Glucose represses connexin36 in insulin-secreting cells

Abstract

IntroductionThe homeostasis of multicellular organisms depends on many systems allowing the cells to review the functional state of their neighbors (Spray, 1998). Channels located at gap junctions are one way by which vertebrate cells communicate (Meda, 2000). It has been established that this type of intercellular communication permits coordinated cellular activity, including secretion (Meda, 1996). Gap junctions are specific membrane structures consisting of aggregates of intercellular channels that enable the direct exchange of ions and small metabolites such as second messengers. Intercellular channels result from the association of two hemichannels, named connexons, which are separately contributed by two adjacent cells. Each connexon is an assembly of six transmembrane connexins, encoded by a family of genes with more than 20 members (Sohl and Willecke, 2004).We and others have demonstrated that connexin36 (Cx36; 36 kDa) is the sole connexin isoform expressed in insulinproducing ␤-cells of rat and mouse pancreatic islets (SerreBeinier et al., 2000;Theis et al., 2004). In order to evaluate the contribution of Cx36 to the control of insulin secretion, the Cx36 content has been artificially modified in insulin-secreting cells Le Gurun et al., 2003). These experiments provided evidence that Cx36 has to be expressed at a very precise level in order to maintain a normal insulin secretion. Recently, Leite et al. have suggested that, during the maturation process of neonatal islet, the gain of capacity of the islet to secrete insulin was associated with an increase in Cx36 expression levels. Therefore, variations of the endogenous Cx36 expression levels could have major repercussions on ␤-cell function. Considering the fundamental implication of glucose on gene regulation in ␤-cells, we investigated the effect of glucose on the Cx36 levels in insulin-producing cells. Early morphological studies have demonstrated that glucose induces a remodeling of the gap junctions aggregates in pancreatic islets (In't Veld et al., 1986;Meda et al., 1980). Here, we have demonstrated for the first time that glucose induces a time-and dose-dependent decrease of Cx36 mRNA and protein content in several insulin-secreting cell lines and adult pancreatic rat islets.In ␤-cells, glucose metabolism generates fluctuations in the ATP/ADP ratio, leading to oscillations of membrane potential and cytoplasmic calcium concentrations, which then trigger insulin release (Deeney et al., 2000). Thus, ␤-cell stimulation is associated with regular Ca 2+ oscillations (Lenzen et al., 2000). Surprisingly, glucose-induced Ca 2+ oscillations are synchronized among the thousands of ␤-cells that form each pancreatic islet (Jonkers and Henquin, 2001), probably because of a diffusible regulator (Meda, 1996). These oscillations become more regular and prominent in the presence of ␤-cell

Functional significance of repressor element 1 silencing transcription factor (REST) target genes in pancreatic beta cells

“…Cell line and mouse islet isolation The rat insulinoma cell line INS-1E was maintained in RPMI 1640 medium, as previously described [15]. Islets of Langerhans of adult C57BL/6 male mice, weighing 25 to 30 g, were isolated and cultured as previously described [15].…”

“…Islets of Langerhans of adult C57BL/6 male mice, weighing 25 to 30 g, were isolated and cultured as previously described [15].…”

“…RNA isolation and northern blotting RNA isolation and northern blot analysis were performed as previously described [15]. Transcripts levels were revealed using specific probes for rat Cx36 (also known as Gja9), β-actin [11], rat Munc18-1 (also known as Stxbp1), Syntaxin 1A and Rab3a.…”

“…Real-time RT-PCR Quantitative RT-PCR was performed using a kit (SYBR Premix Ex Taq PCR Kit TaKaRa; Axon Lab, Le Mont-sur-Lausanne, Switzerland) in a Lightcycler (Roche Diagnostics, Mannheim, Germany), as previously described [15]. cDNAs were amplified using specific primers (ESM Table 3).…”

“…Western blotting Western blots were performed as previously described [15]. Specific protein levels were revealed with polyclonal rabbit antibodies against: CX36 [11], synaptosomal-associated protein (SNAP) 25 (kindly provided by H. Hirling, EPFL, Lausanne, Switzerland) and SYT IV.…”