed of red or white fast-twitch fibers. After excision and partitioning, muscles were placed in liquid nitrogen until frozen and were stored at -80°C until assessed for incorporation of tritiated phenylalanine into trichloroacetic acid (TCA)-precipitable extracts (25) and corrected for the specific radioactivity of the perfusion medium. Protein determinations were conducted using the bicinchoninic acid (BCA) assay (Sigma, St. Louis, MO). This procedure allowed for the determination of rates of muscle protein synthesis in the presence of insulin and/or a specific inhibitor of insulin signal transduction between the lean and obese Zucker rats.

Phenylalanine concentrations in the perfusates were quantified with high-performance liquid chromatography after TCA precipitation and dabsylation (17, 25). These concentrations were used to establish perfusate specific radioactivity. The precise time that each muscle or portion of muscle was placed into liquid nitrogen was recorded to establish rates of protein synthesis on an hourly basis. Rates of protein synthesis are reported as nanomoles of phenylalanine incorporated per gram wet weight of muscle per hour and were calculated by use of the methods of Garlick et al. (10).

Rapamycin Experiments

For the second portion of the study, lean (n = 6) and obese (n = 6) Zucker rats were perfused as before, but the medium was with or without insulin (20,000 µU/ml) and/or with or without rapamycin (1 µM). This concentration of rapamycin was ~5 times higher than the concentrations used in studies observing an attenuation of muscle protein synthesis in vitro (4). Thus limbs were perfused with four possible perfusion medium combinations: control (no treatment), rapamycin only, insulin only, or insulin plus rapamycin. Treatments were counterbalanced within groups for equal representation of all possibilities. These procedures enabled us to examine the effect of inhibited insulin signal transduction through a rapamycin-sensitive pathway on rates of muscle protein synthesis and to examine how rates were influenced by the addition of insulin.

p70S6k Assay

Activation of the rapamycin-sensitive pathway results in the activation of p70S6k (4); therefore, we examined the effectiveness of insulin and/or rapamycin on the activity of this important kinase. A portion (~150 mg) of the red or white gastrocnemius was homogenized on ice in 400 µl of buffer containing 25 mM HEPES, 4 mM EDTA, 25 mM benzamidine, 1 µM concentrations of leupeptin and pepstatin, 0.15 mM aprotinin, and 2 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 15,000 g at 4°C for 1 h. After centrifugation, the supernatant was taken, protein content in the supernatant was assayed with BCA, and 1 µg of protein was applied to a 4-8% discontinuous polyacrylamide gel. After electrophoresis, the gel was blotted to Immobilon paper by means of a semidry method (Multiphor II, Amersham-Pharmacia-Biotech), and immunoblotted with a p70S6k antibody (SC-230) obtained from Santa Cruz Biotechnology (Santa Cruz, CA). In addition, activity of p70S6k was indirectly assessed in the presence of rapamycin by apparent mobility with a gradient polyacrylamide gel (4-12%). Blotting was conducted as before. Because phosphorylation of p70S6k is necessary for enzyme activity, the active phosphorylated form of p70S6k is less mobile on a gradient gel (4). The blots were visualized with enhanced chemiluminescence on Kodak film and digitized on a Hewlett-Packard scanner. For comparisons of protein content between groups, analyses were conducted with image software from the National Institutes of Health. Both red and white gastrocnemius muscles of all animals were represented on the same blot so that direct comparisons could be made. Data are presented as arbitrary densitometric units of area under the curve. Kinase activity was visually determined by protein migration.

Statistics

Analysis of variance was used to compare means for rates of muscle protein synthesis within each muscle with or without insulin and/or rapamycin between lean and obese Zucker rats. Differences among means were considered significant when P < 0.05. When f ratios were significant, a Student-Newman-Keuls test was used to compare means. All data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All lean (n = 16) and obese (n = 16) animals were studied at ~12 wk of age. Body weights, as expected with this strain of rats, were significantly different (P < 0.05) for obese and lean rats (595 ± 22 and 434 ± 14 g, respectively). Although body weights were higher in obese animals, the hindlimb wet weights of the obese animals were significantly lower (P < 0.05) than those of the lean animals for soleus (123 ± 4 vs. 145 ± 7 mg, respectively) and extensor digitorum longus (120 ± 10 vs. 149 ± 8 mg, respectively).

Influence of Insulin on Muscle Protein Synthesis

In the first portion of the study, we examined in situ rates of muscle protein synthesis in lean (n = 10) and obese (n = 10) Zucker rats with and without insulin. Although the obese Zucker rat is considered insulin resistant with respect to muscle glucose regulation compared with its lean littermate, it was not known how acute insulin administration might influence muscle protein synthesis in these animals. Results from this portion of the study demonstrated that muscle protein synthesis was significantly higher (P < 0.05) in red and white gastrocnemius from obese rats in the presence of insulin compared with that without insulin (Fig. 1). Furthermore, rates of muscle protein synthesis were higher (P < 0.05) with insulin in red and white muscle of obese rats compared with those of lean rats with or without insulin. Rates of muscle protein synthesis were not different from those of lean animals with or without insulin in red or white muscle (P > 0.05), and they were similar to muscle protein synthesis of obese animals without insulin.


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Fig. 1.   Rates of muscle protein synthesis during a bilateral hindlimb preparation with (filled bars) and without (open bars) insulin in 12-wk-old lean (n = 10) and obese (n = 10) Zucker rats. F, phenylalanine. Red gastrocnemius muscle protein synthesis is represented on the left, white gastrocnemius muscle protein synthesis on the right. * Different from others within muscle (P < 0.05).

The Rapamycin-Sensitive Signal Transduction Pathway and Muscle Protein Synthesis

The rapamycin-sensitive pathway has been implicated as a regulatory step of protein synthesis in vitro, likely involving p70S6k. However, results from our studies in situ using rapamycin demonstrated that rates of muscle protein synthesis were not different with or without rapamycin or insulin in the predominantly red gastrocnemius of lean rats (Fig. 2). Unlike in lean animals, rates of muscle protein synthesis were significantly lower (P < 0.05) with rapamycin than in control (no treatment) with obese red gastrocnemius. As with the first portion of this study, muscle protein synthesis in the presence of insulin was higher than in control in obese muscle (P < 0.05). Rates of protein synthesis in red gastrocnemius were significantly higher (P < 0.05) with insulin and rapamycin compared with control and were similar to those with insulin alone. Therefore, rapamycin had an inhibitory effect on muscle protein synthesis in obese red gastrocnemius, but this inhibition was overcome by the presence of insulin.


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Fig. 2.   Rates of protein synthesis in red gastrocnemius during a bilateral hindlimb preparation of 12-wk-old lean (n = 6; open bars) and obese (n = 6; filled bars) Zucker rats in the presence of medium only (none), rapamycin only (rap), insulin only (insulin), or rapamycin plus insulin (rap+ins). * Different from none within phenotype (P < 0.05).

Unlike in red gastrocnemius, protein synthesis in white gastrocnemius was reduced (P < 0.05) in the presence of rapamycin in both phenotypes compared with controls (Fig. 3). The addition of insulin to rapamycin in white muscle of lean rats yielded rates of protein synthesis that were similar to those with rapamycin alone. Unlike in lean animals, rates of muscle protein synthesis in the presence of this inhibitor and insulin in obese rats were similar to those with insulin alone. When combined, our data suggest that mechanisms of insulin signal transduction with respect to protein synthesis may be genetic strain specific.


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Fig. 3.   Rates of protein synthesis in white gastrocnemius during a bilateral hindlimb preparation of 12-wk-old lean (n = 6; open bars) and obese (n = 6; filled bars) Zucker rats in the presence of medium only (none), rapamycin only (rap), insulin only (insulin), or rapamycin plus insulin (rap+ins). * Different from none within phenotype (P < 0.05).

p70S6k Content and Activity

There was a tendency for an overall reduction of p70S6k in muscle of obese Zucker rats (Fig. 4). Content of p70S6k was significantly lower in obese rats compared with lean rats in red (P < 0.05) but not in white muscle (P > 0.05). Because it has been demonstrated that this kinase must be phosphorylated to be active, we used relative migration of p70S6k as an apparent marker of kinase activity (4). Relative migration of this protein was farther during gradient gel electrophoresis when tissues were perfused with rapamycin or with rapamycin plus insulin in lean Zucker rats compared with no treatment or insulin alone (Fig. 5). Similar migration patterns, yielding multiple bands of p70S6k, were observed for insulin and for no treatment. These results suggest that the dosage of rapamycin (1 µM) was effective for inhibition of p70S6k, even in the presence of insulin, for both red and white lean tissues. In addition, the migration of p70S6k in the presence of insulin alone of obese Zucker muscle, as represented in Fig. 5, was comparable with that in lean muscle with insulin, suggesting that our maximal dose of insulin was sufficient to stimulate p70S6k in obese tissues. Furthermore, obese tissues had similar patterns of migration without insulin, with rapamycin, and with rapamycin plus insulin.


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Fig. 4.   p70S6k content of red and white muscle from 12-wk-old lean (n = 6; open bars) and obese (n = 6; filled bars) Zucker rats. * Different from lean within fiber type (P < 0.05).



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Fig. 5.   A representative immunoblot for the apparent mobility of p70S6k from red and white muscle of lean and obese Zucker rats during gradient gel electrophoresis. This qualitative blot contains lanes from individual lean and obese animals without treatment (none), with insulin, with rapamycin plus insulin, and with rapamycin. Because phosphorylation (P) is prerequisite to p70S6k activity, an elevated phosphorylation state resulting from insulin signal transduction should attenuate migration relative to p70S6k in the presence of rapamycin. The observation that migration distances were similar and were represented by a single band for p70S6k in the presence of rapamycin only or rapamycin plus insulin suggests that this kinase was inhibited by rapamycin in our system.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The obese Zucker rat is resistant to insulin for muscle glucose disposal compared with its lean littermate (3, 12, 24), a condition likely caused by attenuated insulin signal transduction (1, 5, 27). This study examined whether an impaired insulin action for glucose regulation affected other actions, namely muscle protein synthesis, by this important metabolic hormone. To our knowledge, this is the first study to demonstrate the important finding that an acute administration of insulin results in augmented rates of muscle protein synthesis in obese Zucker rats, an effect that was not observed in lean rats. We also demonstrated that the augmented insulin action on protein synthesis in obese rats persisted in the presence of rapamycin, which had been shown to attenuate protein synthesis in vitro (4, 11, 21, 26). Our results suggest that obese Zucker rats exhibit insulin sensitivity with respect to muscle protein synthesis and that this adaptation does not involve a rapamycin-sensitive pathway.

At this time, we cannot be certain why obesity and/or chronic hyperinsulinemia facilitate(s) an adaptation for an enhanced insulin action on muscle protein synthesis. We speculate that in the case of the obese Zucker rat, it may be a compensatory mechanism for the preservation of muscle mass in the face of elevated muscle degradation. To support this notion, data obtained from hindlimbs demonstrated that muscle mass is significantly smaller in obese Zucker rats compared with that in their lean littermates. Thus we propose that, in an attempt to maintain muscle mass, the atrophied muscles of obese Zucker rats have adapted by becoming sensitive to insulin's action on muscle protein synthesis. Furthermore, this improved insulin sensitivity for muscle protein homeostasis may be directed specifically toward synthesis and not toward the prevention of proteolysis. We believe, although it was not directly measured, that in addition to muscle glucose disposal, obese Zucker rats have become resistant to insulin's antiproteolytic actions, because in the prevailing state of hyperinsulinemia, a significant reduction of muscle mass occurred despite an enhanced insulin action on protein synthesis.

The present study did not observe an enhanced insulin action in muscle of lean rats in situ, regardless of fiber type, which is unlike results obtained in vitro in rat muscle (4) or in various cell lines (10, 19, 22). The finding that insulin does not have an effect on muscle in normal rats is similar to that of other in situ studies using hindlimb preparations (7-9). The apparent contrast between studies may be the result of the methodological approaches that were used. Our in situ studies examined rates of muscle protein synthesis in tissues with and without insulin during perfusion periods of <1 h. The length of time for our perfusion protocol was based on work by others (15, 22), demonstrating that rates of muscle protein synthesis were attenuated after 1 h of insulin deprivation. Thus our perfusion protocol should not elicit an augmented insulin action on muscle protein synthesis in otherwise normal tissues. In a study demonstrating an effect of insulin on rat muscle protein synthesis in vitro (4), muscles were incubated for <= 3 h with or without insulin. Therefore, it is likely that, at least in part, the differences of muscle protein synthesis with and without insulin observed in that study were a result of a methodologically imposed insulin deprivation and not an enhanced insulin effect that may be observed in vivo. We thought that it was important for us to examine insulin's effect on muscle protein synthesis under conditions where prolonged insulin deprivation was not a factor and, furthermore, whether or not the rapamycin-sensitive pathway contributed to the augmented insulin sensitivity observed in the present study.

In addition to evidence that obese Zucker rats are insulin sensitive for muscle protein synthesis, the present study shows that a rapamycin-sensitive pathway is not necessary for this enhanced insulin effect. Several reports (4, 11, 21, 26) have demonstrated that a rapamycin-sensitive pathway is important for the stimulation of protein synthesis by insulin in vitro. In general, those studies observed an attenuating effect of insulin on protein synthesis in the presence of rapamycin. Similarly, we observed a rapamycin-induced attenuation of muscle protein synthesis that was not normalized by insulin in white tissues of lean rats. We must note, however, that we did not observe an enhanced insulin action on protein synthesis in lean rats, which might be explained by inherent differences of methodological approaches between studies. Furthermore, rapamycin attenuated rates of muscle protein synthesis in red muscle of obese animals but not in similar tissues of lean animals. More work is necessary to examine these phenotypic and fiber type differences. Unlike the previous studies (4, 11, 21, 26), the present study demonstrated that when an augmented insulin action for muscle protein synthesis was observed, as in the obese Zucker rat, rapamycin did not ameliorate its effect. These results suggest that the rapamycin-sensitive pathway is important for the regulation of protein synthesis, but adaptations of signaling mechanisms that result in an enhanced insulin action for muscle protein synthesis operate independently of a rapamycin-sensitive pathway in the obese Zucker rat.

Further support for this speculation may be found in our analysis of p70S6k. In lean animals, there is a similar apparent phosphorylation of p70S6k, a marker of rapamycin-sensitive pathway activation, with and without insulin, but the addition of insulin had no apparent effect on muscle protein synthesis. The apparent activation of this kinase in the untreated condition (without insulin) may have been caused by the presence of amino acids in our perfusion medium. Amino acids have been shown to activate this kinase in vitro, and this activation is sensitive to rapamycin (23). The present study demonstrated that the addition of rapamycin attenuated muscle protein synthesis in these animals, and this attenuation persisted in the presence of insulin. Together, our results suggest that a rapamycin-sensitive pathway influences muscle protein synthesis, at least in a basal state, in lean muscle.

In obese animals, we observed an insulin-mediated phosphorylation of this important kinase, which was accompanied by an elevation of muscle protein synthesis. Taken alone, this would imply that a rapamycin-sensitive pathway is necessary for muscle protein synthesis in these tissues. However, the addition of rapamycin and insulin together, which resulted in reduced kinase activity, resulted in augmented rates of synthesis that were similar to those with insulin alone. These results suggest that the activation of muscle protein synthesis by insulin operates via a mechanism that is independent of a rapamycin-sensitive pathway. It is surprising and exciting to note that we observed an augmented insulin action on muscle protein synthesis even though the obese Zucker rat is otherwise considered insulin resistant. Although we are uncertain of the specific pathways responsible for this enhanced insulin sensitivity for muscle protein synthesis, it is apparent that specific adaptations have occurred in the obese Zucker rat that do not require a rapamycin-sensitive pathway.

In summary, we present data showing that obese Zucker rats exhibit insulin sensitivity for muscle protein synthesis relative to nonobese littermates. This enhanced insulin action may be a mechanism to offset, albeit inadequately, the muscle atrophy that occurs in the hindlimbs of these animals. We also examined the contribution of the rapamycin-sensitive pathway for this enhanced effect of insulin, because this pathway has been demonstrated to be a key modulator of muscle protein synthesis in vitro. When insulin does not enhance muscle protein synthesis, as in lean animals, an attenuation of muscle synthesis by rapamycin is not overcome by insulin. However, when insulin augments muscle protein synthesis, as in obese animals, this insulin action is still observed in the presence of rapamycin. Therefore, although others have shown that a rapamycin-sensitive pathway is of importance for the regulation of protein metabolism (4, 11, 21, 26), we conclude that the enhanced insulin action on muscle protein synthesis of obese animals does not occur via a rapamycin-sensitive pathway.


    ACKNOWLEDGEMENTS

The authors would like to thank Ed Tapscott, Mike Lang, Susan Price, and Brian Roberts of the Department of Biochemistry at East Carolina University School of Medicine, and Aaron Roland of the Department of Geriatrics at the University of Arkansas for Medical Sciences, for expert technical assistance during the completion of this project.


    FOOTNOTES

Address for reprint requests and other correspondence: J. D. Fluckey, Nutrition, Metabolism and Exercise Laboratory, Department of Geriatrics, University of Arkansas for Medical Sciences, Biomedical Research Center, B120, SLOT 748, 4301 W. Markham, Little Rock, AR 72205 (E-mail: fluckeyjamesd{at}exchange.uams.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 2 June 1999; accepted in final form 16 February 2000.


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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 279(1):E182-E187
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