Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic b cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H 2 O 2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADH F, indicated its *20% decrease. Matrix NAD + F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD + F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPH F (iNAP1 fluorescence monitoring) at the expense of matrix NADH F , was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13 Cincorporation from 13 C-L-glutamine into 13 C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH F (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I I F site contribution, but a lower superoxide fraction ascribed to the Complex III site III Qo. Thus, the diminished matrix NADH F /NAD + F decreased Complex I flavin site I F superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic b cells on GSIS, due to the decreasing matrix NADH F /NAD + F (NADPH F /NADP + F) at increasing cytosolic NADPH F levels. The developed innovative methods enable real-time NADH/NAD + and NADPH/ NADP + monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic b cells. Antioxid. Redox Signal. 33, 789-815.

Psilocybin, psilocin, baeocystin, norbaeocystin, and aeruginascin are tryptamines structurally similar to the neurotransmitter serotonin. Psilocybin and its pharmacologically active metabolite psilocin in particular are known for their psychoactive effects. These substances typically occur in most species of the genus Psilocybe (Fungi, Strophariaceae). Even the sclerotia of some of these fungi known as “magic truffles” are of growing interest in microdosing due to them improving cognitive function studies. In addition to microdosing studies, psilocybin has also been applied in clinical studies, but only its pure form has been administrated so far. Moreover, the determination of tryptamine alkaloids is used in forensic analysis. In this study, freshly cultivated fruit bodies of Psilocybe cubensis were used for monitoring stability (including storage and processing conditions of fruiting bodies). Furthermore, mycelium and the individual parts of the fruiting bodies (caps, stipes, and basidiospores) were also examined. The concentration of tryptamines in final extracts was analyzed using ultra‐high‐performance liquid chromatography coupled with mass spectrometry. No tryptamines were detected in the basidiospores, and only psilocin was present at 0.47 wt.% in the mycelium. The stipes contained approximately half the amount of tryptamine alkaloids (0.52 wt.%) than the caps (1.03 wt.%); however, these results were not statistically significant, as the concentration of tryptamines in individual fruiting bodies is highly variable. The storage conditions showed that the highest degradation of tryptamines was seen in fresh mushrooms stored at −80°C, and the lowest decay was seen in dried biomass stored in the dark at room temperature.

Wild type mitochondrial isocitrate dehydrogenase (IDH2) was previously reported to produce oncometabolite 2-hydroxyglutarate (2HG). Besides, mitochondrial deacetylase SIRT3 has been shown to regulate the oxidative function of IDH2. However, regulation of 2HG formation by SIRT3mediated deacetylation was not investigated yet. We aimed to study mitochondrial IDH2 function in response to acetylation and deacetylation, and focus specifically on 2HG production by IDH2. We used acetylation surrogate mutant of IDH2 K413Q and assayed enzyme kinetics of oxidative decarboxylation of isocitrate, 2HG production by the enzyme, and 2HG production in cells. The purified IDH2 K413Q exhibited lower oxidative reaction rates than IDH2 WT. 2HG production by IDH2 K413Q was largely diminished at the enzymatic and cellular level, and knockdown of SIRT3 also inhibited 2HG production by IDH2. Contrary, the expression of putative mitochondrial acetylase GCN5L likely does not target IDH2. Using mass spectroscopy, we further identified lysine residues within IDH2, which are the substrates of SIRT3. In summary, we demonstrate that 2HG levels arise from non-mutant IDH2 reductive function and decrease with increasing acetylation level. The newly identified lysine residues might apply in regulation of IDH2 function in response to metabolic perturbations occurring in cancer cells, such as glucose-free conditions.