Study of the 1,5-anhydroglucitol transporters

Details

Supervisors

Veiga da Cunha, Maria

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité spécialisée: nutrition humaine

Abstract

Neutropenia and neutrophil dysfunction found in G6PC3 and in glucose-6-phosphate transporter G6PT/SLC37A4 deficiencies are due to the accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), an inhibitor of hexokinase formed from 1,5-anhydroglucitol (1,5-AG), an abundant polyol present in blood. 1,5-AG is reabsorbed in the kidney tubules by a still not characterized Na+/glucose dependent transporter, suggested to be SGLT4 or SGLT5. This transporter is inhibited by glucosuria either due to diabetes or to the SGLT2 inhibitors that are commonly used to treat diabetes, which leads to a decrease in concentration of 1,5-AG in blood. Consequently, my host laboratory successfully repurposed the usage of empagliflozin, one of the available SGLT2 inhibitors, to treat the neutropenia and neutrophil dysfunction in G6PC3 and in G6PT deficient patients. The purpose of this master thesis was to study the transport properties of SGLT4 and SGLT5, the two active transporters that are still confusingly suggested to transport 1,5-AG. Our hope is to better understand the role of these transporters on the renal reabsorption of 1,5-AG and on its entry in neutrophils. Using lentiviral vector technology, I was able to create HEK293T cell lines that overexpress in a reproducible way mouse and human SGLT4 and SGLT5. These model cell lines allowed me to set up robust transport-activity assays in live cells. I was able to show that both mouse and human SGLT4 and SGLT5 can transport mannose, while only SGLT5 is able to transport 1,5-AG (and fructose). I characterized in detail the substrate specificity of both transporters which indicated that SGLT5 evolved to become a less specific mannose transporter than SGLT4, which likely explains its capacity to transport 1,5-AG. Together, these results suggest that SGLT5 is most likely the renal 1,5-AG transporter in vivo, and not SGLT4 as often described in the literature. The establishment of SGLT5 as the renal 1,5-AG transporter, together with the identification by my host laboratory of a very rare SGLT5 variant (Arg401His) in a G6PC3-deficient patient, that responded particularly well to the empagliflozin treatment, led us to create other SGLT5 overexpressing cell lines. These overexpressed the Arg401His as well as other SGLT5 variants previously associated with lower blood 1,5-AG concentrations. They enabled us to characterize their transport for 1,5-AG and to study in detail how SGLT5 variants impacted the transport of 1,5-AG. Another aspect of my research project consisted on investigating the possibility that the SGLT2-inhibitors might lack specificity and also act directly on SGLT5 by the direct inhibition of 1,5-AG renal reabsorption and/or its entry in neutrophils. This is an interesting question with a potential therapeutic outcome.