Downregulation of the mitochondrial tRNA-derived fragment mt-tRF-Leu^TAA enhances skeletal muscle insulin sensitivity

The mitochondrial tRNA-derived fragment mt-tRF-Leu^TAA couples mitochondrial metabolism to insulin secretion. While its role in pancreatic β-cell function is well established, its broader impact on multiorgan glucose homeostasis remains unclear. In insulin target tissues, the presence, regulation, and mechanism of action of mt-tRF-Leu^TAA are entirely unexplored. This study addresses this gap by investigating the impact of diet, nutritional status, and diabetes on mt-tRF-Leu^TAA regulation and by assessing its role in insulin sensitivity. We examined mt-tRF-Leu^TAA levels in different insulin target tissues, including skeletal muscle, liver, and epididymal white adipose tissue, of rodents under physiological and pathological conditions. In skeletal muscle myotubes, we combined subcellular fractionation, antisense oligonucleotide-mediated knockdown, and glucose uptake assays to determine mt-tRF-Leu^TAA's mitochondrial localization and its influence on insulin sensitivity. mt-tRF-Leu^TAA levels in mouse skeletal muscle decreased twofold in response to fasting. In myotubes, this tRNA fragment was enriched in mitochondria, and its downregulation enhanced glucose uptake. While the levels of mt-tRF-Leu^TAA remained unchanged in insulin target tissues of diabetic mice, we observed a skeletal muscle-specific downregulation of mt-tRF-Leu^TAA in young adult rats exhibiting insulin hypersensitivity. This study identifies mt-tRF-Leu^TAA as a candidate regulator of skeletal muscle insulin response. By modulating both insulin secretion and action, mt-tRF-Leu^TAA appears to play a notable role in systemic metabolic control and may represent a promising target for diabetes treatment.<b>NEW & NOTEWORTHY</b> Fasting downregulates levels of mt-tRF-Leu^TAA in skeletal muscle. While this small RNA fragment is enriched in the mitochondria of myotubes, inhibition of mt-tRF-Leu^TAA in myotubes enhances insulin-mediated glucose uptake. Consistently, mt-tRF-Leu^TAA is also downregulated in the skeletal muscle of insulin-hypersensitive rats. Together, these findings highlight mt-tRF-Leu^TAA as a key metabolic regulator influencing both insulin secretion and action.