In vivo and in vitro regulation of thyroid leukemia inhibitory factor (LIF): marker of hypothyroidism.

Several cytokines regulate thyroid function and may be involved in the pathogenesis of thyroid disorders, including euthyroid sick syndrome. Leukemia inhibitory factor (LIF), a neuroimmune pleiotropic cytokine, was measured to assess its role in hypothalamic-pituitary-thyroid function. Mean circulating serum LIF levels in 10 hypothyroid patients [TSH, 23+/-0.5 mIU/L (mean+/-SEM); free T4, 0.77+/-0.1 ng/dL] was 0.29+/-0.04 ng/mL, 145% higher (P < 0.04) than in 20 normal subjects (LIF, 0.20+/-0.02 ng/mL; TSH, 2.23+/-0.21 mIU/L; free T4, 1.23+/-0.04 ng/dL) but was not different from those in 10 hyperthyroid patients (LIF, 0.21+/-0.03 ng/mL; TSH, 0.01+/-0.00 mIU/L; free T4, 3.63+/-0.51 ng/dL). Serum LIF concentrations linearly correlated with serum TSH in the 40 samples (r = 0.58, P < 0.001). When T4 (1-8 microg/kg x day) was administered to cynomolgus monkeys with methimazole-induced hypothyroidism, serum T4 and T3 levels increased appropriately, and TSH and LIF concentrations decreased. When methimazole was given alone, both serum TSH (146+/-30 mIU/L) and LIF (8.84+/-0.49 ng/mL) were markedly induced. When methimazole together with T4 (>2 microg/kg x day) was administered, both serum TSH (7.5+/-1.2 mIU/L) and LIF (6.22+/-0.31 ng/mL) were lowered (P < 0.01). Monkey serum LIF levels and log TSH levels also correlated (r = 0.72, P < 0.01). Cultured thyroid carcinoma cells produced LIF (9.2 ng/10(6) cells/48 h). TSH (100 mIU/mL) and interleukin (IL)-6 (10 nmol/L) stimulated in vitro LIF secretion from the cells by 170+/-12% (P < 0.05) and 261+/-8% (P < 0.05), respectively. Dexamethasone (1 micromol/L) inhibited basal LIF concentration by 83% (P < 0.05), whereas TSH and IL-6 stimulated LIF by 52% (P = 0.04) and 42% (P = 0.03), respectively. However, using Northern blot analysis, we could not observe induction of LIF mRNA by TSH, suggesting that LIF regulation by TSH may be due to stimulation of secretion. The results show that the thyroid gland is a source of LIF production; TSH, IL-6, and glucocorticoid influence thyroid cell LIF expression. The correlation between TSH and LIF suggests that LIF may participate in the physiologic regulation of hypothalamic-pituitary-thyroid function.     

Glucocorticoid induces apoptosis in rat leydig cells.

The aim of the present study was to investigate whether glucocorticoid induces apoptosis in rat Leydig cells. To determine whether there are developmental differences in glucocorticoid sensitivity, Leydig cells were isolated at distinct stages of their differentiation [mesenchymal-like progenitors (PLC), immature Leydig cells (ILC), and adult Leydig cells (ALC)] from 21-, 35-, and 90-d-old Sprague Dawley rats, respectively. Glucocorticoid induction of apoptosis was evaluated after both in vitro and in vivo exposures. In the first set of experiments, PLC, ILC, and ALC were treated with 100 nM corticosterone (CORT) for either 4 or 24 h in vitro and then assessed for labeling with the apoptotic marker annexin V. PLC exposed to CORT had levels of annexin V-fluorescein isothiocyanate labeling that were unchanged relative to control values at both time points (P > 0.05). In contrast, CORT-treated ILC and ALC had increased frequencies of apoptosis: in ALC, a 22.1 +/- 1.7% incidence after 4 h and 30.5 +/- 2.3% after 24 h compared with 7.4 +/- 0.8% in untreated controls (P < 0.05). Similar trends were observed for ILC. Ultrastructural analysis confirmed that the increase in annexin V labeling was associated with characteristic signs of apoptosis, including nuclear fragmentation and formation of apoptotic bodies. A second line of experiments examined whether apoptosis was evident in purified Leydig cells after administration of CORT in vivo. Male rats were subjected to bilateral adrenalectomy and were treated with CORT by ip injection twice daily at doses ranging from 2.5-7.5 mg/100 g BW starting 3 d after surgery. The frequency of Leydig cell apoptosis was measured at 12, 24, 48, and 72 h after the first injection. Administration of the 2.5-mg dose raised circulating CORT 5-10 times above normal basal concentrations, and LH levels sampled at these times were not altered in the treated animals. Increased Leydig cell apoptosis was measurable after 24 h of treatment, with an incidence of 21.1 +/- 1.8% in ALC compared with 5.7 +/- 0.8% in untreated controls (P < 0.05). Sharp reductions in immunocytochemical staining intensity were observed in the treated animals for a Leydig cell marker, 11beta-hydroxysteroid dehydrogenase, which occurred concurrently with decreased serum T levels. This was consistent with the hypothesis that CORT-mediated induction of apoptosis leads to declines in Leydig cell numbers, thereby affecting T production. These results suggest that excessive exposure to CORT initiates apoptosis in rat Leydig cells, potentially contributing to suppression of circulating T levels during stress and other conditions in which glucocorticoid concentrations are elevated.     

Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells

Exposure of humans to bisphenol A (BPA), a monomer in polycarbonate plastics and a constituent of resins used in food packaging and dentistry, is significant. In this report exposure of rats to 2.4 microg/kg.d (a dose that approximates BPA levels in the environment) from postnatal d 21-35 suppressed serum LH (0.21 +/- 0.05 ng/ml; vs. control, 0.52 +/- 0.04; P < 0.01) and testosterone (T) levels (1.62 +/- 0.16 ng/ml; vs. control, 2.52 +/- 0.21; P < 0.05), in association with decreased LHbeta and increased estrogen receptor beta pituitary mRNA levels as measured by RT-PCR. Treatment of adult Leydig cells with 0.01 nm BPA decreased T biosynthesis by 25% as a result of decreased expression of the steroidogenic enzyme 17alpha-hydroxylase/17-20 lyase. BPA decreased serum 17beta-estradiol levels from 0.31 +/- 0.02 ng/ml (control) to 0.22 +/- 0.02, 0.19 +/- 0.02, and 0.23 +/- 0.03 ng/ml in rats exposed to 2.4 microg, 10 microg, or 100 mg/kg.d BPA, respectively, from 21-35 d of age (P < 0.05) due to its ability to inhibit Leydig cell aromatase activity. Exposures of pregnant and nursing dams, i.e. from gestation d 12 to postnatal d 21, decreased T levels in the testicular interstitial fluid from 420 +/- 34 (control) to 261 +/- 22 (P < 0.05) ng/ml in adulthood, implying that the perinatal period is a sensitive window of exposure to BPA. As BPA has been measured in several human populations, further studies are warranted to assess the effects of BPA on male fertility.     

Phthalate-induced Leydig cell hyperplasia is associated with multiple endocrine disturbances

The possibility that exposures to environmental agents are associated with reproductive disorders in human populations has generated much public interest recently. Phthalate esters are used most commonly as plasticizers in the food and construction industry, and di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate in the environment. Daily human exposure to DEHP in the U.S. is significant, and occupational and clinical exposures from DEHP-plasticized medical devices, e.g., blood bags, hemodialysis tubing, and nasogastric feeding tubes, increase body burden levels. We investigated the effects of chronic exposures to low environmentally relevant DEHP levels on testicular function. Our data show that prolonged exposures to this agent induced high levels of the gonadotropin luteinizing hormone and increased the serum concentrations of sex hormones [testosterone and 17beta-estradiol (E2)] by >50%. Increased proliferative activity in Leydig cells was evidenced by enhanced expression of cell cycle proteins, as determined by RT-PCR. The numbers of Leydig cells in the testis of DEHP-treated rats were 40-60% higher than in control rats, indicating induction of Leydig cell hyperplasia. DEHP-induced elevations in serum testosterone and E2 levels suggest the possibility of multiple crosstalks between androgen, estrogen, and steroid hormone receptors, whereas the presence of estrogen receptors in nonreproductive tissues, e.g., cardiovascular system and bones, implies that the increases in serum E2 levels have implications beyond reproduction, including systemic physiology. Analysis of the effects of phthalate exposures on gonadotropin and steroid hormone levels should form part of overall risk assessment in human populations.     

Estrogen receptor-alpha gene deficiency enhances androgen biosynthesis in the mouse Leydig cell

Leydig cells, which produce the primary male steroid hormone testosterone (T), express the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, and have the capacity to convert testosterone to the natural estrogen 17beta-estradiol. Thus, Leydig cells are subject to estrogen action. The development of transgenic mice that are homozygous for targeted deletion of genes encoding the ER subtypes provides an opportunity to examine the role of estrogen in Leydig cell function. In this study androgen biosynthesis was analyzed in Leydig cells from mice that were homozygous for targeted deletion of the ERalpha gene (alphaERKO). T production by alphaERKO Leydig cells was 2-fold higher than that in wild-type (WT) cells. Serum T levels were accordingly higher in alphaERKO compared with WT mice (5.1 +/- 1.1 vs. 2.2 +/- 0.4 ng/ml; P 相似文献   

DEHP对妊娠大鼠肺泡巨噬细胞脂质过氧化反应及肺组织OPN表达的影响

目的观察邻苯二甲酸二(2-乙基已基)酯(DEHP)对妊娠期大鼠肺泡巨噬细胞(AM)内脂质过氧化反应及肺组织骨桥蛋白(OPN)表达的影响。方法 24只SD大鼠随机分为四组,每组6只。从孕第1天开始至子鼠出生后第21天,对照组(NC)给予玉米油0.5 mL/d,其余三组给予玉米油和10、100、750 mg/(kg.d)DEHP混合物0.5 mL灌胃染毒,分别为低、中、高剂量组。HE、Masson三色染色光镜下观察孕鼠肺组织的形态学改变,采用免疫组织化学染色法测定肺组织中的OPN。用含10%小牛血清的RPMI-1640细胞培养液分离、培养肺泡AM,并测定其超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GSH-PX)、一氧化氮合酶(NOS)的活性及丙二醛(MDA)、NO含量。结果高剂量组、中剂量组GSH-PX、SOD活性明显低于NC组,MDA、NO含量及NOS活性显著高于NC组(P均<0.05);HE染色高剂量组显示肺间质增厚、肺间质细胞增多,肺泡数目减少;Masson三色染色显示,低剂量组肺细小动脉以及肺间质均见有蓝色的胶原纤维沉积,中剂量组和高剂量蓝色胶原纤维沉积更为显著;免疫组化法显示NC组孕鼠肺组织OPN表达主要见于支气管、肺泡上皮细胞内;低剂量组大鼠肺细小动脉内皮细胞、平滑肌细胞以及周围巨噬细胞均见有OPN的表达,中剂量组和高剂量表达更强。结论 DEHP可使妊娠期大鼠肺泡AM内脂质过氧化反应增强,并促进肺组织内OPN过表达。     

Leukemia inhibitory factor reduces contractile function and induces alterations in energy metabolism in isolated cardiomyocytes

Interleukin (IL)-6 related cytokines may be involved in the pathophysiology of heart failure. Leukemia inhibitory factor (LIF) is an IL-6 related cytokine, and elevated levels of LIF have been found in failing hearts. The aim of our study was to investigate how LIF may influence isolated cardiomyocytes. Adult cardiomyocytes were isolated from male Wistar rat hearts and treated with 1 nM LIF for 48 h. Contractile function was measured using a video-edge detection system. Fractional shortening was reduced at 0.25 Hz in LIF treated cells (7.4% +/- 0.5%) compared to control cells (9.0% +/- 0.7%). Gene expression analysis showed that expression of the mitochondrial ATP-synthase F(1) alpha subunit was reduced in cells exposed to LIF. The activity of the enzyme was also reduced in these cells (0.10 +/- 0.05 mumol/min per mg protein) compared to controls (1.23 +/- 0.40 mumol/min per mg protein). The levels of ATP and creatine phosphate were reduced by 15.0% +/- 3.0% and 11.2% +/- 2.7% in LIF treated cells. LIF increased both (3)H-deoxyglucose uptake and lactate levels, suggesting an increase in anaerobic energy metabolism. Beta-oxidation of (14)C-oleic acid was increased by 51.2% +/- 14.1% following LIF treatment, but no changes were found in cellular uptake or oxidation of (14)C-oleic acid to CO(2). In conclusion, LIF induces contractile dysfunction and changes in energy metabolism in isolated cardiomyocytes.     

Transdermal testosterone gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men

Testosterone (T) therapy for hypogonadal men should correct the clinical abnormalities of T deficiency, including improvement of sexual function, increase in muscle mass and strength, and decrease in fat mass, with minimal adverse effects. We have shown that administration of a new transdermal T gel formulation to hypogonadal men provided dose proportional increases in serum T levels to the normal adult male range. We now report the effects of 180 days of treatment with this 1% T gel preparation (50 or 100 mg/day, contained in 5 or 10 g gel, respectively) compared to those of a permeation-enhanced T patch (5 mg/day) on defined efficacy parameters in 227 hypogonadal men. In the T gel groups, the T dose was adjusted up or down to 75 mg/day (contained in 7.5 g gel) on day 90 if serum T concentrations were below or above the normal male range. No dose adjustment was made with the T patch group. Sexual function and mood changes were monitored by questionnaire, body composition was determined by dual energy x-ray absorptiometry, and muscle strength was measured by the one repetitive maximum technique on bench and leg press exercises. Sexual function and mood improved maximally on day 30 of treatment, without differences across groups, and showed no further improvement with continuation of treatment. Mean muscle strength in the leg press exercise increased by 11 to 13 kg in all treatment groups by 90 days and did not improve further at 180 days of treatment. Moderate increases were also observed in arm/chest muscle strength. At 90 days of treatment, lean body mass increased more in the 100 mg/day T gel group (2.74 +/- 0.28 kg; P = 0.0002) than in the 50 mg/day T gel (1.28 +/- 0.32 kg) and T patch groups (1.20 +/- 0.26 kg). Fat mass and percent fat were not significantly decreased in the T patch group, but showed decreases in the T gel groups (50 mg/day, -0.90 +/- 0.32 kg; 100 mg/day, - 1.05 +/- 0.22 kg). The increase in lean mass and the decrease in fat mass were correlated with the changes in average serum T levels attained after transdermal T replacement. These beneficial effects of T replacement were accompanied by the anticipated increases in hematocrit and hemoglobin but without significant changes in the lipid profile. The increase in mean serum prostate-specific antigen levels (within the normal range) was correlated with serum levels of T. The greatest increases were noted in the 100 mg/day T gel group. Skin irritation was reported in 5.5% of subjects treated with T gel and in 66% of subjects in the permeation-enhanced T patch group. We conclude that T gel replacement improved sexual function and mood, increased lean mass and muscle strength (principally in the legs), and decreased fat mass in hypogonadal men with less skin irritation and discontinuation compared with the recommended dose of the permeation-enhanced T patch.     

Elevated plasma fibrinogen level predicts suboptimal response to therapy with both single- and double-bolus eptifibatide during percutaneous coronary intervention.

OBJECTIVES: This study sought to determine the factors associated with suboptimal platelet inhibition (PI) with single- and double-bolus eptifibatide during percutaneous coronary intervention (PCI). BACKGROUND: Although PI > or = 95% measured 10 min after glycoprotein IIb/IIIa inhibitor therapy is associated with improved outcomes following PCI, this level of PI often is not achieved. METHODS: We prospectively studied 150 patients undergoing PCI with single-bolus eptifibatide (180 microg/kg) (n = 100) and double-bolus eptifibatide (180 microg/kg administered 10 min apart) (n = 50) followed by standard infusion (2 microg/kg/min). Measuring platelet aggregation at baseline and at 10 min and 30 to 45 min after eptifibatide bolus, patients were classified as optimal responders (OPT) (> or =95% PI) or suboptimal responders (sub-OPT) (<95% PI) based on 10-min PI after final bolus. RESULTS: Suboptimal PI was achieved in 61% of patients with single-bolus eptifibatide and in 36% with double-bolus eptifibatide. In the single-bolus group, sub-OPT had higher fibrinogen levels (324 +/- 85 mg/dl vs. 259 +/- 49 mg/dl, p = 0.0002), platelet counts (221 +/- 70 vs. 186 +/- 47, p = 0.008), and white blood cell counts (7.7 +/- 2.3 vs. 6.6 +/- 1.9, p = 0.02). In the double-bolus group, sub-OPT also had higher fibrinogen levels (324 +/- 68 mg/dl vs. 278 +/- 53 mg/dl, p = 0.01) and were more likely to be smokers (38.9% vs. 9.4%, p = 0.01). Multivariable analysis showed that fibrinogen level was the only independent predictor of suboptimal PI, with fibrinogen cutoffs at 375 and 325 mg/dl predicting suboptimal PI (single-bolus: 100% and 90.0%, respectively; double-bolus: 100% and 60%, respectively) with both doses. CONCLUSIONS: During PCI, both single- and double-bolus eptifibatide provide suboptimal PI in a substantial proportion of patients. A fibrinogen level >375 mg/dl is a strong predictor of suboptimal PI.     

In vitro stimulation of adenosine 3',5'-monophosphate in unilateral undescended testes of humans by follicle-stimulating hormone and luteinizing hormone.

The in vitro effects of FSH and LH on cAMP generation by unilateral undescended testes and contralateral descended testes were investigated. Biopsy tissue was preincubated at 37 C for 5 min in 1 ml Krebs-Ringer bicarbonate buffer (KRB) containing 10 mM aminophylline. Half of each specimen was frozen in liquid nitrogen to assess basal levels of cAMP. The other half was incubated for 20 min at 37 C in 1 ml fresh KRB containing aminophylline and FSH (10 microgram) or LH (10 microgram). The mean concentration of cAMP in the descended testes was 1.03 +/- 0.04 pmol/mg protein, whereas in the undescended testes the level was 1.98 +/- 0.07 pmol/mg protein (P less than or equal to 0.01). After the 20-min incubation period with FSH the concentrations of cAMP in the descended and undescended testes were 10.85 +/- 0.86 pmol/mg protein and 11.21 +/- 1.1 mol/mg protein, respectively. Incubation with LH resulted in the generation of equivalent levels of cAMP in both the descended (2.76 +/- 0.30 pmol/mg protein) and undescended (3.0 +/- 0.17 pmol/mg protein) testes. Although marked histological differences were observed in the germinal epithelium, the appearance and numbers of Leydig and Sertoli cells were comparable. The data suggest that the Sertoli cell is the primary receptor for FSH. Leydig and Sertoli cell functions of the undescended testes, at least in terms of cAMP generation, are similar to normal descended testes.     

Antiplatelet effects of abciximab, tirofiban and eptifibatide in patients undergoing coronary stenting

OBJECTIVES: We sought to investigate whether abciximab, tirofiban and eptifibatide achieve comparable antiplatelet effects with coronary stenting. BACKGROUND: The glycoprotein (GP) IIb/IIIa antagonists abciximab, tirofiban and eptifibatide differ in chemical structure, binding site and pharmacokinetics. METHODS: Sixty patients undergoing coronary stenting were randomly assigned to abciximab (bolus 0.25 mg/kg body weight, infusion 10 microg per min for 12 h), tirofiban (bolus 10 microg/kg, infusion 0.15 microg/kg per min for 72 h) or eptifibatide (bolus 180 microg/kg, infusion 2 microg/kg per min for 72 h). We took serial blood samples to analyze platelet function by using flow cytometry, turbidimetric aggregometry and the rapid platelet-function assay (RPFA). RESULTS: As assessed by RPFA, platelet aggregation after 2 h of infusion was reduced to 5.9 +/- 7.8% (mean +/- SD) of baseline by abciximab, to 5.0 +/- 5.4% by tirofiban and to 7.8 +/- 7.1% by eptifibatide (p = 0.42). Turbidimetric aggregometry with adenosine diphosphate stimulation yielded similar results, whereas percent inhibition of platelet aggregation after thrombin receptor stimulation was 45.8 +/- 16.8% with abciximab, 51.3 +/- 17.6% with tirofiban and 52.9 +/- 14.8% with eptifibatide (p = 0.37). Tirofiban and eptifibatide maintained their level of platelet inhibition during infusion. Flow cytometry revealed that the reduction in the monocyte-platelet interaction by abciximab, tirofiban and eptifibatide was not significantly different (20.0 +/- 21.9%, 23.8 +/- 18.2% and 21.0 +/- 19.8%, respectively; p = 0.87). CONCLUSIONS: Abciximab, tirofiban and eptifibatide, at currently recommended doses, achieved similar levels of inhibition of platelet aggregation and a similar reduction in the platelet-monocyte interaction.     

The hypothalamic-pituitary-testicular axis in thyrotoxicosis.

The hypothalamic-pituitary-testicular axis was evaluated in seven men with thyrotoxicosis due to Graves' disease. Loss of libido and decreased potency were present in 71% and 56%, respectively. All patients had normal testicular volume (25 ml in all) and gynecomastia was detected in two of seven patients. Total sperm counts were less than 40 million in four of the five men tested. There was an inverse correlation between basal serum 17 beta-estradiol (E2) levels and total sperm count (r = -0.87; P less than 0.05). Mean (+/- SE) total testosterone (T) and E2 levels (1008 +/- 104 ng/100 ml and 104 +/- 16 pg/ml) were significantly higher than in normal men (P less than 0.05). Free T (13.6 +/- 2.4 ng/100 ml) was indistinguishable from normal (15.3 +/- 1.5 ng/100 ml). The mean (+/- SE) response of serum T to hCG administration was blunted (80 +/- 40%) compared to controls (193 +/- 19%; P less than 0.02). Basal plasma LH levels (15.5 +/- 1.5 mIU/ml) were significantly higher (P less than 0.05) than in normal men (9.1 +/- 0.6 mIU/ml) and hyperresponded to 100 microgram LRH iv in five of seven patients. Basal plasma FSH levels and the FSH response to LRH were normal. These results suggest that men with hyperthyroidism have 1) partial Leydig cell failure, 2) impairment of spermatogenesis, and 3) blunting of the feedback effects of E2.     

Leukemia inhibitory factor antagonizes gonadotropin induced-testosterone synthesis in cultured porcine leydig cells: sites of action

In the present report, the action of leukemia inhibitory factor (LIF) on testicular steroid hormone formation was studied. For this purpose, the direct effects of LIF were evaluated on basal and human (h)CG-stimulated testosterone synthesis by cultured, purified Leydig cells isolated from porcine testes. LIF reduced (more than 60%) hCG-stimulated testosterone synthesis. This inhibitory effect was exerted in a dose- and time-dependent manner. The maximal and half-maximal effects were obtained with, respectively, 10 ng/ml (0.5 nM ) and 2.5 ng/ml (0.125 nM ) of LIF after a 48-h treatment of the Leydig cells. Such an effect of the cytokine was not a cytotoxic effect, because it was reversible and Leydig cells recovered most of their steroidogenic activity after the removal of LIF. Considering the sites of action of LIF in inhibiting gonadotropin-stimulated testosterone formation, it was shown that LIF significantly (P < 0.002) reduced, in a comparable range (about 60% decrease), testosterone synthesis stimulated with LH/hCG or with pharmacological agents that enhance cAMP levels (cholera toxin, forskolin, and PG E2), and testosterone synthesis stimulated with 8-bromo-cAMP. Such an observation indicates that the antigonadotropic action of the cytokine is exerted in a predominant manner at a step (or steps) located beyond cAMP formation. Furthermore, incubation of Leydig cells with 22R-hydroxycholesterol (5 microg/ml, 2 h), a cholesterol substrate derivative that does not need an assisted process to be delivered to the inner mitochondrial membrane, reversed most of the inhibitory effect of LIF on the steroid hormone formation. Such results indicate that LIF acts by reducing cholesterol substrate availability in the mitochondria. Consequently, LIF action was tested on steroidogenic acute regulatory protein and PBR (peripheral benzodiazepine receptor) shown to be potentially involved in such a cholesterol transfer. LIF reduced, in a dose- and time-dependent manner, LH/hCG-induced steroidogenic acute regulatory protein messenger RNA levels. The maximal inhibitory effect was obtained with 6.6 ng/ml of LIF after 48 h of treatment. In contrast, LIF had no effect on PBR messenger RNA expression or PBR binding. This inhibitory effect of LIF on Leydig cell steroidogenesis is probably exerted via an auto/paracrine action of the cytokine. Indeed, by immunohistochemistry, LIF and LIF receptor proteins were identified in Leydig and Sertoli cells but not in other testicular cell types, except for LIF receptor in spermatogonia. Furthermore, the presence of LIF and its receptor in Leydig cells at the neonatal and adult periods suggests that the inhibitory effect of LIF on androgen formation reported here probably occurs in both the fetal and the adult Leydig cell populations during testicular development.     

Short-term oral ingestion of Ginkgo biloba extract (EGb 761) reduces malondialdehyde levels in washed platelets of type 2 diabetic subjects

We have recently reported that ingestion of Ginkgo biloba extract (EGb 761) (a) significantly reduced collagen-induced platelet aggregation and thromboxane B2 (TXB2) production in both non-diabetic individuals as well as those with type 2 diabetes mellitus (T2DM), (b) significantly reduced platelet malondialdehyde (MDA), an index of lipid peroxidation, in non-diabetic subjects. In the present study we report that ingestion of EGb 761 (120 mg daily for 3 months), significantly decreased platelet MDA-thiobarbituric acid reacting substances (TBARS) (41 +/- 9 pmol/10(7) platelets versus 30 +/- 11 pmol/10(7) platelets) (p < 0.005) in T2DM subjects with normal cholesterol levels (total cholesterol, 164 +/- 22 mg/dl; age, 54 +/- 9 years; BMI, 35.0 +/- 8.8 kg/m2, n = 12). In T2DM subjects with high cholesterol (total cholesterol, 218 +/- 15 mg/dl; age, 52 +/- 5 years; BMI, 36.2 +/- 6.6 kg/m2, n = 7), EGb 761 ingestion reduced the platelet TBARS from 29 +/- 9 to 22 +/- 9 pmol/10(7) platelets (p < 0.04). Because ingestion of EGb 761 did not alter platelet counts it is concluded that EGb 761, probably due to the flavonoid fraction, reduced the TBARS by inhibiting cyclooxygenase (COX)-1-mediated arachidonic acid oxygenation or by reducing the arachidonic acid pool. This is likely to lead to a reduction of platelet hyperactivity, a significant contributor to the development of cardiovascular disease in T2DM patients. Because of other reported beneficial properties of EGb 761, such as stimulation of pancreatic beta-cell function in T2DM subjects with pancreatic exhaustion, it appears that T2DM subjects might benefit from ingesting EGb 761 as a dietary supplement.     

Influence of growth hormone on glucose-induced glucose uptake in normal men as assessed by the hyperglycemic clamp technique

To determine whether physiological increments in circulating GH concentrations influence glucose-induced glucose uptake (GIGU), two-step sequential hyperglycemic clamp (plasma glucose, 6 and 14 mmol/L) studies were performed in six normal subjects with and without GH infusion (40 ng/kg.min). The latter resulted in serum GH levels of 15 +/- 1 (+/- SE) microgram/L. Infusion of somatostatin (250 micrograms/h during step 1 and 750 micrograms/h during step 2) together with a replacement dose of insulin (1.1 pmol/kg.min) resulted in serum insulin levels comparable to basal levels in both studies. The GIGU ([3-3H]glucose), assessed as the difference between steps 2 and 1 glucose utilization during the final 60 min of each step (150 min) was markedly impaired during GH infusion (with GH, 1.1 +/- 0.2 mg/kg.min; without GH, 3.1 +/- 0.3 mg/kg.min; P less than 0.001). Moreover, the percent increase in glucose uptake was considerably reduced during hypersomatotropinemia (with GH, 44 +/- 9%; without GH, 97 +/- 11%; P less than 0.01). In the GH infusion as well as control studies, endogenous glucose production (EGP) was similar at the two levels of glycemia, whereas GH infusion approximately doubled EGP [2.3 +/- 0.2 vs. 1.1 +/- 0.3 mg/kg.min and 2.0 +/- 0.4 vs. 1.1 +/- 0.4 mg/kg.min (step 1 and 2, respectively)]. We conclude that moderate hypersomatotropinemia for several hours is characterized by impaired GIGU as well as augmented EGP.     

Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in nonhuman primates

The relative antithrombotic effectiveness of targeting glycoprotein (GP) Ib-dependent versus GPIIb-IIIa-dependent platelet interactions has been determined in baboons by measuring thrombus formation after infusing comparable antihemostatic doses of anti-von Willebrand factor (vWF) monoclonal antibody (MoAb) BB3-BD5, anti-GPIb MoAb AP1, and anti- GPIIb-IIIa MoAb LJ-CP8 under conditions of arterial and venous flow (shear rates of 750 to 1,000 seconds-1 and 100 seconds-1, respectively). Thrombus formation was quantified as 111In-platelet deposition and 125I-fibrin accumulation on segments of collagen-coated tubing interposed in chronic exteriorized arteriovenous (AV) shunts for 40 minutes. In vitro, anti-vWF MoAb BB3 BD5 (IgG) and anti-GPIb MoAb AP1 [IgG or F(ab)2 fragments] inhibited ristocetin-induced platelet aggregation (IC50 50 nmol/L and 1 mumol/L, respectively), but neither of these MoAbs blocked platelet aggregation induced by adenosine diphosphate (ADP) (P > .5). Conversely, anti-GPIIb-IIIa MoAb LJ-CP8 inhibited platelet aggregation induced by ADP (IC50 1 mumol/L, but failed to block ristocetin-induced platelet aggregation (P > .5). In vivo, the intravenous infusion of anti-vWF MoAb BB3 BD5 or anti-GPIIb- IIIa MoAb LJ-CP8 into baboons at doses that abolished corresponding agonist-induced aggregation ex vivo (bolus injections of 0.5 mg/kg and 10 mg/kg, respectively) prolonged template bleeding times from baseline values of 4.0 +/- 0.3 minutes to > 27 +/- 4 minutes, and to > 26 +/- 4 minutes, respectively (P < .001 in both cases), without affecting the peripheral platelet count (P > .5). However, injection of anti-GPIb MoAb AP1 [10 mg/kg as IgG or 1 mg/kg as F(ab)2 fragments] produced immediate irreversible thrombocytopenia (< 40,000 platelets/microL). Anti-GPIIb-IIIa MoAb LJ-CP8 abolished platelet deposition and fibrin accumulation on collagen segments under both arterial and venous flow conditions (P < .01 in all cases), whereas MoAb BB3 BD5 produced minimal inhibition of platelet deposition and no decrease in fibrin accumulation at arterial shear rates and undetectable antithrombotic outcomes at low shear. Thus, inhibiting GPIIb-IIIa-dependent platelet recruitment abrogates both thrombus formation and platelet hemostatic function at both venous and arterial shear rates. By contrast, interfering with GPIb-vWF-dependent platelet interactions abolishes platelet hemostatic function without producing corresponding antithrombotic effects.     

The collection, preservation and function of peripheral blood hematopoietic cells in dogs.

Semicontinuous flow centrifugation (SFC) was employed in canines to obtain adequate numbers of cells for autologous marrow repopulation following supralethal cyclophosphamide administration (100 mg per kg). Four cycles using a 225 ml bowl and 30 ml per minute flow rate were carried out for procurement. Collections averaged 8.4 +/- 0.4 x 10(9) (n = 30) leukocytes. Mononuclear cells (MNC) comprised 75 +/- 2% of the population and granulocyte colony-forming units (CFU-C) totaled 1.9 +/- 0.09 x 10(5) colonies per collection. Eighty-six percent of mononuclear cells were "T" cells in the 30 to 90 second fraction compared to 49% at 150 to 120 seconds. CFU-C fractionation revealed a peak at 30-210 seconds and a second peak at 120-210 seconds. Following programed freezing and rapid thawing 77.7 +/- 11.4% of CFU-C were recovered. Using a dose of 1 x10(9) MNC per kg for reinfusion, marrow repopulation and clinical recovery occurred in four out of four dogs. It was concluded that 1) SFC was effective for obtaining adequate numbers of peripheral blood stem cells for autologous marrow repopulation. 2) A rapid thawing and direct transfusion technique appears satisfactory for administration. 3) Some separation of "T", "B" and CFU-C peripheral blood components is possible by centrifugation.     

An observational study of reduction of insulin resistance and prevention of development of type 2 diabetes mellitus in women with polycystic ovary syndrome treated with metformin and diet

Our first specific aim in an observational study of 431 nondiabetic women with polycystic ovary syndrome (PCOS), aged >or=20 years and with >or=11 months follow-up on metformin diet, was to prospectively assess relationships between pretreatment glucose and insulin resistance (IR) and the development of type 2 diabetes mellitus (T2DM) or gestational diabetes (GD). Our second specific aim was to determine whether development of T2DM and GD was independently associated with lesser reduction of IR on metformin diet when compared with women who remained free of T2DM and GD. Women with body mass index <25 kg/m(2) and those with body mass index >or=25 kg/m(2) were, respectively, instructed in a 2000- or 1500-cal/d, high-protein (26% of calories), low-carbohydrate (44%) diet, with 30% of calories as fat and a polyunsaturate-saturate ratio of 2:1. Three groups of women with PCOS were categorized: (a) 17 with no previous GD, who developed T2DM on metformin diet (mean +/- SD follow-up, 49 +/- 33 months), (b) 401 with no previous GD and free of T2DM on metformin diet (follow-up, 38 +/- 25 months), and (c) 13 with either previous GD or GD on metformin diet (follow-up, 38 +/- 25 months). On metformin diet, women who developed T2DM vs those who remained free of T2DM had higher pretreatment glucose (odds ratio [OR], 1.09; 95% confidence interval [CI], 1.03-1.16; P = .003) and homeostasis model assessment of insulin resistance (HOMA-IR) (OR, 1.22; 95% CI, 1.04-1.42; P = .01), and less reduction of HOMA-IR (OR, 0.82; 95% CI, 0.72-0.92; P = .0008). On metformin diet, women either with previous GD or who developed GD vs those who remained free of T2DM had less reduction of HOMA-IR (OR, 0.88; 95% CI, 0.78-0.99; P = .03). By repeated-measures analysis, on metformin diet, women who did not develop T2DM had reduction in HOMA-IR (P < .0001), with the slope of this curve different (P = .002) from the unchanged IR exhibited by women who developed T2DM and different (P = .017) from an increased IR slope (P = .049) in women who had GD. In women with PCOS, pretreatment glucose and IR, and lesser reduction in IR on metformin diet were associated with T2DM and GD.     

Effect of cimetidine and omeprazole on aspirin- and taurocholate-induced gastric mucosal damage in the rat.

The effect of varying doses of cimetidine and omeprazole, on acute gastric mucosal lesions produced by topical aspirin (200 mg/kg; 1 ml/100 g) in 0.175 M HCl or topical sodium taurocholate (40 mM; 1 ml/100 g) in 0.175 M HCl was studied in the pylorus ligated rat. Preliminary studies revealed dose dependent inhibition of acid secretion with both cimetidine and omeprazole. Intraperitoneal cimetidine 50 mg/kg and 100 mg/kg given before topical acidified aspirin reduced mucosal lesions from a control score of 23.7 +/- 3.5 (mean +/- SEM) in which 83% of stomachs contained lesions, to 6.9 +/- 2.2 and 3.5 +/- 2.1 respectively (p less than 0.05), 36% and 27% of stomachs containing lesions. A dose of 250 mg/kg failed to reduce lesion score significantly (17.8 +/- 4.9) and 64% of stomachs contained lesions. Taurocholate-induced lesion score was reduced from a control value of 32.7 +/- 4.3 in which 97% of stomachs contained lesions to 10.8 +/- 3.3 and 15.5 +/- 3.8 by cimetidine 10 mg/kg and 25 mg/kg respectively (p less than 0.05), 62% and 68% of stomachs containing lesions. Cimetidine 50 mg/kg and 100 mg/kg failed to significantly reduce mucosal damage. Intraduodenal omeprazole (1.25 to 50 mumol/kg) given before topical acidified aspirin or taurocholate failed to reduce mucosal lesions, as did intragastric omeprazole 5 mumol/kg and 50 mumol/kg given before acidified aspirin. Cimetidine showed cytoprotective properties over a specific dose range beyond which the effect was lost despite continuing suppression of acid secretion. Omeprazole exhibited no cytoprotective activity.     

Mitochondrial cholesterol availability during gonadotropin-induced Leydig cell desensitization

In order to define the early lesion (before pregnenolone formation) of the androgen biosynthetic pathway induced by human CG (hCG) or LH in the Leydig cell, we initially have optimized the use of aminoglutethimide to obtain maximal and sustained inhibition of steroidogenesis in vivo and in vitro. Aminoglutethimide inhibited Leydig cell steroidogenesis in vitro at a dose of 100 micrograms/ml. The minimal serum concentration of aminoglutethimide necessary for maximal inhibition of testosterone in vivo was also 100 micrograms/ml (1 h after the ip injection of 20 mg aminoglutethimide). However, testosterone levels were normal 12 h later, coincident with a marked fall in the serum aminoglutethimide levels. The t 1/2 of the circulating aminoglutethimide was 5 +/- 0.7 h on the first day of treatment but was reduced to 3.0 +/- 0.4 and 2.25 +/- 0.35 h at 2 and 3 days of treatment. At the dose eliciting maximal and sustained steroid inhibition (60 mg/day) aminoglutethimide was able to prevent the estradiol-dependent late steroidogenic lesion (after pregnenolone formation) induced by 1 microgram hCG, with no effect on the early lesion (before pregnenolone formation) caused by 10 micrograms hCG. The aminoglutethimide-induced in vivo accumulation of cholesterol in the inner mitochondrial membrane (by 50%) was associated with an increase in the production of testosterone and pregnenolone by the Leydig cell when subsequently incubated in vitro. Similar increases in the steroidogenic capacity were observed after initial exposure of Leydig cells to aminoglutethimide in vitro, even after acid wash to remove the surface-bound endogenous LH. The steroidogenic cholesterol was also increased in desensitized Leydig cells (by 50-70%); however, the conversion of cholesterol to pregnenolone was substantially blocked in animals with the early lesion. Our findings define the requirement of increasing high levels of aminoglutethimide to inhibit cholesterol metabolism and provide a dose schedule suitable for studies on cholesterol availability and inhibition of steroidogenesis in the rat. These results support our proposal that the early lesion observed in desensitized Leydig cells is due to inhibition of the side-chain cleavage activity rather than to a decrease in the amount of metabolically available cholesterol.