Congenic and bioinformatics analyses resolved a major-effect Fob3b QTL on mouse Chr 15 into two closely linked loci

We previously identified a Chr 15 quantitative trait locus (QTL) Fob3b in lines of mice selected for high (Fat line) and low (Lean line) body fat content that represent an unique model of polygenic obesity. Here we genetically dissected the Fob3b interval by analyzing the phenotypes of eight overlapping congenic lines and four F[down]2 congenic intercrosses and prioritized candidates by bioinformatics approaches. Analyses revealed that the Fob3b QTL consists of at least two separate linked QTLs Fob3b1 and Fob3b2. They exhibit additive inheritance and are linked in coupling with alleles originating from the Lean line, decreasing obesity-related traits. In further analyses, we focused on Fob3b1 because it had a larger effect on obesity-related traits than Fob3b2. e.g., the difference between homozygotes for adiposity index (ADI) percentage was 1.22 and 0.77% for Fob3b2, respectively. A set of bioinformatics tools was used to narrow down positional candidates from 85 to 4 high-priority Fob3b1 candidates. A previous single Fob3b QTL was therefore resolved into another two closely linked QTLs, confirming the fractal nature of QTLs mapped at low resolution. The interval of the original Fob3b QTL was narrowed from 22.39 to 4.98 Mbp for Fob3b1 and to 7.68 Mbp for Fob3b2, which excluded the previously assigned candidate squalene epoxidase (Sqle) as the causal gene because it maps proximal to refined Fob3b1 and Fob3b2 intervals. A high-resolution map along with prioritization of Fob3b1 candidates by bioinformatics represents an important step foward to final identification of the Chr 15 obesity QTL

DEPTOR cell-autonomously promotes adipogenesis, and its expression is associated with obesity

DEP domain-containig mTOR-interacting protein (DEPTOR) inhibits the mechanistic target of rapamty-cin (mTOR), but its in vivo functions are unknown. Previous work indicates that Deptor is part of the Fob3a quantitative trait locus (QTL) linked to obesity/leanness in mice, with Deptor expression being elevated in white adipose tissue (WAT) of obese animals. This relation is unexpected, considering the positive role of mTOR inadipogenesis. Here, we dissected the Fob3a QTL and show that Deptor is the highest-priority candidate promoting WAT expansion in this model. Consistently, transgenic mice overexpressing DEPTOR accumulate more WAT. Furthermore, in humans, DEPTOR expression in WAT correlates with the degree ofobesity. We show that DEPTOR is induced by glucocrtioids during adipogenesisand that its overexpression promotes, while its suppression blocks, adipogenesis. DEPTOR activates the proadipogenic Akt/PKB-PRAR-[ni] axisby dampening mTORC1-mediated feedback inhibition of insulin signaling. These results establish DEPTOR as a new regulator of adipogenesis

Systems View of Deconditioning During Spaceflight Simulation in the PlanHab Project: The Departure of Urine 1 H-NMR Metabolomes From Healthy State in Young Males Subjected to Bedrest Inactivity and Hypoxia

We explored the metabolic makeup of urine in prescreened healthy male participants within the PlanHab experiment. The run-in (5 day) and the following three 21-day interventions [normoxic bedrest (NBR), hypoxic bedrest (HBR), and hypoxic ambulation (HAmb)] were executed in a crossover manner within a controlled laboratory setup (medical oversight, fluid and dietary intakes, microbial bioburden, circadian rhythm, and oxygen level). The inspired O₂ (FᵢO₂) fraction next to inspired O₂ (PᵢO₂) partial pressure were 0.209 and 133.1 ± 0.3 mmHg for the NBR variant in contrast to 0.141 ± 0.004 and 90.0 ± 0.4 mmHg (approx. 4,000 m of simulated altitude) for HBR and HAmb interventions, respectively. ¹H-NMR metabolomes were processed using standard quantitative approaches. A consensus of ensemble of multivariate analyses showed that the metabolic makeup at the start of the experiment and at HAmb endpoint differed significantly from the NBR and HBR endpoints. Inactivity alone or combined with hypoxia resulted in a significant reduction of metabolic diversity and increasing number of affected metabolic pathways. Sliding window analysis (3 + 1) unraveled that metabolic changes in the NBR lagged behind those observed in the HBR. These results show that the negative effects of cessation of activity on systemic metabolism are further aggravated by additional hypoxia. The PlanHab HAmb variant that enabled ambulation, maintained vertical posture, and controlled but limited activity levels apparently prevented the development of negative physiological symptoms such as insulin resistance, low-level systemic inflammation, constipation, and depression. This indicates that exercise apparently prevented the negative spiral between the host's metabolism, intestinal environment, microbiome physiology, and proinflammatory immune activities in the host

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.

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 (FiO2) and partial pressure of inspired O2 (PiO2) 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

Hypoxia and inactivity related physiological changes precede or take place in absence of significant rearrangements in bacterial community structure: The PlanHab randomized trial pilot study.

We explored the assembly of intestinal microbiota in healthy male participants during the randomized crossover design of run-in (5 day) and experimental phases (21-day normoxic bed rest (NBR), hypoxic bed rest (HBR) and hypoxic ambulation (HAmb) in a strictly controlled laboratory environment, with balanced fluid and dietary intakes, controlled circadian rhythm, microbial ambiental burden and 24/7 medical surveillance. The fraction of inspired O2 (FiO2) and partial pressure of inspired O2 (PiO2) 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; ~4000 m simulated altitude), respectively. A number of parameters linked to intestinal environment such as defecation frequency, intestinal electrical conductivity (IEC), sterol and polyphenol content and diversity, indole, aromaticity and spectral characteristics of dissolved organic matter (DOM) were measured (64 variables). The structure and diversity of bacterial microbial community was assessed using 16S rRNA amplicon sequencing. Inactivity negatively affected frequency of defecation and in combination with hypoxia increased IEC (p < 0.05). In contrast, sterol and polyphenol diversity and content, various characteristics of DOM and aromatic compounds, the structure and diversity of bacterial microbial community were not significantly affected over time. A new in-house PlanHab database was established to integrate all measured variables on host physiology, diet, experiment, immune and metabolic markers (n = 231). The observed progressive decrease in defecation frequency and concomitant increase in IEC suggested that the transition from healthy physiological state towards the developed symptoms of low magnitude obesity-related syndromes was dose dependent on the extent of time spent in inactivity and preceded or took place in absence of significant rearrangements in bacterial microbial community. Species B. thetaiotamicron, B. fragilis, B. dorei and other Bacteroides with reported relevance for dysbiotic medical conditions were significantly enriched in HBR, characterized with most severe inflammation symptoms, indicating a shift towards host mucin degradation and proinflammatory immune crosstalk

Hypoxia and inactivity related physiological changes precede or take place in absence of significant rearrangements in bacterial community structure: The PlanHab randomized trial pilot study.

We explored the assembly of intestinal microbiota in healthy male participants during the randomized crossover design of run-in (5 day) and experimental phases (21-day normoxic bed rest (NBR), hypoxic bed rest (HBR) and hypoxic ambulation (HAmb) in a strictly controlled laboratory environment, with balanced fluid and dietary intakes, controlled circadian rhythm, microbial ambiental burden and 24/7 medical surveillance. The fraction of inspired O2 (FiO2) and partial pressure of inspired O2 (PiO2) 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; ~4000 m simulated altitude), respectively. A number of parameters linked to intestinal environment such as defecation frequency, intestinal electrical conductivity (IEC), sterol and polyphenol content and diversity, indole, aromaticity and spectral characteristics of dissolved organic matter (DOM) were measured (64 variables). The structure and diversity of bacterial microbial community was assessed using 16S rRNA amplicon sequencing. Inactivity negatively affected frequency of defecation and in combination with hypoxia increased IEC (p < 0.05). In contrast, sterol and polyphenol diversity and content, various characteristics of DOM and aromatic compounds, the structure and diversity of bacterial microbial community were not significantly affected over time. A new in-house PlanHab database was established to integrate all measured variables on host physiology, diet, experiment, immune and metabolic markers (n = 231). The observed progressive decrease in defecation frequency and concomitant increase in IEC suggested that the transition from healthy physiological state towards the developed symptoms of low magnitude obesity-related syndromes was dose dependent on the extent of time spent in inactivity and preceded or took place in absence of significant rearrangements in bacterial microbial community. Species B. thetaiotamicron, B. fragilis, B. dorei and other Bacteroides with reported relevance for dysbiotic medical conditions were significantly enriched in HBR, characterized with most severe inflammation symptoms, indicating a shift towards host mucin degradation and proinflammatory immune crosstalk

Intestinal Metagenomes and Metabolomes in Healthy Young Males: Inactivity and Hypoxia Generated Negative Physiological Symptoms Precede Microbial Dysbiosis

We explored the metagenomic, metabolomic and trace metal makeup of intestinal microbiota and environment in healthy male participants during the run-in (5 day) and the following three 21-day interventions: normoxic bedrest (NBR), hypoxic bedrest (HBR) and hypoxic ambulation (HAmb) which were carried out within a controlled laboratory environment (circadian rhythm, fluid and dietary intakes, microbial bioburden, oxygen level, exercise). The fraction of inspired O2 (FiO2) and partial pressure of inspired O2 (PiO2) were 0.209 and 133.1 ± 0.3 mmHg for the NBR and 0.141 ± 0.004 and 90.0 ± 0.4 mmHg (~4,000 m simulated altitude) for HBR and HAmb interventions, respectively. Shotgun metagenomes were analyzed at various taxonomic and functional levels, 1H- and 13C -metabolomes were processed using standard quantitative and human expert approaches, whereas metals were assessed using X-ray fluorescence spectrometry. Inactivity and hypoxia resulted in a significant increase in the genus Bacteroides in HBR, in genes coding for proteins involved in iron acquisition and metabolism, cell wall, capsule, virulence, defense and mucin degradation, such as beta-galactosidase (EC3.2.1.23), α-L-fucosidase (EC3.2.1.51), Sialidase (EC3.2.1.18), and α-N-acetylglucosaminidase (EC3.2.1.50). In contrast, the microbial metabolomes, intestinal element and metal profiles, the diversity of bacterial, archaeal and fungal microbial communities were not significantly affected. The observed progressive decrease in defecation frequency and concomitant increase in the electrical conductivity (EC) preceded or took place in absence of significant changes at the taxonomic, functional gene, metabolome and intestinal metal profile levels. The fact that the genus Bacteroides and proteins involved in iron acquisition and metabolism, cell wall, capsule, virulence and mucin degradation were enriched at the end of HBR suggest that both constipation and EC decreased intestinal metal availability leading to modified expression of co-regulated genes in Bacteroides genomes. Bayesian network analysis was used to derive the first hierarchical model of initial inactivity mediated deconditioning steps over time. The PlanHab wash-out period corresponded to a profound life-style change (i.e., reintroduction of exercise) that resulted in stepwise amelioration of the negative physiological symptoms, indicating that exercise apparently prevented the crosstalk between the microbial physiology, mucin degradation and proinflammatory immune activities in the host

Weekly defecation rates as a direct measure of constipation due to increased retention times of organic matter in intestinal system throughout the PlanHab experiment.

<p>Weekly defecation rates as a direct measure of constipation due to increased retention times of organic matter in intestinal system throughout the PlanHab experiment.</p

CONSORT flowchart of participants recruitment and the cross-over designed PlanHab study trial flow.

<p>Interventions: normoxic bed rest (NBR), hypoxic bed rest (HBR) and hypoxic ambulation (HAmb).</p

Schematic overview of the detected changes in microbial communities.

<p>The congruency of four statistical tests was used to detect significant differences in the structure of microbial community. The additional four statistical tests were used to congruently identify the first responding taxa over the course of the PlanHab experiment. The end-points of the PlanHab experiment with the significant changes are shown (p<0.05, FDR corrected). NBR–normoxic bed rest, HBR–hypoxic bed rest, HAmb–hypoxic ambulatory.</p