Glycogen Storage Capacity Research Articles

Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions.

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace primary human hepatocytes as a new source of functional hepatocytes in various medical applications. However, the hepatic functions of HLCs are still low and it takes a long time to differentiate them from human iPS cells. Furthermore, HLCs have very low proliferative capacity and are difficult to be passaged due to loss of hepatic functions after reseeding. To overcome these problems, we attempted to develop a technology to dissociate, cryopreserve, and reseed HLCs in this study. By adding epithelial-mesenchymal transition inhibitors and optimizing the cell dissociation time, we have developed a method for passaging HLCs without loss of their functions. After passage, HLCs showed a hepatocyte-like polygonal cell morphology and expressed major hepatocyte marker proteins such as albumin and cytochrome P450 3A4 (CYP3A4). In addition, the HLCs had low-density lipoprotein uptake and glycogen storage capacity. The HLCs also showed higher CYP3A4 activity and increased gene expression levels of major hepatocyte markers after passage compared to before passage. Finally, they maintained their functions even after their cryopreservation and re-culture. By applying this technology, it will be possible to provide ready-to-use availability of cryopreserved HLCs for drug discovery research.

Herpetin Promotes Bone Marrow Mesenchymal Stem Cells to Alleviate Carbon Tetrachloride-Induced Acute Liver Injury in Mice.

Herpetin, an active compound derived from the seeds of Herpetospermum caudigerum Wall., is a traditional Tibetan herbal medicine that is used for the treatment of hepatobiliary diseases. The aim of this study was to evaluate the stimulant effect of herpetin on bone marrow mesenchymal stem cells (BMSCs) to improve acute liver injury (ALI). In vitro results showed that herpetin treatment enhanced expression of the liver-specific proteins alpha-fetoprotein, albumin, and cytokeratin 18; increased cytochrome P450 family 3 subfamily a member 4 activity; and increased the glycogen-storage capacity of BMSCs. Mice with ALI induced by carbon tetrachloride (CCl4) were treated with a combination of BMSCs by tail-vein injection and herpetin by intraperitoneal injection. Hematoxylin and eosin staining and serum biochemical index detection showed that the liver function of ALI mice improved after administration of herpetin combined with BMSCs. Western blotting results suggested that the stromal cell-derived factor-1/C-X-C motif chemokine receptor 4 axis and the Wnt/β-catenin pathway in the liver tissue were activated after treatment with herpetin and BMSCs. Therefore, herpetin is a promising BMSC induction agent, and coadministration of herpetin and BMSCs may affect the treatment of ALI.

In silico investigation of molecular networks linking gastrointestinal diseases, malnutrition, and sarcopenia

Malnutrition (MN) is a common primary or secondary complication in gastrointestinal diseases. The patient's nutritional status also influences muscle mass and function, which can be impaired up to the degree of sarcopenia. The molecular interactions in diseases leading to sarcopenia are complex and multifaceted, affecting muscle physiology, the intestine (nutrition), and the liver at different levels. Although extensive knowledge of individual molecular factors is available, their regulatory interplay is not yet fully understood. A comprehensive overall picture of pathological mechanisms and resulting phenotypes is lacking. In silico approaches that convert existing knowledge into computationally readable formats can help unravel mechanisms, underlying such complex molecular processes. From public literature, we manually compiled experimental evidence for molecular interactions involved in the development of sarcopenia into a knowledge base, referred to as the Sarcopenia Map. We integrated two diseases, namely liver cirrhosis (LC), and intestinal dysfunction, by considering their effects on nutrition and blood secretome. We demonstrate the performance of our model by successfully simulating the impact of changing dietary frequency, glycogen storage capacity, and disease severity on the carbohydrate and muscle systems. We present the Sarcopenia Map as a publicly available, open-source, and interactive online resource, that links gastrointestinal diseases, MN, and sarcopenia. The map provides tools that allow users to explore the information on the map and perform in silico simulations.

Conjugated Linoleic Acid Treatment Attenuates Cancerous features in Hepatocellular Carcinoma Cells.

Background A growing number of hepatocellular carcinoma (HCC), and recurrence frequency recently have drawn researchers' attention to alternative approaches. The concept of differentiation therapies (DT) relies on inducing differentiation in HCC cells in order to inhibit recurrence and metastasis. Hepatocyte nuclear factor 4 alpha (HNF4α) is the key hepatogenesis transcription factor and its upregulation may decrease the invasiveness of cancerous cells by suppressing epithelial-mesenchymal transition (EMT). This study aimed to evaluate the effect of conjugated linoleic acid (CLA) treatment, natural ligand of HNF4α, on the proliferation, migration, and invasion capacities of HCC cells in vitro. Materials and Method. Sk-Hep-1 and Hep-3B cells were treated with different doses of CLA or BIM5078 [1-(2′-chloro-5′-nitrobenzenesulfonyl)−2-methylbenzimidazole], an HNF4α antagonist. The expression levels of HNF4a and EMT related genes were evaluated and associated to hepatocytic functionalities, migration, and colony formation capacities, as well as to viability and proliferation rate of HCC cells. Results In both HCC lines, CLA treatment induced HNF4α expression in parallel to significantly decreased EMT marker levels, migration, colony formation capacity, and proliferation rate, whereas BIM5078 treatment resulted in the opposite effects. Moreover, CLA supplementation also upregulated ALB, ZO1, and HNF4α proteins as well as glycogen storage capacity in the treated HCC cells. Conclusion CLA treatment can induce a remarkable hepatocytic differentiation in HCC cells and attenuates cancerous features. This could be as a result of HNF4a induction and EMT inhibition.

Mitochondrial depletion of glutaredoxin 2 induces metabolic dysfunction-associated fatty liver disease in mice

Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that facilitates glutathionylation/de-glutathionylation of target proteins. The main variants of Grx2 are the mitochondrial Grx2a and the cytosolic Grx2c. The aim of this study was to investigate the specific role of mitochondrial Grx2 in vivo using a mitochondrial Grx2 depleted (mGD) mouse model. mGD mice displayed an altered mitochondrial morphology and functioning. Furthermore, the lack of Grx2 in the mitochondrial compartment is responsible for increased blood lipid levels under a normal diet, a metabolic dysfunction-associated fatty liver disease (MAFLD) phenotype and a decreased glycogen storage capacity. In addition, depleting Grx2a leads to an alteration in abundance and in glutathionylation pattern of different mitochondrial enzymes, highlighting the selective role of Grx2 in the regulation of metabolic pathways. Overall, our findings identify the involvement of mitochondrial Grx2a in the regulation of cell metabolism and highlight a previously unknown association between Grx2 and MAFLD.

Fermented Deer Blood Ameliorates Intense Exercise-Induced Fatigue via Modulating Small Intestine Microbiota and Metabolites in Mice.

Intense and excessive exercise-induced fatigue has become an important health issue and can damage intestinal health. Deer blood, as a food byproduct with nutritional value, has been found to restore physical strength. However, little is known about the antifatigue effect of fermented deer blood (FDB) on intense exercise mice. The purpose of the present study is to investigate the antifatigue effect of FDB, and whether this effect is correlated with the altered small intestinal microbiota and metabolites in exercise mice. In this study, 5-week-old male C57BL/6J mice are given treadmill exercise with or without FDB supplementation (30 and 150 mg/kg/d) for 3 weeks. FDB significantly reduces metabolic byproduct accumulation, liver and intestinal damage, and enhances glycogen storage and antioxidant capacity in intense exercise mice. Moreover, FDB restructures the small intestinal microbiota by increasing the abundance of probiotics and butyric acid producing bacteria and decreasing the abundance of pathogenic bacteria. FDB also regulates the levels of metabolites involved in TCA cycle and amino acid metabolism in urine and small intestine content. Correlation analysis shows that FDB-modulated microbiota is highly associated with its antifatigue effect. FDB may ameliorate fatigue and intestinal injury through targeting small intestinal microbiota.

Online monitoring of hiPSC expansion and hepatic differentiation in 3D culture by dielectric spectroscopy.

Hepatocyte-like cells derived from human-induced pluripotent stem cells (hiPSC-HLC) are expected to have important applications in drug screening and regenerative medicine. However, hiPSC-HLC are difficult to produce on a large-scale to obtain relevant numbers for such applications. The aim of this study was to implement a novel integrated strategy for scalable production of hiPSC-HLC and demonstrate the applicability of dielectric spectroscopy to monitor hiPSC expansion/differentiation processes. We cultured hiPSC as three-dimensional (3D) aggregates in stirred-tank bioreactors (STB) operated in perfusion with an in situ capacitance probe. Dissolved oxygen concentration and dilution rate were controlled along the process and after 5 days of cell expansion, the hepatic differentiation was integrated in sequential steps for 28 days. The hiPSC were able to grow as 3D aggregates and the expression of hepatic markers and albumin production after differentiation confirmed that hepatocyte differentiation improved when compared to 2D culture. These hiPSC-HLC exhibited functional characteristics of hepatocytes including glycogen storage and drug metabolization capacity. Our results also show a good correlation between the cell permittivity measured online and the aggregate biovolume measured by standard offline methods, demonstrating for the first time the potential of dielectric spectroscopy to monitor hiPSC expansion and differentiation in STB.

GlgA plays an important role in the induction of hydrosalpinx byChlamydia muridarum

While glycogen synthase A deficiency can reduce the growth and proliferation of Chlamydia muridarum, the effect of glycogen synthase A on the pathogenic process of C. muridarum remains unclear. To characterize the effect of glycogen synthase A deficiency on the pathogenicity of C. muridarum in the genital tract, BALB/c mice were intravaginally inoculated with wild-type, plasmid-free and glycogen synthase A-deficient C. muridarum, and the genital tract tissue was isolated to assess the severity of hydrosalpinx and the levels of oviduct dilatation at day 60 after infection. The glycogen storage capacity and in vitro infection ability of different C. muridarum strains were analyzed by periodic acid-Schiff staining and quantification of progeny elementary body(EB) formation. The tissue homogenate was used to determine the recovery of different C. muridarum strains. The results show that glycogen synthase A-deficient C. muridarum induced reduction of hydrosalpinx and attenuated the extent of oviduct dilatation in mice, and exhibited reduced growth and proliferation in the mouse lower genital tract. In addition, glycogen synthase A point mutations at different sites reduced the glycogen storage capacity and in vitro infectivity of C. muridarum to different degrees. Glycogen synthase A deficiency also reduced the host inflammatory reaction and ascending infection of C. muridarum.

Casein fortified diet reversed the diabetes like changes induced by 4G connected mobile phone radiation in mice

Aim: Mobile phone usage is increasing day by day and has become an indispensable part of our life. However, people are constantly being exposed to the harmful electromagnetic radiation emitted from mobile phones and the remedy for the same is yet indiscernible. In our current report, we have investigated the role of a high casein diet (HCD) in ameliorating the pancreatic and hepatotoxic effects of mobile phone radiation. Materials and Methods: Mice (n=24) were divided into four groups and maintained as control groups (Gr-A: normal diet; Gr-D: HCD) and mobile phone radiation (SAR value: 1.5 W/ kg at 1g body wt.) exposed groups, irradiated for 3 hours/ day for 90 days (Gr-B: normal diet; Gr-C: HCD). The normal diet contained 5% casein while HCD contained 20% casein. After the tenure of radiation exposure terminates, animals were sacrificed; the pancreatic, hepatic tissues, and blood were collected for the histology and serum isolation respectively. Periodic acid- Schiff staining was performed to assess the glycogen content of hepatic tissue and hematoxylin-eosin staining for general histological analysis. The serum was used to estimate the fasting glucose level. Observations: Increased fasting serum glucose level and shrunken pancreatic islets on radiation exposure may indicate the diabetes progression. Loss of glycogen storage capacity by the liver was observed in radiation exposed group which are suggestive of compromised hepatic functions. Histo-architectural analysis of the liver revealed severely distorted hepatic cells with prominent occurrences of karyorrhexis and karyolysis, irregular and enlarged sinusoidal spaces upon radiation exposure. HCD countermanded these abnormal manifestations of the electromagnetic radiation to a considerable extent. Inference: Thus, electromagnetic radiation from the mobile phone may be responsible for the impairment of glucose metabolism and thereby enhancing the progression of diabetes in mice. In contrast, HCD was prudent in combating these degenerative alterations of mobile phone radiation.

Differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules

A variety of approaches have been developed for the derivation of hepatocyte-like cells from pluripotent stem cells. Currently, most of these strategies employ step-wise differentiation approaches with recombinant growth-factors or small-molecule analogs to recapitulate developmental signaling pathways. Here, we tested the efficacy of a small-molecule based differentiation protocol for the generation of hepatocyte-like cells from human pluripotent stem cells. Quantitative gene-expression, immunohistochemical, and western blot analyses for SOX17, FOXA2, CXCR4, HNF4A, AFP, indicated the stage-specific differentiation into definitive endoderm, hepatoblast and hepatocyte-like derivatives. Furthermore, hepatocyte-like cells displayed morphological and functional features characteristic of primary hepatocytes, as indicated by the production of ALB (albumin) and α-1-antitrypsin (A1AT), as well as glycogen storage capacity by periodic acid-Schiff staining. Together, these data support that the small-molecule based hepatic differentiation protocol is a simple, reproducible, and inexpensive method to efficiently drive the differentiation of human pluripotent stem cells towards a hepatocyte-like phenotype, for downstream pharmacogenomic and regenerative medicine applications.

Gain of function AMP-activated protein kinaseγ3 mutation (AMPKγ3R200Q) in pig muscle increases glycogen storage regardless of AMPK activation

Chronic activation of AMP‐activated protein kinase (AMPK) increases glycogen content in skeletal muscle. Previously, we demonstrated that a mutation in the ryanodine receptor (RyR1R615C) blunts AMPK phosphorylation in longissimus muscle of pigs with a gain of function mutation in the AMPK γ3 subunit (AMPK γ3R200Q); this may decrease the glycogen storage capacity of AMPK γ3R200Q + RyR1R615C muscle. Therefore, our aim in this study was to utilize our pig model to understand how AMPK γ3R200Q and AMPK activation contribute to glycogen storage and metabolism in muscle. We selected and bred pigs in order to generate offspring with naturally occurring AMPK γ3R200Q, RyR1R615C, and AMPK γ3R200Q + RyR1R615C mutations, and also retained wild‐type littermates (control). We assessed glycogen content and parameters of glycogen metabolism in longissimus muscle. Regardless of RyR1R615C, AMPK γ3R200Q increased the glycogen content by approximately 70%. Activity of glycogen synthase (GS) without the allosteric activator glucose 6‐phosphate (G6P) was decreased in AMPK γ3R200Q relative to all other genotypes, whereas both AMPK γ3R200Q and AMPK γ3R200Q + RyR1R615C muscle exhibited increased GS activity with G6P. Increased activity of GS with G6P was not associated with increased abundance of GS or hexokinase 2. However, AMPK γ3R200Q enhanced UDP‐glucose pyrophosphorylase 2 (UGP2) expression approximately threefold. Although UGP2 is not generally considered a rate‐limiting enzyme for glycogen synthesis, our model suggests that UGP2 plays an important role in increasing flux to glycogen synthase. Moreover, we have shown that the capacity for glycogen storage is more closely related to the AMPK γ3R200Q mutation than activity.

3D evaluation model for drug hepatotoxicity testing on HepG2 cells and its application in drug safety evaluation

3D in vitro toxicity testing model was developed by magnetic levitation method for culture of the human hepatoma cell line HepG2 and applied to evaluate the drug hepatotoxicity. After formation of stable 3D structure for HepG2 cells, their glycogen storage capacity under 2D and 3D culture conditions were detected by immunohistochemistry technology, and the mRNA expression levels of phase Ⅰ and Ⅱ drug metabolism enzymes, drug transporters, nuclear receptors and liver-specific marker albumin(ALB) were compared between 2D and 3D culture conditions by using RT-PCR method. Immunohistochemistry results showed that HepG2 cells had abundant glycogen storage capacity under 3D culture conditions, which was similar to human liver tissues. The mRNA expression levels of major drug metabolism enzymes, drug transporters, nuclear receptors and ALB in HepG2 cells under 3D culture conditions were up-regulated as compared with 2D culture conditions. For drug hepatotoxicity evaluation, the typical hepatotoxic drug acetaminophen(APAP), and most reported drugs Polygonum multiflorum Thunb.(Chinese name He-shou-wu) and Psoraleae corylifolia L.(Chinese name Bu-gu-zhi) were selected for single dose and repeated dose(7 d) exposure. In the repeated dose exposure test, 3D HepG2 cells showed higher sensitivity. This established 3D HepG2 cells model with magnetic levitation 3D culture techniques was more close to the human liver tissues both in morphology and functions, so it was a better 3D hepatotoxicity evaluation model.

Metabolic Disturbances in Children with Chronic Liver Disease

Introduction: Liver disease results in complex pathophysiologic disturbances affecting nutrient digestion, absorption, distribution, storage, and use. This article aimed to present a classification of metabolic disturbances in chronic liver disease in children? Materials and Methods: In this review study databases including proquest, pubmedcentral, scincedirect, ovid, medlineplus were been searched with keyword words such as” chronic liver disease" ” metabolic disorder””children” between 1999 to 2014. Finally, 8 related articles have been found. Results: Metabolic disorder in this population could be categorized in four set: 1)carbohydrates, 2)proteins,3) fats and 4)vitamins. 1) Carbohydrates: Children with CLD are at increased risk for fasting hypoglycemia, because the capacity for glycogen storage and gluconeogenesis is reduced as a result of abnormal hepatocyte function and loss of hepatocyte mass. 2) Proteins: The liver’s capacity for plasma protein synthesis is impaired by reduced substrate availability, impaired hepatocyte function, and increased catabolism. This results in hypoalbuminemia, leading to peripheral edema and contributing to ascites. Reduced synthesis of insulin-like growth factor (IGF)-1 and its binding protein IGF-BP3 by the chronically diseased liver results in growth hormone resistance and may contribute to the poor growth observed in these children. 3) Fats: There is increased fat oxidation in children with end-stage liver disease in the fed and fasting states compared with controls, which is probably related to reduced carbohydrate availability. The increased lipolysis results in a decrease in fat stores, which may not be easily replenished in the setting of the fat malabsorption that accompanies cholestasis. Reduced bile delivery to the gut results in impaired fat emulsification, and hence digestion. The products of fat digestion are also poorly absorbed, because bile is also required for micelle formation. In various liver diseases, there may be reduced bile production by inadequately functioning hepatocytes, reduced hepatocyte excretion into the bile canaliculus (as in PFIC), or obstruction to biliary flow. The circulating bile salt pool may be depleted secondary to treatment with binding agents, such as cholestyramine, which is often prescribed for pruritus in cholestatic patients. Pancreatic insufficiency may further exacerbate fat malabsorption in certain cholestatic liver diseases. Vitamins: Fat malabsorption occurs in cholestatic disorders, and one must also consider any accompanying fat-soluble vitamin and essential fatty acid deficiencies.Breastfed infants with CLD are at high risk for vitamin D and K deficiencies. Conclusion: The clinician should proactively evaluate, treat, and re-evaluate response to treatment of nutritional deficiencies. Because a better nutritional state is associated with better survival before and after LT, aggressive nutritional management is an important part of the care of these children.

Isolation of multilineage progenitors from mouse brain

Stem cells are unique cell populations with the ability to undergo self-renewal and differentiation. These cells have been identified in a wide range of tissues and possess varied differentiation potentials. Tissue-specific stem cells have typically been thought to have limited differentiation capabilities. We show here that fibroblast-like cells isolated from mouse brain possess cross-germ layer differentiation abilities. These cells were found to express typical mesenchymal stem cell markers (CD44, CD29, and CD105) and were able to be passaged more than 50 times. When treated under defined conditions, the brain-derived cells were able to generate many different cell types including adipocytes, osteocytes, astrocytes, neurons, and even hepatocyte-like cells. The hepatocyte-like cells not only expressed liver cell-specific markers, but also exhibited the capacity for glycogen storage and low-density lipoprotein uptake. These results demonstrate the existence of cells in the brain with three-germ-layer differentiation potential.

Human decidua-derived mesenchymal stromal cells differentiate into hepatic-like cells and form functional three-dimensional structures

Previously, we have shown that human decidua-derived mesenchymal stromal cells (DMSC) are mesenchymal stromal cells (MSC) with a clonal differentiation capacity for the three embryonic layers. The endodermal capacity of DMSC was revealed by differentiation into pulmonary cells. In this study, we examined the hepatic differentiation of DMSC. DMSC were cultured in hepatic differentiation media or co-cultured with murine liver homogenate and analyzed with phenotypic, molecular and functional tests. DMSC in hepatic differentiation media changed their fibroblast morphology to a hepatocyte-like morphology and later formed a 3-dimensional (3-D) structure or hepatosphere. Moreover, the hepatocyte-like cells and the hepatospheres expressed liver-specific markers such as synthesis of albumin (ALB), hepatocyte growth factor receptor (HGFR), α-fetoprotein (AFP) and cytokeratin-18 (CK-18), and exhibited hepatic functions including glycogen storage capacity and indocyanine green (ICG) uptake/secretion. Human DMSC co-cultured with murine liver tissue homogenate in a non-contact in vitro system showed hepatic differentiation, as evidenced by expression of AFP and ALB genes. The switch in the expression of these two genes resembled liver development. Indeed, the decrease in AFP and increase in ALB expression throughout the co-culture were consistent with the expression pattern observed during normal liver organogenesis in the embryo. Interestingly, AFP and ALB expression was significantly higher when DMSC were co-cultured with injured liver tissue, indicating that DMSC respond differently under normal and pathologic micro-environmental conditions. In conclusion, DMSC-derived hepatospheres and DMSC co-cultured with liver homogenate could be suitable in vitro models for toxicologic, developmental and pre-clinical hepatic regeneration studies.

Yolk sac carbohydrate levels and gene expression of key gluconeogenic and glycogenic enzymes during chick embryonic development

Glycogen and glucose concentrations (mg/g of tissue) and amounts (mg) were determined in the yolks of fertile eggs on the day of set and in the yolk sac (YS) and liver of broiler chick embryos between 11 and 21 embryonic days of age (E). On the day of set, the yolk contained 50 mg of glucose (0.31% of yolk) but did not contain glycogen. During incubation, the amount of glucose in the YS increased from 20 mg on E11 to 60 mg on E19. A parallel increase in YS and liver glycogen concentrations (mg/g) during the last week of incubation implied a similar capacity for glycogen synthesis per gram of tissue. However, due to its larger size, the YS capacity for glycogen storage far exceeded that of the liver, which stored less than 12 mg of glycogen up to E19, as compared with more than 200 mg in the YS. Between E19 and 21, liver and YS glycogen amounts decreased by 10 mg and 100 mg, respectively. These results indicated that the YS is a glycogenic and perhaps gluconeogenic organ. We therefore evaluated the gene expression of glycogen synthase and glycogen phosphorylase as well as gluconeogenic enzymes (fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase) in the YS membrane and liver by real-time reverse-transcription PCR. Although the YS membrane and liver displayed different patterns of mRNA abundance, the high abundance of fructose 1,6-bisphosphatase mRNA in the YS membrane between E11 and 15, and the expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, supported the postulated gluconeogenic abilities of the YS membrane and indicated its role in providing glucose to the embryo. Thus, glucose is probably synthesized in the YS, stored in the form of glycogen, and toward hatch, the YS may have the potential to transfer 10 times more glycogen-derived glucose to the embryo as compared with the liver. As such, the YS may play a major role in the synthesis and storage of glucose and its supply to the chick embryo toward hatch.

혼합곡 식이의 지구력 향상 효과

본 연구에서는 4주간 혼합곡 식이를 공급하여 treadmill을 통한 운동시간과 glycogen 저장능력 변화를 관찰함으로써 지구력 개선에 대한 평가를 하였다. 7주령 SD rats은 정상군 (Nor, 정상식이, n=6), 대조군(Con, 20% 정상식이＋80% milled rice, n=6), GⅠ(혼합곡 식이 Ⅰ, n=6), GⅡ(혼합곡 식이 Ⅱ, n=6), GⅢ(혼합곡 Ⅲ, n=6)와 GⅣ(혼합곡 식이 Ⅳ, n=6) 으로 임의 군배정을 하였다. 지구력 시험은 24일간의 적응 훈련과 혼합곡식이 공급을 한 후 실시하였다. 운동 지속 시간과 glycogen 저장능력은 혼합곡식이 공급에 의해서 증가하였다. 더욱이 25 min 운동 그리고/또는 탈진 시까지의 운동 후에서 혈액 내 무기질인, CPK와 lactate의 농도는 혼합곡 투여에 의하여 감소하였으나, GOP, GTP, lactate LDH의 농도는 군간 차이는 보이지 않았다. 본 연구에서는 4주간의 혼합곡 식이가 운동 지속 시간과 glycogen 장 능력이 증가되었으며, 피로물질인 무기질인, CPK와 lactate의 농도가 감소시킴으로써 지구력 개선에 도움을 줄 것으로 생각된다.

Bovine sire selection based on maintenance energy affects muscle fiber type and meat color of F1 progeny1

A total of 42 F(1) Red Angus progeny from sires divergent in maintenance energy (ME(M)) EPD were analyzed to determine whether selecting for sire ME(M) would alter end-product meat quality. Data from animals were grouped based on the divergence of the ME(M) EPD of their sire from the Red Angus Association-reported breed average and defined as either high or low, the assumption being that high-ME(M) cattle are less efficient because their maintenance requirements represent a larger proportion of their dietary intake. Steer progeny (n = 7) from the high group produced bottom round steaks with a greater a* (redness) color value (P = 0.02) after 5 d in a simulated retail display when compared with bottom round steaks from the low group (n = 18). Bottom round steaks from the high group had a greater b* (yellowness) color value at d 1 (P = 0.03) and d 5 (P = 0.01) of retail display. Samples from the biceps femoris were taken at 12 mo (from both steers and heifers) and 15 mo (from steers only) of age for fiber type proportion analysis. At 12 mo of age, steers from the low group had more type I fibers (P = 0.02), whereas steers from the high group had more type IIb fibers (P = 0.01). Furthermore, samples from steers in the low group at 15 mo had more type I fibers (P = 0.02), and steers from the high group maintained more type IIb fibers (P = 0.02). No changes in fiber type proportions were observed between the high- and low-ME(M) EPD heifers (n = 17). Relative mRNA abundance of genes involved in the synthesis, storage, and breakdown of glycogen were analyzed as a variable important for meat quality, but no statistical differences were observed. At 12 mo age, glycogenin (glyc) was negatively correlated with the proportion of type IIa fibers (r = -0.32 and P = 0.12) as well as with the proportion of type IIb fibers (r = -0.42 and P = 0.03) in the biceps femoris of the steers. In samples taken from the biceps femoris at 15 mo age, glyc was negatively correlated with the proportion of type IIa fibers (r = -0.42 and P = 0.03) in the steers. This indicates that relative mRNA expression of glyc may serve as a marker of muscle glycogen storage capacity in steers. Thus, selection for efficient Red Angus beef cattle based on sire ME(M) EPD does not adversely affect meat quality in F(1) progeny, based on the variables assessed in this study. Furthermore, selection for progeny from low-ME(M) EPD sires may improve fresh meat quality within Red Angus beef cattle.

Differentiation of human embryonic stem cells along a hepatic lineage

The limited availability of hepatic tissue suitable for the treatment of liver disease and drug discovery research advances the generation of hepatic-like cells from alternative sources as a valuable approach. In this investigation we exploited a unique hepatic differentiation approach to generate hepatocyte-like cells from human embryonic stem cells (hESCs). hESCs were cultured for 10–20 days on collagen substrate in highly defined and serum free hepatocyte media. The resulting cell populations exhibited hepatic cell-like morphology and were characterized with a variety of biological endpoint analyses. Real-time PCR analysis demonstrated that mRNA expression of the ‘stemness’ marker genes NANOG and alkaline phosphatase in the differentiated cells was significantly reduced, findings that were functionally validated using alkaline phosphatase activity detection measures. Immunofluorescence studies revealed attenuated levels of the ‘stemness’ markers OCT4, SOX2, SSEA-3, TRA-1-60, and TRA-1-81 in the hepatic-like cell population. The hepatic character of the cells was evaluated additionally by real-time PCR analyses that demonstrated increased mRNA expression of the hepatic transcription factors FOXA1, C/EBPα, and HNF1α, the nuclear receptors CAR, RXRα, PPARα, and HNF4α, the liver-generated plasma proteins α-fetoprotein, transthyretin, transferrin, and albumin, the protease inhibitor α-1-antitrypsin, metabolic enzymes HMGCS2, PEPCK, and biotransformation enzymes CYP3A7, CYP3A4, CYP3A5, and CYP2E1. Indocyanine green uptake albumin secretion and glycogen storage capacity further confirmed acquisition of hepatic function. These studies define an expeditious methodology that facilitates the differentiation of hESCs along a hepatic lineage and provide a framework for their subsequent use in pharmacological and toxicological research applications requiring a renewable supply of human hepatocytes.

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two-dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell-specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three-dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen-coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver-cell-specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver-specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real-time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well-preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF-4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re-established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO-1-positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes.