1. Introduction
Growing attention has been paid to the role of diet, particularly macronutrients, affecting composition and metabolic activity of the human gut microbiota, thereby possibly affecting health, as recently reviewed, for example, by Conlon and Bird [
1]. The prevalence of harmful and pathogenic gut microbes has been associated with intestinal disease such as obesity or gastric cancer [
2]. Dietary means have proven to be efficient in preserving a healthy gut microbiota population [
1], both in terms of short- and long-term effects [
3].
Several bacteria such as species of the
Lactobacillus or
Bifidobacterium genera [
4], or
Faecalibacterium prausnitzii [
5] have shown beneficial effects on the health of humans and animals, and are recognized as biomarkers of intestinal health. On the other hand, the
Enterobacteriaceae family for example is rather considered to be detrimental for both the human and animal host because of their pathogenic members (e.g., enterotoxic
Escherichia coli and
Shigella) [
6,
7]. For example, activation of the innate immune response has been associated with the presence of lipopolysaccharides (LPS, plasma endotoxins) located on the outer membrane of these gram-negative bacteria [
8,
9]. Considering this, Myles
et al. [
10] could demonstrate that dietary fat consumption may lead to an increased colonic permeability to gut microbial products such as LPS, which, in turn, may cause immune dysregulation with colonic and systemic inflammation. The role of dietary fat in metabolic endotoxemia is a central issue, which may partly explain the high rate of chronic diseases in Western countries [
11,
12] as Western-style diets are usually composed of food ingredients rich in fat content [
13]. On the other hand, Western-style diets are commonly very poor in dietary fiber content [
13]. Dietary fibers are defined as the edible part of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine. They include polysaccharides, oligosaccharides, lignin and associated plant substances [
14]. Major end-products of dietary fiber fermentation are volatile fatty acids including acetate, propionate and butyrate, which reduce the colonic pH, thereby preventing the growth and activity of a variety of pathogenic bacteria [
1]. In particular, butyrate serves as main source of metabolic energy for the colonocytes, is supportive in maintaining mucosal integrity, controls intestinal inflammation and endorses genomic stability [
15].
Though, assessing the impact of diet on the gut microbiota composition, metabolic activity and related processes remains difficult due to restrictions in using human models and the obvious physiological differences between rodents and humans [
16]. On the other hand, primate models have stringent ethical restrictions for experimentation compared with mice or pigs [
17]. Therefore, the pig as human-sized, omnivorous animal with analogous digestive processes [
4] was used as model in the present study. Two different diets, referred to as low-fat/high-fiber (LF), or high-fat/low-fiber (HF), were used to assess their effect on gut microbiota composition and production of microbial metabolites. In addition their effect on the development of digestive organs, and on different biochemical parameters in pigs’ blood serum was determined. This comparative study using LF and HF diets represents an alternative approach to previous studies where, e.g., standard pig diets high in fat, or genetically obese (mini-) pigs were used [
18,
19,
20]. The results of this study are expected to provide more evidence, if and under which conditions the pig can serve as human model to assess the effect of dietary modulations on gut microbiota, biochemical markers, and development of digestive organs.
4. Discussion
The trophic effect of high-fiber diets on gastrointestinal tract development of growing pigs such as higher stomach and colon weights has been reported repeatedly (e.g., [
45,
46]). Besides bulking effects, the trophic action of fiber has been attributed to its fermentability, resulting in the production of SCFA [
47], thereby stimulating epithelial cell proliferation. In rats, for example, colonic infusion of SCFA showed a trophic effect throughout the intestinal tract [
47]. Similarly, Scheppach
et al. [
48] found
in vitro raised cell proliferation in human cecal mucosa following incubation with SCFA. Furthermore, higher liver weight as observed for pigs of the LF treatment has been associated with increased basal metabolic rate of the animal [
46]. In addition, the higher crude protein content in the LF diet (244.3 g/kg·DM) compared to the HF diet (210.2 g/kg·DM) results in higher amounts of nitrogenous compounds being processed by the liver, thus causing a hypertrophic effect on the liver [
49]. In line with this finding, increased serum urea concentrations in LF pigs with average concentrations at the upper end of reference values [
50], especially at the second and third sampling date, point towards an increased metabolic rate in the liver. It has to be mentioned that high protein diets are increasingly recommended as major management strategy for weight control in overweight and obese individuals, particularly if combined with exercise [
51,
52,
53], since they appear to be effective regarding reductions in appetite, body mass, fat mass and retention of lean mass, at least in the short term [
54,
55]. In the LF pigs, GPT concentrations were rather elevated in comparison to reference values [
50], though still within normal range. Generally, this parameter suggests muscle and liver cell injury, and is increased in most cases of non-alcoholic fatty liver disease in humans [
56]. Elevated levels of liver GPT have also been reported by Peters and Harper [
57] upon feeding protein-rich (over 35% protein) diets to rats, similar to the conditions in the LF treatment.
The observed increase both in serum GPT concentrations and liver weight suggests a rather detrimental effect on the health status of the pigs compared to the HF treatment. On the other hand, concentrations of CRP, an acute-phase marker for systemic inflammation, decreased with time in the LF treatment. This suggests an improved inflammatory status of the liver. Ismail
et al. [
58] observed higher CRP concentrations in obese humans, linking the chronic low-grade inflammation associated with obesity. Higher BW often results from rather unhealthy nutrition such as consumption of the HF diet, which tended to increase backfat thickness of these pigs compared to the LF pigs. Yet, no increase in CRP concentration was observed for the HF pigs, eventually due to the limited length of the experiment, not allowing the development of a real obesity in HF pigs. Furthermore, despite higher glucose values observed in the HF compared to LF pigs, concentrations were within reference values according to Nerbas [
50]. Concerning lipoproteins, one might expect rather reduced LDL levels upon feeding the LF diet due to the high fiber content. However, a lowering effect has especially been ascribed to the consumption of soluble fiber fractions (e.g., [
59]) while wheat bran, representing the main constituent of the fiber fraction in this diet, mainly consists of insoluble fiber [
60].
Dietary effects on gut microbiota composition revealed lower numbers of total bacteria in cecal digesta of the LF pigs. Similarly, Varel
et al. [
61] observed an initial suppression of the porcine gut microbiota in rectal samples upon feeding a high-fiber diet, followed by an adaption process to the diet with a re-establishment of the microbiota. Moreover, numbers of
Enterobacteriaceae were lower in the LF in comparison to HF pigs. These gram-negative bacteria are known to induce inflammation through the LPS located on the outer membrane [
9]. Here, higher colonic SCFA concentrations in LF pigs, possibly accompanied by a subsequent decrease in luminal pH, might have created an inhospitable environment, e.g., for some acid-sensitive bacteria strains of
E. coli, which would explain the lower abundance of
Enterobacteriaceae in LF pigs. Similarly, Smith
et al. [
62] found a decrease in the
Enterobacteriaceae population in association with higher concentrations of total SCFA in the colon of finisher pigs when feeding barley-based diets compared to oat-based diets. Correspondingly, higher numbers of
Enterobacteriaceae were found in feces of European children consuming a typical Western diet high in animal protein, sugar, starch and fat but low in fiber compared to children from rural Africa living on more vegetarian diets low in fat and animal protein and rich in starch, fiber, and plant polysaccharides [
7]. Still, the different environmental conditions as well as their genetic background have to be considered when comparing humans from different continents.
According to the results of the present study, there were higher values of
Bacteroidetes (
Bacteroides group,
Prevotella spp.) in cecum and colon of the HF pigs. Although differences varied between 0.4 and 0.5 log
10 only, results pointed towards different proliferation of these bacteria as influenced by diet composition. As recently reviewed by Tjalsma
et al. [
63], the genus
Bacteroides spp., especially enterotoxigenic
Bacteroides fragilis, is among bacterial groups possibly being associated with a predisposition for the development of colorectal cancer. In
in vitro studies, pure cultures of
Bacteroides strains incubated with human feces stimulated the production of fecapentenes, which are assumed to be co-carcinogens or mutagens [
64]. Dietary fat stimulates bile flow, which, in turn, promotes the growth of
Bacteroides species [
65]. This might possibly be an explanation for the enhanced proliferation of this species in cecal and colonic samples of the HF compared to the LF pigs in this study. Furthermore, in a study with human subjects by Wu
et al. [
3], the
Bacteroides enterotype was highly associated with animal protein, several amino acids, and saturated fats, which suggests that meat consumption according to a Western diet is characteristic for this enterotype. Within this regard, Williams
et al. [
66] recently investigated the impact of solubilized wheat arabinoxylans (AX) added to red meat diets fed to pigs. A counteracting potential of AX could be observed, characterized by reduced protein fermentation and lower microbial production of toxic end products such as ammonia in cecum and colon, while, e.g., the
Bacteroides fragilis group was relatively high in pigs fed the red meat diet devoid of AX. The results of the present study correspond with those of a study with pigs by Yan
et al. [
19], where a high-fat diet resulted in higher proportions of
Bacteroides in cecal samples compared to a low-fat diet. On the other hand, there were fewer numbers of
Bacteroides in genetically obese mini-pigs rather than lean pigs [
20], thus pointing towards differences in microbiota composition induced by diet effects and genetically induced obesity.
According to Feng
et al. [
67], dietary fat consumption increased proportion of
Prevotella in the cecum of growing pigs compared to a basal diet. This result is in line with the present study, yet not expected, since the
Prevotella genus has mainly been associated with fiber degradation in pigs, as for example reported by Wang
et al. [
68] feeding cellulose-supplemented high-fat diets. Similarly, Zhang
et al. [
69] reported enrichment of the microbiota in obese humans with
Prevotellaceae, a bacterial group known as potential source of LPS. Schwiertz
et al. [
70] observed a higher proportion of
Bacteroidetes in the microbiota of obese humans. On the other hand, De Filippo
et al. [
7] found a significant proliferation of
Bacteroidetes and
Prevotella in the gut microbiota of children from a rural African village and hypothesized that this could be due to high fiber intake, thereby maximizing metabolic energy extraction from ingested plant polysaccharides. Thus, compared to studies with the pig as animal model, human studies might be less consistent due to varying environmental conditions, genetics, differences in diet composition, and restrictions in the design and standardization of experiments with human subjects. In this respect, Pedersen
et al. [
71] assessed changes in the gut microbiota during development of obesity in cloned
vs. non-cloned pigs. However, the authors did not observe less inter-individual differences in cloned pigs.
With regard to the rather beneficial members of the gut microbiota,
Faecalibacterium prausnitzii is a prominent butyrate forming bacteria and dominant member of the subgroup
C. leptum, being predominant in the colonic microbiota of healthy humans [
5]. No differences between treatments were observed for
F. prausnitzii in the present study, although the
C. leptum group, including several butyrate producing members [
72], showed higher values in cecal samples of the LF treatment. This could be in part due to generally higher abundance of
F. prausnitzii in feces and lower numbers in mucus, with varying implantation along the gastrointestinal tract [
73].
There were higher numbers of bifidobacteria in cecum and colon of the LF pigs than in the HF animals. Similarly, there is also evidence from human studies that dietary fiber promotes growth of bifidobacteria (e.g., [
74]). Enhanced proliferation of bifidobacteria is considered to be beneficial for the host, as these bacteria produce lactate, which lowers the pH and, in turn, may be metabolized by butyrate producers [
75]. Apparently, there exists a relationship between lower numbers of bifidobacteria and overweight and obesity both in adults [
70] and children [
76] compared to normal weight persons. Interestingly, compared to a standard diet, a high-fat diet also reduced cecal
Bifidobacterium numbers in mice, which negatively correlated with circulating LPS concentrations and thus metabolic endotoxemia [
11].
The metabolic activity of the gut microbiota is characterized by the production of various microbial metabolites. In the present study, consumption of the LF diet, supplemented with wheat and wheat bran, resulted in increased acetate and butyrate concentrations in cecum and colon. Lower amounts of acetate and butyrate found in the HF pigs correspond with results of Yan
et al. [
19], who measured lower concentrations of acetate, propionate and butyrate in cecal samples of pigs fed a high-fat diet (17.5% swine grease), compared to pigs fed a low-fat diet (5% swine grease). Possibly, this could be ascribed to an inhibitory effect of fat on fermentation activity, as observed in ruminants [
77]. Propionate did not significantly differ between diets in the present study, though a numerically higher concentration of propionate was observed in the cecum of HF pigs. Since the genera
Bacteroides and
Prevotella are known propionate producers [
69] their higher abundance in HF pigs corresponds to this increase in propionate concentrations.