RESUMO
PURPOSE: To investigate the collagen and elastin architecture at the junction of the human cornea and trabecular meshwork (TM). METHODS: The cornea, TM, and ciliary body (CB) tendons of unfixed human corneal buttons were imaged with an inverted 2-photon excited fluorescence microscope (FluoView FV-1000; Olympus, Central Valley, PA). The laser (Ti:sapphire) was tuned to 850 nm for 2-photon excitation. Backscatter signals of second harmonic generation and autofluorescence were collected through a 425/30-nm emission filter and a 525/45-nm emission filter, respectively. The second harmonic generation signal corresponds to collagen fibers, and the autofluorescence signal corresponds to elastin-containing tissue. Tissue structure representations were obtained through software-generated reconstructions of consecutive and overlapping (z-stack) images through a relevant sample depth. RESULTS: Collagen-rich CB tendons insert into the cornea between Descemet membrane (DM) and posterior stroma along with elastin fibers originating from the TM. The CB tendons directly abut DM, and their insertion narrows as they course centrally in the cornea, giving a wedge appearance to these parallel collagen fibers. Approximately 260 µm centrally from the edge of DM, the CB tendons fan out and merge with pre-DM collagen. As the CB tendons enter the cornea, they form a dense collagenous comb-like structure orthogonal to the edge of DM and supported by a delicate elastin network of interwoven fibers originating from the TM. CONCLUSIONS: Two-photon excited fluorescence microscopy has improved our understanding of the peripheral corneal architecture. CB tendon insertions in this region may contribute to the radial tears encountered when preparing DM endothelial keratoplasty grafts.
Assuntos
Colágeno/metabolismo , Córnea/diagnóstico por imagem , Elastina/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica , Malha Trabecular/diagnóstico por imagem , Córnea/metabolismo , Bancos de Olhos , Humanos , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Transmissão , Tendões/metabolismo , Doadores de Tecidos , Malha Trabecular/metabolismoRESUMO
Hepatic glucose production (HGP) is required to maintain normoglycemia during fasting. Glucagon is the primary hormone responsible for increasing HGP; however, there are many additional hormone and metabolic factors that influence glucagon sensitivity. In this study we report that the bioactive lipid lysophosphatidic acid (LPA) regulates hepatocyte glucose production by antagonizing glucagon-induced expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Treatment of primary hepatocytes with exogenous LPA blunted glucagon-induced PEPCK expression and glucose production. Similarly, knockout mice lacking the LPA-degrading enzyme phospholipid phosphate phosphatase type 1 (PLPP1) had a 2-fold increase in endogenous LPA levels, reduced PEPCK levels during fasting, and decreased hepatic gluconeogenesis in response to a pyruvate challenge. Mechanistically, LPA antagonized glucagon-mediated inhibition of STAT3, a transcriptional repressor of PEPCK. Importantly, LPA did not blunt glucagon-stimulated glucose production or PEPCK expression in hepatocytes lacking STAT3. These data identify a novel role for PLPP1 activity and hepatocyte LPA levels in glucagon sensitivity via a mechanism involving STAT3.
Assuntos
Glucagon/metabolismo , Gluconeogênese , Hepatócitos/metabolismo , Lisofosfolipídeos/metabolismo , Fosfatidato Fosfatase/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Glucagon/administração & dosagem , Glucose/biossíntese , Camundongos , Camundongos Knockout , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Fator de Transcrição STAT3/genéticaRESUMO
PURPOSE: To investigate the architecture and distribution of collagen and elastin in human limbal conjunctiva, Tenon's capsule, and sclera. METHODS: The limbal conjunctiva, Tenon's capsule, and sclera of human donor corneal buttons were imaged with an inverted two-photon excited fluorescence microscope. No fixation process was necessary. The laser (Ti:sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of second harmonic generation (SHG) and autofluorescence (AF) were collected through a 425/30-nm and a 525/45-nm emission filter, respectively. Multiple, consecutive, and overlapping (z-stack) images were acquired. Collagen signals were collected with SHG, whereas elastin signals were collected with AF. RESULTS: The size and density of collagen bundles varied widely depending on depth: increasing from conjunctiva to sclera. In superficial image planes, collagen bundles were <10 µm in width, in a loose, disorganized arrangement. In deeper image planes (episclera and superficial sclera), collagen bundles were thicker (near 100 µm in width) and densely packed. Comparatively, elastin fibers were thinner and sparse. The orientation of elastin fibers was independent of collagen fibers in superficial layers; but in deep sclera, elastin fibers wove through collagen interbundle gaps. At the limbus, both collagen and elastin fibers were relatively compact and were distributed perpendicular to the limbal annulus. CONCLUSIONS: Two-photon excited fluorescence microscopy has enabled us to understand in greater detail the collagen and elastin architecture of the human limbal conjunctiva, Tenon's capsule, and sclera.
Assuntos
Colágeno/metabolismo , Túnica Conjuntiva/citologia , Elastina/metabolismo , Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Esclera/citologia , Cápsula de Tenon/citologia , Adulto , Idoso , Cadáver , Túnica Conjuntiva/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Esclera/metabolismo , Cápsula de Tenon/metabolismoRESUMO
Lipid deposition in the liver is associated with metabolic disorders including fatty liver disease, type II diabetes, and hepatocellular cancer. The enzymes acetyl-CoA carboxylase 1 (ACC1) and ACC2 are powerful regulators of hepatic fat storage; therefore, their inhibition is expected to prevent the development of fatty liver. In this study we generated liver-specific ACC1 and ACC2 double knockout (LDKO) mice to determine how the loss of ACC activity affects liver fat metabolism and whole-body physiology. Characterization of LDKO mice revealed unexpected phenotypes of increased hepatic triglyceride and decreased fat oxidation. We also observed that chronic ACC inhibition led to hyper-acetylation of proteins in the extra-mitochondrial space. In sum, these data reveal the existence of a compensatory pathway that protects hepatic fat stores when ACC enzymes are inhibited. Furthermore, we identified an important role for ACC enzymes in the regulation of protein acetylation in the extra-mitochondrial space.