Metabolic origin of δ15N values in nitrogenous compounds from Brassica napus L. leaves

Nitrogen isotope composition (δ15N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ15N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ15N. Here, we investigated the δ15N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ15N between nitrogenous compounds (up to 30 ) and the content in (15N enriched) nitrate had a clear influence on leaf δ15N. Using a simple steady-state model of day metabolism, we suggest that the δ15N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme-catalysed reactions in primary nitrogen metabolism. δ15N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ15N value of leaf nitrogenous compounds

Sphingomonas wittichii RW1 gene reporters interrogating the dibenzofuran metabolic network highlight conditions for early successful development in contaminated microcosms.

In order to better understand the fate and activity of bacteria introduced into contaminated material for the purpose of enhancing biodegradation rates, we constructed Sphingomonas wittichii RW1 variants with gene reporters interrogating dibenzofuran metabolic activity. Three potential promoters from the dibenzofuran metabolic network were selected and fused to the gene for enhanced green fluorescent protein (EGFP). The stability of the resulting genetic constructions in RW1 was examined, with plasmids based on the broad-host range vector pME6012 being the most reliable. One of the selected promoters, upstream of the gene Swit_4925 for a putative 2-hydroxy-2,4-pentadienoate hydratase, was inducible by growth on dibenzofuran. Sphingomonas wittichii RW1 equipped with the Swit_4925 promoter egfp fusion grew in a variety of non-sterile sandy microcosms contaminated with dibenzofuran and material from a former gasification site. The strain also grew in microcosms without added dibenzofuran but to a very limited extent, and EGFP expression indicated the formation of consistent small subpopulations of cells with an active inferred dibenzofuran metabolic network. Evidence was obtained for competition for dibenzofuran metabolites scavenged by resident bacteria in the gasification site material, which resulted in a more rapid decline of the RW1 population. Our results show the importance of low inoculation densities in order to observe the population development of the introduced bacteria and further illustrate that the limited availability of unique carbon substrate may be the most important factor impinging growth.

Metabolic effects of insulin and IGFs on gilthead sea bream (Sparus aurata) muscle cells.

Primary cultures of gilthead sea bream myocytes were performed in order to examine the relative metabolic function of insulin compared with IGF-I and IGF-II (insulin-like growth factors, IGFs) at different stages in the cell culture. In these cells, the in vitro effects of insulin and IGFs on 2-deoxyglucose (2-DG) and L-alanine uptake were studied in both myocytes (day 4) and small myotubes (day 9). 2-DG uptake in gilthead sea bream muscle cells was increased in the presence of insulin and IGFs in a time dependent manner and along with muscle cell differentiation. On the contrary, L-alanine uptake was also stimulated by insulin and IGFs but showed an inverse pattern, being the uptake higher in small myocytes than in large myotubes. The results of preincubation with inhibitors (PD-98059, wortmannin, and cytochalasin B) on 2-DG uptake indicated that insulin and IGFs stimulate glucose uptake through the same mechanisms, and evidenced that mitogenesis activator protein kinase (MAPK) and PI3K-Akt transduction pathways mediate the metabolic function of these peptides. In the same way, we observed that GLUT4 protein synthesis was stimulated in the presence of insulin and IGFs in gilthead sea bream muscle cells in a different manner at days 4 or 9 of the culture. In summary we describe here, for the first time, the effects of insulin and IGFs on 2-DG and L-alanine uptake in primary culture of gilthead sea bream muscle cells. We show that both MAPK and PI3K-Akt transduction pathways are needed in order to control insulin and IGFs actions in these cells. Moreover, changes in glucose uptake can be explained by the action of the GLUT4 transporter, which is stimulated in the presence of insulin and IGFs throughout the cell culture.

Effect of sex and prior exposure to a cafeteria diet on the distribution of sex hormones between plasma and blood cells

It is generally assumed that steroid hormones are carried in the blood free and/or bound to plasma proteins. We investigated whether blood cells were also able to bind/carry sex-related hormones: estrone, estradiol, DHEA and testosterone. Wistar male and female rats were fed a cafeteria diet for 30 days, which induced overweight. The rats were fed the standard rat diet for 15 additional days to minimize the immediate effects of excess ingested energy. Controls were always kept on standard diet. After the rats were killed, their blood was used for 1) measuring plasma hormone levels, 2) determining the binding of labeled hormones to washed red blood cells (RBC), 3) incubating whole blood with labeled hormones and determining the distribution of label between plasma and packed cells, discounting the trapped plasma volume, 4) determining free plasma hormone using labeled hormones, both through membrane ultrafiltration and dextrancharcoal removal. The results were computed individually for each rat. Cells retained up to 32% estrone, and down to 10% of testosterone, with marked differences due to sex and diet (the latter only for estrogens, not for DHEA and testosterone). Sex and diet also affected the concentrations of all hormones, with no significant diet effects for estradiol and DHEA, but with considerable interaction between both factors. Binding to RBC was non-specific for all hormones. Estrogen distribution in plasma compartments was affected by sex and diet. In conclusion: a) there is a large non-specific RBC-carried compartment for estrone, estradiol, DHEA and testosterone deeply affected by sex; b) Prior exposure to a cafeteria (hyperlipidic) diet induced hormone distribution changes, affected by sex, which hint at sex-related structural differences in RBC membranes; c) We postulate that the RBC compartment may contribute to maintain free (i.e., fully active) sex hormone levels in a way similar to plasma proteins non-specific binding.

Metabolic gene expression changes in astrocytes in Multiple Sclerosis cerebral cortex are indicative of immune-mediated signaling.

Emerging as an important correlate of neurological dysfunction in Multiple Sclerosis (MS), extended focal and diffuse gray matter abnormalities have been found and linked to clinical manifestations such as seizures, fatigue and cognitive dysfunction. To investigate possible underlying mechanisms we analyzed the molecular alterations in histopathological normal appearing cortical gray matter (NAGM) in MS. By performing a differential gene expression analysis of NAGM of control and MS cases we identified reduced transcription of astrocyte specific genes involved in the astrocyte-neuron lactate shuttle (ANLS) and the glutamate-glutamine cycle (GGC). Additional quantitative immunohistochemical analysis demonstrating a CX43 loss in MS NAGM confirmed a crucial involvement of astrocytes and emphasizes their importance in MS pathogenesis. Concurrently, a Toll-like/IL-1β signaling expression signature was detected in MS NAGM, indicating that immune-related signaling might be responsible for the downregulation of ANLS and GGC gene expression in MS NAGM. Indeed, challenging astrocytes with immune stimuli such as IL-1β and LPS reduced their ANLS and GGC gene expression in vitro. The detected upregulation of IL1B in MS NAGM suggests inflammasome priming. For this reason, astrocyte cultures were treated with ATP and ATP/LPS as for inflammasome activation. This treatment led to a reduction of ANLS and GGC gene expression in a comparable manner. To investigate potential sources for ANLS and GGC downregulation in MS NAGM, we first performed an adjuvant-driven stimulation of the peripheral immune system in C57Bl/6 mice in vivo. This led to similar gene expression changes in spinal cord demonstrating that peripheral immune signals might be one source for astrocytic gene expression changes in the brain. IL1B upregulation in MS NAGM itself points to a possible endogenous signaling process leading to ANLS and GGC downregulation. This is supported by our findings that, among others, MS NAGM astrocytes express inflammasome components and that astrocytes are capable to release Il-1β in-vitro. Altogether, our data suggests that immune signaling of immune- and/or central nervous system origin drives alterations in astrocytic ANLS and GGC gene regulation in the MS NAGM. Such a mechanism might underlie cortical brain dysfunctions frequently encountered in MS patients.

An overview of the metabolic differences between Bradyrhizobium japonicum 110 bacteria and differentiated bacteroids from soybean (Glycine max) root nodules: An in vitro 13C-and 31P-nuclear magnetic resonance spectroscopy study

Bradyrhizobium japonicum is a symbiotic nitrogen-fixing soil bacteria that induce root nodules formation in legume soybean (Glycine max.). Using 13C- and 31P-nuclear magnetic resonance (NMR) spectroscopy, we have analysed the metabolite profiles of cultivated B.japonicum cells and bacteroids isolated from soybean nodules. Our results revealed some quantitative and qualitative differences between the metabolite profiles of bacteroids and their vegetative state. This includes in bacteroids a huge accumulation of soluble carbohydrates such as trehalose, glutamate, myo-inositol and homospermidine as well as Pi, nucleotide pools and intermediates of the primary carbon metabolism. Using this novel approach, these data show that most of the compounds detected in bacteroids reflect the metabolic adaptation of rhizobia to the surrounding microenvironment with its host plant cells.

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: An electron microscopy and P-31 and C-13 nuclear magnetic resonance study

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using C-13- and P-31-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N-2 fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

Thyroid function in the elderly and sex hormones in elderly men: reference intervals and associations with health

Aims: The aims were to create clinically feasible reference intervals for thyroidstimulating hormone (TSH) and free thyroxine (FT4) and to analyze associations between thyroid function and self-rated health, neuropsychiatric symptoms, depression and dementia in the elderly. The second aim was also to establish reference intervals for sex hormones and to analyze associations between sex hormone levels and self-rated health, symptoms, depression and dementia in elderly men. Subjects and methods: The study population comprised 1252 subjects aged 65 years or over, living in the municipality of Lieto, south-western Finland. Self-rated health, life satisfaction, symptoms, depression, and dementia were assessed with specific questions, clinical examination and tools such as the Zung Self-report Depression Scale and the Mini-Mental State Examination. Independent variables were dichotomized, and associations of these variables with TSH, FT4 or sex hormone levels were assessed. Levels of TSH and FT4 in thyroid disease–free women and women treated with thyroxine were also compared. Results: Elevated concentrations of thyroid peroxidase antibodies (TPOAb) or thyroglobulin antibodies (TgAb) were found to have a marked effect on the upper reference limit for TSH among women, who were thyroid antibody positive more higher than suggested in several recent guidelines. After age adjustment, there were no associations between TSH levels and self-rated health, life satisfaction, or most neuropsychiatric symptoms in the thyroid disease-free population. Although women with thyroxine treatment for primary hypothyroidism had far higher TSH levels than thyroid disease-free women, there were no differences between thyroid-disease free women and women with stable thyroxine treatment regarding self-rated health, life satisfaction or symptoms. Age had a significant positive association with luteinizing hormone (LH), follicle 2 practice, one range in men aged 65 years or over can be used for T, E2 and FSH measured with the AutoDelfia method, but two separate reference intervals should be used for fT, LH and SHBG. After adjustment for age, higher levels of T and fT were associated with better self-rated health (SRH) in the reference population. After adjustment for age and body mass index (BMI), there were no associations between sex hormone concentrations and self-rated health, life satisfaction or most symptoms in concentration. Conclusion: Age-specific reference intervals were derived for thyroid function and sex hormones based on comprehensive data from a community-dwelling population with a high participation rate. The results do not support the need to decrease the upper reference limit for TSH or to lower the optimal TSH target in levothyroxine treatment in older adults, as recommended in recent guidelines. Older age or being overweight symptoms among elderly men. The associations of single symptoms with T levels were inconsistent among elderly men, although the association of low T level with diagnosed depression might be clinically significant.

Metabolic connectivity as index of verbal working memory.

Positron emission tomography (PET) data are commonly analyzed in terms of regional intensity, while covariant information is not taken into account. Here, we searched for network correlates of healthy cognitive function in resting state PET data. PET with [(18)F]-fluorodeoxyglucose and a test of verbal working memory (WM) were administered to 35 young healthy adults. Metabolic connectivity was modeled at a group level using sparse inverse covariance estimation. Among 13 WM-relevant Brodmann areas (BAs), 6 appeared to be robustly connected. Connectivity within this network was significantly stronger in subjects with above-median WM performance. In respect to regional intensity, i.e., metabolism, no difference between groups was found. The results encourage examination of covariant patterns in FDG-PET data from non-neurodegenerative populations.

Inflammatory and metabolic responses to high-fat meals with and without dairy products in men.

Postprandial inflammation is an important factor for human health since chronic low-grade inflammation is associated with chronic diseases. Dairy products have a weak but significant anti-inflammatory effect on postprandial inflammation. The objective of the present study was to compare the effect of a high-fat dairy meal (HFD meal), a high-fat non-dairy meal supplemented with milk (HFM meal) and a high-fat non-dairy control meal (HFC meal) on postprandial inflammatory and metabolic responses in healthy men. A cross-over study was conducted in nineteen male subjects. Blood samples were collected before and 1, 2, 4 and 6 h after consumption of the test meals. Plasma concentrations of insulin, glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, TAG and C-reactive protein (CRP) were measured at each time point. IL-6, TNF-α and endotoxin concentrations were assessed at baseline and endpoint (6 h). Time-dependent curves of these metabolic parameters were plotted, and the net incremental AUC were found to be significantly higher for TAG and lower for CRP after consumption of the HFM meal compared with the HFD meal; however, the HFM and HFD meals were not different from the HFC meal. Alterations in IL-6, TNF-α and endotoxin concentrations were not significantly different between the test meals. The results suggest that full-fat milk and dairy products (cheese and butter) have no significant impact on the inflammatory response to a high-fat meal.

Long-Lasting Metabolic Imbalance Related to Obesity Alters Olfactory Tissue Homeostasis and Impairs Olfactory-Driven Behaviors.

Obesity is associated with chronic food intake disorders and binge eating. Food intake relies on the interaction between homeostatic regulation and hedonic signals among which, olfaction is a major sensory determinant. However, its potential modulation at the peripheral level by a chronic energy imbalance associated to obese status remains a matter of debate. We further investigated the olfactory function in a rodent model relevant to the situation encountered in obese humans, where genetic susceptibility is juxtaposed on chronic eating disorders. Using several olfactory-driven tests, we compared the behaviors of obesity-prone Sprague-Dawley rats (OP) fed with a high-fat/high-sugar diet with those of obese-resistant ones fed with normal chow. In OP rats, we reported 1) decreased odor threshold, but 2) poor olfactory performances, associated with learning/memory deficits, 3) decreased influence of fasting, and 4) impaired insulin control on food seeking behavior. Associated with these behavioral modifications, we found a modulation of metabolism-related factors implicated in 1) electrical olfactory signal regulation (insulin receptor), 2) cellular dynamics (glucorticoids receptors, pro- and antiapoptotic factors), and 3) homeostasis of the olfactory mucosa and bulb (monocarboxylate and glucose transporters). Such impairments might participate to the perturbed daily food intake pattern that we observed in obese animals.

Role of Connexins and Pannexins in the Pancreas.

The pancreas produces enzymes with a digestive function and hormones with a metabolic function, which are produced by distinct cell types of acini and islets, respectively. Within these units, secretory cells coordinate their functioning by exchanging information via signals that flow in the intercellular spaces and are generated either at distance (several neural and hormonal inputs) or nearby the pancreatic cells themselves (inputs mediated by membrane ionic-specific channels and by ionic- and metabolite-permeant pannexin channels and connexin "hemichannels"). Pancreatic secretory cells further interact via the extracellular matrix of the pancreas (inputs mediated by integrins) and directly with neighboring cells, by mechanisms that do not require extracellular mediators (inputs mediated by gap and tight junction channels). Here, we review the expression and function of the connexins and pannexins that are expressed by the main secretory cells of the exocrine and endocrine pancreatic cells. Available data show that the patterns of expression of these proteins differ in acini and islets, supporting distinct functions in the physiological secretion of pancreatic enzymes and hormones. Circumstantial evidence further suggests that alterations in the signaling provided by these proteins are involved in pancreatic diseases.

Novel Roles of HCG/LH Signalling in the Mouse Mammary Gland and its Tumorigenesis.

Breast cancer is the most common cancer in women, and its development is intimately related to hormonal factors, but how hormones affect breast physiology and tumorigenesis is not sufficiently known. Pregnancy elicits long-term protection from breast cancer, but during the first ten years after pregnancy, breast cancer risk is increased. In previous studies, there has been conflicting data on the role of human chorionic gonadotropin (HCG) and the functionality of its receptor in extragonadal tissues. The aim of this study was to elucidate the role of chronically elevated HCG in mouse physiology. We have created a transgenic (TG) mouse model that overexpresses HCG. HCG is similar to lutenizing hormone (LH), but is secreted almost solely by the placenta during pregnancy. HCG and LH both bind to the LH receptor (LHR). In the current study, mammary gland tumors were observed in HCG TG mice. We elucidated the role of HCG in mammary gland signalling and the effects of LHR mediated signalling in mouse mammary gland gene expression. We also studied the effects of HCG in human breast epithelial cell cultures. Several endocrine disturbances were observed in HCGβ TG female mice, resulting in precocious puberty, infertility, obesity and pituitary and mammary gland tumors. The histology of the mammary gland tumors of HCGβ TG females resembled those observed in mouse models with activated Wnt/β-catenin signalling pathway. Wnts are involved in stem cell regulation and tumorigenesis, and are hormonally regulated in the mammary gland. We observed activated β-catenin signalling and elevated expression of Wnt5b and Wnt7b in TG tumors and mammary glands. Furthermore, we discovered that HCG directly regulates the expression of Wnt5b and Wnt7b in the mouse mammary gland. Pharmacological treatment with HCG also caused upregulation of several Wnt-pathway target genes in ovariectomized wild type (WT) mice in the presence of physiological concentrations of estradiol and progesterone. In addition, differential expression of several metabolic genes was observed, suggesting that HCG affects adipocyte function or glucose metabolism. When WT mice were transplanted with LHR deficient or wild type WT mammary epithelium, differential expression of several genes affecting the Wnt-signalling pathway was observed in microarray analysis. Diminished expression of several genes associated with LHR function in other tissues, such as the ovary, was observed in mammary glands deficient of epithelial LHR. In cultured human mammary epithelial cells HCG upregulated the expression of WNT5B, WNT7B similar to mouse, suggesting that the observations found are relevant in human physiology. These studies suggest that HCG/LHR signalling affects gene expression in non-gonadal tissues, and that Wnt-signalling is regulated by HCG/LH in human and mouse mammary glands.