Metabolism Trafficking Research Articles

Cell identity dynamics and insight into insulin secretagogues when employing stem cell-derived islets for disease modeling.

Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.

Iron-mediated oxidative stress induces PD-L1 expression via activation of c-Myc in lung adenocarcinoma.

Introduction: The PD-1/PD-L1 axis is hijacked by lung adenocarcinoma (LUAD) cells to escape immune surveillance. PD-L1 expression in LUAD is affected, among others, by the metabolic trafficking between tumor cells and the tumor microenvironment (TME). Methods: Correlation between PD-L1 expression and iron content within the TME was established on FFPE LUAD tissue samples. The effects of an iron rich microenvironment on PD-L1 mRNA and protein levels were assessed in vitro in H460 and A549 LUAD by using qPCR, western blot and flow citometry. c-Myc knockdown was performed to validate the role of this transcription factor on PD-L1 expression. The effects of iron-induced PD-L1 on T cell immune function was assessed by quantifying IFN-γ release in a co-colture system. TCGA dataset was used to analyse the correlation between PD-L1 and CD71 mRNA expression in LUAD patients. Results: In this study, we highlight a significant correlation between iron density within the TME and PD-L1 expression in 16 LUAD tissue specimens. In agreement, we show that a more pronounced innate iron-addicted phenotype, indicated by a higher transferrin receptor CD71 levels, significantly correlates with higher PD-L1 mRNA expression levels in LUAD dataset obtained from TCGA database. In vitro, we demonstrate that the addition of Fe3+ within the culture media promotes the significant overexpression of PD-L1 in A549 and H460 LUAD cells, through the modulation of its gene transcription mediated by c-Myc. The effects of iron lean on its redox activity since PD-L1 up-regulation is counteracted by treatment with the antioxidant compound trolox. When LUAD cells are co-cultured with CD3/CD28-stimulated T cells in an iron-rich culture condition, PD-L1 up-regulation causes the inhibition of T-lymphocytes activity, as demonstrated by the significant reduction of IFN-γ release. Discussion: Overall, in this study we demonstrate that iron abundance within the TME may enhance PD-L1 expression in LUAD and, thus, open the way for the identification of possible combinatorial strategies that take into account the iron levels within the TME to improve the outcomes of LUAD patients treated with anti-PD-1/PD-L1-based therapies.

Intracellular Reactive Oxygen Species Mediate the Therapeutic Effect of Induced Pluripotent Stem Cells for Acute Kidney Injury.

Aims Treatment for acute kidney injury (AKI) is challenging. Induced pluripotent stem cells (iPSCs) have great therapeutic potential. This study sought to determine whether iPSCs attenuate AKI and the role of reactive oxygen species (ROS). Results We intravenously injected isogenic iPSCs into mice 2 h after renal ischemia-reperfusion injury (IRI). The cells were selectively trafficked to ischemia/reperfusion-injured kidney where they decreased kidney ROS and inflammatory cytokines and improved kidney function and morphology. Pretreating the cells with ROS inhibitors before administration decreased iPSC engraftment and abolished the protective effect of iPSCs. In contrast, pretreating iPSCs with hydrogen peroxide increased iPSC engraftment and therapeutic effect. Although the intravenously administered iPSCs trafficked to the IRI kidney, the cells did not differentiate into proximal or distal tubular epithelial cells. In vitro, the capabilities of the iPSC-released substances to promote proliferation and decrease apoptosis of renal epithelial cells were increased by ROS pretreatment of iPSCs. Moreover, pretreatment of the iPSCs with ROS inhibitor had the opposite effect. Similarly, moderate concentrations of ROS increased while ROS inhibitors decreased iPSC mobility, adhesion to the extracellular matrix, and mitochondrial metabolism. Innovation and Conclusion. iPSCs decreased renal ischemia/reperfusion injury mainly through iPSC-released substances. The therapeutic effect, mitochondrial metabolism, mobility, and kidney trafficking of iPSCs were ROS dependent.

Reprogramming Yeast Metabolism from Alcoholic Fermentation to Lipogenesis

Engineering microorganisms for production of fuels and chemicals often requires major re-programming of metabolism to ensure high flux toward the product of interest. This is challenging, as millions of years of evolution have resulted in establishment of tight regulation of metabolism for optimal growth in the organism's natural habitat. Here, we show through metabolic engineering that it is possible to alter the metabolism of Saccharomyces cerevisiae from traditional ethanol fermentation to a pure lipogenesis metabolism, resulting in high-level production of free fatty acids. Through metabolic engineering and process design, we altered subcellular metabolic trafficking, fine-tuned NADPH and ATP supply, and decreased carbon flux to biomass, enabling production of 33.4 g/L extracellular free fatty acids. We further demonstrate that lipogenesis metabolism can replace ethanol fermentation by deletion of pyruvate decarboxylase enzymes followed by adaptive laboratory evolution. Genome sequencing of evolved strains showed that pyruvate kinase mutations were essential for this phenotype.

Abstract 434: A Diet Rich in Oleic Acid, as Primary Lipid Source, Attenuates the Progressive Functional Decline in Hypertrophied Hearts During Pathological Stress

Two major dietary long chain fatty acids (LCFA), oleate and palmitate, display differing capacities to induce transcriptional activation of metabolic genes, lipid trafficking, substrate metabolism, and contractile function in the failing heart. Perfusing isolated failing rat hearts with the unsaturated LCFA, oleate, improves contractile function and restores intracellular lipid dynamics vs. failing rat hearts perfused with the saturated LCFA, palmitate. We therefore investigated whether chronically feeding rats a diet of either high-oleate (as triolein) or high-palmitate (as tripalmitin) could alter the development of heart failure in response to chronic pressure overload induced by transverse aortic constriction (TAC). Following TAC or sham surgery rats were randomized to a diet rich in either triolein or tripalmitin as primary fat source (65% carbohydrate, 20% protein and 15% fat (210 Kcal/kg)). TAC induced similar increases in LV mass independent of diet, however rats fed tripalmitin showed a significant reduction in survival and a decline in ejection fraction and fractional shortening as well as LV dilation, evident at 6 and 10 weeks post TAC (Figure). In contrast, triolein diet attenuated declining systolic function and LV dilation (Figure) and eliminated the otherwise expected elevation of plasma BNP, as seen with tripalmitin diet (50%, P<0.05) after 14 weeks of TAC, indicating differential effects of these fats on both the heart and whole body metabolism and physiology during cardiac stress. The results suggest that a diet rich in oleic acid affords benefits in attenuating the development of cardiac decompensation independent of changes in hypertrophic growth.

Abstract 1489: Differential gene expression profiles between normal cervical tissue and high-grade squamous intraepithelial lesion expressing p16 and high-risk HPV E6/E7 mRNA

Abstract Background: HPV is necessary but not sufficient for development of uterine cervix carcinoma. There are genetic factors that influence the progression of premalignant lesions. Gene expression profile differences between healthy and premalignant lesion could contribute to identify potential biomarkers for each step of cervical tumor progression. Design: A female patient with an initial diagnosis of low grade squamous intraepithelial lesion (SIL) was invited to participate in the study. During follow-up a high grade SIL was observed by colposcopy, and two samples were obtained from the lesion: one sample was processed for histopathologic diagnosis, and additionally stained by IHC with p16 antibody; the other sample, along with a matched sample of healthy tissue taken distantly from the lesion were stored in a nucleic acids preservation reagent for subsequent analysis of gene expression. Differences in gene expression between healthy tissue and high grade SIL tissue were assessed in triplicate using the cDNA gene expression Illumina microarray Human-HT-12-V4. A statistical p value was computed comparing expression levels from the tissue lesion and healthy tissue. Genes with a fold-change ≥ 2 and a p value <0.05 were regarded as differentially expressed. Additionally, two cervix swabs were obtained for detection of HPV DNA by PCR, and for assessment of high risk HPV E6/E7 mRNA expression by RT-PCR. Results: Histopathological evaluation of the lesion confirmed a high grade squamous intraepithelial lesion, with positive p16 staining. HPV-18 DNA was detected and E6/E7 HPV 18 mRNA expression was confirmed. Gene expression analysis detected 33 overexpressed genes, including TMEM45A, SPRR2B, KRT16 and RBP1, and 30 underexpressed genes, including SPARCL1, GCNT3, TMPRSS11b and ESR1, in high grade SIL compared to healthy uterine cervical tissue. Overexpressed genes belonged to important cellular pathways, such as queratinization, defensins, cytoskeletal signaling, cellular adhesion and extracellular matrix remodeling. Underexpressed genes belonged to metabolic pathways, cell trafficking, and chemokine signaling, among others. Conclusions: Microarray analysis identified differential expression between healthy cervical tissue and high grade SIL with confirmed HPV-18 transforming infection. Differentially expressed genes belonged to important cellular pathways. Further studies are required to evaluate the usefulness of these genes as risk markers for progression. Citation Format: Ines Benedetti, Lia Barrios, Niradiz Reyes. Differential gene expression profiles between normal cervical tissue and high-grade squamous intraepithelial lesion expressing p16 and high-risk HPV E6/E7 mRNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1489.

Coral lipid bodies as the relay center interconnecting diel-dependent lipidomic changes in different cellular compartments

Lipid bodies (LBs) in the coral gastrodermal tissues are key organelles in the regulation of endosymbiosis and exhibit a diel rhythmicity. Using the scleractinian Euphyllia glabrescens collected across the diel cycle, we observed temporally dynamic lipid profiles in three cellular compartments: host coral gastrodermal cells, LBs, and in hospite Symbiodinium. Particularly, the lipidome varied over time, demonstrating the temporally variable nature of the coral–Symbiodinium endosymbiosis. The lipidome-scale data highlight the dynamic, light-driven metabolism of such associations and reveal that LBs are not only lipid storage organelles but also act as a relay center in metabolic trafficking. Furthermore, lipogenesis in LBs is significantly regulated by coral hosts and the lipid metabolites within holobionts featured predominantly triacylglycerols, sterol esters, and free fatty acids. Given these findings through a time-varied lipidome status, the present study provided valuable insights likely to be crucial to understand the cellular biology of the coral–Symbiodinium endosymbiosis.

Acyl CoA synthetase-1 links facilitated long chain fatty acid uptake to intracellular metabolic trafficking differently in hearts of male versus female mice

RationaleAcyl CoA synthetase-1 (ACSL1) is localized at intracellular membranes, notably the mitochondrial membrane. ACSL1 and female sex are suggested to indirectly facilitate lipid availability to the heart and other organs. However, such mechanisms in intact, functioning myocardium remain unexplored, and roles of ACSL1 and sex in the uptake and trafficking of fats are poorly understood. ObjectiveTo determine the potential for ACSL1 and sex-dependent differences in metabolic trapping and trafficking effects of long-chain fatty acids (LCFA) within cardiomyocytes of intact hearts. Methods and results13C NMR of intact, beating mouse hearts, supplied 13C palmitate, revealed 44% faster trans-sarcolemmal uptake of LCFA in male hearts overexpressing ACSL1 (MHC-ACSL1) than in non-transgenic (NTG) males (p<0.05). Acyl CoA content was elevated by ACSL1 overexpression, 404% in males and 164% in female, relative to NTG. Despite similar ACSL1 content, NTG females displayed faster LCFA uptake kinetics compared to NTG males, which was reversed by ovariectomy. NTG female LCFA uptake rates were similar to those in ACSL1 males and ACSL1 females. ACSL1 and female sex hormones both accelerated LCFA uptake without affecting triglyceride content or turnover. ACSL1 hearts contained elevated ceramide, particularly C22 ceramide in both sexes and specifically, C24 in males. ACSL1 also induced lower content of fatty acid transporter-6 (FATP6) indicating cooperative regulation with ACSL1. Surprisingly, ACSL1 overexpression did not increase mitochondrial oxidation of exogenous palmitate, which actually dropped in female ACSL1 hearts. ConclusionsACSL1-mediated metabolic trapping of exogenous LCFA accelerates LCFA uptake rates, albeit to a lesser extent in females, which distinctly affects LCFA trafficking to acyl intermediates but not triglyceride storage or mitochondrial oxidation and is affected by female sex hormones.

The effects of a single bolus of Capsosiphon fulvescenssupplement on a postprandial oxidative stress induced by a challenge of high‐fat formula in healthy subjects

The postprandial state is a dynamic period of metabolic trafficking, biosynthesis, and oxidative metabolism of absorbed nutrients. Considering the fact that most people eat several times a day, postprandial period‐induced chronic disease can be attributed to an exaggerated metabolic and oxidative imbalance. This study was investigated to test whether Capsosiphon fulvescens supplement can attenuate the oxidative stress and imbalance in lipid metabolism which was induced by feeding a bolus of high‐fat, high‐carbohydrate meal in healthy subjects. In a double‐blinded, randomized, and crossover design, thirty six subjects (18 females and 18 males) were enrolled in the trial and underwent three 6‐h oral lipid tolerance tests: control, low dose Capsosiphon fulvescens supplement, and high dose Capsosiphon fulvescens supplement. Treatment visits were scheduled approximately 1 week apart. Ethics approved for the trial was obtained from the institution review board of Ewha Womans University. The Capsosiphon fulvescens supplement was standardized with pheophorbide A. Plasma and urine samples were collected at 0, 30, 60, 120, and 360 min after the consumption of oral lipid tolerance test formula with Capsosiphon fulvescenssupplement or placebo. In plasma, glucose, insulin, triglycerides, oxidized low‐density lipoprotein, malondialdehyde, and reactive oxygen species were measured and area under the curve (AUC), Cmax, and Tmas of each parameter were calculated. Homeostatic model assessment insulin resistance (HOMA‐IR) was also calculated. Together, these data suggest that Capsosiphon fulvescens is an excellent antioxidant and may exert beneficial effects on postprandial metabolic overload.

Abstract 4024: Ten degrees of separation: novel genes affecting outcome in ovarian cancer.

Abstract Objective: To identify genomic predictors of overall survival of women with high grade serous ovarian cancer based on best separation overall survival Kaplan-Meier curves Methods: Comprehensive data analysis of the TCGA dataset of high grade serous epithelial ovarian cancer was carried out with a “training” (n= 375) and “validation” (n= 188) data sets. The R-code was written specifically to identify all genes with high expression that gave the greatest predictive potential for patient survival. The top ten cases with lowest sums of the log rank test p-values obtained for training and validation sets were chosen to be included with Kaplan-Meier survival curves for analysis. Results: The ten genes with high expression that had the smallest p-values in their respective order were: SLC6A1, LIPK, EHBP1, SUSD5, PEX3, SLC22A3, RABGEF1, PPM2C, KIAA1219, and GALNT10. The range of p-value sum ranged from 1.77E-3 to 2.09E-3. The lowest p-value was for SLC6A1, which encodes for GABA Transporter-1 that removes GABA from extracellular to intracellular space. Interestingly, four of the 10 genes predictive for poor outcome are directly involved in cell metabolism: LIPK, PPM2C, PEX3, and GALNT10. The median OS of the high gene expression group ranged from 38 to 48 months. The median OS of the low gene expression group ranged from 55-70 months. The median OS difference of high gene vs. low gene expression groups ranged from 15-25 months. EHBP1 gene, involved in endocytic trafficking of transferrin into endosomes and GLUT4 into adipocytes, had the greatest difference in median OS between high and low expressions at 25 months (p= 2.16E-3). Finally, low expression of PPM2C compared to its high expression was significantly associated with &amp;gt; 5 year survival (10.1% vs. 1.1%, p=0.001) and no recurrence in both training and validation sets (12.5% vs. 2.2%, p=0.04). PPM2C encodes for pyruvate dehydrogenase-phosphatase 1 that catalyzes reactivation of the alpha subunit of the E1 component of the pyruvate dehydrogenase complex. Pyruvate dehydrogenase complex in the active form transforms pyruvate from glycolysis to acetyl-CoA in the mitochondria. To evaluate whether similar findings are noted in other cancers, we also examined the TCGA data for breast, lung, colon cancer, and glioblastoma. PPM2C is amplified by 7% in invasive breast cancer, and leads to an overall worse survival (68.9 months vs. 129.5 months, p = 0.037). Conclusion: Genes involved in cell metabolism, neurotransmitter functioning, and endocytic trafficking are highly predictive of outcome in high grade serous ovarian cancer. High PPM2C expression and gene amplification leads to worsening overall survival in ovarian and invasive breast cancer, respectively. These pathways likely reflect novel and important therapeutic targets. Citation Format: Behrouz Zand, Cristina Ivan, Chad V. Pecot, Rajesha Rupaimoole, Heather J. Dalton, Justin Bottsford-Miller, Wei Hu, Alpa M. Nick, Anil K. Sood. Ten degrees of separation: novel genes affecting outcome in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4024. doi:10.1158/1538-7445.AM2013-4024

The role of glia in neuronal recovery following anoxia: In vitro evidence of neuronal adaptation

We investigated the effects of 3 h of anoxia on metabolism of neurons and astrocytes, using a robust cell-based model system that mimics closely the living tissue milieu, i.e., in 3D neural aggregates cultured in bioreactors. Cells were incubated simultaneously with [1- 13C]glucose and [1,2- 13C]acetate; and, the gliotoxin fluorocitrate (FC) was used for glial tricarboxylic acid (TCA) cycle inhibition to assess the role of astrocytes for neuronal metabolism after oxygen deprivation. Results show that culture viability was not compromised by exposure to anoxia with and without FC. Interaction between astrocytes and glutamatergic neurons was altered due to anoxia: labeling in glutamine from [1- 13C]glucose was decreased, whereas that in glutamate from [1,2- 13C]acetate was increased. In contrast, GABA labeling was not affected by anoxia. It was shown that anoxia did not affect astrocytic capacity to synthesize glutamine in the reoxygenation period. The selective action of FC on astrocytes was confirmed. However, the presence of small amounts of glutamate and GABA labeled from acetate indicated residual activity of the glial TCA cycle. Although major metabolic changes were found due to FC-treatment, the intracellular pool of GABA was kept unchanged. Overall, our data clearly confirm that the glutamate–glutamine cycle depends on astrocytic TCA cycle activity and that mitochondrial impairment of astrocytes will ultimately stop metabolic trafficking between astrocytes and glutamatergic neurons. Additionally, our data suggest a metabolic independence of GABAergic neurons from astrocytes even after situations of complete oxygen depletion.

Integration of Epidemiologic and Other Types of Data Into Dietary Reference Intake Development

This article will examine issues in the use of epidemiologic and other types of data in dietary reference intake development, and offer an approach toward integration. In1994, the framework to determine revised dietary reference intakes (DRIs) was meant to incorporate the concept of risk reduction of chronic disease (Institute of Medicine, 1994). However, of the 19 estimated average requirements (EARs) that were established, none were able to be set on the basis of a chronic disease end point because of inadequacies in the available data. Adequate intake levels were partially determined by using disease end points for five nutrients: calcium (fracture rates), vitamin D (fracture rates), fluoride (dental carries), potassium (hypertension, kidney stones), and fiber (coronary heart disease). Using chronic disease end points was found to be difficult for setting individual nutrient DRIs for a number of reasons: 1. Chronic diseases have long latencies in their development, which results in almost inevitable inconsistencies in the data and makes them difficult and expensive to study. 2. Chronic diseases have numerous linkages to genetic and environmental factors. Risk of a chronic disease can never be brought to zero or reach 100% by any single nutritional intervention (Trumbo, 2008). The notion of attributable risk assumes that each factor is independent of every other factor, which is almost certainly not true. 3. Foods are complex entities. Attributing a chronic disease (e.g., cancer) linkage of a food (e.g., vegetables) to a specific nutrient (e.g., carotene) contained in that food is fraught with uncertainty. 4. Many studies on nutrients and chronic disease prevention were conducted as secondary prevention studies, rather than primary prevention studies. Extrapolation of the results of a secondary prevention study to primary prevention of a disease cannot be done and the results cannot, therefore, be translated into a public health recommendation. 5. Intervention studies on nutrients and chronic disease prevention have generally used single nutrients or a small number of combined nutrients. Such studies are expensive to conduct, and few have incorporated dose responses, which are a necessary component in DRI development. Because of the nature of the enumerated issues above, it can be concluded that prospective cohort studies can and do play a key and important role in initial hypothesis formation (as well as a confirmatory role) of chronic disease-nutrient linkages. However, such studies alone cannot yield definitive answers because of the uncertainties already mentioned that arise from long-term latency periods and from the multiple etiologic causative or modifying factors in chronic disease development. Results from prospective cohort studies (and, less importantly, observational epidemiologic studies) must be combined with evidence from experimental, animal, and small-scale, randomized, controlled clinical studies, which have used verified biomarkers of effect (i.e., biomarkers in the pathway of, or predictive of, chronic disease development) as end points (Institute of Medicine, 2008). Such biomarkers as effectors or predictors of chronic disease would have previously been demonstrated in prospective cohort and clinical intervention studies. Verified biomarkers of effect require an understanding of metabolic trafficking and can be enormously useful for decreasing the cost of studies, for providing early answers, and for studying dose-response relationships. In addition, such biomarkers should be safe to use in large-scale studies and in public health interventions. The interplay between the various types of studies will be an essential part of future DRI development. Thus, the results from several different types of studies (experimental, epidemiologic, and clinical) should be moving in the same direction with consistency, to be useful for determining DRIs for individual nutrients. In most cases, it would not be necessary to perform large-scale, randomized, and controlled nutrient-intervention trials. This gold standard type of study should be applied very selectively and only for questions of extreme public health relevance, and after a synthesis of data from all of the other various types of studies has been performed. Systematic reviews, to include meta-analysis, should always be used to complete such a synthesis (Lichtenstein et al., 2008), both in the process of designing costly intervention trials and during the DRI development process itself.

Storage problems in lysosomal diseases

Biochemical disorders in lysosomal storage diseases consist of the interruption of metabolic pathways involved in the recycling of the degradation products of one or several types of macromolecules. The progressive accumulation of these primary storage products is the direct consequence of the genetic defect and represents the initial pathogenic event. Downstream consequences for the affected cells include the accumulation of secondary storage products and the formation of histological storage lesions, which appear as intracellular vacuoles that represent the pathological hallmark of lysosomal storage diseases. Relationships between storage products and storage lesions are not simple and are still largely not understood. Primary storage products induce malfunction of the organelles where they accumulate, these being primarily, but not only, lysosomes. Consequences for cell metabolism and intracellular trafficking combine the effects of primary storage product toxicity and the compensatory mechanisms activated to protect the cell. Induced disorders extend far beyond the primarily interrupted metabolic pathway.

Astrocyte Oxidative Metabolism and Metabolite Trafficking after Fluid Percussion Brain Injury in Adult Rats

Despite various lines of evidence pointing to the compartmentation of metabolism within the brain, few studies have reported the effect of a traumatic brain injury (TBI) on neuronal and astrocyte compartments and/or metabolic trafficking between these cells. In this study we used ex vivo ¹³C NMR spectroscopy following an infusion of [1-¹³C] glucose and [1,2-¹³C₂] acetate to study oxidative metabolism in neurons and astrocytes of sham-operated and fluid percussion brain injured (FPI) rats at 1, 5, and 14 days post-surgery. FPI resulted in a decrease in the ¹³C glucose enrichment of glutamate in neurons in the injured hemisphere at day 1. In contrast, enrichment of glutamine in astrocytes from acetate was not significantly decreased at day 1. At day 5 the ¹³C enrichment of glutamate and glutamine from glucose in the injured hemisphere of FPI rats did not differ from sham levels, but glutamine derived from acetate metabolism in astrocytes was significantly increased. The ¹³C glucose enrichment of the C3 position of glutamate (C3) in neurons was significantly decreased ipsilateral to FPI at day 14, whereas the enrichment of glutamine in astrocytes had returned to sham levels at this time point. These findings indicate that the oxidative metabolism of glucose is reduced to a greater extent in neurons compared to astrocytes following a FPI. The increased utilization of acetate to synthesize glutamine, and the acetate enrichment of glutamate via the glutamate-glutamine cycle, suggests an integral protective role for astrocytes in maintaining metabolic function following TBI-induced impairments in glucose metabolism.

Macromolecular diffusion characteristics of ageing human Bruch's membrane: Implications for age-related macular degeneration (AMD)

Macromolecular species such as retinal binding protein, transferrin, ceruloplasmin, etc., released by the fenestrated choroidal capillaries must diffuse across Bruch's membrane for interaction with the basal membranes of the retinal pigment epithelium (RPE) for delivery of essential metabolites to the neural retina. The patency of this pathway through ageing Bruch's was examined by quantifying the diffusional flux of a 21.2 kDa fluorescein-isothiocyanate labelled dextran. Dextran flux measurements across Bruch's membrane from the macular region of the human fundus showed a highly significant decrease ( p < 0.001) with ageing of donor such that diffusional transport in the ninth decade was about 6.5% of that in the first decade of life. Peripheral regions also showed a highly significant decline ( p < 0.001) but ageing changes were considerably slowed in comparison to the macula with diffusional rates in the ninth decade being about 44% of that in the first decade. Peripheral samples from AMD donors displayed diffusional rates that were lower than the control population. The age-related decline in macromolecular diffusion across Bruch's membrane suggests that in the elderly, the patency of the conducting pathways may be compromised and in the more advanced ageing of Bruch's associated with AMD, the metabolic trafficking of carrier proteins may be severely impaired.

Isolation of symbiosomes and the symbiosome membrane complex from the zoanthid Zoanthus robustus

A. Kazandjian, V.A. Shepherd, M. Rodriguez-Lanetty, W. Nordemeier, A.W.D. Larkum and R.G. Quinnell. 2008. Isolation of symbiosomes and the symbiosome membrane complex from the zoanthid Zoanthus robustus. Phycologia 47: 294–306. DOI: 10.2216/07-23.1The zoanthid Zoanthus robustus was used as a model organism to develop procedures for isolating pure symbiosomes and symbiosome membranes. The symbiosome is comprised of a zooxanthella (Symbiodinium sp.) cell that divides rarely and is separated from the host gastrodermal cytoplasm by a symbiosome multimembrane complex. Devising a method to isolate membranes at the interface between the symbiotic partners is a critical first step in characterising the molecular components involved in the metabolic trafficking necessary to sustain an effective symbiosis. After zoanthid gastrodermal cells were extracted, symbiosomes were released by mechanical disruption, recovered by centrifugation, and then purified using discontinuous sucrose gradient centrifugation. The material forming the membrane complex around symbiosomes proved highly resistant to disruption. Methods used to dissociate this interface from symbionts included (1) Triton X-100 detergent solubilisation, (2) osmotic shock with mechanical disruption, and (3) vigorous mechanical disruptions, where powerful shearing forces were used, combined with a series of sucrose density gradient centrifugation steps. The lipophilic styryl fluorochrome FM 1-43, at a concentration of 30 µM, selectively labelled the symbiosome membrane complex, both for isolated symbiosomes and those in hospite. Other cell membranes, including plasma membranes, endoplasmic reticulum, tonoplast, and organelle membranes, were not visibly labelled at this concentration. The selective labelling of the symbiosome membrane complex remained stable even after long exposure times (3 h). At 30 µM concentration, FM 1-43 also labelled symbiosome membrane fragments isolated using methods (1), (2) and (3). Method (3) proved to be the most effective in producing a fraction enriched in FM-143-labelled membrane material, which we call a symbiosome membrane complex. Transmission electron microscopy, together with confocal and conventional epifluorescence microscopy of the FM 1-43-stained preparations, was used to validate the purity of symbiosome preparations and to infer the complexity of the symbiosome membrane complex. This membrane complex has regions where the membranes contributed by the alga are appressed, and punctate regions whose function remains unclear.

Gene expression in elongating and gastrulating embryos from ruminants

In ruminants, more than 30% of the embryonic losses observed after artificial insemination (AI) have an early origin, coincident with a marked elongation of the trophoblast which occurs before implantation. Several observations provide clear evidence that early elongation of the conceptus relies on cell multiplication, cell growth and cell shape remodeling. Recent results indicating an intense multiplication of a non-fully differentiated trophoblast, which still expresses some epiblast genes, has to be considered at the onset of elongation. It has also been shown in the last two years that general metabolism and protein trafficking are characteristic of the onset of elongation whereas cellular interactions, cell to cell signaling and cell adhesion become predominant at the end of elongation. Accordingly, expression of most of the single genes identified so far increases during elongation and is related to the establishment of embryo-maternal exchanges before implantation. However, not much is known of what controls the induction of the elongation process or the coordinated development of the embryonic and extra-embryonic tissues. This review highlights new information on this developmental phase and summarizes the views on the complex cross-talk among molecules which might govern conceptus development and lead to successful implantation.

Lymphatic Endothelial Cell Behavior: Emerging Roles in Transport, Metabolism, and Lymphocyte Trafficking

Lymphatic Endothelial Cell Behavior: Emerging Roles in Transport, Metabolism, and Lymphocyte Trafficking

Phosphatidylinositol-transfer protein and its homologues in yeast

Yeast Sec14p acts as a phosphatidylinositol/phosphatidylcholine-transfer protein in vitro. In vivo, it is essential in promoting Golgi secretory function. Products of five genes named SFH1-SFH5 (Sec Fourteen Homologues 1-5) exhibit significant sequence homology to Sec14p and together they form the Sec14p family of lipid-transfer proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism and membrane trafficking.

Kinetics of endothelin‐induced inhibition and glucose permeability of astrocyte gap junctions

Gap junctions contribute to important functions of communicating glial cells in brain physiology and pathology. Endothelins (ETs), a vasoactive family of peptides present in the brain, have been described as potent inhibitors of astrocyte gap junctional communication. Through dye-coupling studies we demonstrate here that this inhibition occurs rapidly and then successively reverses and returns to control levels after 90 min of continuous ET1 or ET3 exposure. In addition, long-term exposure of cells to ET3, which acts mainly on ETB receptors, also desensitized the acute action of ET1, which was previously shown to act through either ETA or ETB receptor sites, or both. The gap junction blocker carbenoxolone did not show any time-dependent desensitization and was fully effective also in cultures treated with ETs for prolonged times. The ETs inhibitory effects were partially prevented when blocking pertussis toxin-sensitive G-proteins, chelating intracellular Ca2+, or omitting extracellular Ca2+. We further show that ETs modulate gap junction-mediated transfer of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-Y1)amino]-2-deoxyglucose (2-NBDG), a fluorescent glucose molecule, indicating a role of astrocyte gap junction coupling in metabolic trafficking and suggesting the importance of these peptides in the control of intercellular diffusion of energetic compounds. These findings might have particular relevance in early tissue reactions after various cerebral injuries, which commonly involve increased cerebral ET levels.