Different Hypothalamic Mechanisms Control Decreased Circulating Thyroid Hormone Levels in Infection and Fasting-Induced Non-Thyroidal Illness Syndrome in Male Thyroid Hormone Action Indicator Mice.

Background: Non-Thyroidal Illness Syndrome (NTIS) caused by infection or fasting is hallmarked by reduced circulating thyroid hormone (TH) levels. To better understand the role of local TH-action in the development of NTIS, we assessed tissue-specific changes of TH signaling in Thyroid Hormone Action Indicator (THAI) mice. Methods: NTIS was induced in young adult THAI mice by bacterial lipopolysaccharide (LPS)-administration or by 24 or 48 hours' fasting. Tissue-specific TH-action was assessed by the detection of changes of the Luciferase reporter of THAI mice with quantitative polymerase chain reaction along with tissue-specific examination of regulators of TH metabolism and signaling. Age dependence of revealed alterations of hypothalamic TH-action was also studied in 1-year-old male THAI mice. Results: LPS-treatment increased TH-action in the hypothalamic arcuate nucleus-median eminence (ARC-ME) region preceded by an increase of type 2 deiodinase (D2) expression in the same region and followed by the suppression of proTrh expression in the hypothalamic paraventricular nucleus (PVN). In contrast, LPS decreased both TH-action and D2 activity in the pituitary at both ages. Tshß expression and serum free thyroxine (fT4) and free triiodothyronine (fT3) levels decreased in LPS-treated young adults. Tshß expression and serum fT4 levels were not significantly affected by LPS treatment in aged animals. In contrast to LPS treatment, TH-action remained unchanged in the ARC-ME of 24 and 48 hours fasted animals accompanied with a modest decrease of proTrh expression in the PVN in the 24-hour group. Tshß expression and fT3 level were decreased in both fasted groups, but the fT4 decreased only in the 48 hours fasted animals. Conclusions: Although the hypothalamo-pituitary-thyroid (HPT) axis is inhibited both in LPS and fasting-induced NTIS, LPS achieves this by centrally inducing local hyperthyroidism in the ARC-ME region, while fasting acts without affecting hypothalamic TH signaling. Lack of downregulation of Tshß and fT4 in LPS-treated aged THAI mice suggests age-dependent alterations in the responsiveness of the HPT axis. The LPS-induced tissue-specific hypo-, eu-, and hyperthyroidism in different tissues of the same animal indicate that under certain conditions TH levels alone could be a poor marker of tissue TH signaling. In conclusion, decreased circulating TH levels in these two forms of NTIS are associated with different patterns of hypothalamic TH signaling.

Expression of glucagon-like peptide 1 receptor in neuropeptide Y neurons of the arcuate nucleus in mice.

Glucagon-like peptide 1 (GLP-1) and its agonists exert anorexigenic effect at least partly via acting on GLP-1 receptors (GLP-1R) in the arcuate nucleus (ARC). While the anorexigenic, proopiomelanocortin (POMC) neurons of the ARC were shown previously to express GLP-1R, the putative GLP-1R-content of the orexigenic, neuropeptide Y (NPY) neurons remained so far undetected. As GLP-1R is abundant in the ventromedial ARC, where NPY neurons are located; here, we address the possibility that GLP-1 can act directly on the orexigenic NPY system via GLP-1R. Double-labeling immunocytochemistry and in situ hybridization were performed on tissues of adult male mice to detect GLP-1R in NPY neurons. In double-immunolabeled preparations, GLP-1R-immunoreactivity was observed in NPY neurons and in axons ensheathing the majority of NPY neurons. Ultrastructural studies confirmed that GLP-1R-immunoreactivity is associated with the outer membrane of NPY perikarya as well as with axons forming symmetric type, inhibitory synapses on NPY-containing neurons. Double-labeling in situ hybridization experiments demonstrated the expression of GLP-1R mRNA in approximately 20% of NPY mRNA-containing neurons of the ARC. In summary, our data demonstrate the presence of GLP-1R protein and mRNA in NPY neurons of ARC and also reveal the innervation of NPY neurons by GLP-1R-containing inhibitory neurons. These observations suggest that GLP-1 signaling can influence NPY neurons both directly and indirectly. Furthermore, GLP-1 signaling on energy homeostasis appears to involve both direct and indirect effects of GLP-1 on the orexigenic NPY neurons, in addition to the previously known effects via the anorexigenic POMC neuronal system.

Application of BisANS fluorescent dye for developing a novel protein assay.

In many biology- and chemistry-related research fields and experiments the quantification of the peptide and/or protein concentration in samples are essential. Every research environment has unique requirements, e.g. metal ions, incubation times, photostability, pH, protease inhibitors, chelators, detergents, etc. A new protein assay may be adequate in different experiments beyond or instead of the well-known standard protocols (e.g. Qubit, Bradford or bicinchoninic acid) in related conceptions. Based on our previous studies, we developed a novel protein assay applying the 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (BisANS) fluorescent dye. This molecule has several advantageous properties related to protein detection: good solubility in water, high photostability at adequate pH, quick interaction kinetics (within seconds) with proteins and no exclusionary sensitivity to the chelator, detergent and inhibitor ingredients. The protocol described in this work is highly sensitive in a large spectrum to detect protein (100-fold diluted samples) concentrations (from 0.28 up to more than 100 µg/mL). The BisANS protein assay is valid and applicable for quantification of the amount of protein in different biological and/or chemical samples.

Triterpenes from the Mushroom Hypholoma lateritium: Isolation, Structure Determination and Investigation in Bdelloid Rotifer Assays.

Twelve compounds (1⁻12) were isolated from the methanol extract of brick cap mushroom (Hypholoma lateritium (Schaeff.) P. Kumm.). The structures of the compounds were elucidated using extensive spectroscopic analyses, including NMR and MS measurements. Lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (1) and 8-hydroxy-13-oxo-9E,11E-octa-decadienoic acid (2) were identified as new natural products, together with ten known compounds, from which 3ß-hydroxyergosta-7,22-diene (4), demethylincisterol A2 (5), cerevisterol (6), 3ß-O-glucopyranosyl-5,8-epidioxyergosta-6,22-diene (7), fasciculol E (9), and uridine (12) were identified in this species for the first time. The isolated triterpenes (1, 3⁻11) were investigated for their toxicity in vivo using bdelloid rotifer assays. Most of the examined steroids in general showed low toxicity, although the effects of the compounds varied in a wider range from the non-toxic lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (1) to the significantly toxic cerevisterol (6), with substantial dependence in some cases on the presence of nutrient in the experimental environment.

Redox Modulating Factors Affect Longevity Regulation in Rotifers.

Rotifers are microinvertebrate models to study the phylogenetically based mechanisms of aging. Our study aimed to develop a physiological system with electron deprivation via a chemical electron carrier/acceptor pair together with extreme caloric restriction (ECR). Middle-aged Philodina acuticornis rotifers were treated with combinations of phenazine methosulfate (PMS, electron carrier) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT, electron acceptor) for a period of 72 hours under total food deprivation (preselection). The ability of XTT to be reduced was confirmed both in vitro (with NADH) and in vivo (with live rotifers). Subsequently, the respective electron acceptor alone at a lower dose was administered in combination with ECR for several months on preselected survivors. We found that the longevity of rotifers markedly increased (4×) after PMS/XTT/total food deprivation preselection followed by XTT/ECR treatment. Ascorbic acid in equivalent concentrations caused similar but less pronounced tendencies. The synergistic effect of chemical electron deprivation and ECR caused delayed aging and the development of an outstanding phenotype that we refer to as "super rotifers," characterized by increased longevity and retained reproductive ability compared with normal middle-aged individuals. The presented model provides new insights into the connection between redox modulation and age-related features in vivo.

Biological Activities of Four Adaptogenic Plant Extracts and Their Active Substances on a Rotifer Model.

Rotifers have been widely used as well-characterized models of aging, since their multiorgan character makes them suitable as in vivo toxicological and lifespan models. Here we report the assessment of four adaptogenic plants and their extracts for the first time in this model. The effects on rotifer viability of extracts and characteristic active markers of Panax ginseng, Withania somnifera, Leuzea carthamoides, and Rhodiola rosea were tested in vivo. The crude extracts were nontoxic to Philodina acuticornis bdelloid rotifers; however, the pure substances of the plants influenced negatively the viability. Ginsenoside Rb1 and secondary metabolites of Withania somnifera exerted deleterious effect on the animals. The aglycone tyrosol and cinnamyl alcohol (from Rhodiola rosea) were more toxic than their glycosides salidroside and rosavin. Although the 20-OH-ecdysone and ajugasterone C (from Leuzea carthamoides) are chemically very similar, the latter was less toxic.

Exceptional in vivo catabolism of neurodegeneration-related aggregates.

Neurodegenerative diseases are linked to a systemic enzyme resistance of toxic aggregated molecules and their pathological consequences. This paper presents a unique phenomenon that Philodina acuticornis, a bdelloid rotifer, is able to catabolize different types of neurotoxic peptide and protein aggregates (such as beta-amyloids /Aß/, alpha-synuclein, and prion) without suffering any damage. P. acuticornis is capable of using these aggregates as an exclusive energy source (i.e., as 'food', identified in the digestive system and body) in a hermetically isolated microdrop environment, increasing their survival. As regards Aß1-42, five other bdelloid rotifer species were also found to be able to perform this phenomenon. Based on our experiments, the Aß1-42-treated bdelloid rotifers demonstrate significantly increased survival (e.g. mean lifespan = 51 ± 2.71 days) compared to their untreated controls (e.g. mean lifespan = 14 ± 2.29 days), with similar improvements in a variety of phenotypic characteristics. To our knowledge, no other animal species have so far been reported to have a similar capability. For all other microscopic species tested, including monogonant rotifers and non-rotifers, the treatment with Aß1-42 aggregates proved to be either toxic or simply ineffective. This paper describes and proves the existence of an unprecedented in vivo catabolic capability of neurotoxic aggregates by bdelloid rotifers, with special focus on P. acuticornis. Our results may provide the basis for a new preclinical perspective on therapeutic research in human neurodegenerative diseases.

In vivo screening of diterpene alkaloids using bdelloid rotifer assays.

The group of diterpene alkaloids contains numerous compounds with complex chemistry and diverse pharmacological activities. Beside toxicity, these compounds possess activity on the cardiovascular system, tumor cell lines and nervous system. The pharmacological properties have been described using in vitro and in vivo techniques; however, the bioactivities of many compounds have not thoroughly been studied. Here we report on the in vivo evaluation of ten diterpene alkaloids using bdelloid rotifer assays. Napelline exerted toxic effects on rotifers, while wide tolerance range was observed for other investigated compounds. Weak toxicity of songorine is supported by our experiment. Toxicological data for senbusine A, senbusine C, septentrioidine and hetisinone are reported for the first time.

Novel in vivo experimental viability assays with high sensitivity and throughput capacity using a bdelloid rotifer.

Rotifers have been used in biological research as well-characterized models of aging. Their multi-organ characters and their sensitivity for chemicals and environmental changes make them useful as in vivo toxicological and lifespan models. Our aim was to create a bdelloid rotifer model to use in high-throughput viability and non-invasive assays. In order to identify our species Philodina acuticornis odiosa (PA), 18S rDNA-based phylogenetic analysis was carried out and their species-specific morphological markers identified. To execute the rotifer-based experiments, we developed an oil-covered water-drop methodology adapted from human in vitro fertilization techniques. This enables toxicological observations of individual one-housed rotifers in a closed and controllable micro-environment for up to several weeks. Hydrogen peroxide (H2O2) and sodium azide (NaN3) exposures were used as well-understood toxins. The toxicity and survival lifespan (TSL), the bright light disturbance (BLD) the mastax contraction frequency (MCF) and the cellular reduction capacity (CRC), indices were recorded. These newly developed assays were used to test the effects of lethal and sublethal doses of the toxins. The results showed the expected dose-dependent decrease in indices. These four different assays can either be used independently or as an integrated system for studying rotifers. These new indices render the PA invertebrate rotifer model a quantitative system for measuring viability, toxicity and lifespan (with TSL), systemic reaction capacity (with BLD), organic functionality (with MCF) and reductive capability of rotifers (with CRC), in vivo. This novel multi-level system is a reliable, sensitive and replicable screening tool with potential application in pharmaceutical science.