γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus.

BACKGROUND: The association between diabetes mellitus (DM) and the increased risk and progression of cholangiocarcinoma (CCA) has been reported with unclear underlying mechanisms. Previous studies showed that γ-aminobutyric acid (GABA) B2 receptor (GABBR2) was upregulated in CCA cells cultured in high glucose (HG) conditions. Roles of GABA receptors in CCA progression have also been studied, but their association with DM and hyperglycemia in CCA remains unclarified. AIM: To investigate the effects of hyperglycemia on GABBR2 expression and the potential use of GABBR2 as a CCA therapeutic target. METHODS: CCA cells, KKU-055 and KKU-213A, were cultured in Dulbecco Modified Eagle's Medium supplemented with 5.6 mmol/L (normal glucose, NG) or 25 mmol/L (HG) glucose and assigned as NG and HG cells, respectively. GABBR2 expression in NG and HG cells was investigated using real-time quantitative polymerase chain reaction and western blot. Expression and localization of GABBR2 in CCA cells were determined using immunocytofluorescence. GABBR2 expression in tumor tissues from CCA patients with and without DM was studied using immunohistochemistry, and the correlations of GABBR2 with the clinicopathological characteristics of patients were analyzed using univariate analysis. Effects of baclofen, a GABA-B receptor agonist, on CCA cell proliferation and clonogenicity were tested using the MTT and clonogenic assays. Phospho-kinases arrays were used to screen the affected signaling pathways after baclofen treatment, and the candidate signaling molecules were validated using the public transcriptomic data and western blot. RESULTS: GABBR2 expression in CCA cells was induced by HG in a dose- and time-dependent manner. CCA tissues from patients with DM and hyperglycemia also showed a significantly higher GABBR2 expression compared with tumor tissues from those with euglycemia (P < 0.01). High GABBR2 expression was significantly associated with a poorer non-papillary histological subtype but with smaller sizes of CCA tumors (P < 0.05). HG cells of both tested CCA cell lines were more sensitive to baclofen treatment. Baclofen significantly suppressed the proliferation and clonogenicity of CCA cells in both NG and HG conditions (P < 0.05). Phospho-kinase arrays suggested glycogen synthase kinase 3 (GSK3), ß-catenin, and the signal transducer and activator of transcription 3 (STAT3) as candidate signaling molecules under the regulation of GABBR2, which were verified in NG and HG cells of the individual CCA cell lines. Cyclin D1 and c-Myc, the common downstream targets of GSK3/ß-catenin and STAT3 involving cell proliferation, were accordingly downregulated after baclofen treatment. CONCLUSION: GABBR2 is upregulated by HG and holds a promising role as a therapeutic target for CCA regardless of the glucose condition.

High glucose enhances the aggressiveness of lung adenocarcinoma via activating epidermal growth factor receptor/signal transducer and activator of transcription 3 pathways.

Epidemiological studies revealed hyperglycemia as a poor prognostic factor for lung adenocarcinoma with unclear molecular mechanisms. The present study thus aimed to investigate the effects of high glucose on the progression of lung adenocarcinoma and its underlying mechanisms. Lung adenocarcinoma cell lines, A549 and RERF-LC-KJ, were cultured in 5.6 mM glucose (normal glucose; NG) or 25 mM glucose (high glucose; HG) resembling euglycemia and hyperglycemia. Cells were examined for proliferation by the MTT assay, and migration-invasion using Transwell. The expressions of signaling proteins in epidermal growth factor receptor (EGFR) pathways and their downstream targets were investigated using Western blots. The effects of diabetes mellitus (DM) and hyperglycemia on lung adenocarcinoma growth in vivo were studied in streptozotocin-induced diabetic BALB/cAJcl-Nu/Nu mice and their nondiabetic counterparts. High glucose significantly promoted proliferation, migration, and invasion of lung adenocarcinoma cells compared with those in normal glucose (P<.05). Western blot analyses showed the increased ratio of pEGFR/EGFR in cells cultured in high glucose and subsequently activated the signal transducer and activator of transcription 3 (STAT3). Epithelial-mesenchymal (EMT) markers were also altered in lung adenocarcinoma cells in high glucose conditions, corresponding with increased migration and invasion abilities. Erlotinib, an EGFR inhibitor, significantly reversed high glucose-induced aggressive phenotypes confirming high glucose-enhancing lung adenocarcinoma progression via the activation of EGFR. DM and hyperglycemia also promoted the growth of lung adenocarcinoma xenografts in vivo in which erlotinib significantly suppressed the growth of tumors (P<.05) suggesting EGFR inhibitor as an effective therapeutic agent for lung adenocarcinoma with DM.

Localization of Diacylglycerol Kinase ζ in the Kidney of Adult Rats: Its Dominant Expression in Collecting Tubules and its Selective Lack in Proximal Tubules / Localización de Diacilglicerol Quinasa ζ en el Riñón de Ratas Adultas: su Expresión Dominante en Túbulos Colectores y su Carencia Selectiva en Túbulos Proximales

SUMMARY: Diacylglycerol kinase (DGK) exerts balancing the intracellular level between two-second messengers, diacylglycerol and phosphatidic acid, by its phosphorylation activity. DGK ζ is often localized in cell nuclei, suggesting its involvement in the regulation of intranuclear activities, including mitosis and apoptosis. The present immunohistochemical study of rat kidneys first revealed no detection levels of DGK ζ -immunoreactivity in nuclei of most proximal tubule epithelia in contrast to its distinct occurrence in cell nuclei of collecting and distal tubules with the former more dominant. This finding suggests that DGK ζ is a key factor regulating vulnerability to acute kidney injury in various renal tubules: its low expression represents the high vulnerability of proximal tubule cells, and its distinct expression does the resistance of collecting and distal tubule cells. In addition, this isozyme was more or less localized in nuclei of cells forming glomeruli as well as in endothelial nuclei of peritubular capillaries and other intrarenal blood vessels, and epithelial nuclei of glomerular capsules (Bowman's capsules) and renal calyces, including intrarenal interstitial cells.

La diacilglicerol quinasa (DGK) ejerce el equilibrio del nivel intracelular entre dos segundos mensajeros, diacilglicerol y ácido fosfatídico, por su actividad de fosforilación. La DGK ζ a menudo se localiza en los núcleos celulares, lo que sugiere su participación en la regulación de las actividades intranucleares, incluidas la mitosis y la apoptosis. El presente estudio inmunohistoquímico en riñones de rata no reveló niveles de detección de inmunorreactividad de DGK ζ en los núcleos de la mayoría de los epitelios de los túbulos proximales, en contraste a la detección en los núcleos celulares de los túbulos colectores y distales, siendo el primero más dominante. Este hallazgo sugiere que DGK ζ es un factor clave que regula la vulnerabilidad a la lesión renal aguda en varios túbulos renales: su baja expresión representa la alta vulnerabilidad de las células del túbulo proximal, y su expresión distinta hace a la resistencia de las células del túbulo colector y distal. Además, esta isoenzima estaba más o menos localizada en los núcleos de las células que forman los glomérulos, así como en los núcleos endoteliales de los capilares peritubulares y otros vasos sanguíneos intrarrenales, y en los núcleos epiteliales de las cápsulas glomerulares (cápsulas de Bowman) y los cálices renales, incluidas las células intersticiales intrarrenales.

Hyperglycemia Alters O-GlcNAcylation Patterns of Hepatocytes in Mice Treated With Hepatoxic Carcinogen.

BACKGROUND/AIM: Diabetes mellitus (DM) is an established risk for hepatocellular carcinoma (HCC), with unclarified mechanisms. This study investigated the effects of hyperglycemia on O-GlcNacylation in hepatocytes and its associations with hepatocarcinogenesis. MATERIALS AND METHODS: Mouse and human HCC cell lines were used in an in vitro model of hyperglycemia. Western blotting was used to determine the effects of high glucose on O-GlcNacylation in HCC cells. Twenty 4-week-old C3H/HeNJcl mice were randomized into four groups: non-DM control, non-DM plus diethylnitrosamine (DEN), DM, and DM plus DEN. DM was induced using intraperitoneal injection of a single high dose of streptozotocin. DEN was used to induce HCC. All mice were euthanized at week 16 after DM induction, and the liver tissues were histologically examined using hematoxylin and eosin, and immunohistochemistry. RESULTS: High glucose increased O-GlcNacylated proteins in mouse and human HCC cell lines compared with those cultured at normal glucose concentration. Mice with hyperglycemia or DEN treatment had increased O-GlcNacylated proteins in hepatocytes. No gross tumors were evident at the end of the experiment but hepatic morbidity was observed. Mice with hyperglycemia and DEN treatment showed greater histological morbidity in their livers, i.e. increased nuclear size, hepatocellular swelling and sinusoidal dilatation, compared with mice in the DM group or treated with DEN alone. CONCLUSION: Hyperglycemia increased O-GlcNAcylation in both in vitro and animal models. Increased O-GlcNAcylated proteins may be associated with hepatic histological morbidities which then promote HCC development in carcinogen-induced tumorigenesis.

Localization of phosphatidylinositol phosphate 5 kinase γ, phospholipase ß3 and diacylglycerol kinase ζ in corneal epithelium in comparison with conjunctival epithelium of mice.

Based on the theory that the phosphoinositide (PI) signal is involved in the physiology of cornea and conjunctiva, we examined the localization in the mouse anterior ocular epithelia of immunoreactivities for phosphatidylinositol 4-phosphate 5-kinase (PIP5K), phospholipase C (PLC) and diacylglycerol kinase (DGK), enzymes that work sequentially in PI cycle. Immunoreactivity for PIP5Kγ in the corneal epithelium, including the limbus, was distinct in adults in contrast to faint or negligible immunoreactivity in the conjunctival epithelium in neonatal mice. This adult localization pattern was first recognized at the postnatal time of eyelid opening. Immunoreactivity for PLCß3 was rather equally distinct throughout the entire corneal and conjunctival epithelia in adults. DGKζ-immunoreactive nuclei were mainly localized in the basal half domain of the corneal epithelium but in both basal and apical domains of the conjunctival epithelium in adults. This nuclear immunoreactivity was at weak or negligible levels in the peripheral and limbus cornea and in a considerable portion of the bulbar conjunctival epithelium continuous with the limbus. The adult patterns for PLCß3 and DGKζ were already present at birth. The present findings suggest the following possibilities on the functional significance of the three enzyme molecules. PIP5Kγ is involved in cornea-specific functions such as bright-field vision, including corneal transparency, and in the stability of epithelial junctions, for which there seems to be a much higher requirement in the corneal epithelium than in the conjunctival epithelium. PLCß3 is involved from birth in as-yet undefined functions exerted ubiquitously from birth in both corneal and conjunctival epithelia. DGKζ is involved in regulation from birth of the transcription in epithelial cells, including apoptosis as well as regulation of mitosis of epithelial cells in both cornea and conjunctiva, with the transcription involvement more apparent in the conjunctiva, although it does not work in stem cells of the corneal limbus.

Mitochondrial Localization of CB1 in Progesterone-producing Cells of Ovarian Interstitial Glands of Adult Mice.

Localization of cannabinoid receptor type 1 (CB1) immunoreactivity on mitochondrial membranes, at least their outer membranes distinctly, was detected in progesterone-producing cells characterized by mitochondria having tubular cristae and aggregations of lipid droplets in ovarian interstitial glands in situ of adult mice. Both immunoreactive and immunonegative mitochondria were contained in one and the same cell. Considering that the synthesis of progesterone is processed in mitochondria, the mitochondrial localization of CB1 in the interstitial gland cells suggests the possibility that endocannabinoids modulate the synthetic process of progesterone in the cells through CB1.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α confined to the surface of lipid droplets and adjacent narrow cytoplasm in progesterone-producing cells of in situ ovaries of adult mice.

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) produced by phosphatidylinositol phosphate 5 kinase (PIP5K) plays not only as a precursor of second messengers in the phosphoinositide signal transduction, but also multiple roles influencing a variety of cellular activities. From this viewpoint, the present study attempted to localize PIP5Kα in the ovaries in situ of adult mice. PIP5Kα-immunoreactivity was confined to the surfaces of lipid droplets (LDs) and their adjacent cytoplasm in progesterone-producing cells of the interstitial glands, corpora lutea and theca interna. The LDs often contained membranous tubules/lamellae along their surfaces and within their interior whose membranes were continuous with those delineating LDs composed of a monolayer of phospholipids and were partially PIP5Kα-immunoreactive. Although granulosa cells of healthy-looking follicles were immunonegative, as the atresia progressed, PIP5Kα-immunoreactivity first appeared in sparsely dispersed dot forms in mural cells of the follicular epithelia, and then were dominant in almost all mural cells that remained after desquamation of the antral cells. The present study provides evidence suggesting that PI(4,5)P2 locally synthesized by PIP5K in LDs is involved in the lipid transfer between lipid droplets (LDs) and the endoplasmic reticulum, which eventually regulates ovarian progesterone production through control of multiple dynamic activities of LDs. It is also suggested that PIP5Kα and PI(4,5)P2 are implicated in the modulation of programmed cell death and/or acquiring the ability of progesterone production in some follicular cells surviving atresia.

Identification in immuno-electron microscopy of solitary multi-polar peripheral neurons of adult Opisthorchis viverrini by antibody against rat diacylglycerol kinase / Identificación por microscopía imuno-electrónica de las neuronas periféricas multipolares solitarias del Opisthorchis viverrini adulto mediante anticuerpo contra la diacilglicerol kinasa de ratas

By utilizing the antibody for rat DGKz a substantial number of immunopositive cells were found in the OV (Opisthorchis viverrini). The immunopositive cells appeared solitarily and they were distributed rather symmetrically to the longitudinal axis of the OV. Some of them were located in close proximity to internal organs such as uterus, ovary, testes, vitelline glands and guts. The immunostained cells extended tapering processes horizontally or obliquely to the OV longitudinal axis. In immuno-electron microscopy, the immunopositive cells were characterized by intensely immunostained mitochondria and weakly immunostained cytoplasm and immunonegative chromatin-poor nucleus. Vacuoles of various sizes without the immunoreactivity were also contained in the cells. Thin cellular processes without the immunoreactivity were found to enclose thinly the entire surfaces of the immunostained cells and processes, and they were in continuity with the interstitial partition-like processes which contained nuclei and aggregation of microfibrils at some distance from the cytoplasmic envelopes. The present finding suggests the possibility that the immunostained cells were peripheral neurons enveloped by peripheral glia and that the glia are of mesenchymal origin because of their cytoplasmic continuity to the interstitial partition-like processes. The motor or sensory nature of the neurons remains to be elucidated.

Mediante el uso del anticuerpos DGK para rata se determinó un número considerable de células inmunopositivas en el Opisthorchis viverrini (OV). Las células inmunopositivas aparecían solitarias y se distribuían simétricamente al eje longitudinal de la OV. Algunas estaban ubicadas en las proximidades de los órganos internos como el útero, ovarios, testículos, glándulas vitelinas e intestino. Las células inmunoteñidas extendían sus procesos horizontalmente u oblicuamente al eje longitudinal de la OV. Por microscopía inmunoelectrónica, las células inmunopositivas se caracterizaron por presentar mitocondrias intensamente teñidas, citoplasma con tinción débil e inmunonegatividad en núcleos pobres en cromatina. También se observó en las células, vacuolas de diversos tamaños sin inmunorreactividad. Se encontraron procesos celulares sin inmunorreactividad para cerrar finamente todas las superficies de las células y procesos, y se continuaron con los procesos de partición intersticiales que contenían núcleos y agregación de microfibrillas a cierta distancia de las envolturas citoplásmicas. El presente hallazgo sugiere la posibilidad de que las células inmunoteñidas son neuronas periféricas envueltas por glia periférica y que la glía presenta origen mesenquimal debido a su continuidad citoplasmática con los procesos de partición intersticiales. La naturaleza motora o sensorial de las neuronas aún no se ha dilucidado.

Localization of vesicular inhibitory amino acid transporter (VIAAT) in the submandibular salivary gland throughout the postnatal development of mice / Localización del transportador vesicular de aminoácidos inhibidores (VIAAT) en la glándula salival submandibular durante el desarrollo postnatal de ratones

According to recent studies, it is highly possible that the occurrence of vesicular inhibitory amino acid transporter (VIAAT) is a good marker of GABA-signaling not only in the brain, but also in extra-brain tissue cells containing GABA and GAD. In view of this, the present study was attempted to localize VIAAT-immunoreactivity in the submandibular gland of mice. In the present study, the submandibular glands of male mice at various postnatal developmental stages were examined for detailed localization of VIAAT-immunoreactivity in immunohistochemistry at light microscopic level. The immunoreactivity for VIAAT was localized in epithelial cells of proximal and distal excretory ducts with the striated portion more intensely immunopositive at young postnatal stages. No significant immunoreactivity was seen in the acinar cells throughout the postnatal development. In addition, the immunoreactivity for VIAAT was detected in the salivary parasympathetic ganglionic neurons, but not in any nerve fibers surrounding the glandular cells. Furthermore, VIAAT-immunoreactivity was found in smooth muscle cells forming the outermost layer of intralobular arterioles. From the present findings, it is possible that GABA plays roles as paracrine and autocrine regulators in the saliva secretion as well as the gland development.

Según estudios recientes, es altamente posible que la aparición del transportador vesicular de aminoácidos inhibidores (VIAAT) sea un buen marcador de señalización de GABA no sólo en el cerebro, sino también en células de tejido extra-cerebrales que contienen GABA y GAD. En el presente estudio se intentó localizar inmunoreactividad a VIAAT en la glándula submandibular de ratones. En el presente estudio, se examinaron las glándulas submandibulares de ratones machos en las distintas etapas del desarrollo postnatal para la localización detallada de inmunoreactividad a VIAAT inmunohistoquímicamente a nivel de microscopía óptica. La inmunorreactividad para VIAAT se localizó en las células epiteliales de los conductos excretores proximal y distal, con mayor intensidad en la porción estriada en las etapas tempranas. No se observó inmunoreactividad significativa en las células acinares durante el desarrollo postnatal. Además, se detectó la inmunoreactividad para VIAAT en las neuronas ganglionares parasimpáticas salivales, pero no en las fibras nerviosas que rodean las células glandulares. Además, la inmunoreactividad a VIAAT se encuentra en las células del músculo liso que forman la capa más externa de las arterias interlobulillares. En base a estos hallazgos, es posible que GABA tenga una función como regulador autocrino y paraparacrino en la secreción de saliva, así como en el desarrollo de la glándula.