ChREBP is activated by reductive stress and mediates GCKR-associated metabolic traits

Abstract

Common genetic variants in glucokinase regulator (GCKR), which encodes GKRP, a regulator of hepatic glucokinase (GCK), influence multiple metabolic traits in genome-wide association studies (GWASs), making GCKR one of the most pleiotropic GWAS loci in the genome. It is unclear why. Prior work has demonstrated that GCKR influences the hepatic cytosolic NADH/NAD⁺ ratio, also referred to as reductive stress. Here, we demonstrate that reductive stress is sufficient to activate the transcription factor ChREBP and necessary for its activation by the GKRP-GCK interaction, glucose, and ethanol. We show that hepatic reductive stress induces GCKR GWAS traits such as increased hepatic fat, circulating FGF21, and circulating acylglycerol species, which are also influenced by ChREBP. We define the transcriptional signature of hepatic reductive stress and show its upregulation in fatty liver disease and downregulation after bariatric surgery in humans. These findings highlight how a GCKR-reductive stress-ChREBP axis influences multiple human metabolic traits.

Keywords: ChREBP; FGF21; GCK; GCKR; MLIXPL; NAD(+); NADH; fatty liver disease; gastric bypass surgery; metabolism; reductive stress; trigylcerides.

Figure 1.. Increased cytosolic NADH/NAD⁺ induces hepatic transcriptional changes.

(A-B) Schematic of the effects of LbNOX, EcSTH, and ethanol on NADH and NAD⁺ and α-hydroxybutyrate in the liver. (C) A combination of ethanol gavages, hepatic LbNOX and EcSTH expression raises or lowers hepatic NADH/NAD⁺ as measured by circulating α-hydroxybutyrate. LbNOX/EtOH data was previously reported in reference 1. (D) Hepatic RNAseq demonstrates approximately 160 transcripts are NADH/NAD⁺ responsive. (E) Examples of NADH/NAD⁺ responsive genes include Fgf21, Acaca, and Pnpla3. Values denote mean +/− s.e.m unless otherwise noted. n= 4–8 (C), 3–5 (D), or 4 (E) per group. * p < 0.05, ** p <0.01; *** p < 0.001.

Figure 2:. ChREBP mediates NADH/NAD+-dependent hepatic transcriptional changes:

(A) Overrepresentation analysis of NADH/NAD⁺ sensitive transcripts identifies ChREBP-related pathways (B) Transcript abundance of ChREBP canonical targets G6pc, Pklr, Acly, Fasn and Me1 in the context of +/−LbNOX +/−ethanol gavages (top row), EcSTH/GFP (middle row), and ChREBP WT/KO +/− ethanol gavage (bottom row) experiments. (C) Mean z-score of gene expression of NADH/NAD⁺-responsive genes in different mouse experiments. (D) Overlap of NADH/NAD⁺-responsive genes and ChREBP/ethanol responsive genes. (E) Chea3 transcription factor enrichment analysis (TFEA) of 1632 transcription factors in the overlapping (N=116 genes) and (F) non-overlapping NADH/NAD+ and ChREBP/EtOH responsive gene sets. Data are mean +/− s.e.m unless otherwise noted. n=3–5ewawd.* p < 0.05, ** p <0.01; *** p < 0.001.

Figure 3:. Elevated cytosolic NADH/NAD⁺ activates ChREBP.

Relative (A) final lactate/pyruvate ratio, (B) αHB, (C) ChREBP mRNA in primary hepatocytes from n=3 mice with different clamped media lactate/pyruvate ratios. (D) Design of a ChREBP/luciferase reporter construct. (E) ChREBP expression in 239T cells, with (F) relative luciferase activity with and without ChREBP and (G) with different glucose concentrations. (H) Effect of LbNOX expression on media lactate/pyruvate ration and (I) ChREBP activity. (J) Effect of EcSTH on media lactate/pyruvate levels and (K) ChREBP activity in HEK 293T cells. P values were determined using unpaired, Student’s t test (F, H, I, K, L) or one-way ANOVA (G). Data are mean +/− s.e.m unless otherwise noted. * p < 0.05, ** p <0.01; *** p < 0.001. n=3 for all experiments except for H (6) and J (7).

Figure 4:. Metabolic perturbations of ChREBP activity correlate with intracellular phospho esters.

(A) Luciferase activity of ChREBP reporter system after transfection with different metabolic enzymes. (B) PCA of intracellular metabolites. Correlation of intracellular (C) Glyceradehyde-3-phosphate, (D) Fructose 1,6-bisphopshate, (E) NADH, and (F) Glycerol-3-phosphate. (G) Xylulose-5-phosphate, (H) Glucose-6-phosphate. (I) Intracellular NADH/NAD⁺ are linked to F1,6BP, GAP, and G3P via the dehydrogenases GAPDH and GPDH. Data are mean +/− s.e.m unless otherwise noted. n=4 for all experimental groups.

Figure 5:. Effect of GCKR and GCK on ChREBP and NADH/NAD⁺.

Effect of GCKR overexpression on (A) media lactate/pyruvate ratio and (B) ChREBP transcript abundance in HepaRG cells. (C) Effect of GCK expression with and without LbNOX on ChREBP reporter activity in HEK 293Ts. (D) Preranked GSEA of the NADH/NAD⁺ response gene set in RNA-Seq with TT versus CC genotypes in human liver biopsies and (E) human liver organoids with different GCKR gneotypes. Data are mean +/− s.e.m unless otherwise noted. * p < 0.05, ** p <0.01; *** p < 0.001. n=3 (A,B,D), 4 (C), or 9 (E).

Figure 6:. EcSTH and ChREBP influence GCKR-associated metabolic traits.

Circulating FGF21 is influenced by hepatic EcSTH (A) and ethanol via ChREBP (B) as are hepatic triglycerides (C and D). E (top): Serum triglyceride species Z score by genotype and gavage, (bottom) p and b value of effect of GCKR rs1260326 on triglyceride species taken from reference. * indicates TAG species formally linked to GCKR genetic variation in human GWAS studies. (F-H) Specific TAG species associated with GCKR rs1260326 in humans and the influence of alcohol and ChREBP on its abundance. (I) Overlap of GCKR, FGF21, or MLXIPL influenced traits from Type 2 Diabetes Knowledge Portal. Data are mean +/− s.e.m unless otherwise noted. * p < 0.05, ** p <0.01; *** p < 0.001.

Figure 7:. αHB, FGF21, serum triglycerides, and the transcriptional signature of hepatic reductive stress is increased in patients with fatty liver disease

(A) Serum αHB, (B) FGF21, and (C) triglycerides are elevated in patients with NAFLD compared to healthy controls. p values calculated from Welch’s t-test. (D) The transcriptional signature of reductive stress is upregulated in multiple human liver genomic data sets of patients with NAFLD. In the box and whisker plots the horizontal line represents the median, the top and bottom of the box are the 75^th and 25^th percentile, respectively, and the top/bottom error bars reflect the largest/smallest value within 1.5 times of the interquartile range beyond the 75^th/25^th percentile. *** p < 0.001. (E) Proposed model describing how hepatic NADH/NAD+ influences metabolic traits.