The effect of oral essential amino acids on incretin hormone production in youth and ageing

Abstract Background The effect of substantive doses of essential amino acids (EAA) on incretin and insulin production, and the impact of age upon this effect, is ill‐defined. Methods A 15‐g oral EAA drink was administered to young (N = 8; 26 ± 4.4 years) and older (N = 8; 69 ± 3.8 years) healthy volunteers. Another group of younger volunteers (N = 9; 21 ± 1.9 years) was given IV infusions to achieve equivalent plasma amino acids (AA) profiles. Plasma AA, insulin, glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic peptide (GIP) were quantified over 2 hours. Results In younger recruits, EAA‐induced rapid insulinaemia and aminoacidaemia with total amino acids(AA), EAA and branched chain amino acids (BCAA) matched between oral and IV groups. Insulin peaked at 39 ± 29 pmol L−1 at 30 minutes following oral feeding compared to 22 ± 9 pmol L−1 at 60 minutes following IV feeding (P: NS). EAA peaked at 3395 μmol L−1 at 45 minutes during IV infusion compared to 2892 μmol L−1 following oral intake (Feeding effect: P < 0.0001. Oral vs IV feeding: P: NS). There was an 11% greater increase in insulin levels in the 120 minutes duration of the study in response to oral EAA as opposed to IV EAA. GIP increased following oral EAA (452 pmol L−1 vs 232 pmol L−1, P < 0.05). Age did not impact insulin or incretins production. Conclusion Postprandial rises in EAA levels lead to rapid insulinaemia which is higher with oral compared with IV EAA, that is attributed more to GIP and unaffected by age. This finding supports EAA, on their own or as part of high‐protein meal, as nutritive therapeutics in impaired glycaemia and ageing.


| INTRODUC TI ON
Oral glucose has been shown to stimulate greater insulin release compared to a comparable glucose challenge given intravenously 1 (measured as the difference between insulin attributed to oral vs IV glucose load). This difference is attributed to the secretion of incretin hormones such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP)-known as the "incretin effect," produced from the L and K cells respectively, and accounts for up to 65% of the amount of insulin secreted following oral glucose ingestion. 1,2 Orally ingested lipids have also been shown to induce the incretin effect due to postprandial elevations in both GLP-1 and GIP. 3 The extent to which these incretin hormones are produced in response to oral intake of dietary proteins and amino acids (AA) remains poorly defined. Nonetheless, it has been shown that consumption of a mixture of oral amino acids (AA) stimulates GLP-1 and GIP secretion. 4 Moreover, in the only study assessing the true incretin effects of AA, Lindgren et al 5 reported an incretin effect of ~25% attributed mainly to rises in GIP (rather than GLP-1) following a small dose (6.5 g) of mixed (ie, essential AA [EAA] and non-EAA) vs comparable I.V AA administration. Since the estimated daily protein requirement for a 70 kg adult is 0.8 g kg −1 d −1 , 6 studies to investigate the impact of more typical AA meal intake of >10 g of AA (~20 g protein) on incretin hormones production is important. Moreover, determining the effects of EAA vs NEAA on insulin production is important in relation to determining the key AA drivers of the incretin hormone productions.
Ageing of the gut and gut-brain axis is a burgeoning area of research. Ageing is associated with reduced energy intake, protein malnutrition (in some instances) and impaired anabolic responsiveness to protein foods [7][8][9][10][11][12] ; likely contributing to deleterious shifts in body composition-engendering sarcopenia, age-related insulin resistance and metabolic ill-health. 13 These factors are facilitated by age-related changes in gastrointestinal (GI) physiology in response to mixed meal [14][15][16] -which could play an important role in nutrient deficiency and predisposition to age-related sarcopenia. Yet the effects of EAA alone on incretin and insulin hormone production, and in relation to age, remains poorly defined. Crucially, while oral lipid and glucose intake did not lead to differences in either L and K cells responses in younger and older people, 17 older individuals exhibited higher levels of GIP (not GLP-1) compared to younger subjects after oral protein consumption. 18 In the postabsorptive state, mobilization of NEAA (mainly glutamate and alanine) from skeletal muscle represent the main mechanism via which splanchnic tissues are provided with fuel at the expense of negative muscle protein balance. 19 During the fed state, splanchnic tissues rely on enteral extraction of glutamine and alanine with most EAA appearing in the systemic circulation. 20 Therefore, EAA, due to their lesser influence on splanchnic tissue gluconeogenesis, could be regarded as an important nutraceutical strategy for the regulation of muscle and glucose metabolism. In addition, this later fact makes EAA a suitable testing mixture to allow more accurate estimation of protein-derived incretin secretion. Also, since EAA are the primary drivers of muscle protein synthesis [21][22][23] while incretin hormones are critical to glucose metabolism, studies investigating the effects of oral vs intravenous EAA in relation to incretin hormones are relevant to promoting healthy ageing. The aim of this study was thus to investigate: (a) the extent oral EAA could induce increases in incretin and insulin hormone production, and (b) if ageing impacts upon these postulated EAA-induced effects.  Table 1 for the subjects' characteristics.

| Study design
All groups reported to the study sites following overnight fasting  warmed to room temperature in the morning of the study. The stability of the EAA components of the solution following overnight stay was tested beforehand. The composition of the EAA beverage was lhistidine: 1.21 g; l-isoleucine: 1.73 g; l-leucine: 3.59 g; l-lysine: 3.07 g; l-methionine: 0.95 g; l-phenylalanine: 0.91 g; l-threonine: 1.13 g; ltryptophan: 0.48 g and l-valine 1.86 g. 24  were removed and volunteers were offered a snack. They remained on site for monitoring as described above. See Figure 1 for the schematic representation of the study protocol.  The incretin effect was calculated as the difference between insulin responses after oral and intravenous EAA stimulation, relative to the response seen after oral EAA ingestion.

| Power of calculation and statistical analysis
The sample size was prospectively determined with a power of calculation and taking a population (inter-and intraindividual) variance of 15% (based on previous laboratory data on insulin and AA F I G U R E 2 Plasma total AA (A), EAA (B) and BCAA (C) concentrations after oral vs IV EAA, demonstrated with AUC in (D), (E) and (F), respectively. a Greater than respective baseline (P < 0.05). Main effect of feeding, P < 0.0001; effect of feeding type, NS; Interaction, P < 0.05. Values are expressed as mean ± SD. Measured by 2-way ANOVA. AA, amino acids; BCAA, branched-chain amino acids; EAA, essential amino acids concentrations on young and old individuals) and CV of laboratory techniques also of 15% to detect differences in feeding modes with 80% confidence at the 5% significance level. The analysis was conducted using Prism 7 (GraphPad). Data are presented as mean ± SD.

Normality of distribution was tested using D'Agostino and Pearson
Omnibus normality tests. Comparison between two measures made at times before and after feeding or same time point between two groups were made via repeated measures 2-way ANOVA with Bonferroni post-test analysis to determine significance. Change from baseline was calculated as the ratio between time point measurement and calculated average baseline value. The net incremental area under the response curve (AUC) was calculated for each individual separately and presented as a two-group comparison.

| D ISCUSS I ON
In this study, we report that 15 g of orally consumed EAA increased plasma insulin concentrations which are higher following oral compared with IV EAA challenge. We are not aware of any other study that examined such an effect in relation to EAA. This rise and fall in insulin was matched by a significant rise followed by a fall in GIP.
GLP-1 also had risen significantly from baseline but the fact that insulin levels fell despite the continued elevation of GLP-1 concentration points to lack of causal effect. The difference in insulin concentration with oral vs IV is likely to be attributed the incretin hormones (and likely GIP) was ~11% calculated as total AUC over 120 minutes.
This effect is EAA-related since no gluconeogenic source was infused in our study. It is also important to highlight the use of a dose Our results show that providing 15 g of EAA stimulates glucagon production, although the rise overall was not significantly different from baseline. Glucagon stimulation was previously reported following mixed AA and whey protein. 30,31 It was suggested that AA and glucagon regulate each other in a feedback loop that involves α-cells and hepatocytes. 32 In comparison to AA, orally or intravenously delivered glucose suppresses glucagon secretion, which is more pronounced with the latter mode of delivery. 2  has proven to be a better predictor for HbA1c compared to fasting blood glucose. 34 Therefore, although our study was performed in normoglycaemic individuals, we believe that optimising AA mixtures could represent a feeding strategy for individuals with impaired glycaemia. For example, a previous study has shown that consumption of whey protein prior to a standard breakfast was associated with a significant reduction in postprandial glucose, driven by increased in post prandial GLP-1 and insulin concentration compared with control. 35 The amount of 15 g of EAA in our study also ensures that a sufficient meal portion is delivered per meal in a daily AA requirement of 24-56 g of protein per day for a 70 kg adult. In addition to this antiglycaemic property showed in our study, EAA have the advantage of superior "anabolic profile" compared to other dietary protein mixes. 36 We acknowledge that the lack of cross-over comparison could be a potential source for variability between the two young groups.
We also acknowledge the fact that the physiological process of digestion and absorption is subject to both intra and inter-individual variations and that a cross-over design may have been more powerful. However, we think the well-matched plasma appearance of EAA across the groups and the greater capture of biological variance are validatory and study strengths.
We did not detect age-related differences in gut hormone responses to EAA. This finding suggests that, despite adverse reports on ageing related gut physiology, 37-39 the function of gut incretin secretion, at least in response to EAA, remains largely intact with ageing. 18 Nonetheless, older age did appear to be related to a slower return (albeit not significant) of incretin and insulin to baseline following oral consumption (Y.O and O.O groups); we suspect this may be due to increased rates of clearance of EAA in the young. In contrast, AA profiles were also similar between both age groups indicating that digestion, absorption and splanchnic extraction of EAA is minimally affected by the ageing process.
Therefore, nutritional strategies aimed at improving health performance and skeletal muscle health in the older population should focus on the quality of diet and appetite enhancement. Future directions could aim at further scrutinising the mechanisms by which EAA stimulate L cells and whether this could be translated into more potent stimulation (ie, higher GLP-1 concentration) as a result of EAA mixture consumption.

| CON CLUS ION
We have shown that EAA are able to induce rises in incretins and insulin hormones. There was a demonstrable differential effect of EAA on insulin production when given orally and matched with an intravenous equivalent. Our results also suggest that an EAA-induced effect is delivered through hormonal rises likely related to GIP. Our results add to those from a series of studies previously proved that the "incretin effect" exists following consumption of glucose, mixed AA and lipid beverage. In particular relation to AA-induced incretin hormone production, we suggest using EAA gives more accurate estimate in this regards. Our results suggest that the process of physiological ageing does not adversely affect EAA-induced incretin hormone release. Hence (although it was not directly estimated in this study), we predict that associated incretin effect in elderly population is likely to remain intact. Further research is needed to inform clinically or nutritionally meaningful intervention especially that could elucidate the mechanisms via which L and K cells sense EAA in the gut and EAA-mediated hormones release in both youth and ageing.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest in relation to this manuscript.

E TH I C A L A PPROVA L
The study was performed according to the Declaration of Helsinki and was approved by The University of Nottingham Ethics Committee and Hamilton Integrated Research Ethics Board. All participants gave written informed consent following full explanation of the rationale for and conduct of the study, in addition to the procedures involved.

DATA ACCE SS I B I LIT Y
The data that support the findings of this study are available from the corresponding author (PJA) upon reasonable request.