Low Glucose Medium Research Articles

Heme deficiency in skeletal muscle exacerbates sarcopenia and impairs autophagy by reducing AMPK signaling

Heme serves as a prosthetic group in hemoproteins, including subunits of the mammalian mitochondrial electron transfer chain. The first enzyme in vertebrate heme biosynthesis, 5-aminolevulinic acid synthase 1 (ALAS1), is ubiquitously expressed and essential for producing 5-aminolevulinic acid (ALA). We previously showed that Alas1 heterozygous mice at 20–35 weeks (aged-A1+/−s) manifested impaired glucose metabolism, mitochondrial malformation in skeletal muscle, and reduced exercise tolerance, potentially linked to autophagy dysfunction. In this study, we investigated autophagy in A1+/−s and a sarcopenic phenotype in A1+/−s at 75–95 weeks (senile-A1+/−s). Senile-A1+/−s exhibited significantly reduced body and gastrocnemius muscle weight, and muscle strength, indicating an accelerated sarcopenic phenotype. Decreases in total LC3 and LC3-II protein and Map1lc3a mRNA levels were observed in aged-A1+/−s under fasting conditions and in Alas1 knockdown myocyte-differentiated C2C12 cells (A1KD-C2C12s) cultured in high- or low-glucose medium. ALA treatment largely reversed these declines. Reduced AMP-activated protein kinase (AMPK) signaling was associated with decreased autophagy in aged-A1+/−s and A1KD-C2C12s. AMPK modulation using AICAR (activator) and dorsomorphin (inhibitor) affected LC3 protein levels in an AMPK-dependent manner. Our findings suggest that heme deficiency contributes to accelerated sarcopenia-like defects and reduced autophagy in skeletal muscle, primarily due to decreased AMPK signaling.

RAB17 promotes endometrial cancer progression by inhibiting TFRC-dependent ferroptosis

Studies have indicated that RAB17 expression levels are associated with tumor malignancy, and RAB17 is more highly expressed in endometrial cancer (EC) tissues than in peritumoral tissues. However, the roles and potential mechanisms of RAB17 in EC remain undefined. The present study confirmed that the expression of RAB17 facilitates EC progression by suppressing cellular ferroptosis-like alterations. Mechanistically, RAB17 attenuated ferroptosis in EC cells by inhibiting transferrin receptor (TFRC) protein expression in a ubiquitin proteasome-dependent manner. Because EC is a blood-deprived tumor with a poor energy supply, the relationship between RAB17 and hypoglycemia was investigated. RAB17 expression was increased in EC cells incubated in low-glucose medium. Moreover, low-glucose medium limited EC cell ferroptosis and promoted EC progression through the RAB17-TFRC axis. The in vitro results were corroborated by in vivo studies and clinical data. Overall, the present study revealed that increased RAB17 promotes the survival of EC cells during glucose deprivation by inhibiting the onset of TFRC-dependent ferroptosis.

In Vitro Simulated Ketogenic Diet Inhibits the Proliferation and Migration of Liver Cancer Cells by Reducing Insulin Production and Down-regulating FOXC2 Expression.

Ketogenic diet (KD) may benefit patients with liver cancer, but the underlying mechanism of its anti-cancer effect remains an open issue. This work aimed to explore the influence of simulated KD on the proliferation and migration of cultured hepatoma cells. The low-glucose medium supplemented with β-hydroxybutyrate (BHB-Glow) was utilized to simulate clinical KD treatment. Western blot was utilized for detecting the expression of glycolysis-related proteins, Seahorse XF96 for oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), and ELISA for insulin content. Expression of FOXC2 in liver cancer cells was analyzed by bioinformatics and qPCR. Cell Count Kit-8 (CCK-8) testing kit was utilized for testing cell viability. KD treatment significantly reduced the expression of glycolysis-related proteins in Huh-7 cells, inhibited insulin production in β islet cells, reduced ECAR, and increased OCR. FOXC2 was significantly up-regulated in Huh-7 cell line, and sh-FOXC2 hindered the proliferation and migration of Huh-7 cells. The exogenous addition of insulin promoted the malignant progression of Huh-7 cells. Together, the medium simulating KD environment strengthened the protection of liver cancer cells by reducing insulin production and down-regulating FOXC2 expression. This study confirmed through in vitro cell experiments that KD could inhibit the proliferation and migration of liver cancer cells by targeting down regulation of insulin and FOXC2 expression, providing new theoretical basis for the treatment of liver cancer patients.

Evaluating Osteogenic Cell Differentiation Efficacy in the Presence of Polylactide Samples With Varied Compositions for Bone Grafting: In Vitro Study.

In oral implantology, surgeons often confront the need to improve alveolar bone quality and volume before implantation in patients with bone defects. Whereas guided bone regeneration with titanium meshes is a clinical gold standard for bone augmentation, mesh removal pre-implantation presents a drawback. This study explores biodegradable scaffolds as an alternative. The research investigates the impact of various compositions of customized bone-grafting scaffolds on proliferation and osteogenic differentiation processes in vitro. Plates (10 × 10 × 0.5 mm) were fabricated from polylactide (PLA), PLA with 15% hydroxyapatite nanoparticles (PLA/HA), and polylactide with glycolic acid copolymers (PLGA 60:40 and 85:15). Gingival fibroblasts assessed the influence of experimental samples on proliferation and osteogenic differentiation in a low-glucose medium. Osteogenic differentiation was induced, and alizarin red staining measured extracellular matrix calcification via spectrophotometry. Active proliferation of gingival fibroblasts occurred along scaffold edges during cultivation. Although cells proliferated with experimental samples, rates were lower than control cells. PLA/HA showed higher alizarin red staining intensity, indicating enhanced matrix calcification. Experimental samples (PLA, PLA/HA, PLGA 85:15, PLGA 60:40) supported cell proliferation at lower rates than control. PLA/HA demonstrated increased matrix calcification. Biodegradable membranes were nontoxic, suggesting potential for bone augmentation.

The PAH1-encoded phosphatidate phosphatase of Yarrowia lipolytica differentially affects gene expression and lipid biosynthesis

Yarrowia lipolytica is a model oleaginous yeast with a strong capacity for lipid accumulation, yet its lipid metabolic pathways and regulatory mechanisms remain largely unexplored. The PAH1-encoded phosphatidate (PA) phosphatase governs lipid biosynthesis by its enzymatic activity and regulating the transcription of genes involved in phospholipid biosynthesis. In this work, we examined the effect of the loss of Pah1 (i.e., pah1Δ) on cell metabolism in cells growing in low- and high-glucose media. Multi-omics analyses revealed the global effect of the pah1Δ mutation on lipid and central carbon metabolism. Lipidomics analyses showed that the pah1Δ mutation caused a massive decrease in the masses of triacylglycerol (TAG) and diacylglycerol (DAG), and these effects were independent of glucose concentration in the media. Conversely, phospholipid levels declined in low-glucose media but increased in high-glucose media. The loss of Pah1 affected the expression of genes involved in key pathways of glucose metabolism, such as glycolysis, citric acid cycle, oxidative phosphorylation, and the pentose phosphate pathway, and these effects were more pronounced in high-glucose media. In lipid biosynthesis, the genes catalyzing phosphatidylcholine (PC) synthesis from phosphatidylethanolamine (PE) were upregulated within the CDP-DAG pathway. In contrast, PC synthesis through the Kennedy pathway was downregulated. The ethanolamine branch of the Kennedy pathway that synthesizes PE was also upregulated in pah1Δ. Interestingly, we noted a massive increase in the levels of lysophospholipids, consistent with the upregulation of genes involved in lipid turnover. Overall, this work identified novel regulatory roles of Pah1 in lipid biosynthesis and gene expression.

Ketomimetic Medium Promotes Metastatic Disposition and Chemoresistance in Breast Cancer Cells through Hypersialylation and Lipid Synthesis.

Although metastasis accounts for the vast majority of cancer-related fatalities, the triggers for the metastatic transformation of breast cancer (BC) cells remain unknown. Recent evidence suggests that a common feature of invasive and resistant cells could be their metabolic state. However, attempts to control metabolic state via nutrient intake, e.g., ketogenic or low carbohydrate diets, have shown inconsistent results with respect to improving chemotherapy efficacy and curbing metastasis. Aiming to decode the molecular mechanisms that alter cell phenotype upon nutrient alteration, we study how a ketomimetic (ketone body-rich, low glucose) medium affects Doxorubicin (DOX) susceptibility and invasive disposition of BC cells. We quantified glycocalyx sialylation and found an inverse correlation with DOX-induced cytotoxicity and DOX internalization. These measurements were coupled with single-cell metabolic imaging, bulk migration studies, and transcriptomic and metabolomic analyses to map the mechanisms involved in ketone body-driven BC cell metabolic maneuvering. Our findings revealed that a ketomimetic medium enhances chemoresistance and invasive disposition of BC cells via two main oncogenic pathways: hypersialylation and lipid accumulation. We propose that the crosstalk between these pathways leads to synthesis of the glycan precursor UDP-GlcNAc, which leads to advancement of a metastatic phenotype in BC cells under ketomimetic conditions.

Abstract 1032: Development Of A Bio-hybrid Endovascular Stent-graft That Enables Immunosuppression Free Islet Cell Transplantation

Introduction: Islet cell transplantation has had limited clinical success freeing type 1 diabetics from exogenous insulin. Currently, islet cell transplants are performed via infusion of islet cells into the recipient's portal vein. Approximately 60% of transplanted cells die within 3 days of transplantation due to ischemic injury. Significant immunosuppression is needed to avoid rejection but is also toxic to islet cells. We have developed a novel endovascular biologic stent-graft that provides cellular immunoisolation while preventing ischemic injury through endovascular deployment (Fig 1). Hypothesis: We hypothesize islet cells seeded within the semipermeable biohybrid endovascular graft will survive and secrete insulin in an in-vitro model of perfusion. Methods: The semipermeable cell chamber was made from ePTFE with a pore size of 0.22 um, and was seeded with islet cells harvested from BALB/c mice. A normothermic machine perfusion circuit was used to model intravascular deployment. Devices were exposed to varying glucose concentrations and insulin was detected by ELISA. Results: There were significantly higher levels of insulin secretion after exposure to high vs low glucose media (p = 0.005). At hour 3 of low glucose (2.8 mM), the average insulin concentration was 76 ± 14 mIU/ml, and at hour 3 of high glucose (28 mM), the average insulin concentration was 134 ± 30 mIU/ml (Fig 2). The rate of insulin secretion was significantly higher in high vs low glucose conditions (6759 ± 503 mIU/h vs. 5344 ± 144 mIU/hr, p = 0.008). Conclusions: The endovascular biohybrid device enables islet cell function and survival. In-vivo studies are needed to study the immune interactions.

Comparing the effect of using 2-Mercaptoethanol in the cell culture medium of different cell passages of human mesenchymal stem cells.

Preparing a suitable cell culture medium that supports the biological needs of the growing cells is crucial to enhancing the success rate of any in vitro and in vivo experiments and minimizing undesirable interferences. Mesenchymal stem cells ( MSCs) which are powerful regenerative stem cells require being grown in proper culture media to preserve their stemness and therapeutic properties. MSCs are usually grown in Dulbecco's Modified Eagle low glucose Medium (DMEM low glucose) which contains 5.6 mmol/L of glucose and is supplemented with Fetal Bovine Serum (FBS), antibiotics, and 2-Mercaptoethanol. The addition of 2-Mercaptoethanol to the cell culture medium was proposed long ago and has continued to be used until now. Despite the positive effects of adding 2-Mercaptoethanol in the cell culture medium, its use is still controversial and needs continuous updates to limit its interference with experimental treatments. Herein, we found that 2-Mercaptoethanol is beneficial to enhancing the proliferation and survival of MSCs at higher passage numbers while its effect is negligible for earlier passages. This concise study provides updates regarding the suitable time to add 2-Mercaptoethanol which can minimize its intermeddling with the experimental design and treatments.

Abstract A037: Metabolic maneuvering of glycocalyx hypersialylation is a smart and healthy way of curbing metastatic potential in breast cancer

Abstract Metastatic breast cancer is currently the most deadly and incurable stage of breast cancer. A recent metabolic screen has identified sialic acid as a key player in progression of breast cancer metastasis. In the present study, we metabolically target the hypersialylation of metastatic breast cancer cells using appropriately designed nutritionary interventions. Our rationales are the following: (a) The aberrantly sialylated, hyperbranched glycocalyx promotes metastasis by facilitating cellular migration and immune evasion. (b) As hypersialylation relies heavily on enhanced sialic acid biosynthesis from the oncogenic glycolytic surge and preferential dietary uptake of glucose by cancer cells, it stands to reason that dietary changes can impact the extent of sialylation of the glycocalyx. Considering these points, we hypothesized that it was possible to control the degree of glycocalyx sialylation in breast cancer cells using an appropriate nutritionary regimen, which will in turn modulate the progression to invasive phenotype. The goal is to harness the metabolism of cancer cells themselves to curb their metastatic potential. Using a specific ketone-body forming metabolite called 3-hydroxybutyrate (HB) and lowering of glucose concentration, the cell culture medium was modified to mimic ketogenic diet. The response of metastatic breast cancer cells and normal breast epithelial cells was then monitored. The two main measures of cellular response were: cell surface sialylation via fluorescence microscopy and fluorescence lifetime imaging (FLIM) of intracellular NADH as a marker of metabolic index. We are currently investigating the consequences of nutritionary interventions on metastatic potential of breast cancer cells by measuring their impact on cellular proliferation, clonogenicity and migratory abilities. We have observed that the main metabolite of ketogenic diet, HB, tends to protect metastatic breast cancer cells from DOX treatment when supplemented in a low-glucose (LG) medium. However, the extent of increase in cell surface sialylation is very modest in comparison to that in normal breast epithelial cells. This differential increase in sialylation implies that a ketogenic diet might significantly promote the proliferation of healthy cells over the proliferation of metastatic breast cancer cells. FLIM measurements indicate that the chemotherapeutic, Doxorubicin tends to induce an initial preference for glycolysis in invasive breast cancer cells. While an HB rich LG media by itself promotes glycolysis in these cells, it suppresses any further Doxorubicin induced onset of glycolysis pointing to a protective mechanism against the chemotherapeutic. Through this ongoing study, we aim to decipher the right set of nutrients that would “deshield” the breast cancer cells and lower their metastatic disposition. The ultimate goal of our research is to determine a paradigm of drugs and diet that metastatic breast cancer cells are most responsive to, and that would translate into a viable remedy for metastatic breast cancer with minimal side-effects. Citation Format: Mohini Kamra, Yuan-I Chen, Paula C Delgado, Tim Yeh, Amy Brock, Sapun H Parekh. Metabolic maneuvering of glycocalyx hypersialylation is a smart and healthy way of curbing metastatic potential in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A037.

Pyruvate maintains and enhances the pro-inflammatory response of microglia caused by glucose deficiency in early stroke.

Pro-inflammatory microglia mainly rely on glycolysis to maintain cytokine production during ischemia, accompanied by an increase in inducible nitric oxide synthase (iNOS) and monocarboxylate transporter 1 (MCT1). The role of energy metabolism in the pro-inflammatory response of microglia is currently unclear. In this study, we tested the response of microglia in mice after cerebral ischemia and simulated an energy environment in vitro using low glucose culture medium. The research results indicate that the expression levels of iNOS and arginase 1 (ARG1) increase in the ischemic mouse brain, but the upregulation of MCT1 expression is mainly present in iNOS positive microglia. In microglia exposed to low glucose conditions, iNOS and MCT1 levels increased, while ARG1 levels decreased. Under the same conditions, knocking down MCT1 in microglia leads to a decrease in iNOS levels, while overexpression of MCT1 leads to the opposite result. The use of NF-κB inhibitors reduced the expression levels of iNOS and MCT1 in microglia. In summary, our data indicate that pyruvate maintains and enhances the NF-κB regulated pro-inflammatory response of microglia induced by low glucose.

Lactate-induced histone lactylation by p300 promotes osteoblast differentiation.

Lactate, which is synthesized as an end product by lactate dehydrogenase A (LDHA) from pyruvate during anaerobic glycolysis, has attracted attention for its energy metabolism and oxidant effects. A novel histone modification-mediated gene regulation mechanism termed lactylation by lactate was recently discovered. The present study examined the involvement of histone lactylation in undifferentiated cells that underwent differentiation into osteoblasts. C2C12 cells cultured in medium with a high glucose content (4500 mg/L) showed increases in marker genes (Runx2, Sp7, Tnap) indicating BMP-2-induced osteoblast differentiation and ALP staining activity, as well as histone lactylation as compared to those cultured in medium with a low glucose content (900 mg/L). Furthermore, C2C12 cells stimulated with the LDH inhibitor oxamate had reduced levels of BMP-2-induced osteoblast differentiation and histone lactylation, while addition of lactate to C2C12 cells cultured in low glucose medium resulted in partial restoration of osteoblast differentiation and histone lactylation. These results indicate that lactate synthesized by LDHA during glucose metabolism is important for osteoblast differentiation of C2C12 cells induced by BMP-2. Additionally, silencing of p300, a possible modifier of histone lactylation, also inhibited osteoblast differentiation and reduced histone lactylation. Together, these findings suggest a role of histone lactylation in promotion of undifferentiated cells to undergo differentiation into osteoblasts.

The generation of senescent-like CD4+ EMRA T cells in T2D and their contribution to poor COVID-19 vaccine responses

Abstract CD4+ T cells are essential for protection from viral pathogens, such as SARS-CoV-2. However, an increase in the dysfunction CD4+ EMRA subset is likely to hinder the immune response towards viruses. We show here that CD4+ EMRAs are increased with elevated blood glucose, such as people living with T2D, which alters mitochondrial function and causes the differentiation of CD4+ T cells, reducing the immune response to COVID-19 vaccination. CD4+ T cells were examined for senescence, their insulin dynamics, and mitochondrial function after in vitro culture of high and low glucose media, with or without rotenone or mitoQ. Serum samples were used to assess circulating inflammation and IgG antibodies to SARS-CoV-2. People living with T2D had increased expression of CD4+ EMRA T cells, the appearance of which correlated with increasing blood glucose values. The T2D cohort showed a reduced mitochondrial membrane potential and increased mtROS production. These results were mimicked using high glucose media which accelerated CD4+ T cell differentiation and reduced MMP. People living with T2D (non-hyperglycaemic and hyperglycaemic) had altered expression of inflammatory mediators. CD4+ EMRA cells did not respond to COVID-19 peptides, and people with T2D had a reduced T cell and antibody response to SARS-CoV-2 S1 spike protein. We have shown that senescent-like CD4+ EMRA influence the viral response in SARS-CoV-2 and that CD4+ EMRAs may arise from faulty mitochondrial dynamics due to increased environmental glucose. Further study is required to determine the direct link increased glucose has with CD4+ EMRA formation.

Abstract 13757: Suppression of Ischemia-Hypoxia Reperfusion Injury in Human iPS Cell-Derived Cardiomyocytes Through Downregulation of EGR1 Mediated by miR-124-3p

Background: Ischemic heart diseases are one of the leading causes of death globally. Although timely revascularization is performed, some cases may still result in heart failure due to ischemia-hypoxia reperfusion (IH/R) injury. Early growth response 1 (EGR1) was reported to involved in reperfusion injury, but its specific role in IH/R-induced apoptosis in human cardiomyocytes (CMs) remains unexplored. Hypothesis: EGR1 is involved in the exacerbation of the IH/R-mediated apoptosis in human CMs, and miR-124-3p acts as an antagonist of EGR1. Methods: Human induced pluripotent stem cell (hiPSC)-derived CMs were cultured in serum-free low-glucose medium with a hypoxic chamber. EGR1 expression and the extent of apoptosis were evaluated by qRT-PCR, Western blotting (WB), and immunofluorescent assay (IFA). RNA-seq was conducted to identify specific pathways. SiRNA targeting EGR1 and miR-124-3p mimic were used to downregulate EGR1 under IH/R. Results: qRT-PCR (Control vs IH/R: 1.000 ± 0.000 vs 5.749 ± 2.947; P<0.01) and WB (0.037 ±0.038 vs 0.953 ± 0.306; P<0.0001) analyses revealed higher expression of EGR1 in IH/R group. IFA demonstrated a higher ratio of cleaved Caspase-3-positive apoptotic cells in IH/R group (0.202 ±0.206 vs 0.377 ± 0.252; P<0.001). RNA-seq analysis indicated significant downregulation of pathways related to cell survival in IH/R. The siRNA downregulated EGR1 successfully, as confirmed by qRT-PCR, WB, and IFA (IH/R Water vs IH/R siRNA: 0.612 ±0.307 vs 0.257 ±0.122; P<0.01), which led to the suppression of apoptosis (0.288 ±0.143 vs 0.142 ±0.049; P<0.05). miR-124-3p mimic downregulated EGR1 under IH/R conditions, as shown by PCR (IH/R Water vs IH/R miRNA mimic: 3.731 ±1.126 vs 2.568 ±0.959; P<0.0001) and WB (0.947 ±0.438 vs 0.478 ±0.277; P<0.05). IFA proved the suppression of EGR1 by miR-124-3p mimic (0.414 ±0.101 vs 0.062 ±0.064; P<0.0001), which subsequently reduced CM apoptosis (0.234 ±0.127 vs 0.092 ±0.047; P<0.001). Conclusion: EGR1 participate in exacerbating apoptosis mediated by IH/R in human CMs. The downregulation of EGR1 suppresses IH/R-induced apoptosis. Treatment with miR-124-3p targeting EGR1 may potentially serve as a novel therapeutic approach for cardioprotection in the future.

Promoting the Anti-Tumor Activity of Radiotherapy on Lung Cancer through a Modified Ketogenic Diet and the AMPK Signaling Pathway

Taking advantage of the characteristics of high metabolic heterogeneity of tumor cells, the modified ketogenic diet (KD) combined with radiotherapy was used to investigate and analyze the radiosensitivity of a lung cancer model from the perspective of energy metabolism. Different concentrations of glucose and βhydroxybutyrate (βHB) were used at the cellular level to simulate the level of ketone bodies. A cell counting kit was used to detect the effect of different concentrations of glucose (2.78mM, 5.56mM, 12.5mM, and 25mM) and βHB (0mM, 5mM, and 10mM) combined with radiotherapy on the proliferation of LLC cells. Flow cytometry was used to detect tumor cell cycle and apoptosis. Immunofluorescence was used to detect the expression of γH2AX, a DNA damage marker, and western blot was used to detect the expression of AMPK and ρ-AMPK. At the animal level, C57BL/6J female mice were used to establish a transplanted tumor model of lung cancer, and fed with different fat ratio diets combined with radiotherapy. The volume, tumor size, blood glucose level, blood ketone level, survival time and safety of the mice were monitored and observed. The LLC cells were treated with different concentrations of glucose and βHB. The results showed that the survival rate of LLC cells decreased significantly with the increase of irradiation dose when the glucose concentration was 5.56mM and 2.78mM; However, the survival rate of cells in low glucose medium added with βHB was significantly lower than that of the control group, and the survival rate of LLC decreased significantly with the extension of culture time after irradiation (p < 0.001). After irradiation, LG (low glucose) group, LG+βHB 5mM group and LG+βHB 10mM group had a significantly higher proportion of G2 phase, and a significantly higher proportion of early and late phase than the control group. γH2AX foci were detected in LG group, LG +βHB 5mM group and LG +βHB 10mM group at 2h and 24h after radiotherapy, which were significantly higher than those in the control group (p < 0.05). The median survival time was 38 days in the PT group, 55 days in the PT+RT group, 41 days in the 45F group, and not reached in the 45F+RT group. HE staining showed no tumor metastasis and toxic side effects in liver and kidney. The expression of ρ-AMPK/AMPK in the combined treatment group was higher than that in the other groups. The expression of ρ-AMPK/AMPK in RT, 45F and combined treatment group was higher than that in PT group. The expression of ρ-AMPK/AMPK in RT group was higher than that in 45F group, and the difference was statistically significant (p < 0.01). Modified ketogenic diet can enhance the anti-tumor effect of radiotherapy in LLC tumor-bearing mice by reducing glucose and increasing the energy supply ratio from fat.

MiR-100-5p activation of the autophagy response through inhibiting the mTOR pathway and suppression of cerebral infarction progression in mice.

In recent years, the association between microRNAs (miRNAs) and autophagy in cerebral infarction (CI) has attracted increasingly more attention. The mammalian target of the rapamycin (mTOR) pathway is a key protein regulating the autophagy response. miR-100-5p can bind to the mTOR protein, but its role in CI remains unclear yet. This experiment aims to clarify the role of miR-100-5p in CI. Bioinformatics analysis was performed to screen differentiated expressed functional genes between CI tissue and normal tissue specimens. In vivo experiments: the mouse model of CI was established by middle cerebral artery occlusion (MCAO) methods, After being treated with miR-100-5p-overexpressing lentivirus, the amount of terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)-positive fluorescence and the fluorescent expression level of mTOR protein were significantly inhibited in the CI region. Western blotting analysis showed that miR-100-5p inhibited the protein expression level of phosphorylated mTOR and total mTOR and enhanced the expression of autophagy-related proteins Beclin, microtubule-associated protein light chain 3II (LC-3II), and autophagy-related gene 7 (ATG-7). For in vitro experiment, after the BV-2 cells were successfully infected with the control lentivirus and miR-100-5p-overexpression lentivirus, they were stimulated with 1% hypoxia and low-glucose medium in a tri-gas incubator for 24 h. It was found that miR-100-5p could significantly lower the protein expression level of phosphorylated mTOR and total mTOR, and increase the expression of the Beclin, LC-3II, ATG-7 autophagy related proteins. miR-100-5p promotes the autophagy response through binding to mTOR protein, thereby inhibiting apoptosis and delaying the progression of CI.

Identification and Validation of Genes Related to RNA Methylation Modification in Diabetic Retinopathy

Purpose To identify and validate the differentially expressed genes related to RNA methylation modification in diabetic retinopathy. Methods The data sets GSE12610 and GSE111465 related to diabetic retinopathy in the Gene Expression Omnibus were selected. The R software package was used to identify differentially expressed genes related to RNA methylation modification in diabetic retinopathy. Protein-protein interaction network was constructed to explore the interactions between proteins and predict proteins. Then, Gene Ontology annotation analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to analyze the potential enrichment pathways and clarify the biological functions of these genes. In addition, the correlation between them and immune cells was visualized, and receiver operating characteristic curves were drawn to evaluate the diagnostic performance of each one of them for diabetic retinopathy. To verify the differentially expressed genes, the mRNA expression of rat retinal vascular endothelial cells cultured in low and high glucose medium separately were detected by RT-qPCR. Results The expression of Lrpprc, Nsun4, Nsun6 and Trdmt1 were significantly up-regulated in diabetic retinopathy samples, while the expression of Cbll1, Hnrnpc, Mettl3 and Wtap were significantly down-regulated. Differentially expressed genes were mainly enriched in the RNA-methylation-medication pathways and biological function. The results of immune infiltration analysis proved that eosinophils aggregated more in diabetic group, while T cells follicular helper aggregated more in normal samples. These genes of Cbll1 (AUC = 0.986), Hnrnpc (AUC = 0.819), Lrpprc (AUC = 0.806), Mettl3 (AUC = 0.917), Nsun4 (AUC = 0.819), Nsun6 (AUC = 0.819), Trdmt1 (AUC = 0.972) and Wtap (AUC = 0.972) were respectively used as the diagnostic basis of diabetic retinopathy. According to the RT-qPCR results, the expression of Mettl3 was significantly down-regulated (p < 0.0005) in cells cultured in high glucose, while Trdmt1 (p < 0.05), Nsun4 (p < 0.05) and Nsun6 (p < 0.05) were significantly up-regulated. Conclusion Differentially expressed genes such as Mettl3, Nsun4, Nsun6, and Trdmt1 should be conducted to explore, and the role of RNA methylation in the process of diabetic retinopathy would be revealed in-depth.

Influence of Media Composition on the Level of Bovine Satellite Cell Proliferation.

It is predicted that already in 2040, 35% of requirements for meat will be provided by in vitro production. Recreating the course of myogenesis in vitro, and thus resembling a structure of muscle tissue, is the basis for research focusing on obtaining cultured meat and requires providing relevant factors supporting the proliferation of satellite cells-being precursors of skeletal muscles. The present work aimed to develop the composition of the medium that would most effectively stimulate the proliferation of bovine satellite cells (BSCs). The modeling and optimization methods included the measurements of the synergistic, co-stimulatory effect of three medium components: the amount of glucose, the type of serum (bovine or horse), and the amount of mitogenic factor-bFGF. Additionally, the qPCR analyses determined the expression of genes involved in myogenesis, such as Pax7 and Myogenic Regulatory Factors, depending on the level of the tested factor. The results showed significant positive effects of serum type (bovine serum) and mitogenic factor (addition of 10 ng/mL bFGF) on the proliferation rate. In turn, qPCR analysis displayed no significant differences in the relative expression level of Pax7 genes and MRF factors for both factors. However, a statistically higher Pax7 and Myf5 gene expression level was revealed when a low glucose medium was used (p < 0.05). In conclusion, the components of the medium, such as bovine serum and the addition of a mitogenic factor at the level of 10 ng/mL, ensure a higher proliferation rate of BSCs and lower glucose content ensured the expression of crucial genes in the self-renewal of the satellite cell population.

NMR-Based Metabolic Profiling of the Effects of α-Ketoglutarate Supplementation on Energy-Deficient C2C12 Myotubes.

Skeletal muscle is closely linked to energy metabolism, but it is inevitably deprived of energy. Cellular differentiation is an essential and energy-demanding process in skeletal muscle development. Much attention has been paid to identifying beneficial factors that promote skeletal muscle satellite cell differentiation and further understanding the underlying regulatory mechanisms. As a critical metabolic substrate or regulator, α-ketoglutarate (AKG) has been recognized as a potential nutritional supplement or therapeutic target for skeletal muscle. We have previously found beneficial effects of AKG supplementation on the proliferation of C2C12 myoblasts cultured under both normal and energy-deficient conditions and have further elucidated the underlying metabolic mechanisms. However, it remains unclear what role AKG plays in myotube formation in different energy states. In the present study, we investigated the effects of AKG supplementation on the differentiation of C2C12 myoblasts cultured in normal medium (Nor myotubes) and low glucose medium (Low myotubes) and performed NMR-based metabonomic profiling to address AKG-induced metabolic changes in both Nor and Low myotubes. Significantly, AKG supplementation promoted myotube formation and induced metabolic remodeling in myotubes under normal medium and low glucose medium, including improved energy metabolism and enhanced antioxidant capacity. Specifically, AKG mainly altered amino acid metabolism and antioxidant metabolism and upregulated glycine levels and antioxidase expression. Our results are typical for the mechanistic understanding of the effects of AKG supplementation on myotube formation in the two energy states. This study may be beneficial for further exploring the applications of AKG supplementation in sports, exercise, and therapy.

Mitochondrial reactive oxygen species promote mitochondrial damage in high glucose-induced dysfunction and apoptosis of human dental pulp cells

Background/purposeHigh glucose (HG)-induced aberrant proliferation, apoptosis and odontoblastic differentiation of dental pulp cells (DPCs) have been implicated in the pathogenesis of impaired diabetic pulp healing; however, the underlying mechanism remains unclear. This study aimed to investigate the role of mitochondrial reactive oxygen species (mtROS) and mitochondria in HG-induced dysfunction and apoptosis of DPCs. Materials and methodsHuman DPCs (hDPCs) were cultured in a low-glucose, high-glucose, mannitol, and MitoTEMPO medium in vitro. Methylthiazol tetrazolium assay, Annexin V-FITC/PI staining and scratch-wound assay were used to analyze cell proliferation, apoptosis and migration, respectively. Alkaline phosphatase staining and alizarin red S staining were used to evaluate cell differentiation. DCF-DA staining, MitoSOX staining, MitoTracker Red staining, JC-1 staining, and adenosine triphosphate (ATP) kit assay were performed to investigate total ROS and mtROS generation, mitochondrial density, mitochondrial membrane potential (MMP), and ATP synthesis, respectively. Quantitative PCR assay was performed to detect the mRNA expression of mitochondrial biogenesis- and dynamics-related markers. Transmission electron microscopy was used to observe the mitochondrial ultrastructure. ResultsHG augmented the production of total ROS and mtROS, and triggered mitochondrial damage in hDPCs, as reflected by decreased mitochondrial density, depolarized MMP, reduced ATP synthesis, altered mRNA expression of mitochondrial biogenesis- and dynamics-related markers, and abnormal mitochondrial ultrastructure. Supplementation of MitoTEMPO alleviated the mitochondrial damage and reversed the aberrant proliferation, apoptosis, migration and odontoblastic differentiation of HG-stimulated hDPCs. ConclusionHG triggers mitochondrial damage via augmenting mtROS generation, resulting in the inhibited proliferation, migration, and odontoblastic differentiation of hDPCs and enhanced their apoptosis.

Dual-Functional Nanoplatform Based on Bimetallic Metal-Organic Frameworks for Synergistic Starvation and Chemodynamic Therapy.

Tumor microenvironment (TME)-responsive chemodynamic therapy (CDT) mediated by nanozymes has been extensively studied in oral squamous cell carcinoma. However, the low catalytic efficiency due to insufficient H2O2 in the TME is still a major challenge for its clinical translation. Herein, we present an antitumor nanoplatform based on a Mn-Co organometallic framework material (MnCoMOF), which shows peroxidase-like (POD-like) activity, loaded with glucose oxidase (GOx@MnCoMOF), demonstrating the ability of H2O2 self-supply and H2O2 conversion to toxic hydroxyl radicals. The encapsulated GOx efficiently catalyzes glucose into gluconic acid and H2O2 at the tumor site, which can cut off the energy supply to inhibit tumor growth and produce a large amount of H2O2 and acid to compensate for their lack in the tumor microenvironment. The POD-like activity of MnCoMOF can convert H2O2 into hydroxyl radicals and eliminate tumor cells. The nanoplatform exhibits enhanced tumor cell cytotoxicity in a high-glucose medium compared with a low-glucose medium, illustrating sufficient generation of H2O2 from glucose by GOx. The in vivo results indicate that GOx@MnCoMOF has excellent antitumor efficacy and can remodel the immune-suppressive tumor microenvironment. In conclusion, the GOx@MnCoMOF nanoplatform possesses dual enzymatic activities, i.e., POD-like and glucose oxidase, to achieve improved tumor-suppressive efficiency through synergistic starvation and chemodynamic therapy, thus providing a new strategy for the clinical treatment of oral cancer.