External Glucose Concentration Research Articles

Nutritional compensation of the circadian clock is a conserved process influenced by gene expression regulation and mRNA stability.

Compensation is a defining principle of a true circadian clock, where its approximately 24-hour period length is relatively unchanged across environmental conditions. Known compensation effectors directly regulate core clock factors to buffer the oscillator's period length from variables in the environment. Temperature Compensation mechanisms have been experimentally addressed across circadian model systems, but much less is known about the related process of Nutritional Compensation, where circadian period length is maintained across physiologically relevant nutrient levels. Using the filamentous fungus Neurospora crassa, we performed a genetic screen under glucose and amino acid starvation conditions to identify new regulators of Nutritional Compensation. Our screen uncovered 16 novel mutants, and together with 4 mutants characterized in prior work, a model emerges where Nutritional Compensation of the fungal clock is achieved at the levels of transcription, chromatin regulation, and mRNA stability. However, eukaryotic circadian Nutritional Compensation is completely unstudied outside of Neurospora. To test for conservation in cultured human cells, we selected top hits from our fungal genetic screen, performed siRNA knockdown experiments of the mammalian orthologs, and characterized the cell lines with respect to compensation. We find that the wild-type mammalian clock is also compensated across a large range of external glucose concentrations, as observed in Neurospora, and that knocking down the mammalian orthologs of the Neurospora compensation-associated genes CPSF6 or SETD2 in human cells also results in nutrient-dependent period length changes. We conclude that, like Temperature Compensation, Nutritional Compensation is a conserved circadian process in fungal and mammalian clocks and that it may share common molecular determinants.

HyfF subunit of hydrogenase 4 is crucial for regulating FOF1 dependent proton/potassium fluxes during fermentation of various concentrations of glucose.

Escherichia coli anaerobically ferment glucose and perform proton/potassium exchange at pH 7.5. The role of hyf (hydrogenase 4) subunits (HyfBDF) in sensing different concentrations of glucose (2g L-1 or 8g L-1) via regulating H+/K+ exchange was studied. HyfB, HyfD and HyfF part of a protein family of NADH-ubiquinone oxidoreductase ND2, ND4 and ND5 subunits is predicted to operate as proton pump. Specific growth rate was optimal in wild type and mutants grown on 2g L-1 glucose reaching ~ 0.8h-1. It was shown that in wild type cells proton but not potassium fluxes were stimulated ~ 1.7 fold reaching up to 1.95mmol/min when cells were grown in the presence of 8g L-1 glucose. Interestingly, cells grown on peptone only had similar proton/potassium fluxes as grown on 2g L-1glucose. H+/K+ fluxes of the cells grown on 2g L-1 but not 8g L-1 glucose depend on externally added glucose concentration in the assays. DCCD-sensitive H+ fluxes were tripled and K+ fluxes doubled in wild type cells grown on 8g L-1 glucose compared to 2g L-1 when in the assays 2g L-1glucose was added. Interestingly, in hyfF mutant when cells were grown on 2g L-1glucose and in 2g L-1 assays DCCD-sensitive fluxes were not determined compared to wild type while in hyfD mutant it was doubled reaching up to 0.657mmol/min. In hyf mutants DCCD-sensitive K+ fluxes were stimulated in hyfD and hyfF mutants compared to wild type but depend on external glucose concentration. DCCD-sensitive H+/K+ ratio was equal to ~ 2 except hyfF mutant grown and assayed on 2g L-1glucose while in 8g L-1 conditions role of hyfB and hyfD is considered. Taken together it can be concluded that Hyd-4 subunits (HyfBDF) play key role in sensing glucose concentration for regulation of DCCD-sensitive H+/K+ fluxes for maintaining proton motive force generation.

Glucose-responsive oral insulin delivery platform for one treatment a day in diabetes

Glucose-responsive oral insulin delivery platform for one treatment a day in diabetes

Effect of Glucose on Morphofunctional Properties Chondrocytes in Vitro

Articular cartilage is a highly specialized dense connective tissue, and can be considered as a composite gel with a relatively low content (5%) of cells, chondrocytes, embedded in the extracellular matrix. Chondrocytes are the only cell type in articular cartilage and are responsible for the biosynthesis and catabolism of the extracellular matrix. Osteoarthritis, the most common cartilage disease, has many independent risk factors, among which is diabetes mellitus, which allows us to hypothesize that different glucose concentrations have a huge effect on the morfunctional properties of chondrocytes in general and on the formation of osteoarthritis in particular. Despite numerous studies, the question of the effect of glucose on cartilage function is still open. In this regard, the study of morphofunctional changes in chondrocytes under the influence of various glucose concentrations is an urgent problem. The following results were obtained: an increase in the concentration of glucose in cell culture has a positive effect on cell viability and proteoglycan synthesis, but at an external glucose concentration of 25 мМ, cells die, while the synthesis of proteoglycans remains at a high level. The higher the concentration of glucose in the nutrient medium, the larger the cell size, which is probably due to hypertrophy of chondrocytes. In the future, the results obtained will be useful for understanding the process of hypertrophy and identifying ways to control it, as well as for a detailed study of other biochemical processes.

Synthetic enzyme-based nanoparticles act as smart catalyst for glucose responsive release of insulin

BackgroundEarlier studies have attempted to create electronic free insulin delivery systems using different glucose sensing mechanism, no successful clinical translation as hitherto been made. This study aimed to assess the faster responsiveness of the insulin release from this enzyme based nanoparticles which is a self-regulated insulin delivery system constructed by loading with insulin, enzyme glucose oxidase into hyaluronic acid and 2-nitroimidazole forming enzyme-based nanoparticles which works in accordance to the blood glucose level. Materials and methodEnzyme-based nanoparticles were prepared by ionic gelation method. Insulin content in the nanoparticles kept for stability study was estimated by human insulin enzyme based immunosorbent assay. In in-vitro studies; different concentrations of glucose were taken and the release study of insulin was recorded. ResultsThis enzyme-based nanoparticles were having average diameter of around 193 nm and stability studies showed that nanoparticles were stable upto 30 days at 4 °C. In-vitro studies showed the release of insulin from nanoparticle conjugates which was effectively correlated with the external glucose concentration created where different concentrations of glucose taken thus facilitating the stabilization of blood glucose levels in the hyperglycemia state which was achieved within 10 min. (400 mg/dL) wherein drug release rate remarkably increased in hyperglycemia state and no specific changes or small amount of release was observed in normoglycemia state (100 mg/dL). ConclusionOverall, this preliminary study of this enzyme-based nanoparticles formulation showed excellent rapid responsiveness towards hyperglycemia which might act as a potential biomimetic system in triggering the release of insulin in sustained manner.

Bridging substrate intake kinetics and bacterial growth phenotypes with flux balance analysis incorporating proteome allocation

Empirical kinetic models such as the Monod equation have been widely applied to relate the cell growth with substrate availability. The Monod equation shares a similar form with the mechanistically-based Michaelis-Menten kinetics for enzymatic processes, which has provoked long-standing and un-concluded conjectures on their relationship. In this work, we integrated proteome allocation principles into a Flux Balance Analysis (FBA) model of Escherichia coli, which quantitatively revealed potential mechanisms that underpin the phenomenological Monod parameters: the maximum specific growth rate could be dictated by the abundance of growth-controlling proteome and growth-pertinent proteome cost; more importantly, the Monod constant (Ks) was shown to relate to the Michaelis constant for substrate transport (Km,g), with the link being dependent on the cell’s metabolic strategy. Besides, the proposed model was able to predict glucose uptake rate at given external glucose concentration through the size of available proteome resource for substrate transport and its enzymatic cost, while growth rate and acetate overflow were accurately simulated for two E. coli strains. Bridging the enzymatic kinetics of substrate intake and overall growth phenotypes, this work offers a mechanistic interpretation to the empirical Monod law, and demonstrates the potential of coupling local and global cellular constrains in predictive modelling.

DEVELOPMENT OF "KERR EFFECT" DEVICES TO MEASURE URINE GLUCOSE LEVELS OF DIABETES MELITUS (DM) PATIENTS

<p>The development of the "Kerr Effect" device has been carried out to measure urine glucose levels of Diabetes Mellitus (DM) patients by applying an external electric field of two parallel plate capacitors. The purpose of this research was to design and construct a new "Kerr Effect" device to measure urine glucose levels of DM patients, to determine the effects of external electric fields and glucose concentrations on light polarization. The measurement of the polarization changes was done by emitting polarized light through a glucose standard solution sample and urine samples of DM patients. In this measurement, the sample was irradiated with an external electric field (0 kV / m - 80 kV / m) of parallel plate capacitors, and the changes of light polarization in the sample were observed. The obtained data of the measurement have been collected and plotted as a graph. The results showed that as the electric field increased the changes in polarization have a tendency to increase. In addition, the amount of glucose concentration in the urine also affected the magnitude of the change in the rotational polarization angle of the light. Therefore, the changes in the rotational polarization angle increase linearly due to the influence of the external electric field and the concentration of glucose in the urine.</p>

A microfluidic device for inferring metabolic landscapes in yeast monolayer colonies.

Microbial colonies are fascinating structures in which growth and internal organization reflect complex morphogenetic processes. Here, we generated a microfluidics device with arrays of long monolayer yeast colonies to further global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined boundary conditions. We observed the emergence of stable glucose gradients using fluorescently labeled hexose transporters and quantified the spatial correlations with intra-colony growth rates and expression of other genes regulated by glucose availability. These landscapes depended on the external glucose concentration as well as secondary gradients, for example amino acid availability. This work demonstrates the regulatory genetic networks governing cellular physiological adaptation are the key to internal structuration of cellular assemblies. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development and morphogenesis in more complex systems.

Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor.

Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR-TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose-binding protein with a circularly permuted variant of the monomeric fluorescent protein T-Sapphire. Here, we provide more details on iGlucoSnFR-TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2-10mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4-2mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7-2.5mM in cortical neurons from awake mice.

A voltage-dependent depolarization induced by low external glucose in neurons of the nucleus of the tractus solitarius: interaction with KATP channels.

Neurons from the brainstem nucleus of the tractus solitarius (NTS) participate in the counter-regulatory mechanisms in response to hypoglycaemia. ATP-sensitive potassium (KATP ) channels are expressed in NTS neurons, and are partially open at rest in normoglycaemic 5mM glucose. In normoglycaemic conditions, most NTS neurons depolarize in response to low external glucose (0.5mM), via a voltage-dependent mechanism. Conversely, most NTS neurons incubated in hyperglycaemic 10mM glucose do not respond to low glucose due to a more positive resting membrane potential caused by the closure of KATP channels following increased intracellular metabolic ATP. Our findings show that in hyperglycaemic conditions, NTS neurons failed to sense rapid changes in external glucose, which could be related to hypoglycaemia-associated autonomic failure. The nucleus of the tractus solitarius (NTS) is an integrative centre for autonomic counter-regulatory responses to hypoglycaemia. KATP channels link the metabolic status of the neuron to its excitability. Here we investigated the influence of KATP channels on the membrane potential of NTS neurons in normo- and hyperglycaemic external glucose concentrations, and after switching to a hypoglycaemic concentration, using in vitro electrophysiological recordings in brainstem slices. We found that in normoglycaemic (5mM) glucose, tolbutamide, a KATP channel antagonist, depolarized the membrane of most neurons, and this effect was observed in more hyperpolarized neurons. All neurons hyperpolarized after pharmacological activation of KATP channels. Most NTS neurons depolarized in the presence of low glucose (0.5mM), and this effect was only seen in hyperpolarized neurons. The effect of glucose was caused by a cationic current with a reversal potential around -50mV. In the presence of hyperglycaemic glucose (10mM), neurons were more depolarized, and fewer neurons responded to KATP blockage. Application of 0.5mM glucose solution to these neurons depolarized the membrane only in more hyperpolarized neurons. We conclude that NTS neurons present with KATP channels open at rest in normoglycaemic conditions, and their membrane potential is affected by extracellular glucose. Moreover, NTS neurons depolarize the membrane in response to the application of a low glucose solution, but this effect is occluded by membrane depolarization triggered by KATP blockage. Our data suggest a homeostatic regulation of the membrane potential by external glucose, and a possible mechanism related to the hypoglycaemia-associated autonomic failure.

(Invited) Artificial on-Skin Pancreas Fabricated with Smart Hybrid Hydrogel for Self-Regulated Insulin Delivery

Diabetes mellitus is the sixth leading cause of death. Traditional insulin injection not only involves pain, inconvenience, infection and nerve damage, but also is difficult to achieve persistent glycemic control. Current “closed-loop” delivery systems are either electronics-derived or protein-based, leading to the concerns of electrical failure or immunetoxicity. Phenylboronic acid (PBA), which can reversibly bind with glucose, is an attractive candidate for glucose sensing and self-regulated insulin delivery as it does not have the denaturing and immunetoxicity concerns. A totally synthetic smart PBA gel exhibiting supreme glucose-sensitivity at physiological pH and temperature was developed. [1] The release of insulin can be self-regulated by the skin layer formed on the gel surface and the insulin release rate was well corresponding to the external glucose concentration. [2] Recently, a catheter-combined device incorporating this smart gel has been successfully validated for mouse diabetes. [3] In order to deliver insulin in a glucose-responsive, painless, convenient and safe way, we developed an electronics-free, enzyme-free smart artificial on-skin pancreas, which is a microneedles (MNs) patch fabricated with smart hybrid hydrogel and silk fibroin. [4] The semi-interpenetrating network hydrogel was fabricated using PBA derivative for glucose sensing, N-isopropylacrylamide and silk fibroin for mechanical robustness. After skin insertion, the hybrid hydrogel in the tip region of MNs patch will continuously monitor the glucose level. At normoglycemic condition, the skin layer formed on the MNs surface will effectively block the release of insulin. However, with the increase of glucose concentration, the binding of PBA with glucose leads to the hydration of the hybrid MNs, and thus allows the release of insulin at hyperglycemia condition (Fig.1A). The hybrid hydrogel formulation was optimized by systematically investigating key parameters that may affect the gel properties, e.g. monomer concentration, silk fibroin proportion, crosslinking percentage, etc. The optimized hybrid hydrogel formed micro-porous and inter-connected structure, which enabled smooth and sustained release of loaded insulin (Fig. 1B). The physicochemical properties including swelling, stability, and glucose-responsive insulin release were explored. Furthermore, two-layer MNs were fabricated using micro-molding technology with hybrid hydrogel in the tip region and silk fibroin as the base layer. An insulin reservoir was attached to enhance the amount of loaded insulin. The morphology, gel & insulin distribution, mechanical strength of the MNs patch were characterized. The MNs patch penetrated the skin effectively, and could release insulin corresponding to the external glucose concentration (Fig. 1C). This novel on-skin pancreas is patient-friendly, possible to achieve persistent glycemic control, durable and disposable, which is promising to be the next-generation therapy for diabetes. R e ferences: [1]. A. Matsumoto et al., Chem. Commun., 2010, 46, 2203-2205. [2]. A. Matsumoto et al., Angew. Chem. Int. Ed., 2012, 51(9), 2124-2128 [3]. A. Matsumoto et al., Sci. Adv., 2017, 3(11), eaaq0723. [4]. A. Matsumoto and S. Chen, Japanese patent, No. 2018-053817, patent pending. Figure 1

A FRET flow cytometry method for monitoring cytosolic and glycosomal glucose in living kinetoplastid parasites.

The bloodstream lifecycle stage of the kinetoplastid parasite Trypanosoma brucei relies solely on glucose metabolism for ATP production, which occurs in peroxisome-like organelles (glycosomes). Many studies have been conducted on glucose uptake and metabolism, but none thus far have been able to monitor changes in cellular and organellar glucose concentration in live parasites. We have developed a non-destructive technique for monitoring changes in cytosolic and glycosomal glucose levels in T. brucei using a fluorescent protein biosensor (FLII12Pglu-700μδ6) in combination with flow cytometry. T. brucei parasites harboring the biosensor allowed for observation of cytosolic glucose levels. Appending a type 1 peroxisomal targeting sequence caused biosensors to localize to glycosomes, which enabled observation of glycosomal glucose levels. Using this approach, we investigated cytosolic and glycosomal glucose levels in response to changes in external glucose or 2-deoxyglucose concentration. These data show that procyclic form and bloodstream form parasites maintain different glucose concentrations in their cytosol and glycosomes. In procyclic form parasites, the cytosol and glycosomes maintain indistinguishable glucose levels (3.4 ± 0.4mM and 3.4 ± 0.5mM glucose respectively) at a 6.25mM external glucose concentration. In contrast, bloodstream form parasites maintain glycosomal glucose levels that are ~1.8-fold higher than the surrounding cytosol, equating to 1.9 ± 0.6mM in cytosol and 3.5 ± 0.5mM in glycosomes. While the mechanisms of glucose transport operating in the glycosomes of bloodstream form T. brucei remain unresolved, the methods described here will provide a means to begin to dissect the cellular machinery required for subcellular distribution of this critical hexose.

964: Effects of external glucose concentration on the contractility of human pregnant myometrium in vitro

964: Effects of external glucose concentration on the contractility of human pregnant myometrium in vitro

Characterization of the transport activity of SGLT2/MAP17, the renal low-affinity Na+-glucose cotransporter.

The cotransporter SGLT2 is responsible for 90% of renal glucose reabsorption, and we recently showed that MAP17 appears to work as a required β-subunit. We report in the present study a detailed functional characterization of human SGLT2 in coexpression with human MAP17 in Xenopus laevis oocytes. Addition of external glucose generates a large inward current in the presence of Na, confirming an electrogenic transport mechanism. At a membrane potential of -50 mV, SGLT2 affinity constants for glucose and Na are 3.4 ± 0.4 and 18 ± 6 mM, respectively. The change in the reversal potential of the cotransport current as a function of external glucose concentration clearly confirms a 1:1 Na-to-glucose transport stoichiometry. SGLT2 is selective for glucose and α-methylglucose but also transports, to a lesser extent, galactose and 3-O-methylglucose. SGLT2 can be inhibited in a competitive manner by phlorizin (Ki = 31 ± 4 nM) and by dapagliflozin (Ki = 0.75 ± 0.3 nM). Similarly to SGLT1, SGLT2 can be activated by Na, Li, and protons. Pre-steady-state currents for SGLT2 do exist but are small in amplitude and relatively fast (a time constant of ~2 ms). The leak current defined as the phlorizin-sensitive current in the absence of substrate was extremely small in the case of SGLT2. In summary, in comparison with SGLT1, SGLT2 has a lower affinity for glucose, a transport stoichiometry of 1:1, very small pre-steady-state and leak currents, a 10-fold higher affinity for phlorizin, and an affinity for dapagliflozin in the subnanomolar range.

Cooperative roles of glucose and asparagine-linked glycosylation in T-type calcium channel expression.

T-type calcium channels are key contributors to neuronal physiology where they shape electrical activity of nerve cells and contribute to the release of neurotransmitters. Enhanced T-type channel expression has been causally linked to a number of pathological conditions including peripheral painful diabetic neuropathy. Recently, it was demonstrated that asparagine-linked glycosylation not only plays an essential role in regulating cell surface expression of Cav3.2 channels, but may also support glucose-dependent potentiation of T-type currents. However, the underlying mechanisms by which N-glycosylation and glucose levels modulate the expression of T-type channels remain elusive. In the present study, we show that site-specific N-glycosylation of Cav3.2 is essential to stabilize expression of the channel at the plasma membrane. In contrast, elevated external glucose concentration appears to potentiate intracellular forward trafficking of the channel to the cell surface, resulting in an increased steady-state expression of the channel protein at the plasma membrane. Collectively, our study indicates that glucose and N-glycosylation act in concert to control the expression of Cav3.2 channels, and that alteration of these mechanisms may contribute to the altered expression of T-type channels in pathological conditions.

Fabrication of Biobased Polyelectrolyte Capsules and Their Application for Glucose-Triggered Insulin Delivery.

To enhance the glucose sensitivity and self-regulated release of insulin, biobased capsules with glucose-responsive and competitive properties were fabricated based on poly(γ-glutamic acid) (γ-PGA) and chitosan oligosaccharide (CS) polyelectrolytes. First, poly(γ-glutamic acid)-g-3-aminophenylboronic acid) (γ-PGA-g-APBA) and galactosylated chitosan oligosaccharide (GC) were synthesized by grafting APBA and lactobionic acid (LA) to γ-PGA and CS, respectively. The (γ-PGA-g-APBA/GC)5 capsules were then prepared by layer-by-layer (LBL) assembly of γ-PGA-g-APBA and GC via electrostatic interaction. The size and morphology of the particles and capsules were investigated by DLS, SEM, and TEM. The size of the (γ-PGA-g-APBA/GC)5 capsules increased with increasing glucose concentration due to the swelling of the capsules. The capsules could be dissociated at high glucose concentration due to the breaking of the cross-linking bonds between APBA and LA by the competitive reaction of APBA with glucose. The encapsulated insulin was able to undergo self-regulated release from the capsules depending on the glucose level and APBA composition. The amount of insulin release increased with incubation in higher glucose concentration and decreased with higher APBA composition. Moreover, the on-off regulation of insulin release from the (γ-PGA-g-APBA/GC)5 capsules could be triggered with a synchronizing and variation of the external glucose concentration, whereas the capsules without the LA functional groups did not show the on-off regulated release. Furthermore, the (γ-PGA-g-APBA/GC)5 capsules are biocompatible. These (γ-PGA-g-APBA/GC)5 with good stability, glucose response, and controlled insulin delivery are expected to be used for future applications to glucose-triggered insulin delivery.

Relationship between patterns of autonomous activity and external glucose concentration of culture medium

Relationship between patterns of autonomous activity and external glucose concentration of culture medium

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time.

We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of protein concentrations in single functional cells, and enables the distributions of concentrations across a cell population to be measured. This could be useful in investigating several cellular processes that are mediated by proteins, especially where changes in protein concentration in a single cell in response to changes in the extracellular chemical environment are subtle and rapid and may be smaller than the variability across a cell population.

Bio-inspired synthetic nanovesicles for glucose-responsive release of insulin.

A new glucose-responsive formulation for self-regulated insulin delivery was constructed by packing insulin, glucose-specific enzymes into pH-sensitive polymersome-based nanovesicles assembled by a diblock copolymer. Glucose can passively transport across the bilayer membrane of the nanovesicle and be oxidized into gluconic acid by glucose oxidase, thereby causing a decrease in local pH. The acidic microenvironment causes the hydrolysis of the pH sensitive nanovesicle that in turn triggers the release of insulin in a glucose responsive fashion. In vitro studies validated that the release of insulin from nanovesicle was effectively correlated with the external glucose concentration. In vivo experiments, in which diabetic mice were subcutaneously administered with the nanovesicles, demonstrate that a single injection of the developed nanovesicle facilitated stabilization of the blood glucose levels in the normoglycemic state (<200 mg/dL) for up to 5 days.

The Glucose Concentration‐Dependency of Spontaneous Activity in a Cultured Neuronal Network

SUMMARYCultured rat hippocampal network on a multielectrode array (MEA) dish is a useful model for analyzing network electrical dynamics and its developmental changes. Neurons autonomously form a complex network on an MEA dish and spontaneous electrical activity is often observed without any input from the external world. The spontaneous activity is generated by synaptic interactions between neurons and reflects an internal biochemical state of a whole neuronal network. The origin of electrical activity is electrochemical potential generated by active transport of ions, which requires catabolism of ATP. We elucidated the relationship between spontaneous electrical activity and external glucose concentration. The spontaneous activity changed transiently, depending on glucose concentrations. The number of electrical spikes in spontaneous activity increases depending on the concentration of external glucose concentration. Interestingly, this increase is not only suppressed but turn to decrease. In the case of glucose concentration is 17.56 mM, the number of activity is the most, and then it decreases in the case of glucose concentration is more than 20 mM. In addition, the decrease of neuronal activity at a high glucose concentration is not influenced by the blockade of inhibitory synaptic activity. These results suggest that a cultured neuronal network has optimal glucose concentration 17.56 mM, which corresponds to the concentration of glucose in a culture medium.