Opposite Effects of Diabetes on Insulin Stimulation

In Vivo Insulin Signaling in the Myocardium ofStreptozotocin-Diabetic Rats: Opposite Effects ofDiabetes on Insulin Stimulation of Glycogen Synthaseand c-Fos*

PING H. WANG†, ABDULRAOF ALMAHFOUZ‡, FRANCESCO GIORGINO¶,KAREN C. MCCOWEN, AND ROBERT J. SMITH

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

ABSTRACTDiabetes induced by streptozotocin (STZ) in laboratory rats leads

to impaired glucose metabolism in the heart and changes in myocar-dial contractile protein isoform expression and cardiac function. Thepurpose of this study was to investigate in vivo insulin signalingresponses in the myocardium of STZ-diabetic rats. Insulin rapidlystimulated tyrosine phosphorylation of the insulin receptor, insulinreceptor substrate-1 (IRS-1) and, to a lesser extent, IRS-2 in normaland diabetic myocardium. In diabetic rats, there was 2-fold higherinsulin receptor content and insulin-stimulated receptor tyrosinephosphorylation in comparison with control rats. IRS-1 tyrosine phos-phorylation also increased in STZ diabetes in spite of a decrease inIRS-1 content, resulting in a 4-fold higher ratio of phosphorylated to

total IRS-1. This was associated with 2-fold higher IRS-1 precipitablephosphatidylinositide 3-kinase activity in diabetic animals. Insulinstimulation of glycogen synthase activity was significantly dimin-ished in STZ diabetes, consistent with resistance to insulin in a stepdownstream from phosphatidylinositide 3-kinase activation. Underthe same experimental conditions, there was a marked increase ininsulin stimulation of myocardial c-fos messenger RNA content indiabetic animals in comparison with controls. These data demon-strate altered early steps in insulin signaling in STZ-diabetic ratmyocardium. Consequent oppositely directed disturbances in growthregulatory and glucose regulatory responses to insulin may contributeto the development of myocardial functional abnormalities in thismodel of diabetes. (Endocrinology 140: 1141–1150, 1999)

INSULIN has important regulatory effects on myocardialmetabolism and growth. In cardiac muscle, metabolic

responses to insulin include the stimulation of glucose up-take (1), glycolysis (2), and glycogen synthesis (3). Althoughfatty acids normally serve as the principal fuel source in themyocardium, the utilization of glucose via anaerobic glyco-lytic pathways becomes an important source of metabolicenergy when oxygen availability is limited during periods ofischemia (4, 5). In uncontrolled diabetes mellitus, diminishedglucose uptake and metabolism secondary to insulin defi-ciency and/or insulin resistance may contribute to the de-velopment of myocardial dysfunction, particularly whenthere is coexistent ischemia (6). In addition to its effects on

myocardial glucose metabolism, insulin regulates cardiacmuscle growth. Specific effects of insulin observed in myo-cardial tissue and cultured cardiomyocytes include the stim-ulation of amino acid transport (7) and protein synthesis (8,9), the inhibition of protein degradation (10), and possibly thestimulation of DNA synthesis (11).

The biological actions of insulin in all target tissues aremediated by high affinity membrane-spanning insulin re-ceptors (12). Insulin binding to the insulin receptor a-subunitactivates a tyrosine kinase intrinsic to the cytoplasmic por-tion of the transmembrane b-subunit, which then undergoesautophosphorylation on tyrosine residues (13) and phos-phorylates nonreceptor proteins, including insulin receptorsubstrate-1 (IRS-1)1 (13), IRS-2 (14), and other substrates (15–17). These initial tyrosine phosphorylation events ultimatelytransmit the insulin signal to a branching series of intracel-lular pathways that regulate cellular metabolism, growthand differentiation. Substantial evidence indicates that theactivation of the enzyme phosphatidylinositide (PI) 3-kinase,through its association with tyrosine phosphorylated IRS-1,is essential for insulin effects on glucose uptake and glycogensynthesis (18, 19). Similarly the proto-oncogenes c-fos andc-jun have been identified as intermediates in pathways thatultimately lead to insulin-stimulated tissue growth re-sponses (20, 21). Both c-fos and c-jun have been shown to beinvolved in the regulation of cardiac muscle growth anddifferentiation by other stimuli (22, 23) and, thus, insulinstimulation of growth responses in cardiac muscle is likelyto involve c-fos and c-jun.

Treatment of rats with the b-cell toxin streptozotocin (STZ)

Received May 1, 1998.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: [email protected].

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

† Current address: Departments of Medicine and Biological Chem-istry, Division of Endocrinology, Diabetes, and Metabolism, Universityof California, Irvine, California 92697-4086.

‡ Current address: Department of Medicine, King Faisal SpecialistHospital and Research Center, P.O. Box 3354, Riyadh 11211, SaudiArabia.

¶ Current address: Istituto di Clinica Medica, Endocrinologia e Malat-tie Metaboliche, University of Bari School of Medicine, Bari, Italy I-70124.

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results in a diabetic state characterized by both insulin de-ficiency and insulin resistance (24). In skeletal muscle, al-though there is resistance to the effects of insulin on glucoseuptake and glycogen synthesis in STZ-diabetes (25), there isa paradoxical and yet unexplained increase in insulin-stim-ulated tyrosine phosphorylation of the insulin receptorb-subunit and IRS-1 (26, 27). Abnormalities in cardiac musclehave been demonstrated in STZ-diabetes, including defectsin the glucose transport system (28) and changes in contrac-tile protein isoform expression that may be related to com-promised cardiac contractile function (29). However, little isknown about the insulin signaling responses that may belinked to these abnormalities in the myocardium of diabeticrats.

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

Materials and MethodsAnimals

Male Sprague Dawley rats weighing approximately 200 g were pur-chased 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 animalswere subsequently allowed free access to standard rat chow and water,tail vein blood glucose was determined after 5 days with an AccuChecksystem (Boehringer Mannheim, Indianapolis, IN), and rats with non-fasting whole blood glucose .16.7 mm were selected for further study.These diabetic animals were either maintained untreated, or treatedtwice 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 withamobarbital after an overnight fast. A laparotomy was performed, anda 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 specifiedtime period, the cardiac ventricles were rapidly excised and frozen inliquid nitrogen. In longer duration experiments on proto-oncogene ex-pression, which involved up to 60 min insulin stimulation, 2.22 m glu-cose (2 ml in normal controls and 1 ml in diabetics) was given scimmediately after insulin injection to prevent hypoglycemia. Thesedoses of glucose were able to maintain stable blood glucose levels for atleast 2 h. All animal procedures performed in this study were approvedby the Joslin Diabetes Center Institutional Animal Care and UseCommittee.

Immunoprecipitation and immunoblotting

The frozen ventricles were powdered in a stainless steel mortar andpestle with liquid nitrogen and homogenized for 30 sec with a Polytron(Brinkmann Instruments, Westbury, NY) in ice-cold buffer containing 50mm Tris (pH 7.4), 10 mm sodium pyrophosphate, 10 mm sodium r-ni-trophenylphosphate, 2 mm EDTA, 2 mm phenylmethylsulfonyl fluoride(PMSF), 6 mg/ml leupeptin, 2 mm sodium orthovanadate, and 1% (vol/vol) Triton-X100. The homogenates were incubated on ice for 45 min andthen centrifuged at 55,000 3 g for 1 h at 4 C. Protein concentrations inthe resulting supernatants were determined by the Bradford methodusing BSA as a standard.

For direct immunoblotting studies, equal amounts of solubilizedventricular proteins (200–500 mg protein/lane) were separated by 7%SDS-PAGE. The resolved proteins were electrophoretically transferredto nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH)using a transfer buffer containing 192 mm glycine, 20% (vol/vol) meth-anol, 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 with5 g/dl BSA and 0.05% (vol/vol) Nonidet P-40 (NP-40) at 37 C for 2 h toreduce nonspecific binding, and then incubated overnight at 4 C withantiphosphotyrosine, antiinsulin receptor, or anti-IRS-1 antibodies. Af-ter washing twice with TNA buffer containing 0.05% NP-40 and oncewith TNA buffer containing 0.1% (vol/vol) Tween-20, the filters wereincubated at room temperature for 1 h with [125I]Protein A (1 mCi/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. Specificprotein bands were identified and quantified either by autoradiographyand densitometric analysis, or with a phosphorimaging system (Mo-lecular Dynamics, Inc., Sunnyvale, CA).

For sequential immunoprecipitation and immunoblotting studies,solubilized tissue proteins (1–5 mg) were incubated for 2 h at 4 C withantiinsulin receptor, anti-IRS-1, anti-IRS-2, or anti-Shc antibodies asindicated. The immunocomplexes were adsorbed to Protein A-Sepha-rose 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 bufferat pH 7.8 containing 150 mm NaCl, 1% NP-40, 1 mm sodium orthovana-date, and 1 mm PMSF. The washed immunoprecipitates were resus-pended in Laemmli buffer with 100 mm dithiothreitol, heated for 5 minat 100 C, and resolved by SDS-PAGE. After transfer onto nitrocellulosemembranes, the resulting blots were sequentially incubated with block-ing buffer and antiphosphotyrosine antibody, and specific tyrosinephosphorylated protein bands were identified and quantified with an-tiphosphotyrosine antibody and [125I]Protein A as described above fordirect immunoblotting studies.

The polyclonal antiphosphotyrosine and antirat insulin receptor an-tibodies 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 Dia-betes Center, Boston, MA) (14), and anti-Shc antibodies were from Up-state Biotechnology (Lake Placid, NY).

PI-3 kinase assay

IRS-1 associated PI-3 kinase activity was analyzed as previously de-scribed (31). In brief, ventricular muscle from basal or insulin-stimulatedstates 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 mmPMSF, and 6 mg/ml leupeptin. The homogenates were centrifuged, andthe supernatant incubated with anti-IRS-1 antibody at 4 C overnight. Theimmunocomplexes were precipitated after adsorption with Protein A-agarose beads and extensively washed. The activity of PI 3-kinase thatcoprecipitated with IRS-1 was determined by incubation of the resus-pended pellets with [g-32P]ATP and phosphatidylinositol, followed byseparation of the labeled PI-3 product by TLC, autoradiography, andlaser densitometry.

Glycogen synthase activity

Glycogen synthase activity was measured by a modification of apublished method based on the measurement of [14C]uridine diphos-phoglucose (UDPG) incorporation into glycogen (3). Frozen myocar-dium was pulverized in liquid nitrogen and homogenized with a Poly-tron homogenizer for 15 sec in a buffer containing 100 mm NaF and 10mm EDTA, pH 7.4. The homogenates were cleared by centrifugation at2,000 3 g for 20 min at 4 C, and glycogen synthase activity was deter-mined using aliquots of the supernatants. Equal amounts of the solublemyocardial proteins were reacted at 30 C for 30 min with a mixturecontaining 50 mm Tris, pH 7.8, 25 mm NaF, 20 mm EDTA, 10 mg/mlglycogen, 25 mm UDPG, and tracer [14C]UDPG with either 6 or 0.3 mmglucose-6-phosphate (G6P). The reaction products were blotted ontopieces of filter paper, washed three times with 66% (vol/vol) ethanol,and air dried. The amount of [14C]UDPG incorporated into glycogen wasdetermined by liquid scintillation counting, and the activity of glycogensynthase was calculated as the ratio of the G6P-independent form (mea-sured with 0.3 mm G6P) vs. the G6P-dependent form (measured with 6mm G6P).

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Northern blotting

The levels of c-fos and c-jun messenger RNA (mRNA) were assessedby Northern blotting. Total RNA from ventricular muscle was extractedessentially as described by Chomczynski and Sacchi (32). The quality ofRNA was assessed by the 260/280 absorbance ratio, and the majority ofthe RNA samples used in this study showed A260/A280 ratios $ 1.7.Equal amounts of RNA (15 mg/lane) were electrophoresed in 1.2%agarose-formaldehyde gels, transferred to Genescreen membranes (Du-Pont NEN, Boston, MA) by overnight blotting in 10 3 SSC, UV cross-linked and hybridized overnight at 42 C with [32P] labeled probes (0.5 3106 cpm/ml) in hybridization buffer containing 25 mm Tris, pH 7.4, 1 mNaCl, 10 g/dl dextran sulfate, 50% formamide, 1% SDS, and 50 mg/mlsalmon 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 bymultiprime random labeling with Klenow fragments. The filters werewashed twice at room temperature (1% SDS and 1 3 SSC, 30 min), onceat 50 C (1% SDS and 0.2 3 SSC, 20 min), twice at 60 C (0.1% SDS and0.2 3 SSC, 20 min), air dried, and analyzed by autoradiography and laserscanning densitometry.

Statistical analysis

Data are presented as mean 6 sem, and statistical significance wasevaluated by Student’s t test.

ResultsCharacteristics of experimental animals

The plasma glucose and body weight determinations inthe experimental animals are summarized in Table 1. Controlrats gained an average of 74.5 g and maintained normalblood glucose levels during the course of the study. In con-trast, untreated diabetic rats lost an average of 26.3 g by theend of study, and their blood glucose levels were signifi-cantly higher than in the controls (19.8 6 0.8 vs. 4.4 6 0.2 mm,P , 0.001). After insulin deficiency was corrected with in-sulin replacement (3–9 U Ultralente, twice daily) for 5 days,fasting blood glucose levels were markedly reduced intreated diabetic rats (8.6 6 1.6 mm) and, compared with theuntreated diabetic rats, significant weight gain was observed(41.0 vs. 226.3 g, P , 0.001).

Insulin-stimulated protein tyrosine phosphorylation in vivo

To investigate initial events in insulin signaling in themyocardium, overnight-fasted control rats were anesthe-tized and given a bolus injection of insulin into the inferiorvena cava. At defined time points, the cardiac ventricles wererapidly removed, frozen in liquid nitrogen, and homoge-nized in a buffer containing nonionic detergent and inhibi-tors of proteolysis and dephosphorylation. The solubilized

myocardial proteins were resolved by SDS-PAGE, and ty-rosine phosphoproteins were identified by immunoblottingwith phosphotyrosine antibody. In vivo insulin injection ledto rapid tyrosine phosphorylation of a 98-kDa protein bandand two additional bands migrating at approximately 170–180 kDa. Figure 1 shows that tyrosine phosphorylation ofthese three protein bands was stimulated by insulin injectionat a dose as low as 0.2 U/kg body wt and was maximal withinsulin at doses $ 1.0 U/kg. For subsequent studies, insulinwas administered at the supramaximal dose of 10 U/kg bodywt. Tyrosine phosphorylation of the myocardial proteinsreached a maximum within one minute after in vivo insulininjection (data not shown), as previously demonstrated forother tissues (26, 27).

The identities of the three insulin-stimulated tyrosinephosphoproteins were confirmed by immunoprecipitationwith specific antibodies. Preparations of myocardial proteinswere first immunoprecipitated with antiinsulin receptor, an-ti-IRS-1, or anti-IRS-2 antibodies, and the resulting immu-noprecipitates were resolved by SDS-PAGE and immuno-blotted with antiphosphotyrosine antibody. As shown in Fig.2, sequential immunoprecipitation and immunoblotting con-firmed that the 98-kDa band represents the insulin receptorb-subunit, whereas the 170 kDa and 180 kDa bands, respec-tively, correspond to IRS-1 and IRS-2. It should be noted thatthe similar intensities of the insulin-stimulated IRS-1 andIRS-2 bands in Fig. 2 do not necessarily reflect the relativeabundance of these tyrosine phosphorylated proteins in themyocardium because immunoprecipitation was performedwith different antibodies. In further studies comparing con-trol and diabetic rats, we focused on the insulin receptor and170 kDa IRS-1 bands because they demonstrated the mostmarked and consistent insulin-stimulated tyrosine phos-phorylation. Insulin stimulation of tyrosine phosphorylationof the 180 kDa IRS-2 band assessed by direct phosphoty-rosine antibody blotting was variable, as is evident in Fig. 1.

To determine the effects of STZ-diabetes on insulin recep-

FIG. 1. Dose-response effects of insulin on the tyrosine phosphory-lation of myocardial proteins. Insulin at the indicated doses wasinjected in vivo via the inferior vena cava, and cardiac ventriculartissue was removed after 1 min. Solubilized myocardial proteins wereresolved by 7% SDS-PAGE, and tyrosine phosphoproteins were de-tected by immunoblotting with antiphosphotyrosine antibody. Eachlane represents an individual rat.

TABLE 1. Animal characteristics

GroupBody Wt (g) Blood glucose

(mM)

Initial Final Day 5 Day 11

Control 201.4 6 4 275.9 6 3 4.4 6 0.2Untreated diabetes 196.6 6 3 170.3 6 8a 19.8 6 0.8 16.2 6 1.1Treated diabetes 197.6 6 4 238.6 6 11b 20.7 6 0.6 8.6 6 1.6b

Rats labeled “Treated diabetes” were given 3–9 U of Ultralenteinsulin SC twice daily from days 5 through 10. Blood glucose wasmeasured in the overnight fasted state 5 days after streptozotocininjection and at the time animals were killed.

N 5 16 rats/group, aP , 0.001 vs. control, bP , 0.001 vs. diabetes.

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tor and IRS-1 tyrosine phosphorylation, control, untreateddiabetic, and diabetic rats treated twice daily for 5 days withsc insulin were fasted overnight and given a bolus injectionof either 10 U insulin/kg body wt or vehicle. After 1 min, thecardiac ventricles were removed and frozen in liquid nitro-gen, myocardial proteins were solubilized, and tyrosinephosphoproteins were identified by phosphotyrosine anti-body immunoblotting. Basal tyrosine phosphorylation ofIRS-1 was significantly decreased in untreated diabetic com-pared with control rats (31.8 6 7.9% lower in diabetic thancontrol, P , 0.05), and mean basal phosphorylation of theinsulin receptor also was lower in diabetic myocardium(32.0 6 13.4% lower in diabetes), although this difference wasnot significant (P 5 0.098, data not shown). We previouslyhave reported that overnight fasting and insulin withdrawalin treated diabetic animals results in basal receptor and IRS-1phosphorylation similar to that in untreated diabetics (26).Following acute insulin stimulation, tyrosine phosphoryla-tion of IRS-1 and the insulin receptor b-subunit was mark-edly increased in untreated diabetic compared with controlanimals (Fig. 3). This is similar to the increase of in vivoinsulin-stimulated IRS-1 and receptor tyrosine phosphory-lation in STZ-diabetic rats reported for skeletal muscle andliver (26, 27). In diabetic animals treated with insulin for 5days, acute insulin-stimulated IRS-1 and insulin receptortyrosine phosphorylation returned to levels similar to thosein the nondiabetic controls (Fig. 3).

To investigate a potential alternative pathway of insulinsignaling, tyrosine phosphorylation of the Shc proteins (33)was determined in the myocardium of control and diabeticrats. Myocardial protein extracts prepared 10 min after theinfusion of insulin or vehicle as described above were sub-jected to immunoprecipitation with anti-Shc antibody fol-lowed by immunoblotting with antiphosphotyrosine anti-

body. As shown in Fig. 4, basal tyrosine phosphorylation ofthe 52 and 46 kDa Shc isoforms was evident, but there wasno stimulation following insulin injection in control or dia-betic rats. Phosphorylation of the 66 kDa Shc isoform wasessentially undetectable in all animals.

To further define the changes in early steps in insulinreceptor signaling in diabetic myocardium, the abundanceand tyrosine phosphorylation of the insulin receptor b-sub-unit and IRS-1 were quantified by densitometric analysis ofimmunoblots from multiple control, untreated STZ-diabeticand treated diabetic rats, and the overall stoichiometry ofphosphorylation of these proteins was determined. Receptorphosphorylation, assessed by immunoblotting with an-tiphosphotyrosine antibody, increased 2-fold in diabeticmyocardium (Fig. 5A). After diabetic rats received insulinreplacement therapy for 5 days to correct insulin deficiencyand hyperglycemia, the level of receptor tyrosine phosphor-ylation stimulated by insulin returned to that of the controls.The total myocardial content of insulin receptors, deter-mined by immunoblotting with insulin receptor antibody,also increased 2-fold in diabetic myocardium (Fig. 5B), andthe amount of receptor protein was restored to the controllevel after the diabetic rats received insulin replacement ther-apy. Therefore, the ratio of receptor tyrosine phosphoryla-tion/receptor content was not altered in the myocardium ofuntreated or treated diabetic rats (Fig. 5C). The amount oftyrosine phosphorylated insulin receptor increased in dia-betes, but the overall stoichiometry of receptor autophos-phorylation remained unchanged.

Similar quantitative analysis of IRS-1 demonstrated that itsphosphorylation also was increased approximately 2-fold indiabetic myocardium and returned to the control level afterthe diabetic rats had received insulin replacement therapy(Fig. 5D). However, the content of IRS-1 decreased in diabeticrats, and insulin replacement therapy failed to restore thelevel of IRS-1 protein back to that of the controls (Fig. 5E).Thus, the amount of tyrosine phosphorylated IRS-1 per totalamount of IRS-1 increased 4-fold in the myocardium of un-treated diabetic rats and was persistently (5-fold) increasedin the myocardium of insulin-treated diabetic rats (Fig. 5F).The basis for the increased IRS-1 tyrosine phosphorylation indiabetic myocardium is not known, but it could be a con-sequence 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 downstreamfrom IRS-1 tyrosine phosphorylation is the activation of theenzyme PI 3-kinase, resulting from the noncovalent bindingof its regulatory subunit to phosphotyrosine residues inIRS-1 (31). To determine whether the changes in receptorb-subunit and IRS-1 tyrosine phosphorylation in the diabeticmyocardium affect subsequent steps in the insulin signalingpathway, the activity of PI 3-kinase associated with IRS-1 wasanalyzed in protein extracts from cardiac ventricular muscleof control and diabetic rats obtained 10 min after stimulationby in vivo insulin injection. Solubilized proteins from ven-tricular muscle were subjected to immunoprecipitation with

FIG. 2. Identification of insulin-stimulated tyrosine phosphoproteinsin myocardium. Proteins solubilized from the myocardium of ratsunder basal conditions or stimulated with insulin in vivo for 2 minwere immunoprecipitated with antiinsulin receptor, anti-IRS 1, oranti-IRS 2 antibodies, and then immunoblotted with antiphosphoty-rosine antibody.


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IRS-1 antibody, and PI 3-kinase activity in the reconstitutedpellets was assayed by measuring the transfer of [32P] fromATP to the 3-position of phosphatidylinositol (31). Followinginsulin injection, 3-phosphoinositol formation was markedlyincreased in IRS-1 immunoprecipitates from control and di-abetic myocardium (representative data from control ratsshown in Fig. 6A). Quantitative analysis of multiple exper-iments demonstrated that the level of insulin-stimulated PI3-kinase activity was increased 2-fold in untreated diabeticcompared with control myocardium (P , 0.01) and returnedto the level of the controls in insulin-treated diabetic rats (Fig.6B). In the absence of insulin stimulation, basal IRS-1-asso-ciated PI 3-kinase activity was barely detectable in controland diabetic animals, reflecting the low insulin levels fol-lowing overnight fasting. In a subset of animals (n 5 4), basalPI 3-kinase was quantified and shown to be decreased by85.3 6 5.2% (P , 0.001) in diabetic rats in comparison withcontrols). Thus, both the absolute level of insulin-stimulatedIRS-1-associated PI 3-kinase activity, and the fold effect ofinsulin on PI 3-kinase were higher in the STZ-diabetic rats.These data demonstrate that an increase in insulin stimula-tion of IRS-1-associated PI 3-kinase activity in diabetic myo-cardium parallels the increase in IRS-1 tyrosine phosphor-ylation. The correction of hyperglycemia by insulinreplacement therapy restores both insulin-stimulated PI 3-ki-

nase activity, and IRS-1 tyrosine phosphorylation to the con-trol level.

Glycogen synthase activity

Significant resistance to the effects of insulin on glucoseregulatory pathways has previously been noted in insulintarget tissues of STZ-diabetic rats (24, 28, 35, 36). To assessin vivo insulin effects on a downstream glucose regulatorypathway in this study, the conversion of glycogen synthasefrom the G6P-dependent D form to the active G6P-indepen-dent I form following insulin administration in vivo wasdetermined in myocardium from control and STZ-diabeticrats. For these and subsequent studies on c-fos and c-junexpression (see below), an additional series of STZ-diabeticrats was prepared. The expected increase in insulin-stimu-lated receptor and IRS-1 tyrosine phosphorylation was con-firmed, and the general characteristics of the animals (bloodglucose levels, body wt) were similar to those of the pre-ceding group of rats used for the tyrosine phosphorylationand PI 3-kinase studies (data not shown). Insulin stimulationof glycogen synthase activity, as assessed by the percentagein the G6P-independent I form reached a maximum within10 min of in vivo injection of insulin. A comparison of basaland 10-min insulin-stimulated glycogen synthase activitiesin normal and diabetic myocardium is shown in Fig. 7. In-sulin increased the percentage of I form glycogen synthaseapproximately 2-fold in the normal myocardium (P , 0.001),whereas insulin had no effect on the percentage of I formglycogen synthase in diabetic myocardium (P 5 0.501). Thus,stimulation of glycogen synthase by insulin was markedlyimpaired in diabetic myocardium, confirming the results ofprevious studies on glycogen synthase regulation in STZ-diabetes (3). Because tyrosine phosphorylation of the insulinreceptor and IRS-1 was increased in the myocardium of di-abetic rats under the same experimental conditions, thesedata indicate that STZ-diabetes leads to oppositely directedchanges in the proximal insulin signaling pathways and themore distal glycogen synthase pathway. Glycogen synthesisregulation is believed to be downstream from and mediatedby IRS-1 tyrosine phosphorylation (37), and these findingstherefore support the concept that the site of resistance toinsulin that leads to compromised glycogen synthase activity

FIG. 3. Comparison of in vivo insulin-stimulated tyrosine phosphorylation ofmyocardial proteins in control, un-treated diabetic, and treated diabeticrats. In the treated diabetic group, in-sulin was administered twice daily for 5days as described in Materials andMethods. Animals in all groups werefasted overnight, given an acute bolusinjection of insulin (10 U/kg body wt) viathe inferior vena cava and, after 1 min,the cardiac ventricles were removedand frozen in liquid nitrogen. Myocar-dial proteins were solubilized, resolvedby SDS-PAGE, and immunoblottedwith antiphosphotyrosine antibody.Each lane represents myocardial pro-teins extracted from an individual rat.

FIG. 4. Determination of insulin effects on Shc tyrosine phosphory-lation in control and diabetic rats. After an overnight fast, animalswere anesthetized and given an injection of insulin (10 U/kg body wt)via the inferior vena cava. After 10 min, the cardiac ventricles wereremoved and frozen in liquid nitrogen. Shc tyrosine phosphorylationin myocardial protein extracts was determined by immunoprecipita-tion with anti-Shc antibody followed by immunoblotting with an-tiphosphotyrosine antibody.


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in this model of diabetes is downstream from insulin receptorcatalyzed IRS-1 tyrosine phosphorylation.

Expression of c-fos and c-jun

To investigate downstream insulin signaling effects ongrowth regulatory pathways in the diabetic myocardium,mRNA levels for the c-fos and c-jun proto-oncogenes weredetermined. Insulin or vehicle was injected in vivo via theinferior vena cava, cardiac ventricular muscle was removedat various time points, and total RNA was extracted forNorthern blotting analysis with specific c-fos or c-jun probes.As noted in the Materials and Methods section, insulin-in-duced hypoglycemia was prevented in these experiments,

which involved longer periods of insulin stimulation, by thesc injection of a 2.22 m glucose solution. Figure 8 showsrepresentative Northern blotting data, which demonstrate amodest increase in c-fos and little or no change in c-junmRNA 30 min after insulin injection in overnight fastedcontrol rats (left panel). In STZ-diabetic rats (right panel), amarked augmentation of c-fos mRNA and an apparent moremodest increase in c-jun mRNA is evident 30 min after in-sulin injection. The results of quantitative densitometric anal-ysis of myocardial c-fos and c-jun mRNA from multiple ratsstudied 15, 30, or 60 min after insulin injection are shown inFig. 9. The expression of c-fos was increased by approxi-mately 40% in the normal myocardium 30 min after insulin

FIG. 5. Quantitation of insulin-stimulated insulin receptor b-subunit and IRS-1 tyrosine phosphorylation, protein content, and phosphory-lation/protein in the myocardium of control and diabetic rats. Diabetic animals were either untreated or treated with Ultralente insulin for 5days as described in Materials and Methods. After an overnight fast, an acute bolus of insulin (10 U/kg body wt) was injected into the venacava and, after 2 min, the cardiac ventricles were removed and frozen in liquid nitrogen. Tyrosine phosphorylation of the insulin receptor wasdetermined by immunoblotting with antiphosphotyrosine antibody (A), the total amount of insulin receptor b-subunit was determined byimmunoblotting 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 ofIRS-1 was determined by immunoblotting with anti-IRS-1 antibody (E), and the ratio of phosphorylation/IRS-1 was determined for eachindividual rat (F). Data represent mean 6 SEM for 9 animals (insulin receptor) or 16 animals (IRS-1) in each group: *, P , 0.001 vs. controland 5-day insulin-treated diabetes; **, P , 0.001 vs. control.


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injection (P , 0.001). In the normal controls, there was nodifference in c-fos mRNA levels between 30 and 60 min ofinsulin stimulation (P 5 0.11). In the basal (unstimulated)state, there was a modest increase of c-fos mRNA in themyocardium of diabetic compared with control rats (74%above control, P , 0.001). Insulin injection in the diabetic ratsled to a much greater (6- to 7-fold) and more rapid increase(peak at 15 to 30 min) in c-fos mRNA levels in comparisonwith control rats. The content of c-jun mRNA was not alteredby insulin in control rats, and basal levels of c-jun mRNAwere similar in diabetic and control rats. The mean insulin-stimulated c-jun mRNA levels were higher in diabetic ratmyocardium 15 and 30 min after stimulation, but these val-ues did not reach statistical significance.

To determine whether correction of hyperglycemia and

insulin deficiency can reverse the augmentation of insulin-stimulated c-fos expression in diabetic myocardium, a subsetof diabetic rats was given insulin replacement therapy asdescribed earlier. The basal c-fos mRNA levels were similarin treated and untreated diabetic rats (P 5 0.335), but theincrease in insulin-stimulated c-fos expression observed inuntreated diabetic rats was completely reversed after 5 daysof insulin replacement therapy (Fig. 10, left panel). The meanvalues for c-jun mRNA showed a similar decrease to thecontrol 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 statisticallysignificant (Fig. 10, right panel). These data demonstrate anaugmentation of insulin effects on c-fos and possibly c-jungene expression in the myocardium of STZ-diabetic rats thatcorrelates with the observed increases in receptor and IRS-1tyrosine phosphorylation. In contrast to the resistance to

FIG. 6. Activation of IRS-1 associatedPI-3 kinase by insulin in normal anddiabetic myocardium. After in vivo in-sulin stimulation, myocardial proteinswere immunoprecipitated with anti-IRS-1 antibody, and PI 3-kinase activ-ity in the pellet was determined by mea-suring the incorporation of [32P] into the3-position of phosphatidylinositol. A,Representative thin layer chromato-gram demonstrating basal and insulin-stimulated 3-phosphoinositol formation(designated PIP) in IRS-1 immunopre-cipitates from normal rat heart. B,Quantitation of insulin-stimulated PI3-kinase activity from multiple experi-ments in control and diabetic rats. Di-abetic animals were either untreated,or treated with Ultralente insulin for 5days as described in Materials andMethods. Data represent mean 6 SEMfor 16 animals in each group: *, P ,0.001 vs. control and 5-day insulin-treated diabetes.

FIG. 7. Myocardial glycogen synthase activity in control and diabeticrats. Insulin (10 U/kg body wt) (solid bars) or vehicle (open bars) wasinjected into the inferior vena cava, the heart was removed after 10min, and the activity of glycogen synthase was measured in a solublemyocardial protein fraction in the presence of 0.3 mM G6P (I form) and6 mM G6P (total I 1 D forms) as described in Materials and Methods.Data represent the I form of glycogen synthase as % of total from fourseparate experiments analyzed in triplicate: *, P , 0.001 vs. basal.

FIG. 8. Northern blots of c-fos and c-jun mRNA in control and diabeticmyocardium. The cardiac ventricles were removed 30 min after in vivoinjection of insulin (10 U/kg body wt) or vehicle, and total RNA wasextracted and analyzed by Northern blotting. Each lane contains 15mg of total myocardial RNA from an individual rat.


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insulin effects on glycogen synthase activation, STZ-diabetesresults in increased insulin effects on proto-oncogeneexpression.

Discussion

This study provides the first characterization of early sig-naling responses to insulin in the myocardium of normal andSTZ-diabetic rats in vivo. Insulin stimulation of tyrosinephosphorylation of the insulin receptor and its substrateIRS-1 was demonstrated in the normal rat heart and shownto be significantly augmented in diabetic myocardium.Downstream responses, including activation of PI-3 kinaseand stimulation of c-fos and possibly c-jun gene expressionby insulin also were increased in diabetic myocardium,whereas insulin activation of glycogen synthase was atten-uated. These results suggest that the induction of STZ-dia-betes may have opposite effects on distal glucose regulatoryand growth regulatory pathways of insulin signaling in car-diac muscle.

Although this represents the first demonstration of oppo-site effects on c-fos expression and glycogen synthase acti-vation in the myocardium, and the first characterization ofthese opposite effects of STZ-diabetes in any tissue within asingle experimental study, previous work suggests that dif-ferential effects of diabetes on glucose regulatory and growthregulatory pathways also exist in other tissues. For example,published studies have shown impaired insulin-mediatedglucose metabolism in skeletal muscle (24, 36) as well ascardiac muscle (3, 28) of STZ-diabetic rats. Work from ourgroup and others has demonstrated increased insulin-stim-ulated receptor and IRS-1 phosphorylation in skeletal muscleand liver of diabetic rats (26, 27), and augmented insulinstimulation of c-fos expression has been observed in adiposetissue in STZ-diabetes (38). A human patient with a rare formof extreme insulin resistance and acromegalic features hasbeen described with resistance to the effects of insulin onglucose transport in cultured fibroblasts, but normal insulineffects on amino acid uptake (39). Thus, selective resistanceto the effects of insulin on glucose regulatory pathways butnot other pathways can occur in both experimental animalsand humans.

Tyrosine phosphorylation of the insulin receptor and IRS-1represent initial steps of insulin signal transduction. Becausemutations in the insulin receptor gene affecting tyrosine res-idues or the tyrosine kinase domain are associated with de-creased insulin effects on both glycogen synthase activityand thymidine incorporation (37), it appears that the auto-phosphorylation of the insulin receptor and activation of itsintrinsic tyrosine kinase initiate insulin signaling events thatlead to glucose regulatory and growth regulatory pathways(40). In this study, because tyrosine phosphorylation of theinsulin receptor and IRS-1 was not decreased in the diabeticmyocardium, it can be concluded that the site of insulinresistance responsible for decreased glycogen synthase ac-tivation is likely to involve step(s) distal to the insulin re-ceptor and IRS-1. Current evidence suggests that insulinstimulation of glycogen synthase involves a PI 3-kinase de-pendent pathway (18), and our data demonstrated an aug-mented insulin effect on PI-3 kinase activation in diabeticmyocardium, further suggesting that the insulin resistance inSTZ-diabetes involves signaling intermediates downstreamfrom PI 3-kinase. Although there is little information aboutnegative feedback mechanisms in the postreceptor insulinsignaling cascade, it is possible that resistance to insulin at adistal glucose-regulatory step might somehow disrupt a neg-ative feedback response and contribute to reciprocal en-hancement of insulin signaling at initial and intermediatesteps of the signaling pathway.

In the diabetic myocardium, increased insulin-stimulatedc-fos expression correlated with enhanced tyrosine phos-phorylation of the insulin receptor and IRS-1. Maximumtyrosine phosphorylation of the myocardial insulin receptorand IRS-1 occurred within one minute after in vivo insulininjection, whereas the level of c-fos mRNA was not fullystimulated until 15–30 min later. Thus, in the diabetic myo-cardium, the augmentation of insulin-stimulated c-fos geneexpression is likely to be a response secondary to increasedinsulin receptor kinase activity and possibly secondary toincreased IRS-1 tyrosine phosphorylation. An absence of in-sulin-stimulated Shc tyrosine phosphorylation in control anddiabetic rats indicates that this alternative insulin receptorsubstrate is not mediating the increased effects of insulin on

FIG. 9. Time-course of the effects of in-sulin on mRNA levels of c-fos (left panel)and c-jun (right panel) in control (openbars) and diabetic (solid bars) myocar-dium. Cardiac ventricles were removedat the indicated times after in vivo in-jection of insulin (10 U/kg body wt), andtotal RNA was extracted and analyzedby Northern blotting. The levels of c-fosand c-jun mRNA were quantitated bylaser densitometry. Data representpooled results from three to four exper-iments in triplicate: *, P , 0.001 vs. 0time control, **, P , 0.001 vs. control(nondiabetic) animals at the indicatedtime points.


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c-fos mRNA. The augmentation of c-fos expression in thediabetic rats (6- to 7-fold) was substantially greater than theincrease in receptor and IRS-1 tyrosine phosphorylation (ap-proximately 2-fold), suggesting that myocardial insulin sig-naling might have been amplified between receptor phos-phorylation and the steps that directly control c-fostranscription. Our data differ from a previous study (38),which reported a more marked insulin effect on c-fos ex-pression in the myocardium of normal rats and no change inSTZ-diabetes. The methodology in this previous study dif-fered from ours in the use of CO2 asphyxiation instead ofamobarbital anesthesia, and absence of a glucose infusionmethod to prevent insulin-induced hypoglycemia before theremoval of tissues for c-fos analysis. It is possible that theseor other factors may have led to an augmented effect ofinsulin on c-fos expression in the myocardium of controlanimals, such that it was not possible to visualize an increasein diabetes. In contrast to c-fos, we observed a much smallerincrease in insulin-stimulated c-jun mRNA in diabetic myo-cardium, which did not reach statistical significance. It haspreviously been shown that the time course of c-fos phos-phorylation in response to insulin differs from that of c-jun(21). This finding and our results indicate that insulin mayregulate c-fos and c-jun through distinct pathways.

The STZ-diabetic rat is characterized by significant insulininsufficiency and hyperglycemia. A previous study from ourlaboratory, which used phlorizin to normalize blood glucosein STZ-diabetic rats, suggested that increased IRS-1 phos-phorylation in skeletal muscle was the consequence of in-sulin insufficiency, rather than hyperglycemia (26). Otherstudies have shown that hyperglycemia, not a lack of insulin,appears to be responsible for decreased insulin actions onglucose metabolism in diabetic animals (41). Based on thesereports, it is possible that augmented c-fos expression andperhaps other growth responses in diabetic myocardium arethe result of insulin insufficiency, whereas diminished gly-cogen synthesis in response to insulin is the consequence ofhyperglycemia.

A number of cardiac abnormalities have been described inboth diabetic patients and animal models of experimentaldiabetes. In addition to accelerated vascular occlusive dis-ease (42), it has been noted that significant myocardial con-

tractile dysfunction may be present in humans and experi-mental animals with diabetes in the absence ofatherosclerotic lesions (43–45). There is also evidence forincreased vulnerability of the myocardium to injury duringischemia (45). Both abnormal glucose metabolism and aber-rant growth responses in the myocardium in diabetes couldcontribute to the development of these functional abnormal-ities. The changes of glucose regulatory and growth regu-latory pathways described in this study may thus have rel-evance to pathological responses in the myocardium in thediabetic state. Resistance to insulin effects on glucose regu-latory pathways might limit myocardial glucose metabolism,which could lead to functional and cellular loss during pe-riods of ischemia-related anaerobic metabolism in diabeticheart (4). In considering the potential significance of in-creased insulin-stimulated c-fos expression in diabetic myo-cardium, increased proto-oncogene expression has been as-sociated with changes in myocardial contractile proteinisoform expression in response to pressure overload (22, 23)and in transgenic animals overexpressing proto-oncogenes(46). Limited experimental data suggest that similar isoformswitching may occur in diabetic myocardium. In STZ-dia-betic rats, there is markedly diminished expression of crea-tine kinase-B compared with creatine kinase-M (29), andthere also appears to be an increase in the content of the bisoform of myosin heavy chain (47). In comparison with thea isoform of myosin heavy chain, the b isoform has lowerATPase activity and a slower shortening velocity (48), and itsincreased expression could thus contribute to altered con-tractile properties of the diabetic myocardium. In futurestudies, it will be important to determine whether the ob-served increases in tyrosine phosphorylation of the insulinreceptor and IRS-1, activation of PI 3-kinase, and expressionof c-fos have a role in causing altered contractile proteinisoform expression and diminished contractile properties ofthe heart in diabetes.

In summary, we have demonstrated significant alterationsin insulin receptor signaling in vivo at initial, intermediate,and distal signaling steps in diabetic myocardium. We spec-ulate that the oppositely directed changes in glucose regu-latory and growth regulatory pathways that we have ob-served may contribute to the development of ventricular

FIG. 10. The effects of chronic (5 days)insulin therapy on myocardial c-fos andc-jun expression. Insulin (10 U/kg bodywt) or vehicle (Basal) was injected intothe inferior vena cava of overnightfasted rats, and myocardium was iso-lated 30 min later for RNA extraction.The expression of c-fos and c-jun in con-trol (open bars), untreated diabetic (sol-id bars), and 5-day insulin-treated dia-betic (hatched bars) rats was assessedby Northern blotting and quantitatedby laser densitometry. Data representpooled results from three experimentsin triplicate: *, P , 0.001 vs. control and5-day insulin-treated diabetic.


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dysfunction in STZ-diabetic rats. The alterations of insulinsignaling in diabetic myocardium are not irreversible be-cause insulin replacement therapy corrected most of the ab-normalities investigated in this study. Whether or not similarsignaling changes occur in human diabetes remains to bedetermined.

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Opposite Effects of Diabetes on Insulin Stimulation

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