Effects of ghrelin on nocturnal melatonin secretion in sheep: An in vitro and in vivo approach.

Recent studies confirmed that pineal melatonin (MEL) secretion is regulated by ghrelin (GHRL) in seasonally reproductive sheep. The first in vivo experiment investigated whether the effect of GHRL on nocturnal secretion of MEL in sheep is mediated by type 2 serotonin receptors. Sheep ( = 16) were intravenously injected with GHRL (2.5 µg/kg of BW) and meta-Chlorophenylpiperazine (mCPP; a mixed agonist of 5-HT2B/5-HT2C receptors; 1 mg/kg BW), either combined or individually, during the short-day (SDS) and long-day (LDS) seasons. Blood samples were collected at 15-min intervals for 4 h. The second in vitro experiment examined the effect of GHRL (10 ng/mL) on MEL secretion by pineal gland (PG) explants incubated for 5 h. The expression levels and/or concentrations of tryptophan 5-hydroxylase 1 (TPH1), aralkylamine N-acetyltransferase (AA-NAT), and the phosphorylated form of AA-NAT (p31T-AA-NAT) were determined at selected time points during the SDS and LDS seasons. The experiments demonstrated that GHRL reduced MEL secretion ( < 0.01) during the SDS season. Administration of mCPP or a combination of GHRL + mCPP stimulated MEL secretion ( < 0.01) regardless of the season. Furthermore, GHRL regulated nightly MEL secretion in a TPH1-independent manner. However, during the SDS season, GHRL reduced p31T-AA-NAT expression and the AA-NAT concentration ( < 0.01) and inhibited MEL secretion ( < 0.001), whereas during the LDS season, GHRL had no effect on MEL secretion or on the expression of the examined enzymes. These findings indicate that GHRL directly and indirectly affects PG activity in sheep and that the photoperiod modulates the effects of GHRL.

Inhibition of blood platelet adhesion by phenolics' rich fraction of Hippophae rhamnoides L. fruits.

Beneficial influence of fruits on human health may be their ability to prevent the hyperactivation of blood platelets and cardiovascular disorders. Effects of the phenolic fraction from Hippophae rhamnoides fruit on different stages of blood platelet activation (platelet adhesion and aggregation) were studied in vitro. We also examined effects of the H. rhamnoides fraction on metabolism of thiol groups, which plays an important role in platelet functions. The effects of the H. rhamnoides fraction on adhesion of blood platelets to collagen and fibrinogen were determined with Tuszynski's and Murphy's method. The platelet aggregation was determined with turbidimetry. The action of the H. rhamnoides fraction on the level of thiol groups in platelet proteins and a level of glutathione (GSH) in platelets was estimated with 5,5'-dithio-bis(2-nitro-benzoic acid). The tested fraction of H. rhamnoides (0.5 - 50 µg/ml; 30 min of the incubation time 30 min) inhibited blood platelets adhesion to collagen and fibrinogen. The effect of the tested fraction on blood platelet adhesion depended on concentration of fraction. In presence of the highest tested concentration which was 50 µg/ml, inhibition of platelet adhesion for thrombin-activated platelets was about 55%. On the other hand, tested plant fraction had no anti-aggregatory properties. Our results showed anti-adhesive properties of phenolic fraction from H. rhamnoides fruit and we suggest that it may be beneficial for prevention of cardiovascular diseases.

Phenomenon of leptin resistance in seasonal animals: the failure of leptin action in the brain.

The core of the leptin resistance hypothesis promulgated several years ago to explain obesity as a result of environmental causes consists of 2 tenets: the extinction of leptin-induced intracellular signaling downstream of leptin binding to the long form of the neuronal receptor LTRb in the hypothalamus and the impedance to leptin entry imposed at the blood-brain barrier (BBB). A recent comprehensive investigation concluded that a central leptin insufficiency associated with obesity can be attributed to a decreased efficiency of BBB leptin transport and not to leptin insensitivity within the hypothalamus. Interestingly, anorectic leptin's effects are counteracted in some individuals by a natural resistance associated with hyperleptinemia, which is related to changes in hypothalamic sensitivity to leptin (eg, due to malnutrition, obesity, or seasonal variations due to day-length-dependent reproduction changes). In sheep, it has been observed that the hypothalamus is resistant to leptin in some periods, which is related to the adaptation of these animals to annual changes in energy supply and demand. However, a broad range of ambiguities exists regarding the implications that the intracellular signaling of signal transducer and activator of transcription-2/suppressor of cytokine signaling 3 (STAT2/SOCS3) imparts central leptin resistance. Furthermore, several plausible alternative possibilities have been proposed, such as compensatory functional and anatomic reorganizations in the appetite regulating network, rearrangements in the afferent hormonal feedback signaling involved in weight homeostasis, and modifications in leptin transport to the hypothalamus across the BBB. Taken together, these observations suggest that the contention that impaired intracellular signaling downstream of leptin entry into the appetite regulating network expedites environmentally induced obesity remains unsubstantiated and requires further evidence. Furthermore, pregnancy decreases hypothalamic sensitivity to leptin (or other unknown mechanisms), and lactation can also alter the appetite-suppressing central activity of leptin. The objective of this review was to offer an approach to understanding (1) how information regarding nutritional status is transmitted to and interpreted within the hypothalamus in animals, with special attention on seasonally breeding animals and (2) whether central leptin resistance and/or leptin insufficiency in the hypothalamus favors the development of obesity.

Influence of season and nutritional status on the direct effects of leptin, orexin-A and ghrelin on luteinizing hormone and growth hormone secretion in the ovine pituitary explant model.

The aim of this study was to examine whether leptin (anorexigenic peptide), orexin-A, and ghrelin (orexigenic peptides) could directly (ie, independently of hypothalamic influences) affect the secretion of luteinizing hormone (LH) and growth hormone (GH) by adenohypophyseal (AP) explants obtained from normally fed or fasted (48 h) ewes during the breeding and nonbreeding seasons. In addition, a specific ovine super leptin antagonist (SLAN-3) was used to assess the interactions between leptin and ghrelin and/or orexin-A. Pituitary glands from 16 ovariectomized Polish Longwool ewes that had received estradiol-releasing subcutaneous implants were collected in the breeding (November; n = 8) and nonbreeding (May; n = 8) seasons. The AP explants were incubated for 240 min in a gas-liquid interface and treated with leptin (50 ng/mL), ghrelin (100 ng/mL), orexin-A (100 ng/mL), and SLAN-3 (500 ng/mL) with orexin-A or ghrelin. Treatments with leptin and SLAN-3 + orexin-A increased (P < 0.05) LH concentrations in the cultures of AP explants from fasted animals in the breeding season. Orexin-A increased (P < 0.05) LH secretion by AP explants from both fasted and fed animals in the breeding season. Ghrelin stimulated (P < 0.05) GH secretion by AP explants collected from fasted animals in nonbreeding season and from normally fed ewes in both seasons. Leptin decreased (P < 0.05) GH secretion by AP explants collected from fasted ewes in both seasons and from nonfasted ewes in the breeding season. However, the treatment with SLAN-3 + ghrelin resulted in greater (P < 0.05) GH concentrations compared with leptin treatment of AP explants from fasted ewes in the breeding season and from normally fed ewes in nonbreeding season. In summary, leptin, orexin-A, and ghrelin exerted direct effects on AP secretory function in an ex situ model and both the reproductive season and nutritional status of the animals impinged on the direct effects of the peptides on LH and GH release. Specifically, orexin-A was more potent than leptin in directly stimulating LH secretion in cycling ewes, whereas ghrelin and leptin generally had opposing effects on the secretory function of somatotrophs in sheep.

Seasonal changes in the interactions among leptin, ghrelin, and orexin in sheep.

The adaptation of the physiology of an animal to changing conditions of light and food availability is evident at the behavioral and hormonal levels. Melatonin, leptin, ghrelin, and orexin, which exhibit rhythmic secretion profiles under ad libitum feeding conditions, are sensitive to changes in daylength, forming a tight web of interrelationships in the regulation of energy balance. The aim of this study was to determine the effects of central injections of leptin, ghrelin, and orexin on the reciprocal interactions among these hormones and the influence of photoperiod on these responses. Twenty-four ovariectomized and estradiol-implanted ewes were used in a replicated switchback design. The ewes were assigned randomly to 1 of 6 treatment groups, and the treatments were infused into their third ventricles 3 times at 0, 1, and 2 h, with 0 h being at dusk. The treatments were as follows: 1) control, Ringer-Locke buffer; 2) leptin, 0.5 µg/kg BW; 3) ghrelin, 2.5 µg/kg BW; 4) orexin B, 0.3 µg/kg BW; 5) leptin antagonist, 50 µg/kg BW, then ghrelin, 2.5 µg/kg BW; and 6) leptin antagonist, 50 µg/kg BW, then orexin B, 0.3 µg/kg BW. Blood samples (5 mL) were collected at 15-min intervals for 6 h. The administration of leptin increased (P < 0.05) plasma concentrations of melatonin during short-day (ShD) photoperiods and decreased (P < 0.05) them during long-day (LD) photoperiods, whereas ghrelin decreased (P < 0.05) melatonin concentrations during ShD photoperiod, and orexin had no effect (P > 0.1). Leptin attenuated (P < 0.05) ghrelin concentrations relative to the concentration in controls during ShD. The plasma concentrations of orexin were reduced (P < 0.05) after leptin infusions during LD and ShD photoperiods; however, ghrelin had the opposite effect (P < 0.05) on orexin concentration. Orexin increased (P < 0.05) ghrelin concentrations during LD. Ghrelin and orexin concentrations were increased (P < 0.05) after leptin antagonist infusions. Our data provide evidence that the secretion of leptin, ghrelin, and orexin are seasonally dependent, with relationships that are subject to photoperiodic regulation, and that leptin is an important factor that regulates ghrelin and orexin releases in sheep.

Seasonal effects of central leptin infusion and prolactin treatment on pituitary SOCS-3 gene expression in ewes.

Suppressors of cytokine signalling (SOCS) negatively regulate cytokine-induced signalling pathways and may be involved in leptin and prolactin (PRL) interactions. Herein, we examined the effect of PRL on SOCS-3 mRNA expression in pituitary explants and investigated whether leptin could modify the expression of SOCS-3 mRNA in pituitary explants. In the first experiment, we used pituitaries isolated from 16 ewes decapitated in March, May, July and October (four per month). Tissues were cut into 50 mg explants, which were treated with control or medium containing PRL (100 or 300 ng/ml). Incubation was maintained for different time intervals: 0, 60, 120, 180, 240 or 300 min. Real-time PCR was used to measure SOCS-3 mRNA levels. In the second study, we used 24 ewes surgically fitted with third ventricle cannulas (12 were used during the long-day period, and 12 were used during the short-day (SD) period). Each ewe was administered an i.c.v. injection of Ringer-Locke buffer or leptin (0.5 or 1.0 µg/kg body weight). Explants of anterior pituitaries were collected and snap frozen 1 h after injection. Semi-quantitative expression of SOCS-3 mRNA was performed using reverse transcription-PCR. PRL stimulated SOCS-3 expression in the pituitaries collected in March (P<0.05) and May (P<0.01 and P<0.05 for lower and higher doses respectively), inhibited SOCS-3 expression in pituitaries collected in July (P<0.01) and had no effect in pituitaries collected in October. Treatment with leptin increased SOCS-3 expression during the SDs in a dose-dependent manner (P<0.01). The results demonstrated that photoperiod may be involved in leptin and PRL effects on SOCS-3 expression in sheep.

Seasonal effects of central leptin infusion on secretion of melatonin and prolactin and on SOCS-3 gene expression in ewes.

Recent studies have demonstrated photoperiodic changes in leptin sensitivity of seasonal mammals. Herein, we examined the interaction of season (long days (LD) versus short days (SD)) and recombinant ovine leptin (roleptin) on secretion of melatonin and prolactin (PRL) and on mRNA expression of suppressor of cytokine signaling-3 (SOCS-3) in the medial basal hypothalamus (MBH) in sheep. Twenty-four Polish Longwool ewes, surgically fitted with third ventricle (IIIV) cannulas, were utilized in a replicated switchback design involving 12 ewes per season. Within-season and replicate ewes were assigned randomly to one of three treatments (four ewes/treatment) and infused centrally three times at 0, 1 and 2 h beginning at sunset. Treatments were 1) control, Ringer-Locke buffer; 2) L1, roleptin, 0.5 microg/kg BW; and 3) L2, roleptin, 1.0 microg/kg BW. Jugular blood samples were collected at 15-min intervals beginning immediately before the start of infusions and continued for 6 h. At the end of blood sampling, a washout period of at least 3 days elapsed before ewes were re-randomized and treated with one of the treatments described above (four ewes/treatment). Ewes were then killed and brains were collected for MBH processing. Leptin treatments increased (P<0.001) circulating leptin concentrations compared with controls during both seasons in a dose-dependent manner. Overall, mean plasma concentrations of melatonin were greater (P<0.001) during LD than SD. However, leptin treatments increased melatonin concentrations during SD in a dose-dependent manner and decreased it during LD. Similarly, plasma concentrations of PRL were greater (P<0.001) during LD than SD. However, unlike changes in melatonin, circulating PRL decreased (P<0.001) in response to leptin during LD. Semi-quantitative PCR revealed that leptin increased (P<0.001) SOCS-3 expression in the MBH region during LD in a dose-dependent manner. Data provide evidence that secretion of photoperiodic hormones such as melatonin and PRL are inversely regulated by leptin during SD and LD. However, the increase in expression of SOCS-3 in the MBH during LD compared with SD fails to fully explain these effects.

Leptin as a nutritional signal regulating appetite and reproductive processes in seasonally-breeding ruminants.

Photoperiod and nutrition both exert major influences on reproduction. Thus, it seems axiomatic that seasonal rhythms in ovulation are influenced by nutrition. In this context, leptin is one of the most important hormonal signals involved in the control of energy homeostasis, feeding behavior and reproductive function in mammals. However, the number of published investigations establishing a functional interaction between leptin and photoperiodism in seasonal breeders is limited. In common with most seasonally-breeding mammals, sheep exhibit robust circannual cycles in body weight and reproduction, which are driven mainly by changes in day-length. Recently, attention has focused on the role of leptin in this process, particularly in its roles as a major peripheral signal controlling appetite, melatonin and prolactin secretion. The purpose herein is to review current concepts in the overall biology of leptin, to summarize its influence on the hypothalamic-pituitary axis, and to highlight recent developments in our understanding of its interaction with season in regulating appetite, body weight and reproduction in seasonally-breeding mammals. The latter observations may be important in delineating states of leptin resistance and obesity in humans.

[The review of the European Union documents relating to contamination of aflatoxins in food]. / Przeglad dokumentów Unii Europejskiej dotyczacych zanieczyszczenia zywnosci aflatoksynami.

In the following review, Polish and European Union legislation, concerning maximum level of aflatoxins in foodstuffs, was reported. In this moment, no specific requirements exist but according to Food and Nutrition Law all kind of food ought to be free from aflatoxins. Practically, maximum aflatoxins level should be below the detection limit of official analytical method (thin layer chromatography)--5 micrograms/kg and 0.05 microgram/l for milk. Commission Directive 98/53/EC laying down the sampling methods and the methods of analysis for aflatoxins in foodstuffs and Commission Regulation No 152/98 setting maximum levels for aflatoxins in foodstuffs were presented. Also, Ministry of Health Draft Regulation, concerning maximum levels of contamination in food is prepared in accordance with EU regulation, was reported.

[Determination of natamycin residues in ripening cheeses by high-performance liquid chromatography]. / Oznaczanie pozostalosci natamycyny w serach dojrzewajacych metoda wysokosprawnej chromatografii cieczowej.

The purpose of the study was to determine the usefulness of high performance liquid chromatography (HPLC) for natamycin determination in routine control of ripening cheeses. In the method the antibiotic is extracted from the studied sample with a 2:1 methanol/water solution, freezing of contaminants at -18 degrees C and determination of HPLC using a RP C8 column and UV detection. In case of low concentrations of the antibiotic the extract was condensed by extraction to solid phase (SPE). In the study of the fortified samples the basic analytical parameters of the method were tested (determinability, repeatability, recovery) and its usefulness in the 0.05-0.4 mg/kg concentration range (by SPE) and above 0.5 mg/kg of cheese was checked. Recovery was from 87 to 98%, repeatability was 1.3-7.3% and determinability was 0.5 mg/kg (using SPE 0.05 mg/kg). The obtained results demonstrated a good usefulness of HPLC for routine control of natamycin content in ripening cheeses. It seems recommendable to apply HPLC for natamycin determination in ripening cheese by sanitary-epidemiological stations.

[Determination of antibiotic residues and other inhibitors in milk--quality assurance system]. / Oznaczanie zawartosci antybiotyków i innych substancji hamujacych w mleku--system zapewniania jakosci.

In the methods used widely for the determination of antibiotic residues in food product of animal origin the antibiotic-sensitivity of microorganisms is used. An advantage of microbiological methods is their high detection rate, but they are not specific. The test strains are not selectively sensitive and are inhibited by many antibiotics. False positive results of these tests may be due to the presence in tissues or milk of natural substance, e.g. enzymes or compounds of external origin--detergents or other drugs, e.g. sulphonamides. The microbiological tests--plate and tube STD and Polutest, as well as the enzymatic Penzym test, all recommended in the Polish Standards PN-91/A-86033 for milk control for the detection of antibiotic residues were compared. The authors describe the requirements to be met by the systems of quality ensuring during determination of antibiotics and other inhibitors in milk. The requirements to be met by the methods of this determination are discussed, together with the principles of intralaboratory and interlaboratory quality controls.

[Residues of antibiotics and other inhibitory substances in milk]. / Pozostalosci antybiotyków i innych substancji hamujacych w mleku.

The aim of the study was determination of the contamination of raw milk, milk for consumption and powdered milk with antibiotics and inhibitory substances. The study was carried out in the years 1990-1993 with the assistance of the Province Sanitary-Epidemiological Stations using methods agreeing with the Polish Norms 91/A-86033: the enzymatic test Penzym, the microbiological tests S.STD Polulest and the plate method. In all, 16334 samples were tested. Depending on the method used the number of positive samples was: in raw milk: from 13.1 to 22.4% in milk for consumption: from 10.5 to 19.5% in powdered milk: from 12.9 to 18.2%. These results point out that the health quality of milk is insufficient, both milk for processing and milk in the market failing to meet the standards. The source of the seem to be inadequate hygienic conditions during milking and, especially, failure to meet the necessary requirements for keeping the time period during which milk is not suitable for consumption after treatment with antibiotics.

[Therapeutic assessment of the evening primrose]. / Ocena zdrowotna nasion z wiesiolka.

In the light of a survey of literature data the chemical composition of described of evening primrose seeds and of the oil obtained from them which has a high content of unsaturated fatty acids, mainly linolic and gamma-linolenic acid. The therapeutic value of the preparations of evening primrose and their effect on the metabolism are discussed. The criteria of the therapeutic quality of the seeds are given.