Roles Of The Circadian Clock In The Metformin Pharmacological Effects

Metformin as the first-line drug for the type 2 diabetes mellitus treatment has long been known to reduce blood glucose levels through repressing hepatic glucose production.Mounting evidence shows therapeutic benefits of metformin to numerous diseases,for instance,metformin is known to prevent fatty liver by improving lipid metabolism and repressing steatosis.The circadian clock as an endogenous time-keeping mechanism controls 24-hour physiological and behavioral processes of living organisms on the Earth.The transcription-translation-based autoregulatory feedback loops underpin the circadian clock mechanism.Circadian misalignment leads to numerous diseases,such as sleep disorders,obesity and diabetes,cardiovascular disease,pulmonary fibrosis immune disease,cancers,and epilepsy.A recent study observed that low-dose metformin binds to lysosomal membrane protein PEN2 and then activates AMPK.PEN2 is one of the subunits of γ-secretase,and its protein structure and amino acid sequence are highly conserved among humans,mice,and zebrafish.However,the chronopharmacological study of metformin is still in its early fancy,and the role of the circadian clock in metformin pharmacology is still unexplored.In order to address these questions,the following experiments were conducted in this thesis project.Zebrafish possess only one copy psenen encoding Pen2.psenen is rhythmically expressed under DD(constant darkness)and LD(light and dark)conditions,while Pen2 protein displays rhythmicity in the adult fish liver under LD condition.Sequence search revealed that the psenen promoter harbors RRE(retinoic acid-related orphan receptor(ROR)-binding elements),E-box and CCAAT enhancer.The circadian clock regulates psenen via RRE and E-box.Interestingly,Cebpb also regulates psenen transcription.We found that Pen2 binds to Atp6ap1a and Atp6ap1b,respectively,consistent with that PEN2 binds to ATP6AP1 in mice.Hence,Pen2 and ATP6AP1 also contribute to the AMPK phosphorylated pathway in zebrafish.We treated zebrafish larvae with different concentrations of metformin for 1 hour,we observed that the phosphorylated AMPK level does not change significantly when using higher than 4 mM metformin.Thus,we selected 4 mM as the minimum concentration of metformin for drug treatment.Under DD condition,larvae treated with 4 mM metformin at CT0/120 hpf and CT12/132 hpf display 1.8-4.0 h phase delay of locomotor rhythms.Further,4 mM metformin treatment at CTO resulted in the up-regulation of tefa,per2 and cry1aa,while 4 mM metformin treatment at CT12 led to the up-regulation of clocka,bmal2/arntll,and nr1d2a but the down-regulation of nfil3,per1b and cebpb.RNA-seq-based transcriptome analysis revealed that 4 mM metformin at CT12 resulted in the up-regulation of acot15,apobb.2,and scp2b,potentially promoting fatty acid β-oxidation in peroxisomes,and preventing the development of the fatty liver.In summary,we investigated the relationship between the circadian clock and the metformin pharmacological effects.The circadian clock regulates the rhythmic expression of psenen encoding Pen2(metformin-binding protein)via RRE and E-box;low-dose metformin treatment results in phase delay of larval zebrafish locomotor rhythms and alters the expression patterns of key circadian clock genes.We also observed that low-dose metformin up-regulates acot15,apobb.2,and scp2b in zebrafish,thereby promoting fatty transport and metabolism and blocking the occurrence of fatty liver.Together,these results shed light on the circadian roles in the metformin-mediated reduction of glucose and fatty acids,provide insights into clinical use of metformin,and facilitate the investigation of metformin chronopharmacology.