Increased insulin-like growth factor 1 concentrations in a population of non-diabetic cats with overweight/obesity.

Feline hypersomatotropism (HST) is typically associated with diabetes mellitus (DM), whereas HST without concurrent DM has only been reported in a few cases. Weight gain may be observed in cats with HST. The aims of this study were to evaluate circulating insulin-like growth factor-1 (IGF-1) in non-diabetic cats with overweight/obesity, to screen this population for the presence of HST, and to assess whether there is a correlation between body weight/body condition score (BCS) and serum IGF-1 concentration in overweight/obese cats. In this prospective study, 80 overweight/obese cats from referral centers in Buenos Aires (Argentina) were evaluated. Serum IGF-1 was measured as part of the routine tests for overweight/obesity. Non-diabetic cats were included in the study if they had a BCS>6/9. Twenty-nine cats were classified as overweight (BCS 7/9), whereas 51 were classified as obese (BCS 8-9/9). Median serum IGF-1 concentrations of cats with BCS 7/9, 8/9, and 9/9 were 570 ng/ml (range 123-1456 ng/ml), 634 ng/ml (range 151-1500 ng/ml), and 598 ng/ml (range 284-2450 ng/ml), respectively. There was a positive linear correlation between serum IGF-1 concentrations and body weight (r= 0.24, 95% CI 0.01-0.44 P=0.03), and between IGF-1 and BCS (r= 0.27, 95% CI 0.08-0.44 P=0.004). In total, 8.75% (95% confidence interval 3.6-17.2%) of the cats with overweight/obesity had IGF-1 concentrations >1000 ng/ml. Pituitary enlargement was detected on computed tomography in 4/7 cases. These seven cats showed varying degrees of phenotypic changes consistent with acromegaly. A proportion of 8.75 % of overweight/obese non-diabetic cats from referral centers in Buenos Aires had serum IGF-1 concentration in a range consistent with HST in diabetic cats. Likewise, 5% of overweight/obese cats were likely to be diagnosed with HST, supported by evidence of pituitary enlargement. Serum IGF-1 concentrations were positively correlated with body weight and BCS in this population of cats. This study highlights the relevance of screening different populations of non-diabetic cats to increase the detection of HST/acromegaly.

Fenofibrate treatment for severe hypertriglyceridemia in dogs.

Lipid disorders are relatively common in dogs. Hyperlipidemia can be primary or secondary to other diseases. In humans, fenofibrate is used to control hypertriglyceridemia. In dogs, there are no studies evaluating fenofibrate in hypertriglyceridemia. The aim of the study was to evaluate the safety and efficacy of fenofibrate to control severe hypertriglyceridemia in dogs. A total of 124 dogs (n = 124) with severe hypertriglyceridemia (>300 mg/dL, 3.39 mmol/L) were randomly distributed in the fenofibrate group (n = 64) and the diet group (n = 60). Dogs of the fenofibrate group were treated with fenofibrate (10 mg/Kg) once daily. Dogs of the diet group were treated with low-fat diet (10%). Serum triglycerides (TGs), total cholesterol (TC), liver enzymes, and creatine kinase concentrations were evaluated, before and after 1 mo of medical or dietary treatment. Triglyceride concentrations were reduced with fenofibrate (P < 0.001), and 85.93% of the dogs normalized their levels. Triglyceride concentrations also decreased with low-fat diet (P < 0.001), but only 26.6% of the dogs normalized their levels. Triglyceride concentrations were reduced with fenofibrate (P < 0.01) and with low-fat diet (P < 0.01). Of the cases with hypercholesterolemia, 53.7% and 50% of the dogs normalized their TC concentrations, with fenofibrate and diet, respectively. No significant adverse effects were observed (3% showed diarrhea). Fenofibrate was safe and effective in reducing and normalizing TG concentrations in dogs with severe hypertriglyceridemia, regardless of the cause of hyperlipidemia. The low-fat diet was effective in reducing, but not normalizing, TG concentrations. Fenofibrate and low-fat diet were effective in reducing TC concentrations. This is the first study evaluating fibrates in dogs with severe hypertriglyceridemia and comparing results with a low-fat diet.

High glucose-induced changes in steroid production in adrenal cells.

BACKGROUND: Increased activity of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in enhanced adrenocorticotropin (ACTH) and serum glucocorticoid levels, has been described in patients with diabetes mellitus and in animal models of this disease; however, altered steroid production by adrenocortical cells could result from local changes triggered by increased reactive oxygen species (ROS), induced in turn by chronic hyperglycaemia. Experiments were designed (1) to analyse the effects of incubating murine adrenocortical cells in hyperglycaemic media on the generation of oxidative stress, on steroid synthesis and on its modulation by the activity of haeme oxygenase (HO); and (2) to evaluate the effect of antioxidant treatment on these parameters. METHODS: Y1 cells were incubated for 7 days with either normal or high glucose (HG, 30 mmol/L) concentrations, with or without antioxidant treatment. Parameters of oxidative stress and expression levels of haeme oxygenase-1 (HO-1), nitrite levels, L-arginine uptake and progesterone production were determined. RESULTS: HG augmented ROS and lipoperoxide production, decreasing glutathione (GSH) levels and increasing antioxidant enzymes and HO-1 expression. Basal progesterone production was reduced, while a higher response to ACTH was observed in HG-treated cells. The increase in HO-1 expression and the effects on basal steroid production were abolished by antioxidant treatment. Inhibition of HO activity increased basal and ACTH-stimulated steroid release. Similar results were obtained by HO-1 gene silencing while the opposite effect was observed in Y1 cells overexpressing HO-1. CONCLUSIONS: HG induces oxidative stress and affects steroid production in adrenal cells; the involvement of HO activity in the modulation of steroidogenesis in Y1 cells is postulated.

Induction of nitric oxide synthase and heme oxygenase activities by endotoxin in the rat adrenal cortex: involvement of both signaling systems in the modulation of ACTH-dependent steroid production.

The present study was designed to investigate the effect of lipopolysaccharide (LPS) on the expression levels and activities of the nitric oxide synthase (NOS) and heme oxygenase (HO) systems in the rat adrenal gland. Both enzymatic activities were significantly increased in this tissue after in vivo treatment with LPS. The concurrent induction of the HO-1, NOS-1, and NOS-2 gene products was also detected as both mRNAs and protein levels were augmented by this treatment in a time-dependent way. A significant interaction between both signaling systems was also demonstrated as in vivo blockage of NOS activity with N(G)-nitro-L-arginine methyl ester (L-NAME) resulted in a significant reduction in HO expression and activity levels, while an increase in NOS activity was observed when HO was inhibited by Sn-protoporphyrin IX (Sn-PPIX). As both NOS and HO activities have been previously involved in the modulation of adrenal steroidogenesis, we investigated the participation of these signaling systems in the adrenal response to LPS. Our results showed that acute stimulation of steroid production by ACTH was significantly increased when either NOS or HO activities were inhibited. We conclude that adrenal NOS and HO can be induced by a non-lethal dose of endotoxin supporting a modulatory role for these activities in the adrenal response to immune challenges.

Concurrent hyperadrenocorticism and diabetes mellitus in dogs.

Hyperadrenocorticism (HAC) and diabetes mellitus (DM) are two diseases that can occur concurrently in dogs. The objective of this study was to evaluate the coexistence of HAC and DM, and the risk factors involved that could contribute to the development of DM in dogs with HAC. A total of 235 dogs with HAC were studied and, according to their fasting glycemia, they were divided into three groups: <5.6mmol/L, between 5.6 and 10.08mmol/L and >10.08mmol/L. The following parameters were evaluated: age, gender, cause of HAC, body condition, glycemia, total cholesterol, triglycerides, urinary cortisol:creatinin ratio (UCCR) and survival time. A 13.61% concurrence of HAC and DM was observed. Dogs with a fasting glycemia >5.6mmol/L, with dislipemia, with Pituitary-Dependent Hyperadrenocorticism, UCCR >100×10-6 and non-castrated females showed a higher risk of developing DM. The development of DM in dogs with HAC reduces the survival time.

Studies with insulin and insulin-like growth factor-I show that the increased labeling of phosphatidylinositol-3,4-bisphosphate is not sufficient to elicit the diverse actions of epidermal growth factor on MA-10 Leydig tumor cells.

In a recent publication we showed that addition of mouse epidermal growth factor (mEGF) to MA-10 Leydig tumor cells rapidly leads to an increase in the incorporation of [3H]inositol-derived radioactivity into an unusual lipid that was identified as phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2). Other ligands that are known to bind to MA-10 cells, such as hCG and arginine vasopressin, however, did not elicit this effect. Inasmuch as mEGF modulates the differentiated functions of MA-10 cells in a number of ways, our findings raised the possibility that PI-3,4-P2 may be an intracellular mediator of these actions of mEGF. In an attempt to answer this question, we set out to determine if other ligands increase the labeling of PI-3,4-P2 in MA-10 cells prelabeled with [3H]inositol, and if such ligands mimic the diverse biological actions of mEGF on these cells. The experiments presented herein show that insulin, insulin-like growth factor-I, and transforming growth factor-alpha increase the labeling of PI-3,4-P2 in MA-10 cells, but only transforming growth factor-alpha mimics the actions of mEGF on the differentiated functions of MA-10 cells. We conclude that an increase in the labeling of PI-3,4-P2 is not sufficient to elicit these actions of mEGF.

Cyclic adenosine 3',5'-monophosphate negatively regulates clusterin gene expression in Leydig tumor cell lines.

The clusterin protein and its messenger RNA were identified in many tissues including testis. In this report, we demonstrate the expression of clusterin gene in four Leydig tumor cell lines, including mouse MA-10 and I-10 and rat R2C and LC-540. When the cells were incubated with 0.1 mM 8-bromo-cAMP or (Bu)2cAMP for 17 h, an unexpected, profound suppression of clusterin mRNA accumulation was observed. A 60-70% decrease in clusterin mRNA was observed in MA-10 and R2C cells, 10% in I-10 cells, and no apparent change in LC-540 cells. The inhibitory effect of cAMP was specific to the clusterin gene, since in the same cells cholesterol side-chain cleavage enzyme mRNA was drastically elevated in MA-10 and I-10 cells while alpha-tubulin mRNA levels were not changed in all four cell lines. The reduction could be detected as early as 4 h, and was evident at 17 h after cAMP administration. Removal of cAMP from culture media at 17 h prevented the decline of clusterin mRNA. The suppression of clusterin gene expression can also be demonstrated by treatment with human CG or forskolin, which were known to elevate intracellular cAMP levels. Our observations suggest: 1) cAMP negatively regulates clusterin gene expression in two Leydig tumor cell lines, MA-10 and R2C; 2) The inhibitory effect of cAMP on clusterin gene expression is probably acting through the protein kinase A pathway; and 3) The four Leydig tumor cell lines respond differently to cAMP in the expression of clusterin and side-chain cleavage genes.

Nitric oxide inhibits Leydig cell steroidogenesis.

Testicular macrophages as well as endothelial cells, which are intimately associated with Leydig cells, constitute a potential source of paracrine nitric oxide (NO) in the testis. In the present study, we investigated the effect of NO donors on MA-10 murine Leydig tumor cell line and rat Leydig cell steroidogenesis. We show that NO donors inhibit human CG-induced steroidogenesis in both type of cells. We also studied NO mechanism of action. Contrary to what is observed in many other systems, NO inhibitory effect on Leydig cell steroidogenesis is not mediated by cyclic GMP (cGMP) because NO fails to increase cGMP production, and cGMP analogs do not reproduce NO effect. NO does not modify the production of cAMP, the main second messenger that mediates gonadotropin action. When we studied NO effect over the steroidogenic pathway in MA-10 cells, we found that NO was inhibiting the conversion of cholesterol to pregnenolone. Taken together these results show an inhibitory effect of NO donors on Leydig cell steroidogenesis, and suggest that NO can be directly inhibiting cholesterol side-chain cleavage enzyme (cytochrome P450scc) as it does with other heme proteins, including different cytochromes P450.

Alterations in intracellular messengers mobilized by gonadotropin-releasing hormone in an experimental ovarian tumor.

Cells derived from an experimental luteinized ovarian tumor are more sensitive to GnRH endocrine action than control luteal cells. In an attempt to understand the possible causes of the differential sensibility to GnRH action, we examined the number and affinity of GnRH receptors and the second messenger response to GnRH stimulation in both tissues. For GnRH receptor studies membranes were obtained from 4- to 6-week-old ovarian tumors (luteoma) and ovaries from prepubertal rats treated with 25 IU PMSG and 25 IU hCG (SPO) and were incubated with [125I]Buserelin. The number of GnRH receptors were increased in luteoma compared with that in SPO ovaries; dissociation constants were similar in both tissues. GnRH stimulation of second messenger release was assessed in cells obtained from luteoma and SPO ovaries by collagenase treatment. Buserelin (100 ng/ml) induced a significant 35% calcium increase in SPO cells, as determined by the fura-2 method; in luteoma cells no response was observed after buserelin stimulation, although a calcium transient was induced by thapsigargin (0.5 microM), an inhibitor of Ca2+-adenosine triphosphatase associated with the endoplasmic reticulum. The effect of buserelin on inositol phosphates was evaluated after incubation of luteoma and SPO cells with [3H]myoinositol for 48 h. Buserelin induced a 400% increase in inositol trisphosphate in SPO cells. Again, luteoma cells did not respond to buserelin stimulation, although NaF (10 mM), an activator of G proteins coupled to phospholipase C, induced an 800% increase in inositol trisphosphate. Although the number of GnRH receptors is augmented in luteoma cells, justifying an increased endocrine response, neither inositol phosphates nor intracellular calcium were released by a GnRH analog, indicating the uncoupling of GnRH receptors from phospholipase C. These data provide evidence that the transformation of the ovary into a luteoma implies the acquisition of novel characteristics in the GnRH receptor second messenger-generating system.

Calcium homeostasis in Trypanosoma cruzi amastigotes: presence of inositol phosphates and lack of an inositol 1,4,5-trisphosphate-sensitive calcium pool.

The permeabilization of Trypanosoma cruzi amastigotes with digitonin allowed the study of Ca2+ fluxes between intracellular organelles in situ. In addition, fura-2 was used to determine the cytosolic Ca2+ concentration in the intact cells. When amastigotes were permeabilized in a reaction medium containing MgATP, succinate and 3.5 microM Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level, a range which correlates favorably with that detected in the intact cells with fura-2. The presence of 1 microM FCCP strongly decreased the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably represented by the endoplasmic reticulum, was completely inhibited by 500 microM vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased and the Ca2+ set point was increased to about 0.8 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and of a large extramitochondrial Ca2+ pool, no IP3-sensitive or thapsigargin-sensitive Ca2+ release could be detected in either amastigotes or epimastigotes.

Inhibin/activin subunits and activin receptor are co-expressed in Leydig tumor cells.

The expression of genes encoding inhibin/activin subunits and activin receptor was examined in four cultured Leydig tumor cells (MA-10, I-10, R2C, and LC-540). Inhibin alpha-subunit gene was highly expressed in Leydig tumor cell lines except LC-540. Both inhibin beta-A- and beta-B-subunit mRNAs were present in low levels. The 6.5-kb beta-A-subunit mRNA was detected in MA-10, R2C and LC-540 cells, and not in I-10 cells. The expression of the two species of beta-B-subunit mRNA is cell specific. In MA-10 and I-10 cells, 4.4-kb beta-B-subunit mRNA was the predominant species, while in R2C and LC-540 cells both 4.4-kb and 3.3-kb mRNA were present in equal quantities. By contrast, two species (6 and 3 kb) of activin receptor ActRII mRNA were identified in equal intensity in all four Leydig tumor cell lines. Addition of cAMP derivative to MA-10 cells at 0.1 mM for 17 h or 1 mM for 5 h produced a two-fold increase in inhibin alpha-subunit mRNA levels, and small or no significant change in inhibin beta-B-subunit and ActRII mRNAs. However, a 70-80% reduction in inhibin beta-A-subunit mRNA was observed by 1 mM cAMP for 5 h. We concluded that: (1) the inhibin/activin subunit genes and activin receptor gene are co-expressed in Leydig tumor cell lines, and (2) the three inhibin/activin subunit genes are expressed differently, while the activin receptor gene is expressed identically in the four cell lines.

Mitochondrial biosynthesis of cholesterol in Leydig cells from rat testis.

The subcellular location of some enzymes responsible for cholesterol biosynthesis was studied in metrizamide-purified rat Leydig cells. The highest activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA reductase), a key regulatory enzyme in the cholesterol pathway, was associated with highly enriched mitochondrial fractions with recovery of 62% of the total activity and was located on the inner membrane. A significant part of the activity (35%) was also present in the cytoplasm. The activity of this enzyme in the other subcellular fractions was negligible. The HMG-CoA synthase activity was also found almost entirely in the mitochondria (90%). Otherwise no detectable activity of HMG-CoA lyase was present in the subcellular fractions studied. Furthermore, cholesterol may be synthesized from acetyl-CoA inside the mitochondrion, since a significant incorporation (90%) of [14C]acetyl-CoA into digitonin-precipitable sterols was observed in this organelle and only 10% in the cytoplasmic fraction. The evidence strongly suggests that much of the cholesterol biosynthesis that takes place in Leydig cells is carried out within the mitochondria.

Effect of a second injection of human chorionic gonadotropin on the desensitized Leydig cells.

Experimental evidence has demonstrated that multiple doses of LH will increase the steroidogenic capacity of Leydig cells. This work was undertaken with the aim of defining the effect of a second hCG administration on the desensitized state, measuring binding of the gonadotropin and the steroidogenic capacity of rat testes. A single injection of 200 IU hCG induced a sharp increase of plasma testosterone which was still evident 24 h later. A second peak was observed at 72 h. The in vivo refractoriness of Leydig cells between 24 and 72 h after the single injection was proved by the fact that a second administration of hCG, 2 h before sacrifice, did not induce any increase in plasma testosterone. A second administration of 200 IU hCG, 48 h after the first injection, showed a similar pattern but on the 5th day there was an increased stimulation of testosterone production with respect to that obtained after a single dose of hCG. The in vitro studies on testicular binding capacity and steroidogenic responsiveness showed that the second administration of hCG, 48 h after the first injection, maintained the testicular binding capacity at the lowest level and the 'adenylate cyclase desensitization' but restored the steroidogenic capacity to even supramaximal values, compared to normal rats, 3 days after this second hCG administration. These results would support a dissociation between receptor loss and maximal testosterone synthesis as well as possibly indicating an alternative pathway different from the classical.

Regulation of the cholesterol ester cycle and progesterone synthesis by juvenile hormone in MA-10 Leydig tumor cells.

We had previously reported that juvenile hormone III (JH III) and the JH analogue 2-(4-phenoxy phenoxy)-ethoxytetrahydropyran exert inhibitory effects on progesterone synthesis by blocking cAMP production in hCG-stimulated MA-10 Leydig tumor cells. In the present study, the effects of JH analogue upon the biosynthetic pathway of progesterone synthesis have been examined. Our results demonstrated that JH analogue inhibited progesterone production even in the presence of 20-hydroxycholesterol or 25-hydroxycholesterol. Furthermore, although JH analogue inhibited pregnenolone production in hCG-stimulated MA-10 cells the activity of the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was unaffected. These data suggest that JH analogue might inhibit the steroidogenic pathway in Leydig tumor cells by inhibiting the activity of the cholesterol side chain cleavage (CSCC) enzymatic complex. The JH analogue was also evaluated for inhibitory actions on cholesterol availability. An important effect of this compound was the interference with the cellular process of plasma membrane cholesterol internalization. Moreover, JH analogue inhibited not only the use of cholesterol ester for steroid biosynthesis under Bt2cAMP stimulation, but also the cholesterol ester hydrolase (CEH) activity in MA-10 Leydig tumor cells.

Biosynthesis of bufadienolides in toads. V. The origin of the cholesterol used by toad parotid glands for biosynthesis of bufadienolides.

Sodium (1-14C)acetate and (5-3H) mevalonate were incubated with Bufo arenarum toad parotid gland and liver tissues. Both labelled compounds were incorporated into cholesterol produced by liver while the incubations with parotid gland produced no labelled cholesterol. Low and high density lipoproteins isolated from toad plasma were iodinated and used for binding studies. Membrane preparations of parotid gland showed high affinity binding sites for 125I-LDL and 125I-HDL. In addition, while colchicine inhibits the in vitro uptake of (3H)cholesteryl linoleate-LDL into parotid gland tissue an opposite effect was seen with (3H)cholesteryl linoleate-HDL. The above mentioned results would support the hypothesis that the cholesterol used by the parotid gland for the biosynthesis of bufadienolides would be produced in the liver, transported by the circulating lipoproteins and incorporated by the glands by a receptor-mediated mechanism.

[Regulation of testicular steroidogenesis by nitric oxide]. / Regulación de la esteroidogenesis testicular por oxido nítrico.

Testicular macrophages as well as endothelial cells, which are intimately associated with Leydig cells, constitute a potential source of paracrine nitric oxide (NO). In the present study, we investigated the effect of NO donors on MA-10 murine Leydig tumor cell line and rat Leydig cell steroidogenesis. We observed that NO donors, reversibly inhibit hCG-induced steroidogenesis in both types of cells. We also studied NO mechanism of action. Contrary to what is observed in many other systems, NO inhibitory effect on Leydig cell steroidogenesis is not mediated by cGMP, as NO fails to increase cGMP production and cGMP analogs do not reproduce NO effect. NO does not modify the production of cAMP, the main second messenger that mediates gonadotropin action. When we studied NO effect over the steroidogenic pathway in MA-10 cells, we found that NO is inhibiting the conversion of cholesterol to pregnenolone. Taken together these results show an inhibitory effect of NO donors on Leydig cell steroidogenesis and suggest that NO can be directly inhibiting cholesterol side-chain cleavage enzyme (cytochrome P-450 scc) as it does with other heme proteins, including different cytochromes P-450.

Involvement of glucagon-like peptide 1 in the glucose homeostasis regulation in obese and pituitary-dependent hyperadrenocorticism affected dogs.

The incretin glucagon-like peptide 1 (GLP-1) enhances insulin secretion. The aim of this study was to assess GLP-1, glucose and insulin concentrations, Homeostatic Model Assessment (HOMA insulin sensitivity and HOMA ß-cell function) in dogs with pituitary-dependent hyperadrenocorticism (PDH), and compare these values with those in normal and obese dogs. The Oral Glucose Tolerance Test was performed and the glucose, GLP-1 and insulin concentrations were evaluated at baseline, and after 15, 30, 60 and 120 minutes. Both basal concentration and those corresponding to the subsequent times, for glucose, GLP-1 and insulin, were statistically elevated in PDH dogs compared to the other groups. Insulin followed a similar behaviour together with variations of GLP-1. HOMA insulin sensitivity was statistically decreased and HOMA ß-cell function increased in dogs with PDH. The higher concentrations of GLP-1 in PDH could play an important role in the impairment of pancreatic ß-cells thus predisposing to diabetes mellitus.

Estudio de la expresión del receptor de vitamina D en la ontogenia del testículo y en tumores de células de leydig: posible interacción con el sistema histaminérgico

RESUMEN La vitamina D (VD), un esteroide pleiotrópico, ha sido relacionada con la función reproductiva masculina, pero aún no se ha estudiado la expresión de su receptor (RVD) en el desarrollo testicular. RVD regula la expresión de componentes del sistema histaminérgico, y la histamina (HA) modula la esteroidogénesis en células de Leydig (CL). Se ha relacionado a la deficiencia de VD con múltiples patologías, entre ellas cáncer. Los tumores de células de Leydig (TCL) son los más frecuentes del intersticio testicular, y al malignizar no responden a radio/quimioterapia. VD fue descripta como tratamiento para varios tumores, pero se desconoce su aplicación en TCL. Por lo expuesto, hemos estudiado la expresión de RVD en la ontogenia de testículo de rata, evaluando su correlación con los niveles de testosterona séricos (T) y el contenido de HA; y además evaluamos la expresión de RVD en testículo humano fetal, neonatal, prepuberal, TCL e hiperplasia de CL. En testículo de rata, se observó un aumento en la expresión de RVD en CL con la edad, en línea con el incremento de T, y en contraposición con la disminución del contenido de HA, lo cual fue consistente con la reducción en los niveles de la enzima que cataliza su síntesis, HDC. Esto sugiere que la VD podría ejercer una función en el desarrollo testicular normal, ya sea en forma directa sobre las CL o mediante la regulación de la expresión de componentes del sistema histaminérgico (HDC y/o receptores de HA). Por su parte, el TCL humano presentó sobreexpresión de RVD y HDC. Considerando que las hormonas esteroideas se encuentran aumentadas en esta patología y funcionan como factores de crecimiento, si el calcitriol pudiera modular la esteroidogénesis podría tener una aplicación terapéutica.

ABSTRACT Vitamin D (VD) is a steroid hormone traditionally related to bone health. However, several authors have associated VD with reproduction and steroidogenesis in males. The presence ofVD receptor (VDR) and the enzymes involved in its activation had been reported in several cell types of the testes. Until now, nobody has studied RVD expression during testicular development. In addition, VDR in association with its co-activators or co-repressors, regulates the expression of several genes, including those related to the histaminergic system. Previously, we demonstrated that histamine (HA) can modulate steroidogenesis in Leydig cells (LC) in a concentration-dependent manner. Also, we observed a decrease in the endogenous HA content, consistent with the previously described decrease of HDC (histidine decarboxylase, the enzyme responsible of HA synthesis) levels, during LC ontogeny. Epidemiologic studies strongly suggest that a relationship exists between VD deficiency and multiple pathologies, particularly cancer. Leydig cell tumors (LCT) are rare endocrine tumors ofunknown etiology, which originate in the testicular interstitium. The incidence worldwide is 1-3% in adults and 4% in prepubertal boys, but recent publications indicate that these figures have been increasing. While usually benign, approximately 10% of LCT in adults become malignant and do not respond to chemo or radiotherapy. It is imperative to deeply investigate the biology of LCT, to identify new therapeutic targets. The potential role of calcitriol (1a,25(OH)2-vitamin-D3) in cancer treatment has been described for several types of tumors, but it remains unexplored in LCT. Thus, as a first step, it is worth evaluating VDR expression in LCT.In view of the aforecited evidence, herein we studied VDR expression during the rat testicular ontogeny, evaluating a possible correlation withserum testosterone (T) levels in blood, endogenous levels of HA and the previously described HDC expression levels. We also analized VDR expression in human testes corresponding to three different stages of development (fetal, neonatal andjuvenile), in LCT and in LC hyperplasia. Methods: Rat testes of different ages (7, 21, 35, 90 y 240 days), human fetal, neonatal and pre pubertal testes, a human LCT and a human LC hyperplasia; were used for detection of VDR by immunohistochemistry. Results: In the rat testes, VDR expression increased with age in LC, in line with the increase in serum testosterone; and in contrast with the decrease in the endogenous content of HA and HDC levels. Likewise, we detected an increase in VDR expression with age in the human testes samples. LCT presentedVDR and HDC overexpression. We also detected VDR in LC hyperplasia. Conclusions: Given that VDR testicular expression increases with age in LC, as well as testosterone serum levels, it is reasonable to speculate thatVD may play a role in normal testicular development, either acting directly on LC or by regulating one of more components of the histaminergic system (HDC or HA receptors). Considering that VDR is overexpressed in LCT, and that steroids are increased in this pathology (and act like growth factors); if calcitriol could modulate steroidogenesis, it could have a therapeutic role.

Inhibition of goiter growth and of cyclic AMP formation in rat thyroid by 2-iodohexadecanal.

INTRODUCTION: Thyroid autoregulation has been related to intraglandular content of an unknown putative iodocompound. The thyroid is capable of producing different iodolipids such as 6-iodo-deltalactone (ILdelta) and 2-iodohexadecanal (2-IHDA). Data from different laboratories have shown that these iodolipids inhibit several thyroid parameters. ILdelta has an antigoitrogenic action but no data about the action of 2-IHDA on this parameter has been published. OBJECTIVES: to study the action of 2-IHDA on methimazole (MMI)-induced goiter and analyze if this compound can cause the involution of preformed goiter. RESULTS: Administration of MMI to rats during 10 days increased thyroid weight by 112%. This effect was significantly inhibited by the simultaneous injection of 20mug/day of 2-IHDA (51% vs. MMI) while iodine or non iodinated hexadecanal were without action. Thyroidal proliferating cell nuclear antigen (PCNA) content was increased by MMI while 2-IHDA decreased this value (control: 100%; MMI: 190+/-11; MMI+2-IHDA: 134+/-10). Serum TSH was increased after MMI administration and 2-IHDA did not modify this parameter (control: 1.89+/-0.10; MMI: 8.19+/-0.93ng/ml; MMI+2-IHDA: 7.38+/-0.72). Treatment with MMI increased thyroidal cAMP content (control: 16.1+/-0.82, MMI: 42.4+/-4.6 fmol/mg protein) while injection of 2-IHDA significantly decreased this value (22.3+/-2.0). Goiter prevention by 2-IHDA was also observed at 30 days of treatment reducing total number of cells (51% inhibition) and epithelial height (81% inhibition). Goiter involution was induced after withdrawal of MMI and injection with 2-IHDA, KI or saline. 2-IHDA led to a reduction of 74.5% in thyroid weight after 3 days while spontaneous involution (saline) was only of 32%. KI failed to alter this value. This significant involution was accompanied by a reduction in the number of cells (66%). Administration of the iodolipids did not produce significant changes in several serum parameters such as total T(3) and T(4), cholesterol, transaminases, urea and creatinine. CONCLUSION: 2-Iodohexadecanal, as 6-iodo-deltalactone, prevents goiter growth in rats and opens a potential therapeutic application of iodolipids.

Epidermal growth factor increases the labeling of phosphatidylinositol 3,4-bisphosphate in MA-10 Leydig tumor cells.

Previous studies from this laboratory have shown that mouse epidermal growth factor (mEGF) modulates the hormonal responsiveness of MA-10 Leydig tumor cells without affecting cell multiplication. In an attempt to characterize the intracellular signaling systems activated by mEGF in this cell type, we examined its effects on the labeling of phosphatidylinositols in cells that had been preincubated with different radioactive precursors. Here we report that exposure of MA-10 cells to mEGF, but not other ligands that affect their differentiated function, results in an increase in the labeling of an unusual phosphatidylinositol that does not appear to be present in unstimulated cells. This phosphatidylinositol has been identified as phosphatidylinositol 3,4-bisphosphate.