Our diet primarily consists of a macronutrient mixture. Carbohydrates, lipids and proteins add up to 51.8%, 32.8% and 15.4%, respectively, of the total food-derived energy in the Western diet [1
]. These numbers show that proteins resemble a relatively small amount of the total food energy compared to the other macronutrients, mainly because they are not primarily dedicated to energy delivery. However, proteins are involved in many, if not all, biological processes (e.g., cell growth and differentiation) and are frequently referred to as “the building blocks of the human body” [2
]. According to the World Health Organization, the world population is projected to reach more than 9 billion by 2050 accompanied by an essential doubling of the food demand. This urges the need for sustainable and novel nutrients, such as proteins. Prior to human consumption, these novel proteins need to be studied and declared safe for consumption.
After consumption of food, the digestive process starts in the oral cavity. Within the mouth, chewing results in smaller pieces, and amylases start breaking down the carbohydrates [4
]. However, the digestive process of proteins starts in the stomach [5
]. During digestion, enzymes are secreted as zymogens, an enzymatically inactive form, by chief cells [2
], which secrete pepsinogen upon stimulation by, e.g., gastrointestinal hormones [6
]. These gastrointestinal hormones are secreted after ingestion of a meal. The amount that is secreted depends on meal composition and calories. The acidic environment denaturizes proteins and transforms the zymogen pepsinogen into its active form, pepsin. The gastric phase is followed by the small intestinal phase, consecutively passing through the duodenum, jejunum and ileum. The small intestine exists of a heterogeneous cell populations. Four major cell types can be distinguished, of which the absorptive enterocytes represent approximately 90% of the total number. The remaining 10% consists of mucus-producing goblet cells, enteroendocrine cells and antimicrobial peptide-producing Paneth cells [7
]. The intestinal epithelium is covered by mucus produced by goblet cells. The mucus contains a high number of different enzymes, antimicrobial peptides, immune factors, growth factors and many more constituents. Mucins feed the microbiota and protect the epithelium from the microbiota, and slow down the diffusion rate of the nutrients [8
]. Besides these four major cell types, there are immune players present such as macrophages, dendritic cells, microfold-cells (M-cells) and Peyer’s patches [10
In the duodenum, the liver and pancreas secrete bile and pancreatic juice, respectively [11
]. Bile is composed of bile salts (derivatives of cholesterol) and plays a major role in digestion. After emulsification, the lipids can be hydrolyzed within the micelle [2
]. The pancreatic juice contains a mixture of bicarbonate and proteases which play an important role in protein digestion. Bicarbonate alkalizes the acidic food bolus coming from the stomach. The resulting neutral environment (pH = 6.5–7.5) is ideal for the activation of the proteases secreted by the pancreas [12
]. These proteases are secreted as zymogens and are activated upon enzymatic cleavage [13
]. The pancreatic proteases can be divided into endopeptidases (hydrolyzing peptides bonds in the interior of the amino acid chain) and exopeptidases (releasing the amino acids from either end of the amino acid chain). Next to these enzymes, the small intestinal brush border located at the epithelial apical membrane contains a number of brush border enzymes, among others, carboxypeptidase, aminopeptidase N, tripeptidase and dipeptidase [14
]. These carboxypeptidase and aminopeptidase are able to completely digest a protein into a mixture of peptides and amino acids.
The duodenum is followed by the jejunum where the majority of absorption processes take place. Just before absorption, protein digestion is finalized by the microbiota and intestinal brush border harboring enzymes. The digestive products of proteins are di- and tripeptides and amino acids [15
]. Compared to the large intestine, the small intestine harbors a relatively low density of microbiota due to the rapid flow and anti-microbial substances secreted by Paneth cells [16
]. Peptides and amino acids can be taken up by the microbiota, depending on the bacteria’s transporter expressions. Once taken up, peptides and amino acids can be used for the synthesis of bacterial cell components or catabolized via other pathways. Their mode of action can either beneficially or detrimentally affect the host [17
]. The microbiota also possess proteinases and peptidases, which might cooperate with the human proteases [18
]. This review thoroughly discusses the absorption mechanisms of peptides and amino acids by the enterocytes. The ileum, the final part of the small intestine, completes nutrient and bile salts absorption. After this absorption, the eventual bioavailability is determined by effective transport over the basolateral membrane, allowing the nutrients to become systemically available [19
]. The efficient transcellular transport of peptides occurs via cooporation between the apical and basolateral transporters [20
The small intestine is followed by the large intestine, which harbors primarily a wide spectrum of microorganisms and reabsorbs liquids and salts. Absorption of food components in this segment is, however, limited and will not be taken into account in this review.
To study the intestinal absorption processes of proteins and their derivatives, human intestinal cell lines are commonly used. We here discuss human intestinal di- and tripeptide and amino acid transporter-mediated uptake and efflux, and the presence of these transporters in frequently used commercially available human intestinal cell lines. Additional attention is paid to their capability to mimic human intestinal absorption processes using advanced cell culturing methods. Furthermore, we will discuss innovations in intestinal in vitro models for studying human intestinal absorption.
3. Intestinal Di- and Tripeptide and Amino Acid Transporters
Proteins differ in absorption rate, depending on multiple factors such as amino acid composition (side chains, groups at N- and C-terminus), protein origin and processing (e.g., heating) [23
]. The transporters responsible for the uptake of di- and tripeptides and amino acids belong to the solute carrier (SLC) and cadherin (CDH) gene families.
Currently, four apical peptide transporters are known; SLC15A1
(peptide transporter 1, PEPT1), SLC15A3
(peptide histidine transporter 2, PHT2), SLC15A4
(peptide histidine transporter 1, PHT1), are members of the peptide transporter family (PTR; SLC15
) and CDH17
(human peptide transporter 1, HPT-1) a member of the 7D cadherins family [26
]. There is only one peptide transporter known to be present at the basolateral membrane, referred to as the basolateral peptide transporter. The encoding gene of the basolateral peptide transporter is, to our knowledge, unknown. PEPT1 is expressed in the human duodenum, jejunum and ileum and located at the brush border membrane [28
]. Herrera-Ruiz et al. [27
] showed the highest PEPT1 expression in the duodenum, decreasing in the jejunum and ileum and no expression in the colon. PHT2 and HPT-1 expressions were not region specific. Abidi et al. [29
] investigated in vivo absorption of di-peptides and demonstrated that the jejunum showed the highest transport activity followed by the ileum and duodenum.
Free amino acids are absorbed in the small intestine, primarily in the proximal jejunum [24
]. An overview of the diverse characteristics of the transport proteins present in the intestine is summarized in Table 1
As most of the transporters depend on Na+
, maintaining their ion gradients is of utmost importance. To prevent the loss of the proton gradient, the Na+
exchanger 3 (NHE3), encoded by SLC9A3
, plays a key role. NHE3 is an antiporter transporting Na+
-ions into the cell in exchange for a proton [30
]. Furthermore, ion-channels, ATPases and exchangers are involved in maintaining the desired ion gradients [31
]. For the apical transporter system b0,+
to become active, dimer formation between rBAT and b0,+
AT is essential. For the basolateral amino acid transporter systems y+
L, L and Asc, formation of a dimer with the 4F2 cell surface antigen heavy chain (4F2hc) is needed. 4F2hc is encoded by SLC3A2
and forms disulfide bonds with the amino acid transporter to direct it to the plasma membrane and assist in the proper assembly for the transporter to become active [32
The transporter expression is regulated via different signaling pathways, involving the kinases general control nonderespressible 2/activating transcription factor 4 (GCN2/ATF4) and mammalian target of rapamycin (mTOR). These pathways are triggered via constant monitoring of the intracellular amino acid pool [29
], where GCN2 and ATF4 are activated during amino acid starvation and mTOR upon amino acid abundance. These pathways have been described recently in more depth by Jewell et al. [35
] and Taylor et al. [36
]. Therefore, we do not elaborate on the intracellular regulation of amino acid transporters in this review.
The most abundant intestinal peptide transporters within the human intestinal tract, PEPT1 and PHT2, are absent in parent HT-29 cells [62
], whereas PEPT2 and PHT1 are present [81
]. HT-29 parent cells are highly sensitive to changes in culture medium. Lindley et al. [82
] investigated the effects of different media and demonstrated that culturing in galactose-rich medium resulted in the expression of all four transporters. HT-29 cells are capable of absorbing taurine at a 5-fold higher activity compared to Caco-2 cells, whereas β-alanine was transported at similar rates. This indicates that the system β is present in these cells [83
]. Via gene expression analysis and immunostaining, the presence of apical transporter systems N (SN2), b0,+
(rBAT), ASC and X−AG
and the basolateral transporter systems L (LAT2), Xc−
L has been reported [76
]. However, Oda et al. [86
] were unable to stain for LAT2, whereas staining for the basolateral-associated subunit 4F2hc was successful. By contrast, Kekuda et al. [76
] and Bourgine et al. [76
] did show the presence of LAT2.
HT-29/MTX cells are capable of transporting the dipeptide (Gly-Pro) at a similar magnitude as Caco-2 monolayers, proving the expression of apical peptide transporters [87
]. However, to our knowledge, there is no specification as to which intestinal peptide transporters are present in HT29/MTX cells. HT29/MTX cells are capable of transporting phenylalanine, although the rate is lower compared to Caco-2 cells. Therefore, Hilgendorf et al. [87
] postulated that the relevant amino acid transporters had lower expression levels.
9. Advanced Culturing Conditions and Future Perspectives
While studying di- and tripeptide and amino acid absorption in cell models, it is important to have a model that is as physiological relevant as possible, especially when such a model is being used as screening tool for novel proteins. Showing the presence of a functional barrier, brush border enzymes and di- and tripeptide and amino acid transporters is considered to be essential—not only from the absorption point of view—as cell models are considered a relevant tool to screen for health effects as well. Commonly studied topics are focused on the prevention and recovery of barrier disruption [91
]. Furthermore, cell models are a useful tool to study molecular pathways, such as the amino acid sensing pathway. Caco-2 and HT-29 cells have been shown to be responsive to changes in amino acid availability via the amino acid response pathway or its associated genes [92
]. Unfortunately, to our knowledge, there is no literature directly connecting amino acid availability to specific peptide and amino acid transporter expression in the cell lines described. Closing this gap of knowledge will provide opportunities to optimize culture conditions by regulating transporter expression. Overall, availability of physiological relevant intestinal models will improve screening methods for novel nutrients and drug candidates.
In an attempt to improve the physiological relevance of cell lines, co-cultures of different epithelial cell lines have been proposed to provide a better representation of the small intestine. Verhoeckx et al. [97
] concluded that a co-culture of Caco-2 and HT-29 provided a more physiological relevant cell population compared to monocultures. This is because the Caco-2 cells provide the barrier function and absorptive enterocyte population, whereas the HT-29 cells present the mucus producing goblet cells. Unfortunately, different growth rates between the cell lines result in HT-29 overgrowth, which could be partially overcome by adapting their ratios. Pan et al. [98
] concluded that a 9:1 ratio of Caco-2:HT-29 is ideal. In line with this way of thinking, Hidalgo et al. [87
] performed a transport study using the amino acid phenylalanine and dipeptide glycine-proline over a Caco-2:HT-29/MTX co-culture at different ratios. Phenylalanine transport decreased as the amount of HT-29/MTX in the co-culture increased, whereas dipeptide transport was not affected compared to the Caco-2 monoculture. These collective data suggest that the attempt to obtain a heterogeneous cell population was successful. However, it did affect the actual absorption, and the model needs constant adaptations as cell growth changes after cell passaging.
Kim et al. [99
] developed a more complex in vitro system with Caco-2 cells in which the cells were positively stained for mucin 2, present in goblet cells. Research groups are developing 3-dimensional culturing systems. These models combine more physiological relevant parameters such as shear stress and microbiota [99
]. Kim et al. [99
] established a model mimicking the peristaltic movement of the intestine, through which they concluded that addition of flow altered the rate of differentiation and spontaneous formation of villi. Furthermore, they introduced microbial co-cultures on fully differentiated Caco-2 cells, while leaving the cells viable. Shah et al. [100
] developed a microfluidic device, bringing together flow and microbial interaction. Just like Kim et al. [99
], they concluded that cells can be co-cultured with microbial compounds in a controlled manner. Another innovative model, recently developed by Trietsch et al., consists of 3-dimensional tube-like structures of Caco-2 cells that form a tight barrier without the presence of a membrane and has proven to be a suitable tool to screen for effects on the intestinal barrier in a high-throughput manner [102
]. These microfluidic devices will most likely become widely accepted, as they are easy to reproduce and are economically attractive [103
]. As these novel models are still in the developmental stage, the effect of these alterations on the presence of peptide and amino acid transporters has yet to be determined.
The next step is combining different organs in the same system, thereby enabling the possibility to study organ cross-talk. The development of multi-organ platforms is accompanied by biological and technical challenges such as balancing in vivo complexity with simplicity, scaling of organs and inter-compartment sealing [105
]. After nutrient absorption by the enterocytes, the liver may be the first organ which comes in contact with these compounds [106
]. In addition, Peyer’s Patches may be encountered, facilitating the generation of the immune response within the mucosa. Though, combining the immune system with a gut-microfluidics platform is an ongoing challenge to address. Therefore, systems mimicking the gut-liver axis will have great value to study the steps after absorption. An example of such a model was developed by Choe et al. In their model, Caco-2 cells and HepG2 cells were co-cultured, and cell activity, intestinal barrier formation and the first pass metabolism of the drug Apigenin was assessed. They concluded that the cells could successfully be co-cultured; however, metabolic activity was shown to be enhanced in both cell types by evaluating P450 enzyme family activity, and the absorptive properties of Caco-2 cells changed as cell junctions were tighter and the absorptive permeability decreased. Furthermore, they concluded that their gut-liver-model better resembled the in vivo pharmacokinetics of Apigenin compared to a gut-monoculture [107
]. Similar to the above-mentioned microfluidic intestinal models, these models are still in the developmental stage, and peptide and amino acid transporters have, as of yet, not been investigated.
Besides the effect of flow as introduced by microfluidic devices, the role of the extracellular matrix providing a 3-dimensional cell culturing is being widely studied. For this, hydrogels are highly suitable, hydrophilic polymeric materials that swell in contact with water and, depending on the proteins incorporated, provide a stiff material suitable for 3-dimensional cell growth. Most often studied in this regard is Matrigel®
, which is composed of basement membrane proteins that are cytocompatible and can be formed into desired structures [108
]. Addition of an extracellular matrix was shown to improve culture conditions supportive for maintaining proliferation and differentiation [109
]. This allows cells to develop into mini-guts, also call organoids, as was demonstrated by McCracken et al. [110
], who cultured stem cells on Matrigel®
-coated wells and showed differentiation towards a small human intestine phenotype [111
]. Addition of an extracellular matrix in non-organoid culture showed improvement in transporter expression levels in Caco-2 cells [51
], suggesting the added value of the matrix in performing protein absorption assays.
In the future, physiologically improved cell culture models will have great potential for a broad range of applications. We foresee that in the future, organoid cultures will play a role in personalized medicine and nutritional intervention. Within the same time frame, more complex microfluidic devices are being developed using traditional cell lines e.g., Caco-2 cells. As the Caco-2 cells are well-defined, effects of changes in the micro-environment (e.g., 3D-culture, shear stress) on parameters (e.g., cell differentiation and barrier formation) can be effectively studied using these cells. However, the development of these novel 3-dimensional culture models is still in its infancy, and the effect on peptide and amino acid transporters has yet to be investigated in more detail. Besides cell differentiation, the microbiota play an important role in the health of an individual. We expect an interplay between organoid cultures, personal microbiota and microfluidic devices in the near future. As more complex culture systems are currently being developed, we stress the importance of an extensive description of all characteristics that researchers can think of when performing experiments with intestinal epithelial cells, in order to bring the field forward.
The final hurdle is the in vivo extrapolation of experimentally obtained data, for which different options can be considered. Often, in vitro results cannot be directly applied to predict human responses; therefore, it is important to build a consistent and reliable in vitro to in vivo extrapolation method. Such method uses, for example, mathematical modeling as shown by Fredlund et al. [112
] or an actual comparison between in vitro and in vivo data as shown by Lennernäs et al. [113
]. Yet, most knowledge is obtained from drug disposition studies, whereas a large gap still exists for nutrient absorption although proteins have been studied as drug delivery systems, such as casein [114
]. Eventually, the integration of information gained from complex in vitro models into computational algorithms that incorporate human-specific physiological parameters will facilitate production of sustainable, novel nutrients.