Molecular Mechanisms of Atherosclerosis in Metabolic Syndrome
Role of Reduced IRS2-Dependent Signaling
Objective— The mechanisms underlying accelerated atherosclerosis in metabolic syndrome (MetS) patients remain poorly defined. In the mouse, complete disruption of insulin receptor substrate-2 (Irs2) causes insulin resistance, MetS-like manifestations, and accelerates atherosclerosis. Here, we performed human, mouse, and cell culture studies to gain insight into the contribution of defective Irs2 signaling to MetS-associated alterations.
Methods and Results— In circulating leukocytes from insulin-resistant MetS patients, Irs2 and Akt2 mRNA levels inversely correlate with plasma insulin levels and HOMA index and are reduced compared to insulin-sensitive MetS patients. Notably, a moderate reduction in Irs2 expression in fat-fed apolipoprotein E-null mice lacking one allele of Irs2 (apoE−/−Irs2+/−) accelerates atherosclerosis compared to apoE-null controls, without affecting plaque composition. Partial Irs2 inactivation also increases CD36 and SRA scavenger receptor expression and modified LDL uptake in macrophages, diminishes Akt2 and Ras expression in aorta, and enhances expression of the proatherogenic cytokine MCP1 in aorta and primary vascular smooth muscle cells (VSMCs) and macrophages. Inhibition of AKT or ERK1/2, a downstream target of RAS, upregulates Mcp1 in VSMCs.
Conclusions— Enhanced levels of MCP1 resulting from reduced IRS2 expression and accompanying defects in AKT2 and Ras/ERK1/2 signaling pathways may contribute to accelerated atherosclerosis in MetS states.
- insulin resistance/metabolic syndrome
- extracellular signal-regulated kinase (ERK)
The metabolic syndrome (MetS) is defined by the presence of at least 3 of the following abnormalities: abdominal obesity, glucose intolerance, hypertension, low HDL-cholesterol levels, or hypertriglyceridemia.1,2 Patients with MetS and type-2 diabetes mellitus (T2DM) have 2 to 5 times higher risk of atherosclerosis, a chronic inflammatory disease that results from interactions between modified lipoproteins and cells of the arterial wall, including endothelial, immune, and vascular smooth muscle cells (VSMCs).3–5 Among the different cardiovascular risk factors that precipitate atherosclerosis and associated cardiovascular disease (CVD), T2DM and MetS are becoming the most relevant given that the prevalence of these metabolic diseases is expected to increase by 165% in the next 40 years, representing the health plague of the 21st century.2 Importantly, the incidence of CVD increases when T2DM and the MetS coexist.1,2 Population aging and acquisition of sedentary lifestyle patterns (eg, obesity and physical inactivity) are major driving forces behind these metabolic diseases. A number of alterations in endothelial cells, VSMCs, and platelets have been identified which may accelerate atherosclerosis, plaque instability, and thrombus formation in T2DM/MetS patients, however the underlying mechanisms remain ill defined.2,6,7 Many MetS patients display insulin resistance (IR), which seems to play a pivotal role in the development of both atherogenic dyslipidemia and T2DM.1 Therefore, IR is an attractive target for prevention of CVD. However, whether IR management per se might reduce CVD risk remains unknown.
On binding to the insulin receptor (INS-R), insulin exerts its action through insulin-receptor substrate proteins (IRS1–4).8 Studies in genetically-modified mice have highlighted a major role of IRS2 in β-cell function, glucose and insulin homeostasis, and atherosclerosis development. First, Irs2-null mice (Irs2−/−) display a T2DM/MetS-like phenotype, including hyperglucemia, hyperinsulinemia, IR and hypertension.9–11 Second, global Irs2-deficiency in apoE-null mice (apoE−/−Irs2−/−) aggravates atherosclerosis compared to apoE−/− counterparts with intact Irs2.12,13 Doubly deficient apoE−/−Irs2−/− mice exhibit hyperinsulinemia, IR, and increased glucose intolerance and a positive correlation between circulating insulin levels and atherosclerotic burden,12 consistent with findings in Lepob/ob:LDLr−/− and Lepob/ob: apoE−/− mice, two additional models of IR-dependent accelerated atherosclerosis.14 It is noteworthy that although whole body Irs2 ablation aggravated atherosclerosis,12,13 the effects of macrophage-specific defective insulin signaling are controversial. Indeed, Ins-r–deficient bone-marrow transplant enhanced atherosclerosis in LDLr−/− mice,15 however macrophage-specific inactivation of Ins-r or Irs2 decreased atherosclerosis in apoE−/− mice.13 The reasons for these controversial findings remain to be established.
The two main downstream effectors of IRS proteins are the phosphatidylinositol-3 kinase (PI-3K)/V-akt murine thymoma viral oncogene homolog kinase (AKT) and the Ras/Raf/ERK pathways.8 Marked reductions in Ins-r, Irs2, and Akt2 gene expression have been reported in pancreatic islets from T2DM patients.16 Here we have tested the hypothesis that impaired IRS2 signaling is a mechanism contributing to accelerated atherosclerosis in MetS states. To this end, we have analyzed IRS2 signaling in white mononuclear blood cells (WMBCs) from MetS patients with and without IR. Additionally, we have studied apoE-null mice with an intact Irs2 gene (apoE−/−) or lacking one allele of Irs2 (apoE−/− Irs2+/− mice), and primary VSMCs and macrophages derived from these animals.
Materials and Methods
Expanded Materials and Methods can be found in supplemental material (available online at http://atvb.ahajournals.org).
In compliance with institutional guidelines, subjects were informed of the present study and all agreed to participate. The study was carried out in accordance with the Helsinki Declaration, and the Ethical Committee of the University Clinic of Navarra approved the protocol. The study was performed with samples from 55 unrelated individuals referred to our institution for routine medical work-up after 12 hours of overnight fasting. Clinical screenings were based on medical history, physical examination, and routine analytic tests. Subjects were diagnosed with MetS when 3 or more of the AHA/NHLBI criteria for defining this condition were present.1 The following criteria were adopted. Central obesity: waist circumference ≥102 cm in men and ≥88 cm in women; hypertriglyceridemia: triglycerides ≥1.7 mmol/L or use of medication to reduce triglycerides; low HDL cholesterol: HDL cholesterol <1.03 mmol/L in men and <1.3 mmol/L in women or use of medication to increase HDL cholesterol; high blood pressure: systolic blood pressure (SBP) ≥130 mm Hg, diastolic blood pressure (DBP) ≥85 mm Hg, or use of antihypertensive medication; high fasting glucose: glucose ≥5.55 mmol/L or use of medication to reduce glucose.
IR was diagnosed when the homeostasis model assessment (HOMA) index (fasting glucose [mmol/L]×fasting insulin [μU/mL]/22.5) was equal or greater than the median in normal-weight subjects plus 2.5 standard deviations (HOMA ≥3.3). Using this criterium, we identified 30 patients with IR within the studied population.
Mice and Diets
Care of animals was in accordance with institutional guidelines. Irs2−/− (C57BL/6J)10 and apoE−/− (C57BL/6J, Charles River Lyon, France) mice were crossbred to generate apoE−/−Irs2+/− mice. Genotyping was done by polymerase chain reaction (PCR) as described.10,12 After weaning, male mice were maintained on a low-fat (control) standard diet (2.8% fat; Panlab, Barcelona, Spain) or placed on an atherogenic diet (10.8% total fat, 0.75% cholesterol, S8492-E010, Ssniff, Germany) for the indicated periods of time.
Irs2 and Akt2 Expression Is Decreased in WMBCs and Inversely Correlates With Insulin Levels and HOMA Index in Insulin-Resistant MetS Patients
The demographic and clinical characteristics of the MetS patients included in our studies are summarized in supplemental Table I (available online at http://atvb.ahajournals. org). Patients were classified as insulin-sensitive or insulin-resistant based on the HOMA index (see methods). No significant differences in gender distribution, age, and frequency of cardiovascular medications were observed between both groups. However, insulin-resistant patients presented higher body mass index (BMI), waist circumference, DBP, HOMA index, and plasma levels of insulin, oxidized LDL (oxLDL), and metalloproteinase-9 (MMP-9), and lower plasma HDL-cholesterol levels as compared with insulin-sensitive patients. No significant differences in the remaining parameters were noted between the 2 groups of patients. Thus, IR is associated with higher risk of CVD in our cohort of MetS patients.
Given that total genetic ablation of Irs2 in fat-fed apoE−/− mice produces IR and accelerates atherosclerosis12,13 and that IRS proteins signal in part through the PI3K/AKT pathway,8 we examined Irs2 and Akt expression in WMBCs from both groups of patients. Quantitative real-time PCR (qPCR) analysis revealed reduced Irs2 and Akt2 mRNA levels in insulin-resistant versus insulin-sensitive subjects, although only differences in Akt2 reached statistical significance (Figure 1A). We also found increased expression of Irs1 in insulin-resistant versus insulin-sensitive individuals (P=0.029), whereas Akt1 and Akt3 were expressed at similar level in both groups of patients (Figure 1A). Correlation studies demonstrated a significant and positive bivariate correlation between Irs2 and Akt2 expression in all the MetS patients (r2=0.66, P<0.001, Figure 1B), which remained highly significant after controlling for age and sex (r2=0.63, P<0.001), and when analyzing separately insulin-sensitive (r2=0.69, P<0.001) and insulin-resistant (r2=0.46, P<0.001) patients.
In insulin-resistant patients, mRNA expression levels of Irs2 and Akt2 in WMBCs exhibited a statistically significant inverse correlation with plasma insulin levels (Figure 1C and 1E) and the HOMA index (Figure 1D and 1F). These associations were not observed in insulin-sensitive patients (Irs2 versus insulin: r2=0.039, P=0.342; Irs2 versus HOMA: r2=0.051, P=0.278; Akt2 versus insulin: r2=0.02, P=0.500; Akt2 versus HOMA: r2=0.022, P=0.484). Additional association studies are presented in supplemental Tables II through V. These results indicate that hyperinsulinemia and IR in MetS patients are associated with downregulation of Irs2 and its downstream effector Akt2 in WMBCs.
Partial Inactivation of Irs2 in ApoE−/− Mice Combined With Severe Hypercholesterolemia Produces Increased Glucose Intolerance and Mild Hyperinsulinemia and Accelerates Atherosclerosis
We next sought to generate an animal model with moderate reductions in Irs2 expression to determine whether this alteration observed in insulin-resistant MetS patients accelerates atherosclerosis. To this end, we partially inactivated the Irs2 gene in apoE−/− mice, which spontaneously develop hypercholesterolemia and complex atherosclerotic lesions resembling those observed in humans, which can be accelerated by a high-fat cholesterol-rich diet.17 Mice received either standard chow or were challenged for 3 months with a high-fat cholesterol-rich atherogenic diet. Under either dietary regimen, partial disruption of Irs2 did not influence circulating glucose levels (Figure 2A and 2B). Similarly, fasting plasma insulin levels did not differ between apoE−/− and apoE−/−Irs2+/− mice fed control diet (Figure 2A). However, on a high-fat diet, a trend toward increased fasting plasma insulin was observed in apoE−/−Irs2+/− mice compared with apoE−/− mice (1.00±0.17 versus 0.65±0.14 μg/dL, respectively, P=0.074; Figure 2B). Glucose tolerance measured by the area under the curve (AUC) was similar in apoE−/−Irs2+/− and apoE−/− mice fed control diet (Figure 2C), whereas, fat-fed apoE−/−Irs2+/− mice were more glucose intolerant than apoE−/− counterparts on a high-fat diet (P<0.05, Figure 2D). Under both dietary regimens, glucose-stimulated insulin secretion was similar in both groups of mice, as revealed by the AUC (Figure 2C and 2D). Likewise, body weight did not differ statistically between control and fat-fed apoE−/−Irs2+/− and apoE−/− mice (data not shown), thus excluding obesity as a principal factor in the aforementioned metabolic differences developed by apoE−/−Irs2+/− mice.
Circulating levels of total cholesterol (Total-C), HDL-C, and triacylglycerides (TAG) were indistinguishable between apoE−/− and apoE−/−Irs2+/− mice fed either control (Figure 2E) or atherogenic (Figure 2F) diet. As expected, levels of plasma Total-C and TAG in fat-fed mice were increased in comparison to prediet values. These findings suggest that lipid metabolism in apoE−/− mice is unaffected by deletion of one allele of Irs2.
As shown in Figure 3A, oil red O staining revealed similar extent of atherosclerosis in the aortic arch of apoE−/− and apoE−/−Irs2+/− mice fed control diet (Total-C ≈300 mg/dL, cf. Figure 2E). In contrast, atherosclerosis burden was significantly increased in fat-fed apoE−/−Irs2+/− mice (Total-C>550 mg/dL, cf. Figure 2F, postdiet values) receiving the atherogenic diet for 2 months (aortic root, supplemental Figure IA) and 3 months (aortic arch, Figure 3A). We also found that neointimal accumulation of Mac3-immunoreactive macrophages, SMα -actin-immunoreactive VSMCs, and collagen was undistinguishable when comparing apoE−/− and apoE−/−Irs2+/− mice fed either standard chow or atherogenic diet (Figure 3B, and supplemental Figure IB).
We next investigated the consequences of reduced Irs2 expression on the uptake of modified LDL by macrophages, a key event in atherosclerosis.18 Macrophages from apoE−/− Irs2+/− mice exhibited a 30% increase in AlexaFluor488-acLDL uptake compared to apoE−/− controls (Figure 3C), coinciding with higher mRNA levels of CD36 and SRA (Figure 3D), the two main scavenger receptors involved in the uptake of modified LDLs by neointimal macrophages.
Effect of Partial Irs2 Inactivation on Aortic Expression of Genes Related to Insulin Signaling
To further investigate the underlying mechanisms by which partial inactivation of Irs2 in apoE−/− mice aggravates diet-induced atherosclerosis, we used a pathway-focused RT-PCR array which profiles the expression of 84 genes related to insulin action (supplemental Table VI and expanded Materials and Methods). We analyzed atheroma-rich aortic tissue obtained from mice maintained for 2 months on a high-fat diet. The array analysis revealed changes in the expression of 14 genes in aorta of apoE−/−Irs2+/− versus apoE−/− mice (3 with P=0.06 and 11 with at least P<0.05) (supplemental Figure II). As expected, Irs2 mRNA expression was significantly reduced in apoE−/−Irs2+/− aorta. Among the altered genes, several are related with glucose and lipid metabolism (G6pc, G6pc2, LDLr) whereas others encode kinases and phosphatases (Dusp14, Nck1, Prkc), transcription factors (Cebpa, Klf10), and scaffold protein genes (Shc1). Interestingly, apoE−/−Irs2+/− aorta exhibited reduced Akt2, Rras, and Hras1 mRNA levels. These results clearly demonstrate that the Irs2/Akt2/Ras insulin-signaling effector pathway is downregulated in aortic tissue of fat-fed apoE−/−Irs2+/− mice.
Both Partial Irs2 Genetic Inactivation and Pharmacological Inhibition of AKT or ERK1/2 Enhance Mcp1 Expression
Atherosclerosis, T2DM, and IR are characterized by chronic inflammation in different tissues.3–5,19,20 qPCR revealed increased expression of the proinflammatory cytokine Mcp1 in atherosclerotic plaque-rich aorta of apoE−/−Irs2+/− mice as well as in primary cultures of VSMCs and macrophages obtained from these animals versus apoE−/− controls (Figure 4A). Given the results of our expression studies in aorta showing reduced mRNA level of Akt2, Hras 1, and Rras (supplemental Figure II), we sought to assess whether defective signaling through AKT2 or ERK1/2 (a downstream effector of RAS signaling) might be linked to the increased Mcp1 expression associated with reduced expression of Irs2. Indeed, pharmacological inhibition of either AKT (inhibitor VIII) or ERK1/2 (U0126) increased Mcp1 mRNA expression in cultures of rat VSMCs (Figure 4B). Western blot analysis confirmed the inhibition of AKT and ERK, as indicated by reduced accumulation of phosphorylated (active) AKT1/2 and ERK1/2 (Figure 4B, pAKT1/2, pERK1/2) in treated VSMCs. Moreover, inhibitor VIII and U0126 markedly reduced, respectively, the phosphorylation of p70S6K (which is triggered on AKT activation) and of serum-inducible c-Fos upregulation (which depends on ERK1/2 activation; Figure 4C), thus providing functional validation of the efficacy of these drugs in our experimental settings.
Irs2-null mice develop T2DM- and MetS-like alterations (eg, IR, hyperinsulinemia, glucose intolerance, hypertension, moderate hyperlipidemia).9–11 Recently, we and others reported that total ablation of Irs2 accelerates atherosclerosis in severely hypercholesterolemic apoE−/− mice.12,13 Novel findings in the present study include: (1) the demonstration that increased risk of CVD in insulin-resistant versus insulin-sensitive MetS patients is associated with reduced expression of Irs2 in WMBCs, and that a moderate reduction in Irs2 expression is sufficient to accelerate atherosclerosis in severely hypercholesterolemic apoE−/−Irs2+/− mice, which display characteristics of MetS and IR; (2) the identification in aorta of changes in the expression of insulin-related genes potentially involved in augmented atherosclerosis in apoE−/− Irs2+/− mice, including the downregulation of Akt2 and Ras; and (3) the identification of defective IRS2/AKT and IRS2/RAS/ERK1/2 signaling as mechanisms of upregulating the expression of proatherogenic MCP1.
Reduced expression of Ins-r, Irs2, and Akt2 has been reported in pancreatic β-cells of T2DM/IR patients.16 However, our study is the first to analyze the expression of these signaling molecules in WMBCs from insulin-resistant and insulin-sensitive patients (Figure 1A). Notably, in accordance with the notion that AKT acts downstream of IRS2, we found a direct correlation between the level of Irs2 and Akt2 mRNA, both when considering all MetS patients (Figure 1B) and when analyzing separately insulin-sensitive and insulin-resistant subjects. We also found that levels of Irs2 and Akt2 mRNA were inversely correlated with plasma insulin levels and the HOMA index only in insulin-resistant patients (Figure 1C through 1F). Thus, hyperinsulinemia and IR in humans are associated with the downregulation of Irs2 and Akt2 in cell types which play major roles in the pathogenesis of T2DM and atherosclerosis. We also found similar level of Akt1 and Akt3 in both groups of MetS patients and higher Irs1 expression in insulin-resistant subjects, perhaps as a compensatory mechanism, a possibility which deserves further investigation. Additional studies are also warranted to clarify the relative contribution of defined subpopulations of WMBCs in the establishment of the observed differences between insulin-sensitive and insulin-resistant MetS patients.
We and others have previously reported that fat-fed apoE−/− mice with complete deficiency for IRS-2 exhibit features of MetS (severe dyslipidemia, hyperinsulinemia, glucose intolerance, IR) and accelerated atherosclerosis.12,13 To address whether a moderate reduction in Irs2 expression (comparable to that which we observed in the WMBCs of insulin-resistant patients with MetS) might have pathological consequences in an animal model, we generated apoE−/− Irs2+/− mice which lack only one allele of Irs2. In response to high-fat feeding, these mice developed severe hypercholesterolemia (Total-C>550 mg/dL), higher glucose intolerance, and a trend toward hyperinsulinemia as compared to similarly hypercholesterolemic apoE−/− controls (Figure 2). This MetS-like phenotype of fat-fed apoE−/−Irs2+/− mice was associated with significantly enhanced aortic atherosclerosis, without changes in plaque composition (eg, macrophage, vascular smooth muscle cell [VSMC], and collagen content; Figure 3, and supplemental Figure I). Notably, none of the differences caused by partial Irs2 ablation were observed when mice were fed standard chow (Total-C <300 mg/dL), suggesting that reduced Irs2 expression can accelerate atherosclerosis only when combined with other features of MetS such as severe hypercholesterolemia. Interestingly, IR combined with severe hypercholesterolemia (>700 mg/dL) was also associated with severe atherosclerosis in fat-fed liver Ins-r–deficient mice.21 We also noted higher expression of the scavenger receptors SRA and CD36 and increased uptake of acLDL in apoE−/−Irs2+/− versus apoE−/− macrophages (Figure 3C and 3D). Similar findings have been previously reported for Ins-r–deficient and ob/ob mouse macrophages.15,22 Thus, increased macrophage uptake of modified LDLs through upregulation of SRA and CD36 appears to contribute to accelerated atherosclerosis in different MetS murine models.
As fat-fed apoE−/−Irs2+/− mice seemed an appropriate model to investigate how impaired insulin-dependent signaling promotes atherosclerosis, we used aorta from these mice to investigate the consequences of reduced Irs2 on the expression of 84 genes implicated in insulin-signaling using a qPCR array (supplemental Table VI and supplemental Figure II). As expected, our gene profiling study confirmed a significant reduction in Irs2 mRNA in atheroma-rich aorta from fat-fed apoE−/−Irs2+/− versus apoE−/− mice. The expression of Akt2 and Ras was also significantly diminished in aorta of fat-fed apoE−/−Irs2+/− mice, indicating a correlation between a moderate reduction of Irs2 expression and the downregulation of 2 major pathways which mediate insulin signaling. Remarkably, insulin-dependent IRS2/PI3K/AKT and IRS1/ERK1/2 signaling are impaired in macrophages from db/db and ob/ob diabetic mice,22,23 and in human adipocytes from T2DM patients,24 respectively. It is also noteworthy that Akt2 ablation in the mouse causes major alterations in glucose homeostasis and insulin sensitivity, leading to IR and T2DM.25,26 Therefore, our results extend these findings to the cardiovascular system by suggesting a mechanistic link between impaired AKT2- and RAS-dependent signaling in aortic tissue and accelerated atherosclerosis in fat-fed apoE−/−Irs2+/− mice.
The proatherogenic role of MCP1 has been firmly established.27,28 Thus, the upregulation of Mcp1 mRNA observed in aorta of fat-fed apoE−/−Irs2+/− mice (Figure 4A) may accelerate atherosclerosis in this model. Mcp1 expression was also higher in primary VSMCs and macrophages from apoE−/− Irs2+/− mice (Figure 4A). Moreover, this cytokine is upregulated in adipocytes exhibiting IR (IR-3T3-L1 and ob/ob adipocytes)29 and in platelets from diabetic patients.30 Our finding that pharmacological inhibition of AKT2 or ERK1/2 upregulates Mcp1 mRNA in rat VSMCs (Figure 4B) is consistent with the notion that accelerated atherosclerosis in MetS conditions is attributable, at least in part, to defective IRS2-AKT2 and IRS2-RAS/ERK1/2 pathways and the resulting upregulation of Mcp1 in various cell types involved in atherothrombosis (eg, VSMCs, macrophages, platelets, adipocytes). However, additional studies are needed to firmly establish causal relationships between human MetS states, Mcp1 upregulation, and dysfunctional IRS2-AKT2- and IRS2-RAS/ERK1/2–dependent signaling.
In summary, our studies demonstrate that WMBCs from MetS patients with IR display diminished expression of Irs2 and its downstream effector Akt2 compared to insulin-sensitive MetS patients, suggesting a mechanistic link between reduced IRS2 expression and human metabolic diseases. Indeed, the moderate reduction of Irs2 expression achieved in fat-fed apoE−/−Irs2+/− mice is sufficient to produce MetS-like symptoms and accelerate atherosclerosis. Our studies with aortic tissue, primary VSMCs, and macrophages demonstrate that partial Irs2 inactivation impairs AKT2- and Ras/ERK1/2-dependent signaling, leading to augmented MCP1 expression and enhanced CD36 and SRA scavenger receptor expression and macrophage acLDL uptake beyond hypercholesterolemia. These findings highlight defective IRS2-associated AKT2- and Ras/ERK1/2-dependent signaling as a potential mechanism underlying accelerated atherosclerosis in MetS/IR states.
We thank Núria Ruiz for help with statistical analysis and M. J. Andrés-Manzano for help preparing figures.
Sources of Funding
This work was supported by grants from the Spanish Ministry of Education and Science and the European Regional Development Fund (SAF2004-03057, SAF2007-62110, SAF2007-62533), Instituto de Salud Carlos III (RECAVA, grants RD06/0014/0008, RD06/0014/0021, CIBERDEM), Generalitat Valenciana (GV/2007/164), Sociedad Española de Cardiología (Beca Novartis 2006), European Union (InGenious HyperCare, grant LSHM-CT-2006-037093), and from the agreement between the Foundation for Applied Medical Research (FIMA) and “UTE project CIMA”. H.G.-N. received salary support from the European Union (Marie Curie fellowship MEIF-CT-2005-024393). M.V.-C. was a fellow from the Regional Government of Valencia and from Instituto Danone.
Original received April 30, 2008; final version accepted August 30, 2008.
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