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. Author manuscript; available in PMC: 2014 May 20.
Published in final edited form as: Cell Metab. 2006 Apr;3(4):247–256. doi: 10.1016/j.cmet.2006.02.010

Myeloid lineage cell-restricted insulin resistance protects apolipoproteinE-deficient mice against atherosclerosis

Julia Baumgartl 1, Stephanie Baudler 1, Maximilian Scherner 2, Vladimir Babaev 3, Liza Makowski 4, Jill Suttles 5, Marcia McDuffie 6, Sergio Fazio 3, C Ronald Kahn 7, Gökhan S Hotamisligil 4, Wilhelm Krone 2, MacRae Linton 3, Jens C Brüning 1,*
PMCID: PMC4027059  NIHMSID: NIHMS418144  PMID: 16581002

Summary

Inflammatory processes play an important role in the pathogenesis of vascular diseases, and insulin-resistant diabetes mellitus type 2 represents an important risk factor for the development of atherosclerosis. To directly address the role of insulin resistance in myeloid lineage cells in the development of atherosclerosis, we have created mice with myeloid lineagespecific inactivation of the insulin receptor gene. On an ApoE-deficient background, MphIRKO mice developed smaller atherosclerotic lesions. There was a dramatic decrease in LPS-stimulated IL-6 and IL-1β expression in the presence of macrophage autonomous insulin resistance. Consistently, while insulin-resistant IRS-2-deficient mice on an ApoE-deficient background display aggravated atherosclerosis, fetal liver cell transplantation of IRS-2–/–ApoE–/– cells ameliorated atherosclerosis in Apo-E-deficient mice. Thus, systemic versus myeloid cell-restricted insulin resistance has opposing effects on the development of atherosclerosis, providing direct evidence that myeloid lineage autonomous insulin signaling provides proinflammatory signals predisposing to the development of atherosclerosis.

Introduction

Type 2 diabetes mellitus represents a frequent endocrine disease, affecting more than 5% of Western populations (Engelgau et al., 2004). Besides the direct consequences on glucose metabolism, insulin resistance is associated with a number of cardiovascular risk factors, such as obesity, dyslipoproteinemia, hypertension, and accelerated atherosclerosis (Reaven, 2004). Diabetic patients and individuals with the metabolic syndrome show an increased rate of both myocardial infarction and stroke (Reaven, 2004). Conversely, consequent insulin treatment of patients suffering from acute myocardial infarction improves their survival (Malmberg et al., 1995).

Beside the well-characterized cardiovascular risk factors associated with insulin resistance, insulin receptors are expressed and functional in many cell types implicated in the pathogenesis of atherosclerosis, including endothelial cells, vascular smooth muscle cells, and macrophages (Baudler et al., 2003). Through the use of Cre-loxP-mediated conditional inactivation of the insulin receptor in vascular endothelial cells, we could demonstrate that functional insulin signal transduction in this cell type is required for expression of vasoactive mediators and may play a role in maintaining vascular tone and regulation of insulin sensitivity to dietary salt intake (Vicent et al., 2003).

Consistent with the clinical association of insulin resistance, type 2 diabetes and atherosclerosis mouse models of insulin resistance such as insulin receptor substrate (IRS)-2-deficient mice exhibit increased neointima formation after vessel injury and increased neointima lipid accumulation on an ApoE-deficient background (Kubota et al., 2003; Clough et al., 2005).

Considerable recent evidence has demonstrated that inflammatory processes, in particular macrophages and foam cells play a key role in the pathogenesis of atherosclerosis (Libby, 2002). However, the direct role of insulin signaling in myeloid lineage cells remains controversial. Insulin acutely stimulates expression of tumor necrosis factor (TNF)-α in immortalized murine macrophages and enhances LPS-stimulated transcription of TNF-α, interleukin (IL)-6, and IL-1β, characterizing insulin as a proinflammatory signal in macrophages (Holan and Minowada, 1992; Iida et al., 2001). On the other hand, insulin negatively regulates NFκB-activation and the generation of reactive oxygen species in monocytes, pointing to a role as an anti-inflammatory signal (Dandona et al., 2001). A role for insulin in the regulation of macrophage lipid metabolism has also been suggested from its transcriptional control of hormone sensitive lipase and ACAT and the translational control of CD36-expression (Liang et al., 2004; O'Rourke et al., 2002). Taken together, there is experimental evidence that insulin through its receptors is involved in the regulation of key processes in macrophages implied in the development of atherosclerosis. To directly address the role of insulin signaling in myeloid lineage cells, we have used two models of myeloid cell-restricted insulin resistance, i.e., macrophage-specific insulin receptor knockout mice and mice lacking IRS-2 in hematopoetic cells by adoptive transfer of IRS-2–/–ApoE–/– fetal liver cells (FLCs) in irradiated ApoE–/– mice. Moreover, we have compared the effect of myeloid cell-restricted IRS-2 deficiency to body-wide IRS-2 deficiency on the development of atherosclerosis on an ApoE-deficient background.

Results

Generation of MphIRKO mice

To specifically inactivate the insulin receptor gene in myeloid lineage cells, mice carrying an insulin receptor floxed allele (IRflox) were bred with mice expressing the Cre recombinase under control of the Lysozyme M promoter. Double-heterozygous mice were intercrossed to obtain IRflox/flox and IRflox/flox LysCre+/–, i.e., MphIRKO mice. Inactivation of the IR in mice carrying the LysMCre transgene and homozygous for the IRflox allele by Western blot analysis of thioglycollate (TG)-elicited macrophages of control and MphIRKO mice was >90% (Figure 1A). Similarly, recombination of the IRflox allele occurred with 90% efficiency in TG macrophages from MphIRKO mice (Figure 1A). This parallels the percentage of macrophages obtained by this procedure as analyzed by FACS analysis using anti-CD19 and anti-MAC1 antibodies (Figure 1A). To investigate the specificity of the myeloid lineage-restricted insulin receptor knockout in MphIRKO mice, we performed Western blot analyses on tissue extracts from a variety of different nonmyeloid lineage cells such as white adipose tissue, liver, skeletal muscle, kidney, and brain. This analysis revealed unaltered insulin receptor expression in these tissues of MphIRKO mice (Figure 1B). Taken together, we achieved efficient, macrophage-specific inactivation of the insulin receptor gene in MphIRKO mice.

Figure. 1. Effective and selective myeloid lineage-specific inactivation of the insulin receptor gene in MphIRKO–mice.

Figure. 1

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A) PCR analysis of Cre-mediated recombination in thioglycollate-elicited macrophages from IRflox/flox (WT) and IRflox/floxLysMCre+/– (KO) mice. The upper-right panel shows the percentage of macrophages (MAC1 positive, CD19 negative) present in peritoneal lavage after thioglycollate treatment by FACS analysis using CD19 and MAC1-antibodies. The lower panel shows the expression of the insulin receptor b subunit in thioglycollate-elicited macrophages from four individual wild-type and knockout mice. The lower panel shows a Western blot analysis against the chemokine receptor CCR-1, serving as a loading control.

B) Western blot analysis of the insulin receptor b subunit in white adipose tissue (WAT), liver, brain, skeletal muscle, and kidney of IRflox/flox (WT) and IRflox/flox LysMCre+/– (KO) mice.

Myeloid lineage-specific deletion of the insulin receptor gene had no impact on growth and development of MphIRKO mice. Both growth curves of control and MphIRKO mice were indistinguishable as were plasma leptin and insulin concentrations and white adipose tissue mass (data not shown). Moreover, glucose-tolerance tests and insulin-tolerance tests revealed unaltered glucose homeostasis in these animals (data not shown).

Amelioration of atherosclerotic lesions in MphIRKO/ApoE–/– mice

To analyze the impact of macrophage insulin signaling on the development of atherosclerosis, we crossed the MphIRKO mice on an ApoE-deficient background. Also IRflox/floxApoE–/– and IRflox/floxLysMCre+/–ApoE–/– mice were indistinguishable with respect to body weight and glucose metabolism (data not shown).

Atherosclerotic lesion size was quantified in the descending aorta of male IRflox/flox ApoE–/– and IRflox/floxLysMCre+/–2ApoE–/– mice at the age of 5 months after 4 months of exposure to a high-cholesterol diet. This analysis revealed a significant ~50% reduction in lesion size in the presence of macrophage-specific insulin resistance (Figure 2A). Importantly, also under high-cholesterol diet conditions there was no difference between IRflox/floxApoE–/– and IRflox/floxLysMCre+/–ApoE–/– mice with respect to plasma glucose, insulin, and cholesterol concentrations (Figure 2B). Moreover, lipoprotein profiles did not differ depending on macrophage insulin receptor expression, again ruling out major metabolic alterations in the presence of macrophage-specific insulin resistance leading to the reduction of atherosclerosis (Figure 2C).

Figure 2. Reduced atherosclerosis in ApoE-deficient MphIRKO mice.

Figure 2

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A) Representative Sudan IV stainings of male ApoE–/– IRflox/flox and ApoE–/–IRflox/floxLysMCre+/– mice under high-cholesterol diet and quantitation of lesion size in the descending aorta of male ApoE–/–IRflox/flox (control) and ApoE–/–IRflox/floxLysMCre+/– (MphIRKO) mice. Data represent the mean of six to nine animals of each genotype (*p < 0.05 in unpaired Student's t test).

B) Plasma glucose, insulin, and cholesterol concentrations were determined in male ApoE–/–IRflox/flox (control) and ApoE–/–IRflox/floxLysMCre+/– (MphIRKO) mice exposed to high-cholesterol diet at the age of 5 months after overnight fasting. Data represent the mean ± SEM of six to nine animals of each genotype.

C) Lipoprotein profiles as determined by FPLC analysis from pooled sera of male ApoE–/–IRflox/flox (red line) and ApoE–/–IRflox/flox LysMCre +/– (green line) mice.

Unaltered metabolism of modified LDL particles in MphIRKO mice

Since macrophages play an important role in the metabolism of modified LDL particles, we next determined the expression and function of scavenger receptors in the macrophages of MphIRKO mice. Uptake of oxidized LDL is mainly mediated by the scavenger receptor CD36. Recently, it has been demonstrated that macrophage insulin signaling regulates protein expression of CD36 through translational control mechanisms (Liang et al., 2004). Therefore, we determined both expression of CD36 mRNA by Northern blot analysis and CD36 protein expression by Western blot analysis in thioglycollate-elicited primary macrophages and in immortalized control and IR-deficient macrophage cell lines. In order to obtain an in vitro culture system for the analysis of insulin receptor function in macrophages, we immortalized macrophage cell lines by retroviral transduction of bone marrow precursor cells from IRflox/flox mice. FACS analysis of these cell lines revealed a macrophage phenotype as assessed by staining with anti-MAC1 and F4/80 antibodies, while the cells were negative for the B-cell marker CD19 (data not shown). To circumvent potential problems arising from differences between cell lines obtained from individual control and MphIRKO mice, we decided to acutely inactivate the insulin receptor gene in immortalized macrophages derived from IRflox/flox mice. Therefore, these cells were cultured overnight in the presence of a recombinant fusion protein of the Cre-recombinase and the protein transduction domain of the HIV-Tat protein. In vitro transduction of the IRflox/flox cells resulted in complete recombination of the IRflox allele and abolishment of IR expression, as assessed by Western blot analysis (data not shown).

Analysis of CD36 mRNA and protein expression revealed no alterations in insulin receptor-deficient macrophages (Figure 3A). Since it had been demonstrated that in other tissues, such as adipose and cardiac muscle, insulin is capable of acutely stimulating translocation of CD36 to the plasma membrane (Luiken et al., 2002), we determined the expression of CD36 on the plasma membrane by FACS analysis. When plasma membrane CD36 expression was analyzed, cell surface expression of CD36 was indistinguishable between control and IR-deficient macrophages (Figure 3B). Also, cell surface expression of the alternative scavenger receptor CD204 was unaltered in the presence of macrophage-specific insulin resistance (data not shown). Moreover, analysis of mRNAs of key regulators of LDL metabolism in macrophages, such as a lipoprotein lipase and the reverse cholesterol transporters ABCA-1 and ABCG-2, revealed unaltered expression in insulin receptor-deficient macrophages (data not shown). Consistent with these findings, we found unaltered degradation of oxidized LDL particles in TG macrophages from MphIRKO mice (Figure 3C). Consistent with the unaltered expression of CD36 and CD204, these data indicate that uptake of modified LDL particles is not altered in the presence of macrophage-specific insulin resistance.

Figure 3. Unaltered metabolism of modified LDL particles in insulin-resistant macrophages.

Figure 3

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A) Northern blot analysis of CD36 in IRflox/flox (WT) and Cre-transduced IRflox/flox (KO) immortalized macrophage cell lines. Cells had been left either untreated or were stimulated with 100 nM insulin for 24 hr. The upper-right panel shows a Western blot analysis of CD36 expression in thioglycollate-elicited macrophages of four individual ApoE–/–IRflox/flox (WT) and IRflox/floxLysMCre+/– (KO) mice. The lower panel shows Western blot analysis of CD36 expression in IRflox/flox (WT) and Cre-transduced IRflox/flox (KO) immortalized macrophage cell lines.

B) FACS analysis of CD36 surface expression in control and IR-deficient immortalized macrophage cell lines.

C) Specific uptake of oxidized LDL cholesterol in thioglycollate-elicited macrophages from IRflox/flox (WT) and IRflox/floxLysMCre+/– (KO) mice. Data represent the mean ± SEM of four to six animals of each genotype.

D) Cholesterol efflux in thioglycollate-elicited macrophages from control and IR-deficient mice. Cholesterol efflux was determined in the absence (open bars) and in the presence of 10 μg/ml ApoA1 (closed bars). Data represent the mean ± SEM of four to six animals of each genotype.

Moreover, analysis of cholesterol efflux in cells isolated from control and MphIRKO mice revealed no difference in basal acetylated LDL efflux and that after ApoA-1 stimulation (Figure 3D). Taken together, metabolism of modified LDL particles remains unaltered in the presence of macrophage-specific insulin resistance.

Impaired inflammatory response in IR-deficient macrophages

To further analyze the mechanism for decreased atherosclerosis in MphIRKO mice, and to assess the inflammatory capacity of control and IR-deficient macrophages, we determined mRNA expression of TNF-α, MCP-1, IL-6 and IL-1β under basal conditions and after LPS-stimulation. This analysis revealed that in the basal state, both control and IR-deficient macrophages express very little mRNA of these genes (Figure 4A). In contrast, LPS potently stimulated expression of TNF-α, MCP-1, IL-6, and IL-1β mRNA in IRflox/flox macrophages (Figure 4A). In IR-deficient macrophages LPS stimulated expression of TNF-α and MCP-1 to similar extend as compared to control cells but almost completely failed to stimulate expression of IL-6 and IL-1β (Figure 4A). These data indicate that macrophage autonomous insulin signaling is required for selective LPS-stimulated transcription of proinflammatory cytokines such as IL-6 and IL-1β, while insulin resistance in these cells does not influence LPS ability to induce transcription of TNF-α and MCP-1.

Figure 4. Impaired inflammatory response in insulin-resistant macrophages.

Figure 4

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A) Northern blot analysis of MCP1, TNF-α, IL-1β, and IL-6 expression in IRflox/flox immortalized macrophage cell lines treated without (WT) or with HNTC-Cre (KO), which have been cultured either in the absence or presence of 10 ng/ml LPS for 24 hr. The lower panel shows the densitometric quantitation of mRNA-expression obtained in three independent experiments. Signal intensity detected in unstimulated WT cells was set as 1. Data represent the mean ± SEM (**p < 0.01 in unpaired Student's t test).

B) Amount of the indicated cytokines released into the culture medium by control or IR-deficient immortalized macrophages, which had been cultured either in the absence or presence of 10 ng/ml LPS for 3 days. Data represent the mean ± SEM obtained in three independent experiments (**p < 0.01 in unpaired Student's t test).

C) Amount of the indicated cytokines released into the culture medium by TG-elicited macrophages from control (WT) and MphIRKO-(KO) mice. Cells have been cultured in the absence or presence of 10 ng/ml LPS for 3 days. Data represent the mean ± SEM obtained from cells of four mice of each genotype, each cell line assayed in triplicate (*p < 0.05 in unpaired Student's t test).

To further test the functional significance of altered interleukin transcription, we determined the release of the cytokines tested above into the supernatant of cultured cells. Under basal conditions the levels of TNF-α, MCP-1 and IL-6 were below the detection limit of the ELISA assays (Figure 4B). On the other hand, LPS potently stimulated the release of MCP-1 and TNF-α in control and IR-deficient macrophages, although there was a tendency toward decreased release of TNF- α in IR-deficient macrophages compared to control cells (954 + 75 ng/ml versus 1137 + 58 ng/ml; p = 0.09). Strikingly, while LPS potently stimulated the release of IL-6 into the supernatant of control cells, it completely failed to do so in IR-deficient macrophages (Figure 4B). To confirm these findings in a second macrophage system, TG-elicited macrophages from control and MphIRKO mice were analyzed for their capacity to release TNF-α and IL-6 in response to LPS stimulation. Consistent with the results obtained in the immortalized IR-deficient cell lines, LPS-stimulated IL-6 release was significantly reduced in cells derived from MphIRKO mice, while TNF-α release remained unaltered (Figure 4C). Taken together, these results of a significantly reduced capacity of LPS-stimulated IL-6 release in the presence of macrophage autonomous insulin resistance.

Uncoupling of systemic versus hematopoetic cell-restricted insulin resistance in atherosclerosis

To directly address the apparent difference between the clinical association of systemic insulin resistance and atherosclerosis on one hand and the protection from atherosclerosis as a consequence of myeloid-specific insulin receptor deletion on the other hand, we decided to employ another mouse model for both systemic and myeloid-specific insulin resistance. Since IRS-2 represents the major insulin receptor substrate protein expressed in macrophages, mice lacking the insulin receptor substrate (IRS)-2 in all tissues throughout development via conventional gene targeting were backcrossed on an ApoE-deficient background. Male IRS-2-deficient mice exhibited significantly increased fasting blood glucose concentrations and severely impaired glucose tolerance also on an ApoE-deficient background (data not shown) as previously described for other backgrounds (Kubota et al., 2000). Also, when exposed to high-cholesterol diet, male IRS-2 knockout mice demonstrated severe glucose intolerance (Figure 5A). On the other hand, plasma cholesterol and triglyceride concentrations were indistinguishable between control and IRS-2-deficient mice on an ApoE-deficient background when exposed to high-cholesterol diet (Figure 5C).

Figure 5. Metabolic characterization of IRS-2-deficient mice on an ApoE-deficient background.

Figure 5

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A) Glucose-tolerance tests were performed at the age of 12 weeks in IRS-2+/+ApoE–/– (square) and IRS-2–/–ApoE–/– (diamond) male mice. Data represent the mean ± SEM of 10–14 animals of each genotype.

B) Glucose-tolerance tests in male ApoE-deficient mice which have been reconstituted after lethal irradiation with either IRS-2+/+ApoE–/– (square) or IRS-2–/–ApoE–/– (diamond) fetal liver cells. Glucose-tolerance test was performed 10 weeks after transplantation. Data represent the mean ± SEM of 12–13 animals in each group.

C) Plasma triglyceride and cholesterol concentrations in the different mutant mice. Concentrations of triglyceride and cholesterol were determined in serum obtained from IRS-2+/+ApoE–/– and IRS-2–/–ApoE–/– mice at the age of 12 weeks, after 9 weeks of exposure to Western diet. Plasma cholesterol and triglyceride concentrations in irradiated and reconstituted IRS/ApoE-deficient mice were determined 11 weeks after transfer, having been exposed for 8 weeks to high-cholesterol diet. Data represent the mean ± SEM of 8–12 mice in each group.

To directly restrict the same molecular mechanism of insulin resistance - namely IRS-2 deficiency - to hematopoetic cells, we performed transfers of IRS-2+/+ApoE–/– (controls) and IRS-2–/–ApoE–/– fetal liver cells into lethally irradiated ApoE–/– recipients. Thereby, only hematopoetic cells lack functional IRS-2 expression, whereas IRS-2 expression in nonhematopoetic cells remains unaltered on an ApoE-deficient background. Physiological characterization of reconstituted ApoE-deficient mice revealed, that parameters reflecting glucose and lipid metabolism were indistinguishable between mice, which received either control or IRS-2-deficient fetal liver cells (Figures 5B and 5C).

Analysis of atherosclerotic lesions in IRS-2+/+ApoE–/– and IRS-2–/–ApoE–/– mice revealed that abdominal lesion area increased 1.7fold in IRS-2–/–ApoE–/– mice compared to IRS-2+/+ApoE–/– mice (Figure 6A). Similarly, lesion area in the aortic arch increased from 32% in IRS-2+/+ApoE–/– mice to 52% in IRS-2–/–ApoE–/– mice (Figure 6B) and lesion area in cross-sections of the aortic root increased from 150.000 to 250.000 μm2 (Figure 6C). Taken together, these data indicate that systemic insulin resistance and impaired glucose tolerance due to IRS-2 deficiency aggravate atherosclerosis in ApoE-deficient mice.

Figure 6. Aggravated atherosclerosis in IRS-2–/–ApoE–/– mice.

Figure 6

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A) Lesion surface area in the abdominal aorta was determined in 4-month-old IRS-2+/+ApoE–/– and IRS-2–/–ApoE–/– mice after 12 weeks of exposure to high-cholesterol diet. The left panel demonstrates an example of the lipid-stained lesions, the graph demonstrates a quantitative assessment of lesion size in 12–22 animals in each group. (p < 0.05 in unpaired Student's t test).

B) Quantification of lipid-positive lesions of the aortic arch as percentage of surface. The left panel demonstrates exemplary pictures, the graph on the right side presents the quantitative assessment of lesion size in 12–20 mice of each genotype. (p < 0.05 in unpaired Student's t test).

C) Cross-section analysis of atherosclerotic lesions in the aortic root. Left panel demonstrates a representative result obtained in IRS-2+/+ApoE–/– and IRS-2–/–ApoE–/– mice. The graph shows the quantitative assessment of lesion area in these animals for 10–15 animals in each genotype. (p < 0.05 in unpaired Student's t test).

On the other hand, analysis of atherosclerotic lesions in the reconstituted ApoE-deficient mice revealed a significant reduction in plaque formation in the aortic arch and in the aortic root of mice receiving IRS-2-deficient FLC (Figures 7A and 7B). Taken together, these data directly uncouple the role of systemic versus hematopoetic lineage-restricted insulin resistance in the development of atherosclerosis by demonstrating that the same genetic defect when present systemically aggravates atherosclerosis while when only restricted to hematopoetic cells protects from the development of atherosclerosis.

Figure 7. Adoptive transfer of IRS-2+/+ApoE–/– and IRS-2–/–ApoE–/– fetal liver cells protects against atherosclerosis.

Figure 7

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Male ApoE-deficient mice were lethally irradiated at the age of 6–8 weeks and reconstituted the following day by iv injection of fetal liver cells obtained from male IRS-2+/+ApoE–/– and IRS-2–/–ApoE–/– embryos. Mice were allowed to recover under normal chow diet for 2 weeks and then exposed to high-cholesterol diet for 12 weeks before analysis of atherosclerosis.

A) Quantification of lipid-positive lesions of the aortic arch as percentage of surface. The left panel demonstrates exemplary pictures, the graph on the right side presents the quantitative assessment of lesion size in 17–22 mice of each genotype. (p < 0.05 in unpaired Student's t test).

B) Cross-section analysis of atherosclerotic lesions in the aortic root. Left panel demonstrates a representative result, the graph shows the quantitative assessment of lesion area in these animals for 14–15 animals in each genotype. (p < 0.05 in unpaired Student's t test).

Discussion

In summary, our data provide direct evidence for a proinflamma-tory role of cell autonomous insulin action in macrophages. The reduced capacity of IR-deficient macrophages to express IL-6 and IL-1β and to secrete IL-6 provides a potential mechanism by which myeloid lineage-specific insulin resistance may attenuate development of atherosclerosis in ApoE-deficient mice. Moreover, these data are consistent with previous studies showing enhanced development of atherosclerosis in control and ApoE-deficient mice after injection of IL-6 and protection from bacterial and high fat diet-induced atherosclerosis in ApoE+/– mice by deletion of the IL1 receptor gene (Chi et al., 2004; Huber et al., 1999). Although the animals examined in the present study had only a ~90% contribution of C57Bl6J alleles and since genetic background heterogeneity can influence atherosclerosis studies, protection from atherosclerosis in two independent models (MphIRKO mice and IRS-2–/–ApoE–/– transplantation) supports the notion that myeloid-restricted insulin resistance protects from the development of atherosclerosis.

These findings may seem somewhat surprising in view of the fact that insulin-resistant patients with type 2 diabetes exhibit an increased risk for the development of atherosclerosis. However, this likely relates to the multifactorial nature of atherosclerosis. First, it is not clear to what extend macrophages in diabetes exhibit insulin resistance. In humans it remains controversial whether IR-expression in monocytes is decreased in diabetic patients. While decreased insulin binding to circulating monocytes in both diabetic and obese insulin-resistant patients has been described, others studies revealed unaltered binding but impaired internalization of insulin receptors in these cells (Kelly and Fantus, 1995); (Mandarino et al., 1984); (Trischitta et al., 1989). Studies have also demonstrated impaired insulin and IGF-1-stimulated IRS-2 tyrosine phosphorylation in a murine model of diet induced obesity and in genetically obese and insulin-resistant ob/ob mice and impaired insulin-stimulated IRS-2 phosphorylation after incubation of cultured macrophages in the presence of elevated insulin concentrations (Hartman et al., 2004; Liang et al., 2004). Nevertheless, in patients with type 2 diabetes, hyperinsulinemia might overcome reduced macrophage insulin signaling leading to constant proinflammatory signaling predisposing for the development atherosclerosis. Alternatively, although macrophage-autonomous insulin resistance provides a protective factor for the development of atherosclerosis, insulin resistance in other tissues leading to elevated blood pressure, decreased concentrations of HDL-cholesterol and increased concentrations of VLDL-cholesterol may override this protective effect. Indeed this notion is supported by the IRS-2-deficient mouse models used in the present study, unraveling the contrasting effect of systemic versus hematopoetic cell-restricted insulin resistance in the development of atherosclerosis. Thus, further analysis of interactions between these multiple molecular mechanisms engaged in insulin's proinflammatory action in macrophages may help to develop novel therapeutic approaches for the treatment of atherosclerosis associated with insulin resistance and type 2 diabetes mellitus.

Experimental procedures

Animals and genotyping

For disruption of the insulin receptor in myeloid lineage cells, IRflox mice (generated on a hybrid C57BL/6J/129sv background and backcrossed for two generations on a C57BL/6J background) were bred with mice expressing the Cre recombinase under control of the lysozyme (Lys) M promoter (back-crossed for more than seven generations on a C57/bl6J background) ((Bruning et al., 1998; Clausen et al., 1999). Animals were housed in a virus-free facility on a 12 hr light/dark cycle (0700 on/1900 off) and were fed a standard rodent chow or, when indicated, a diet containing 5% cholesterol (Teklad 88051, Harlan Winkelmann Inc., Borchen, Germany). For analysis of atherosclerosis development, IRflox/flox LysMCre mice (s.a.) were crossed with ApoE-deficient mice on a C57BL/6J background obtained from Jackson laboratory. Analysis of polymorphic markers revealed the expected frequency of ~90% homozygous C57BL/6J alleles in the resulting offspring (data not shown). IRS-2-deficient mice on a mixed C57BL6J/129sv background (Kubota et al., 2000) were backcrossed on a C57BL/6J background for four generations and then intercrossed with ApoE-deficient mice on a C57BL/6J background. IRS-2+/–ApoE+/– mice were intercrossed to yield IRS-2+/–ApoE–/– mice, which were further intercrossed to yield littermates, which were IRS-2+/+ApoE–/– or IRS-2–/–ApoE–/–. Genotyping was performed by PCR using genomic DNA isolated from tail tips as previously described (Bruning et al., 1998). All protocols for animal use and euthanasia were reviewed by the animal care committee of the University of Cologne, approved by local government authorities and were in accordance with NIH-guidelines.

Genome scanning

Using DNA prepared from ten randomly selected experimental mice, 56 mapped microsatellite loci ((Dietrich et al., 1996); http://carbon.wi.mit.edu:8000/cgi-bin/mouse/index), previously determined to be polymorphic in a C57BL/6J X 129X1/SvJ (formerly 129/SvJ) cross, were analyzed using methods adapted from Dietrich (Dietrich et al., 1992). Briefly, PCR amplification from genomic DNA was performed using fluorochrome-labeled primers purchased from Research Genetics, Inc. (Huntsville, AL) or synthesized from published oligonucleotide sequences (Integrated DNA Technologies, Coral-ville, IA). Products were separated using a 4% agarose gel and scored by comparison to control C57BL/6J, 129X1, and F1 DNA samples. Detailed amplification protocols, locus list, and allele sizes are available on request.

Fetal liver cell transfers

Embryos were obtained by intercrossing IRS-2+/–ApoE–/– mice and typed for male gender as previously described (Bruning et al., 2000). IRS-2+/+ApoE–/– and IRS-2–/–ApoE–/– FLC were isolated from male embryos on day 16.5 postconception. For FLC transfer, single-cell suspensions of fetal livers were prepared, and 106 FLC in HBSS in a volume of 300 μl were injected i.v. into 8-week-old ApoE–/–C57BL/6 mice (24) that had been lethally irradiated (900 rad) the previous day (Baudler et al., 2005). Recipients were treated with antibiotics in the drinking water after irradiation; the mix of antibiotics was changed every 2 weeks (kanamycin sulfate, neomycin sulfate, and bacitracin, or penicillin, streptomycin sulfate, and gentamycin sulfate; 30 mg/liter each) and exposed to high-cholesterol diet starting 2 weeks after transplantation.

Quantification of atherosclerosic lesions

Quantification of atherosclerotic lesions was performed by determining lesion surface area in the aortic arch and in the total abdominal aorta as indicated in the figure legends. Additionally, when indicated, lesions were quantified in cross-sections obtained at the level of the aortic valve. Procedures were performed as previously described (Makowski et al., 2001).

Cell lines and primary cultured cells

To immortalize control and IR-deficient macrophage cell lines with inducible deletion of the insulin receptor, IRflox/flox mice were sacrificed and bone marrow cells were isolated. Cells were transduced with a v-myc/v-raf-expressing retrovirus as previously described (Mukhopadhyay et al., 2001). Cell lines derived from IRflox/flox mice were transduced with a recombinant fusion protein of the Cre-recombinase and the HIV-Tat protein transduction domain (HNTC), allowing for efficient fusion protein-mediated recombination and IR inactivation. HNTC transduction was performed as previously described (Peitz et al., 2002).

Immunoprecipitations and Western blot analysis

Tissues were removed and homogenized in homogenization buffer (50 mM HEPES [pH 7.4], 1% Triton X-100, 50 mM sodium pyrophosphate, 0.1 M sodium fluoride, 10 mM EDTA, 10 mM sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mM benzamidine, and 2 mM PMSF) with a Polytron homogenizer. Samples were allowed to solubilize for 30 min on ice, and particulate matter was removed by centrifugation at 42,000 rpm for 1 hr at 4°C in a Sorvall centrifuge. Immunoprecipitations and Western blot of insulin signaling proteins were performed as previously described (Bruning et al., 1998).

Northern blot

Total RNA from macrophages was prepared using Trizol RNA extraction reagent according to the manufacturer's guidelines. RNA gels, blots, and hybridizations with radiolabeled cDNA-probes were performed as previously described (Bruning et al., 1998).

Analytical procedures

Blood glucose values were determined from whole venous blood using an automatic glucose monitor (Glucometer Elite, Bayer). Insulin and leptin levels in serum were measured by ELISA using mouse standards according to manufacturer's guidelines (Crystal Chem., Downers Grove, IL). Serum cholesterol and triglyceride determinations were performed on a Beckmann CX7 analyzer. Lipoprotein profiles were determined by FPLC analysis as previously described (Makowski et al., 2001).

Glucose-tolerance and insulin-tolerance tests were performed on animals that had been fasted overnight for 16 hr. Animals were injected with either 2 g/kg body weight of glucose or 0.75 U/kg body weight of human regular insulin (Novo Nordisk Pharmaceuticals Inc., Princeton, NJ) into the peritoneal cavity. Glucose levels were determined in blood collected from the tail tip immediately before and 15, 30, 60, and 120 min after the injection.

Cytokine Release

Primary macrophages or immortalized macrophage cell lines were incubated with the indicated reagents for 24 hr. Supernatants were collected after 72 hr of treatment and cytokine levels were determined by commercially available ELISA systems (TNF-α, IL-6, IL-1β, and MCP-1 from R&D, Minneapolis, MN).

Cholesterol efflux and degradation

For the analysis of cholesterol efflux, acLDL particles were incubated for 30 min with 3H-labeled cholesterol to incorporate the radiolabel into the particles. Cells were loaded with 3H-acLDL particles for 24 hr, rested overnight without tritiated or acLDL particles in the medium, and then efflux to increasing doses of apoA1 was induced for 4 hr. For the analysis of degradation, apoB particles were labeled with 125I. Unlabeled LDL and 125I-LDL particles were oxidized for 18 to 24 hr. Cells were loaded with 10 μg/ml of labeled oxLDL and 70 μg/ml unlabeled oxLDL. Degradation and efflux was calculated as previously described (Makowski et al., 2001).

Acknowledgments

We wish to thank Takashi Kadowaki for providing IRS-2-deficient mice. We are grateful to Gisela Schmall for excellent secretarial assistance, to Frank Edenhofer for kindly providing the recombinant HNTC protein, and to Irmgard Förster for providing the LysM-Cre-transgenic mice. The study was supported by funds of the Center of Molecular Medicine Cologne (CMMC to J.C.B. and W.K.), the Deutsch Forschungsgemeinschaft (DFG) (1492-3/1 to J.C.B.), and the National Institutes of Health (NIH) (NRSA F32 HL075970-01 to L.M.).

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