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Multicenter Study
. 2021 Apr;160(5):1725-1740.e2.
doi: 10.1053/j.gastro.2020.12.008. Epub 2020 Dec 10.

Integrated Multiomics Reveals Glucose Use Reprogramming and Identifies a Novel Hexokinase in Alcoholic Hepatitis

Veronica Massey  1 , Austin Parrish  2 , Josepmaria Argemi  3 , Montserrat Moreno  4 , Aline Mello  5 , Mar García-Rocha  6 , Jose Altamirano  7 , Gemma Odena  1 , Laurent Dubuquoy  8 , Alexandre Louvet  8 , Carlos Martinez  6 , Anna Adrover  6 , Silvia Affò  4 , Oriol Morales-Ibanez  4 , Pau Sancho-Bru  4 , Cristina Millán  4 , Edilmar Alvarado-Tapias  9 , Dalia Morales-Arraez  5 , Juan Caballería  10 , Jelena Mann  11 , Sheng Cao  12 , Zhaoli Sun  13 , Vijay Shah  12 , Andrew Cameron  13 , Phillipe Mathurin  8 , Natasha Snider  14 , Càndid Villanueva  15 , Timothy R Morgan  16 , Joan Guinovart  6 , Rajanikanth Vadigepalli  2 , Ramon Bataller  17
Affiliations
Multicenter Study

Integrated Multiomics Reveals Glucose Use Reprogramming and Identifies a Novel Hexokinase in Alcoholic Hepatitis

Veronica Massey et al. Gastroenterology. 2021 Apr.

Erratum in

  • Correction.
    [No authors listed] [No authors listed] Gastroenterology. 2021 Sep;161(3):1075. doi: 10.1053/j.gastro.2021.06.068. Epub 2021 Jul 1. Gastroenterology. 2021. PMID: 34332936 No abstract available.

Abstract

Background & aims: We recently showed that alcoholic hepatitis (AH) is characterized by dedifferentiation of hepatocytes and loss of mature functions. Glucose metabolism is tightly regulated in healthy hepatocytes. We hypothesize that AH may lead to metabolic reprogramming of the liver, including dysregulation of glucose metabolism.

Methods: We performed integrated metabolomic and transcriptomic analyses of liver tissue from patients with AH or alcoholic cirrhosis or normal liver tissue from hepatic resection. Focused analyses of chromatin immunoprecipitation coupled to DNA sequencing was performed. Functional in vitro studies were performed in primary rat and human hepatocytes and HepG2 cells.

Results: Patients with AH exhibited specific changes in the levels of intermediates of glycolysis/gluconeogenesis, the tricarboxylic acid cycle, and monosaccharide and disaccharide metabolism. Integrated analysis of the transcriptome and metabolome showed the used of alternate energetic pathways, metabolite sinks and bottlenecks, and dysregulated glucose storage in patients with AH. Among genes involved in glucose metabolism, hexokinase domain containing 1 (HKDC1) was identified as the most up-regulated kinase in patients with AH. Histone active promoter and enhancer markers were increased in the HKDC1 genomic region. High HKDC1 levels were associated with the development of acute kidney injury and decreased survival. Increased HKDC1 activity contributed to the accumulation of glucose-6-P and glycogen in primary rat hepatocytes.

Conclusions: Altered metabolite levels and messenger RNA expression of metabolic enzymes suggest the existence of extensive reprogramming of glucose metabolism in AH. Increased HKDC1 expression may contribute to dysregulated glucose metabolism and represents a novel biomarker and therapeutic target for AH.

Keywords: Alcoholic Liver Disease; Metabolomics; Therapeutic Targets.

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Conflict of interest statement

Conflicts of interest

The authors disclose no conflicts.

Figures

Figure 1.
Figure 1.
Schematic work flowchart of human samples analyses. (A) Liver metabolomics and RNA-seq were performed by using a cohort of normal liver fragments from resection for liver metastasis (n = 15), livers from patients with AH (n = 12), and livers from patients with AC (n = 10). (B) Liver ChIP-seq data were retrieved from a previous study (Database of Genotypes and Phenotypes phs001807). Data from a validation cohort of patients with AH (n = 15) and control individuals (n = 7) were retrieved from previous study (GSE28619). (D) Real-time PCR validation and serum HKDC1 measurements were performed in a cohort of normal liver tissue (n = 13) and from patients with AH (n = 69), AC (n = 26), HCV-infected patients (n = 26), and patients with NASH (n = 37). dbGAP, Database of Genotypes and Phenotypes; RP/UPLC-MS/MS, reversed phase/ultraperformance liquid chromatography coupled to tandem mass spectrometry.
Figure 2.
Figure 2.
The hepatic metabolome is altered in patients with AH. Metabolomics was performed at Metabolon, Inc, as described in the Materials and Methods section. (A) Plot of the Principal Component Analysis of the hepatic metabolome of normal liver tissue (control), AC, and AH. (B) Venn diagram representing the metabolites found to be significantly changed by multiple comparison testing between AH and control, between AC and control, and between AH and AC. (C) Heatmap showing the normalized expression of all metabolites detected by metabolomics analysis of the liver tissue. Annotation indicates which metabolites were significantly changed in patients with AH compared to either control individuals or patients with AC (yellow), only significantly changed in patients with AH compared to control individuals (gray), and only significantly changed in patients with AH compared to patients with AC (blue).
Figure 3.
Figure 3.
Heatmap showing normalized mRNA expression levels of enzymes involved in intermediate metabolism. RNA-seq of human liver tissue was performed as described in the Materials and Methods section. A subset of genes involved in intermediate metabolism pathways is shown. The color scale represents sample Z-score. Statistical significance was determined by using the DESeq2 package and was adjusted for multiple comparisons. Statistical significance is reported as adjusted P values compared to control.
Figure 4.
Figure 4.
Integrated analysis of metabolomics and RNA-seq. (A) Metabolic map showing significant changes in gene expresses (boxes) and intermediates (text) of glucose metabolism in patients with AH compared to normal control samples. (B) Representative plots showing correlations between genes and metabolites for control (gray) and AH (orange) samples.
Figure 5.
Figure 5.
Increased hepatic HKDC1 mRNA expression and serum HKDC1 levels in patients with AH are associated with higher incidence of AKI and decreased short-term survival. (A) Representative pictures of HKDC1 immunostaining (original magnification, ×200) in control livers and in livers from patients with AH, ALD cirrhosis, and non-ALD cirrhosis. (B) Gene expression of HKDC1 was analyzed in control (n = 13), AH (n = 69), HCV (n = 26), NASH (n = 37), and compensated AC liver tissue (n = 26). Data are shown as mean ± standard error of the mean. *P < .05 compared to control samples, #P < .05 compared to the other groups. (C) High HKDC1 mRNA expression (≥32-fold of control) is associated with the development of AKI during hospitalization in patients with AH. (D) Kaplan-Meier curve showing the 90-day survival in patients with AH with high (>32-fold of control) or low (<32-fold of control) hepatic HKDC1 expression. (E) Fasting serum levels of HKDC1 were evaluated in samples from healthy control individuals (n = 14), heavy drinkers (n = 10), and in patients with AH (n = 46), HCV (n = 17), NASH (n = 12), and compensated AC (n = 22). Data are shown as mean ± standard error of the mean; *P < .05 compared to all other groups. (F) High HKDC1 serum level (>2900 ng/mL) is associated with the development of AKI during hospitalization in patients with AH. (G) Kaplan-Meier curve showing the 90-day survival in patients with AH with high (>2900 ng/mL) or low (<2900 ng/mL) serum HKDC1 level. (H–J) Rat primary hepatocytes were treated with either AdCMV-HKDC1 or AdCMV-GFP as described in the Materials and Methods section. Cells were serum- and glucose-starved overnight followed by incubation with 30 mmol/L glucose for 4 hours. Hexokinase activity (H), G6P levels (I), and glycogen content (J) were determined as described in the Materials and Methods section. Data represent the mean for 3 independent experiments ± standard error; *P < .05 compared to the AdCMV-GFP–treated cells in medium plus glucose.
Figure 6.
Figure 6.
Genomic view of histone ChIP-seq data showing increased activity in HKDC1 regulatory regions. Data from ChIP-seq in livers from patients with AH (n = 7) and normal livers (n = 5) is presented in the genomic context of the HKDC1 gene. H3K27ac and H3K4me1 were used to mark the activity and location of enhancers, respectively. H3K4me3 was used to verify the activity of the promoter. The H3K27me3 mark indicates the presence of gene silencing. Shadow squares indicate the detected peaks by MACS2.
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