Inositol polyphosphates contribute to cellular circadian rhythms: Implications for understanding lithium's molecular mechanism.

Most living organisms maintain cell autonomous circadian clocks that synchronize critical biological functions with daily environmental cycles. In mammals, the circadian clock is regulated by inputs from signaling pathways including glycogen synthase kinase 3 (GSK3). The drug lithium has actions on GSK3, and also on inositol metabolism. While it is suspected that lithium's inhibition of GSK3 causes rhythm changes, it is not known if inositol polyphosphates can also affect the circadian clock. We examined whether the signaling molecule inositol hexaphosphate (IP6 ) has effects on circadian rhythms. Using a bioluminescent reporter (Per2::luc) to measure circadian rhythms, we determined that IP6 increased rhythm amplitude and shortened period in NIH3T3 cells. The IP6 effect on amplitude was attenuated by selective siRNA knockdown of GSK3B and pharmacological blockade of AKT kinase. However, unlike lithium, IP6 did not induce serine-9 phosphorylation of GSK3B. The synthesis of IP6 involves the enzymes inositol polyphosphate multikinase (IPMK) and inositol pentakisphosphate 2-kinase (IPPK). Knockdown of Ippk had effects opposite to those of IP6 , decreasing rhythm amplitude and lengthening period. Ipmk knockdown had few effects on rhythm alone, but attenuated the effects of lithium on rhythms. However, lithium did not change the intracellular content of IP6 in NIH3T3 cells or neurons. Pharmacological inhibition of the IP6 kinases (IP6K) increased rhythm amplitude and shortened period, suggesting secondary effects of inositol pyrophosphates may underlie the period shortening effect, but not the amplitude increasing effect of IP6 . Overall, we conclude that inositol phosphates, in particular IP6 have effects on circadian rhythms. Manipulations affecting IP6 and related inositol phosphates may offer a novel means through which circadian rhythms can be regulated.