Serum and cecal metabolic profile of the insulin resistant and dyslipidemic p47phox knockout mice

Abstract Involvement of NOX-dependent oxidative stress in the pathophysiology of metabolic disorders as well as in the maintenance of metabolic homeostasis has been demonstrated previously. In the present study, the metabolic profile in p47phox–/– and WT mice fed on a chow diet was evaluated to assess the role of metabolites in glucose intolerance and dyslipidemia under altered oxidative stress conditions. p47phox–/– mice displayed glucose intolerance, dyslipidemia, hyperglycemia, insulin resistance (IR), hyperinsulinemia, and altered energy homeostasis without any significant change in gluconeogenesis. The expression of genes involved in lipid synthesis and uptake was enhanced in the liver, adipose tissue, and intestine tissues. Similarly, the expression of genes associated with lipid efflux in the liver and intestine was also enhanced. Enhanced gut permeability, inflammation, and shortening of the gut was evident in p47phox–/– mice. Circulating levels of pyrimidines, phosphatidylglycerol lipids, and 3-methyl-2-oxindole were augmented, while level of purine was reduced in the serum. Moreover, the cecal metabolome was also altered, as was evident with the increase in indole-3-acetamide, N-acetyl galactosamine, glycocholate, and a decrease in hippurate, indoxyl sulfate, and indigestible sugars (raffinose and melezitose). Treatment of p47phox–/– mice with pioglitazone, marginally improved glucose intolerance, and dyslipidemia, with an increase in PUFAs (linoleate, docosahexaenoic acid, and arachidonic acid). Overall, the results obtained in p47phox–/– mice indicate an association of IR and dyslipidemia with altered serum and cecal metabolites (both host and bacterial-derived), implying a critical role of NOX-derived ROS in metabolic homeostasis. Graphical Abstract


Introduction
Type 2 diabetes, a complex and heterogeneous metabolic disorder, is associated with alterations in the endocrine, inflammatory, genetic, environmental factors, and gut microbiome [1]. Role of ROS and RNS (reactive oxygen and nitrogen species) has been implied in the pathophysiology of metabolic perturbations [2][3][4][5][6] as well as in the maintenance of metabolic homeostasis [7][8][9].
NOX-mediated ROS production limits lipopolysaccharide (LPS)-induced acute in vivo inflammatory response [22]. p47 phox-/mice [23] mutations in p47 phox or gp91 phox [24] in humans, predispose them to chronic granulomatous disease (CGD) due to compromised anti-microbial defense, hyperinflammation, and enhanced susceptibility to infections [25]. Oxidative stress has a pivotal role in impacting the metabolic profile, host inflammatory response, and immune defense [26][27][28]. Recently, the bacterial-derived metabolites are being recognized as important messengers from the gut with profound effects on host metabolic regulation [29]. The present study explored the metabolic perturbations in mice lacking p47 phox . We observed glucose intolerance and dyslipidemia in p47 phox-/mice along with perturbed serum and cecal metabolome, suggesting a critical role of p47 phox -derived ROS in metabolic homeostasis.

Materials and methods
Mice and diet C57BL/6 (WT, wild type) and p47 phox knockout (p47 phox-/-, Jackson Laboratory, Bar Harbor, ME; 004742) age matched, 12-13 weeks old, male mice on C57BL/6J background were bred and maintained in individually ventilated cages (IVC) (Tecniplast, Buguggiate, Italy) at 24 ± 2 C. Animals were fed on a chow diet (1320, Altromin, Lage, Germany) with water ad libitum. All procedures were approved by CSIR-CDRI Institutional Animal Ethics Committee in accordance with CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines (IAEC/2014/43). Anti-diabetic pioglitazone (10 mg/kg) was administered to p47 phox-/mice orally for 5 weeks [30]. Body weight was measured weekly from day 0 to the completion of study at 5 weeks.
Tolerance tests D-Glucose (2 g/kg), insulin (0.6 IU/kg, human insulin R, Eli Lilly, Indianapolis, IN), or sodium pyruvate (2 g/kg) were administered to the 6 h fasted mice by the intraperitoneal (i.p.) route to perform glucose tolerance test (GTT), insulin tolerance test (ITT), or pyruvate tolerance test (PTT) tolerance test, respectively. Blood glucose levels were measured after the administration of glucose, insulin, or pyruvate at regular time intervals (0, 15, 30, 60, and 120 min) using Accu-Chek glucometer (Roche Diagnostics, Mumbai, India) and the area under the curve (AUC) was calculated as described previously [31].

Body composition analysis
Mice were allowed limited horizontal and vertical movements and radio frequency pulses were applied at a distinct static magnetic field to analyze the body composition (fat and lean mass) using echo MRI (Magnetic Resonance Imaging, E26-226-RM Echo MRI LLC, Houston, TX) [20].

Indirect calorimetry
Unrestrained, conscious mice were kept individually in the Oxymax CLAMS (Comprehensive lab animal monitoring system, Columbus Instruments, Columbus, OH) with six stations and food and water ad libitum for metabolic measurements [32]. After acclimatization of animals and pre-calibration of system, carbon dioxide produced (VCO 2 , ml/kg/h), oxygen consumed (VO 2 , ml/ kg/h), energy expenditure (kcal/h; heat production), RER (respiratory exchange ratio, VCO 2 /VO 2 , ml/kg/min), and metabolic rates (basal metabolic ratio, BMR and resting metabolic ratio, RMR) were determined over three days period along with the food and water intake. The locomotor activity was measured during the course of a given time interval as the number of times the mice break the infrared beam in the X and Y directions [33].

Gut permeability assay
The in vivo gut permeability was assessed using fluorescein isothiocyanate (FITC)-labeled dextran (4 kDa) [34] which was administered orally to four hours fasted mice (0.8 mg/ml in 200 ml PBS, phosphate-buffered saline) followed by removal of both food and water. After four hours of administering the tracer, blood was collected retro-orbitally and fluorescence intensity was measured in the serum (at an excitation wavelength of 493 nm and an emission wavelength of 518 nm).

Serum biochemistry
Mice were fasted for six hours; blood was collected from the retro-orbital plexus and serum separated. Circulating lipids such as total cholesterol (TC), triglycerides (TGs), non-esterified fatty acids (NEFA), and lowand high-density lipoproteins (LDL and HDL) were estimated in the serum using kits (Randox, Crumlin, UK) [35]. b-hydroxybutyrate was also estimated using Randox kit. Levels of leptin and adiponectin were estimated using ELISA kits from R&D Systems (Minneapolis, MN). Insulin and total bile acids (TBAs) were measured using the kit from Crystal Chem (Elk Grove Village, IL) and C-reactive protein (CRP) levels using the kit from Abcam (Cambridge, UK). Indices of insulin resistancehomeostatic model assessment for insulin resistance (HOMA-IR), insulin sensitivityquantitative insulin sensitivity check index (QUICKI), and beta cell functionality homeostasis model assessment of b-cell function (HOMA-B) were calculated as per the formulae used by other investigators from fasting blood glucose and serum insulin [36].

Total nitrite estimation
Mice were sacrificed and the tissues (liver, epididymal white adipose tissue (eWAT), and small intestine) retrieved after collection of blood and feces. Liver, eWAT, intestine, and feces (50 mg each) were homogenized in 500 ml of hypotonic TKM buffer (25 mM Tris-HCl (pH 7.4), 5 mM KCl, 2 mM MgCl 2 , and 1% NP-40) and then sonicated. Samples were centrifuged at 15,000Âg for 20 min at 4 C and the supernatant was collected. Griess reagent was used to estimate the total nitrite levels (nitrate and nitrite) in the serum (100 ll) and tissue homogenates by using pre-activated cadmium pellets to reduce nitrate to nitrite and using sodium nitrite as a standard as described previously [37].

Tissue biochemistry
Liver tissue, small intestine, and fecal samples (50 mg each) were processed as described previously for the estimation of TC, TGs, and free fatty acids (FFAs) using the Randox kit [31].

Hematoxylin and eosin (HE) staining
Adipose tissue was formalin fixed for 72 h and then processed in graded concentrations of ethanol and then xylene followed by liquid paraffin infiltration [20]. Paraffin embedded tissue blocks were prepared, sectioned into 5 lm thin serial slices and HE stained. Upright microscope (DM5000, Leica Microsystems, Wetzlar, Germany) in bright field mode was used to capture the images. Adipocytes area was calculated using Adiposoft plugin in Fiji software.

Alcian blue staining
Small intestine and colonic tissues were formalin fixed, paraffin embedded and 5 lm thin sectioned slices were stained with 1% Alcian blue (AB) solution (in 3% acetic acid, pH 2.5) and counterstained with 0.1% nuclear Fast Red solution for morphological analysis of acid mucosubstances and acetic mucins [38]. Percent AB stained area, villi and crypt lengths were calculated using Image J software (Bethesda, MD).

Real-time PCR
Quantitative gene expression analysis was performed by using SYBR Green and LightCycler 480II Real-Time PCR system (Roche Applied Science, Indianapolis, IN) as described previously [39]. Briefly, total RNA was extracted using TRIZOL reagent followed by cDNA synthesis using RevertAid first strand cDNA synthesis kit as per the manufacturer's protocol. The primers used in the present study are listed in Table S1. RPL10 was used as a reference gene for normalization in the small intestinal tissue and 18S rRNA in the liver and eWAT to calculate the expression of the candidate genes. Relative fold change was calculated from mean normalized gene expression for p47 phox-/mice as compared to WT mice.

Metabolomics analysis
Lyophilized serum (100 ml) and cecal contents (100 mg) were suspended in methanol (200 ml), water (50 ml), and methyl tert-butyl ether (MTBE, 870 ml) and vortexed to extract the metabolites for an hour as described earlier [40]. Briefly, water (250 ml) was added, samples were centrifuged to separate the aqueous (lower) and organic (upper) layers, and both the layers were vacuum dried. Samples were reconstituted in 15% methanol (50 ml), kept on ice, vortexed, centrifuged, supernatant collected, and then subjected to metabolomic analysis using the LC-MS platform. The data were acquired on an orbitrap fusion mass spectrometer (Thermo Scientific, Waltham, MA) equipped with heated electrospray ionization (HESI) source in both positive and negative modes as per the previously described settings [40]. The extracted metabolites were separated on UPLC ultimate 3000 maintained at 40 C temperature using the HSS T3 column (100 Â 2.1 mm i.d., 1.7 mm, waters) as previously described [40]. To monitor retention time shift, signal variation, and drift in mass error, a pool (prepared by collecting 10 ml from each sample) quality control (QC) sample was run after every five samples. The acquired data were processed using Progenesis QI software (Water Corporation, Milford, MA) using the default setting. Retention time alignment, feature detection, elemental composition prediction, and database search were performed using the untargeted metabolomics workflow of Progenesis QI. Metabolite identification was done as described previously [40]. Metabolomics data were normalized by sum and pareto scaled followed by multivariate analysis. Relative foldchange was calculated for each sample with respect to the control (WT) for differential analysis and was logtransformed for the heat map analysis.

Statistical analysis
Data have been presented as mean ± SEM. An independent unpaired Student's t-test was used for the comparisons using GraphPad Prism 8 software (La Jolla, CA). Differences were considered statistically significant at p < 0.05.

Availability of data and materials
All data used in this study are present in the main text and supplementary materials.

Results
p47 phox-/mice display systemic metabolic perturbations p47 phox-/mice fed on chow diet were glucose intolerant ( Figure 1(A,B)) and hyperglycemic (Figure 1(C)) as compared to WT. These knockout mice also displayed systemic insulin resistance (IR) (Figure 1(D,E)) and hyperinsulinemia ( Figure 1(C)). The gluconeogenesis as evident by PTT was not altered in p47 phox-/as compared to WT mice ( Figure 1(G,H)). p47 phox-/mice were dyslipidemic with increased circulating levels of TC, TGs, LDL, and NEFA ( Figure 1(F)). The serum HDL levels were comparable to WT mice. We observed an imbalance in adipokines levels in p47 phox-/mice with increased circulating leptin and adiponectin levels as compared to WT. The serum TBAs and CRP were enhanced in p47 phox-/mice with unaltered b-hydroxybutyrate levels ( Figure 1(I)).
The fat mass (Fig. S1C) and liver weight ratio (Fig. S1D) were augmented in insulin-resistant and dyslipidemic p47 phox-/mice as compared to WT with unaltered lean mass (Fig. S1C) and body weight (Fig.  S1A, B). The weight of other tissues (eWAT, BAT (brown adipose tissue), heart, spleen, kidney, small intestine, cecum, and colon) of p47 phox-/mice was comparable to WT (Fig. S1D). Gut shortening was observed with a significant decrease in the length of small intestine and colon in p47 phox-/mice (Fig. S1E). The knockout mice also displayed altered energy homeostasis with a decrease in VCO 2 , VO 2 , heat production, and metabolic rates (BMR and RMR) despite unaltered RER, food or water consumption, and physical activity (as measured by Xand Z-axis movements, Fig. S2A-I).
p47 phox-/mice exhibit altered hepatic lipids and glucose metabolism Hepatic TC and TG levels were significantly enhanced in the p47 phox-/mice while FFA levels were comparable to WT mice (Figure 2(A)). The gene expression of transcription factors and enzymes involved in hepatic lipid synthesis (PPARc, FAS, and ACC1) was increased, with no alteration in SREBP-1c, SREBP-2, and HMGCR, while CYP7A1, enzyme involved in the bile acid synthesis was reduced (Figure 2(B)). The expression of CPT-1, involved in the transportation of fatty acids into the mitochondria for beta-oxidation, was significantly increased in the liver of p47 phox-/mice, while the expression of PPARa and ACC2 remained unaltered (Figure 2(C)). The expression of genes involved in the lipid uptake (CD36), lipolysis (LPL), and efflux (ABCG5 and ABCG8) was also increased in the liver of p47 phox-/mice as compared to WT (Figure 2(D,E)).
Gene expression of Akt2, involved in the insulin signaling, was reduced in p47 phox-/mice (Fig. S3A). Expressions of other genes involved in glucose and insulin homeostasis (Glut2, AMPKa, PTP-1B, and FGF21) and adipokines (leptin and adiponectin) were comparable in the p47 phox-/and WT mice (Fig. S3A, B). Gluconeogenic genes, PEPCK and G6PC were not altered in the liver of p47 phox-/mice with a marginal increase in expression of PC (Fig. S3C) which is agreement to the PTT data. The expression of inflammatory cytokine, TNFa was increased in the liver of p47 phox-/mice along with IR and dyslipidemia (Fig. S3E).
p47 phox-/mice have altered lipid and glucose metabolism in the adipose tissue Histological analysis of adipose tissue revealed an increase in the size of adipocytes in p47 phox-/mice as compared to WT mice (Figure 3(A)) along with skewed adipocytes area frequency distribution and increase in the mean adipocytes area (Figure 3(B)). The expression of lipid synthesis genes (SREBP-1c, FAS, and ACC1) was enhanced in adipose tissue of p47 phox-/mice; while, the expression of genes involved in lipid oxidation (PPARa and ACC2) remained unaltered (Figure 3(C,D)). The expression of genes regulating lipid uptake (CD36 and LPL) was augmented in the adipose tissue of p47 phox-/mice (Figure 3(E)). The expression of gluconeogenic genes (PEPCK, G6PC) and inflammatory gene (TNFa) was unaltered in the adipose tissue of p47 phox-/mice (Fig. S3D, F).  The barrier functionality of the gut was assessed by in vivo FITC-dextran gut permeability assay, which showed enhanced gut permeability in p47 phox-/mice (Figure 4(A)). It was also evident by the percent AB stained area in intestinal (Figure 4(D,E)) and colonic tissues (Figure 4(G,H)). The intestinal villi length ( Figure  4(F)) was more in p47 phox-/mice, as compared to WT without any change in the colonic crypt length ( Figure  4(I)). In the small intestine of p47 phox-/mice, gene expression of tight junction proteins, Claudin-2 and occludin was increased, but the expression ZO-1 was not changed. The expression of Muc-5AC was significantly increased, while Muc-2 and Reg3c (an antimicrobial peptide) remain unaltered in the small intestinal tissue of p47 phox-/mice (Figure 4(B)). The expression of LXRa, HMGCR, and CYP7A1 was higher in the small intestine of p47 phox-/mice (Figure 4(C)). Expression of the lipid uptake genes was also augmented (SR-1B, FFAR1, and FFAR2) without any change in the expression of CD36, NPC1L1, FABP-1, LDLR, and ApoE (Figure 4(L)) in the small intestine of p47 phox-/mice. In addition, expression of ABCG8 expression was increased with no change in the ABCG5 and ABCA1 genes in the small intestine of p47 phox-/mice ( Figure  4(J)). In the intestinal tissue samples of p47 phox-/mice, amount of TC, TG, and FFA was similar while TC and FFA levels were higher in the fecal samples in comparison to the WT mice (Figure 4(K)).
Further, the total nitrite levels in p47 phox-/mice were not altered in the serum, liver, adipose tissue, intestine, and stool samples (Fig. S4A). The gene expression of NOX enzymes remained unaltered in p47 phox-/mice in the liver with a marginal increase in NOX2 in adipose tissue (Fig. S4B, C). nNOS expression was enhanced in the liver and small intestine of p47 phox-/mice with decreased eNOS in adipose tissue (Fig. S4D-F).
p47 phox-/mice display partial improvement in glucose tolerance and dyslipidemia after treatment with pioglitazone The glucose intolerance was marginally (p < 0.05) improved in the p47 phox-/mice after the treatment with anti-diabetic drug, pioglitazone ( Figure 6(A,B)) with no effect on the blood glucose levels (Figure 6(C)). Also, the pioglitazone treatment had no effect on the gluconeogenesis as assessed by PTT (Fig. S6A, B). Dyslipidemia in p47 phox-/mice was improved by pioglitazone with decrease in TC, LDL, and NEFA levels and unaltered TG and HDL. Also, the CRP and TBA levels were unaltered upon pioglitazone treatment in p47 phox-/mice ( Figure 6(C)). The liver and kidney weight ratios were reduced with an increase in BAT weight ratio in p47 phox-/mice following pioglitazone volcano plot of differential metabolites (p < 0.05) and (F) heat map of differential metabolites. Data are represented as mean ± SEM (n ! 3). Ã p < 0.05, ÃÃ p < 0.01, ÃÃÃ p < 0.001, and ÃÃÃÃ p < 0.0001 vs. p47 phox-/-. # p < 0.05 vs. p47 phox-/in the IPGTT curve. See also Figure S6. treatment (Fig. S6G) despite unaltered body weight and food consumption (Fig. S6C-F).
The PCA score plots of pioglitazone treated and untreated p47 phox-/mice displayed marginal separations ( Figure 6(D)), further the differential metabolites were visualized through volcano plots (Figure 6(E)). Polyunsaturated fatty acids (PUFAs), linoleate, docosahexaenoic acid, and arachidonic acid were increased after the pioglitazone treatment. Thyrotropin-releasing hormone, levothyroxine, purine metabolites (methyladenosine and xanthosine), anthranilate were reduced, while riboflavin, amino acid metabolites, and PGE 1 were enhanced upon pioglitazone treatment. The carbohydrate metabolites, glycerol-3-phosphate, N-acetylneuraminate were enhanced with decrease in fructose-6phosphate ( Figure 6(F)). This suggests that pioglitazone treatment only marginally improved the glucose intolerance and dyslipidemia in p47 phox-/mice and that other factors like inflammation and bacterial associated metabolites seem to be the major drivers of metabolic perturbations in the knockout mice.
Mice treated with sodium nitrite (50 mg/l, NaNO 2 ) in drinking water ad libitum for 5 weeks exhibited reduction in the glucose intolerance observed in the untreated p47 phox-/mice (Fig. S7A), improved VCO 2 , heat production, and overall metabolic homeostasis (Fig. S7B, C). The circulating blood glucose, TC and TG levels were reduced in nitrite treated p47 phox-/mice (Fig. S7D). However, the circulating HDL, NEFA, CRP, and TBAs levels, and the relative liver, heart, spleen, and kidney weight ratios remain unaltered (Fig. S7D, E).

Discussion
Excess ROS production seems to play an important role in the metabolic perturbations [2,16] and enhanced activity of NOX enzymes in the major metabolic organs has been observed in the dietary or genetic models of IR/obesity [2,3,11,41,42]. HFD-fed male p47 phox-/mice were protected against IR, inflammation [10], skeletal muscle IR [41], and adipose tissue dysfunction [11]. NOX4 -/mice also exhibited IR, adipose tissue hypertrophy, inflammation, hypoxia, and diet-induced obesity [13]. On the contrary, basal ROS was found crucial for maintaining glucose and insulin homeostasis [8,[14][15][16][17] suggesting a dual role of NOX-derived ROS. Previous studies from our lab demonstrated reductions in ROS levels in the LFD and HFD-fed p47 phox-/mice were associated with augmented lipids, altered insulin signaling, and expression of key metabolic genes [20].
We observed enhanced fat mass and altered energy homeostasis in p47 phox-/mice without any change in the body weight and food intake as was previously observed in a study conducted on NOX2 -/mice fed on chow or 45% HFD for 3 or 9 months [41]. On the other hand, NOX2 -/mice (gp91 phox subunit deletion) fed on 60% HFD for 14 weeks displayed weight gain without any change in the food intake [11]. NOX2 -/mice fed on chow or 60% HFD for 13 or 18 weeks with hyperphagia exhibited weight gain, but no change in energy homeostasis [19]. Similarly, chow or HFD-fed male p47 phox-/mice for 13 weeks weighed more as compared to WT mice with similar fat mass [18]. Most of the studies with different outcomes have used diverse diets, different sources of dietary fat (lard or vegetable oils), and protocols.
Previously conducted studies did not observe the change in the glucose tolerance in chow [19,43] or HFD-fed NOX2 -/mice [19], although hyperinsulinemia was observed [18,19]. Female and male p47 phox-/mice kept for 13 weeks on HFD, displayed protection against diet-induced steatosis, hyperglycemia, reduced serum lipids, but exhibited hyperinsulinemia and imbalance in adipokines [18]. In addition, male p47 phox-/mice had enhanced body weight and adipocyte size, while, female p47 phox-/mice were resistant to HFD-induced obesity and adipose tissue hypertrophy [18]. In yet another study, p47 phox-/mice were protected against HFD-induced adiposopathy and skeletal muscle IR, but did not exhibit alterations in the circulating insulin levels and lipid profile [11,41]. NOX2 -/mice kept on chow and 60% HFD diet for 18 weeks displayed enlarged liver with higher lipid accumulation [19] as observed by us in the chow-fed p47 phox-/mice, suggesting that reduction in ROS exacerbates IR by promoting steatosis and inflammation. These studies prompted us to assess the metabolic status of adult p47 phox-/mice fed on a normal chow diet. Chow-fed p47 phox-/mice were insulin resistant with higher circulating insulin levels. We observed an increase in the size of adipocytes with unaltered eWAT weight, as was also observed earlier [11]. On the contrary, NOX2 -/mice had reduced eWAT weight with no change in the adipocyte size [19]. Metabolic syndrome and obesity are commonly associated with elevated leptin and decreased adiponectin levels [44][45][46]. Ronis et al. like our study also observed imbalanced adipokines in p47 phox-/mice along with IR and dyslipidemic phenotype [18]. Transcription factor profile data in p47 phox-/mice corroborate the biochemical and functional analysis with increased expression of lipid synthesis, oxidation, uptake and efflux genes in liver, adipose tissue, and intestine. On the contrary, hepatic SREBP-1c and FAS expression was decreased in the chow and HFD fed NOX2 -/mice [19].
Interestingly, in the present study, p47 phox-/mice also exhibited perturbed metabolic profile along with changes in the gut functions.
RONS, produced by the cellular components of innate immune system, are crucial for maintaining the bacterial homeostasis in the gut [26,27,47,48] and the systemic metabolic homeostasis [20,31,35,40,[48][49][50]. In fact, mutations in subunits of NOX enzymes, compromised the ROS production leading to CGD phenotypes in rodents like humans [23,24] compromised anti-microbial defense [25]. p47 phox-/mice are prone to infections [43] and exhibit enhanced inflammation. Interestingly, gut microbiota depletion by antibiotic intervention makes them susceptible to infections and sepsis [27], survival of p47 phox-/mice following treatment with antibiotics was adversely affected, we therefore, could not perform the desired studies. Our preliminary and limited analysis however showed alterations in the gram-negative bacteria in p47 phox-/mice with increased S24-7 and Akkermansia and decreased Prevotella (16S rRNA gene sequencing data has been deposited in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) under accession number PRJNA827678). Interestingly, similar observations have been also reported in db/db mice [51] which is a commonly used diabetic model. Surprisingly, these diabetic mice have mutations in p47 phox [21] suggesting an association of gut bacteria with enhanced inflammation, IR, and dyslipidemia in p47 phox-/mice. Gut length shortening along with enhanced gut permeability in the insulin resistant and dyslipidemic p47 phox-/mice has also been observed in other obese mice models [52][53][54]. The increase in the intestinal villi length as observed by us in p47 phox-/mice, has been linked with alteration in the absorption of nutrients [55]. Metabolic endotoxemia due to disruption in the gut barrier enhances the availability of microbial products in circulation [52,56,57] drive metabolic perturbations [56] in the p47 phox-/mice.
The intricate interplay between the host and the gut derived metabolites is important [58] for the characterization of biochemical processes in the insulin resistant and dyslipidemic p47 phox-/mice. Untargeted serum metabolomics conducted by us revealed enhanced pyrimidine metabolism in insulin-resistant and dyslipidemic p47 phox-/mice, as has been observed in diabetic patients [58]. Amino acid metabolite, anthranilate has been reported to be down-regulated in diabetic dogs [59]. We observed decrease in the serum anthranilate, carnitine, and N-acetyl cysteine in p47 phox-/mice. Others have also found reduction in the carnitine levels in obese subjects, thus, hampering the transportation of fatty acids into the mitochondria, leading to lipid accumulation [60]. N-acetyl cysteine plays a role in alleviating obesity and cardiovascular disorders [60][61][62]. Glycolysis and Kreb's cycle intermediates were decreased in p47 phox-/mice, glycolytic intermediates instead of entering the TCA cycle, push fatty acids synthesis [63]. Bis(2-ethylhexyl) phthalate, a toxic plasticizer is a common pollutant in the drinking water, has been linked with hypothyroidism and obesity in rodents and humans [64][65][66] was more in the serum of p47 phox-/mice. Altered lipid composition is well reported in the diabetic and obese subjects [67], PC lipid species which improve glucose homeostasis and insulin sensitivity [68,69] were decreased in p47 phox-/mice. Enhanced HFD-induced fatty liver state has been linked with a decrease in the hepatic PC and PE lipids [70,71]. In p47 phox-/mice, PGs levels were enhanced, previous study in the obese individuals has shown their association with the gut microbiota dysbiosis via endotoxins, inflammation, and altered adipose tissue homeostasis [72]. 3-Methyl-2-oxindole, a tryptophan metabolite produced in the gut was increased in p47 phox-/mice [73] suggesting an altered gut microbiota metabolism.
Cecal metabolomics revealed alterations in the various microbial metabolites in p47 phox-/mice. Indole metabolites, indoxyl sulfate, and indole-3-acetamide, are derived from tryptophan by the activity of gut microbial enzymes [74,75] and have been linked with the chronic kidney disease [76]. Hippurate, an early urinary biomarker of diabetes [77] and a bacterial cometabolite associated with high microbial richness [78,79], was reduced in p47 phox-/mice. N-acetyl galactosamine was increased in p47 phox-/mice which seems to be involved in the pathogenesis of diabetes [80]. Raffinose and melezitose, indigestible sugars [81] improve IR, and obesity [82][83][84] were less in the p47 phox-/mice. Enhanced bile acid levels in p47 phox-/in circulation and cecal contents correlate with the decrease in the expression of hepatic CYP7A1. Bile acids have been positively associated with IR [85,86] and dyslipidemia in both serum and cecum of p47 phox-/mice. Pioglitazone, a PPARc agonist and commonly used antidiabetic drug marginally improved the glucose intolerance and dyslipidemia in p47 phox-/mice with increased PUFA (poly-unsaturated fatty acids; linoleate, docosahexaenoic acid, and arachidonic acid) levels which have shown to have a beneficial effect on obesity and related comorbidities [87,88]. Although, the microbiota-associated metabolites found altered in p47 phox-/mice were not normalized by pioglitazone treatment, which might be the reason for partial recovery in IR and dyslipidemia. Nitrite, a strong oxidant is known for its beneficial effects in diabetic conditions [89], improves the metabolic homeostasis in redox imbalanced p47 phox-/mice. The findings suggest toward the importance of gut microbiota derived/associated metabolites in the perturbed metabolic homeostasis in p47 phox-/mice. It is therefore important to understand the interplay between, oxidative stress, genetics and, the host and microbial interactions in metabolic disorders.
In the present study, we observed inflammation, gut shortening and gut barrier disruption in the chow fed insulin resistant and dyslipidemic p47 phox-/mice. Pyrimidines, PG lipids and 3-methyl-2-oxindole were increased in the serum, while indole-3-acetamide, Nacetyl galactosamine, and glycocholate were enhanced in the cecal samples. Hippurate, indoxyl sulfate, and indigestible sugars (raffinose and melezitose) were however reduced along with the alteration in the bile acid metabolism. Overall, the results obtained suggest critical role of NOX mediated regulation of host-bacterial-derived metabolites and gut permeability, in the maintenance of metabolic homeostasis.