Evidence for a neuromuscular circuit involving hypothalamic interleukin-6 in the control of skeletal muscle metabolism.

Hypothalamic interleukin-6 (IL6) exerts a broad metabolic control. Here, we demonstrated that IL6 activates the ERK1/2 pathway in the ventromedial hypothalamus (VMH), stimulating AMPK/ACC signaling and fatty acid oxidation in mouse skeletal muscle. Bioinformatics analysis revealed that the hypothalamic IL6/ERK1/2 axis is closely associated with fatty acid oxidation- and mitochondrial-related genes in the skeletal muscle of isogenic BXD mouse strains and humans. We showed that the hypothalamic IL6/ERK1/2 pathway requires the α2-adrenergic pathway to modify fatty acid skeletal muscle metabolism. To address the physiological relevance of these findings, we demonstrated that this neuromuscular circuit is required to underpin AMPK/ACC signaling activation and fatty acid oxidation after exercise. Last, the selective down-regulation of IL6 receptor in VMH abolished the effects of exercise to sustain AMPK and ACC phosphorylation and fatty acid oxidation in the muscle after exercise. Together, these data demonstrated that the IL6/ERK axis in VMH controls fatty acid metabolism in the skeletal muscle.

Regulation of Energy Expenditure and Brown/Beige Thermogenic Activity by Interleukins: New Roles for Old Actors.

Obesity rates and the burden of metabolic associated diseases are escalating worldwide Energy burning brown and inducible beige adipocytes in human adipose tissues (ATs) have attracted considerable attention due to their therapeutic potential to counteract the deleterious metabolic effects of nutritional overload and overweight. Recent research has highlighted the relevance of resident and recruited ATs immune cell populations and their signalling mediators, cytokines, as modulators of the thermogenic activity of brown and beige ATs. In this review, we first provide an overview of the developmental, cellular and functional heterogeneity of the AT organ, as well as reported molecular switches of its heat-producing machinery. We also discuss the key contribution of various interleukins signalling pathways to energy and metabolic homeostasis and their roles in the biogenesis and function of brown and beige adipocytes. Besides local actions, attention is also drawn to their influence in the central nervous system (CNS) networks governing energy expenditure.

Divergent responses to thermogenic stimuli in BAT and subcutaneous adipose tissue from interleukin 18 and interleukin 18 receptor 1-deficient mice.

Brown and beige adipocytes recruitment in brown (BAT) or white adipose tissue, mainly in the inguinal fat pad (iWAT), meet the need for temperature adaptation in cold-exposure conditions and protect against obesity in face of hypercaloric diets. Using interleukin18 (Il18) and Il18 receptor 1- knockout (Il18r1-KO) mice, this study aimed to investigate the role of IL18 signaling in BAT and iWAT activation and thermogenesis under both stimuli. Il18-KO, extremely dietary obesity-prone as previously described, failed to develop diet-induced thermogenesis as assessed by BAT and iWAT Ucp1 mRNA levels. Overweight when fed standard chow but not HFD, HFD-fed Il18r1-KO mice exhibited increased iWAT Ucp1 gene expression. Energy expenditure was reduced in pre-obese Il18r1-KO mice and restored upon HFD-challenge. Cold exposure lead to similar results; Il18r1-KO mice were protected against acute body temperature drop, displaying a more brown-like structure, alternative macrophage activation and thermogenic gene expression in iWAT than WT controls. Opposite effects were observed in Il18-KO mice. Thus, Il18 and Il18r1 genetic ablation disparate effects on energy homeostasis are likely mediated by divergent BAT responses to thermogenic stimuli as well as iWAT browning. These results suggest that a more complex receptor-signaling system mediates the IL18 adipose-tissue specific effects in energy expenditure.

Energy Balance Regulating Neuropeptides Are Expressed through Pregnancy and Regulated by Interleukin-6 Deficiency in Mouse Placenta.

The placenta produces a number of signaling molecules including metabolic and reproductive hormones as well as several inflammatory mediators. Among them, Interleukin-6 (IL-6), a well-known immune and metabolic regulator, acts peripherally modulating metabolic function and centrally increasing energy expenditure and reducing body fat. IL-6 interacts with key hypothalamic neuropeptidergic systems controlling energy homeostasis such as those producing the orexigenic/anabolic: neuropeptide Y (NPY) and agouti-related peptide (AgRP) and anorectic/catabolic neuropeptides: proopiomelanocortin (POMC) and cocaine and amphetamine regulated transcript (CART). Human and rat placenta have been identified as source of these neuropeptides, but their expression and regulation in murine placental tissues remain unknown. Therefore, placental mRNA levels of IL-6, NPY, AgRP, POMC, and CART at different pregnancy stages (gestational days 13, 15, and 18) were analyzed by real time PCR, as were the effect of IL-6 deficiency (IL-6 knockout mice) on their placental expression. Our results showed that placenta-derived neuropeptides were regulated by gestational age and IL-6 throughout the second half of mouse pregnancy. These data suggest that IL-6 may participate in the fine tune control of energy balance during pregnancy by extending its action as a metabolic signal to the main organ at the fetomaternal interface: the placenta.

Interleukin 6 deficiency modulates the hypothalamic expression of energy balance regulating peptides during pregnancy in mice.

Pregnancy is associated with hyperphagia, increased adiposity and multiple neuroendocrine adaptations. Maternal adipose tissue secretes rising amounts of interleukin 6 (IL6), which acts peripherally modulating metabolic function and centrally increasing energy expenditure and reducing body fat. To explore the role of IL6 in the central mechanisms governing dam's energy homeostasis, early, mid and late pregnant (gestational days 7, 13 and 18) wild-type (WT) and Il6 knockout mice (Il6-KO) were compared with virgin controls at diestrus. Food intake, body weight and composition as well as indirect calorimetry measurements were performed in vivo. Anabolic and orexigenic peptides: neuropeptide Y (Npy) and agouti-related peptide (Agrp); and catabolic and anorectic neuropeptides: proopiomelanocortin (Pomc), corticotrophin and thyrotropin-releasing hormone (Crh and Trh) mRNA levels were determined by in situ hybridization. Real time-PCR and western-blot were used for additional tissue gene expression and protein studies. Non-pregnant Il6-KO mice were leaner than WT mice due to a decrease in fat but not in lean body mass. Pregnant Il6-KO mice had higher fat accretion despite similar body weight gain than WT controls. A decreased fat utilization in absence of Il6 might explain this effect, as shown by increased respiratory exchange ratio (RER) in virgin Il6-KO mice. Il6 mRNA levels were markedly enhanced in adipose tissue but reduced in hypothalamus of mid and late pregnant WT mice. Trh expression was also stimulated at gestational day 13 and lack of Il6 blunted this effect. Conversely, in late pregnant mice lessened hypothalamic Il6 receptor alpha (Il6ra), Pomc and Crh mRNA were observed. Il6 deficiency during this stage up-regulated Npy and Agrp expression, while restoring Pomc mRNA levels to virgin values. Together these results demonstrate that IL6/IL6Ra system modulates Npy/Agrp, Pomc and Trh expression during mouse pregnancy, supporting a role of IL6 in the central regulation of body fat in this physiological state.

The test chemical selection procedure of the European Centre for the Validation of Alternative Methods for the EU Project ReProTect.

The selection of reference compounds is crucial for a successful in vitro test development in order to proof the relevance of the test system. This publication describes the criteria and the selection strategy leading to a list of more than 130 chemicals suitable for test development within the ReProTect project. The presented chemical inventory aimed to support the development and optimization of in vitro tests that seek to fulfill ECVAM's criteria for entering into the prevalidation. In order to select appropriate substances, a primary database was established compiling information from existing databases. In a second step, predefined selection criteria have been applied to obtain a comprehensive list ready to undergo a peer review process from independent experts with industrial, academic and regulatory background. Finally, a peer reviewed chemical list containing 13 substances challenging endocrine disrupter tests, additional 50 substances serving as reference chemicals for various tests evaluating effects on male and female fertility, and finally 61 substances were identified as known to provoke effects on the early development of mammalian offspring. The final list aims to cover relevant and specific mode/site of actions as they are known to be relevant for various substance classes. However, the recommended list should not be interpreted as a list of reproductive toxicants, because such a description requires proven associations with adverse effects of mammalian reproduction, which are subject of regulatory decisions done by involved competent authorities.

Culturing cells without serum: lessons learnt using molecules of plant origin.

To successfully grow cells in serum-free medium is an interesting challenge to cell biology. The use of such media for in vitro cell culture work would be a key contribution to the 3Rs concept, enabling the avoidance of the use of animals and animal products at all stages of the experiment. In addition, numerous problems related to virus infections transmitted by animal serum would be avoided, thus increasing reproducibility. Prolifix is a new reagent of plant origin. It contains a molecule (GCR 1003) that has an activity similar to that of the mitogenic molecules in serum and could be suitable to substitute serum in culture medium. Two epithelial cell lines, LLC-PK1 and Caco-2, were progressively adapted to a special culture medium containing 10% Prolifix in the absence of serum. After adaptation, cell cultures were characterised. We found that these reagents of plant origin could be promising alternatives to serum. However, more work and effort is needed to improve cell adaptation, cell attachment, growth rates as well as freezing/thawing protocols.

New insights into the mechanisms involved in renal proximal tubular damage induced in vitro by ochratoxin A.

The mycotoxin ochratoxin A is a contaminant of human and animal food products. It is a potent nephrotoxin known to damage the proximal tubule. The aim of this work was to investigate the effects of ochratoxin A on a porcine renal proximal tubular epithelial cell line (LLC-PK1), and to identify sensitive endpoints revealing damage at the epithelial barrier level and at the molecular level. Cells exposed for 24 h to 5-10 microM ochratoxin indicated a clear damage to the intactness of the epithelial barrier, as shown by measurements of trans-epithelial resistance and zonula occludens-1 protein expression. At the mitochondrial level we observed alterations of the normal functions, such as an increase of the membrane potential, the formation of straight extensions, and the formation of giant mitochondria. At higher ochratoxin concentrations (50 microM), at which cytotoxicity assays revealed a significant toxicity, alterations of the cytoskeleton organization and induction of apoptosis were evident. In addition, we analyzed the expression of genes by using a cDNA macroarray. Our data indicate that ochratoxin-induced nephrotoxicity can be detected at the barrier and at the mitochondrial level at rather low concentrations, at which conventional cytotoxicity assays are unable to reveal toxic effects.

Comparison of the sensitivity of different toxicological endpoints in Caco-2 cells after cadmium chloride treatment.

The human colorectal adenocarcinoma cell line Caco-2 is a widely used in vitro model of the intestinal barrier. Cadmium chloride (CdCl(2)) is a highly toxic metal compound, ubiquitous in the biosphere, able to enter the food chain and to reach the intestinal epithelium, causing structural and functional damages. The aim of this work was to characterise cadmium toxicity in Caco-2 cells and, in particular, to compare the sensitivity of different endpoints revealing damage both on the epithelial barrier and at the cellular or molecular level. After 24-h exposure of the cells to CdCl(2), lactate dehydrogenase (LDH) leakage showed cadmium-induced cell toxicity, significant from 25 microM CdCl(2) and above, and analysis of different cell death pathways indicated the presence of necrosis after treatment with 50 microM CdCl(2). At the molecular level, we observed an increase in the protective protein heat shock protein 70 (HSP70), starting at 10 microM CdCl(2). At the barrier level, transepithelial electrical resistance (TEER) decreased while paracellular permeability (PCP) significantly increased after the treatment, showing an EC(50) of 6 and 16 microM CdCl(2), respectively, and indicating the loss of barrier integrity. In conclusion, our data reveal that CdCl(2) toxicity in Caco-2 cells can be detected at the barrier level at very low concentrations; also, HSP70 was shown to be a sensitive marker for detecting in vitro cadmium-induced toxicity.

Long-term in vitro toxicity models: comparisons between a flow-cell bioreactor, a static-cell bioreactor and static cell cultures.

In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects. The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse, Integra, Wallisellen, Switzerland) and a static cell bioreactor system (CELLine CL 6-well, Integra), and to compare them with the use of conventional cell culture flasks. A human cell line, Int 407, was exposed to cadmium chloride (CdCl(2); 10-(7-)10-(8)M) for 4 weeks. Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay. In addition, total protein determination and cadmium uptake measurements were performed. The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl(2) concentration. However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl(2). Expression of heat-shock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl(2) concentration applied and the system used. In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies. It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available.

Inducción de adenocarcinomas mamarios por progestagenos en ratones BALB/c. Progresión del tumor hormono-dependiente al autónomo / Progestin-induced mammary adenocarcinomas in BALB/c mice. Progression from hormone-dependent to autonomous tumors

We have developed an experimental model in which the administration of progestins induces mammary tumors in female virgin BALB/c mice. In this paper we review the morphological and biological features of progestin-induced tumors, such as estrogen receptor (ER) and progesterone receptor (PR) patterns of expression, hormone dependence and epidermal growth factor receptors (EGF-R) we also examine our data concerning the systemic effects of medroxyprogesterone acetate (MPA) as regards its stimulating EGF synthesis in salivary glands and its subsequent increase in serum. This growth factor seems to play an important role in the induction of mammary tumors. Direct MPA proliferative effects mediated by PR were demonstrated using primary cultures of progestin-dependent (PD) mammary tumors. Antiprogestins inhibited cell growth beyond control values, suggesting that PR are involved in cell proliferation even in the absence of the ligand. Progesterone-independent (PI) tumors expressing high levels of PR and ER are also inhibited by estrogen or antiprogestin treatment, suggesting that PR are involved in the control of autonomous tumor growth. Estrogen-resistant variants may be selected which may revert to an estrogen-sensitive phenotype after several transplants in untreated mice. The similarities between the tumors obtained with this model and human breast cancer as regards morphological features, evolution and the regulation of growth control converts this model into a useful tool to explore the mechanisms related with acquisition of hormone independence and autonomous tumor growth.

Inducción de adenocarcinomas mamarios por progestagenos en ratones BALB/c. Progresión del tumor hormono-dependiente al autónomo / Progestin-induced mammary adenocarcinomas in BALB/c mice. Progression from hormone-dependent to autonomous tumors

We have developed an experimental model in which the administration of progestins induces mammary tumors in female virgin BALB/c mice. In this paper we review the morphological and biological features of progestin-induced tumors, such as estrogen receptor (ER) and progesterone receptor (PR) patterns of expression, hormone dependence and epidermal growth factor receptors (EGF-R) we also examine our data concerning the systemic effects of medroxyprogesterone acetate (MPA) as regards its stimulating EGF synthesis in salivary glands and its subsequent increase in serum. This growth factor seems to play an important role in the induction of mammary tumors. Direct MPA proliferative effects mediated by PR were demonstrated using primary cultures of progestin-dependent (PD) mammary tumors. Antiprogestins inhibited cell growth beyond control values, suggesting that PR are involved in cell proliferation even in the absence of the ligand. Progesterone-independent (PI) tumors expressing high levels of PR and ER are also inhibited by estrogen or antiprogestin treatment, suggesting that PR are involved in the control of autonomous tumor growth. Estrogen-resistant variants may be selected which may revert to an estrogen-sensitive phenotype after several transplants in untreated mice. The similarities between the tumors obtained with this model and human breast cancer as regards morphological features, evolution and the regulation of growth control converts this model into a useful tool to explore the mechanisms related with acquisition of hormone independence and autonomous tumor growth.(Au)

Regularización hormonal de los adenocarcinomas mamarios inducidos por acetato de medroxiprogesterona estudios "in vivo" e "in vitro" / Hormonal regulation of the mammary adenocarcinoma induced by medroxiprogesterone acetate: studies \"in vivo\" and \"in vitro\"

En nuestro modelo experimental el acetato de medroxiprogesterona induce adenocarcinomas de mama en hembras vírgenes de la cepa BALB/c. Estos tumores, con receptores para progesterona y estrógenos, originaron líneas de crecimiento hormono-dependientes (HD) o independiente (HI)..La progesterona y el MPA estímulan el crecimiento de las HD y los estrógenos inhiben el crecimiento de las HD y HI. En este trabajo demostramos que cultivos primarios de tumores HD conservan la misma sensibilidad hormonal que los tumores parenterales: la proliferación celular aumenta con concentraciones de MPA 10-9-10-7 M y es inhibida con estrógenos 10-9 o 10-7 M aun en presencia de MPA. Estos resultados sugerirían que las hormonas actúan directamente sobre las células tumorales. En experimentos "in vivo" demostraron que animales tratados con progesterona también desarrollaron adenocarcinomas de mama aunque la incidencia fue menor que en los tratados con MPA. Curiosamente se observó que la mayoría de los adenocarcinomas de mama inducidos por progesterona eran lobulillares y III mientras que en los tratados con MPA la mayoría fue ductal y HD. También se demostró que la ovariectomía y la sialoadenectomía disminuyen el poder carcinogénico del MPA sin alterar el patrón morfológico: 70 por ciento ductal y HD

Regularización hormonal de los adenocarcinomas mamarios inducidos por acetato de medroxiprogesterona estudios "in vivo" e "in vitro" / Hormonal regulation of the mammary adenocarcinoma induced by medroxiprogesterone acetate: studies "in vivo" and "in vitro"

En nuestro modelo experimental el acetato de medroxiprogesterona induce adenocarcinomas de mama en hembras vírgenes de la cepa BALB/c. Estos tumores, con receptores para progesterona y estrógenos, originaron líneas de crecimiento hormono-dependientes (HD) o independiente (HI)..La progesterona y el MPA estímulan el crecimiento de las HD y los estrógenos inhiben el crecimiento de las HD y HI. En este trabajo demostramos que cultivos primarios de tumores HD conservan la misma sensibilidad hormonal que los tumores parenterales: la proliferación celular aumenta con concentraciones de MPA 10-9-10-7 M y es inhibida con estrógenos 10-9 o 10-7 M aun en presencia de MPA. Estos resultados sugerirían que las hormonas actúan directamente sobre las células tumorales. En experimentos "in vivo" demostraron que animales tratados con progesterona también desarrollaron adenocarcinomas de mama aunque la incidencia fue menor que en los tratados con MPA. Curiosamente se observó que la mayoría de los adenocarcinomas de mama inducidos por progesterona eran lobulillares y III mientras que en los tratados con MPA la mayoría fue ductal y HD. También se demostró que la ovariectomía y la sialoadenectomía disminuyen el poder carcinogénico del MPA sin alterar el patrón morfológico: 70 por ciento ductal y HD