Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • In this present study the availability

    2022-06-24

    In this present study, the availability of commercial GLUT and SGLT of course directed against different epitopes, enabled us to screen for members of the SGLT family and compare the expression patterns of GLUTs and SGLTs in the different regions of the rat lens. Based on these regional and subcellular localisation studies with antibodies against different epitopes, it became apparent that we needed to update and revise the profile of glucose transporters reported in Merriman-Smith et al., 2003, Merriman-Smith et al., 1999. Using a combination of RT-PCR and western blotting, we show for the first time that SGLT1 and SGLT2, but not SGLT3 is expressed in the rat lens. Initially, western blotting of crude membrane fractions of the rat lens indicated SGLT1 to be expressed in the outer cortex, inner cortex and core, while SGLT2 expression was only detected in the inner cortex and core. However, immunohistochemistry revealed that SGLT2 was the only isoform localized to the fiber cell membranes and this was only evident in the lens core. Since the primary fiber cells that eventually constitute the lens core, are laid down early in gestation (Berman, 1991, Lang, 1997), the labelling of SGLT2 specifically in the core, and not the cortical fiber cells, suggests that SGLT2 may only be required early on during lens development to support the growth and metabolic demands of primary fiber cells. Re-screening for members of the GLUT family using antibodies directed against different epitopes of the GLUT protein, revealed that GLUT1 expression was not in fact restricted to the epithelium, as originally reported by Merriman-Smith et al., but is in fact expressed in the membranes of fiber cells throughout the outer cortex, inner cortex and core of the lens. Western blotting and immunohistochemistry revealed strong bands for GLUT1 in the epithelium and fiber cells of the rat lens which were localized to the membranes throughout the entire lens. Unlike the expression pattern of GLUT3, which was restricted to the inner cortex and core regions, GLUT1 appears to be the most predominant glucose transporter isoform since it was expressed in all regions of the rat lens. Furthermore it was the only transporter expressed in the epithelium and outer cortex. The identification of GLUT1 in the rat lens is consistent with the findings of Bassnett et al., which identified GLUT1 in the membrane proteome of mouse fiber cells using mass spectrometry-based shotgun proteomics (Bassnett et al., 2009). While GLUT3 and SGLT2 were not detected by this method, we cannot however exclude their expression from the lens as they may be present at lower abundance and masked by more abundant membrane proteins such as connexins, AQPO, Lim2 and cadherin 2 (Cdh2) (Bassnett et al., 2009). Another interesting finding to emerge was the inability of the antibody directed against the N-terminal epitope of protein to detect GLUT1 in fiber cells of the rat lens. This absence of N-terminal labelling may reflect truncation of the N-terminus, a lack of an N-terminus due to a GLUT1 splice variant or another possibility, such as an occlusion of the epitope binding site. We cannot find evidence of GLUT1 splice variants in the literature and the occlusion of the epitope binding site appears unlikely given that the antibody is able to bind to the N-terminus of GLUT1 in the epithelium. This leaves truncation of the N-terminus as the most likely explanation for the loss of signal of the GLUT1 protein in the fiber cells. Post translational modifications of lens proteins are a normal feature of the process of fiber cell differentiation and include modifications such as truncation, oxidation, deamidation, acetylation, phosphorylation, and glycosylation (Yanshole et al., 2013). The consequence of an N-terminal truncation in lens fiber cells for GLUT1 is unknown. A search of the literature indicates that the loss of the N-terminus is unlikely to affect GLUT1 function. Several studies have shown that replacement of the N terminus tail does not affect the targeting of GLUT to the plasma membrane (Inukai et al., 1995, Sakyo et al., 2007) indicating that the N-terminal truncation of GLUT1 we observe in our study may occur as a normal process of epithelial to fiber cell differentiation. Moreover, it has been demonstrated that loss of the C-terminus is also not critical for GLUT1 function (Verhey et al., 1993), but rather, the intracellular middle loop and the region encompassing the membrane spanning domains 7–12, along with N- and O-glycosylation of GLUT1 appear to play crucial roles in GLUT1 transport function and trafficking (Inukai et al., 1995).