Congenital Hypothyroidism is Associated With Impairment of the Leptin Signaling Pathway in the Hypothalamus in Male Wistar Animals in Adult Life.

The goal of this study is to investigate whether congenital hypothyroidism induced by MMI during gestation (G) or gestation plus lactation (GL) would affect the leptin action upon body weight control on hypothalamus. Six to eight pups per group were killed at 90 days of age. For statistical analysis one-way ANOVA followed by the Holm-Sìdak post hoc test was used. Hypothyroidism resulted in a significant increase in leptin serum levels in G 20% and GL 25% (p<0.04). There was a significant expression decrease of OBR in G 45% and GL 63%; pSTAT3 in G 56% and GL 51%; pERK in G 50% and GL 48%; POMC in G 41% and GL 46% (p<0.04), while a significant increase was assigned to SOCS3 in G 52% and GL 170% (p<0.04) protein expression. We can conclude that hypothyroxinemia condition in rats on adulthood results in impairment of the leptin signaling pathway via ObRb-STAT3 in the hypothalamus, which is likely to be involved in the leptin resistance.

Unravelling purinergic regulation in the epididymis: activation of V-ATPase-dependent acidification by luminal ATP and adenosine.

KEY POINTS: In the epididymis, elaborate communication networks between epithelial cells are important with respect to establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa, which is essential for male fertility. Proton secretion by epididymal clear cells is achieved via the proton pumping V-ATPase located in their apical membrane. In the present study, we dissect the molecular mechanisms by which clear cells respond to luminal ATP and adenosine to modulate their acidifying activity via the adenosine receptor ADORA2B and the pH-sensitive ATP receptor P2X4. We demonstrate that the hydrolysis of ATP to produce adenosine by ectonucleotidases plays a key role in V-ATPase-dependent proton secretion, and is part of a feedback loop that ensures acidification of the luminal compartment These results help us better understand how professional proton-secreting cells respond to extracellular cues to modulate their functions, and how they communicate with neighbouring cells. ABSTRACT: Cell-cell cross-talk is crucial for the dynamic function of epithelia, although how epithelial cells detect and respond to variations in extracellular stimuli to modulate their environment remains incompletely understood. In the present study, we used the epididymis as a model system to investigate epithelial cell regulation by luminal factors. In the epididymis, elaborate communication networks between the different epithelial cell types are important for establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa. In particular, clear cells (CCs) secrete protons into the lumen via the proton pumping V-ATPase located in their apical membrane, a process that is activated by luminal alkalinization. However, how CCs detect luminal pH variations to modulate their function remains uncharacterized. Purinergic regulation of epithelial transport is modulated by extracellular pH in other tissues. In the present study, functional analysis of the mouse cauda epididymis perfused in vivo showed that luminal ATP and adenosine modulate the acidifying activity of CCs via the purinergic ADORA2B and P2X4 receptors, and that luminal adenosine content is itself regulated by luminal pH. Altogether, our observations illustrate mechanisms by which CCs are activated by pH sensitive P2X4 receptor and ectonucleotidases, providing a feedback mechanism for the maintenance of luminal pH. These novel mechanisms by which professional proton-secreting cells respond to extracellular cues to modulate their functions, as well as how they communicate with neighbouring cells, might be translatable to other acidifying epithelia.

Leptin and leptin receptor are expressed only in clear cells of rat epididymis epithelia / La leptina y el receptor de leptina se expresan solamente en células claras del epitelio epidídimario de rata

SUMMARY: Leptin is a 16 kilodaltons hormone secreted by adipose tissue and in the past few years it has been related to the reproductive system regulation. Leptin and its receptor (OBR) have been described in several reproductive organs and in different species but, in epididymis, there is still a lack of information. The aim of this work is to establish if leptin and its receptor are present on epididymis and where the production is occurring. At mRNA level the cauda portion showed a high expression of leptin (p<0.025) and OBRa (p<0.002) while at protein level the OBR expression was lower in cauda region (p<0.025) and leptin was not detected. The ratio between OBRa and OBRb was higher in both regions despite its total amount. By immunohistochemistry leptin and OBR were detected on epididymis epithelia, restricted to clear cells (CC). After efferent duct ligation (EDL) a decrease on leptin staining on CC was observed, suggesting that despite of epididymis production, most of leptin source may probably come from testis. Our results show that leptin and OBR, both mRNA and protein, are present on epididymis and exclusively in CC, suggesting that this tissue is responsive to the hormone and may have an important role on CC regulation.

RESUMEN: La leptina es una hormona de 16 kilodaltons secretada por el tejido adiposo y se ha relacionado en los últimos años con la regulación del sistema reproductivo. La leptina y su receptor (OBR) se han reportado en varios órganos reproductores y en diferentes especies sin embargo, en el epidídimo aún falta información. El objetivo de este trabajo fue establecer si la leptina y su receptor están presentes en el epidídimo y donde se produce. A nivel de ARNm la porción de cauda mostró una alta expresión de leptina (p <0,025) y OBRa (p <0,002) mientras que a nivel de proteína la expresión de OBR fue menor en la región de la cauda epididimaria (p <0,025) y no se detectó leptina. La relación entre OBRa y OBRb fue mayor en ambas regiones a pesar de su cantidad total. Por inmunohistoquímica se detectaron leptina y OBR en el epitelio del epidídimo restringido a células claras (CC). Después de la ligadura del conducto deferente (EDL) se observó una disminución en la tinción de leptina en CC, lo que sugiere que a pesar de la producción del epidídimo, la mayor parte de la fuente de leptina puede provenir probablemente del testículo. Nuestros resultados mostraron que la leptina y OBR, mRNA y proteína, están presentes en el epidídimo y exclusivamente en CC, lo que sugiere que este tejido es sensible a la hormona y puede tener un papel importante en la regulación CC.

Hormonal and testicular changes in rats submitted to congenital hypothyroidism in early life.

The goal of this study was to evaluate the influence of hypothyroidism induced by MMI, during gestation (G) or gestation plus lactation (GL) on testis and its relation with leptin in rats. Six to eight pups were killed at 90 days of age. For statistical analysis One-way ANOVA followed by the Holm-Sìdak post hoc test was used. Hypothyroidism resulted in a significant reduction in LH, FSH and testosterone and an increase in leptin serum levels (p < 0.04). There was a significant decrease in StAR, AR, FSHR, LHR, pSTAT3 and SOCS3 (p < 0.04) protein expression and in the fertility parameters (p < 0.04). We can conclude that hypothyroidism is associated with reduction of steroidogenesis and spermatogenesis leading to a low fertility potential in these animals. This outcome could be a consequence of low pituitary stimulus and testicular response and probably are not related with leptin hormone since its signaling pathway is down-regulated in the testis.

Perinatal malnutrition programs gene expression of leptin receptors isoforms in testis and prostate of adult rats.

The aim of this paper was to evaluate if maternal malnutrition during lactation programs the expression of leptin receptor isoforms in the testes and prostate ventral lobe of adult rats. At delivery, Wistar rats were separated into 3 groups: control group (C) with free access to a standard laboratory diet containing 22% protein; protein-energy restricted group (PER) with free access to an isoenergy and protein-restricted diet containing 8% protein; and energy-restricted group (ER) receiving standard laboratory diet in restricted quantities, which were calculated according to the mean ingestion of the PER group. All animals were sacrificed at 90 days of age. Both PER and ER groups presented low body weight from the first days after birth, however, while the ER group reached the control weight around day 80, the body weight of PER group was significantly lower compared to controls until the day the animals were killed. In relation to tissue weight, only the relative testis weight of the ER group presented an alteration compared to the control group (p<0.03). There was also no alteration in the leptin serum levels among the groups. The main leptin receptors isoforms, OBRa and OBRb were significantly increased in the testis (OBRa: C=0.71±0.10; PER=1.14±0.17; ER=1.92±0.70, p<0.0007, OBRb: C=0.87±0.04; PER=1.20±0.05; ER=1.44±0.17, p<0.001) and prostate (OBRa: C=0.70±0.18; PER=1.30±0.14; ER=1.65±0.22, p<0.014, OBRb: C=0.77±0.14; PER=1.16±0.04; ER=1.30±0.13, p<0.027) of both malnourished groups. However, the testis OBRc (C=1.52±0.06; PER=1.35±0.23; ER=3.50±0.72, p<0.023) and OBRf (C=1.31±0.12; PER=1.66±0.27; ER=3.47±0.55, p<0.009) and prostate OBRc (C=0.48±0.13; ER=1.18±0.34, p<0.01) and OBRf (C=0.73±0.15; PER=0.99±0.11; ER=1.83±0.30, p<0.016) isoforms were significantly increased only in the ER group. The results presented here show for the first time that both testis and prostate leptin receptor isoforms gene expression are programmed by perinatal malnutrition. These data further stress the importance of monitoring maternal and neonatal status, as well as other pathophysiological situations, to combat the appearance of long-term diseases.

Glutamine supplementation prevents collagen expression damage in healthy urinary bladder caused by radiotherapy.

OBJECTIVE: Patients who have had pelvic radiotherapy as part of their cancer therapy may develop subsequent urinary bladder effects such as hyperactive bladder, incontinence, and dysuria. Therefore, the goal of this study was to evaluate whether glutamine supplementation could prevent collagen expression damage in healthy urinary bladder caused by radiotherapy. METHODS: Fifteen adult Wistar rats were separated into a control group that received food and water ad libitum (C group), an irradiated group that received a single pelvic radiation dose of 1164 cGy (I group), and an irradiated group supplemented with l-glutamine every day during the entire experimental period (0.65 g/kg of body weight; I+G group). All animals were sacrificed 15 d after irradiation. The extracellular matrix and muscle were quantified by a morphometric method. Picro Sirius Red was used to visualize the different collagen types. Reverse transcription-polymerase chain reaction and immunohistochemistry were used to determine collagen type I and III expressions. RESULTS: The extracellular matrix (C group 36.84±4.37, I group 31.64±5.00, I+G group 35.53±2.60, P=0.0001), muscle (C group 36.43±6.15, I group 29.39±7.08, I+G group 31.38±3.14, P=0.0001), and gene expressions of collagen type I (C group 1.067±0.31, I group 0.579±0.17, I+G group 1.816±0.66, P=0.0009) and type III (C group 0.99±0.28, I group 0.54±0.13, I+G group 1.07±0.28, P=0.0080) were decreased in the I group. Apart from muscle, glutamine supplementation prevented these alterations. Immunohistochemistry and Picro Sirius Red showed similar results. CONCLUSION: Supplementation with l-glutamine seems to prevent bladder wall damage in relation to extracellular matrix volumetric density and collagen expression. These results suggest that glutamine supplementation could be efficient in protecting healthy tissues from the adverse effects of radiotherapy.

Metabolic programming of lipid profile and reproductive organs weight by leptin treatment on early life.

PURPOSE: To evaluate whether the neonatal leptin treatment during the first days of life can program the male reproductive organs weight and the lipid profile. METHODS: At birth 6 dams were divided into 2 groups: Leptin - each pup was injected with 50microL of recombinant rat leptin (80ng/g BW, sc), for the first 10 d of lactation; Control - each pup received the same volume of saline. After weaning, all pups received unlimited access to food until 190 days of age when they were killed. Values are given as mean + or - SEM of 6 animals and Test t Student was used to analyze the results. RESULTS: The leptin treatment resulted in a significant increase in body weight (Control= 411.8 + or - 16.31; Leptin= 481.8 + or - 11.29, p=0.005) and food consumption (Control= 25.32 + or - 0.09; Leptin= 32.42 + or - 0.15, p=0.0001) and a significant reduction in triglycerides levels (Control= 540.0 + or - 117.9; Leptin= 93.25 + or - 15.21, p=0.006) and in the weight of hypothalamus (Control= 0.234 + or - 0.016; Leptin= 0.154 + or - 0.015, p=0.007), pituitary (Control= 0.104 + or - 0.0120; Leptin= 0.033 + or - 0.012, p=0.003), testis (Control= 3.75 + or - 0.055; Leptin= 3.19 + or - 0.10, p=0.002) and prostate (Control=1.641 + or - 0.1389; Leptin= 0.91 + or - 0.07, p=0.001). CONCLUSION: Leptin treatment on the first days of life can program the reproductive organs weight and the lipid profile of the progeny.

Metabolic programming of lipid profile and reproductive organs weight by leptin treatment on early life / Programação metabólica do perfil lipídico e peso dos órgãos do sistema reprodutor pelo tratamento com leptina nos primeiros dias de vida

PURPOSE: To evaluate whether the neonatal leptin treatment during the first days of life can program the male reproductive organs weight and the lipid profile. METHODS: At birth 6 dams were divided into 2 groups: Leptin - each pup was injected with 50μL of recombinant rat leptin (80ng/g BW, sc), for the first 10 d of lactation; Control - each pup received the same volume of saline. After weaning, all pups received unlimited access to food until 190 days of age when they were killed. Values are given as mean ± SEM of 6 animals and Test t Student was used to analyze the results. RESULTS: The leptin treatment resulted in a significant increase in body weight (Control= 411.8±16.31; Leptin= 481.8±11.29, p=0.005) and food consumption (Control= 25.32±0.09; Leptin= 32.42±0.15, p=0.0001) and a significant reduction in triglycerides levels (Control= 540.0±117.9; Leptin= 93.25±15.21, p=0.006) and in the weight of hypothalamus (Control= 0.234±0.016; Leptin= 0.154±0.015, p=0.007), pituitary (Control= 0.104±0.0120; Leptin= 0.033±0.012, p=0.003), testis (Control= 3.75±0.055; Leptin= 3.19±0.10, p=0.002) and prostate (Control=1.641±0.1389; Leptin= 0.91±0.07, p=0.001). CONCLUSION: Leptin treatment on the first days of life can program the reproductive organs weight and the lipid profile of the progeny.

OBJETIVO: Avaliar se o tratamento neonatal com leptina durante os primeiros dias de vida poderia programar o peso dos orgãos do sistema reprodutor masculino e o perfil lipídico. MÉTODOS: Ao nascimento seis ratas-mãe foram distribuídas em dois grupos: Leptina - cada filhote recebeu 50μL de leptina recombinante (80ng/gPC, SC) nos primeiros 10 dias de lactação; Controle - cada filhote recebeu o mesmo volume de salina. Após o desmame, todos os filhotes tiveram acesso ilimitado a ração até 190 dias de idade quando foram mortos. Os dados são expressos como média ± erro padrão e foram analisados pelo teste t Student. RESULTADOS: O tratamento com leptina resultou em aumento significativo no peso corporal (Control= 411.8±16.31; Leptin= 481.8±11.29, p=0.005) e consumo alimentar (Control= 25.32±0.09; Leptin= 32.42±0.15, p=0.0001) e redução significativa nos níveis séricos de triglicerídeos (Control= 540.0±117.9; Leptin= 93.25±15.21, p=0.006), no peso do hipotálamo (Control= 0.234±0.016; Leptin= 0.154±0.015, p=0.007), hipófise (Control= 0.104±0.0120; Leptin= 0.033±0.012, p=0.003), testículo (Control= 3.75±0.055; Leptin= 3.19±0.10, p=0.002) e próstata (Control=1.641±0.1389; Leptin= 0.91±0.07, p=0.001). CONCLUSÃO: O tratamento com leptina nos primeiros dias de vida pode programar o peso dos órgãos do sistema reprodutor e o perfil lipídico da prole.

Metabolic programming of ovarian angiogenesis and folliculogenesis by maternal malnutrition during lactation.

OBJECTIVE: To evaluate whether maternal malnutrition during lactation programs ovarian folliculogenesis and the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and its receptors KDR, Flt-1, and FGFR. DESIGN: Experimental study. SETTING: University-based research laboratory. ANIMAL(S): Adult female rats from a urogenital research laboratory. INTERVENTION(S): Six rat dams randomly assigned to the following groups: control group (C), with free access to a standard laboratory diet containing 23% protein; and a protein-energy-restricted group (PER), with free access to an isoenergy and protein-restricted diet containing 8% protein. After weaning, the female pups had free access to the standard laboratory diet until 90 days of age, when they were sacrificed at the proestrum stage. MAIN OUTCOME MEASURE(S): Quantification of ovarian follicles, vessels, and expression of growth factors and their receptors. RESULT(S): Maternal malnutrition during lactation caused a significant reduction in the number of primordial (C = 6.60 +/- 0.24, PER = 5.20 +/- 0.20), primary (C = 5.80 +/- 0.66, PER = 4.00 +/- 0.31), and Graafian follicles/section (C = 2.18 +/- 0.29, PER = 1.08 +/- 0.37), in KDR (C = 0.22 +/- 0.04, PER = 0.09 +/- 0.01), Flt-1 (C = 0.28 +/- 0.05, PER = 0.12 +/- 0.02), and FGFR mRNA expression (C = 0.34 +/- 0.05, PER = 0.13 +/- 0.05) and in the vessel density of follicles (C = 17.26 +/- 2.30, PER = 9.96 +/- 0.97). CONCLUSION(S): Maternal malnutrition during lactation programs the follicular development by a reduction of VEGF and FGF mRNA receptors expression, probably from a direct action on the follicular development or a reduction in vasculature resulting in a decreased delivery of folliculotrophic substances in PER animals.

Maternal protein-energy and energy-restricted diets during lactation possibly could program folliculogenesis and the ovarian expression of leptin and its different isoform receptors in rats.

Both protein-energy and energy-restricted diets during lactation program the ovarian function of the rat offspring, leading to a reduction of folliculogenesis, possibly as the consequence of the altered expression of leptin and its isoform receptor genes.