RESUMEN
CONTEXT: Although growing evidence points toward a role of lipotoxicity in the development of hyperandrogenesis, the main feature of polycystic ovary syndrome, few studies directly assessed this association in vivo in humans, and none targeted the ovarian milieu. OBJECTIVE: The main objective of this study was to correlate follicular fluid (FF) T levels with lipids, lipid metabolites, and inflammation markers. DESIGN: This was a cross-sectional study. SETTING: Recruitment was performed in two fertility clinics at one private and one academic center. PARTICIPANTS: Eighty women requiring in vitro fertilization were recruited during one of their scheduled visit at the fertility clinic. All women aged between 18 and 40 years with a body mass index between 18 and 40 kg/m(2) were invited to participate. INTERVENTION(S): There were no interventions. MAIN OUTCOME MEASURE(S): At the time of oocyte aspiration, FF was collected and analyzed for total T, lipids [nonesterified fatty acids (NEFAs) plus triglycerides], NEFA metabolites (acylcarnitines; markers of ineffective NEFAs ß-oxidation), and inflammatory marker composition. The hypothesis being tested was formulated before the data collection. RESULTS: FF T levels were significantly correlated with FF levels of lipids (r = 0.381, P = .001; independently of IL-6), acylcarnitines (r ≥ 0.255, all P = .008; not independently of lipids), and IL-6 (r = 0.300, P = .009, independently of lipids). Additionally, FF lipid levels were significantly and strongly correlated with acylcarnitines (r ≥ 0.594; all P < .001). CONCLUSIONS: These results suggest that ovarian androgen production is related to intraovarian exposure to lipids, independently of inflammation and mainly through ineffective NEFA ß-oxidation (as shown by higher acylcarnitine levels). Inflammation is also associated with intraovarian androgenesis, independently of lipids.
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Andrógenos/biosíntesis , Fertilización In Vitro/métodos , Líquido Folicular/química , Metabolismo de los Lípidos/fisiología , Lípidos/análisis , Adulto , Femenino , HumanosRESUMEN
Central retinal artery occlusion (CRAO) is an uncommon eye disorder, but one that typically produces severe and irreversible vision loss in the affected eye. The retina has a dual blood supply, with the retinal circulation supplying the inner layers and the choroidal circulation supplying the outer layers. In CRAO, vision loss results from cell death in the inner retinal layers despite relative sparing of the outer layers. If supplemental oxygen is provided, however, oxygen from the choroidal circulation may diffuse in adequate quantity to the inner layers of the retina to maintain retinal function and restore vision. In some patients this can be achieved with normobaric hyperoxia; in others, hyperbaric oxygen (HBO2) may be required. The challenge is to provide the supplemental oxygen early enough after the onset of vision loss to prevent irreversible damage to the retina. In experimental models of complete CRAO, the ischemic time window before permanent retinal damage occurs is just over 90 minutes; in the clinical setting where occlusion may be incomplete, return of vision may be achieved even after delays of eight to 24 hours. In patients with a clinical picture of CRAO who present within 24 hours of vision loss, supplemental oxygen should be started immediately at the highest possible fraction of inspired oxygen (FiO2). If vision is not quickly restored, emergent HBO2 should be undertaken if feasible. If the patient responds to HBO2, follow-up treatment with supplemental oxygen should be customized to maintain retinal viability until the obstructed retinal artery recanalizes, which typically occurs within the first 72 hours. This paper reviews the pertinent literature on CRAO and HBO2 and provides a treatment algorithm.
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Algoritmos , Oxigenoterapia Hiperbárica/métodos , Oclusión de la Arteria Retiniana/terapia , Ceguera/etiología , Ceguera/terapia , Medicina Basada en la Evidencia , Humanos , Oxigenoterapia Hiperbárica/economía , Terapia por Inhalación de Oxígeno/métodos , Oclusión de la Arteria Retiniana/complicacionesRESUMEN
Idiopathic sudden sensorineural hearing loss (ISSHL) is the newest indication approved by the Undersea and Hyperbaric Medical Society's Hyperbaric Oxygen Therapy Committee. Idiopathic sudden sensorineural hearing loss appears to be characterized by hypoxia in the perilymph and therefore the scala tympani and the organ of Corti. A review of the literature reveals more than 100 publications evaluating the use of hyperbaric oxygen (HBO2) for the treatment of ISSHL, including eight randomized controlled trials. The best and most consistent results are obtained when HBO2 is initiated within two weeks of symptom onset and combined with corticosteroid treatment. The average hearing gain is 19.3 dB for moderate hearing loss and 37.7 dB for severe cases. This improvement brings hearing deficits from the moderate/severe range into the slight/no impairment range. This is a significant gain that can markedly improve a patient's quality of life, both clinically and functionally.
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Pérdida Auditiva Sensorineural/terapia , Pérdida Auditiva Súbita/terapia , Oxigenoterapia Hiperbárica/métodos , Corticoesteroides/uso terapéutico , Animales , Terapia Combinada/métodos , Pérdida Auditiva Sensorineural/etiología , Pérdida Auditiva Súbita/etiología , Humanos , Guías de Práctica Clínica como Asunto , Estudios Prospectivos , Ensayos Clínicos Controlados Aleatorios como Asunto , Estudios RetrospectivosRESUMEN
Hyperbaric oxygen therapy (HBOT) is a primary or adjunctive therapy for a variety of medical disorders including some involving the eye. This paper is the first comprehensive review of HBOT for ocular indications. The authors recommend the following as ocular indications for HBOT: decompression sickness or arterial gas embolism with visual signs or symptoms, central retinal artery occlusion, ocular and periocular gas gangrene, cerebro-rhino-orbital mucormycosis, periocular necrotizing fasciitis, carbon monoxide poisoning with visual sequelae, radiation optic neuropathy, radiation or mitomycin C-induced scleral necrosis, and periorbital reconstructive surgery. Other ocular disorders that may benefit from HBOT include selected cases of ischemic optic neuropathy, ischemic central retinal vein occlusion, branch retinal artery occlusion with central vision loss, ischemic branch retinal vein occlusion, cystoid macular edema associated with retinal venous occlusion, post-surgical inflammation, or intrinsic inflammatory disorders, periocular brown recluse spider envenomation, ocular quinine toxicity, Purtscher's retinopathy, radiation retinopathy, anterior segment ischemia, retinal detachment in sickle cell disease, refractory actinomycotiC lacrimal canaliculitis, pyoderma gangrenosum of the orbit and refractory pseudomonas keratitis. Visual function should be monitored as clinically indicated before, during, and after therapy when HBOT is undertaken to treat vision loss. Visual acuity alone is not an adequate measure of visual function to monitor the efficacy of HBOT in this setting. Ocular examinations should also include automated perimetry to evaluate the central 30 degrees of visual field at appropriate intervals. Interpretation of the literature on the efficacy of HBOT in treating ocular disorders is complicated by several factors: frequent failure to include visual field examination as an outcome measure, failure to adequately address the interval from symptom onset to initiation of HBOT, and lack of evidence for optimal treatment regimens for essentially all ocular indications. Because some ocular disorders require rapid administration of HBOT to restore vision, patients with acute vision loss should be considered emergent when they present. Visual acuity should be checked immediately, including vision with pinhole correction. If the patient meets the criteria for emergent HBOT outlined in the paper, normobaric oxygen should be started at the highest inspired oxygen fraction possible until arrangements can be made for HBOT.
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Oftalmopatías/terapia , Oxigenoterapia Hiperbárica , Intoxicación por Monóxido de Carbono/terapia , Enfermedad de Descompresión/terapia , Embolia Aérea/terapia , Fascitis Necrotizante/terapia , Gangrena Gaseosa/terapia , Humanos , Mucormicosis/terapia , Necrosis/terapia , Traumatismos por Radiación/terapia , Oclusión de la Arteria Retiniana/terapia , Oclusión de la Vena Retiniana/terapia , Esclerótica/patología , Trastornos de la Visión/terapiaRESUMEN
Our understanding of the mechanisms by which sleep deteriorates with age almost exclusively stems from comparisons of young and elderly subjects. The present study investigated the different effects of a 25-h sleep deprivation on the recovery sleep initiated in the morning (when circadian sleep propensity decreases) of young (20-39 y) and middle-aged subjects (40-60 y). Middle-aged subjects showed a steeper increase in the duration of wakefulness during daytime recovery sleep than the young subjects. Slow-wave sleep (SWS) and EEG slow-wave activity (SWA: spectral power between 0.5-4.5 Hz) were potentiated in both groups following sleep deprivation. However, the rebound of SWS and SWA was significantly less pronounced in the middle-aged than in the young. This reduction in homeostatic recuperative drive in middle-aged subjects might account for the decrease in their ability to maintain sleep when they have to recuperate at an abnormal circadian phase. These results helps to understand the increase in complaints related to shift work and jet lag in the middle years of life.
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Envejecimiento/fisiología , Ritmo Circadiano/fisiología , Privación de Sueño/fisiopatología , Sueño/fisiología , Adulto , Encéfalo/fisiología , Encéfalo/fisiopatología , Electroencefalografía , Femenino , Homeostasis/fisiología , Humanos , Luz , Masculino , Persona de Mediana Edad , Polisomnografía , Sueño REM/fisiología , Factores de Tiempo , Vigilia/fisiologíaRESUMEN
Postmenopausal women (PMW) commonly believe that hormone replacement (HR) leads to weight gain, and fear of weight gain and/or an actual increase in weight is one of the principle reasons evoked for the discontinuation of HR. However, the potential effects of physiologic HR on body composition have yet to be separated from the effects of lifestyle or aging. Therefore, we examined the effect of short-term hormone replacement and age on alterations in weight, body composition, and energy balance. A prospective study of 28 healthy PMW aged 45 to 55 years (younger PMW, studies completed n = 18) and 70 to 80 years (older PMW, studies completed n = 15) was conducted. The last menstrual period was more than 12 months previously. The women had a body mass index (BMI) less than 30 kg/m(2) and were taking no medication. Subjects were studied at baseline, after 1 month of transdermal estrogen (Estraderm, 50 microg/day) administration (E2), followed by a further month of transdermal estrogen with progesterone (100 mg per vagina twice daily) for the final 7 days (E2 + P). Anthropometric measurements and energy assessments were performed at each visit. Physiologic HR was achieved in each subject, and there was no difference between levels achieved in older and younger women. Resting energy expenditure and activity level were positively correlated with fat-free mass (P <.0001), while energy intake was not. Resting energy expenditure was lower in older compared with younger PMW when adjusted for fat-free mass (P <.005). Energy intake was also lower in the older PMW when corrected for fat-free mass (P <.0001); as was activity level (P <.05). There was no effect of hormonal treatment on any of the parameters measured. Changes in weight from baseline for E2 (0.37 +/- 0.25 and 0.61 +/- 0.27 kg in younger and older) and E2 + P (0.11 +/- 0.38 and 0.28 +/- 0.31 kg) were not statistically significant. In addition, there was no difference in BMI, fat mass, fat-free mass, total body water, or waist-to-hip ratio (WHR) between groups or with hormonal treatment. In conclusion, short-term transdermal HR is not associated with significant changes in weight or other anthropometric measures in younger or older PMW. These studies confirm the decrease in energy expenditure that occurs with aging, but indicates that there is no effect of HR on resting energy expenditure.
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Composición Corporal , Metabolismo Energético , Terapia de Reemplazo de Estrógeno , Posmenopausia/fisiología , Anciano , Anciano de 80 o más Años , Envejecimiento/fisiología , Esquema de Medicación , Estradiol/administración & dosificación , Estradiol/uso terapéutico , Femenino , Humanos , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
The steroidogenic acute regulatory (StAR) protein is indispensable for maximal trophic hormone-stimulated steroidogenesis by the adrenal gland, testis, and ovary. Recently, our laboratory developed an in vitro primary culture system of porcine granulosa-luteal cells that retain responsiveness to LH and show LH and insulin [or insulin-like growth factor (IGF-I)] synergy in stimulating StAR messenger RNA accumulation. Here, we examine the mechanisms subserving this LH-insulin (IGF-I) augmentation. We corroborate LH's amplification of insulin as well as IGF-I-stimulated granulosa-luteal cell progesterone and cAMP accumulation (P < 0.001). Insulin or IGF-I elevated LH receptor transcript accumulation, and LH did not alter this effect. To determine the hormonal responsiveness of StAR promoter, truncated regions of the -1423 to +130 bp upstream sequence of the porcine gene were ligated into a firefly luciferase reporter plasmid. Transient transfection of the StAR plasmid containing the full-length porcine 5'-flanking region of StAR (pStAR1423/luc) showed superadditive stimulation by LH and insulin or IGF-I after 24 h. LH, but not insulin or IGF-I alone, stimulated pStAR1423/luc activity. Deletion of the proximal putative steroidogenic factor-1 (-48 to -41) site abolished hormonally driven StAR promoter activity. A stable cAMP analog, 8-bromo-cAMP (1 mM), and insulin/IGF-I also evoked supraadditive StAR promoter expression. To further explore the role of cAMP in LH-insulin (or IGF-I) actions, we cotransfected a Rous sarcoma virus (RSV)-driven minigene encoding the heat-stable inhibitor of the cAMP-dependent protein kinase (RSV/PKI) or a mutant plasmid (RSV/PKImut) along with the pStAR1423/luc promoter construct. Cotransfection of PKI, but not PKImut, with pStAR1423/luc significantly attenuated LH's stimulation of luciferase activity and also reduced the magnitude of the transcriptional amplification exerted by LH and insulin or IGF-I. In corollary analyses of the protein kinase A (PKA) pathway, cotransfection of full-length pStAR1423/luc and a complementary DNA encoding a constitutively activated PKA catalytic subunit elevated basal and insulin (or IGF-I)-stimulated StAR promoter expression. LH and insulin (or IGF-I) also augmented steady state StAR transcript levels, as assessed by homologous RT-PCR, and StAR protein concentrations, as evaluated by Western blotting. Together, these investigations document a significant role for insulin or IGF-I in enhancing LH-stimulated progesterone and cAMP biosynthesis and endogenous StAR message and protein accumulation and in augmenting cAMP-PKA-dependent transcriptional activation of the exogenous StAR promoter.
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Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/metabolismo , Insulina/farmacología , Células Lúteas/metabolismo , Hormona Luteinizante/farmacología , Fosfoproteínas/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sinergismo Farmacológico , Femenino , Factor I del Crecimiento Similar a la Insulina/farmacología , Luciferasas/genética , Progesterona/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptores de HL/genética , Proteínas Recombinantes de Fusión , Porcinos , TransfecciónRESUMEN
OBJECTIVE: To determine whether all cases of oculopharyngeal muscular dystrophy (OPMD) among Bukhara Jews share the same founder mutation. BACKGROUND: Autosomal dominant OPMD is caused by a (GCG)8-13 repeat expansion in the polyadenylation binding protein 2 (PABP2) gene. The disease has a worldwide distribution but is particularly prevalent in Bukhara Jews and in French Canadians, in whom it was introduced by three sisters in 1648. METHODS: We established the size of the PABP2 mutation in 23 Bukhara Jewish patients belonging to eight unrelated families. In all families, we constructed haplotypes for the carrying chromosomes composed of the alleles for eight chromosome 14q polymorphic markers. RESULTS: All patients share a (GCG)9 PABP2 mutation and a four-marker haplotype. Furthermore, a shared intron single nucleotide polymorphism (SNP) in the PABP2 gene 2.6Kb from the mutation was not observed in 22 families with (GCG)9 mutations from nine different countries. The smaller size of the chromosomal region in linkage disequilibrium around the mutation in Bukhara Jews, as compared with French Canadians, suggests a founder effect that occurred more than 350 years ago. Based on the Luria-Delbrück corrected "genetic clock," we estimate that the mutation appeared or was introduced once in the Bukhara Jewish population between AD 872 and 1512 (mean, AD 1243). CONCLUSION: OPMD among Bukhara Jews is the result of a shared, historically distinct, PABP2 (GCG)9 mutation that likely arose or was introduced in this population at the time they first settled in Bukhara and Samarkand during the 13th or 14th centuries.
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Judíos/genética , Distrofias Musculares/genética , Mutación/genética , Proteínas de Unión al ARN/genética , Ligamiento Genético/genética , Genotipo , Humanos , Proteínas de Unión a Poli(A) , Uzbekistán/etnologíaRESUMEN
UNLABELLED: Increasing evidence suggests that aging is associated with dynamic changes in the hypothalamic and pituitary components of the reproductive axis that are independent of changes in gonadal hormone secretion. This study was designed to determine the effect of age on GnRH pulse frequency in women in the absence of gonadal feedback using gonadotropin free alpha-subunit (FAS) and LH as neuroendocrine markers of endogenous GnRH secretion. All studies were performed in healthy, euthyroid postmenopausal women (PMW) during daytime hours. The impact of sampling interval and duration on assessment of pulse frequency in PMW was first examined in 10 women with a mean age of 61.6 +/- 8 yr (mean +/- SD), in whom blood was sampled every 5 min for 12 h. Each 5-min series was then reduced to simulate a 10-min series and then a 15-min series for pulse analysis, and the effect of 8 h compared with 12 h of sampling was determined. To define the changes in the frequency and amplitude of pulsatile hormone secretion with aging, 11 younger (45-55 yr) and 11 older (70-80 yr) PMW were then studied over 8 h at a 5-min sampling interval. In the initial series, the mean interpulse intervals (IPIs) for FAS were 53.8 +/- 3.6, 69.2 +/- 3.9, and 87.6 +/- 7.3 min at sampling intervals of 5, 10, and 15 min, respectively (P < 0.0005). The LH IPI also increased progressively with sampling intervals of 5, 10, and 15 min (54.4 +/- 2.5, 70.4 +/- 2.3, and 91.1 +/- 4.4 min; P < 0.0001). At the 5-min sampling interval, the calculated number of pulses/24 h was not different between a 12-h series compared with an 8-h series for either FAS or LH. In the second series of studies, the older PMW had lower gonadotropin levels (LH, 86.5 +/- 8.8 vs. 51.3 +/- 7.7 IU/L, P < 0.01; FSH, 171.6 +/- 16.9 vs. 108.2 +/- 10.5 IU/L, P < 0.005; FAS, 1021.5 +/- 147.4 vs. 425.6 +/- 89.6 ng/L, P < 0.005, in younger and older PMW, respectively) despite no differences in estrone or estradiol levels. The older PMW also demonstrated a slower FAS pulse frequency compared with their younger counterparts, as reflected in an increased FAS IPI (52.6 +/- 3.1 and 70.6 +/- 5.9 min; P < 0.002). The difference in IPIs between younger and older PMW was not statistically significant for LH (65.4 +/- 5.6 and 71.8 +/- 6.6 min for younger and older PMW, respectively). FAS pulse amplitude was decreased in older PMW compared with younger PMW (431.7 +/- 66.2 vs. 224.6 +/- 81.9 ng/L; P < 0.01), whereas the decrease in LH pulse amplitude with age was of borderline statistical significance (23.2 +/- 3.1 vs. 15.9 +/- 2.1 IU/L; P = 0.09). IN CONCLUSION: 1) the use of a 5-min sampling interval and measurement of FAS as the primary marker of GnRH pulse generator activity indicate that GnRH pulse frequency in younger PMW is faster than previously reported, but not increased over that seen in the late follicular phase and midcycle surge in women with intact ovarian function; and 2) the marked decrease in FAS pulse frequency with age provides evidence of age-related changes in the hypothalamic component of the reproductive axis that are independent of changes in gonadal function.
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Ciclos de Actividad , Envejecimiento/fisiología , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Hormona Luteinizante/metabolismo , Posmenopausia/fisiología , Anciano , Envejecimiento/sangre , Índice de Masa Corporal , Estradiol/sangre , Estrona/sangre , Femenino , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/sangre , Hormona Liberadora de Gonadotropina/sangre , Humanos , Hormona Luteinizante/sangre , Persona de Mediana Edad , Posmenopausia/sangreRESUMEN
OBJECTIVE: Leptin is a hormone which is secreted by adipocytes and appears to influence the reproductive axis. Previous studies have demonstrated higher leptin levels in relation to body fat mass in women compared to men, higher levels in normally cycling compared to postmenopausal women, and a decrease in leptin levels with increased age. The purpose of this study was to determine whether oestrogen replacement with or without progesterone increases serum leptin levels in postmenopausal women, independently of changes in body fat, and to determine if ageing affects leptin levels at baseline or in response to hormone replacement. PATIENTS: Twenty-one healthy postmenopausal women on no hormone replacement were studied at baseline, after 1 month of oestrogen (E2: estraderm 50 microg/day) and after a further month of oestrogen and 7 days of progesterone (P: progesterone 100 mg per vagina bid) designed to achieve physiological hormone levels. Subjects included 11 younger (45-55 years) and 10 older (70-80 years) postmenopausal women. RESULTS: The relationship between leptin and the absolute fat mass (% body fat x weight [kg]) at baseline was not different between the younger and older postmenopausal women. The adequacy of physiological hormone replacement was confirmed in all subjects. Despite the absence of an effect of hormone replacement on weight, body mass index (BMI), % and absolute fat mass (bioimpedance) or waist-hip ratio, there was an increase in serum leptin levels with hormone replacement (15.4 +/- 1.7, 17.6 +/- 1.7, and 18.1 +/- 1.6 microg/l; mean +/- SEM at baseline, with E2, and with E2 + P, respectively; P < 0.001 vs. baseline) for the group as a whole. An increase in leptin with hormonal treatment was seen in both the younger (15.1 +/- 2.1, 18.1 +/- 2.4, and 18.5 +/- 1.9 microg/l; P < 0.01) and the older (15.7 +/- 2.8, 17.0 +/- 2.5, 17.7 +/- 2.8 microg/l; P = 0.06) postmenopausal women. CONCLUSIONS: (1) Short-term physiological oestrogen replacement increases serum leptin levels in postmenopausal women independently of changes in fat mass; and (2) physiological progesterone replacement does not influence leptin levels in postmenopausal women.
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Estradiol/uso terapéutico , Terapia de Reemplazo de Estrógeno , Leptina/sangre , Posmenopausia/sangre , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Composición Corporal , Índice de Masa Corporal , Femenino , Humanos , Persona de Mediana Edad , Progesterona/uso terapéuticoRESUMEN
Expert and definitive airway management is fundamental to the practice of emergency medicine. In critically ill patients, rapid sedation and paralysis, also known as rapid-sequence intubation, is used to facilitate endotracheal intubation in order to minimize aspiration, airway trauma, and other complications of airway management. An alternative method of emergent endotracheal intubation, intubation minus paralysis, is performed without the use of neuromuscular blocking agents. The present study compared complications of these two techniques in the emergency setting. Sixty-seven intubations minus paralysis were prospectively compared with 166 rapid-sequence intubations. Complications were greater in number and severity in the nonparalyzed group and included aspiration (15%), airway trauma (28%), and death (3%). None of these difficulties were observed in the rapid-sequence group (P < .0001). These results show that rapid-sequence intubation when compared with intubation minus paralysis significantly reduces complications of emergency airway management and should be made available to emergency physicians trained in its use.
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Urgencias Médicas , Mortalidad Hospitalaria , Intubación Intratraqueal/efectos adversos , Laringe/lesiones , Bloqueantes Neuromusculares/administración & dosificación , Neumonía por Aspiración/etiología , Adulto , Sedación Consciente , Servicio de Urgencia en Hospital , Humanos , Louisiana , Evaluación de Procesos y Resultados en Atención de Salud , Estudios Prospectivos , Factores de RiesgoRESUMEN
Insulin-like growth factor I (IGF-I) and the gonadotropin, FSH, can synergize to stimulate progesterone production in primary cultures of maturing human, rat, and pig granulosa cells. These trophic hormones act by increasing the activity and production of proteins and their gene transcripts essential to sterol uptake, delivery, and utilization in steroidogenesis. We previously observed that FSH and IGF-I interact synergistically to promote the accumulation of steroidogenic acute regulatory protein (StAR) messenger RNA and protein in granulosa cells. Here we investigate potential mechanisms of IGF-I synergy with FSH and the protein kinase A (PKA) pathway in activating the porcine StAR gene promoter. To this end, we first cloned 1423 bp of the porcine StAR promoter upstream of the transcriptional start site using PCR and created 5'-deletional constructs coupled to a cytoplasmically targeted firefly luciferase reporter gene. FSH, 8-bromo-cAMP, and transient transfection of the protein kinase A (PKA) catalytic subunit (driven by the Rous sarcoma virus promoter) were used to activate the PKA effector pathway. All three agonists alone stimulated StAR promoter-driven luciferase activity in primary cultures of granulosa cells after 4-h treatment. IGF-I significantly augmented PKA pathway agonist activation of the StAR promoter, whereas IGF-I had no effect alone. Binding experiments with 125I-labeled ovine FSH-20 in IGF-I (100 ng/ml)-treated granulosa cells showed that FSH binding affinity and receptor number were unchanged by IGF-I treatment. However, IGF-I augmented FSH-stimulated, but not forskolin-stimulated, cAMP accumulation. Analysis of 5'-deletion constructs of the StAR promoter revealed three regions of stimulatory activity within the -139-bp fragment upstream of the transcriptional start site as well as another potentially inhibitory region upstream (-1115 to 905). Elimination of the putative SF-1 site (-48 to -41) virtually abolished StAR promoter responsiveness. In summary, our data indicate that IGF-I can act via two post FSH-binding mechanisms to augment FSH/PKA pathway-mediated StAR gene promoter transactivation: at the level of cAMP accumulation and distal to cAMP production and PKA activation.
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Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Hormona Folículo Estimulante/fisiología , Regulación de la Expresión Génica , Células de la Granulosa/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/fisiología , Fosfoproteínas/genética , Regiones Promotoras Genéticas , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Secuencia de Bases , Dominio Catalítico , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sinergismo Farmacológico , Femenino , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Datos de Secuencia Molecular , Fosfoproteínas/biosíntesis , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Ovinos , Porcinos , TransfecciónRESUMEN
Insulin-like growth factor I (IGF-I) and the gonadotropin, FSH, can synergize to stimulate progesterone production in primary cultures of maturing granulosa cells. These trophic hormones increase low density lipoprotein (LDL) receptor binding and internalization, and the utilization of LDL-borne cholesterol by granulosa cells. To determine whether and how IGF-I and FSH control the genomic expression of the LDL receptor, we evaluated their individual and concerted effects on LDL receptor messenger RNA (mRNA) accumulation, stability, and gene promoter activity in first passage monolayer (serum-free) cultures of porcine granulosa cells. Ribonuclease protection assays revealed that LDL receptor mRNA accumulation was increased by human recombinant IGF-I (100 ng/ml), FSH (25 ng/ml NIDDK oFSH-20), or their combination by 2.2-, 2.6-, and 4.6-fold, respectively (P < 0.01). Hormonally stimulated LDL receptor mRNA accumulation was suppressed by 54-75% by the concurrent addition of LDL substrate (50 microg/ml). The combination of FSH and IGF-I significantly prolonged the message half-life, even in the presence of LDL. Using a combination of rapid amplification of cDNA 5'-ends, PCR with adapter-ligated genomic DNA, Southern hybridization, and DNA sequencing, we isolated 1076 bp of the porcine LDL receptor gene upstream of the coding region. In transient transfection assays, with a pLDLR1076/luciferase plasmid construct, FSH, FSH plus IGF-I, or 8-bromo-cAMP (1 mM) treatment (but not IGF-I alone) increased luciferase reporter gene activity by 10- to 23-fold in porcine granulosa cells. Over time in serum-free culture, the basal activity of the LDL receptor gene promoter increased and eventually surpassed hormone-stimulated effects, but was suppressed by LDL substrate (by 75%) at 24 h. The foregoing stimulatory hormone effects and sterol repression were localized to a 116-bp region in the porcine promoter between -255 and -139 upstream of the translational start site. We conclude that the combination of FSH and IGF-I can induce accumulation of LDL receptor mRNA in cultured granulosa cells even in the presence of sterol negative feedback and can do so mechanistically by a combination of promoter activation and increased mRNA stability.
Asunto(s)
Codón Iniciador/química , Hormona Folículo Estimulante/farmacología , Regulación de la Expresión Génica , Células de la Granulosa/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , ARN Mensajero/metabolismo , Receptores de LDL/genética , Esteroles/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Retroalimentación , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Progesterona/metabolismo , Regiones Promotoras Genéticas , Ovinos , Porcinos , TransfecciónRESUMEN
The goals of this study were to determine whether women with idiopathic hypogonadotropic hypogonadism (IHH) respond to pulsatile GnRH replacement therapy with exaggerated glycoprotein free alpha-subunit (FAS) levels, as reported in GnRH-deficient men, and to determine whether this pattern is unique to congenital GnRH deficiency or is also characteristic of patients with hypogonadotropic hypogonadism caused by other factors. GnRH was administered i.v. at a physiologic frequency and dose (75-100 ng/kg.bolus) to women with IHH (n = 11; n = 6 with anosmia); acquired GnRH deficiency secondary to treatment for cranial tumors (AHH; n = 7); and secondary hypothalamic amenorrhea (HA; n = 8). Results were compared with 24 normal cycling women. Gonadotropins, sex steroids, and FAS levels were measured in samples drawn daily across induced or normal menstrual cycles in patients or normal women, respectively. Samples were drawn at the same time of day and were collected 45 min after a GnRH bolus in patients. All women ovulated in response to pulsatile GnRH. There were no differences in the patterns of LH or gonadal steroid secretion between any of the patient groups (IHH, AHH, and HA). The patterns of LH and FSH secretion in the induced patient cycles were not different from normal women, with the exception of lower midcycle FSH levels in IHH women (P < 0.002). However, the daily dynamic secretion of FAS was exaggerated in IHH (compared with AHH, HA, and normal) women (P < 0.002). The increase in FAS levels in IHH was dependent on cycle stage, with the greatest difference observed during the early (P < 0.005) and midfollicular phase (P < 0.05) and the early luteal phase (P < 0.05). There was no difference in FAS between groups during the late follicular phase, at the midcycle, or in the midluteal and late luteal phase. This exaggerated FAS response to GnRH replacement in IHH was demonstrated in repeat cycles in two patients. Conclusions are: 1) Women with IHH respond to pulsatile GnRH replacement with an exaggerated secretion of FAS, which seems to be modified by gonadal factors; 2) this exaggerated FAS response, which is similar to that seen in GnRH-deficient men, is unique to congenital GnRH deficiency, and it is not observed in patients with acquired or secondary hypogonadotropic hypogonadism, suggesting that IHH patients may be missing a factor, in addition to GnRH, which normally restrains FAS secretion; and 3) the FAS response may prove to be a useful marker to distinguish constitutional delay of puberty from congenital GnRH deficiency.
Asunto(s)
Hormonas Glicoproteicas de Subunidad alfa/sangre , Hormona Liberadora de Gonadotropina/deficiencia , Hormona Liberadora de Gonadotropina/uso terapéutico , Hipogonadismo/sangre , Hipogonadismo/tratamiento farmacológico , Adolescente , Adulto , Amenorrea , Neoplasias Encefálicas/complicaciones , Esquema de Medicación , Estradiol/sangre , Femenino , Hormona Folículo Estimulante/sangre , Hormona Liberadora de Gonadotropina/sangre , Humanos , Hipogonadismo/fisiopatología , Hormona Luteinizante/sangre , Masculino , Ciclo Menstrual , Progesterona/sangre , Valores de ReferenciaRESUMEN
To investigate the coordinate developmental expression of low-density lipoprotein (LDL) receptor, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, sterol carrier protein 2 (SCP2), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) enzyme messages throughout the pig estrous cycle, RNase protection analysis was performed using homologous (partially cloned) porcine sequences. Total RNA was isolated from ovarian tissues from unstimulated prepubertal gilts and gilts stimulated with eCG (Day -3) and hCG (Day 0) to induce follicular growth and ovulation. Specific transcripts (relative to 18S rRNA) were quantified in immature ovaries, preovulatory follicles (> or = 5 mm), corpora lutea (CL), and corpora albicantia. As an index of steroidogenesis, tissue progesterone content (per microgram protein) was low in the unstimulated ovary and preovulatory follicles, and it began to increase 4 days post-hCG, peaked at 12 days, and returned to preovulatory concentrations by 20 days post-hCG. HMG-CoA reductase mRNA was expressed at low levels and did not change significantly throughout the estrous cycle. The amount of LDL receptor mRNA increased approximately 6-fold after eCG stimulation and was expressed at similar concentrations in both preovulatory follicles and functional CL. Expression of SCP2 mRNA did not differ among the four tissue types but tended to be highest in midcycle (Day 12) CL compared other stages of CL (p = 0.007). StAR mRNA expression was minimal in unstimulated ovaries, was higher in preovulatory follicles (p = 0.014), and then rose again in CL (p = 0.009 compared with unstimulated ovary). P450scc mRNA concentrations were low in unstimulated ovaries, increased in preovulatory follicles (p = 0.044), and increased further in CL (p = 0.001 compared with preovulatory follicles). P450scc and StAR mRNA levels correlated with progesterone levels (r = +0.37, p = 0.025, and r = +0.71, p < 0.001, respectively). The expression of LDL receptor, StAR, and P450scc messages showed a dramatic decline by Day 20 post-hCG (p = 0.002, p = 0.003, p = 0.006, respectively, compared with CL) corresponding with functional regression of the CL. In summary, P450scc and StAR message expression are coordinately amplified during the pig follicular and luteal phase, whereas LDL receptor message after an initial increase is expressed at constitutively high levels, thus indicating a differential regulation of ovarian sterol-metabolizing genes during the steroidogenic life of the follicle and CL.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Expresión Génica , Ovario/metabolismo , Proteínas de Plantas , Esteroles/biosíntesis , Porcinos/genética , Animales , Proteínas Portadoras/genética , Gonadotropina Coriónica/farmacología , Femenino , Hidroximetilglutaril-CoA Reductasas/genética , Ovario/química , Ovario/crecimiento & desarrollo , Inducción de la Ovulación , Fosfoproteínas/genética , Progesterona/biosíntesis , ARN Mensajero/análisis , ARN Mensajero/metabolismo , ARN Ribosómico 18S/análisis , Receptores de LDL/genéticaRESUMEN
The transfer of cholesterol from the outer to the inner mitochondrial membrane, where side-chain cleavage occurs to form pregnenolone, is a crucial event in the regulation of steroidogenesis and recently has been demonstrated to be mediated by steroidogenic acute regulatory protein (StAR). We generated a partial porcine StAR complementary DNA (280 bp) by RT-PCR and used the corresponding antisense riboprobe to quantify the control of StAR gene expression by FSH and insulin-like growth factor I (IGF-I) in hormonally responsive swine granulosa cells, which typically manifest synergistic steroidogenic stimulation by these two dominant intrafollicular regulators. RNase protection assays were implemented to investigate the time course of the actions of FSH (100 ng/ml), IGF-I (100 ng/ml), and FSH plus IGF-I on StAR messenger RNA accumulation in serum-free cultures granulosa cells. Treatment with FSH (1.6-fold) or IGF-I (2.7-fold) alone had a small but consistent stimulatory effect on StAR message accumulation (corrected for 18S ribosomal RNA in each lane) at 48 h, whereas only IGF-I stimulated StAR protein expression (at least 6-fold as assessed by Western blot). Notably, the combined effect of FSH plus IGF-I was strongly synergistic and already significant by 24 h and maximal at 48 h (P < 0.001). Protein kinase A agonist, 8-bromoadenosine 3',5'-cAMP (8-bromo-cAMP) (1 mM) alone elicited a 3.5-fold increase in StAR message and more than 3.7-fold increase in StAR protein expression by 48 h. The combination of IGF-I and FSH or 8-bromo-cAMP evoked a 26- to 40-fold (P < 0.001) synergistic rise in StAR message accumulation. StAR protein also showed a similar synergistic pattern of expression driven by IGF-I and FSH or 8-bromo-cAMP, namely a greater than 56- to 60-fold increase. In summary, two distinct first messenger regulatory molecules, FSH and IGF-I, interact synergistically to induce amplification of StAR messenger RNA and protein expression in serum-free monolayer cultures of immature (swine) granulosa cells.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/agonistas , Hormona Folículo Estimulante/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Fosfoproteínas/biosíntesis , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Secuencia de Bases , Células Cultivadas , Sinergismo Farmacológico , Femenino , Datos de Secuencia Molecular , Fosfoproteínas/genética , ARN Mensajero/análisis , PorcinosAsunto(s)
Arquitectura y Construcción de Instituciones de Salud/economía , Promoción de la Salud/economía , Departamentos de Hospitales/economía , Administración de Línea de Producción , Costos y Análisis de Costo , Emprendimiento , Arquitectura y Construcción de Instituciones de Salud/tendencias , Renta , Michigan , Afiliación Organizacional , Técnicas de PlanificaciónRESUMEN
In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/agonistas , Hormona Folículo Estimulante/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/enzimología , Hidroximetilglutaril-CoA Reductasas/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Animales , Secuencia de Bases , ADN Complementario/química , ADN Complementario/genética , Sinergismo Farmacológico , Femenino , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Homología de Secuencia , PorcinosRESUMEN
Recent studies of the function of adrenal "incidentalomas" have revealed that a proportion of those tumors secrete cortisol insufficiently to produce overt clinical Cushing s syndrome, but that their autonomous cortisol production can suppress the hypothalamo-pituitaryadrenal (HPA) axis to various degrees; this needs to be recognized to avoid acute adrenal insufficiency after adrenalectomy. Several diagnostic approaches have been utilized to identify the partially autonomous cortisol-secreting adenomas. It has been suggested that a lack of normal suppression of cortisol (> 140 nmol/L) on the morning after 1-mg oral dexamethasone at bedtime would identify most functional autonomous cortisol-secreting tumors. Based on this criterion, approximately 18% of published cases of incidentalomas would secrete cortisol autonomously. However, other tests indicating alterations of the HPA axis, such as abnormal adrenal iodocholesterol uptake or decreased plasma levels of dehydroepiandrosterone sulfate (DHAS), were found to be present in up to 79%-86% of incidentalomas. This is illustrated by the description of three patients with incidentalomas with plasma cortisol levels < 140 nmol/L in 2 of 3 patients after 1-mg dexamethasone overnight; however, various degrees of HPA axis suppression were demonstrated by an i.v. dexamethasone (4-mg) suppression test, decreased plasma DHAS levels and unilateral adrenal iodocholesterol uptake. After laparoscopic adrenalectomy, the response of plasma cortisol to 250 mug i.v. of ACTH (1-24) was subnormal in 2 of 3 patients and was restored to normal within 2 months. We conclude that the criterion of a plasma cortisol level > 140 nmol/L, after an overnight 1-mg dexamethasone suppression test, underestimates the incidence of partially autonomous cortisol-secreting adrenal adenomas. The literature on this subject is reviewed, and recommendations for evaluation and treatment are presented.
RESUMEN
Studies were undertaken to identify intracellular mediators of prolactin inhibition of glucocorticoid-induced apoptosis in Nb2 lymphoma cells. A short-term assay was implemented that quantitates fragmented DNA released from the genome by reaction with diphenylamine. Induction and inhibition of internucleosomal DNA cleavage (indicative of apoptosis) was verified by agarose gel electrophoresis of extracted cellular DNA. Synchronized Nb2 cells (G0/G1) exhibited increased DNA fragmentation after 4-hr incubation with dexamethasone (DEX) (25-100 nM) which was inhibited by ovine prolactin (oPRL) (0.1-1 ng/ml), the glucocorticoid receptor antagonist, RU486 (500 nM), and the nuclease inhibitor, aurintricarboxylic acid (100 microM). Signals previously implicated in prolactin induction of mitogenesis in Nb2 cells were investigated for their role in prolactin inhibition of apoptosis including: protein kinase C activation, arachidonic acid metabolism, polyamine production, tyrosine phosphorylation, and extracellular calcium. Protein kinase C agonists, phorbol-12-myristate-13-acetate, and 1,2-dioctanoyl-sn-glycerol, +/- the calcium ionophore, A23187 (200 nM), did not mimic oPRL inhibition of DEX-induced DNA fragmentation. Protein kinase C inhibitors, gossypol and quercetin, did not block prolactin action. Arachidonic acid did not mimic prolactin protection against DEX-induced DNA fragmentation. Inhibitors of arachidonic acid metabolism, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and indomethacin did not block prolactin action. The polyamine, spermine, inhibited DEX-induced DNA fragmentation at 1.5 to 2.5 mM. However, inhibition of polyamine synthesis with alpha-difluoromethyl ornithine or methylglyoxal bis(guanylhydrazone) did not inhibit prolactin action. Prolactin action was not blocked by inhibitors of tyrosine kinase activation, genistein and tyrphostin-47. On the other hand, pervanadate, a potent tyrosine phosphatase inhibitor, consistently inhibited DEX-induced DNA fragmentation. Prolactin action and DEX-induced apoptosis both occurred in calcium-free PBS. In summary, protein kinase C activation and eicosanoid production do not appear to mediate this prolactin action. Although spermine could block DNA fragmentation, blockade of the polyamine cascade did not inhibit prolactin action, suggesting that polyamines do not mediate this prolactin effect. While inhibitors of tyrosine kinase activation did not block prolactin action, tyrosine phosphatase inhibition in the presence of basal tyrosine kinase activity mimicked prolactin action, suggesting tyrosine phosphorylation participation in the anti-apoptotic effect. Extra-cellular calcium was not required for prolactin or DEX action.