Dysregulation of cellular metabolism within the gut-brain axis is associated with behavioural changes in chronic intestinal inflammation

Abstract

Background: Inflammatory bowel disease (IBD) is a chronic debilitating condition significantly affecting patient quality of life. Although the exact aetiology remains unknown, accumulating evidence has shown that disruption of the gut-brain axis may be related to the occurrence and development of chronic intestinal inflammation. Psychological disorders are highly prevalent in patients with IBD. However, an association between altered behaviour and dysregulated metabolic pathways within the gut-brain axis is yet to be explored.

Methods: Metabolic multiplexed phenotyping system involving indirect calorimetry and flow-through respirometry monitors was used to assess energy metabolism in Winnie mice with spontaneous chronic colitis and C57BL/6 littermates. Depressive and anxiety-like behaviours were evaluated with light dark, open field, grooming, elevated plus maze, and forced swimming tests. To investigate underlying mechanisms of the metabolic changes in Winnie mice, glycolysis/gluconeogenesis, fatty acid ß-oxidation, tricarboxylic acid cycle and oxidative phosphorylation gene expressions were determined by transcriptome analysis using high-throughput sequencing of mRNA extracted from the distal colon and brain samples.

Results: Our findings showed that energy metabolism and spontaneous activity were reduced in Winnie mice corresponding to alterations in the expression of cellular metabolism-associated genes in the distal colon. Winnie mice displayed depressive and anxiety-like behaviours reflecting downregulation of glycolysis/gluconeogenesis, fatty acid ß-oxidation, tricarboxylic acid cycle and oxidative phosphorylation in the distal colon and brain. Subsequent analyses showed pro-inflammatory cytokine expression was upregulated in the Winnie mouse brain.

Conclusions: These data provide evidence that the dysregulation of cellular metabolism within the gut-brain axis underlies changes in behaviour and energy metabolism in chronic intestinal inflammation.

Keywords: Cellular metabolism; Chronic colitis; Depression; Energy metabolism; Gut-brain axis; Inflammatory bowel disease (IBD).

Fig. 1

Decreased energy metabolism in Winnie mice. The Promethion system for indirect calorimetry was used to evaluated energy metabolism in C57BL/6 and Winnie mice (n=14-16/group). (A) Sum ofconsumed water (g). (B) Total number of drinking bouts. (C)Sum ofconsumed food (g). (D) Total number offood bouts.(E)Rate of oxygen consumption (VO2, mL/kg/min/body weight). (F) Rate of carbon dioxide emission (VCO2, mL/kg/min/body weight). (G)Respiratory quotient. (H)Energy expenditure (EE, kcal/kg/body weight).(I)Glucose oxidation (GOX, g/kg/body weight).(J)Lipid oxidation (LOX, g/kg/body weight). Data are expressed as mean±SEM. *p<0.05,***p<0.001 when compared to C57BL/6 mice

Fig. 2

DEGs associated with glycolysis and gluconeogenesis and FAO in the Winnie distal colon. KEGG pathways showing upregulated and downregulated genes involved in (A)glycolysis and gluconeogenesis and (B)FAO in the distal colon transcriptome of Winnie (n=6) compared to C57BL/6 (n=7) mice. Data are presented as significance scores of gene expression values between Winnie and C57BL/6 mice. Green (dotted line circle) indicates genes that are downregulated (glycolysis and gluconeogenesis: EC:2.7.1.11, Pfk9; EC:5.1.3.3, galM; EC:3.1.3.9, G6pc; EC:4.2.1.11, Eno; EC:1.1.1.27, Ldh; EC:1.1.1.1, Adh6; EC:1.2.1.3, Aldh2; EC:4.1.1.32, Pck, FAO: EC:CPT1, Cpt1; EC:1.3.8.7, Acadm; EC:1.3.8.9, Acadvl; EC:1.3.8.5, Acadsb; EC:1.3.8.6, Gcdh; EC:2.3.1.16, Hadhb; EC:5.3.3.8, Eci; EC:1.1.1.1, Aldh2; EC:1.2.1.3, Adh; EC:1.14.14.80, Cyp4a10. Red (solid line circle) indicates genes that are upregulated (glycolysis and gluconeogenesis: EC:2.7.1.1, Hk2; EC:2.7.1.147, Adpgk; EC:1.2.1.12, Gapdh; EC:2.7.2.3 Pgk1; EC:5.4.2.2, Pgm; EC:3.1.3.80, Minpp1; EC:5.4.2.1, Pgam; EC:5.4.2.4, Bpgm; EC:2.7.1.40, Pkm; EC:1.1.1.2, Akr1a1; EC:1.2.1.5, Aldh3b2; FAO: EC:6.2.1.3, Acsl; EC:1.3.8.8, Acadl; EC:2.3.1.9, Acat1). Heat map representation of upregulated genes (red), downregulated genes (blue), and unchanged genes (white) associated with (A^I) glycolysis and gluconeogenesis and(B^I) FAO in distal colons from Winnie (n=6) and C57BL/6 (n=7) mice determined by RNA-Seq. The homology in the expression of target genes associated with (A^II) glycolysis and gluconeogenesis and (B^II) FAO metabolism between Winnie mice and IBD patients compared to their respective uninflamed controls

Fig. 3

DEGs associated with TCA cycle and OXPHOS in the Winnie distal colon. KEGG pathways showing upregulated and downregulated genes involved in (C) TCA cycle and (B)OXPHOS in the distal colon transcriptome of Winnie (n=6) compared to C57BL/6 (n=7) mice. Data are presented as significance scores of gene expression values between Winnie and C57BL/6 mice. Green (dotted line circle) indicates genes that are downregulated (TCA: EC:4.1.1.32, Pepck; EC:1.2.4.2, Ogdh; EC:6.2.1.5, Suclg2 andEC:6.2.1.4, Sucla2; OXPHOS: EC:7.1.1.2 (NADH dehydrogenase (ubiquinone)), Ndufa1, Ndufa2, Ndufa4, plus Nduf1b subcomplex, Ndufb2, Ndufb5, QCR6, Cox6b, Cox7A, Cox8, Cox17, EC: 7.1.1.9, cytochrome c oxidase cbb3-type subunit I, ccoN, EC:7.1.2.2 ATP synthase, F-type H+-transporting ATPase subunit a, ATPF0A, Atp5e (epsilon), AtpeV0A, Atp6N, AtpeV0E, Atp6H;and EC3.6.1.1, Ppa1). Red (solid line circle) indicates genes that are upregulated (TCA: EC:6.4.1.1, Pcx; EC:4.2.1.3, Aco;EC:1.1.1.42,Idh;EC 1.1.1.37, Mdh; OXPHOS: NADH dehydrogenase Nduf Fe-S protein/flavoprotein complex, mitochondria, Ndufs8, Ndufa10; Cytochrome c oxidase, Cox4, Cox15; F-type ATPAse, ATP5A1 (alpha), ATP5G (c)I; and V-type ATPase, Atp6B. Heat map representation of upregulated genes (red), downregulated genes (blue), and unchanged genes (white) associated with (A^I) TCA cycle and (B^I) OXPHOS in distal colons from Winnie (n=6) and C57BL/6 (n=7) mice determined by RNA-Seq. The homology in the expression of target genes associated with (A^II) TCA and (B^II) OXPHOS metabolism between Winnie mice and IBD patients compared to their respective uninflamed controls

Fig. 4

Reduced spontaneous physical activity and increased sleep behaviour inWinnie mice. Spontaneous physical activity and sleep behaviour measured in C57BL/6 and Winnie mice (n=16/group) during the dark cycle (12h). (A) Wheel distance travelled (m). (B) Wheel speed (m/s). (C) Percentage of wheel running time (wheel running time %). (D) Percentage of time spent in ambulatory locomotion outside of the wheel (ambulatory locomotion time %). (E)Duration of sleep (h). (F) Percentageof sleeping time (sleep time %). (G) Percentage of still time (still time %). (H)Percentage of quiet time without sleeping (quiet time without sleeping %). ***p<0.001 when compared to C57BL/6 mice

Fig. 5

Winnie mice exhibit increased depressive and anxiety-like behaviours. The light-dark test (LDT), open field test (OFT), elevated plus maze (EPM) test, and forced swim test (FST) were used to measure depressive and anxiety-like behaviour in C57BL/6 and Winnie mice (n=16/group).Winnie mice exhibited higher levels of depressive and anxiety-like behaviours when compared to C57BL/6 mice as measured by : (A)time spent in the white box (LDT), (B)number of entries into the white box (LDT), (C)time spent in the black box, (D)number of pokes from the black box (LDT): (E)time spent in the centre, (F)time spent in the corners, (G)grooming time, (H)number of groomings: (I)time in the open arm: (J)swimming mobility time, (K)swimming immobility time. *p<0.05, **p<0.01,***p<0.001 when compared to C57BL/6 mice

Fig. 6

DEGs related to glycolysis and gluconeogenesis and FAO in the Winnie brain. Pathview plots for the (A)glycolysis and gluconeogenesis and (B)FAO pathways rendered from the KEGG database. Data are presented as significance scores for analysis of gene expressions associated with these metabolic pathways between the brains of Winnie mice and C57BL/6 littermates (n=10/group). Green (dotted line circle) represents downregulated genes (glycolysis and gluconeogenesis: EC:5.3.1.1, Tpi1; EC:1.2.1.12, Pgk1; EC:5.4.2.4, Bpgm; EC:5.4.2.11, Pgam; EC:1.2.4.1 Pdha1; EC:1.1.1.27, Ldha; EC:1.2.1.3, Aldh3a1; EC:1.2.1.5Aldh3b1; EC:1.1.1.1, Adh 1/7; FAO: EC:1.1.1.35,Hadh; EC:2.3.1.16, Acaa2 or Hadhb; EC:1.1.1.1, Adh1; EC:1.2.1.3; Aldh2). Red (solid line circle) represents the upregulated genes (glycolysis and gluconeogenesis: EC:2.7.1.1, Hk2, FAO: Cpt1; EC:6.2.1.3, Acsl; and EC:1.14.14.80, Cyp4A). Heat map representation of upregulated genes (red), downregulated genes (blue), and unchanged genes (white) associated with (A^I) glycolysis and gluconeogenesis and (B^I) FAO in distal colons from Winnie (n=10) and C57BL/6 (n=10) mice determined by RNA-Seq

Fig. 7

DEGs related to TCA cycle and OXPHOS in the Winnie brain. Pathview plots for the (A)TCA cycle and (B)OXPHOS pathways rendered from the KEGG database. Data presented as significance scores for analysis of gene expressions associated with these metabolic pathways between the brains of Winnie mice and C57BL/6 littermates (n=10/group). Green (dotted line circle) represents downregulated genes for TCA: EC:1.2.1.4,Pdha, EC:1.3.5.1, Sdha, EC:6.2.1.4 and EC:6.2.1.5,Sucla2; OXPHOS: EC7.1.1.2 NADH ubiquinone oxidoreductase supernumerary subunits(Nduf),Ndufs4, Ndufs5, Ndufv3, Ndufa1, Ndufa2, Ndufa5, Ndufa6, Ndufa7,Ndufab1, Ndufab12, Ndufab13, Ndufb2, Ndufb3, Ndufb6, Ndufb7, Ndufab1, Ndufa12, Ndufa13, Ndufb2, Ndufb3, Ndufb6, Ndufb7, Ndufb11, Ndufc1, Ndufc2; EC1.3.5.1, Sdhc; EC7.1.1.8, fbcH, Cyt1, QCR6, QCR7, QCR8, QCR9, QCR10; EC7.1.1.9,Cox4, Cox5a, Cox5b, Cox6a, Cox6b, Cox7a, Cox7b, Cox8; EC7.1.2.2, Atpfoa; EC7.2.2.19 Atp4a, Atpef1d, Atp5f1e, Atp50, Atp5mc1, Atp5me, Atp5mf,Atp5mg; and EC 3.6.1.1, Ppa1. Heat map representation of upregulated genes (red), downregulated genes (blue), and unchanged genes (white) associated with (A^I) TCA cycle and (B^I) OXPHOS in distal colons from Winnie (n=10) and C57BL/6 (n=10) mice determined by RNA-Seq

Fig. 8

Inflammatory cytokine expression in Winnie mice brains. High-throughput RNA-Seq of mRNA was performed to determine inflammatory cytokine transcripts in brains from Winnie when compared to C57BL/6 mice (n=10/group). (A)Pathview plot for the IBD pathway from the KEGG database. Data presented as significance scores for cytokine gene expression analysis between the brains fromWinnie mice and C57BL/6 littermates. Green (dotted line circle) represents downregulated gene expression (Il22, Il12a, Il12rb2). Red (solid line circle) represents upregulated gene expression (Tgfb1,Smad2, Il2rγ, Il5, Il21, Il23a, Il12rb1, Tlr5, Tlr2, Tnf). (B)Heat map representation of inflammatory cytokine gene expression in brains from C57BL/6 and Winnie mice. Red indicates upregulated genes, blue signifies downregulated genes, and white denotes unchanged genes