Acute changes in the metabolome following resistance exercise combined with intake of different protein sources (cricket, pea, whey)

doi:10.21203/rs.3.rs-2949302/v1

Introduction

Separately, both exercise and protein ingestion have been shown to alter the blood and urine metabolome. This study goes a step further and examines changes in the metabolome derived from blood, urine and muscle tissue extracts in response to resistance exercise combined with ingestion of three different protein sources.

Methods

In an acute parallel study, 52 young males performed one-legged resistance exercise (leg extension, 4 x 10 repetitions at 10 repetition maximum) followed by ingestion of either cricket (insect), pea or whey protein (0.25 g protein/kg fat free mass). Blood and muscle tissue were collected at baseline and three hours after protein ingestion. Urine was collected at baseline and four hours after protein ingestion.

Results

Nuclear magnetic resonance (NMR)-based metabolomics resulted in the annotation and quantification of 25 metabolites in blood, 35 in urine and 21 in muscle tissue. Changes in the muscle metabolome after combined exercise and protein intake indicated effects related to the protein source ingested. Muscle concentrations of leucine, methionine, glutamate and myo-inositol were higher after intake of whey protein compared to both cricket and pea protein. The blood metabolome revealed changes in a more ketogenic direction three hours after exercise reflecting that the trial was conducted after overnight fasting. Urinary concentration of trimethylamine N-oxide was significantly higher after ingestion of cricket than pea and whey protein.

Conclusion

The blood, urine and muscle metabolome showed different and supplementary responses to exercise and ingestion of the different protein sources, and in synergy the summarized results provided a more complete picture of the metabolic state of the body.

Protein ingestion

NMR-based metabolomics

blood metabolome

muscle metabolome

urine metabolome

human metabolome

Insect protein

During the last decades, metabolomics has been established as a commonly employed approach in nutrition research. Based on the analyses of biofluids such as urine and blood, numerous investigations have reported the application of metabolomics in the study of dietary interventions with various food items (Bertram, 2023; Gibbons et al., 2015). Metabolomics studies focused on examining the acute effects of protein intake have mainly been conducted on blood samples (Skov et al., 2019; Thøgersen et al., 2021a; Thøgersen et al., 2021b), while acute metabolomics studies on urine are scarce. Nevertheless, Madrid-Gambin et al. (2018) employed nuclear magnetic resonance (NMR)-based metabolomics to elucidate changes in urinary metabolites in 1-h intervals immediately after consumption of different pulses including lentils, chickpeas, and beans. The study revealed major differences between the protein sources in the urinary metabolite profile in the time range between 2 and 6 h after ingestion (Madrid-Gambin et al., 2018) and thereby demonstrated that the urine metabolite profile is sensitive to acute changes related to protein ingestion.

Metabolomics has also been introduced in sport science to expand our knowledge on the metabolic response to exercise (Khoramipour et al., 2022). In a study reported by Pechlivanis et al. (2010b), male subjects performed three sets of two 80 m maximal runs separated by either 10 s or 1 min of rest. Urine samples collected before and after the exercise were analyzed by NMR-based metabolomics and the results revealed that NMR-based metabolomics could discriminate between pre and post exercise samples as changes were detected in lactate, pyruvate, hypoxanthine, intermediates of the Krebs cycle, amino acids, and products of branched-chain amino acid (BCAA) catabolism. Also, they reported changes in urinary metabolites in the urine collected in the resting period after the two sprint intervals (Pechlivanis et al., 2010). Another study analyzed blood samples collected from male subjects after a resistance exercise session and showed increases in serum 2-hydroxybutyrate, 2-oxoisocaproate, 3-hydroxyisobutyrate, alanine, hypoxanthine, lactate, pyruvate and succinate (Berton et al., 2017). Collectively, the present evidence indicates that NMR-based metabolomics might be a valuable tool for examining the endogenous metabolic effects of exercise. Nevertheless, to the best of our knowledge, no studies have yet employed metabolomics in the examination of combined effects of exercise and protein intake. This is despite it is well-documented that protein intake supports muscle growth when performing resistance exercise training (Devries and Phillips, 2015; Morton et al., 2015; Tang et al., 2009; Tipton et al., 1999).

Metabolomics may also be conducted on other sample matrices than biofluids. In sport science, the muscle metabolome is relevant, but NMR-based analyses of the human muscle metabolome are sparse. However, recently profound perturbations in muscle metabolites during chronic limb threatening ischemia was successfully unveiled by NMR-based analyses, and the results demonstrated that severe changes in oxygen and energy supply to the muscle are reflected in the muscle metabolome (Khattri et al., 2021).. Nevertheless, there is a lack of studies using NMR-based metabolomics which have collected samples simultaneously from the skeletal muscle, urine, and blood to give a more complete elucidation of changes in the human body’s metabolic state in response to the combined stimuli from exercise and protein ingestion.

While a sample matrix such as urine has clear advantages in being easily accessible and pain-free to sample, other samples such as muscle biopsies are clearly less easily accessible and involve discomfort during sampling. It therefore seems relevant to establish which information that can be obtained from different sample matrices. However, few studies have made a thorough comparison of the urine, blood, and muscle metabolome in acute studies. Therefore, the aim of the present study was to use NMR-based metabolomics to i) elucidate the acute changes in the urine, blood and muscle metabolome following resistance exercise training combined with protein intake and ii) to investigate potential differential acute changes in the metabolome after ingestion of either whey, pea, or cricket (insect) protein. Whey (milk protein) represents the golden standard in sports nutrition, while pea and insect represent two emerging alternative protein sources that potentially could become important future products in protein supplements and protein fortification. To the best of our knowledge, this is the first study to investigate the combined effect of exercise and protein intake on the human metabolome and report a direct comparison of the urine, blood, and muscle metabolome collected simultaneously from the same individuals.

Experimental protocol

This study presents an analysis of urine, blood and muscle tissue samples collected from the trial by Lanng et al. (2022). A detailed description of the experimental protocol can be found elsewhere (Lanng et al., 2022). Briefly, a randomized single-blinded parallel study was conducted. The protocol consisted of one experimental day where the subjects were randomly assigned to ingest either pea protein, insect protein or whey protein. Fifty-two healthy young normal to overweight (BMI between 18.5 and 30 kg/m²) males between 18 and 30 years were recruited to participate in the trial. Exclusion criteria were allergy against shellfish or house-dust mites (later changed to all types of allergies due to an unexpected incidence, see section “Adverse event”), medication or health issues conflicting with the experiment, smoking, resistance exercise training more than once per month, and endurance training at moderate to high intensity more than 2 hours per week. All subjects gave their informed consent to participate before the experiment. The trial complied with the Declaration of Helsinki and was approved by The Central Denmark Region Committees on Health Research Ethics (journal nr. M-2019-291-19) and registered at ClinicalTrial.gov (ID NCT04633694).

Before the experimental day, the participants underwent a familiarization session where they were familiarized with the knee-extension exercise and their 10-repetition maximum (RM) was determined. The participants were told to avoid any strenuous exercise the day prior to the experimental day. All participants were instructed to eat their habitual energy-sufficient diet the day before the experiment corresponding to a Danish mixed diet. Analyses of the dietary records showed no difference in energy intake or macronutrient content between the experimental groups.

After overnight fasting from 10 pm, the participants arrived in the laboratory at Department of Public Health, Aarhus University. Firstly, baseline blood, spot urine and muscle biopsy samples were obtained. Thereafter, the participants performed a warm-up consisting of 5 min of light aerobic exercise on a stationary bicycle (Monark 928E, Varberg, Sweden) followed by one-legged resistance exercise with either the dominant or the non-dominant leg determined by randomization. The resistance exercise protocol included a warm-up set with 10 repetitions in a knee-extension machine (Technogym SpA, Gambettola, Italy) with a load corresponding to 50% of the estimated 10 RM. Then the participants performed the resistance exercise protocol, including five sets of 10 repetitions at 10 RM with 2 min rest between sets. The last set was performed to voluntary muscle failure. Following the resistance exercise, each participant ingested the assigned protein beverage, either pea, cricket or whey protein. All the protein beverages contained 0.25 g protein per kilo fat free mass dissolved in 400 mL water. Three hours after protein ingestion, a blood sample and a muscle biopsy were obtained from the exercised-leg. Urine samples were collected at the beginning (baseline) and corresponding four hours after protein ingestion and exercise from all participants and frozen immediately after collection.

Muscle Biopsies

Muscle biopsies were obtained from the thigh (vastus lateralis), approximately midway between the iliac crest to the patellar plateau using a Bergström needle with suction under local anesthesia. The samples were dissected free from adipose and connective tissue and immediately frozen in liquid nitrogen and stored at – 80 ^oC until further analysis.

Preparation of muscle, urine, and serum samples for proton nuclear magnetic resonance (¹H -NMR) spectroscopy

Blood samples were centrifuged at 1200 × g for 10 min at 4°C 30–60 min after collection and stored at -80 ^oC. For each sample, 325 µL of buffer (IVDr-designated phosphate buffer purchased from Bruker Biospin) was added to 325 µL of serum sample. Thereafter 600 µL was transferred to a 5 mm NMR tube for analysis.

Urine samples were thawed at room temperature and centrifuged 10 min at 2000 g. Then, 900 µL urine were mixed with 100 µL phosphate buffer (1.5 M KH₂PO₄, 2 mM NaN₃, 6 mM TSP in D₂O, pH = 7.4). The mix was vortexed and 600 µL transferred to 5 mm NMR tubes for analysis.

Muscle tissue (~ 10–20 mg) was lyophilized and grinded. Each muscle sample was added 300 µL ice-cold methanol, the sample was whirlmixed and placed on ice. Thereafter, 300 µL ice-cold CHCl₃ was added to the sample, which was then whirlmixed thoroughly and placed on ice for 10 min. Subsequently, 300 µL water was added to the sample, it was mixed thoroughly and placed at 4 ^oC for overnight separation. Next morning, the samples were centrifuged for 30 min at min. 1400x g at 4 ^oC. The upper methanol-phase was transferred to a 1.5 mL eppendorff tube, dried by vacuum centrifuge for approximately three hours. The sample was prepared for NMR analysis by adding 150 µL D₂O, 8 µL H₂O and 12 µL D₂O with 0.05% trimethylsilylpropanoic acid (TSP) and transferred to 3 mm NMR tubes.

Quantification of metabolites in muscle, urine and blood by ¹H-NMR spectroscopy

NMR spectroscopic analyses were performed on a 600 MHz Bruker Avance III spectrometer operating at a 600.13 MHz ¹H frequency and quipped with a 5 mm ¹H-optimized double resonance broad banded probe (Bruker BioSpin, Rheinstetten, Germany). The muscle and urine samples were analyzed by 1D Nuclear Overhauser Effect Spectroscopy (NOESY). Thereafter, muscle and urine metabolites were identified and quantified using TSP as reference using Chenomx NMR Suite 8.13 (Chenomx Inc., Edmonton, Canada). The metabolite concentrations in the muscle samples were thereafter normalized to the wet weight of the muscle analyzed, while the urine metabolites were normalized to the creatinine concentration.

Blood samples were analyzed via a Bruker SampleJet set to 4°C and performed using the in vitro diagnostics research (IVDr) tool (Avance IVDr, IVDr methods, User manual, version 3, Bruker). Four experiments were applied in automation, 1D Nuclear Overhauser Effect Spectroscopy (NOESY), 2D J-resolved experiment (JRES), Carr − Purcell − Meiboom − Gill (CPMG), and difference spectroscopy (DIFF), which facilitated the automated quantification by B.I.Quant-PS (version 2.0, Bruker BioSpin, Rheinstetten, Germany) for the automatic quantification of serum metabolites.

Statistics

For the statistical analysis and visualization of data, GraphPad Prism, version 9.2.0 (GraphPad Software, San Diego, California, USA) was used. Outliers were defined as values which exceeded the mean value within the group ± two standard deviations. Metabolomics data were analyzed using a mixed-effects analysis to examine the effect of the time (baseline vs post), protein (cricket, pea, whey), and time x protein interaction. If the mixed-effects analysis showed a significant difference, Tukey’s multiple comparisons post hoc tests were performed to analyze the differences between groups. P-values below 0.05 were considered statistically significant.

In blood, a total of 25 metabolites were annotated and quantified, while 35 metabolites were annotated and quantified in urine, and 21 in muscle. A total of 14 metabolites were found in both urine and blood, four in both blood and muscle, and four metabolites were present in all three sample types (Fig. 1, Table 1, 2, 3). The metabolites quantified in both urine and blood were 3-hydroxybutyrate, acetate, acetoacetate, acetone, citrate, creatinine, formate, glucose, histidine, N,N-dimethylglycine, phenylalanine, succinate, trimethylamine N-oxide (TMAO) and tyrosine. The metabolites annotated in both blood and muscle were creatine, glutamine, isoleucine and leucine. The four metabolites annotated in all three sample types were alanine, glycine, lactate, and valine.

Table 1

Blood metabolites determined at baseline and 4 hours after protein ingestion and exercise (post).
	Cricket						Pea						Whey						P-values
	Baseline			Post			Baseline			Post			Baseline			Post			Time		Protein	Time x protein
3-Hydroxybutyric acid	0.078	±	0.034	0.192	±	0.116	0.109	±	0.049	0.254	±	0.193	0.102	±	0.04	0.168	±	0.101	0.0004	0.3219		0.4905
Acetic acid	0.031	±	0.013	0.02	±	0.006	0.042	±	0.021^c	0.026	±	0.015	0.023	±	0.007^b	0.017	±	0.007	< 0.0001	0.0027		0.1443
Acetoacetic acid	0.011	±	0.006	0.029	±	0.021	0.014	±	0.01	0.033	±	0.026	0.008	±	0.005	0.03	±	0.023	< 0.0001	0.5346		0.8929
Acetone	0.031	±	0.008	0.047	±	0.017	0.031	±	0.014	0.047	±	0.025	0.031	±	0.012	0.045	±	0.022	< 0.0001	0.9296		0.8527
Alanine	0.425	±	0.083	0.401	±	0.058	0.449	±	0.08	0.423	±	0.069	0.407	±	0.061	0.417	±	0.073	0.1589	0.4879		0.166
Citric acid	0.181	±	0.035	0.193	±	0.03	0.178	±	0.036	0.182	±	0.031	0.173	±	0.033	0.175	±	0.029	0.3522	0.382		0.7065
Creatine	0.01	±	0.008^c	0.01	±	0.008^c	0.007	±	0.007	0.007	±	0.007	0.003	±	0.003^a	0.003	±	0.003^a	0.9989	0.0042		0.9645
Creatinine	0.091	±	0.01	0.092	±	0.009	0.1	±	0.016	0.093	±	0.016	0.091	±	0.017	0.092	±	0.012	0.5601	0.4581		0.0349
Dimethylsulfone	0.011	±	0.003	0.011	±	0.003	0.011	±	0.003	0.01	±	0.003	0.009	±	0.002	0.009	±	0.003	0.0125	0.0988		0.8832
Formic acid	0.029	±	0.009	0.03	±	0.006	0.028	±	0.005	0.029	±	0.005	0.032	±	0.006	0.027	±	0.012	0.4659	0.7619		0.166
Glucose	5.294	±	0.362	5.395	±	0.336^bc	5.322	±	0.315	5.061	±	0.240^a	5.361	±	0.397	4.864	±	0.369^a	< 0.0001	0.0716		0.0006
Glutamine	0.795	±	0.074	0.84	±	0.069	0.802	±	0.07	0.802	±	0.097	0.821	±	0.075	0.847	±	0.089	0.0586	0.3082		0.293
Glycine	0.279	±	0.046	0.267	±	0.033	0.297	±	0.051	0.276	±	0.032	0.285	±	0.047	0.253	±	0.024	0.0078	0.2361		0.5944
Histidine	0.086	±	0.013	0.085	±	0.008	0.08	±	0.01	0.081	±	0.011	0.081	±	0.009	0.077	±	0.008	0.5219	0.0815		0.46
Isoleucine	0.06	±	0.011	0.059	±	0.012	0.061	±	0.011	0.059	±	0.012	0.057	±	0.012	0.061	±	0.013	0.9908	0.9335		0.5886
Lactic acid	1.976	±	0.32	1.904	±	0.318	2.129	±	0.448	2.122	±	0.433	1.844	±	0.266	1.974	±	0.225	0.7908	0.0494		0.4761
Leucine	0.135	±	0.023	0.127	±	0.023	0.124	±	0.025	0.127	±	0.02	0.12	±	0.018	0.139	±	0.022	0.1554	0.6172		0.0092
N,N-Dimethylglycine	0.007	±	0.001	0.007	±	0.001	0.008	±	0.001	0.007	±	0.001	0.008	±	0.002	0.007	±	0.001	0.0763	0.3673		0.1926
Phenylalanine	0.058	±	0.007	0.056	±	0.01	0.055	±	0.006	0.05	±	0.008	0.057	±	0.01	0.049	±	0.008	0.0002	0.2051		0.0886
Pyruvic acid	0.081	±	0.019	0.066	±	0.015	0.094	±	0.029	0.066	±	0.013	0.079	±	0.023	0.068	±	0.014	< 0.0001	0.4141		0.0797
Sarcosine	0.006	±	0.003	0.007	±	0.003	0.006	±	0.003	0.007	±	0.003	0.006	±	0.003	0.008	±	0.003	0.055	0.92		0.5491
Succinic acid	0.005	±	0.002	0.011	±	0.008	0.005	±	0.003	0.013	±	0.01	0.004	±	0.001	0.01	±	0.007	0.0002	0.5521		0.8223
Trimethylamine N-oxide	0.023	±	0.015	0.031	±	0.015	0.022	±	0.016	0.025	±	0.016	0.022	±	0.011	0.023	±	0.011	0.1052	0.5576		0.4059
Tyrosine	0.056	±	0.006	0.052	±	0.006	0.054	±	0.009	0.049	±	0.008	0.052	±	0.005	0.046	±	0.007	< 0.0001	0.0873		0.7526
Valine	0.288	±	0.06	0.28	±	0.041	0.26	±	0.029	0.273	±	0.031	0.27	±	0.041	0.254	±	0.025	0.4503	0.2274		0.0788
Concentrations (mM) are provided as mean ± standard deviation. The results were analyzed by using a mixed effects model (REML), and significant p-values are shown in bold. Pairwise comparisons were analyzed by Tukey's multiple comparisons test, and values significantly different for cricket, pea or whey are marked by different superscript letters.

Table 2

Urine metabolites determined at baseline and 4 hours after protein ingestion and exercise (post).
	Cricket						Pea						Whey						P-value
	Baseline			Post			Baseline			Post			Baseline			Post			Time	Protein	Time x protein
2-Furoylglycine	0.023	±	0.014	0.012	±	0.02	0.027	±	0.024	0.017	±	0.012	0.021	±	0.019	0.02	±	0.019	0.0571	0.6227	0.4903
3-Aminoisobutyrate	0.292	±	0.138	0.27	±	0.125	0.325	±	0.222	0.423	±	0.258^c	0.285	±	0.164	0.262	±	0.134^b	0.3305	0.1337	0.1043
3-Hydroxybutyrate	0.124	±	0.073	0.147	±	0.083	0.119	±	0.057	0.134	±	0.104	0.173	±	0.17	0.161	±	0.168	0.7127	0.3493	0.8249
Acetate	0.136	±	0.058	0.142	±	0.042	0.134	±	0.066	0.152	±	0.079	0.133	±	0.04	0.117	±	0.039	0.7381	0.4311	0.369
Acetoacetate	0.234	±	0.11	0.267	±	0.156	0.238	±	0.143	0.395	±	0.612	0.231	±	0.119	0.493	±	1.008	0.1581	0.6916	0.6761
Acetone	0.08	±	0.048	0.087	±	0.047	0.062	±	0.041	0.084	±	0.041	0.088	±	0.061	0.065	±	0.033	0.8118	0.6639	0.1202
Alanine	0.375	±	0.132	0.362	±	0.075	0.363	±	0.12	0.395	±	0.221	0.354	±	0.097	0.357	±	0.11	0.9324	0.7812	0.8467
Allantoin	0.156	±	0.052	0.166	±	0.078	0.136	±	0.078	0.123	±	0.059	0.128	±	0.069	0.138	±	0.071	0.9581	0.1686	0.6557
Betaine	0.181	±	0.113	0.178	±	0.06	0.152	±	0.106	0.155	±	0.062	0.194	±	0.105	0.162	±	0.074	0.5415	0.4164	0.6902
Carnosine	0.586	±	0.619	0.659	±	0.57	0.532	±	0.43	0.839	±	0.616	0.763	±	0.707	0.808	±	0.8	0.2326	0.6248	0.6761
Citrate	3.329	±	1.573	3.572	±	1.559	2.481	±	1.383	3.143	±	1.317	3.26	±	1.828	3.18	±	1.633	0.383	0.2555	0.6117
Creatine	0.897	±	0.29	0.789	±	0.181	0.787	±	0.37	0.812	±	0.269	0.875	±	0.327	0.88	±	0.349	0.7036	0.7351	0.4268
Dimethylamine	0.618	±	0.039	0.668	±	0.056^b	0.616	±	0.055	0.606	±	0.056^a	0.654	±	0.07	0.633	±	0.071	0.6854	0.1153	0.0063
Formate	0.333	±	0.149	0.317	±	0.138	0.248	±	0.166	0.276	±	0.147	0.283	±	0.151	0.275	±	0.132	0.9084	0.2824	0.8535
Fumarate	0.005	±	0.004	0.003	±	0.005	0.007	±	0.006	0.006	±	0.007	0.004	±	0.005	0.003	±	0.004	0.352	0.0688	0.9269
Glucose	0.491	±	0.135	0.487	±	0.127	0.503	±	0.141	0.492	±	0.129	0.513	±	0.118	0.507	±	0.11	0.734	0.8437	0.9831
Glycerophospho-choline	2.46	±	1.642	2.288	±	1.829	1.519	±	0.959	1.739	±	0.863	1.718	±	1.159	1.955	±	1.209	0.6931	0.1069	0.6915
Glycine	1.581	±	0.35	1.571	±	0.477	1.57	±	0.596	1.967	±	1.054	1.579	±	0.686	1.537	±	0.536	0.3887	0.3968	0.2958
Glycylproline	0.602	±	0.213	0.714	±	0.227^b	0.528	±	0.219	0.523	±	0.154^a	0.588	±	0.298	0.531	±	0.193	0.7067	0.0618	0.2957
Guanidoacetate	0.549	±	0.16	0.5	±	0.128	0.494	±	0.19	0.464	±	0.129	0.434	±	0.119	0.457	±	0.091	0.4494	0.183	0.3947
Hippuric acid	1.53	±	0.735	1.1	±	0.55	1.251	±	0.834	1.226	±	0.741	1.075	±	0.487	0.996	±	0.648	0.1476	0.2967	0.4048
Histidine	1.154	±	0.218	1.209	±	0.438	1.159	±	0.454	1.356	±	0.795	1.494	±	0.7	1.227	±	0.511	0.9713	0.4647	0.2114
Hypoxanthine	0.279	±	0.238	0.214	±	0.076	0.195	±	0.091	0.245	±	0.185	0.284	±	0.235	0.182	±	0.122	0.2625	0.8249	0.1693
Lactate	0.627	±	0.803	0.196	±	0.045	0.691	±	1.366	0.711	±	1.282	0.441	±	0.569	0.209	±	0.086	0.2291	0.1888	0.5888
N,N-Dimethylglycine	0.059	±	0.023	0.067	±	0.021	0.068	±	0.023	0.065	±	0.02	0.068	±	0.024	0.068	±	0.034	0.7175	0.7341	0.5787
Phenylalanine	1.044	±	0.305	1.085	±	0.397^c	0.92	±	0.504	0.997	±	0.595	0.709	±	0.37	0.677	±	0.311^a	0.6788	0.0144	0.752
Succinate	0.043	±	0.015	0.043	±	0.023	0.031	±	0.014	0.038	±	0.018	0.042	±	0.02	0.044	±	0.013	0.3775	0.0911	0.6446
Trigonelline	0.076	±	0.042	0.073	±	0.048	0.09	±	0.048	0.072	±	0.026	0.089	±	0.046	0.082	±	0.051	0.3002	0.6115	0.7933
Trimethylamine	0.022	±	0.011	0.026	±	0.012	0.019	±	0.012	0.019	±	0.012	0.019	±	0.013	0.022	±	0.008	0.2728	0.2651	0.8424
Trimethylamine N-oxide	0.865	±	0.584	0.989	±	0.558^bc	0.607	±	0.234	0.558	±	0.191^a	0.627	±	0.328	0.6	±	0.342^a	0.7669	0.0045	0.5381
Tyrosine	0.192	±	0.048	0.205	±	0.044	0.221	±	0.069	0.247	±	0.151	0.199	±	0.076	0.2	±	0.052	0.3392	0.3346	0.7442
Urea	63.64	±	28.352	66.78	±	21.597	53.042	±	29.461	67.012	±	21.848	62.113	±	18.1	61.784	±	26.382	0.2572	0.6949	0.456
Valine	0.066	±	0.021	0.068	±	0.019	0.056	±	0.01	0.061	±	0.022	0.065	±	0.019	0.06	±	0.011	0.7764	0.2044	0.5634
Trans-Aconitic acid	0.305	±	0.279	0.499	±	0.324	0.306	±	0.285	0.407	±	0.249	0.325	±	0.217	0.433	±	0.359	0.0231	0.8218	0.7761
τ-Methylhistidine	0.391	±	0.186	0.396	±	0.121	0.332	±	0.146	0.366	±	0.099	0.407	±	0.177	0.345	±	0.161	0.7582	0.5324	0.3281
Relative concentrations normalized to creatinine are provided as mean ± standard deviation. Results were analyzed by using a mixed effects model (REML), and significant p-values are shown in bold. Pairwise comparison were analyzed by Tukey's multiple comparisons test and values significantly different for cricket, pea or whey are marked by different superscript letters.

Table 3

Muscle metabolites determined at baseline and 3 hours after protein ingestion and exercise (post).
	Cricket																		P-value
	Baseline			Post			Baseline			Post			Baseline			Post			Time	Protein	Time x protein
Adenosine diphosphate	0.252	±	0.059	0.231	±	0.109	0.26	±	0.119	0.255	±	0.078	0.26	±	0.079	0.294	±	0.096	0.167	0.3286	0.0972
Alanine	0.093	±	0.022	0.091	±	0.054	0.118	±	0.044	0.096	±	0.036	0.107	±	0.04	0.131	±	0.056	0.542	0.0983	0.113
Arginine	0.023	±	0.008	0.02	±	0.012^c	0.025	±	0.009	0.021	±	0.008	0.021	±	0.006	0.024	±	0.006^a	0.402	0.3325	0.1948
Carnitine	0.267	±	0.086	0.232	±	0.12	0.23	±	0.106	0.215	±	0.089	0.274	±	0.107	0.305	±	0.118	0.453	0.4197	0.1034
Carnosine	0.339	±	0.074	0.385	±	0.177	0.377	±	0.168	0.377	±	0.154	0.331	±	0.114	0.45	±	0.234	0.253	0.1228	0.0371
Choline	0.06	±	0.029	0.074	±	0.041^c	0.083	±	0.056	0.084	±	0.052	0.071	±	0.03	0.122	±	0.051^a	0.036	0.1734	0.005
Creatine	0.757	±	0.125	0.677	±	0.41	0.688	±	0.279	0.627	±	0.242	0.709	±	0.232	0.825	±	0.384	0.923	0.893	0.0161
Creatine phosphate	1.146	±	0.328	1.125	±	0.508	1.247	±	0.622	1.224	±	0.361	1.17	±	0.467	1.472	±	0.577	0.251	0.1527	0.0882
Ethanol	0.029	±	0.051	0.035	±	0.08	0.069	±	0.181	0.281	±	0.66	0.029	±	0.074	0.072	±	0.25	0.252	0.4087	0.3592
Glutamate	0.134	±	0.047	0.122	±	0.062	0.137	±	0.068	0.105	±	0.041^c	0.144	±	0.061	0.168	±	0.069^b	0.279	0.4156	0.0075
Glutamine	0.097	±	0.038	0.074	±	0.036	0.076	±	0.038	0.073	±	0.031	0.08	±	0.04	0.098	±	0.037	0.425	0.2312	0.0231
Glycine	0.032	±	0.007	0.029	±	0.013	0.04	±	0.018	0.032	±	0.013	0.035	±	0.012	0.039	±	0.021	0.762	0.1409	0.2767
Homocysteine	0.047	±	0.024^b	0.043	±	0.019	0.079	±	0.039^a	0.069	±	0.034	0.066	±	0.028	0.064	±	0.029	0.214	0.0149	0.4409
Isocitrate	0.08	±	0.023	0.09	±	0.051	0.1	±	0.051	0.098	±	0.049	0.092	±	0.038	0.117	±	0.068	0.522	0.2806	0.1342
Isoleucine	0.006	±	0.002	0.004	±	0.002	0.005	±	0.002	0.004	±	0.002	0.005	±	0.002	0.006	±	0.002	0.395	0.5674	0.0969
Lysine	0.016	±	0.005	0.015	±	0.006	0.017	±	0.009	0.014	±	0.005	0.016	±	0.005	0.019	±	0.006	0.592	0.3498	0.0102
Lactate	0.275	±	0.161	0.201	±	0.136	0.176	±	0.083	0.213	±	0.101	0.267	±	0.192	0.324	±	0.229	0.867	0.9556	0.0011
Leucine	0.011	±	0.003	0.01	±	0.004	0.011	±	0.005	0.01	±	0.003	0.011	±	0.003	0.015	±	0.01	0.694	0.4362	0.006
Methionine	0.059	±	0.027	0.068	±	0.045^c	0.081	±	0.06	0.083	±	0.054	0.068	±	0.033	0.121	±	0.046^a	0.06	0.1964	0.0018
Valine	0.017	±	0.005	0.016	±	0.007	0.018	±	0.007	0.014	±	0.005	0.016	±	0.005	0.018	±	0.005	0.744	0.8551	0.1192
myo-Inositol	0.08	±	0.026	0.07	±	0.027	0.079	±	0.038	0.058	±	0.025^c	0.087	±	0.054	0.096	±	0.043^b	0.576	0.5627	0.0117
Relative concentrations normalized to sample mass are provided as mean ± standard deviation. Results were analyzed by using a mixed effects model (REML), and significant p-values are shown in bold. Pairwise comparison were analyzed by Tukey's multiple comparisons test, and values significantly different for cricket, pea or whey protein are marked by different superscript letters.

Blood metabolites

The mixed effects model analysis of the individual metabolites in blood revealed an effect of time (baseline vs. post) for 3-hydroxybutyric acid, acetic acid, acetoacetic acid, acetone, dimethylsulfone, glucose, glycine, phenylalanine, pyruvic acid, succinic acid, and tyrosine (Table 1). Thus, the concentrations of 3-hydroxybutyric acid, acetoacetic acid and acetone were higher in the post intervention blood sample than at baseline. In contrast, the concentrations of acetic acid and phenylalanine and tyrosine were lower in the post intervention blood samples than in the baseline samples.

In addition, the statistical analysis showed an effect of protein source (cricket, pea, whey) in plasma concentrations of acetic acid, creatine, lactic acid, and a time x protein interaction effect regarding the changes in the metabolites creatinine, glucose, and leucine.

Pairwise comparisons showed that the concentration of acetic acid was increased after intake of pea protein compared to whey protein, while the creatine concentration was increased after intake of cricket protein compared to whey protein. The glucose concentration increased after ingestion of cricket protein compared to after ingestion of pea and whey protein. Unexpectedly, the concentration in creatine was higher at baseline in the group who afterwards ingested cricket protein compared to the group who afterwards ingested whey protein.

Urine metabolites

Of the 35 quantified urine metabolites, a higher concentration of trans-aconitic acid was found after the intervention than at baseline (Table 2). Moreover, an effect of protein source on the concentration of phenylalanine and TMAO was observed, and cricket protein ingestion resulted in a higher TMAO concentration compared to both pea and whey protein ingestion. Pea protein ingestion increased the concentration of phenylalanine compared to whey protein. For dimethylamine, a time x protein interaction effect was found, and intake of cricket protein increased the concentration of dimethylamine compared to pea protein.

Muscle metabolites

In total, 21 metabolites were annotated and quantified in the muscle tissue extracts (Table 3). Mixed-effects analysis revealed a higher concentration of choline after the intervention compared to baseline. In addition, significant time x protein interaction effects were found for choline, glutamate, lysine, leucine, methionine, and myo-inositol. Pairwise comparisons showed that whey protein ingestion increased the concentration of choline and methionine in the muscle compared to ingestion of cricket protein. In addition, whey protein intake increased the concentration of glutamate and myo-inositol compared to ingestion of pea protein.

The main finding of this study is that NMR-based metabolomics can be used to give a more complete elucidation of the metabolic state of the body in response to a stimulus when blood, urine and muscle tissue samples are collected simultaneously. In addition, NMR-based metabolomics was able to detect differential responses to the ingestion of different types of protein. In the skeletal muscle, the concentrations of methionine, glutamate, and myo-inositol were higher after intake of whey compared to cricket or pea protein ingestion. Moreover, the blood metabolome revealed changes to a more ketogenic state three hours after exercise.

In blood, NMR-based metabolomics enabled the detection and quantification of 25 metabolites, while 35 metabolites were quantified in urine and 21 in muscle tissue. This number of metabolites is similar to earlier reports on blood (Jiménez et al., 2018) and urine (Lima et al., 2020). In contrast to NMR metabolomics applied on urine and blood, studies of the muscle metabolome are sparse, but a study employing ¹H NMR spectroscopy on methanol/chloroform/water extracts of mice muscle tissue reported that that were able to detect a total of 38 metabolites (Bruno et al., 2018).

When comparing the urine, blood, and muscle metabolome in the present study, it was evident that the different sample types provided different supplementary information. For blood, the most evident effects of combining protein intake and resistance exercise included increases in 3-aminobutyric acid, acetoacetic acid, acetone, and succinic acid concentrations and a decreased concentration of pyruvic acid. Acetoacetic acid and acetone are ketone bodies produced in the liver during reduced carbohydrate availability and serve as an alternative fuel source for peripheral tissues, including the brain, heart, and skeletal muscle. Ketone bodies are oxidized as a fuel source during exercise and are reported to be markedly elevated during the post-exercise recovery period (Evans et al., 2017). The increase in ketone bodies indicates a more ketogenic metabolic state of the body which aligns with that the participants were tested after overnight fasting, performed energy demanding exercise and only ingested a relatively small bolus of protein during the experimental day. The increase in succinic acid concentration in blood is most likely related to an increase in the flow of the Krebs cycle. A possible increase in the Krebs cycle flow may be related to the requirement for phosphocreatine resynthesis, a critical energy reserve during resistance exercise. The observed decrease in pyruvic acid is opposite to a previous study where an increase in serum pyruvic acid was observed during the first 60 min after a resistance exercise bout (Berton et al., 2017). However, the divergent findings may be ascribed to we analyzed a tissue sample collected three hours after exercise in contrast to one hour post exercise in Berton et al (2017). Moreover, an immediate increase in pyruvic acid occurs during intense exercise as a result of the high energy demands required, causing the mitochondria to amplify anaerobic metabolism instead of oxidizing pyruvate. As such, pyruvic acid is increased in a short term-period after intense exercise (⁓60 min), but this might be followed by a subsequent decrease once the mitochondria’s ability to oxidize pyruvate is reestablished.

An effect of protein source was identified on leucine concentration in the blood as leucine was higher four hours after ingestion of whey protein, whereas the leucine concentration did not differ from baseline after cricket and pea protein ingestion. This result most likely reflects the higher leucine content in the whey protein compared to the other sources (cricket 0.98 g/100g, pea 1.19 g/100g, whey 1.62 g /100g). This reasoning is supported by our previous analyses showing that whey protein resulted in higher plasma levels of leucine 20, 40 and 60 minutes after protein ingestion compared to the two other protein sources (Lanng et al., 2022).

While the human muscle metabolome has been analyzed using gas chromatography-mass spectrometry (GC-MS) (Sato et al., 2018) and capillary electrophoresis-mass spectrometry (CE-MS) (Saoi et al., 2019), NMR-based analyses of the human muscle metabolome are sparse. However, recently changes in the NMR-derived human muscle metabolome during chronic limb threatening ischemia was reported (Khattri et al., 2021) showing that at least 14 hydrophilic metabolites were altered during ischemia. The present study revealed interactions between time and protein sources for some of the identified muscle metabolites including myo-inositol, glutamate, lysine, leucine, and methionine. Myo-inositol was increased after whey protein ingestion compared to after the ingestion of pea protein. Whether the higher myo-inositol found after whey protein intake originates directly from the protein source or is a result of an endogenous production after whey protein ingestion, remains unknown. However, intriguingly, myo-inositol has been shown to be able to reverse insulin resistance in persons with metabolic syndrome as it’s derivate shows insulin-like actions in vivo (Paul et al., 2016). Thus, the effect of whey protein ingestion on muscular myo-inositol could indicate an accompanying beneficial effect which may have positive impact on insulin resistance. The amino acids; glycine and methionine, showed higher muscle concentrations following ingestion of whey protein compared to after ingestion of cricket or pea protein. This discovery might be related to whey protein providing a higher supply and bioavailability of these amino acids. This is supported by higher serum concentrations of glycine and methionine in the immediate period following ingestion of whey protein compared to the two other protein sources (Lanng et al., 2022). Nevertheless, Western blotting analyses of muscle tissue showed no significant difference between the protein sources in protein expression of signaling parameters related to activation of the mTORC1 signaling pathway important for enhancing myofibrillar protein synthesis (Lanng et al., 2022). Thus, whether these higher amino acid concentrations measured in the blood and muscle tissue after whey protein ingestion exert any physiological role remains unknown.

Several NMR analyses of human urine collected after exercise have been reported, including studies exploring the effect of endurance exercise bicycling (Enea et al., 2010; Kistner et al., 2020; Krug et al., 2012; Ma et al., 2015; Miccheli et al., 2009; Mukherjee et al., 2014; Neal et al., 2013; Pechlivanis et al., 2010a; Pechlivanis et al., 2015). In addition, few studies have explored the effect of endurance exercise combined with resistance exercise (Cronin et al., 2018; Pellegrino et al., 2022; Wang et al., 2015), while to our knowledge only one study has explored effects of resistance exercise on the urinary metabolome (Sheedy et al., 2014). In the current study, several urine metabolites were found to be changed by the intervention compared to baseline values. Thus, the concentration of trans-aconitic acid was higher in post intervention samples. In contrast, Cronin et al., found that endurance and resistance exercise combined with ingestion of whey protein increased trans-aconitic acid compared to whey protein alone (Cronin et al., 2018). In another study by Kistner et al., trans-aconitic acid was also increased 15–30 minutes after finalizing endurance exercise in adults (Kistner et al., 2020). The authors speculated that this was due to the ingestion of wheat in the controlled breakfast before the exercise intervention (Kistner et al., 2020) as the presence of trans-aconitic acid has been reported in some plants such as soybean and wheat (Yuhara et al., 2015). In line, we speculate that trans-aconitic acid also originates from the protein sources in the present study. Cricket protein ingestion resulted in higher levels of dimethylamine and glycylproline in the urine compared to ingestion of pea protein and increased phenylalanine concentrations compared to ingestion of whey protein. The concentration of TMAO in the urine was also increased after exercise combined with ingestion of cricket protein compared to exercise combined with ingestion of either pea or whey protein. In contrast, three other studies have found a decreased concentration of TMAO in urine after exercise (Ma et al., 2015; Mukherjee et al., 2014; Pechlivanis et al., 2015), while two other studies similar to our findings have reported a rise in TMAO after exercise (Cronin et al., 2018; Jang et al., 2018). Several studies have reported that ingestion of fish (Schmedes et al. 2016; Wang et al. 2022) and meat (Dhakal et al., 2022; Thøgersen et al., 2020; Wang et al., 2019) increased urinary excretion of TMAO. Nevertheless, TMAO and dimethylamine have also been proposed as potential biomarkers of pulse consumption (chickpeas, lentils, and bean) (Madrid-Gambin et al., 2017). In addition, recently plant-based diets (Mediterranean, vegetarian and vegan) have been reported to increase TMAO levels (Lombardo et al., 2022). Furthermore, correlations between levels of TMAO in urine and blood, and ingestion of selected food groups including fish, vegetables, and whole-grain products and have also been shown (Costabile et al., 2021). From this, it is evident that the use of TMAO as a simple biomarker is limited, as it seems to be associated with ingestion of a wide variety of different foods and different studies report different responses to exercise.

In conclusion, the present study demonstrated that the human blood, urine, and muscle metabolomes provide supplementary information about the endogenous response to a combined intervention with resistance exercise and a protein bolus. We show that the human blood metabolome after overnight fasting followed by resistance exercise, a small bolus of protein and further 3 hours of recovery reflected that the body was in a more ketogenic state than when measured in morning prior to exercise and protein intake. In addition, the protein source ingested affected the muscle metabolome as reflected in the concentration of myo-inositol and some amino acids. In urine, TMAO was found to be increased after ingestion cricket protein compared to pea and whey protein. Nevertheless, it remains questionable whether TMAO is a potential urinary biomarker for insect protein ingestion.

Acknowledgement

This research was supported by grants from A.P. Møller Fonden, Denmark and Beckett Fonden, Denmark (#19-2-4811). The present study was part of Sofie Kaas Lanng’s Ph.D. project, which was funded by Centre for Innovative Food Research (CiFOOD), Aarhus University and HCB’s EliteForsk grant (#6161-00016B). NMR data were generated though accessing research infrastructure at Aarhus University, including FOODHAY (Food and Health Open Innovation Laboratory, Danish Roadmap for Research Infrastructure). The authors thank Janni Mosgaard Jensen and Gitte Kaiser Hartvigsen for technical support during the experimental days.

Conflicts of Interest Statement

This study was funded by the A.P. Møller Fonden, Copenhagen, Denmark and Beckett Foundation, Denmark (19-2-4811). The present study was part of Sofie Kaas Lanng’s PhD project, which was funded by Centre for Innovative Food Research (CiFOOD), Aarhus University and HCB’s Elite Research grant (6161-00016B). NMR data were generated though accessing research infrastructure at Aarhus University, including FOODHAY (Food and Health Open Innovation Laboratory, Danish Roadmap for Research Infrastructure).

Author contributions

SKL, MO, MH and HCB designed the experiment. SKL, MO, FTJ and TR conducted the experiments. SKL analysed the samples and performed the statistical analysis. SKL and HCB wrote the manuscript while all authors have read, edited, and approved the manuscript.

Ethical statement

All procedures performed in studies involving human participants were in accordance with the ethical standards the Declaration of Helsinki and the study was approved by The Central Denmark Region Committees on Health Research Ethics (journal nr. M-2019-291-19). Informed consent was obtained from all individual participants included in the study.

Data Availability

Data reported in this paper will be made available upon request.

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No competing interests reported.

Acute changes in the metabolome following resistance exercise combined with intake of different protein sources (cricket, pea, whey)

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and methods

Experimental protocol

Muscle Biopsies

Preparation of muscle, urine, and serum samples for proton nuclear magnetic resonance (¹H -NMR) spectroscopy

Quantification of metabolites in muscle, urine and blood by ¹H-NMR spectroscopy

Statistics

Results

Blood metabolites

Urine metabolites

Muscle metabolites

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1

Acute changes in the metabolome following resistance exercise combined with intake of different protein sources (cricket, pea, whey)

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and methods

Experimental protocol

Muscle Biopsies

Preparation of muscle, urine, and serum samples for proton nuclear magnetic resonance (1H -NMR) spectroscopy

Quantification of metabolites in muscle, urine and blood by 1H-NMR spectroscopy

Statistics

Results

Blood metabolites

Urine metabolites

Muscle metabolites

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1

Preparation of muscle, urine, and serum samples for proton nuclear magnetic resonance (¹H -NMR) spectroscopy

Quantification of metabolites in muscle, urine and blood by ¹H-NMR spectroscopy