The novel GCK variant p.Val455Leu associated with hyperinsulinism is susceptible to allosteric activation and is conducive to weight gain and the development of diabetes.

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 [S0.5]: 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.

Inheritance of rare functional GCKR variants and their contribution to triglyceride levels in families.

Significant resources have been invested in sequencing studies to investigate the role of rare variants in complex disease etiology. However, the diagnostic interpretation of individual rare variants remains a major challenge, and may require accurate variant functional classification and the collection of large numbers of variant carriers. Utilizing sequence data from 458 individuals with hypertriglyceridemia and 333 controls with normal plasma triglyceride levels, we investigated these issues using GCKR, encoding glucokinase regulatory protein. Eighteen rare non-synonymous GCKR variants identified in these 791 individuals were comprehensively characterized by a range of biochemical and cell biological assays, including a novel high-throughput-screening-based approach capable of measuring all variant proteins simultaneously. Functionally deleterious variants were collectively associated with hypertriglyceridemia, but a range of in silico prediction algorithms showed little consistency between algorithms and poor agreement with functional data. We extended our study by obtaining sequence data on family members; however, functional variants did not co-segregate with triglyceride levels. Therefore, despite evidence for their collective functional and clinical relevance, our results emphasize the low predictive value of rare GCKR variants in individuals and the complex heritability of lipid traits.

Drp1 guarding of the mitochondrial network is important for glucose-stimulated insulin secretion in pancreatic beta cells.

Mitochondria form a tubular network in mammalian cells, and the mitochondrial life cycle is determined by fission, fusion and autophagy. Dynamin-related protein 1 (Drp1) has a pivotal role in these processes because it alone is able to constrict mitochondria. However, the regulation and function of Drp1 have been shown to vary between cell types. Mitochondrial morphology affects mitochondrial metabolism and function. In pancreatic beta cells mitochondrial metabolism is a key component of the glucose-induced cascade of insulin secretion. The goal of the present study was to investigate the action of Drp1 in pancreatic beta cells. For this purpose Drp1 was down-regulated by means of shDrp1 in insulin-secreting INS1 cells and mouse pancreatic islets. In INS1 cells reduced Drp1 expression resulted in diminished expression of proteins regulating mitochondrial fusion, namely mitofusin 1 and 2, and optic atrophy protein 1. Diminished mitochondrial dynamics can therefore be assumed. After down-regulation of Drp1 in INS1 cells and spread mouse islets the initially homogenous mitochondrial network characterised by a moderate level of interconnections shifted towards high heterogeneity with elongated, clustered and looped mitochondria. These morphological changes were found to correlate directly with functional alterations. Mitochondrial membrane potential and ATP generation were significantly reduced in INS1 cells after Drp1down-regulation. Finally, a significant loss of glucose-stimulated insulin secretion was demonstrated in INS1 cells and mouse pancreatic islets. In conclusion, Drp1 expression is important in pancreatic beta cells to maintain the regulation of insulin secretion.

Glucose-induced dissociation of glucokinase from its regulatory protein in the nucleus of hepatocytes prior to nuclear export.

The glucose phosphorylating enzyme glucokinase regulates glucose metabolism in the liver. Glucokinase activity is modulated by a liver-specific competitive inhibitor, the glucokinase regulatory protein (GRP), which mediates sequestration of glucokinase to the nucleus at low glucose concentrations. However, the mechanism of glucokinase nuclear export is not fully understood. In this study we investigated the dynamics of glucose-dependent interaction and translocation of glucokinase and GRP in primary hepatocytes using fluorescence resonance energy transfer, selective photoconversion and fluorescence recovery after photobleaching. The formation of the glucokinase:GRP complex in the nucleus of primary hepatocytes at 5 mmol/l glucose was significantly reduced after a 2 h incubation at 20 mmol/l glucose. The GRP was predominantly localized in the nucleus, but a mobile fraction moved between the nucleus and the cytoplasm. The glucose concentration only marginally affected GRP shuttling. In contrast, the nuclear export rate of glucokinase was significantly higher at 20 than at 5 mmol/l glucose. Thus, glucose was proven to be the driving-force for nuclear export of glucokinase in hepatocytes. Using the FLII2Pglu-700mu-delta6 glucose nanosensor it could be shown that in hepatocytes the kinetics of nuclear glucose influx, metabolism or efflux were significantly faster compared to insulin-secreting cells. The rapid equilibration kinetics of glucose flux into the nucleus facilitates dissociation of the glucokinase:GRP complex and also nuclear glucose metabolism by free glucokinase enzyme. In conclusion, we could show that a rise of glucose in the nucleus of hepatocytes releases active glucokinase from the glucokinase:GRP complex and promotes the subsequent nuclear export of glucokinase.

Characterization of two MODY2 mutations with different susceptibility to activation.

Glucokinase plays a key role in glucose sensing in pancreatic beta cells and in liver metabolism. Heterozygous inactivating glucokinase mutations cause the autosomal dominantly inherited MODY2 subtype of maturity-onset diabetes of the young. The goal of this study was to elucidate the pathogenicity of the recently described glucokinase mutants L304P and L315H, located in an alpha-helix and connecting region, respectively, at the outer region of the large domain of glucokinase. Both mutants showed wild-type-like cytosolic localization, but faster protein degradation in insulin-secreting MIN6 cells. However, strongly reduced nuclear/cytoplasmic localization of the mutants was observed in primary hepatocytes suggesting reduced interaction with the liver specific glucokinase regulatory protein. Both mutants displayed a significantly lowered glucokinase activity compared to the wild-type protein. Even though the L315H protein showed the lowest enzymatic activity, this mutant was very sensitive to allosteric activation. The endogenous activator fructose-2,6-bisphosphatase evoked an increase in glucokinase activity for both mutants, but much stronger for L315H compared to L304P. The synthetic activator RO281675 was ineffective against the L304P mutant. Expression of the mutant proteins evoked loss of glucose-induced insulin secretion in MIN6 cells. Administration of RO281675 increased insulin secretion, however, only for the L315H mutant. Thus, a glucokinase activator drug therapy may help MODY2 patients not in general, but seems to be a useful strategy for carriers of the L315H glucokinase mutation.

Collagen Remodeling Plays a Pivotal Role in Endothelial Corneal Dystrophies.

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.

MiD51 Is Important for Maintaining Mitochondrial Health in Pancreatic Islet and MIN6 Cells.

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.

Cytosolic and mitochondrial Ca2+ concentrations in primary hepatocytes change with ageing and in consequence of an mtDNA mutation.

Mitochondrial Ca2+ flux is crucial for the regulation of cell metabolism. Ca2+ entry to the mitochondrial matrix is mediated by VDAC1 and MCU with its regulatory molecules. We investigated hepatocytes isolated from conplastic C57BL/6NTac-mtNODLtJ mice (mtNOD) that differ from C57BL/6NTac mice (controls) by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase, resulting in functional and morphological mitochondrial adaptations. Mice of both strains up to 12 months old were compared using mitochondrial GEM-GECO1 and cytosolic CAR-GECO1 expression to gain knowledge of age-dependent alterations of Ca2+ concentrations. In controls we observed a significant increase in glucose-induced cytosolic Ca2+ concentration with ageing, but only a minor elevation in mitochondrial Ca2+ concentration. Conversely, glucose-induced mitochondrial Ca2+ concentration significantly declined with ageing in mtNOD mice, paralleled by a slight decrease in cytosolic Ca2+ concentration. This was consistent with a significant reduction of the MICU1 to MCU expression ratio and a decline in MCUR1. Our results can best be explained in terms of the adaptation of Ca2+ concentrations to the mitochondrial network structure. In the fragmented mitochondrial network of ageing controls there is a need for high cytosolic Ca2+ influx, because only some of the isolated mitochondria are in direct contact with the endoplasmic reticulum. This is not important in the hyper-fused elongated mitochondrial network found in ageing mtNOD mice which facilitates rapid Ca2+ distribution over a large mitochondrial area.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and small chemical activators affect enzyme activity of activating glucokinase mutants by distinct mechanisms.

Glucokinase (GK), a monomeric glucose-phosphorylating enzyme characterised by high structural flexibility, acts as a glucose sensor in pancreatic beta cells and liver. Pharmaceutical efforts to control the enzyme are hampered by an incomplete understanding of GK regulation. We investigated GK characteristics of wild-type and activating S64Y and G68V mutant proteins in the presence of various combinations of the synthetic activators RO-28-1675 and compound A, the endogenous activator fructose-2,6-bisphosphatase (FBPase-2), and the inhibitor mannoheptulose. S64Y impedes formation of a turn structure that is characteristic for the inactive enzyme conformation, and complex formation with compound A induces collision with the large domain. G68V evokes close contact of connecting region I and helix α13 with RO-28-1675 and compound A. Both mutants showed higher activity than the wild-type at low glucose and were susceptible to further activation by FBPase-2 and RO-28-1675, alone and additively. G68V was less active than S64Y, but was activatable by compound A. In contrast, compound A inhibited S64Y, and this effect was even more pronounced in combination with mannoheptulose. Mutant and wild-type GK showed comparable thermal stability and intracellular lifetimes. A GK-6-phosphofructo-2-kinase (PFK-2)/FBPase-2 complex predicted by in silico protein-protein docking demonstrated possible binding of the FBPase-2 domain near the active site of GK. In summary, activating mutations within the allosteric site of GK do not preclude binding of chemical activators (GKAs), but can alter their action into inhibition. Our postulated GK-PFK-2/FBPase-2 complex represents the endogenous principle of activation by substrate channelling which permits binding of other small molecules and proteins.

Precise expression of Fis1 is important for glucose responsiveness of beta cells.

Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucose-stimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein.