mM Of Glucose Research Articles

Novel mitochondria-targeted therapy AP39 limits mitochondrial dysregulation and diastolic dysfunction in 2D & 3D human models of diabetic cardiomyopathy

Abstract Introduction Mitochondrial dysregulation has been implicated in many complications of diabetes. Mitochondrial-targeted therapies AP39 and SS31 are renoprotective, but their cardioprotective potential in diabetes has not been fully resolved. Purpose We investigated AP39 and SS31 in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-hCM) and endothelial cells (iPSC-hEC) independently in 2D culture, as well as collectively in a 3D cardiac organoid (iPSC-hCO) model also comprising fibroblasts (iPSC-hFB), under type 2 diabetes (T2D)-like conditions. Methods Cells (iPSC-hCM, iPSC-hEC, iPSC-hFB; foreskin-2 cell line) were exposed to either 5.55mM glucose (control) or T2D conditions (20mM glucose plus fatty acids, endothelin-1 and cortisol) ± AP39 (50nM) or SS31 (1µM) for 48h. Cardiac organoids were subjected to 5.55mM glucose (control) or T2D for 4 days, with AP39 (50nM) or SS31 (1µM) added on day 2. Data are expressed as mean±SEM (fold) with n=4-6 from 2-3 independent experiments. One-way ANOVA with Dunnett`s post hoc analysis was performed with P<0.05 considered significant. Results 2D human cells: After 48h, T2D increased mitochondrial superoxide in iPSC-hCM (2.4±0.1 fold vs control) and iPSC-hEC (3.2±0.29 fold vs control), both P<0.05 (measured by MitoSOX). This was blunted by both AP39 (to 1.1±0.17) and SS31 (to 1.5±0.17) in T2D iPSC-hCM (P<0.05), with similar impact in iPSC-hEC, to 2.9±0.26 and 1.3±0.16 fold, both P<0.05. T2D increased cell death in both iPSC-hCM (1.6±0.09 fold vs control) and iPSC-hEC (1.6±0.12 fold, both P<0.05 measured by propidium iodide staining. Interestingly, AP39 selectively blunted cell death in T2D iPSC-hCM (to 1.1±0.06, P<0.05), mimicked by SS31 in T2D iPSC-hEC only (to 1.0±0.10, P<0.05). Further, T2D-induced mitochondrial hyperpolarisation in iPSC-hCM (1.5±0.09 fold vs control, assessed using TMRM), was only protected by AP39 (1.2±0.06 fold, P<0.05), but not SS31. 3D human cardiac organoids: After 4 days, T2D increased total contraction duration (1.6±0.10 fold control), time to peak (1.7±0.11) and relaxation duration (1.5±0.13, indicative of diastolic dysfunction), assessed by Olympus IX71 with a DP72 camera. All 3 contractile parameters were attenuated with AP39 (to 1.5±0.06, 1.3±0.09 and 1.0±0.04 fold, respectively) or SS31 (to 1.1±0.05, 1.2±0.15 and 1.2±0.12 fold, respectively), all P<0.05. T2D increased mitochondrial superoxide (2.7±0.16 fold control), blunted by AP39 (to 1.8±0.16) and SS31 (to 1.9±0.17), both P<0.05. Cellular injury measured by LDH was evident in T2D iPSC-hCO (2.1±0.23 fold control) was limited by AP39 (to 1.2±0.09) and SS31 (to 1.1±0.07), both P<0.05. Conclusion Our results highlight the cardioprotective potential of AP39 and SS31 to limit mitochondrial dysregulation, cell death and contractile dysfunction in human in vitro models of T2D. These findings may facilitate their clinical progression as therapeutic options for diabetic cardiomyopathy.

α-MnO2 with a cryptomelane structure for the non-enzymatic glucose electrooxidation in a neutral medium

A high demand for non-enzymatic glucose sensors and continuous glucose monitoring systems encourages the development of stable and active catalysts for the enzymeless anodic oxidation of glucose in the neutral medium. In regard to this reaction, we analyze the prospects of alpha manganese dioxide with a cryptomelane structure synthesized via an aqueous chemical route. To provide various specific surface area, the hydrated α-MnO2 oxide is annealed in a wide temperature range from 60 to 700 °C. Furthermore, we investigate the influence of the oxide annealing temperature on the amount of crystal water, chemical composition, morphology, electrochemical recharging behavior, electrocatalytic activity and stability in the glucose oxidation carried out in an isotonic NaCl-based phosphate-buffered saline (pH 7.40). To solidly determine potentials of the irreversible cathodic dissolution of α-MnO2 as well as O2/Cl2 evolution on α-MnO2, a rotating ring-disk electrode is applied. For the most active α-MnO2 sample with the 100 μg cm−2geo loading the glucose oxidation current of ca. 40 μA cm−2geo after 1 h at +0.8 V (SCE) for 7 mM of glucose is achieved. The linear concentration dependence in a physiological glucose range (1–30 mM) with the sensitivity of ca. 17.8 μA mM−0.5 cm−2geo at 25 °C is observed. Finally, glucose selectivity of α-MnO2 in relation to fructose, galactose, xylose, ribose, urea, lactic acid, acetone, ascorbic acid and paracetamol is revealed.

Interleukin-1 receptor-dependent and -independent caspase-1 activity in retinal cells mediated by receptor interacting protein 2.

Inflammation and cell death play an important role in the pathogenesis of diabetic retinopathy. Previously we observed sustained activation of pro-inflammatory caspase-1 in retinas of diabetic animals and patients. In this study, we aimed to look at mechanisms underlying chronic caspase-1 activation in vitro and in vivo. Non-diabetic and diabetic wild type and IL-1 receptor (IL-1R1) knockout mice were used for in vivo experiments. Diabetes was induced using STZ (streptozotocin). Human Müller cells were used for in vitro studies. Cells were treated with either 5mM or 25mM glucose or interleukin-1beta (IL-1β) in the presence or absence of IL-1 receptor antagonist (IL-1ra) or siRNA against RIP2 (receptor interacting protein-2) for up to 96h. Outcome measurements to assess Müller cell functions included measurements of caspase-1 activity using a fluorescence peptide substrate, production of IL-1β by Elisa, and cell death using trypan blue exclusion assays. Our in vivo results demonstrate that caspase-1 activation progresses from an IL-1R1 independent mechanism at 10weeks of diabetes to an IL-1R1 dependent mechanism at 20weeks indicating that feedback through IL-1R1 is crucial for sustained caspase-1 activity in retinas of mice. A similar hyperglycemia-mediated caspase-1/IL-1β/IL-1R1 feedback signaling was detected in vitro in human Müller cells which was prevented by treatment with IL-1ra. Our data also indicate that hyperglycemia induces caspase-1 activation initially but IL-1β sustains caspase-1 activation via caspase-1/IL-1β/IL-1R1 feedback and we identified RIP2 as mediator for both hyperglycemia- and IL-1β-induced caspase-1 activation. Activation of caspase-1/IL-1β/IL-1R1 feedback signaling caused Müller cell death which was prevented by RIP2 knockdown. We conclude that any intervention in caspase-1/IL-1β/IL-1R1 feedback signaling presents novel therapeutic options for the treatment of diabetic retinopathy.

12239 Effect Of Nano2, A Donor Of Nitric Oxide, On Glucose Mediated Oxidative Stress In Pancreaticb-cells

Abstract Disclosure: K.N. Islam: None. R.K. Islam: None. M.B. Rashid: None. B. Smith: None. Diabetes is a leading cause of morbidity and mortality. Elevation of oxidative stress is a common cause in diabetic patients because of higher levels of blood sugar concentration. Nitric oxide (NO) is a gasotransmitter that has many health benefits for many disease conditions. NO also has an antioxidant property. The pancreatic β-cells are insulin-secreting cells. The present study was undertaken to investigate the effects of NaNO2, a donor of NO on glucose-induced oxidative stress in pancreatic β-cells (HIT) from a Chinese hamster. First, we determined the effects of dose responses of glucose (25, 50, 75, 100, and 150 mM) on the induction of oxidative stress as it was measured by the formation of thiobarbituric acid reactive substances (TBARS) in serum starved β-cells. We observed marked oxidative stress in the serum starved β-cells by the treatment with 75 mM of glucose. Later, we tested the effects of NaNO2 on glucose-induced oxidative stress in serum starved β-cells, and we found that NaNO2 markedly inhibited glucose-induced oxidative stress in serum starved β -cells. Treatment with NaNO2 caused significant induction of cell viability and a marked reduction of cellular cytotoxicity in β-cells under serum starvation incubated without or with glucose. In addition, NaNO2 treatment of β-cells antagonized expression levels of inflammatory cytokines, TNF-α and inhibited the activation of the transcription factor, NF-κB signaling pathway. Our findings suggest that NO or NO donors may be used as therapy to cure diabetes via inhibiting oxidative stress thereby decreasing blood sugar levels in diabetic patients. Presentation: 6/3/2024

7880 The Role Of PTHrP In Glucose Metabolism And Insulin Secretion

Abstract Disclosure: I.M. Max-Harry: None. N. Kantake: None. C.S. Nunemaker: None. T.J. Rosol: None. Insulin and glucagon are the two most important hormones for blood glucose homeostasis. Identifying the key players in their regulation will contribute greatly towards combatting types 1 and 2 diabetes. Parathyroid Hormone-related protein (PTHrP), a protein expressed by pancreatic beta cells, has been implicated in various developmental and physiological roles, but particularly, PTHrP increases beta-cell proliferation and insulin secretion in pancreatic islets. Incubating mouse pancreatic islets and Min6 cells with 10nM exogenous PTHrP showed a significant increase in both insulin secretion (for islets, PTHrP: 865.4ng/ml ± 77.66, con: 498.5ng/ml ± 40.63, p=0.024, n=4) and glucagon secretion (PTHrP: 20.87pmol/l ± 3.776, con: 8.798pmol/l ± 1.616, p= 0.068, n=3) after 24hours. In order to further investigate the effects observed, min6 cells were transiently transfected to overexpress PTHrP, and a glucose-stimulated insulin secretion (GSIS) assay was carried out with 2mM and 20mM glucose. The results showed a reduction in basal insulin secretion at 2mM glucose and an increase in insulin secretion at 20mM glucose for the PTHrP transfected cells. PTHrP improved the stimulation index of Min6 cells (PTHrP: 2.279 ± 0.2056, con: 1.659 ± 0.1386, p= 0.03, n=6) and enhanced their sensitivity to glucose. Preliminary proliferation assays showed that cells transfected with PTHrP have increased proliferation and a protection from cell death when incubated in serum-free media. This study, in addition to our previously published research showing that a lack of intact PTHrP leads to islet dysfunction, revealed that PTHrP may be crucial to appropriate glucose sensitivity of beta cells. Further studies are being carried out to understand the mechanism behind these effects and the genes/proteins involved in the regulation of insulin secretion by PTHrP. Presentation: 6/1/2024

7700 Two Years Of Testosterone Therapy Reduces Glycemia In Males At High Risk Of Type 2 Diabetes Without Affecting Beta Cell Function: Results From A Randomised Controlled Trial And Cellular Model

Abstract Disclosure: M. Umapathysivam: None. S. Nawaz: None. K. Robledo: None. C.A. Allan: None. K. Bracken: None. M. Grossmann: None. D.J. Handelsman: None. W.J. Inder: None. D. Jesudason: None. B. Stuckey: None. B. Yeap: None. A. Januszewski: None. A. Jenkins: None. A. Conway: None. B. Hastoy: None. G.A. Wittert: Consulting Fee; Self; I-Nova. Grant Recipient; Self; Lawley pharmaceuticals, Bayer, Weight Watchers, Eli Lilly & Company. Speaker; Self; Bayer, Inc., Amgen Inc, Besin Health Care. Background: In the Testosterone for the prevention of type 2 diabetes (T4DM) Study, treatment with testosterone (T)-undecanoate (1000 mg IM 3 monthly) vs placebo decreased fat mass and reduced the risk of T2D by 40% after 2 years. However, the mechanism(s) by which T therapy prevents or reverts T2D remain unclear. Aim: To assess (1) the impact of T treatment on insulin resistance (IR) and beta-cell function in the T4DM study and (2) the in-vitro effect of graded T exposure on glucose stimulated insulin secretion (GSIS) from the human beta-cell line EndoC-βH1. Methods: (1) T4DM study: Men (n= 1007) from the T4DM study aged, 50-74 years, waist circumference (WC) >95cm, with prediabetes, or newly diagnosed T2D (by OGTT) and serum testosterone (T) ≤ 14 nmol/L (chemiluminescent assay) treated with T-undecanoate (1000 mg IM 3 monthly) or placebo for 2 years. Men were included if they had fasting bloods at baseline, weeks 18, 66, and 102 (N=743). We compared the change in glycated albumin, and derived measures of beta-cell function (HOMA2-B) and insulin resistance (HOMA-IR) using fasting glucose and c-peptide in T and placebo treated men using linear regression models.(2) EndoC-βH1 were incubated with T 5nM, 10nM and 15nM for 20 min and 15nM T for 2-days. Insulin secretion and content were measured at baseline (1mM glucose) and after 20 min exposure 20mM glucose. Results: Treatment with T compared to placebo reduced glycated albumin (-3.4 umol/L; 95%CI:1.4-5.4, p<0.001) maximally at week 66, along with C-peptide (corrected for baseline C-peptide and change in fasting glucose) (-0.12 nmol/L;-0.22,-0.02; p=0.01), HOMA2-B (-6.1; 95%CI: -16, 3.7; p=0.2) and HOMA-IR (-0.20; 95%CI: -0.59,0.20; p=0.3). In-vitro, acute exposure of EndoC-βH1 to 5nM and 10nM of T had no effect on GSIS, and 15nM T reduced GSIS (<20%, p=0.002). T, at 15nM for 2 days was without effect on GSIS. EndoC-βH1 insulin content was not altered at any concentration or timeframe of T exposure. Conclusion: In men with pre-diabetes or early T2D, T treatment reduces glycemia without evidence either in vivo or in-vitro of an affect to increase insulin secretion. Presentation: 6/1/2024

Characterization of Electrochemically Active Bacteria Utilizing Redox Response in Microbial Electrolysis Cell

This study focuses on micro-electrochemical screening to select microbial strains capable of directly transfer electrons to the working electrode dependent on specific enzymatic machinery. The main objective of this work is to select and identify promising strains for allow the bioelectrolysis production of hydrogen. To achieve this goal, microbial composites (artificial biofilms), have been developed using Escherichia coli CGE1 from LCPME. CNRS Fransh, Pseudomonas putrifisciens (CIP 69.13) (CIP, Collection Institut Pasteur, Fransh). Shewanella oneidensis MR-1 (ATCC 700550), and Thiobacillus denitrificans, from (ATCC, American Type Culture Collection), each one enclosed in a matrix carbon nanotubes and protamine matrix, forming an artificial biofilm on buckypaper. Cyclic Voltammetry (CV) measurements were performed over a potential range of +0.4 to -0.7V at 5mV/s under 30°C, using a saturated KCl Ag/AgCl reference electrode and a stainless-steel grid counter electrode. For E. coli and P. putrifisciens, the measurement focused on the oxidation of 20mM glucose, while the former bacteria were growth with and without O2. For S. oneidensis and T. denitrificans the focus was on the reduction of fumarate and 20 mM of NaNOH3+, respectively. As results, E. coli and P. putrifisciens species show no notable electrochemical activity, with no signal of glucose oxidation, due to the absence of type C cytochromes in the cytoplasmic membrane, unlike S. oneidensis and T. denitrificans, that demonstrate a direct electron transfer.

Paper chip-based colorimetric biosensor for glucose by using CuFe2O4@GO as nanozyme

The development of nanomaterials with notable enzyme-like activity is of great significance to the field of bioanalysis. Herein, a nanomaterial composed of copper ferrite nanoparticles (CuFe2O4 NPs) and graphene oxide (GO) was successfully prepared (CuFe2O4@GO). The obtained CuFe2O4@GO was proved to have peroxidase-like (POD-like) activity, which could catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to generate oxidized TMB (ox-TMB) in the presence of H2O2 with an obvious color change. On this basis, a colorimetric biosensor using a UV–vis spectrometer as readout and a paper chip-based colorimetric biosensor using a smartphone as readout was constructed for glucose detection, respectively, as glucose oxidase (GOD) could oxidize glucose to form H2O2. The UV–vis spectrometer-based approach exhibits a linear range of 0 to 1000 μM and a limit of detection (LOD) of 0.79 μM for glucose. The smartphone-based method shows a linear range of 0.1 to 50 mM and a LOD of 0.04 mM for glucose. Moreover, the fabricated colorimetric biosensors have good selectivity towards glucose and the sample matrixes cannot affect the detection of glucose. Finally, the concentration of glucose in human serum samples was successfully detected using two different ways, and the results are close to the clinical values. The proposed method embraces several good merits. Firstly, the colorimetric biosensors combining UV–vis spectrometer and smartphone offer precise results, which could effectively prevent the occurrence of false events. Secondly, paper-based microfluidic analytical device (μPAD) as analytical platform and smartphone as readout provides a versatile approach for constructing sensitive, cost-effective, and simple colorimetric biosensor for the visual monitoring of glucose. Lastly, except for glucose detection, this work also shows great application prospects for other small molecules detection that could generate H2O2.

The Self-Powered Thermoelectric System Driving an Electric Field for Raman-Spectrum Glucose Quantitative Analysis

For a long time, blood glucose monitoring has been a challenging issue in the field of medicine. Although it is possible to visit a hospital for regular blood tests to track blood sugar concentration, the physiological reactions triggered by blood collection might lead to feeling weak, dizzy, or even vomiting. Therefore, especially for diabetic patients, they need to frequently monitor their blood sugar concentration in their daily lives to ensure their health. Currently, the primary methods for blood glucose testing still rely on invasive techniques, such as using fingertip glucose meters and other methods which require skin puncture to obtain blood samples. Although these methods provide a quick assessment of current blood glucose concentration, they come with the pain and discomfort of blood collection process and the potential risk of infection. As a result, in recent years, many researchers have been dedicated to developing non-invasive blood glucose monitoring methods. However, accurately measuring glucose concentration inside the human body remains a challenging task. One of the primary issues is the presence of strong noise and background noise. To avoid this challenge, we have developed an innovative detection platform that is combines surface-enhanced Raman spectroscopy (SERS) and external electric field sensing technology. With the electromagnetic effect, we add external electric field in our platform to excite more the surface plasmon resonance. By using this method, we can achieve signal amplification to detect lower concentrations of glucose. Now, we can detect down to 0.001mM of glucose. We also exhibit self-powered electric field system, using the flexible themoelctric device to combine our platform. By the property of self-powered, we success to create a self-powered electric field system. We focus on driving themoelectric through the temperature different between body temperature and environment. The approach is aimed at generating power from the human body, providing a self-powered thermoelectric system for Raman-spectrum glucose sensors. This innovative approach holds promise for advancing non-invasive blood glucose monitoring and benefiting a larger population of diabetes patients in the future.

Abstract We095: Using Human Cardiac Organoids to Elucidate the Cardioprotective Potential of Mitochondrial-Targeted Therapies in a Preclinical Model of Diabetic Cardiomyopathy

Introduction: Mitochondrial dysregulation is implicated in many complications of type 2 diabetes (T2D). Mitochondrial-targeted therapies SkQ1, AP39 and SS31 limit renal T2D complications. We sought to obtain the first evidence of the cardioprotective potential of SkQ1, AP39 and SS31 in T2D, using a human induced pluripotent stem cell (iPSC)-derived 3D multicellular cardiac organoid model of diabetic cardiomyopathy. Methods: Human cardiac organoids (foreskin-2 cell-line) generated from human iPSC-derived cardiomyocytes, cardiac fibroblasts and endothelial cells were subjected to 5.55mM glucose (control) or T2D-like conditions (20mM glucose, fatty acids, endothelin-1 and cortisol) for 4 days with supplementation of either SkQ1 (50nM), AP39 (50nM) or SS31 (1µM) on day 2. On day 4, endpoint parameters were evaluated in cardiac organoids, including contractile function (captured via Olympus IX71), mitochondrial superoxide levels (MitoSOX assay), mitochondrial membrane potential (TMRM assay), and cellular injury (lactate dehydrogenase; LDH). Data are expressed as mean±SEM (fold control) with n =4-6 from 2-3 independent experiments. One-way ANOVA with Dunnett`s post hoc analysis was performed with P<0.05 considered significant. Results: After 4 days, T2D-conditions increased total contraction duration (1.55±0.10-fold control), time-to-peak (1.71±0.11-fold control, indicative of systolic dysfunction) and relaxation duration (1.46±0.13-fold control, indicative of diastolic dysfunction) in cardiac organoids. All 3 functional parameters were attenuated by AP39 (to 1.15±0.06, 1.32±0.09 and 1.04±0.04-fold control, respectively), or by SS31 (to 1.10±0.05, 1.12±0.15 and 1.09±0.12-fold control, respectively; all P<0.05), but not SkQ1. T2D-conditions increased mitochondrial superoxide (2.68±0.16-fold control), which was blunted by all 3 interventions, SkQ1 to 2.00±0.29, AP39 to 1.82±0.16 and SS31 to 1.89±0.17 (all P<0.05). Cellular injury was evident in T2D cardiac organoids (2.07±0.23-fold control), which was again limited by SkQ1 (to 1.06±0.11), AP39 (to 1.18±0.09) or SS31 (to 1.08±0.07), all P<0.05. Conclusion: These findings in human cardiac organoids highlight the cardioprotective potential of mitochondrial-targeted therapies to limit T2D-induced cardiac dysfunction, mitochondrial dysregulation and cellular injury, potentially facilitating clinical progression of candidates such as AP39 and SS31 as new therapies for patients with diabetic cardiomyopathy.

Enhanced non-enzymatic multicomponent detection via one-step hydrothermal synthesis of widely dispersed Zn-SnO2 nanoparticles on nitrogen-doped reduced graphene oxide

ABSTRACT Herein, we report a new type of non-enzymatic nanostructured biosensor designed for the concurrent determination of dopamine, cholesterol, and glucose in human blood samples. The prepared biosensor using low-temperature one-pot hydrothermal processing is a Zn-doped SnO2-dispersed nitrogen-doped reduced graphene oxide (N-rGO)/Zn-SnO2 nanocomposite. This modified electrode allows direct electron transfer and thus offers electrochemical techniques to detect target molecules in analytes without the use of enzymes. The biosensor displayed good voltammetric responses within the human reference range: 20–70 nM for dopamine, 1–8 mM for cholesterol, and 1–10 mM for glucose. It also showed excellent reusability and can be restored by washing with ethanol. Due to the use of Zn-doped SnO2 nanoparticles for enhanced catalytic activity and N-rGO for high conductivity and active sites, sensitive and selective detection can be achieved. This hybrid composite demonstrates excellent catalytic activity, repeatability, stability, and clinical comparability for real-time measurement of dopamine, cholesterol, and glucose simultaneously.

Glucose-Stimulated Mucus Secretion by Goblet Cells Mitigates Intestinal Barrier Dysfunction in a Rat Model of Mesenteric Ischemia/Reperfusion Injury

BackgroundSuperior mesenteric ischemia/reperfusion (I/R) causes barrier dysfunction and facilitates bacterial translocation (BT) in the small intestine, which can even lead to systemic sepsis. Our previous research showed that luminal administration of glucose and its anaerobic glycolytic metabolites exerted cytoprotective effects on epithelial cells and ameliorated I/R-induced BT in the liver and spleen. Notably, the reduction of BT occurs over the whole intestinal tract, not only restricted in the ligated glucose-containing loop. ObjectivesIn this study, we hypothesized that local jejunal glucose-contacting might confer on the remote intestinal epithelium regeneration potential, fortify their barrier function and goblet cell secretory activity. MethodsTwo 10-cm jejunal segments were isolated in Wistar rats. One segment was ligatured at both ends and infused with Krebs buffer containing 0- or 50-mM glucose (local loop), whereas the adjacent segment was left unaltered and not exposed to glucose (remote loop). The rats then underwent either a sham operation or I/R challenge by occlusion of the superior mesenteric artery for 20 min, followed by reperfusion for 1 h. ResultsEnteral addition of glucose in the local jejunum loop alleviated ischemia-induced barrier defects, histopathological scores, cell death, and mucosal inflammation (myeloperoxidase and inflammatory cytokine production) in the remote jejunum. After ischemia, goblet cells in the remote jejunum showed cavitation of mucin granules and low MUC2 expression. Local addition of glucose enhanced MUC2 synthesis and stimulated a jet-like mucus secretion in the remote jejunum, which was accompanied by the restoration of crypt activity. ConclusionsOur results showed local enteral glucose effectively mitigates I/R-induced barrier dysfunction, suggesting that local glucose-stimulated mucus secretion by remote goblet cells may serve to mitigate mucosal inflammation and BT. We provide a more precise barrier protection role of enteral glucose upon I/R challenge, presenting new opportunities for future therapeutic potential.

Glucose influences endometrial receptivity to embryo implantation through O-GlcNAcylation-mediated regulation of the cytoskeleton.

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycemia on these processes remains poorly understood. Here, we show that physiological levels of glucose (5 mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17 mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in cocultures at 17 mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17 mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarized epithelium. Two-dimensional (2-D) and three-dimensional (3-D) morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of Rho (ras homologous)A-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5 mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signaling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes.NEW & NOTEWORTHY Understanding how glucose regulates endometrial function will support preconception guidance and/or the development of targeted interventions for individuals living with diabetes wishing to embark on pregnancy. We found that glucose can influence endometrial epithelial cell receptivity to embryo implantation by regulating posttranslational modification of proteins involved in the maintenance of cell polarity. Impaired or inappropriate endometrial receptivity could contribute to fertility and/or early pregnancy complications caused by poor glucose control.

Nimbolide protects against diabetic cardiomyopathy by regulating endoplasmic reticulum stress and mitochondrial function via the Akt/mTOR pathway

Nimbolide has been demonstrated to possess protective properties against gestational diabetes mellitus and diabetic retinopathy. However, the role and molecular mechanism of nimbolide in diabetic cardiomyopathy (DCM) remain unknown. Diabetes was induced in rats via a single injection of streptozotocin (STZ) and then the diabetic rats were administered nimbolide (5 mg/kg and 20 mg/kg) or dimethyl sulfoxide daily for 12 weeks. H9c2 cardiomyocytes were exposed to high glucose (25 mM glucose) to mimic DCM in vitro. The protective effects of nimbolide against DCM were evaluated in vivo and in vitro. The potential molecular mechanism of nimbolide in DCM was further explored. We found that nimbolide dose-dependently decreased blood glucose and improved body weight of diabetic rats. Additionally, nimbolide dose-dependently improved cardiac function, alleviated myocardial injury/fibrosis, and inhibited endoplasmic reticulum (ER) stress and apoptosis in diabetic rats. Moreover, nimbolide dose-dependently improved mitochondrial function and activated the Akt/mTOR signaling. We consistently demonstrated the cardioprotective effects of nimbolide in an in vitro model of DCM. The involvement of ER stress and mitochondrial pathways were further confirmed by using inhibitors of ER stress and mitochondrial division. By applying a specific Akt inhibitor SC66, the cardioprotective effects of nimbolide were partially blocked. Our study indicated that nimbolide alleviated DCM by activating Akt/mTOR pathway. Nimbolide may be a novel therapeutic agent for DCM treatment.

Vitamin D supplementation affects bone marrow-derived mesenchymal stem cells differentiation into insulin-producing cells.

Background this study was conducted to assess the effects of vitamin D on differentiation of bone marrow- derived mesenchymal stem cells (BM-MSCs) into insulin producing cells (IPCs). Method BM-MSCs were isolated from femur and tibia of rats and incubated in low (LG) or high glucose (HG) (5mM or 25mM), or high glucose DMEM media supplemented with vitamin D (0.2nM) (HGD) for 14 days. Cells viability was analysis by MTT assay. Differentiation of SCs was confirmed using measuring genes expression level of pdx1 and insulin, and insulin secretion, glucose stimulated insulin secretion, and insulin content by ELISA method. Results Cell viability was significantly higher in HGD than LG (p < 0.05) in day 3, also, in HG and HGD than LG (p < 0.001), and HGD vs. HG (p < 0.001) in day 7. Pdx1 and insulin level was markedly higher in HGD than LG (p < 0.05 and p < 0.01). pdx1 expression was markedly higher in HGD (p < 0.05) than LG, also insulin expression the HG (p < 0.05), and HGD (p < 0.01) groups compared to the LG group. Insulin release at 5mM glucose was notably higher in the HGD group compared to LG (p < 0.05), and at 25mM glucose, both HG and HGD showed significant increases vs. LG (p < 0.05 and p < 0.01, respectively). Insulin content was significantly higher in both 5mM and 25mM glucose for HG and HGD vs. LG (p < 0.01 and p < 0.001, respectively). In conclusion, treatment BM-MSCs with vitamin D could increase their differentiation into IPCs and it can be considered as a potential supplementary agent in enhancing differentiation SCs into insulin generating cells.

Enrichment and Characterization of Sheep Caecum Mixed Lactic Acid Bacteria as Potential Direct-fed Microbials (DFMs) in Livestock

Abstract Lactic Acid Bacteria (LAB) have been widely used as feed supplements in the form of live bacteria or Direct-fed microbials (DFMs) which are expected to have a positive impact on livestock productivity. However, each LAB strain has different characteristics so its ability to influence its host livestock will also be different. This research aims to characterize the LAB enrichment process obtained from the caecum of sheep. The sheep caecum LAB underwent re-inoculated while being enriched in MRS broth medium. After obtaining the LAB isolates, characterization was carried out in the form of pH measurements, total plate count, bacteriocin production, and bacterial kinetic growth. The results showed that LAB could be grown from the digesta of sheep caecum as seen from the decrease in the average pH of the growth medium reaching pH 4.14 with a bacterial colony count of 6,01 log CFU/ml. The grown LAB had a Ks value of 0.24 μM glucose and a μmax value of 2.69 OD. Apart from that, the clear zone test showed a positive result. In conclusion, Enrichment of mixed LAB cultures from sheep caecum has decreased environmental pH by 33%. Sheep caecum mixed LAB culture has easy growth characteristics and antimicrobial activity so it can be a suitable alternative DMF candidate. Further research focusing on the application of this LAB strain is needed to be able to determine the effect of bacteria on livestock.

Gut Epithelial Barrier Function is Impacted by Hyperglycemia and Secondary Bile Acids in Vitro: Possible Rescuing Effects of Specific Pectins.

Gut epithelial barrier disruption is commonly observed in Western diseases like diabetes and inflammatory bowel disease (IBD). Enhanced epithelial permeability triggers inflammatory responses and gut microbiota dysbiosis. Reduced bacterial diversity in IBD affects gut microbiota metabolism, altering microbial products such as secondary bile acids (BAs), which potentially play a role in gut barrier regulation and immunity. Dietary fibers such as pectin may substitute effects of these BAs. The study examines transepithelial electrical resistance of gut epithelial T84 cells and the gene expression of tight junctions after exposure to (un)sulfated secondary BAs. This is compared to the impact of the dietary fiber pectin with different degrees of methylation (DM) and blockiness (DB), with disruption induced by calcium ionophore A23187 under both normal and hyperglycemic conditions. Unsulfated lithocholic acid (LCA) and deoxycholic acid (DCA) show a stronger rescuing effect, particularly evident under 20mM glucose levels. DM19 with high DB (HB) and DM43HB pectin exhibit rescuing effects under both glucose conditions. Notably, DM19HB and DM43HB display higher rescue effects under 20mM glucose compared to 5mM glucose. The study demonstrates that specific pectins such as DM19HB and DM43HB may serve as alternatives for preventing barrier disruption in the case of disturbed DCA metabolism.

Characterization of glucose/non-glucose-tolerant β-glucosidases from the metatranscriptome in compost

Functional microbial communities producing β-glucosidases differentially regulate the expression of glucose/non-glucose-tolerant β-glucosidases in response to carbon catabolite repression (CCR), which is a common phenomenon. To better understand their biological roles in a composting environment, four representative β-glucosidase genes were cloned from the metatranscriptome of the compost for expression and characterization. BGLA contains conserved sites Trp168 and Leu173, associated with glucose tolerance. In the presence of 100 mM glucose, BGLA's hydrolysis activity increases by 80%. BGLB also contains Trp168 and Leu173; however, its hydrolysis activity is significantly inhibited by glucose. On the contrary, Trp168 of BGLC was replaced by Phe, yet its hydrolysis activity increased by 20% in the presence of 25 mM glucose. However, BGLD did not exhibit any enzymatic activity. Molecular docking and dynamic simulations revealed the structural basis of glucose tolerance. Metatranscriptomic analysis of the four genes showed that functional microbial communities upregulated or downregulated gene expression in response to different CCR environments to maintain overall carbon metabolic balance. These findings demonstrate that the enzymatic activity characteristics of individual glucose/non-glucose-tolerant β-glucosidases are consistent with their transcriptional regulation under CCR in the complex composting environment.

Bifunctional oxidase-peroxidase mimicking Fe–Ce MOF on paper-based analytical devices to intensify luminol chemiluminescence: Application for measuring different sugars with a smartphone readout

This study presents a potent paper-based analytical device (PAD) for quantifying various sugars using an innovative bi-nanozyme made from a 2-dimensional Fe/Ce metal-organic framework (FeCe-BTC). The MOF showed excellent bifunctional peroxidase-oxidase activities, efficiently catalyzing luminol's chemiluminescence (CL) reaction. As a peroxidase-like nanozyme, FeCe-BTC could facilitate the dissociation of hydrogen peroxide (H2O2) into hydroxyl radicals, which then oxidize luminol. Additionally, it was also discovered that when reacting with H2O2, the MOF turns into a mixed-valence MOF, and acts as an oxidase nanozyme. This activity is caused by the generated Ce4+ ions in the structure of MOF that can directly oxidize luminol. The MOF was directly synthesized on the PAD and cascaded with specific natural enzymes to establish simple, rapid, and selective CL sensors for the measurement of different sugars. A cell phone was also used to record light intensities, which were then correlated to the analyte concentration. The designed PAD showed a wide linear range of 0.1–10 mM for glucose, fructose, and sucrose, with detection limits of 0.03, 0.04, and 0.04 mM, respectively. It showed satisfactory results in food and biological samples with recovery values ranging from 95.8 to 102.4 %, which makes it a promising candidate for point-of-care (POC) testing for food control and medicinal purposes.

A novel bifunctional electrochemical nanosensor for simultaneous detection of glucose and insulin based on NiO/Co3O4@CuAl LDH-MWCNT nanocomposite-modified carbon paste electrode

The appropriate development of electrocatalytic nanomaterials is a significant step for constructing high-quality non-enzymatic electrochemical sensors. In this work, we presented effective copper aluminum layered double hydroxide (CuAl LDH)-based nanoparticles with two consecutive optimization steps through nickel oxide/cobalt oxide (NiO/Co3O4) merging and subsequent assembly with multi-walled carbon nanotubes (MWCNT). The obtained nanostructures were characterized by using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction pattern (XRD), and field emission scanning electron microscope images (FE-SEM). The synthesized nanomaterials were used as a carbon paste electrode (CPE) material for simultaneous dual-signal evaluation of glucose and insulin. The response of the modified electrode was investigated by some electrochemical approaches, including cyclic voltammetry (CV), electrical impedance (EI), differential pulse voltammetry (DPV), and amperometric titration. The NiO/Co3O4 CuAl LDH@MWCNT electrode displayed multi-functional properties through two well-defined separated peaks toward glucose and insulin detection with a linear range of 0.01–26.5 mM and 0.02–70.0 nM, respectively. The detection limit values were obtained as 2.8 µM for glucose and 17.3 pM for insulin. The sensor showed good tolerance to common interfering agents, excellent sensitivity, and appropriate stability during 30 days of dry storage at room temperature. This sensor was also used for the quantification of glucose and insulin in human serum samples, obtaining the recovery rate of 101.0 %-103.7 % and 98.0 %-106.0 %, respectively, with all the RSD values below 3.0 %. This study represented an efficient simultaneous sensing platform for glucose and insulin detection, which warrants its operational application in clinical samples.