Effects of atherogenic diet supplemented with fermentable carbohydrates on metabolic responses and plaque formation in coronary arteries using a Saddleback pig model.

Fermentable carbohydrates are gaining interest in the field of human nutrition because of their benefits in obesity-related comorbidities. The aim of this study was to investigate the influence of fermentable carbohydrates, such as pectin and inulin, in an atherogenic diet on metabolic responses and plaque formation in coronary arteries using a Saddleback pig model. Forty-eight healthy pigs aged five months were divided into four feeding groups (n = 10) and one baseline group (n = 8). Three feeding groups received an atherogenic diet (38% crisps, 10% palm fat, and 2% sugar with or without supplementation of 5% pectin or inulin), and one group received a conventional diet over 15 weeks. Feed intake, weight gain, body condition score, and back fat thickness were monitored regularly. Blood and fecal samples were collected monthly to assess the metabolites associated with high cardiovascular risk and fat content, respectively. At the end of 15 weeks, the coronary arteries of the pigs were analyzed for atherosclerotic plaque formation. Independent of supplementation, significant changes were observed in lipid metabolism, such as an increase in triglycerides, bile acids, and cholesterol in serum, in all groups fed atherogenic diets in comparison to the conventional group. Serum metabolome analysis showed differentiation of the feeding groups by diet (atherogenic versus conventional diet) but not by supplementation with pectin or inulin. Cardiovascular lesions were found in all feeding groups and in the baseline group. Supplementation of pectin or inulin in the atherogenic diet had no significant impact on cardiovascular lesion size. Saddleback pigs can develop naturally occurring plaques in coronary arteries. Therefore, this pig model offers potential for further research on the effects of dietary intervention on obesity-related comorbidities, such as cardiovascular lesions, in humans.

Introduction

The increasing incidence of obesity has become a major global health concern as it is well known that obesity is associated with an elevated risk of cardiovascular disease [1,2]. Various animal models have been developed to investigate the link between obesity and cardiovascular diseases [3-8]. Rodent and lagomorpha models can provide important insights into the influence of metabolism, such as fat metabolism, on the development of atherosclerotic lesions [9-11]. However, rodent and lagomorpha models are limited because atherosclerotic plaques must be artificially induced. In addition, induced plaques occur in arteries different from those in humans [4,11].

In contrast, pig models have attracted interest due to the similarity of their cardiovascular anatomy and physiology with that of humans [12-15]. Moreover, due to similarities in human and pig digestive systems, pig models are advantageous for understanding potential dietary implications in humans [16]. The dietary spectrum of pigs is more similar to that of humans than those of rodents and lagomorpha [13]. Furthermore, the body size of pigs offers advantages in terms of relevant blood and tissue sampling [16]. Previous pig models have used cholesterol with or without the addition of cholic acid to induce the formation of atherosclerotic plaques [17,18]. In Rapacz pigs, familial predisposition to atherosclerosis has been described [19-21]. Furthermore, transgenic pig models have been also used [22]. These studies showed that atherosclerotic plaques developed at similar predilection sites in pigs and humans [13,17-22]. Interestingly, pigs can also spontaneously develop atherosclerotic lesions [23,24]. Using spontaneously occurring lesions in pigs enables the feeding of a typical western-style diet with high amounts of saturated fatty acids, sugar, and salt [25-27].

Recently, various strategies have been developed to counteract obesity-related cardiovascular risk factors. Several studies in humans have shown that fermentable carbohydrates such as pectin and inulin have health benefits in obesity comorbidities [28,29]. In this context the effects of fermentable carbohydrates have been also confirmed by the extensive meta-analysis by Zhou et al. [30].

In the small intestine, pectin and inulin are not digested by endogenous enzymes. Instead, these carbohydrates are available in the large intestine for fermentation by microbiota. The resulting fermentation products are short-chain fatty acids (SCFAs) such as acetate and propionate [31-33]. Consequently, pectin and inulin may lead to a shift in the composition of the microbiota, such as an increase in bifidobacteria [34-39]. Furthermore, a number of positive effects are known from SCFAs, such as immune and inflammation regulation [40]. Studies in humans and pigs have reported a cholesterol-lowering effect of pectin and inulin supplementation [41-43]. Moreover, supplementing high-fat diets with inulin slowed body weight gain in pigs [44]. In addition, inulin supplementation increased satiety by influencing glucagon-like peptides, thereby reducing total energy intake in humans [45,46].

This study aimed to investigate whether fermentable carbohydrates such as pectin and inulin can mitigate cardiovascular risk factors in pigs as a model for obese humans. We hypothesized that SCFA modulation by pectin and inulin will lead to changes in lipid metabolism and may reduce the spontaneous formation of atherosclerotic plaques in pigs fed a western-style diet.

Materials and methods

Animals and housing

Forty-eight healthy Saddleback pigs owned by the Institute of Animal Nutrition, Nutrition Diseases and Dietetics, Leipzig University, were included in this study. The animal sample included 21 female and 27 castrated male pigs from five litters with the same sire. The pigs were aged five months and had a mean (± SD) body weight (BW) of 97.5 ± 9.36 kg. The median [25th / 75th] percentiles of body condition score (BCS) were 3.13 [3.0 / 3.5] out of five, and the mean (± SD) back fat thickness (BFT) was 20.3 ± 2.08 cm at the beginning (t0) of the study. Pigs were housed in the same stable and separated in one pen per group according to the allotted treatment. The ambient temperature was 16–18°C, and the humidity was 60–75%. The pigs were bedded with wood shavings. Water was provided ad libitum by using an automatic watering system. Animals were adapted to the general experimental environment for at least three weeks. During the adaptation period, pigs were fed the same conventional diet. The project was approved by the Ethics Committee for Animal Rights Protection of the Leipzig District Government (no. TVV 04/20) in accordance with German legislation for animal rights and welfare. Supporting information on the project are provided in a supplementary file (.

Study design

The pigs were randomly divided into four feeding groups of ten each (atherogenic diet = AD, atherogenic diet + 5% pectin = ADp, atherogenic diet + 5% inulin = ADi, and conventional diet = CD) and one baseline group (BL; n = 8). After the adaptation period, BL pigs were slaughtered to obtain baseline values of the coronary artery samples. The remaining groups were fed treatment diets for 15 weeks. At the end of the feeding period, pigs were slaughtered for sample collection (January 29 to February 8, 2021).

Feed management

During the feeding period of 15 weeks, the three groups (AD, ADp, and ADi) received an atherogenic diet that contained high amounts of saturated fatty acids, sugar, and salt. For the ADp group, 5% pectin (agroPECT-A 100/70, agro Food Solution GmbH, Werder, Germany) was added to the atherogenic diet. In the ADi group, 5% inulin (Orafti® IPS; Beneo GmbH, Tienen, Belgium) was added according to literature [36,43,44,54]. The feed was provided ad libitum. The nutrient composition of the different diets is shown in .

Health monitoring and morphometric measurements

The health status of each animal was examined in two-day intervals by clinical examination, including evaluations of general behavior, feed and water intake, fecal quality, breathing rate, and body temperature. In addition, blood tests (blood count and chemistry) were performed before and after the feeding period. During the feeding period, the daily feed intake per group was recorded. BW was measured weekly using a portable electronic scaling system (Minipond 21; Baumann Waagen und Maschinenbau GmbH, Thiersheim, Germany). BCS was evaluated weekly using a scale from 0 to 5 [47,48]. BFT was obtained monthly by transcutaneous ultrasound measurements (Portable Ultrasonic Diagnostic System A6V, SonoScape Co., Shenzhen, China) at six measurement points according to the ABC-6-method [49].

Blood sampling

Blood samples were collected at the beginning of the study (t0; October 8–13, 2020) via a single puncture of the left or right jugular vein. Follow-up blood samples of the four feeding groups were collected after one (t1; November 11, 2020), two (t2; December 17, 2020), and three months (t3; January 21, 2021) of experimental diet feeding. For blood chemistry and metabolome analyses, serum tubes (Monovette® Z; Sarstedt AG & Co. KG, Nümbrecht, Germany) containing a coagulation activator was used. Blood count tubes containing EDTA (Monovette® K3E (1.6 mg EDTA/mL); Sarstedt AG & Co. KG) were analyzed immediately after sampling. Serum tubes were centrifuged after 30 min of clotting at room temperature, and the serum was frozen in multiple aliquots of 1 mL at −80°C until analysis.

Fecal sampling

Fecal samples were collected at the same time points (t0–t3) as the blood samples. Rectal feces were collected from each animal. Pooled fecal samples were prepared for each feeding group and analyzed directly.

Slaughtering and sampling of coronary arteries

The pigs were stunned using electrical stunning equipment (TGB 200; Hubert Haas, Neuler, Germany). Blood was withdrawn immediately after electrical stunning by severing the brachiocephalic trunk and jugular vein. Pigs were slaughtered in accordance with European and German law [Council Regulation (EC) No 1099/2009 of 24 September 2009, Tierschutz-Schlachtverordnung, § 4 Tierschutzgesetz]. After slaughter, the hearts were separated in toto for subsequent sampling and washed with isotonic saline (NaCl, Carl Roth GmbH + Co. KG, Karlsruhe, Germany). The left anterior descending branch of the left coronary artery (LAD) was manually flushed with isotonic saline, and four 2 mm segments of each animal were removed 3 mm after the bifurcation of the left main stem of the coronary artery, where the artery got divided into the left circumflex and the LAD. The 1st and 3rd LAD segments per animal were shock-frozen in liquid nitrogen and stored at -80°C until frozen sections were prepared. The 2nd and 4th LAD segments were fixed in 10% neutral-buffered formalin (Sigma-Aldrich, St. Louis, USA) for at least two days to prepare polyethylene glycol sections.

Analysis

Diets

All diets were analyzed for crude nutrient and fiber fractions. Dry matter (DM) was determined after oven-drying (103°C). Crude nutrient contents were assayed using the Weende system [50]. Crude fiber (CF), ash free neutral detergent fiber (NDF), and ash free acid detergent fiber (ADF) were analyzed using ANKOM A220® (ANKOM Technology, Salzwedel, Germany) according to Van Soest, Robertson, & Lewis (1991) [51]. The content of nitrogen-free extracts (NFE) was calculated as follows:

NFE = DM – (CA + CP + CL + CF); (CA = crude ash, CP = crude protein, CL = crude lipid). Starch was quantified polarimetrically (VDLUFA III, 7.2.1 Erg. 2012), and sugar content was determined gravimetrically (VDLUFA III, 7.1.3, 1976; LKS mbH, Lichtenwalde, Germany). Sodium was analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES) according to DIN EN ISO 11885:2009–09 (LKS mbH). The analyzed values were used to calculate the metabolizable energy (ME) content in DM [52]: ME (MJ/kg DM) = 0.021503 × CP (g/kg) + 0.032497 × CL (g/kg)– 0.021071 × CF (g/kg) + 0.016309 × starch (g/kg) + 0.014701 × organic residue (g/kg); organic residue = organic fraction–(CP + CL + starch + CF). The nutrient composition of each diet is shown in .

Serum parameters of liver and lipid metabolism

Serum triglyceride (TG), bile acids (BA), hepatic triglyceride lipase (LIPC), cholesterol (CHOL), alkaline phosphatase (ALP), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH) and amylase (AMYL) levels were analyzed using an automated chemistry analyzer (Roche Cobas C311, Roche Diagnostic GmbH, Mannheim, Germany). Blood counts were evaluated in all pigs before and after the 15-week feeding period using an ADVIA 120 (Siemens Healtheneers, Dreieich, Germany).

1H Nuclear magnetic resonance (NMR) spectroscopy of serum samples

Metabolomic analysis was performed using NMR spectroscopy (Lifespin GmbH, Regensburg, Germany). Serum samples were thawed at room temperature and inverted five-fold; 350 μL serum and 350 μL aqueous buffer (H2O p.A., 0.1 g/L NaN3, 0.067 mol/L Na2HPO4, 0.033 mol/L NaH2PO4 (pH-value: 7.15 ± 0.05), 5% D2O, 6 mM pyrazine as an internal standard for quantification) were mixed. Then, 600 μL of the mixture was transferred to a 5 mm NMR tube (Bruker Corporation, Billerica, USA). The samples were kept at 4°C prior to measurement and analyzed within 24 h.

NMR spectra of the serum were recorded at 310 K using an AVANCE NEO spectrometer (Bruker Corporation) operating at a proton frequency of 600 MHz. Spectra were recorded using a NOESY pulse sequence with water presaturation “noesygppr1d” in Bruker notation. For each sample, 16 subsequent scans were collected with a 10 s relaxation delay, an acquisition time of 2.75 s, 96 k data points, and a spectral width of 30 ppm.

The spectra were processed, and 102 blood serum metabolites were quantified using Lifespin’s proprietary profiling software version 1.4 (Lifespin GmbH). The concentration of any NMR-measured metabolite was obtained as a signal integral of non-overlapping resonances or a cluster of partly overlapping resonances. The metabolite resonances were identified according to chemical shift assignments using Lifespin´s proprietary substance reference database (Lifespin GmbH).

Fat content of the pooled fecal samples

The amount of CL was determined in pooled fecal samples from the four feeding groups (AD, ADp, ADi, and CD) and at each sampling point (t0–t3). The analysis of CL in feces was performed analogously to the analysis of CL in the diets.

Histological examination of the LAD

To prepare frozen artery sections, a freezing microtome (CM 1859, Leica Biosystems Nussloch GmbH, Nussloch, Germany, -25°C) was used. The segments were aligned in embedding medium (Epredia™ Neg-50™ Frozen Section Medium, Thermo Fisher Scientific, Waltham, USA) and fixed with a freezing spray (Shandon Enviro-Tech Freezing Spray, ThermoFisher Scientific). Cross sections with a layer thickness of 7 μm were prepared.

To prepare PEG sections according to Mulisch and Welsch (2015) [53], the segments were first dehydrated and then embedded in polyethylene glycol (PEG 1500, Merck KGaA, Darmstadt, Germany). After embedding, sections with a layer thickness of 4 μm were prepared with a rotary microtome (HM 335 E, Microm International GmbH, Walldorf, Germany). The arterial sections were stained by Movat`s Pentachrome to quantify the size of the vascular lesions. Based on the histological data, the ratio between the plaque and vessel area was calculated. Additionally, LAD sections were stained with Oil red to visualize lipids and with Von Kossa staining to detected vascular calcification. An immunohistochemical analysis was performed to identify macrophages using a specific mouse anti pig macrophages antibody (MCA2317GA, BIO-RAD, Hercules, USA). To detected smooth muscle cells a mouse anti human actin alpha (smooth muscle) antibody (MCA5781GA, BIO-RAD), previously applied for porcine tissue was used.

Statistics

Data analysis was performed using commercial statistical software (SPSS Statistics 27.0, IBM, New York, USA and STATISTICA 14.0, TIBCO, Palo Alto, USA). All data were checked for normal distribution using the Shapiro–Wilk test and for variance homogeneity using the Levene test. The datasets of BWs, BCS, BFT, CHOL and GGT were normally distributed and homogeneous. Repeated measures ANOVAs were used for these parameters, and as a post hoc test, the Tukey HSD test was applied. As the data sets were not normally distributed, the Kruskal–Wallis test with Bonferroni correction was performed (TG, BA, LIPC, AST, LDH, AMYL, ALP and plaque size). The chi-square test was used to evaluate plaque frequency per group. The level of significance was set at P < 0.05. Data are shown as medians and [25th / 75th] percentiles for TG, BA, CHOL, and plaque size or mean values ± standard deviation (mean ± SD) for BW, BCS, and BFT.

Further statistical analyses were conducted using R (R version 4.0.2. 2021, R Core Team). Principal component analysis (PCA) was performed on the serum NMR data to show that the groups could be separated by metabolites. In addition, NMR data were analyzed using partial least squares-discriminant analysis (PLS-DA). A default seven-fold cross-validation strategy and a permutation test (200 permutations) were performed to avoid overfitting PLS-DA.

Results

Feed intake and morphometric measurements

The total feed intake was similar between the different feeding groups (3680 ± 360 kg). All pigs showed significant increases in BW, BCS, and BFT. However, there were no significant group differences in BW gain (P = 0.82), BCS (P = 0.99), or BFT (P = 0.42) (). Original data of BW, BCS and BFT are provided in a supplementary table (.

Fecal fat content

The CL content in the DM of pooled fecal samples () increased after diet change (t1) in the groups fed atherogenic diets (AD, ADp, and ADi).

Serum parameters of liver and lipid metabolism

Serum parameters were significantly different between the feeding groups ( and ). For example, significantly higher concentrations of TGs and BAs were determined at times t1 and t2 in pigs in the AD, ADp, and ADi groups than in the CD group. The same significant group difference between the pigs fed the atherogenic diet and the pigs fed the conventional diet was observed for LIPC at time t1. The concentration of CHOL was significantly higher in the AD group than in the CD group at time t1. The other parameters (ALP, AST, GGT, LDH and AMYL) without treatment related effects are provided in a supplementary table (. Original data of serum analysis are also provided in a supplementary table (.

Triglycerides, bile acids, hepatic triglyceride lipase, and cholesterol concentrations in serum.

Box plots show the concentration of TG, BA, LIPC and CHOL at the time point before the feed change (t0) and time points 1–3 months after the feed changes (t1–t3) for all feeding groups (AD, ADp, ADi, CD). TG, triglycerides; LIPC, hepatic triglyceride lipase; BA, bile acids; CHOL, cholesterol; AD, group fed atherogenic diet (n = 10); ADp, group fed atherogenic diet + pectin (n = 10); ADi, group fed atherogenic diet + inulin (n = 10); CD, group fed conventional diet (n = 10);°*, outliers and extreme values.

Multivariate data analyses of serum 1H NMR data

First, unsupervised PCA was performed on the centered and scaled to unit variance serum NMR data to determine the general structure of each dataset. The PCA score plots () showed that the metabolite profiles of the AD groups (AD, ADp, and ADi) differed significantly from the CD group and could be clearly distinguished at t1, t2, and t3 but not at t0.

PCA analysis of the 1H NMR spectra of serum samples.

Data present the time point before the feed change (t0) and time points 1–3 months after the feed changes (t1–t3) for all feeding groups (AD, ADp, ADi, CD).

Additionally, supervised PLS-DA with mean-centered and unit variance NMR data was performed. A permutation test (200 permutations) was performed to confirm the reliability of the models. PLS-DA score plots () revealed a separation among the four feeding groups for t1 (Q = 0.29, RX = 0.38, RY = 0.4, pQ = 0.005) and t2 (Q = 0.59, RX = 0.48, RY = 0.81, pQ = 0.005). At t3 (Q = 0.9, RX = 0.41, RY = 0.97, pQ = 0.005), only ADs (including AD, ADp and ADi) and CD could be distinguished. At time point t0, a visual separation is possible; however, it can be classified as overfitting based on the performance indicators (Q = -0.02, pQ = 0.09).

PLS-DA analysis of 1H NMR spectra of serum samples.

Data present the time point before the feed change (t0) and the time points 1–3 months after feed changes (t1–t3) for all feeding groups (AD, ADp, ADi, CD).

In total, 102 metabolites were identified in this study. In particular, metabolites related to fat metabolism, including cholesterol, fatty acid residues, and saturated carbon chain elements, were more abundant in the AD, ADp, and ADi groups than in the CD group.

Plaque formation

Moderate plaque formation in the LAD was found in the baseline group and in the four feeding groups (). All plaques detected in the LAD sections represent very early lesion stages of atherosclerosis without calcification, high amounts of lipids, invasion of macrophages or differences in stainable smooth muscle cells. In the ADp group, significantly more animals had plaques (10/10 animals affected) than in the AD group (5/10 animals affected, ). Plaque sizes did not differ significantly between the groups (P = 0.33, Kruskal-Wallis test with Bonferroni correction). Additionally, no association was found between the plaque frequency per group and plaque size. The smallest plaque was observed in the baseline group. Original data of plaque formation are provided in a supplementary table (.

Representative images of LAD cross-sections of all groups.

Movat`s Pentachrome staining (frozen). Scale bar 500 μm. a BL. b AD. c ADp. d ADi. e CD.

Discussion

In our study, we developed a pig model under well-defined feeding and housing conditions as an atherogenic dietary model for humans. Dietary modeling using pigs is advantageous compared to that using other laboratory animals due to factors such as similarities in food spectra, digestive systems, and the manifestation of coronary heart disease. The atherogenic diet used in this study was comparable to a typical western-style human diet. Analyses of several parameters relevant to evaluating cardiovascular and cardiometabolic health were performed. We hypothesized that supplementation of an atherogenic diet with two different fermentable carbohydrates, pectin and inulin, has beneficial effects on metabolism and plaque formation.

As expected, feed intake was high in all feeding groups, and the amount of feed intake was comparable between the groups. Significant increases in BW, BCS, and BFT were observed in all the groups. However, despite differences in energy content between atherogenic diets (17.2–17.8 MJ ME/kg DM) and the conventional diet (14.1 MJ ME/kg DM) increase in BW, BCS and BFT did not significantly differ between the four feeding groups. One reason for this difference may be the incomplete digestion of fat, as fecal fat content increased after diet change (t1) in the groups fed the atherogenic diets.

Independent of supplementation with pectin and inulin, significant changes in blood parameters were observed after one month of feeding with the atherogenic diet. PCA showed a clear clustering into the groups fed the atherogenic diet (AD, ADp, Adi) and the conventionally fed group (CD) after the feed change (t1–t3). These effects were also observed in blood parameters such as TG, BA, LIPC and CHOL.

PLS-DA did not allow a clear metabolome-based distinction between the four feeding groups for t1 and t2 because of the small differences in circulating metabolites between the AD, ADp, and ADi groups. Additionally, at time t3, no separation was possible in the PLS-DA between the groups fed an atherogenic diet (AD, ADp, and ADi). These results were also supported by the blood levels of TG, BA, LIPC and CHOL at time t3, which did not show significant differences between these groups. Therefore, NMR spectroscopy for metabolome analyses has been demonstrated to be a powerful tool for expanding the density of data in veterinary nutrition research and should be considered in future studies. However, our results might be somewhat confounded by the fact that the pigs were kept on wood shavings per ethical considerations, and the intake of wood shavings could not be excluded.

Interestingly, supplementation with pectin or inulin did not affect atherogenic diet-induced changes in blood parameters such as cholesterol. These findings are in contrast to those obtained by other authors, who reported beneficial effects of pectin on circulating cholesterol in pigs and humans [42,43,54,55]. It is tempting to speculate that 5% of pectin used in the current study was too low. Similar pectin levels have been used in other pig studies, showing cholesterol-lowering effects [43,54]. It is possible that the high-energy intake of pigs in the present study overlapped with the effects of pectin and inulin. Another factor could be the duration of the study, which lasted 15 weeks. Other studies have been conducted over a shorter feeding period (10–12 weeks), showing cholesterol-lowering effects for pectin [54] and lower BW gain for inulin [44] in pigs.

Interestingly, all groups, including the baseline group, developed moderate lesions in the coronary arteries. It can be speculated that the Saddleback pigs in our study may have a predisposition to plaque development. A familial disposition for plaque formation is also known for other pig breeds, such as Rapacz pigs [20,21]. However, the etiology in Rapacz pigs is hypercholesterolemia. In contrast, in this study, the cholesterol levels of all pigs remained within the normal range [56] despite significant increases in cholesterol after feeding the atherogenic diet. Beside these findings, it must be emphasized that the plaques represent very early lesion stages of atherosclerosis.

An unexpected finding was related to the higher lesion rate per group in the coronary arteries of the ADp group with pectin supplementation (10/10 animals affected) compared with the atherogenic diet without supplementation (5/10 animals affected). Interestingly, pectin supplementation induced smaller lesions in the coronary arteries than the atherogenic diet alone or the atherogenic diet supplemented with inulin. It remains unclear whether a higher lesion rate or smaller lesions are more beneficial. In contrast, the effect of inulin on plaque formation in coronary arteries was similar to that in pigs fed an atherogenic diet without fermentable carbohydrates. The plaques in the coronary arteries of the pectin-supplemented group were similar in size to those of the conventionally fed group. The baseline group, which was 15 weeks younger than the other groups, had the smallest plaques in their coronary arteries. Because the baseline group was slaughtered earlier, the younger age is probably the reason for the smallest plaques in this group. Therefore, age may be a critical factor in plaque formation in the coronary arteries. This indicates that Saddleback pigs are capable of developing vascular lesions within a comparatively young age. In the present study, the pigs were five months old at the beginning of the feeding study. However, it is possible that earlier or longer administration or higher doses of pectin and inulin would have exerted more beneficial effects on the vascular system and metabolism. In particular, to investigate the progression of lesion development, changes during a longer supplementation period should be the focus of future studies.

Conclusion

Our findings indicate that Saddleback pig models are very useful for studying the effects of diets on atherosclerosis because these pigs develop spontaneous vascular lesions in their coronary arteries in early life. In contrast to our hypothesis, pectin and inulin supplementation of an atherogenic diet did not show any major beneficial effects on atherosclerosis and cardiometabolic risk factors under the applied conditions. In human medicine, in addition to supplementation with pectin, inulin, or other fermentable carbohydrates, energy restriction is indispensable.

ARRIVE guideline.

(DOCX)

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Original data of BW, BCS and BFT.

(XLSX)

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Serum parameters of liver and lipid metabolism.

(DOCX)

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Original data of serum analysis.

(XLSX)

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Original data of plaque formation in the LAD.

(XLSX)

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30 May 2022

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Reviewer #1: The current manuscript describes a porcine model of coronary artery atherosclerosis and the effects of the fermentable carbohydrates, pectin or inulin on disease after 15 weeks feeding with a Western-style diet. While the Western-style diet increased serum lipid levels, surprisingly no apparent change in the incidence of atherosclerotic lesions in the LAD was noted compared to the control diet group, although lesion size appeared to increase in the atherogenic diet group. This raises questions on the viability of this experimental model for the testing potential anti-atherogenic interventions. Also, key information is missing regarding the measurement of atherosclerosis in the LAD artery including at what anatomical site/region of the LAD artery was atherosclerosis incidence and lesion size measured and was this site/region uniform for all measurements across the different animals. Also, it would be helpful for lesion size to be expressed in the more conventional mean +/- SD format and shown as a scatter plot with the individual values of lesion size for each animal provided, as well as an accompanying stats analysis. It is also important for further analysis to be performed regarding the stage and phenotype of the lesions formed. For example, are the lesion early fatty streaks or have they progressed to a more advanced state. Therefore, histological assessment of macrophage, collagen, SMC and other indices of lesion status should be performed. While lesion size is one important measurement, lesion inflammatory status and stability are also critical readouts when assessing the utility of this porcine model of atherosclerosis. If as suspected, the lesions are early in nature, then perhaps 15 weeks atherogenic diet is insufficient time to drive the development of advanced atherosclerotic lesions in a larger animal such as the Saddleback pig and hence it is difficult to ascertain the impact of potential anti-atherogenic interventions such as pectin and inulin. Have the authors performed prior studies examining the impact of the time of feeding of an atherogenic diet on atherosclerosis development in this model? If not this appears a sensible first step in model development and validation.

Several other aspects warrant attention:

1. What is the rationale for the choice of 5% pectin and inulin?

2. What was the rationale for 15 weeks of atherogenic diet? This appears short for a larger animal such as a pig.

3. The large tables of data are difficult to analyze and digest the data. The additional use of figures to show key data sets would be beneficial.

4. A rationale for the choice of Saddleback pigs would be beneficial. How does this model compare to other porcine models of atherosclerosis?

Reviewer #2: Major Comments:

1) The Introduction should highlight clinical trials in humans that have investigated fermentable carbohydrates and cardiovascular risk (eg Bocheng Xu et al, 2022).

2) A further detailed investigation into the atherogenic plaques need to be performed. Are the plaques stable/unstable? This can be easily done by immunohistochemistry investigating smooth muscle cell content, macrophage/immune cells, and fibrosis?

3) A more detailed analysis on why pectin was more effective in reducing plaque than inulin? The hypothesis is not supported in the study and needs further clarification.

Minor Comments:

1) In the introduction line 57, "provoked" plaques is not the right term to use, please ammend.

2) Statistical significances in the study in particular Table 5 is relatively confusing as to which group or timepoint it refers to. Please explicitly state significance values and group compared to.

**********

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18 Jul 2022

Response letter

“Effects of atherogenic diet supplemented with fermentable carbohydrates on metabolic responses and plaque formation in coronary arteries using a Saddleback pig model”

Manuscript Submission No.: PONE-D-22-07717

Summary

We would like to thank the Associate Editor and the Reviewers for a thorough analysis of the submitted work as well as precise and constructive comments which helped to better organize and polish the manuscript.

These contributions were expressed more clearly throughout the manuscript via:

• additional figures to illustrate the results of serum parameter analyses

• a more detailed description of the coronary artery sampling procedure

• additional information and further investigations on the phenotype of vascular lesions

• information about the statistical evaluation of plaque size

1) Response to the Editor`s Report

Comment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_ sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/ PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: Thank you for this comment. We checked that our manuscript meets PLOS ONE's style requirements by using your English Editing Service to improve the manuscript.

Comment 2: To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

Response: Thank you very much for this response. The experiment was approved by the Ethics Committee for Animal Rights Protection of the Leipzig District Government (no. TVV 04/20) in accordance with German legislation for animal rights and welfare (line 114−117). The licensing authority also considered the burden on the animals to be low due to the blood and fecal samples taken once a month during the experiment. All methods such as blood sampling, fecal sampling and slaughtering has been described in the chapter “materials and methods” (line 156−188). During the experiment, great importance was attached to animal welfare. In addition to occupation with the bedding material (wood shavings), the pigs were provided with new organic manipulable material twice a week like chewing ropes, biting woods and a stimulus rod.

In addition, strict inclusion and exclusion criteria were established prior to the study, which are described as follows in supplementary file 1 (S1 File. ARRIVE guideline):

Including criteria:

- Breed: Saddle back pigs (5 litters, same sire)

- Age: 5 Month

- Health status: Healthy (examined by clinical examination and blood check)

Excluding criteria:

- A score sheet was developed to establish the criteria for excluding animals from the study. The score was developed specifically for pigs and modified based on the results for pain assessment in pigs by Ison et al. (2016).

- Scored parameter: general behaviour, condition score, feed and water intake, quality of faeces, breathing rate, body temperature, limbs, injuries (especially tail, ears and flanks), umbilical hernia, symptoms of gastric ulcera

- Score > 0 to 1 is reached for one parameter �  animals were intensively observed (clinical examination three times a day)

- Score > 1 is reached for more than one parameter or a score of 3 is reached for one parameter �  the animals are excluded of the study and undergo further diagnostic examinations (e.g. further lameness diagnostics) and, if necessary, lege artis medical treatment (e.g. pain therapy or antibiosis).

- If a complete recovery of the animals is possible, they are included in the trial again.

- If individual animals cannot be recovered and included in the trial, these animals are euthanised to avoid further pain and suffering.

As we described in the manuscript, the pigs were slaughtered by a qualified butcher in accordance with European and German law [Council Regulation (EC) No 1099/2009 of 24 September 2009, Tierschutz-Schlachtverordnung, § 4 Tierschutzgesetz] (line 173−177). We added a detailed description of the slaughtering process as follows in supplementary file 1 (S1 File. ARRIVE guideline):

Slaughtering process

Pigs were adapted and familiar with manual handling. All animals underwent a veterinary examination immediately before slaughtering and all pigs were diagnosed to be clinically healthy. Slaughter of the pigs took place on the same farm where the pigs were kept during the experiment (distance from the stable to the slaughterhouse: 200 m), which excluded long transportation stress. The pigs were individually stunned with an electric stunning system according to European and German law [Council Regulation (EC) No 1099/2009 of 24 September 2009, Tierschutz-Schlachtverordnung, § 4 Tierschutzgesetz] (TGB 200; Hubert Haas, Neuler, Germany, brain and brain-heart perfusion, minimum current of 1.3 A within the 1st second, 250 V, alternating current with 50−100 Hz, perfusion duration with 1.3 A for at least 4 seconds, data were recorded by the stunning system) by the qualified butcher and then sacrificed by blood withdrawal within 10 seconds (knife with a blade length of at least 12 cm, stabbing direction in the jugular fossa 2−3 cm in front of the sternal apex in the direction of the opposite scapula, cut length of 2−3 cm, opening of the brachiocephalic trunk and jugular vein, blood loss of at least 3−4 L in 30 seconds). Thereafter, the carcasses were examined by an official veterinarian and declared edible. The carcasses were processed by regional butchers.

We hope that we have considered your comments in an adequate way.

Comment 3: There are concerns about the model, statistical analysis, controls and the assessment of plaque morphology and composition. Please assess the reviewer comments and provide a strong rationale for the model, present data in form of additional figures or tables and perform additional assessment of plaque morphology and composition.

Response: We appreciate the effort of the associate editor as well as of the reviewers for highlighting flaws in our submission. The comments and concerns associated to the review have been carefully inspected by us. Based on the review, the manuscript has been refined and adapted. We added a figure for each of the serum parameters (Triglycerides [TG], hepatic triglyceride lipase [LIPC], bile acids [BA], and cholesterol [CHOL] line 336−343). As the visualization of the statistic is complex, we divided Table 5 into sub tables (5a-5d, line 345−387) to provide information about significances.

2) Response to Reviewer 1

Comment 1: The current manuscript describes a porcine model of coronary artery atherosclerosis and the effects of the fermentable carbohydrates, pectin or inulin on disease after 15 weeks feeding with a Western-style diet. While the Western-style diet increased serum lipid levels, surprisingly no apparent change in the incidence of atherosclerotic lesions in the LAD was noted compared to the control diet group, although lesion size appeared to increase in the atherogenic diet group. This raises questions on the viability of this experimental model for the testing potential anti-atherogenic interventions.

Response: The authors confirm that the lesions were only very moderate. At this lesion stage, the plaques were of minor significance in terms of health consequences for the pigs. Nevertheless, the authors consider the formation of spontaneous plaques without the induction by feeding cholesterol and cholic acids relevant to mention. Although of minor significance, group-related differences were detected in plaque formation.

The influencing aspects such as duration of feeding period, levels of pectin and inulin in the diet as well as age of the pigs were addressed in the discussion as follows: “Because the baseline group was slaughtered earlier, the younger age is probably the reason for the smallest plaques in this group. Therefore, age may be a critical factor in plaque formation in the coronary arteries. This indicates that Saddleback pigs are capable of developing vascular lesions within a comparatively young age. In the present study, the pigs were five months old at the beginning of the feeding study. However, it is possible that earlier or longer administration or higher doses of pectin and inulin would have exerted more beneficial effects on the vascular system and metabolism. In particular, to investigate the progression of lesion development, changes during a longer supplementation period should be the focus of future studies.” (line 517−526)

Comment 2: Also, key information is missing regarding the measurement of atherosclerosis in the LAD artery including at what anatomical site/region of the LAD artery was atherosclerosis incidence and lesion size measured and was this site/region uniform for all measurements across the different animals.

Response: The arterial section for the histological analyses was obtained from the same site of each animal. The section was excised 3 mm after the bifurcation of the left main stem of the coronary artery, where the artery gets divided into the left circumflex (LCX) and the left anterior descending (LAD). From each animal four 2 mm segments of the LAD were gained. The 1st and 3rd segments were fixed to prepare the PEG sections; the 2nd and 4th segments were snap frozen for the preparation of the frozen sections. We included a more detailed description in the manuscript (“materials and methods”, “slaughtering and sampling of coronary arteries”, line 180−188).

Comment 3: Also, it would be helpful for lesion size to be expressed in the more conventional mean +/- SD format and shown as a scatter plot with the individual values of lesion size for each animal provided, as well as an accompanying stats analysis.

Response: As data of lesion size were not normally distributed, the authors decided to use median and [25th / 75th] percentile to present the data. However, the expression in mean ± SD does not change the outcome. For statistical evaluation of plaque size Kruskal-Wallis test with Bonferroni correction was used (P = 0.33). We included this information in the manuscript (“results”, “plaque formation”, line 433−434). As there were no significant group differences in plaque size, the authors decided to present only the plaque sizes of the plaque-positive pigs. We provided the single values in a supplementary file (S5 Table. Original data of plaque formation in the LAD).

Comment 4: It is also important for further analysis to be performed regarding the stage and phenotype of the lesions formed. For example, are the lesion early fatty streaks or have they progressed to a more advanced state. Therefore, histological assessment of macrophage, collagen, SMC and other indices of lesion status should be performed. While lesion size is one important measurement, lesion inflammatory status and stability are also critical readouts when assessing the utility of this porcine model of atherosclerosis.

Response: We agree that lesion inflammatory status and plaque stability are critical readouts to evaluate the progress of atherogenesis. However, all lesions detected in the LAD sections represented very early lesion stages of atherosclerosis. None of the pigs had advanced lesions with fibrous caps or lipid cores. Additionally, the area of atherosclerotic lesions in relation to the area of arterial tissue was on average below 6% in all groups of pigs, which confirm that the atherosclerotic changes in the blood vessel were moderate intima thickening. Moreover, staining of the LAD sections with Oil red illustrates that only one pig had detectable amounts of lipids in the lesions. Analysis of Von Kossa staining which usually visualize vascular calcification, revealed no calcification spots in the LAD of pigs. As suggested by the reviewers, we added additional immunohistochemical analyses. First, we tried to record macrophages by using a specific mouse anti pig macrophages antibody (MCA2317GA, BIO-RAD), but could not detect any macrophages in the LAD lesions. Staining of smooth muscle cells performed with a mouse anti human actin alpha (smooth muscle) antibody (MCA5781GA, BIO-RAD), which was used for porcine tissue before, showed stainable smooth muscle cells but no differences between the feeding groups. Based on the current data, we suppose that feeding pigs for 15 weeks with an atherogenic diet can only induce very early forms of atherosclerotic lesions. We therefore conclude that a longer feeding period is required for the development of more pronounced vascular lesions with a detectable inflammatory status. Authors added the respective information in the manuscript (“material and methods” line 266−272, “results” line 429−431 and “discussion” line 504−506).

Comment 5: If as suspected, the lesions are early in nature, then perhaps 15 weeks atherogenic diet is insufficient time to drive the development of advanced atherosclerotic lesions in a larger animal such as the Saddleback pig and hence it is difficult to ascertain the impact of potential anti-atherogenic interventions such as pectin and inulin.

Response: The authors agree with this comment. We discussed this point, especially the duration of the experiment, in the discussion line 517−526 (see comment 1 above).

Comment 6: Have the authors performed prior studies examining the impact of the time of feeding of an atherogenic diet on atherosclerosis development in this model? If not this appears a sensible first step in model development and validation.

Response: Thank you for this comment. This was one idea of the experiment. We want to use this study to be the basis of further research.

Comment 7: What is the rationale for the choice of 5% pectin and inulin?

Response: The used dosages or lower dosages for pectin and inulin were already fed in other studies in pigs and the chosen levels have been already linked to positive effects [36,43,44,54]. We added this information in the manuscript (“materials and methods”, “feed management”, line 132−133)

Comment 8: What was the rationale for 15 weeks of atherogenic diet? This appears short for a larger animal such as a pig.

Response: The authors acknowledged this comment. The duration is an issue of concern. The experiment was planned for a longer time period, starting the experiment with 5-month-old pigs and mean body weight of 97.5 kg. Because of the high body weight increase during the experiment, pigs started to change their behavior in the 14th week of the experiment by decreasing their activity level.

Therefore, it was not possible to use these pigs for a longer time period due to reasons of animal welfare. Furthermore, it could not be excluded that these changes in activity level may have an impact on the outcome. Consequently, we decided to stop the experiment after 15 weeks. Authors would like to emphasize that all pigs already reached a BCS between 4 and 5 on a scale 0 to 5 at this timepoint.

Comment 9: The large tables of data are difficult to analyze and digest the data. The additional use of figures to show key data sets would be beneficial.

Response: We added a figure for each serum parameter (Triglycerides [TG], hepatic triglyceride lipase [LIPC], bile acids [BA], and cholesterol [CHOL]). As you also mentioned, the presentation of the statistic is complex. Therefore, we provide tables to give information about significances. However, it is very difficult to show all P values separately. We hope that by providing data in sub tables (line 345−387) and figures (Fig 1: Triglycerides, bile acids, hepatic triglyceride lipase, and cholesterol concentrations in serum, line 336−343) data presentation becomes clearer.

Comment 10: A rationale for the choice of Saddleback pigs would be beneficial. How does this model compare to other porcine models of atherosclerosis?

Response: We compared the commonly used porcine models of atherosclerosis in the introduction (line 65−72). Previous studies used cholesterol and cholic acids to induce the formation of atherosclerotic plaques, familiar predisposed pigs or genetically modified animals [17−22]. However, it is also known that pigs can develop plaques spontaneously [23−24]. Authors would like to emphasize that we want to use pigs which can develop plaques spontaneously to get deeper insight about the consequences of consuming typical western style diets.

3) Response to Reviewer 2

Comment 1: The Introduction should highlight clinical trials in humans that have investigated fermentable carbohydrates and cardiovascular risk (eg Bocheng Xu et al, 2022).

Response: Thank you for this information. Indeed, we did not acknowledge this paper. However, we have included the information about the interesting meta-analysis of Xu et al. [30] in the introduction (line 77−78).

Comment 2: A further detailed investigation into the atherogenic plaques need to be performed. Are the plaques stable/unstable? This can be easily done by immunohistochemistry investigating smooth muscle cell content, macrophage/immune cells, and fibrosis?

Response: The reviewers are completely right, that lesion inflammatory status and plaque stability are critical readouts to evaluate the progress of atherogenesis. However, all lesions detected in the LAD sections represented very early lesion stages of atherosclerosis. None of the pigs had advanced lesions with fibrous caps or lipid cores. Additionally, the area of atherosclerotic lesions in relation to the area of arterial tissue was on average below 6% in all groups of pigs, which confirm that the atherosclerotic changes in the blood vessel were moderate intima thickening. Moreover, staining of the LAD sections with Oil red illustrates that only one pig had detectable amounts of lipids in the lesions. Analysis of Von Kossa staining which usually visualize vascular calcification, revealed no calcification spots in the LAD of pigs. As suggested by the reviewers, we added additional immunohistochemical analyses. First, we tried to record macrophages by using a specific mouse anti pig macrophages antibody (MCA2317GA, BIO-RAD), but could not detect any macrophages in the LAD lesions. Staining of smooth muscle cells performed with a mouse anti human actin alpha (smooth muscle) antibody (MCA5781GA, BIO-RAD), which was used for porcine tissue before, showed stainable smooth muscle cells but no differences between the feeding groups. Based on the current data, we suppose that feeding pigs for 15 weeks with an atherogenic diet can only induce very early forms of atherosclerotic lesions. We therefore conclude that a longer feeding period is required for the development of more pronounced vascular lesions with a detectable inflammatory status. Authors added the respective information in the manuscript (“material and methods” line 266−272, “results” line 429−431 and “discussion” line 504−506).

Comment 3: A more detailed analysis on why pectin was more effective in reducing plaque than inulin? The hypothesis is not supported in the study and needs further clarification.

Response: The authors speculated that the production of short chain fatty acids (SCFA) in the large intestine by adding pectin or inulin to the diet may play a significant role. The analyses of SCFA are currently under investigation.

Comment 4: In the introduction line 57, "provoked" plaques is not the right term to use, please ammend.

Response: The authors agree to your point. We have changed the word "provoked" into "induced" in the introduction (line 57).

Comment 5: Statistical significances in the study in particular Table 5 is relatively confusing as to which group or timepoint it refers to. Please explicitly state significance values and group compared to.

Response:

We added a figure for each serum parameter (Triglycerides [TG], hepatic triglyceride lipase [LIPC], bile acids [BA], and cholesterol [CHOL]). As you also mentioned, the presentation of the statistic is complex. Therefore, we provide tables to give information about significances. However, it is very difficult to show all P values separately. We hope that by providing data in sub tables (line 345−387) and figures (Fig 1: Triglycerides, bile acids, hepatic triglyceride lipase, and cholesterol concentrations in serum, line 336−343) data presentation becomes clearer.

12 Sep 2022

Effects of atherogenic diet supplemented with fermentable carbohydrates on metabolic responses and plaque formation in coronary arteries using a Saddleback pig model

PONE-D-22-07717R1

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Reviewer #1: All comments have been addressed

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Reviewer #1: Partly

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Reviewer #1: Yes

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27 Sep 2022

PONE-D-22-07717R1

Effects of atherogenic diet supplemented with fermentable carbohydrates on metabolic responses and plaque formation in coronary arteries using a Saddleback pig model

Dear Dr. Vervuert:

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Table 1

Composition of atherogenic and conventional experimental diets.

Atherogenic diet		Conventional diet
Percentage	Components	Percentage	Components
101817.52.5238102	WheatOatsSoy bean mealVitamins and Minerals1Fiber supplement2Crisps3Palm fat4Sugar	43.538142.52	WheatOatsSoy bean mealVitamins and Minerals1Fiber supplement2

Data are presented as component percentages. 1Troumix®M1, Trouw Nutrition Deutschland GmbH, Burgheim, Germany; 2FaserSpezial 2.0, Trouw Nutrition Deutschland GmbH; 3By-product of snack industry, feedstuff according to Regulation (EU) No. 575/2011; 4BEWI-SPRAY® 99L, BEWITAL agri GmbH & Co. KG, Südlohn-Oeding, Germany.

Table 2

Nutrients and calculated metabolizable energy levels of experimental diets.

Crude nutrients in DM (%)	AD	AD+ 5 % pectin	AD+ 5 % inulin	CD
CA	5.27	5.42	5.02	4.77
CL	27.2	25.7	25.2	3.40
CP	15.1	14.7	14.2	19.0
CF	7.22	7.94	7.34	7.13
NDF	11.7	10.7	10.7	21.7
ADF	5.60	5.56	5.45	9.46
NFE	39.2	39.9	41.9	55.4
Starch	34.4	34.3	32.4	45.5
Sugar	6.71	6.16	10.8	3.35
Sodium	0.34	0.35	0.34	0.14
ME (MJ/kg DM) [51]	17.8	17.2	17.3	14.1

The data are presented as percentages of DM. DM, dry matter; AD, atherogenic diet; CD, conventional diet; CA, crude ash; CL, crude lipid; CP, crude protein; CF, crude fiber; NDF, ash-free neutral detergent fiber; ADF, ash-free acid detergent fiber; NFE, nitrogen-free extracts; ME, metabolizable energy.

Table 3

Increases in BW, BCS, and BFT in the four feeding groups during the observation period.

Parameter in (%)	Group AD	Group ADp	Group ADi	Group CD
BW	61.2 ± 16.3	58.1 ± 12.0	62.1 ± 11.8	58.9 ± 8.60
BSC	32.1 ± 13.9	33.8 ± 6.30	33.6 ± 11.2	33.0 ± 12.3
BFT	110 ± 41.9	90.9 ± 45.3	109 ± 53.1	82.3 ± 44.7

Data are presented as mean ± SD. Significant differences between feeding groups (n = 10) were identified by P values ≤ 0.05, using repeated measures ANOVA with Tukey’s HSD. AD, group fed atherogenic diet; ADp, group fed atherogenic diet + pectin; ADi, group fed atherogenic diet + inulin; CD, group fed conventional diet; BW, body weight; BCS, body condition score; BFT, back fat thickness. The observation period was 15 weeks.

Table 4

Crude lipid content in pooled fecal samples of each dietary group (AD, ADp, ADi, CD) at each sampling point (t0–t3).

CL in DM (%)	Groups	t0 (start)	t1 (1 month)	t2 (2 months)	t3 (3 months)
	AD	6.27	24.6	17.5	21.1
	ADp	6.44	24.2	25.3	17.9
	ADi	7.21	21.8	18.5	20.9
	CD	5.98	4.10	4.81	4.14

Data are presented as percentages (in DM) for one pool of ten animals per group. CL, crude lipid; DM, dry matter; AD, group fed an atherogenic diet (n = 10); ADp, group fed an atherogenic diet + pectin (n = 10); ADi, group fed an atherogenic diet + inulin (n = 10); CD, group fed a conventional diet (n = 10).

Table 5

a: Triglycerides concentrations in serum (mmol/L) of all groups at each sampling point (t0–t3).

b: Bile acids concentrations in serum (μmol/L) of all groups at each sampling point (t0–t3). c: Hepatic triglyceride lipase concentrations in serum (mmol/L) of all groups at each sampling point (t0–t3). d: Cholesterol concentrations in serum (mmol/L) of all groups at each sampling point (t0–t3).

Groups	t0	t1	t2	t3
BL	0.38[0.28 / 0.49]	n/a	n/a	n/a
AD	0.34▲[0.33 / 0.44]	0.73#, a[0.62 / 0.82]	0.66#, a[0.52 / 1.21]	0.40▲[0.29 / 0.53]
ADp	0.39▲[0.28 / 0.50]	0.76#, a[0.62 / 0.95]	0.62▲#, ab[0.38 / 0.71]	0.43▲[0.27 / 0.60]
Adi	0.32▲[0.30 / 0.41]	0.63#■, a[0.44 / 0.82]	0.76■, a[0.54 / 1.17]	0.43▲#[0.26 / 0.63]
CD	0.30[0.25 / 0.34]	0.29b[0.25 / 0.30]	0.31b[0.24 / 0.35]	0.29[0.25 / 0.32]

Triglycerides concentrations are presented as medians and [25th / 75th] percentiles in mmol/L. ▲#■Different symbols indicate significant differences within a row (time-point differences in a group). abLowercase letters indicate significant effects within a column (group differences at one timepoint). Significant differences are identified by P values ≤ 0.05 using Kruskal–Wallis test with Bonferroni correction. BL, baseline group (n = 8); AD, group fed atherogenic diet (n = 10); ADp, group fed atherogenic diet + pectin (n = 10); ADi, group fed atherogenic diet + inulin (n = 10); CD, group fed conventional diet (n = 10); n/a, not available.

Groups	t0	t1	t2	t3
BL	5.25[3.83 / 5.70]	n/a	n/a	n/a
AD	4.70▲[3.68 / 6.40]	21.2#, a[17.2 / 23.5]	15.8#, a[11.0 / 27.9]	11.3▲#[7.73 / 17.6]
ADp	4.85▲[3.88 / 5.70]	17.4#, a[14.0/ 21.2]	8.55▲#, ab[7.08 / 15.3]	11.8#[7.60 / 16.4]
ADi	5.95▲[4.25 / 11.8]	14.7#, ab[7.48 / 22.3]	13.0▲#, a[11.7 / 15.0]	12.6▲#[6.30 / 13.5]
CD	5.85[5.00 / 8.30]	7.70b[6.38 / 11.1]	6.55b[4.38 / 10.5]	8.65[6.25 / 11.3]

Bile acids concentrations are presented as medians and [25th / 75th] percentiles in μmol/L. ▲#■Different symbols indicate significant differences within a row (time-point differences in a group). abLowercase letters indicate significant effects within a column (group differences at one timepoint). Significant differences are identified by P values ≤ 0.05 using Kruskal–Wallis test with Bonferroni correction. BL, baseline group (n = 8); AD, group fed atherogenic diet (n = 10); ADp, group fed atherogenic diet + pectin (n = 10); ADi, group fed atherogenic diet + inulin (n = 10); CD, group fed conventional diet (n = 10); n/a, not available.

Groups	t0	t1	t2	t3
BL	5.00[4.25 / 5.00]	n/a	n/a	n/a
AD	4.00▲[4.00 / 4.00]	6.50#, a[6.00 / 7.00]	6.50#, ab[5.00 / 7.50]	5.00▲#[5.00 / 5.25]
ADp	4.00▲[4.00 / 4.00]	6.00#■, a[6.00 / 7.25]	7.00■, a[6.00 / 7.25]	5.00▲#[5.00 / 5.00]
ADi	4.00▲[4.00 / 4.00]	6.00#, a[5.75 / 6.25]	6.00#, ab[5.00 / 6.25]	4.50▲[4.00 / 5.00]
CD	4.00▲[4.00 / 4.25]	5.00#, b[5.00 / 5.00]	6.00#, b[5.00 / 6.00]	5.00▲#[4.00 / 5.25]

Hepatic triglyceride lipase concentrations are presented as medians and [25th / 75th] percentiles in mmol/L. ▲#■Different symbols indicate significant differences within a row (time-point differences in a group). abLowercase letters indicate significant effects within a column (group differences at one timepoint). Significant differences are identified by P values ≤ 0.05 using Kruskal–Wallis test with Bonferroni correction. BL, baseline group (n = 8); AD, group fed atherogenic diet (n = 10); ADp, group fed atherogenic diet + pectin (n = 10); ADi, group fed atherogenic diet + inulin (n = 10); CD, group fed conventional diet (n = 10); n/a, not available.

Groups	t0	t1	t2	t3
BL	2.37[2.18 / 2.58]	n/a	n/a	n/a
AD	2.47[2.27 / 2.66]	2.47a[2.33 / 2.53]	2.32[2.10 / 2.49]	2.49a[2.17 / 2.71]
ADp	2.24▲#[2.04 / 2.31]	2.40▲, ab[2.21 / 2.51]	2.43▲[2.34 / 2.51]	2.14#, ab[1.96 / 2.35]
ADi	2.17▲[1.98 / 2.35]	2.24▲#, ab[2.14 / 2.37]	2.35▲#[2.21 / 2.52]	2.38#, a[2.24 / 2.48]
CD	2.27▲[2.09 / 2.36]	2.10▲#, b[1.87 / 2.33]	2.14▲#[1.92 / 2.29]	1.88#, b[1.75 / 2.09]

Cholesterol concentrations are presented as medians and [25th / 75th] percentiles in mmol/L. ▲#■Different symbols indicate significant differences within a row (time-point differences in a group). abLowercase letters indicate significant effects within a column (group differences at one timepoint). Significant differences are identified by P values ≤ 0.05 using repeated measures ANOVA with Tukey HSD. BL, baseline group (n = 8); AD, group fed atherogenic diet (n = 10); ADp, group fed atherogenic diet + pectin (n = 10); ADi, group fed atherogenic diet + inulin (n = 10); CD, group fed conventional diet (n = 10); n/a, not available.

Table 6

Parameters of LAD plaque formation in all groups.

Group	Percentage of pigs within a group showing vascular lesions in LAD (%)	Plaque size in plaque-positive pigs (μm2)
BL	63ab	35061[22930 / 71273]
AD	50a	157062[73901 / 198848]
ADp	100b	40207[21287 / 181180]
ADi	70ab	128539[34196 / 212237]
CD	70ab	46162[14275 / 139486]

Data show the plaque frequency per group (%) and plaque size (μm2) of the plaque-positive pigs per group, expressed as medians and [25th / 75th] percentiles.

abLowercase letters indicate significant effects within the columns. Significant differences between the groups (BL, n = 8; AD, ADp, ADi, CD, n = 10) were identified using the chi-squared test (P ≤ 0.05). LAD, left anterior descending branch of the left coronary artery; BL, baseline group; AD, group fed atherogenic diet; ADp, group fed atherogenic diet + pectin; ADi, group fed atherogenic diet + inulin; CD, group fed conventional diet.