High glucose concentrations induce TNF-α production through the down-regulation of CD33 in primary human monocytes.

BACKGROUND: CD33 is a membrane receptor containing a lectin domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that is able to inhibit cytokine production. CD33 is expressed by monocytes, and reduced expression of CD33 correlates with augmented production of inflammatory cytokines, such as IL-1ß, TNF-α, and IL-8. However, the role of CD33 in the inflammation associated with hyperglycemia and diabetes is unknown. Therefore, we studied CD33 expression and inflammatory cytokine secretion in freshly isolated monocytes from patients with type 2 diabetes. To evaluate the effects of hyperglycemia, monocytes from healthy donors were cultured with different glucose concentrations (15-50 mmol/l D-glucose), and CD33 expression and inflammatory cytokine production were assessed. The expression of suppressor of cytokine signaling protein-3 (SOCS-3) and the generation of reactive oxygen species (ROS) were also evaluated to address the cellular mechanisms involved in the down-regulation of CD33. RESULTS: CD33 expression was significantly decreased in monocytes from patients with type 2 diabetes, and higher levels of TNF-α, IL-8 and IL-12p70 were detected in the plasma of patients compared to healthy donors. Under high glucose conditions, CD33 protein and mRNA expression was significantly decreased, whereas spontaneous TNF-α secretion and SOCS-3 mRNA expression were increased in monocytes from healthy donors. Furthermore, the down-regulation of CD33 and increase in TNF-α production were prevented when monocytes were treated with the antioxidant α-tocopherol and cultured under high glucose conditions. CONCLUSION: Our results suggest that hyperglycemia down-regulates CD33 expression and triggers the spontaneous secretion of TNF-α by peripheral monocytes. This phenomenon involves the generation of ROS and the up-regulation of SOCS-3. These observations support the importance of blood glucose control for maintaining innate immune function and suggest the participation of CD33 in the inflammatory profile associated with type 2 diabetes.

Impact of biotin supplemented diet on mouse pancreatic islet ß-cell mass expansion and glucose induced electrical activity.

Biotin supplemented diet (BSD) is known to enhance ß-cell replication and insulin secretion in mice. Here, we first describe BSD impact on the islet ß-cell membrane potential (Vm) and glucose-induced electrical activity. BALB/c female mice (n ≥ 20) were fed for nine weeks after weaning with a control diet (CD) or a BSD (100X). In both groups, islet area was compared in pancreatic sections incubated with anti-insulin and anti-glucagon antibodies; Vm was recorded in micro dissected islet ß-cells during perfusion with saline solutions containing 2.8, 5.0, 7.5-, or 11.0 mM glucose. BSD increased the islet and ß-cell area compared with CD. In islet ß-cells of the BSD group, a larger ΔVm/Δ[glucose] was found at sub-stimulatory glucose concentrations and the threshold glucose concentration for generation of action potentials (APs) was increased by 1.23 mM. Moreover, at 11.0 mM glucose, a significant decrease was found in AP amplitude, frequency, ascending and descending slopes as well as in the calculated net charge influx and efflux of islet ß-cells from BSD compared to the CD group, without changes in slow Vm oscillation parameters. A pharmacological dose of biotin in mice increases islet insulin cell mass, shifts islet ß-cell intracellular electrical activity dose response curve toward higher glucose concentrations, very likely by increasing KATP conductance, and decreases voltage gated Ca2+ and K+ conductance at stimulatory glucose concentrations.

Neurons and satellite glial cells in adult rat lumbar dorsal root ganglia express connexin 36.

Previous studies have shown that following peripheral nerve injury there was a downregulation of the gap junction protein connexin 36 (Cx36) in the spinal cord; however, it is not known whether Cx36 protein is expressed in the dorsal root ganglia (DRGs), nor if its levels are altered following peripheral nerve injuries. Here we address these aspects in the adult rat lumbar DRG. Cx36 mRNA was detected using qRT-PCR, and Cx36 protein was identified in DRG sections using immunohistochemistry (IHC) and immunofluorescence (IF). Double staining revealed that Cx36 co-localizes with both anti-ß-III tubulin, a neuronal marker, and anti-glutamine synthetase, a satellite glial cell (SGC) marker. In neurons, Cx36 staining was mostly uniform in somata and fibers of all sizes and its intensity increased at the cell membranes. This labeling pattern was in contrast with Cx36 IF dots mainly found at junctional membranes in islet beta cells used as a control tissue. Co-staining with anti-Cx43 and anti-Cx36 showed that whereas mostly uniform staining of Cx36 was found throughout neurons and SGCs, Cx43 IF puncta were localized to SGCs. Cx36 mRNA was expressed in normal lumbar DRG, and it was significantly down-regulated in L4 DRG of rats that underwent sciatic nerve injury resulting in persistent hypersensitivity. Collectively, these findings demonstrated that neurons and SGCs express Cx36 protein in normal DRG, and suggested that perturbation of Cx36 levels may contribute to chronic neuropathic pain resulting from a peripheral nerve injury.

CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer.

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10-6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5-10, p = 3.3 x 10-6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.

Connexin43 is expressed in mouse fetal ovary.

Developmental studies have shown that connexin43 (Cx43) is expressed in the ovary from the first day of life and throughout the rest of postnatal development. In both mouse embryonic ovaries and testes, target-directed deletion of Cx43 gene induces a significant decrease in germinal cells, but the exact mechanism determining this reduction remains unknown. Moreover, recently we found that Cx43 is abundantly expressed in mouse testes from the earliest stages of its fetal development. In the present work we investigate whether Cx43 transcript and protein are expressed in mouse embryonic ovaries. Total RNA was analyzed with specific Cx43 oligonucleotides in RT-PCR studies. A Cx43 PCR product was detected in ovaries at 16.5 and 18.5 days postcoitum (dpc). Bands of 43-45 kDa, characteristic of Cx43, were detected in immunoblots of total homogenates of ovaries at 14.5 and 18.5 dpc. Cell type-specific expression of Cx43 was investigated using double-labeled sections incubated with specific antibodies against Cx43 and the enzyme 3beta-hydroxysteroid dehydrogenase (3betaHSD) or a germ cell nuclear antigen (GCNA1), which are cell markers of steroidogenic and germinal cells, respectively. At 18.5 dpc, Cx43 was found in conglomerates of 3betaHSD-positive cells. Cx43 was also localized at homocellular junctions between parenchyma pregranulosa cells, and at heterocellular junctions between pregranulosa and germinal cells. At these two latter localizations, Cx43 was traced back to 12.5 dpc. In conclusion, this study demonstrates for the first time that from the earliest stages of embryonic ovary development, Cx43 is expressed in principal cell types involved in control of female fertility. These data suggest that the gap junctions formed with Cx43 between somatic and germinal cells may be necessary for prenatal expansion of germinal cells at initial stages of fetal gonadal development.

Connexin 36, a key element in pancreatic beta cell function.

The prevalence of diabetes at a global scale has markedly increased during the last three decades. Diabetes is a chronic disease that includes a group of metabolic disorders, in which high serum glucose levels is a common factor. Insulin is the only hormone that decreases serum glucose levels. Therefore, it is relevant to deepen our understanding of cell mechanisms that regulate insulin production and release. Insulin is produced in pancreatic islet beta cells. They are excitable cells and most of them are electrically coupled through gap junction channels. Connexin 36 (Cx36) has been identified at junctional membranes of islet beta cells in both rodents and humans. Co-localization of Cx36 with Cx30.2 has been recently identified. Functional studies in Cx36 deficient mice have provided direct evidence that Cx36 gap junction channels are necessary for the synchronization of [Ca(2+)]i oscillations in islet beta cells. The latter allows for the generation of insulin pulses in a single perfused islet. Moreover, Cx36 deficient mice were found to have altered serum insulin pulse dynamics and to be glucose intolerant. In addition, Cx36 has been recently identified as an early gene that is specifically expressed in embryonic beta cells, whose transcript and protein are upregulated in unison with the main wave of beta cell differentiation. In conclusion, Cx36 is critical for endocrine pancreatic function and may represent a molecular target for future prevention and treatment of diabetes. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Linajes madurativos de las células que regeneran al hígado / Maturative lineages of cells that regenerate the liver

Resumen Diferentes linajes de las células hepáticas participan en la regeneración del hígado y los hepatocitos guiescentes pueden responder ante un daño hepático leve con división celular moderada, mientras gue las células troncales y progenitoras responderán para una amplia restitución del parénquima hepático severamente dañado. Ocho linajes madurativos se sitúan dentro del hígado con diferente grado de madurez. Cuatro de ocho subpoblaciones gue presentan marcadores para células troncales o progenitoras se ubican en nichos intrahepáticos como los canales de Hering y la vena porta, estructuras en las gue pueden continuar su diferenciación si se recibe algún estímulo. Recientemente se describió un nuevo nicho entrahepático con una composición celular heterogénea ubicado entre los grandes duetos biliares y en el páncreas. Las células gue se encuentran en estos compartimentos expresan marcadores tempranos de células troncales y se consideran como precursores de las células troncales hepáticas.

Abstract Different cellular lineages participate in liver regeneration. Quiescent hepatocytes may respond to a mild liver injury with moderate cell division. Meanwhile, stem and progenitor cells are responsible for the large restitution of a severe damaged organ. Eight matu rational lineages are in the liver, each one with a different maturity grade. Four subpopulations out of eight express stem or progenitor cell markers are found within intrahepatic niches such as the canals of Hering and the portal vein. Each structure is able to continue its differentiation if a stimuli is received. Recently, a new extra-hepatic stem niche with a heterogeneous cellular composition was found between the large biliary ducts and the pancreas. Stem or progenitor cells contained in these compartments express early stem markers and are considered as a precursor of hepatic stem cells.

Biophysical evidence that connexin-36 forms functional gap junction channels between pancreatic mouse beta-cells.

Connexin-36 (Cx36) is the only gap junction protein that has been unambiguously identified in rodent pancreatic beta-cells. However, properties of gap junction channel unitary currents between beta-cells remain unrevealed. To address whether Cx36 forms functional channels in beta-cells, we characterized biophysical properties of macro- and microscopic junctional currents recorded from dual whole cell voltage clamp isolated pairs of dispersed mouse beta-cells. Electrical coupling was recorded in 80% of cell pairs with a junctional conductance (g(j)) of 355 +/- 45 pS (n = 20). Transjunctional voltage dependence was identified in three of seven cell pairs with high-input membrane resistances. Normalized steady-state g(j) (Gj) and transjunctional-voltage relation were well described by a two-state Boltzmann equation [maximal conductance (Gmax) = 1.0, voltage-insensitive conductance (Gmin) = 0.3 and 0.28, voltage gating sensitivity (A) = 0.21 and 0.23, and voltage at which one-half of the initial voltage-dependent conductance was reached (Vo) = -85 and 87 mV for negative and positive potentials, respectively]. Halothane reversibly uncoupled beta-cell pairs, and, during recovery, unitary conductances of 5-10 pS were recorded while using patch pipettes containing mainly CsCl. Although these properties are similar to those previously described for Cx36 channels in mammalian cell systems, we found that beta-cell junctional currents were insensitive to quinine. Cx36 transcript and protein expression in islets and freshly dispersed cell preparations was confirmed by RT-PCR and immunofluorescence. In conclusion, biophysical properties of junctional channels between beta-cells are similar but not identical to those previously described for homomeric Cx36 channels. Cell type-specific mechanisms that may account for these differences are discussed.