Glycogen Formation Research Articles

Dihydroorotase MoPyr4 is required for development, pathogenicity, and autophagy in rice blast fungus

Dihydroorotase (DHOase) is the third enzyme in the six enzymatic reaction steps of the endogenous pyrimidine nucleotide de novo biosynthesis pathway, which is a metabolic pathway conserved in both bacteria and eukaryotes. However, research on the biological function of DHOase in plant pathogenic fungi is very limited. In this study, we identified and named MoPyr4, a homologous protein of Saccharomyces cerevisiae DHOase Ura4, in the rice blast fungus Magnaporthe oryzae and investigated its ability to regulate fungal growth, pathogenicity, and autophagy. Deletion of MoPYR4 led to defects in growth, conidiation, appressorium formation, the transfer and degradation of glycogen and lipid droplets, appressorium turgor accumulation, and invasive hypha expansion in M. oryzae, which eventually resulted in weakened fungal pathogenicity. Long-term replenishment of exogenous uridine-5’-phosphate (UMP) can effectively restore the phenotype and virulence of the ΔMopyr4 mutant. Further study revealed that MoPyr4 also participated in the regulation of the Pmk1-MAPK signaling pathway, co-localized with peroxisomes for the oxidative stress response, and was involved in the regulation of the Osm1-MAPK signaling pathway in response to hyperosmotic stress. In addition, MoPyr4 interacted with MoAtg5, the core protein involved in autophagy, and positively regulated autophagic degradation. Taken together, our results suggested that MoPyr4 for UMP biosynthesis was crucial for the development and pathogenicity of M. oryzae. We also revealed that MoPyr4 played an essential role in the external stress response and pathogenic mechanism through participation in the Pmk1-MAPK signaling pathway, peroxisome-related oxidative stress response mechanism, the Osm1-MAPK signaling pathway and the autophagy pathway.

A fungal protein organizes both glycogen and cell wall glucans

Glycogen is a glucose storage molecule composed of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies, we focused on the pathogenic yeast Cryptococcus neoformans, which causes ~150,000 deaths per year worldwide. We identified two proteins that influence formation of both glycogen and the cell wall: glycogenin (Glg1), which initiates glycogen synthesis, and a protein that we call Glucan organizing enzyme 1 (Goe1). We found that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and β-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.

Natural history study of hepatic glycogen storage disease type IV and comparison to Gbe1ys/ys model.

BackgroundGlycogen storage disease type IV (GSD IV) is an ultrarare autosomal recessive disorder that causes deficiency of functional glycogen branching enzyme and formation of abnormally structured glycogen termed polyglucosan. GSD IV has traditionally been categorized based on primary hepatic or neuromuscular involvement, with hepatic GSD IV subclassified as discrete subtypes: classic (progressive) and nonprogressive.MethodsTo better understand the progression of liver disease in GSD IV, we present clinical and histopathology data from 23 patients from around the world and characterized the liver involvement in the Gbe1ys/ys knockin mouse model.ResultsWe propose an alternative to the established subtype-based terminology for characterizing liver disease in GSD IV and recognize 3 tiers of disease severity: (i) "severe progressive" liver disease, (ii) "intermediate progressive" liver disease, and (iii) "attenuated" liver disease. Analysis of liver pathology revealed that risk for liver failure cannot be predicted from liver biopsy findings alone in individuals affected by GSD IV. Moreover, analysis of postmortem liver pathology from an individual who died over 40 years after being diagnosed with nonprogressive hepatic GSD IV in childhood verified that liver fibrosis did not regress. Last, characterization of the liver involvement in a mouse model known to recapitulate the adult-onset neurodegenerative form of GSD IV (Gbe1ys/ys mouse model) demonstrated hepatic disease.ConclusionOur findings challenge the established subtype-based view of GSD IV and suggest that liver disease severity among patients with GSD IV represents a disease continuum.Trial registrationClinicalTrials.gov NCT02683512FundingNone.

147 The interaction between growth and immunity: Insights into the immune response of finisher pigs with divergent feed efficiency

Abstract This work evaluated the immune response of finisher pigs with divergent feed efficiency to identify immune pathways that were associated with efficiency. Blood gene expression data from a previous trial (Liu et al., 2019) were re-analyzed with novel methods that quantified changes in biological pathways and associated them with animal performance. Data were quality control checked and aligned to the Sus scrofa reference genome (Sscrofa11.1) using Kallisto. The dataset had 32 samples from eight pigs with high efficiency (HE) or low efficiency (LE) that had been injected with lipopolysaccharide (LPS). Blood samples were taken before challenge, and at 2, 6, and 24 h after challenge. After normalization and evaluation of differential gene expression, activation or inhibition of pathways was quantified using a topology-based pathway activation method and a customization of the Reactome database. Pathways were associated with feed efficiency using a regularized regression algorithm to identify immune pathways that were associated with efficiency. For statistical analysis, RNA integrity number, feed efficiency and time post LPS challenge were included in the model. At 2 h post LPS, HE pigs showed greater activation in pathways related to heat stress and creatine metabolism, but inhibition of pathways related to lysine and threonine catabolism, and immune resolving mediators (P-adj < 0.01), compared with the response in LE pigs. At 6 h post challenge there was a striking difference in cell cycle response, whereby cell cycle was activated in HE pigs but inhibited in LE pigs (P-adj < 0.01). The biggest difference in immune response was at 24h, with a strong response to LPS still evident in HE pigs, but a diminishing response in LE pigs. High efficiency pigs had activation in pathways related to oxidative stress, the metabolism of tryptophan, serine, tyrosine, threonine, glycogen, vitamin B5, and fibrin clot formation (P-adj < 0.01), which were not evident in LE pigs. The regression of pathways with feed efficiency showed that vitamin B5 metabolism was positively associated with gain:feed, whilst calcium-dependent signaling was negatively associated with gain:feed at multiple timepoints post-challenge. High efficiency pigs appeared to expand leukocyte populations at 6 h post LPS challenge, which was not evident in LE pigs. By 24 h, the response of LE pigs to LPS was minimal, but HE pigs still showed a strong response with greater utilization of energy, amino acids, and more oxidative stress. Low efficiency pigs appeared to recover faster from the LPS challenge, which was partially explained by differences in vitamin B5 utilization and calcium homeostasis. These findings confirm differences in the immune response of HE and LE pigs and provide insights related to amino acid, vitamin B5, and calcium homeostasis, which could help optimize the immune response of HE pigs. The novel analysis methods provided additional insights, which complement the original findings (T1).

Studi of In Vivo Antidiabetic Activity of Namnam Leaves (Cynometra cauliflora) Extract in Sprague Dawley Rat

Various treatments for Type 2 Diabetes Mellitus (T2DM) aim to alleviate hyperglycemia. Although natural products have been extensively utilized, their mechanism of action and efficacy as antidiabetic agents, particularly for T2DM, has not been extensively assessed in vivo. In this research, the leaves of the Namnam (Cynometra cauliflora) were extracted and tested for their activity as an antidiabetic agent. Sprague Dawley rats aged 3.5-4 months, weighing 200-250 g, were used as the experimental model, with a total of 30 individuals. The rats were induced with a high-fat diet and 30% sucrose until their blood glucose concentration reached ≥ 120 mg/dL. Subsequently, the rats were divided into four groups (groups 1, 2, 3, and 4). Over 21 days, changes in blood glucose, triacylglycerol, glycogen, and blood plasma insulin levels were assessed. The results demonstrated that the methanol extract of Namnam leaves (NLME) effectively reduced blood glucose levels by 23-34%, decreased plasma triglyceride levels by 13-30%, and increased liver glycogen levels by 68-96% compared to the control group (Diabetes). Among all the parameters assessed, NLME exhibited similar performance to metformin, a commonly prescribed diabetes medication (p<0.05). Furthermore, the study revealed that NLME exerted antidiabetic effects, particularly for T2DM, by promoting liver glycogen formation, enhancing insulin secretion control, facilitating glucose absorption by muscles, and restricting fat metabolism in the blood

GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways

BackgroundCyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear.ResultsHere, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains.ConclusionsThe developed approach, based on parallel 13C tracer studies with GC–MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments.

Bredemolic acid restores glucose utilization and attenuates oxidative stress in palmitic acid-induced insulin-resistant C2C12 cells.

Objective. Due to insulin resistance and oxidative stress that are associated with type 2 diabetes mellitus (T2DM), T2DM has become a prevalent metabolic disorder that presents various side effects. However, alternative antidiabetic treatment has commonly been used in treating diabetes mellitus in diabetic patients. In our previous studies, bredemolic acid has been reported as an antidiabetic agent that improves glucose uptake, ameliorates insulin resistance, and oxidative stress in the liver, heart, kidney, and skeletal muscle of prediabetic rats. However, these effects have not been validated in vitro. Therefore, this study was aimed to investigate the effects of bredemolic acid on insulin-mediated glucose utilization, lipid peroxidation, and the total antioxidant capacity (TOAC) in palmitic acid-induced insulin-resistant C2C12 skeletal muscle cells in vitro. Methods. Insulin resistance was induced in the skeletal muscle cells after 4 h of exposure to palmitic acid (0.5 mmol/l). Different cell groups were incubated in culture media DMEM supplemented with fetal calf serum (10%), penicillin/streptomycin (1%), and L-glutamine (1%) and then treated with either insulin (4 µg/ml) or bredemolic acid (12.5 mmol/l) or with both. Thereafter, the cells were seeded in 24- or 96-well plates for determination of the cell viability, glucose utilization, glycogen formation, and antioxidant capacity. Results. The results showed that bredemolic acid significantly improved TOAC and promoted glucose utilization via attenuation of lipid peroxidation and increased glycogen formation in the insulin-resistant cells, respectively. Conclusion. This study showed that bredemolic acid restored the insulin resistance through improved glucose utilization, glycogen formation, and TOAC in the skeletal muscle cells.

A comparative approach on the impact of diet and physical activity on young people between 19 and 26 years

Introduction. In the literature, nutrition is considered to be the basis for the energy supply in all the forms of physical activity. Energy needs depend on gender, age, body weight and possibly the type of the physical activity. Inadequate energy intake can lead to weight loss, fatigue, lack of concentration and no athletic performance. The constant physical activity involves the consumption of carbohydrates, useful for the formation and storage of muscle glycogen. The development of healthy eating and physical activity behaviors in childhood and adolescence are very important for adult life. That is why an early intervention can lead to healthy behaviors and it can prevent the occurrence of overweight and obesity. Objectives. The purpose of this study was to demonstrate how important a controlled diet is correlated with regular physical activity, in the normal development of young people aged between 19 and 26 years. Conclusions. The attitude is the one that should lead to a food program with a balanced diet that ensures good health, without increasing the percentage of fat, and to cause a weight loss for the young people who want to lose weight. Keywords: impact, diet, physical activity, young people,

MO036DAPAGLIFLOZIN RESCUES THE RENAL PHENOTYPE OF GLYCOGEN STORAGE DISEASE TYPE IB

Abstract Background and Aims Glycogenosis I type b (GsdI-b) is a rare metabolic disease and immune disorder characterized by hepato-renal glycogen accumulation caused by a deficiency in the Glucose-6-phosphate transporter (G6PT). G6PT transports glucose-6-phosphate (G6P) from cytoplasm to endoplasmic reticulum (ER) where a G6Pase catalyses the hydrolysis of G6P in glucose and phosphate. G6PT deficiency lead to impaired glucose homeostasis, myeloid disfunction and long-term risk of hepatocellular adenomas. No causal therapy is so far available for GSDI-b patients besides a dietary approach to control glycemia and the use of Granulocyte Colony-Stimulating Factor (GCSF) to improve neutropenia. Over time, these supports increase the chronicity of GSDI-b with some complications. A mouse model recapitulating the GDSI-b has been recently generated by inducing G6PT suppression after tamoxifen injection. Here, we characterized the renal phenotype of TM-G6PT-/- mice model focusing on the molecular mechanisms that lead to renal dysfunction. Finally, we evaluated the efficiency of Dapagliflozin, a selective inhibitor of SGLT2, on kidney functions in terms of therapeutic effect. Method Machine learning approach to computer based evaluation of renal morphology was used to analyze the renal sections from TM-G6PT-/- treated with or without dapagliflozin. Results: G6PT is expressed in all renal zones and a severe downregulation of G6PT mRNA expression in whole kidney of TM-G6PT-/- mice can be observed. TM-G6PT-/- mice show tubular vacuolization and overall cellular dysfunction of PT due to a high glycogen accumulation. TM-G6PT-/- mice manifest glycosuria, phosphaturia and polyuria associated with a down regulation of main transporters of PT cells. The urine concentrating defect is due to a primarily role of G6PT in CNT/CD cells confirmed by a downregulation of AQP2, main water channel along CD segments. This mouse model recapitulates the human GSD-Ib renal phenotype characterized by a disfunction of PT but also CNT/CD cells. In order to evaluate whether targeting the glucose metabolism would improve the renal phenotype of these mice we limited glucose flux across the apical membrane of PT cells, applying the SGLT2-inhibitor dapagliflozin to reduce new glycogen formation. After one month of treatment, Dapagliflozin prevents glycogen accumulation in TM-G6PT-/- mice and ameliorates the main dysregulated markers of PT function. This finding was paralleled by an improvement of the histological features of kidney morphology in dapagliflozin treated TM-G6PT-/- mice. Conclusion Our data provide evidence that treatment with dapagliflozin ameliorates intracellular glycogen storage and improves the renal functions in TM-G6PT-/- mice.

Triterpene betulin may be involved in the acute effects of pulp and paper mill effluent on testis physiology in zebrafish

Pulp and paper mill effluent can cause changes in the morphology and energy metabolism in the zebrafish (Danio rerio) testis. Betulin, a naturally occurring triterpene is commonly present in this type of effluent and is suspected of being involved in these effects. The aim of this study was to compare the effects pulp and paper mill effluent and betulin on various aspects of testicular physiology in the zebrafish. This included the in vitro effects of effluent and betulin on testicular lactate content and lactate dehydrogenase (LDH) activity. In addition, the effects of betulin on glucose uptake, glycogen, alanine aminotransferase (ALT), reactive oxygen and nitrogen species formation and oxidative damage in the testes were determined. Furthermore, we compared the effects and mechanism of action of betulin and effluent on calcium homeostasis in testes. In vitro exposure to both effluent and betulin decreased lactate and calcium influx, possibly due to the activation of the sodium‑calcium exchanger (NCX) pump. Additionally, betulin-treated testes had higher reactive oxygen species (ROS) and reduced glutathione (GSH) content, as well as increased glutathione transferase (GST) activity and a tendency towards decreased catalase (CAT) activity. Thus, this study shows that alterations in testis physiology caused by the pulp and paper mill effluent in the testis may be due in part to the actions of betulin.

The multi-target small-molecule inhibitor SB747651A shows in vitro and in vivo anticancer efficacy in glioblastomas

Glioblastoma multiforme is the most common primary brain tumor and among the most lethal types of cancer. Several mono-target small molecule-inhibitors have been investigated as novel therapeutics, thus far with poor success. In this study we investigated the anticancer effects of SB747651A, a multi-target small-molecule inhibitor, in three well characterized patient-derived glioblastoma spheroid cultures and a murine orthotopic xenograft model. Concentrations of 5–10 µM SB747651A reduced cell proliferation, spheroid formation, migration and chemoresistance, while apoptotic cell death increased. Investigation of oncogenic kinase signaling showed decreased phosphorylation levels of mTOR, CREB, GSK3 and GYS1 leading to altered glycogen metabolism and formation of intracellular reactive oxygen species. Expression levels of cancer stemness marker SOX2 were reduced in treated tumor cells and SB747651A treatment significantly prolonged survival of mice with intracranial glioblastoma xenografts, while no adverse effects were observed in vivo at doses of 25 mg/kg administered 5 days/week for 8 weeks. These findings suggest that SB747651A has anticancer effects in glioblastoma. The cancer-related pathophysiological mechanisms targeted by SB747651A are shared among many types of cancer; however, an in-depth clarification of the mechanisms of action in cancer cells is important before further potential application of SB747651A as an anticancer agent can be considered.

Trehalose Ameliorates Seizure Susceptibility in Lafora Disease Mouse Models by Suppressing Neuroinflammation and Endoplasmic Reticulum Stress.

Lafora disease (LD) is one of the progressive and fatal forms of a neurodegenerative disorder and is characterized by teenage-onset myoclonic seizures. Neuropathological changes in LD include the formation of abnormal glycogen as Lafora bodies, gliosis, and neuroinflammation. LD is caused by defects in the gene coding for phosphatase (laforin) or ubiquitin ligase (malin). Mouse models of LD, developed by targeted disruption of these two genes, develop most symptoms of LD and show increased susceptibility to induced seizures. Studies on mouse models also suggest that defective autophagy might contribute to LD etiology. In an attempt to understand the specific role of autophagy in LD pathogenesis, in this study, we fed LD animals with trehalose, an inducer of autophagy, for 3months and looked at its effect on the neuropathology and seizure susceptibility. We demonstrate here that trehalose ameliorates gliosis, neuroinflammation, and endoplasmic reticulum stress and reduces susceptibility to induced seizures in LD animals. However, trehalose did not affect the formation of Lafora bodies, suggesting the epileptic phenotype in LD could be either secondary to or independent of Lafora bodies. Taken together, our results suggest that autophagy inducers can be considered as potential therapeutic molecules for Lafora disease.

RESEARCH OF TO ULTRATHIN STRUCTURE OF CAGES OF DIFFERENT RACES OF SPIRIT YEASTS IN DEPENDENCE ON CONCENTRATION OF DRY SUBSTANCES OF WAS

One of the directions of introduction of resource- and energy-saving technology of alcohol brews into alcohol production is the use of highly concentrated wort from grain raw materials. The use of highly productive strains of alcoholic yeast is the basis of resource- and energy-saving technologies, a way to reduce the cost of ethyl alcohol and increase the profitability of its production. To develop the technology of highly concentrated mash from grain raw materials, it is necessary to select and select the appropriate breeds of yeast and study their morphological and physiological properties. Diagnosis of the physiological state of microorganisms. The influence of wort dry matter concentration on morphological and cytological features of the structure of yeast cells of alcoholic breeds of yeast S. cerevisiae DO–16, DO–11, K–81, XII in the conditions of cultivation on media from starch-containing raw materials was studied. The concentration of dry matter of the wort is 20 and 28%. It was found that the selected race of yeast S. cerevisiae DO–16 synthesizes the largest number of yeast cells at a concentration of 28%. Osmophilic races of S. cerevisiae DO–16 and DO–11 had smaller cell sizes and areas in comparison with thermotolerant and mesophilic races of S. cerevisiae K–81 and XII at a wort concentration of 28.0%. During fermentation, these indicators characterize the increase in the working surface of the yeast in the fermented medium, it allows to accelerate the fermentation process and ensure microbiological purity of the environment, which is especially important for highly concentrated wort. On the basis of morphological and cytological studies of the yeast race S. cerevisiae DO–16, its advantages over the races DO –11, K–81, XII for fermentation of high-concentration wort have been proved. Based on studies of the intracellular structure of the yeast S. cerevisiae DO–16, DO–11, K–81, XII, the relationship between the formation of glycogen in yeast cells and the concentration of DM wort was established. When culturing industrial yeast at a DM concentration of 28%, the glycogen content in the cells of S. cerevisiae DO–16 was significantly higher compared to the studied races, which indicates favorable conditions of the culture medium for this race.

Proteome characteristics of liver tissue from patients with parenteral nutrition-associated liver disease

BackgroundParenteral nutrition (PN)-associated liver disease (PNALD) is a common and life-threatening complication in patients receiving PN. However, its definitive etiology is not yet clear. Therefore, performed proteomic analyses of human liver tissue to explore the same.MethodsLiver tissue was derived and compared across selected patients with (n = 3) /without (n = 4) PNALD via isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-based quantitative proteomics. Bioinformatics analysis was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to explore the mechanisms of PNALD based on differentially expressed proteins (DEPs). The essential proteins that were differentially expressed between the two groups were explored and verified by western blotting.ResultsA total of 112 proteins were found to be differentially expressed, of which 73 were downregulated, and 39 were upregulated in the PNALD group. Bioinformatics analysis showed DEPs to be associated with mitochondrial oxidative phosphorylation (mainly involved in mitochondrial respiratory chain complex I assembly), hepatic glycolipid metabolism (involved primarily in glycogen formation and gluconeogenesis), and oxidative stress (mainly involved in antioxidant change).ConclusionOverall, our results indicated that mitochondrial energy metabolism impairment, hepatic glycolipid metabolism disorder, and excessive oxidative stress injury might explain the comprehensive mechanism underlying PNALD. Moreover, we have provided multiple potential targets for further exploring the PNALD mechanism.

Loss of autophagy in condylar chondrocytes causes increased apoptosis rate in temporomandibular joint osteoarthritis of rats

Objective: To observe the effect of autophagy of condylar chondrocytes on apoptosis in temporomandibular joint osteoarthritis (TMJOA) of rats. Methods: Fourty male 2-month-old SPF SD rats were equally divided into sham group (n=20) and experimental group (n=20). UAC metal prosthesis was cemented to the left incisors of maxilla and mandible of the rats in experimental group rats. After 8 weeks, all rats were sacrificed and the temporomandibular joint was taken. Two groups of rat condylar chondrocytes were extracted and cultured in vitro to the third generation. Immunofluorescence technique was used to detect the levels of collagen Ⅱ and matrix metalloproteinase-13 (MMP-13) in chondrocytes. The level of light chain-3 (LC-3), an autophagy marker of chondrocytes, was detected. Immunohistochemical technique was used to detect the level glycogenin-1, a glycogen formation marker of chondrocyte, was detected. The level of caspase-3, an apoptosis marker of chondrocyte, was also detected. Tunel technique was used to detect the apoptosis rate of the two groups at 72 h. Cracking cell extraction of total protein, Western-blotting (WB) technology to detect the levels of collagen Ⅱ, MMP -13, LC-3, glycogenin-1, caspase-3 and make gray analysis. Results: Compared with sham group, the level of collagen Ⅱ decreased, MMP-13 increased, LC-3 decreased, glycogenin-1 increased and caspase-3 increased in experimental group. The apoptosis rate of chondrocytes in experimentaal group [ (17.3±4.4) %] at 72h was higher than that in control group [ (5.6±2.1) %](t=10.732, P<0.001) .WB bands gray statistical results show that the level of collagen Ⅱ in chondrocytes of experimental group (0.43±0.21) was lower than that of control group (0.71±0.26) (t=2.409, P=0.043) , the level of MMP-13 in chondrocytes of experimental group (0.73±0.31) was higher than that of control group (0.24±0.10) (t=3.364, P=0.010) , the level of LC-3 in chondrocytes of experimental group (0.09±0.04) was lower than that of control group (0.39±0.18) (t=3.638, P=0.007) , the level of glycogenin-1 in chondrocytes of experimental group (0.68±0.30) was higher than that of control group (0.29±0.17) (t=2.529, P=0.035) , the level of caspase-3 in chondrocytes of experimental group (0.19±0.08) was higher than that of control group (0.05±0.02) (t=3.796, P=0.005) . Conclusions: The level of autophagy of condylar chondrocytes in temporomandibular joint of rats decreased, glycogen accumulation increased, the rate of chondrocyte apoptosis increased, and the number of chondrocytes decreased, resulting in degeneration of condylar cartilage tissue.

LEFTY2/endometrialbleeding-associated factor up-regulates Na+Coupled Glucose Transporter SGLT1 expression and Glycogen Accumulation in Endometrial Cancer Cells.

LEFTY2 (endometrial bleeding associated factor; EBAF or LEFTYA), a cytokine released shortly before menstrual bleeding, is a negative regulator of cell proliferation and tumour growth. LEFTY2 down-regulates Na+/H+ exchanger activity with subsequent inhibition of glycolytic flux and lactate production in endometrial cancer cells. Glucose can be utilized not only for glycolysis but also for glycogen formation. Both glycolysis and glycogen formation require cellular glucose uptake which could be accomplished by the Na+ coupled glucose transporter-1 (SGLT1; SLC5A1). The present study therefore explored whether LEFTY2 modifies endometrial SGLT1 expression and activity as well as glycogen formation. Ishikawa and HEC1a cells were exposed to LEFTY2, SGLT1 and glycogen synthase (GYS1) transcript levels determined by qRT-PCR. SGLT1, GYS1 and phospho-GYS1 protein abundance was quantified by western blotting, cellular glucose uptake from 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) uptake, and cellular glycogen content utilizing an enzymatic assay and subsequent colorimetry. As a result, a 48-hour treatment with LEFTY2 significantly increased SGLT1 and GYS1 transcript levels as well as SGLT1 and GYS1 protein abundance in both Ishikawa and HEC1a cells. 2-NBDG uptake and cellular glycogen content were upregulated significantly in Ishikawa (type 1) but not in type 2 endometrial HEC1a cells, although there was a tendency of increased 2-NBDG uptake. Further, none of the effects were seen in human benign endometrial cells (HESCs). Interestingly, in both Ishikawa and HEC1a cells, a co-treatment with TGF-β reduced SGLT1, GYS and phospho-GYS protein levels, and thus reduced glycogen levels and again HEC1a cells had no significant change. In conclusion, LEFTY2 up-regulates expression and activity of the Na+ coupled glucose transporter SGLT1 and glycogen synthase GYS1 in a cell line specific manner. We further show the treatment with LEFTY2 fosters cellular glucose uptake and glycogen formation and TGF-β can negate this effect in endometrial cancer cells.

Malfunction of autophagy in tibial growth plate chondrocytes causes increased apoptosis rate in chronic renal insufficiency rats

Objective: To observe the effect of autophagy of tibial growth plate chondrocyte on apoptosis in chronic renal insufficiency (CRI) rats. Methods: Male 4-week-old SD rats were randomly divided into two groups: (1) Sham group: only the left ureter was exposed (n=10); (2) CRI group: the left ureter was ligated to cause CRI (n=10). The urine from all the rats was collected 6 weeks after the operation and the total protein content was measured. Then all the rats were sacrificed and the concentrations of creatinine and urea nitrogen in intracardiac blood were detected. The proximal tibia were fixed and decalcified to prepare histological sections, and the number of chondrocytes of column cells in the proliferative area of tibia growth plate was observed by saffron O staining. The expression rate of protein Light Chain-3, an autophagy marker of chondrocytes, was detected by immunofluorescence. The apoptosis rate of chondrocytes was detected by the method of TUNEL assay. The level of glycogenin-1, a glycogen formation marker of chondrocyte was detected by immunohistochemistry in chondrocytes. Results: The 24 h urine total protein was higher in CRI group [(163.5±11.3) mg vs (38.6±9.8) mg, t=25.620, P<0.001]. The levels of blood creatinine [(67.3±16.2) μmol/L vs (28.4±11.5) μmol/L, t=5.974, P<0.001] and urea nitrogen [(16.4±6.4) mmol/L vs (4.8±2.0) mmol/L, t=5.198, P<0.001] were higher in CRI group. The number of chondrocytes of column cells in the proliferating area of tibia growth plate was lower in CRI group (4.2±2.1 vs 9.1±3.8, t=3.109, P=0.006). The expression rate of LC-3 protein in chondrocytes of CRI group was lower [(27.2±12.6)% vs (51.4±18.2)%, t=3.457, P=0.003]. The level of glycogenin-1 of chondrocytes in CRI group increased significantly (6.1±2.5 vs 3.5±1.8, t=2.669, P=0.016). The apoptosis rate of chondrocytes in CRI group also increased [(17.2±4.8)% vs (5.1±3.4)%, t=6.505, P<0.001]. Conclusion: Malfunction of autophagy in tibial growth plate chondrocytes causes increased apoptosis rate in CRI rats, which might be caused by the failure of glycogen degradation in chondrocytes.

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies.

BACKGROUND:Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes.OBJECTIVE:For Upcyte® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte® hepatocytes representing proliferating primary-like hepatocytes.METHODS:POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method.RESULTS:The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3(p = 0.0829) might be present. Compared to primary human hepatocyte microsomes, POR activity was considerably lower in all hepatocyte models.CONCLUSION:In summary, our study revealed that POR expression and activity were clearly detectable in all in vitro hepatocyte models with the highest POR expression in cancer cell line HepG2. However, POR activity was lower in tested hepatocyte models when compared to human primary hepatocyte microsomes. Whether this was caused by e.g. polymorphisms or metabolic differences of investigated hepatocyte models will be target for future studies.

Global Sensitivity Analysis of Metabolic Models for Phosphorus Accumulating Organisms in Enhanced Biological Phosphorus Removal

The aim of this study was to identify, quantify and prioritize for the first time the sources of uncertainty in a mechanistic model describing the anaerobic-aerobic metabolism of phosphorus accumulating organisms (PAO) in enhanced biological phosphorus removal (EBPR) systems. These wastewater treatment systems play an important role in preventing eutrophication and metabolic models provide an advanced tool for improving their stability via system design, monitoring and prediction. To this end, a global sensitivity analysis was conducted using standard regression coefficients and Sobol sensitivity indices, taking into account the effect of 39 input parameters on 10 output variables. Input uncertainty was characterized with data in the literature and propagated to the output using the Monte Carlo method. The low degree of linearity between input parameters and model outputs showed that model simplification by linearization can be pursued only in very well defined circumstances. Differences between first and total-order sensitivity indices showed that variance in model predictions was due to interactions between combinations of inputs, as opposed to the direct effect of individual inputs. The major sources of uncertainty affecting the prediction of liquid phase concentrations, as well as intra-cellular glycogen and poly-phosphate was due to 64% of the input parameters. In contrast, the contribution to variance in intra-cellular PHA constituents was uniformly distributed among all inputs. In addition to the intra-cellular biomass constituents, notably PHB, PH2MV and glycogen, uncertainty with respect to input parameters directly related to anaerobic propionate uptake, aerobic poly-phosphate formation, glycogen formation and temperature contributed most to the variance of all model outputs. Based on the distribution of total-order sensitivities, characterization of the influent stream and intra-cellular fractions of PHA can be expected to significantly improve model reliability. The variance of EBPR metabolic model predictions was quantified. The means to account for this variance, with respect to each quantity of interest, given knowledge of the corresponding input uncertainties, was prescribed. On this basis, possible avenues and pre-requisite requirements to simplify EBPR metabolic models for PAO, both structurally via linearization, as well as by reduction of the number of non-influential variables were outlined.

BIOMARKERS OF APOPTOSIS IN GLYCOGEN STORAGE DISEASE IN CHILDREN

Glycogen storage disease (GSD) is a rare form of the pathology in children caused by genetically determined pathological changes of the formation or cleavage of glycogen. Depending on the disorders of functions of enzymes involved in glycogen metabolism, there are known up to 15 GSD types, among them there are isolated liver, muscular and mixed forms. There are presented data of the immunoassay analysis of changes in concentrations of the array of proteins considered as biomarkers of apoptosis: sAPO-1/FAS receptor, sFAS-L, cytochrome C, annexin V, caspase-8, caspase-9 and TNF-α in the serum of GSD with the prevailed liver damage (I, III, VI and IX types). There was established the excess of the concentration of cytochrome C in the serum of GSD children by 2,7 times sAPO-1/FAS receptor - 8,9 times, sFAS-L - 2,5 times, annexin V - 4,8 times and TNF-α - 2,9 times in comparison with reference values. Whereby the excess in the cytochrome C content, sAPO-1/FAS receptor, sFAS-L annexin V and the serum was observed in all patients, that indicating to the higher activity of apoptosis in GSD. In this connection GSD in children can be considered as a form of pathology associated with the pronounced apoptosis that contributes to the progression of structural changes in the liver.