Lineage tracing and resulting phenotype of haemopoietic-derived cells in the pancreas during beta cell regeneration.

AIMS: Transplantation of bone marrow-derived haemopoietic stem cells following streptozotocin (STZ) treatment to induce pancreatic beta cell loss in mice causes the partial regeneration of beta cell mass, with many haemopoietic cells demonstrating endothelial cell markers. This study used genetically tagged haemopoietic lineage-derived cells to determine how endogenous cells are mobilised following beta cell loss and subsequent replacement. METHODS: A double transgenic mouse model, Vav-iCre; R26R-enhanced yellow fluorescent protein (YFP), was used where only haemopoietic lineage cells expressed the Vav1 gene promoter allowing expression of the YFP reporter gene. Between postnatal days 2 and 4 mice were injected with STZ or vehicle (control) and body weight and glycaemia were monitored. Mice were killed between days 10 and 130, and the pancreases were examined by immunofluorescence microscopy. RESULTS: YFP-expressing cells infiltrated the pancreas at all ages, being present around newly forming islets at the pancreatic ducts, and within larger islets. Small numbers of YFP-positive cells (<5%) co-stained for the macrophage markers F4/80 or Mac1, for cytokeratin 19, or for the transcription factor pancreatic and duodenal homeobox 1 (PDX-1), but no co-localisation was seen with insulin or other endocrine hormones. Within islets approximately 30% of YFP-positive cells co-stained for the endothelial cell marker CD31, and following STZ the number of haemopoietic-derived cells, and the proportion that were CD31-positive, both significantly increased after 21 and 40 days, coincident with a partial replacement of beta cells. CONCLUSIONS: Our results suggest that following beta cell loss endogenous haemopoietic-lineage cells contribute to intra-islet angiogenesis, which supports a partial recovery of beta cell mass.

GABAB receptors and glucose homeostasis: evaluation in GABAB receptor knockout mice.

GABA has been proposed to inhibit insulin secretion through GABAB receptors (GABABRs) in pancreatic beta-cells. We investigated whether GABABRs participated in the regulation of glucose homeostasis in vivo. The animals used in this study were adult male and female BALB/C mice, mice deficient in the GABAB1 subunit of the GABABR (GABAB(-/-)), and wild types (WT). Blood glucose was measured under fasting/fed conditions and in glucose tolerance tests (GTTs) with a Lifescan Glucose meter, and serum insulin was measured by ELISA. Pancreatic insulin content and islet insulin were released by RIA. Western blots for the GABAB1 subunit in islet membranes and immunohistochemistry for insulin and GABAB1 were performed in both genotypes. BALB/C mice preinjected with Baclofen (GABABR agonist, 7.5 mg/kg ip) presented impaired GTTs and decreased insulin secretion compared with saline-preinjected controls. GABAB(-/-) mice showed fasting and fed glucose levels similar to WT. GABAB(-/-) mice showed improved GTTs at moderate glucose overloads (2 g/kg). Baclofen pretreatment did not modify GTTs in GABAB(-/-) mice, whereas it impaired normal glycemia reinstatement in WT. Baclofen inhibited glucose-stimulated insulin secretion in WT isolated islets but was without effect in GABAB(-/-) islets. In GABAB(-/-) males, pancreatic insulin content was increased, basal and glucose-stimulated insulin secretion were augmented, and impaired insulin tolerance test and increased homeostatic model assessment of insulin resistance index were determined. Immunohistochemistry for insulin demonstrated an increase of very large islets in GABAB(-/-) males. Results demonstrate that GABABRs are involved in the regulation of glucose homeostasis in vivo and that the constitutive absence of GABABRs induces alterations in pancreatic histology, physiology, and insulin resistance.

Gonadotropins and inhibins along the development of a luteinized rat ovarian tumor.

Luteinized intrasplenic ovarian tumors develop in response to high circulating gonadotropins. The relationship between tumor development, gonadotropins and inhibins was studied. Tumor-bearing animals were sacrificed weekly along the first 6 weeks of development. Inhibins were measured by enzyme-linked immunosorbent assay (ELISA), serum gonadotropins, GH and IGF-1 by RIA. Inhibin subunit mRNAs were determined by Northern blot. Tumor histology was examined. Ovarian grafts grew significantly along development. LH increased ten-fold on week 1; a further significant increment was observed on week 3. FSH peaked on weeks 1 and 2 and fell significantly thereafter. Serum inhibins markedly increased on weeks 3-5. Tumor inhibin A content and mRNA levels for alpha and beta A subunits also increased on week 3. Inverse correlations between inhibins and FSH and direct correlations between inhibins and LH were observed. Tumor inhibin A and IGF-1 contents correlated significantly. Increasing levels of luteinization were observed along tumor development. These luteinized tumors develop mainly in response to LH, since growth continues under FSH inhibition. The active inhibin secretion and the positive correlation between inhibins and LH suggests that LH may be the main driving force behind this production, while growth factors produced by the gonads may also participate in their regulation.

Actions of immunosuppressor drugs on the development of an experimental ovarian tumor.

Immunosuppression has been related to the incidence of tumor apparition, including endocrine tumors. The intrasplenic ovarian tumor (luteoma) is a typical benign endocrine tumor that develops under high gonadotropin stimulation and, from the immunological perspective, is located in a critical organ involved in immune response. To establish if immunosuppression could alter the development of this experimental tumor, the effects of cyclosporin A (CsA) and dexamethasone (Dex) were evaluated. After surgery, tumor-bearing and sham animals were kept without treatment for 4 weeks; thereafter, they were distributed into CsA (25 mg/kg), Dex (0.1 mg/kg), or vehicle (75:25 castor oil:ethanol) groups and were injected on alternate days for 50 days. Body weight was evaluated weekly. Animals were sacrificed after a jugular vein blood sample was obtained. Thymi were weighed. Tumors were measured and placed in formaline for histological studies. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and estradiol were measured by radioimmunoassay. Hematological parameters were determined. CsA induced a significant decrease in survival rates both in tumor-bearing and sham animals (P < 0.01). Dex significantly impaired weight increase in both groups of animals. CsA induced a significant weight loss in sham animals, not observed in tumor-bearing animals. Dex induced thymus weight loss in both groups, whereas CsA induced thymus weight loss only in sham animals. Only Dex induced a decrease in lymphocyte number in both groups. CsA induced an increase in monocyte number only in sham animals. Treatments did not alter LH, FSH, or estradiol, whereas PRL was increased by CsA only in sham rats. Neither Dex nor CsA induced any significant variations in tumor volume, nor did they alter tumor histology. In addition, no visible metastases or alterations in other organs were observed. We conclude that, though immunological parameters were altered by the treatments, immunosuppressor drugs did not condition tumor development. In addition, tumors secrete one or more factor/s that counteract CsA effect.

GABA(B) receptors in anterior pituitary cells. Mechanism of action coupled to endocrine effects.

The activation of pituitary GABA(B) receptors by the specific agonist baclofen inhibits pituitary hormone secretion in vitro. Here we studied the mechanism of action of GABA(B) receptors in rat adenohypophysis. Anterior pituitary cells were obtained by trypsinization and were either plated for hormonal studies and cAMP determination or incubated in FURA 2AM for calcium measurements. Baclofen (BACL: 1 x 10(-5) M) significantly inhibited basal and thyrotropic releasing hormone (TRH)-stimulated (1 x 10(-7) M) PRL secretion in anterior pituitary cells from proestrous rats. In the presence of pertussis toxin (PTX: 150 ng/ml, 20 h), which leads to the uncoupling of the G(i/o)-protein from the receptor, both effects of BACL were abolished while the effect of dopamine (DA: 1 x 10(-8) M), used as an inhibitory control, was reduced from 70 to 25%. PTX also reversed BACL-induced inhibition of gonadotropin-releasing hormone (GnRH)-elicited luteinizing hormone (LH) secretion in anterior pituitary cells from 15-day-old female rats. In addition, though working in a pituitary mixed cell population, in which only some cell types possess GABA(B) receptors, BACL (1 x 10(-5) M) attenuated the forskolin-induced (0.5 microM) increase in cAMP. This effect was prevented by co-incubation with the antagonist 2 hydroxysaclofen and by preincubation with PTX. BACL (5 x 10(-5) M) and DA (5 x 10(-7) M) inhibited basal intracellular calcium concentrations ([Ca(2+)](i)) in pituitary cells and the effect of the latter was significantly stronger. The effect of BACL on [Ca(2+)](i) was abolished after preincubation with PTX. In the presence of the potassium channel blocking agents barium (200 microM and 1 mM) and tetraethylammonium (10 mM), BACL was still able to inhibit [Ca(2+)](i). Blockade of voltage-sensitive calcium channels (VSCC) with either verapamil (5 x 10(-6) M) or nifedipine (1 x 10(-6) M) completely abolished the effect of BACL on [Ca(2+)](i). In the presence of 12.5 mM potassium concentration baclofen significantly inhibited [Ca(2+)](i). In conclusion, our results describe the negative coupling of adenohypophyseal GABA(B) receptors to VSCC through PTX-sensitive G-proteins. These characteristics suggest a resemblance of these receptors to the typical presynaptic GABA(B) sites described in the central nervous system.

Reproductive effects of hexachlorobenzene in female rats.

Hexachlorobenzene (HCB) is a polyhalogenated aromatic hydrocarbon widely distributed in the environment. In animal testing, HCB has been shown to be a reproductive toxin. Previous investigations of the effects of HCB on ovarian function have yielded equivocal results. Thus, the effects of chronic administration of HCB (1 g kg(-1) body wt.) on the ovary and pituitary hormone levels, hepatic and uterine oestradiol receptors, ovarian histopathological changes and oestrus cycle characteristics were investigated in spontaneously cycling rats. Our data demonstrate that HCB treatment, under the conditions of the present study, reduced circulating levels of oestradiol and prolactin without differences in serum concentrations of progesterone. Follicle-stimulating hormone serum levels were elevated. Hexachlorobenzene treatment resulted in irregularity of cycles, characterized mainly as prolonged periods of oestrus with a reduced number of ova recovered. In addition, HCB administration resulted in significantly decreased uterine nuclear oestrogen receptor levels. Histopathological examination revealed degenerative changes of the ovarian follicles and germinal epithelium and increased numbers of atresic follicles.

Development of an experimental ovarian tumor over a year in the rat.

Tumor growth, possible malignant transformation or metastatic propagation and hormonal patterns were evaluated over a year in luteoma induced by introducing an ovary into the spleen of ovariectomized 60 day-old rats. Sham castrated animals had a piece of muscle inserted into the spleen. Jugular blood samples were taken monthly. After a year animals were cycled and decapitated. Troncal blood was collected, autopsies were performed and luteoma were measured and fixed in 10% buffered formalin. Serum LH, FSH, PRL, estradiol and progesterone were measured. Serum inhibin content was determined in one month-old tumors-bearing animals and estrous rats as controls. After one year no external changes in tumor-bearing rats were observed, nor differences in body weight or mortality rates compared to Sham animals. Metastatic propagation was absent. Routine histological examination showed two types of tumors according to either granulosa or luteal cell predomination, tumor type did not determine hormonal patterns. However, a clear relationship between gonadotropin levels and tumor size was established. Low gonadotropins: Small tumors, 18.7% of cases and high gonadotropins: Large tumors, 81.3%. In Sham animals gonadotropins attained castrate levels and remained elevated until the end of the experiment. In the Small group no increases in gonadotropins or estradiol were detected, progesterone and PRL fluctuated. In the Large tumor group LH increased to Sham titers until month 7, then fell to initial levels, FSH augmented significantly as from month three and remained high up to month 5. No variations in either estradiol, progesterone or PRL were observed. Serum inhibin of one month-old tumor-bearing rats was significantly elevated, justifying the lack of FSH increase at this time point. We conclude that these luteoma do not suffer malignant transformation or induce metastases. They appear in two histological types. Tumor size depends on hormonal patterns. The delay in the initial increase and the sharp decrease observed in FSH in animals bearing Large tumors suggest a possible role for inhibin in this regulation.

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.

GnRH receptors and GnRH endocrine effects on luteoma cells.

An ovary implanted into the spleen of an ovariectomized rat develops into a luteinized tumor, growing in response to gonadotrophins. Previously, it was shown that in vivo Buserelin, a gonadotrophin-releasing hormone (GnRH) analog, inhibited tumor growth. To determine if GnRH had a direct effect on tumor cells, the presence of GnRH receptors as well as the endocrine effects of buserelin were studied on tumoral tissue. GnRH receptors were present in luteoma in similar concentrations and dissociation constant (Kd) to control estrous ovaries. In vivo treatment with buserelin did not modify luteoma GnRH receptors. In organ incubations, luteoma secreted significantly higher estradiol and lower progesterone than estrous ovaries; addition of buserelin did not modify steroid secretion. The same difference in basal steroid secretion between luteoma cells and luteal cells superovulated prepubertal ovaries was observed in cell cultures. Although luteinizing-hormone (LH)-stimulated progesterone in both kinds of cells, buserelin significantly inhibited LH-stimulated progesterone only in luteoma cells. These results describe clear differences in basal steroid secretion between tumoral and normal tissue. Furthermore, they show that luteoma possess GnRH receptors similar to those in normal ovarian tissue, and that GnRH analogs have endocrine effects on these cells. Therefore, a direct effect of buserelin on luteoma cells can be postulated.

In vivo interaction of baclofen, TRH and serotonin on PRL and TSH secretion in the developing and adult male and female rats.

Gamma-aminobutyric acid (GABA) is involved in the neural control of hypophyseal hormones, including PRL and TSH. In the present work we investigated the ontogeny of the effect of baclofen, a GABA B agonist, on basal PRL and TSH release and in the presence of releasing stimulus which act at two different levels: TRH, at the hypophyseal level, and serotonin, at the central nervous system. Ages studied were 4, 12, 20, 28-29, 37-38 day-old and adult male and female animals. Rats of each age and sex were separated in groups and each group received two intraperitoneally injections, one 45 minutes after the other: saline-saline, saline-TRH, baclofen-saline, baclofen-TRH, saline-serotonin or baclofen-serotonin. Rats were decapitated 15 minutes after the last injection and serum hormones were measured by RIA. Baclofen (7 mg/kg) significantly elevated basal prolactin levels at 4, 12 and 20 days of age and the stimulating effect increased with age. At 28 days of age baclofen significantly inhibited PRL whereas from 38 days of age onwards it had no effect on basal PRL levels. No sex differences were evident. Interaction of TRH (4 microg/kg) and baclofen on PRL secretion resulted in an additive effect on days 4 and 12, this effect was not observed when baclofen was administered with serotonin (10 mg/kg). In 28 day-old and older animals baclofen completely blunted the PRL releasing effect of TRH or serotonin. Again, no sex differences were observed. With regard to TSH, baclofen did not alter either basal or TRH stimulated TSH secretion regardless of sex and age. The present experiments indicate that GABA B receptors are involved in the regulation of basal and stimulated PRL secretion from the first days of life to adulthood. Receptor activation results in stimulation or inhibition of PRL release depending on the age of the animals and the site of action. This GABA B regulation of PRL secretion is sex independent. In contrast, pituitary GABA B receptors do not seem to be involved in the regulation of TSH secretion.