Effects Of High Glucose Concentrations Research Articles

Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response

The unfolded protein response (UPR) detects increased misfolded proteins and activates protein refolding, protein degradation and inflammatory responses. UPR sensors in the endoplasmic reticulum, IRE1α and PERK, bind and are activated by proteins with unexpected surface hydrophobicity, whereas sensor ATF6 is activated by proteolytic cleavage when released from complexation with protein disulfide isomerases (PDIs). Metabolic dysfunction leading to the formation of misfolded proteins with surface hydrophobicity and disruption of ATF6-PDI complexes leading to activation of UPR sensors remains unclear. The cellular concentration of reactive dicarbonyl metabolite, methylglyoxal (MG), is increased in impaired metabolic health, producing increased MG-modified cellular proteins. Herein we assessed the effect of high glucose concentration and related increased cellular MG on activation status of IRE1α, PERK and ATF6. Human aortal endothelial cells and HMEC-1 microvascular endothelial cells were incubated in low and high glucose concentration to model blood glucose control, with increase or decrease of MG by silencing or increasing expression of glyoxalase 1 (Glo1), which metabolizes MG. Increased MG induced by high glucose concentration activated IRE1α, PERK and ATF6 and related downstream signalling leading to increased chaperone, apoptotic and inflammatory gene expression. Correction of increased MG by increasing Glo1 expression prevented UPR activation. MG modification of proteins produces surface hydrophobicity through arginine-derived hydroimidazolone MG-H1 formation, with related protein unfolding and preferentially targets PDIs and chaperone pathways for modification. It thereby poses a major challenge to proteostasis and activates UPR sensors. Pharmacological decrease of MG with Glo1 inducer, trans-resveratrol and hesperetin in combination, offers a novel treatment strategy to counter UPR-related cell dysfunction, particularly in hyperglycemia associated with diabetes.

Effects of High Glucose Concentrations on HMGB1 Expression in MG-63 Cells

Effects of High Glucose Concentrations on HMGB1 Expression in MG-63 Cells

MOLECULAR ASPECTS OF SOME RETINAL DISEASES

Diabetic retinopathy and age-related macular degeneration are eye diseases most likely to lead to blindness. The success of diagnosis, prevention, treatment and proper management largely depends on a deep knowledge of the molecular mechanisms of these pathologies. Analyzing the available data, it is established that their pathogenesis is complex were retinal neurovascular damage is the main component, which includes inflammatory, ischemic, immune, neurodegenerative changes, taking into account the important role of oxidative stress. The trigger of the pathological process in DR is the deleterious effect of high glucose concentration, and in the case of AMD, a number of metabolic changes occur with age. New data on the biochemical mechanisms of these diseases may be the basis for more effective and modern therapeutic modalities in ophthalmological practice.

Mechanism of Glucose Water as a Neural Injection: A Perspective on Neuroinflammation.

The entrapment of peripheral nerves is associated with chronic neuroinflammation and neuropathic pain, and perineural injection therapy with glucose is emerging as an effective treatment for peripheral entrapment neuropathy. However, the mechanism underlying the pharmacological effect of glucose on nerves remains unclear. One of the hypothesized mechanisms is that glucose reduces neurogenic inflammation. Therefore, we investigated the effects of high glucose concentrations on cytokine-induced neuroinflammation in vitro. Human SH-SY5Y neuronal cells were challenged with 10 ng/mL TNF-α for 16 h and subsequently treated with different glucose concentrations (0–25 mM) for 24 h. Cell viability was evaluated using the diphenyltetrazolium bromide assay, and proinflammatory cytokine levels were assessed using ELISA and quantitative PCR. In addition, mRNA levels of NF-κB and cyclooxygenase-2 were analyzed using quantitative PCR. Exposure to 10 ng/mL TNF-α resulted in decreased viability of SH-SY5Y cells and significant upregulation of IL-6, IL-1β, NF-κB, and cyclooxygenase-2. Subsequent exposure to high glucose levels (25 mM) markedly reduced the upregulation of IL-6, IL-1β, cyclooxygenase-2, and NF-κB, and restored the functional metabolism of SH-SY5Y cells, compared with that of the normal glucose control. Our findings suggest that high glucose concentrations can mitigate TNF-α-induced NF-κB activation, upregulation of proinflammatory cytokines, and metabolic dysfunction.

Sustained Hyperglycemia and Its Relationship with the Outcome of Hospitalized Patients with Severe COVID-19: Potential Role of ACE2 Upregulation.

Chronic hyperglycemia increases the risk of developing severe COVID-19 symptoms, but the related mechanisms are unclear. A mean glucose level upon hospital admission >166 mg/dl correlates positively with acute respiratory distress syndrome in patients with hyperglycemia. The objective of this study was to evaluate the relationship between sustained hyperglycemia and the outcome of hospitalized patients with severe COVID-19. We also evaluated the effect of high glucose concentrations on the expression of angiotensin-converting enzyme 2 (ACE2). We carried out a case-control study with hospitalized patients with severe COVID-19 with and without sustained hyperglycemia. In a second stage, we performed in vitro assays evaluating the effects of high glucose concentrations on ACE2 gene expression. Fifty hospitalized patients with severe COVID-19 were included, of which 28 (56%) died and 22 (44%) recovered. Patients who died due to COVID-19 and COVID-19 survivors had a high prevalence of hyperglycemia (96.4% versus 90.9%), with elevated central glucose upon admission (197.7 mg/dl versus 155.9 mg/dl, p = 0.089) and at discharge (185.2 mg/dl versus 134 mg/dl, p = 0.038). The mean hypoxemia level upon hospital admission was 81% in patients who died due to COVID-19 complications and 88% in patients who survived (p = 0.026); at the time of discharge, hypoxemia levels were also different between the groups (68% versus 92%, p ≤ 0.001). In vitro assays showed that the viability of A549 cells decreased (76.41%) as the glucose concentration increased, and the ACE2 gene was overexpressed 9.91-fold after 72 h (p ≤ 0.001). The relationship between hyperglycemia and COVID-19 in hospitalized patients with COVID-19 plays an important role in COVID-19-related complications and the outcome for these patients. In patients with chronic and/or sustained hyperglycemia, the upregulation of ACE2, and its potential glycation and malfunction, could be related to complications observed in patients with COVID-19.

Hyperglycemia induces miR-26-5p down-regulation to overexpress PFKFB3 and accelerate epithelial–mesenchymal transition in gastric cancer

ABSTRACT Gastric cancer (GC) is one of the most deadly malignancies with high morbidity worldwide. Cancer cells exhibited higher level of glucose catabolism than normal cells to meet the needs for rapid growth. Emerging evidences indicated that hyperglycemia has positive effects on the progression of tumor. As a vital regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) was confirmed to have a higher expression level in tumor tissue and correlated with the prognosis of GC patients. However, the role of PFKFB3 in GC patients with hyperglycemia remains unclear. The data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were utilized to analyze the expression level of PFKFB3 and conducted survival analysis of GC patients. Western blot assay was used to detect gene expression at the protein level. Small interfering RNA (siRNA) transfection assay was conducted to down-regulate the expression of PFKFB3. Cell functional assays were carried out to reflect the ability of cell proliferation and migration. The results indicated that PFKFB3 was significantly upregulated and its overexpression was associated with poor prognosis of GC patients. Besides, hyperglycemia stimulated the higher expression of PFKFB3 along with the enhanced proliferation, migration and epithelial–mesenchymal transition (EMT) in GC cells. Knocking down of PFKFB3 effectively reversed the effects of high glucose concentration on GC malignant phenotype and the opposite results were gained when miR-26-5p was inhibited. Therefore, PFKFB3 down-regulated by miR-26-5p inhibited the malignant phenotype of GC with hyperglycemia.

High glucose concentrations induce oxidative stress by inhibiting Nrf2 expression in rat M\xfcller retinal cells in vitro

Diabetic retinopathy (DR) is a complication of diabetes. Several studies have implicated oxidative stress as a fundamental factor in the progression of the disease. The nuclear factor erythroid-2-related factor 2 (Nrf2) is one of the main regulators of redox homeostasis. Glia Müller cells (MC) maintain the structural and functional stability of the retina. The objective of this study was to evaluate the effect of high glucose concentrations on reactive oxygen species (ROS) production and Nrf2 expression levels in rat MC. MC were incubated with normal (NG; 5 mM) or high glucose (HG; 25 mM) for different times. Incubation with HG increased ROS levels from 12 to 48 h but did not affect cell viability. However, exposure to 3 h of HG caused a transient decrease Nrf2 levels. At that time, we also observed a decrease in the mRNA expression of Nrf2 target genes, glutathione levels, and catalase activity, all of which increased significantly beyond initial levels after 48 h of incubation. HG exposure leads to an increase in the p65 subunit of nuclear factor-κB (NF-kB) levels, and its target genes. These results suggest that high glucose concentrations lead to alteration of the redox regulatory capacity of Nrf2 mediated by NF-kB regulation.

High Glucose Concentrations Impair the Processing and Presentation of Mycobacterium tuberculosis Antigens In Vitro.

Type 2 diabetes is an established risk factor for tuberculosis, but the underlying mechanisms are largely unknown. We established an in vitro model to analyze the effect of high glucose concentrations in antigen processing and presentation in antigen-presenting cells. Human monocyte-derived macrophages (MDMs) were exposed to high (11 mM and 30 mM) and low (5.5 mM) glucose concentrations and infected with Mycobacterium tuberculosis (Mtb). Flow cytometry was used to analyze the effect of high glucose concentrations in histocompatibility complex (MHC) class II molecules (HLA-DR) and co-stimulatory molecules (CD80 and CD86), indispensable for an adequate antigenic presentation and CD4+ T cell activation. HLA-DR and CD86 were significantly decreased by high glucose concentrations compared with low glucose concentrations. Confocal microscopy was used to detect Rab 5 and Lamp-1, proteins involved in the kinetics of antigen processing as early markers, and Rab 7 and cathepsin D as late markers. We observed a delay in the dynamics of the acquisition of Rab 7 and cathepsin D in high glucose concentrations. Moreover, the kinetics of the formation M. tuberculosis peptide–MHC II complexes in MDMs was decreased under high glucose concentrations, reducing their capacity for T cell activation. These findings suggest that high glucose concentrations directly affect antigenic processing, and therefore antigenic presentation.

In vitro effect of high glucose concentrations on erythrocyte morphology assessed by scanning electron microscopy

AimsTo evaluate the in vitro effect of different glucose concentrations and the time series in erythrocyte cell viability and morphology parameters. MethodsDifferent glucose concentrations were assessed, from a normal physiological glucose concentration (5 mM) to a diabetes scenario (100 mM). Cell viability was obtained by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The erythrocyte morphology (diameter, height, and axial ratio) was assessed by scanning electron microscopy (SEM). ResultsA significant decrease in cell viability was observed in erythrocytes with higher glucose concentration (100 mM). There was also a significant increase in the cells’ diameter and height exposed to 100 mM, vs. those cultured with 5, 20, and 45 mM glucose, but only at 24 and 48 h. When comparing the same glucose concentration by a time series, it was observed that erythrocytes from the culture with 45 and 100 mM of glucose had a significant continuous decrease (approximately -0.40 μm) in diameter and height (24, 48, and 72 h), but there was no difference in the axial ratio observed. ConclusionContinuous exposure to high glucose concentrations in diabetes models produced less cell viability and changes in erythrocyte morphology (larger and scrambled cells), which in vivo might impact in microvascular complications.

DYNAMIC BIOREACTOR MODEL TO MIMIC EARLY CARDIAC FIBROSIS IN DIABETES

In clinical diabetic cardiomyopathy, hyperglycemia and dyslipidemia induce tissue injury, activation of cardiac fibroblasts and interstitial and perivascular fibrosis. Myofibroblasts repair the injured tissue by increasing collagen deposition in the cardiac interstitium and suppressing the activity of matrix metalloproteinases. The goal of this study was to find an ideal model to mimic the effect of high glucose concentration on human cardiac fibroblast activation. The profibrotic role of the transforming growth factor-[Formula: see text] (TGF-[Formula: see text]) and the protective modulation of nitric oxide were examined in two-dimensional and three-dimensional cell culture models, as well as tissue engineering models, that involved the use of cardiac fibroblasts cultured within myocardial matrix scaffolds mounted in a bioreactor that delivered biochemical and mechanical stimuli. Results showed that high glucose levels were potent pro-fibrotic stimuli. In addition, high glucose levels in concert with TGF-[Formula: see text] constituted very strong signals that induced human cardiac fibroblast activation. Cardiac fibroblasts cultured within decellularized myocardial scaffolds and exposed to biochemical and mechanical stimuli represented an adequate model for this pathology. In conclusion, the bioreactor platform was instrumental in establishing an in vitro model of early fibrosis; this platform could be used to test the effects of various agents targeted to mitigate the fibrotic processes.

Involvement of nitric oxide synthase/nitric oxide pathway in the regulation of SIRT1–AMPK crosstalk in podocytes: Impact on glucose uptake

The protein deacetylase sirtuin 1 (SIRT1) and adenosine monophosphate-dependent protein kinase (AMPK) play important roles in the development of insulin resistance. In glomerular podocytes, crosstalk between these two enzymes may be altered under hyperglycemic conditions. SIRT1 protein levels and activity and AMPK phosphorylation decrease under hyperglycemic conditions, with concomitant inhibition of the effect of insulin on glucose uptake into these cells. Nitric oxide (NO)-dependent regulatory signaling pathways have been shown to be downregulated under diabetic conditions. The present study examined the involvement of the NO synthase (NOS)/NO pathway in the regulation of SIRT1-AMPK signaling and glucose uptake in podocytes.We examined the effects of NOS/NO pathway alterations on SIRT1/AMPK signaling and glucose uptake using pharmacological tools and a small-interfering transfection approach. We also examined the ability of the NOS/NO pathway to protect podocytes against high glucose-induced alterations of SIRT1/AMPK signaling and insulin-dependent glucose uptake.Inhibition of the NOS/NO pathway reduced SIRT1 protein levels and activity, leading to a decrease in AMPK phosphorylation and blockade of the effect of insulin on glucose uptake. Treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prevented high glucose-induced decreases in SIRT1 and AMPK activity and increased GLUT4 protein expression, thereby improving glucose uptake in podocytes.These findings suggest that inhibition of the NOS/NO pathway may result in alterations of the effects of insulin on glucose uptake in podocytes. In turn, the enhancement of NOS/NO pathway activity may prevent these deleterious effects of high glucose concentrations, thus bidirectionally stimulating the SIRT1-AMPK reciprocal activation loop.

Sulodexide reduces glucose induced senescence in human retinal endothelial cells

Chronic exposure of retinal endothelium cells to hyperglycemia is the leading cause of diabetic retinopathy. We evaluated the effect of high glucose concentration on senescence in human retinal endothelial cells (HREC) and modulation of that effect by Sulodexide. Experiments were performed on HREC undergoing in vitro replicative senescence in standard medium or medium supplemented with glucose 20 mmol/L (GLU) or mannitol 20 mnol/L (MAN). Effect of Sulodexide 0.5 LRU/mL (SUL) on the process of HREC senescence was studied. Glucose 20 mmol/L accelerates senescence of HREC: population doubling time (+ 58%, p < 0.001) β-galactosidase activity (+ 60%, p < 0.002) intracellular oxidative stress (+ 65%, p < 0.01), expression of p53 gene (+ 118%, p < 0.001). Senescent HREC had also reduced transendothelial electrical resistance (TEER) (− 30%, p < 0.001). Mannitol 20 mmol/L used in the same scenario as glucose did not induce HREC senescence. In HREC exposed to GLU and SUL, the senescent changes were smaller. HREC, which became senescent in the presence of GLU, demonstrated higher expression of genes regulating the synthesis of Il6 and VEGF-A, which was reflected by increased secretion of these cytokines (IL6 + 125%, p < 0.001 vs control and VEGF-A + 124% p < 0.001 vs control). These effects were smaller in the presence of SUL, and additionally, an increase of TEER in the senescent HREC was observed. Chronic exposure of HREC to high glucose concentration in medium accelerates their senescence, and that process is reduced when the cells are simultaneously exposed to Sulodexide. Additionally, Sulodexide decreases the secretion of IL6 and VEGF-A from senescent HREC and increases their TEER.

Effects of High Glucose Concentration on Pericyte-Like Differentiated Human Adipose-Derived Mesenchymal Stem Cells.

A pericyte-like differentiation of human adipose-derived mesenchymal stem cells (ASCs) was tested in in vitro experiments for possible therapeutic applications in cases of diabetic retinopathy (DR) to replace irreversibly lost pericytes. For this purpose, pericyte-like ASCs were obtained after their growth in a specific pericyte medium. They were then cultured in high glucose conditions to mimic the altered microenvironment of a diabetic eye. Several parameters were monitored, especially those particularly affected by disease progression: cell proliferation, viability and migration ability; reactive oxygen species (ROS) production; inflammation-related cytokines and angiogenic factors. Overall, encouraging results were obtained. In fact, even after glucose addition, ASCs pre-cultured in the pericyte medium (pmASCs) showed high proliferation rate, viability and migration ability. A considerable increase in mRNA expression levels of the anti-inflammatory cytokines transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) was observed, associated with reduction in ROS production, and mRNA expression of pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and angiogenic factors. Finally, a pmASC-induced better organization of tube-like formation by retinal endothelial cells was observed in three-dimensional co-culture. The pericyte-like ASCs obtained in these experiments represent a valuable tool for the treatment of retinal damages occurring in diabetic patients.

A high glucose concentration is well tolerated by colorectal adenocarcinoma and melanoma cells but toxic to normal human gingival fibroblast: Results of an in vitro investigation

Background: Glucose is associated with weight gain, which increases the risk of cancer. There is insufficient information on the effects of high glucose concentrations on cell lines. This study evaluated the viability patterns of human cancer and normal cell lines treated with glucose.

AMPK and Akt/mTOR signalling pathways participate in glucose-mediated regulation of hepatitis B virus replication and cellular autophagy.

A growing consensus indicates that host metabolism plays a vital role in viral infections. Hepatitis B virus (HBV) infection occurs in hepatocytes with active glucose metabolism and may be regulated by cellular metabolism. We addressed the question whether and how glucose regulates HBV replication in hepatocytes. The low glucose concentration at 5 mM significantly promoted HBV replication via enhanced transcription and autophagy when compared with higher glucose concentrations (10 and 25 mM). At low glucose concentration, AMPK activity was increased and led to ULK1 phosphorylation at Ser 555 and LC3-II accumulation. By contrast, the mTOR pathway was activated by high glucose concentrations, resulting in reduced HBV replication. mTOR inhibition by rapamycin reversed negative effects of high glucose concentrations on HBV replication, suggesting that low glucose concentration promotes HBV replication by stimulating the AMPK/mTOR-ULK1-autophagy axis. Consistently, we found that glucose transporters inhibition using phloretin also enhanced HBV replication via increased AMPK/mTOR-ULK1-induced autophagy. Surprisingly, the glucose analogue 2-deoxy-D-glucose reduced HBV replication through activating the Akt/mTOR signalling pathway also at the low glucose concentrations. Our study reveals that glucose is an important factor for the HBV life cycle by regulating HBV transcription and posttranscriptional steps of HBV replication via cellular autophagy.

MON-207 THE NRF2-HEME OXYGENASE-1 SYSTEM MODULATES AUTOPHAGY AND INHIBITS HIGH GLUCOSE INDUCED APOPTOSIS IN RENAL TUBULAR EPITHELIAL CELLS

Autophagy is a tightly regulated process in which endog­enous cellular proteins aggregate and damaged organelles are degraded by the lysosomal pathway. This process functions to maintain intracellular homeostasis and cell integrity. Its renoprotective role in several animal models, including those used for aging and acute kidney injury, has also been demonstrated. However, The role of autophagy in diabetic nephropathy remains a largely undetermined; thus, its underlying mecha­nisms are presently unclear. In present study, we evaluated the effects of high glucose concentrations on the induction of autophagy in the human renal tubular epithelial cell line, HK-2 cells. We also investigated the ability of Sulforaphane(SFN, nuclear factor E2-related factor 2 (Nrf2) inhibitor) to protect HK-2 cells against apoptosis induced by high glucose levels by targeting autophagy. In the present study, we evaluated the effects of high glucose concentrations on the induction of autophagy in the human renal tubular epithelial cell line, HK-2 cells. We also investigated the ability of Sulforaphane(SFN, nuclear factor E2-related factor 2 (Nrf2) inhibitor) to protect HK-2 cells against apoptosis induced by high glucose levels by targeting autophagy. The HK-2 cells stimulated with a high concentration of glucose for 72h exhibited an increased expression of the autophagic markers, LC3-II and Beclin 1. The level of LC3-II and Beclin 1 in cells treated with SFN was decreased compared to control cells. The level of Caspase-3 activated in tubule cells cultured in high glucose medium was also decreased. One important NRF2 target gene, Heme oxygenase-1(HO-1) is known as a key molecule of NRF2 protective function. To examine whether HO-1 can modulate autophagy and apoptosis in tubule cells like SFN, HK-2 cells cultured in 250 mM glucose medium for 1-2 days were treated with Adenovirus-HO-1 gene for 1 day. HO-1 transfection decreased the expression of Beclin1 and LC3-II associated with a decrease in the expression Caspase-3 with HG for 1-2days compared with mock-treated cells. Reactive Oxygen Species(ROS) are important inducers of autophagy and apoptosis. As expected, an increase of ROS was observed in HK-2 cells cultured in 250 mM glucose medium for 1-3 days. A decrease of ROS was observed in cells treated with SFN. HO-1 and autophagy are induced after high glucose injury to overcome to oxidative stress and serve as adaptive responses to prevent cell death. NRF2-HO-1 overexpression is able to limit ROS generation and oxidative stress during high glucose injury and thereby significantly inhibiting autophagy and apoptosis. This study is the first study to show the effect of NRF2-HO-1 system on high glucose-induced autophagy and apoptosis of tubule cells. Targeting NRF2-HO-1 as a modulator of autophagy may result in novel therapeutic intervention in diabetic nephropathy.

Metformin Effect on Endocan Biogenesis in Human Endothelial Cells Under Diabetic Condition

Endocan is a novel endothelium-derived proteoglycan and may play a role in endothelial cells activity under diabetic conditions. Here, we evaluated the effect of high glucose concentration (30mmol glucose) on endocan level in presence or absence of metformin in human umbilical vein endothelial cells (HUVECs). Cells were incubated with 30mmol glucose for 72h. High glucose content, metformin (2.5 to 500mmol) and compound C (10mmol) effects were assessed on cell viability. HUVECs migration was studied by scratch test. The changes in endocan expression and protein level were evaluated by RT-PCR, ELISA and flow cytometry assays. Griess reaction was used to measure NO levels. Functional activity of endothelial cells was monitored related to lipoprotein lipase activity using Dil-Ac-LDL uptake. p-AMPK/AMPK ratio was assessed by western blotting. Cells viability significantly was reduced under high glucose condition (p<0.05). 30mmol glucose inhibited HUVECs migration, whereas these features were improved by 50mmol metformin (p<0.05). Endocan transcription and protein levels were increased in diabetic HUVECs exposed to metformin (p<0.05). Metformin increased NO production in HUVECs under high glucose condition (p<0.001). Metformin increased LDL uptake capacity under high glucose condition (p<0.05). The addition of compound C blunted these effects. Western blot analysis confirmed the increase of p-AMPK/AMPK ratio in metformin-treated cells. Data demonstrated that metformin could promote angiogenic potential of endothelial cells which its reduction is a main cause in the development of diabetic foot ulcer, probably by the regulation of endocan dynamics under high glucose condition.

High glucose induces early senescence in adipose-derived stem cells by accelerating p16 and mTOR

Introduction: The senescence of stem cells is the primary reason that causes aging of stem cell-containing tissues. Some hypotheses have suggested that high glucose concentration in diabetic patients is the main factor that causes senescence of cells in those patients. This study aimed to evaluate the effects of high glucose concentrations on the senescence of adipose-derived stem cells (ADSCs).&#x0D; Methods: ADSCs were isolated and expanded from human adipose tissues. They were characterized and confirmed as mesenchymal stem cells (MSCs) by expression of surface markers, their shape, and in vitro differentiation potential. They were then cultured in 3 different media- that contained 17.5 mM, 35 mM, or 55 mM of D-glucose. The senescent status of ADSCs was recorded by the expression of the enzyme beta-galactosidase, cell proliferation, and doubling time. Real-time RT-PCR was used to evaluate the expression of p16, p21, p53 and mTOR.&#x0D; Results: The results showed that high glucose concentrations (35 mM and 55 mM) in the culture medium induced senescence of human ADSCs. The ADSCs could progress to the senescent status quicker than those cultured in the lower glucose-containing medium (17.5 mM). The senescent state was related to the up-regulation of p16 and mTOR genes.&#x0D; Conclusion: These results suggest that high glucose in culture medium can trigger the expression of p16 and mTOR genes which cause early senescence in ADSCs. Therefore, ADSCs should be cultured in low glucose culture medium, or normal glucose concentration, to extend their life in vitro as well as in vivo.&#x0D;

Deregulation of phytoene-\u03b2-carotene synthase results in derepression of astaxanthin synthesis at high glucose concentration in Phaffia rhodozyma astaxanthin-overproducing strain MK19

BackgroundA major obstacle to industrial-scale astaxanthin production by the yeast Phaffia rhodozyma is the strong inhibitory effect of high glucose concentration on astaxanthin synthesis. We investigated, for the first time, the mechanism of the regulatory effect of high glucose (> 100 g/L) at the metabolite and transcription levels.ResultsTotal carotenoid, β-carotene, and astaxanthin contents were greatly reduced in wild-type JCM9042 at high (110 g/L) glucose; in particular, β-carotene content at 24–72 h was only 14–17% of that at low (40 g/L) glucose. The inhibitory effect of high glucose on astaxanthin synthesis appeared to be due mainly to repression of lycopene-to-β-carotene and β-carotene-to-astaxanthin steps in the pathway. Expression of carotenogenic genes crtE, pbs, and ast was also strongly inhibited by high glucose; such inhibition was mediated by creA, a global negative regulator of carotenogenic genes which is strongly induced by glucose. In contrast, astaxanthin-overproducing, glucose metabolic derepression mutant strain MK19 displayed de-inhibition of astaxanthin synthesis at 110 g/L glucose; this de-inhibition was due mainly to deregulation of pbs and ast expression, which in turn resulted from low creA expression. Failure of glucose to induce the genes reg1 and hxk2, which maintain CreA activity, also accounts for the fact that astaxanthin synthesis in MK19 was not repressed at high glucose.ConclusionWe conclude that astaxanthin synthesis in MK19 at high glucose is enhanced primarily through derepression of carotenogenic genes (particularly pbs), and that this process is mediated by CreA, Reg1, and Hxk2 in the glucose signaling pathway.

Less is more or more is less: Implications of glucose metabolism in the regulation of the reproductive potential and total lifespan of the Saccharomyces cerevisiae yeast.

Carbohydrates are dietary nutrients that have an influence on cells physiology, cell reproductive capacity and, consequently, the lifespan of organisms. They are used in cellular processes after conversion to glucose, which is the primary source of energy and carbon skeleton for biosynthetic processes. Studies of the influence of glucose on cellular parameters and lifespan of organisms are primarily concerned with the effect of low glucose concentration defined as calorie restriction conditions. However, the effect of high glucose concentration on cell physiology is also very important. Thus, a comparative analysis of the effects of low and high glucose concentration conditions on cell efficiency was proposed with regard to reproductive capacity and total lifespan of the cell. Glucose concentration determines the type of metabolism and biosynthetic capabilities, which in turn, through the regulation on the cell size, may affect the reproductive capacity of cells. This study was conducted on yeast cells of wild-type and mutant strains Δgpa2 and Δgpr1 with glucose signalling pathway impairment. Such an experimental model enabled testing both the role of glucose concentration in the regulation of metabolic changes and the extent to which these changes depend on the extracellular or intracellular glucose concentrations. It has been shown here that calorie/glucose excess connected with changes in cell metabolic fluxes increases biosynthetic capabilities of yeast cells. This leads to an increase in cell dry weight accompanied by the increase in cell size and a simultaneous decrease in the reproductive potential and the overall length of cell life.