Cardiac failure is a major cause of death in patients with type 2 diabetes, but the molecular mechanism that links diabetes to heart failure remains unclear. Insulin resistance is a hallmark of type 2 diabetes, and insulin receptor substrates 1 and 2 (IRS1 and IRS2) are the major insulin-signaling components regulating cellular metabolism and survival. To determine the role of IRS1 and IRS2 in the heart and examine whether hyperinsulinemia causes myocardial insulin resistance and cellular dysfunction via IRS1 and IRS2, we generated heart-specific IRS1 and IRS2 gene double-knockout (H-DKO) mice and liver-specific IRS1 and IRS2 double-knockout (L-DKO) mice. H-DKO mice had reduced ventricular mass; developed cardiac apoptosis, fibrosis, and failure; and showed diminished Akt→forkhead box class O-1 signaling that was accompanied by impaired cardiac metabolic gene expression and reduced ATP content. L-DKO mice had decreased cardiac IRS1 and IRS2 proteins and exhibited features of heart failure, with impaired cardiac energy metabolism gene expression and activation of p38α mitogen-activated protein kinase (p38). Using neonatal rat ventricular cardiomyocytes, we further found that chronic insulin exposure reduced IRS1 and IRS2 proteins and prevented insulin action through activation of p38, revealing a fundamental mechanism of cardiac dysfunction during insulin resistance and type 2 diabetes.
Diabetes promotes cardiac failure and increases patient morbidity and mortality, with two-thirds of patients with type 2 diabetes dying of heart failure (1). In the patient population, 90–95% are type 2 patients with diabetes in whom insulin resistance is the primary contributor to metabolic and cardiac dysfunction (1). Intensive insulin therapy increases the risk of cardiovascular dysfunction and the death rate by twofold in patients with type 2 diabetes (Action to Control Cardiovascular Risk in Diabetes trial) (2). Thus, understanding the mechanisms responsible for insulin action, resistance, and related cardiac dysfunction will be critical for the development of new strategies for treatment of heart failure.
The heart is an insulin-responsive and energy consuming organ that requires a constant fuel supply to maintain intracellular ATP for myocardial contraction (3). Upon binding to the cell surface receptor, insulin activates its receptor tyrosine kinase, which phosphorylates and recruits insulin receptor (IR) substrates (IRS) 1 to 4 (IRS1, IRS4), and other scaffold proteins, including SHC, CBL, APS, SH2B, GAB1, and DOCK1, that trigger downstream signaling cascades, including phosphatidylinositide 3-kinase (PI-3K) and mitogen-activated protein kinases (MAPKs) (4–6). Activation of PI-3K generates phosphatidylinositol(3-5)-triphosphate (PIP3), recruiting the 3-phosphoinositide–dependent protein kinase-1 and -2 (PDK1 and PDK2) and Akt to the plasma membrane, where Akt is activated by PDK1-mediated phosphorylation at T308 and PDK2-mediated phosphorylation at S473 (7,8). Akt phosphorylates downstream targets, including inhibitors of macromolecular synthesis, such as glycogen synthase kinase-3β (Gsk3β, glycogen synthesis), tuberous sclerosis protein-2 (Tsc2), and p70S6K (protein synthesis), and forkhead transcription factor forkhead box class O1 (Foxo1) (gene transcription). Akt phosphorylates Foxo1 at S253 and inhibits transcriptional activity of Foxo1, which regulates a variety of physiological functions such as energy metabolism (9,10), myocardial growth (11–13), and survival (14). Thus, Akt→Foxo1 phosphorylation mediates the action of insulin and serves as an indicator of insulin sensitivity (5,15).
Systemic IRS1-null mice display growth retardation and develop peripheral insulin resistance mainly in skeletal muscle but not diabetes, owing to IRS2-depedent pancreatic β-cell growth and compensatory insulin secretion (16). Systemic IRS2-null mice display metabolic defects in liver, muscle, and adipose tissues but develop diabetes owing to pancreatic β-cell failure (17). We recently demonstrated that deletion of both IRS1 and IRS2 genes in the liver of L-DKO mice prevents activation of hepatic Akt→Foxo1 phosphorylation and results in the development of diabetes (5,18) and that deletion of both IRS1 and IRS2 in cardiac and skeletal muscle causes heart failure and death of animals at the age of 2 to 3 weeks (19). These results indicate that IRS1 and IRS2 are major mediators of insulin action to support physiological functions in many organs.
In this study, we deleted both IRS1 and IRS2 genes exclusively in the hearts of mice and determined cardiac function. We further examined the regulatory mechanisms for the myocardial loss of IRS1 and IRS2 in mouse models with insulin resistance. In particular, we tested the hypothesis that hyperinsulinemia—chronic or prolonged insulin exposure—can result in myocardial insulin resistance by suppressing IRS1 and IRS2.
RESEARCH DESIGN AND METHODS
Procedures used in animal experiments were approved by the Texas A&M Health Science Center Institutional Animal Care and Use Committee. The floxed IRS1 mice (IRS1L/L), IRS2 mice (IRS2L/L), albumin-Cre mice, and liver-specific IRS1 and IRS2 gene double-knockout (L-DKO) mice (albumin-Cre:: IRS1L/L::IRS2L/L) have been previously described (5). MHC-Cre mice were purchased from The Jackson Laboratory (Bar Harbor, ME; stock #9074) to breed to IRS1L/L::IRS2L/L mice, generating heart-specific IRS1 and IRS2 gene double-knockout (H-DKO) mice (MHC-Cre::IRS1L/L::IRS2L/L), H-DKO-het (MHC-Cre::IRS1L/+::IRS2L/+), and control littermates (IRS1L/L::IRS2L/L mice). All mice were on a C57BL/6 and FVB129 Sv mixed background and fed a regular chow diet with 9% of energy derived from fat. The high-fat diet (HFD) consists of 45% calories from fat, 25.6% carbohydrate, and 16.4% protein (Research Diets, Inc., New Brunswick, NJ). C57BL/6 and db/db and mice were purchased from The Jackson Laboratory. Male mice and control littermates were used in all experiments.
Echocardiograms were performed on anesthetized mice using a Visual Sonics Vevo 2100 system equipped with a 40-MHz linear probe, as previously described (12).
Blood biochemistry and metabolic analysis.
Serum from mice fasted overnight were analyzed for insulin, triglycerides, and free cholesterol using commercial kits. Cardiac ATP concentrations were measured with an Enzylight ATP assay kit, as previously described (20).
Hearts were fixed in 10% formaldehyde and stained with hematoxylin and eosin, Masson trichrome, and Oil Red O staining to visualize morphological features, fibrosis, and triglycerides, as previously described (10).
Hearts were fixed in 10% paraformaldehyde and embedded in paraffin. Paraffin-embedded sections (5 μm) were incubated at 60°C for 15 min, dewaxed, and rehydrated. Apoptosis was detected using the TUNEL assay (In situ Cell Death Detection Kit, Roche), according to the manufacturer’s instructions.
Protein analysis and immunoblotting.
The same amount of protein was resolved by SDS-PAGE and transferred to nitrocellulose membrane for Western blot. Signal intensity was measured and analyzed using ImageJ software (National Institutes of Health), as previously described (15).
Primary cardiomyocyte isolation and cell culture.
Primary cultures of neonatal rat ventricular cardiomyocytes (NRVMs) were prepared from hearts of 1- to 2-day-old Sprague-Dawley rat pups, as previously described (21).
Adenovirus infection of cardiomyocytes.
NRVMs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/M199 medium with serum for 48 h and then transfected by adding adenovirus expressing green fluorescent protein (GFP), IRS1, IRS2, GFP-fused wild-type p38 (p38-WT), and dominant-negative p38 (p38-DN). The dose of adenovirus is indicated as multiplicity of infection (MOI). Cells were infected with adenovirus for 8 h, and then fresh DMEM/M199 medium with serum was changed for another 8 h of culturing. Cells were serum-starved for 8 h before the insulin intervention. For MG132 treatment, the proteasome inhibitor was added to cells for 0.5 h before addition of adenovirus. After 8 h of adenovirus infection, cells were washed and fresh DMEM/M199 medium added for another 8 h of culturing before cellular protein lysates were prepared for immunoblotting.
RNA isolation and quantitative real-time PCR analysis.
RNA was extracted with Trizol Reagent (Invitrogen), cDNA synthesis used the SuperScript first-strand synthesis system (Bio-Rad Laboratories, Inc.), and gene expression was measured with the SYBER Green Supermix system (Bio-Rad Laboratories, Inc.), as described previously (15). The PCR primers are listed in the Supplementary Methods.
Results are presented as the mean ± SEM. Data were analyzed by one-way and two-way ANOVA and the Turkey post hoc test to determine P values. P < 0.05 was considered statistically significant, as previously described (5).
Cardiac deletion of IRS1 and IRS2 prevents Akt→Foxo1 phosphorylation and causes cardiac dilation and heart failure in mice.
To assess the role of IRS1 and IRS2 in cardiac function, cellular signaling, and gene expression, we generated H-DKO mice specifically deficient in the IRS1 and IRS2 genes in the heart. In the H-DKO mice, IRS1 and IRS2 proteins were absent in the heart and normally expressed in the skeletal muscle, fat, and liver (Supplementary Fig. 1). Akt phosphorylation at T308 and S473, indicative of PDK1 and PDK2 activation, respectively, and Akt-induced Foxo1 phosphorylation at S253 were completely blocked (Fig. 1A). Phosphorylation of p70 ribosome S6 kinase (p70S6K), a target of mammalian target of rapamycin (mTOR) downstream from PI-3K for protein synthesis, was markedly reduced; however, extracellular signal–related kinase (ERK)1/2, p38, and Jun NH2-terminal kinase (JNK) phosphorylation were unchanged in H-DKO hearts.
H-DKO mice died at the ages of 6 to 8 weeks, without a change in body weight (Fig. 1B and Table 1). In H-DKO hearts, cardiac left ventricular weight was reduced by 20% (P < 0.05; Table 1), and there was global chamber dilatation, with thinned cardiac muscle walls (Fig. 1C). Apoptotic cells were detected in H-DKO hearts but not in control hearts (Fig. 1D). Cardiomyocytes were irregularly arranged as revealed by hematoxylin and eosin staining (Fig. 1E), and interstitial fibrosis was notably increased (Fig. 1F). Systolic parameters for ejection fraction (EF) and fractional shortening (FS), as determined by echocardiography, were reduced by 70 and 60%, respectively, compared with the control (Fig. 1G). There was severe diastolic failure, as indicated by the mitral valve flow velocity (E-to-A ratio), which was not measurable because late (A′) filling waves were not detectable and early (E) filling waves were severely depressed. Isovolumic relaxation time and isovolumic contraction time could not be distinguished from aortic ejection time (Table 1). Cardiac ATP content was decreased by 40% (Fig. 1H).
Several metabolic parameters were measured in H-DKO mice at the age of 5 weeks. The blood glucose concentration was slightly reduced (H-DKO: 65 ± 6 mg/dL vs. control: 80 ± 7 mg/dL, n = 6 mice per group; P < 0.05) and serum triglycerides were increased by twofold (H-DKO: 130 ± 12 mg/dL vs. control: 60 ± 5 mg/dL, n = 6–10; P < 0.05). Serum insulin and free cholesterol were unchanged, and results of glucose and insulin tolerance tests in H-DKO mice were within normal reference ranges compared with control mice (data not shown).
Cardiac loss of IRS1 and IRS2 disrupts cardiac metabolic gene expression.
Compared with control, H-DKO hearts had increased expression of the heart failure marker genes ANP, BNP, and β-MHC, by 20-, 18-, and 40-fold, respectively (P < 0.05). Expression of the fibrosis marker gene TGF-β1 and collagen I was significantly increased by 2.5-fold each; however, expression levels of α-MHC, IRS1, and IRS2 were markedly reduced (Fig. 1I). We measured expression of genes that regulate glucose and lipid metabolism: H-DKO hearts exhibited reduced mRNA levels of the glucose transporter genes Glut1, Glut4, and glucokinase (GK) for glycolysis, by 50, 80, and 30%, respectively, compared with control (P < 0.05), and had an eightfold increase of pyruvate dehydrogenase kinase-4 (PDK4), a key enzyme and Foxo1 target gene (22), inhibiting glucose oxidation (P < 0.05; Fig. 1I). Lipid synthesis and oxidation genes were decreased in H-DKO hearts. For example, H-DKO hearts had marked reductions in the expression of genes encoding fatty acid synthase (Fasn), acetyl-CoA carboxylase (ACC1), very long-chain acyl-CoA dehydrogenase (VLCAD) for lipid metabolism, and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), which controls mitochondrial biogenesis and lipid oxidation (Fig. 1I). Expression of mitochondrial gene transcription factor (Tfam), a key nuclear gene controlling mitochondrial biosynthesis was significantly reduced by 50%, whereas expression of nuclear respiratory factor (NRF1) was unchanged. Moreover, the heme oxygenase-1 (Hmox-1) that catalyzes degradation of heme, a key component of mitochondria, was increased by sixfold (Fig. 1I), suggesting mitochondrial dysfunction in H-DKO hearts. In addition, expression of ryanodine receptor 2 (RyR2) and sarcoplasmic reticulum ATPase (Serca2A), which govern calcium release and intake from the sarcomere to cytosol, were markedly decreased (Fig. 1I), indicating a calcium mishandling in H-DKO hearts. Taken together, H-DKO hearts disrupted the Akt→Foxo1 signaling cascade, markedly induced apoptosis, impaired metabolic, mitochondrial, and calcium-handling gene expression, and also decreased cardiac ATP production.
Cardiac loss of IRS1 and IRS2 protein and activation of p38 in HFD and db/db mice.
To test the hypothesis that cardiac loss of IRS1 and IRS2 is involved in diabetes and/or insulin resistance, we determined protein levels of IRS1 and IRS2 in the hearts of HFD mice and db/db mice, which have been reported to have cardiac dysfunction (12,23). The HFD mice exhibited hyperinsulinemia after 4 months of HFD treatment, whereas db/db mice exhibited hyperinsulinemia and hyperglycemia, as we previously reported (5,10). IRS1 and IRS2 protein levels and Akt phosphorylation were reduced, whereas p38 phosphorylation was increased in the hearts of HFD and db/db mice, compared with control (Fig. 2A and Supplementary Fig. 2). Echocardiographic analysis indicated that the cardiac function was significantly impaired in HFD and db/db mice: EF and FS were reduced by 18 and 17%, respectively, in HFD-mice; EF and FS were reduced by 36 and 38% in db/db mice, respectively, compared with control (Fig. 2A).
Degradation of IRS1 and IRS2 diminishes AKT→Foxo1 phosphorylation and enhances p38 in L-DKO hearts.
We further examined the cardiac signaling and function in another mouse model we recently developed—L-DKO mice (deficient in hepatic IRS1 and IRS2 genes), which developed insulin resistance and exhibited a reduction in serum triglycerides (L-DKO: 34.7 ± 2.9 mg/dL vs. control: 49.8 ± 4.8 mg/dL, n = 6; P < 0.05), distinct from the HFD and db/db mice (5). L-DKO mice at the age of 6 months exhibited a 17-fold increase in the serum insulin level compared with control (L-DKO: 4.49 ± 0.26 ng/mL vs. control: 0.26 ± 0.03 ng/mL, n = 6; P < 0.05) but displayed a normal blood glucose concentration in the overnight fasting state (L-DKO: 67 ± 6.7 mg/dL vs. control: 61 ± 3 mg/dL, n = 6; P = 0.85). We examined phosphorylation/activation of Akt and MAP kinases in the heart of these mice. In L-DKO hearts, IRS1 and IRS2 proteins were reduced by 70 and 60%, respectively, compared with control (P < 0.05; Fig. 2B and Supplementary Fig. 2). There was reduced phosphorylation of Akt at S473 and phosphorylation of Foxo1 at S253 by at least 50% each (P < 0.05). There was a significant increase in p38 and JNK phosphorylation by 2.1- and 2.5-fold, respectively, whereas ERK1/2 phosphorylation was unchanged. These results suggest cardiac insulin resistance occurred in L-DKO mice, potentially through downregulation of IRS1 and IRS2.
Cardiac dysfunction in L-DKO mice.
Echocardiographic analysis revealed that L-DKO hearts developed systolic cardiac dysfunction, as demonstrated by a 40% reduction in EF and a 20% reduction in FS, as well as diastolic dysfunction, as indicated by a 15% increase in isovolumic relaxation time and a 24% decrease in the E-to-A ratio, an indicator of early and late ventricular filling velocity (Fig. 2C and Table 1). The L-DKO heart had a reduction in ATP content by 20% compared with control (P < 0.05; Fig. 2D), demonstrated irregularly arranged cardiomyocytes (Fig. 2E), and exhibited mild fibrosis (Fig. 2F).
Expression of heart failure markers and energy metabolism genes in L-DKO hearts.
In L-DKO hearts, the level of mRNAs encoding BNP and β-MHC was significantly increased by 1.5- and 3.8-fold, respectively, compared with control, whereas α-MHC was reduced. Expression of the fibrosis maker gene TGF-β1 and collagen I gene was mildly increased (Fig. 2G), whereas expression of IRS1 and IRS2 mRNA was not changed. L-DKO hearts exhibited mRNA levels of Glut1, Glut4, and GK that were reduced by 50, 40, and 15%, respectively, compared with control (P < 0.05). Lipid metabolism genes, such as Fasn, ACC1, and VLCAD, were decreased by 50% each, Pgc1α was reduced by 30% (P < 0.05), and PDK4 exhibited a 3.5-fold increase (P < 0.05) (Fig. 2G). L-DKO hearts exhibited markedly accumulated triglycerides, as revealed by Oil Red O staining, using db/db heart as control (Fig. 2H). Expression of Tfam and NRF1 was unchanged, Hmox-1 was increased by 2.2-fold, and expression of Serca2A was significantly decreased (Fig. 2G). Collectively, these results suggest that L-DKO hearts had reduced glucose and lipid utilization as well as impaired mitochondrial and calcium metabolism gene expression compared with control.
Downregulation of IRS1 and IRS2 results in cardiac dysfunction in H-DKO-het mice.
We next determined whether a reduction of IRS1 and IRS2 is sufficient for cardiac dysfunction by analyzing H-DKO-het mice, which lack one allele each of IRS1 and IRS2. H-DKO-het mice survived normally. Echocardiographic analysis revealed that 6-month-old H-DKO-het mice developed cardiac dysfunction, as demonstrated by an 18% reduction in EF and a 19% reduction in FS (Fig. 2I). The H-DKO-het hearts displayed a 50% reduction in IRS1 and IRS2 protein levels, as well as Akt phosphorylation levels, compared with control (Fig. 2J).
Chronic insulin stimulation degrades IRS1 and IRS2 protein and causes insulin resistance in vitro.
A major characteristic of L-DKO mice is hyperinsulinemia, accompanied by degradation of IRS1 and IRS2 in the heart. Thus, we determined whether insulin would cause myocardial insulin resistance by IRS protein regulation. First, we determined Akt→Foxo1 phosphorylation during different time courses of insulin stimulation of cardiomyocytes. NRVMs were treated with 100 nmol/L insulin for 0.5, 3, or 24 h, and cellular Akt and Foxo1 phosphorylation was determined using Western blot. Insulin treatment for 0.5 h robustly stimulated Akt and Foxo1 phosphorylation, whereas the effect of insulin was attenuated at 24 h (Fig. 3A). In parallel, protein levels of IRS1 and IRS2 were significantly decreased by 40 and 60%, respectively, after chronic insulin treatment (24 h), compared with non–insulin-treated cells (Fig. 3A).
Second, we determined whether chronic insulin exposure results in insulin resistance in cells. NRVMs were pretreated with 100 nmol/L insulin for 24 h (chronic stimulation), and the medium was changed and stimulated with the same dose of insulin for another 0.5 h (acute insulin stimulation). We found that chronic insulin treatment almost completely disrupted the effect of acute insulin stimulation on Akt and Foxo1 phosphorylation (P < 0.05; Fig. 3B), indicating that insulin itself causes insulin resistance by impairing Akt→Foxo1 phosphorylation, which is accompanied by a reduction in IRS1 and IRS2 protein. The effect of chronic insulin on attenuating Akt→Foxo1 phosphorylation was also observed in NRVMs cultured with 25 mmol/L glucose (high-glucose stimulation), in which high-glucose treatment did not significantly change IRS1 and IRS2 protein levels and only mildly altered the effect of acute insulin stimulation on Akt and Foxo1 phosphorylation (P < 0.05; Fig. 3C).
Finally, we determined whether the reduction in IRS1 or IRS2 contributed to chronic insulin resistance in cells. NRVMs were infected with adenovirus expressing the gene encoding GFP, IRS1, or IRS2 and then treated with insulin for 24 h before acute insulin stimulation for 0.5 h. Western blot analysis indicated that impaired Akt→Foxo1 phosphorylation after chronic insulin exposure was largely prevented by overexpression of IRS1 or IRS2 (Fig. 3D). These results support our postulate that inhibition of IRS1 and IRS2 is a primary mediator causing insulin resistance during chronic insulin stimulation.
p38 mediates degradation of IRS1 and IRS2 after chronic insulin stimulation.
In addition to Akt activation, acute insulin exposure (0.5 h) robustly induced ERK1/2 and slightly increased JNK phosphorylation, whereas chronic insulin exposure (24 h) persistently increased p38 phosphorylation (Fig. 4A). We next tested the hypothesis that activation of these kinases after chronic insulin exposure might be involved in the feedback degradation of IRS1 and IRS2, leading to myocardial insulin resistance. NRVMs were treated with the following kinase inhibitors: rapamycin (mTOR inhibitor), PD98059 (ERK1/2 inhibitor), and SB (p38 inhibitor) or SP (JNK inhibitor) for 0.5 h before 24 h of insulin stimulation, in which insulin-induced protein kinase phosphorylation/activation was completely blocked. We noted that SB had no effect on blocking p38 phosphorylation (Fig. 4B), which was consistent with a recent report that SB treatment effectively inhibited p38 activity without altering p38 phosphorylation in cardiomyocytes (24). Among these kinase inhibitors, the p38 inhibitor SB completely prevented chronic insulin-induced IRS1 and IRS2 degradation and rescued Akt→Foxo1 phosphorylation (Fig. 4C and D), suggesting that p38 activation is necessary for chronic insulin-induced IRS1 and IRS2 degradation and insulin resistance.
We next determined whether overexpression of p38 is sufficient for degrading IRS1 and IRS2 protein in cells. Overexpression of p38-WT, rather than p38-DN containing two amino acid mutations in the TGY motif to block phosphorylation at T and Y (TGY→AGF), reduced IRS1 and IRS2 protein levels by at least 80% in NRVMs, with or without 24 h of insulin treatment (Fig. 4E). Moreover, IRS1 and IRS2 protein and the basal level of Foxo1 phosphorylation were reduced by p38 in a dose-dependent manner (Fig. 4F). These results indicate that induction of p38 is necessary and sufficient for degrading IRS1 and IRS2 protein in insulin-signaling cascades.
p38 overexpression prevents the insulin effect on Akt→Foxo1 phosphorylation and metabolic gene expression in vitro.
We next determined whether p38 overexpression causes insulin resistance by preventing insulin-induced Akt→Foxo1 phosphorylation and insulin-mediated gene expression in cells. NRVMs were infected with adenoviruses expressing GFP, p38-WT, and p38-DN and then treated with insulin for 0.5 h for cellular signaling analysis or 18 h for determination of insulin-regulated gene transcription. Compared with the non–insulin-treated control, p38 overexpression prevented the effect of 0.5 h of insulin stimulation on Akt→Foxo1 phosphorylation (Fig. 5A). Insulin treatment for 18 h stimulated expression of genes encoding α-MHC, Glut4, Fasn, and ACC1 by 1.8-, 1.4-, 2.5-, and 2.2-fold, respectively (P < 0.05), while inhibiting expression of genes encoding β-MHC and BNP by 50 and 70%, respectively (P < 0.05, Fig. 5B). These insulin-regulated effects were blocked by overexpression of p38-WT, rather than GFP or p38-DN, supporting the concept that p38 activation causes myocardial insulin resistance. Of note is that p38 overexpression did not block the effect of insulin on suppressing the transcriptional levels of IRS1 and IRS2 (Fig. 5B).
Overexpression of IRS1 or IRS2 prevents p38-induced heart failure and metabolic gene expression.
To determine if maintaining IRS expression is sufficient to reverse gene expression changes induced by p38 activation in cultured cells, we co-overexpressed p38, together with control GFP, IRS1, or IRS2, and analyzed gene transcriptional profiles. The results indicated that overexpression of IRS1 or IRS2 completely or partially rescued β-MHC, α-MHC, and BNP (heart failure genes) and metabolic gene expression such as Glut4 (Fig. 5C).
Chronic insulin treatment prevents the effect of insulin on metabolic gene expression and promotes IRS1 phosphorylation at Ser 636 in vitro.
Similar to p38 overexpression, 24 h of chronic insulin exposure prevented the effect of 18 h of insulin on expression of genes encoding α-MHC, Glut-4, Fasn, and ACC1 (stimulation) and β-MHC and BNP (inhibition). The insulin inhibition on IRS1 and IRS2 gene transcription was maintained after the chronic insulin exposure (Fig. 6A).
IRS1 and IRS2 serine or threonine phosphorylation is believed to mediate IRS degradation, ubiquitination, and insulin resistance (25). Thus, we determined the insulin effect on IRS1 serine phosphorylation at S636 and S612, both of which mediate IRS1 degradation or inhibition (26,27). Insulin treatment for 24 h or p38 overexpression increased phosphorylation at both residues, and p38-DN expression also increased IRS1-S612 phosphorylation (Fig. 6B and C), suggesting that IRS1-S636 may participate in IRS1 inhibition and degradation. We further determined whether the 26S proteasome-activated ubiquitination pathway mediates p38- or chronic insulin-induced IRS1 or IRS2 degradation. Treatment with MG132, a 26S proteasome inhibitor, prevented chronic insulin- or p38-WT–induced degradation of IRS2, rather than IRS1 (Fig. 6D and E). Of note is that MG132 induced IRS1 degradation, the mechanism of which is unknown. These results suggest that IRS1 and IRS2 degradation is mediated by distinct mechanisms in which IRS2 is involved in the 26S proteasome pathway and IRS1 is involved in S636 phosphorylation.
Finally, we determined whether insulin degrades IRS proteins in a dose-dependent manner. Insulin treatment for 24 h resulted in reduced IRS1 and IRS2 in a dose-dependent manner, in which IRS2 had a higher sensitivity for reduction than IRS1 after insulin treatment (Fig. 6F).
In this study, we present three important findings: First, IRS1 and IRS2 have key roles in regulating cardiac homeostasis, energy metabolism, and function. A loss of IRS1 and IRS2 in the heart (H-DKO mice) increases apoptosis; decreases ventricular mass; increases interstitial fibrosis; diminishes myocardial Akt→Foxo1 phosphorylation; impairs cardiac metabolism, calcium handling, and myosin gene expression; and reduces cardiac ATP content, with sudden death of animals beginning at 6 to 8 weeks of age.
Second, a 50% reduction in cardiac IRS1 and IRS2 is sufficient for induction of cardiac dysfunction, and downregulation of both genes occurs in insulin-resistant or type 2 diabetic mouse models (HFD, db/db, and L-DKO mice).
Third, chronic insulin exposure promotes IRS1 and IRS2 degradation, resulting in myocardial insulin resistance, in which p38 activation is necessary and sufficient for the effect of chronic insulin action.
Given that the Akt and MAP kinase pathways are both important for cardiac biology and function and are activated by many stimuli, including mechanical stretch, growth factors, and hormones through a variety of receptor tyrosine kinases or G-protein–coupled receptors (28), it is unclear which endogenous upstream mediators govern the downstream effectors for Akt and MAPK activation (29). In this study, we used H-DKO mice to provide evidence demonstrating that IRS1 and IRS2 are major mediators for the endogenous cardiac Akt→Foxo1 signaling cascade. Importantly, the RAS→MAPK cascades are not dependent on IRS1 and IRS2, suggesting that other IR-associated proteins may be involved in the activation of MAPKs. Thus, we expect that deactivation of Akt resulting from inactivation of PDK1 and PDK2 in H-DKO hearts may mediate the heart failure observed. In fact, cardiac inactivation of PDK1 is sufficient for the development of dilated heart failure with apoptosis and fibrosis (30,31), similar to the phenotype observed in H-DKO hearts. Akt is required for cardiac growth, metabolism, and survival (32), and targets of Akt include p70S6K (protein synthesis), glut4 (glucose transport), and Foxo1 (gene expression) (9,33,34).
Cardiac inactivation of Akt after the loss of IRS1 and IRS2 may serve as a central mechanism for the induction of heart failure, which involves multiple mechanisms (Fig. 7A):
1) Akt inactivation promotes cardiac apoptosis, at least by Foxo1 activation, that promotes cell death (35). The incidence of myocardial apoptosis in the H-DKO heart is relatively small (1%), but a minimal increase can be of considerable biological importance on myocardial homeostasis and morphology. Indeed, Foxo1 deficiency in H-DKO mice prevented early death and cardiac dysfunction (Y.Q. and S.G., unpublished data).
2) Akt inactivation activates Foxo1 resulting in heme deficiency, limiting mitochondrial cofactor biosynthesis and ATP production, as we observed in liver lacking both IRS1 and IRS2 (20). Heme is an essential component for mitochondrial complex III and IV and it is controlled by Hmox-1, a Foxo1 target gene promoting heme degradation (20).
3) PI3K→Akt activation is required for proper calcium handling because blocking PI3K or Akt markedly decreases intracellular calcium transients essential for cardiac contraction (36), which may also involve RyR2 and Serca2A gene expression and regulation.
4) PI3K→Akt activation is required for controlling MHC plasticity and motor gene expression, which is required for normal cardiac contractility. This is controlled by Foxo1 that promotes β-MHC gene expression (Q.Z. and S.G., unpublished data).
5) Inactivation of PDK1 promotes cardiac fibrosis, suggesting that unknown factors released from the myocardium after loss of IRS1 and IRS2 and Akt inactivation may influence cell communications between myocytes and nonmyocytes, resulting in interstitial fibrosis and cardiac failure.
IRS1 and IRS2 also mediate the effect of IGF-1, which can activate PI-3K and MAPK pathways through the IGF-1 receptor. Mice deficient in cardiac and skeletal muscle in both insulin and the IGF-1 receptor, rather than each alone, died of heart failure in the first month of life with cardiac Akt inactivation (37), suggesting that insulin or IGF-1 is cardioprotective for survival. Activation of Foxo1, resulting from inactivation of Akt, may contribute to heart failure because overexpression of a constitutive active Foxo1 in the heart resulted in embryonic lethality at 10.5 days of gestation, with overt heart failure in mice (13), whereas inactivation of Foxo1 in the heart prevented heart failure in HFD-induced insulin-resistant mice (12). Conversely, MAPKs have different roles in cardiac biology. For example, loss of ERK1/2 had no effect on the cardiac growth responses, but overexpression of ERK promoted cardiac hypertrophy in mice (38), loss of cardiac p38 promoted cardiac hypertrophy (39), and cardiac JNK inactivation inhibited cardiac apoptosis and activation inhibited hypertrophic responses in pressure overload (40). Collectively, cardiac PI-3K→Akt→Foxo1 and MAPKs activation have differential roles in the heart, but the IRS1- and IRS2-associated Akt activation is essential for cardiac growth, survival, and function.
In our cell-based studies, chronic high glucose exposure did not significantly alter the IRS1 or IRS2 protein level and minimally reduced insulin-stimulated Akt→Foxo1 phosphorylation. However, chronic insulin exposure results in insulin resistance by decreasing IRS1 and IRS2 at the mRNA and protein levels. Insulin resistance is implicated in the pathogenesis of type 2 diabetes, and insulin serves as an inducer of insulin resistance in liver and adipose tissue (41). In the heart, hyperinsulinemic exposure also exacerbates cardiac systolic dysfunction, caused by pressure overload in mice (42). Diabetic cardiomyopathy is directly related to hyperglycemia, independent of hypertension or coronary atherosclerosis (43), and hyperglycemia is currently believed to promote production of reactive oxygen species, advanced glycation end products, and metabolic perturbations that result in increased glucose toxicity and myocardial injury and apoptosis (21,44). Hyperglycemia and hyperinsulinemia may cause cardiac dysfunction through different mechanisms, in which loss of IRS1 and IRS2 by chronic insulin stimulation may provide a fundamental mechanism for heart failure in insulin resistance and type 2 diabetes. Of note is that the presence of IRS1 or IRS2 alone maintained cardiac function to support animal survival (19).
p38 phosphorylation increases in the heart of insulin-resistant and diabetic mice (HFD, db/db, and L-DKO mice) and is closely associated with downregulation of IRS1 and IRS2 protein levels and Akt inactivation. Recent studies demonstrated that the HFD dramatically increased the death rate of animals, secondary to heart failure resulting from Akt inactivation (12). Importantly, p38 activation is implicated in a wide spectrum of cardiac pathologies, including myocardial infarction, inflammation (45), and metabolic stress (46), in which some insults may be secondary to IRS1 and IRS2 degradation in response to mechanical and metabolic stresses (47). Administration of p38 chemical inhibitors prevented diabetes and pressure overload–induced cardiac dysfunction in mice (46,48), and we expect that p38 inhibition in L-DKO mice would also improve the cardiac dysfunction.
p38 mediates the effect of chronic insulin on promoting insulin resistance by suppressing IRS1 and IRS2. p38 and insulin both promoted IRS2 degradation via a 26S proteasome–dependent ubiquitination pathway, whereas IRS1 degradation in cardiomyocytes was independent of this pathway (Fig. 7A). IRS1-S612 and -S636 phosphorylation reduced PI-3K in skeletal muscle of patients with type 2 diabetes (49), and S636 phosphorylation was also increased by the inflammatory factor tumor necrosis factor-α (26,50). In our studies, p38-DN–induced S612 phosphorylation did not block insulin action, an observation that conceptually supports IRS1-S636 phosphorylation as important in IRS1 degradation; but the coupling mechanism of IRS1 degradation and S636 phosphorylation will necessitate further investigation. Of note, whether p38 can directly interact with IRS1 to phosphorylate S636 has not been validated, because p38 also targets other intracellular protein kinases, such as cAMP-dependent protein kinase, for metabolic regulation (51). Regardless, our data suggest that p38 activation in the myocardium triggers distinct regulatory mechanisms for IRS1 and IRS2 protein degradation, independent of gene transcriptional regulation.
In conclusion, our data indicate IRS1 and IRS2 have major roles in the control of cardiac homeostasis, metabolism, and function, while suppressing cardiac IRS1 and IRS2 may serve as a fundamental mechanism for induction of heart failure (Fig. 7B). We believe that sensitizing myocardial Akt→Foxo1 signaling by integrating insulin therapy and blocking the p38→IRS1/2 signaling cascade will provide new insight in the treatment of heart failure during insulin resistance, type 2 diabetes, or other chronic physiologic stresses.
This work is supported by American Diabetes Association grant JF-7-07-27, American Heart Association grant BGIA-7880040, Faculty Start-up Funds from the Texas A&M University Health Science Center College of Medicine, and National Institutes of Health (NIH) grants R01-DK-095118-01 to S.G. and in part by DK-38712-23 to M.F.W. and HL-68838-06 to D.E.D. This material is the result of work supported with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas.
No potential conflicts of interest relevant to this article were reported.
Y.Q. and Z.X. researched data and wrote the manuscript. Q.Z. and C.T. researched data. R.K. researched data and reviewed and edited the manuscript. H.F. prepared and provided p38 adenovirus. D.E.D. prepared and provided p38 adenovirus and provided IRS1/2-loxP mice. M.F.W. provided IRS1/2-loxP mice and reviewed and edited the manuscript. K.M.B. reviewed and edited the manuscript. S.G. designed and supervised the research and wrote the manuscript. S.G. is the guarantor of this work and as such, had full access to all the data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis.