Francisco J. Valenzuela

Clock gene expression in adult primate suprachiasmatic nuclei and adrenal: is the adrenal a peripheral clock responsive to melatonin?

The circadian production of glucocorticoids involves the concerted action of several factors that eventually allow an adequate adaptation to the environment. Circadian rhythms are controlled by the circadian timing system that comprises peripheral oscillators and a central rhythm generator located in the suprachiasmatic nucleus (SCN) of the hypothalamus, driven by the self-regulatory interaction of a set of proteins encoded by genes named clock genes. Here we describe the phase relationship between the SCN and adrenal gland for the expression of selected core clock transcripts (Per-2, Bmal-1) in the adult capuchin monkey, a New World, diurnal nonhuman primate. In the SCN we found a higher expression of Bmal-1 during the h of darkness (2000-0200 h) and Per-2 during daytime h (1400 h). The adrenal gland expressed clock genes in oscillatory fashion, with higher values for Bmal-1 during the day (1400-2000 h), whereas Per-2 was higher at nighttime (about 0200 h), resulting in a 9- to 12-h antiphase pattern. In the adrenal gland, the oscillation of clock genes was accompanied by rhythmic expression of a functional output, the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase. Furthermore, we show that adrenal explants maintained oscillatory expression of Per-2 and Bmal-1 for at least 36 h in culture. The acrophase of both transcripts, but not its overall expression along the incubation, was blunted by 100 nm melatonin. Altogether, these results demonstrate oscillation of clock genes in the SCN and adrenal gland of a diurnal primate and support an oscillation of clock genes in the adrenal gland that may be modulated by the neurohormone melatonin.

Evidence of a role for melatonin in fetal sheep physiology: direct actions of melatonin on fetal cerebral artery, brown adipose tissue and adrenal gland

Although the fetal pineal gland does not secrete melatonin, the fetus is exposed to melatonin of maternal origin. In the non‐human primate fetus, melatonin acts as a trophic hormone for the adrenal gland, stimulating growth while restraining cortisol production. This latter physiological activity led us to hypothesize that melatonin may influence some fetal functions critical for neonatal adaptation to extrauterine life. To test this hypothesis we explored (i) the presence of G‐protein‐coupled melatonin binding sites and (ii) the direct modulatory effects of melatonin on noradrenaline (norepinephrine)‐induced middle cerebral artery (MCA) contraction, brown adipose tissue (BAT) lypolysis and ACTH‐induced adrenal cortisol production in fetal sheep. We found that melatonin directly inhibits the response to noradrenaline in the MCA and BAT, and also inhibits the response to ACTH in the adrenal gland. Melatonin inhibition was reversed by the melatonin antagonist luzindole only in the fetal adrenal. MCA, BAT and adrenal tissue displayed specific high‐affinity melatonin binding sites coupled to G‐protein (Kd values: MCA 64 ± 1 pm, BAT 98.44 ± 2.12 pm and adrenal 4.123 ± 3.22 pm). Melatonin binding was displaced by luzindole only in the adrenal gland, supporting the idea that action in the MCA and BAT is mediated by different melatonin receptors. These direct inhibitory responses to melatonin support a role for melatonin in fetal physiology, which we propose prevents major contraction of cerebral vessels, restrains cortisol release and restricts BAT lypolysis during fetal life.

Synthesis of nanostructured porous silica coatings on titanium and their cell adhesive and osteogenic differentiation properties

Nanostructured porous silica coatings were synthesized on titanium by the combined sol-gel and evaporation-induced self-assembly process. The silica-coating structures were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and nitrogen sorptometry. The effect of the nanoporous surface on apatite formation in simulated body fluid, protein adsorption, osteoblast cell adhesion behavior, and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) is reported. Silica coatings with highly ordered sub-10 nm porosity accelerate early osteoblast adhesive response, a favorable cell response that is attributed to an indirect effect due to the high protein adsorption observed on the large-specific surface area of the nanoporous coating but is also probably due to direct mechanical stimulus from the nanostructured topography. The nanoporous silica coatings, particularly those doped with calcium and phosphate, also promote the osteogenic differentiation of hBMSCs with spontaneous mineral nodule formation in basal conditions. The bioactive surface properties exhibited by the nanostructured porous silica coatings make these materials a promising alternative to improve the osseointegration properties of titanium dental implants and could have future impact on the nanoscale design of implant surfaces.

Preparation and bioactive properties of novel bone-repair bionanocomposites based on hydroxyapatite and bioactive glass nanoparticles †

Bionanocomposites based on ceramic nanoparticles and a biodegradable porous matrix represent a promising strategy for bone repair applications. The preparation and bioactive properties of bionanocomposites based on hydroxyapatite (nHA) and bioactive glass (nBG) nanoparticles were presented. nHA and nBG were synthesized with nanometric particle size using sol-gel/precipitation methods. Composite scaffolds were prepared by incorporating nHA and nBG into a porous alginate (ALG) matrix at different particle loads. The ability of the bionanocomposites to induce the crystallization of the apatite phase from simulated body fluid (SBF) was systematically evaluated using X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray analysis, and Fourier transform infrared spectroscopy. Both nHA/ALG and nBG/ALG composites were shown to notably accelerate the process of crystallization and growth of the apatite phase on the scaffold surfaces. For short immersion times in SBF, nBG (25%)-based nanocomposites induced a higher degree of apatite crystallization than nHA (25%)-based nanocomposites, probably due to the more reactive nature of the BG particles. Through a reinforcement effect, the nanoparticles also improve the mechanical properties and stability in SBF of the polymer scaffold matrix. In addition, in vitro biocompatibility tests demonstrated that osteoblast cells are viable and adhere well on the surface of the bionanocomposites. These results indicate that nHA- and nBG-based bionanocomposites present potential properties for bone repair applications, particularly oriented to accelerate the bone mineralization process.

Cryptochrome 2 Expression Level Is Critical for Adrenocorticotropin Stimulation of Cortisol Production in the Capuchin Monkey Adrenal

Timely production of glucocorticoid hormones in response to ACTH is essential for survival by coordinating energy intake and expenditure and acting as homeostatic regulators against stress. Adrenal cortisol response to ACTH is clock time dependent, suggesting that an intrinsic circadian oscillator in the adrenal cortex contributes to modulate the response to ACTH. Circadian clock gene expression has been reported in the adrenal cortex of several species. However, there are no reports accounting for potential involvement of adrenal clock proteins on cortisol response to ACTH. Here we explored whether the clock protein cryptochrome 2 (CRY2) knockdown modifies the adrenal response to ACTH in a primate. Adrenal gland explants from adult capuchin monkey (n = 5) were preincubated for 6 h with transfection vehicle (control) or with two different Cry2 antisense and sense probes followed by 48 h incubation in medium alone (no ACTH) or with 100 nm ACTH. Under control and sense conditions, ACTH increased cortisol production, whereas CRY2 suppression inhibited ACTH-stimulated cortisol production. Expression of the steroidogenic enzymes steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase at 48 h of incubation was increased by ACTH in control explants and suppressed by Cry2 knockdown. Additionally, we found that Cry2 knockdown decreased the expression of the clock gene brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) at the mRNA and protein levels. Altogether these results strongly support that the clock protein CRY2 is involved in the mechanism by which ACTH increases the expression of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase. Thus, adequate expression levels of components of the adrenal circadian clock are required for an appropriate cortisol response to ACTH.

Maternal melatonin stimulates growth and prevents maturation of the capuchin monkey fetal adrenal gland.

Abstract.  The primate fetal adrenal reaches a large size relative to body weight followed by a rapid decrease in size in the postnatal period. We tested the hypothesis that maternal melatonin stimulates growth and prevents maturation of the primate fetal adrenal gland. We suppressed maternal melatonin by exposing eight pregnant capuchin monkeys to constant light (LL) from 63% to 90% gestation (term 155 days). Three of these received daily oral melatonin replacement (LL + Mel). Five mothers remaining in light:dark cycle were used as controls. Fetuses were delivered at 90% gestation. The absence of maternal melatonin selectively decreased fetal adrenal weight (Control: 488.8 ± 51.5; LL: 363.2 ± 27.7 and LL + Mel 519 ± 46 mg; P < 0.05 ANOVA) without effecting fetal weight, placental weight or the weight of other fetal tissues. Changes in fetal adrenal size were accompanied by an increase in the levels of Δ5‐3β‐hydroxysteroid dehydrogenase (3β‐HSD) mRNA (Control: 0.8 ± 0.2; LL: 5.2 ± 0.6 and LL + Mel 0.8 ± 0.1; 3β‐HSD/18S‐rRNA; P < 0.05 ANOVA). In vitro we found that maternal melatonin suppression increased basal progesterone production to levels similar to those of the adult adrenal gland (Control: 0.36 ± 0.09; LL 0.99 ± 0.13; LL + Mel 0.18 ± 0.06 and adult: 0.88 ± 0.10 ng/mg of tissue; P < 0.05 ANOVA) but no change in cortisol production. We found an increased production of cortisone (Control: 1.65 ± 0.60; LL: 5.44 ± 0.63; LL + Mel: 2.90 ± 0.38 and adult: 1.70 ± 0.45 ng/mg of tissue; P < 0.05 ANOVA). Collectively, the effects of maternal melatonin suppression and their reversion by maternal melatonin replacement suggest that maternal melatonin stimulates growth and prevents maturation of the capuchin monkey fetal adrenal gland.

Impact of chronodisruption during primate pregnancy on the maternal and newborn temperature rhythms.

Disruption of the maternal environment during pregnancy is a key contributor to offspring diseases that develop in adult life. To explore the impact of chronodisruption during pregnancy in primates, we exposed pregnant capuchin monkeys to constant light (eliminating the maternal melatonin rhythm) from the last third of gestation to term. Maternal temperature and activity circadian rhythms were assessed as well as the newborn temperature rhythm. Additionally we studied the effect of daily maternal melatonin replacement during pregnancy on these rhythms. Ten pregnant capuchin monkeys were exposed to constant light from 60% of gestation to term. Five received a daily oral dose of melatonin (250 µg kg/body weight) at 1800 h (LL+Mel) and the other five a placebo (LL). Six additional pregnant females were maintained in a 14∶10 light:dark cycles and their newborns were used as controls (LD). Rhythms were recorded 96 h before delivery in the mother and at 4–6 days of age in the newborn. Exposure to constant light had no effect on the maternal body temperature rhythm however it delayed the acrophase of the activity rhythm. Neither rhythm was affected by melatonin replacement. In contrast, maternal exposure to constant light affected the newborn body temperature rhythm. This rhythm was entrained in control newborns whereas LL newborns showed a random distribution of the acrophases over 24-h. In addition, mean temperature was decreased (34.0±0.6 vs 36.1±0.2°C, in LL and control, respectively P<0.05). Maternal melatonin replacement during pregnancy re-synchronized the acrophases and restored mean temperature to the values in control newborns. Our findings demonstrate that prenatal melatonin is a Zeitgeber for the newborn temperature rhythm and supports normal body temperature maintenance. Altogether these prenatal melatonin effects highlight the physiological importance of the maternal melatonin rhythm during pregnancy for the newborn primate.

La melatonina reduce la respuesta de cortisol al ACTH en humanos

Adrenal glandswere obtained from 4 patients undergoing unilateral nephrectomy-adrenalectomy for renalcancer. Expression of mRNA MT1 and MT2 melatonin receptors was measured by ReverseTranscriptase Polymerase Chain Reaction (RT-PCR). The effect of melatonin on the response tointravenous (i.v.) ACTH was tested (randomized cross-over, double-blind, placebo-controlledtrial) in eight young healthy males pretreated with dexamethasone (1 mg) at 23:00 h. On thenext day, at 08:00 h, an i.v. line was inserted, at 08:30 h, and after a blood sample, subjectsingested 6 mg melatonin or placebo. At 09:00 h, 1-24 ACTH (Cortrosyn, 1

Impact of Maternal Melatonin Suppression on Amount and Functionality of Brown Adipose Tissue (BAT) in the Newborn Sheep.

In human and sheep newborns, brown adipose tissue (BAT) accrued during fetal development is used for newborn thermogenesis. Here, we explored the role of maternal melatonin during gestation on the amount and functionality of BAT in the neonate. We studied BAT from six lambs gestated by ewes exposed to constant light from 63% gestation until delivery to suppress melatonin (LL), six lambs gestated by ewes exposed to LL but receiving daily oral melatonin (12 mg at 1700 h, LL + Mel) and another six control lambs gestated by ewes maintained in 12 h light:12 h dark (LD). Lambs were instrumented at 2 days of age. At 4–6 days of age, they were exposed to 24°C (thermal neutrality conditions) for 1 h, 4°C for 1 h, and 24°C for 1 h. Afterward, lambs were euthanized and BAT was dissected for mRNA measurement, histology, and ex vivo experiments. LL newborns had lower central BAT and skin temperature under thermal neutrality and at 4°C, and higher plasma norepinephrine concentration than LD newborns. In response to 4°C, they had a pronounced decrease in skin temperature and did not increase plasma glycerol. BAT weight in LL newborns was about half of that of LD newborns. Ex vivo, BAT from LL newborns showed increased basal lipolysis and did not respond to NE. In addition, expression of adipogenic/thermogenic genes (UCP1, ADBR3, PPARγ, PPARα, PGC1α, C/EBPβ, and perilipin) and of the clock genes Bmal1, Clock, and Per2 was increased. Remarkably, the effects observed in LL newborns were absent in LL + Mel newborns. Thus, our results support that maternal melatonin during gestation is important in determining amount and normal functionality of BAT in the neonate.

The Role of Placenta in the Fetal Programming Associated to Gestational Diabetes

Gestational diabetes mellitus (GDM) is a human pregnancy disease characterized by elevation of glucose levels (i.e., hyperglycemia) responsible for a several adverse perinatal outcomes included macrosomia, fetal hypoglycemia, requirement of neonatal intensive care and neonatal mortality, among others. Estimation of the epidemiological impact of GDM has indicated that at least 1 out of 10 pregnant woman is being affected by GDM worldwide. In addition, GDM causes not only short-term complication in both mother and fetus, but also is associated with elevated risk for long-term complication such as cardiovascular disease, obesity and diabetes. Even though it is not feasible to exclude the genetic component in the elevated risk for metabolic/cardiovascular disease later in life, the general agreement is that hyperglycemia generates an adaptive response in the fetus addressing to control the glucose level, characterized by hyperinsulinemia. Part of this adaptive response, might also include the elevation in the placental consumption of glucose and enhancement of the feto-placental blood flow, especially in fetus large-for-gestational age (LGA). On the other hand, due to lack of innervation in the placenta, the vascular tone is controlled by the regulation of the synthesis and release of vasoactive substances from the endothelium like vasoactive molecules, nitric oxide, adenosine, prostaglandin, among others. Interestingly, vasoactive molecules may also regulate endothelial proliferation and migration, suggesting that they also affect the vessel formation (i.e., angiogenesis). In this regard, several studies have shown that placenta from GDM is characterized by hypervascularization and elevation in the pro-angiogenic signals including the secretion and activity of the vascular endothelial growth factor (VEGF). In addition, hyperglycemia also generates a status of oxidative stress, where free radicals derived from oxygen (ROS) induces changes in the endothelial cell membranes producing an elevation