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Gluconeogenesis is one of the major mechanisms to maintain hepatic glucose homeostasis and dysregulation of hepatic gluconeogenesis contributes to hyperglycemia in type 2 diabetes. Under fasted conditions, increases in circulating glucagon promote hepatic glucose production through activation of gluconeogenic pathway by HDAC5 and CREB coactivator CRTC2 (Lv et al., 2016; Lv et al., 2017; Qiu et al., 2017). The circadian clock coordinates behavior and metabolism into rhythms not only in the central hypothalamus but also in peripheral tissues (Marcheva et al., 2010; Vollmers et al., 2009). Transcription factor BMAL1 heterodimerizes with CLOCK to activate the expression of Per and Cry, which in turn suppress CLOCK/BMAL1 activity (Reppert & Weaver, 2002). Although BMAL1 knockout mice show fasting hypoglycemia (Rudic et al., 2004), the detailed mechanism of BMAL1 regulation on hepatic gluconeogenesis has not been thoroughly understood. To explore the function of BMAL1 on hepatic gluconeogenesis, we generated liver-specific BMAL1 knockout mice (BMAL1 LKO) by crossing BMAL1 flox/flox mice with AlbuminCre transgenic mice. BMAL1 LKOmice were indistinguishable from their wild-type littermates in body weight and blood glucose levels under ad lib and fasted conditions (Fig. S1A and S1B). However, fasting serum insulin levels were relatively lower in BMAL1 LKO mice compared with WT littermates (Fig. S1C). When challenged with sodium pyruvate, BMAL1 LKOmice showed decreased gluconeogenesis as assayed by pyruvate tolerance test (PTT) (Fig. S1D). Similarly, the expression of endogenous gluconeogenic genes including G6Pase,Pck1andPgc1awasdecreased in the liver (Fig.S1E), suggesting a pro-gluconeogenic effect of BMAL1. Supporting this scenario, adenovirus-mediated BMAL1 expression (AdBMAL1) in primary hepatocytes greatly prompted forskolin (FSK)-induced gluconeogenic gene expression (G6Pase, Pck1), while BMAL1 knockdown mediated by adenovirus encoding BMAL1 RNAi (Ad-BMAL1i) inhibited gluconeogenic gene expression (Figs. 1A, 1B, S1F and S1G). Since hepatic gluconeogenic gene expression is mainly regulated by the CREB/CRTC2 pathway and HDAC5/ FOXO1 pathway (Altarejos & Montminy, 2011; Mihaylova et al., 2011; Wang et al., 2011), we tried to identify which pathway contributes to the effect of BMAL1 on gluconeogenic gene expression. The promoter of G6Pase, a ratelimiting enzyme for gluconeogenesis, contains binding sites of both CREB/CRTC2 (cAMP response element, CRE) and HDAC5/FOXO1 (insulin response element, IRE) (Lan et al., 2007; Liu et al., 2008). Similar to the effect on endogenous G6Pase mRNA expression, Ad-BMAL1 expression increased FSK-induced G6Pase-luc activity in primary hepatocytes (Fig. S2A). In contrast, FSK-induced CRE-luc (composed of 3 tandem copies of CRE but not IRE sites) activity was not influenced by Ad-BMAL1 expression (Fig. S2B). Furthermore, the effect of Ad-BMAL1 expression on G6Pase-luc activity was largely abolished when IRE sites but not CRE sites were mutated, suggesting that BMAL1 specifically regulates HDAC5 pathway (Fig. S2C). Consistent with the results of Ad-BMAL1 expression, knockdown of BMAL1 by Ad-BMAL1i resulted in attenuation of G6Pase-luc activity in primary hepatocytes (Fig. S2D). HDAC5 is phosphorylated by SIK kinases and sequestered in the cytoplasm under basal conditions. Exposure to FSK triggers HDAC5 dephosphorylation and nuclear translocation where they associate with the promoters of gluconeogenic enzymes such as G6Pase (Mihaylova et al., 2011). Based on the ability of BMAL1 to promote HDAC5-mediated gluconeogenic gene expression, we tested whether BMAL1 modulated HDAC5 dephosphorylation upon FSK stimulation. Indeed, Ad-BMAL1 expression in primary hepatocytes greatly enhanced FSK-induced HDAC5 dephosphorylation, while BMAL1 knockdown by Ad-BMAL1i blocked HDAC5 dephosphorylation upon FSK stimulation (Fig. S2E and S2F). In contrast, FSK-induced CREB phosphorylation was not influenced by BMAL1 overexpression or knockdown (Fig. S2E and S2F). Collectively, these data show that BMAL1 promotes gluconeogenic gene expression through HDAC5 pathway. We next tested whether BMAL1 associates with HDAC5. Supporting this idea, we recovered endogenous BMAL1 from immunoprecipitations (IPs) of endogenous HDAC5 in primary hepatocytes (Fig. 1C). We obtained similar results in

BMAL1 functions as a cAMP-responsive coactivator of HDAC5 to regulate hepatic gluconeogenesis