Long-term changes in the diurnal temporal regulation and set points of metabolic parameters associated with chronic maternal overnutrition in rabbits.

Metabolic syndrome (MS) and obesity have become a worldwide epidemic with an alarming prevalence in women of reproductive age. Maternal metabolic condition is considered a risk factor for adverse birth outcomes and long-term MS. In this study, we developed a rabbit model of maternal overnutrition via the chronic intake of a high-fat and carbohydrate diet (HFCD), and we determined the effects of this diet on maternal metabolism and offspring metabolic set points and temporal metabolic regulation in adult life. Before and during pregnancy, the female rabbits that consumed the HFCD exhibited significant changes in body weight, serum levels of analytes associated with carbohydrate and lipid metabolism, levels of liver and kidney damage markers, and liver histology. Our data suggest that rabbits are a valuable model for studying the development of MS associated with the chronic intake of unbalanced diets and fetal metabolic programming. Furthermore, the offspring of overnourished dams exhibited considerable changes in 24-h serum metabolite profiles in adulthood, with notable sexual dimorphism. These data suggest that maternal nutritional conditions due to the chronic intake of an HFCD adversely impact key elements related to the development of circadian rhythmicity in offspring.NEW & NOTEWORTHY Maternal overnutrition previous and during pregnancy leads to long-term changes in the 24-h regulation and setpoint of metabolic profiles of the offspring.

Misadjustment of diurnal expression of core temperature and locomotor activity in lactating rabbits associated with maternal over-nutrition before and during pregnancy.

Metabolic parameters ranging from circulating nutrient levels and substrate utilization to energy expenditure and thermogenesis are temporally modulated by the circadian timing system. During critical embryonic developmental periods, maternal over-nutrition could alter key elements in different tissues associated with the generation of circadian rhythmicity, compromising normal rhythmicity development. To address this issue, we determine whether maternal over-nutrition leads to alterations in the development of circadian rhythmicity at physiological and behavioral levels in the offspring. For this, female rabbits were fed a standard diet (SD) or high-fat and carbohydrate diet (HFCD) before mating and during gestation. Core body temperature and gross locomotor activity were continuously recorded in newborn rabbits, daily measurements of body weight and the amount of milk ingested was carried out. At the end of lactation, tissue samples, including brown adipose tissue (BAT) and white adipose tissue (WAT), were obtained for determining the expression of uncoupling protein-1 (UCP1) and cell death-inducing DNA fragmentation factor-like effector A (CIDEA) genes. HFCD pups exhibited conspicuous differences in the development of the daily rhythm of temperature and locomotor activity compared to the SD pups, including a significant increase in the daily mean core temperature, changes in the time when temperature or activity remains above the average, shifts in the acrophase, decrease in the duration and intensity of the anticipatory rise previous to nursing, and changes in frequency of the rhythms. HFCD pups exhibited a significant increase in BAT thermogenesis markers, and a decrease of these markers in WAT, indicating more heat generation by brown adipocytes and alterations in the browning process. These results indicate that maternal over-nutrition alters offspring homeostatic and chronostatic regulation at the physiological and behavioral levels. Further studies are needed to determine whether these alterations are associated with the changes in the organization of the circadian system of the progeny.

Temporal variations of nucleosides and nucleotides in rabbit milk.

Nucleotides and nucleosides have a preeminent role in physiological and biochemical processes for newborns, the major source of these during early development is the breast milk. Different biomolecules exhibit daily fluctuations in maternal milk that could transfer temporal information that synchronize newborn circadian system. As a first approach, we characterized the diurnal profile of nucleotides and nucleosides contained in maternal milk of rabbits during the first week of lactation. It is possible that some nucleosides, such as adenosine, play a relevant role in setting up the emerging circadian rhythmicity, whereas uridine and guanosine could participate in the maintenance of rhythmicity.

Utility of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as biomarker to predict therapeutic response to methotrexate in rheumatoid arthritis.

The objective of this study was to investigate the usefulness of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in predicting short-term therapeutic response to methotrexate (MTX) in rheumatoid arthritis (RA). Patients with active RA, with Disease Activity Score-28 joints (DAS-28) >3.2, starting oral MTX, were included. We measured at baseline, 3 and 6 mo: DAS-28, Health Assessment Questionnaire-Disability Index (HAQ-DI), patient's perception of disease severity, morning stiffness and pain, as well as modifications in sTREM-1 levels. A reduction in DAS-28 > 1.2 at 3 or 6 mo was considered adequate response. A significant decrease in DAS-28 was observed at 3 and 6 mo. HAQ-DI also decreased at 3 and 6 mo. No significant changes were observed in sTREM-1 levels at 3 or 6 mo. Using as cut-off a baseline value of sTREM-1 levels > 390 pg/ml, we obtained low values of sensitivity (61.5%), specificity (59.3%), positive predictive value (59.3%) and negative predictive value (61.5%) for adequate response to MTX at 3 mo. We found no clinical value of sTREM-1 levels in predicting therapeutic response to MTX in RA. Further studies should evaluate if sTREM-1 levels are predictive for other outcomes, including higher structural damage or good response to biologics.

Participation of the Olfactory Bulb in Circadian Organization during Early Postnatal Life in Rabbits.

Experimental evidence indicates that during pre-visual stages of development in mammals, circadian regulation is still not under the control of the light-entrainable hypothalamic pacemaker, raising the possibility that the circadian rhythmicity that occurs during postnatal development is under the control of peripheral oscillators, such as the main olfactory bulb (MOB). We evaluated the outcome of olfactory bulbectomy on the temporal pattern of core body temperature and gross locomotor activity in newborn rabbits. From postnatal day 1 (P1), pups were randomly assigned to one of the following conditions: intact pups (INT), intact pups fed by enteral gavage (INT+ENT), sham operated pups (SHAM), pups with unilateral lesions of the olfactory bulb (OBx-UNI), and pups with bilateral lesions of the olfactory bulb (OBx-BI). At the beginning of the experiment, from P1-8, the animals in all groups were fed at 11:00, from P9-13 the feeding schedule was delayed 6 h (17:00), and finally, from P14-15 the animals were subjected to fasting conditions. The rabbit pups of the INT, INT+ENT, SHAM and OBx-UNI groups exhibited a clear circadian rhythmicity in body temperature and locomotor activity, with a conspicuous anticipatory rise hours prior to the nursing or feeding schedule, which persisted even during fasting conditions. In addition, phase delays in the nursing or feeding schedule induced a clear phase shift in both parameters. In contrast, the OBx-BI group exhibited atypical rhythmicity in both parameters under entrained conditions that altered the anticipatory component, as well as deficient phase control of both rhythms. The present results demonstrate that the expression of circadian rhythmicity at behavioral and physiological levels during early stages of rabbit development largely depends on the integrity of the main olfactory bulb.

Maternal olfactory cues synchronize the circadian system of artificially raised newborn rabbits.

In European newborn rabbits, once-daily nursing acts as a strong non-photic entraining cue for the pre-visual circadian system. Nevertheless, there is a lack of information regarding which of the non-photic cues are capable of modulating pup circadian system. In this study, for the first time, we determined that the mammary pheromone 2-methylbut-2-enal (2MB2) presented in the maternal milk acts as a non-photic entraining cue. We evaluated the effect of once-daily exposure to maternal olfactory cues on the temporal pattern of core body temperature, gross locomotor activity and metabolic variables (liver weight, serum glucose, triacylglycerides, free fatty acids, cholecystokinin and cholesterol levels) in newborn rabbits. Rabbit pups were separated from their mothers from postnatal day 1 (P1) to P8 and were randomly assigned to one of the following conditions: nursed by a lactating doe (NAT); exposed to a 3-min pulse of maternal milk (M-Milk), mammary pheromone (2MB2), or water (H2O). To eliminate maternal stimulation, the pups of the last three groups were artificially fed once every 24-h. On P8, the rabbits were sacrificed at different times of the day. In temperature and activity, the NAT, M-Milk and 2MB2 groups exhibited clear diurnal rhythmicity with a conspicuous anticipatory rise hours prior to nursing. In contrast, the H2O group exhibited atypical rhythmicity in both parameters, lacking the anticipatory component. At the metabolic level, all of the groups exhibited a diurnal pattern with similar phases in liver weight and metabolites examined. The results obtained in this study suggest that during pre-visual stages of development, the circadian system of newborn rabbits is sensitive to the maternal olfactory cues contained in milk, indicating that these cues function as non-photic entraining signals mainly for the central oscillators regulating the expression of temperature and behavior, whereas in metabolic diurnal rhythmicity, these cues lack an effect, indicating that peripheral oscillators respond to milk administration.

Determining the period, phase and anticipatory component of activity and temperature patterns in newborn rabbits that were maintained under a daily nursing schedule and fasting conditions.

During the last decade, lagomorphs have gained relevance as valuable models for the study of the development of circadian rhythmicity. This relevance is due to both the peculiar behavior of the lactating doe, in which maternal care is limited from 3 to 5 min per day, and the temporal organization that newborn rabbits exhibit during the early stages of development. In this study, we characterized the development of the temporal pattern of core body temperature and locomotor activity of newborn rabbits. This activity was recorded simultaneously for individual newborn rabbits and was maintained under constant light conditions, a 24-h nursing schedule and without access to the lactating doe. In addition, different mathematical algorithms were designed to determine the period, phase and anticipatory component of the time series obtained for the newborn rabbits. During the first two weeks of life, the average gross locomotor activity decreased as age increased; conversely however, the core body temperature exhibited a significant increment during the early stages of postnatal development. The newborn rabbits' circadian patterns of activity and temperature were consolidated as early as the first week of life. Similarly, the acrophase and nadir of both rhythms were settled by postnatal day 5, and the maximum activity consistently occurred approximately 2 h before the animals' maximum body temperature. The anticipation of nursing was evident from postnatal day 2 for both parameters, and the duration and intensity showed changes associated with the stage of development. In addition, the anticipatory component persisted with the same duration and intensity, even when nursing was omitted. The mathematical methods used in this study are suitable for producing unbiased analyses of the time series that are obtained from developing animals in situations during which biological signals generally show variability in frequencies and trends. By using these methods, it was possible to establish that circadian rhythmicity at the behavioral and physiological levels was evident during the first week of age in newborn rabbits. This circadian rhythmicity represents an endogenous rhythm because it persists throughout constant conditions.

Nongenomic uterine relaxing effect of RU 486 (mifepristone) prior to its antiprogesterone activity in the human pregnancy.

The antiprogestin mifepristone (RU 486) is used for termination of pregnancy, as RU 486 blocks the quiescent action of progesterone, increases uterine contractility, sensitizes the myometrium to prostaglandins, and elicits cervical ripening. Since RU 486 represents a class of compound that is structurally related to steroid hormones, some of which possess a nongenomic uterine relaxing effect, we investigated the potential nongenomic relaxing action of RU 486 on the human pregnant myometrium. Myometrial tissues were obtained from pregnant women undergoing elective cesarean section at term and were isometrically recorded. RU 486 caused relaxation on spontaneous contractility and high potassium-induced contractions with lower relaxing efficacy than progesterone. The progesterone receptor-blocking activity of RU 486 did not antagonize the uterine relaxation of progesterone. Moreover, contractions induced by oxytocin or different prostaglandins (PGF(2alpha), PGE(2), and a prostaglandin analogue, misoprostol) were inhibited rather than increased by RU 486. RU 486 induced a rapid and reversible relaxing effect, which was unaffected by inhibitors of protein synthesis and transcription, implying that RU 486 acts through a nongenomic mechanism. This study reveals that RU 486: (i) reduced high potassium-induced contraction and prevented calcium-induced contraction in depolarized tissue; and (ii) relaxed the oxytocin- and prostaglandin-induced contractions, indicating a blockade of voltage- and receptor-operated calcium channels by RU 486. These data show that this antiprogestin may induce a rapid nongenomic antiuterotonic effect prior to its antiprogesterone action.

The modulatory role of androgens and progestins in the induction of vasorelaxation in human umbilical artery.

Sex steroids have been described as protectors of the cardiovascular system and one of their relevant actions is inhibition of vascular tone. However, this information has been derived from animal models. The aim of this study was to investigate the vasorelaxant properties of several progestins and androgens on the vascular tone of human umbilical artery (HUA) to elucidate their potential regulatory role on fetoplacental blood flow. HUA rings, obtained from umbilical cords at vaginal deliveries and cesarean section from term uncomplicated pregnancies, were isometrically recorded and precontracted with either KCl or serotonin. Subsequently, dehydroepiandrosterone, testosterone, progesterone and some of their 5-reduced metabolites were added at different noncumulative concentrations on KCl-induced precontraction. There were significant differences in the vasorelaxing responses to these steroids; excluding 5alpha-pregnandione, the remaining steroids induced concentration-dependent vasorelaxations. In general, androgens were more potent than progestins, with 5beta-dihydrotestosterone being the most potent one. These vasorelaxations remained unaffected by inhibitors of transcription and translation, selective steroid receptor antagonists, a nitric oxide synthase inhibitor or specific blockers of K(+) channels. Interestingly, the serotonin contraction was significantly less sensitive to steroid-induced vasorelaxation. Moreover, the contraction evoked by Ca(2+) in depolarized tissues (by KCl-Ca(2+) free solution) was prevented by steroids. These data, taken together, suggest that sex steroids (particularly androgens) induce an acute (nongenomically-mediated) vasorelaxing effect on the HUA which may be mediated by: (i) a nitric oxide-independent pathway; and/or (ii) a decrease in external Ca(2+) influx by inactivating Ca(2+) channels, but not by activating K(+) channels.

Evidence that 17alpha-estradiol is biologically active in the uterine tissue: antiuterotonic and antiuterotrophic action.

BACKGROUND: 17alpha-Estradiol has been considered as the hormonally inactive isomer of 17beta-estradiol. Recently, nongenomic (smooth muscle relaxation) and genomic (light estrogenic activity) effects of 17alpha-estradiol have been reported, but no reports have yet determined its possible antiestrogenic activity. Therefore, this study investigated: the nongenomic action of 17alpha-estradiol on uterine contractile activity and its potential agonist-antagonist activity on uterine growth. METHODS: Uterine rings from rats were isometrically recorded. Different concentrations (0.2-200 microM) of 17alpha-estradiol were tested on spontaneous contraction and equimolarly compared with 17beta-estradiol. To examine the mechanism of 17alpha-estradiol action, its effect was studied in presence of beta2-antagonist (propranolol), antiestrogens (tamoxifen and ICI 182,780) or inhibitors of protein synthesis (cycloheximide) and transcription (actinomycin D). Moreover, contractions induced by high potassium (KCl) solution or calcium in depolarized tissues by KCl-calcium free solution were exposed to 17alpha-estradiol. Collaterally, we performed an uterotrophic assay in adult ovariectomized rats measuring the uterine wet weight. The administration for three days of 0.3 microM/day/Kg 17beta-estradiol was equimolarly compared with the response produced by 17alpha-estradiol. Antiuterotrophic activity was assayed by administration of 0.3 microM/day/Kg 17beta-estradiol and various doses ratios (1:1, 1:3, 1:5, and 1:100) of 17alpha-estradiol. RESULTS: The estradiol isomers elicited an immediate relaxation, concentration-dependent and reversible on spontaneous contraction. 17alpha-Estradiol presented lower potency than 17beta-estradiol although it did not antagonize 17beta-estradiol-induced relaxation. Relaxation to 17alpha-estradiol was not inhibited by propranolol, tamoxifen, ICI 182,780, cycloheximide or actinomycin D. The KCl contractions were also sensitive to 17alpha-estradiol-induced relaxation and calcium contractions in depolarized tissues were markedly prevented by 17alpha-estradiol, implying a reduction of extracellular calcium influx through voltage-operated calcium channels (VOCCs). Uterotrophic assay detected significant increase in uterine weight using 17alpha-estradiol, which was significantly minor as compared with 17beta-estradiol. 17alpha-Estradiol, at all doses ratios, significantly antagonized the hypertrophic response of 17beta-estradiol. CONCLUSION: 17alpha-Estradiol induces a relaxing effect, which may be independent of the classical estrogen receptor, nongenomic action, apparently mediated by inactivation of VOCCs. 17alpha-Estradiol is also a weak estrogen agonist (uterotrophic response); likewise, 17alpha-estradiol may act as an antiestrogen (antiuterotrophic response). The overall data document a nongenomic relaxing action and a novel antiestrogenic action of 17alpha-estradiol, which are relevant in estrogen-mediated uterine physiology.

Androgens induce relaxation of contractile activity in pregnant human myometrium at term: a nongenomic action on L-type calcium channels.

It has long been accepted that progesterone regulates uterine contractile activity. However, little is known about the role of androgens in female physiology, and their importance and biological function on myometrial contractility so far have received limited attention. In this work, we examined the direct effect of androgens on the contractile activity of the isolated human myometrium. Myometrial biopsies were obtained, with consent, from pregnant women undergoing elective cesarean section at term. Each androgen tested (dehydroepiandrosterone, testosterone, 5alpha- and 5beta-dihydrotestosterone, androsterone, or androstanediol) caused a concentration-dependent inhibition of spontaneous contractile activity; a relaxing effect of these androgens was also observed on the contractions induced by high potassium (KCl) solution. Interestingly, nonpregnant myometrium was also sensitive to androgen-induced relaxation. 5beta-Dihydrotestosterone (5beta-DHT) was dramatically more potent than the other androgens in inducing myometrial relaxation in all preparations. Relaxation response to androgens had very rapid time courses and was affected by neither the specific antiandrogen (flutamide) nor inhibitors of protein synthesis (cycloheximide) and transcription (actinomycin D), implying that androgens act through a nongenomic mechanism. Importantly, 5beta-DHT significantly reduced the increase in intracellular calcium concentration associated with exposure to KCl in human myometrial smooth-muscle cells loaded with Fura-2-AM. The blockade of l-type calcium channels seems to be involved in the nongenomic relaxing action of androgens. These observations demonstrate that androgens may play a crucial role in maintaining pregnancy.

Vasodilating effect of norethisterone and its 5 alpha metabolites: a novel nongenomic action.

Estrogens are generally administered in hormone replacement therapy in combination with synthetic progestins. Studies of cardiovascular risk factors in postmenopausal women have shown a variety of responses according to the molecular structure of the progestin used in hormone replacement therapy schemes. The present study sets out to determine the vasoactive effects of norethisterone and its 5alpha-dihydro (5alpha-norethisterone) and -tetrahydro (3alpha,5alpha-norethisterone and 3beta,5alpha-norethisterone) metabolites in isolated precontracted rat thoracic aorta. The addition of norethisterone and 3alpha,5alpha-norethisterone in rat aorta exhibited a potent, concentration-response inhibition of noradrenaline-induced contraction, while 5alpha- and 3beta,5alpha-norethisterone had very little, if any, vasorelaxing effect. Relaxation to norethisterone and 3alpha,5alpha-norethisterone had very rapid time-courses and it was neither affected by the absence of endothelium nor by the inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester (L-NAME). The addition of specific anti-androgen, anti-progestin and anti-estrogen compounds and protein synthesis inhibitors did not preclude the vasorelaxing effect of norethisterone and its 3alpha,5alpha-reduced metabolite. The results strongly suggest that these effects are not mediated by nuclear sex steroid hormone receptors. The overall data document a novel nongenomic endothelium-independent vasorelaxing action of a 19-nor synthetic progestin and one of its A-ring-reduced derivatives.