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Endocrinology Vol. 140, No. 1 106-111
Copyright © 1999 by The Endocrine Society

ARTICLES

Effects of Streptozocin-Induced Diabetes and Islet Cell Transplantation on Insulin Signaling in Rat Skeletal Muscle1

Jeffrey F. Markuns2, Raffaele Napoli3, Michael F. Hirshman, Alberto M. Davalli4, Bentley Cheatham and Laurie J. Goodyear

Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Dr. Laurie J. Goodyear, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215. E-mail: goodyeal{at}joslab.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Streptozocin-induced diabetes is associated with alterations in insulin signaling in rat skeletal muscle, including increased insulin receptor substrate-1 phosphorylation and phosphotidylinositol 3-kinase activity. In the current study, we determined the effects of streptozocin-induced diabetes and treatment of diabetes by islet cell transplantation on several proximal insulin-activated signaling proteins. Three groups of male Lewis rats (untreated streptozocin-diabetic animals, islet cell-transplanted diabetic rats, and nondiabetic control rats) were studied in the basal state or 30 min after ip insulin injection (20 U/rat). Mixed hindlimb skeletal muscle lysates were used to determine the expression and enzymatic activities of the extracellular regulated kinase 2 (ERK2), p90 ribosomal S6 kinase (RSK2), Akt, and p70 S6 kinase (p70S6k). In all three groups of rats, insulin significantly increased ERK2, RSK2, Akt, and p70S6k activities. There was no effect of diabetes on insulin-stimulated ERK2 activity or ERK2 protein levels. RSK2 expression and insulin-stimulated RSK2 activity were significantly elevated in diabetic rats compared with those in the control animals. Insulin-stimulated Akt activity was also significantly greater in the diabetic animals, but there was no change in protein expression. In contrast, there was a decrease in insulin-stimulated p70S6k activity with no change in protein expression in the diabetic rats. Islet transplantation partially (RSK2) or fully (Akt, p70S6k) normalized these diabetes-induced changes in insulin signaling proteins. We conclude that streptozocin diabetes results in the dysregulation of several critical insulin-activated proteins in rat skeletal muscle, but islet cell transplantation is an effective therapy to partially correct these alterations in insulin signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DIABETES is frequently associated with moderate to severe insulin resistance in skeletal muscle (1). The molecular mechanisms for this impaired insulin action are not entirely clear, although studies using animal models of diabetes have revealed significant defects in both the skeletal muscle glucose transport system (2, 3, 4) and the insulin signaling pathways (5, 6, 7, 8). During the past 2 yr we have made the important observation that transplantation of pancreatic islet cells into streptozocin-diabetic rats can normalize the diabetes-induced defects in the skeletal muscle glucose transport system (9, 10). The effects of normalizing hyperglycemia and hypoinsulinemia by islet cell transplantation on insulin signaling in rat skeletal muscle are not known.

It is now clear that the biological actions of insulin in skeletal muscle and other tissues are mediated through the complex stimulation of multiple signaling cascades (reviewed in Ref. 11). One of these insulin-activated signaling pathways involves stimulation of p21ras and the sequential phosphorylation and activation of the Raf-1 kinase, the mitogen-activated protein (MAP) kinase kinase (MEK), MAP kinase or extracellular regulated kinase (ERK), and the p90 ribosomal S6 kinase (RSK2) (reviewed in Refs. 12, 13, 14). Another important insulin signaling cascade involves insulin receptor substrate-1 (IRS-1)-mediated activation of the phosphotidylinositol (PI) 3-kinase. PI 3-kinase activity is necessary for insulin-stimulated glucose transport in skeletal muscle (15, 16, 17, 18), and there is increasing evidence that this mechanism may involve the activation of Akt (19, 20, 21, 22, 23, 24, 25, 26). The p70 S6 kinase (p70S6k) may also be downstream of PI 3-kinase (27, 28), but is thought to function to regulate protein synthesis, not glucose transport (29, 30).

Studies of insulin signaling in skeletal muscle have demonstrated that streptozocin-induced diabetes causes an increase in the expression of insulin receptors and an increase in total receptor phosphorylation, but a reduced efficiency of phosphorylation (5, 6). Streptozocin-induced diabetes is associated with large increases in insulin-stimulated IRS-1 tyrosine phosphorylation (5, 6) and IRS-1-associated PI 3-kinase activity (7) despite slightly decreased levels of total IRS-1 protein in the muscle (6). Although these initial reports demonstrated an up-regulation of these components of the insulin signaling pathway, a recent study suggested that streptozocin-induced diabetes decreases p70S6k activity, accompanied by an increase in the amount of p70S6k protein (8). These researchers also reported decreases in ERK and RSK2 activities with no change in ERK and RSK2 protein levels (8).

In the current investigation, we studied the effects of streptozocin-induced diabetes on ERK and RSK2 signaling, two components of the MAP kinase signaling cascade, and Akt and p70S6k signaling, two proteins downstream of PI 3-kinase, on insulin-stimulated signaling. To determine whether normalization of hyperglycemia and hypoinsulinemia would reverse the effects of diabetes on these insulin signaling proteins, a separate group of diabetic animals was studied after islet cell transplantation. We show that similar to the up-regulation of IRS-1 and PI 3-kinase signaling, streptozocin-induced diabetes causes increased RSK2 activity and protein expression as well as increased Akt activity. In contrast, diabetes causes a decrease in insulin-stimulated p70S6k activities in skeletal muscle. These alterations in insulin signaling were partially or fully normalized by islet cell transplantation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
RSK2, Akt, and p70S6k antibodies as well as RSK substrate peptide and Akt/PKB-specific substrate peptide were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). [125I]Protein A was obtained from ICN (Costa Mesa, CA). [{gamma}-32P]ATP was purchased from DuPont-New England Nuclear (Boston, MA). Protein A-Sepharose beads were obtained from Pharmacia (Piscataway, NJ). Reagents for protein assays and SDS-PAGE were purchased from Bio-Rad (Rockville Center, NY). All chemiluminescence reagents were obtained from Amersham (Arlington Heights, IL). Streptozocin, myelin basic protein, and all other standard chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Animals
The animal protocol used for this study was approved by the Joslin Diabetes Center institutional animal care and use committee. Male Lewis rats, weighing 150–200 g, were received from Taconic Farms, Inc. (Germantown, NY), and randomly divided into three groups: diabetic, transplanted, and controls. Four weeks after arrival, diabetic rats were given an iv injection of streptozocin (65 mg/kg BW) and were studied 6 weeks later. Islet cell-transplanted rats were injected with streptozocin (65 mg/kg BW) 3–4 days after arrival, received islet cell transplants (10) 2–3 weeks after streptozocin injection, and were studied 8 weeks later. Control rats received vehicle (citrate buffer) injection and were studied at 10 weeks. Thus, all rats were studied between 10–11 weeks after arrival to the Joslin Animal Facility. Rats were considered diabetic if fed tail blood glucose concentrations were greater than 300 mg/dl 2 days after streptozocin injection. Blood glucose concentrations were monitored throughout the protocol using blood samples taken from the tail. On the day of the study, all transplanted animals had fasting plasma glucose concentrations similar to the control values.

For the three treatment groups (control, diabetic, and diabetic-transplanted), fasted rats were studied in the basal state or 30 min after ip insulin injection (20 U/rat). Animals were killed by decapitation, and trunk blood was collected for analysis of plasma glucose concentrations using a glucose analyzer and of plasma insulin concentrations by RIA (31). Quadriceps muscles from both legs were quickly dissected, frozen in liquid N2, pulverized, and stored at -80 C.

Skeletal muscle preparation
Approximately 0.75 g skeletal muscle was homogenized in 3 ml buffer containing 20 mM HEPES, 2 mM EGTA, 50 mM ß-glycerol phosphate, 1 mM dithiothreitol (DTT), 1 mM Na3VO4, 1% Triton X-100, 10% glycerol, 10 µM leupeptin, 3 mM benzamidine, 5 µM pepstatin A, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride (PMSF), and 200 µg/ml soybean trypsin inhibitor, pH 7.4. Samples were rotated end over end for 45 min at 4 C, then centrifuged at 15,500 x g for 1 h. Supernatants were retained, carefully avoiding fat, and assayed for protein concentrations using the Bradford method (32).

RSK2 assay
Aliquots of muscle protein (1 mg) were precleared with protein A-Sepharose beads for 1 h at 4 C. The supernatants were collected and then incubated in 3.5 µg/ml anti-RSK2 polyclonal antibody for 1 h at 4 C. Protein A-Sepharose beads were added, and tubes were incubated for 16 h at 4 C. Pellets were washed three times with homogenization buffer (see above), three times with LiCl buffer (500 mM LiCl, 100 mM Tris, 0.1% Triton X-100, and 1 mM DTT, pH 7.6), and three times with RSK assay buffer (30 mM Tris-HCl, 10 mM MgCl2, 0.1 mM EGTA, and 1 mM DTT, pH 7.4). Beads were incubated in a reaction mixture containing 10 µg RSK substrate peptide (RRRLSSLRA), 500 µM cold ATP, and 10 µCi [{gamma}-32P]ATP for 25 min at 25 C. The reactions were stopped with a solution containing 1% BSA, 1 mM ATP, and 0.6% HCl, spotted in duplicate onto P81 phosphocellulose papers, and extensively washed with 1% phosphoric acid. Papers were dried and counted by Cerenkov counting.

ERK2 assay
Aliquots of protein (250 µg) were incubated overnight with 5 µl anti-ERK2 polyclonal antibody at 4 C. Protein A-Sepharose beads were added, and tubes were incubated for 1 h at 4 C. Pellets were washed twice with nonionic detergent buffer (100 mM NaCl, 10 mM Tris, 2 mM DTT, 1 mM EDTA, 1 mM Na3VO4, 40 µg/ml PMSF, 1% Nonidet P-40, and 0.5% deoxycholate, pH 7.2), twice with high salt buffer (1 M NaCl, 10 mM Tris, 2 mM DTT, 1 mM Na3VO4, 40 µg/ml PMSF, and 0.1% Nonidet P-40, pH 7.2), and once with sodium-Tris buffer (150 mM NaCl and 50 mM Tris, pH 7.2). Beads were resuspended in kinase buffer (200 mM HEPES, 100 mM MgCl2, and 1 mg/ml BSA, pH 7.2) and incubated in a reaction mixture containing 15 µg myelin basic protein as substrate, 50 µM ATP, and 20 µCi [{gamma}-32P]ATP for 10 min with continuous vortexing at 30 C. The reactions were stopped with Laemmli’s sample buffer containing 200 mM DTT. Reaction products were separated by SDS-PAGE, and gels were stained with Coomassie blue, destained with 30% CH3OH-20% CH3COOH, dried, and exposed to film. Specific bands were quantitated by densitometry.

Akt assay
Three micrograms of anti-Akt1 were preincubated with protein G beads in buffer A (20 mM HEPES, 2 mM EGTA, 50 mM ß-glycerol phosphate, 1 mM DTT, 1 mM Na3VO4, 1% Triton X-100, 10% glycerol, 10 µM leupeptin, 3 mM benzamidine, 5 µM pepstatin A, 10 µg/ml aprotinin, and 1 mM PMSF, pH 7.4) for 1 h at room temperature. Aliquots of protein (0.5 mg) were then added and incubated for 16 h at 4 C. Pellets were washed three times with wash buffer [20 mM Tris (pH 7.4), 5 mM EDTA, 10 mM Na4PO3, 100 mM NaF, 2 mM Na3VO4, and 1% Nonidet P-40] and twice with kinase buffer [20 mM Tris (pH 7.4), 10 mM MgCl2, and 1 mM DTT]. Beads were incubated in a reaction mixture containing 30 µM Akt/PKB-specific substrate peptide (RPRAATF), 5 mM ATP, 50 mM Tris, 10 mM MgCl2, 1 mM DTT, 1 µM protein kinase inhibitor (pH 7.4), and 3 µCi [{gamma}-32P]ATP for 30 min at 30 C. The samples were spotted in duplicate onto P81 phosphocellulose papers and extensively washed with 75 mM phosphoric acid. Papers were dried, placed in vials with scintillation fluid, and counted with a Beckman Coulter, Inc. scintillation counter (Fullerton, CA).

Immunoblotting
Aliquots of protein (100–300 µg) were prepared with Laemmli’s sample buffer containing 100 mM DTT. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. For ERK kinase immunoblotting, the nitrocellulose membranes were blocked with Tris-buffered saline containing 100 mM Tris, 1.5 M NaCl, and 0.01% sodium azide (TNA); 5% BSA; and 0.05% Nonidet P-40 for 2 h at 37 C. Membranes were incubated overnight at 4 C with anti-MAPK polyclonal antibody (1:200), washed, and incubated in [125I]protein A (1 µCi/ml) for 1 h at room temperature. Membranes were washed, dried, and exposed to a phosphorimaging cassette, and bands were quantitated using a phosphorimager.

For RSK2 immunoblotting, the nitrocellulose membranes were blocked with TNA, 10% nonfat dry milk (nfdm), 0.05% Tween-20 for 2 h at room temperature. Membranes were incubated overnight with anti-RSK2 polyclonal antibody (1:5000) in TNA, 5% nfdm, 0.05% Tween-20 at 4 C. For Akt immunoblotting, nitrocellulose membranes were blocked in TNA, 5% nfdm, and 0.05% Tween-20 for 1 h at room temperature. Membranes were incubated overnight with anti-Akt1 polyclonal antibody (1 µg/ml) in TNA and 3% nfdm at 4 C. For p70S6k immunoblotting, the nitrocellulose membranes were blocked with TNA, 5% BSA, and 0.05% Nonidet P-40 for 2 h at 37 C. Membranes were incubated overnight with anti-p70S6k polyclonal antibody (1 µg/ml) in TNA, 5% BSA, and 0.05% Nonidet P-40 at 4 C. For these immunoblotting experiments, the membranes were washed and incubated for 1 h at room temperature with 1:2000 IgG/horseradish peroxidase in TBS, 2.5% nfdm, and 0.05% Tween-20. Membranes were washed, blotted dry, and placed in a 1:1 solution of chemiluminescence reagents. Membranes were exposed to film, and bands were quantitated by densitometry.

Statistical analysis
All data are expressed as the mean ± SE. Statistical analysis was performed using the SAS general linear model. Differences are considered significant if P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Animals
Streptozocin injection resulted in a 3- to 4-fold elevation in blood glucose concentrations, which was normalized in the animals transplanted with islet cells (Table 1Go, basal data). The streptozocin-treated rats were also characterized by lower plasma insulin concentrations and reduced body weights (Table 1Go). Insulin concentrations and body weights were partially normalized by islet transplantation. To study insulin signaling in skeletal muscle, half of the rats from each group were injected with 20 U insulin and studied 30 min later. This time point was chosen because our preliminary experiments demonstrated that 30 min of insulin stimulation results in a significant increase in the activity of all the enzymes studied. Table 1Go shows that insulin injection caused a significant decrease in plasma glucose concentrations and a significant increase in plasma insulin concentrations in all groups.


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  		Table 1. Effects of insulin on glucose, insulin, and body weight in control, diabetic, and transplanted rats

 
RSK2 activities and protein levels
Basal RSK2 activities were not different among the control, diabetic, and transplanted animals (Fig. 1Go). In the control rats, insulin significantly increased RSK2 activity by approximately 1.6-fold above basal. In the diabetic rats, the insulin-stimulated increase in RSK2 activity was significantly greater, reaching 2.4-fold above basal. Islet transplantation partially corrected the elevated insulin-stimulated RSK2 activity in the diabetic rats.



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  		Figure 1. RSK2 activity in skeletal muscle from control, diabetic, and islet cell-transplanted rats in the basal fasting state (open bars) or after maximal insulin stimulation (solid bars). The data are expressed as counts normalized to a control sample (unstimulated Chinese hamster ovary cell lysates) that was analyzed with each assay. Results are the mean ± SE (n = 5–8/group). #, Significantly different from the respective basal value (P < 0.0001); *, significantly different from the insulin control value (P < 0.002).

 
RSK2 activity was also assessed by mol wt shift of the RSK2 protein. The representative immunoblot in Fig. 2Go shows that insulin caused a greater mobility shift in the muscle protein lysate from the diabetic rats compared with those in the control and transplanted rats. To estimate the degree of RSK2 mobility shift, the two bands were quantitated by densitometry. The degree of mobility shift was calculated by dividing the density (in arbitrary units) of the upper band by the combined densities of the upper and lower bands. As the upper band should represent the phosphorylated protein, and therefore the more active species, this calculation provides an estimate of RSK2 activity. Similar to the activity assay, these data show that insulin significantly (P < 0.01) increased RSK2 activity in the muscle from control (38.4 ± 2.1 vs. 56.5 ± 4.0 arbitrary units), diabetic (43.5 ± 3.6 vs. 68.1 ± 3.2), and transplanted (37.8 ± 1.5 vs. 63.1 ± 3.2) rats. The insulin-stimulated increase in RSK2 activity was significantly greater in the diabetic muscle (56.5 ± 4.0 vs. 68.1 ± 3.2; P < 0.05), and transplantation partially normalized this increase. To determine whether the increase in RSK2 activity in the diabetic rats was associated with an altered expression of the RSK2 protein, we determined the effects of diabetes on RSK2 levels in the same muscle samples. Diabetes significantly increased RSK2 protein (100 ± 9 vs. 182 ± 23 arbitrary units for control and diabetic muscles, respectively; P < 0.002). The increase in RSK2 protein with diabetes was normalized by islet cell transplantation (111 ± 11 arbitrary units vs. diabetic muscles; P < 0.002).



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  		Figure 2. RSK2 protein in skeletal muscle from control, diabetic, and islet cell-transplanted rats in the basal fasting state (B) or after maximal insulin stimulation (I). This representative phosphorimage shows the mobility shift of the RSK2 protein in response to insulin stimulation, with a greater shift in the skeletal muscle from the diabetic rat. Aliquots of muscle proteins (300 µg) were resolved by SDS-PAGE and immunoblotted with {alpha}RSK2.

 
ERK2 activities and protein levels
Basal ERK2 activities were lower in the diabetic animals vs. controls, and this was normalized in transplanted animals (Fig. 3Go). Insulin increased ERK2 activity in all groups. The insulin-stimulated increase in ERK2 activity was not significantly different in either the diabetic rats or the transplanted rats compared with that in the controls. In addition, diabetes had no effect on basal ERK2 protein levels in the skeletal muscle (188 ± 19 vs. 191 ± 30 vs. 177 ± 25 in arbitrary units for control, diabetic, and transplanted animals, respectively).



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  		Figure 3. ERK2 activity in skeletal muscle from control, diabetic, and islet cell-transplanted rats in the basal fasting state (open bars) or after maximal insulin stimulation (solid bars). The data are expressed as a percentage of the control value in the basal state. Results are the mean ± SE (n = 5–8/group). #, Significantly different from the respective basal value (P < 0.03).

 
Akt activities
Insulin resulted in a significant increase in Akt activity in all three groups of rats (Fig. 4Go). The insulin-stimulated increase in Akt activity was elevated in the diabetic rats compared with that in the control rats. This diabetes-induced increase in Akt activity was fully normalized by islet cell transplantation. Diabetes had no effect on basal Akt protein levels in skeletal muscle (92 ± 12 vs. 82 ± 10 vs. 83 ± 11 in arbitrary units for control, diabetic, and transplanted animals, respectively).



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  		Figure 4. Akt activity in skeletal muscle from control, diabetic, and islet cell-transplanted rats in the basal fasting state (open bars) or after maximal insulin stimulation (solid bars). The data are expressed as counts normalized to a control sample that was analyzed with each assay. Results are the mean ± SE (n = 4–8/group). #, Significantly different from the respective basal value (P < 0.001); *, significantly different from the respective control value (P < 0.03); +, significantly different from the diabetic insulin value (P < 0.002).

 
p70S6k activities and protein expression
The activity of p70S6k was assessed by mol wt shift of the protein. The immunoblot and graph in Fig. 5Go shows that insulin significantly increased p70S6k activity in control, diabetic, and diabetic-transplanted rats. The data, quantitated as described for RSK2 mobility shift, showed that the insulin-stimulated p70S6k activity was significantly lower in the diabetic rats compared with that in the controls. Islet cell transplantation normalized this change in p70S6k mobility shift. To determine whether the decrease in p70S6k activity was due to a decrease in the level of p70S6k expression in the muscle, the combined densities of the upper and lower bands were quantitated by densitometry. The p70S6k protein content among the three groups was not significantly different (26 ± 5 vs. 26 ± 3 vs. 24 ± 2 arbitrary units for control, diabetic, and diabetic-transplanted animals, respectively). Thus, the diabetes-induced decrease in p70S6k activity is not associated with a decrease in the amount of p70S6k protein in the skeletal muscle.



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  		Figure 5. Upper panel, p70S6k protein in skeletal muscle from control, diabetic, and islet cell-transplanted rats in the basal fasting state (B) or after maximal insulin stimulation (I). This representative phosphorimage shows the mobility shift of the p70S6k protein in response to insulin stimulation, with a lesser shift in the skeletal muscle from the diabetic rat. Aliquots of muscle proteins (300 µg) were resolved by SDS-PAGE and immunoblotted with {alpha}p70S6k. Lower panel, p70S6k activity in skeletal muscle from control, diabetic, and islet cell-transplanted rats in the basal fasting state (open bars) or after maximal insulin stimulation (solid bars). The data are calculated by dividing the density (in arbitrary units) of the upper band by the combined densities of the upper and lower bands, expressed as a percentage of phosphorylated protein and normalized to a reference standard used for comparisons among blots. Results are the mean ± SE (n = 5–8/group). #, Significantly different from the respective basal value (P < 0.0001); *, significantly different from the insulin control value (P <= 0.05); +, significantly different from the respective diabetic value (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of islet cell transplantation as a treatment for diabetes may become more common in the future, increasing the need for understanding the basic physiological consequences of this procedure (33). Recently, we have shown that islet cell transplantation in streptozocin-induced diabetic rats normalizes defects in glucose transport and GLUT4 (glucose transporter-4) protein content in skeletal muscle (9, 10). In these studies, islet transplantation corrected the impairment in glucose transporter translocation (9) and the reduction in GLUT4 protein content (10). In the current study, we determined whether there were diabetes-induced changes in distal components of the insulin signaling pathways in skeletal muscle, and then investigated the use of islet cell transplantation to normalize any putative alterations in these insulin signaling molecules. Our results demonstrate that there is a dysregulation of RSK2, Akt, and p70S6k signaling in animals made diabetic by injection of streptozocin, and that islet cell transplantation can partially or fully correct these alterations in insulin signaling.

To study the effects of diabetes and islet cell transplantation on MAP kinase signaling, we measured RSK2 and ERK2 activities and protein expression in the skeletal muscle. Interestingly, we found that insulin-stimulated RSK2 activity and muscle content of RSK2 protein were significantly elevated in streptozocin-diabetes, but that there was no effect of diabetes on ERK2 activity and expression. Thus, although RSK2 is a direct substrate for ERK in this MAP kinase signaling cascade (34), there is differential regulation of these proteins in response to diabetes. Phosphorylation and activation of RSK2 by an additional protein kinase could be one mechanism for the up-regulation of insulin-stimulated RSK2 with diabetes (35, 36). Alternatively, the diabetes-induced increase in RSK2 activity may entirely be a consequence of increased RSK2 expression, suggesting that the primary effect of diabetes is altered transcriptional or translational regulation of RSK2.

Streptozocin-induced diabetic rats are insulin deficient and hyperglycemic and have a reduction in glucose uptake in skeletal muscle (37, 38). Despite the decrease in skeletal muscle glucose uptake with this model of diabetes, these animals display a marked up-regulation in the phosphorylation or activity of molecules that have been implicated in the regulation of insulin-stimulated glucose uptake, including the insulin receptor, IRS-1, and PI 3-kinase (5, 6, 7). Akt is a serine/threonine kinase that can be activated via PI 3-kinase and 3-phosphoinositide dependent kinase (PDK1) (22), and has also been proposed to mediate the stimulation of glucose uptake and GLUT4 translocation in response to insulin (24, 39, 40, 41, 42, 43). Similar to the up-regulation of proximal insulin signaling molecules, in the current study we found that insulin-stimulated Akt activity was increased with streptozocin-diabetes in the rat skeletal muscle. Thus, streptozocin-induced diabetes results in an up-regulation of this insulin signaling cascade from the level of the insulin receptor to Akt. One interpretation of these findings is that there is an uncoupling of all of these signaling molecules with the activation of glucose uptake in skeletal muscle. Alternatively, the increase in these insulin signaling proteins during streptozocin-diabetes may be an attempt by the muscle to compensate for the insulin-resistant state, and the critical defect may lie elsewhere.

The p70S6k is another serine/threonine kinase that is activated by insulin, presumably through PI 3-kinase and PDK1 (22, 44, 45). The p70S6k is not thought to control the glucose transport process, but instead may play an important role in regulating protein synthesis by controlling the translation of numerous messenger RNA transcripts that encode components of the translational apparatus (29, 30). The different physiological consequence of p70S6k activation may explain the opposite effects of streptozocin-diabetes on this signaling molecule. In contrast to the up-regulation of PI 3-kinase and Akt in diabetic skeletal muscle, we found a decrease in p70S6k activity, as assessed by mobility shift. Our results are consistent with a previous investigation that also demonstrated a decrease in p70S6k activity in skeletal muscle from streptozocin-diabetic rats (8). These findings suggest that there is divergence of the insulin signal, perhaps at the level of PDK. Future work is necessary to fully understand the functional consequences of the down-regulation of the p70S6k protein and the up-regulation of the PI 3-kinase and Akt molecules.

Elucidating the relative importance of hyperglycemia vs. hypoinsulinemia will be an important step in determining the cause of defects in insulin signaling proteins. One study has addressed this issue with regard to dysregulation of IRS-1 signaling, suggesting that hypoinsulinemia, and not hyperglycemia, is responsible for the up-regulation of IRS-1 phosphorylation with streptozocin-diabetes (5). These investigators found that normalization of insulin concentrations by daily insulin injections, but not treatment with phlorizin to only lower glucose concentrations, was effective in attenuating the increase in tyrosine phosphorylation of IRS-1 (5). Further support for this hypothesis comes from studies in animal models of type 2 diabetes, in which there is no insulin deficiency. In contrast to the model of streptozocin-induced diabetes, the ob/ob mouse (6, 7) and the Goto-Kakizaki rat (39) are characterized by decreases in IRS-1 tyrosine phosphorylation (6), PI 3-kinase activity (7), and Akt activity (39). All of these studies taken together suggest that the up-regulation of IRS/PI 3-kinase/Akt signaling in streptozocin-diabetic rats is a consequence of insulin deficiency.

In summary, diabetes causes dysregulation of the RSK2, Akt, and p70S6k insulin signaling proteins. Similar to previous studies showing diabetes-induced up-regulation of IRS-1 and PI 3-kinase signaling, RSK2 protein and activity as well as Akt activity were increased. In contrast, diabetes was associated with a significant decrease in p70S6k activity, with no change in the expression of the p70S6k protein. Islet cell transplantation and subsequent normalization of both hyperglycemia and hypoinsulinemia resulted in partial or full correction of these defects in insulin signaling molecules.


   Footnotes
 
1 This work was supported by NIH-NIAMS Grant AR-42238 (to L.J.G.). Back

2 Supported by a predoctoral position under Grant T32-DR07260–21 from the NIDDK, NIH. Back

3 Current address: Federico II University School of Medicine, Via Pansini, 5–80131 Naples, Italy. Back

4 Current address: Instituto Scientifico San Raffaele, Via Olgettina 60, Milan, Italy. Back

Received July 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. DeFronzo R 1988 The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 37:667–687[Medline]
  2. Napoli R, Hirshman MF, Horton ES 1995 Mechanisms and time course of impaired skeletal muscle glucose transport activity in streptozocin diabetic rats. J Clin Invest 96:1–11
  3. Barnard RJ, Youngren JF, Kartel DS, Martin DA 1990 Effects of streptozotocin-induced diabetes on glucose transport in skeletal muscle. Endocrinology 126:1921–1926[Abstract]
  4. Ramlal T, Tastogi S, Vranic M, Klip A, Rastogi S 1989 Decrease in glucose transporter number in skeletal muscle of mildly diabetic (streptozotocin-treated) rats. Endocrinology 125:890–897[Abstract]
  5. Giorgino F, Chen J, Smith RJ, Chen JH 1992 Changes in tyrosine phosphorylation of insulin receptors and a 170,000 molecular weight nonreceptor protein in vivo in skeletal muscle of streptozotocin-induced diabetic rats: effects of insulin and glucose. Endocrinology 130:1433–1444[Abstract]
  6. Saad MJA, Eraki E, Miralpeix M, White MF, Kahn CR 1992 Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance. J Clin Invest 90:1839–1849
  7. Folli F, Saad MJA, Backer JM, Kahn CR 1993 Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus. J Clin Invest 92:1787–1794
  8. Hei YJ, Chen X, Pelech SL, Diamond J, McNeill JH 1995 Skeletal muscle mitogen-activated protein kinases and ribsomal S6 kinases. Diabetes 44:1147–1155[Abstract]
  9. Napoli R, Davalli AM, Hirshman MF, Weitgasser R, Weir GC, Horton ES 1996 Islet transplantation under the kidney capsule fully corrects the impaired skeletal muscle glucose transport system of streptozotocin diabetic rats. J Clin Invest 97:1389–1397[Medline]
  10. Davalli AM, Napoli R, Weitgasser R, Capotorto JV, Hirshman MF, Finegood DT, Bonner-Weir S, Horton ES, Weir GC 1996 Long-term normalization of GLUT-4 protein content in skeletal muscle of diabetic rats following islet transplantation. J Endocrinol 150:255–263[Abstract/Free Full Text]
  11. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142[CrossRef][Medline]
  12. Blenis J 1993 Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci USA 90:5889–5892[Abstract/Free Full Text]
  13. Cano E, Mahadevan LC 1995 Parallel signal processing among mammalian MAPKs. Trends Biochem Sci 20:117–122[CrossRef][Medline]
  14. Marshall CJ 1995 Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179–185[CrossRef][Medline]
  15. Lee AD, Hansen PA, Holloszy JO 1995 Wortmannin inhibits insulin-stimulated but not contraction-stimulated glucose transport activity in skeletal muscle. FEBS Lett 361:51–54[CrossRef][Medline]
  16. Yeh JI, Gulve EA, Rameh L, Birnbaum MJ 1995 The effects of wortmannin on rat skeletal muscle. Dissociation of signaling pathways for insulin- and contraction-activated hexose transport. J Biol Chem 270:2107–2111[Abstract/Free Full Text]
  17. Wojtaszewski JFP, Hansen BF, Urso B, Richter EA 1996 Wortmannin inhibits both insulin- and contraction-stimulated glucose uptake and transport in rat skeletal muscle. J Appl Phyisol 81:1501–1509
  18. Lund S, Holman GD, Schmitz O, Pedersen O 1995 Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin. Proc Natl Acad Sci USA 92:5817–5821[Abstract/Free Full Text]
  19. Shepherd PR, Nave BT, Rincon J, Haigh RJ, Foulstone E, Proud C, Zierath JR, Siddle K, Wallberg-Henriksson H 1997 Involvement of phosphoinositide 3-kinase in insulin stimulation of MAP-kinase and phosphorylation of protein kinase-B in human skeletal muscle: implications for glucose metabolism. Diabetologia 40:1172–1177[CrossRef][Medline]
  20. Burgering BMT, Coffer PJ 1995 Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376:599–602[CrossRef][Medline]
  21. Franke TF, Yang S, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN 1995 The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727–736[CrossRef][Medline]
  22. Cohen P, Alessi DR, Cross DA 1997 PDK1, one of the missing links in insulin signal transduction? FEBS Lett 410:3–10[CrossRef][Medline]
  23. Cross DAE, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA 1995 Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789[CrossRef][Medline]
  24. Kohn AD, Summers SA, Birnbaum MJ, Roth RA 1996 Expression of a constitutively active Akt Ser/Thr kinase in 3T3–L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271:31372–31378[Abstract/Free Full Text]
  25. Stephens L, Anderson K, Stokoe D, Erdjument-Bromage H, Painter GF, Holmes AB, Gaffney PR, Reese CB, McCormick F, Tempst P, Coadwell J, Hawkins PT 1998 Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science 279:710–714[Abstract/Free Full Text]
  26. Andjelkovic M, Alessi DR, Meier R, Fernandez A, Lamb NJ, Frech M, Cron P, Cohen P, Lucocq JM, Hemmings BA 1997 Role of translocation in the activation and function of protein kinase B. J Biol Chem 272:31515–31524[Abstract/Free Full Text]
  27. Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR 1994 Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 14:4902–4911[Abstract/Free Full Text]
  28. Chung J, Grammer TC, Lemon KP, Kazlauskas A, Blenis J 1994 PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature 370:71–75[CrossRef][Medline]
  29. Pause A, Belsham GJ, Gingras A-C, Donze O, Lin T-A, Lawrence Jr JC, Sonenberg N 1994 Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature 371:762–767[CrossRef][Medline]
  30. Lin T-A, Kong X, Haystead TAJ, Pause A, Belsham G, Sonenberg N, Lawrence JCJ 1994 PHAS-1 as a link between mitogen-activated protein kinase and translation initiation. Science 266:653–656[Abstract/Free Full Text]
  31. Soeldner JS, Sloane D 1965 Critical variables in the radioimmunoassay of serum insulin using the double antibody technique. Diabetes 14:771–779[Medline]
  32. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein, utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  33. Weir GC, Bonner-Weir S 1997 Scientific and political impediments to successful islet transplantation. Diabetes 46:1247–1256[Abstract]
  34. Sturgill TW, Ray LB, Erikson E, Maller JL 1988 Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature 334:715–718[CrossRef][Medline]
  35. Chen RH, Sarnecki C, Blenis J 1992 Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol 12:915–927[Abstract/Free Full Text]
  36. Chen RH, Chung J, Blenis J 1991 Regulation of pp90rsk phosphorylation and S6 phosphotransferase activity in Swiss 3T3 cells by growth factor-, phorbol ester-, and cyclic AMP-mediated signal transduction. Mol Cell Biol 11:1861–1867[Abstract/Free Full Text]
  37. Wallberg-Henriksson H, Holloszy JO 1985 Activation of glucose transport in diabetic muscle: responses to contraction and insulin. Am J Physiol 249:C233–C237
  38. Goodyear LJ, Hirshman MF, Knutson S, Horton ED, Horton ES 1988 Effect of exercise training on glucose homeostasis in normal and insulin-deficient diabetic rats. J Appl Physiol 65:844–851[Abstract/Free Full Text]
  39. Krook A, Kawano Y, Song XM, Efendic S, Roth RA, Wallberg-Henriksson H, Zierath JR 1997 Improved glucose tolerance restores insulin-stimulated Akt kinase activity and glucose transport in skeletal muscle from diabetic Goto-Kakizaki rats. Diabetes 46:2110–2114[Abstract]
  40. Summers SA, Birnbaum MJ 1997 A role for the serine/threonine kinase, Akt, in insulin-stimulated glucose uptake. Biochem Soc Trans 25:981–988[Medline]
  41. Cong LN, Chen H, Li Y, Zhou L, McGibbon MA, Taylor SI, Quon MJ 1997 Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11:1881–1890[Abstract/Free Full Text]
  42. Ueki K, Yamamoto-Honda R, Kaburagi Y, Yamauchi T, Tobe K, Burgering BMT, Coffer PJ, Komuro I, Akanuma Y, Yazaki Y, Kadowaki T 1998 Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J Biol Chem 273:5315–5322[Abstract/Free Full Text]
  43. Calera MR, Martinez C, Liu H, Jack AKE, Birnbaum MJ, Pilch PF 1998 Insulin increases the association of akt-2 with Glut4-containing vesicles. J Biol Chem 273:7201–7204[Abstract/Free Full Text]
  44. Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Hemmings BA, Thomas G 1998 Phosphorylation and activation of p70s6k by PDK1. Science 279:707–710[Abstract/Free Full Text]
  45. Alessi DR, Kozlowski MT, Weng QP, Morrice N, Avruch J 1998 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol 8:69–81[CrossRef][Medline]




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