CP: Collagen Peptide;  Gly: Glycine; Pro: Proline; Hyp: Hydroxyproline; Pro-Hyp: Prolylhydroxyproline; Hyp-Gly: Hydroxyprolylglycine; DNFB: 2,4- Dinitrofluorobenzene; V: Vehicle; HIF-1: Hypoxia-Inducible Factor 1; DAMPs: Damage-Associated Molecular Patterns; PAMPs: Pathogen-Associated Molecular Patterns

Collagen is the most abundant extracellular matrix protein in animal tissues, and genes encoding for multiple types of collagen has been identified in human and animals. Collagen can be extracted from collagen-rich materials such as skin, bone, and fish scales as gelatin using hot water, and gelatin or its hydrolysate (collagen peptide: CP) is frequently used as a food supplement. CP ingestion has been reported to have beneficial effects on the skin such as reduced skin wrinkles and increased collagen synthesis [1,2]. Although collagen synthesis is increased with CP ingestion, deterioration of a fibrotic disease with CP ingestion was not observed in a study by Dr. Kenji Sato using choline deficiency-induced animal model of liver cirrhosis [3]. Beneficial effects were also observed for bone [4] and joints [5]. However, the effective dose of a supplement will change depending on the amount of collagen obtained from the daily diet; the adequate ingestion of a nutrient from the diet results in supplemental ingestion of this nutrient having little or no beneficial health effect. Therefore, the effective dose of a nutrient in clinical trials could change depending on the dietary habits, as well as the genetic background and life style, of the trial participants.

Type I collagen is the most abundant type of collagen in animalderived foods. The content of collagen in food varies considerably depending on its origin; it is very high in beef tendon, pork chitterlings, chicken sternum cartilage, and fish skin (Table 1) [6].

Table 1: Collagen content in animal-derived food materials [ a) without skin, b) with skin. Modified from [6]].

Using the values of collagen content reported by Noguchi et al. [6], we estimated that Japanese people ingest on average 1.9 g collagen from their daily diet [6].

Clinical trials to investigate the effects of CP supplemental ingestion by Japanese people have been published; placebo-controlled doubleblind trials revealed that daily ingestion of 5 g CP improved the properties of facial skin [7] and suppressed ultraviolet-induced skin erythema [8]. It was also reported that the T-cell-related immune status of Japanese suffering from chronic tiredness was improved by the intake of 10 g CP per day for 8 weeks [9]. Thus, supplemental ingestion of 5 g to 10 g CP by Japanese people, in addition to their daily intake of 1.9 g collagen, delivers beneficial health effects.

The amino acid sequence of collagen in the triple-helical region is represented as -(Gly-X-Y)_n- . The X and Y positions are frequently occupied by proline (Pro) and hydroxyproline (Hyp), respectively, and thus the di-peptide sequences Pro-Hyp and Hyp-Gly are frequently found in collagen. Hyp can be used as a marker for investigation of the digestion and absorption of collagen, because Hyp is not found in other proteins, with for a few exceptions such as elastin.

It is believed that proteins are hydrolyzed into amino acids prior to the absorption of the amino acids from the gastrointestinal tract, and that little peptide absorption occurs during protein digestion [10]. However, it was reported that there is a considerable amount of Hypcontaining peptide in the plasma following the ingestion of gelatin [11]; however, the peptide’s identity remained unknown until 2005. Iwai et al. [12] identified six collagen-derived Hyp-containing oligopeptides in human blood after oral ingestion of CP; the peptideform of Hyp increased and reached a maximal level of 20-60 nmol/mL of plasma after ingestion of 9.4 g-23 g of CP, and was primarily in the form of the dipeptide Pro-Hyp. Another collagen-derived dipeptide, Hyp-Gly, was identified in the blood after ingestion of CP, but its ratio to Pro-Hyp varied extensively between subjects [13]. Recently, 13 oligopeptides, including Pro-Hyp and Hyp-Gly, were accurately measured in the blood of individuals following ingestion of CP by using stable isotope-labeled collagen oligopeptides as internal standards [14]. Hyp-containing oligopeptides likely appear in blood because they are highly resistant to blood proteases and are stable in serum [12,13,15].

These studies indicate that collagen-derived oligopeptides appear in blood at fairly high concentrations and suggest that the beneficial effects of collagen ingestion are mediated by these oligopeptides. Shigemura et al. [16] reported that Pro-Hyp increased the number of fibroblasts that migrated from explanted skin in vitro and that Pro- Hyp enhanced the growth of fibroblasts on collagen gel in a dosedependent manner. Hyp-Gly was more effective than Pro-Hyp at stimulating murine fibroblast proliferation on collagen gel [13]. In vitro treatment of human skin fibroblasts with Pro-Hyp resulted in increased cell proliferation and hyaluronic acid synthesis, probably by elevating hyaluronan synthase 2 mRNA levels [17]. Treatment of murine chondrocyte ATDC5 with Pro-Hyp inhibited differentiation into mineralized chondrocytes in vitro [18], and hyaluronic acid production by the synovium cell line HIG-82 was enhanced by treatment with Pro-Hyp [19]. Furthermore, the size of oil droplets decreased significantly upon treatment with Pro-Hyp during the differentiation of murine pre-adipocyte 3T3-L1 into mature adipocytes in vitro [20]. These in vitro studies can be related with several in vivo animal studies of CP ingestion, including the promotion of cutaneous wound healing in a rat model of pressure ulcer [21], protection from articular cartilage damage induced by excess ingestion of phosphorus in mice [18], and lowering of blood lipids following the ingestion of oil in rats [22].

It therefore seems probable that the biological activities of ingested collagen are mediated, at least partly, by collagen-derived oligopeptides such as Pro-Hyp and/or Hyp-Gly. Transportation of an oligopeptide into a cell can be mediated by proton-coupled oligopeptide transporters PEPT1, PEPT2, PHT1, and PHT2 (Ci1 in mice). In this context, it is noteworthy that the genes encoding these oligopeptide transporters are expressed in Pro-Hyp-responsive pre-adipocytes and their expression profiles change when they differentiate into mature adipocytes [20]. It is not clear at present whether Pro-Hyp exhibits activity in the extracellular space by binding to an unidentified receptor, or by modulating signal transduction pathways after entering a cell. However, it is known that collagen-derived oligopeptides modulate the catalyzing activity of enzymes including angiotensin Iconverting enzyme [23,24] and dipeptidyl peptidase IV [25]. Hypoxiainducible factor 1 (HIF-1) is a ubiquitously expressed key regulator responsible for the induction of multiple genes that facilitate adaptation to low oxygen tension (hypoxia). The stability and subsequent transcriptional function of HIF-1α (a subunit of HIF-1) is controlled partly through hydroxylation of two specific proline residues by a family of 2-oxoglutarate-dependent dioxygenases resulting in the formation of Hyp [26]. Therefore, the working hypothesis of this study is that collagen-derived oligopeptides, after being transported into the cytoplasm, modulate the enzymatic activity of a family of 2-oxoglutarate-dependent dioxygenases. Studies of the activity of 2-oxoglutarate-dependent dioxygenases in Pro-Hypresponsive skin fibroblasts [16,17], chondrocytes [18], synovium cells [19] and adipocytes [20] may reveal novel aspects of collagen-derived oligopeptide functions.

As described above, cells that respond to Pro-Hyp include 1) skin fibroblasts that migrated from explanted skin and proliferated [13,16], 2) chondrocytic cells that differentiate into mineralized chondrocytes in vitro [18], and 3) pre-adipocytes that mature into adipocytes in vitro [20]. Explant culture of a skin sample is an in vitro model of cutaneous wound healing, and differentiation of stem cells plays a pivotal role in the healing process [27]. In the latter two cases, chondrocytic cells and pre-adipocytes respond to Pro-Hyp when they are undergoing differentiation processes. Although information is limited with regard to what cell types respond to Pro-Hyp, it is tempting to speculate that the target cells of Pro-Hyp are precursor cells that differentiate or mature into more differentiated states, since differentiation processes are involved in all three cases, though embryonic stem cells seem not to respond to Pro-Hyp and Hyp-Gly [28].

Peptide-form Hyp has been identified in the urine of growing children [29] and patients with bone tumors [30] or rheumatoid arthritis [31]. These studies suggest that collagen oligopeptides are generated endogenously by extensive degradation of collagen when the turnover of collagen is enhanced. Therefore, it seems likely that endogenously generated oligopeptides and food-derived oligopeptides co-localize in the same tissue and interact with each other. We studied this possibility by using Pro-Hyp and Pro double-labeled with the stable isotopes ¹³C₅ and ¹⁵N [32]. Endogenous Pro-Hyp was identified in inflammatory ear tissue caused by allergic contact dermatitis in mice fed a Hyp-free diet (Figure 1).

Figure 1: Concentrations of Pro-Hyp (A) and Hyp-Gly (B) in the ear of a mouse fed a Hyp-free diet at 9 and 18 days following the induction of contact dermatitis. Values in the same figure not sharing a common letter above the bar are significantly different from each other at p<0.05. Modified from [32]. DNFB: 2,4-dinitrofluorobenzene.

When labeled Pro-Hyp was administered orally to these mice, labeled Pro was detected after 4 h in the ear with chronic inflammation but not in the control ear of the same mouse (Figure 2A). This suggests that ingested Pro-Hyp was cleaved by an enzyme that is expressed or activated in chronically inflamed tissue, generating free Pro that can be incorporated into newly synthesized collagen. On the other hand, when labeled Pro was administered, labeled Pro-Hyp was identified at 4 h in the inflamed ear but not in the control ear, suggesting that orally ingested Pro was incorporated into newly synthesized collagen and that this collagen was degraded, generating Pro-Hyp in the chronically inflamed tissue (Figure 2B). Thus, turnover of tissue collagen and digestion of food-derived collagen may interact with each other in the same tissue through generation of the common dipeptide Pro-Hyp. Accumulation of food-derived Pro-Hyp in the inflammatory tissue may explain the promotion of skin wound healing by ingestion of CP [21].

Figure 2: Identification of ¹³C₅, ¹⁵N-Pro and ¹³C₅,¹⁵N-Pro-Hyp in the ear of a mouse at 4 h after oral administration of ¹³C₅,¹⁵N-Pro-Hyp (A) or ¹³C₅,¹⁵N-Pro (B). Modified from [32]. DNFB: 2,4-dinitrofluorobenzene.

It is noteworthy that the concentration of Hyp-Gly was very low in the ear subjected to contact dermatitis, despite the generation of Pro- Hyp at this site (Figure 1). In contrast, Hyp-Gly was found in the blood of individuals who ingested CP [13]. Hyp-Gly might therefore be produced differentially between two processes: endogenous collagen turnover and digestive cleavage of food-derived collagen.

Collagen-derived oligopeptides are generated in small amounts in normal tissue because of slow collagen turnover. However, invasion by a pathogen or a tissue wound evokes inflammatory responses, and the extracellular matrix, including collagen, is rapidly reorganized. Abundant Pro-Hyp appears in the inflamed tissue, as was shown in the case of chronic inflammation of the skin [32]. This implies that collagen-derived oligopeptides such as Pro-Hyp could provide damage- or pathogen-associated molecular patterns (DAMPs, PAMPs) [33] representing damage to the extracellular matrix. Therefore, the appearance of Pro-Hyp in a tissue could be an extracellular matrixassociated signal to initiate tissue repair by stimulating stem cells or immune cells. This protective response could be a common system throughout the body because collagen is a ubiquitous extracellular matrix component. Further studies on Pro-Hyp may reveal novel aspects of the interactions between tissue homeostasis and this animalderived nutrient.