HPLC Confirms Creatine Uptake in Rainbow Trout

Creatine is a vital molecule that helps cells store and use energy, and it relies on a special transporter protein (called SLC6A8 or CT1) to get into cells. A research team discovered that rainbow trout (Oncorhynchus mykiss) have two versions of the gene responsible for making this transporter, which share about 69% of their genetic code. When the team tagged the two resulting proteins (Slc6a8a and Slc6a8b) with fluorescent markers, they could see that both end up sitting in the cell membrane, exactly where a transporter needs to be to do its job. Testing with high performance liquid chromatography (HPLC) then confirmed that the cells were indeed taking up creatine. The team published their findings in the journal Fish Physiology and Biochemistry.¹

Why Does Creatine Transport Matter, and What Makes Fish Different from Mammals in How They Handle It?

In humans, faults in the gene responsible for making the CT1 transporter can cause serious health problems, including a condition called creatine deficiency syndrome, which is associated with developmental delays and intellectual disability.^2-5 In mice, research has shown that a working creatine transporter is also vital for a healthy immune system, affecting how certain immune cells (macrophages) behave, and losing the transporter disrupts the immune response more broadly.^6,7

In fish and other aquatic vertebrates, however, much less is known about how creatine transport works. Fish have evolved their own metabolic strategies to suit life in water, and it turns out their creatine system works quite differently from that of mammals. In mammals, the two steps involved in making creatine happen in different organs. In fish, this separation doesn't exist. Instead, the key enzymes needed to produce creatine are found in high levels in muscle tissue, suggesting that fish make creatine locally in their muscles rather than shipping it in from elsewhere.^8,9 Fish and mammals also handle certain building-block molecules differently, all of which points to fish having their own distinct, muscle-based way of producing creatine.⁸

What Did the Researchers Find Regarding the Two Versions of the Creatine Transporter Gene in Rainbow Trout?

The researchers found that the duplication of the two versions of the gene in question happened deep in the evolutionary history of the trout, in an ancestor shared by all bony fish, before the split between fish and land-dwelling vertebrates. Most vertebrates eventually lost one of the two copies, but fish kept both.¹

Looking at where and when each gene is active, the team found clear differences: both versions were highly active in the heart, but Slc6a8a was the dominant version across most body tissues, while Slc6a8b was notably more active in muscle and in the embryonic cell line used in the study. The activity patterns of both genes also lined up closely with key enzymes involved in creatine production and use, suggesting that fish have developed their own distinct way of managing creatine levels.¹

On a structural level, both proteins had the same basic architecture: 12 segments threading through the cell membrane, along with certain chemical features known to be critical for function. Slc6a8a, however, had an extra piece on its outer surface not seen in Slc6a8b, adding an additional site for a type of chemical modification called glycosylation, which can affect how a protein behaves.¹

“This study,” write the authors of the paper,¹ “provides the first characterization of the duplicated slc6a8 genes in bony fish and suggests their sub-functionalization during teleost evolution possibly reflecting functional specialization of the paralogs. We hypothesize that one variant may primarily mediate creatine uptake, whereas the other could contribute to creatine release in distinct tissues under certain physiological conditions.”

References

1. Borchel, A.; Müller-Eigner, A.; Görs, S. et al. Fuelled by Creatine: Exploring Two Copies of the Creatine Transporter SLC6A8 Gene in Rainbow Trout. Fish Physiol Biochem. 2026, 52 (3), 96. DOI: 10.1007/s10695-026-01723-y
2. 2.Salomons, G. S.; van Dooren, S. J.; Verhoeven, N. M. et al. X-Linked Creatine-Transporter Gene (SLC6A8) Defect: A New Creatine-Deficiency Syndrome. Am J Hum Genet. 2001, 68 (6), 1497-1500. DOI: 10.1086/320595
3. 3.Rosenberg, E. H.; Almeida, L. S.; Kleefstra, T. et al. High Prevalence of SLC6A8 Deficiency in X-Linked Mental Retardation. Am J Hum Genet. 2004, 75 (1), 97-105. DOI: 10.1086/422102
4. 4.Santacruz, L.; Jacobs, D. O. Structural Correlates of the Creatine Transporter Function Regulation: The Undiscovered Country. Amino Acids 2016, 48 (8), 2049-2055. DOI: 10.1007/s00726-016-2206-3
5. 5.Abdennadher, M.; Inati, S. K.; Rahhal, S. et al. Characterization of Seizures and EEG Findings in Creatine Transporter Deficiency Due to SLC6A8 Mutation. Am J Med Genet A 2024,194 (2), 337-345. DOI: 10.1002/ajmg.a.63418
6. 6.Ji, L.; Zhao, X.; Zhang, B. et al. Slc6a8-Mediated Creatine Uptake and Accumulation Reprogram Macrophage Polarization via Regulating Cytokine Responses. Immunity 2019, 51 (2), 272-284.e7. DOI: 10.1016/j.immuni.2019.06.007
7. 7.Samborska, B.; Roy, D. G.; Rahbani, J. F. et al. Creatine Transport and Creatine Kinase Activity is Required for CD8⁺ T Cell Immunity. Cell Rep. 2022, 38 (9), 110446. DOI: 10.1016/j.celrep.2022.110446
8. 8.Borchel, A.; Verleih, M.; Rebl, A. et al. Creatine Metabolism Differs Between Mammals and Rainbow Trout (Oncorhynchus mykiss). Springerplus 2014, 3, 510. DOI: 10.1186/2193-1801-3-510
9. Borchel, A.; Verleih, M.; Kühn, C. et al. Evolutionary Expression Differences of Creatine Synthesis-Related Genes: Implications for Skeletal Muscle Metabolism in Fish. Sci Rep. 2019, 9 (1), 5429. DOI: 10.1038/s41598-019-41907-6