Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes.

The endocrine cells of the rat pancreatic islets of Langerhans, including insulin-producing beta-cells, turn over every 40-50 days by processes of apoptosis and the proliferation and differentiation of new islet cells (neogenesis) from progenitor epithelial cells located in the pancreatic ducts. However, the administration to rats of islet trophic factors such as glucose or glucagon-like peptide 1 for 48 h results in a doubling of islet cell mass, suggesting that islet progenitor cells may reside within the islets themselves. Here we show that rat and human pancreatic islets contain a heretofore unrecognized distinct population of cells that express the neural stem cell-specific marker nestin. Nestin-positive cells within pancreatic islets express neither the hormones insulin, glucagon, somatostatin, or pancreatic polypeptide nor the markers of vascular endothelium or neurons, such as collagen IV and galanin. Focal regions of nestin-positive cells are also identified in large, small, and centrolobular ducts of the rat pancreas. Nestin-positive cells in the islets and in pancreatic ducts are distinct from ductal epithelium because they do not express the ductal marker cytokeratin 19 (CK19). After their isolation, these nestin-positive cells have an unusually extended proliferative capacity when cultured in vitro (approximately 8 months), can be cloned repeatedly, and appear to be multipotential. Upon confluence, they are able to differentiate into cells that express liver and exocrine pancreas markers, such as alpha-fetoprotein and pancreatic amylase, and display a ductal/endocrine phenotype with expression of CK19, neural-specific cell adhesion molecule, insulin, glucagon, and the pancreas/duodenum specific homeodomain transcription factor, IDX-1. We propose that these nestin-positive islet-derived progenitor (NIP) cells are a distinct population of cells that reside within pancreatic islets and may participate in the neogenesis of islet endocrine cells. The NIP cells that also reside in the pancreatic ducts may be contributors to the established location of islet progenitor cells. The identification of NIP cells within the pancreatic islets themselves suggest possibilities for treatment of diabetes, whereby NIP cells isolated from pancreas biopsies could be expanded ex vivo and transplanted into the donor/recipient.

Simultaneous indirect activity measurements of GH and PRL genes in the same, living mammosomatotrope.

Dynamic intracellular processes in endocrine cells are usually controlled by the coordinated modulation of two or more functionally related genes. Attempts to gain a more complete understanding of these processes would be facilitated greatly by a method enabling activity measurements of two genes at the same time. Here we describe how we developed such a system and used it to determine indirectly whether individual, living pituitary cells could concurrently express both the growth hormone (GH) and prolactin (PRL) genes. Our results demonstrate that coexpression of these genes is indeed possible. Moreover, our findings provide a general paradigm for future "real-time" analysis of other interrelated genes involved in the regulation of endocrine processes.

Transforming growth factor beta1 is a paracrine inhibitor of prolactin gene expression.

We have shown previously that rat mammotropes produce an activity that suppresses PRL gene expression by neighboring mammotropes. Here, we tested the hypothesis that this mammotrope-derived inhibitor is transforming growth factor-beta1 (TGFbeta1). To this end, we pursued a two-pronged strategy wherein we added exogenous TGFbeta1 to primary cultures of anterior pituitary cells transfected with a rat PRL-luc construct. Measurement of luciferase activity by luminometry of extracts revealed that administration of TGFbeta1, over a range of doses shown by others to be secreted by cultures of pituitary cells, caused a significant (P < 0.05) suppression of PRL gene expression. In contrast, immunoremoval of secreted TGFbeta1 led to an elevation of PRL promoter-driven reporter activity in these cultures. In a subsequent study, we repeated these experiments with a single cell model in an attempt to determine the demographics of the cellular responses. Accordingly, we transfected (via microinjection) individual mammotropes with the rat PRL-luc construct; exposed them to TGFbeta1, its neutralizing antibody, or respective controls; and then assessed PRL gene expression in "real-time" by quantification of photons emitted by the living cells after exposure to the substrate luciferin. Our results revealed that 1) TGFbeta1 inhibited PRL gene expression in all mammotrope studied; 2) only a subgroup of mammotropes (approximately 23%) was relieved of TGFbeta1 inhibition by antibody treatment; and 3) the growth factor exerted its inhibitory effect via a paracrine, as opposed to an autocrine, mechanism. These findings identify TGFbeta1 as the paracrine agent that exerts a tonic inhibitory influence over PRL gene expression in mammotropes.

Pituitary function in the acute phase response in domestic farm animals: cytokines, prostaglandins, and secretion of ACTH.

Contained in this report is a review of available data on pituitary cytokines in domestic species of agricultural importance. The concept is advanced that the pituitary gland is essential to appropriate generation of host defense mechanisms and thus should be considered among other tissues contributing to innate immunity. The functions of these intrapituitary cytokines, principally IL-6, are discussed in the context of potential regulation of the pituitary-adrenal axis (ACTH secretion) via intrapituitary PGE2 generation during the acute-phase response to infectious/inflammatory stimuli. Data from other species are cited as appropriate for comparative purposes and elaboration of proposed mechanisms. However, the scope of the review is not intended to comprehensively cover the vast literature on proinflammatory cytokines and prostaglandins generated peripherally and centrally during host responses to inflammatory stimuli.

Effects of cellular interactions on calcium dynamics in prolactin-secreting cells.

Signals derived from other pituitary cells can have a dramatic effect on PRL gene expression and secretion by mammotropes. However, the intracellular mechanisms by which these effects are manifested on the target cell remain unexplored. Inasmuch as calcium is a key modulator of both gene expression and hormone export in mammotropes, we evaluated the effects of cell to cell contact vs. specific cellular interactions on calcium dynamics within these cells. This was accomplished by digital-imaging fluorescence microscopy of fura-2 in pituitary cells that were isolated in culture (singles) or adjoining one other cell (doublets). After calcium imaging, we then subjected cells to immunocytochemistry for PRL. Doublets were further categorized into mammotropes attached to another mammotrope (M-M) or to a nonmammotrope (M-nonM). We then calculated and compared Mean [Ca2+]i values as well as Oscillation Indices (which reflect the oscillatory behavior of cells) in singles and doublets and found that they were not different (P > 0.05). However, the phenotype of the adjoining cell had a profound influence on both of these calcium parameters, such that the presence of one mammotrope could consistently decrease (P < 0.05) the Mean [Ca2+]i value (39.17 +/- 3.83 vs. 56.24 +/- 5.56 in M-nonM) and Oscillation Index (10.19 +/- 1.76 vs. 21.21 +/- 3.73 in M-nonM) of its neighboring counterpart. A more detailed analysis of oscillatory patterns in these cells revealed that nonoscillators were more abundant in M-M (23%) than in M-nonM (12%) doublets. Taken together, our results indicate that PRL-secreting cells convey a signal that dampens the oscillatory behavior of neighboring mammotropes. Thus, it appears that it is the phenotype rather than the physical presence of a neighbor that controls intercellular regulation of calcium dynamics among mammotropes.

Cytokines in the hypophysis: a comparative look at interleukin-6 in the porcine anterior pituitary gland.

Interleukin-6 (IL-6) is a multifunctional cytokine produced by a variety of cell types in tissues of both the immune and endocrine systems. Among the major functions described for IL-6 are its role in the maturation of B cells to high-output antibody-producing cells and its contribution to the acute physiological responses to infection and inflammation, notably production of hepatic acute phase proteins and activation of the hypothalamic-pituitary-adrenal axis. In addition to these better known functions, IL-6 recently has been found within the pituitary of laboratory rats and also in the human pituitary. In rats, pituitary IL-6 mRNA is upregulated by peripheral exposure to bacterial endotoxin. However, the role of anterior pituitary IL-6 in host responses to infection and inflammation remains uncertain, although it may regulate pituitary hormone secretion. The following brief review summarizes the information available concerning cytokine production within the anterior pituitary of species of domestic livestock. To our knowledge, experiments conducted in our laboratory evaluating regulation of IL-6 mRNA expression and secretion from the porcine anterior pituitary provide most of the data in domestic species confirming the presence of IL-6 in the pituitary. Our data indicate that IL-6 mRNA is present in cultured porcine anterior pituitary cells and that the pituitary directly responds to stimulation with bacterial endotoxin by increasing secretion of IL-6. Furthermore, endotoxin-induced upregulation of IL-6 mRNA expression and secretion appears to be dependent upon production of cyclooxygenase products of arachidonic acid metabolism.

Octylphenol (OP), an environmental estrogen, stimulates prolactin (PRL) gene expression.

A growing concern about "endocrine disrupters" and their impact on estrogen-dependent-phenomena led us to investigate the effects of OP, an environmental estrogen, on PRL gene expression. To this end, we found that OP stimulates the PRL gene to a similar degree as estradiol (E2), although E2 is a 1000-fold more potent in this regard. We then sought to confirm that OP exerts its effects through E2-receptors. Accordingly, we treated cells with tamoxifen (TAM), an anti-estrogen, and found that it blunted the response induced by OP, suggesting that E2-receptors mediated this response. Finally, using "real time" measurements of gene expression in living cells, we found that a large fraction of mammotropes were OP-responsive. These results clearly demonstrate that OP affects PRL gene expression in a manner identical to that of E2. Inasmuch as PRL subserves an obligatory role in the regulation of lactation as well as in neonatal development, we believe that environmental estrogens such as OP have the potential to adversely affect the maternal-infant unit by altering PRL gene expression.

Effect of endotoxin on interleukin-6 secretion and messenger ribonucleic acid in porcine anterior pituitary cells.

The pleiotropic cytokine interleukin-6 (IL-6) is produced in and secreted from anterior pituitary (AP) cells of a number of species. Bacterial endotoxin (END) may enhance the transcription of IL-6 and its secretion from the AP. In the studies presented here, we evaluated pig AP cells for the presence of IL-6 mRNA. In addition, because we had observed previously that END stimulated the secretion of prostaglandin E2 from cultured porcine AP cells, the effects of the inhibition of END-stimulated cyclooxygenase products on IL-6 mRNA abundance and the secretion of IL-6 were evaluated. In the first experiment, RNA was extracted from cultured pig AP cells that had been treated with END for 0.5 or 1 hr and subjected to reverse transcription followed by polymerase chain reaction and hybridization after Southern transfer. Bands of expected amplified product size, corresponding to IL-6, were observed only from cells treated with END, although specific hybridization was observed from both control and END-treated wells. In the next experiment, RNA was extracted from cultured AP cells treated with END or END in the presence of the cyclooxygenase inhibitor indomethacin (IND). Amplification of the expected product could be observed from all cultured cells except those treated with IND. However, hybridization data indicated that IND did not eliminate IL-6 mRNA entirely. Next, we measured IL-6 secretion from cultured AP cells exposed to END or END and IND. Treatment with END stimulated IL-6 secretion (P < 0.001) above controls, whereas IND blocked END stimulation of IL-6 secretion (P < 0.001). Finally, using immunostaining, we confirmed the presence of CD14, an END receptor, in cultured pig AP cells. These studies clearly establish the presence of IL-6 mRNA and secretion of the cytokine from cultured porcine AP cells. In addition, END stimulates the secretion of IL-6, perhaps through cells expressing CD14, and END-stimulated IL-6 secretion appears to be mediated by products of the cyclooxygenase pathway.

Intercellular communication: relative importance of cellular adhesion and paracrine signaling to hormonal gene expression.

Although there is a consensus that a cell's microenvironment can have a dramatic influence on its ability to express a particular gene, the relative contribution of physical interaction (cell to cell adhesion) and paracrine signaling to this phenomenon has been difficult to discern. Here, we addressed this problem in mammotropes by making "real-time" measurements of prolactin (PRL) gene expression followed by immunocytochemistry (for post facto identification of a neighbor's phenotype). Our results show that it is the nature (phenotype) rather than the physical presence of a neighboring cell that dictates the degree to which the PRL gene is expressed.

Effects of corticotropin-releasing hormone, lysine vasopressin, oxytocin, and angiotensin II on adrenocorticotropin secretion from porcine anterior pituitary cells.

The aim of this study was to determine the ability of corticotropin-releasing hormone (CRH), lysine vasopressin (LVP), oxytocin (OT), and angiotensin II (AII) to stimulate adrenocorticotropin (ACTH) secretion from porcine anterior pituitary (AP) cells in vitro and to evaluate the role of protein kinase C (PKC) in the interaction between CRH and LVP. In this study, porcine AP cells were enzymatically and mechanically dispersed, cultured (150,000 cells/well) for 4 d, and then challenged with doses of various neuropeptides for 3 hr. CRH (10(-7)-10(-10) M) was the most potent of the peptides tested in stimulating ACTH release from porcine AP cells. In fact, none of the other peptides consistently affected ACTH concentrations relative to basal levels. However, LVP potentiated CRH action, even though by itself, it failed to stimulate ACTH production. Neither OT or AII potentiated CRH-stimulated ACTH release from porcine AP cells. To determine whether the inter-action between CRH and LVP was regulated partially by the protein Kinase C (PKC) pathway, we challenged AP cells in a 30-min incubation with 10(-7) M staurosporine (ST), a treatment predicted to decrease PKC activity. Then, cells were washed and challenged with 10(-9) M LVP, 10(-9) M CRH, and 10(-9) M CRH + LVP. Treatment with ST decreased (P < 0.05) CRH + LVP-stimulated ACTH release. To further demonstrate an interaction between protein kinase A (PKA) and PKC transduction pathways in the observed synergism between CRH and LVP to enhance ACTH secretion, we also challenged AP cells with 10(-7) M phorbol 12, 13-myristate acetate (PMA) and 5 microM forskolin (FOR) for 3 hr. This treatment was predicted to enhance PKA and PKC activities, respectively, and thereby enhance ACTH concentrations. Challenging cells with FOR + PMA enhanced (P < 0.001) ACTH release above basal concentrations, but more important, it increased (P < 0.001) ACTH concentration above that elicited by either drug given alone. Taken together, our in vitro studies support the conclusion that CRH is the principal regulator of ACTH secretion in the pig. In contrast to the results in most other species evaluated, vasopressin alone did not affect ACTH release. However, LVP can enhance the effectiveness of CRH in releasing ACTH, and this enhancement appears to rely, at least in part, on the activation of the PKC signal transduction pathway.