High Concentration Of D-glucose Research Articles

Investigating the Effects of Simultaneous Administration of Melatonin and Atorvastatin against High Glucose-Induced Oxidative Stress and Apoptosis in Cultured Chondrocytes.

Osteoarthritis (OA) is a prevalent joint disorder categorized into phenotypic subtypes, including those associated with age, traumatic events, and metabolic syndrome. In the aging population, type 2 diabetes mellitus (T2DM) and osteoarthritis (OA) frequently coexist. This can result in higher rates of disability and a greater financial burden. This study aimed to investigate the protective effects of melatonin and atorvastatin together against oxidative stress and apoptosis induced by high glucose in C28I2 human chondrocytes. After being pretreated for 6 hours with melatonin (10 and 100 μM) and atorvastatin (0.01 and 0.1 μM), C28I2 cells were exposed to a high concentration of D-glucose (75 mM) for 72 hours. The impact of a high D-glucose concentration (75 mM), with or without melatonin and/or atorvastatin, on cell viability, intra-cellular ROS generation, lipid peroxidation level, antioxidant activities, and the expression of proteins, including Bax, Bcl-2, and caspase-3, was analyzed. Melatonin and atorvastatin combination effectively inhibited high glucose-induced cytotoxicity, ROS production, and MDA and mitochondrial membrane potential levels. The combination of melatonin and atorvastatin was more successful in reducing ROS production compared to each of the drugs alone. Melatonin, but not atorvastatin, reversed high glucose-induced alteration in the catalase activity. Furthermore, the combination of melatonin and atorvastatin significantly enhanced the ability of each medication to lower the expression of pro-apoptotic protein Bax. The combination of melatonin and atorvastatin exerted greater protective effects against hyperglycemia-induced toxicity in chondrocytes.

Regulation role of miR-204 on SIRT1/VEGF in metabolic memory induced by high glucose in human retinal pigment epithelial cells.

To examine the regulatory role of microRNA-204 (miR-204) on silent information regulator 1 (SIRT1) and vascular endothelial growth factor (VEGF) under high-glucose-induced metabolic memory in human retinal pigment epithelial (hRPE) cells. Cells were cultured with either normal (5 mmol/L) or high D-glucose (25 mmol/L) concentrations for 8d to establish control and high-glucose groups, respectively. To induce metabolic memory, cells were cultured with 25 mmol/L D-glucose for 4d followed by culture with 5 mmol/L D-glucose for 4d. In addition, exposed in 25 mmol/L D-glucose for 4d and then transfected with 100 nmol/L miR-204 control, miR-204 inhibitor or miR-204 mimic in 5 mmol/L D-glucose for 4d. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect miR-204 mRNA levels. SIRT1 and VEGF protein levels were assessed by immunohistochemical and Western blot. Flow cytometry was used to investigate apoptosis rate. It was found that high glucose promoted miR-204 and VEGF expression, and inhibited SIRT1 activity, even after the return to normal glucose culture conditions. Upregulation of miR-204 promoted apoptosis inhibiting SIRT1 and increasing VEGF expression. However, downregulation of miR-204 produced the opposite effects. The study identifies that miR-204 is the upstream target of SIRT1 and VEGF, and that miR-204 can protect hRPE cells from the damage caused by metabolic memory through increasing SIRT1 and inhibiting VEGF expression.

Synergistic effect of metformin and vitamin D3 on osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells under high d-glucose conditions

IntroductionVitamin D3 plays a vital role in bone health, with low levels of vitamin D3 being related to skeletal fragility, fractures, and metabolic disorders such as diabetes. Metformin is known as an antihyperglycemic agent for regulating blood sugar. A correlation between diabetes mellitus and osteoporosis is attracting considerable interest, and research to find the prevention and treatment is gradually being studied. In this study, we investigated the effect of metformin and vitamin D3 on osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (AT-MSCs) under high d-glucose concentrations and optimized by combining vitamin D3 and metformin in the process. MethodsROS production of AT-MSCs under high d-glucose conditions was measured by DCFH-DA assay. The differentiated AT-MSCs were analyzed by Alizarin Red S staining and optical density measurement. The investigation involved the examination of osteogenic master genes' expressions using quantitative reverse transcription polymerase chain reaction (qRT-PCR) techniques. ResultsInterestingly, the results have shown that human AT-MSCs will exhibit high ROS accumulation and low osteogenic differentiation capabilities, indicated by low calcium deposition, as well as low expression of indicative genes such as ALP, Runx-2 under high d-glucose conditions. The combination of vitamin D3 and metformin remarkedly accelerated the osteogenic differentiation of AT-MSCs under high d-glucose concentrations more effectively than the administration of either agent. ConclusionsThis study partially explains an aspect of an in vitro model for pre-clinical drug screening for osteoporosis-related diabetic pathological mechanisms, which can be applied for further research on the prevention or treatment of osteoporosis in diabetic patients.

Production of d-glucaric acid with phosphoglucose isomerase-deficient Saccharomyces cerevisiae

d-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of d-glucose to d-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of d-glucose to biomass, and to increase d-glucaric acid yield, the four step d-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High d-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13C-glucose confirmed conversion of d-glucose to d-glucaric acid. In batch bioreactor cultures with pulsed d-fructose and ethanol provision 1.3 g d-glucaric acid L−1 was produced. The d-glucaric acid titer (0.71 g d-glucaric acid L−1) was lower in nitrogen limited conditions, but the yield, 0.23 g d-glucaric acid [g d-glucose consumed]−1, was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield d-glucaric acid yeast strains.

Ultrastructural features mirror metabolic derangement in human endothelial cells exposed to high glucose

High glucose-induced endothelial dysfunction is the early event that initiates diabetes-induced vascular disease. Here we employed Cryo Soft X-ray Tomography to obtain three-dimensional maps of high d-glucose-treated endothelial cells and their controls at nanometric spatial resolution. We then correlated ultrastructural differences with metabolic rewiring. While the total mitochondrial mass does not change, high d-glucose promotes mitochondrial fragmentation, as confirmed by the modulation of fission–fusion markers, and dysfunction, as demonstrated by the drop of membrane potential, the decreased oxygen consumption and the increased production of reactive oxygen species. The 3D ultrastructural analysis also indicates the accumulation of lipid droplets in cells cultured in high d-glucose. Indeed, because of the decrease of fatty acid β-oxidation induced by high d-glucose concentration, triglycerides are esterified into fatty acids and then stored into lipid droplets. We propose that the increase of lipid droplets represents an adaptive mechanism to cope with the overload of glucose and associated oxidative stress and metabolic dysregulation.

Partial replacement of d-glucose with d-allose ameliorates peritoneal injury and hyperglycaemia induced by peritoneal dialysis fluid in rats.

Peritoneal dialysis (PD) is a crucial dialysis method for treating end-stage kidney disease. However, its use is restricted due to high glucose-induced peritoneal injury and hyperglycaemia, particularly in patients with diabetes mellitus. In this study, we investigated whether partially replacing d-glucose with the rare sugar d-allose could ameliorate peritoneal injury and hyperglycaemia induced by peritoneal dialysis fluid (PDF). Rat peritoneal mesothelial cells (RPMCs) were exposed to a medium containing d-glucose or d-glucose partially replaced with different concentrations of d-allose. Cell viability, oxidative stress and cytokine production were evaluated. Sprague-Dawley (SD) rats were administrated saline, a PDF containing 4% d-glucose (PDF-G4.0%) or a PDF containing 3.6% d-glucose and 0.4% d-allose (PDF-G3.6%/A0.4%) once a day for 4 weeks. Peritoneal injury and PD efficiency were assessed using immuno-histological staining and peritoneal equilibration test, respectively. Blood glucose levels were measured over 120 min following a single injection of saline or PDFs to 24-h fasted SD rats. In RPMCs, the partial replacement of d-glucose with d-allose increased cell viability and decreased oxidative stress and cytokine production compared to d-glucose alone. Despite the PDF-G3.6%/A0.4% having a lower d-glucose concentration compared to PDF-G4.0%, there were no significant changes in osmolality. When administered to SD rats, the PDF-G3.6%/A0.4% suppressed the elevation of peritoneal thickness and blood d-glucose levels induced by PDF-G4.0%, without impacting PD efficiency. Partial replacement of d-glucose with d-allose ameliorated peritoneal injury and hyperglycaemia induced by high concentration of d-glucose in PDF, indicating that d-allose could be a potential treatment option in PD.

Ferulic acid attenuates high glucose-induced MAM alterations via PACS2/IP3R2/FUNDC1/VDAC1 pathway activating proapoptotic proteins and ameliorates cardiomyopathy in diabetic rats

BackgroundDiabetic cardiomyopathy (DCM) is one of the severe complications of diabetes with no known biomarkers for early detection. Mitochondria-associated endoplasmic reticulum membranes (MAM) are less studied subcellular targets but an emerging area for exploration in metabolic disorders including DCM. We herein studied the role of MAMs and downstream mitochondrial functions in DCM. We also explored the efficacy of ferulic acid (FeA) against DCM via modulation of MAM and its associated signaling pathway. MethodsThe H9c2 cardiomyoblast cells were incubated with high concentration (33 mM) of d-glucose for 48 h to create a high glucose ambience in vitro. The expression of various critical proteins of MAM, mitochondrial function, oxidative phosphorylation (OxPhos) and the genesis of apoptosis were examined. The rats fed with high fat/high fructose/streptozotocin (single dose, i.p.) were used as a diabetic model and analyzed the insulin resistance and markers of cardiac hypertrophy and apoptosis. ResultsHigh glucose conditions caused the upregulation of MAM formation via PACS2, IP3R2, FUNDC1, and VDAC1 and decreased mitochondrial biogenesis, fusion and OxPhos. The upregulation of mitochondria-driven SMAC-HTRA2-ARTS-XIAP apoptosis and other cell death pathways indicate their critical roles in the genesis of DCM at the molecular level. The diabetic rats also showed cardiomyopathy with increased heart mass index, TNNI3K, troponin, etc. FeA effectively prevented the high glucose-induced MAM alterations and associated cellular anomalies both in vitro and in vivo. ConclusionHigh glucose-induced MAM distortion and subsequent mitochondrial dysfunctions act as the stem of cardiomyopathy. MAM could be explored as a potential target to treat diabetic cardiomyopathy. Also, the FeA could be an attractive nutraceutical agent for diabetic cardiomyopathy.

Crystal structure of metagenomic β-glycosidase MeBglD2 in complex with various saccharides.

Metagenomic MeBglD2 is a glycoside hydrolase family 1 (GH1) β-glycosidase that has β-glucosidase, β-fucosidase, and β-galactosidase activities, and is highly activated in the presence of monosaccharides and disaccharides. The β-glucosidase activity of MeBglD2 increases in a cellobiose concentration-dependent manner and is not inhibited by a high concentration of D-glucose or cellobiose. Previously, we solved the crystal structure of MeBglD2 and designed a thermostable mutant; however, the mechanism of substrate recognition of MeBglD2 remains poorly understood. In this paper, we report the X-ray crystal structures of MeBglD2 complexed with various saccharides, such as D-glucose, D-xylose, cellobiose, and maltose. The results showed that subsite - 1 of MeBglD2, which contained two catalytic glutamate residues (a nucleophilic Glu356 and an acid/base Glu170) was common to other GH1 enzymes, but the positive subsites (+ 1 and + 2) had different binding modes depending on the type of sugar. Three residues (Glu183, Asn227, and Asn229), located at the positive subsites of MeBglD2, were involved in substrate specificity toward cellobiose and/or chromogenic substrates in the presence of additive sugars. The docking simulation of MeBglD2-cellobiose indicated that Asn229 and Trp329 play important roles in the recognition of + 1 D-glucose in cellobiose. Our findings provide insights into the unique substrate recognition mechanism of GH1, which can incorporate a variety of saccharides into its positive subsites. KEY POINTS: •Metagenomic glycosidase, MeBglD2, recognizes various saccharides •Structures of metagenomic MeBglD2 complexed with various saccharides are determined •MeBglD2 has a unique substrate recognition mechanism at the positive subsites.

The impairment of osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells under high D-glucose concentrations

Type 2 diabetic patients have an increased risk of developing serious long-term complications such as cardiovascular disease, retinopathy, nephropathy, neuropathy, diabetic foot, and osteoporosis. However, the correlation between hyperglycaemia and osteoporosis has not yet been fully clarified. In this research, we investigated the effect of different high D-glucose concentrations (25, 50, and 100 mM) on osteogenic differentiation of human non-diabetic and diabetic adipose tissue-derived mesenchymal stem cells (nAT-MSCs and dAT-MSCs). The differentiated cells were qualified by Alizarin Red S staining and quantified by measuring the absorbance at 482 nm. The expressions of osteogenic master genes were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. Interestingly, the osteogenic differentiation and the expression of osteogenic-specific genes (Runx-2 and ALP) decreased with an increase of D-glucose concentration. Our work contributes to the understanding of the relationship between hyperglycaemia and osteoporosis and support the role of stem cells in developing new medicine or therapeutic treatment for preventing diabetic complications.

Enhancing biosynthesis of 2'-Fucosyllactose in Escherichia colithrough engineering lactose operon for lactose transport and α -1,2-Fucosyltransferase for solubility.

2'-Fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk and one of the most actively studied human milk oligosaccharides (HMOs). When 2'-FL is produced through biological production using a microorganism, like Escherichia coli, d-lactose is often externally fed as an acceptor substrate for fucosyltransferase (FT). When d-glucose is used as a carbon source for the cell growth and d-lactose is transported by lactose permease (LacY) in lac operon, d-lactose transport is under the control of catabolite repression (CR), limiting the supply of d-lactose for FT reaction in the cell, hence decreasing the production of 2'-FL. In this study, a remarkable increase of 2'-FL production was achieved by relieving the CR from the lac operon of the host E. coliBL21 and introducing adequate site-specific mutations into α-1,2-FT (FutC) for enhancement of catalytic activity and solubility. For the host engineering, the native lac promoter (Plac ) was substituted for tac promoter (Ptac ), so that the lac operon could be turned on, but not subjected to CR by high d-glucose concentration. Next, for protein engineering of FutC, family multiple sequence analysis for conserved amino acid sequences and protein-ligand substrate docking analysis led us to find several mutation sites, which could increase the solubility of FutC and its activity. As a result, a combination of four mutation sites (F40S/Q150H/C151R/Q239S) was identified as the best candidate, and the quadruple mutant of FutC enhanced 2'-FL titer by 2.4-fold. When the above-mentioned E. coli mutant host transformed with the quadruple mutant of futC was subjected to fed-batch culture, 40 g l-1 of 2'-FL titer was achieved with the productivity of 0.55 g l-1 h-1 and the specific 2'-FL yield of 1.0 g g-1 dry cell weight.

Improvement of endothelial function by Gunnera tinctoria extract with antioxidant properties

BackgroundGunnera tinctoria has been collected by Mapuche-Pewenche people for food and medicinal purposes. The high polyphenol content of methanolic extract from G. tinctoria leaves with chemical constituents such as ellagic acid and quercetin derivatives suggests its application to prevent endothelial dysfunction and oxidative stress. The aim of this study was to provide evidence of the protective effect of this extract on endothelial function by reducing oxidative stress induced by high d-glucose and H2O2, as well as by stimulating nitric oxide (NO) levels in human umbilical vein endothelial cells (HUVECs).ResultsA methanolic extract with a high content of polyphenols (520 ± 30 mg gallic acid equivalents/g dry extract) was obtained from G. tinctoria leaves. Its main constituent was ellagic acid. The results of Ferric reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays of the extract confirmed its antioxidant activity by inhibition pathway of radical species. The incubation of HUVECs with the extract decreased the apoptosis and reactive oxygen species (ROS) synthesis induced by high extracellular concentration of d-glucose or hydrogen peroxide. The extract increased endothelial NO levels and reduced vasoconstriction in human placental vessels.ConclusionsThis study provides evidence about the antioxidant and endothelial protective properties of methanolic G. tinctoria leaf extract. The extract improves the availability of NO in HUVECs, inhibiting the production of ROS and vasoconstriction.

Benzyl Isothiocyanate Produced by Garden Cress (Lepidium sativum) Prevents Accumulation of Hepatic Lipids.

We determined the physiological effects of glucotropaeolin-rich lyophilized garden cress sprout powder (GC) administered to fasting and nonfasting mice. High-performance liquid chromatography analysis revealed glucotropaeolin (57.4±1.1 mg/g dry weight) as a major phytochemical constituent of GC. Decreasing tendency in body weight and feeding efficiency ratio were detected in the group of mice fed 0.05% (w/w) GC (GC0.05). Nonfasting mice exhibited significantly lower liver weights that were unchanged after fasting. Decreased total lipid (TL) and triglyceride (TG) levels in the liver were detected in the nonfasted GC0.01 and GC0.05 groups, but only in TLs of the fasted groups. The levels of plasma TGs and nonesterified fatty acids of the GC0.05 group, which remained unchanged during nonfasting, decreased after fasting. To determine its effects on the accumulation of lipids in the liver, the glucotropaeolin aglycone, benzyl isothiocyanate (BITC), was added to the liver-derived HepG2 human cell line cultured in a medium containing a high concentration of D-glucose (4,500 mg/L D-glucose) (HG group) or 1 mM oleic acid (SO group). Toxicity was not detected when cells were treated with as much as 5 μM BITC; however, lipid accumulation was inhibited by BITC in a concentration-dependent manner in the HG groups. The same effect was observed when 2 μM BITC was added to the diet of the SO groups. These results suggest that moderate levels of GC or BITC are useful for reducing liver and plasma TGs.

2103-P: Glucose-Dependent Insulinotropic Polypeptide (GIP) Protects against Cytokine-Induced Cell Death and Exerts Both Insulinotropic and Glucagonotropic Effects in Human Islets

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is secreted from enteroendocrine K cells following food intake and potentiates glucose-stimulated insulin secretion from β-cells. Recent studies suggest that GIP also has glucagonotropic properties under euglycemic and hypoglycemic conditions in humans. GIP may therefore be exploited as a safeguard against hypoglycemia in patients with type 1 diabetes (T1D). However, a direct glucagonotropic effect of GIP on human α-cells has not yet been investigated. In this study, we investigated the effects of GIP on glucagon and insulin secretion in isolated human islets during 30-min exposures to low (2 mM) and high (20 mM) D-glucose concentrations. We further investigated the potency of GIP to protect human islets from cell death induced by a 48-h exposure to pro-inflammatory cytokines (50 U/mL interleukin-1β + 1,000 U/mL interferon-γ) known to contribute to islet destruction in T1D. GIP (2.5 nM) significantly (P=0.048, n=4) increased glucagon secretion by 48% at 2 mM glucose in isolated human islets as compared to 2 mM glucose alone. At 20 mM glucose, GIP did not affect glucagon secretion, but potentiated insulin secretion by 45% (P=0.015, n=4). Further, cytokine-induced human islet cell death was decreased by 50% by co-treatment with GIP (P=0.001, n=3). Our findings demonstrate that GIP protects against cytokine-induced islet death and possesses glucagonotropic and insulinotropic effects on human islets at low and high glucose, respectively. These findings support GIP as a bifunctional blood glucose-stabilizer that might be exploited to treat T1D. Disclosure A. Jørgensen: None. M. Egeskov: None. F.K. Knop: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Mundipharma International, Novo Nordisk A/S, Sanofi. Consultant; Self; Carmot Therapeutics, Inc., Eli Lilly and Company, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Gubra, Novo Nordisk A/S, Sanofi, Zealand Pharma A/S. Speaker’s Bureau; Self; AstraZeneca, Lupin Pharmaceuticals, Inc., Merck Sharp & Dohme Corp., Norgine B.V., Novo Nordisk A/S. J. Størling: None.

Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications.

Lignocellulosic biomass yields after hydrolysis, besides the hexose D-glucose, D-xylose, and L-arabinose as main pentose sugars. In second generation bioethanol production utilizing the yeast Saccharomyces cerevisiae, it is critical that all three sugars are co-consumed to obtain an economically feasible and robust process. Since S. cerevisiae is unable to metabolize pentose sugars, metabolic pathway engineering has been employed to introduce the respective pathways for D-xylose and L-arabinose metabolism. However, S. cerevisiae lacks specific pentose transporters, and these sugars enter the cell with low affinity via glucose transporters of the Hxt family. Therefore, in the presence of D-glucose, utilization of D-xylose and L-arabinose is poor as the Hxt transporters prefer D-glucose. To solve this problem, heterologous expression of pentose transporters has been attempted but often with limited success due to poor expression and stability, and/or low turnover. A more successful approach is the engineering of the endogenous Hxt transporter family and evolutionary selection for D-glucose insensitive growth on pentose sugars. This has led to the identification of a critical and conserved asparagine residue in Hxt transporters that, when mutated, reduces the D-glucose affinity while leaving the D-xylose affinity mostly unaltered. Likewise, mutant Gal2 transporter have been selected supporting specific uptake of L-arabinose. In fermentation experiments, the transporter mutants support efficient uptake and consumption of pentose sugars, and even co-consumption of D-xylose and D-glucose when used at industrial concentrations. Further improvements are obtained by interfering with the post-translational inactivation of Hxt transporters at high or low D-glucose concentrations. Transporter engineering solved major limitations in pentose transport in yeast, now allowing for co-consumption of sugars that is limited only by the rates of primary metabolism. This paves the way for a more economical second-generation biofuels production process.

Efficient, D-glucose insensitive, growth on D-xylose by an evolutionary engineered Saccharomyces cerevisiae strain.

Optimizing D-xylose consumption in Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. An evolutionary engineering approach was used to elevate D-xylose consumption in a xylose-fermenting S. cerevisiae strain carrying the D-xylose-specific N367I mutation in the endogenous chimeric Hxt36 hexose transporter. This strain carries a quadruple hexokinase deletion that prevents glucose utilization, and allows for selection of improved growth rates on D-xylose in the presence of high D-glucose concentrations. Evolutionary engineering resulted in D-glucose-insensitive growth and consumption of D-xylose, which could be attributed to glucose insensitive D-xylose uptake via a novel chimeric Hxt37 N367I transporter that emerged from a fusion of the HXT36 and HXT7 genes, and a down regulation of a set of Hxt transporters that mediate glucose sensitive xylose transport. RNA sequencing revealed the downregulation of HXT1 and HXT2 which, together with the deletion of HXT7, resulted in a 21% reduction of the expression of all plasma membrane transporters genes. Morphological analysis showed an increased cell size and corresponding increased cell surface area of the evolved strain, which could be attributed to genome duplication. Mixed strain fermentation of the D-xylose-consuming strain DS71054-evo6 with the D-glucose consuming CEN.PK113-7D strain resulted in decreased residual sugar concentrations and improved ethanol production yields compared to a strain which sequentially consumes D-glucose and D-xylose.

The pleiotropic molecule NGF regulates the in vitro properties of fibroblasts, keratinocytes, and endothelial cells: implications for wound healing.

Nerve growth factor (NGF) is recognized as a pleiotropic molecule, exerting a variety of biological effects on different cell types and pathophysiological conditions, and its role in tissue wound healing has been recently highlighted. However, the preferential cellular target of NGF is still elusive in the complex cellular and molecular cross talk that accompanies wound healing. Thus, to explore possible NGF cellular targets in skin wound healing, we investigated the in vitro NGF responsiveness of keratinocytes (cell line HEKa), fibroblasts (cell line BJ), and endothelial cells (cell line HUVEC), also in the presence of adverse microenvironmental conditions, e.g., hyperglycemia. The main results are summarized as follows: 1) NGF stimulates keratinocyte proliferation and HUVEC proliferation and angiogenesis in a dose-dependent manner although it has no effect on fibroblast proliferation; 2) NGF stimulates keratinocyte but not fibroblast migration in the wound healing assay; and 3) NGF completely reverts the proliferation impairment of keratinocytes and the angiogenesis impairment of HUVECs induced by high d-glucose concentration in the culture medium. These results contribute to better understanding possible targets for the therapeutic use of NGF in skin repair.

Curcumin protects against diabetic cardiomyopathy by promoting autophagy and alleviating apoptosis

The effects of curcumin on regulating cardiac apoptosis and autophagy were analyzed in diabetic models both in vivo and in vitro. In vivo, experimental diabetes was induced in mice by low-dose STZ injection combined with a high-fat diet. In vitro, cultured H9c2 cardiomyoblasts were exposed to high d-glucose concentrations combined with palmitate. Our results showed that apoptosis was increased and autophagy was suppressed in the hearts of diabetic mice, which was ameliorated by curcumin treatment, ultimately improving cardiac function. Moreover, the inhibition of autophagy exacerbated apoptotic death in cardiac cells under diabetic condition. Curcumin activated AMPK and JNK1, which phosphorylated Bcl-2 and Bim and subsequently disrupted their interactions with Beclin1, thereby promoting autophagy and alleviating apoptosis respectively. In addition, AMPK-mediated inhibition of mTORC1 pathway likely played a role in regulating autophagy by curcumin under diabetic condition. Our study suggests that curcumin protects against diabetic cardiomyopathy by modulating the crosstalk between autophagic and apoptotic machinery. Modulation of autophagy may be an effective strategy for the treatment of cardiovascular diseases associated with diabetes.

Whole transcriptome sequencing reveals biologically significant RNA markers and related regulating biological pathways in cardiomyocyte hypertrophy induced by high glucose.

Cardiomyocyte hypertrophy is a physiological adaptation used in an attempt to augment or preserve cardiac function for short periods. Long-term cardiomyocyte hypertrophy often progresses to heart failure. Previous studies have presented comprehensive mechanisms underlying cardiomyocyte hypertrophy, such as signaling pathways, marker genes, and marker miRNAs or lncRNAs. However, the mechanism in RNA level is still unclear. In this study, we used the whole transcriptome technology on cardiomyocety hypertrophy cells, which were cultured with a high concentration of d-glucose. Many differentially expressed markers, including genes, lncRNAs, miRNAs, and circRNAs were identified. Further quantitative real-time PCR verified the highly specific expressed genes, such as Eid1, Timm8b, Mrpl50, Dusp18, Abrc1, Klf13, and Igf1. Moreover, the functional pathways were also enriched with the differentially expressed lncRNA, miRNA, and circRNA. Our study gives new insights into cardiomyocyte hypertrophy and makes great progress in comprehending its mechanism.

High glucose concentration suppresses a SIRT2 regulated pathway that enhances neurite outgrowth in cultured adult sensory neurons

In peripheral nerve under hyperglycemic conditions high flux of d-glucose through the polyol pathway drives an aberrant redox state contributing to neurodegeneration in diabetic sensorimotor polyneuropathy (DSPN). Sirtuins, including SIRT2, detect the redox state via the NAD+/NADH ratio to regulate mitochondrial function via, in part, AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). In adult dorsal root ganglia (DRG) sensory neurons mitochondrial dysfunction has been proposed as an etiological factor in dying-back neuropathy in diabetes. We tested the hypothesis that a high concentration of d-glucose depleted SIRT2 expression via enhancement of polyol pathway activity. We posited that this would lead to impaired mitochondrial function and suppression of neurite outgrowth in cultured sensory neurons. The use of dominant negative mutants or neurons from SIRT2 knockout (KO) mice to block SIRT2 signaling revealed that neurons derived from control or type 1 diabetic rodents required SIRT2 for optimal neurite outgrowth. Over-expression of WT-SIRT2 elevated neurite outgrowth in normal and diabetic cultures. SIRT2 protein isoforms 2.1 and 2.2 were reduced by 20–30% in DRG of type 1 diabetic mice (p < .05). After 72 h exposure to high d-glucose (25 mM vs 5 mM) cultured sensory neurons showed a significant 2-fold (p < .05) decrease in SIRT2 expression, P-AMPK, levels of respiratory Complexes II/III and respiratory capacity. DRG neurons expressed aldose reductase and the aforementioned deficits were prevented by treatment with aldose reductase inhibitors (lidorestat or sorbinil) or sorbitol dehydrogenase inhibitor (SDI-158). In cultures derived from type 1 diabetic rats treatment with SDI-158 elevated expression of SIRT2, P-AMPK/PGC-1α and neurite outgrowth (p < .05). SIRT2 KO neurons exhibited deficits in the LKB-1/AMPK/PGC-1α pathway and mitochondrial function. In cultured neurons the SIRT2 pathway enhances axonal outgrowth and this signaling axis encompassing activation of AMPK/PGC-1α is impaired in DSPN, in part, due to enhanced polyol pathway activity caused by hyperglycemia.

Metabolome analysis-based design and engineering of a metabolic pathway in Corynebacterium glutamicum to match rates of simultaneous utilization of d-glucose and l-arabinose

Backgroundl-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to d-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as d-glucose, leading to a preferential utilization of d-glucose over pentoses. In order to simultaneously utilize both d-glucose and l-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis.ResultsCorynebacterium glutamicum ATCC 31831 utilized d-glucose and l-arabinose simultaneously at a low concentration (3.6 g/L each) but preferentially utilized d-glucose over l-arabinose at a high concentration (15 g/L each), although l-arabinose and d-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for d-glucose and l-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of l-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of d-glucose and l-arabinose (15 g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized d-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of l-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of d-glucose and l-arabinose.ConclusionsMetabolome analysis identified potential bottlenecks in d-glucose and l-arabinose metabolism and was then applied to the following rational metabolic engineering. Manipulation of only two genes enabled simultaneous utilization of d-glucose and l-arabinose at the same rate in metabolically engineered C. glutamicum. This is the first report of rational metabolic design and engineering for simultaneous hexose and pentose utilization without inactivating the phosphotransferase system.