The role of salt-inducible kinase in the control of hepatic gluconeogenesis

  • Kashyap Patel

    Student thesis: Doctoral ThesisDoctor of Philosophy


    Regulation of hepatic gluconeogenesis by hormones insulin and glucagon is central to glucose homeostasis. Recent work has proposed that amongst the salt inducible kinase isoforms (SIK1, 2 and 3), members of the AMPK-related kinase family, the SIK2 isoform may play a role as signalling mediator in the control of insulin- and glucagon-regulated hepatic gluconeogenesis. However, the mechanisms of the hormonal-regulation of SIK2 in liver remain controversial, with much of the data based on the studies in non-hepatic tissues/cells. Therefore, the exact molecular regulation of SIK2 by these hormones in the liver required robust and intensive molecular/biochemical research coupled to physiological readout (e.g. gluconeogenesis).
    My studies with phosphopeptide mapping by mass spectrometry followed by verification with well-characterised phospho-specific antibodies revealed that SIK2 was phosphorylated on Ser343, Ser358, Thr484 and Ser587 in response to glucagon and fasting but not following insulin treatment or refeeding in primary hepatocytes and liver in vivo, respectively. Unexpectedly, fasting- or glucagon-stimulated phosphorylation of SIK2 (individually or in combination) did not directly modulate kinase activity of SIK2 and its subcellular localisation. These findings collectively questioned the role of SIK2 in hepatic gluconeogenesis.
    Thus to establish the role of SIK2 in vivo, liver-specific SIK2 knock-out mice were generated and analysed (in collaboration with Dr Marc Foretz, Institut Cochin, Paris). Surprisingly, the liver-specific SIK2 knock-out mice displayed normal blood glucose levels and gluconeogenic gene expression in both fasted and refed states compared to the control mice. The primary hepatocytes from these liver-specific SIK2 knock-out mice had unaltered basal and Bt2-cAMP-stimulated glucose production and gluconeogenic gene expression compared to the control hepatocytes. These suggest that SIK2 is either not involved in hepatic gluconeogenesis or its loss is compensated by other SIK isoforms in the liver. This may indicate a possible redundancy amongst SIK isoforms for their role in hepatic gluconeogenesis.
    To test this hypothesis, highly selective pan SIK inhibitors (HG-9-91-01 and KIN112) were characterised and used to study the importance of SIKs activity in hepatic glucose production employing mouse primary hepatocytes. Incubation of hepatocytes with SIK inhibitors significantly reduced CRTC2, CRTC3 and HDAC4 phosphorylation and robustly increased gluconeogenic gene expression and glucose production. The effects of HG-9-91-01 on hepatic gluconeogenesis were validated as SIK-specific, using HG-9-91-01-resistant SIK mutants, LKB1-null hepatocytes (cells where SIK activity was already depleted) and also AMPKa1/a2-null hepatocytes.
    These experiments have proposed a novel insulin-independent pathway of gluconeogenesis suppression, in which SIK isoforms collectively work together as a ‘molecular gatekeeper’ to continually suppress hepatic gluconeogenesis. This inhibition is released following glucagon/fasting by inhibiting SIKs through a mechanism yet to be elucidated but likely to be dependent on phosphorylation of SIKs on multiple residues.
    Importantly and contrary to the previous studies, my data demonstrates that SIK2 is clearly not the only or the dominant SIK isoform that regulates hepatic gluconeogenesis and glucagon regulates phosphorylation of four specific residues on SIK2, while insulin does not induce phosphorylation of SIK2 in liver. These data inform on how glucagon signals to these important kinases, but more work is required to decipher how phosphorylation actually changes SIKs function at the molecular and biochemical level and how such phosphorylation effects downstream targets and biological processes.
    Date of Award2014
    Original languageEnglish
    SupervisorKei Sakamoto (Supervisor) & Calum Sutherland (Supervisor)

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