Protein metabolism is dysregulated in individuals with metabolic disturbances such as obesity or T2DM. These individuals typically have increased fasting levels of various AAs, including BCAAs and AAAs [6,7,8, 11, 12, 15]. Other affected AAs with higher concentrations include alanine, glutamic acid and proline, while glycine, serine and glutamine levels are generally lower. These changes are attributed to fat deposition, especially visceral fat accumulation [16, 18] and IR, which promotes muscle protein breakdown and impacts liver gluconeogenesis [8, 9, 38]. Many overweight or moderately obese individuals with normal blood glucose levels are not diagnosed as prediabetics and for this reason they commonly show a delay to proceed to dietary and lifestyle interventions that could prevent metabolic disease progression. The altered AAs metabolism before T2DM diagnosis may predict diabetes onset up to 10 years in advance, especially in those with high visceral fat [9, 12].

In the present study, fasting concentrations of 24 AAs, as well as their postprandial levels at 5 time points following the consumption of plant protein-enriched wheat biscuits with different AAs compositions, were measured in plasma samples from NW and OW subjects. Subjects were apparently healthy, with no apparent underlying conditions other than a BMI > 25 kg/m2 in the overweight/obese group. Health status was confirmed through the assessment of basic biochemical parameters, including fasting glucose, insulin, lipid profile, and liver function indices. Significant differences between the two study groups (NW and OW) were observed for a number of AAs, both in fasting as well as in the postprandial state. In the fasting state, serum concentrations of methionine, tryptophan, and tyrosine were significantly higher in OW compared to NW subjects. Significantly higher fasting concentrations of these AAs in obese subjects have been also recorded in other studies [13, 15, 40]. Fasting isoleucine concentrations were higher in OW compared to NW subjects with a p value approaching statistical significance (p = 0.053); a larger sample size may have revealed a significant difference, as suggested by findings in similar studies [12, 13, 15]. It has been shown that isoleucine, methionine and AAAs have a significant positive correlation with visceral fat as well as subcutaneous fat area [13]. Elevated circulating BCAAs levels have been associated with incomplete oxidation of fatty acids and glucose, potentially contributing to disrupted glucose homeostasis [18, 41]. It has been previously shown that AAAs transport in cells is impaired by the chronic elevations in BCAAs in subjects with overweight/obesity [15].

NW individuals had significantly higher fasting values of glutamine (p < 0.05) as well as higher values of glycine compared to OW individuals, for which although the difference did not reach statistical significance (p = 0.059), this trend aligns with findings from previous studies comparing similar populations[14, 15]. Higher levels of glutamine, glycine and serine have been also recorded in other studies and have been negatively correlated with body fat and specifically visceral fat, due to the enhancement of oxidation of free fatty acids in the adipose tissue [13, 18, 24]. Decrease in plasma glycine concentration has been correlated with higher visceral fat and insulin resistance [12, 14]. Elevated levels of BCAAs, which are common among obese individuals, lead in glycine depletion and strategies targeting in lower BCAAs concentrations can help to restore glycine levels [11, 24].

In this study the postprandial AAs responses of NW and OW subjects following the ingestion of two biscuits differing in their amino acid composition were evaluated. Significant differences were observed between the two groups in the postprandial responses of certain AAs, as measured by the iAUC. Overall, higher postprandial AAs responses were found in NW, compared to the OW group. More specifically, in the OW group, iAUC, after the ingestion of BCAAsB was significantly lower for alanine, asparagine, glutamine, serine and threonine compared to NW. Regarding the ArgB, significantly lower postprandial responses were found for alanine, glutamine, glycine and threonine, in OW group, whereas iAUC of taurine was significantly higher in this group. For the CB, although the changes were less pronounced compared to the protein enriched biscuits in both study groups, significant differences were observed for hydroxyproline, methionine and ornithine. Similarly with the other two biscuit samples, the NW group had significantly higher iAUCs, compared to OW.

Alanine, glutamine and threonine exerted significantly lower iAUC in OW subjects after ingestion of both plant protein-enriched biscuits (BCAAsB and ArgB). Alanine is a key amino acid involved in hepatic gluconeogenesis due to its ability to convert to pyruvate [11]. As a key gluconeogenic precursor, a greater increase in postprandial alanine provides the body with more capacity to generate glucose, thereby reducing the risk of hypoglycemia [23]. Regarding glutamine, it has been linked with increased circulating levels of GLP-1 and GIP postprandially in both NW and OW individuals. This suggests that glutamine helps in suppressing postprandial glycemia through stimulation of insulin secretion [42, 43]. Individuals with normal body weight seem to more effectively extract and absorb glutamine from the same food product, and through its action on glucose and appetite regulation could contribute to enhanced satiety and thus aid in long-term weight management [12]. Threonine is an essential amino acid that, along with aspartic acid and methionine, aid in fat digestion by the liver, reducing fat accumulation and supporting liver function. Threonine is a precursor of serine and glycine and thus indirectly enhances insulin sensitivity and blood glucose regulation [11, 44].

Lower postprandial glycine levels for all three biscuits were observed in OW individuals. Glycine has been strongly correlated with insulin secretion and interacts closely with BCAAs metabolism, with its circulating levels being inversely associated with BMI [24, 45]. The alteration of glycine response has been also observed in individuals with obesity and is linked with higher risk of T2DM development. According to Alves et al., [11] an explanation for the significantly lower postprandial glycine levels can be a decreased intestinal absorption of glycine in subjects with overweight/obesity. It has been proposed that elevated BCAAs levels in obesity may contribute to glycine depletion, as the body attempts to detoxify excess ammonia (NH₃) produced during BCAAs transamination. This process is often impaired in individuals with obesity, leading to further metabolic disruption. The efficient BCAAs transamination supports proper protein utilization and prevents the accumulation of BCAAs in circulation—a condition often observed in obesity and metabolic disorders. In contrast, impaired transamination can lead to elevated plasma BCAAs levels, which are associated with IR, mitochondrial dysfunction, and altered metabolic signaling [11, 24, 46,47,48].

Regarding the taurine, a higher postprandial response was observed in OW subjects after the ingestion of ArgB. This may be attributed to the higher ingested amounts of cysteine and methionine through this biscuit, which are precursors of taurine. Taurine is an amino acid which is involved in many biological and physiological functions in the body. It has been shown that in the condition of obesity the levels of taurine are depleted. Supplementation of taurine leads to increased plasma taurine and adiponectin levels in humans while reducing inflammatory and oxidative markers [49].

After consumption of BCAAsB a significantly lower postprandial serine response was observed in OW individuals compared to NW. Higher levels of available serine have been linked with enhanced insulin sensitivity [18]. Regarding asparagine, its postprandial circulating levels were also found to be significantly lower in the OW group. Asparagine is another glucogenic amino acid, with its by-product oxaloacetate used in the tricarboxylic acid cycle for glucose production. Asparagine plays a crucial role in cell catabolism and antioxidant capacity [18].

Considering the findings of the present study alongside relevant literature, individuals with overweight/obesity may absorb and utilize AAs less efficiently than their lean counterparts [7]. As a result, subjects with overweight/obesity are expected to have a lower availability of AAs, which are mainly responsible for the stimulation of protein synthesis (anabolic response), along with the minimization of catabolic response in the body postprandially [9, 50]. Lean individuals generally exhibited more pronounced postprandial responses for most of the AAs measured, even in the cases that their iAUC values were not significantly different from those with overweight/obesity. This consistent trend may indicate physiological differences between groups that could become statistically significant with larger sample size. The small sample size—typical of acute postprandial crossover studies—may limit the power to detect smaller but biologically relevant differences (Table 5). According to the available literature, larger differences between pre- and postprandial amino acid concentrations after ingesting a mixed meal have been correlated with greater IR [51]. In the OW group of subjects significantly higher values of fasting serum insulin, HOMA-IR and HOMA-β indexes were present [15].

Specific AAs could act as metabolic signals involved in appetite regulation, either through the stimulation of gut-derived satiety hormones or via central mechanisms. Table 6 provides an overview of AAs physiological role and proposed mechanisms of action, highlighting their relevance in postprandial metabolism. It is assumed that generally higher postprandial concentrations of several AAs which was observed in the NW group may contribute to more effective signaling for appetite control. This trend is consistent with previous literature suggesting a greater sensitivity to nutrient signaling in lean individuals [47, 52,53,54]. For instance, the iAUC of the glutamine, which was significantly higher in the NW group for both protein-enriched biscuits, has been associated with increased GLP-1 secretion, a gut peptide secreted by enteroendocrine cells as AAs and small peptides enter in the small intestine. The elevated postprandial GLP-1 levels have been associated with slower rate of gastric emptying, enhanced satiety and reduced hunger [42, 43, 55]. The postprandial threonine response following the ingestion of both BCAAs-enriched and L-arg-enriched biscuits was significantly lower in the OW group compared to the NW group. Elevated postprandial circulating threonine levels have been associated with increased satiety and reduced subsequent ad libitum food intake. These findings underscore the complexity of protein-induced satiety, which may rely on indirect and complex signaling pathways rather than a direct action of circulating AAs on the brain [54, 56]. In individuals with overweight or obesity, this attenuated amino acid response may contribute to reduced gut hormone secretion and impaired central signaling, potentially leading to impaired appetite regulation. As a result, this population may face greater challenges in effectively managing body weight due to decreased sensitivity to postprandial metabolic signals [47].

The results of the present study showed that subjects with overweight/obesity have lower, less pronounced AAs responses after the consumption of the same plant protein-based snack compared to normalweight and different AAs composition causes different postprandial AAs responses which can further affect metabolic dysregulation in the condition of overweight/obesity. However, it must be mentioned that plant protein structures differ from animal proteins due to different peptide sequences, leading to distinct secondary and tertiary structures and consequently different digestibility and functional properties [70, 71].

A limitation of the present study is the relatively small sample size with only 15 participants in each group, occurred after further analysis of existing obtained samples. This constraint limits the robustness of the conclusions and reduces the possibility of detecting statistically significant differences across the examined parameters. However, it must be noted that this is a demanding postprandial protocol and it is difficult to recruit a large number of participants. Another important limitation that may have influenced the outcomes of the present study is the level of participants’ compliance with the study protocol. Specifically, participants were required to adhere to standardized guidelines on the evening prior to each test day and throughout the entire experimental period (i.e., between study sessions). Variations in adherence to these instructions, including dietary intake, physical activity, or other lifestyle factors, may have introduced variability in the postprandial responses observed.

These findings suggest that the amino acid composition of foods influences postprandial amino acid metabolism differently in NW and OW individuals. This highlights the potential for further research in food science towards the development of functional foods with specific amino acid profiles as part of dietary strategies for weight management supporting improvements in metabolic outcomes such as insulin sensitivity, body composition and circulating amino acid profiles.