Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation,cytokine production and increased cell proliferation

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.     

Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.     

Adding value to cocoa (Theobroma cacao L.) germplasm information with domestication history and admixture mapping

A sound understanding of crop history can provide the basis for deriving novel genetic information through admixture mapping. We confirmed this, by using characterization data from an international collection of cocoa, collected 25 years ago, and from a contemporary plantation. We focus on the trees derived from three centuries of admixture between Meso-American Criollo and South American Forastero genomes. In both cacao sets of individuals, linkage disequilibrium extended over long genetic distances along chromosome regions, as expected in populations derived from recent admixture. Based on loose genome scans, genomic regions involved in useful traits were identified. Fifteen genomic regions involved in seed and fruit weight variation were highlighted. They correspond to ten previously identified QTLs and five novel ones. Admixture mapping can help to add value to genetic resources and thus, help to encourage investment in their conservation. Maria Marcano and Tatiana Pugh contributed equally to this work.     

Photoreceptor disc morphogenesis: The classical evagination model prevails

Vision begins in photoreceptor outer segments with light captured by opsins in continually synthesized disc membranes. The process by which rod photoreceptor discs are formed has been controversial. In this issue, Ding et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201508093) show conclusively that rod discs are formed by plasma membrane evagination.The vertebrate retina contains two types of photoreceptors, rod cells and cone cells, whose outer segments initiate phototransduction under night and daytime conditions, respectively. The outer segments of these cells lack ER, Golgi, and mitochondria and are filled with hundreds to a few thousand flattened membrane organelles, called photoreceptor discs, which are loaded with the molecular machinery of phototransduction. The structural organization of outer segments differs between rods and cones. Although cone outer segments contain “open” discs that are infoldings of the plasma membrane, rod outer segments possess “closed” discs that are completely separated from the plasma membrane.In 1967, in a paper that has been cited nearly 800 times, Richard Young reported the seminal finding that rod and cone outer segments are continually renewed (Young, 1967). Young’s classic experiment was elegantly simple: he injected [³H]methionine into a rat, mouse, and frog and performed autoradiograms of the excised retina on various days after the injection. He observed that the radiolabeled band moved along the outer segment as time after injection increased and ultimately disappeared at the apex of the cell (Fig. 1, republished from Young, 1967). (As Young was at the University of California, Los Angeles, this result was given the memorable moniker of “the UCLA marching band.”) Young’s seminal insight that outer segments are continually rebuilt posed a problem that has challenged photoreceptor cell biologists ever since: How are rod disc membranes initially formed? In this issue, Ding et al. present a compelling resolution to this question. Specifically, their work differentiates between currently competing models to determine whether rod discs are formed by evagination of plasma membrane at the base of the outer segment or by fusion of intracellular vesicles transported to the outer segment.Open in a separate windowFigure 1.Photoreceptor outer segments are continually renewed. Rats were injected with [³H]methionine, and radioautographs of photoreceptor cells were performed on various days after the injection. As time after injection increases (images 2–7), the radiolabel components are displaced from the inner segment along the outer segment toward the apex of the cell, revealing that the outer segment is continually renewed (figure republished from Young, 1967).The classic hypothesis of disc morphogenesis is that they are formed by evagination of basal outer segment plasma membrane (Steinberg et al., 1980). This hypothesis is based largely on evidence that one surface of the most basal discs of rods is open to the extracellular space, as shown by EM (Carter-Dawson and LaVail, 1979; Steinberg et al., 1980), with lipophilic dye fluorescence (Laties et al., 1976), and by analysis of membrane capacitance (Rüppel and Hagins, 1973). In addition, rods and cones might be expected to share a common machinery of disc formation. Because most cone discs are well established by EM, lipophilic dye imaging, and electrophysiology to be continuous with the plasma membrane, nascent rod discs would seem likely to also be part of the plasma membrane. Thus, according to the classic hypothesis, new discs in both photoreceptor types are formed from outgrowths (evaginations) of the plasma membrane at the outer segment base. In both photoreceptor types, discs would begin life with one face exposed to the extracellular space, but at some point after formation, rod discs would pinch off from the outer segment plasma membrane to become self-contained and fully separated from the plasma membrane, whereas cones discs remain open. On the contrary, the vesicle fusion hypothesis postulates that nascent discs are born completely internalized in rods. Photoreceptor outer segments are now understood to be the plus end of a modified primary cilium (Bloodgood, 2009) and are joined to their inner segments by a narrow ciliary tube called the connecting cilium. This realization, combined with evidence of vesicles in the connecting cilium seen in electron micrographs, has been taken to support the model that vesicles are actively transported through the connecting cilium and generate nascent discs by membrane fusion at the base of the outer segment (Chuang et al., 2007, 2015).Ding et al. (2015) addressed these competing hypotheses with two distinct approaches. First, they treated sections of retinas of mice perfused with a membrane-staining mixture of tannic acid and uranyl acetate and performed EM. Because tannic acid penetrates intact membranes poorly, this treatment distinguishes between membranes exposed to the extracellular space and intracellular membrane structures. The researchers found that, like the plasma membrane, a small number of basal rod discs were intensely stained by tannic acid, whereas the staining of fully internalized discs was weak, confirming that newly formed rod discs are open to the extracellular space. Consistently and strikingly, EM analysis also revealed a single basal disc face (approximately five to seven discs north of the most basal disc) that is contiguous with the plasma membrane. Second, Ding et al. (2015) performed EM with an immunogold-tagged antibody raised against an intracellular epitope of peripherin, a protein that plays an essential role in disc stacking (Arikawa et al., 1992; Goldberg, 2006). Quantification of gold particle counts showed that the peripherin antibody closely associated intracellularly with the edges of fully internalized discs but was negligibly associated with the surface of nascent discs identified as facing the extracellular space, suggesting that peripherin redistributes along the rod disc edge upon its separation from the plasma membrane and enclosure into the outer segment. Finally, Ding et al. (2015) performed experiments using the fixation techniques reported by other investigators and demonstrated that artifacts of tissue fixation were responsible for the erroneous interpretation that basal discs are fully internalized and for the evidence supporting the vesicular fusion hypothesis.Other tools, such as superresolution microscopy of living rods stained with lipophilic dyes or fluorescent antibodies raised against epitopes on the extracellular face of the rod plasma membrane, could further test aspects of the evagination model of disc formation. Nonetheless, the work of Ding et al. (2015) unequivocally shows that basal rod discs are open to the extracellular space and provides a new system and conceptual framework for the investigation of the fundamental biological mechanism of plasma membrane evagination. As outer segment discs exhibit a specialized composition of lipids and phototransduction proteins, further work will also focus on how disc lipids and proteins are transported from the inner segment to the basal outer segment. The current hypotheses about such transport include (a) vesicular transport through the connecting cilium followed by fusion with the outer segment plasma membrane; (b) directed transport through the connecting cilium membrane after vesicle fusion at the base of the connecting cilium in the inner segment; and (c) exocytotic release from the inner segment followed by endocytotic capture in the outer segment. As the molecular details of disc formation and specialization become clearer, Richard Young’s “UCLA marching band” (Young, 1967) will continue to have a broad conceptual impact on the cell biology of photoreceptor development and cilia.     

The atmospheric chemistry of trace gases and particulate matter emitted by different land uses in Borneo

We report measurements of atmospheric composition over a tropical rainforest and over a nearby oil palm plantation in Sabah, Borneo. The primary vegetation in each of the two landscapes emits very different amounts and kinds of volatile organic compounds (VOCs), resulting in distinctive VOC fingerprints in the atmospheric boundary layer for both landscapes. VOCs over the Borneo rainforest are dominated by isoprene and its oxidation products, with a significant additional contribution from monoterpenes. Rather than consuming the main atmospheric oxidant, OH, these high concentrations of VOCs appear to maintain OH, as has been observed previously over Amazonia. The boundary-layer characteristics and mixing ratios of VOCs observed over the Borneo rainforest are different to those measured previously over Amazonia. Compared with the Bornean rainforest, air over the oil palm plantation contains much more isoprene, monoterpenes are relatively less important, and the flower scent, estragole, is prominent. Concentrations of nitrogen oxides are greater above the agro-industrial oil palm landscape than over the rainforest, and this leads to changes in some secondary pollutant mixing ratios (but not, currently, differences in ozone). Secondary organic aerosol over both landscapes shows a significant contribution from isoprene. Primary biological aerosol dominates the super-micrometre aerosol over the rainforest and is likely to be sensitive to land-use change, since the fungal source of the bioaerosol is closely linked to above-ground biodiversity.     

A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival.

A cadherin family member, prCAD, was identified in retina cDNA by subtractive hybridization and high throughput sequencing. prCAD is expressed only in retinal photoreceptors, and the prCAD protein is localized to the base of the outer segment of both rods and cones. In prCAD(-/-) mice, outer segments are disorganized and fragmented, and there is progressive death of photoreceptor cells. prCAD is unlikely to be involved in protein trafficking between inner and outer segments, since phototransduction proteins appear to be correctly localized and the light responses of both rods and cones are only modestly compromised in prCAD(-/-) mice. These experiments imply a highly specialized cell biological function for prCAD and suggest that localized adhesion activity is essential for outer segment integrity.     

Probing luminal negative charge in the type 3 ryanodine receptor

In this study, we have investigated block of potassium (K(+)) current by neomycin, a large polycation, from the luminal face of the type 3 ryanodine receptor (RyR3). Previous studies have shown that neomycin is an open channel blocker of RyR2 that interacts with negatively charged residues in the mouth of the conduction pathway to partially occlude it. In the current study, we have used neomycin as a probe to investigate proposed negatively charged regions in the luminal pore mouth of RyR3. Luminal neomycin induces concentration- and voltage-dependent partial block to a subconductance state in RyR3. Blocking parameters calculated in this study show that neomycin has a higher affinity for RyR3 than RyR2, but block may occur at the same site within the pore mouth. The change in affinity may be due to altered negative charge density at the site of interaction.