Simone Baltrusch

Fragestellung Der kongenitale Hyperinsulinismus (CHI) ist eine seltene Erkrankung, die durch schwere postnatale Hypoglykämie gekennzeichnet ist. Dem Krankheitsbild liegen häufig Mutationen in den Genen ABCC8 und KCNJ11 zugrunde, welche für den ATP-sensitiven Kaliumkanal kodieren. Die fokale Form der Erkrankung weist Regionen mit einer unkontrollierten Insulinausschüttung auf. Ziel dieser Studie war es, isolierte Inseln aus Pankreasteilresektionen von CHI-Kindern in vivo zu untersuchen. Methodik Fokale und gesunde Inseln wurden durch Kollagenaseverdau aus Proben von 2 – 12 Monate alten Kindern (n=4) isoliert. Die Inseln wurden auf PCA-Objektträgern kultiviert, um Einzelzellanalysen zu ermöglichen. Insulin und Glukagon Genexpression wurde durch RT-PCR, Lokalisation durch Immunfluoreszenz und Freisetzung im Überstand durch ELISA bestimmt. Die intrazelluläre Calciumkonzentration wurde mittels FURA untersucht. Ergebnisse Fokale versus gesunde Inseln zeigten eine 5-fach höhere Insulin- und 3-fach höhere Glucagonsekretion. Die Genexpression beider Hormone war ebenfalls signifikant erhöht. Fokale versus gesunde Inseln wiesen im intrazellulären Calcium keine Glucose Responsivität auf. Nur fokale Inseln von 2 Monate alten Kindern hatten einen hohen intrazellulären Calciumspiegel. Nur gesunde Inseln von 12 Monate alten Kindern präsentierten Calciumoszillationen vergleichbar adulten Inseln. Fokale Inseln enthielten unterschiedliche Anteile an Insulin und Glukagon positiven Zellen und solche, die beide Hormone produzieren. Schlussfolgerung Der Schweregrad der CHI Manifestation scheint vom Entwicklungsstadium des endokrinen Pankreas abzuhängen. Daher sollten Marker der frühen und späten Pankreasentwicklung weiter analysiert werden, um auch das Vorliegen der Insulin und Glukagon doppelt positiven Zellen erklären zu können. Die Untersuchung älterer und insbesondere geheilter CHI-Patienten könnte Ansatzpunkte zu neuen Therapieansätzen für den Diabetes mellitus Typ 2 geben. Article published online: 02 May 2023 © 2023. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

Fragestellung Der mitochondriale oxidative Stoffwechsel ist für die Glucose-stimulierte Insulinsekretion der Beta-Zelle vital. Für seine Funktionalität ist die ständige Adaptation der Mitochondrien-Morphologie durch Fusions- und Teilungsprozesse notwendig. Der Einsatz der Fluoreszenzmikroskopie ermöglicht die Visualisierung der Mitochondrien-Dynamik. Alle Parameter des Prozesses zu variieren, stößt aber an experimentelle Grenzen. Ziel dieser Studie war es, die Veränderung des Mitochondrien-Netzwerkes in Beta-Zellen auf Basis der numerischen Modellierungsmethode „Zellulärer Automat“ räumlich zu simulieren. Methodik Die Expression des dynamin-related protein 1 (Drp1) als zentraler Teilungsfaktor der Mitochondrien wurde in MIN6-Beta-Zellen mittels shRNA reduziert. Das mitochondriale Netzwerk wurde durch Transfektion mit pTaqBFP-Mito in der Fluoreszenzmikroskopie sichtbar gemacht und versus Kontrollzellen hinsichtlich Größe und Sphärizität der Mitochondrien in der Software Imaris analysiert. Im Zellulären Automaten wurde die Mitochondrien-Bewegung durch einen Random Walk dargestellt und die Prozesse der Fusion und Teilung als stochastisches Ereignis modelliert. Ergebnisse Unter Drp1-Reduktion vergrößerten sich die Mitochondrien in den Beta-Zellen signifikant. Damit einhergehend nahm die Sphärizität ab. Die Nachbildungen der zerteilten und fusionierten Strukturen im Zellulären Automaten entsprachen mit geringen Abweichungen, trotz der Vernachlässigung der weiteren realbiologischen Prozesse, den experimentellen Ergebnissen. In Abhängigkeit der Drp1-beeinflussten Parameter entstanden aggregierte, stark fusionierte Mitochondrien-Netzwerke oder zerteilte Strukturen, mit vielen kleinen, eher rundlichen Mitochondrien. Die Trennungswahrscheinlichkeit und die Fusionswahrscheinlichkeit gerade getrennter Mitochondrien konnten als entscheidende Faktoren für die Strukturausbildung identifiziert werden. Schlussfolgerung Die Weiterentwicklung dieses Zellulären Automaten ist aufgrund der erreichten guten Annäherung an die realen Prozesse der Beta-Zelle vielversprechend, Veränderungen des Mitochondrien-Netzwerkes zu modellieren und darüber Funktionsstörungen im Diabetes mellitus zu verstehen. Article published online: 02 May 2023 © 2023. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

Aims/hypothesis: The mammalian enzyme glucokinase (GK), expressed predominantly in liver and pancreas, plays an essential role in carbohydrate metabolism. Monogenic GK disorders emphasise the role of GK in determining the blood glucose set point. Methods: A family with congenital hyperinsulinism (CHI) was examined for GCK gene variants by Sanger sequencing. A combined approach, involving kinetic analysis (also using GK activators and inhibitors), intracellular translocation assays, insulin secretion measurements and structural modelling, was used to investigate the novel variant compared with known variants. Results: We report on the novel gain-of-function GCK variant p.Val455Leu (V455L), inherited as an autosomal dominant trait in a German family with CHI and concomitant obesity (fasting blood glucose 2.1 mmol/l, BMI 45.0 kg/m², HOMA-IR 1.5 in an adult female family member); one male family member developed type 2 diabetes until age 35 years (with fasting glucose 2.8–3.7 mmol/l, BMI 38.9 kg/m², HOMA-IR 4.6). Kinetic characterisation of the V455L variant revealed a significant increase in glucose affinity (glucose concentration at which reaction rate is half its maximum rate [S_0.5]: mutant 2.4 ± 0.3 mmol/l vs wild-type 7.6 ± 1.0 mmol/l), accompanied by a distinct additive susceptibility to both the endogenous activator fructose 2,6-bisphosphatase and the synthetic allosteric activator RO-28-1675. The effect of RO-28-1675 was more pronounced when compared with the previously known GK variants V455M and V455E. Binding to the inhibitor glucokinase regulatory protein was unimpaired for V455L and V455E but was reduced for V455M, whereas mannoheptulose inhibited all GK variants and the wild-type enzyme. Structural analyses suggested a role for residue 455 in rearrangements between the inactive and active conformations of GK and also in allosteric activation. Comparison with V455M and V455E and an overview of activating GK variants provided a context for the novel sequence aberration in terms of altered GK enzyme characteristics caused by single amino acid changes. Conclusion/interpretation: We provide new knowledge on the structure–function relationship of GK, with special emphasis on enzyme activation, potentially yielding fresh strategic insights into breaking the vicious circle of fluctuating blood glucose levels and the attendant risk of long-lasting metabolic changes in both CHI and type 2 diabetes. Graphical

Damage and regeneration naturally occur in the peripheral nervous system. The neurotropic B vitamins thiamine (B1), pyridoxine (B6), and cobalamin (B12) are key players, which maintain the neuronal viability in different ways. Firstly, they constantly protect nerves against damaging environmental influences. While vitamin B1 acts as a site‐directed antioxidant, vitamin B6 balances nerve metabolism, and vitamin B12 maintains myelin sheaths. However, nerve injury occurs at times, because of an imbalance between protective factors and accumulating stress and noxae. This will result in the so‐called Wallerian degeneration process. The presence of vitamins B1, B6, and B12 paves the way out to the following important regeneration by supporting the development of new cell structures. Furthermore, vitamin B1 facilitates the usage of carbohydrates for energy production, whereas vitamin B12 promotes nerve cell survival and remyelination. Absence of these vitamins will favor permanent nerve degeneration and pain, eventually leading to peripheral neuropathy.

Purpose: To elucidate the collagen structure in the Descemet membrane (DM) of the human cornea and to characterize its rearrangement in patients with endothelial corneal dystrophies. Methods: Corneas from nine human donors and dystrophic DMs removed from 16 affected eyes of 13 patients by endothelial keratoplasty (DMEK) were investigated using a correlative RT-qPCR and label-free two-channel multiphoton microscopy (MPM) setup. Although collagen formation was visualized by second harmonic generation, the cellular structure was determined by autofluorescence. Results: The DM of the human donor cornea was characterized by a consistent pattern of fine hexagonal collagen structures that form a supportive scaffold for the endothelial cells. Accordingly, network-forming collagens (8A1 and 8A2) but less fibrillar collagens (only 1A2) were expressed. DMEK resulted in significant (P < 0.0001) improvement of best-corrected visual acuity. In the removed dystrophic DMs, MPM analyses revealed collagen rearrangement in addition to loss of endothelial cells and the development of guttae. MPM analyses of the whole patient's DM demonstrated this collagen remodeling in its entirety and facilitated correlation to Scheimpflug corneal tomography. In most DMs a unique honeycomb collagen network was identified, with distinct bundles surrounding the guttae and correlating with expression of fibrillar collagens (1A1). Conversely, some DMs showed either reduced collagen on MPM and RT-qPCR analysis or diffuse thickening and storage of extracellular matrix. Conclusions: The collagen structure of the DM and its adaptive remodeling in endothelial corneal dystrophies has been characterized for the first time here and will facilitate individual therapeutic approaches.

Background: Mitochondrial dynamics are important for glucose-stimulated insulin secretion in pancreatic beta cells. The mitochondrial elongation factor MiD51 has been proposed to act as an anchor that recruits Drp1 from the cytosol to the outer mitochondrial membrane. Whether MiD51 promotes mitochondrial fusion by inactivation of Drp1 is a controversial issue. Since both the underlying mechanism and the effects on mitochondrial function remain unknown, this study was conducted to investigate the role of MiD51 in beta cells. Methods: Overexpression and downregulation of MiD51 in mouse insulinoma 6 (MIN6) and mouse islet cells was achieved using the pcDNA expression vector and specific siRNA, respectively. Expression of genes regulating mitochondrial dynamics and autophagy was analyzed by quantitative Real-Time PCR, glucose-stimulated insulin secretion by ELISA, and cellular oxygen consumption rate by optode sensor technology. Mitochondrial membrane potential and morphology were visualized after TMRE and MitoTracker Green staining, respectively. Immunofluorescence analyses were examined by confocal microscopy. Results: MiD51 is expressed in insulin-positive mouse and human pancreatic islet and MIN6 cells. Overexpression of MiD51 resulted in mitochondrial fragmentation and cluster formation in MIN6 cells. Mitochondrial membrane potential, glucose-induced oxygen consumption rate and glucose-stimulated insulin secretion were reduced in MIN6 cells with high MiD51 expression. LC3 expression remained unchanged. Downregulation of MiD51 resulted in inhomogeneity of the mitochondrial network in MIN6 cells with hyperelongated and fragmented mitochondria. Mitochondrial membrane potential, maximal and glucose-induced oxygen consumption rate and insulin secretion were diminished in MIN6 cells with low MiD51 expression. Furthermore, reduced Mfn2 and Parkin expression was observed. Based on MiD51 overexpression and downregulation, changes in the mitochondrial network structure similar to those in MIN6 cells were also observed in mouse islet cells. Conclusion: We have demonstrated that MiD51 plays a pivotal role in regulating mitochondrial function and hence insulin secretion in MIN6 cells. We propose that this anchor protein of Drp1 is important to maintain a homogeneous mitochondrial network and to avoid morphologies such as hyperelongation and clustering which are inaccessible for degradation by autophagy. Assuming that insulin granule degradation frequently suppresses autophagy in beta cells, MiD51 could be a key element maintaining mitochondrial health.