Table 1.
SNP rs3743123 reduces hCx36 coupling between adjacent cells.
Fig 1.
SNP rs3743123 alters the distribution of Cx36 at the cell membrane.
A, In HeLa cells stably transfected with the wild type form of Cx36 (left) the protein shows a spotted distribution (green) at the cell membrane. After transfection of the SNP rs3743123 variant (right) the spotted distribution of the protein alternates with regions of continuous membrane staining. Scale bar, 20 μm. B, Freeze-fracture electron microscopy revealed polygonal and array-shaped gap junction plaques (arrow heads) in HeLa cells transfected with either the WT or variant form of Cx36 (right). Scale bar, 85 nm. C, Distribution of different gap junction patterns (polygonal, linear, array shaped) and D, numbers of particles (connexons) per plaque in HeLa cells transfected with the wild type (n = 37) and SNP rs3743123 forms of Cx36 (n = 48). Images and mean + SEM data are from three independent clones stably expressing either the wild type or the SNP rs3743123 form of the protein.
Fig 2.
SNP rs3743123 does not alter the stability of Cx36 mRNA.
Exposure to 5 μg/ml actinomycin D, revealed comparable levels of Cx36 mRNA at different time points in 3 independent clones of HeLa cells stably transfected with either the wild type (black symbols) or the SNP rs3743123 form of hCx36 (red symbols).
Fig 3.
RIP-hCx36SNPrs3743123 mice display β-cell loss and impaired glucose control over time.
A, Immunofluorescence images of islets of RIP-hCx36rs3743123 and RIP-hCx36WT mice. In young mice, islets display a normal morphology with α cells in the periphery and β cells at the center. Aging does not alter the islets of RIP-hCx36WT mice but determines loss of organization of α and β cells in RIP-hCx36rs3743123 mice. Scale bar 10 μm. B, Number of β cells per islet section in islets of RIP-hCx36WT and RIP-hCx36rs3743123 mice. Compared to 1 month old littermates, 5 month old RIP-hCx36rs3743123 mice display a decrease of β cells per islet section. Data show means + SEM of islets of 3–9 mice per group. C, Glycaemia curve of RIP-hCx36WT and RIP-hCx36rs3743123 mice. RIP-hCx36 rs3743123 show increase in blood glucose with aging. D, Area under the entire glycaemia curve. *P ≤ 0.05**P ≤ 0.01***P ≤ 0.001**** P ≤ 0.0001 (Student t-test with Welch’s correction).
Fig 4.
SNP rs3743123 modify hCx36 expression and its distribution at the beta cell membrane.
A, The volume density (Vv) of Cx36 decreases postnatally in RipβglohCx36 SNP rs3743123 mice. B, The numeric density (Nv) of Cx36 is reduced with time in RIP-hCx36rs3743123 mice. C, The length of the Cx36 plaques was also decreased postnatally in these mice. Data show means + SEM of islets of three mice per group. *P ≤ 0.05**P ≤ 0.01***P ≤ 0.001**** P ≤ 0.0001 versus 1 month old litters.