Hypoxia and Inactivity Related Physiological Changes (Constipation, Inflammation) Are Not Reflected at the Level of Gut Metabolites and Butyrate Producing Microbial Community: The PlanHab Study

Abstract

We explored the assembly of intestinal microbiota in healthy male participants during the run-in (5 day) and experimental phases [21-day normoxic bed rest (NBR), hypoxic bedrest (HBR)], and hypoxic ambulation (HAmb) in a strictly controlled laboratory environment, balanced fluid, and dietary intakes, controlled circadian rhythm, microbial ambiental burden, and 24/7 medical surveillance. The fraction of inspired O₂ (F_iO₂) and partial pressure of inspired O₂ (P_iO₂) were 0.209 and 133.1 ± 0.3 mmHg for NBR and 0.141 ± 0.004 and 90.0 ± 0.4 mmHg for both hypoxic variants (HBR and HAmb; ~4,000 m simulated altitude), respectively. A number of parameters linked to intestinal transit spanning Bristol Stool Scale, defecation rates, zonulin, α₁-antitrypsin, eosinophil derived neurotoxin, bile acids, reducing sugars, short chain fatty acids, total soluble organic carbon, water content, diet composition, and food intake were measured (167 variables). The abundance, structure, and diversity of butyrate producing microbial community were assessed using the two primary bacterial butyrate synthesis pathways, butyryl-CoA: acetate CoA-transferase (but) and butyrate kinase (buk) genes. Inactivity negatively affected fecal consistency and in combination with hypoxia aggravated the state of gut inflammation (p < 0.05). In contrast, gut permeability, various metabolic markers, the structure, diversity, and abundance of butyrate producing microbial community were not significantly affected. Rearrangements in the butyrate producing microbial community structure were explained by experimental setup (13.4%), experimentally structured metabolites (12.8%), and gut metabolite-immunological markers (11.9%), with 61.9% remaining unexplained. Many of the measured parameters were found to be correlated and were hence omitted from further analyses. The observed progressive increase in two immunological intestinal markers suggested that the transition from healthy physiological state toward the developed symptoms of low magnitude obesity-related syndromes was primarily driven by the onset of inactivity (lack of exercise in NBR) that were exacerbated by systemic hypoxia (HBR) and significantly alleviated by exercise, despite hypoxia (HAmb). Butyrate producing community in colon exhibited apparent resilience toward short-term modifications in host exercise or hypoxia. Progressive constipation (decreased intestinal motility) and increased local inflammation marker suggest that changes in microbial colonization and metabolism were taking place at the location of small intestine.

Keywords: butyrate producing community; constipation; gut metabolites; human intestinal microbiome; hypoxia; inactivity; inflammation; noncommunicable diseases.

Figure 1

Changes in Bristol stool scale values (A) and retention time (as time between particular defecations) (B) during run-in (week 1) and subsequent 3-week experimental phase.

Figure 2

Changes in immunological markers present in fecal samples: zonulin (A), α₁-antitrypsin (B), eosinophil-derived neurotoxin (EDN) (C) and bile acids (D) during run-in (week 1) and subsequent 3-week experimental phase.

Figure 3

Variation in water content (A), pH (B), and total soluble organic carbon (TSOC) in fecal samples (C) during run-in (week 1) and subsequent 3-week experimental phase.

Figure 4

Changes in concentration of C1–C6 short chain fatty acids (SCFA) present in fecal samples during run-in (week 1) and subsequent 3-week experimental phase: total SCFA (A), acetic acid (B), propionic acid (C), iso-butyric acid (D), n-butyric acid (E), iso-valeric acid (F), n-valeric acid (G), and n-capric acid (H).

Figure 5

Changes in molecular weight indices (MWI) of fecal samples during run-in (week 1) and subsequent 3-week experimental phase: Sr = ratio of absorption slopes between 275–295 and 350–400 nm slope (A), E2:E3 = ratio between absorption coefficients at 250 nm and at 365 nm (B), a (300 nm) = Napierian absorption coefficient at 300 nm (C), a (255 nm) = Napierian absorption coefficient at 255 nm (D), Total a = integrated absorbance between 250 nm and 450 nm (E) and S = spectral slope of absorbance from 300 to 700 nm (F).

Figure 6

A schematic representation of closest matches of buk (A) and but (B) gene sequences describing butyrate producing communities in 54 PlanHab samples.

Figure 7

NM-MDS ordination showing a host-specific grouping of butyrate communities based on combined but and buk gene datasets for all participants in experimental variants: NBR (blue), HBR (red), HAmb (yellow). The time spent in experiment is designated by darker colors. Stress_buk+but = 0.18. Only OTUs represented by at least 50 sequences were used in analyses. The numbers designate sample groupings according to participants.

Figure 8

Abundance of butyrate producing community members as estimated by two approaches, qPCR (A,B), and band intensities (C,D) during run-in (week 1) and subsequent 3-week experimental phase. Units are scaled to represent % of total bacterial microbial community (A,B). Band intensities of PCR products (C,D) were categorized according to band intensity classes (0–5) (Vital et al., 2015).