Skeletal muscle and adipose tissue represent primary targets of insulin action and contribute significantly towards the homeostatic control of circulating blood glucose.  This important physiological function relies upon insulin’s ability to induce a rapid stimulation of glucose uptake across the blood-facing membranes of these tissues, a process critically dependent upon the translocation of the insulin-regulated glucose transporter, GLUT4, from specialised intracellular storage vesicles to the plasma membrane.  The precise mechanism by which GLUT4 transporters are mobilised is far from clear but recent work, including that from the Hundal lab, has shown that activation of the serine/threonine kinase, protein kinase B (PKB, also known as Akt) is a crucial for allowing insulin to promote an increase in glucose transport.  The hormonal activation of PKB can be disrupted by agents implicated in the pathogenesis of insulin resistance and Type II diabetes (e.g. saturated fatty acids, ceramide) and results in a substantial loss in insulin-stimulated glucose uptake and a failure to promote glycogen accretion (Fig 1).  Thus our work in this area not only aims to define the signalling proteins involved in the regulation of glucose uptake/utilisation but also hopes to gain novel insight into the mechanisms of insulin resistance.

Fig 1.  The binding of insulin to its receptor activates the classical insulin signalling cascade involving IRS proteins and PI3 kinase which results in the activation of PKB (aka Akt).  Activated PKB phosphorylates and inactivates GSK3 thereby alleviating its inhibitory input to glycogen synthase thus promoting glycogen synthesis.  Another consequence of PKB activation is phosphorylation of a Rab GAP (AS160), which is known to be associated with GLUT4-containing vesicles and whose phosphorylation is considered important in supporting the translocation of GLUT4 to the plasma membrane in response to insulin.  PKB activation can become dysregulated in response to an increase in intracellular ceramide as well as increased extracellular availability of free fatty acids such as palmitate, which can be used for de novo ceramide synthesis.  This loss in PKB activation results in impaired insulin-stimulated glucose transport and glycogen synthesis.

 

 

 

Glucose transport in skeletal muscle is also acutely regulated in response to exercise and stress stimuli. The mechanism by which these stimuli instigate a rapid increase in glucose uptake remains poorly understood, although the stimulation in both cases appears, like insulin, to involve an increase in the number of cell surface transporters. We are currently attempting to elucidate the signalling pathways responsible for activating glucose uptake in response to cellular stresses (aresenite, heat shock etc) and are particularly keen to determine whether the hormonal and stress mediated increases in sugar uptake involve the participation of common intracellular signalling molecules.

 

A second major area of research activity concerns the molecular regulation of the System A amino acid transporter.  System A (of which three isoforms have described SNAT1, 2 and 4) mediates the Na+-dependent uptake of small neutral amino acids (AAs) including key intermediary metabolites such as alanine, glutamine and serine as well as the essential AAs methionine and threonine.  The transporter develops an outwardly-directed concentration gradient of its AA substrates and is upregulated in contracting skeletal muscle and in liver during prolonged fasting, becoming the rate-determining step in hepatic alanine metabolism (for gluconeogenesis and ureagenesis) under these conditions. Upregulation of System A within specific brain regions is also closely associated with the central responses to dietary depletion of single amino acids and there is considerable evidence for regulation of System A by growth factors (including insulin), extracellular pH and cell volume both in vitro (cell culture) and in vivo.  A major feature of System A is its upregulation in response to amino acid limitation, a process referred to as adaptive regulation, which involves an increase in SNAT2 mRNA expression and cell surface abundance of the SNAT2 transporter protein. This upregulation can be attenuated by resupply of a single substrate amino acid (e.g. alanine or glutamine), but also by select non-substrate amino acids such as tyrosine.  The ability of these amino acids to repress System A expression implies the existence of proteins that “sense” amino acid availability and that are capable of transducing the change in availability to a signal event that culminates in the altered SNAT2 gene expression (Fig 2).  We are currently assessing whether SNAT2 itself operates as a “transceptor” (that responds to availability of its own substrates) as well as trying to identify other novel proteins that may be involved in amino acid sensing. Establishing how these putative sensors relay changes in amino acid availability to the transcriptional machinery that regulates System A gene expression represents a major investigative goal.     

 

Fig 2,  The SNAT2 amino acid transporter is responsible for mediating the Na-dependent uptake of short chain neutral amino acids (AAs) such as alanine, glutamine, threonine etc.  SNAT2 is extensively regulated by hormones such as insulin, changes in cellular osmolarity and changes in amino acid availability.  SNAT2 undergoes adaptive upregulation in response to cellular amino acid deprivation, a response that can be repressed by resupply of substrate AAs and by select non-substrate AAs.  It is possible that SNAT2 may be capable of sensing availability of its own substrates and thus operates as a “transceptor” that modifies the activity of signalling molecules/transcription factors which regulate SNAT2 gene expression.  The ability of select non-substrates to repress the adaptive response implies an additional AA sensor/signalling pathway exists for regulating gene expression.