An accelerated mouse model for atherosclerosis and adipose tissue inflammation

Male LDLR^-/- and ApoE^-/- mice, both on a C57BL/6 J background were purchased from Charles River Laboratories (Sulzfeld, Germany). At 9 weeks of age LDLR^-/- mice were placed on either a high-fat diet enriched with 0.15% cholesterol (HFC) containing 60 kcal% fat (primarily lard) and 20 kcal% carbohydrates with 6.8 kcal% deriving from sucrose (D01120401; Research Diets Inc., New Brunswick, NJ, USA), a sucrose-enriched high-fat diet with 0.15% cholesterol (HFSC) consisting of 58 kcal% fat (primarily lard) and 28 kcal% carbohydrates (with 17.5 kcal% from sucrose; D09071704, Research Diets Inc), [33] or a low-fat control diet (LF, 10 kcal% fat; D12450B; Research Diets Inc.) for up to 20 weeks. Further details about composition of the used diets are presented as Additional file 1: Table S1. In addition, ApoE^-/- mice were fed HFSC for 16 weeks. LDLR^-/- mice on LF served as a negative control for adipose tissue inflammation and insulin resistance.

All mice were housed in a specific pathogen-free facility on a 12-hour light/dark cycle with free access to food and water. Food intake and weight gain were monitored throughout the studies. To determine food intake, diets were weighed before and after food change and difference was calculated as estimated food intake. Blood was drawn after a 3-hour fasting period immediately before mice were sacrificed. Gonadal adipose tissue and liver samples were collected and immediately snap frozen in liquid nitrogen. The study protocols were approved by the Austrian Federal Ministry for Science and Research and followed the guidelines on accommodations and care of animals formulated by the European Convention for Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes.

Plasma triglyceride and cholesterol concentration was analyzed using an automated analyzer (Falcor 350, A. Menarini Diagnostics, Florence, Italy). Enzyme-linked immunosorbent assay kits were used to determine plasma insulin (Mercodia AB, Uppsala, Sweden) and osteopontin levels (R&D Systems, Minneapolis, MN USA). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as an index for insulin resistance [34]. Insulin sensitivity was assessed by insulin tolerance test after a 5-hour fasting period. Briefly, intraperitoneal injection of recombinant human insulin aspart (Novo Nordisk A/S, Denmark) at a dose of 0.75 U/kg body weight was given to HFC- and HFSC-fed LDLR^-/- mice and HFSC-fed ApoE^-/- mice. Blood glucose concentrations were determined before and 30, 60, 90 and 120 minutes after insulin injection. Glucose tolerance test was performed after a 6-hour fasting period and blood glucose was measured before and 15, 45, 75, 105 and 135 minutes after intraperitoneal injection of 20% glucose (0.75 g/kg body weight).

To analyze plasma lipoproteins 12 μL of EDTA-treated plasma were injected by autosampler into an Agilent 1200 HPLC instrument equipped with a Superose 6 10/300 gel-filtration FPLC column which separates the intact lipoproteins by size. An in-line assay for total cholesterol (Infinity Cholesterol reagent, Thermo Scientific) was performed at 37°C using a post-column reaction. Reaction products were monitored in real-time at 500 nm and the data analyzed using Agilent Chemstation software.

Tissue samples were homogenized in TRIzol reagent (Life Technologies, Carlsbad, CA, USA) and RNA was isolated according to the manufacturer’s protocol. One microgram of total RNA was treated with DNase I and transcribed to cDNA using Superscript II and random hexamer primers (all Invitrogen) as described [35]. Gene expression of F4/80 (Emr1, Mm00802530_m1), MCP-1 (Ccl2, Mm00441242_m1), TNFα (Tnf, Mm00443258_m1), IL-6 (Il6, Mm00446190_m1), osteopontin (Spp1, Mm00436767_m1), adiponectin (Adipoq, Mm00456425_m1), fatty acid synthase (Fasn, Mm00662319_m1), acetyl-CoA carboxylase (Acaca, Mm01304289_m1), stearoyl-CoA desaturase 1 (Scd1, Mm00772290_m1), CD3 (Cd3e, Mm01179194_m1), CD8 (Cd8a, Mm01182108_m1), FoxP3 (Foxp3, Mm00475162_m1), CD19 (Cd19, Mm00515420_m1), fatty acid binding protein 4 (FABP4; Fabp4, Mm00445878_m1), leptin (Lep, Mm00434759_m1), and peroxisome proliferator-activated receptor γ (PPARγ; Pparg, Mm01184322_m1) was analyzed by quantitative real-time RT-PCR on an ABI Prism 7000 cycler using assays-on-demand kits (TaqMan® Gene Expression Assay, Life Technologies) and normalized to ubiquitin C mRNA (Ubc, Mm01198158_m1).

Liver tissue was embedded in OCT and samples were cut into 5-μm section, and stained with hematoxylin-eosin. To further evaluate hepatic lipid content with Oil Red O cryosections (7 μm) were fixed in 60% isopropanol for 5 min and stained with 0.5% Oil Red-O (Sigma-Aldrich) in 60% isopropanol for 20 min, and finally counterstained with hematoxylin. Sections were analyzed with standard light microscopy, and relative areas of lipid accumulation (expressed as percentage Oil Red O staining) were quantified using ImageJ software. A minimum of 5 independent fields per sample was evaluated.

Atherosclerotic plaque formation was determined using en face technique as described [36]. Briefly, thorax of sacrificed mice was opened and aorta was cleaned by removing fat and connective tissue. Then the aorta was excised 2 mm above aortic root and below iliac bifurcation, opened longitudinally and pinned to silicone plates with acupuncture needles (asia-med, Suhl, Germany) and fixed overnight in 4% paraformaldehyde, 5% sucrose, 20 μM EDTA (pH 7.4). Atherosclerotic plaques were stained with Sudan IV for 15 minutes and destained with 75% ethanol. Pictures were taken with a Sony Z-1000 camera and atherosclerotic lesion area was assessed by a person blinded to the samples by using ImageJ software.

Data are given as means ± SEM. Effects of dietary treatment within LDLR^-/- mice were analyzed by one-way ANOVA using Dunnett’s multiple comparisons test. Comparisons between LDLR^-/- and ApoE^-/- mice both fed HFSC were assessed by unpaired two-tailed Student t test. A P value of ≤0.05 was considered statistically significant.

In order to establish an appropriate mouse model for cardio-metabolic disease, we placed male LDLR^-/- mice either on a low-fat control diet (LF) or two different high-fat diets, namely a common high-fat diet containing 0.15% cholesterol (HFC) or a high-fat diet rich in sucrose with 0.15% cholesterol (HFSC). In addition, ApoE^-/- mice which were used as a positive control for atherosclerotic plaque formation were fed HFSC. Baseline fasting glucose and insulin concentration before diet start did not significantly differ between LDLR^-/- and ApoE^-/- mice (Additional file 1: Table S2). LDLR^-/- mice fed both HFSC and HFC developed significant diet-induced obesity compared to LF-fed controls with no significant difference between diets (Figure 1A). Also ApoE^-/- mice on HFSC showed marked obesity (Figure 1A). Gonadal adipose tissue weight was not significantly altered between HFSC and HFC-fed LDLR^-/- mice as well as LDLR^-/- compared to ApoE^-/- mice both on HFSC (Additional file 2: Figure S1).

After dietary treatment for 12 weeks fasting plasma insulin and HOMA-IR in LDLR^-/- mice was higher in the HFSC and HFC group compared to LF. Moreover, HFSC feeding significantly increased fasting insulin level compared to HFC-fed LDLR^-/- mice indicating pronounced insulin resistance in animals on HFSC (Figure 1B). Correspondingly, HOMA-IR in LDLR^-/- mice on HFSC was significantly increased compared to the HFC-fed group (Figure 1C). ApoE^-/- mice developed significantly less pronounced insulin resistance than LDLR^-/- mice both on HFSC (Figure 1C). Testing whole body insulin tolerance did not show significant differences between HFSC and HFC group (Additional file 2: Figure S1) while glucose tolerance was impaired in HFSC-fed LDLR^-/- mice as illustrated by significantly increased glucose levels in response to a glucose tolerance test at the 15-minute time point (Figure 1D). After 20 weeks on diet, however, there were no differences in fasting insulin levels, HOMA-IR and glucose tolerance in LDLR^-/- mice on HFSC and HFC (Figure 1E-G) while body weight did not increase further.

Compared to LF-fed LDLR^-/- mice, both groups on HFC and HFSC developed hypertriglyceridemia and hypercholesterolemia (Figure 2A,B). Fasting triglyceride levels tended to be higher (P = 0.07) and total cholesterol was significantly elevated in LDLR^-/- mice on HFSC compared to those on HFC (Figure 2A,B).

Fractionation of plasma lipoproteins by FPLC revealed markedly increased cholesterol in VLDL and LDL fractions of obese LDLR^-/- mice on both high-fat diets compared to lean LF-fed LDLR^-/- mice after 16 and 20 weeks of feeding (Figure 3A,B). Lipoprotein profile of HFSC-fed LDLR^-/- mice showed elevated VLDL (P = 0.013) and LDL (P = 0.04) cholesterol compared to HFC-fed group after 16 but not 20 weeks of diet (Figure 3A,B). In addition, cholesterol in the VLDL and LDL fractions was significantly lower (P ≤ 0.001) in HFSC-fed ApoE^-/- mice than HFSC-fed LDLR^-/- mice as shown in Figure 3A.

Cytokines secreted by adipose tissue, so-called adipokines, induce local and systemic low-grade inflammation in obesity. In obese adipose tissue macrophages represent the main source of inflammatory adipokines although other cell types, such as lymphocytes, adipocytes, and pre-adipocytes may contribute. To investigate the dietary impact on obesity-associated adipose tissue inflammation in LDLR^-/- mice we next analyzed expression of inflammatory adipokines and estimated accumulation of macrophages, T cells and B cells in gonadal adipose tissue. After dietary treatment for 16 weeks mRNA expression of Emr1, the gene encoding macrophage marker F4/80, as well as mRNA levels of the inflammatory genes for TNFα (Tnf), IL-6 (Il6), MCP-1 (Ccl2) and osteopontin (Spp1) were significantly higher in adipose tissue of obese LDLR^-/- mice on both high-fat diets compared to lean control group on LF (Figure 4). In contrast gene expression of the anti-inflammatory and insulin-sensitizing adiponectin (AdipoQ) as well as expression of typical genes related to adipocyte function such as those for PPARγ (Pparg), FABP4 (Fabp4) and fatty acid synthase (FAS, Fasn) was significantly lower in obese LDLR^-/- mice on both high-fat diets compared to LF-fed group after 16 weeks of feeding (Figure 4F and Additional file 3: Figure S4). Adipose tissue expression of the marker genes specific for pan-T cells (Cd3e), cytotoxic (Cd8a), and regulatory T cells (Foxp3) as well as B cells (Cd19) revealed no significant obesity-induced alterations after both high-fat diets compared to lean LF-fed LDLR^-/- mice (Additional file 4: Figure S2). In LDLR^-/- mice adipose tissue inflammation was significantly higher than in ApoE^-/- mice fed the same diet (Figure 4).

Notably, after 16 weeks of feeding adipose tissue inflammation was more pronounced in LDLR^-/- mice on HFSC than on HFC as indicated by significantly higher mRNA levels of the genes encoding macrophage marker F4/80, TNF-α, and osteopontin (Figure 4A,B,E). Moreover, the marked increase of osteopontin mRNA levels in adipose tissue was accompanied by a borderline significant increase of osteopontin plasma levels in the HFSC-fed group only (P = 0.07), while osteopontin plasma levels in HFC-fed LDLR^-/- mice were equal to animals on LF (Figure 4G). After 20 weeks of feeding, all mice showed similar osteopontin plasma levels (Figure 4G).

After 20 weeks on diet, adipose tissue inflammation remained more pronounced in high-fat diet-fed LDLR^-/- mice compared to LF while adipose tissue inflammation in the HFSC and HFC group did not remain significantly different from each other suggesting a delayed peak of inflammation in the HFC group (Figure 4).

In order to characterize impact of HFSC compared to HFC feeding on metabolic phenotype in LDLR^-/- mice in more detail, we next determined hepatic lipid accumulation and inflammation after treatment with indicated diets. LDLR^-/- mice on both high fat diets (HFSC or HFC) developed extensive hepatic steatosis, compared to LF-fed lean controls after 16 as well as 20 weeks of feeding (Additional file 5: Figure S3). Hepatic lipogenic gene expression for FAS (Fasn) and acetyl-CoA carboxylase (ACC; Acaca) but not stearoyl-CoA desaturase 1 (SCD1; Scd1) were elevated in HFSC- compared to HFC-fed LDLR^-/- mice after 16 weeks of treatment (Figure 5A-C).

In addition, hepatic inflammation was significantly elevated in obese LDLR^-/- mice on both high-fat diets compared to the LF group as shown by increased mRNA expression of the genes for macrophage marker F4/80, MCP-1 and TNFα after 16 weeks of feeding (Figure 5D-F). This high-fat diet-induced increase of hepatic inflammation was even more pronounced after 20 weeks of diet. Of note, HFSC feeding of LDLR^-/- mice for 16 weeks induced a significant higher expression of osteopontin and TNFα compared to HFC-fed animals (Figure 5F, G). These differences in inflammatory gene expression evened out after 20 weeks of feeding suggesting that using a sucrose-enriched high-fat diet accelerates some features of fatty liver inflammation.

To next determine if HFSC feeding affects atherosclerosis development, atherosclerotic lesion area was quantified in the entire aorta by en face analysis. After 16 weeks of dietary treatment, HFSC-fed LDLR^-/- mice achieved similar levels of atherosclerotic lesion area as HFSC-fed ApoE^-/- mice which we used as a positive control (Figure 6A). In addition, atherosclerotic lesion formation was significantly increased in LDLR^-/- mice fed HFSC compared to LDLR^-/- mice fed HFC for 16 weeks (Figure 6A,C). Only after 20 weeks of dietary treatment, LDLR^-/- mice on HFSC and HFC achieved similar levels of atherosclerotic lesion areas (Figure 6B).