Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimulator glucose-6-phosphate (G6P) and by dephosphorylation through inactivation of GS kinase-3 with insulin. The relative importance of these two regulatory mechanisms in controlling GS is not established, mainly due to the complex interplay between multiple phosphorylation sites and allosteric effectors. Here we identify a residue that plays an important role in the allosteric activation of GS by G6P. We generated knockin mice in which wild-type muscle GS was replaced by a mutant that could not be activated by G6P but could still be activated normally by dephosphorylation. We demonstrate that knockin mice expressing the G6P-insensitive mutant display an ∼80% reduced muscle glycogen synthesis by insulin and markedly reduced glycogen levels. Our study provides genetic evidence that allosteric activation of GS is the primary mechanism by which insulin promotes muscle glycogen accumulation in vivo.

The potent insulin-sensitizing effects of peroxisome proliferator-activated receptor g (PPARg) agonists are well established. However, it is still a matter of intense debate as to which tissue(s) represent the most critical sites of action for PPARg agonists, and what the relevant target genes are that ultimately mediate the improvements in insulin sensitivity. The cell type with the highest levels of PPARg is the adipocyte, and as such the adipocyte is an excellent candidate cell to look for critical mediators of PPARg agonist action. Adiponectin, an adipocyte-specific secretory protein, is upregulated in response to PPARg agonist exposure, and its serum levels consequently increase significantly. Genetic, pharmacological and clinical studies have demonstrated potent insulin-sensitizing effects of adiponectin. Here, we summarize the evidence that implicates adiponectin as a critical mediator of PPARg-agonist-mediated improvements in insulin sensitivity, particularly in the context of PPARg-agonistmediated enhancements of hepatic insulin sensitivity.

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating glycogen synthase kinase (GSK) 3 through phosphorylation. Insulin also promotes glucose uptake and glucose 6-phosphate (G-6- P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphorylation of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knockin mice in which wild-type GSK3α and -β genes are replaced with mutant forms (GSK3α/βS21A/S21A/S9A/S9A), which are nonresponsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3α/βS21A/S21A/S9A/S9Amice, glycogen content in different muscles from these mice was similar compared with wild-type mice. Basal and epinephrine-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knockin mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G-6- P, the rate of [14C]glucose incorporation into glycogen, and an increase in total glycogen content were similar between wild-type and GSK3 knockin mice. Injection of glucose containing 2-deoxy-[3H]glucose and [14C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knockin mice. These results suggest that insulin-mediated inhibition of GSK3 is not a rate-limiting step in muscle glycogen synthesis in mice. This suggests that allosteric regulation of GS by G-6- P may play a key role in insulin-stimulated muscle glycogen synthesis in vivo.

Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type 11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

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Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating GSK3. Insulin also promotes glucose uptake and glucose‐6‐phosphate (G6P) production, which allosterically activates GS. The aims of this study were (1) to determine if the inhibition of GSK3 is necessary for insulin‐mediated muscle glycogen synthesis in vivo and (2) to identify the molecular mechanism by which G6P regulates GS. We used knock‐in (KI) mice in which GSK3α and β genes are replaced with constitutively‐active forms (GSK3α/βS21A/S21A/S9A/S9A). Insulin‐induced dephosphorylation and activation of GS was abolished in GSK3 KI mice, however muscle glycogen content from these mice was not reduced. Injection of [3H]2‐deoxyglucose and [14C]glucose resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between WT and GSK3 KI. These results provided genetic evidence that inhibition of GSK3 and subsequent dephosphorylation of GS are not essential for muscle glycogen synthesis in vivo, indicating that allosteric regulation of GS by G6P may play a key role in this process. We next mutated putative G6P binding residues of GS and expressed these mutants in HEK293 cells and activity assessed. We identified that Arg582/586 residues are responsible for G6P‐mediated activation of muscle GS. Characterization of these mutants will allow us to determine the role that G6P regulates GS and glycogen levels in vivo.