An obesogenic feedforward loop involving PPARγ, acyl-CoA binding protein and GABAA receptor

Abstract

Acyl-coenzyme-A-binding protein (ACBP), also known as a diazepam-binding inhibitor (DBI), is a potent stimulator of appetite and lipogenesis. Bioinformatic analyses combined with systematic screens revealed that peroxisome proliferator-activated receptor gamma (PPARγ) is the transcription factor that best explains the ACBP/DBI upregulation in metabolically active organs including the liver and adipose tissue. The PPARγ agonist rosiglitazone-induced ACBP/DBI upregulation, as well as weight gain, that could be prevented by knockout of Acbp/Dbi in mice. Moreover, liver-specific knockdown of Pparg prevented the high-fat diet (HFD)-induced upregulation of circulating ACBP/DBI levels and reduced body weight gain. Conversely, knockout of Acbp/Dbi prevented the HFD-induced upregulation of PPARγ. Notably, a single amino acid substitution (F77I) in the γ2 subunit of gamma-aminobutyric acid A receptor (GABA_AR), which abolishes ACBP/DBI binding to this receptor, prevented the HFD-induced weight gain, as well as the HFD-induced upregulation of ACBP/DBI, GABA_AR γ2, and PPARγ. Based on these results, we postulate the existence of an obesogenic feedforward loop relying on ACBP/DBI, GABA_AR, and PPARγ. Interruption of this vicious cycle, at any level, indistinguishably mitigates HFD-induced weight gain, hepatosteatosis, and hyperglycemia.

Fig. 1. PPARγ transcription factor regulates the expression of ACBP.

A Heatmap representation of correlation (R) between ACBP mRNA and mRNA of several genes in the human liver, subcutaneous white adipose tissue (scWAT), and visceral white adipose tissue (viscWAT) (*p < 0.05). B Correlation plots of PPARG and ACBP mRNA in liver and WAT from human (liver: n = 179, visceral WAT: n = 355), mouse (liver: n = 179, epididymal WAT: n = 56), and rat (liver: n = 207, epididymal WAT: n = 47) extracts. Correlation plots of PPARG and ACBP mRNA in subcutaneous white adipose tissue (n = 442), skeletal muscle (n = 304), and the aggregate of all tissues (n = 7172) from human origin. C Venn diagram representation includes transcription factors (TFs) the targets of which are upregulated in bovine adipocytes, murine hepatocytes, and human leukocytes when their donors receive a high-fat diet (left). Significance of the upregulation of PPARγ target genes in each of the three datasets (right). D Chromatin immunoprecipitation sequencing (ChIP-seq) signals of PPARγ and H3K4me3 (Acbp promoter, mouse liver, n = 3). The black line corresponds to the peaks called per MACS. E Silencing of TFs encoding human ACBP in HepG2 cells. F Cytofluorometric peaks quantifying ACBP after silencing the unrelated negative control, ACBP, or PPARG (siUNR, siACBP, or siPPARG). G Heatmap representation of cytofluorometric ACBP protein levels upon silencing various TFs encoding ACBP in HepG2 cells (n = 3; one-way ANOVA). For statistical analyses (A, B) p values and R were calculated by Pearson and Spearman correlations respectively. See also Fig. S1.

Fig. 2. The effects of PPARγ-modulating agents depend on ACBP function.

A Cytofluorometric measurement of ACBP protein after treatment with PPARγ agonists (Ctr: vehicle, Rosi: rosiglitazone, GW1929, S26948, Eda: edaglitazone) (n = 6) in control (siUNR) or PPARG-silenced (siPPARG) HepG2 cells (n = 5) (B) (MFI: mean fluorescence intensity normalized to control). C Representative immunoblot images of PPARγ, ACBP, and β-actin proteins in control (shUNR) and Acbp-knocked down (shAcbp) Hep55.1c cells after treatment with vehicle or Rosi (48 h), densitometric quantification (n = 3) (D, E). F Pparg and Acbp mRNA expression measurements in liver extracts obtained from mice receiving Rosi or vehicle (5 days) (n = 10 to 13 mice per condition). G Plasma ACBP concentration (n = 7 to 12 mice per condition), and H body weight measurements from mice receiving Rosi or vehicle (5 days) (n = 7 to 8 mice per condition). I Liver representative immunoblot images of FASN, PPARγ, ACBP, and β-actin proteins from ACBP-control (ubi:Acbp WT) or ACBP knockout (ubi:Acbp KO) mice receiving vehicle or Rosi (5 days), densitometric quantification (n = 4 to 8 mice per condition) (J–L). M Body weight measurements from mice administrated with Rosi or vehicle (2 months) (n = 4 to 6 mice per condition). Results are displayed as whisker plots (with each dot representing one in vitro biological replicate or one single mouse) including the mean ± SEM. For statistical analyses, p values (indicating statistical comparisons with the control condition) were calculated by a two-tailed unpaired Student’s t-test. For statistical analysis p values were calculated by two-tailed unpaired Student’s t-test (G, H, J–L) applying Welch correction (F), one-way ANOVA (A, B), or two-way ANOVA (D, E, M). MFI mean fluorescence intensity, a.u. arbitrary units, kDa kilodaltons, sh short-hairpin, ubi ubiquitous, ns non-significant. See also Fig. S2.

Fig. 3. Pharmacological and dietary PPARγ manipulations regulate the expression of Acbp.

A Liver representative immunoblot images of PPARγ, ACBP, FASN, and β-actin proteins from mice receiving control (Vehicle), or RXRα agonist Bexarotene (Bex), densitometric quantification (n = 4 to 9 mice per condition) (B). C Liver representative immunoblot images of PPARγ, ACBP, FASN, and β-actin proteins from mice receiving control (Vehicle), or RXRα antagonist HX531 drugs (5 days), densitometric quantification (n = 4 to 8 mice per condition) (D). E Qualitative α-PPARγ chromatin immunoprecipitation (ChIP) analysis from liver extracts obtained from mice receiving regular-chow (RCD) or high-fat diet (HFD). ChIP PCR products in the case of DNA templates originated from chromatin samples that have been precipitated with a PPARγ-specific antibody (α-PPARγ). No product in chromatin samples precipitated with negative isotype control (IgG). No ChIP PCR product in the α-PPARγ sample originating from the whole-body PPARγ knockout mice (ubi:Cre PPARγ KO) (n = 3 per condition). F Quantitative analysis of ChIP Real-Time PCR (n = 4 to 8 mice per condition). G, H Liver and K, L epididymal white adipose tissue (eWAT) Pparg and Acbp mRNA expression measurements obtained from mice receiving RCD or HFD (6 weeks) (n = 7 to 13 mice per condition). I Liver representative immunoblot images of FASN, PPARγ, ACBP, and β-actin proteins from mice receiving RCD or HFD (6 weeks), densitometric quantification (n = 5 mice per condition) (J). Results are displayed as whisker plots (with each dot representing one single mouse) including the mean ± SEM. For statistical analysis p values were calculated by two-tailed unpaired Student’s t-test (G, H, K, L) applying Welch correction (B, D, J), or two-way ANOVA (F). kDa kilodaltons, a.u. arbitrary units, bp base pairs, ns non-significant. See also Fig. S3.

Fig. 4. Liver-specific PPARγ knockout yields decreased ACBP levels.

A Pparg mRNA expression (n = 5 to 15 mice per condition) and B, C protein level measurements (n = 4 to 7 mice per condition) in liver extracts obtained from control (PPARγ WT) or liver-specific CRISPR/Cas9-mediated PPARγ-knockdown mice (PPARγ KD) receiving regular-chow (RCD) or high-fat diet (HFD). For statistical analysis (C) p values were calculated comparing HFD groups to the corresponding RCD group for each genetic background (PPARγ WT, PPARγ KD). D Acbp mRNA expression (n = 5 to 13 mice per condition), E plasma ACBP concentration (n = 5 to 15 mice per condition), and F body weight gain measurements in control or PPARγ KD mice rendered obese (HFD, 2 months) (n = 9 to 10 mice per condition). G Representative hematoxylin eosin (HE) images of control or PPARγ KD livers (n = 15 to 25 mice per condition) obtained from mice receiving RCD or HFD, hepatosteatosis score quantification (bar: 50 μm, ND non-detected) (H). I Fasted blood glucose concentration from control or liver-PPARγ KD mice receiving RCD or HFD (n = 9 to 10 mice per condition). Results are displayed as whisker plots (with each dot representing one single mouse) including the mean ± SEM. For statistical analysis p values were calculated by two-tailed unpaired Student’s t-test (C), two-way ANOVA (A, D–F, I), or Mann–Whitney test (H). kDa kilodaltons, a.u. arbitrary units, ns non-significant, ND non-detected. See also Fig. S4.

Fig. 5. Neutralization or genetic ablation of ACBP results in decreased PPARγ.

A ACBP-neutralizing antibody (α-ACBP) or isotype control (Iso) was administered in vivo by intraperitoneal injection in mice fed with regular-chow (RCD) or high-fat diet (HFD). B Acbp mRNA expression measurement in liver extracts obtained from mice receiving Iso or α-ACBP in RCD or HFD feeding regimens (n = 5 to 9 mice per condition). C Plasma ACBP concentration measurement in Iso—or α-ACBP – treated mice (n = 8 to 10 mice per condition). D, F Liver, epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT) representative immunoblot images of PPARγ and β-actin proteins from mice receiving Iso or α-ACBP (RCD or HFD), densitometric quantification (RCD: n = 5 to 10 mice per condition, HFD: n = 4 to 10 mice per condition) (E, G). H Fasted blood glucose concentration from mice receiving Iso or α-ACBP (RCD or HFD) (n = 5 to 10 mice per condition). I, J eWAT and BAT representative immunoblot images of PPARγ and β-actin proteins from adipocyte-specific ACBP knockout murine model (adipo: Acbp KO) compared to control (adipo: Acbp WT), densitometric quantification (n = 5 to 7 mice per condition) (K, L). Results are displayed as whisker plots (with each dot representing one single mouse) including the mean ± SEM. For statistical analysis p values were calculated by two-tailed unpaired Student’s t-test (C, E, G, K, L) or two-way ANOVA (B, H). a.u. arbitrary units, ns non-significant, kDa kilodaltons. See also Fig. S5.

Fig. 6. Metabolic effects of GABA_AR – ACBP compromised signaling.

A Co-immunoprecipitation (co-IP) describing the physical interaction between the ACBP and the GABA_AR γ2 subunit in liver extracts from mice subjected to a high-fat diet (n = 3 per condition). B Liver representative immunoblot images of GABA_AR γ2 subunit and β-actin proteins from mice receiving regular-chow (RCD) or high-fat diet (HFD) (1 month), densitometric quantification (n = 5 to 9 mice per condition) (C). D Plasma ACBP concentration measurement from WT and F77I mice fed with RCD or HFD (1 month) (n = 5 to 7 mice per condition). E Liver representative immunoblot images of PPARγ and ACBP proteins from WT and F77I mice receiving HFD (1 month), densitometric quantification (n = 5 mice per condition) (F). G Representative HE images of liver sections, H hepatosteatosis score quantification from WT or F77I mice after 1 month of HFD (bar: 50 μm, ND non-detected) (n = 3 to 5 mice per condition). I Body weight measurement from WT and F77I mice fed with RCD or HFD (n = 8 to 23 mice per group). J Heatmap representation of fatty acid, lipid, and carbohydrate plasma concentrations depicted as Area log2-fold change (Area Log2FC) from WT or F77I mice fed with RCD or HFD (1 month) (n = 3 to 13 mice per condition) followed by quantification of representative lipid metabolism-related metabolites (K). Results are displayed as whisker plots (with each dot representing one single mouse) including the mean ± SEM. For statistical analysis p values were calculated by two-tailed unpaired Student’s t-test (F, K) applying Welch correction (C), two-way ANOVA (D, I), or Mann–Whitney test (H). kDa kilodaltons, a.u. arbitrary units, ns non-significant, ND non-detected. See also Fig. S6.