The transcriptional corepressor CtBP2 serves as a metabolite sensor orchestrating hepatic glucose and lipid homeostasis

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  Fig. 1. Unraveling CtBP2 cistrome leads to an identification of CtBP2/FoxO1 complex. a–c CtBP2 ChIP-seq analysis in normal mouse liver tissues (6-h fasted). a Gene ontology analysis. Upper: biological process, lower: BioCyc pathway. b ChIP-seq peaks at representative metabolic gene loci. c Motif analysis of CtBP2 binding sites. The motifs enclosed by a rectangle are CtBP2-binding motifs enriched in the ChIP-seq. Prediction of known transcription factors targeted by CtBP2 based on sequence similarity is shown below. The statistical evaluation was performed based on the specific algorithm85. d Mutation of the PSDL motif in FoxO1(Mut: PSDL > PSAS) diminishes CtBP2/FoxO1 interaction in HEK293 cells. e, f Primary hepatocytes were stimulated with vehicle or 2 μM forskolin (fsk) after knockdown (e) or overexpression (f) of CtBP2. e The effect of CtBP2 and/or FoxO1 knockdown on G6pc expression (n = 5, biologically independent cells). The y-axis scale is expanded in the inset to show the data in the absence of fsk. The p values are as follows: 0.011 for sh-cont vs sh-CtBP2, 0.036 for sh-CtBP2 vs sh-FoxO1, 0.022 for sh-cont/fsk vs sh-FoxO1/fsk, 1.6 × 10−4 for sh-cont/fsk vs sh-CtBP2/fsk, 9.4 ×…

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  Fig. 2. CtBP2 confers metabolite-sensing capabilities to FoxO1. a The NADH-sensing capability of CtBP2/FoxO1 complex. Increasing concentrations of NADH (0, 0.5, 5, 50 μM) were added to the HEK293 cell lysates expressing indicated proteins. b Direct binding of recombinant CtBP2 protein to oleoyl-CoA and NADH analyzed by MST (n = 3 or 4 for each point). c Computer-assisted structural analysis of CtBP2 with palmitoyl-CoA. Palmitoyl-CoA is localized at the interface of CtBP2 adopting dimeric configuration. d The fatty acyl-CoA-sensing capability of CtBP2/FoxO1 complex. Increasing concentrations of oleoyl-CoA (0, 50, 150, 500 μM) were added to the HEK293 lysates. e The thermal shift induced by NADH and oleoyl-CoA binding in DSF. Recombinant CtBP2 were mixed with either NADH (20 μM) or oleoyl-CoA (20 μM) and thermal stability was monitored on a temperature gradient. f The effect of cytosolic redox state on CtBP2/FoxO1 complex. HEK293 cells expressing FLAG-FoxO1 and CtBP2 were incubated with different ratios of lactate and pyruvate for 1 h. g HEK293 cells expressing indicated proteins were incubated with control BSA or palmitate-BSA conjugate (200 μM) for 30 min. h Schematic diagram showing the…

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  Fig. 3. CtBP2 is markedly inactivated in the liver of obesity. Liver homogenates from mice: a–d, l normal mice, e, h genetically obese mice, f, i diet-induced obese mice (12–16 weeks on high-fat diet(HFD), RD: regular diet) and human liver autopsy specimens (g) were subjected to co-immunoprecipitation to examine endogenous CtBP2/FoxO1 complex (a–g) or CtBP2/SREBP1 complex (h, i, l, n = 3 or 4 biological independent animals as shown in the blots for each experimental group). The densitometric quantification is shown to the right of each blot. a–d CtBP2/FoxO1 complex in normal mice in response to either a fasting-feeding cycle (a 24-h-fasted or fed ad libitum), glucagon (b 30 min after injection, 200 μg/kg, 3-h food withdrawal, p = 1.1 × 10−3), insulin (c 30 min after injection, 0.4 U/kg, 3-h food withdrawal, p = 6.2 × 10−3), or glucose (d 30 min after injection, 2 g/kg, overnight fasted, p = 9.5 × 10−3). e–i CtBP2/FoxO1 complex in mouse models of obesity (e, f p = 1.3 × 10−5 and 4.1 × 10−4, respectively) or in human obesity (liver autopsy samples. Body mass index (BMI) for each subject was indicated above the blot, g p = 0.022), or CtBP2/SREBP1 complex in mouse models of obesity (h, i p =…

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  Fig. 4. Dissociation of CtBP2 from FoxO1 occurs in the nucleus. a CtBP2 recruitment to promoter regions of gluconeogenic genes (n = 4, biologically independent animals, p values are as follows: 0.011 for G6pc and 2.4 × 10−6 for Pck1). HFD diet-induced obese mice. b Sequential ChIP (re-ChIP) in primary hepatocytes. FLAG-CtBP2 and FoxO1 were exogenously expressed, the chromatin was first enriched by FLAG (CtBP2) immunoprecipitation and the eluents were further immunoprecipitated with either control IgG or anti-FoxO1 antibody (n = 3, biologically independent cells, p = 2.5 × 10−3). c FoxO1 recruitment to promoter regions of gluconeogenic genes (n = 4, biologically independent animals). Liver tissues were collected following a 6-h fast (a, c). d FoxO1-DsRed and/or CtBP2-GFP were expressed in hepa1-6 cells and treated with vehicle or 100 nM insulin for 30 min. The yellow bars indicate 10 μm. e The forced cytoplasmic localization of FoxO1 markedly diminishes CtBP2/FoxO1 interaction. Either the wild-type FoxO1 (WT) or the mutant FoxO1 (△DB) was exogenously expressed in HEK293 cells, and the CtBP2/FoxO1 complex was co-immunoprecipitated. Data are expressed as the mean ± SEM. * and ** denote p <…

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  Fig. 5. CtBP2 deficiency in liver leads to obesity-associated metabolic disturbances. a Liver-specific CtBP2-deficient mice (KO) maintained on a regular chow diet (a–h) or an MCD diet for a week (h, i) (6–8 weeks of age, a, c, e–i: 6-h fasted, b, d: overnight fasted) were analyzed along with their wild-type controls (WT). a CtBP1 and CtBP2 expression at protein levels in either liver or adipose tissues. b Glucose tolerance test (GTT) (n = 16 for WT and n = 8 for KO, biologically independent animals, p values are as follows: 4.3 × 10−3 for 0 min, 4.4 × 10−4 for 15 min, 2.2 × 10−4 for 30 min, 2.0 × 10−6 for 60 min, 2.6 × 10−5 for 90 min, 8.1 × 10−3 for 120 min). c Gluconeogenic gene expression (n = 19 for WT and n = 9 for KO, biologically independent animals, p = 2.5 × 10−4 and 1.0 × 10−3 for G6pc and Pck1, respectively). d Pyruvate tolerance test (PTT) (n = 12 for WT and n = 6 for KO, biologically independent animals, p values are as follows: 1.2 × 10−3 for 0 min, 1.9 × 10−3 for 15 min, 3.1 × 10−3 for 30 min, 3.5 × 10−4 for 60 min, 1.9 × 10−5 for 90 min, 1.4 × 10−4 for 120 min). e Liver triglyceride (TG) content (n = 11 for WT and n = 9 for KO, biologically independent animals, p =…

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  Fig. 6. CtBP2 gain of function in liver ameliorates diabetes and hepatic steatosis in obese mice. CtBP2 was exogenously expressed in the liver of diet-induced obese mice by adenoviral transduction (AdCtBP2), along with its control (AdGUS). Animals were analyzed after 3–7 days of transduction (a–d, h overnight fasted, e–g, i 6-h fasted). a Blood glucose levels (n = 4 for GUS/HFD, n = 5 CtBP2/HFD, and n = 5 for GUS/lean, biologically independent animals, p = 7.8 × 10−4). HFD high-fat diet, RD regular diet. b Glucose tolerance test (GTT, n = 10, biologically independent animals, p -values are 8.8 × 10−3 for 0 min, 1.0 × 10−8 for 15 min, 1.4 × 10−4 for 30 min). c Pyruvate tolerance test (PTT, n = 9 for GUS, n = 10 for CtBP2, biologically independent animals, p values are 7.6 × 10−6 for 0 min, 2.5 × 10−9 for 15 min, 1.1 × 10−7 for 30 min, 2.6 × 10−6 for 60 min, 4.9 × 10−5 for 90 min, 3.0 × 10−4 for 120 min). d Gene expression in liver (n = 4 for GUS, n = 5 for CtBP2, biologically independent animals, p = 0.015). e Representative hematoxylin and eosin stained sections of liver. Yellow bars indicate 100 μm. f Liver triglyceride (TG) content (n = 10, biologically independent animals, p = 6.6 ×…

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  Fig. 7. The structure–function relationships in CtBP2 reveals critical coupling of metabolite-sensing with transcriptional regulation. a Either wild-type CtBP2 (WT), CtBP2 mutated in its Rossmann fold (mut) or a control gene (GUS) was expressed in the liver of diet-induced obese (HFD) mice by adenoviral transduction and the expression of metabolic genes was analyzed (n = 7, biologically independent animals, 6-h fasted, p values for GUS vs WT/WT vs mut are follows: 8.6 × 10−4/5.4 × 10−4 (G6pc), 1.1 × 10−3/0.048 (Scd1), 4.0 × 10−3/3.3 × 10−3 (Fasn), and 2.9 × 10−3/8.2 × 10−4 (Srebf1c), respectively). b Heatmap showing gene expression levels examined by RNA-seq. c–e Gene ontology analysis of RNA-seq data. Biological process (c), cellular component (d), molecular function (e). f Three-dimensional principal component analysis. Pink: GUS, blue: wild-type CtBP2, green: mutant CtBP2. g The CtBP2/FoxO1 complex formation was analyzed using purified recombinant proteins in vitro. Increasing concentrations of oleoyl-CoA (0, 15, 50 μM) with indicated concentrations of NADH (0 or 10 μM) were included. The p values are as follows: 9.0 × 10−3 for control vs NADH+/oleoyl-CoA 0 μM, 0.021 for…

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  Fig. 8. Dimerization is a key step for CtBP2 activation. a Screening of mutations to stabilize the CtBP2 dimerization based on the computer-assisted structural analysis. y-axis: △stability, x-axis: △affinity. Red rectangle shows promising mutations to potentially promote CtBP2 dimerization. b Examination of dimer formation by co-immunoprecipitation of differentially tagged CtBP2 (interaction between FLAG-CtBP2 and CtBP2-HA, n = 3 biologically independent cells for each group, p = 1.3 × 10−3). c Cell lysates were obtained from HEK293 cells expressing either wild-type (WT) CtBP2 or ALA201HIS mutant (A201H) CtBP2 with FLAG-FoxO1, and the CtBP2/FoxO1 complex was detected in the presence of the increasing concentrations of oleoyl-CoA (0, 50, 150, 500 μM). d–g Adenovirus-mediated expression of either GUS, wild-type CtBP2 (WT), or ALA201HIS mutant CtBP2 (A201H) in diet-induced obese mice (3 days after transduction, overnight fasted). d, e Blood glucose levels (d n = 16, biologically independent animals, p = 5.9 × 10−6 and 0.012 for GUS vs WT and WT vs A201H, respectively) and liver triglyceride content (e n = 8, biologically independent animals, p = 7.5 × 10−3 and 4.7 × 10−3 for GUS vs WT and…