The mitochondrial tRNA-derived fragment, mt-tRF-Leu^TAA, couples mitochondrial metabolism to insulin secretion.

The contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-Leu <sup>TAA</sup> ) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-Leu <sup>TAA</sup> , on mitochondrial metabolism and pancreatic islet functions. We used antisense oligonucleotides to reduce mt-tRF-Leu <sup>TAA</sup> levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-Leu <sup>TAA</sup> inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-Leu <sup>TAA</sup> silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. Our study unveils a modulation of mt-tRF-Leu <sup>TAA</sup> levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-Leu <sup>TAA</sup> interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-Leu <sup>TAA</sup> in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-Leu <sup>TAA</sup> impairs insulin secretion of rat and human pancreatic β-cells. Our findings indicate that mt-tRF-Leu <sup>TAA</sup> interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion

Bimodal Effect on Pancreatic β-Cells of Secretory Products From Normal or Insulin-Resistant Human Skeletal Muscle

OBJECTIVE: Type 2 diabetes is characterized by insulin resistance with a relative deficiency in insulin secretion. This study explored the potential communication between insulin-resistant human skeletal muscle and primary (human and rat) beta-cells. RESEARCH DESIGN AND METHODS: Human skeletal muscle cells were cultured for up to 24 h with tumor necrosis factor (TNF)-alpha to induce insulin resistance, and mRNA expression for cytokines was analyzed and compared with controls (without TNF-alpha). Conditioned media were collected and candidate cytokines were measured by antibody array. Human and rat primary beta-cells were used to explore the impact of exposure to conditioned media for 24 h on apoptosis, proliferation, short-term insulin secretion, and key signaling protein phosphorylation and expression. RESULTS: Human myotubes express and release a different panel of myokines depending on their insulin sensitivity, with each panel exerting differential effects on beta-cells. Conditioned medium from control myotubes increased proliferation and glucose-stimulated insulin secretion (GSIS) from primary beta-cells, whereas conditioned medium from TNF-alpha-treated insulin-resistant myotubes (TMs) exerted detrimental effects that were either independent (increased apoptosis and decreased proliferation) or dependent on the presence of TNF-alpha in TM (blunted GSIS). Knockdown of beta-cell mitogen-activated protein 4 kinase 4 prevented these effects. Glucagon-like peptide 1 protected beta-cells against decreased proliferation and apoptosis evoked by TMs, while interleukin-1 receptor antagonist only prevented the latter. CONCLUSIONS: Taken together, these data suggest a possible new route of communication between skeletal muscle and beta-cells that is modulated by insulin resistance and could contribute to normal beta-cell functional mass in healthy subjects, as well as the decrease seen in type 2 diabetes

siRNA-Mediated Reduction of Inhibitor of Nuclear Factor-κB Kinase Prevents Tumor Necrosis Factor-α–Induced Insulin Resistance in Human Skeletal Muscle

OBJECTIVE—Proinflammatory cytokines contribute to systemic low-grade inflammation and insulin resistance. Tumor necrosis factor (TNF)-α impedes insulin signaling in insulin target tissues. We determined the role of inhibitor of nuclear factor-κB kinase (IKK)β in TNF-α–induced impairments in insulin signaling and glucose metabolism in skeletal muscle

Effect of Testosterone on Insulin Stimulated IRS1 Ser Phosphorylation in Primary Rat Myotubes—A Potential Model for PCOS-Related Insulin Resistance

Polycystic ovary syndrome (PCOS) is characterized by a hyperandrogenic state and frequently develops skeletal muscle insulin resistance. We determined whether testosterone adversely affects insulin action by increasing serine phosphorylation of IRS-1(636/639) in differentiated rat skeletal muscle myotubes. The phosphorylation of Akt, mTOR, and S6K, downstream targets of the PI3-kinase-IRS-1 complex were also studied.Primary differentiated rat skeletal muscle myotubes were subjected to insulin for 30 min after 16-hour pre-exposure to either low (20 ng/ml) or high (200 ng/ml) doses of testosterone. Protein phosphorylation of IRS-1 Ser(636/639), Akt Ser(473), mTOR-Ser(2448), and S6K-Thr(389) were measured by Western blot with signal intensity measured by immunofluorescence.Cells exposed to 100 nM of insulin had increased IRS-1 Ser(636/639) and Akt Ser(473) phosphorylation. Cells pre-exposed to low-dose testosterone had significantly increased insulin-induced mTOR-Ser(2448) and S6K-Thr(389) phosphorylation (p<0.05), and further increased insulin-induced IRS-1 Ser(636/639) phosphorylation (p = 0.042) compared to control cells. High-dose testosterone pre-exposure attenuated the insulin-induced mTOR-Ser(2448) and S6K-Thr(389) phosphorylation.The data demonstrated an interaction between testosterone and insulin on phosphorylation of intracellular signaling proteins, and suggests a link between a hyperandrogenic, hyperinsulinemic environment and the development of insulin resistance involving serine phosphorylation of IRS-1 Ser(636/639). These results may guide further investigations of potential mechanisms of PCOS-related insulin resistance

Lymphocyte-Derived Exosomal MicroRNAs Promote Pancreatic β Cell Death and May Contribute to Type 1 Diabetes Development.

Type 1 diabetes is an autoimmune disease initiated by the invasion of pancreatic islets by immune cells that selectively kill the β cells. We found that rodent and human T lymphocytes release exosomes containing the microRNAs (miRNAs) miR-142-3p, miR-142-5p, and miR-155, which can be transferred in active form to β cells favoring apoptosis. Inactivation of these miRNAs in recipient β cells prevents exosome-mediated apoptosis and protects non-obese diabetic (NOD) mice from diabetes development. Islets from protected NOD mice display higher insulin levels, lower insulitis scores, and reduced inflammation. Looking at the mechanisms underlying exosome action, we found that T lymphocyte exosomes trigger apoptosis and the expression of genes involved in chemokine signaling, including Ccl2, Ccl7, and Cxcl10, exclusively in β cells. The induction of these genes may promote the recruitment of immune cells and exacerbate β cell death during the autoimmune attack. Our data point to exosomal-miRNA transfer as a communication mode between immune and insulin-secreting cells

A circular RNA generated from an intron of the insulin gene controls insulin secretion.

Fine-tuning of insulin release from pancreatic β-cells is essential to maintain blood glucose homeostasis. Here, we report that insulin secretion is regulated by a circular RNA containing the lariat sequence of the second intron of the insulin gene. Silencing of this intronic circular RNA in pancreatic islets leads to a decrease in the expression of key components of the secretory machinery of β-cells, resulting in impaired glucose- or KCl-induced insulin release and calcium signaling. The effect of the circular RNA is exerted at the transcriptional level and involves an interaction with the RNA-binding protein TAR DNA-binding protein 43 kDa (TDP-43). The level of this circularized intron is reduced in the islets of rodent diabetes models and of type 2 diabetic patients, possibly explaining their impaired secretory capacity. The study of this and other circular RNAs helps understanding β-cell dysfunction under diabetes conditions, and the etiology of this common metabolic disorder

A circular RNA generated from an intron of the insulin gene controls insulin secretion

Fine-tuning of insulin release from pancreatic β-cells is essential to maintain blood glucose homeostasis. Here, we report that insulin secretion is regulated by a circular RNA containing the lariat sequence of the second intron of the insulin gene. Silencing of this intronic circular RNA in pancreatic islets leads to a decrease in the expression of key components of the secretory machinery of β-cells, resulting in impaired glucose- or KCl-induced insulin release and calcium signaling. The effect of the circular RNA is exerted at the transcriptional level and involves an interaction with the RNA-binding protein TAR DNA-binding protein 43 kDa&nbsp;(TDP-43). The level of this circularized intron is reduced in the islets of rodent diabetes models and of type 2 diabetic patients, possibly explaining their impaired secretory capacity. The study of this and other circular RNAs helps understanding β-cell dysfunction under diabetes conditions, and the etiology of this common metabolic disorder

In Vitro Proliferation of Adult Human Beta-Cells

A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1) analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide