Combined Intermittent & Sustained Hypoxia & High-Fat Diet

Introduction

Obesity hypoventilation syndrome (OHS) is an excessive phenotype of obstructive sleep apnea (OSA) and is characterised by morbid weight problems, daytime hypoventilation, and sleep-disordered respiratory. Sleep-disordered inhaling OHS is additional characterised by each sleep-related hypoventilation (inflicting sustained hypoxia [SH]) along with repetitive closure of the higher airway that characterizes OSA, which, in flip, causes intermittent hypoxia [IH]).¹ OHS impacts practically a million people within the US and is related to a better danger for cardiometabolic illness and early mortality when in comparison with people with solely morbid weight problems or OSA.^2–5

IH, that includes OSA has been proven to selectively activate NF-kB-dependent transcription and downstream proinflammatory pathways, whereas SH triggers Hypoxia Inducible Factor (HIF-1α) and downstream pathways.⁶ However, extreme OSA could cause activation of HIF-1α. A attainable rationalization is that sufferers with extreme OSA could also be uncovered to adequate cumulative intervals of sustained hypoxia throughout sleep to activate (HIF-1α) through Ca²⁺/calmodulin kinase-dependent pathway.⁷ Such a variable response to IH versus SH could also be accountable for the extra extreme pathogenesis of irritation and cardiometabolic dysfunction noticed in OHS when in comparison with that in OSA. Besides the direct results of IH and SH on mobile pathways, IH seen in OSA can result in intestine dysbiosis and alteration within the intestine barrier perform.⁸ Both intestine dysbiosis and intestine barrier dysfunction brought on by a shift within the microbiota profile can result in systemic irritation and consequent cardiometabolic derangements.^9–13 We know that the spatial oxygen gradient (PO₂) between the epithelial lining and the intestine lumen determines the composition of intestine microbiota. When host-tissue oxygen ranges are diminished in illness states, resembling IH of OSA, there’s a additional discount of luminal oxygen ranges that causes a shift within the intestine microbiota composition.⁸ The impact of a mixture of SH and IH on the intestine microbiota is much less clear.

A significant factor influencing the intestine microbiota is dietary fats content material. High-fat weight-reduction plan (HFD) was causally associated to intestine dysbiosis and manifested by a rise in Firmicutes:Bacteroidetes (F:B) ratio. Furthermore, the interactive results of IH and high-fat weight-reduction plan on the intestine microbiome and cardiovascular morbidity (eg, hypertension) has been beforehand studied.^14–18 In a rat mannequin, high-fat weight-reduction plan together with IH led to intestine dysbiosis and hypertension, whereas IH with a traditional weight-reduction plan didn’t trigger hypertension.¹⁴ Fecal transplantation from hypertensive rats with intestine dysbiosis to normoxic and normotensive rats led to hypertension when uncovered to IH. This research demonstrated that there’s a causal relationship between intestine dysbiosis and cardiometabolic derangement (hypertension) and that there could also be an interactive impact between IH and high-fat weight-reduction plan.^14,19 Understanding that there are vital variations within the proinflammatory versus adaptive response of IH versus SH, respectively, we got down to examine the interactive impact of mixed intermittent and sustained hypoxia and high-fat weight-reduction plan on proximal colonic microbiota and colonic gene expression sample. To deal with this purpose, we carried out a pilot research of mice subjected to a 2×2 factorial design of a mixture of oxygen derangements ([IH+SH] or normoxia) and dietary circumstances (high-fat weight-reduction plan or regular fats weight-reduction plan). We hypothesized that the mixture of intermittent and sustained hypoxia (IH+SH) and high-fat weight-reduction plan will trigger better diploma of intestine dysbiosis than high-fat weight-reduction plan alone. A secondary speculation was that the mixture of (IH+SH) and excessive -at weight-reduction plan may have better affect on colonic epithelial transcriptome than the impact of excessive fats alone.

Materials and Methods

Protocol

All mice used on this work had been saved within the University of Arizona Animal Care, Specific Pathogen Free facility, and dealt with in accordance with the college’s tips and accepted by the University of Arizona Institutional Animal Care and Use Committee (IACUC) (Gillian Paine, Protocol # 13–431 and 07–029). All mice had advert libitum entry to meals, water, and 12/12 h day/night time gentle cycle (gentle cycle was 7:00 am to 7:00 pm and darkish cycle was 7:00 pm to 7:00 am). The research was carried out with 16, three-week outdated, male C57BL/6 mice that had been initially obtained from The Jackson Laboratory (Sacramento, CA). All mice had been housed in identical space to standardize the sound surroundings and decrease exterior variations. Mice had been randomly assigned to 4 experimental teams for 4 weeks – completely different combos of weight-reduction plan (regular weight-reduction plan [ND] or high-fat weight-reduction plan [HFD]) and oxygen therapies (normoxia [NM] and intermittent hypoxia+sustained hypoxia [IH+SH]). Experimental group designations had been as follows: ND and NM, HFD and NM, ND and IH+SH, and HFD and IH+SH. The high-fat chow was comprised of 60% kcal from fats, 21% kcal from carbohydrates and 19% kcal from protein. Normal chow was comprised with 18% kcal from fats, 59% kcal from carbohydrates and 23% kcal from protein. During their sleep cycle (from 7:00 am to 7:00 pm), mice within the hypoxia teams had been uncovered to a mixture of IH and SH. IH consisted of 10 cycles per hour for 3 hours (starting from 20.95% FiO₂ to 9% FiO₂), adopted by 6 hours of persistent hypoxia (9% FiO₂), then one other 3 hours of intermittent hypoxia.

The rationale of our protocol and the sequential method we adopted (IH-SH-IH) displays the pathophysiology of OHS that’s characterised by sustained hypoxemia (SH) of alveolar hypoventilation in addition to the intermitted hypoxemia (IH) of the coexistent obstructive sleep apnea (OSA) occasions in distinction to the intermittent hypoxemia with out sustained hypoxemia of easy OSA.^2,20–22 Observations by us and different investigators of the hypoxia signature in people with OHS revealed IH through the early portion of the night time marked by occasions of OSA adopted by sustained hypoxia (SH) of sluggish wave sleep after which intermittent hypoxia (IH) of REM sleep.^22,23 Our rationale for such diploma of hypoxemia is in line with reviews of diploma of hypoxemia in sufferers with OHS.²⁴ Moreover, in numerous animal research, Farre et al discovered that there was certainly a variety of hypoxic stimulus noticed in numerous research that ranged from a FiO₂ nadir of 4–15%.²⁵ In our research, we selected to make use of the mid-point of such research at 9%. Moreover, the commonest paradigms of intermittent hypoxia when utilized to mice used nadir values within the vary 50–70%.^26–30 Our paradigm was once more on the midpoint of this vary that has been utilized by different investigators and goes in favor of the pondering that such low nadirs (50–70%) in mice mimic the hypoxemic occasions in sufferers since they really correspond to PaO₂ values much like these in sufferers with OSA.²⁵ The oxygen focus management was maintained by a computer-regulated move of gasoline circulated by means of a sealed commercially designed chamber (OxyCycler A44XO, BioSpherix, Parish, NY). During the 12-hour darkish cycle, mice within the hypoxia teams had been uncovered to normoxia within the animal facility exterior the chambers. Hypoxia publicity was constantly monitored and recorded through inside oxygen sensors constructed into the computer-regulated Oxycycler machine and periodically cross-verified with exterior oxygen sensors (ISO-OXY-2, World Precision Instruments). A management NM group was uncovered to a steady move of room air. Normoxia and hypoxia therapies had been maintained throughout the research. Each mouse was weighed each 3 days. At the tip of 4 weeks, after euthanasia, small sections of tissue from proximal colon had been collected. The proximal segments of the colon had been collected and had been frozen at −80°C till analyzed.

Complete Blood Count

Blood collected from cardiac puncture on the time of euthanasia was submitted for full blood depend (CBC) to University of Arizona Animal Care Pathology Services Laboratory which makes use of the HEMAVET Multispecies Hematology Analyzer, a quantitative, automated multispecies hematology analyzer for in vitro diagnostic use.

DNA and RNA Extraction

Both DNA for adherent microbiota evaluation and RNA for microarray gene expression analyses had been purified from 25–30 mg of collected tissues utilizing AllPrep DNA/RNA mini Kit (Qiagen). Tissue was homogenized in TissueLyser II (MoBio) disruptor at 30Hz for 10 min in a chilly room. After the primary spherical, the plate orientation was modified and one other cycle of 10 min at 30Hz was carried out. Lysis buffer for DNA/RNA extraction was supplemented with Reagent DX (Qiagen), to forestall foaming of samples throughout mechanical disruption. The lysis step was adopted by the DNA and RNA extraction as described within the producer’s protocol. The integrity of RNA was confirmed utilizing Agilent Bioanalyzer 2100 (min. RIN=9.0) and focus assessed with Nanodrop (Thermo Fisher).

Microbiome Analysis

The hypervariable V4 area of the 16S rRNA gene was amplified from every pattern utilizing 806R and 515F primers.³¹ Each PCR (remaining quantity 40µ) contained 2x MyFi Mix (BioLine), 0.5µM V4-specific primers prolonged with Illumina adapter, sequencing primer binding websites and the reverse primer with a pattern distinctive barcode. The biking circumstances had been 95°C for two min, adopted by 35 cycles of 95°C for 30 seconds, 50°C for 45 seconds, 74°C for 30 seconds, after which adopted with a remaining extension at 72°C for five min. Amplicons had been quantified utilizing Quant-iT PicoGreen dsDNA Assay Kit in response to the producer’s protocol (Thermo Fisher Scientific). Pooled sequencing library was created by combining 240 ng of DNA from every pattern. Pooled library was cleaned utilizing ExtremelyClean PCR Clean-Up Kit (MoBio). Then, the library was quantified towards a regular curve utilizing the qPCR-based KAPA Library Quantification Kit for Illumina platforms (KAPA). The library was diluted to a focus of 4nM, denatured with 0.2N NaOH and additional diluted to a focus 7pM. Due to the restricted sequence range amongst 16S rRNA amplicons, 5% of the PhiX management library (Illumina) created from phiX174 was added and the library was subjected to the paired-end sequencing utilizing 2 x 151bp MiSeq Reagent Kit V2 (Illumina) utilizing customized primers.³¹ Sequencing was carried out on the Illumina MiSeq (SN M03190, with the MiSeq Control Software v 2.5.0.5). The reads had been processed, and knowledge analyzed as we described beforehand.^32,33 Briefly, de-multiplexing was carried out utilizing idemp script (https://github.com/yhwu/idemp). Filtering, dereplication, pattern inference, chimera identification, and merging of paired-end reads was achieved with a reference-free Divisive Amplicon Denoising Algorithm 2 (Dada2, model 1.18.0)³⁴ package deal in RStudio (model 1.3.1093 with R model 4.0.3). The ASVs taxonomy was assigned utilizing RDP classifier towards SILVA database launch 132³⁵ (https://www.arb-silva.de/documentation/release-132/). The vegan package deal³⁶ (model 2.5.7) was used as a software for range evaluation, ordination strategies, for the evaluation of dissimilarities, and statistical evaluation. The obtained outcomes had been visualized with ggplot2³⁷ (2D plots, model 3.3.3) and plotly³⁸ (3D plots, model 4.9.3) packages.

Microarray Analysis of Colonic Gene Expression Profile

Amplified and biotinylated sense-stranded DNA targets had been generated from complete RNA remoted from particular person mice in every experimental group (n = 4 in every therapy group) utilizing GeneChip^® WT PLUS Reagent package (Affymetrix) and hybridized to GeneChip^® Mouse Gene 2.0 ST Arrays (Affymetrix). Gene expression evaluation was carried out utilizing GeneSpring GX 14.9 software program (Agilent Technologies, Santa Clara, CA). Data had been processed utilizing the RMA16 summarization algorithm and normalized towards the imply of management samples (NM, ND). Gene ontology (GO) practical annotation evaluation was carried out both with GeneSpring or utilizing the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8 on-line software.³⁹

Statistical Analysis

Complete blood depend knowledge had been analyzed utilizing one-way ANOVA adopted by Fisher’s least vital distinction (LSD) pairwise comparability take a look at (Prism 9.1.2; GraphPad). Statistical evaluation of gene expression statistical was carried out utilizing built-in instruments, together with normalized t-test or two-way ANOVA, in each circumstances with Benjamini–Hochberg a number of testing correction (GeneSpring GX, Agilent). Gene ontology (GO) practical annotation evaluation was carried out both with GeneSpring or utilizing the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8 on-line software (Nature Protocols 2009; 4(1):44 and Nucleic Acids Res. 2009;37(1):1).^39,40 More detailed outcomes of the analyses, together with uncooked and normalized expression values, could be seen on the National Center for Biotechnology Information Gene Expression Omnibus microarray depository website (www.ncbi.nlm.nih.gov; GEO accession no. pending).

Results

Hematological Evidence of Exposure to Hypoxia

To confirm that each one mice within the hypoxia group skilled adequate publicity to hypoxic circumstances, we analyzed their blood collected on the time of euthanasia for attribute physiological response utilizing CBC. All parameters examined confirmed vital distinction between teams (ANOVA, p<0.002; (Figure 1). In no case did HFD alone have an effect on the CBC profile. Intermittent and sustained hypoxia, no matter weight-reduction plan, considerably elevated crimson blood depend (RBC), hemoglobin focus (Hb), hematocrit (HCT), imply corpuscular quantity (MCV), imply corpuscular hemoglobin (MCH), and crimson cell distribution width (RDW). Interestingly, in mice fed HFD, hypoxia (IH + SH) additional elevated RBC, HCT, and RDW values, whereas it decreased MCH (Figure 1).

Characterization of Colonic Mucosal Microbiota

We first conducted a permutational multivariate analysis of variance using ADONIS tool in vegan using 16S profiling data from the mucosal adherent microbiota in the proximal and distal segments of the colon. This tool is directly analogous to MANOVA (multivariate analysis of variance) and allows for identification of the main drivers of community differences in multifactorial ecological experiments. ADONIS test indicated that diet and oxygen status were the most significant variables, while segment did not contribute to the observed differences (Table 1). Thus, we targeted subsequent microbiota and gene expression analyses on the proximal colon alone. Richness evaluation indicated HFD considerably decreased microbial range as in comparison with mice on regular weight-reduction plan, no matter the oxygen standing (Figure 2). IH+SH, nonetheless, reversed the consequences of HFD to a small extent (Figure 2), suggesting that intermittent and sustained hypoxia modulated the affect of HFD on the mouse intestine microbial range. The β-diversity evaluation and ordination utilizing non-metric multidimensional scaling (NMDS) confirmed that in ND mice, IH+SH had a minor affect on microbial composition (Figure 3), with solely two unclassified genera from the Muribaculaceae household that handed DESeq2 evaluation (corrected p<0.05, cutoff twofold; Supplementary Data-A). Genus-level evaluation confirmed that weight-reduction plan was probably the most vital variable shaping mucosal colonic microbiota, with each HFD teams clustering intently collectively and separated from ND teams (Supplementary Data-B). However, in HFD-fed mice, IH+SH extra significantly influenced the ordination of HFD_IH+SH group in comparison with HFD_NM, suggesting that intermittent and sustained hypoxia modulates the adherent mucosal microbiota in mice fed high-fat weight-reduction plan (Figure 3). In phylum-level taxonomic evaluation, HFD induced an enlargement of Firmicutes at the price of Bacteroidetes and different phyla, and this phenomenon was additional exacerbated by hypoxia in HFD_IH+SH group (Figure 4A). This was particularly mirrored within the Firmicutes:Bacteroidetes ratio which elevated from 5.89 ± 1.19 in HFD-NM to 22.6 ± 5.73 in HFD-IH+SH (Mean ± SD; p=0.029; Figure 4B).

Table 1 Permutational Multivariate Analysis of Variance (ADONIS) of the Murine Mucosal Microbiota in the Proximal and Distal Segment of the Colon

Figure 2 Richness (number of amplicon sequence variants; ASVs) in colonic mucosal microbiome is modulated by diet and the oxygen status. The lower and upper hinges represent the first and third quartiles, respectively. The middle line represents the median value. Kruskal–Wallis rank sum test followed by Dunn’s multiple comparison test were used for statistical comparisons (P value indicated). Abbreviations: HFD, high-fat diet; ND, normal diet; IH+SH, intermittent and sustained hypoxia; NM, normoxia.

Figure 3 Bray–Curtis-based non-metric multidimensional (NMDS) scaling plot of dissimilarities between all four experimental groups. Analysis of similarities (ANOSIM) was used to test the differences between groups. ADONIS for beta diversity distances was used to determine the factor (diet or oxygen status) with the strongest impact on dissimilarities between groups. Abbreviations: HFD, high-fat diet; ND, normal diet; IH+SH, intermittent and sustained hypoxia; NM, normoxia.

Figure 4 Phylum-level taxonomic analysis of the four experimental groups. (A) Bar graph depicting the effects of diet and combined intermittent and sustained hypoxia on the relative abundance of phyla in the colonic mucosa. (B) The effects of diet and combined intermittent and sustained hypoxia on Firmicutes:Bacteroidetes ratio in the colonic mucosa. Kruskal–Wallis rank sum test followed by Dunn’s multiple comparison test were used for statistical comparisons (P value indicated). Abbreviations: HFD, high-fat diet; ND, normal diet; IH+SH, intermittent and sustained hypoxia; NM, normoxia.

Taxonomic analysis at the genus level confirmed the effects of high-fat diet and their modulation by intermittent and sustained hypoxia (Figure 5A). Stringent pairwise evaluation with DESeq2 (corrected p<0.05, cutoff twofold) between ND_NM and HFD_NM (the impact of high-fat weight-reduction plan in normoxia) recognized a number of ASVs with genera representing the households of Lachnospiraceae (A2, Acetifactor, Eisenbetgiella, Lachnospiraceae_NK4A136_group), Ruminococcaceae (Oscillibacter, Ruminiclostridium, Ruminococcaceae_UCG-009), Akkermansiaceae (Akkermansia), Rikenellaceae (Alistipes), Anaeroplasmataceae (Anaeroplasma), Streptococcaceae (Lactococcus), and Erisipelotrichaceae (Turicibacter) (Figure 5B). In high-fat diet-fed mice, intermittent and sustained hypoxia (HFD_IH+SH vs HFD_NM) considerably elevated the relative abundance of 4 ASVs from Lachnospiraceae household representing two genera: A2 and Lachnospiraceae_NK4A136_group), in addition to three unclassified ASVs from the Muribaculaceae household (Figure 5C). One ASV from this household was considerably decreased within the HFD_IH+SH group (Figure 5C). An in depth abstract of microbial neighborhood adjustments between the experimental teams are offered in Supplementary Data-C.

Figure 5 Genus-level taxonomic analysis of the four experimental groups. (A) Bar graph depicting the effects of diet and combined intermittent and sustained hypoxia on the relative abundance of specific genera in the colonic mucosa. Genera with relative abundance less than 0.1% were filtered out for figure clarity. (B) DESeq2 analysis (corrected p<0.05, cutoff twofold) of the effect of HFD in normoxic mice (HFD_NM vs ND_NM). Genus name indicated on top, and color indicates the respective family. NA=lack of classification of the ASV at the genus level. (C) Analogous: DESeq2 analysis of the modulating effects of intermittent and sustained hypoxia in mice fed HFD on mucosal microbiota.

Transcriptional Response of the Host to IH, SH, and HFD

Two-way ANOVA was performed to investigate how each variable (diet, oxygen status, or combination of the two) contributed to most significant changes in gene expression. Similar to microbiota, diet was demonstrated to have the most impact with 1702 transcript cluster IDs (TIDs) significantly altered (corrected p<0.05) in contrast to the 39 TIDs associated with oxygen status (Figure 6) solely. Furthermore, solely three TIDs are concerned with the mixed affect of weight-reduction plan and oxygen standing (Figure 6).

Figure 6 Diet is the dominant factor shaping colonic transcriptome. Two-way ANOVA analysis was performed on samples filtered to include genes with raw intensity >50 at least in 3/16 samples. Diet had the most impact with 1702 transcript cluster IDs (TIDs; corrected p<0.05), while 39 TIDs were associated with the oxygen status. Three TIDs were identified as affected by the combined influence of diet and oxygen status. Normoxia-normal diet (NM-ND), normoxia-high-fat diet (NM-HFD), intermittent and sustained hypoxia-normal diet (IH+SH-ND) and intermittent and sustained hypoxia-high-fat diet (IH+SH-HFD).

When compared to baseline controls (ND_NM), HFD_IH+SH treatment showed a profound effect on colonic transcriptomic profile with a skewed proclivity for gene upregulation (Figure 7A and B). The set of genes that had been distinctive, annotated, statistically completely different between ND_NM and HFD_IH+SH had been analyzed with the DAVID gene ontology (GO) annotation software underneath excessive stringency (minimal 10 genes per class, EASE rating <0.05). As proven in (Figure 8A), a number of the molecular perform GO classes overrepresented on this dataset had been protein binding, RNA and DNA binding, hydrolases, kinases, but additionally microtubule binding, actin-, and lipid binding. Actin binding proteins could also be concerned with tight junction meeting and thus with intestinal permeability regulation. On the opposite hand, lipid binding could mirror an affect of HFD on lipid metabolism, particularly in peroxidation, fats absorption and adipogenesis. GO organic processes evaluation indicated adjustments in expression of genes related in cell cycle/division/proliferation, transcription, membrane transport, lipid metabolism, and red-ox, amongst others (Figure 8B).

Figure 7 Transcriptiomic differences in colonic gene expression in healthy (ND-NM) mice and a model of intermittent and sustained hypoxia (HFD-IH+SH). (A) Volcano plot illustrating upregulated (in red) and downregulated genes (in blue) (B) Histogram depicting skewed pattern of regulation with majority of genes upregulated in HFD-IH+SH.

Figure 8 Gene ontology (GO) analysis utilizing DAVID with focus is on (A) molecular function and (B) biological process. Number of transcript cluster IDs (TIDs) for each GO category is plotted (bars) along with p-value (dashed line).

Considering that diet is a factor potentially easier to modify as a part of lifestyle change in patients with OSA, we also focused our analysis on the effects of HFD in normoxic vs hypoxic conditions. Although HFD had significant impact on both IH+SH- and NM-treated mice, microarray analysis data demonstrated that while HFD influence in the NM group resulted in significant change of 251 TIDs (HFD_NM vs ND_NM; Figure 9A), HFD utilized in hypoxic circumstances (HFD_IH+SH vs HFD_NM) led to altered expression of 1804 genes (Figure 9B). Interestingly, the units of genes dysregulated by HFD in NM or IH+SH states had been very discordant, each in quantity and within the ascribed perform (Figure 10). GO evaluation of the gene units regulated by HFD solely in NM or IH+SH indicated that whereas underneath NM, HFD had vital affect on notable processes such because the downregulation of bile acid biosynthesis and brown adipocyte differentiation, the affected organic processes had been extra expansive within the HFD_IH+SH group, with many distinctive GO classes noticed. One of them, prominently overrepresented on this experimental group was cell–cell adhesion (Figure 10). Among the dysregulated genes on this class had been genes coding for identified members of the apical junctional complexes (eg, claudins, nectins), or adhesion molecules (eg, CD164) (Figure 11). Interestingly, Claudin 2 (Cldn2) and spleen-associated tyrosine kinase (Syk), each related to tight junction dysfunction and elevated mucosal permeability had been considerably up regulated within the HFD_IH+SH group, whereas claudin 4 (Cldn4), a barrier-forming claudin solely expressed in luminal epithelial cells, was downregulated by HFD in hypoxic mice.

Figure 9 Intermittent and sustained hypoxia modulates colonic transcriptomic response to high-fat diet. (A) 251 transcript cluster IDs (TIDs) identified as differentially regulated by HFD in normoxic (NO) conditions (corr. p<0.05) and plotted in all four experimental groups. (B) 1804 TIDs’s identified as differentially regulated by HFD in intermittent and sustained hypoxia (IH+SH) conditions (corr. p<0.05) and plotted in all four experimental groups.

Figure 10 Intermittent and sustained hypoxia modulates colonic transcriptomic response to high-fat diet – gene ontology analysis. Venn diagram depicts the minimal overlap among the genes significantly regulated by high-fat diet (HFD) in normoxic mice (NM; in orange) and mice under intermittent and sustained hypoxia (IH+SH; in blue) treatment. Gene ontology (GO) analysis was performed with transcript cluster IDs (TIDs) exclusively regulated by HFD in NM (145) and in mice treated with IH+SH (1698). Ease score threshold of p<0.05 was chosen and calculated per number of GO-annotated TIDs. Out of a diverse group of 242 biological process categories within 1698 TIDs, only 18 most significant are shown for clarity and cell–cell adhesion category is highlighted in red.

Figure 11 Intermittent and sustained hypoxia affects expression of genes associated with cell adhesion in high-fat diet-fed mice. Genes identified by gene set analysis (GSA) focused on a subset of 89 genes within the cell–cell adhesion category, which were significantly different (moderated t-test, p<0.05) between mice that underwent intermittent and sustained hypoxia (IH+SH) and fed normal diet (ND) or high-fat diet (HFD). Of the 20 genes shown, asterisk denoted three genes with very low expression (<30 raw intensity data) in all eight experimental mice, and which may not be of biological relevance. Data was clustered using hierarchical clustering algorithm using normalized intensity values, with Euclidean distance metrics and Ward’s linkage rule.

Discussion

Certain general observations can be made from our study. First, while high-fat diet was the major driving force in changing the microbiota composition at the family and genera levels, intermittent hypoxia combined with sustained hypoxia modulates the effect of high-fat diet on the colonic mucosal microbiome. Second, intermittent hypoxia combined with sustained hypoxia accentuated the increase in Firmicutes:Bacteroidetes ratio affected by HFD alone. Finally, and more importantly, IH+SH combined with HFD altered the expression of proteins associated with cell–cell interactions, actin binding, and lipid binding, thus suggesting a dysregulation of the intestinal barrier function and lipid metabolism.

Most experiments in this area of study investigated the impact of intermittent hypoxia (with or without high-fat diet) on the gut microbiome but not the effects of sustained hypoxia.^14,41 Recently, Zhen et al⁴² carried out an experiment in mice evaluating the affect of intermittent hypoxia, sustained hypoxia, and mixture of intermittent and sustained hypoxia on a number of cardiometabolic variables when in comparison with normoxia. Mice experiencing a mixture of intermittent and sustained hypoxia confirmed a rise in systolic blood strain, proper ventricular systolic blood strain, hepatic oxidative stress, low-density lipoprotein, and really low-density lipoprotein. There was no noticed change in glucose disposal. However, the investigators didn’t research the interactive impact of high-fat weight-reduction plan and hypoxia on this experiment (mice had been fed a daily chow weight-reduction plan advert libitum). More importantly, they didn’t research the impact of such influences on intestine dysbiosis. It is conceivable, that intestine dysbiosis could have mediated the impact of the mixture of intermittent and sustained hypoxia and high-fat weight-reduction plan on such cardiometabolic adjustments. In our research, we demonstrated the interactive results of the mixture of intermittent and sustained hypoxia with high-fat weight-reduction plan in inflicting intestine dysbiosis that has beforehand been proven to trigger hypertension by Durgan et al.¹⁴ In the research by Durgan et al, high-fat weight-reduction plan and intermittent hypoxia led to vital alterations of the intestine microbiota together with decreases in bacterial taxa which might be identified to supply the short-chain fatty acid butyrate. The causal affect of such intestine dysbiosis on hypertension was demonstrated by transplantation of the dysbiotic cecal contents from hypertensive rats (experiencing intermittent hypoxia and high-fat weight-reduction plan) to recipient normotensive rats (receiving intermittent hypoxia and regular chow weight-reduction plan) that resulted in hypertension much like that of the donor.

The Firmicutes:Bacteroidetes (F:B) ratio is a well-established attribute of intestine dysbiosis. In the research by Durgan et al, the Firmicutes:Bacteroidetes ratio of rats receiving high-fat weight-reduction plan and intermittent hypoxia tended to be decrease than rats on a high-fat weight-reduction plan alone. However, in our research, we discovered that mice receiving a mixture of intermittent and sustained hypoxia along with high-fat weight-reduction plan manifested an almost fourfold enhance in Firmicutes:Bacteroidetes ratio when in comparison with that affected by HFD alone. We acknowledge nonetheless that within the research by Durgan et al, they subjected their animals to a shorter interval (2 weeks) versus our 4-week protocol and that their mannequin (rats) versus our murine mannequin could have contributed to such variations.^14,19 Nevertheless, our longer protocol recognized an adversarial interactive impact of the mixture of intermittent and sustained hypoxia that was not noticed with intermittent hypoxia alone.

Interestingly, one of many options noticed in animal fashions with intestine dysbiosis is impaired epithelial barrier and elevated intestinal permeability (leaky intestine), which is believed to contribute to a state of low-grade systemic irritation that constitutes a cornerstone within the pathophysiology of many comorbid ailments and persistent sicknesses.^43–46 Moreno-Indias et al⁴⁷ demonstrated low-grade endotoxemia throughout restoration from intermittent hypoxia in mice. Moreover, Ganesh et al⁴⁸ demonstrated that rats uncovered to HFD-IH confirmed lack of epithelial goblet cells, thinning of the mucus barrier, and decrease ranges of brief chain fatty acids (primarily acetate) within the cecum. Short chain fatty acids function a nutrient supply for epithelial cells and assist keep the integrity of cell–cell adhesion and intestine barrier permeability. Supplementation with pre- and pro- biotics in these rats prevented goblet cell loss and mucosal thinning. Replacement of acetate by steady infusion for two weeks prevented IH-induced systemic hypertension.^19,48

Our colonic gene expression evaluation indicated that along with genes related to lipid metabolism, HFD and IH+SH synergized to control the expression of genes related to epithelial barrier perform. Barrier-forming claudin 4 (Cldn4)^49,50 identified to be downregulated in ulcerative colitis,⁵¹ collagenous colitis,⁵² and irritable bowel syndrome⁵³ was additionally downregulated in HFD_IH+SH group in our research. Concurrently, expression of claudin 2, identified for its affiliation with leaky epithelia and upregulated in intestinal irritation was elevated within the HFD_IH+SH group.⁵⁴ Similarly, spleen-associated tyrosine kinase (SYK), which not directly controls epithelial barrier, was upregulated in HFD_IH+SH mice.^55,56 Admittedly, not all genes recognized on this GO class help the notion about faulty barrier. Nectins, that are identified for his or her affiliation with adherens and tight junctions, and which help barrier formation had been upregulated within the HFD_IH+SH group.⁵⁷ Thus, future research should decide whether or not the online impact of the noticed adjustments certainly interprets to impaired barrier perform and elevated epithelial permeability.

To our data, our experiment is the primary to look at the affect of the mixture of intermittent and sustained hypoxia and the interactive impact of such publicity with HFD on the colonic mucosal microbiome and host tissue gene expression profile. We additionally used the colonic mucosa tissue as a substitute of the fecal samples to look at the microbiome. Our research has limitations. First, we uncovered the mice in our experiment to intermittent hypoxia with out intermittent hypercapnia.^{15,16,58–60} There is powerful proof that intermittent hypercapnia performs an vital function in shaping the intestine microbiota. However, a number of research utilized solely intermittent hypoxia protocols and concluded a shift within the intestine microbiota profile.^41,47,61 Second, our host gene expression findings of altered intestinal permeability and cell–cell adhesion weren’t confirmed by permeability assay (FITCH-conjugated dextran assay) to verify the luminal translocation of molecules into the bloodstream. Third, we didn’t measure cardiometabolic outcomes of the mice within the numerous circumstances of our research. The efficiency of such measures would have enabled us to raised join the hypoxia exposures with cardiometabolic outcomes in relation to intestine dysbiosis and fats consumption. Fourth, we didn’t apply an intermittent hypoxia alone or a sustained hypoxia alone arm in our research to instantly evaluate the differential results of such publicity. Instead, we in contrast findings from our research with that of others who can carry out the same experiment with intermittent hypoxia alone and high-fat weight-reduction plan.¹⁴ Nevertheless, though the addition of the IH alone and SH alone teams would have offered better perception, they’d have elevated the variety of teams and the complexity of our research and wouldn’t have detracted something away from the comparisons inside our present research design which stay legitimate. We plan to conduct further research performing head-to-head comparisons of intermittent and sustained hypoxia. Finally, our pattern measurement was small which resulted in underpowered outcomes.

Conclusion

High-fat weight-reduction plan throughout mixed intermittent and sustained hypoxia causes better intestine dysbiosis and probably adversarial adjustments in colonic epithelial transcriptome than high-fat weight-reduction plan throughout normoxia. The latter adjustments are suggestive of impaired intestine barrier perform which will type the idea for low systemic irritation and cardiometabolic illness. These findings can shed extra gentle on novel therapeutic interventions focusing on the intestine microbiome as an adjuvant therapy in sufferers with weight problems hypoventilation syndrome to mitigate metabolic issues and systemic irritation related to sleep-disordered respiratory and dietary fats consumption.

Acknowledgments

Dr Parthasarathy reviews analysis grants funded by the NIH (OT2-HL-161847-01, OT2-HL-156812, OT2-HL-158287, R25HL126140, C06OD028307, U01HL128954, UG3HL140144), PCORI (CER‐2018C2‐13262, DI-2018C2-13161), CDC (CDC-OT21-2103 (Subcontract #: CTR056154)) and Office of Minority Health (Department of Health and Human Services; CT-HD-22-089), Foundations (ASMF-169-SR-17) and Industry (Philips, Whoop. Inc.). He is a site-PI or co-investigator on analysis funded by DOD (W81XWH-14-1-0570, W81XWH20C0051, W81XWH2110025).

Disclosure

SP is a guide for Jazz Pharmaceuticals, Inc., receives royalty from UpToDate, Inc., and has a patent that was licensed by SaiOx, Inc. (US20160213879A1). SP reviews receiving grants to establishment from the next entities: Sergey Brin Family Foundation (Verily Life Sciences, Inc.), Philips-Respironics, Inc., WHOOP, Inc., Sommetrics, Inc., and Regeneron, Inc. These conflicts are unrelated to this manuscript. The creator reviews no different conflicts of curiosity on this work.

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