Human skeletal muscle mass accounts for up to 35% of total body weight (Janssen et al., 2000) and serves vital functions such as generating force and thus enabling locomotion. Additionally, skeletal muscle metabolism comprises numerous metabolic processes including energy storage (Hultman et al., 1971), energy consumption and endocrine functioning (Hoffmann and Weigert, 2017). Thus, the preservation or even increase of skeletal muscle mass and strength are of fundamental importance to sustain and improve many physiological functions including a healthy aging process with the longest possible maintenance of activities of daily living (Wang et al., 2020). Resistance exercise in combination with protein ingestion is known to be a potent stimulator of skeletal muscle protein synthesis and the timely coordinated combination of both has been demonstrated to augment net muscle protein synthesis (Burd et al., 2009; Wirth et al., 2020).

Previous research indicated that the beneficial effects of protein supplementation (in particular whey protein (Moore et al., 2011; Areta et al., 2013; Morton et al., 2018; Hamarsland et al., 2019)) on muscle protein synthesis are predominantly mediated by the essential amino acid leucine, which augments intramuscular muscle protein synthesis via the mechanistic target of rapamycin (mTOR) complex and its downstream regulators (Drummond and Rasmussen, 2008; Moberg et al., 2016). Additionally, via exercise-induced growth factor secretion, the stimulation of the PI3k-Akt pathway has repeatedly been shown to induce protein synthesis via activation of its downstream targets (Bodine et al., 2001). Independently of mTOR activity, resistance exercise can also activate the mitogen-activated protein kinase (MAPK) pathway. Previous researchers (Moore et al., 2011) speculated that MAPK might facilitate the stimulation of myofibrillar protein synthesis by regulating proteins which mediate the initiation and elongation stages of mRNA translation (Wang et al., 2001; Roux et al., 2007).

Based on clinical and preclinical studies on the acute and longitudinal muscular responses to resistance exercise and protein consumption, whey protein (Moore et al., 2011; Areta et al., 2013; Hamarsland et al., 2019), soy (Darren et al., 2006; Tang et al., 2009), casein (Tipton et al., 2004; Tang et al., 2009) or collagen peptides (Jendricke et al., 2019; Kirmse et al., 2019; Zdzieblik et al., 2021) have been receiving increased attention over the past 2 decades.

Collagen peptides (CP) in combination with resistance training have previously been shown to improve muscle function and increase fat-free mass beyond the levels of resistance training alone (Zdzieblik et al., 2015; Jendricke et al., 2019; Kirmse et al., 2019), although some studies found less pronounced effects on muscle protein synthesis (Oikawa et al., 2020) and muscle mass compared to whey protein (Jacinto et al., 2022). In fact, however, Jacinto et al. (2022) did not find significant group differences with regard to performance parameters, which is practically relevant for the effectiveness of training. To date, little is known about the underlying mechanisms on a cellular level responsible for these observations. While CPs are rich in amino acids such as proline, glycine and arginine (Zdzieblik et al., 2015), the amount of e.g., leucine is low, which might lead to the conclusion about limited anabolic potential. Yet, in vitro data by Kitakaze et al. (2016) have indicated that the collagen derived dipeptide hydroxyprolyl-glycine promotes activation of the PI3K-Akt-mTOR signaling pathway. In a recent clinical investigation, Oertzen-Hagemann and others (2019) analyzed the skeletal muscle proteome before and after 12 weeks of resistance training in combination with CP supplementation. The authors demonstrated that anabolic signaling pathways were upregulated with CP combined with resistance training, as indicated by a higher upregulation of the MAPK pathway in response to CP supplementation compared to placebo intake (Oertzen-Hagemann et al., 2019).

Based on these findings (Kitakaze et al., 2016; Oertzen-Hagemann et al., 2019), the present study aimed to investigate the acute effects of CP in combination with resistance exercise on gene expression in pathways known to be activated in muscular remodeling, such as MAPK, mTOR and PI3K-Akt. Specifically, this study examined acute changes in gene expression in relation to a single high-load resistance exercise. Investigating these short-term responses is crucial for understanding basic physiological mechanisms of CP action in combination with resistance exercise.

All participants successfully completed all aspects of the study (n = 15 CP, n = 15 PLA). There were no differences between groups for any anthropometric or exercise related measurements (Table 2).

The main purpose of the present study was to systematically investigate changes in gene expression following an acute bout of high-load resistance exercise combined with specific collagen peptides compared to placebo. This acute study design was chosen, since it best reflects the temporal dynamics of gene expression. The main findings indicated a significantly upregulated gene expression in key myocellular pathways (PI3K-Akt, MAPK) involved in protein synthesis and muscle hypertrophy following the ingestion of collagen peptides compared to placebo.

The PI3K-Akt pathway can be activated by several precursors including IGF-1 and regulates multiple intracellular processes including muscle hypertrophy (Glass, 2010) and collagen synthesis (Yokoyama et al., 2012). As a serine-threonine protein kinase, Akt is capable of inducing myofibrillar protein synthesis and inhibit the upregulation of muscle atrophy markers such as MuRF-1 or Atrogin-1 via phosphorylation of FOXO (Glass, 2010). In addition to the inhibition of atrophy-related markers, PI3K-Akt has been demonstrated to block myostatin and thus stimulate differentiation and skeletal protein synthesis via distinct physiological mechanisms.

Previous research shows that the phosphorylation levels of Akt and PI3K-Akt are significantly elevated following an acute bout of resistance exercise (Dreyer et al., 2008; Camera et al., 2010; Horii et al., 2020) and that this response can be augmented by protein supplementation (Dreyer et al., 2008; Camera et al., 2015; Lane et al., 2017). Besides being stimulated by whey protein (Lane et al., 2017), Akt protein has recently been shown to be upregulated following the ingestion of CP in combination with chronic resistance exercise training (12 weeks) (Oertzen-Hagemann et al., 2019). The results of the present study complement these previous findings and demonstrated that PI3K and Akt mRNA are significantly upregulated following an acute bout of high-load exercise when ingesting a CP supplement compared to placebo. After inspection of relevant genes which might be involved in PI3K-Akt pathway upregulation following CP, angiopoietin-1 seems to play key roles as important activators of the PI3k signaling (Figure 4). In previous animal models, McClung et al. (2015) demonstrated that muscle cell derived angiopoietin-1 is involved in promoting myoblast differentiation and myotube formation. These findings are supported by studies from Mofarrahi et al. (2015) confirming strong positive associations of angiopoietin-1 and muscle regeneration following fiber injury. Interestingly, previous findings point towards the notion that the PI3K-Akt pathway plays an essential role in the expression of type I collagen induced by TGF-beta (Yokoyama et al., 2012). Although speculative, it might be assumed that the improvement in extracellular connective tissue might help to facilitate an enhanced contractile force transmission from the muscle to tendons or bones (Holwerda and van Loon, 2021). Indeed, such improvements in muscle strength and force have repeatedly been demonstrated in previous experiments following prolonged CP intake in combination with resistance training (Zdzieblik et al., 2015; Jendricke et al., 2019).

The mechanistic target of rapamycin (mTORC1) signaling pathway is generally considered as an important regulator of translation initiation (Bodine et al., 2001; Hoppeler, 2016). Besides the stimulatory effects of resistance exercise on the mTOR pathway (Drummond et al., 2009; Dreyer et al., 2010), numerous studies have also found favorable effects of protein supplementation manifested as an augmentation of mTOR phosphorylation and activation (Farnfield et al., 2009; Farnfield et al., 2012; Kakigi et al., 2014). Despite a visual increase, the present mRNA expression data demonstrated similar findings to these previous results, however, no statistical group by time effects were identified. A potential explanation for these results may lay within the temporal upregulation of the investigated pathways. Previous researchers confirmed that Akt acts as a precursor of mTOR by phosphorylating (and thus inhibiting) TSC2, a known suppressor of mTOR (Inoki et al., 2002). Given the present upregulation of PI3K-Akt gene expression at 4 h post exercise combined with CP intake, it may be speculated that mTOR upregulation peaked between 4 and 24 h and was thus missed due to the specific timing of sampling in the current study. Additionally, it could be speculated that the previously observed effects of longitudinal CP supplementation combined with resistance training on fat-free mass (Zdzieblik et al., 2015; Jendricke et al., 2019; Kirmse et al., 2019; Jendricke et al., 2020; Zdzieblik et al., 2021) can be explained via the stimulation of pathways other than the mTOR pathway (e.g., MAPK). The hypothesis of a potential convergence of mTOR and MAPK pathways to promote enhancements in anabolic signaling has been formulated by previous authors (Burd et al., 2010) although this aspect warrants further investigation. The exact mechanisms by which CP induce upregulations in gene expression remain largely unknown (Figure 5). Within the physiological chain of how upregulated gene expression pathways might facilitate physiologically relevant effects, Kitakaze et al. (2016) showed an increased upregulation of ribosomal S6 kinase which might facilitate increases in mRNA translation via eukaryotic initiation factor 3. Further studies are, however, needed to elucidate the physiological mechanisms in this field.

The MAPK-pathway has been shown to be involved in myofibrillar muscle protein synthesis as well as indirectly in collagen synthesis (Kimoto et al., 2004). A study by Holm et al. (2009) has shown that several steps in the MAPK pathway such as ERK1/2 or p38 are upregulated after exercise in an intensity-dependent manner and may therefore be reflective of the magnitude of mechanical stress applied to the muscle fiber (Martineau and Gardiner, 2001). Besides exercise-induced stimulation of the MAPK pathway (Williamson et al., 2003; Tannerstedt et al., 2009) there is conflicting evidence regarding the effects of protein feeding. In an early study by Karlsson et al. (2004) the authors found that the intake of branched-chained amino acids did not significantly augment the phosphorylation of several proteins found within the MAPK pathway (e.g., ERK1/2, p38) compared to resistance exercise alone. This is supported by a study from Moore and others (2011) indicating no effect of protein feeding alone but only when exercise was performed. Conversely, following a long-term trial with 12-weeks of resistance training and concomitant ingestion of CP, Oertzen-Hagemann et al. (2019) performed muscle proteome analyses and found that the MAPK pathway was overrepresented in the CP group compared to PLA. Although changes in muscle proteome and gene expression are not necessarily inter-related, these findings were confirmed by the present data, showing a significantly higher upregulation of MAPK mRNA in CP compared to PLA at 4 h post-exercise.

Interestingly, besides angiopoietin-1, upregulations of the MAPK pathway following CP ingestion seem to be driven by TGF-β-1 (Figure 6). TGF-β-1 is a multifunctional cytokine being involved in various cellular processes including type I collagen expression (Ismaeel et al., 2019) or myofibroblast transdifferentiation (Fan et al., 1999). In an earlier study by Heinemeier et al. (2003), the authors conclude that TGF-β-1 may play an important role in collagen formation after mechanical loading of tendon structures. As the PI3K-Akt and MAPK pathways are strongly inter-related, functional effects of extracellular matrix might facilitate optimized contractile force transmissions seen with long-term CP supplementation (Zdzieblik et al., 2015; Jendricke et al., 2019). This link, however, needs to be confirmed in future studies.

Although speculative, it may be argued that MAPK might be involved in mediating the effects on fat-free mass previously reported following 12 weeks of combined resistance training and CP supplementation (Zdzieblik et al., 2015; Jendricke et al., 2019; Jendricke et al., 2020; Zdzieblik et al., 2021). Such changes in fat-free mass might be explained by increases in either contractile or non-contractile elements. Generally, it must be mentioned that most studies focusing on intramuscular signaling following resistance exercise and protein supplementation investigated changes on the protein level. Therefore, further studies are needed which investigate the link between upregulated gene expression and myocellular as well as ECM protein synthesis. Conclusively, it needs to be mentioned that the findings from the present study are valid for this specific collagen peptide composition but might not necessarily be transferred to other collagen peptide ingredients.

The main objective was to examine important gene expression pathways involved in muscular remodeling. The choice of these specific pathways was based on previous experiments (Oertzen-Hagemann et al., 2019) highlighting increased upregulations of proteins associated with protein metabolism and contractile fibers. It is important to note that the anabolic response to nutrition and exercise is highly dynamic and the observed acute changes are not necessarily predictive of long-term adaptations. Moreover, the current study investigated young and recreationally active males and the acute responses in healthy individuals cannot readily be transferred to responses in clinical cohorts. Nevertheless, this might also be of high clinical relevance for patients suffering from sarcopenia or diseases associated with muscular atrophy. To obtain a high level of standardization, individuals in this study were fasted (except CP and PLA intake) between the baseline and 4 h biopsy to exclude other mediating factors resulting from additional nutritional protein sources which might influence the results obtained. Conclusively, future studies are needed which validate these findings in further study populations including gender, age and training status as well as long-term effects including pre-exercise meals.

The present study provides evidence for an increased upregulation of gene expression in the PI3K-Akt and MAPK pathways at 4 h following acute high-load resistance exercise combined with ingestion of CP compared to PLA. These results demonstrate for the first time that CP directly increase gene expression signaling of anabolic pathways in human skeletal muscle in response to an acute bout of resistance exercise. However, further studies, are needed to investigate the effects of combined resistance exercise and CP supplementation on PI3K-Akt and MAPK post-translational activation or miRNA regulation.