A Kir6.2 mutation causing severe functional effects in vitro produces neonatal diabetes without the expected neurological complications

AIMS/HYPOTHESIS: Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects. METHODS: The patients (currently aged 2, 8 and 20 years) developed diabetes shortly after birth. The two younger patients attempted transfer to sulfonylurea therapy but were unsuccessful (up to 1.1 mg kg(-1) day(-1)). They remain insulin dependent. None of the patients displayed neurological symptoms. Functional properties of wild-type and mutant channels were examined by electrophysiology in Xenopus oocytes. RESULTS: Heterozygous (het) and homozygous L164P K(ATP) channels showed a marked reduction in channel inhibition by ATP. Consistent with its predicted location within the pore, L164P enhanced the channel open state, which explains the reduction in ATP sensitivity. HetL164P currents exhibited greatly increased whole-cell currents that were unaffected by sulfonylureas. This explains the inability of sulfonylureas to ameliorate the diabetes of affected patients. CONCLUSIONS/INTERPRETATION: Our results provide the first demonstration that mutations such as L164P, which produce a severe reduction in ATP sensitivity, do not inevitably cause developmental delay or neurological problems. However, the neonatal diabetes of these patients is unresponsive to sulfonylurea therapy. Functional analysis of PNDM mutations can predict the sulfonylurea response

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.Peer reviewe

A longitudinal study of muscle rehabilitation in the lower leg after cast removal using magnetic resonance imaging and strength assessment

Magnetic resonance imaging (MRI) was used to investigate muscle rehabilitation following cast immobilization. The aim was to explore MRI as an imaging biomarker of muscle function. Sixteen patients completed an eight-week rehabilitation programme following six weeks of cast immobilization for an ankle fracture. MRI of the lower leg was performed at two-week intervals for 14 weeks. Total volume and anatomical cross-sectional areas at 70% of the distance from lateral malleolus to tibial tuberosity (ACSA) were measured for tibialis anterior (TA), medial and lateral gastrocnemius (GM and GL) and soleus (SOL). Pennation angle of muscle fascicules was measured at the same position in GM. Fractional fat/water contents and T2 relaxation times before and after exercise were calculated. Strength was measured as maximum isometric torque developed in plantar- and dorsi-flexion. Torque increased by (mean [SD]) 1.10 (0.32) N m day−1 in males, 0.74 (0.43) N m day−1 in females in plantar-flexion (0.9% of final strength per day), and 0.36 (0.15) N m day−1 in males, 0.28 (0.19) N m day−1 in females in dorsi-flexion (1.1% per day). Neither difference between males and females was significant. Volume and ACSA of muscles recovered by week 14 apart from SOL which was still 6.8% smaller (p = 0.006) than the contralateral leg. T2 peaked at the end of the cast period for TA and SOL, and at week 8 for GM before returning to baseline. Pennation angle recovered rapidly following cast removal. Quantitative MRI can generate markers of muscle biomechanics and indicates that many of these return to baseline within eight weeks of remobilization

Reversible changes in pancreatic islet structure and function produced by elevated blood glucose

Diabetes is characterized by hyperglycaemia due to impaired insulin secretion and aberrant glucagon secretion resulting from changes in pancreatic islet cell function and/or mass. The extent to which hyperglycaemia per se underlies these alterations remains poorly understood. Here we show that β-cell-specific expression of a human activating KATP channel mutation in adult mice leads to rapid diabetes and marked alterations in islet morphology, ultrastructure and gene expression. Chronic hyperglycaemia is associated with a dramatic reduction in insulin-positive cells and an increase in glucagon-positive cells in islets, without alterations in cell turnover. Furthermore, some β-cells begin expressing glucagon, whilst retaining many β-cell characteristics. Hyperglycaemia, rather than KATP channel activation, underlies these changes, as they are prevented by insulin therapy and fully reversed by sulphonylureas. Our data suggest that many changes in islet structure and function associated with diabetes are attributable to hyperglycaemia alone and are reversed when blood glucose is normalized

The origin of defects induced in ultra-pure germanium by Electron Beam Deposition

The creation of point defects in the crystal lattices of various semiconductors by subthreshold events has been reported on by a number of groups. These observations have been made in great detail using sensitive electrical techniques but there is still much that needs to be clarified. Experiments using Ge and Si were performed that demonstrate that energetic particles, the products of collisions in the electron beam, were responsible for the majority of electron-beam deposition (EBD) induced defects in a two-step energy transfer process. Lowering the number of collisions of these energetic particles with the semiconductor during metal deposition was accomplished using a combination of static shields and superior vacuum resulting in devices with defect concentrations lower than $10^{11}\,$cm$^{-3}$, the measurement limit of our deep level transient spectroscopy (DLTS) system. High energy electrons and photons that samples are typically exposed to were not influenced by the shields as most of these particles originate at the metal target thus eliminating these particles as possible damage causing agents. It remains unclear how packets of energy that can sometimes be as small of 2eV travel up to a $\mu$m into the material while still retaining enough energy, that is, in the order of 1eV, to cause changes in the crystal. The manipulation of this defect causing phenomenon may hold the key to developing defect free material for future applications.Comment: 18 pages, 9 figure

Mutations at the same residue (R50) of Kir6.2 (KCNJ11) that cause neonatal diabetes produce different functional effects

Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive K(+) channel (K(ATP) channel), are a common cause of neonatal diabetes. We identified a novel KCNJ11 mutation, R50Q, that causes permanent neonatal diabetes (PNDM) without neurological problems. We investigated the functional effects this mutation and another at the same residue (R50P) that led to PNDM in association with developmental delay. Wild-type or mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes. Both mutations increased resting whole-cell currents through homomeric and heterozygous K(ATP) channels by reducing channel inhibition by ATP, an effect that was larger in the presence of Mg(2+). However the magnitude of the reduction in ATP sensitivity (and the increase in the whole-cell current) was substantially larger for the R50P mutation. This is consistent with the more severe phenotype. Single-R50P channel kinetics (in the absence of ATP) did not differ from wild type, indicating that the mutation primarily affects ATP binding and/or transduction. This supports the idea that R50 lies in the ATP-binding site of Kir6.2. The sulfonylurea tolbutamide blocked heterozygous R50Q (89%) and R50P (84%) channels only slightly less than wild-type channels (98%), suggesting that sulfonylurea therapy may be of benefit for patients with either mutation

Mice expressing a human KATP channel mutation have altered channel ATP sensitivity but no cardiac abnormalities

AIMS/HYPOTHESIS: Patients with severe gain-of-function mutations in the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, have neonatal diabetes, muscle hypotonia and mental and motor developmental delay-a condition known as iDEND syndrome. However, despite the fact that Kir6.2 forms the pore of the cardiac K(ATP) channel, patients show no obvious cardiac symptoms. The aim of this project was to use a mouse model of iDEND syndrome to determine whether iDEND mutations affect cardiac function and cardiac K(ATP) channel ATP sensitivity. METHODS: We performed patch-clamp and in vivo cine-MRI studies on mice in which the most common iDEND mutation (Kir6.2-V59M) was targeted to cardiac muscle using Cre-lox technology (m-V59M mice). RESULTS: Patch-clamp studies of isolated cardiac myocytes revealed a markedly reduced K(ATP) channel sensitivity to MgATP inhibition in m-V59M mice (IC(50) 62 μmol/l compared with 13 μmol/l for littermate controls). In vivo cine-MRI revealed there were no gross morphological differences and no differences in heart rate, end diastolic volume, end systolic volume, stroke volume, ejection fraction, cardiac output or wall thickening between m-V59M and control hearts, either under resting conditions or under dobutamine stress. CONCLUSIONS/INTERPRETATION: The common iDEND mutation Kir6.2-V59M decreases ATP block of cardiac K(ATP) channels but was without obvious effect on heart function, suggesting that metabolic changes fail to open the mutated channel to an extent that affects function (at least in the absence of ischaemia). This may have implications for the choice of sulfonylurea used to treat neonatal diabetes

ATP-Sensitive Potassium Channels Exhibit Variance in the Number of Open Channels below the Limit Predicted for Identical and Independent Gating

In small cells containing small numbers of ion channels, noise due to stochastic channel opening and closing can introduce a substantial level of variability into the cell's membrane potential. Negatively cooperative interactions that couple a channel's gating conformational change to the conformation of its neighbor(s) provide a potential mechanism for mitigating this variability, but such interactions have not previously been directly observed. Here we show that heterologously expressed ATP-sensitive potassium channels generate noise (i.e., variance in the number of open channels) below the level possible for identical and independent channels. Kinetic analysis with single-molecule resolution supports the interpretation that interchannel negative cooperativity (specifically, the presence of an open channel making a closed channel less likely to open) contributes to the decrease in noise. Functional coupling between channels may be important in modulating stochastic fluctuations in cellular signaling pathways

Phonon-assisted radiofrequency absorption by gold nanoparticles resulting in hyperthermia

It is suggested that in gold nanoparticles (GNPs) of about 5 nm sizes used in the radiofrequency (RF) hyperthermia, an absorption of the RF photon by the Fermi electron occurs with involvement of the longitudinal acoustic vibrational mode (LAVM), the dominating one in the distribution of vibrational density of states (VDOS). This physical mechanism helps to explain two observed phenomena: the size dependence of the heating rate (HR) in GNPs and reduced heat production in aggregated GNPs. The argumentation proceeds within the one-electron approximation, taking into account the discretenesses of energies and momenta of both electrons and LAVMs. The heating of GNPs is thought to consist of two consecutive processes: first, the Fermi electron absorbs simultaneously the RF photon and the LAVM available in the GNP; hereafter the excited electron gets relaxed within the GNP's boundary, exciting a LAVM with the energy higher than that of the previously absorbed LAVM. GNPs containing the Ta and/or Fe impurities are proposed for the RF hyperthermia as promising heaters with enhanced HRs, and GNPs with rare-earth impurity atoms are also brought into consideration. It is shown why the maximum HR values should be expected in GNPs with about 5-7 nm size.Comment: proceedings at the NATO Advanced Research workshop FANEM-2015 (Minsk, May 25-27, 2015). To be published in the final form in: "Fundamental and Applied NanoElectroMagnetics" (Springer Science + Business Media B.V.