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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-1007
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5551-5558
Copyright © 2005 by The Endocrine Society

Effects of the Rapid-Acting Insulin Analog Glulisine on Cultured Human Skeletal Muscle Cells: Comparisons with Insulin and Insulin-Like Growth Factor I

Theodore P. Ciaraldi, Susan A. Phillips, Leslie Carter, Vanita Aroda, Sunder Mudaliar and Robert R. Henry

Veterans Affairs San Diego HealthCare System (T.P.C., L.C., V.A., S.M., R.R.H.) and Departments of Medicine (T.P.C., L.C., V.A., S.M., R.R.H.) and Pediatrics (S.A.P.), University of California-San Diego, La Jolla, California 92093

Address all correspondence and requests for reprints to: Dr. Theodore P. Ciaraldi, Department of Medicine (9111G), University of California-San Diego, La Jolla, California 92093. E-mail: tciaraldi{at}ucsd.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The insulin analog LysB3,GluB29-insulin (glulisine) displays accelerated in vivo bioavailability compared with native insulin.

Objective: Biological properties of this rapid-acting insulin analog were compared with the actions of native insulin and IGF-I.

Design: The effects of the hormones on hormone binding, glucose uptake, and thymidine uptake were evaluated in cultured human skeletal muscle cells.

Setting: This study was performed at a Veterans Administration hospital for patient characterization and tissue biopsies; in vitro studies were performed in a research laboratory.

Patients or Other Participants: Skeletal muscle tissue was obtained from nondiabetic (n = 13) and type 2 diabetic (n = 14) subjects.

Intervention: Cultured skeletal muscle cells were treated acutely (15–90 min) or chronically (16 h) with varying concentrations of hormones.

Main Outcome: The main study outcomes were measures of sensitivity (concentration required to attain 50% displacement of specific [125I]insulin or [125I]IGF-I bound and sensitivity (EC50) and potency (maximal response) for hormone binding and biological responses.

Results: Insulin and glulisine were comparable in their ability to displace insulin binding. Neither insulin nor glulisine competed efficiently for IGF-I binding. Insulin, glulisine, and IGF-I were equipotent in the stimulation of glucose uptake. Maximal stimulation of phosphorylation of Akt was greatest for IGF-I, whereas sensitivities were similar to those for glucose uptake. Sensitivities were comparable in muscle cells from nondiabetic and type 2 diabetic subjects. Stimulation of [3H]thymidine uptake was most responsive to IGF-I; insulin and glulisine were equally less effective, with sensitivities approximately 1–2% of that for IGF-I. Stimulation of p42/44 MAPK phosphorylation reflected the behavior of thymidine uptake.

Conclusions: Although altered pharmacokinetics of glulisine can have therapeutic advantages, glulisine is indistinguishable from native insulin at the skeletal muscle level.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
STRUCTURALLY MODIFIED INSULINS have proven to be useful tools in increasing the understanding of mechanisms of insulin signaling and in the development of more effective therapeutics for the treatment of diabetes. Insulin analogs have permitted mapping of the sites on the insulin molecule involved in hexamer association and dissociation, interaction with the insulin receptor, and cross talk with the IGF-I receptor (1). Important information revealed by such studies includes the facts that the C terminus of the B chain is not crucial for binding to the insulin receptor (2), but does play a role both in recognition by the IGF-I receptor (3) and self-association (1, 2). Two insulin analogs modified in this region, LysB28,ProB29-insulin (lispro) and AspB28-insulin (aspart), display reduced self-association, leading to faster dissociation of hexamers and more rapid bioavailability (4, 5, 6), yet with properties comparable to those of native insulin with regard to receptor recognition (3, 7) and biological responses (7, 8, 9). This feature has been exploited in the use of lispro and aspart as rapid-acting insulins, as part of the approach to mimic normal postprandial patterns of insulin secretion (5, 6, 10). Conversely, the addition of positively charged residues to the C terminus, such as in GlyA21,ArgB31,ArgB32-insulin (glargine), results in a slowly dissociating insulin, with prolonged pharmacokinetics (11, 12).

Insulin is a pleiotropic hormone, regulating metabolism as well as aspects of growth and development through activation of divergent signaling pathways (13). Selection between metabolic and mitogenic responses to insulin is influenced by several factors. Among these are cross talk with the strongly mitogenic IGF-I receptor (14) and residency time on the insulin receptor (15, 16), the later leading to prolonged activation of the receptor tyrosine kinase activity and sustained phosphorylation of specific substrates (15, 17). Each of these properties can be influenced by modification of the C terminus of the B chain (3, 15), and there are a number of insulin analogs that display disproportionately greater mitogenic than metabolic activities compared with native insulin (7, 18, 19, 20). A new rapid-acting insulin analog, LysB3,GluB29-insulin (glulisine), has been shown, at high concentrations (500 nM), to be similar to or somewhat more effective than human insulin in stimulating tyrosine phosphorylation of insulin receptor substrate-2 (IRS-2) (21) and to have a greater antiapoptotic action in a ß-cell line (22). The current investigation was designed to investigate the potentially selective potency of glulisine for metabolic and mitogenic responses in a model of the major insulin target tissue, cultured human skeletal muscle cells, compared with insulin and IGF-I as primary stimulators of metabolism and mitogenesis, respectively. Muscle cells from both nondiabetic and type 2 diabetic subjects were studied to evaluate effects in normal vs. insulin-resistant muscle.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Experimental subjects

Thirteen healthy nondiabetic subjects and 14 patients with type 2 diabetes participated in these studies. Glucose tolerance was determined from a 75-g oral glucose tolerance test. Insulin action was determined by a 3-h hyperinsulinemic (300 mU/m2·min euglycemic (5.0–5.5 mM) clamp (23). Patients with type 2 diabetes had their medication withheld on the morning of the biopsy. Subject characteristics are summarized in Table 1Go. Although the diabetic group was significantly more obese than the nondiabetic group, this difference did not influence the activities measured in vitro, because there was no relationship between body mass index and rates of glucose uptake measured in cultured muscle cells (not shown). Impairments of the maximally insulin-stimulated glucose disposal rate confirmed the insulin resistance of the diabetic group (Table 1Go). The experimental protocol was approved by the Committee on Human Investigation of University of California-San Diego. Informed written consent was obtained from all subjects after explanation of the protocol.


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  		TABLE 1. Subject characteristics

 
Materials

Human biosynthetic insulin and IGF-I were obtained from Calbiochem (La Jolla, CA). Glulisine (LysB3,GluB29-insulin) was provided by Aventis Pharma (Frankfurt au Main, Germany). Cell culture materials were purchased from Irvine Scientific (Irvine, CA), except for skeletal muscle basal medium, which was obtained from BD Clonetics Corp. (San Diego, CA). Fetal bovine serum was purchased from Gemini (Calabasas, CA). Roche (Indianapolis, IN) supplied the BSA (Cohn fraction V). [125I]Insulin and [125I]IGF-I, 2-[1,2-3H]deoxy-D-glucose, L-[1-14C]glucose, and [3H]thymidine were purchased from NEN Life Science Products (Boston, MA). Polyclonal antibodies against pSer473-Akt and phospho-MAPK were purchased from Cell Signaling Technology (Beverly, MA). Polyclonal antibodies against Akt1/2 and MAPK were purchased from Cell Signaling Technology and Upstate Biotechnology, Inc. (Charlottesville, VA), respectively. The antirabbit Ig conjugated to horseradish peroxidase was purchased from Amersham Biosciences (Arlington Heights, IL), and the SuperSignal chemiluminescence substrate was purchased from Pierce Chemical Co. (Rockford, IL).

Cell culture

Biopsy of the vastus lateralis muscle was performed according to previously described procedures (24). Human skeletal muscle cells were isolated and grown in culture as described in detail previously (25). When myoblasts attained 80–90% confluence, the growth medium was changed to {alpha}-MEM supplemented with 2% fetal bovine serum and antibiotics to induce fusion to multinucleated myotubes. The medium was changed every other day during cell fusion. All studies were performed on cells after one passage. Due to the limited number of cells that could be propagated from a biopsy, not all measurements were performed in each subject.

Insulin and IGF-I binding assays

Hormone binding assays were performed by the modification of a method described previously (25). Fully differentiated human skeletal muscle cells were washed four times with reaction buffer, then incubated with reaction buffer and [125I-Tyr-A14]insulin (final concentration, 67 pM) or [125I-Tyr-A14]IGF-I (final concentration, 39 pM) for 4 h at 12 C in the absence or presence of varying concentrations of unlabeled hormone. Results were calculated based on the displacement of specific insulin/IGF-I binding normalized to protein concentration.

Glucose uptake assay

Glucose uptake measurements were made using a previously described procedure (26). Cells were incubated with hormones for 75–90 min at 37 C in a 5% CO2 incubator before washing and the uptake assay. An aliquot of the suspension was removed for protein analysis using the Bradford method (27). The uptake of L-glucose was used to correct each sample for the contribution of diffusion. Dose-response curves were calculated as a function of the basal (no insulin added) sample in each set of cells.

Thymidine incorporation into DNA

A modification of the method described by Berhanu et al. was employed (28). Confluent cells were treated in serum-free (0.1% BSA) {alpha}-MEM with varying concentrations of hormones for 16 h in a 37 C, 5% CO2 incubator. The medium was replaced with {alpha}-MEM/0.1% BSA, pH 7.4, together with ligands, and [3H]thymidine (0.5 µCi) was added to each well. The cells were incubated for 1 h at 37 C, washed, and then solubilized. Aliquots were removed for scintillation counting and protein determination by the Bradford method. Results are presented as a percentage of the total thymidine added incorporated into DNA for each individual set of cells and normalized to cell protein.

Intracellular signal transduction

Cells were washed free of medium and then treated with varying concentrations of ligands in serum-free medium for 15 min at 37 C. Reactions were terminated by washing with 4 C PBS, and cells were lysed in a buffer containing 20 mM Tris-HCl, 145 mM sodium chloride (NaCl), 10% glycerol, 5 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 200 µM sodium orthovanadate, 200 µM phenylmethylsulfonylfluoride, 1 µM leupeptin, 1 µM pepstatin, and 10 µg/ml aprotinin, pH 7.5. After assay for protein content, extracts were stored at –70 C before additional analysis. Proteins were separated by SDS-PAGE on 10% gels. Western blot analysis was performed by the method reported by Burnette (29), as described previously (25). Proteins were visualized with the SuperSignal Western Blot Detection Kit (Pierce Chemical Co.) and exposed to autoradiograph film. The intensities of the phosphorylated bands were quantified by scanning laser densitometry using ScanAnalysis software (BioSoft, Cambridge, UK). Membranes were stripped and reprobed with antibodies against total specific protein (Akt or MAPK).

Statistical analysis

Statistical analysis was preformed using the StatView SE+ program (Abacus Concepts, Inc., Berkeley, CA). All data are expressed as the mean ± SEM. For all variables, statistical significance was tested with two-tailed Student’s t test, using paired analysis when appropriate. Significance was accepted at P < 0.05. Results, presented as a percentage or fold change, were obtained from paired comparisons with the appropriate control for each set of cells from that single individual. Ligand sensitivities (EC50 values) were determined from log-logit transformations of individual dose-response curves.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Hormone binding

Insulin and IGF-I binding were assayed under conditions where primarily association with the cell surface receptors was measured. Because we have previously shown that the quantity and affinities of insulin and IGF-I receptors are similar in myotubes from nondiabetic and type 2 diabetic subjects (30), the current studies were performed in nondiabetic cells.

Glulisine displaced specifically bound insulin as efficiently as insulin (Fig. 1AGo). There were no differences in the affinities of insulin and glulisine for the insulin receptor (Table 2Go). IGF-I was recognized by the insulin receptor, but only at high hormone concentrations and with 1–2% the affinity of insulin or glulisine (Fig. 1AGo and Table 2Go).



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  		FIG. 1. Comparison of affinities of human insulin, glulisine, and IGF-I for insulin and IGF-I receptors on human skeletal muscle cells. Binding was measured for 4 h at 12 C with [125I]insulin (67 pM) or [125I]IGF-I (39 pM) in the presence of increasing concentrations of unlabeled peptides. The results are presented as the displacement of initial specific hormone binding, normalized to protein. A, Insulin binding to cells from nondiabetic subjects (n = 7–8). B, IGF-I binding to cells from nondiabetic subjects (n = 3). Results are the average ± SEM. Each subject was studied in triplicate.

 

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  		TABLE 2. Affinities of human insulin, glulisine, and IGF-I for insulin and IGF-I receptors on human skeletal muscle cells from nondiabetic subjects

 
IGF-I bound to its receptor with high affinity (Fig. 1BGo and Table 2Go). Insulin and glulisine were equally poor at competing for IGF-I binding (Fig. 1BGo); neither hormone displaced more than 10–15% of the specifically bound IGF-I.

Metabolic actions: glucose uptake

Stimulation of glucose uptake in cultured myotubes was evaluated as an acute metabolic response to the hormones. Glucose uptake was impaired in diabetic myotubes in both the absence (19.8 ± 2.7 vs. 12.2 ± 2.1 pmol/mg protein·min, nondiabetic vs. diabetic, P < 0.05) and presence of the hormones. Nondiabetic cells responded similarly to all three hormones (Fig. 2Go); there were no significant differences in the magnitude of the maximal stimulation due to insulin and glulisine while the response to IGF-I was slightly greater (Fig. 2Go). Insulin and glulisine were also equally potent for stimulation of glucose uptake in myotubes from diabetic subjects (Fig. 2Go). In diabetic cells, the relative response to IGF-I was increased compared with those to insulin and glulisine (Fig. 2Go; P < 0.025, by paired analysis).



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  		FIG. 2. Concentration dependency of human insulin, glulisine, and IGF-I stimulation of deoxyglucose uptake in human skeletal muscle cells. Results are presented as dose-response curves calculated as a function of the basal (no added insulin) activity in each individual set of cells. A, Muscle cells from nondiabetic subjects (n = 10–11). B, Type 2 diabetic muscle cells (n = 9–10). Results are the average ± SEM.

 
Full dose-response curves permitted evaluation of responses at more physiological hormone levels. Muscle cells were most sensitive to IGF-I (Fig. 2Go) and were equally sensitive to insulin and glulisine. Hormone sensitivities for this response were similar in nondiabetic and diabetic myotubes (Table 3Go).


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  		TABLE 3. Sensitivities of human insulin, glulisine, and IGF-I for glucose uptake and [3H]thymidine uptake in human skeletal muscle cells

 
Mitogenic actions: [3H]thymidine uptake

Thymidine uptake, as measured in fused, differentiated myotubes, is a response to chronic hormone exposure that involves mechanisms distinct from acute metabolic effects. Unlike glucose uptake, basal [3H]thymidine uptake was similar in nondiabetic and diabetic muscle cells (not shown). Although both insulin and glulisine were able to stimulate thymidine uptake, significant effects were apparent only at higher hormone levels (Fig. 3Go). The dose-response curves for insulin and the analog were superimposable (Fig. 3Go). The maximal responses to insulin and glulisine were approximately half that of the response to IGF-I (P < 0.01).



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  		FIG. 3. Concentration dependency of human insulin, glulisine, and IGF-I stimulation of [3H]thymidine uptake in human skeletal muscle cells. Results are presented as dose-response curves calculated as a function of the basal (no added insulin) activity in each individual set of cells. A, Muscle cells from nondiabetic subjects (n = 9–12). B, Type 2 diabetic muscle cells (n = 8–10). Results are the average ± SEM.

 
Sensitivities of insulin and glulisine for stimulation of thymidine uptake were similar and only about 10% of that for IGF-I (Table 3Go). This behavior was the same in nondiabetic and diabetic myotubes.

Intracellular signaling

Two different steps in receptor kinase signaling cascades were analyzed to compare the actions of insulin, glulisine, and IGF. Although not producing a complete picture of insulin/IGF-I signaling, these two steps were selected as key, regulated events in different pathways. Serine phosphorylation of the serine kinase Akt/protein kinase B has been shown to play a role in mediating acute metabolic responses to insulin (reviewed in Ref.31). Activation of the ras-MAPK pathway is involved in insulin and IGF-I regulation of gene expression, growth, and mitogenesis (32, 33). Although greatly differing treatment times were employed in the current studies for the stimulation of glucose uptake (75–90 min) and thymidine uptake (16 h), hormone stimulation of the phosphorylation of key signaling intermediates was transient. Preliminary studies revealed that 15 min of cell exposure to hormones attained maximal stimulation of phosphorylation of both Akt and MAPK, so this time was employed to treat cells before extraction.

Insulin, glulisine, and IGF-I all stimulated serine phosphorylation of Akt in a dose-dependent manner (Fig. 4Go). The maximal responses to insulin and glulisine were identical, as were the dose-response curves. Both the sensitivities (EC50, ~0.5 nM) and maximal responses to insulin and glulisine were similar in nondiabetic and diabetic muscle cells (Fig. 4Go). As was the case with glucose uptake stimulation, IGF-I generated a greater maximal response for AKT phosphorylation and did so with a higher sensitivity (EC50, ~0.1 nM).



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  		FIG. 4. Effects of human insulin, glulisine, and IGF-I on Akt serine phosphorylation in human skeletal muscle cells. Cells were treated with the indicated concentrations of ligands for 15 min at 37 C before cell extraction. Proteins were detected by Western blotting with a phosphospecific antibody. A, Representative autoradiogram. B, Dose responses in cells from nondiabetic subjects (n = 4–6). C, Dose responses in cells from type 2 diabetic subjects (n = 4–6). Results are the average ± SEM.

 
Appreciable phosphorylation of MAPK was observed in the absence of added hormones, most likely due to the lack of serum starvation before hormone treatment. The greatest response was to IGF-I, double that seen with insulin and glulisine (Fig. 5Go), similar to the differences for stimulation of [3H]thymidine uptake. The sensitivity series for stimulation of MAPK phosphorylation was IGF-I>>insulin=glulisine. Once again, insulin and glulisine were indistinguishable for both sensitivity and potency.



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  		FIG. 5. Effects of human insulin, glulisine, and IGF-I on MAPK phosphorylation in human skeletal muscle cells. Cells were treated with the indicated concentrations of ligands for 15 min at 37 C before cell extraction. Proteins were detected by Western blotting with a phosphospecific antibody. A, Representative autoradiogram. Values from the 42- and 44-kDa bands were combined for the purpose of quantitation. Dose responses in cells from nondiabetic subjects (n = 4–6). C, Dose responses in cells from type 2 diabetic subjects (n = 4–6). Results are the average ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the search for ways to duplicate the kinetic action profile of insulin in the circulation, insulin analogs have proven very useful (10). For example, GlyA21,ArgB31,ArgB32-insulin (glargine) is poorly soluble at physiological pH, so upon sc injection, glargine precipitates in adipose tissue and is then slowly released into the circulation (34). This delayed bioavailability makes glargine useful as a long-acting insulin (12). Conversely, AspB28-insulin (aspart) and LysB28,ProB29-insulin (lispro), compared with native insulin, have a reduced ability to self-associate and appear more rapidly in the circulation (4, 5); both analogs are commonly used as fast-acting insulins.

The concept of long- and fast-acting insulin analogs has a different meaning at the level of the target cell, relating, rather, to the time of insulin receptor occupancy or the rate of dissociation from the receptor (16). Insulin analogs that have longer residency times on the receptor, such as AspB10,HisA8,LeuB25-insulin, display relatively greater mitogenic potencies than their metabolic activities (7, 15). Some intermediate responses to insulin, such as inhibition of protein degradation, appear to be linked to the processing and degradation of the hormone (35, 36). Thus, at the cellular level, it is also possible to define long- and short-acting insulins. That the pharmacokinetic and cellular durations of action of insulin analogs can be distinct properties is illustrated by glargine, aspart, and lispro, whose affinities for and dissociation from the insulin receptor are similar to those for native insulin (7).

Another determinant of the balance between metabolic and mitogenic responses to insulin analogs is cross talk with the IGF-I receptor (14). In cells and tissues in which IGF-I receptors are present in considerable excess over insulin receptors, it is the affinity of an insulin analog for the IGF-I receptor, rather than residency time on the insulin receptor, that is most closely correlated with mitogenic potency (7). Aspart, lispro, and insulin are equally poorly recognized by the IGF-I receptor and show the same balance of metabolic and mitogenic actions (7).

LysB3,GluB29-insulin (glulisine) is a new insulin analog that also displays rapid bioavailability (37, 38). At the cell level, the binding and processing of glulisine have been shown to be equivalent to those of insulin (21). The potencies of glulisine for metabolic (glucose uptake) and mitogenic (5-bromo-2'-deoxyuridine uptake) responses are also comparable to those of insulin. The similarities of biological responses to glulisine and insulin extend to effects on phosphorylation of a number of molecules downstream of the insulin receptor (21, 39). Despite these similarities, when studied in vitro, glulisine proved to be poor at stimulating tyrosine phosphorylation of IRS-1, but was equal to or slightly more effective than insulin with regard to tyrosine phosphorylation of IRS-2 (21). However, when C57BL/6 mice were injected with insulin or glulisine, there were similar increases in IRS-1 and IRS-2 tyrosine phosphorylation in both skeletal muscle and liver (39). To understand more about the relationships among insulin receptor occupancy, signaling pathways, and final responses, we compared the actions of biosynthetic human insulin, glulisine, and IGF-I over a range of concentrations, including physiological levels. Given the importance of cell type to relative hormone actions (8, 40), we studied glulisine in a cell system in which the ratio of insulin to IGF-I receptors was similar to that in a major insulin target tissue, skeletal muscle (30). We and others have shown that cultured human skeletal muscle myotubes reflect many of the properties of mature skeletal muscle (25), and that muscle cells cultured from type 2 diabetic subjects display defects in glucose uptake (26, 41), glycogen synthase (41, 42), and insulin signaling (43, 44), which are also seen in vivo.

The insulin receptor of human skeletal muscle cells recognizes glulisine with the same affinity as insulin itself. Conversely, both insulin and glulisine are poorly recognized by the IGF-I receptor. Insulin and glulisine are approximately 0.1–0.2% as effective as IGF-I in competing for labeled IGF-I binding. This result is consistent with the finding in K6 myoblasts, a rat cardiomyocyte cell line primarily expressing IGF-I receptors (45), in which insulin and glulisine were similar in their abilities to stimulate IGF-I receptor autophosphorylation (21).

Insulin and glulisine were fully comparable with regard to both potency and sensitivity for their abilities to stimulate glucose uptake, a major metabolic response. Additional studies will be needed to determine whether this equivalency exists for the regulation of protein and fat metabolism. The abilities of insulin, glulisine, and IGF-I to stimulate serine phosphorylation of Akt were similar to their effects on glucose uptake, suggesting that the hormones are acting through a common pathway. The differences in final responsiveness of Akt phosphorylation to IGF-I compared with insulin and glulisine are greater than those for glucose uptake stimulation, suggesting that lower levels of Akt phosphorylation are sufficient to fully stimulate uptake, supporting the importance of comparing actions at more physiological hormone concentrations. These results on glucose uptake and Akt phosphorylation are in general agreement with those obtained in K6 cardiac myoblasts (21), although those data were obtained at higher hormone concentrations (500 nM), at which cross talk with the IGF-I receptor is likely.

The actions of insulin to stimulate [3H]thymidine uptake as an indicator of mitogenic effects were consistent with previous reports (30, 46), with reduced sensitivities and final potencies compared with IGF-I, suggesting that in human myotubes, much of this action of insulin, and glulisine, because it behaves identically to insulin, is occurring through the IGF-I receptor. Mitogenic signaling by peptide hormones involves phosphorylation and activation of p42/44 MAPK. The effects of insulin, glulisine, and IGF-I on MAPK phosphorylation generally paralleled those on [3H]thymidine uptake, with IGF-I displaying the greatest potency, whereas insulin and glulisine were equally low.

Although the current results in human skeletal myotubes and a recent report studying intact mice (39) indicate no differences between insulin and glulisine with regard to potencies for the responses tested, the results obtained and confirmed in multiple muscle cell lines showed a reduced ability of glulisine to stimulate tyrosine phosphorylation of IRS-1, but a comparable action on IRS-2 phosphorylation (21). One potential source of differences between the studies may be the experimental systems employed. In fully differentiated human skeletal muscle cells, the ratio of insulin receptors to IGF-I receptors (1:3–4) is similar to that in skeletal muscle tissue (30). Meanwhile, the K6 myoblasts (45), rat cardiomyocytes, and human myocytes employed by Rakatzi et al. (21) all predominantly express IGF-I receptors. The physiological significance of the preferential phosphorylation of IRS-2 in response to glulisine treatment of muscle cells is uncertain. At the concentration studied (500 nM), the augmented tyrosine phosphorylation of IRS-2 due to glulisine did not result in any difference in downstream responses compared with human insulin (21). This lack of difference was evident for Akt phosphorylation as well as for stimulation of glucose uptake and cell proliferation. Potential explanations for this behavior include the possibility that phosphorylated IRS-2 can substitute for IRS-1, or that the low amount of phosphorylated IRS-1 with glulisine is sufficient to maximally stimulate these responses. Several lines of evidence suggest that IRS-2 may be of secondary importance in skeletal muscle. First is the fact that the amount of insulin-stimulated phosphotidylinositol 3-kinase activity associated with IRS-2 in skeletal muscle is modest (10%) compared with that associated with IRS-1 (43, 47). Second, IRS-2 is not necessary for insulin stimulation of muscle glucose uptake (48), whereas in other tissues the role of IRS-2 in glucose transport regulation is highly cell specific (49). In agreement with these findings, it was reported that reduction of IRS-2 expression by small interfering RNA-mediated gene silencing had no effect on insulin stimulation of glucose uptake, glucose transporter translocation, or phosphorylation of Akt1 (50). In addition, diabetes develops in the IRS-2 knockout mouse due not to peripheral insulin resistance, but, rather, to a failure of the ß-cell to compensate by increasing insulin production (51). The literature suggests that IRS-2 plays a significant role in ß-cell function (52, 53). Indeed, these tissue-specific differences in the importance of IRS-2 were highlighted by the work of Rakatzi et al. (22), who found enhanced ß-cell survival with glulisine compared with insulin.

One message resulting from the current study is that cell context is important in considering the relative metabolic and mitogenic actions of insulin analogs (40). Relative contents of insulin and IGF-I receptors and intracellular processing of hormone analogs (8) can influence the results. Although insulin and glulisine appear identical at the level of the muscle cell, this relationship could vary between cell type, and other work, in adipocytes and hepatocytes, for example, is required to address this issue. An example of such cell specificity would be an enhanced antiapoptotic action of glulisine on the ß-cell (22), which could aid in preserving ß-cell mass and function, improving glycemic control beyond effects in muscle.


   Footnotes
 
This work was supported by a grant from the Medical Research Service, Department of Veterans Affairs, and the Veterans Affairs San Diego HealthCare System; research (to T.P.C.) and mentor-based fellowship (to R.R.H.) grants from the American Diabetes Association; National Institutes of Health Grants R01-DK-58291 (to R.R.H.) and K08-DK-61987 (to S.A.P.); a research grant from Aventis Pharma; and Grant MO1-RR-00827 from the General Clinical Research Branch, Division of Research Resources, National Institutes of Health.

First Published Online July 19, 2005

Abbreviation: IRS, Insulin receptor substrate.

Received May 6, 2005.

Accepted July 7, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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