High Glucose Concentrations Research Articles

Human HDL subclasses modulate energy metabolism in skeletal muscle cells

In addition to its antiatherogenic role, HDL reportedly modulates energy metabolism at the whole-body level. HDL functionality is associated with its structure and composition, and functional activities can differ between HDL subclasses. Therefore, we studied if HDL2 and HDL3, the two major HDL subclasses, are able to modulate energy metabolism of skeletal muscle cells. Differentiated mouse and primary human skeletal muscle myotubes were used to investigate the influences of human HDL2 and HDL3 on glucose and fatty uptake and oxidation. HDL-induced changes in lipid distribution and mRNA expression of genes related to energy substrate metabolism, mitochondrial function, and HDL receptors were studied with human myotubes. Additionally, we examined the effects of apoA-I and discoidal, reconstituted HDL particles on substrate metabolism. In mouse myotubes, HDL subclasses strongly enhanced glycolysis upon high and low glucose concentrations. HDL3 caused a minor increase in ATP-linked respiration upon glucose conditioning but HDL2 improved complex I–mediated mitochondrial respiration upon fatty acid treatment. In human myotubes, glucose metabolism was attenuated but fatty acid uptake and oxidation were markedly increased by both HDL subclasses, which also increased mRNA expression of genes related to fatty acid metabolism and HDL receptors. Finally, both HDL subclasses induced incorporation of oleic acid into different lipid classes. These results, demonstrating that HDL subclasses enhance fatty acid oxidation in human myotubes but improve anaerobic metabolism in mouse myotubes, support the role of HDL as a circulating modulator of energy metabolism. Exact mechanisms and components of HDL causing the change, require further investigation.

Culture Method and Regulation Mechanisms of Stages of C2C12 Cell Line Myogenesis

INTRODUCTION: C2C12 is an immortalized mouse myoblast cell line actively used as in vitro experimental models to study skeletal muscle metabolism in biomedical research. C2C12 cells can differentiate into myocytes under appropriate culture conditions. It is important to understand the specifics of each stage of myogenesis and its regulatory mechanisms for the targeted exposure and development of medical drugs, taking into account the mechanisms of pathogenesis of various diseases.
 AIM: To describe the method of culturing С2С12 cell line and to study regulatory mechanisms of С2С12 cell line myogenesis.
 MATERIALS AND METHODS: The study was conducted on C2C12 cell line. Differentiation of myoblasts was induced in the nutrient medium containing 2% horse serum. The differentiation stages were evaluated by studying the cells on the 1st, 4th and 7th days. Cells before differentiation were used as a comparison group. At each differentiation stage, the myoblast fusion index (IM) was evaluated with preliminary staining of the cells by Romanowsky–Giemsa method. The cell differentiation mechanism was evaluated by the level of myosin, a-actin, myogenic differentiation protein (MyoD), myogenin (MyoG) using Western blot method.
 RESULTS: On the 1st day of C2C12 cells culturing, formation of myotubes was observed (IM = 0.15 ± 0.05), on the 4th day — fusion of myoblasts with the formation of binucleated cells accompanied by an increase in the amount of MyoD and α-actin protein (IM = 0.44 ± 0.14). By the 7th day of differentiation, the fusion of cells increased with the formation of myotubes containing more than two nuclei; the content of MyoD and α-actin did not differ from the control, and the amount of MyoG and myosin increased (IM = 0.77 ± 0.04).
 CONCLUSION: The described method of culturing C2C12 cell line in Dulbecco’s Modified Eagle’s Medium with a high glucose content (4500 mg/l), containing 2% horse serum, L-glutamine (4 mМ), 100 Un/ml and 100 µg/ml of penicillin and streptomycin, is suitable for the formation of muscle cells, where MyoD and MyoG participate in the regulation of the increase in the amount of specific muscle proteins — α-actin and myosin.

Effects of feeding mycotoxin-contaminated diets and the use of an adsorbent on the intake, digestibility, rumen fermentation, nitrogen balance and physiological parameters in the blood of Nellore steers

The study objective was to investigate the effects of mycotoxin contaminated diets and the use of an adsorbent (yeast cell wall extract; YCWE) on the intake, digestibility, rumen fermentation, nitrogen balance and physiological parameters in the blood of Nellore steers. Eight rumen-cannulated Nellore steers (initial BW = 417 ± 42 kg) were used in a 4 × 4 replicated Latin square design. A 2 × 2 factorial treatment structure was used to investigate the effects of mycotoxin contamination of the diet, the use of a mycotoxin adsorbent and their interactions. The dietary contamination consisted of: (1) diet without mycotoxins (CTRL), and (2) control diet added mycotoxins (MYCOT). The second factor was: (1) absence (YCWE–) or (2) presence (YCWE+) of YCWE. Mycotoxin contamination negatively affected dry matter intake during the first week of treatment application (P = 0.05) and tended to lower intake during the sampling period (P = 0.08). MYCOT resulted in lower (P = 0.03) digestibility of neutral detergent fiber and tended to lower (P = 0.08) ruminal pH compared with CTRL. There was MYCOT × YCWE interaction on the proportion of isovalerate (P < 0.01) and trend on the proportion of valerate (P = 0.08) in the rumen. There was also an effect of MYCOT (P = 0.04) and a trend of YCWE (P = 0.10) on the proportion of isobutyrate. Animals received MYCOT treatments had higher N excretion in the urine (P = 0.01), and consequently lower N retention (P = 0.05). There was MYCOT × YCWE interaction effect (P = 0.04) on the serum glucose concentration, where the MYCOT YCWE– treatment had a higher glucose concentration than the CTRL YCWE–, and the use of YCWE in MYCOT diet resulted in a concentration similar to CTRL treatments. The use of YCWE resulted in higher (P = 0.03) serum concentration of total proteins and lower (P < 0.01) proportion of ceruloplasmin. Animals fed mycotoxins had a lower (P < 0.01) proportion of IgA. MYCOT × YCWE effects (P < 0.01) on hemoglobin concentration and hematocrit were observed, where MYCOT had lower concentrations than the control and the use of YCWE in the contaminated diet resulted in values similar to CTRL. Animals fed mycotoxins tended to have lower (P = 0.08) number of lymphocytes and had lower (P = 0.02) lymphocytes: neutrophils ratios. This study suggests that mycotoxins may have negative impacts on certain physiological parameters that can influence health and performance of beef cattle, and the use of YCWE can mitigate some of these negative changes.

Novel Fluorometric Assay of Antiglycation Activity Based on Methylglyoxal-Induced Protein Carbonylation.

Advanced glycation end products (AGEs), which can have multiple structures, are formed at the sites where the carbonyl groups of reducing sugars bind to the free amino groups of proteins through the Maillard reaction. Some AGE structures exhibit fluorescence, and this fluorescence has been used to measure the formation and quantitative changes in carbonylated proteins. Recently, fluorescent AGEs have also been used as an index for the evaluation of compounds that inhibit protein glycation. However, the systems used to generate fluorescent AGEs from the reaction of reducing sugars and proteins used for the evaluation of antiglycation activity have not been determined through appropriate research; thus, problems remain regarding sensitivity, quantification, and precision. In the present study, using methylglyoxal (MGO), a reactive carbonyl compound to induce glycation, a comparative analysis of the mechanisms of formation of fluorescent substances from several types of proteins was conducted. The analysis identified hen egg lysozyme (HEL) as a protein that produces stronger fluorescent AGEs faster in the Maillard reaction with MGO. It was also found that the AGE structure produced in MGO-induced in HEL was argpyrimidine. By optimizing the reaction system, we developed a new evaluation method for compounds with antiglycation activity and established an efficient evaluation method (HEL-MGO assay) with greater sensitivity and accuracy than the conventional method, which requires high concentrations of bovine serum albumin and glucose. Furthermore, when compounds known to inhibit glycation were evaluated using this method, their antiglycation activities were clearly and significantly measured, demonstrating the practicality of this method.

Curcumin Attenuates Ferroptosis and Ameliorates Erectile Function in Diabetic Rats by Activating Nrf2/HO-1 Pathway

Previous studies have shown that curcumin has a therapeutic effect on diabetic erectile dysfunction (ED), but the exact mechanism of action remains unclear. In order to ascertain the involvement of ferroptosis in diabetic ED and explore the underlying mechanism by which curcumin mitigates ferroptosis in penile endothelial cells under conditions of high-glucose stimulation, we conducted an investigation utilizing a rat model of diabetes induced by a high-fat diet and streptozotocin injection. The 40 male Sprague–Dawley rats were randomly divided into four groups: blank control, diabetes control, ferroptosis inhibitor treatment, and curcumin treatment. After 8 weeks, the erectile function of all rats was evaluated through electrical stimulation of the cavernous nerve. Histological and molecular changes of the cavernous body were analyzed using western blot and immunohistochemistry. Penile endothelial cells were then treated with appropriate concentrations of high glucose and curcumin to explore the mechanism of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling in curcumin’s ability to alleviate ferroptosis in endothelial cells stimulated by high glucose. According to research, diabetic rats experience a decrease in erectile function, damaged endothelial cells, and ferroptosis in tissues. However, curcumin treatment has been shown to be effective in improving erectile function and related tissue and molecular changes in diabetic rats. Furthermore, in vitro experiments have confirmed that curcumin can inhibit the occurrence of ferroptosis in penile endothelial cells that are stimulated by high glucose through Nrf2/HO-1 signaling. Curcumin has been found to improve the occurrence of penile ED in diabetic penile endothelial cells. This is achieved by inhibiting the level of ferroptosis, and the mechanism behind this improvement may be linked to the upregulation of the expression level of Nrf2/HO-1.

Effects of Glucose Levels on Inflammation and Amino Acid Utilization in Lipopolysaccharide-Induced Bovine Mammary Epithelial Cells.

Glucose and amino acids are important sources of nutrients in the synthetic milk of dairy cows, and understanding the fate of amino acids is essential to optimize the utilization of amino acids in milk protein synthesis, thereby reducing nutrient inefficiencies during lactation. The purpose of this study was to investigate the effects of LPS and different concentrations of glucose on (1) the expression of inflammatory factors and genes, (2) the glucose metabolism, and (3) amino acid utilization in BMECs. The results showed that there was an interaction (LPS × glucose, p < 0.05) between LPS and glucose content in the inflammatory cytokine genes (IL-6 and TNF-α) and the inflammatory regulatory genes (CXCL2, CXCL8, and CCL5). With the addition of LPS, the HG + LPS group caused downregulated (p < 0.05) expression of IL-6 and TNF-α, compared with the LG + LPS group. Interestingly, compared with the LG + LPS group, the HG + LPS group upregulated (p < 0.05) the expression of CXCL2, CXCL8, and CCL5. LPS supplementation increased (p = 0.056) the consumption of glucose and GLUT1 gene expression (p < 0.05) and tended to increase (p = 0.084) the LDHA gene expression of BMECs under conditions of different concentrations of glucose culture. High glucose content increased (p < 0.001) the consumption of glucose and enhanced (p < 0.05) the GLUT1, HK1, HK2, and LDHA gene expression of BMECs with or without LPS incubation, and there was an interaction (LPS × glucose, p < 0.05) between LPS and glucose concentrations in GLUT1 gene expression. In this study, LPS enhanced (p < 0.05) the consumption of amino acids such as tryptophan, leucine, isoleucine, methionine, valine, histidine, and glutamate, while high levels of glucose decreased (p < 0.01) consumption, except in the case of tyrosine. For histidine, leucine, isoleucine, and valine consumption, there was an interaction (LPS × glucose, p < 0.05) between LPS and glucose levels. Overall, these findings suggest that relatively high glucose concentrations may lessen the LPS-induced BMEC inflammatory response and reduce amino acid consumption, while low glucose concentrations may increase the demand for most amino acids through proinflammatory responses.

The addition of Aspergillus flavus on composting process of cotton waste for cultivation of paddy straw mushroom [Volvariella volvacea (Bull.) Singer 1951

Paddy straw mushroom [Volvariella volvacea (Bull.) Singer 1951] is a tropical edible mushroom and commonly cultivated in Indonesia. Composting process for preparing substrate plays an important role during mushroom cultivation. The purpose of this study is to investigate the effect of the addition of Aspergillus flavus, the lignocellulolytic fungi in the composting process of cotton waste for cultivation of V. volvacea. The result showed that there was an increase in temperature of compost of 27-58°C and pH of 7-8 for seven days observation. The concentration of cellulose, hemicelluloses and lignin was decreased from the 2nd days of the composting process in both the addition and without the addition of A. flavus. The highest concentration of glucose was 0.089 mg/mL without addition of A. flavus and 0.122 mg/mL with the addition of A. flavus. The highest concentration of xylose was 0.070 mg/mL without addition of A. flavus and 0.173 mg/mL with the addition of A. flavus. The fresh weight of the fruiting body of V. volvacea was 11.153 g and 9.395 g with the addition of A. flavus and without the addition of A. flavus in the composting process, respectively.

Long non-coding RNA PANDA drives diabetic vascular dysfunction by promoting endothelial senescence and oxidative damage

Abstract Background Accumulation of reactive oxygen species and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. Long non-coding RNAs (lncRNAs) are emerging as important players in the pathogenesis of cardiovascular disease. Recent work has shown that PANDA, a newly identified lncRNA, is a key regulator of cellular senescence and apoptosis. Purpose To investigate the role of lncRNA PANDA in diabetic vascular disease. Methods Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5 mmol/L) and high glucose concentrations (HG, 25 mmol/L). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection while a scrambled RNA was used as a negative control. Expression of PANDA was assessed by real time PCR. PANDA RNA immuno-precipitation (RIP) was performed to check its binding to relevant transcriptional factors (NF-YA). Beta-galactosidase staining was used to detect endothelial senescence while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. Finally, RNAs sequencing (RNA-seq) and bioinformatic analysis (network perturbation amplitude, NPA) were leveraged to unveil transcriptional changes upon PANDA depletion. Finally, the ex vivo effect of PANDA siRNA on endothelial function was assessed in aortic rings from diabetic mice. Results PANDA expression was significantly increased in HAECs exposed to HG as compared to NG (Fig. 1A). We found that under HG conditions PANDA sequesters the transcription factor NF-YA, thus inhibiting the expression of NF-YA-dependent pro-survival and anti-aging genes (Fig. 1B). Indeed, silencing of PANDA in HG-treated HAECs restored the expression of anti-apoptotic genes Bcl-X(L) and Bcl-2 while blunting senescence-related genes such as p53 and p16INK (Fig. 1C). PANDA depletion also abolished HG-induced cellular apoptosis and senescence, as assessed by caspase-3 activity assay and b-gal staining (Fig. 1D-E), while improving endothelial migration and tube formation (Fig.1F-G). Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 (HMOX1) as the top-ranking transcript in HG-treated cells (Fig.1H). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation (Fig. 1I). Of interest, PANDA levels were increased in aortas from diabetic mice (Fig. 2A) while depletion of PANDA rescued endothelial dysfunction (Fig. 2B). Conclusions Hyperglycemia-induced upregulation of PANDA drives endothelial senescence while impairing angiogenic properties. PANDA depletion in HG-treated HAECs rescues maladaptive transcriptional changes by restoring expression of the antioxidant gene HMOX1. Of note, targeting PANDA in the diabetic vasculature was able to rescue endothelial dysfunction. Our results indicate PANDA as a novel molecular target in the setting of diabetic vascular disease.

Histone Mono-methylation by SETD7 Orchestrates Angiogenic Response in Diabetic Limb Ischemia

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes (DM) and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in DM patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in this setting. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the histone methyltransferase SETD7 - favours an open chromatin thus enabling gene transcription. Purpose: To investigate whether SETD7-dependent epigenetic changes modulate angiogenic response in diabetes. Methodology: Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G, (Figure 1). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle (Figure 2). Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls (Figure 2) Conclusion: Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD.

Role of methyltransferase Set7 in obesity-related endothelial activation: functions beyond histone methylation

Abstract Introduction Obesity is associated with environmental changes such as hyperglycemia, hyperinsulinemia, inflammation, and elevated free fatty acids, all of which compromise endothelial function1,2. We have previously reported that in individuals with type 2 diabetes, lysine methyltransferase Set7 is involved in hyperglycemia-induced endothelial damage through monomethylation of histone 3 at lysine 4 (H3K4me1)3. Apart from such histone-driven epigenetic modulation, Set7-induced methylation of non-histone proteins is emerging as a novel mechanism underlying the (dys)regulation of many biological processes4. Purpose To investigate the role of Set7-related endothelial damage in cells exposed to different obesity-associated stimuli and to determine its impact on the protein methylome in these settings. Methods Human aortic endothelial cells (HAECs) were exposed to high concentrations of glucose, insulin, TNFa, or fatty acids (palmitate or oleate). Set7 overexpression, silencing, and inhibition studies were also performed in these cells. Gene and protein expression were assessed by RT-PCR and immunoblotting respectively, whereas production of the reactive oxygen species (ROS) was measured by electron spin resonance spectroscopy. Specific antibodies were used for the enrichment of lysine mono-, di-, and tri-methylated proteins and LC-MS/MS proteomic analysis was carried out for their identification. Results Each obesity model in HAECs increased the expression of nearly all the inflammatory markers tested and, except for TNFa, they also increased the expression of Set7. Significant changes in the global levels of H3K4me1, as well as of methylated proteins were also observed in these cells. Studies overexpressing a mutant form of Set7, which is lacking the SET domain, revealed that Set7 methyltransferase activity is required to define the protein methylome in HAECs. Similar results were obtained after Set7 silencing and inhibition. Despite no differences were observed in the production of ROS when modulating Set7 expression in HAECs, overexpression of an active form of Set7 and its silencing/inhibition resulted in a consistent up- and down-regulation, respectively, of several inflammatory mediators and adhesion molecules. Finally, proteomics analysis identified, for the first time, the protein methylome of endothelial cells, comprising almost 2,700 methylated proteins involved in essential biological processes such as RNA metabolism and cytoskeleton. Conclusions Our results indicate that Set7 may contribute to inflammatory changes in response to obesogenic insults. Moreover, our findings uncover a dual role of methyltransferase Set7 in governing the methylation of both histone and non-histone proteins in endothelial cells. These preliminary findings open a new field of research aiming to further elucidate the molecular mechanisms underlying the modulation of the protein methylome in the endothelium and its role in endothelial damage.

Biomineralized ceria nanoparticles target the heart to improve diabetic cardiac remodeling by regulating mitochondrial oxidative stress and decreasing excessive mitophagy

Abstract Background An altered state of cardiomyocytes due to high glucose concentrations results in cell hypertrophy, leading to diabetic cardiomyopathy (DCM).The molecular regulatory process of mitochondrial dyshomeostasis and mitophagy, oxidative stress, and reactive oxygen species (ROS) production in diabetes is still not fully understood. Mitochondrial dyshomeostasis and mitophagy could be critical factors in the heart remodeling that occurs in diabetes. Purpose Our transcriptome sequencing of heart tissue revealed upregulation of the gene encoding the transferrin receptor (TfR) in the diabetic rat (Figure 1A). We then validated in vitro that high glucose increased TfR protein expression in the HL-1 cell line (Figure 1B). Herein, we developed a novel therapeutic ceria nanoparticle via biomineralization using transferrin (Tf) protein as a temple and assessed its targeted therapy for DM-induced cardiac remodeling via Tf-TfR mediated endocytosis. Method We synthesized Tf protein-based ceria NPs (CeO2@Tf) via biomineralization. Then the physicochemical and biological properties of NPs were characterized. Cellular uptake, cytotoxicity test, intracellularmitochondrial membrane potential (MMP), and ROS were verified with mouse cardiomyocytes HL-1 cells line. Afterward, the male C57BL/6N mice were randomly divided into control, DM, DM+Metformin (Met), and DM+NPs groups with eight weeks of treatment. Heart structure and function were measured by echocardiographic. The heart tissues were stained with hematoxylin, eosin (H&amp;E), and Masson's trichrome stain to observe morphological changes and fibrosis. Western blot was used to determine mitochondrial homeostasis regulation-related proteins and mitophagy pathway. Results TEM and X-ray photoelectron spectroscopy characterized the properties successfully. MTT assay shows that HL-1 cell viabilities were not influenced by CeO2@Tf at 200 µM.High-glucose treatment led to increased ROS and decreased intracellular MMP in HL-1 cells. DM mice cardiac transcriptome sequencing results showed upregulation of transferrin receptor gene expression. Echocardiographic showed increased LAD and abnormal diastolic and systolic functions. All these abnormal changes were improved in the DM+ Met and DM+NPs groups. Our investigations revealed proteins linked to the PINK1-Parkin signaling pathway (Figure 2A-B) and found that the level of mitophagy in the diabetic group was overly high, which was improved after the application of CeO2@Tf. We observed the mitochondrial autophagosomes through transmission electron microscopy (Figure 2C). The autophagosomes in the diabetic group increased significantly and improved after treatment with CeO2@Tf. Conclusions These consistent biochemical and histopathological results suggest that the biomimetic mineralization of CeO2@Tf could inhibit oxidative stress and improve mitochondrial function in vitro induced by high-glucose and in vivo caused by DM, alleviating cardiac remodeling in DM mice.

Abstract 176: The Pathological Role of Aldose Reductase in Isolated Cardiomyocytes Undergoing Hypoxia / Reoxygenation After Prior Exposure to High Glucose Concentrations

Hyperglycemia increases the vulnerability of the cardiovascular system to ischemia/reperfusion (IR) injury. There are currently no effective therapeutic strategies. We hypothesized that hyperglycemia would increase glucose flux through the polyol pathway. Aldose reductase (AR) catalyzes the first step of this pathway by converting glucose to sorbitol. This reaction generates more oxidative stress due to the depletion of NADPH and osmotic stress caused by sorbitol accumulation, making the heart more susceptible to IR injury. This study aimed to better understand the injury mechanism in cardiomyocytes after high glucose exposure and IR injury. Confluent cardiomyocytes were exposed for 24 hours to 25, 50, or 100 mM glucose or 25 mM glucose with 25 or 75 mM sucrose concentrations added. The non-metabolizable sugar sucrose was used as an osmolarity control. Epalrestat, an AR inhibitor, was added at 0, 1, or 10 μM per well (n = 18 per group) during glucose or sucrose exposure. Cells were then subjected to 24 hours of hypoxic conditions (glucose-, sucrose- and serum-free media; 0.01% O 2 , 37 °C; hypoxic chamber) or normoxic conditions (complete media; 21% O 2 , 37 °C), followed by a 2-hour reoxygenation period in normoxic conditions. Cell injury was quantified by measurement of lactate dehydrogenase (LDH) release. Data were normalized to 25 mM glucose after control/normoxic conditions without AR inhibitor. A three-way ANOVA was conducted to compare groups, followed by the Holm-Šidák test. Increasing glucose concentrations lead to increased cell damage after hypoxia/reoxygenation. Adding the AR inhibitor during the glucose exposure period reduced LDH release after hypoxia/reoxygenation in a dose-dependent manner. The addition of sucrose did not increase cell injury. The AR inhibitor did not affect cells exposed to sucrose. The polyol pathway may play a dominant role in increased cell injury by hyperglycemia after hypoxia/reoxygenation.

Dose-dependent anti-hyperglycemic & anti-dyslipidemic potential of aqueous leaves extract of Typha elephantina in-vivo and in-vitro

Diabetes mellitus is among the fundamental causes of illness and millions of deaths around the globe are directly attributed to it each year. Current antidiabetic medications often lack sustained glycemic control and carry significant risks of side effects. As a result, the use of plant-based treatments has gained popularity. In this experimental study, we evaluated the aqueous extracts (LQE) of Typha elephantina (also known as Elephant grass) leaves collected from freshwater marshes, for their potential anti-hyperglycemic and anti-hyperlipidemic antioxidant effects in healthy streptozotocin caused diabetic-mice. We employed glucose adsorption tests at different glucose levels and glucose diffusion tests to assess the in-vitro antidiabetic action of plant extract. For the in-vivo trail, we measured fasting blood glucose (FBG), glucose tolerance (GTT), as well as long-term anti-diabetic, anti-hyperlipidemic, and antioxidant activities. Our results from the glucose diffusion test indicated that the extract was highly effective at both low glucose concentrations (5 mmol L) and high glucose concentrations (100 mmol L). However, the glucose-diffusion ability reached its peaked at an excessively high dosage of the aqueous extract, suggesting a dose-related effect. Similarly, we observed that high doses of TEL.AQ extracts (400 mg/kg body weight) significantly reduced blood glucose levels in healthy mice during the glucose tolerance test (GTT) at 3 h and fasting blood glucose studies (FBG) at 6 h. Furthermore, the high-dose TEL.AQ extract effectively reduced liver-related serum markers and blood-glucose concentration (BGC) in severely chronic diabetic rats. The extract dosage also influenced lipid profile, conjugate and unconjugated bilirubin levels, cholesterol, triglycerides, HDL, and total bilirubin levels. Additionally, after administering a high extract dose, we observed considerable improvement in the liver homogenate markers CAT, POD, and SOD. In contrast, the extract at a low dosage(100 mg/kg),showed minimal, while a moderate dose (200 mg/kg), yielded promising results.

Colorimetric detection of glucose with smartphone-coupled µPADs: harnessing machine learning algorithms in variable lighting environments

Recent advances in microfluidic paper-based analytical devices (µPADs) have shown immense potential for point-of-care testing (POCT) in resource-constrained settings, especially for monitoring glucose levels in patients with diabetes. However, the practical adoption of smartphone-coupled µPADs has been limited due to the variability in ambient lighting conditions, which affects the colorimetric detection. In this study, we have developed a machine learning-based algorithm together with a flash/no-flash technic, adaptable to various µPADs and capable of accurately monitoring plasma glucose levels across a wide range (25 µM to 30 mM), addressing issues related to environmental illumination variations, and shooting angles.The algorithm is implemented in an Android application, Gluco estimator. It utilizes images captured by smartphones and incorporates intelligent feature engineering. The XYZ color space is adopted for device-independent analysis. KI and TMB color indicators are used to enhance resolution for low and high glucose concentrations, respectively. The EBC classifier, combined with a handcrafted feature set, exhibits outstanding performance, achieving an accuracy of 95% and 91% for TMB and KI, respectively. Additionally, multiple linear regression (MLR) yields high reliability among regression models, with R2 values of 0.95 at 0.25 µM to 3 mM and 0.97 at 3–30 mM. This work represents a significant stride toward realizing a portable, high-resolution, and reliable smartphone-coupled µPAD in uncontrolled environments.

Effect of shortening the dry period on colostrum and milk quality, blood parameters and some reproductive parameters in high-producing Holstein cows of different body condition score.

This study aimed to evaluate the effect of shortening the dry period in high-yielding cows of different body condition scores (BCS). We report colostrum and milk quality, some serum metabolites, BCS changes, and some reproductive parameters with measurements being made over the first two months of lactation. Cows were grouped based on the length of the dry period (normal: about 50 d and short: about 28 d) and BCS (moderate: 2.75 to 3.5 and high ≥ 3.5). Short dry period decreased colostrum volume and, in combination with high BCS only, caused a decrease in milk production. Short dry period moderate BCS cows had the highest serum insulin concentration on day 14 after calving and highest glucose concentration on day 28, but neither differed significantly when measured over the whole period. By contrast, short dry period cows had significantly lower concentrations of non-esterified fatty acids and beta-hydroxybutyrate measured over the whole period. Post-partum loss of BCS was less in short and especially so in the short, moderate BCS group. Following a synchronization protocol at 35 d postpartum. The cows with a short dry period and moderate BCS had lower open days, days to first postpartum estrus and services per conception. It was concluded that short dry periods and moderate BCS had a positive influence on serum metabolites, BCS changes and reproductive parameters.

Periodontitis therapy with iron alginate and glucose oxidase based on the Fenton reaction

Periodontitis is a frequent chronic inflammatory disease brought on by bacterial infection in the biofilm that forms on the teeth. It can cause the gingival recession and tooth loss, gravely jeopardizing the oral health of people. Current therapies still suffer from issues like bleeding, pain, injured gums, limited treatment effectiveness, a lengthy treatment cycle, and bacterial resistance when using a combination of surgical intervention and antibiotic therapy. Herein, a simple one-step cross-linking process was used to create glucose oxidase (GOD) loaded upon iron alginate (FeAlg/GOD), which has a strong anti-biofilm activity, good biocompatibility, and was successfully used to treat periodontitis caused by biofilms. The results demonstrated that in the presence of a high glucose concentration in the gingival crevicular fluid, FeAlg/GOD could catalyze the conversion of glucose into gluconic acid and hydrogen peroxide. The produced hydrogen peroxide could convert Fe3+ to Fe2+ when it got to the deep periodontal pockets. Following that, the Fenton reaction was activated, producing large amounts of hydroxyl radical (OH). In conclusion, oral plaque biofilms were removed by the FeAlg/GOD. Additionally, successful in vivo treatment was accomplished in rat's experimental periodontitis models, as evidenced by a markedly reduced inflammatory response, decreased bone loss, and made healthier gum tissue. Overall, our biocompatible and antibacterial FeAlg/GOD study presents a novel strategy for effectively speeding periodontal tissue repair and eliminating oral pathogens to tackle the grave threat of biofilm-associated illnesses.

The concentration of glucose in the media influences the susceptibility of stallion spermatozoa to ferroptosis.

Although common in many commercial extenders, supraphysiological concentrations of glucose in the media may be detrimental to stallion spermatozoa. In this study, we present evidence that these elevated glucose levels may predispose spermatozoa to ferroptosis. Stallion spermatozoa depend on oxidative phosphorylation as their major source of ATP; however, the metabolism of these cells is complex and a great degree of metabolic plasticity exists. The composition of the media in which the spermatozoa are extended, or exposed to in the mare's reproductive tract, exerts a profound effect on sperm function and may even accelerate cell demise. Recent research indicates that high concentrations of glucose in the media, although common in commercial extenders, may be detrimental. To determine if supraphysiological glucose concentration may induce or predispose to ferroptosis (a caspase-independent form of programmed cell death, triggered by oxidative stress), stallion spermatozoa were incubated under different concentrations of glucose, 67 mM (HG) or 1 mM plus 10 mM pyruvate (LG-HP), in the presence or absence of known inductors of ferroptosis. Furthermore, we developed a single-cell flow metabolic assay to identify different metabolic phenotypes in spermatozoa. Storage and incubation of spermatozoa under high glucose concentrations led to an increase in the percentage of necrotic spermatozoa (P < 0.0001). Moreover, ferroptosis was induced more intensely in sperm in media with high glucose concentrations (P < 0.0001). Finally, we observed that induction of ferroptosis modified two proteins (oxoglutarate dehydrogenase and superoxide dismutase 2) in spermatozoa incubated in media containing 67 mM glucose but not in media containing 1 mM glucose and 10 mM pyruvate. The composition of the media, especially the concentration of glucose, exerts a major impact on the functionality and life span of the spermatozoa. The results reported here may pave the way for improvements in existing semen extenders.

Betaine regulates steroidogenesis, endoplasmic reticulum stress response and Nrf2/HO-1 antioxidant pathways in mouse Leydig cells under hyperglycaemia condition

We studied the effects of betaine on steroidogenesis, endoplasmic reticulum stress and Nrf2 antioxidant pathways of mice Leydig cells under hyperglycaemia conditions. Leydig cells were grown in low and high glucose concentrations (5 mM and 30 mM) in the presence of 5 mM of betaine for 24 h. Gene expression was determined using a real-time PCR method. The protein levels were determined by Western blot analysis. The testosterone production was evaluated by the ELISA method. Cellular contents of reduced and oxidised glutathione were measured by colorimetric method. Hyperglycaemia caused impaired steroidogenesis and ERS in Leydig cells associated with the down-regulation of 3β-HSD, StAR, P450scc, LH receptor and increased expression of GRP78, CHOP, ATF6 and IRE1. Betaine could improve cell viability, attenuate the ERS, and restore testosterone production in Leydig cells under hyperglycaemia conditions. Betaine can protect Leydig cells against the adverse effects of hyperglycaemia by regulating steroidogenesis, antioxidants, and ERS.

Krüppel-like factor 4 promotes autophagy in macrophages under high glucose concentration by inhibiting the AKT/mTOR signaling pathway

Background: Diabetic atherosclerosis (AS) is the main cause of disability and death in diabetes. In the progression of AS, autophagic activity plays an important role. Krüppel-like factor 4 (KLF4) is a member of the zinc finger protein transcription factor family and is believed to play a protective role in the pathogenesis of atherosclerosis. This study aimed to explore the role of KLF4 in diabetic atherosclerosis and the autophagic mechanism. Methods: A diabetic mouse model was established and the expression level of KLF4 protein in the aorta of the mice was detected after a high-fat diet. The effects of KLF4 on cholesterol content, apoptosis, autophagy-related proteins, and the AKT/mTOR signaling pathway of THP-1 macrophages were also evaluated. Results: The expression level of KLF4 protein in the aorta of diabetic mice was decreased. Meanwhile, overexpression of KLF4 in THP-1 macrophages significantly decreased cholesterol accumulation, increased beclin-1 expression, decreased P62 expression, enhanced LC3 fluorescence intensity decreased cell apoptosis and p-mTOR and p-AKT expression were decreased under the condition of high glucose. After the reduction of KLF4 expression, the result is reversed. Conclusion: KLF4 induces autophagy by inhibiting the AKT/mTOR pathway and alleviates cholesterol deposition in THP-1 macrophages under high glucose concentration.

OYSTER MUSHROOM (PLEUROTUS OSTREATUS) ETHANOLIC EXTRACT PROTECTS YEAST CELLS FROM HYPER- AND HYPO-GLYCEMIC STRESS

Glucose is the primary source of carbon and energy. Cells possess a sophisticated mechanism for sensing glucose and responding to it appropriately. However, hyperglycemia represents the predominant risk factor for the development of diabetes. On the other hand, nutrient starvation, including glucose, may lead to the development of malnutrition. Noteworthy, glucose restriction has been associated with significantly delayed aging. Thus, controlled glucose intake is vital for healthy living. The presented study shows the effect of the oyster mushroom Pleurotus ostreatus ethanolic extract on fission yeast Schizosaccharomyces pombe undergoing glucose-derived stress. Fruiting bodies of P. ostreatus were used for ethanolic extraction and its anti-hyper- and hypo-glycemic stress activity was investigated. Compared were cells treated with ethanolic extract of P. ostreatus (1 or 2%) with untreated control. Different glucose concentrations in growth media were used to analyze its impact on growth intensity, generation time (gt), metabolic activity, ROS generation, MDA content, and cell death and apoptosis of S. pombe cells. Glucose deprivation or its over-supplementation resulted in growth retardation and prolonged cell doubling time (gt) which was significantly adjusted by the oyster mushroom extract. Interestingly, oyster mushroom extract reduced excessive ROS and formed MDA that were produced as a consequence of glycemic stress. Unexpectedly, the extract was able to increase the metabolic activity of cells supplemented with high glucose content and thereby protected them from death. Taken together, our study suggests that growth media supplementation with the ethanolic extract of P. ostreatus protects S. pombe cells from glucose-mediated stress through alleviation of oxidative stress and enhancement of metabolic activity.