PUFA Changes in White Adipose Tissue during Hibernation in Common Hamsters.

AbstractHibernators save energy during winter by expressing torpor bouts characterized by strongly reduced body temperature and metabolic rate. Polyunsaturated fatty acids (PUFAs), specifically n-6 PUFAs, are known to positively affect hibernation performance and thereby energy savings predominantly in fat-storing hibernators. Accordingly, hibernators usually retain PUFAs and mobilize monounsaturated fatty acids (MUFAs) or saturated fatty acids (SFAs) during hibernation. In food-storing common hamsters (Cricetus cricetus), however, we previously found that PUFA proportions in white adipose tissue (WAT) decreased during winter, indicating that individuals did mobilize PUFAs. To further investigate these patterns, we analyzed PUFA changes in WAT during hibernation as well as hibernation performance in free-ranging and captive common hamsters with lower prehibernation PUFA proportions compared to those in the previous study. Under controlled conditions, total PUFAs, n-6 PUFAs, and SFAs increased while n-3 PUFAs and MUFAs decreased during hibernation. Higher prehibernation n-6 PUFA proportions resulted in fewer torpor bouts and less time spent in torpor. In free-ranging hamsters, n-6 PUFAs increased while n-3 PUFAs and SFAs decreased during winter. Prehibernation n-6 PUFA proportions, however, did not affect hibernation performance. In summary, these results indicate that the mobilization or retention of n-6 PUFAs during hibernation could depend on their availability in WAT or in the diet before the onset of the hibernation period.

Effects of dietary fatty acids on the social life of male Guinea pigs from adolescence to adulthood.

Dietary intake of polyunsaturated fatty acids (PUFAs) or saturated fatty acids (SFAs) differently modulates neurophysiological and behavioral functions in response to altered hypothalamic-pituitary-adrenal (HPA)-axis activity and an individual's development. In this context, an individual's social environment, including social interactions and social hierarchies, is closely related to hormone concentrations and possibly interacts with dietary fatty acid effects. We investigated if dietary supplementation with walnut oil (high in PUFAs) and coconut fat (high in SFAs), compared to a control group, affects body mass gain, cortisol and testosterone concentrations, plasma fatty acids, and social behavior in male domestic guinea pigs from adolescence to adulthood. For analyses of cortisol and testosterone concentrations, social interactions were included as covariates in order to consider effects of social behavior on hormone concentrations. Our results revealed that SFAs increased escalated conflicts like fights and stimulated cortisol and testosterone concentrations, which limited body mass gain and first-year survival. PUFAs did not remarkably affect social behavior and hormone concentrations, but enabled the strongest body mass gain, which probably resulted from an energetic advantage. Neither sociopositive nor agonistic behaviors explained age-specific differences in hormone concentrations between groups. However, a high number of subdominant individuals and lower testosterone concentrations were related to increased cortisol concentrations in adult PUFA males. Our findings demonstrate the importance of dietary fatty acids regarding behavioral and endocrine developmental processes and adaptations to the social environment by modulating HPA-axis function and body homeostasis.

Inhibition of Lipid Accumulation in Skeletal Muscle and Liver Cells: A Protective Mechanism of Bilirubin Against Diabetes Mellitus Type 2.

Ectopic lipid accumulation in skeletal muscle and liver drives the pathogenesis of diabetes mellitus type 2 (DMT2). Mild hyperbilirubinaemia has been repeatedly suggested to play a role in the prevention of DMT2 and is known for its capacity to shape an improved lipid phenotype in humans and in animals. To date, the effect of bilirubin on lipid accumulation in tissues that are prone to ectopic lipid deposition is unclear. Therefore, we analyzed the effect of bilirubin on lipid accumulation in skeletal muscle and liver cell lines. C2C12 skeletal mouse muscle and HepG2 human liver cells were treated with physiological concentrations of free fatty acids (FFA) (0.5 mM and 1 mM) and unconjugated bilirubin (UCB) (17.1 and 55 µM). The intracellular presence of UCB upon exogenous UCB administration was confirmed by HPLC and the lipid accumulation was assessed by using Nile red. Exposure of both cell lines to UCB significantly reduced lipid accumulation by up to 23% (p ≤ 0.001) in HepG2 and by up to 17% (p ≤ 0.01) in C2C12 cells at 0.5 and 5 h under hypoglycaemic conditions. Simultaneously, UCB slightly increased FFA uptake in HepG2 cells after 0.5 and 5 h and in C2C12 cells after 12 h as confirmed by gas chromatographic analyses of the remaining FFA content in the incubation media. The effects of UCB on lipid accumulation and uptake were abolished in the presence of higher glucose concentrations. Monitoring the uptake of a radiolabeled glucose analogue [18F]FDG: (2-deoxy-2-[18F]fluoro-D-glucose) into both cell types further indicated higher glucose consumption in the presence of UCB. In conclusion, our findings show that UCB considerably decreases lipid accumulation in skeletal muscle and liver cells within a short incubation time of max. 5 h which suggests that mildly elevated bilirubin levels could lower ectopic lipid deposition, a major key element in the pathogenesis of DMT2.

Dietary fatty acids sex-specifically modulate guinea pig postnatal development via cortisol concentrations.

Early ontogenetic periods and postnatal maturation in organisms are sex-specifically sensitive to hypothalamic-pituitary-adrenal (HPA)-axis activities, related glucocorticoid secretions, and their effects on energy balance and homeostasis. Dietary polyunsaturated (PUFAs) and saturated (SFAs) fatty acids potentially play a major role in this context because PUFAs positively affect HPA-axis functions and a shift towards SFAs may impair body homeostasis. Here we show that dietary PUFAs positively affect postnatal body mass gain and diminish negative glucocorticoid-effects on structural growth rates in male guinea pigs. In contrast, SFAs increased glucocorticoid concentrations, which positively affected testes size and testosterone concentrations in males, but limited their body mass gain and first year survival rate. No distinct diet-related effects were detectable on female growth rates. These results highlight the importance of PUFAs in balancing body homeostasis during male's juvenile development, which clearly derived from a sex-specific energetic advantage of dietary PUFA intakes compared to SFAs.

Effects of food store quality on hibernation performance in common hamsters.

Hibernating animals can adjust torpor expression according to available energy reserves. Besides the quantity, the quality of energy reserves could play an important role for overwintering strategies. Common hamsters are food-storing hibernators and show high individual variation in hibernation performance, which might be related to the quality of food hoards in the hibernacula. In this study, we tested the effects of food stores high in fat content, particularly polyunsaturated fatty acids (PUFAs), on hibernation patterns under laboratory conditions. Control animals received standard rodent pellets only, while in the other group pellets were supplemented with sunflower seeds. We recorded body temperature during winter using subcutaneously implanted data loggers, documented total food consumption during winter, and analysed PUFA proportions in white adipose tissue (WAT) before and after the winter period. About half of the individuals in both groups hibernated and torpor expression did not differ between these animals. Among the high-fat group, however, individuals with high sunflower seeds intake strongly reduced the time spent in deep torpor. PUFA proportions in WAT decreased during winter in both groups and this decline was positively related to the time an individual spent in deep torpor. Sunflower seeds intake dampened the PUFA decline resulting in higher PUFA levels in animals of the high-fat group after winter. In conclusion, our results showed that common hamsters adjusted torpor expression and food intake in relation to the total energy of food reserves, underlining the importance of food hoard quality on hibernation performance.

Reproductive performance and gestational effort in relation to dietary fatty acids in guinea pigs.

BACKGROUND: Dietary saturated (SFAs) and polyunsaturated (PUFAs) fatty acids can highly affect reproductive functions by providing additional energy, modulating the biochemical properties of tissues, and hormone secretions. In precocial mammals such as domestic guinea pigs the offspring is born highly developed. Gestation might be the most critical reproductive period in this species and dietary fatty acids may profoundly influence the gestational effort. We therefore determined the hormonal status at conception, the reproductive success, and body mass changes during gestation in guinea pigs maintained on diets high in PUFAs or SFAs, or a control diet. RESULTS: The diets significantly affected the females' plasma fatty acid status at conception, while cortisol and estrogen levels did not differ among groups. SFA females exhibited a significantly lower body mass and litter size, while the individual birth mass of pups did not differ among groups and a general higher pup mortality rate in larger litters was diminished by PUFAs and SFAs. The gestational effort, determined by a mother's body mass gain during gestation, increased with total litter mass, whereas this increase was lowest in SFA and highest in PUFA individuals. The mother's body mass after parturition did not differ among groups and was positively affected by the total litter mass in PUFA females. CONCLUSIONS: While SFAs reduce the litter size, but also the gestational effort as a consequence, PUFA supplementation may contribute to an adjustment of energy accumulations to the total litter mass, which may both favor a mother's body condition at parturition and perhaps increase the offspring survival at birth.

Sex-specific effects of dietary fatty acids on saliva cortisol and social behavior in guinea pigs under different social environmental conditions.

BACKGROUND: Unbalanced dietary intakes of saturated (SFAs) and polyunsaturated (PUFAs) fatty acids can profoundly influence the hypothalamic-pituitary-adrenal (HPA)-axis and glucocorticoid secretions in relation to behavioral performances. The beneficial effects of higher dietary PUFA intakes and PUFA:SFA ratios may also affect social interactions and social-living per se, where adequate physiological and behavioral responses are essential to cope with unstable social environmental conditions. METHODS: Effects of diets high in PUFAs or SFAs and a control diet were investigated in male and female guinea pigs after 60 days of supplementation. Plasma fatty acid patterns served as an indicator of the general fatty acid status. HPA-axis activities, determined by measuring saliva cortisol concentrations, social behaviors, and hierarchy ranks were analyzed during group housing of established single-sexed groups and during challenging social confrontations with unfamiliar individuals of the other groups. RESULTS: The plasma PUFA:SFA ratio was highest in PUFA supplemented animals, with female levels significantly exceeding males, and lowest in SFA animals. SFA males and females showed increased saliva cortisol levels and decreased aggressiveness during group housing, while sociopositive behaviors were lowest in PUFA males. Males generally showed higher cortisol increases in response to the challenging social confrontations with unfamiliar individuals than females. While increasing cortisol concentrations were detected in control and PUFA animals, no such effect was found in SFA animals. During social confrontations, PUFA males showed higher levels of agonistic and sociopositive behaviors and also gained higher dominance ranks among males, which was not detected for females. CONCLUSIONS: While SFAs seemingly impaired cortisol responses and social behaviors, PUFAs enabled adequate behavioral responses in male individuals under stressful new social environmental conditions. This sex-specific effect was possibly related to a general sex difference in the n-3 PUFA bioavailability and cortisol responses, which may indicate that males are more susceptible to changing environmental conditions, and shows how dietary fatty acids can shape social systems.

Melatonin: Free Radicals and Metabolites Resulting by Emission and Consumption of Solvated Electrons (eaq-): Reaction Mechanisms.

BACKGROUND/AIM: Melatonin not only regulates circadian rhythm, but also induces apoptosis in tumor cells. Hence, elucidation of the basic reaction mechanisms of melatonin and its metabolites is a matter of interest. MATERIAL AND METHODS: Melatonin dissolved in a mixture of water/ethanol=40/60 form associates (unstable complexes). For simulation of biological processes, melatonin was excited by UV light into the singlet state. RESULTS: By using monochromatic UV light (λ=254 nm) melatonin ejects solvated electrons (eaq (-)), a part of which is scavenged by melatonin in ground state contained in the associates. Consequently, with increase of melatonin concentration a decrease of the determined quantum yield of emitted eaq (-), Q(eaq (-)), is obtained. The complex molecular structure of melatonin contains functional groups which can emit eaq (-), as well such consuming eaq (-). As a succession of these processes various types of metabolites are generated, as well as degradation products, with lower molecular weight, are formed. CONCLUSION: Not melatonin per se, but the ejected eaq (-) and thereby resulting various metabolites are responsible for different biological properties of melatonin.

Corticosterone metabolites originating as a consequence of solvated electron (e(-)aq) emission.

Corticosterone in water-ethanol solution can eject "solvated electrons" (eaq(-)) when excited into the singlet state by monochromatic UV-light (λ=254 nm). As a consequence of this process free radicals and H(+) ions were also generated. Hence, the objectives of this study were to determine the quantum yield, Q, at different corticosterone concentrations, and elucidate the fate of the generated free radicals and the involved reaction mechanisms. Because of the formation of associates, which consume a part of the emitted eaq(-), the Q decrease with increase of cortisone concentration. Additionally the H(+) ions scavenge and convert a part of the ejected eaq(-) into H-atoms. In comparsion with progesterone, the Q of corticosterone is much higher. Evidently, this effect is due to the two OH groups of corticosterone, which act as intense emission centres for eaq(-). Thereby, the generated free radicals from corticosterone lead to formation of metabolites, which were analyzed by combination of liquid-chromatography with mass spectrometry (LC/MS) method. Two of them were identified: 5α-pregnan-3α, 21-diol-11, 20-dione and 20ß-dihydroxycortisone. Both have the same mass number of 348.230. To explain the involved, rather complicated processes, a probable reaction mechanism is suggested.

Formation of sex hormone transients resulting from attack of free radicals.

BACKGROUND: Transients of the sex hormones testosterone (TES) and estrone (E1) exhibit an impact on the carcinogenesis of most prostate and breast cancer types. For elucidation of involved reaction mechanisms, in vitro, experiments using γ-ray for generation of attacking hormone transients and UV-light (λ=254 nm) for excitation of hormone molecules were applied. Materials and Methods. Experiments in vitro (Escherichia coli AB1157) incubated with TES and E1, individually as well as in mixture with vitamin C (electron donor), were performed under γ-irradiation in water-alcohol (40/60) medium for clarifying-up the reaction mechanism. The hormone degradation/regeneration processes were studied by high performance liquid chromatography analysis. RESULTS: Independently of hormone molecular structure, the determining factor for the biological properties, such as carcinogenity, were found to be based on the hormone transients. The biological ability of these, however, depends on the chemical properties of the species attacking the corresponding hormone. Hormone degradation can be, at least partly, converted into hormone regeneration by electron transfer from an electron donor (e.g. vitamin C), when available during the period of status nascendi of the hormone radicals.

Photo-induced regeneration of hormones by electron transfer processes: Potential biological and medical consequences.

Based on the previous results concerning electron transfer processes in biological substances, it was of interest to investigate if hormone transients resulting by e.g. electron emission can be regenerated.The presented results prove for the first time that the hormone transients originating by the electron emission process can be successfully regenerated by the transfer of electrons from a potent electron donor, such as vitamin C (VitC). Investigations were performed using progesterone (PRG), testosterone (TES) and estrone (E1) as representatives of hormones. By irradiation with monochromatic UV light (λ=254 nm) in a media of 40% water and 60% ethanol, the degradation as well as the regeneration of the hormones was studied with each hormone individually and in the mixture with VitC as a function of the absorbed UV dose, using HPLC. Calculated from the obtained initial yields, the determined regeneration of PRG amounted to 52.7%, for TES to 58.6% and for E1 to 90.9%. The consumption of VitC was determined in the same way.The reported results concerning the regeneration of hormones by the transfer of electrons from an electron donor offer a new, promising method for the therapy with hormones. As a consequence of the regeneration of hormones, a decreased formation of carcinogenic metabolites is expected.

The effect of progesterone on the electron emission and degradation of testosterone.

Based on recent findings that hormones can emit electrons () from their excited singlet state in polar media, it was of importance to study a possible mutual interaction of progesterone (PRG) and testosterone (TES) in this respect. Hormones of highest purity were dissolved in an air-free mixture of 40% triply distilled water and 60% ethanol, because the hormones are unsoluble in water. As energy source for substrate excitation in singlet state served a monochromatic UV-light (254 nm), the emitted electrons were scavenged by chloroethanol, whereby the quantum yield of produced Cl⁻ ions, Q (Cl⁻), is equal to Q(e⁻(aq)). Hormone degradation initiated by the electron emission was studied by HPLC method, using a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm, 5 µm). The quantum yield of emitted e⁻(aq), Q(e⁻(aq)), from TES was 3.6 times higher than that from PRG, which is explained by the different molecular structures of the hormones. Observed 2nd and 3rd maxima of electron emission indicate the ability of TES and PRG products to also eject e⁻(aq), but with lower yield. It can be stated that a part of the emitted electrons from TES are consumed by PRG⁺ leading to a partial regeneration of hormone. The present results offer a deeper insight in the biological behavior of hormones.

Electron emission and product analysis of estrone: progesterone interactions studied by experiments in vitro.

Recent studies showed that hormones like progesterone, testosterone, etc. can eject [Formula: see text] (solvated electrons). By means of electron transfer processes via the brain, the hormones communicate with other biological systems in the organism. The present study proves that also estrone is able to emit electrons. Their yield strongly depends on the concentration of the hormone, temperature and on the absorbed energy. The metabolites resulting from this process are likewise able to generate electrons, however with much smaller yields. The formation of the estrone metabolites is studied by HPLC-analyses. In vitro experiments with MCF-7 cells demonstrate the distinct effect of progesterone on the carcinogenity of estrone metabolites. Probable reaction mechanisms for explanation of the observed effects are postulated.

Metabolite formation of 17alpha-hydroxyprogesterone as a consequence of e⁻(aq)-emission and progesterone effect regarding cancer.

Based on previous investigations on several hormones, 17α-hydroxyprogesterone (17α-HOPRG) was studied in respect to cancer initiation by its metabolites resulting from electron emission. The emission of electrons (e⁻(aq)) from its singlet excited state of 17α-HOPRG and HPLC-analysis of products were studied. Possible carcinogenicity of metabolites originating from 17α-HOPRG and the effect of progesterone (PRG) in this respect were studied in vitro. The results showed that 17α-HOPRG is very sensitive towards oxygen. The highest Q(e⁻(aq)) values were obtained by dissolution and UV-irradiation of substrate in airfree media. 17α-HOPRG metabolites showed a strong anticancer activity, which is, however, lower compared to that of PRG-metabolites. Mixture of both hormones, 17α-HOPRG and PRG, in respect to carcinogenicity showed a synergistic effect of PRG on 17α-HOPRG. Reaction mechanisms are presented.

17beta-estradiol acting as an electron mediator: experiments in vitro.

BACKGROUND: The present work reports on the effect of oxidizing (OH, O(2)(*-)) and reducing free radicals (e(-)(aq), H) on 17beta-estradiol (17betaE2) in respect to breast cancer initiation. The objectives of the study were based on the following premise: the ability of 17betaE2 to emit electrons (e(-)(aq)) as well as to transfer them to other biological systems. Thereby, the resulting transient hormone products are leading to the formation of metabolites, some of which may initiate the neoplastic process. The present work considers the effect of the simultaneously generated oxidizing and reducing free radicals on the carcinogenic properties of the 17betaE2 metabolites. MATERIALS AND METHODS: Water-soluble 17betaE2 with incorporated 2-hydroxypropyl-beta-cyclodextrin (HBC) in various aqueous media (pH ~7.4), saturated with air, N(2)O or argon, as well as HBC alone, were exposed to the action of free radicals produced by gamma-ray. Escherichia coli bacteria (AB 1157) were used as a model for living systems. RESULTS: From the survival curves obtained under different conditions, the derived DeltaD(37) values (representing the radiation dose at which N/N(0)=0.37; N/N(0) ratio: N(0)=starting number of colonies, N=number after irradiation treatment) illustrate that 17betaE2 as well as HBC act as very powerful scavengers of OH and O(2)(*-) radicals. On the other hand, 17betaE2 and HBC intermediates resulting from attack of the reducing species (e(-)(aq), H) have strong anticancer properties. CONCLUSION: It is stated that DeltaD(37) values strongly depend on the reactivity of the individual free radicals. Oxidizing free radicals lead to positive DeltaD(37) values, illustrating the strongly pronounced radiation protecting ability of the systems. On the contrary, the primary reducing free radicals result in negative DeltaD(37) values, indicating anticancer effect.

The 4-hydroxyestrone: Electron emission, formation of secondary metabolites and mechanisms of carcinogenesis.

4-Hydroxyestrone (4-OHE(1)), a typical cancer-inducing metabolite, originating from 17beta-estradiol (17beta-E2), was chosen as a model for the studies. The aim was to get a deeper insight in the mechanisms of its ability to initiate cancer. It was found, that 4-OHE(1) can eject electrons (e(aq)(-)), when excited in the singlet state by monochromatic UV-light (lambda=254 nm) in polar media (water:ethanol=40:60 vol.%). The quantum yield Q(e(aq)(-)), determined for various 4-OHE(1) concentrations, is found to be as high as that previously observed for 17beta-E2. It decreases with increasing substrate concentration, but it is enhanced at higher temperature. The ability of 4-OHE(1) to eject as well as to consume and to transfer electrons to other biological systems, classifies it as an electron mediator, similar to 17beta-E2. The 4-OHE(1) transients resulting of the electron emission process are leading to the formation of secondary metabolites. Surprisingly, it was established that the secondary metabolites possess likewise the ability to eject as well as to consume electrons. Hence, they behave similar like 17beta-E2. However, the structure of the secondary formed metabolites, which determinates their biological properties and carcinogenity, depends on the nature of the available reaction partners involved in their formation. A probable reaction mechanism explaining the subject matter is discussed.

Electron emission from photo-excited testosterone in water-ethanol solution.

Testosterone (TES; 4-androstene-17beta-ol-3-on) is found for the first time to eject electrons from its singlet excited state in water-ethanol solvent mixture. This ability was very recently also observed for 17beta-estradiol (17betaE2) and progesterone (PRG)/1/. With increasing TES-concentration, the yield of solvated electrons (e(s)(-)) is decreasing, because of "associate" formation. At higher absorbed UV-doses (lambda=254 nm) the e(s)(-) yield is passing a sharp maximum by formation of TES-ethanol adducts, which are able likewise to emit electrons when excited. At prolonged irradiation the resulting photolytic products of TES-ethanol adducts are also able to emit electrons. The capability of the hormones: 17betaE2, PRG and TES to eject electrons and the resulting metabolites, some of which can induce cancer, is discussed.

Photo-induced electron emission from 17beta-estradiol and progesterone and possible biological consequences.

It was established for the first time, that the sexual hormones 17beta-estradiol (17betaE2) and progesterone (PRG) are able to emit electrons from their excited single state in water-ethanol mixtures. The yield of the "solvated electrons" (e(s)(-)) depends on the substrate concentration, the ratio of water-alcohol-mixtures and the temperature. The e(s)(-) yield obtained from 17betaE2 is by two orders of magnitude higher than this of PRG. The possible relationship of the resulting hormone transients from 17betaE2 leading via specific metabolites to breast cancer is discussed.