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First published online 15 April 2003
doi: 10.1242/jcs.00442

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Lentivirus-mediated transduction of connexin cDNAs shows level- and isoform-specific alterations in insulin secretion of primary pancreatic ß-cells

¹ Department of Morphology, University of Geneva Medical School, 1211 Geneva 4, Switzerland
² Department of Genetics and Microbiology, University of Geneva Medical School, 1211 Geneva 4, Switzerland

View larger version (61K): [in a new window]   Fig. 1. Insulin-secreting cells are efficiently transduced using lentiviral vectors. (A) Lentiviral vectors that contained a self-inactivating deletion (SIN) in the LTR and the post-transcriptional regulatory element of the woodchuck hepatitis virus (Wpre) used the cytomegalovirus promoter (CMV) to drive the expression of either GFP or a Cx (32, 36 or 43) cDNA. (B) GFP fluorescence was analysed by FACS two days after transduction of RIN2A cells with a viral vector containing GFP coding sequence. The analysis revealed a marked increase in cell fluorescence, indicating efficient transduction of the GFP cDNA. The proportion of transduced cells (80%) was given by dividing the number of cells found in the region indicated by the double-headed arrow by the total cell number. Open area, uninfected controls; filled area, infected cells. (C) Monolayers of uninfected RIN cells and cells infected with lentiviral vectors encoding Cx32, Cx36 and Cx43 were immunostained using antibodies against these three Cxs. Whereas no Cx36 was detected in control cultures, this protein was abundantly expressed after transduction of the cognate cDNA. Similarly, Cx32 and Cx43 were detectable between most RIN cells 2 days after infection by the lentiviral vectors. Bar, 10 µm. (D) Western blots revealed Cx32, Cx36 and Cx43 in membrane extracts of transduced cells. By contrast, no specific band was detected in wild-type uninfected RIN2A cells. Membrane extracts of liver, Min6 cells and heart served as positive controls for Cx32, Cx36 and Cx43, respectively. Lanes containing membrane extracts of positive controls were loaded with 10 µg protein; other lanes were loaded with 1 µg protein. (E) Contrasting with the situation in wild-type RIN cells, which show no gap junctions (not shown), large gap junctional plaques were detected by freeze-fracture electron microscopy at membrane interfaces of RIN2A cells transduced with a lentiviral vector coding for Cx32, Cx36 or Cx43.

View larger version (73K): [in a new window]   Fig. 2. Lentiviral vectors allow for over-expression of connexins in primary ß-cells. (A) Immunofluorescence labelling of monolayer cultures showed that uninfected primary rat ß-cells expressed little Cx36 but no detectable Cx32 and Cx43. (B) After transduction with a lentiviral vector coding for one Cx isoform, Cx32, Cx43 and Cx36 were easily detected at the cell membrane. Some infected cells also showed a cytoplasmic labelling of variable intensity. The red background is due to the Evan's blue counterstaining of the cultures. Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 3. Transfer of Lucifer Yellow or neurobiotin between primary islet cells depends on the connexin isoform. (A) Individual islet cells of monolayer clusters were microinjected with 5% Lucifer Yellow. Microinjections resulted in an intercellular transfer of the tracer that was limited between wild-type uninfected cells and cells transduced for Cx36. By contrast, this transfer was much larger between cells transduced for either Cx43 or Cx32. Asterisks indicate the injected cell. Bar, 20 µm. Bars show the extent of Lucifer Yellow transfer. Data are expressed as the percentage of cells showing no coupling (0) or transfer of the dye from the injected cell to first (1st) or third and more order of neighbours (3rd). Data from three experiments were pooled. (B) Individual islet cells of monolayer clusters were also microinjected with 5% neurobiotin and the tracer was detected by FITC-streptavidin staining. Transfer of neurobiotin was restricted between wild-type uninfected cells but extended through several orders of adjacent cells after transduction of a vector coding for either Cx36 or Cx43. Asterisks indicate the injected cell. Bar, 20 µm. Bars show the extent of neurobiotin transfer. Data are expressed as the percentage of cells showing no coupling (0) or transfer of the dye from the injected cell to first (1st) or third and more orders of neighbours (3rd). Data from three experiments were pooled.

View larger version (44K): [in a new window]   Fig. 4. Connexins are differently expressed in pseudo-islets made of non-infected and transduced islet cells. (A) Two days after transduction with a lentiviral vector coding for GFP, numerous primary islet cells exhibited a high fluorescence in monolayer cultures. When allowed to re-aggregate spontaneously into three-dimensional pseudo-islets, most of these cells still exhibited GFP fluorescence 5 days after the infection. Bars, 22 µm in monolayers and 10 µm in pseudo-islets. (B) Immunostaining confirmed the expression of Cx36 in uninfected pseudo-islets and increased levels of this connexin after transduction. Lentiviral vectors also allowed the formation of pseudo-islets expressing either Cx32 or Cx43. Bar, 10 µm. (C) Bars show the percentage of cells within pseudo-islets featuring GFP fluorescence or immunofluorescence staining for Cx32, Cx36 or Cx43 after infection with the lentiviral vectors. 43 pseudo-islets were scored in four experiments.

View larger version (101K): [in a new window]   Fig. 5. Gap junctions form between transduced pseudo-islets made of non-infected and transduced islet cells. (A) Minute gap junctional plaques (arrowheads) associated with linear particle arrays were detected by freeze-fracture electron microscopy at membrane interfaces of non-infected primary ß-cells. In ß-cells transduced with a lentiviral vector coding for Cx32, Cx36 or Cx43, larger gap junctional plaques were seen between endocrine cells. (B) When assessed by western blotting, extracts of control, uninfected pseudo-islets confirmed the native expression of Cx36, whose levels were increased markedly after transduction with a lentiviral vector coding for Cx36. Samples of pancreatic islets and Min6 cells served as positive controls, whereas heart was used as a negative control. All lanes were loaded with 40 µg protein.

View larger version (18K): [in a new window]   Fig. 6. Uninfected and GFP-transduced pseudo-islets show similar secretion patterns. (A) Pseudo-islets were perfused with glucose concentrations ranging from 2.8 mM to 16.8 mM in the absence or presence of 1 mM IBMX and 5 µM FSK. Infected (open squares) and uninfected (grey squares) pseudo-islets showed a similar secretion pattern: they did not increase insulin release for glucose concentrations <11.2 mM but did significantly increase insulin release in response to 16.8 mM glucose, and released even more insulin when glucose was associated with drugs increasing the intracellular concentration of cAMP. Data are means minus the s.e.m. of three experiments. (B) Total insulin content [ng per 50 pseudo-islets (pi)] was similar in uninfected pseudo-islets (grey columns) and in pseudo-islets infected with lentiviral vectors coding for GFP (open columns). (C) Pseudo-islets expressing GFP (open columns) functioned as uninfected pseudo-islets (grey columns), i.e. they showed increased insulin secretion when the glucose concentration was raised from the basal value of 5.6 mM to the stimulatory concentration of 16.8 mM and even more when 1 mM IBMX plus 5 µM FSK were added to the perfusing medium. Data are means plus s.e.m. of three experiments.

View larger version (27K): [in a new window]   Fig. 7. Control and Cx-transduced pseudo-islets show similar insulin content but different secretion patterns. (A) Total insulin content was similar in control pseudo-islets (solid columns; uninfected and GFP-transduced pseudo-islets were pooled) and in pseudo-islets infected with lentiviral vectors coding for Cx32 (horizontal hatched columns), Cx36 (diagonal hatched columns) or Cx43 (vertical hatched columns). Data are means plus s.e.m. of four experiments. (B) Pseudo-islets expressing Cx43 (vertical hatched columns) increased their insulin secretion in response to glucose similarly to the control pseudo-islets (solid columns)–i.e. they increased their insulin secretion when the glucose concentration was raised from the basal value of 5.6 mM to the stimulatory concentration of 16.8 mM. By contrast, pseudo-islets transduced for either Cx36 (diagonal hatched columns) or Cx32 (horizontal hatched columns) did not significantly raise their insulin release in response to the glucose increase. All connexin-transduced pseudo-islets increased their insulin secretion when exposed to increased intracellular cAMP concentrations. Data are means plus s.e.m. of three experiments, each testing in parallel all the four different groups of pseudo-islets. (C) When the secretion data were expressed as a percentage of the normalized control value (100%), it was apparent that expression of Cx43 did not alter insulin secretion in response to glucose. By contrast, glucose-induced insulin secretion was significantly reduced (P<0.005) by over-expression of either Cx32 or Cx36. The secretion induced by glucose plus IBMX and FSK was lowered in all Cx-transduced pseudo-islets. Data are means plus s.e.m. of three experiments.

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