Leptin gene and protein expression in the trophoblast and uterine tissues during early pregnancy and the oestrous cycle of pigs.

Leptin is a 16-kDa protein hormone encoded by the obese (ob) gene and acts on receptors in the hypothalamus to regulate food intake and energy balance. The identification of leptin and its receptor mRNAs and proteins in human and mouse endometrium and placental trophoblast has attracted attention to the potential role of leptin in implantation. Thus, the aim of this study was to compare the expression levels of porcine leptin mRNA and protein in endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10 - 12 and 14 - 16) as well as during two stages of pregnancy respondent to the beginning (days 14 - 16) and the end (days 30 - 32) of the implantation process, and in trophoblast during both periods of pregnancy. Leptin gene and protein expression in myometrium, and leptin mRNA expression in endometrium was more pronounced in the mid- and late-luteal phases of the cycle in comparison to studied periods of pregnancy, whereas leptin protein concentration in endometrium was either enhanced on days 30 - 32 of pregnancy in relation to days 14 - 16 of the cycle or there were no changes between pregnancy and luteal phase of the cycle. On days 30 - 32 of pregnancy, expression of the leptin gene in the endometrium, and of the leptin gene and protein in the myometrium was more pronounced in comparison to the earlier stage of pregnancy. Moreover, leptin gene expression in porcine trophoblast increased during the beginning of the implantation process compared to days 30 - 32 of pregnancy, while the protein concentration decreased on days 14 - 16 of pregnancy. In conclusion, the finding of leptin gene and protein expression in porcine endometrium, myometrium and trophoblast indicates that locally synthesised leptin can participate in the control of pig reproduction. The fluctuation of the hormone concentration during pregnancy and changes in its level between pregnancy and the oestrous cycle may indicate leptin's involvement in the implantation process.     

Long form of leptin receptor gene and protein expression in the porcine trophoblast and uterine tissues during early pregnancy and the oestrous cycle

Leptin, the product of the OB gene, is a 16-kDa polypeptide of 146 amino acid residues produced mainly by adipocytes that regulates metabolism and reproduction. The actions of leptin are mediated mainly via the long form of the leptin receptor (OB-Rb). The identification of leptin and OB-Rb mRNAs and proteins in human and mouse endometrium, and placental trophoblast suggests that leptin may be involved in the implantation process. Thus, the aim of this study was to compare the expression levels of porcine OB-Rb mRNA and protein in the endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10-12 and 14-16, respectively) as well as during two stages of pregnancy respondent to the beginning of the implantation process (days 14-16) and the post-implantation period (days 30-32), and in trophoblast during both periods of pregnancy. OB-Rb gene expression in endometrium during the examined stages of pregnancy and the mid- and late-luteal phases of the cycle was at the same level. In contrast, in myometrium leptin receptor gene expression decreased on days 14-16 of pregnancy compared to both phases of the cycle, and on days 30-32 of pregnancy in relation to late-luteal phase. OB-Rb protein expression in the tissues was lower during the examined stages of pregnancy in comparison to the mid- and late-luteal phases of the cycle. In trophoblast, OB-Rb mRNA and protein expression was higher on days 30-32 than during days 14-16 of pregnancy. In conclusion, our results might suggest that leptin can participate in the control of pig reproduction by exercising its action at the uterine and trophoblast level and have a direct effect on these organ during both the luteal phase of the cycle and early pregnancy. Moreover, changes in OB-Rb gene and protein expression in tissues of pig reproductive tract strongly suggest that their sensitivity to leptin varies throughout luteal phase of the cycle and early pregnancy.     

Expression of leptin and its receptor genes in the ovarian follicles of cycling and early pregnant pigs

Leptin is a polypeptide hormone produced primarily by adipocytes. It has been implicated in the regulation of satiety and energy homeostasis. Leptin has been suggested to play a role in reproduction based on its involvement in the regulation of the hypothalamic–pituitary–gonadal axis via endocrine, paracrine and/or autocrine pathways. The aim of the present study was to localize the cellular distribution of leptin and the long isoform of leptin receptor (OB-Rb) genes in porcine ovarian antral follicles and to compare the expression levels of leptin and OB-Rb mRNAs in porcine granulosa cells (GC), theca interna (TIC) and theca externa (TEC) cells during the luteal phase of the estrous cycle and in early pregnancy. The expression of leptin and OB-Rb genes was detected in GC, TIC and TEC. Significantly higher levels of leptin gene expression in GC were observed during the mid- and late-luteal phases of the cycle than on days 30 to 32 of pregnancy. On days 14 to 16 of pregnancy, leptin mRNA expression was higher than that on days 14 to 16 of the cycle. The expression of the OB-Rb gene in GC and TEC increased during pregnancy in comparison with the analyzed luteal phases of the cycle. Our results validate the hypothesis that locally produced leptin plays a role in the regulation of porcine reproduction at the ovarian level and exerts a direct effect on porcine follicles. The differences in OB-Rb gene expression in porcine GC and theca cells also suggest that their sensitivity to leptin varies in the ovaries of pregnant and cyclic pigs.     

Plasma leptin-binding activity and hypothalamic leptin receptor expression during pregnancy and lactation in the rat

Leptin, the 16-kDa peptide hormone product of the ob gene, regulates body weight via the hypothalamus but also influences several aspects of reproductive function. Results of previous studies have suggested that pregnancy is a state of leptin resistance, because food consumption remains stable or increases despite a progressive rise in plasma leptin across most of gestation. In the present study, we assessed whether this apparent leptin resistance during rat pregnancy was due to either increased plasma leptin-binding activity and/or reduced expression of hypothalamic leptin receptor. Plasma leptin increased from 2.2 +/- 0.4 ng/ml before pregnancy to a maximum at midgestation (4.2 +/- 0.8 ng/ml on Day 12) and then fell by Day 22 and remained low throughout lactation. Despite the higher plasma leptin levels in pregnancy, food consumption increased from a minimum of 13.6 +/- 0.5 g/day before pregnancy to a peak of 21.9 +/- 0.6 g/day on Day 19, then fell before parturition (11.9 +/- 0.4 g/day on Day 22). At least part of the increase in plasma leptin during pregnancy was attributable to a marked increase (P < 0.001) in plasma leptin-binding activity between diestrus and late pregnancy, which then fell after birth but remained at midpregnancy levels to at least Day 12 of lactation. Hypothalamic expression of mRNA encoding the long form of the leptin receptor (Ob-Rb) was elevated in early pregnancy (Day 7) but returned to prepregnancy levels by midgestation and remained stable thereafter. The results of this study confirm that pregnancy in the rat is a state of relative leptin resistance, which is due primarily to increased plasma leptin-binding activity rather than to changes in hypothalamic Ob-Rb expression.     

Gestational profile of leptin messenger ribonucleic acid (mRNA) content in the placenta and adipose tissue in the rat, and regulation of the mRNA levels of the leptin receptor subtypes in the hypothalamus during pregnancy and lactation

Serum leptin levels were significantly increased during rat gestation. Our data showed that leptin mRNA levels in both the adipose tissue and placenta were higher as pregnancy progressed, suggesting a role for both tissues in the hyperproduction of leptin. This paradoxical increase in leptin concentration during gestation suggests that a physiological state of leptin resistance may exist at the hypothalamic level that may explain the hyperphagia observed in pregnant rats. In order to study this issue further, levels of the mRNA encoding the different leptin receptor isoforms were determined in the hypothalamus of pregnant and nonpregnant rats. We found a specific reduction of the mRNA levels encoding the leptin receptor isoform Ob-Rb in the hypothalamus of pregnant rats compared to nonpregnant animals, suggesting that during pregnancy the hypothalamus shows a physiological resistance to the high levels of leptin due, at least in part, to a decrease in the expression of the long, biologically active form of the leptin receptor (Ob-Rb). During lactation, serum leptin levels returned to values observed in nonpregnant rats. In the hypothalami of these animals, Ob-Rb mRNA content was similar to that observed in nonpregnant rats, but we found an increased expression of some of the short forms of the leptin receptor (Ob-Re and Ob-Rf). This could contribute to induction of the hyperphagia present during lactation. These data provide new insights into the adaptive mechanisms that take place during pregnancy and lactation in order to meet increased metabolic requirements.     

Age-related variations of leptin receptor expression in the growth plate of spine and limb: gender- and region-specific changes

Leptin is a potent growth-stimulating factor of bone. The effects of leptin on bone growth differ significantly between axial and appendicular regions. Gender differences of leptin function have also been suggested in normal pubertal development. To explore the mechanisms underlying these effects, we investigated the spatial and temporal expressions of the active form of the leptin receptor (Ob-Rb) in the tibial and spinal growth plates of the female and male rats during postnatal development. The 1-, 4-, 7-, 12- and 16-week age stages are representative for early life, puberty and early adulthood after puberty, respectively. Quantitative real-time PCR was used for Ob-Rb mRNA examination and comparison. The spatial location of Ob-Rb was determined by immunohistochemical analysis. There were gender- and region-specific differences in Ob-Rb mRNA expression in the growth plate. Mainly cytoplasm staining of Ob-Rb immunoreactivity was observed in the spinal and tibial growth plate chondrocytes of both genders. Spatial differences of region- and gender-related Ob-Rb expression were not observed. Ob-Rb immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in early life stage and during puberty. After puberty, staining was mainly located in the late proliferative and hypertrophic chondrocytes. The results of Ob-Rb HSCORE analysis were similar to those obtained from quantitative real-time PCR. Our study indicated direct effects on the chondrocytes of the growth plate in different development stages. The region-specific expression patterns of Ob-Rb gene might be one possible reason for contrasting phenotypes in limb and spine. Different Ob-Rb expression patterns might partly contribute to age- and gender- related differences in trabecular bone mass.     

Leptin and connective tissue growth factor in advanced glycation end-product-induced effects in NRK-49F cells

Previously, we showed that Janus kinase 2 (JAK2) is important in advanced glycation end-product (AGE)-induced effects in renal interstitial (NRK-49F) fibroblasts. Leptin is a JAK2-activating cytokine via the long form leptin receptor (Ob-Rb). Leptin and connective tissue growth factor (CTGF) may be involved in renal fibrosis. However, the relationship between leptin and CTGF in terms of AGE-induced effects remains unknown. Thus, the effects of AGE (150 microg/ml) and leptin on mitogenesis, CTGF and collagen expression in NRK-49F cells were determined. We found that leptin and AGE increased mitogenesis and type I collagen protein expression at 3 and 7 days, respectively. AGE increased leptin mRNA and protein expression at 2-3 days. AGE increased CTGF mRNA and protein expression at 3-5 days. AG-490 (JAK2 inhibitor) abrogated AGE-induced leptin mRNA and protein expression at 2-3 days. AG-490 and Ob-Rb anti-sense oligodeoxynucleotides (ODN) abrogated AGE-induced CTGF mRNA and protein expression at 3-5 days. AG-490 and CTGF anti-sense ODN abrogated AGE-induced mitogenesis and collagen protein expression at 7 days. Additionally, leptin dose (0.2-1 microg/ml) and time (1-2 days)-dependently increased CTGF protein expression. AG-490 abrogated leptin (1 microg/ml)-induced CTGF protein expression at 2 days. AG-490 and CTGF anti-sense ODN abrogated leptin-induced mitogenesis and collagen protein expression at 3 days. We concluded that AGE induced JAK2 to increase leptin while leptin induced JAK2 to increase CTGF-induced mitogenesis and type I collagen protein expression in NRK-49F cells. Additionally, AGE-induced mitogenesis and type I collagen protein expression were dependent on leptin-induced CTGF.     

Effect of alcohol on the content of sex steroid receptors in the hypothalamus and hypophysis of male rats

In experimental dipsomania model (formation of physical dependence by method of intensive alcoholization) we have studied receptor binding of testosterone (T) and estradiol (E2) in the hypothalamus and pituitary body of mature male rats. Administration (at 10 and 16 h) of 25% ethanol-saline solution at a dose of 7.5 g/kg of body weight in the course of 5 days significantly decreased serum T level but did not change serum LH and FSH levels. Essential reduction of the nuclear androgen receptors in the preoptic-anterior hypothalamic area (POA), mediobasal hypothalamus (MBH) and adenohypophysis was noted in alcohol-treated rats. Unlike androgen receptors the number of the nuclear E2-binding sites in PaO was significantly increased in these males. Thus the results of the present paper demonstrate that multiple administration of ethanol stipulates deficit of serum T, androgen receptors in MBH and pituitary body that possibly results in separation of negative feedback mechanism between the gonads and pituitary body. Increase of specific binding of E2 to nuclear receptors in PoA might appear to explain feminization of alcohol-treated rats.     

Developmental patterns of serum leptin levels, leptin gene expression in adipose tissue and Ob-Rb gene expression in hypothalamus of Erhualian and Large White pigs

~~Developmental patterns of serum leptin levels, leptin gene expression in adipose tissue and Ob-Rb gene expression in hypothalamus of Erhualian and Large White pigs~~     

Localization and role of leptin in the thyroid gland of the lizard Podarcis sicula (Reptilia, Lacertidae)

Leptin, the product of the ob gene, is a hormone secreted by adipocytes that regulates food intake and energy expenditure. The hypothalamus-pituitary-thyroid axis is markedly influenced by the metabolism status, being suppressed during food deprivation. The present study was designed to ascertain whether (1) lizard thyroid gland expresses the long form of leptin receptor (Ob-Rb) and (2) the leptin administration affects the thyroid gland activity in this species (and to verify whether leptin plays a similar role in reptiles as observed in the other vertebrates). The presence of leptin receptor in the thyroid gland of Podarcis sicula was demonstrated by immunohistochemical technique (avidin-biotin-peroxidase complex--ABC method). The role of leptin in the control of thyroid gland activity was studied in vivo using light microscopy (LM) technique coupled to a specific radioimmunoassay for thyroid-stimulating hormone (TSH) and thyroid hormones (T4 and T3). Leptin (0.1 mg/100 g body wt)/day increased T4 and T3 release for 3 days but decreased the plasma concentration of TSH; using LM clear signs of stimulation in the thyroid gland were observed. These findings suggest that systemic administration of leptin stimulates the morphophysiology of the thyroid gland in the lizard through a direct mechanism involving Ob-Rb.     

Rat hypothalamic proopiomelanocortin messenger RNA is unaffected by adrenalectomy.

The negative feedback control of hypothalamic cortocotrophin releasing factor (CRF) and anterior pituitary proopiomelanocortin (POMC) by corticosteroids is well understood. However, less is known about the mechanisms that regulate POMC gene expression in the arcuate nuclei in the medial basal hypothalamus (MBH). Using a sensitive and specific S1 endonuclease protection assay, we have examined the effect of adrenalectomy on POMC mRNA in the rat MBH and pituitary. Our results show that adrenalectomy does not change POMC mRNA levels in the MBH at 7 or 14 days post surgery. The neurointermediate lobe of the pituitary was similarly unaffected by adrenalectomy, while in the anterior lobe, POMC mRNA increased 7-10 fold at both time points, effects that were prevented by dexamethasone treatment. We conclude that while POMC mRNA in the anterior lobe of the pituitary is regulated by plasma glucocorticoids, in the MBH and neurointermediate lobe, it is not.     

Pregnancy-induced hyperphagia is associated with increased gene expression of hypothalamic agouti-related peptide in rats

Pregnancy is characterized by an increase in food intake that, in turn, produce a positive energy balance in order to face the considerable metabolic demands associated with the challenge of reproduction. Since hypothalamus is a key brain region involved in many peripheral signals and neuronal pathways that control energy homeostasis and food intake, we investigated if during pregnancy the increase in food intake is mediated by stimulating orexigenic and/or inhibiting anorexigenic neural pathways. We examined hypothalamic gene expressions of Ob-Rb, NPY, AgRP, POMC, MC4-R, and preproorexins in pregnant Wistar rats at day 19 of gestation. Food intake and body weight were increased progressively during the pregnancy. Visceral fat mass depots and serum leptin levels were also increased when compared with virgin animals. No differences were found in mRNA expression of Ob-Rb, POMC, MC4-R, NPY or preproorexin between virgin and pregnant animals. However, pregnancy produced a selective increase in AgRP mRNA levels. These results indicate that the positive energy balance that occurred during pregnancy can hardly be explained by changes in Ob-Rb despite hyperleptinemia associated with pregnancy. The enhanced expression of AgRP suggests the involvement of this neuropeptide in mediating pregnancy-associated hyperphagia.     

Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus.

Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.     

Influence of stress of a maternal organism on the turnover of catecholamines in the brain of early postnatal rats

Effects of prenatal stress (daily 1-h-long immobilization of pregnant females at the 15th–21st days of pregnancy) on the formation of sex-related dimorphism of the turnover of noradrenaline (NA) and dopamine (DA) in the preoptic area (POA) of the brain and mediobasal hypothalamus (MBH) were studied in 10-day-old rats. Sex-related differences of the turnover of a functional NA pool in the POA and DA pool in the MBH were demonstrated in intact control rats: a higher rate of the monoamine turnover was observed in males. Prenatal stress abolished these sex-related differences and, at the same time, induced such differences in the DA turnover in the POA. It is supposed that prenatal stress-evoked early modifications of sex-related dimorphism of the catecholamine turnover in the brain can result in the development of remote disturbances in the neuroendocrine control of reproduction and adaptation.     

Leptin directly stimulates catecholamine secretion and synthesis in cultured porcine adrenal medullary chromaffin cells.

Leptin, a protein encoded by the ob gene, is an adipose tissue-derived signaling factor involved in body weight homeostasis. The hypothalamus is a major site of central action for leptin. However, mounting evidence indicates expression of leptin receptor mRNA in various peripheral organs including the adrenal medulla. Therefore, we investigated the effects of leptin on catecholamine secretion and synthesis in cultured porcine adrenal medullary chromaffin cells. We initially confirmed the expression of leptin receptor (Ob-Rb) mRNA in cultured porcine adrenal medullary cells. Murine recombinant leptin (>==50 nM) strongly induced the release of both epinephrine (E) and norepinephrine (NE) from chromaffin cells. Removal of external Ca(2+) significantly suppressed these effects. Also, leptin (>==1 nM) enhanced nicotine-induced increases in E- and NE. Leptin (1, 10, 100 nM) significantly increased tyrosine hydroxylase (TH) (a rate-limiting enzyme in the biosynthesis of catecholamine) mRNA levels in a concentration-dependent manner. Furthermore, leptin (1, 10, 100 nM) significantly induced increases in cAMP levels, suggesting that the stimulatory effects on TH mRNA are mediated, at least in part, by the cAMP/protein kinase A pathway. These results indicate that leptin directly stimulates catecholamine release and synthesis, which in turn may potentiate the anti-obesity effects of leptin.