Galactose is one of the three main monosaccharides consumed in the human diet, typically via lactose consumption from dairy products. It is widely thought that after intestinal absorption galactose is exclusively metabolised in the liver (or kidney) via enzymes of the Leloir pathway (i.e., galactokinase [GALK]. galactose-1-phosphate uridyltransferase [GALT], and UDP-galactose 4′-epimerase [GALE])(Gonzalez & Betts, 2019). However, there is evidence from animal studies that galactose uptake can occur in skeletal muscle. Furthermore, our recent unpublished observations suggest that GALK, GALT and GALE are expressed in human skeletal muscle and galactose may directly stimulate glycogen synthesis in skeletal muscle. Collectively, these data question the current perception that galactose requires conversion in the liver before it can be utilized, and points towards skeletal muscle as a previously unrecognised site for galactose metabolism.
Accordingly, the main objective of this project is to characterise galactose metabolism in skeletal muscle. A range of approaches may be taken to investigate this exciting/novel area. This could include determination of gene expression (RT-qPCr), protein expression (Western blotting) and localisation (immunohistochemistry) and enzymatic activity (KinaseGlo detection, radioactivity-based and fluorometric assays) of GALT, GALK and GALE in human skeletal muscle biopsy samples. Furthermore, mechanisms of galactose uptake (i.e., membrane transport) and its subsequent metabolic fate (e.g., oxidation, glycogen storage) in response to physiological stimuli such as insulin and/or muscle contraction could be studied in rodent skeletal muscle, ex-vivo (for experimental model see prior work from 2nd supervisor - Lai et al, 2010). Regardless of the approach, the research aims to make new scientific discoveries that could have fundamental implications for our understanding of dietary carbohydrate metabolism.
Techniques that could be used in pursuit of the proposed objective include:
Gene expression - RT-qPCr
Protein expression - Western blotting
Protein localisation – Immunohistochemistry
Cell culture, retroviral overexpression and genetic knockdown using CRISPR-Cas9
Enzyme activity assays - KinaseGlo detection and fluorometric assays
Skeletal muscle incubation model and substrate uptake/incorporation assays