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Pancreatic polypeptide (PP) is a major agonist for neuropeptide Y4 receptors (NPY4R). While NPY4R has been identified in various tissues, the cells on which it is expressed and its function in those cells has not been clearly delineated. Here we report that NPY4R is present in all somatostatin‐containing cells of tissues that we tested, including pancreatic islets, duodenum, hippocampus, and hypothalamus. Its agonism by PP decreases somatostatin secretion from human islets. Mouse embryonic hippocampal (mHippo E18) cells expressed NPY4Rs and their activation by PP consistently decreased somatostatin secretion. Furthermore, central injection of PP in mice induced c‐Fos immunoreactivity in somatostatin‐containing cells in the hippocampus compared with PBS‐injected mice. In sum, our results identify PP as a pivotal modulator of somatostatin secretion.NPY4Rs were expressed on somatostatin‐containing cells of tissues. PP inhibited somatostatin secretion from human islets and cells. Central injection of PP activated somatostatin‐containing cells in the hippocampus. Our results identify PP as a pivotal modulator of somatostatin secretion. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus (T2DM) results from insulin resistance and β-cell dysfunction, in the setting of hyperglucagonemia. Glucagon is a 29 amino acid peptide hormone, which is secreted from pancreatic α cells: excessively high circulating levels of glucagon lead to excessive hepatic glucose output. We investigated if α-cell numbers increase in T2DM and what factor (s) regulate α-cell turnover. Leprdb/Leprdb (db/db) mice were used as a T2DM model and αTC1 cells were used to study potential α-cell trophic factors. Here, we demonstrate that in db/db mice α-cell number and plasma glucagon levels increased as diabetes progressed. Insulin treatment (EC50 = 2 nM) of α cells significantly increased α-cell proliferation in a concentration-dependent manner compared to non-insulin-treated α cells. Insulin up-regulated α-cell proliferation through the IR/IRS2/AKT/mTOR signaling pathway, and increased insulin-mediated proliferation was prevented by pretreatment with rapamycin, a specific mTOR inhibitor. GcgR antagonism resulted in reduced rates of cell proliferation in αTC1 cells. In addition, blockade of GcgRs in db/db mice improved glucose homeostasis, lessened α-cell proliferation, and increased intraislet insulin content in b cells in db/db mice. These studies illustrate that pancreatic α-cell proliferation increases as diabetes develops, resulting in elevated plasma glucagon levels, and both insulin and glucagon are trophic factors to α-cells. Our current findings suggest that new therapeutic strategies for the treatment of T2DM may include targeting α cells and glucagon. [ABSTRACT FROM AUTHOR]

Heparan sulfate proteoglycans (HSPGs) are important modulators for optimizing signal transduction of many pathways, including the Wnt pathways. We demonstrate that HSPG glycosaminoglycan levels increased with increasing metastatic potential of melanoma cells. Previous studies have demonstrated that Wnt5A increases the invasiveness of melanoma cells. We further demonstrate that HSPGs potentiate Wnt5A signaling, since enzymatic removal of the HSPG backbone resulted in a decrease in cellular Wnt5A levels, an increase in secreted Wnt5A in cell media, a decrease in downstream signaling, and ultimately, a decrease in invasiveness. Specifically, syndecan 1 and syndecan 4 expression correlated to Wnt5A expression and melanoma malignancy. Knockdown of syndecan 1 or 4 caused decreases in cell invasion, which could be restored by treating the cells with recombinant Wnt5A. These data indicate that syndecan 1 and 4 correlate to increased metastatic potential in melanoma patients and are an important component of the Wnt5A autocrine signaling loop, the activation of which leads to increased metastasis of melanoma. [ABSTRACT FROM AUTHOR]

We have previously shown that Wnt5A and ROR2, an orphan tyrosine kinase receptor, interact to mediate melanoma cell motility. In other cell types, this can occur through the interaction of ROR2 with the cytoskeletal protein filamin A. Here, we found that filamin A protein levels correlated with Wnt5A levels in melanoma cells. Small interfering RNA (siRNA) knockdown of WNT5A decreased filamin A expression. Knockdown of filamin A also corresponded to a decrease in melanoma cell motility. In metastatic cells, filamin A expression was predominant in the cytoplasm, which western analysis indicated was due to the cleavage of filamin A in these cells. Treatment of nonmetastatic melanoma cells with recombinant Wnt5A increased filamin A cleavage, and this could be prevented by the knockdown of ROR2 expression. Further, BAPTA-AM chelation of intracellular calcium also inhibited filamin A cleavage, leading to the hypothesis that Wnt5A/ROR2 signaling could cleave filamin A through activation of calcium-activated proteases, such as calpains. Indeed, WNT5A knockdown decreased calpain 1 expression, and by inhibiting calpain 1 either pharmacologically or using siRNA, it decreased cell motility. Our results indicate that Wnt5A activates calpain-1, leading to the cleavage of filamin A, which results in a remodeling of the cytoskeleton and an increase in melanoma cell motility.Journal of Investigative Dermatology (2009) 129, 1782–1789; doi:10.1038/jid.2008.433; published online 29 January 2009 [ABSTRACT FROM AUTHOR]

This article focuses on a clinical study on patients with fibrodysplasia ossificans progressiva (FOP). The article contains an introduction, materials and methods, results, conclusions, as well as charts and graphs. The study extends knowledge of dysregulated bone morphogenetic protein signaling and enhanced osteogenic differentiation of connective tissue progenitor cells.

We and others have reported that taste cells in taste buds express many peptides in common with cells in the gut and islets of Langerhans in the pancreas. Islets and taste bud cells express the hormones glucagon and ghrelin, the same ATP-sensitive potassium channel responsible for depolarizing the insulin-secreting β cell during glucose-induced insulin secretion, as well as the propeptide-processing enzymes PC1/3 and PC2. Given the common expression of functionally specific proteins in taste buds and islets, it is surprising that no one has investigated whether insulin is synthesized in taste bud cells. Using immunofluorescence, we demonstrated the presence of insulin in mouse, rat, and human taste bud cells. By detecting the postprocessing insulin molecule C-peptide and green fluorescence protein (GFP) in taste cells of both insulin 1-GFP and insulin 2-GFP mice and the presence of the mouse insulin transcript by in situ hybridization, we further proved that insulin is synthesized in individual taste buds and not taken up from the parenchyma. In addition to our cytology data, we measured the level of insulin transcript by quantitative RT-PCR in the anterior and posterior lingual epithelia. These analyses showed that insulin is translated in the circumvallate and foliate papillae in the posterior, but only insulin transcript was detected in the anterior fungiform papillae of the rodent tongue. Thus, some taste cells are insulin-synthesizing cells generated from a continually replenished source of precursor cells in the adult mammalian lingual epithelium.

The microscopic image analysis of pancreatic Islet of Langerhans morphology is crucial for the investigation of diabetes and metabolic diseases. Besides the general size of the islet, the percentage and relative position of glucagon-containing alpha-, and insulin-containing beta-cells is also important for pathophysiological analyses, especially in rodents. Hence, the ability to identify, quantify and spatially locate peripheral, and "involuted" alpha-cells in the islet core is an important analytical goal. There is a dearth of software available for the automated and sophisticated positional quantification of multiple cell types in the islet core. Manual analytical methods for these analyses, while relatively accurate, can suffer from a slow throughput rate as well as user-based biases. Here we describe a newly developed pancreatic islet analytical software program, Pancreas++, which facilitates the fully automated, non-biased, and highly reproducible investigation of islet area and alpha- and beta-cell quantity as well as position within the islet for either single or large batches of fluorescent images. We demonstrate the utility and accuracy of Pancreas++ by comparing its performance to other pancreatic islet size and cell type (alpha, beta) quantification methods. Our Pancreas++ analysis was significantly faster than other methods, while still retaining low error rates and a high degree of result correlation with the manually generated reference standard.

Type 2 diabetes mellitus (T2DM) results from insulin resistance and β-cell dysfunction, in the setting of hyperglucagonemia. Glucagon is a 29 amino acid peptide hormone, which is secreted from pancreatic α cells: excessively high circulating levels of glucagon lead to excessive hepatic glucose output. We investigated if α-cell numbers increase in T2DM and what factor (s) regulate α-cell turnover. Lepr(db)/Lepr(db) (db/db) mice were used as a T2DM model and αTC1 cells were used to study potential α-cell trophic factors. Here, we demonstrate that in db/db mice α-cell number and plasma glucagon levels increased as diabetes progressed. Insulin treatment (EC50 = 2 nM) of α cells significantly increased α-cell proliferation in a concentration-dependent manner compared to non-insulin-treated α cells. Insulin up-regulated α-cell proliferation through the IR/IRS2/AKT/mTOR signaling pathway, and increased insulin-mediated proliferation was prevented by pretreatment with rapamycin, a specific mTOR inhibitor. GcgR antagonism resulted in reduced rates of cell proliferation in αTC1 cells. In addition, blockade of GcgRs in db/db mice improved glucose homeostasis, lessened α-cell proliferation, and increased intra-islet insulin content in β cells in db/db mice. These studies illustrate that pancreatic α-cell proliferation increases as diabetes develops, resulting in elevated plasma glucagon levels, and both insulin and glucagon are trophic factors to α-cells. Our current findings suggest that new therapeutic strategies for the treatment of T2DM may include targeting α cells and glucagon.

Heparan sulfate proteoglycans (HSPGs) are important modulators for optimizing signal transduction of many pathways, including the Wnt pathways. We demonstrate that HSPG glycosaminoglycan levels increased with increasing metastatic potential of melanoma cells. Previous studies have demonstrated that Wnt5A increases the invasiveness of melanoma cells. We further demonstrate that HSPGs potentiate Wnt5A signaling, since enzymatic removal of the HSPG backbone resulted in a decrease in cellular Wnt5A levels, an increase in secreted Wnt5A in cell media, a decrease in downstream signaling, and ultimately, a decrease in invasiveness. Specifically, syndecan 1 and syndecan 4 expression correlated to Wnt5A expression and melanoma malignancy. Knockdown of syndecan 1 or 4 caused decreases in cell invasion, which could be restored by treating the cells with recombinant Wnt5A. These data indicate that syndecan 1 and 4 correlate to increased metastatic potential in melanoma patients and are an important component of the Wnt5A autocrine signaling loop, the activation of which leads to increased metastasis of melanoma.

Cytoplasmic expression of claudin-1 in metastatic melanoma cells correlates to increased migration, and increased secretion of MMP-2 in a PKC dependent manner, whereas claudin-1 nuclear expression is found in benign nevi. Melanoma cells were transfected with a vector expressing CLDN-1 fused to a nuclear localization signal (NLS). Despite significant nuclear localization of claudin-1, there was still transport of claudin-1 to the cytoplasm. Phorbol ester treatment of cells transfected with NLS-claudin-1 resulted in an exclusion of claudin-1 from the nucleus, despite the NLS. To ascertain whether PKC or PKA were involved in this translocation, we mutated the putative phosphorylation sites within the protein. We found that mutating the PKC phosphorylation sites to mimic a non-phosphorylated state did not cause a shift of claudin-1 to the nucleus of the cells, but mutating the PKA sites did. Mutations of either site to mimic constitutive phosphorylation resulted in cytoplasmic claudin-1 expression. Stable claudin-1 transfectants containing non-phosphorylatable PKA sites exhibited decreased motility. These data imply that subcellular localization of claudin-1 can be controlled by phosphorylation, dicating effects on metastatic capacity.