Low-glucose Group Research Articles

Effect of short-term fasting on the cisplatin activity in human oral squamous cell carcinoma cell line HN5 and chemotherapy side effects

BackgroundKetogenic interventions like short-term fasting show potential as complementary therapies to enhance the effectiveness of chemotherapy for cancer. However, the specific effects of fasting on head and neck squamous cell carcinoma (HNSCC) cells and healthy oral mucosa cells during these treatments are not well understood. This study investigates whether short-term fasting can differentially impact HNSCC cell survival and viability compared to healthy keratinocytes while undergoing standard chemotherapy regimens.MethodsThis study investigated the effects of fasting on cell viability in HN5 cell line and healthy oral keratinocyte cells. The HN5 cell line, derived from human tongue squamous cell carcinoma, and primary human keratinocytes isolated from the basal layer of gingival epithelium were divided into three groups: (1) control, (2) treated with the standard chemotherapeutic agent cisplatin, and (3) treated with cisplatin under fasting conditions achieved through 48-hour glucose restriction mimicking the blood glucose levels of fasted individuals. Cell proliferation was assessed at 48 and 72 h using the MTT assay, a colorimetric method based on mitochondrial dehydrogenase activity. Flow cytometry analysis with specific apoptosis and necrosis markers distinguished between early and late apoptotic, necrotic, and viable cells.ResultsCell viability in HN5 and healthy keratinocyte cells decreased in cisplatin with low glucose groups compared to cisplatin and control groups. The same results were observed for healthy keratinocyte cells; only a decrease in cell viability in cisplatin groups compared to control groups was observed, which was not statistically significant. Cell apoptosis in HN5 and healthy keratinocyte cells increased in cisplatin with low glucose groups compared to cisplatin and control groups. In healthy keratinocyte cells, the cisplatin with low glucose group showed an impressive increase in necrosis, late apoptosis, and early apoptosis and a significant decrease in live cells compared with other groups.ConclusionThis study revealed that short-term fasting chemotherapy significantly improved HNSCC cell line apoptosis and necrosis.

The role and mechanism of action of miR-483-3p in mediating the effects of IGF-1 on human renal tubular epithelial cells induced by high glucose

This study aimed to elucidate the influence of miR-483-3p on human renal tubular epithelial cells (HK-2) under high glucose conditions and to understand its mechanism. Human proximal tubular epithelial cells (HK-2) were exposed to 50 mmol/L glucose for 48 h to establish a renal tubular epithelial cell injury model, denoted as the high glucose group (HG group). Cells were also cultured for 48 h in a medium containing 5.5 mmol/L glucose, serving as the low glucose group. Transfection was performed in various groups: HK-2 + low glucose (control group), high glucose (50 mM) (HG group), high glucose + miR-483-3p mimics (HG + mimics group), high glucose +miR-483-3p inhibitor (HG + inhibitor group), and corresponding negative controls. Real-time quantitative polymerase chain reaction (qPCR) assessed the mRNA expression of miR-483-3p, bax, bcl-2, and caspase-3. Western blot determined the corresponding protein levels. Proliferation was assessed using the CCK-8 assay, and cell apoptosis was analyzed using the fluorescence TUNEL method. Western blot and Masson’s staining were conducted to observe alterations in cell fibrosis post miR-483-3p transfection. Furthermore, a dual-luciferase assay investigated the targeting relationship between miR-483-3p and IGF-1. The CCK8 assay demonstrated that the HG + mimics group inhibited HK-2 cell proliferation, while the fluorescent TUNEL method revealed induced cell apoptosis in this group. Conversely, the HG + inhibitor group promoted cell proliferation and suppressed cell apoptosis. The HG + mimics group upregulated mRNA and protein expression of pro-apoptotic markers (bax and caspase-3), while downregulating anti-apoptotic marker (bcl-2) expression. In contrast, the HG + inhibitor group showed opposite effects. Collagen I and FN protein levels were significantly elevated in the HG + mimics group compared to controls (P < 0.05). Conversely, in the HG + inhibitor group, the protein expression of Collagen I and FN was notably reduced compared to the HG group (P < 0.05). The dual luciferase reporter assay confirmed that miR-483-3p could inhibit the luciferase activity of IGF-1’s 3′-UTR region (P < 0.05). miR-483-3p exerts targeted regulation on IGF-1, promoting apoptosis and fibrosis in renal tubular epithelial cells induced by high glucose conditions.

Important nutrient sources and carbohydrate metabolism patterns in the growth and development of spargana

BackgroundSparganosis is a worldwide food-borne parasitic disease caused by spargana infection, which infects the muscle of frogs and snakes as well as many tissues and organs in humans. There are currently no viable treatments for sparganosis. Understanding spargana’s nutrition source and carbohydrate metabolism may be crucial for identifying its energy supply and establishing methods of treatment for sparganosis.MethodsUsing an amino acid analyzer and nutrient concentration detection kits, we assessed nutrient concentrations in the muscles of Fejervarya limnocharis and Pelophylax plancyi infected or not infected with spargana. Quantitative polymerase chain reaction (PCR) was used to quantify the major enzymes involved in five glucose metabolism pathways of spargana developing in vivo. We also used quantitative PCR to assess key enzymes and transcriptome sequencing to explore the regulation of carbohydrate metabolic pathways in vitro in response to different 24-h food treatments.ResultsInfected muscle tissues had considerably higher concentrations of glucogenic and/or ketogenic amino acids, glucose, and glycogen than non-infected muscle tissues. We discovered that the number of differentially expressed genes in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was larger in low-glucose than in other dietary groups. We examined differences in the expression of genes producing amino acid transporters, glucose transporters, and cathepsins in spargana grown in various nutritional environments. In the normal saline group, only the major enzymes in the tricarboxylic acid cycle (TCA), glycogenesis, and glycogenolysis pathways were expressed. The l-glutamine group had the greatest transcriptional levels of critical rate-limiting enzymes of gluconeogenesis and glycogenesis. Furthermore, the low-glucose group had the highest transcriptional levels of critical rate-limiting enzymes involved in the TCA, glycolytic, and glycogenolysis pathways. Surprisingly, when compared to the in vitro culturing groups, spargana developing in vivo exhibited higher expression of these critical rate-limiting enzymes in these pathways, with the exception of the pentose phosphate pathway.ConclusionsSpargana have a variety of nutritional sources, and there is a close relationship between nutrients and the carbohydrate metabolism pathways. It takes a multi-site approach to block nutrient absorption and carbohydrate metabolism pathways to provide energy to kill them.Graphical

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.

Effects of glucose and osmotic pressure on the proliferation and cell cycle of human chorionic trophoblast cells.

This study investigated the effects of glucose and osmotic pressure on the proliferation and cell cycle of trophoblast cells. HTR8/SVneo cells were treated with 0 (no glucose), 1 (low glucose), 5 (normal), and 25 mmol/L (high glucose) glucose. In addition, the cells were treated with 5 mmol/L glucose (normal) and 5 mmol/L glucose + 20 mmol/L mannitol (mannitol). The cell morphology and proliferation were determined by microscopy and a cell counting kit-8 assay. The cell cycle and apoptosis were examined by flow cytometry. The cell number was relatively decreased and morphological changes were intermediate in the high-glucose group compared with the low-glucose groups. The proportion of cells in the G2/M phase was higher in the low-glucose group than in the other groups, and it was lower in the G1 phase and higher in the S phase in the high-glucose group than in the other groups. Compared with 24 h, cell proliferative activity was restored to a certain extent after 48 h in the high-glucose group. In summary, the blood glucose concentration might influence the proliferation of trophoblast cells. A high-glucose environment inhibited initial cell proliferation, which could be moderately restored after self-regulation. Furthermore, the proliferation of trophoblasts was not affected by the osmotic pressure.

Dynamic elastic modulus assessment of the early degeneration model of an intervertebral disc in cynomolgus monkeys with one strike loading

ObjectiveDisc degeneration has long been associated with excessive mechanical loading or acute disc injury. Our goal is to perform a shock load on the functional units of the cynomolgus monkey intervertebral disc and analyze the degree of degeneration of the intervertebral disc through image analysis and comprehensive analysis. The organ model establishes a standard organ culture model and a non-invasive biomechanical evaluation protocol close to the early degeneration of the human intervertebral disc. MethodsAfter modeling, the cynomolgus monkey intervertebral discs were collected and loaded into the dynamic mechanical culture system. The physiological group was loaded with 10% high compressive deformation load for one second, the injury group was punctured with annulus fibrosus, the model group was loaded with 20–50% high compressive deformation, and the nutritional components were a high-glucose group and low-glucose group. After day 3 (short term) and day 10 (long term), samples were collected to analyze cell viability, histomorphology, image analysis for imaging and biomechanical changes. ResultsBoth the injury group and the 30–50% strain model group showed signs of early degeneration, including decreased instantaneous compressive stiffness, percent change in gray value, decreased cell viability, AF fissure, and percent increase in dynamic elastic modulus. The glucose-restricted group also showed signs of early disc degeneration in long-term cultures. ConclusionThis study shows that a single shock load can induce early degeneration of healthy cynomolgus monkey intervertebral discs, and 30% strain may be the nociceptive threshold for early degeneration of healthy intervertebral discs. More importantly, a non-invasive biomechanical evaluation scheme of Percentage change in dynamic modulus of elasticity is established, which solves the key scientific problem of how to non-invasively, quantitatively and sensitively detect the development process of early intervertebral disc degeneration and its degree of degeneration in an in vitro organ model.

Long Non-Coding RNA GDAR Regulates Ovine Granulosa Cells Apoptosis by Affecting the Expression of Apoptosis-Related Genes

Short-term dietary supplementation of ewes during the luteal phase can increase fertility, most probably by stimulating glucose uptake by the follicles. However, the molecular mechanism of glucose regulation of follicular development has not yet been clarified, especially the further study of long non-coding RNA (lncRNA) in determining fertility during follicular development. We generated granulosa cell (GC) models of different doses of glucose (0, 2.1, 4.2, 8.4, 16.8 and 33.6 mM), and observed that the highest cell viability was recorded in the 8.4 mM group and the highest apoptosis rates were recorded in the 33.6 mM group. Therefore, a control group (n = 3, 0 mM glucose), a low glucose group (n = 3, add 8.4 mM glucose), and a high glucose group (n = 3, add 33.6 mM glucose) of GCs were created for next whole genomic RNA sequencing. In total, 18,172 novel lncRNAs and 510 annotated lncRNAs were identified in the GCs samples. Gene Ontology indicated that differentially expressed lncRNAs associated with cell apoptosis were highly enriched. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of lncRNA target genes found that the apoptosis pathway and the p53 signaling pathway were both enriched. Furthermore, we focused on the function of a lncGDAR and verified that lncGDAR could influence cell apoptosis in GC development through affecting the mRNA and protein expression of apoptosis-related markers. These results provide the basis for further study of the lncRNA regulation mechanism in nutrition on female fertility.

Effect of human decidua mesenchymal stem cells-derived exosomes on the function of high glucose-induced senescent human dermal fibroblasts and its possible mechanism

Effect of human decidua mesenchymal stem cells-derived exosomes on the function of high glucose-induced senescent human dermal fibroblasts and its possible mechanism

The role and mechanism of NORAD in proliferation and apoptosis of mouse mesangial cells induced by high glucose

Objective: To uncover the role and mechanism of non-coding RNA-activated by DNA damage (NORAD) in proliferation and apoptosis of mouse mesangial cells induced by high glucose. Methods: SV40-MES-13 cells were divided into high glucose group and low glucose group according to the concentration of glucose in the culture medium. After 24 hours of treatment, SV40-MES-13 cells were transfected with si-NORAD and si-Toll-like receptor (TLR4), and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of NORAD and TLR4. NORAD, TLR4 overexpression plasmids and small interfering RNA were transfected into SV40-MES-13 cells respectively. Cell counting kit (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry were performed to determine the results of cell proliferation and apoptosis. Luciferase activity and their mRNA levels were detected after cotransfection of pGL3-NORAD WT and miR-520h mimics or pGL3-TLR4 WT and miR-520h mimics. After cotransfection of si-NORAD and miR-520h inhibitors, TLR4 overexpression plasmids and miR-520h mimics, and cotransfection of si-TLR4 and miR-520h inhibitors, the levels of NORAD, miR-520h and TLR4, as well as cell proliferation were detected to verify the regulatory relationship among them. Results: NORAD and TLR4 were upregulated in SV40-MES-13 cells in high glucose group (1.65±0.08 vs 1.00±0.06, P=0.003; 1.96±0.09 vs 1.01±0.07, P=0.001). Overexpression of NORAD and TLR4 promoted the proliferative ability (1.34±0.04 vs 0.85±0.04, P=0.001; 1.33±0.02 vs 0.82±0.03, P<0.001) and inhibited the apoptosis in SV40-MES-13 cells (0.45±0.03 vs 0.94±0.06, P=0.001; 0.51±0.05 vs 0.99±0.03, P=0.001). The dual-luciferase reporter gene assay showed that miR-520h was confirmed to bind to NORAD and TLR4. The expression of NORAD and miR-520h affected each other, and miR-520h mimics reduced the expression of TLR4. The expression of TLR4 in cotransfected NORAD small interference RNA and miR-520h inhibitor was higher than that in NORAD small interference RNA alone (0.74±0.03 vs 0.55±0.03, P=0.014). Cotransfection of miR-520h mimics and TLR4 overexpression plasmids increased cell proliferation compared with miR-520h mimics alone (0.73±0.01 vs 0.61±0.02, P=0.007). Cotransfection of miR-520h inhibitors and TLR4 small interference RNA reduced cell proliferation compared with miR-520h inhibitors alone (1.31±0.04 vs 1.55±0.04, P=0.013). Conclusions: The regulatory loop NORAD/miR-520h/TLR4 promotes the proliferative ability and inhibits apoptosis in glomerular mesangial cells induced by high glucose.

Upregulation of long non-coding RNA SNHG16 promotes diabetes-related RMEC dysfunction via activating NF-κB and PI3K/AKT pathways

Diabetic retinopathy (DR) is a severe diabetes-induced eye disease, in which its pathological phenomena basically include abnormal proliferation, migration, and angiogenesis of microvascular endothelial cells in the retina. Long non-coding RNAs (lncRNAs) have been proven to be important regulators in various biological processes, but their participation in DR remains largely undiscovered. In the present study, we aimed to unveil the role of lncRNA small nucleolar RNA host gene 16 (SNHG16) in regulating the functions of human retinal microvascular endothelial cells (hRMECs) under a high-glucose (HG) condition. We found that SNHG16 expression was significantly upregulated in hRMECs treated with HG. Functionally, SNHG16 could facilitate hRMEC proliferation, migration, and angiogenesis. Moreover, SNHG16 was associated with nuclear factor κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Mechanistically, SNHG16 could promote hRMEC dysfunction by sequestering microRNA (miR)-146a-5p and miR-7-5p to act as a competing endogenous RNA (ceRNA) with interleukin-1 receptor-associated kinase 1 (IRAK1) and insulin receptor substrate 1 (IRS1). In conclusion, our results illustrated the potential role of SNHG16 in facilitating hRMEC dysfunction under HG treatment, providing a novel approach for DR therapy.

Ketogenic Diet: Impact on Cellular Lipids in Hippocampal Murine Neurons

Background: The mechanism of action of the ketogenic diet (KD), an effective treatment for pharmacotherapy refractory epilepsy, is not fully elucidated. The present study examined the effects of two metabolites accumulating under KD—beta-hydroxybutyrate (ßHB) and decanoic acid (C10) in hippocampal murine (HT22) neurons. Methods: A mouse HT22 hippocampal neuronal cell line was used in the present study. Cellular lipids were analyzed in cell cultures incubated with high (standard) versus low glucose supplemented with ßHB or C10. Cellular cholesterol was analyzed using HPLC, while phospholipids and sphingomyelin (SM) were analyzed using HPTLC. Results: HT22 cells showed higher cholesterol, but lower SM levels in the low glucose group without supplements as compared to the high glucose groups. While cellular cholesterol was reduced in both ßHB- and C10-incubated cells, phospholipids were significantly higher in C10-incubated neurons. Ratios of individual phospholipids to cholesterol were significantly higher in ßHB- and C10-incubated neurons as compared to controls. Conclusion: Changes in the ratios of individual phospholipids to cholesterol in HT22 neurons suggest a possible alteration in the composition of the plasma membrane and organelle membranes, which may provide insight into the working mechanism of KD metabolites ßHB and C10.

Lysine acetylation participates in boar spermatozoa motility and acrosome status regulation under different glucose conditions

Efficient artificial insemination (AI) with liquid preserved boar semen is essential for the swine industry. Glucose is the most widely utilized energy source in refrigerated boar semen extenders. However, the relationship between glucose concentration and spermatozoa quality is not fully understood. In the present study, boar spermatozoa were used as a model to investigate the impact of different glucose concentrations on spermatozoa motility, mitochondrial activity, and acrosome integrity at physiological temperature (37 °C) and during refrigeration (17 °C). The proportion of progressively motile spermatozoa and mitochondrial activity in the high glucose group were significantly lower than in the low glucose group when incubated at 37 °C for 3 h or 17 °C for 3 d, but not at 17 °C for 7 d. Lysine acetylation is a reversible post-translational modification that plays a crucial role in spermatozoa function. Our results show that spermatozoa protein acetylation levels were higher in the high glucose group than in the low glucose group. The proportions of progressively motile and acrosome-intact spermatozoa were higher in acetyltransferase inhibitor (WM-1119)-treated spermatozoa than in the control. Spermatozoa acetyl-CoA concentration, which is directly linked to acetylation, was significantly higher in the high glucose group than in the low glucose group. Taken together, spermatozoa motility and acrosome integrity can be altered by changing the concentration of glucose in the extender. High glucose concentration-induced lysine acetylation participates in the regulation of boar spermatozoa motility and acrosome integrity during preservation. These results can provide insights into spermatozoa preservation and AI in the swine industry.

MiR-124-3p promotes BMSC osteogenesis via suppressing the GSK-3β/β-catenin signaling pathway in diabetic osteoporosis rats.

The purpose of this study is to investigate miRNAs' effects, targeting the Wnt signaling pathway, on osteogenic differentiation to provide new targets for diabetic osteoporosis treatments. Twelve male rats were divided into a normal rat group (NOR group) and a model rat group (MOD group). Cluster analysis of differentially expressed miRNAs and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed. Primary rat bone marrow mesenchymal stem cells (BMSCs) were divided into a high-glucose group and a low-glucose group, and osteogenic differentiation was induced. Alkaline phosphatase (ALP) staining and Alizarin Red staining were used for pathological analysis of the cells. Western blot analysis was used to measure GSK-3β, β-catenin, p-β-catenin, c-Myc, and CyclinD1 expression. Immunofluorescence (IF) was used to analyze the effect of GSK-3β inhibitor (CHIR99021) on β-catenin and CyclinD1 expressions levels in BMSCs. A total of 428 differentially expressed miRNAs were found between the NOR and MOD groups. KEGG analysis showed that the target genes were mostly enriched in signaling pathways, including PI3K-Akt, focal adhesion, AGE-RAGE, HIF-1, and Wnt. qPCR verification demonstrated that miR-124-3p exhibited the greatest difference in expression level. In BMSCs, miR-124-3p overexpression could reverse the inhibited expression of BMSC osteogenic markers, including Alpl, Bglap, and Runx2, induced by high glucose. Western blot analysis revealed that the transfection of miR-124-3p mimics could further reverse the upregulated p-β-catenin and GSK-3β levels and the downregulated c-Myc and CyclinD1 levels induced by high glucose. IF results revealed that BMSCs treated CHIR99021 under high glucose showed the reduced GSK-3β and increased β-catenin and CyclinD1 expression levels. Our research highlighted miRNAs' important roles in regulating the Wnt pathway and provided new information for the diagnosis and treatment of diabetic osteoporosis.

Low glucose enhanced metformin’s inhibitory effect on pancreatic cancer cells by suppressing glycolysis and inducing energy stress via up-regulation of miR-210-5p

ABSTRACT To explore mechanisms underlying the discrepancy in anti-tumor effects of metformin on pancreatic cancer cells PANC-1 under different glucose conditions. We cultured PANC-1 cells in 25 mM and 5 mM glucose media, then treated with or without metformin. It showed that metformin significantly inhibited proliferation and viability, induced apoptosis of PANC-1 cells, which was more pronounced in low-glucose than in high-glucose group. Metformin up-regulated the expression of miR-210-5p in low glucose, but not in high glucose. miR-210-5p mimic inhibited the viability of PANC-1 cells and further enhanced the inhibitory effect of metformin. miR-210-5p down-regulated the expression of PFKFB2, a predicted target gene of miR-210-5p, reduced the activity of PFK1 and LDH. Metformin significantly inhibited the expression of phosphorylation-PFKFB2(p-PFKFB2) in the low-glucose group and inhibited the LDH activity both in the low and high glucose groups, thus inhibiting anaerobic glycolysis and inducing energy stress. Cells in the high glucose group could make a compensatory adaptation to the energy stress induced by metformin through increasing glucose consumption. However, due to the limited glucose supply and high dependence on anaerobic glycolysis of cells in the low glucose group, they couldn’t make effective adaptive compensation. Therefore, cells in the low-glucose group were more vulnerable to the toxicity of metformin. In conclusion, the enhanced inhibitory effect of metformin on PANC-1 cells cultured in low glucose may be due to the up-regulation of the expression of miR-210-5p, then inhibiting anaerobic glycolytic flux and inducing energy stress via repressing the expression of p-PFKFB2 and activity of LDH. Abbreviations PC: pancreatic cancer; DM: diabetes mellitus; PFKFB2: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase2; PFK1: phosphofructokinases; LDH: lactate dehydrogenase; F-2,6-BP: fructose 2,6-bisphosphate

Metabolic status is associated with the recovery of milk somatic cell count and milk secretion after lipopolysaccharide-induced mastitis in dairy cows

Infections of the mammary gland in dairy cows are commonly accompanied by reduced milk production and feed intake and poor milk quality. The metabolic status of early-lactating cows is known to affect immune response to pathogens and imposed immune challenges. We investigated the extent to which metabolic status before an intramammary lipopolysaccharide (LPS) challenge (LPS-CH) is associated with immune response, milk production, and feed intake and the recovery thereof. In 15 Holstein cows, weekly blood sampling and daily recording of dry matter intake, milk yield, milk composition, and body weight (to calculate energy balance) was started immediately after parturition. In wk 4 after parturition, cows underwent an intramammary LPS-CH (50 μg of LPS into 1 quarter). Blood and milk samples were taken in parallel at 30- and 60-min intervals, respectively, until 10 h after the LPS application. Plasma concentrations of glucose, nonesterified fatty acids, β-hydroxybutyrate (BHB), cortisol, and insulin were analyzed. In milk, serum albumin, IgG concentration, somatic cell count (SCC), and lactate dehydrogenase (LDH) activity were determined. Dry matter intake and milk yield were recorded for an additional 6 d. Milk of the LPS-treated quarter was sampled at every milking for 8 d after the challenge. Based on plasma glucose concentrations in wk 1 to 4 after parturition before the LPS-CH, cows were retrospectively grouped into a high-glucose group (HG; 3.34-3.93 mmol/L, n = 7) and a low-glucose group (LG; 2.87-3.31 mmol/L, n = 8). Data were evaluated using mixed models with time, group, and time × group interaction as fixed effects and cow as repeated subject. Glucose was lower and BHB was higher in LG compared with HG before LPS-CH, whereas dry matter intake, energy balance, and SCC did not differ. During LPS-CH, SCC and LDH increased similarly in HG and LG, body temperature increased less in HG, and BHB and nonesterified fatty acids were higher in LG compared with HG. Dry matter intake declined in both groups during the day of the LPS-CH but recovered to prechallenge values faster in HG. Milk yield recovered within 2 d after the LPS-CH with no differences in morning milkings, whereas evening milk yield increased faster in HG. During 8 d after LPS-CH, SCC, LDH, IgG, and serum albumin in milk were lower in HG compared with LG. In conclusion, the level of circulating glucose and BHB concentrations in cows was associated with metabolic responses during an LPS-CH as well as the recovery of udder health and performance thereafter.

The effects of different concentrations of glucose on glucose sensors and GLP-1 secretion in the enteroendocrine cell line STC-1

Glucose triggers glucagon-like peptide (GLP)-1 secretion from L cells involving several glucose sensors including sodium-glucose transporter (SGLT)1, glucose transporter (GLUT)2, and sweet taste receptors (STRs). This study investigated the effects of different glucose concentrations on GLP-1 secretion, intracellular concentrations of Ca2+ and cAMP, glucose uptake, and protein levels of SGLT1, GLUT2, and STRs in STC-1 cells. Low glucose (5.6 mM) increased GLP-1 secretion, intracellular Ca2+ concentration, and SGLT1 protein level compared with glucose-free group. GLP-1 secretion and intracellular Ca2+ concentration triggered by low glucose were inhibited by the SGLT1 inhibitor. GLP-1 secretion or intracellular Ca2+ concentration in high-glucose (25, 100, 200 mM) groups was significantly higher than that of low-glucose group. Elevation of cAMP level was observed in concentration-dependent manner, and decreased glucose uptake was observed in 100 or 200 mM glucose group. High glucose increased protein levels of STRs and GLUT2 in comparison to low-glucose group. GLP-1 secretion and intracellular levels of Ca2+ and cAMP triggered by high glucose were inhibited in the presence of the GLUT2 or STR inhibitor. These results suggest that SGLT1 is dominantly responsible for GLP-1 secretion triggered by low glucose, and that STRs and GLUT2 are involved in GLP-1 secretion induced by high glucose.

Epigenome and transcriptome study of moringa isothiocyanate in mouse kidney mesangial cells induced by high glucose, a potential model for diabetic-induced nephropathy.

Moringa isothiocyanate (MIC-1) is a bioactive constituent found abundantly in Moringa oleifera which possesses antioxidant and anti-inflammation properties. However, epigenome and transcriptome effects of MIC-1 in kidney mesangial cells challenged with high glucose (HG), a pre-condition for diabetic nephropathy (DN) remain unknown. Herein, we examined the transcriptome gene expression and epigenome DNA methylation in mouse kidney mesangial cells (MES13) using next-generation sequencing (NGS) technology. After HG treatment, epigenome and transcriptome were significantly altered. More importantly, MIC-1 exposure reversed some of the changes caused by HG. Integrative analysis of RNA-Seq data identified 20 canonical pathways showing inverse correlations between HG and MIC-1. These pathways included GNRH signaling, P2Y purigenic receptor signaling pathway, calcium signaling, LPS/IL-1-mediated inhibition of RXR function, and oxidative ethanol degradation III. In terms of alteration of DNA methylation patterns, 173 differentially methylation regions (DMRs) between the HG group and low glucose (LG) group and 149 DMRs between the MIC-1 group and the HG group were found. Several HG related DMRs could be reversed by MIC-1 treatment. Integrative analysis of RNA-Seq and Methyl-Seq data yielded a subset of genes associated with HG and MIC-1, and the gene expression changes may be driven by promoter CpG status. These genes include Col4a2, Tceal3, Ret, and Agt. In summary, our study provides novel insights related to transcriptomic and epigenomic/CpG methylomic alterations in MES13 upon challenged by HG but importantly, MIC-1 treatment reverses some of the transcriptome and epigenome/CpG methylome. These results may provide potential molecular targets and therapeutic strategies for DN.

The Impact of Serum Glucose on the Predictive Value of Serum Lactate for Hospital Mortality in Critically Ill Surgical Patients.

Background Lactate has been widely used as a risk indicator of outcomes in critically ill patients due to its ready measurement and good predictive ability. However, the interconnections between lactate metabolism and glucose metabolism have not been sufficiently explored, yet. In this study, we aimed to investigate whether glucose levels could influence the predictive ability of lactate and design a more comprehensive strategy to assess the in-hospital mortality of critically ill patients. Methods We analyzed the clinical data of 293 critically ill patients. The primary outcome was in-hospital mortality. The logistic regression analysis and the area under the receiver operating characteristic curve (AUROC) were applied to evaluate the predictive ability of lactate in association with glucose. Results The lactate level showed significant association with in-hospital mortality, and its predictive ability was also comparable to other prognostic scores such as the SOFA score and APACHE II score. We further divided 293 patients into three groups based on glucose levels: low-glucose group (<7 mmol/L), medium-glucose group (7-9 mmol/L), and high-glucose group (>9 mmol/L). The lactate level was associated with in-hospital mortality in the low- and high- glucose groups, but not in the medium-glucose group, whereas the SOFA score and APACHE II score were associated with in-hospital mortality in all three glucose groups. The AUROC of lactate in the medium-glucose group was also the lowest among the three glucose groups, indicating a decrease in its predictive ability. Conclusions Our findings demonstrated that the predictive ability of lactate to assess in-hospital mortality could be influenced by glucose levels. In the medium glucose level (i.e., 7-9 mmol/L), lactate was inadequate to predict in-hospital mortality and the SOFA score; the APACHE II score should be utilized as a complementation in order to obtain a more accurate prediction.

Transcriptome analysis identified a novel 3-LncRNA regulatory network of transthyretin attenuating glucose induced hRECs dysfunction in diabetic retinopathy

BackgroundDiabetic retinopathy (DR) is the leading cause of blindness in the working age population. Transthyretin (TTR) showed a significantly decreased concentration in DR patients and exerted a visual protective effect by repressing neovascularization. This work intended to identify long non coding RNAs (lncRNAs) and explore their potential mechanism underlying the protective role of TTR.MethodsTranscriptome of human retinal endothelial cells (hRECs) treated with low glucose (LG), high glucose (HG) or high glucose with 4 μM TTR (HG + TTR) was conducted. Differentially expressed lncRNAs, mRNAs and TTR related lncRNAs and mRNA were acquired. Functional annotation and Gene Set Enrichment Analysis were applied to analyse TTR affected pathways and processes. Weighted gene co-expression network analysis (WGCNA) was implemented to obtain hub modules and genes. LncRNA-mRNA regulatory networks were constructed based on cis, trans and competing endogenous RNAs acting mode. QRT-PCR was conducted to validate the expression of lncRNAs in aqueous humor and serum samples from 30 DR patients and 10 normal controls.ResultsRNA-sequencing of hRECs treated with low glucose (LG), high glucose (HG) or high glucose with 4 μM TTR (HG + TTR) was conducted. 146,783 protein-coding transcripts, 12,403 known lncRNA transcripts and 1184 novel non-coding transcripts were characterized. A total of 11,407 differentially expressed mRNAs (DE-mRNAs), 679 differentially expressed lncRNAs (DE-lncRNAs) in HG group versus LG group, 6206 DE-mRNAs and 194 DE-lncRNAs in HG + TTR versus HG group were obtained, respectively. 853 TTR-mRNAs and 48 TTR-lncRNAs were acquired, and functionally involved in cell cycle, apoptosis, inflammation signalling pathway, response to oxidative stress, neovascularization and autophagy. The WGCNA analysis identified a hub module of 133 genes, with the core function of oxidative stress response, angiogenesis, MAPK pathway, cell proliferation and apoptosis. After qRT-PCR validation, a 3-lncRNA regulatory network was proposed. At last, lncRNAs MSTRG.15047.3 and AC008403.3 showed significantly relative higher expression levels in both aqueous humor and serum samples, compared with normal controls, and FRMD6-AS2 was significantly down-regulated.ConclusionsTTR regulated mRNAs and biological processes including oxidative stress, inflammation signalling and autophagy. A 3-lncRNA regulatory network was characterized underlying TTR repressing neovascularization, and showed potential diagnostic performance in DR.

Effect of miR-137 on the proliferation and apoptosis of endothelial cells regulated by AKT2 gene

To assess the effect of miR-137 on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) induced by high glucose and its mechanism. HUVECs cells were divided into low-glucose group (5.5 mmol/L glucose-treated cells), high-glucose group (33.36 mmol/L glucose-treated cells), anti-NC group (cells treated with 33.36 mmol/L glucose after anti-NC transfection) and anti-miR-137 group (cells treated with 33.36 mmol/L glucose after anti-miR-137 transfection). After 48 hours, qRT-PCR was used to determine the expression of miR-137. CCK-8 assay and flow cytometry were used to detect cell proliferation and apoptosis rate, respectively. The targeting relationship between miR-137 and AKT2 was validated by dual fluorescence reporter gene detection system and AKT2 protein expression after overexpression or inhibition of miR-137. High glucose could significantly up-regulate the expression of miR-137 in HUVECs cells, and the expression of miR-137 in HUVECs cells transfected with miR-137 inhibitor was significantly decreased (P<0.05). High glucose can significantly inhibit HUVECs cell proliferation and induce apoptosis, while inhibition of miR-137 expression can weaken the effect of high glucose on HUVECs cell proliferation inhibition and apoptosis promotion (P<0.05). Inhibiting AKT2 expression could weaken the inhibitory effect of miR-137 inhibitor on HUVECs cell proliferation and apoptosis (P<0.05). Inhibiting the expression of miR-137 gene can attenuate the proliferation inhibition and apoptosis promotion of HUVECs induced by high glucose, and the mechanism is related to activating the expression of AKT2.