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. 2024 Sep 27;20(9):e1012565.
doi: 10.1371/journal.ppat.1012565. eCollection 2024 Sep.

Altered hepatic metabolic landscape and insulin sensitivity in response to pulmonary tuberculosis

Mrinal K Das  1 , Ben Savidge  1 , John E Pearl  1 , Thomas Yates  2   3 , Gareth Miles  4 , Manish Pareek  1   3   5 , Pranabashis Haldar  1   3   6 , Andrea M Cooper  1   3
Affiliations

Altered hepatic metabolic landscape and insulin sensitivity in response to pulmonary tuberculosis

Mrinal K Das et al. PLoS Pathog. .

Abstract

Chronic inflammation triggers development of metabolic disease, and pulmonary tuberculosis (TB) generates chronic systemic inflammation. Whether TB induced-inflammation impacts metabolic organs and leads to metabolic disorder is ill defined. The liver is the master regulator of metabolism and to determine the impact of pulmonary TB on this organ we undertook an unbiased mRNA and protein analyses of the liver in mice with TB and reanalysed published data on human disease. Pulmonary TB led to upregulation of genes in the liver related to immune signalling and downregulation of genes encoding metabolic processes. In liver, IFN signalling pathway genes were upregulated and this was reflected in increased biochemical evidence of IFN signalling, including nuclear location of phosphorylated Stat-1 in hepatocytes. The liver also exhibited reduced expression of genes encoding the gluconeogenesis rate-limiting enzymes Pck1 and G6pc. Phosphorylation of CREB, a transcription factor controlling gluconeogenesis was drastically reduced in the livers of mice with pulmonary TB as was phosphorylation of other glucose metabolism-related kinases, including GSK3a, AMPK, and p42. In support of the upregulated IFN signalling being linked to the downregulated metabolic functions in the liver, we found suppression of gluconeogenic gene expression and reduced CREB phosphorylation in hepatocyte cell lines treated with interferons. The impact of reduced gluconeogenic gene expression in the liver was seen when infected mice were less able to convert pyruvate, a gluconeogenesis substrate, to the same extent as uninfected mice. Infected mice also showed evidence of reduced systemic and hepatic insulin sensitivity. Similarly, in humans with TB, we found that changes in a metabolite-based signature of insulin resistance correlates temporally with successful treatment of active TB and with progression to active TB following exposure. These data support the hypothesis that TB drives interferon-mediated alteration of hepatic metabolism resulting in reduced gluconeogenesis and drives systemic reduction of insulin sensitivity.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Mycobacterium tuberculosis alters the immunometabolic landscape of the liver.
(a) Changes in lipid and liver enzyme profiles of adult, drug-naïve TB patients compared to healthy controls, derived from biochemical parameters reported in 15 published articles (details in S1 Data). Number of subjects per parameter are reported in parentheses next to the bar. (b) Experimental design to show days of organ harvest and analysis post aerosol Mtb infection of mice. (c) Bacterial load in lung and liver post infection (n = 10). (d) Immunohistochemical staining of CD45 expression in the lung and liver of 42 day infected mice. (e) K-means cluster analysis of significantly differentially expressed genes (padj <0.05) with the top 5 non redundant pathways listed beside the corresponding clusters (n = 4 mice/group). (f) Cluster analysis of significantly differentially expressed proteins (FDR <0.1) by proteomics analysis with the top 5 non redundant pathways listed beside the corresponding clusters (n = 3 mice/group). For (e)-(f) male mice were used.
Fig 2
Fig 2. Aerosol infection of Mycobacterium tuberculosis shows tissue specific metabolism changes.
(a) Livers (in house), lung (GSE137092) and blood (GSE137092) RNAseq data was compared between infected (42 day) and uninfected mice (n = 4–5) for non redundant pathways enriched by upregulated gene (FC 1.5, padj <0.05; upregulated genes in liver n = 520; lung n = 2862; blood n = 1207). (b) Non redundant pathways enriched by downregulated gene (FC 1.5, padj<0.05, downregulated genes in liver n = 163; lung n = 3450; blood n = 664). Pathways uniquely enriched in liver are highlighted as bold. (c) Ligand-receptor analysis shows interactions between lung, liver and blood of 42 day infected mice. Colour of the line represents each tissue and thickness represents number of genes. The outer arc covering each tissue represents number of overall interactions covering the specific tissue with others. The middle arch represents the number of the receptors in the covering tissue that have ligands in the same and other 2 tissues. The inner most arc represents number of ligands of the covering tissue that have receptors in same and other 2 tissues. (d) Inflammatory cytokine gene expression (fold change in colour-scale, gene not quantified due to below threshold in the transcriptomics data matrix i.e., <1 cpm are presented in grey). (e) Interferon gamma mRNA level quantified using RT-PCR analysis. (f) Western blot analysis of interferon signalling proteins in the bulk liver tissue of uninfected and infected mice. (g) Histochemical analysis of liver tissue of uninfected and 42 day infected mice of phospho-Stat-1 (magenta) in CD45 (green) positive and negative cells, nuclear stain (DAPI-blue) and pseudo-DAB in brown. Panels (f) and (g) are representative of 2 independent experiments (n = 5–6 per group per experiment).
Fig 3
Fig 3. Pulmonary TB suppresses gluconeogenic gene expression in the liver.
(a) Hierarchical clustering of genes involved in glycolytic and gluconeogenic gene expression. (b) Single cell RNAseq analysis of liver cells from GSE192742 to deconvolute the glycolytic and gluconeogenic genes from Fig 3a. (green boxes: upregulated, red boxes: downregulated). (c) Differential expression of gluconeogenic and glycolytic genes in the livers of aerosol Mtb infected mice (Tuberculosis_21D and Tuberculosis _42D) compared to the differential expression of the same genes in acute (day 8) and chronic (day 30) LCMV clone 13 infection (LCMV_day 8 and LCMV_day 30) and of these genes in an acute model of sepsis (CLP_sepsis). The colour of each node represents fold changes and the fold changes that are statistically significant padj <0.05 are represented with bold black margin.
Fig 4
Fig 4. Mtb infection suppresses phosphorylation of CREB, a gluconeogenesis controlling transcription factor and interferon treatment compromises gluconeogenesis gene expression and protein phosphorylation in the hepatocyte.
(a) Phosphorylation of proteins involved in glucose metabolism in the livers of uninfected (UN) and aerosol Mtb infected mice (21D inf, 42D inf) (blot shows n = 3, with combined intensity measurements (n = 6) shown in panels at right of blots). (b) cAMP level in livers of uninfected (UN) and Mtb aerosol infected (42D) mice. (c) Hepatocyte cell line HepG2, was cultured with IFN-γ (1200u/ml), IFN-β (1000U/ml) or a combination of both (IFN-γβ) for 16hrs and interferon signalling mRNA i.e., Irf1 and Mx1 and gluconeogenic mRNA i.e., G6pc quantified using qRT-PCR (n = 6). (d) Hepatocyte cell line, HepG2 was left untreated (control) or was treated with gluconeogenesis inducer forskolin (FSK) for 16 hrs in the absence (control) or presence of a combination of IFNγ (1200u/ml) and IFNβ (1000U/ml) (IFNγβ) and G6pc gene expression was quantified. (e) Hepatocyte cell line, HepG2 was incubated with IFN-γ (1200u/ml), IFN-β (1000U/ml) or a combination of both (IFN-γβ) for 16hrs and the level of phosphorylated CREB determined by Western bot (n = 3 in blot shown), intensity of phosphorylation band n = 6. For western blot and RT-PCR at least 2 independent experiments (n = 3 from each) were used for quantification. Anova and Student’s t-test were used for calculation of band intensity in western blot and mRNA relative level in RT-PCR. *<0.05, **<0.01, ***<0.001, ****<0.0001.
Fig 5
Fig 5. Mtb infection and TB progression are associated with insulin resistance.
(a, b and c) Mice were left uninfected (control, blue symbols) or aerosol infected with Mtb (42d Inf, red symbols), fasted and then treated with either (a) pyruvate, (b) glucose or (c) insulin and the level of glucose in the blood determined over time (n = 6–8 from 2 independent experiment, age and gender matched mice) (a-c, multiple t-test with comparison between uninfected and infected for each time point from 2 independent experiments combined). (d) Western blot and densitometry quantification of phosphorylation of proteins in the livers of uninfected (No Inf) or aerosol Mtb infected (Inf) insulin injected mice (n = 5–10, from 2 independent experiment, age and gender matched mice, Student’s t-test was used for densitometric analysis). (e, f) Reanalysis of metabolomics data (Veiling et. al. 2019), comparing the BCAA related metabolite pattern (PCA, PC2 score) in plasma between healthy controls (HC) and untreated TB patients (TB) (e) and over time from baseline (0w) to 28 weeks of treatment (28w) and healthy controls (HC) (f). (g, h) Reanalysis of metabolomics data (GC6-74), from exposed individuals who either did (progressor, red symbols) or did not (non-progressor blue symbols) progress to TB over 24 months. (g) Direct comparison of BCAA related metabolite pattern (PCA, PC2 score) for both plasma and serum between progressor and non-progressor. (h) Correlation analysis between the time to progression to TB disease (left, progressor) or to discharge (right, non-progressor) and changes over time of BCAA-related metabolic pattern (PCA, PC2 score) in serum samples. (e-h, comparisons between groups- Mann Whitney (e, g) or Kruskal Wallis test (f) and Spearman correlation (ρ).
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