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Endocrinology Vol. 140, No. 3 1141-1150
Copyright © 1999 by The Endocrine Society

ARTICLES

In Vivo Insulin Signaling in the Myocardium of Streptozotocin-Diabetic Rats: Opposite Effects of Diabetes on Insulin Stimulation of Glycogen Synthase and c-Fos1

Ping H. Wang2, Abdulraof Almahfouz3, Francesco Giorgino4, Karen C. McCowen and Robert J. Smith

Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Robert J. Smith, M.D., Research Division, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215. E-mail: smithr{at}joslab.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Diabetes induced by streptozotocin (STZ) in laboratory rats leads to impaired glucose metabolism in the heart and changes in myocardial contractile protein isoform expression and cardiac function. The purpose of this study was to investigate in vivo insulin signaling responses in the myocardium of STZ-diabetic rats. Insulin rapidly stimulated tyrosine phosphorylation of the insulin receptor, insulin receptor substrate-1 (IRS-1) and, to a lesser extent, IRS-2 in normal and diabetic myocardium. In diabetic rats, there was 2-fold higher insulin receptor content and insulin-stimulated receptor tyrosine phosphorylation in comparison with control rats. IRS-1 tyrosine phosphorylation also increased in STZ diabetes in spite of a decrease in IRS-1 content, resulting in a 4-fold higher ratio of phosphorylated to total IRS-1. This was associated with 2-fold higher IRS-1 precipitable phosphatidylinositide 3-kinase activity in diabetic animals. Insulin stimulation of glycogen synthase activity was significantly diminished in STZ diabetes, consistent with resistance to insulin in a step downstream from phosphatidylinositide 3-kinase activation. Under the same experimental conditions, there was a marked increase in insulin stimulation of myocardial c-fos messenger RNA content in diabetic animals in comparison with controls. These data demonstrate altered early steps in insulin signaling in STZ-diabetic rat myocardium. Consequent oppositely directed disturbances in growth regulatory and glucose regulatory responses to insulin may contribute to the development of myocardial functional abnormalities in this model of diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN has important regulatory effects on myocardial metabolism and growth. In cardiac muscle, metabolic responses to insulin include the stimulation of glucose uptake (1), glycolysis (2), and glycogen synthesis (3). Although fatty acids normally serve as the principal fuel source in the myocardium, the utilization of glucose via anaerobic glycolytic pathways becomes an important source of metabolic energy when oxygen availability is limited during periods of ischemia (4, 5). In uncontrolled diabetes mellitus, diminished glucose uptake and metabolism secondary to insulin deficiency and/or insulin resistance may contribute to the development of myocardial dysfunction, particularly when there is coexistent ischemia (6). In addition to its effects on myocardial glucose metabolism, insulin regulates cardiac muscle growth. Specific effects of insulin observed in myocardial tissue and cultured cardiomyocytes include the stimulation of amino acid transport (7) and protein synthesis (8, 9), the inhibition of protein degradation (10), and possibly the stimulation of DNA synthesis (11).

The biological actions of insulin in all target tissues are mediated by high affinity membrane-spanning insulin receptors (12). Insulin binding to the insulin receptor {alpha}-subunit activates a tyrosine kinase intrinsic to the cytoplasmic portion of the transmembrane ß-subunit, which then undergoes autophosphorylation on tyrosine residues (13) and phosphorylates nonreceptor proteins, including insulin receptor substrate-1 (IRS-1)1 (13), IRS-2 (14), and other substrates (15, 16, 17). These initial tyrosine phosphorylation events ultimately transmit the insulin signal to a branching series of intracellular pathways that regulate cellular metabolism, growth and differentiation. Substantial evidence indicates that the activation of the enzyme phosphatidylinositide (PI) 3-kinase, through its association with tyrosine phosphorylated IRS-1, is essential for insulin effects on glucose uptake and glycogen synthesis (18, 19). Similarly the proto-oncogenes c-fos and c-jun have been identified as intermediates in pathways that ultimately lead to insulin-stimulated tissue growth responses (20, 21). Both c-fos and c-jun have been shown to be involved in the regulation of cardiac muscle growth and differentiation by other stimuli (22, 23) and, thus, insulin stimulation of growth responses in cardiac muscle is likely to involve c-fos and c-jun.

Treatment of rats with the ß-cell toxin streptozotocin (STZ) results in a diabetic state characterized by both insulin deficiency and insulin resistance (24). In skeletal muscle, although there is resistance to the effects of insulin on glucose uptake and glycogen synthesis in STZ-diabetes (25), there is a paradoxical and yet unexplained increase in insulin-stimulated tyrosine phosphorylation of the insulin receptor ß-subunit and IRS-1 (26, 27). Abnormalities in cardiac muscle have been demonstrated in STZ-diabetes, including defects in the glucose transport system (28) and changes in contractile protein isoform expression that may be related to compromised cardiac contractile function (29). However, little is known about the insulin signaling responses that may be linked to these abnormalities in the myocardium of diabetic rats.

The purpose of this study was to examine initial steps of insulin signaling in the myocardium of normal and STZ-diabetic rats in vivo. In addition, we correlated these findings with determinations of the effects of diabetes on insulin-stimulated glycogen synthase activity, as an example of a distal glucose-regulatory response, and c-fos/c-jun expression, as downstream responses linked to growth regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats weighing approximately 200 g were purchased from Taconic Farms, Inc. (Germantown, NY). To induce diabetes, rats were fasted overnight and injected with STZ in pH 4.5 citrate buffer (65–100 mg/kg body wt, ip) as previously described (26). All animals were subsequently allowed free access to standard rat chow and water, tail vein blood glucose was determined after 5 days with an AccuCheck system (Boehringer Mannheim, Indianapolis, IN), and rats with nonfasting whole blood glucose >16.7 mM were selected for further study. These diabetic animals were either maintained untreated, or treated twice daily with 3–9 U sc Ultralente recombinant human insulin (Eli Lilly & Co., Indianapolis, IN) for five additional days. Untreated diabetic, insulin-treated diabetic and control rats then were anesthetized with amobarbital after an overnight fast. A laparotomy was performed, and a bolus of regular insulin (10 U/kg body wt) or vehicle (0.9 g/dl NaCl, 0.1 g/dl BSA) was injected via the inferior vena cava. After a specified time period, the cardiac ventricles were rapidly excised and frozen in liquid nitrogen. In longer duration experiments on proto-oncogene expression, which involved up to 60 min insulin stimulation, 2.22 M glucose (2 ml in normal controls and 1 ml in diabetics) was given sc immediately after insulin injection to prevent hypoglycemia. These doses of glucose were able to maintain stable blood glucose levels for at least 2 h. All animal procedures performed in this study were approved by the Joslin Diabetes Center Institutional Animal Care and Use Committee.

Immunoprecipitation and immunoblotting
The frozen ventricles were powdered in a stainless steel mortar and pestle with liquid nitrogen and homogenized for 30 sec with a Polytron (Brinkmann Instruments, Westbury, NY) in ice-cold buffer containing 50 mM Tris (pH 7.4), 10 mM sodium pyrophosphate, 10 mM sodium {rho}-nitrophenylphosphate, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride (PMSF), 6 µg/ml leupeptin, 2 mM sodium orthovanadate, and 1% (vol/vol) Triton-X100. The homogenates were incubated on ice for 45 min and then centrifuged at 55,000 x g for 1 h at 4 C. Protein concentrations in the resulting supernatants were determined by the Bradford method using BSA as a standard.

For direct immunoblotting studies, equal amounts of solubilized ventricular proteins (200–500 µg protein/lane) were separated by 7% SDS-PAGE. The resolved proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH) using a transfer buffer containing 192 mM glycine, 20% (vol/vol) methanol, and 0.02% SDS. The filters were incubated in TNA buffer (10 mM Tris, pH 7.8, 0.9 g/dl NaCl, 0.01 g/dl sodium azide) supplemented with 5 g/dl BSA and 0.05% (vol/vol) Nonidet P-40 (NP-40) at 37 C for 2 h to reduce nonspecific binding, and then incubated overnight at 4 C with antiphosphotyrosine, antiinsulin receptor, or anti-IRS-1 antibodies. After washing twice with TNA buffer containing 0.05% NP-40 and once with TNA buffer containing 0.1% (vol/vol) Tween-20, the filters were incubated at room temperature for 1 h with [125I]Protein A (1 µCi/ml, ICN, Costa Mesa, CA), washed twice more with TNA buffer plus 0.05% NP-40, once with TNA buffer plus 0.1% Tween-20, and dried. Specific protein bands were identified and quantified either by autoradiography and densitometric analysis, or with a phosphorimaging system (Molecular Dynamics, Inc., Sunnyvale, CA).

For sequential immunoprecipitation and immunoblotting studies, solubilized tissue proteins (1–5 mg) were incubated for 2 h at 4 C with antiinsulin receptor, anti-IRS-1, anti-IRS-2, or anti-Shc antibodies as indicated. The immunocomplexes were adsorbed to Protein A-Sepharose beads (Pharmacia Biotech, Inc., Piscataway, NJ) for 1 h at 4 C, pelleted by centrifugation, and washed 3 times at 4 C in Tris-HCl buffer at pH 7.8 containing 150 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, and 1 mM PMSF. The washed immunoprecipitates were resuspended in Laemmli buffer with 100 mM dithiothreitol, heated for 5 min at 100 C, and resolved by SDS-PAGE. After transfer onto nitrocellulose membranes, the resulting blots were sequentially incubated with blocking buffer and antiphosphotyrosine antibody, and specific tyrosine phosphorylated protein bands were identified and quantified with antiphosphotyrosine antibody and [125I]Protein A as described above for direct immunoblotting studies.

The polyclonal antiphosphotyrosine and antirat insulin receptor antibodies used in these studies have been described previously (26). Specific anti-IRS-1 antibodies were generated in our laboratory (30), anti-IRS-2 antibodies were obtained from Morris F. White (Joslin Diabetes Center, Boston, MA) (14), and anti-Shc antibodies were from Upstate Biotechnology (Lake Placid, NY).

PI-3 kinase assay
IRS-1 associated PI-3 kinase activity was analyzed as previously described (31). In brief, ventricular muscle from basal or insulin-stimulated states was homogenized in a buffer containing HEPES 50 mM, pH 7.5, 150 mM NaCl, 10 mM NaF, 2 mM EDTA, 10 mM sodium pyrophosphate, 1 mM MgCl2, 1 mM CaCl2, 10% (vol/vol) glycerin, 1% NP-40, 2 mM PMSF, and 6 µg/ml leupeptin. The homogenates were centrifuged, and the supernatant incubated with anti-IRS-1 antibody at 4 C overnight. The immunocomplexes were precipitated after adsorption with Protein A-agarose beads and extensively washed. The activity of PI 3-kinase that coprecipitated with IRS-1 was determined by incubation of the resuspended pellets with [{gamma}-32P]ATP and phosphatidylinositol, followed by separation of the labeled PI-3 product by TLC, autoradiography, and laser densitometry.

Glycogen synthase activity
Glycogen synthase activity was measured by a modification of a published method based on the measurement of [14C]uridine diphosphoglucose (UDPG) incorporation into glycogen (3). Frozen myocardium was pulverized in liquid nitrogen and homogenized with a Polytron homogenizer for 15 sec in a buffer containing 100 mM NaF and 10 mM EDTA, pH 7.4. The homogenates were cleared by centrifugation at 2,000 x g for 20 min at 4 C, and glycogen synthase activity was determined using aliquots of the supernatants. Equal amounts of the soluble myocardial proteins were reacted at 30 C for 30 min with a mixture containing 50 mM Tris, pH 7.8, 25 mM NaF, 20 mM EDTA, 10 mg/ml glycogen, 25 µM UDPG, and tracer [14C]UDPG with either 6 or 0.3 mM glucose-6-phosphate (G6P). The reaction products were blotted onto pieces of filter paper, washed three times with 66% (vol/vol) ethanol, and air dried. The amount of [14C]UDPG incorporated into glycogen was determined by liquid scintillation counting, and the activity of glycogen synthase was calculated as the ratio of the G6P-independent form (measured with 0.3 mM G6P) vs. the G6P-dependent form (measured with 6 mM G6P).

Northern blotting
The levels of c-fos and c-jun messenger RNA (mRNA) were assessed by Northern blotting. Total RNA from ventricular muscle was extracted essentially as described by Chomczynski and Sacchi (32). The quality of RNA was assessed by the 260/280 absorbance ratio, and the majority of the RNA samples used in this study showed A260/A280 ratios >= 1.7. Equal amounts of RNA (15 µg/lane) were electrophoresed in 1.2% agarose-formaldehyde gels, transferred to Genescreen membranes (DuPont NEN, Boston, MA) by overnight blotting in 10 x SSC, UV cross-linked and hybridized overnight at 42 C with [32P] labeled probes (0.5 x 106 cpm/ml) in hybridization buffer containing 25 mM Tris, pH 7.4, 1 M NaCl, 10 g/dl dextran sulfate, 50% formamide, 1% SDS, and 50 µg/ml salmon sperm DNA. Full-length rat c-fos and c-jun complentary DNAs (from Bruce Spiegelman, Dana-Farber Cancer Institute, Boston, MA) were used as templates to generate specific radiolabeled probes by multiprime random labeling with Klenow fragments. The filters were washed twice at room temperature (1% SDS and 1 x SSC, 30 min), once at 50 C (1% SDS and 0.2 x SSC, 20 min), twice at 60 C (0.1% SDS and 0.2 x SSC, 20 min), air dried, and analyzed by autoradiography and laser scanning densitometry.

Statistical analysis
Data are presented as mean ± SEM, and statistical significance was evaluated by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of experimental animals
The plasma glucose and body weight determinations in the experimental animals are summarized in Table 1Go. Control rats gained an average of 74.5 g and maintained normal blood glucose levels during the course of the study. In contrast, untreated diabetic rats lost an average of 26.3 g by the end of study, and their blood glucose levels were significantly higher than in the controls (19.8 ± 0.8 vs. 4.4 ± 0.2 mM, P < 0.001). After insulin deficiency was corrected with insulin replacement (3–9 U Ultralente, twice daily) for 5 days, fasting blood glucose levels were markedly reduced in treated diabetic rats (8.6 ± 1.6 mM) and, compared with the untreated diabetic rats, significant weight gain was observed (41.0 vs. -26.3 g, P < 0.001).


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  		Table 1. Animal characteristics

 
Insulin-stimulated protein tyrosine phosphorylation in vivo
To investigate initial events in insulin signaling in the myocardium, overnight-fasted control rats were anesthetized and given a bolus injection of insulin into the inferior vena cava. At defined time points, the cardiac ventricles were rapidly removed, frozen in liquid nitrogen, and homogenized in a buffer containing nonionic detergent and inhibitors of proteolysis and dephosphorylation. The solubilized myocardial proteins were resolved by SDS-PAGE, and tyrosine phosphoproteins were identified by immunoblotting with phosphotyrosine antibody. In vivo insulin injection led to rapid tyrosine phosphorylation of a 98-kDa protein band and two additional bands migrating at approximately 170–180 kDa. Figure 1Go shows that tyrosine phosphorylation of these three protein bands was stimulated by insulin injection at a dose as low as 0.2 U/kg body wt and was maximal with insulin at doses >= 1.0 U/kg. For subsequent studies, insulin was administered at the supramaximal dose of 10 U/kg body wt. Tyrosine phosphorylation of the myocardial proteins reached a maximum within one minute after in vivo insulin injection (data not shown), as previously demonstrated for other tissues (26, 27).



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  		Figure 1. Dose-response effects of insulin on the tyrosine phosphorylation of myocardial proteins. Insulin at the indicated doses was injected in vivo via the inferior vena cava, and cardiac ventricular tissue was removed after 1 min. Solubilized myocardial proteins were resolved by 7% SDS-PAGE, and tyrosine phosphoproteins were detected by immunoblotting with antiphosphotyrosine antibody. Each lane represents an individual rat.

 
The identities of the three insulin-stimulated tyrosine phosphoproteins were confirmed by immunoprecipitation with specific antibodies. Preparations of myocardial proteins were first immunoprecipitated with antiinsulin receptor, anti-IRS-1, or anti-IRS-2 antibodies, and the resulting immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine antibody. As shown in Fig. 2Go, sequential immunoprecipitation and immunoblotting confirmed that the 98-kDa band represents the insulin receptor ß-subunit, whereas the 170 kDa and 180 kDa bands, respectively, correspond to IRS-1 and IRS-2. It should be noted that the similar intensities of the insulin-stimulated IRS-1 and IRS-2 bands in Fig. 2Go do not necessarily reflect the relative abundance of these tyrosine phosphorylated proteins in the myocardium because immunoprecipitation was performed with different antibodies. In further studies comparing control and diabetic rats, we focused on the insulin receptor and 170 kDa IRS-1 bands because they demonstrated the most marked and consistent insulin-stimulated tyrosine phosphorylation. Insulin stimulation of tyrosine phosphorylation of the 180 kDa IRS-2 band assessed by direct phosphotyrosine antibody blotting was variable, as is evident in Fig. 1Go.



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  		Figure 2. Identification of insulin-stimulated tyrosine phosphoproteins in myocardium. Proteins solubilized from the myocardium of rats under basal conditions or stimulated with insulin in vivo for 2 min were immunoprecipitated with antiinsulin receptor, anti-IRS 1, or anti-IRS 2 antibodies, and then immunoblotted with antiphosphotyrosine antibody.

 
To determine the effects of STZ-diabetes on insulin receptor and IRS-1 tyrosine phosphorylation, control, untreated diabetic, and diabetic rats treated twice daily for 5 days with sc insulin were fasted overnight and given a bolus injection of either 10 U insulin/kg body wt or vehicle. After 1 min, the cardiac ventricles were removed and frozen in liquid nitrogen, myocardial proteins were solubilized, and tyrosine phosphoproteins were identified by phosphotyrosine antibody immunoblotting. Basal tyrosine phosphorylation of IRS-1 was significantly decreased in untreated diabetic compared with control rats (31.8 ± 7.9% lower in diabetic than control, P < 0.05), and mean basal phosphorylation of the insulin receptor also was lower in diabetic myocardium (32.0 ± 13.4% lower in diabetes), although this difference was not significant (P = 0.098, data not shown). We previously have reported that overnight fasting and insulin withdrawal in treated diabetic animals results in basal receptor and IRS-1 phosphorylation similar to that in untreated diabetics (26). Following acute insulin stimulation, tyrosine phosphorylation of IRS-1 and the insulin receptor ß-subunit was markedly increased in untreated diabetic compared with control animals (Fig. 3Go). This is similar to the increase of in vivo insulin-stimulated IRS-1 and receptor tyrosine phosphorylation in STZ-diabetic rats reported for skeletal muscle and liver (26, 27). In diabetic animals treated with insulin for 5 days, acute insulin-stimulated IRS-1 and insulin receptor tyrosine phosphorylation returned to levels similar to those in the nondiabetic controls (Fig. 3Go).



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  		Figure 3. Comparison of in vivo insulin-stimulated tyrosine phosphorylation of myocardial proteins in control, untreated diabetic, and treated diabetic rats. In the treated diabetic group, insulin was administered twice daily for 5 days as described in Materials and Methods. Animals in all groups were fasted overnight, given an acute bolus injection of insulin (10 U/kg body wt) via the inferior vena cava and, after 1 min, the cardiac ventricles were removed and frozen in liquid nitrogen. Myocardial proteins were solubilized, resolved by SDS-PAGE, and immunoblotted with antiphosphotyrosine antibody. Each lane represents myocardial proteins extracted from an individual rat.

 
To investigate a potential alternative pathway of insulin signaling, tyrosine phosphorylation of the Shc proteins (33) was determined in the myocardium of control and diabetic rats. Myocardial protein extracts prepared 10 min after the infusion of insulin or vehicle as described above were subjected to immunoprecipitation with anti-Shc antibody followed by immunoblotting with antiphosphotyrosine antibody. As shown in Fig. 4Go, basal tyrosine phosphorylation of the 52 and 46 kDa Shc isoforms was evident, but there was no stimulation following insulin injection in control or diabetic rats. Phosphorylation of the 66 kDa Shc isoform was essentially undetectable in all animals.



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  		Figure 4. Determination of insulin effects on Shc tyrosine phosphorylation in control and diabetic rats. After an overnight fast, animals were anesthetized and given an injection of insulin (10 U/kg body wt) via the inferior vena cava. After 10 min, the cardiac ventricles were removed and frozen in liquid nitrogen. Shc tyrosine phosphorylation in myocardial protein extracts was determined by immunoprecipitation with anti-Shc antibody followed by immunoblotting with antiphosphotyrosine antibody.

 
To further define the changes in early steps in insulin receptor signaling in diabetic myocardium, the abundance and tyrosine phosphorylation of the insulin receptor ß-subunit and IRS-1 were quantified by densitometric analysis of immunoblots from multiple control, untreated STZ-diabetic and treated diabetic rats, and the overall stoichiometry of phosphorylation of these proteins was determined. Receptor phosphorylation, assessed by immunoblotting with antiphosphotyrosine antibody, increased 2-fold in diabetic myocardium (Fig. 5AGo). After diabetic rats received insulin replacement therapy for 5 days to correct insulin deficiency and hyperglycemia, the level of receptor tyrosine phosphorylation stimulated by insulin returned to that of the controls. The total myocardial content of insulin receptors, determined by immunoblotting with insulin receptor antibody, also increased 2-fold in diabetic myocardium (Fig. 5BGo), and the amount of receptor protein was restored to the control level after the diabetic rats received insulin replacement therapy. Therefore, the ratio of receptor tyrosine phosphorylation/receptor content was not altered in the myocardium of untreated or treated diabetic rats (Fig. 5CGo). The amount of tyrosine phosphorylated insulin receptor increased in diabetes, but the overall stoichiometry of receptor autophosphorylation remained unchanged.



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  		Figure 5. Quantitation of insulin-stimulated insulin receptor ß-subunit and IRS-1 tyrosine phosphorylation, protein content, and phosphorylation/protein in the myocardium of control and diabetic rats. Diabetic animals were either untreated or treated with Ultralente insulin for 5 days as described in Materials and Methods. After an overnight fast, an acute bolus of insulin (10 U/kg body wt) was injected into the vena cava and, after 2 min, the cardiac ventricles were removed and frozen in liquid nitrogen. Tyrosine phosphorylation of the insulin receptor was determined by immunoblotting with antiphosphotyrosine antibody (A), the total amount of insulin receptor ß-subunit was determined by immunoblotting with antiinsulin receptor antibody (B), and the ratio of phosphorylation/receptor was determined for each individual rat (C). Similarly, tyrosine phosphorylation of IRS-1 was determined by immunoblotting with antiphosphotyrosine antibody (D), the total amount of IRS-1 was determined by immunoblotting with anti-IRS-1 antibody (E), and the ratio of phosphorylation/IRS-1 was determined for each individual rat (F). Data represent mean ± SEM for 9 animals (insulin receptor) or 16 animals (IRS-1) in each group: *, P < 0.001 vs. control and 5-day insulin-treated diabetes; **, P < 0.001 vs. control.

 
Similar quantitative analysis of IRS-1 demonstrated that its phosphorylation also was increased approximately 2-fold in diabetic myocardium and returned to the control level after the diabetic rats had received insulin replacement therapy (Fig. 5DGo). However, the content of IRS-1 decreased in diabetic rats, and insulin replacement therapy failed to restore the level of IRS-1 protein back to that of the controls (Fig. 5EGo). Thus, the amount of tyrosine phosphorylated IRS-1 per total amount of IRS-1 increased 4-fold in the myocardium of untreated diabetic rats and was persistently (5-fold) increased in the myocardium of insulin-treated diabetic rats (Fig. 5FGo). The basis for the increased IRS-1 tyrosine phosphorylation in diabetic myocardium is not known, but it could be a consequence of the enhanced total receptor phosphorylation, which is expected to correlate with receptor kinase activity (34).

Activation of PI-3 kinase
An important insulin signaling response downstream from IRS-1 tyrosine phosphorylation is the activation of the enzyme PI 3-kinase, resulting from the noncovalent binding of its regulatory subunit to phosphotyrosine residues in IRS-1 (31). To determine whether the changes in receptor ß-subunit and IRS-1 tyrosine phosphorylation in the diabetic myocardium affect subsequent steps in the insulin signaling pathway, the activity of PI 3-kinase associated with IRS-1 was analyzed in protein extracts from cardiac ventricular muscle of control and diabetic rats obtained 10 min after stimulation by in vivo insulin injection. Solubilized proteins from ventricular muscle were subjected to immunoprecipitation with IRS-1 antibody, and PI 3-kinase activity in the reconstituted pellets was assayed by measuring the transfer of [32P] from ATP to the 3-position of phosphatidylinositol (31). Following insulin injection, 3-phosphoinositol formation was markedly increased in IRS-1 immunoprecipitates from control and diabetic myocardium (representative data from control rats shown in Fig. 6AGo). Quantitative analysis of multiple experiments demonstrated that the level of insulin-stimulated PI 3-kinase activity was increased 2-fold in untreated diabetic compared with control myocardium (P < 0.01) and returned to the level of the controls in insulin-treated diabetic rats (Fig. 6BGo). In the absence of insulin stimulation, basal IRS-1-associated PI 3-kinase activity was barely detectable in control and diabetic animals, reflecting the low insulin levels following overnight fasting. In a subset of animals (n = 4), basal PI 3-kinase was quantified and shown to be decreased by 85.3 ± 5.2% (P < 0.001) in diabetic rats in comparison with controls). Thus, both the absolute level of insulin-stimulated IRS-1-associated PI 3-kinase activity, and the fold effect of insulin on PI 3-kinase were higher in the STZ-diabetic rats. These data demonstrate that an increase in insulin stimulation of IRS-1-associated PI 3-kinase activity in diabetic myocardium parallels the increase in IRS-1 tyrosine phosphorylation. The correction of hyperglycemia by insulin replacement therapy restores both insulin-stimulated PI 3-kinase activity, and IRS-1 tyrosine phosphorylation to the control level.



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  		Figure 6. Activation of IRS-1 associated PI-3 kinase by insulin in normal and diabetic myocardium. After in vivo insulin stimulation, myocardial proteins were immunoprecipitated with anti-IRS-1 antibody, and PI 3-kinase activity in the pellet was determined by measuring the incorporation of [32P] into the 3-position of phosphatidylinositol. A, Representative thin layer chromatogram demonstrating basal and insulin-stimulated 3-phosphoinositol formation (designated PIP) in IRS-1 immunoprecipitates from normal rat heart. B, Quantitation of insulin-stimulated PI 3-kinase activity from multiple experiments in control and diabetic rats. Diabetic animals were either untreated, or treated with Ultralente insulin for 5 days as described in Materials and Methods. Data represent mean ± SEM for 16 animals in each group: *, P < 0.001 vs. control and 5-day insulin-treated diabetes.

 
Glycogen synthase activity
Significant resistance to the effects of insulin on glucose regulatory pathways has previously been noted in insulin target tissues of STZ-diabetic rats (24, 28, 35, 36). To assess in vivo insulin effects on a downstream glucose regulatory pathway in this study, the conversion of glycogen synthase from the G6P-dependent D form to the active G6P-independent I form following insulin administration in vivo was determined in myocardium from control and STZ-diabetic rats. For these and subsequent studies on c-fos and c-jun expression (see below), an additional series of STZ-diabetic rats was prepared. The expected increase in insulin-stimulated receptor and IRS-1 tyrosine phosphorylation was confirmed, and the general characteristics of the animals (blood glucose levels, body wt) were similar to those of the preceding group of rats used for the tyrosine phosphorylation and PI 3-kinase studies (data not shown). Insulin stimulation of glycogen synthase activity, as assessed by the percentage in the G6P-independent I form reached a maximum within 10 min of in vivo injection of insulin. A comparison of basal and 10-min insulin-stimulated glycogen synthase activities in normal and diabetic myocardium is shown in Fig. 7Go. Insulin increased the percentage of I form glycogen synthase approximately 2-fold in the normal myocardium (P < 0.001), whereas insulin had no effect on the percentage of I form glycogen synthase in diabetic myocardium (P = 0.501). Thus, stimulation of glycogen synthase by insulin was markedly impaired in diabetic myocardium, confirming the results of previous studies on glycogen synthase regulation in STZ-diabetes (3). Because tyrosine phosphorylation of the insulin receptor and IRS-1 was increased in the myocardium of diabetic rats under the same experimental conditions, these data indicate that STZ-diabetes leads to oppositely directed changes in the proximal insulin signaling pathways and the more distal glycogen synthase pathway. Glycogen synthesis regulation is believed to be downstream from and mediated by IRS-1 tyrosine phosphorylation (37), and these findings therefore support the concept that the site of resistance to insulin that leads to compromised glycogen synthase activity in this model of diabetes is downstream from insulin receptor catalyzed IRS-1 tyrosine phosphorylation.



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  		Figure 7. Myocardial glycogen synthase activity in control and diabetic rats. Insulin (10 U/kg body wt) (solid bars) or vehicle (open bars) was injected into the inferior vena cava, the heart was removed after 10 min, and the activity of glycogen synthase was measured in a soluble myocardial protein fraction in the presence of 0.3 mM G6P (I form) and 6 mM G6P (total I + D forms) as described in Materials and Methods. Data represent the I form of glycogen synthase as % of total from four separate experiments analyzed in triplicate: *, P < 0.001 vs. basal.

 
Expression of c-fos and c-jun
To investigate downstream insulin signaling effects on growth regulatory pathways in the diabetic myocardium, mRNA levels for the c-fos and c-jun proto-oncogenes were determined. Insulin or vehicle was injected in vivo via the inferior vena cava, cardiac ventricular muscle was removed at various time points, and total RNA was extracted for Northern blotting analysis with specific c-fos or c-jun probes. As noted in the Materials and Methods section, insulin-induced hypoglycemia was prevented in these experiments, which involved longer periods of insulin stimulation, by the sc injection of a 2.22 M glucose solution. Figure 8Go shows representative Northern blotting data, which demonstrate a modest increase in c-fos and little or no change in c-jun mRNA 30 min after insulin injection in overnight fasted control rats (left panel). In STZ-diabetic rats (right panel), a marked augmentation of c-fos mRNA and an apparent more modest increase in c-jun mRNA is evident 30 min after insulin injection. The results of quantitative densitometric analysis of myocardial c-fos and c-jun mRNA from multiple rats studied 15, 30, or 60 min after insulin injection are shown in Fig. 9Go. The expression of c-fos was increased by approximately 40% in the normal myocardium 30 min after insulin injection (P < 0.001). In the normal controls, there was no difference in c-fos mRNA levels between 30 and 60 min of insulin stimulation (P = 0.11). In the basal (unstimulated) state, there was a modest increase of c-fos mRNA in the myocardium of diabetic compared with control rats (74% above control, P < 0.001). Insulin injection in the diabetic rats led to a much greater (6- to 7-fold) and more rapid increase (peak at 15 to 30 min) in c-fos mRNA levels in comparison with control rats. The content of c-jun mRNA was not altered by insulin in control rats, and basal levels of c-jun mRNA were similar in diabetic and control rats. The mean insulin-stimulated c-jun mRNA levels were higher in diabetic rat myocardium 15 and 30 min after stimulation, but these values did not reach statistical significance.



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  		Figure 8. Northern blots of c-fos and c-jun mRNA in control and diabetic myocardium. The cardiac ventricles were removed 30 min after in vivo injection of insulin (10 U/kg body wt) or vehicle, and total RNA was extracted and analyzed by Northern blotting. Each lane contains 15 µg of total myocardial RNA from an individual rat.

 


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  		Figure 9. Time-course of the effects of insulin on mRNA levels of c-fos (left panel) and c-jun (right panel) in control (open bars) and diabetic (solid bars) myocardium. Cardiac ventricles were removed at the indicated times after in vivo injection of insulin (10 U/kg body wt), and total RNA was extracted and analyzed by Northern blotting. The levels of c-fos and c-jun mRNA were quantitated by laser densitometry. Data represent pooled results from three to four experiments in triplicate: *, P < 0.001 vs. 0 time control, **, P < 0.001 vs. control (nondiabetic) animals at the indicated time points.

 
To determine whether correction of hyperglycemia and insulin deficiency can reverse the augmentation of insulin-stimulated c-fos expression in diabetic myocardium, a subset of diabetic rats was given insulin replacement therapy as described earlier. The basal c-fos mRNA levels were similar in treated and untreated diabetic rats (P = 0.335), but the increase in insulin-stimulated c-fos expression observed in untreated diabetic rats was completely reversed after 5 days of insulin replacement therapy (Fig. 10Go, left panel). The mean values for c-jun mRNA showed a similar decrease to the control level after 5 days of insulin treatment in diabetic rats, although neither the increase in c-jun mRNA with diabetes, nor the decrease with insulin treatment were statistically significant (Fig. 10Go, right panel). These data demonstrate an augmentation of insulin effects on c-fos and possibly c-jun gene expression in the myocardium of STZ-diabetic rats that correlates with the observed increases in receptor and IRS-1 tyrosine phosphorylation. In contrast to the resistance to insulin effects on glycogen synthase activation, STZ-diabetes results in increased insulin effects on proto-oncogene expression.



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  		Figure 10. The effects of chronic (5 days) insulin therapy on myocardial c-fos and c-jun expression. Insulin (10 U/kg body wt) or vehicle (Basal) was injected into the inferior vena cava of overnight fasted rats, and myocardium was isolated 30 min later for RNA extraction. The expression of c-fos and c-jun in control (open bars), untreated diabetic (solid bars), and 5-day insulin-treated diabetic (hatched bars) rats was assessed by Northern blotting and quantitated by laser densitometry. Data represent pooled results from three experiments in triplicate: *, P < 0.001 vs. control and 5-day insulin-treated diabetic.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides the first characterization of early signaling responses to insulin in the myocardium of normal and STZ-diabetic rats in vivo. Insulin stimulation of tyrosine phosphorylation of the insulin receptor and its substrate IRS-1 was demonstrated in the normal rat heart and shown to be significantly augmented in diabetic myocardium. Downstream responses, including activation of PI-3 kinase and stimulation of c-fos and possibly c-jun gene expression by insulin also were increased in diabetic myocardium, whereas insulin activation of glycogen synthase was attenuated. These results suggest that the induction of STZ-diabetes may have opposite effects on distal glucose regulatory and growth regulatory pathways of insulin signaling in cardiac muscle.

Although this represents the first demonstration of opposite effects on c-fos expression and glycogen synthase activation in the myocardium, and the first characterization of these opposite effects of STZ-diabetes in any tissue within a single experimental study, previous work suggests that differential effects of diabetes on glucose regulatory and growth regulatory pathways also exist in other tissues. For example, published studies have shown impaired insulin-mediated glucose metabolism in skeletal muscle (24, 36) as well as cardiac muscle (3, 28) of STZ-diabetic rats. Work from our group and others has demonstrated increased insulin-stimulated receptor and IRS-1 phosphorylation in skeletal muscle and liver of diabetic rats (26, 27), and augmented insulin stimulation of c-fos expression has been observed in adipose tissue in STZ-diabetes (38). A human patient with a rare form of extreme insulin resistance and acromegalic features has been described with resistance to the effects of insulin on glucose transport in cultured fibroblasts, but normal insulin effects on amino acid uptake (39). Thus, selective resistance to the effects of insulin on glucose regulatory pathways but not other pathways can occur in both experimental animals and humans.

Tyrosine phosphorylation of the insulin receptor and IRS-1 represent initial steps of insulin signal transduction. Because mutations in the insulin receptor gene affecting tyrosine residues or the tyrosine kinase domain are associated with decreased insulin effects on both glycogen synthase activity and thymidine incorporation (37), it appears that the autophosphorylation of the insulin receptor and activation of its intrinsic tyrosine kinase initiate insulin signaling events that lead to glucose regulatory and growth regulatory pathways (40). In this study, because tyrosine phosphorylation of the insulin receptor and IRS-1 was not decreased in the diabetic myocardium, it can be concluded that the site of insulin resistance responsible for decreased glycogen synthase activation is likely to involve step(s) distal to the insulin receptor and IRS-1. Current evidence suggests that insulin stimulation of glycogen synthase involves a PI 3-kinase dependent pathway (18), and our data demonstrated an augmented insulin effect on PI-3 kinase activation in diabetic myocardium, further suggesting that the insulin resistance in STZ-diabetes involves signaling intermediates downstream from PI 3-kinase. Although there is little information about negative feedback mechanisms in the postreceptor insulin signaling cascade, it is possible that resistance to insulin at a distal glucose-regulatory step might somehow disrupt a negative feedback response and contribute to reciprocal enhancement of insulin signaling at initial and intermediate steps of the signaling pathway.

In the diabetic myocardium, increased insulin-stimulated c-fos expression correlated with enhanced tyrosine phosphorylation of the insulin receptor and IRS-1. Maximum tyrosine phosphorylation of the myocardial insulin receptor and IRS-1 occurred within one minute after in vivo insulin injection, whereas the level of c-fos mRNA was not fully stimulated until 15–30 min later. Thus, in the diabetic myocardium, the augmentation of insulin-stimulated c-fos gene expression is likely to be a response secondary to increased insulin receptor kinase activity and possibly secondary to increased IRS-1 tyrosine phosphorylation. An absence of insulin-stimulated Shc tyrosine phosphorylation in control and diabetic rats indicates that this alternative insulin receptor substrate is not mediating the increased effects of insulin on c-fos mRNA. The augmentation of c-fos expression in the diabetic rats (6- to 7-fold) was substantially greater than the increase in receptor and IRS-1 tyrosine phosphorylation (approximately 2-fold), suggesting that myocardial insulin signaling might have been amplified between receptor phosphorylation and the steps that directly control c-fos transcription. Our data differ from a previous study (38), which reported a more marked insulin effect on c-fos expression in the myocardium of normal rats and no change in STZ-diabetes. The methodology in this previous study differed from ours in the use of CO2 asphyxiation instead of amobarbital anesthesia, and absence of a glucose infusion method to prevent insulin-induced hypoglycemia before the removal of tissues for c-fos analysis. It is possible that these or other factors may have led to an augmented effect of insulin on c-fos expression in the myocardium of control animals, such that it was not possible to visualize an increase in diabetes. In contrast to c-fos, we observed a much smaller increase in insulin-stimulated c-jun mRNA in diabetic myocardium, which did not reach statistical significance. It has previously been shown that the time course of c-fos phosphorylation in response to insulin differs from that of c-jun (21). This finding and our results indicate that insulin may regulate c-fos and c-jun through distinct pathways.

The STZ-diabetic rat is characterized by significant insulin insufficiency and hyperglycemia. A previous study from our laboratory, which used phlorizin to normalize blood glucose in STZ-diabetic rats, suggested that increased IRS-1 phosphorylation in skeletal muscle was the consequence of insulin insufficiency, rather than hyperglycemia (26). Other studies have shown that hyperglycemia, not a lack of insulin, appears to be responsible for decreased insulin actions on glucose metabolism in diabetic animals (41). Based on these reports, it is possible that augmented c-fos expression and perhaps other growth responses in diabetic myocardium are the result of insulin insufficiency, whereas diminished glycogen synthesis in response to insulin is the consequence of hyperglycemia.

A number of cardiac abnormalities have been described in both diabetic patients and animal models of experimental diabetes. In addition to accelerated vascular occlusive disease (42), it has been noted that significant myocardial contractile dysfunction may be present in humans and experimental animals with diabetes in the absence of atherosclerotic lesions (43, 44, 45). There is also evidence for increased vulnerability of the myocardium to injury during ischemia (45). Both abnormal glucose metabolism and aberrant growth responses in the myocardium in diabetes could contribute to the development of these functional abnormalities. The changes of glucose regulatory and growth regulatory pathways described in this study may thus have relevance to pathological responses in the myocardium in the diabetic state. Resistance to insulin effects on glucose regulatory pathways might limit myocardial glucose metabolism, which could lead to functional and cellular loss during periods of ischemia-related anaerobic metabolism in diabetic heart (4). In considering the potential significance of increased insulin-stimulated c-fos expression in diabetic myocardium, increased proto-oncogene expression has been associated with changes in myocardial contractile protein isoform expression in response to pressure overload (22, 23) and in transgenic animals overexpressing proto-oncogenes (46). Limited experimental data suggest that similar isoform switching may occur in diabetic myocardium. In STZ-diabetic rats, there is markedly diminished expression of creatine kinase-B compared with creatine kinase-M (29), and there also appears to be an increase in the content of the ß isoform of myosin heavy chain (47). In comparison with the {alpha} isoform of myosin heavy chain, the ß isoform has lower ATPase activity and a slower shortening velocity (48), and its increased expression could thus contribute to altered contractile properties of the diabetic myocardium. In future studies, it will be important to determine whether the observed increases in tyrosine phosphorylation of the insulin receptor and IRS-1, activation of PI 3-kinase, and expression of c-fos have a role in causing altered contractile protein isoform expression and diminished contractile properties of the heart in diabetes.

In summary, we have demonstrated significant alterations in insulin receptor signaling in vivo at initial, intermediate, and distal signaling steps in diabetic myocardium. We speculate that the oppositely directed changes in glucose regulatory and growth regulatory pathways that we have observed may contribute to the development of ventricular dysfunction in STZ-diabetic rats. The alterations of insulin signaling in diabetic myocardium are not irreversible because insulin replacement therapy corrected most of the abnormalities investigated in this study. Whether or not similar signaling changes occur in human diabetes remains to be determined.


   Footnotes
 
1 This study was supported in part by grants from the Adler Foundation and the National Institutes of Health (DK-43038, DK-48503, and Diabetes and Endocrinology Research Center Grant DK-36836). P.H.W. was supported by a fellowship award from Juvenile Diabetes Foundation International, F.G. by fellowships from the Juvenile Diabetes Foundation International and the Mary K. Iacocca Foundation, and K.C.M. by an American Diabetes Association Mentor-Based Fellowship (to R.J.S.). Back

2 Current address: Departments of Medicine and Biological Chemistry, Division of Endocrinology, Diabetes, and Metabolism, University of California, Irvine, California 92697-4086. Back

3 Current address: Department of Medicine, King Faisal Specialist Hospital and Research Center, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Back

4 Current address: Istituto di Clinica Medica, Endocrinologia e Malattie Metaboliche, University of Bari School of Medicine, Bari, Italy I-70124. Back

Received May 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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