Numerous illnesses, including neurological and mental disorders, have been associated with mitochondrial dysfunction. Disruptions in mitochondrial respiration and energy production have been linked to dysmetabolism of the tryptophan (Trp)‐kynurenine (KYN) pathway, which produces a diverse array of bioactive metabolites. Kynurenic acid (KYNA) is a putative neuroprotectant. The exact mechanisms through which Trp‐KYN metabolic dysregulation affects mitochondrial function remain largely unclear. This study investigates the impact of the genetic deletion of kynurenine aminotransferase (KAT) enzymes, which are responsible for KYNA synthesis, on mitochondrial function, specifically mitochondrial respiration and ATP synthesis, and its potential role in neuropsychiatric pathology. CRISPR/Cas9‐induced knockout mouse strains kat1 ^−/− , kat2 ^−/− , and kat3 ^−/− were generated. Eight‐to‐ten‐week‐old male mice were used, and cerebral and hepatic respiration, complex I‐ and II‐linked oxidative phosphorylation (CI and CII OXPHOS), and complex IV (CIV) activity were measured using high‐resolution respirometry. Mitochondrial membrane potential changes were measured with Fluorescence‐Sensor Blue and safranin dye. KAT knockout mice exhibited significantly lower cerebellar respiration (CI OXPHOS, CII OXPHOS, and CIV activity) compared to wild‐type mice. Lower baseline respiration and attenuated OXPHOS activities were observed in the hippocampus and striatum, particularly in kat2 ^−/− and kat3 ^−/− mice. Non‐neuronal tissues showed reduced CIV activity, while ADP‐stimulated CI and CII OXPHOS remained unchanged. The deletion of the KAT genes significantly impairs mitochondrial respiration and ATP synthesis, potentially contributing to pathogenesis. This study highlights the importance of KYNA in mitochondrial function, offering new insights into potential therapeutic targets for various disorders. Targeting the KYN pathway could mitigate mitochondrial dysfunction in a variety of diseased conditions. image

Mitochondria govern energy transfer, redox balance, and cell fate. Tryptophan catabolism generates kynurenines (KYNs) that can tune mitochondrial function, with growing evidence that G protein-coupled receptor 35 (GPR35), aryl hydrocarbon receptor (AhR), and N-methyl-D-aspartate receptors (NMDA receptors) link extracellular cues to adenosine 5 prime triphosphate (ATP) maintenance, calcium (Ca²⁺) handling, mitophagy, and inflammasome control. In parallel, quinolinic acid (QA)-driven de novo nicotinamide adenine dinucleotide (NAD⁺) synthesis connects KYN flux to tricarboxylic acid (TCA) cycle activity and sirtuin programs across tissues. Key gaps remain: receptor pharmacology is rarely integrated with NAD⁺ economics and respiration, and clinical workflows still lack single-run assays that quantify both kynurenine and TCA nodes. We therefore integrate receptor proximal signaling, QA-driven NAD⁺ supply, and unified liquid chromatography–mass spectrometry (LC-MS) measurement into one translational framework spanning kynurenic acid (KYNA), KYN, 3-hydroxykynurenine (3-HK), and QA, using mitochondrial endpoints as the common readout. We synthesize evidence for mitochondrial GPR35 signaling that preserves ATP, AhR programs that tune oxidative defenses and mitophagy, and NMDA receptor antagonism that limits excitotoxic stress. These mechanisms are linked to QA-dependent NAD⁺ biogenesis and alpha ketoglutarate control points, then aligned with chromatography and ionization choices suited to routine LC-MS workflows. This receptor to organelle framework couples KYN flux to respiratory control and provides a practical roadmap for standardized single-run LC-MS panels. It can strengthen target validation in ischemia, neurodegeneration, psychiatry, and oncology while improving biomarker qualification through harmonized analytics and decision-grade readouts.

Our aim was to develop a method to detect extramitochondrial Ca²⁺ movement and O₂ fluxes simultaneously. Using High-Resolution FluoRespirometry, we also tested whether mitochondrial permeability transition pore (mPTP) inhibition or anoxia affects the mitochondrial Ca²⁺ flux. Ca²⁺ movement evoked by CaCl₂ or anoxia was assessed with CaGreen-5N dye using Blue-Fluorescence-Sensor in isolated liver mitochondria, liver homogenates and duodenal biopsies. Exogenous CaCl₂ (50 µM) resulted in an abrupt elevation in CaGreen-5N fluorescence followed by a decrease (Ca²⁺ uptake) with simultaneous elevation in O₂ consumption in liver preparations. This was followed by a rapid increase in the fluorescence signal, reaching a higher intensity (Ca²⁺ efflux) than that of the initial CaCl₂-induced elevation. Chelation of Ca²⁺ with EGTA completely abolished the fluorescence of the indicator. After pre-incubation with cyclosporin A, a marked delay in Ca²⁺ movement was observed, not only in isolated liver mitochondria, but also in tissue homogenates. In all samples, the transition to anoxia resulted in immediate increase in the level of extramitochondrial Ca²⁺. The results demonstrate that the CaGreen-5N method is suitable to monitor simultaneous O₂ and Ca²⁺ fluxes, and the opening of mPTP in various biological samples. In this system the duration of stimulated Ca²⁺ fluxes may provide a novel parameter to evaluate the efficacy of mPTP blocker compounds.

Acute myocardial infarction, often associated with ischemia/reperfusion injury (I/R), is a major healthcare issue ranking among the leading causes of death globally. Although kynurenic acid (KYNA), an endogenous tryptophan metabolite, has been previously shown to protect the cardiac tissue against I/R injury, its mechanism of action remains unclear. Therefore, here, we examined whether KYNA administration rescues H9c2 cardiac cells exposed to I/R through the preservation of the structural and functional integrity of the mitochondria. In addition, we assessed whether KYNA-derived agonism on G-protein coupled receptor 35 (GPR35) is involved in the protection of cardiac cells against simulated I/R (SI/R)-induced cellular demise. Our results demonstrated that KYNA attenuated the SI/R-induced calcium overload as well as impairments in the mitochondrial ultrastructure. Furthermore, administration of KYNA was shown to reduce mitochondrial superoxide production and preserve mitochondrial function in cells exposed to SI/R. Activation of the GPR35 receptors using an agonist other than KYNA rescued cardiac cells undergoing SI/R, attenuated the apoptotic activity, and improved various parameters of mitochondrial respiration. The administration of a synthetic GPR35 antagonist in parallel with KYNA attenuated the KYNA-induced cytoprotection. Our findings provide evidence that the protective effect of KYNA against SI/R-induced cardiac cell injury involves mitoprotective mechanisms, acting, at least in part, through the activation of GPR35 receptors.

Skeletal muscle, constituting 40–50% of total body mass, is vital for mobility, posture, and systemic homeostasis. Muscle contraction heavily relies on ATP, primarily generated by mitochondrial oxidative phosphorylation. Mitochondria play a key role in decoding intracellular calcium signals. The endocannabinoid system (ECS), including CB₁ receptors (CB₁Rs), broadly influences physiological processes and, in muscles, regulates functions like energy metabolism, development, and repair. While plasma membrane CB₁Rs (pCB₁Rs) are well-established, a distinct mitochondrial CB₁R (mtCB₁R) population also exists in muscles, influencing mitochondrial oxidative activity and quality control. We investigated the role of mtCB₁Rs in skeletal muscle physiology using a novel systemic mitochondrial CB₁ deletion murine model. Our in vivo studies showed no changes in motor function, coordination, or grip strength in mtCB₁ knockout mice. However, in vitro force measurements revealed significantly reduced specific force in both fast-twitch (EDL) and slow-twitch (SOL) muscles following mtCB₁R ablation. Interestingly, knockout EDL muscles exhibited hypertrophy, suggesting a compensatory response to reduced force quality. Electron microscopy revealed significant mitochondrial morphological abnormalities, including enlargement and irregular shapes, correlating with these functional deficits. High-resolution respirometry further demonstrated impaired mitochondrial respiration, with reduced oxidative phosphorylation and electron transport system capacities in knockout mitochondria. Crucially, mitochondrial membrane potential dissipated faster in mtCB₁ knockout muscle fibers, whilst mitochondrial calcium levels were higher at rest. These findings collectively establish that mtCB₁Rs are critical for maintaining mitochondrial health and function, directly impacting muscle energy production and contractile performance. Our results provide new insights into ECS-mediated regulation of skeletal muscle function and open therapeutic opportunities for muscle disorders and aging.

Background: Fixed‐ratio combinations (FRCs) provide an alternative to intensified conservative insulin treatments (ICTs); however, therapy simplification in patients with high total daily insulin dose (TDD) or high HbA _1c is a debated issue; additionally, its influence on target organ damage (TOD) is less known. Methods: Data were retrospectively collected from patients with Type 2 diabetes, including 58 patients who continued ICT and 104 patients who underwent therapy simplification between January 1, 2017, and January 1, 2023. Patient characteristics and therapy details are at baseline and 3, 6, 12, and 24 months after FRC initiation. Results: HbA _1c significantly decreased in both groups (−0.9% [−1.6%, −0.5%] with ICT vs. −1.3% [−2.1%, −0.3%] with FRC), whereas body weight significantly decreased only after simplification (−1 kg [−4, 1] vs. −5 kg [−7, −2]). Diabetes duration was not associated with therapy efficacy. Significant HbA _1c reduction and FRC dose elevation occurred earlier in patients with an initial HbA _1c > 8.0 % than in those with an initial HbA _1c < 8.0 % . FRC dose was significantly higher at 3 months in patients with a TDD of > 60 U/day than in those with lower TDD. Relative risk reduction with therapy simplification was 72.1%, 50.6%, 32.3%, and 59.7% for hypoglycemia, renal function decline, microalbuminuria, and macrovascular complications, respectively. Risk of retinopathy, neuropathy, and chronic kidney disease did not significantly change with FRCs. Discussion: FRCs are safe and as effective as ICT even in patients with high initial HbA _1c , high TDD, or long diabetes duration. A protective role of FRCs in diabetic ASCVD has been proven, but their protective role in CKD was not observed. Conclusions: The significant improvements in glycemic and weight control, as well as in TODs, suggest that therapy simplification may represent a more favorable approach compared to the continuation of previous ICT even in patients characterized by high baseline TDD and HbA _1c levels.