The global effect of follicle-stimulating hormone and tumour necrosis factor α on gene expression in cultured bovine ovarian granulosa cells

Background Oocytes mature in ovarian follicles surrounded by granulosa cells. During follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. However, most follicles cease growing and undergo atresia or regression instead of ovulating. To investigate the effects of stimulatory (follicle-stimulating hormone; FSH) and inhibitory (tumour necrosis factor alpha; TNFα) factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3–6. Initially dose–response experiments (n = 4) were performed to determine the optimal concentrations of FSH (0.33 ng/ml) and TNFα (10 ng/ml) to be used for the microarray experiments. For array experiments cells were cultured under control conditions, with FSH, with TNFα, or with FSH plus TNFα (n = 4 per group) and RNA was harvested for microarray analyses. Results Statistical analysis showed primary clustering of the arrays into two groups, control/FSH and TNFα/TNFα plus FSH. The effect of TNFα on gene expression dominated that of FSH, with substantially more genes differentially regulated, and the pathways and genes regulated by TNFα being similar to those of FSH plus TNFα treatment. TNFα treatment reduced the endocrine activity of granulosa cells with reductions in expression of FST, INHA, INBA and AMH. The top-ranked canonical pathways and GO biological terms for the TNFα treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. The two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor β signalling, and these were up regulated. Upstream regulator analyses also predicted TNF, interferons γ and β1 and interleukin 1β. Conclusions In vitro, the transcriptome of granulosa cells responded minimally to FSH compared with the response to TNFα. The response to TNFα indicated an active process akin to tissue remodelling as would occur upon atresia. Additionally there was reduction in endocrine function and induction of an inflammatory response to TNFα that displays features similar to immune cells.

A continuum receptor model of hepatic lipoprotein metabolism

A mathematical model describing the uptake of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) particles by a single hepatocyte cell is formulated and solved. The model includes a description of the dynamic change in receptor density on the surface of the cell due to the binding and dissociation of the lipoprotein particles, the subsequent internalisation of bound particles, receptors and unbound receptors, the recycling of receptors to the cell surface, cholesterol dependent de novo receptor formation by the cell and the effect that particle uptake has on the cell's overall cholesterol content. The effect that blocking access to LDL receptors by VLDL, or internalisation of VLDL particles containing different amounts of apolipoprotein E (we will refer to these particles as VLDL-2 and VLDL-3) has on LDL uptake is explored. By comparison with experimental data we find that measures of cell cholesterol content are important in differentiating between the mechanisms by which VLDL is thought to inhibit LDL uptake. We extend our work to show that in the presence of both types of VLDL particle (VLDL-2 and VLDL-3), measuring relative LDL uptake does not allow differentiation between the results of blocking and internalisation of each VLDL particle to be made. Instead by considering the intracellular cholesterol content it is found that internalisation of VLDL-2 and VLDL-3 leads to the highest intracellular cholesterol concentration. A sensitivity analysis of the model reveals that binding, unbinding and internalisation rates, the fraction of receptors recycled and the rate at which the cholesterol dependent free receptors are created by the cell have important implications for the overall uptake dynamics of either VLDL or LDL particles and subsequent intracellular cholesterol concentration. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats

The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-alpha-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver. (C) 2004 Elsevier B.V. All rights reserved.

Differences in glucose-dependent insulinotropic polypeptide hormone and hepatic lipase in subjects of southern and northern Europe: implications for postprandial lipaemia

The aim was to determine in 32 healthy young men from northern and southern Europe whether differences in the secretion of insulin and glucose-dependent insulinotropic polypeptide (GIP) might explain these findings through the actions of these hormones on lipoprotein lipase. In a randomized, single-blind, crossover study the effects of 2 test meals of identical macronutrient composition but different saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) contents were investigated on postprandial GIP, insulin, the ratio of incremental triacylglycerol to apolipoprotein B-48 (a marker of chylomicron size), and the activity of postheparin lipases. Fasting and postprandial GIP concentrations and postheparin hepatic lipase (HL) activities were higher in the southern Europeans (P<0.001 and P<0.02, respectively). Lipoprotein lipase activity after the SFA-rich meal was higher in the northern Europeans (P<0.01). HL activity 9 h after the SFA-rich meal and the area under the curve (AUC) for the postprandial insulin response correlated with the AUC for the postprandial GIP response (r=0.44 (P<0.04) and r=0.46 (P<0.05), respectively). There were no significant differences in chylomicron size between the 2 groups for either meal, but when the groups were combined there was a difference in chylomicron size between the SFA- and MUFA-rich meals (P<0.05), which could be due to the formation of larger chylomicrons after the MUFA-rich meal. The significantly higher GIP and insulin responses and HL activities in southern Europeans may provide an explanation for a previous report of attenuated postprandial triacylglycerol and apolipoprotein B-48 responses in them.

Effects of dietary fatty acid composition on basal and hormonestimulated hepatic lipogenesis and on circulating lipids in the rat

Thirty male rats were randomly assigned to one of three dietary groups in which the source of dietary fat was either a mixed oil, maize oil or fish oil. Effects of dietary fatty acid composition on in virro rates of [U-'4C]glucose incorporation into hepatic total lipids and into hepatic triacylglycerol were measured under basal, insulin (4 nM)-, gastric inhibitory polypeptide (GIP; 6 mi)- and insulin + GIP (4 nM + 6 n ~ ) - stimulated conditions. Effects of the three diets on postprandial plasma triacylglycerol, cholesterol, insulin and GIP concentrations were also measured. The fish-oil diet decreased rates of basal glucose incorporation into hepatic total lipids (P < 0.05) and hepatic triacylglycerol (P < 0.01) compared with the mixed-oil diet. The presence of insulin + GIP in the incubation medium stimulated glucose incorporation into hepatic total lipids in the maize-oil (P < 0.01) and fish-oil groups (P < OW), as well as into hepatic triacylglycerol in the maize-oil group (P < 0.005). In addition, the fish-oil diet decreased postprandial plasma triacylglycerol levels compared with both other dietary groups (P < 0-05 both cases), and the mixed-oil diet markedly increased postprandial plasma insulin levels compared with the other dietary groups (P c 0.001).

Assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1H NMR spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism. In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS

The first application of high field NMR spectroscopy (800 MHz for 1H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional 1H–1H TOCSY and 1H–13C HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids (cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% (glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.

Variation in genes related to hepatic lipid metabolism and changes in waist circumference and body weight.

We analysed single nucleotide polymorphisms (SNPs) tagging the genetic variability of six candidate genes (ATF6, FABP1, LPIN2, LPIN3, MLXIPL and MTTP) involved in the regulation of hepatic lipid metabolism, an important regulatory site of energy balance for associations with body mass index (BMI) and changes in weight and waist circumference. We also investigated effect modification by sex and dietary intake. Data of 6,287 individuals participating in the European prospective investigation into cancer and nutrition were included in the analyses. Data on weight and waist circumference were followed up for 6.9 ± 2.5 years. Association of 69 tagSNPs with baseline BMI and annual changes in weight as well as waist circumference were investigated using linear regression analysis. Interactions with sex, GI and intake of carbohydrates, fat as well as saturated, monounsaturated and polyunsaturated fatty acids were examined by including multiplicative SNP-covariate terms into the regression model. Neither baseline BMI nor annual weight or waist circumference changes were significantly associated with variation in the selected genes in the entire study population after correction for multiple testing. One SNP (rs1164) in LPIN2 appeared to be significantly interacting with sex (p = 0.0003) and was associated with greater annual weight gain in men (56.8 ± 23.7 g/year per allele, p = 0.02) than in women (-25.5 ± 19.8 g/year per allele, p = 0.2). With respect to gene-nutrient interaction, we could not detect any significant interactions when accounting for multiple testing. Therefore, out of our six candidate genes, LPIN2 may be considered as a candidate for further studies.

Long-term cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience.

Whilst not true in all cases, the microbial communities that chronically infect the airways of patients with CF can vary little over a year despite antibiotic perturbation. The species present tended to vary more between than within subjects, suggesting that each CF airway infection is unique, with relatively stable and resilient bacterial communities. The inverse relationship between community richness and disease severity is similar to findings reported in other mucosal infections.

Does bacterial density in cystic fibrosis sputum increase prior to pulmonary exacerbation?

These findings strongly suggest that CFPE do not generally result from increased bacterial density within the airways. Instead, data presented here are consistent with alternative models of pulmonary exacerbation.

Analysis of the bacterial communities present in lungs of patients with cystic fibrosis from American and British centers

The aim of this study was to determine whether geographical differences impact the composition of bacterial communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any, bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was present in the vast majority of patients in both locations, indicating that these are species common to the CF lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF lung were present in both sample groups. The species number per sample was similar, but differences in species presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung infections also are discussed.