1. Introduction
The world’s aging population is rapidly growing, with the number of individuals aged over 60 years projected to reach 2.1 billion by 2050, accounting for about 22% of the total population [
1]. Protein has long been a focus of research in linking nutrition with health among older individuals [
2]. However, approximately 5%-10% of community-dwelling older adults suffer from protein-energy malnutrition [
3], which can lead to sarcopenia [
4], a weakened immune system [
5], and increased rates of morbidity and mortality [
6]. Studies [
7], [
8] have indicated that notable declines occur in circulating amino acid levels during aging, especially for branched-chain amino acids (BCAAs). The deficiency of specific amino acids is considered a major risk factor for many diseases associated with protein deficiency in older adults [
9]. Hence, the resolution of amino acid imbalance in older people demands immediate attention.
Current dietary guidelines emphasize increasing protein intake to combat deficiency but often overlook the importance of amino acid transport in older adults [
10]. Protein intake is always supplied with animal protein, which is generally recommended for its high-quality and abundant BCAAs. However, due to functional declines in the gastrointestinal (GI) tract, studies [
11], [
12] have shown that amino acid utilization from meat or casein decreases in older adults. Moreover, interest in studying plant protein supplementation for older adults is increasing, but it appears that plant-based protein sources may exhibit lower digestibility than animal-based proteins for adults in most current study [
13]. Therefore, older individuals may face a dilemma when selecting the most suitable protein source. As an alternative, amino acids can be directly supplemented. However, previous studies have found that excessive amino acid supplementation can lead to over-appetite and shortened lifespan, with high levels of BCAA intake potentially causing insulin resistance and obesity [
2]. Therefore, increased intake of protein or amino acids through diet alone may not effectively solve amino acid imbalance.
In the GI tract, proteins are initially broken down into peptides and amino acids, which are then absorbed into the blood through amino acid transporters (AATs). Changes in GI enzymes such as pepsin, trypsin, and chymotrypsin during the aging process indicate the release of peptides and amino acids may differ from that in younger individuals [
14], [
15]. However, evidence regarding the changes in GI digestion products during aging is lacking. Recent studies [
16], [
17] have proposed a novel approach for monitoring the digestion patterns of dietary proteins through peptidomics, and this technology has been applied in research related to aging
in vitro. However, the detection of protein digestion products in aged animals has not been thoroughly investigated. The release of amino acids, which are then integrated into the systemic circulation, may be altered during aging. Multiple studies [
18], [
19] have indicated variations in amino acid absorption kinetics in older adults, with low circulation levels of BCAAs. AATs regulate amino acid absorption in the intestines and utilization in different tissues [
20]. However, recent research has only focused on amino acid utilization in muscle, with little attention given to the role of intestinal AATs on amino acid absorption. Nevertheless, there is indirect evidence that aging may lead to changes in gut AATs. Dickinson et al. [
21] found that the post-exercise ingestion of essential amino acids enhanced muscle AAT expression in young individuals, with less significant effects in older adults, suggesting that aging may impact the expression of AATs in the intestine and cause less efficient amino acid transportation. Hence, there is an urgent need to identify key alterations in the process of peptide and amino acid release in older adults in order to improve amino acid deficiencies and enhance protein utilization.
To address these issues, animal and plant protein utilization during aging were compared. We found that animal protein—particularly casein—exhibited the most significant decline in digestibility with age. Subsequently, our investigation revealed that the absorption of amino acids plays a crucial role in the entire process of protein utilization, as supported by the peptide profile analysis of chyme and postprandial amino acid levels in plasma. Furthermore, a proteomic analysis of duodenal epithelial cells uncovered a significant decrease in the expression of large neutral amino acid transporter 2 (LAT2). This downregulation with age, which is observed in the intestinal epithelium of both mice and humans, contributes to the reduced efficiency of large neutral amino acid absorption, especially for BCAAs.
2. Materials and methods
2.1. Meta-analysis
A literature search was conducted in the PubMed database with the subject terms (“animal proteins, dietary” [MeSH] OR “plant proteins, dietary” [MeSH]) AND (“absorption” [MeSH] OR “digestion” [MeSH] OR “metabolism” [MeSH]). Two investigators independently performed the search, limiting the results to English-language publications from January 2019 to January 2024. Of a total of 1072 retrieved documents, 11 were deemed relevant for inclusion. The statistical analysis focused on the difference between the maximum change in blood amino acids pre- and post-meal. This data was analyzed using the standardized mean difference (SMD) method, with 95% confidence intervals (CI) calculated via effect analysis.
2.2. Diet preparation
Adhering to the guidelines of the American Institute of Nutrition (AIN-93G) Growth Purified Diet with consistent energy levels [
22], a total protein content of 20% was maintained for each formulation. The diet included only one of the following protein sources: casein, beef protein, soy protein isolate, or gluten. Table S1 in Appendix A provides detailed compositions of the experimental diets (SYSE, China). The total nitrogen in the diet was determined using the Kjeldahl method with an automated Kjeldahl analyzer (Sonnen, China), using a nitrogen-to-protein conversion factor of 6.25 [
23].
2.3. Animals and experimental design
Male C57BL/6J mice (Vital River, China) were housed in a specific pathogen-free environment with a 12 h:12 h light/dark cycle, including young mice (YM) aged 2-4 months, middle-aged mice (MM) aged 12-14 months, and old mice (OM) aged 24-26 months. The experimental procedures were approved by the Ethics Committee of China Agricultural University (AW52104202-4-1).
2.3.1. Animal study 1
Mice from the three distinct age cohorts (YM, MM, and OM) were randomly allocated into four groups (number of samples (n) = 6 each): the casein group, beef protein group, soy protein group, and gluten group. Each group received a diet containing 20% protein, with the assigned protein source as the sole protein supply. The mice had ad libitum access to food and water. Following an adaptation period of a week, they were transferred from their normal cage to a metabolic cage for fecal collection. By examining the excretion of proteins in feces, the protein with the poorest utilization in aging mice was selected for subsequent research.
2.3.2. Animal study 2
As casein’s digestibility declined most significantly with age, subsequent research focused on the intestinal hydrolysis of this protein. Before euthanasia, animals were fasted overnight (16 h) and gavaged with a casein-purified diet. Following a 2 h post-gavage period (protein could complete the whole intestinal digestion during this time; data not shown), mice were euthanatized with CO
2 for the collection of blood samples, intestine, and chyme. The chyme was collected by gently scraping it from the top to bottom of the stomach and small intestine using forceps and depositing it into a tube. Enterocytes were isolated as previously described [
24]. The duodenum was strung onto a stick with the mucosal surface outward, washed in cold phosphate-buffered saline, and incubated in ethylenediaminetetraacetic acid (EDTA) buffer for 3 min; the stick was then rubbed to isolate and collect cells, which were stored at −80 °C.
2.4. Protein digestibility determinations
The fecal sample collection and apparent total tract digestibility (ATTD) determination were performed according to the method described previously [
25]. In brief, mice were individually housed in stainless steel metabolic cages with a screen-bottom design for 10 d and permitted unrestricted access to food while enabling the collection of excreta. The initial 4 d served as an adaptation period. From day 5 to day 8, daily food intake within 24 h was recorded, and the total weight of feces was measured to determine protein ATTD. From day 9 to day 10, fresh fecal sampling was collected by gently massaging the lower abdomen of the mice and was then stored at −80 °C immediately.
2.5. Determination of amino acids
To prepare the feces samples, it was necessary to first transform the granular, fresh fecal material into powder by adding water, homogenizing, and freeze-drying. Next, a 10 mg powdered fecal sample was combined with 500 µL of water, vortexed, and centrifuged. The resulting supernatant was then collected and freeze-dried. Similarly, for a 20 μL plasma sample, 80 μL of cold methanol was added to precipitate proteins, followed by centrifugation and freeze-drying under identical conditions.
Amino acid analysis was performed using a UPLC-TripleTOF 7600 (AB SCIEX, USA). Chromatographic separation occurred at 45 °C on a BEN Admine column (2.1 mm × 100 mm, 1.7 μL; Waters, USA). Mobile phase A consisted of water with 20 mmol·L−1 ammonium acetate and 0.5% formic acid, while mobile phase B consisted of acetonitrile/water (85:15, v/v) with the same additives. The mobile phase gradient started at 15% B and lasted for 10 min, then shifted to 100% B for 4 min before returning to 15% B within 0.1 min and being equilibrated for 2.9 min. The flow rate was 0.3 mL·min−1 and the injection volume was 5 μL. Data acquisition proceeded in the multiple reaction monitoring (MRM) mode; the ion scan parameters for 20 amino acids and machine parameters are summarized in Table S2 in Appendix A. All data acquisition and analysis were carried out on a SCIEX OS (AB SCIEX, USA).
2.6. Peptidomics profiling
The preparation method for the peptidomics samples of chyme collected from the stomach and ileum was performed according to a previously described method [
26]. NanoLC separation was conducted using a nanoAcquity nano high-performance liquid chromatography (HPLC) system (Waters, USA). The trap column was an Acclaim PepMap 100 (75 μm × 2 mm, C18, 3 μm; Thermo Fisher Scientific, USA), while the analytical column was a homemade setup composed of a 100 μm inner diameter (ID) fused silica capillary (Polymicro, USA) filled with 20 cm of C18 stationary phase (Aqua 3 μm C18 125A; Phenomenex, USA). Mobile phase A consisted of 0.1% formic acid in water, while mobile phase B comprised 0.1% formic acid in acetonitrile. Gradient elution was performed, starting from 1% mobile phase B and increasing linearly to 35% mobile phase B over 65 min. Nanospray electrospray ionization-mass spectrometry (ESI-MS) was performed using a Q-Exactive high-resolution mass spectrometer (Thermo Fisher Scientific) with 70000 mass spectrometry (MS) scan resolution and 17500 tandem mass spectrometry (MS/MS) scan resolution, employing top-10 MS/MS selection. Peptide coverage visualization was analyzed based on the peptide intensity using Peptigram [
27]. Peptide cleavage prediction was analyzed based on the EnzymePredictor tool [
28].
2.7. Determination of pH and digestive enzyme activity
The stomach and jejunum were incised with a small opening, followed by pH measurement of the chyme using a pen acidity meter (Yanlin Laboratories, China) [
29]. For enzyme activity testing, samples of the chyme were processed according to the instructions provided with the pepsin kits (Solarbio, China) and trypsin kits (Abcam, UK).
2.8. In vitro digestion
The GI digestion simulation followed the INFOGEST protocol [
30]. Four
in vitro models were devised to explore changes in older individuals’ protein digestive capacity under varying GI conditions, including the standard for healthy adults (Adult, AD), and three models mimicking the cumulative alterations commonly observed with aging: gastric stage alteration (Older 1, O1), intestinal stage alteration (Older 2, O2), and both gastric and intestinal stage alterations (Older 3, O3). Specific conditions are detailed in Table S3 in Appendix A. The degree of hydrolysis (DH) of casein was determined according to the
o-phthaldialdehyde method, as previously described [
31]. The remaining samples were freeze-dried, followed by microstructural analysis using a scanning electron microscope (SEM; Zeiss, Germany), with an acceleration voltage of 5.00 kV applied and micrographs captured at ×1000 magnification.
2.9. Proteomic profiles
The procedures for proteomic analysis followed previously described methods [
32]. In brief, proteins from duodenal epithelium samples in the YM, MM, and OM groups (
n = 3, each) were extracted and digested using the procedure of the filter-aided sample preparation protocol. Subsequently, data-dependent acquisition (DDA) mass analysis was conducted using trapped ion mobility spectrometry time-of-flight MS (Bruker, Germany). The peptides were then analyzed via liquid chromatography-mass spectrometry utilizing a data-independent acquisition mode (Applied Protein Technology, China). The fast all sequences in A (FASTA) sequence database was queried using Spectronaut 14.4.200727.47784 software (Biognosys, USA) to create the DDA library. Data-independent acquisition data analysis was performed using the same software and spectral library. The duodenal epithelium proteomic data were used in the subsequent bioinformatic analyses. Cluster 3.0 and Java Treeview software were used to perform hierarchical clustering analysis. The identified proteins were compared using the criteria of
p value (
p) < 0.05 and |log
2 fold change| (|log2FC|) > 1 for significant changes; they were then mapped to pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The difference in protein contents between groups is presented as a heatmap. An enrichment analysis was conducted using Fisher’s exact test, with significance determined by a
p < 0.05.
2.10. Volunteers and clinical specimens
All volunteers were males from Beijing Chao-Yang Hospital, Capital Medical University (Beijing, China). Based on their demographic and clinical features, the volunteers were assigned to the young people (YP; (34 ± 1) years), middle-aged people (MP; (47 ± 1) years), and older people (OP; (63 ± 2) years) groups. After providing informed consent, the volunteers underwent a duodenal biopsy from the distal duodenum during a gastroscope. Three biopsy samples were collected from the duodenal mucosa using forceps attached to the endoscope’s tip; two were promptly frozen and stored at −80 °C, while the third was fixed in 10% formalin and embedded in paraffin. The experimental protocol and clinical specimen collection were approved by the Clinical Research Management Committee of Beijing Chao-Yang Hospital, Capital Medical University (LGH-2024-FEI-15) and the Human Research Ethics Committee of China Agricultural University (CAUHR-20240103).
2.11. Western blotting analysis
A Western blot analysis was performed according to a standard method, as described previously [
33]. Protein lysates from duodenal tissues were extracted, quantified, and separated on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The proteins were transferred onto polyvinylidene-difluoride membranes, followed by blocking for 1 h. Antibodies against β-actin (1:1000, #4970; Cell Signaling Technology, USA) or LAT2 (1:500, #MG120503; Abmart, China) were added, followed by incubation overnight at 4 °C. After washing three times with tris-buffered saline with Tween 20 (TBST), membranes were incubated with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:5000, #A0208; Biyuntian, China) or anti-mouse IgG (1:5000, #A0216; Biyuntian, China), and enhanced chemiluminescence was performed (Cytiva, USA).
2.12. Immunofluorescence
Immunofluorescence was performed according to standard protocols, as described previously [
34]. Fresh human tissues were fixed, dehydrated, embedded, sectioned, and conventionally dewaxed. Heat-mediated antigen retrieval was performed with tris-EDTA buffer (pH 9.0). Endogenous peroxidase was excluded, and the sections were treated with 1% Triton-X 100 and blocked for 1 h. Subsequently, the sections were incubated at 4 °C with anti-LAT2 mouse monoclonal antibody (1:50, #M04381; BOSTER, USA). After incubation with goat anti-mouse IgG H&L (1:500, #ab150113; Abcam, UK) and 4',6-diamidino-2-phenylindole (DAPI; Beyotime, China), the sections were observed under a confocal microscope (Zeiss, Germany).
2.13. Statistical analysis
A statistical analysis was performed using SPSS 26.0 (International Business Machines (IBM), USA), with a significance level set at p < 0.05. For clarity, significance levels were denoted by *, **, and *** for p < 0.05, p < 0.01, and p < 0.001, respectively. When the homogeneity test for variance was completed, comparisons among multiple groups were performed using a one-way analysis of variance (ANOVA) followed by a least significant difference (LSD) post hoc test. Figures were generated using GraphPad Prism 8.4.3 (GraphPad Software, USA), and data are presented as means ± standard error of the mean (sem).
3. Results
3.1. Both age and source of protein affected postprandial amino acids levels
To investigate the potential impact of age and protein sources on circulating amino acid availability, a meta-analysis of articles published within the past five years examining blood amino acid levels following protein intake was conducted. This analysis revealed that consuming various dietary proteins led to absorption benefits, resulting in increased postprandial amino acid levels in healthy young adults (
Fig. 1(a)). Notably, older adults also experienced a significant absorption benefit, albeit to a slightly lesser extent compared with young adults (
Fig. 1(b)). In the process of human digestion and absorption, animal protein demonstrated higher amino acid increments compared with other sources, resulting in a higher overall supply of amino acids for the body from animal protein (
Figs. 1(c) and
(d)).
3.2. Total-tract protein bioavailability of mice decreased during aging
As the meta-analysis highlighted differences in bioavailability between animal and plant protein consumption, experiments were conducted using an aging mouse model to explore the utilization of different proteins during aging. The results showed that the bioavailability of casein, beef protein, soy protein, and gluten decreased with age in mice. From YM to OM, the decrease in ATTD was highest for casein (5.4%), followed by beef protein (3.1%), soy protein (2.7%), and gluten (1.7%). Animal protein ATTD began to decline at MM, while plant protein either changed minimally or remained stable (
Fig. 2(b); Table S4 in Appendix A). As ATTD measures protein levels, further analysis of fecal peptide levels was warranted. Peptide compositions were found to be consistent across protein types regardless of age (
Fig. 2(c)), but peptide intensities varied, notably for peptides between 750 to 1250 kDa, whose intensities increased in ion intensity over time (
Fig. 2(d)). Animal protein had higher peptide intensities and molecular weights in feces compared with plant protein during the aging process. An analysis of amino acid profiles showed increased variability (
Fig. 2(e)) and a significant increase in fecal amino acid content with age across all protein types (
Fig. 2(f)), indicating poor absorption of amino acids, most notably for casein. According to these results, ATTD decreased while fecal peptides and free amino acids increased, which implied a reduction in the body’s efficiency in utilizing proteins as age advanced.
3.3. Minimal changes in the release of peptides and amino acids were identified during aging
Due to the significant changes in casein bioavailability during aging, the causes of decreased protein digestion and absorption were investigated. The stomach and ileum mark the beginning and end of the digestive process, respectively. Utilizing the Peptigram tool, qualitative insights into proteins were gained by identifying and mapping peptides. Peptides derived from β-, αs
1-, αs
2-, and κ-casein in gastric (CG) chyme are illustrated in
Fig. 3(a) and Figs. S1(a)-(c) in Appendix A, while peptides from ileal (CI) chyme appear in
Fig. 3(b) and Figs. S1(d)-(f). The peptide patterns across different age groups showed little variation, indicating stable enzyme cleavage sites, as analyzed with the EnzymePredictor tool (Table S5 in Appendix A). However, the ion intensity varied among the age groups. In the stomach, regions 208-224 of β-casein (
Fig. 3(a)), 180-187 of αs
1-casein (Fig. S1(a)), and 38-50 of κ-casein (Fig. S1(c)) showed high peptide intensity in the MM, as did regions 103-109 of αs
2-casein (Fig. S1(b)) in the YM, suggesting better digestion in younger mice. Conversely, in the ileum, regions 95-103 of β-casein (
Fig. 3(b)), 40-46 of αs
1-casein (Fig. S1(d)), 129-138 of αs
2-casein (Fig. S1(e)), and 100-107 of κ-casein (Fig. S1(f)) were abundant in the aged group, indicating incomplete digestion and conversion into amino acids after intestinal digestion in older mice.
Furthermore, the activity of the key protein-digesting enzymes pepsin and trypsin in chyme were assessed. Aged mice exhibited a 60% decrease in pepsin activity (
Fig. 3(c)) compared with their younger counterparts, whereas trypsin activity remained consistent (
Fig. 3(e)). An investigation of GI pH levels showed no significant alterations, indicating that the enzyme activity changes were not pH-dependent (
Figs. 3(d) and
(f)). Further analysis of the impact of reduced pepsin activity on casein digestion involved measuring the DH (
Fig. 3(h)). Despite a general increase in the DH over time, a 60% decrease in pepsin activity notably reduced the DH in the stomach for the O1 and O3 groups compared with the AD group. However, the addition of trypsin compensated for this reduction, restoring the O1 group’s intestinal DH levels to match those of the AD group. Conversely, the O2 group exhibited lower intestinal DH values than the AD group, highlighting trypsin’s essential role in digestion. The SEM images further visually illustrated the structure of casein after
in vitro digestion. The results showed that casein initially formed densely packed polyhedral particles, with consistent sizes across all groups (
Fig. 3(i)). Pepsin hydrolysis created visible fissures in the casein particles of individuals in the AD and O2 groups, while the particles of the other groups retained the initial particle structure (
Fig. 3(j)). Trypsin then transformed these particles into a lamellar network with irregular shapes (
Fig. 3(k)), where the particles of the AD and O1 groups exhibited larger pores, suggesting deeper digestion than in groups with reduced trypsin activity. Despite the differing gastric digestion outcomes between the AD and O1 groups, trypsin addition led to similar structural changes. However, the AD and O2 groups diverged significantly in the final digestion phase, underscoring trypsin’s key role in casein digestion for peptide and amino acid release. Overall, although the degradation of digestion capacity primarily occurred in the stomach during aging, it played a minor role in overall digestion, resulting in a largely unaffected protein-digestion capacity during aging.
3.4. Declined postprandial circulating levels of amino acids were observed during aging
Due to the minimal impact on peptide release from reduced pepsin and unchanged trypsin, the intestinal absorption of amino acids during aging warranted attention. To gain a comprehensive understanding, it was crucial to examine how amino acids changed after absorption. Fecal excretion of amino acids increased following the feeding of mice with different proteins, suggesting insufficient absorption and additional elimination in the intestine. This observation was supported by targeted metabolomics, which revealed altered plasma amino acid profiles after meals. The PCA score plots shown in
Figs. 4(a) and
(b) displayed an initial overlap in the plasma amino acid profiles of the three age groups before meals, with notable separation occurring afterward. Notably, the postprandial blood amino acid levels in the OM were significantly lower than those of younger individuals (
Fig. 4(c)). There were significant age-related declines in neutral amino acids (
Fig. 4(d)) and BCAAs in the plasma (
Fig. 4(e)), which corresponded with increased levels in feces (
Figs. 4(f) and
(g)). Although similar trends were observed in acidic and alkaline amino acids (
Figs. 4(h)-(k)), their changes were less significant. Overall, these results suggested that absorption was impaired during aging, resulting in a decrease in the plasma levels of BCAAs.
3.5. Amino acid absorption impaired due to age-related reduction of LAT2
To reveal the underlying mechanism of intestinal absorption decline, a proteomics analysis of duodenal epithelium was performed. The results, as shown in the Venn diagram, identified 5132 shared proteins from all three groups, and 81, 213, and 120 peptides were unique to YM, MM, and OM, respectively (
Fig. 5(a)). A PCA analysis showed an obvious clustering of all groups, with the YM closer to the MM than to the OM (
Fig. 5(b)). To screen for differentially expressed proteins, up- and down-regulated proteins among the groups were summarized (
Figs. 5(c)-(f)). The results revealed 770 proteins upregulated and 482 downregulated in MM compared with YM. For OM, there were 210 proteins upregulated and 372 downregulated compared with YM. When comparing OM with MM, 246 proteins were upregulated and 478 were downregulated. These findings suggest significant changes in the expression of duodenal proteins with age.
Furthermore, a functional analysis revealed significant down-regulation in the KEGG pathways of protein digestion and absorption (
Fig. 5(g)). Eight key proteins involved in this pathway were notably reduced in the OM, as shown in the heatmap (
Fig. 5(h)), including COLA1, NHRF1, LAT2, ACE2, MEP1B, NEP, DPP4, and BAT1. Then, a Western blot analysis of both mouse and human duodenal epithelium confirmed a significant reduction in LAT2 expression in the old group compared with the middle-aged group and young group in mice (21.4% and 61.9% lower, respectively) and humans (67.8% lower in elderly individuals), as illustrated in
Figs. 5(i) and
(j). During the aging process, the fluorescence intensity of LAT2 in the human duodenum was also decreased (
Fig. 5(k)). Given LAT2’s essential role in transporting large neutral amino acids, its impact on the levels of Trp, Phe, Leu, Gln, Ala, Val, Thr, Ile, Met, Gly, His, Ser, Asn, and Tyr was investigated, and decreased levels were noted (
Fig. 5(l)). Taken together, these findings suggested that protein digestion and absorption was impaired in the aging mice, highlighting LAT2’s pivotal role in amino acid absorption.
4. Discussion
Proteins break down into amino acids within the digestive tract, playing a vital role in physiological functions for older adults [
35]. According to previous research [
36], evaluating the role of protein intake in older people is better done by assessing the bioavailability of amino acids rather than focusing solely on the quantity of protein consumed. This study examined how aging affects the digestion and absorption of animal versus plant protein, pinpointing casein as the protein showing the most significant age-related decline in digestibility. Further investigation into casein revealed that the impact of absorption on the decline of circulating amino acid levels was greater than that of digestion. Notably, duodenal epithelial cells showed a substantial reduction in LAT2 in mice and humans, which was linked to decreased protein absorption efficiency with age. These findings provide new insights into preventing amino acid deficiency and related conditions in older adults.
The availability of circulating amino acids is affected by age and protein source. Older adults exhibit a slower increase in circulating amino acid levels after the consumption of high-protein meals than younger individuals [
19]. Our meta-analysis also confirmed that the postprandial plasma amino acid magnitude decreased in older adults, suggesting that identical protein intake does not guarantee the same effectiveness across different ages. Other research has indicated that older adults might need more protein for optimal health [
37]. In China, where animal and plant protein consumption is almost equal, identifying the more beneficial protein source is vital [
38]. Our findings emphasize the advantages of animal protein over plant protein for amino acid availability. A study [
39] has reported that it is only by consuming larger quantities of plant-based protein that comparable benefits to those of animal-sourced protein can be achieved, which supported our findings. However, another study [
38] has suggested that the source of dietary protein may not be as critical for ensuring the optimal utilization of protein among older adults. To address this contradiction, further studies are needed to clarify the impact of different protein sources on the health of older adults.
During the aging process, proteins from different sources exhibit varying changes in bioavailability. Our study revealed a pronounced decline in the utilization of animal protein in mice with aging, in contrast to the slower changes observed with plant protein. In line with this finding, further research has suggested that this difference may explain why—despite the inherent advantages of animal protein in human digestion and absorption—plant protein has been found to provide comparable benefits to older adults [
40]. Study [
18] using casein as the protein source have shown a faster decline in protein digestion rate and phenylalanine absorption in older individuals. Our findings also highlight casein’s notably low utilization rate among different protein sources. Ultimately, our research points to the significant reduction in animal protein utilization with age as a key contributor to the diminished capacity for protein digestion and absorption observed during aging. However, since animal protein is an important source of essential amino acids, supplementing older individuals with a combination of animal and plant protein may have better effects during the aging process.
Changes in the digestive capacity in the stomach during aging do not significantly reduce protein utilization in older adults. In our study, peptidomics was utilized to analyze the intestinal chyme of aged animals, revealing lower peptide intensity in the gastric chyme. This finding suggests impaired gastric digestion, as evidenced by the decreased pepsin levels. Supporting this, there is evidence that the differences in digestive products at the gastric endpoint are more pronounced than those at the intestinal endpoint under the condition of aging [
41]. Nevertheless, studies [
42], [
43] have shown that gastric proteases play a minimal role in protein digestion compared with pancreatic proteases. When gastric digestive enzyme activity decreases, pancreatic proteases can compensate for the inadequate gastric digestive capacity by hydrolyzing proteins into peptides and free amino acids [
44]. Likewise, a significant decrease in gastric protease activity during the aging process was observed, while pancreatic protease activity in the intestine remained stable, resulting in the protein digestion capacity of older adults being largely unaffected. Similarly, study [
45] showed that there was no significant effect of aging on protein utilization when simulating digestive conditions in older adults. Therefore, although the impaired digestive capacity primarily occurs in the stomach during the aging process, digestive alterations do not affect protein utilization in older adults.
Impaired absorption capacity is the primary cause of decreased circulating amino acids during the aging process. Multiple studies [
18], [
19] have showed that the absorption kinetics of amino acids undergo changes in older individuals. Our study found a decline in the circulating levels of neutral amino acids in the plasma, especially BCAAs. This is supported by previous findings [
8], [
46], [
47], which indicate a more significant impairment in the absorption rate of BCAAs compared with other amino acids. Unlike many other amino acids, BCAAs bypass the liver’s initial metabolic processes and quickly enter circulation, becoming readily available for bodily functions and stimulating muscle protein synthesis in older individuals [
48], [
49]. The reduced absorptive capacity of postprandial BCAAs with aging may be a key factor limiting muscle protein metabolic regulation and contributing to the decline in skeletal muscle function that occurs with age. Overall, impaired amino acid absorption during the aging process is the main cause of declining amino acid utilization. For older adults, direct dietary supplementation of BCAAs and its effects on muscle synthesis require further investigation.
Downregulation of AATs contributes to the reduced absorption of amino acids during the aging process. A crucial role is played by AATs in regulating the levels of amino acids in the body, controlling their absorption and utilization [
20]. Research indicates a direct correlation between changes in AATs and age-related characteristics [
50]. It has been observed that the post-exercise intake of essential amino acids enhances the expression of AATs in young individuals, while its impact is less pronounced in older individuals [
21]. Consequently, aging may potentially affect the function of specific AATs, thereby influencing the biological utilization of amino acids. Our preliminary study has shown that the expression of LAT2 protein, which is responsible for transporting large neutral amino acids, decreases in the intestine as individuals age. In addition, LAT2 serves as the primary transporter protein for BCAAs within the body, contributing to protein synthesis and muscle growth [
51]. Therefore, addressing the decrease in LAT2 levels associated with aging presents a novel approach to enhance amino acid utilization among older adults. Potential inhibitors or modulators of this transporter could provide insights into therapeutic avenues.
In conclusion, this study confirmed, through a meta-analysis and animal experiments, that protein digestion and absorption decreased with aging, particularly affecting casein due to its lower digestibility in mice. Subsequently, we found that the decrease in gastric digestion has minimal consequences for overall protein utilization in aging. Therefore, we further focused on absorption and noted a decrease in postprandial amino acids, particularly observing a decline in BCAAs with age. Moreover, a proteomic analysis of duodenal epithelial cells demonstrated a significant decrease in the expression of LAT2. This finding was validated in both mouse and human intestines, suggesting that LAT2 is a key factor contributing to the decline in age-related protein-utilization efficiency.
Acknowledgments
This research was funded by the National Key Research and Development Program of China (2023YFF1104502) and the Young Elite Scientists Sponsorship Program by China Association for Science and Technology (2022QNRC001). We appreciate the assistance provided by the biological mass spectrometry laboratory from College of Biological Sciences at China Agricultural University on our mass spectrometry experiment.
Compliance with ethics guidelines
Rui Song, Lili Qiu, Liang Zhao, Xiyu Qin, Xiaoxu Zhang, Xiaoxue Liu, Jun Zhou, Mengxiao Hu, Liwei Zhang, Jiaqi Su, Xinjuan Liu, Guang Li, and Xiaoyu Wang declare that they have no conflict of interest or financial conflicts to disclose.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.eng.2024.08.009.
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