Extracellular vesicles from equine mesenchymal stem cells decrease inflammation markers in chondrocytes in vitro.

BACKGROUND: Mesenchymal stem cells (MSCs) have been used therapeutically in equine medicine. MSCs release extracellular vesicles (EVs), which affect cell processes by inhibiting cell apoptosis and regulating inflammation. To date, little is known about equine EVs and their regenerative properties. OBJECTIVES: To characterise equine MSC-derived extracellular vesicles (EVs) and evaluate their effect on equine chondrocytes treated with pro-inflammatory cytokines in vitro. STUDY DESIGN: In vitro experiments with randomised complete block design. METHODS: Mesenchymal stem cells from bone marrow, adipose tissue, and synovial fluid were cultured in vitro. The MSC culture medium was centrifuged and filtered. Isolated particles were analysed for size and concentration (total number of particles per mL). Transmission electron microscopy analysis was performed to evaluate the morphology and CD9 expression of the particles. Chondrocytes from healthy equines were treated with the inflammatory cytokines interleukin (IL)-1ß and tumour necrosis factor-alpha. MSC-derived EVs from bone marrow and synovial fluid cells were added as co-treatments in vitro. Gene expression analysis by real-time PCR was performed to evaluate the effects of EVs. RESULTS: The particles isolated from MSCs derived from different tissues did not differ significantly in size and concentration. The particles had a round-like shape and positively expressed CD9. EVs from bone marrow cells displayed reduced expression of metalloproteinase-13. MAIN LIMITATIONS: Sample size and characterisation of the content of EVs. CONCLUSIONS: EVs isolated from equine bone marrow MSCs reduced metalloproteinase 13 gene expression; this gene encodes an enzyme related to cartilage degradation in inflamed chondrocytes in vitro. EVs derived from MSCs can reduce inflammation and could potentially be used as an adjuvant treatment to improve tissue and cartilage repair in the articular pathologies.

External and internal EGFR-activating signals drive mammary epithelial cells proliferation and viability.

During puberty, the mammary gland undergoes an intense growth, dependent on the interplay between the Epidermal Growth Factor Receptor (EGFR) in the stroma and different mammary epithelial receptors. We hypothesize that EGFR expressed in the mammary epithelium also has a role in puberty and the epithelial cells can self-sustain by EGFR-mediated autocrine signaling. We adopted mammary cell lines from different species, as in vitro model for the epithelium, and we observed that EGFR-signaling positively affects their survival and proliferation. Once deprived of external growth factors, mammary cells still showed strong Erk 1/2 phosphorylation, abolished upon EGFR inhibition, coupled with a further reduction in survival and proliferation. Based on gene expression analysis, three EGFR-ligands (AREG, EREG and HBEGF) are likely to mediate this autocrine signaling. In conclusion, internal EGFR-activating signals sustain mammary epithelial cell proliferation and survival in vitro.

Analysis of mesenchymal cells (MSCs) from bone marrow, synovial fluid and mesenteric, neck and tail adipose tissue sources from equines.

Mesenchymal stem cells (MSCs) have been used in equines as an alternative therapy. A comparative study about the phenotype and in vitro performance of different MSCs tissue sources in adult equines was needed. This study might serve to provide the knowledge to select a valuable harvesting source of MSCs. Bone marrow, synovial and adipose (mesenteric, neck and tail fat) tissues were collected from adult equines. Cell surface markers expression (CD11α/CD18, CD45, CD79α, CD90, CD105 and MHC II) and in vitro differentiation assays were made. In vitro cell migration, cell growth and wound healing capacity tests helped to study their behavior and properties. MSCs phenotype was positively confirmed by the cell surfaces markers and a tri-lineage differentiation profile. Bone marrow cells showed the highest migration capacity, while synovial fluid cells displayed the highest cell growth. Bone marrow cells showed a better wound healing when compared with all the different MSCs. We conclude that bone marrow, synovial and adipose tissue derived from adult equines are a good source for cell therapy but they conserve different functional properties: bone marrow showed an interesting migration and wound healing capacity while synovial fluid cells and their highest cell growth suggest that these MSCs would yield higher cell numbers in a shorter time.

Mammary Stem Cells in Domestic Animals: The Role of ROS.

Reactive oxygen species (ROS) are produced as a natural byproduct of the normal metabolism of oxygen and play significant roles in cell signaling and homeostasis. Although ROS have been involved in pathological processes as diverse as cancer, cardiovascular disease, and aging, they may to exert an effect even in a physiological context. In the central nervous system, stem cells and hematopoietic stem cells are early progenitors that contain lower levels of ROS than their more mature progeny. These different concentrations have been reported to be crucial for maintaining stem cell function. Mammary gland remodeling has been proposed to be organized through the activation and regulation of cells with stemness, either considered real stem cells or primitive precursors. Given the state of oxidative stress in the mammary gland tissue induced by high milk production, in particular in highly productive dairy cows; several studies have focused on the relationship between adult mammary stem cells and the oxidative state of the gland. The oxidative state of the mammary gland appears to be involved in the initial development and metastasis of breast cancer through interference with mammary cancerous stem cells. This review summarizes some links between the mammary stem and oxidative state of the gland.

Bovine Mammary Organoids: A Model to Study Epithelial Mammary Cells.

Bovine mammary organoids are cell aggregates that are produced by an association of a mechanical and an enzymatic dissociation of mammary gland tissue. They provide a useful source to isolate mammary epithelial cells, but can also be frozen as an intermediate dissociation step.Due to the strong cell-cell interactions among epithelial cells, the production and isolation of organoids is an efficient way to remove unwanted cell population of non-epithelial origin like fibroblasts.

Murine and Human Mammary Cancer Cell Lines: Functional Tests.

The biological characterization of mammary cancer cells is a prerequisite that helps the scientist understand some aspect of tumor biology. Once isolated from the tumor, cells are subjected to multiple tests that dissect their ability to growth, migrate, degrade the surrounding stroma, produce 3-dimensional structures and differentiate. Targeted inhibitors, when added to these tests, are used to unravel how specific growth factors, receptors, and intracellular translational pathways promote the ability of mammary tumor cells to achieve their biological behavior. Herein we describe a set of techniques used to put in focus the biological capacities in mammary cancer cells. When the characterization of a biological trait (e.g., proliferation) is assessable by multiple assays, we will limit the description to only one technique, possibly the easier to manage and that requires minimal laboratory equipment.

CD49f+ mammary epithelial cells decrease in milk from dairy cows stressed by overstocking during the dry period.

The work reported in this Research Communication describes the modification in epithelial cell populations during the first and the last month of milking in Holstein Friesian cows that have undergone different management during the dry period, and we report the differential expression of CD49f+ and cytokeratin18+ cell subpopulations. Twenty six cows were randomly divided into 2 balanced groups that were housed at stocking density of either 11 m2 (CTR) or 5 m2 from 21 ± 3 d before the expected calving until calving. Cells collected from milk samples taken in early lactation and late lactation were directly analysed for CD45, CD49f, cytokeratin 14, cytokeratin 18 and cell viability. We observed a differential expression with a significant reduction in CD49f+ (P < 0·01) and cytokeratin 18+ (P < 0·05) cells in early lactation. Differences were still evident in late lactation but were not significant. These observations suggest that mammary epithelial cell immunophenotypes could be associated with different animal management in the dry period and we hypothesise they may have a role as biomarkers for mammary gland function in dairy cows.

Bovine CD49 positive-cell subpopulation remarkably increases in mammary epithelial cells that retain a stem-like phenotype.

We previously proved that adult stem cells reside in the bovine mammary gland and possess an intrinsic potential to generate a functional mammary outgrowth. The aim of this study was to investigate on the immunophenotyping features retained by mammary stem-like cells detected in long term culture. Flow cytometry analysis showed different subpopulations of mammary epithelial cells emerging according to the timing of cell culture. CD49f(+)-cells significantly increased during the culture (p<0.01) and a similar trend was observed, even if less regular, for CD29(+) and ALDH1 positive cell populations. No difference during the culture was observed for CD24 positive cells but after 35 days of culture a subset of cells, CD49f positive, still retained regenerative capabilities in in vivo xenotransplants. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice. These results prove the presence of a multipotent cell subpopulation that retain a strong epithelial induction, confirmed in in vivo xenotransplants with a presumable in vitro expansion of the primitive population of adult mammary stem cells.

Clonogenic assay allows for selection of a primitive mammary epithelial cell population in bovine.

Adult mammary stem cells have been identified in several species including the bovine. They are responsible for the development of the gland and for cyclic remodeling during estrous cycles and pregnancy. Epithelial cell subpopulations exist within the mammary gland. We and others showed previously that the Colony Forming Cell (CFC) assay can be used to detect lineage-restricted mammary progenitors. We carried out CFCs with bovine mammary cells and manually separated colonies with specific morphologies associated with either a luminal or a myoepithelial phenotype. Expression of specific markers was assessed by immunocytochemistry or by flow cytometry to confirm that the manual separation resulted in isolation of phenotipically different cells. When transplanted in recipient immunodeficient mice, we found that only myoepithelial-like colonies gave rise to outgrowths that resembled bovine mammary alveoli, thus proving that adult stem cells were maintained during culture and segregated with myoepithelial cells. After recovery of the cells from the transplanted mice and subsequent progenitor content analysis, we found a tendency to detect a higher progenitor frequency when myoepithelial-like colonies were transplanted. We here demonstrate that bovine adult mammary stem cells can be sustained in short-term culture and that they can be enriched by manually selecting for basal-like morphology.

Generation of Induced Pluripotent Stem Cells from Bovine Epithelial Cells and Partial Redirection Toward a Mammary Phenotype In Vitro.

In contrast to adult stem cells, induced pluripotent stem cells (iPSCs) can be grown robustly in vitro and differentiated into virtually any tissue, thus providing an attractive alternative for biomedical applications. Although iPSC technology is already being used in human biomedicine, its potential in animal production has not been investigated. Herein, we investigated the potential application of iPSCs in dairy production by generating bovine iPSCs and establishing their ability to generate mammary epithelial tissue. iPSCs were derived by retrovirus-mediated expression of murine Oct4, Sox2, Klf4, and c-Myc in mammary epithelium and dermal fibroblasts. The resulting reprogrammed cells stained positive for alkaline phosphatase and showed renewed expression of pluripotency genes, including Lin28, Rex1, Oct4, Sox2, and Nanog. In addition, injection of epithelial- or fibroblast-derived reprogrammed cells into nonobese diabetic (NOD/NOD) mice resulted in the formation of teratomas containing differentiated derivatives of the three germ layers, including cartilage, membranous ossification, stratified squamous epithelial tissue, hair follicles, neural pinwheels, and different types of glandular tissue. Finally, mammary epithelium-derived iPSCs could be induced to differentiate back to a mammary phenotype characterized by epithelial cells expressing cytokeratin 14 (CK14), CK18, and smooth muscle actin (SMA) as a result of treatment with 10 nM progesterone. This study reports for the first time the generation of iPSCs from bovine epithelial cells and demonstrates the potential of using iPSCs technology for generating bovine mammary tissue in vitro.

Bovine mammary epithelial cells retain stem-like phenotype in long-term cultures.

The detection and characterization of bovine mammary stem cells may give a better understanding of the cyclic characteristic of mammary gland development. In turn, this could potentially offer techniques to manipulate lactation yield and for regenerative medicine. We previously demonstrated that adult stem cells reside in the bovine mammary gland and possess an intrinsic regenerative potential. In vitro maintenance and expansion of this primitive population is a challenging task that could make easier the study of adult mammary stem cells. The aim of this study is to investigate this possibility. Different subpopulations of mammary epithelial cells emerge when they are cultured in two defined culture conditions. Specific cell differentiation markers as cytokeratin 18 (CK18) and cytokeratin 14 (CK14) were expressed with significant differences according to culture conditions. Vimentin, a well-known fibroblast marker was observed to increase significantly (P < 0.5) only after day 20. In both conditions, after prolonged culture (25 days) a subset of cells still retained regenerative capabilities. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice as shown by the expression of cytokeratin 14 (CK14), cytokeratin 18 (CK18), p63 (a mammary basal cell layer marker) and Epithelial Cell Adhesion Molecule (EpCAM). We also were able to observe the presence of milk proteins signal in these regenerated structures, which is a specific marker of functional mammary alveoli. Progenitor content was also analyzed in vitro through Colony-Forming Cell (CFC) assays with no substantial differences among culture conditions and time points. These results demonstrate that long-term culture of a multipotent cell subpopulation with intrinsic regenerative potential is possible.

Neuropeptide TLQP-21, a VGF internal fragment, modulates hormonal gene expression and secretion in GH3 cell line.

In the present study we demonstrated that TLQP-21, a biologically active peptide derived from the processing of the larger pro-VGF granin, plays a role in mammotrophic cell differentiation. We used an established in vitro model, the GH3 cell line, which upon treatment with epidermal growth factor develops a mammotrophic phenotype consisting of induction of prolactin expression and secretion, and inhibition of growth hormone. Here we determined for the first time that during mammotrophic differentiation, epidermal growth factor also induces Vgf gene expression and increases VGF protein precursor processing and peptide secretion. After this initial observation we set out to determine the specific role of the VGF encoded TLQP-21 peptide on this model. TLQP-21 induced a trophic effect on GH3 cells and increased prolactin expression and its own gene transcription without affecting growth hormone expression. TLQP-21 was also able to induce a significant rise of cytoplasmic calcium, as measured by Fura2AM, due to the release from a thapsigargin-sensitive store. TLQP-21-dependent rise in cytoplasmic calcium was, at least in part, dependent on the activation of phospholipase followed by phosphorylation of PKC and ERK. Taken together, the present results demonstrate that TLQP-21 contributes to differentiation of the GH3 cell line toward a mammotrophic phenotype and suggest that it may exert a neuroendocrine role in vivo on lactotroph cells in the pituitary gland.

Met receptor acts uniquely for survival and morphogenesis of EGFR-dependent normal mammary epithelial and cancer cells.

Mammary gland development and breast cancer growth require multiple factors both of endocrine and paracrine origin. We analyzed the roles of Epidermal Growth Factor Receptor (EGFR) and Hepatocyte Growth Factor Receptor (Met) in mammary epithelial cells and mammary tumor cells derived from a mutated-ErbB2 transgenic mice. By using highly specific tyrosine kinase inhibitors we found that MCF-10A and NMuMG mammary epithelial cell lines are totally dependent on EGFR activation for their growth and survival. Proliferation and 3D-morphogenesis assays showed that HGF had no role in maintaining mammary cell viability, but was the only cytokine able to rescue EGFR-inhibited mammary cells. Insulin-Like Growth Factor-I (IGF-I), basic-Fibroblast Growth Factor (b-FGF) and Neuregulin, which are well known mammary morphogenic factors, did not rescue proliferation or morphogenesis in these cell lines, following EGFR inhibition. Similarly, ErbB2-driven tumor cells are EGFR-dependent and also display HGF-mediated rescue. Western-blot analysis of the signaling pathways involved in rescue after EGFR inhibition indicated that concomitant ERK1/2 and AKT activation was exclusively driven by Met, but not by IGF-I or b-FGF. These results describe a unique role for EGFR and Met in mammary epithelial cells by showing that similar pathways can be used by tumorigenic cells to sustain growth and resist to EGFR-directed anti-tumorigenic drugs.

Identification of goat mammary stem/progenitor cells.

Goat mammary gland epithelial cells have been used to establish primary and permanent cell lines, but to date, no data have been available regarding mammary stem cells (MaSCs) in this species. The detection and characterization of goat MaSCs is an important task for a better understanding of the cyclic character of mammary gland development, which will also offer the potential for manipulation of lactation yield and persistency. The objective of the present study was to demonstrate that a subpopulation of goat MaSCs resides in the goat mammary gland. Mammary tissue from lactating Saanen goats (Capra hircus) was dissociated and processed to a single-cell suspension. Using an in vitro colony-forming assay, we demonstrated that distinct colony types, which expressed specific lineage markers, arose from unipotent progenitors. Using two different growth media, we showed that the frequencies of caprine clonogenic progenitors differed according to growth conditions. Goat epithelial cells were transplanted under the kidney capsule of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, where they formed organized, bilayered structures. Our results indicate the presence of goat MaSCs in the caprine mammary gland. To our knowledge, these data represent the first description of the tissue hierarchy of the goat mammary gland and demonstrate the regenerative potential of adult goat MaSCs.

Human milk protein production in xenografts of genetically engineered bovine mammary epithelial stem cells.

BACKGROUND: In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer. METHODS AND FINDINGS: We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human ß-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo. CONCLUSIONS: These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.

Epidermal growth factor and hepatocyte growth factor cooperate to enhance cell proliferation, scatter, and invasion in murine mammary epithelial cells.

The development of the mammary gland requires an integrated response to specific growth factors and steroid hormones. Hepatocyte growth factor (HGF) and its tyrosine kinase receptor, MET, are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor (EGFR) and its ligands have also been implicated in mammary gland growth and morphogenesis. Since both cytokines seem to exert a morphogenic program in this tissue, we have investigated the possible concerted action of EGF and HGF on the HC11 cell line, a widely used model of nontumorigenic mammary cells. Western blot analysis indicated that HC11 expressed MET and EGFR, and showed ERK1/2 and AKT activation following HGF or EGF treatment. Analysis by real-time PCR and western blot showed that after an EGF but not HGF or insulin-like growth factor-I treatment, HC11 mammary cells exhibited an increase in MET expression at both the mRNA and protein levels, which was dependent on the AKT pathway. Simultaneous treatment with HGF and EGF increased proliferation, scatter, and invasion as assessed by cell count, cell cycle, scatter, and transwell assays. AKT inhibition did not influence the cooperation on proliferation or invasion after HGF+EGF treatment, while ERK1/2 inhibition abolished MET/EGFR cooperation on proliferation. HGF+EGF treatment increased the duration of ERK1/2 and AKT activation compared to HGF or EGF alone. All these data indicate that a crosstalk between the EGF and HGF pathways in mammary epithelial cells may modulate the development of the mammary gland.

The gastrointestinal hormone ghrelin modulates inhibitory neurotransmission in deep laminae of mouse spinal cord dorsal horn.

Ghrelin is mainly described for its effects on feeding behavior and metabolism. However, central nervous system distribution of its receptor [type 1a GH secretagogue receptor (GHSR)] and modulation of neurotransmission in the hypothalamus suggest broader effects than originally predicted. Systemically administrated ghrelin inhibits inflammatory pain after behavioral observations. Therefore, we investigated the expression and function of type 1a GHSR in mouse spinal cord by molecular biology, biochemistry, histology, and electrophysiology. The mRNA and protein were detected in tissue extracts by RT-PCR and Western blotting. In situ, receptor mRNA and immunoreactivity were localized to cell bodies within the medial aspect of the deep dorsal horn. Patch clamp recordings on laminae IV-VI demonstrated that bath-applied ghrelin (100 nm) induced a strong increase of spontaneous gamma-aminobutyric acid/glycine-mediated current frequency (463 +/- 93% of the control) and amplitude (150 +/- 16% of the control) in about 60% of recorded neurons. Specificity of type 1a GHSR activation was confirmed by the lack of effect of the deacylated form of ghrelin (des-acyl-ghrelin) and after preincubation with the specific receptor antagonist [d-Lys(3)]GHRP-6. In the presence of tetrodotoxin, the effect of the peptide was strongly reduced, mainly indicating an action potential-dependent mechanism. The functional link between ghrelin and pain was confirmed by inhibition in vitro of the c-fos response to capsaicin activation of nociceptive fibers, after quantification of Fos-immunoreactive nuclei in laminae IV-VI. Our results are the first demonstration of the presence of functional type 1a GHSRs in the spinal cord and indicate that ghrelin may exert antinociceptive effects by directly increasing inhibitory neurotransmission in a subset of deep dorsal horn neurons.

Curcuminoid-phospholipid complex induces apoptosis in mammary epithelial cells by STAT-3 signaling.

Curcumin (from the rhizome of Curcuma longa) is well documented for its medicinal properties in Indian and Chinese systems of medicine where it is widely used for the treatment of several diseases. Epidemiological observations are suggestive that curcumin consumption may reduce the risk of some form of cancers and provide other protective biological effects in humans. These biological properties have been attributed to curcuminoids that have been widely studied for their anti-inflammatory, anti-angiogenic, antioxidant, wound healing and anti-cancer effects. In this study we have investigated on the effect of a curcumin phospholipid complex on mammary epithelial cell viability. HC11 and BME-UV cell lines, validated models to study biology of normal, not tumoral, mammary epithelial cells, were used to analyse these effects. We report that curcumin acts on STAT-3 signal pathway to reduce cell viability and increase apoptosis evaluated by the the amount of activated caspase 3. Further it reduces MAPK and AKT activations. JSI-124, a STAT-3 inhibitor (100 nM) was able to block the negative effect of curcumin on cell viability and caspase 3 activation. Finally the negative effect of cucumin on cell viability has been impaired in STAT-3i HC11, where STAT-3 protein was greatly reduced by shRNA-interference. These results indicate that curcumin presents a potential adverse effect to normal mammary epithelial cells and that it has a specific effect on signal trasduction in mammary epithelium.

Leptin enhances STAT-3 phosphorylation in HC11 cell line: effect on cell differentiation and cell viability.

Leptin is produced in the mammary gland by the fat tissue or by the mammary epithelium. The aim of this study was to investigate the role of leptin on mammary epithelial cell differentiation and cell viability. This study was conducted using the mouse mammary epithelial cell line HC11. We show that leptin, synergizes with prolactin to increase beta-casein gene expression during mammary epithelial cell differentiation. This was correlated with increased phosphorylation of the signal transducer and activator of transcription 3 (STAT-3). Inactivating the function of STAT-3 by expression of a short hairpin RNA demonstrated that the effect of leptin on beta-casein expression is mediated by STAT-3. Secondly, cells in which STAT-3 had been inactivated showed increased cell viability compared to controls and were resistant to the negative effect mediated by leptin. Further, leptin triggers apoptosis in mammary epithelial cells cultivated in non-differentiating conditions. Taken together, these results suggest that leptin, by activating STAT-3, may act as a paracrine factor modulating mammary epithelial cell function.

Leptin stimulates growth hormone secretion via a direct pituitary effect combined with a decreased somatostatin tone in a median eminence-pituitary perifusion study.

The aim of this study was to examine the effect of recombinant human leptin on growth hormone (GH) secretion in perifused anterior pituitary slices from adult pigs. Anterior pituitary slices from sows were perifused and treated with recombinant human leptin (10 nM) and GH-releasing hormone (GHRH; 1 nM). In some experiments, pituitary slices were coincubated with stalk median eminence (SME). In a subset of the coincubation experiments, immunoneutralization of endogenous GHRH and somatostatin (SRIH) release was performed with antisera to GHRH and SRIH. Leptin increased GH secretion in pituitary slices alone (up to 100% vs. control at 40 min) as well as in pituitary slices coincubated with SME (up to 122% vs. control at 40 min). A significant difference was observed in GH secretion from pituitary slices when the tissue was coincubated with leptin and GHRH at a low concentration (0.1 nM), but not when GHRH was used at 1 and 10 nM. Furthermore, anti-SRIH antiserum increased GH release from pituitary slices in coincubation experiments with SME. Finally, SRIH secretion was significantly reduced by leptin (down by 35% vs. control from 0 to 30 min of treatment) in cultured SME. These data show that leptin is effective in stimulating GH secretion by acting at two different levels: (1) it stimulates GH secretion directly from pituitary slices, and (2) it reduces SRIH tone from the median eminence and, indirectly, increases GH secretion from the pituitary. These results support the hypothesis that leptin may be an interesting hormonal mediator of growth and related metabolic effects by acting directly on the hypothalamic-pituitary axis.