1. Introduction
Malnutrition results from eating an imbalanced diet that does not supply a healthy amount of one or more nutrients. Among nutrients, proteins play an irreplaceable role. Proteins are macromolecules made up of specific amino acids (AAs) sequences, serving a vast array of functions within the cell. Quantitative and qualitative reduced proteins intake, or an imbalance in protein synthesis and degradation, have been associated with severe depletion of body protein reserve, eventually resulting in malnutrition.
The AAs are fundamental for life, because they are the precursors for protein synthesis, since they are main source of nitrogen for mammals’ metabolism. Unfortunately, about half of the AAs in proteins cannot be synthesized or stored in metazoans, because the genes for their synthesis were lost early in evolution. These AAs are called essential (EAAs), and they must be taken exclusively through food. The other AAs can be synthesized in an autonomous way by the body, and therefore are defined non-essential (NEAAs). To best survive, all organisms must maintain a full complement of the AAs, best if with excess of EAAs [
1,
2,
3].
AAs, resulting from the breakdown of dietary proteins, are absorbed almost completely in the initial tract of the small intestine, crossing the intestinal mucosa and reaching the capillary circulation of the villi. The intestinal mucosa is composed of a mucin layer covering the cells, enterocytes, and the apical junctional complex in between the cells [
4]. Numerous experimental pieces of evidence suggest that alteration of barrier function is a potential pathway for intestinal and extra intestinal inflammation [
5]. Indeed, intestinal mucosa acts not only in digestion and absorption, but also plays an important role as a barrier to toxic and harmful materials and protects from different antigenic and inflammatory reactions. Changes of intestinal barrier function were mainly due to the relaxation of the tight-junction between intestinal epithelial cells [
6]. Different factors causing alterations in gut microbiota, hormones secreted by the enterocytes, and related changes of enzyme synthesis would all damage the intestinal barrier, with the enteric bacteria and endotoxin reinforcing those damages [
7]. Disruption of the gut barrier in several physiological or pathological conditions would lead to altered exchanges of intraluminal solutes into the systemic circulation thus drives the organism to chronic diseases [
8,
9]. Indeed, gut microbiota alterations are also associated either with obesity [
10], cancer [
11], or with systemic inflammation, or even to etiology and pathogenesis of Kawasaki disease in children [
12]. Microbiota modifications through age have been documented, and correlation among different grades of chronic disease and microbiome alterations have also been reported [
13,
14]. Furthermore, a recent pilot study showed that also patients with COVID-19 had persistent alterations in the fecal microbiome during hospitalization associated with severity of disease [
15]. In addition, cumulative evidence shows a linkage between gut microbiota pattern and depression [
16].
Although there is a growing interest and perception about mammalian microbiome and host relationship, most papers have focused mainly on fat and carbohydrate based nutrients, because partially digested proteins may reach, feed, and influence the different components of the microbiome [
10,
11,
17]. Some attention was paid to the interaction between indigested proteins and microbiota [
18], and, above all, to qualitative protein malnutrition. Indeed, qualitative protein malnutrition is not a rare event if we consider, in addition to chronic diseases as aging, cancer, etc., the spread of “junk foods” that invade the world market, or the “do it by yourself” diets, or diets that exclude or are significantly deficient in some macronutrients.
Microbiome and proteins’ biological quality should be peculiarly linked, and this link has been poorly studied, but some studies show a role for peculiar toxic substances derived by metabolic interactions of the microbiome with incomplete digestion products of food proteins, whose roles are becoming more and more significant in different illnesses [
18,
19,
20]. Indeed, different amino acid compositions and digestibility of proteins, which are influenced by source, kinds of thermal processing, and amount of intake, should play a pivotal role in determining microbiota quality and quantities [
21]. Therefore, we studied the effects of a special diet containing as unique nitrogen source a peculiarly balanced mixture of free essential amino acids (EAAs) and non-EAA (NEAAs), further expanding the excess NEAAs usually provided by food proteins, and compared the effects of this perfectly isocaloric diet to laboratory pellet and to control animals fed normal proteins, as a model of qualitative malnutrition. The aim was to determine whether qualitative and not quantitative malnutrition, that is, by diets providing the same nitrogen intake, but with excess in NEAAs and reduced EAAs, could induce alteration of gut epithelium morphology and enzyme as lysozyme, or cytokines as TNFα, TGFβ1, and NF-κB. As markers of altered permeability of gut wall, we measured the levels of zonulin in the epithelium and feces. In addition, the main kinds of microbiome (
Lactobacillus spp.,
Escherichia coli, fecal coliforms,
Mycétes, and
Clostridium spp.) were evaluated. Finally, we observed whether re-feeding of qualitatively malnourished mice with standard diet could reverse the morphological, enzymatic, and microbiota alterations.
4. Discussion
In this study, we have demonstrated that diets providing the same macro and micro nutrients quantities and the same nitrogen content as normal diets, but providing altered EAAs to NEAAs ratios (EAA/NEAA), through excess of NEAAs induce alterations of intestinal epithelium with concomitant altered microbiota relationships. Simultaneously, we found an experimental model of qualitative protein malnutrition capable of inducing quickly sarcopenia-wasting-cachexia syndrome, paralleled by a rapid modification of gut microbiota.
A first result of the present study is that DD-fed mice, although fed by an isocaloric diet and providing the same amount of nitrogen as StD, resulted in rapid body weight reduction, despite the consumption of food being comparable to that of StD-fed animals. These data confirm what emerged in previous studies, where it was shown that the diet rich in NEAAs and deficient EAAs is fully appreciated by animals [
3], and are in contrast with other studies that argued that animals receiving an EAA-deficient or imbalanced diet fail to grow because they they would refuse the diet [
30]. We showed that body weight loss was not due to rejection of diet but, most likely, to the need of qualitatively malnourished mice to recover EAAs and energy production by destroying endogenous proteins, especially muscle ones, as demonstrated by the significant reduction of muscle mass and adipose tissue with increase of serum creatinine concentration.
Adequate nutrition is necessary to maintain the gastrointestinal barrier, because this favors the enterocytes turnover, improving the normal cell division and migration from the crypt to the villi. It is well known that protein malnutrition is associated with villous atrophy, abnormal mucin formation, and impairment in the secretion of IgA during development, adulthood, and ageing [
31,
32]. Numerous studies have focused on quantitative protein malnutrition, finding that it disrupts the normal ecology of the microbiota, impairs host immune response and antibacterial defenses, enhances susceptibility to infection, and leads to mucosal atrophy, altering the gut barrier function and inducing bacterial translocation [
33,
34]. It was demonstrated that diet with reduced content of protein (75%) and vitamins (50%) given to weanling Wistar/NIN male rats for 20 weeks increased intestinal cell apoptosis [
35]. Furthermore, maternal malnutrition altered expression of genes that maintain maternal gut homeostasis, and altered fetal gut permeability, function, and development [
36]. Cancer cachexia is also reported to induce alterations of morphology in small intestine [
37]. In addition, many studies were performed on the intestinal barrier using a number of techniques to evaluate eventually altered function [
38], and a broad range of individual food substances have been tested in Caco-2 monolayers with divergent effects readings the trans epithelial electrical resistance and epithelial flux parameters [
39]. However, these studies and many others, unlike the present study, represent models of quantitative, not qualitative, malnutrition.
Diet containing NEAA in excess quickly change the morphology of epithelium’s cells: the enterocytes, goblet, and Paneth cells. Enterocytes are columnar absorptive cells and have an apical striated border of microvilli. The enterocytes own the apical junctional complex that consists of a network of tight junction proteins and the adherent’s junction [
40] that were anchored in the cell by means of filamentous actin cytoskeleton [
41]. Tight junctions are pivotal elements that govern trans-epithelial transports. These junctions are very dynamic structures, and their dysregulation is involved in development of pathological condition as autoimmune diseases. Intracellular tight junction proteins are called zonulin proteins because they form the
zonula occludens. Zonulin proteins link the cell cytoskeleton to the transmembrane tight junction proteins such as claudins, occludin, and junctional adhesion molecules that are mainly responsible for the intestinal barrier function [
42].
Zonulin was identified as endogenous modulator of tight junction’s integrity in the small intestine, consequently regulating the permeability of the intestinal epithelium, favoring the movement of fluid, macromolecules, microorganisms, and leucocytes between the bloodstream and the intestinal lumen and vice versa. Therefore, dysregulation of zonulin synthesis may contribute to weakening tight junctions and favor disease states as autoimmune diseases, inflammation, and malignant transformation [
43,
44].
Changes in zonulin levels correlated with in vivo increased intestinal permeability [
45]. Reduced expression of zonulin was reported in ulcerative colitis and Crohn’s disease patients, where the pentalaminar structure of tight junction was destroyed [
46]. Zonulin is also altered by toxic substances. Immortalized intestinal cell lines (Caco-2) treated with ethanol (from 0.1 to 10%) induced progressive disruption of tight junction with increased elimination of zonulin by fecal route, and formation of large gaps between the adjacent cells [
47]. Recent data show that zonulin-dependent small intestinal barrier impairment is an early step potentially linked to development of chronic inflammatory diseases [
48]. In light of above evidences, our data show that a diet providing NEAAs in increased ratio of total nitrogen intake, thus lowering EAAs intake, reduces both intracytoplasmic zonulin and increases fecal concentration of zonulin, suggesting the possible alteration of intestinal permeability and a compromised intestinal barrier, possibly correlated to inflammation.
Potentially related to reduced enterocytes zonulin immunoreactivity, we also documented increased inflammatory reaction by TNF-α staining and massive nuclear translocation of NF-κB, and both are relevant findings, since TNF-α exerts a direct effect on gut barrier integrity. Indeed, mice fed with high-fat diet increased TNF-α mRNA expression in the intestine [
49], while patients with Crohn’s disease receiving anti-TNF-α treatment improves gut barrier function [
50]. Furthermore, TNF-α reduced trans-epithelial resistance and Zonulin-1 expression via a NF-κB-dependent pathway in Caco-2 cells [
51]. Additionally, increased activation of NF-κB is detected in cells of tissues affected by chronic inflammation, where it is believed to exert detrimental functions by inducing the expression of pro-inflammatory cytokines that regulate and sustain the inflammatory response and could cause tissue damage [
52]. Intestinal microbiota is believed to regulate the level of NF-κB activity at the epithelial interface and thereby affects the mucosal immune balance [
53,
54]. Effect of TNF-α on NF-κB activation also increased the expression and activity of myosin light chain kinase, which leads to disorganization of tight-junction proteins, thus impairing the intestinal barrier [
55]. The NF-κB pathway, which is activated by alteration of intestinal microbiota, plays an important role in activating host pro-inflammatory responses [
56], and the role of inflammatory pathways appears to be critical in the regulation of gut barrier function [
57], leading to leaky gut syndrome. The harmful effects of pro-inflammatory cytokines on the intestinal barrier can be ameliorated by antibiotic administration, suggesting either gut microbiota changes as causative and some correlation among the innate immune system, gut inflammation, and microbiota profile [
57].
Finally, in animals fed with NEAAs-rich diet, we also observed the depletion of TGF-β1 staining. TGF-β1 is a cytokine synthesized and released by cells with efficient intestinal epithelial barrier function [
58]. Significance of TGF-β1 expression had been previously described in wound healing [
59]. In a healthy gut, a balance between regulatory and inflammation function would be necessary to maintain gut barrier integrity. Within the gut mucosa, TGF-β1 plays a pivotal role in mediating these balanced responses, since it regulates the function of many mucosal cell types in either autocrine and paracrine manner [
60,
61]. Of interest, TGF-β1 promotes the expression of tight junction protein and adhesion molecules, thus improving epithelial barrier integrity: inhibition of TGF-β1 favors gut inflammation [
62]. In addition, it has been proven that mice with selective deletion of TGFβ1 signaling in gut epithelium are more susceptible either to colitis induced by dextran sodium sulphate or to invasive intestinal tumors [
63]. TGF-β levels in a mammal’s gut are directly and indirectly modulated by the microbiota and microbiota-derived products with impacts on the development and functions of immune cell subsets, which in turn regulates microbiota sequestration within the gut lumen [
64]. As predictable by literature, we observed the tendency to decrease in TGF-β1 immunostaining following qualitative protein malnutrition, and a concomitant decrease of zonulin and increased inflammatory state. Therefore, excess in NEAAs, or most probably, insufficient availability of EAAs through diet can severely impair the gut barrier, and so malnutrition could pave the way for serious diseases. Marked rise in TGF-β1 staining observed after refeeding suggest restoration of epithelial barrier integrity consequent to rebalancing EAAs to NEAAs ratios.
Previously, in mice models of intestinal obstruction, pre-treatment with a diet containing arginine (2%) preserved intestinal barrier integrity and reduced bacterial translocation in gut wall [
65]. Additionally, glutamine has been indicated as a possible adjuvant therapy in different gut illnesses [
66]. Our NEAAs-rich diet contained high amounts of arginine (11.8%), and also of glutamine (10.2%); nevertheless, although we have not measured bacterial translocation, we observed a significant alteration of the intestinal mucosa by the loss of zonulin with the stool, suggesting increased permeability of the barrier. An increased supply of arginine and of glutamine, therefore, was not sufficient to protect intestinal barrier integrity when EAAs provided by diets are low. We believe that this discrepancy is due to the fact that our diet contained only 15% EAAs (EAA/NEAA << 0.2), and no study dealing with supplementation of glutamine or arginine has tested this kind of supplementation against EAAs enriched diets (EAA/NEAA ≥ 1). Indeed, in experimental models, we have repeatedly observed that even slight reductions of concentrations of EAAs under the normal content of food proteins (near to 45% or EAA/NEAA < 0.9–0.7) induces proportionally significant alteration both of organ morphology and hematologic parameters [
1,
3].
Goblet cells secrete mucin, a glycoprotein that forms a protective layer of intestinal lumen. Indeed, mucin forms a mucus layer, which keep separate materials in cavities from the intestinal epithelium and acts as a lubricant. However, mucin also constitutes a physical barrier that traps and therefore prevents invasion of pathogenic microorganisms. In addition, goblet cells participate in immune response through nonspecific endocytosis and goblet cell-associated antigen transfers [
67,
68]. Altered mucin production and consequent dysfunction of the mucosal barrier are related to the occurrence of inflammatory diseases such as ulcerative colitis and Crohn’s disease [
69,
70]. We observed that diet providing excess of NEAAs, and so a reduced ratio of EAA/NEAA, decreased goblet cells’ number, hence the following thinning of the mucin layer rapidly exposing intestinal epithelium to a greater risk of inflammation and bacterial attack. This also may result in impairment of intestinal absorption. In effect, the depletion of goblet cells leads to increased adhesion of bacteria to the surface of the epithelium and so reduces digestion and absorption of nutrients, because it alters gut barrier efficiency [
71,
72]. Certainly, the decrease of lysozyme and secretory granules in Paneth cells observed in our mice fed with NEAAs-rich diet contributed to exacerbating any possible inflammatory response due to impairment of the gut barrier. In fact, normality was restored by returning to standard diet. Those findings are of interest, since current evidence suggests that defective Paneth cells may play a key role in promoting gut inflammation and also in Crohn’s disease, by allowing bacterial attachment and invasion [
73].
Gut microbiota is the assembly of microorganisms living in our intestine. The correct composition and functionality of the microbiome is essential for maintaining a “healthy status”. The profile of the gut microbiota is influenced by numerous variables, including requirements of metabolism substrates, microbiological factors, and environmental factors. Based on our findings, however, the variable that may have the greatest impact on the composition of microbiota is adequacy of EAAs provided by diet. Dietary factors, and particularly matching nitrogen needs, can enhance gastrointestinal health by shaping the nature of gut microbiota [
34,
74]. The resident species of gut microbiota may use AAs derived from food proteins or from the host as elements for new protein synthesis; in addition, they drive nutrient metabolism by conversion or fermentation. Of note, gut microbiota can synthesize several nutritionally useful EAAs, and this is a potential regulatory factor in AAs homeostasis [
74], but may produce also uremic toxins [
18]. Indeed, part of the whole protein ingested with the diet is not digested, and peculiarly its content in tryptophan is transformed into toxic indolamine by indolamine 2,3-dioxygenase (IDO) via the kynurenine pathway. This suppresses T cells, activates Treg cells, and promotes inflammatory processes [
75]. In contrast, the free tryptophan contained in the special mixture of EAAs is completely absorbed in the ileum and transported in the blood by albumin [
76]. Keeping tryptophan plasma concentration constant inhibits IDO and consequently blunt endogenous uremic toxin syntheses and systemic inflammation [
77].
In a sample of healthy individuals consuming unbalanced (quantitative) protein diet (high-protein and low-protein diet) for 42 days, reduction mostly in the pool of bacteria belonging to the
Bifidobacteria genus [
78,
79] was reported. In another study, milk-fed piglets after weaning were fed a soybean based diet, and despite the good health status, owing to the buffering effect of the protein, those animals exhibited lowered gastrointestinal pH and a decreased
Lactobacillus-to-
Coliforms ratio [
80,
81]. It is interesting to note that soybean contains NEAAs in excess of EAAs, with the ratio EAA/NEAA being around 30/70 (or <0.43). This is in line with what was highlighted by our data, since diet rich in NEAAs, although providing high amounts of arginine and glutamine, determined an unfavorable ratio between
Lactobacillus spp. and
Coliforms spp., but the rise in number of
Mycétes was by far more relevant. A more balanced refeeding, and peculiarly less reduced EAA/NEAA containing diets restore the predominance of
Lactobacillus spp. over other species. Higher
Lactobacillus-to-
Coliforms ratio indicates a favorable proportion of advantageous
Lactobacillus spp. relative to
Coliforms spp., a population that could include coliform pathogens [
81]. Indeed, it was proposed that
Lactobacillus strains play an important role in development of anti-infectious agents that act luminally and intracellularly in the gastrointestinal tract [
82].
Mycétes spp. reside permanently in the intestine and, in healthy subjects, act synergistically with other microbiota to maintain homeostasis. Correlation between alterations in gut
Mycétes and pathogenesis of inflammatory bowel disease, such as Crohn’s disease and ulcerative colitis, has recently been reviewed [
83,
84]. In addition, also patients with chronic heart failure may have intestinal overgrowth of pathogenic bacteria and
Candida spp. [
85]. Furthermore, also diabetes type 2 may induce significant intestinal
Mycétes overgrowth, increasing intestinal permeability and systemic low-grade inflammation [
86]. In our study,
Mycétes are modified as a consequence of low quality protein intake, but return to their initial state with refeeding with standard proteins. We believe that this finding is of particular importance, as it suggests that low quality protein diet promotes the development of fungi, and also that adequate supplementation of even a poor diet with EAAs (so increasing EAA/NEAA ratio) could be a way to rapidly reduce intestinal inflammatory status. Therefore, modulation of the microbiota and
Mycétes can be considered as a therapeutic approach for treating gut inflammation and related diseases, even in patients with multiple pathologies. On the other hand, we propose that each strategy aiming to obtain the homeostasis of the microbiota should consider carefully modifications and content of
Mycétes.
In summary, the microbiota and intestinal wall represent a peculiar morpho-functional unit, whose characteristics and role are partially unknown. At present, it is difficult to understand how relevant specific alterations of intestinal mucosa would be in determining an alteration of the microbial flora or vice versa. In any case, we provided evidence that damages induced by nutritional deficits, and chiefly by EAAs deficits, that is by a qualitative malnutrition, definitively harm both intestinal wall integrity and equilibrium of microbiota composition.
Study Limitation
A possible limitation of this study is the exclusive application of IHC to support our results. However, as applied in a previous spleen study [
2,
87], this choice was dictated by the fact that the gut also contains heterogeneous cell population (enterocytes, goblet cells, Paneth’s cells, myocytes, etc.). Consequently, histopathological changes and especially the exact location of markers is possible alone with IHC and HC. In contrast, molecular analysis, although much more sensitive in highlighting the presence of proteins and very often used exclusively, does require immediate freezing and homogenization of the sample. As a consequence, it does not take into account the specificities of the proteins location such as the tissue morphology and organization. This is a major limitation in the exclusive use of molecular analysis, which we believe is comparable if not superior to IHC. For these reasons, we believe that our data, even if obtained only by IHC, are worth considering forming the basis for further studies.