Regulatory Role Of Acetyltransferase CBP/p300 In Hepatic Gluconeogenesis And Its Mechanism

Background and ObjectiveType 2 diabetes mellitus(T2DM)is a metabolic disorder characterized by chronic hyperglycemia.The disease has become a global health concern due to its high prevalence and related disability and mortality.Hepatic gluconeogenesis plays an important role in maintaining glucose homeostasis and abnormally enhanced gluconeogenesis is one of the pathological features of T2 DM.Metformin,a commonly used hypoglycemic drug,has been proven to act by regulating hepatic gluconeogenesis.Further in-depth research on the molecular mechanism of hepatic gluconeogenesis will help to discover therapeutic targets for T2 DM that are effective against gluconeogenesis.The CREB binding protein(CBP)and its paralogue p300 are transcriptional coactivators which are essential for a multitude of signaling pathways and energy homeostasis.CBP/p300 functions as a co-activator to enhance the transcriptional activity of CREB,thereby promoting the expression of key gluconeogenic enzymes.However,the regulation mechanism of interaction between CBP/p300 and CREB has not yet been fully elucidated,which will be further addressed in the present study.On the other hand,CBP/p300 belongs to the histone acetyltransferase(HAT)family in addition to transcriptional co-activator.But the role of CBP/p300 HAT domain in regulating energy balance is still unclear.Here,we made use of a recently reported selective inhibitor of CBP/p300 HAT activity A-485 to get a better understanding of its action in metabolic balance,especially in hepatic gluconeogenesis.Contents and Methods1.C57BL/6 mice fed with either normal chow diet(NCD)or high-fat diet(HFD)were intraperitoneally injected with A-485(20 mg/kg)or vehicle for 1 week.Hepatic gluconeogenic and other metabolic changes were then analyzed;2.After liver and white adipose tissue(WAT)were isolated from experimental mice,the expressions of gluconeogenic and lipogenic genes were detected by real-time PCR and Western blot;the effect of A-485 on FOXO1-targeted gluconeogenic gene was verified by Chromatin immunoprecipitation analysis;3.Stromal vascular(SV)cells were isolated from i WAT of C57BL/6 mice for white adipocyte differentiation.The regulatory effect of A-485 on fully differentiated mature adipocytes were observed in vitro;4.In order to verify the role of CBP/p300 HAT in hepatic lipogenesis and gluconeogenesis in vitro,primary mouse hepatocytes were isolated from C57BL/6 mice by a two-step perfusion technique.Real-time PCR and Western blot were used to explore the related molecular mechanism;5.4-week-old db/m and db/db mice were intraperitoneally injected with BT2(40 mg/kg)or vehicle for1 week.Gluconeogenic and other metabolic changes were then analyzed;6.The regulatory effect of BCKDK on hepatic gluconeogenesis was observed by treating primary mouse hepatocytes with BT2 or BCKDK-overexpressing adenovirus.Real-time PCR and Western blot were used to explore the related molecular mechanism;7.Dual-luciferase reporter,immunofluorescence staining,and immunoprecipitation were performed on HepG2 cells to further study the regulatory mechanism of BCKDK on hepatic gluconeogenesis.Results1.A-485 decreased body weight and fat mass in NCD-fed and HFD-fed mice;2.A-485 treatment reduced hepatic glucose production in NCD-fed and HFD-fed mice;3.A-485 lowered fat mass mainly via repressing lipid biosynthesis in WAT.The activity of CBP/p300 HAT domain is indispensable for maintaining normal adipogenesis and lipogenesis in WAT;4.A-485 repressed the expression of hepatic lipogenesis and gluconeogenesis related genes;5.Lowered protein level of FOXO1 was involved in A-485-mediated suppression of gluconeogenesis;6.A-485 deacetylated hepatic FOXO1 and reduces its nuclear abundance;7.A-485 destabilized FOXO1 protein through promoting its ubiquitination;8.A-485 treatment attenuated the binding of FOXO1 to the promoter of G6 pc.9.cAMP increased the protein expression of hepatic BCKDK without changing its m RNA level;10.Inhibition of BCKDK by BT2 repressed hepatic gluconeogenesis and reduced hepatic glucose production in db/db mice;11.Overexpression of BCKDK enhanced hepatic gluconeogenesis,along with increased expressions of gluconeogenic enzymes;12.BT2 inhibited CREB transcriptional activity by dissociating CBP from CREB;13.BT2 destabilized FOXO1 protein through promoting its ubiquitination.Conclusions1.A-485 disrupts lipogenesis in WAT and liver as well as suppresses hepatic gluconeogenesis in C57BL/6 mice,leading to the loss of body weight and improvement of glucose metabolism.A-485-mediated degradation of FOXO1 protein through a proteasome-dependent pathway is involved in the suppression of gluconeogenesis and lipogenesis.2.BCKDK promotes the transcriptional expression of gluconeogenic enzymes by enhancing the interaction between CBP and CREB as well as the stability of FOXO1 protein.