Gestational insulin resistance is mediated by the gut microbiome-indoleamine 2,3-dioxygenase axis

Background and aims: Normal gestation involves reprogramming of maternal gut microbiome (GM) that may contribute to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of GM maternal metabolism interaction. Methods: The GM and plasma metabolome of CD1, NIH Swiss and C57BL/6J mice were analyzed using 16S rRNA sequencing and untargeted LC-MS throughout gestation and postpartum. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM in the process was studied using fecal microbial transplants (FMT). Results: Significant variation in gut microbial alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time-point specific, with species enriched at gestation day 15/19 (G15/19), a point of heightened IR, distinct from those enriched pre- or post-pregnancy. Untargeted and targeted metabolomics revealed elevated plasma kynurenine at G15/19 in all three mouse strains. IDO1, the rate limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine levels effects on IR derive from the GM in both mouse and human pregnancy. Conclusions: GM changes accompanying pregnancy shift IDO1 dependent tryptophan metabolism toward kynurenine production, intestinal inflammation and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1.

Effects of Laboratory Housing Conditions on Core Temperature and Locomotor Activity in Mice

Drug developers worldwide assess compound safety and efficacy using measures that include mouse core temperature andlocomotor activity. Subtle differences in animal housing conditions between institutions can alter these values, impacting scientific rigor and reproducibility. In these studies, adult male NIH Swiss mice were surgically implanted with radiotelemetry probes that simultaneously monitored core temperature and locomotor activity across various housing conditions. In the first study, ambient temperature was varied between 20 °C and 28 °C in groups of singly housed mice. Additional studies held the mice at a constant ambient temperature and examined the effects of cage density (housing animals singly or in groups of 3 or 6), bedding change and provision of nesting material, and the availability of a running wheel on core temperature and locomotor activity. Mice overwhelmingly maintained species-typical core temperatures across all ambient temperatures,across all housing conditions, when bedding was fresh or old, and with or without the provision of cotton squares as nesting material. However, engaging in wheel running and the combination of fresh bedding and cotton squares transiently increased core temperatures beyond the species-typical range. Similarly, the circadian distribution of locomotor activity was significantly disrupted by placing animals in cages with fresh bedding or nesting material, or by performing both of these manipulations concurrently during the light period. These findings suggest that standard husbandry practices and common housing conditions may transiently affect core temperature in adult mice. Furthermore, these practices may have profound and relatively long-lasting effects on motor activity and the regulation of circadian rhythms.

Nitrous Oxide-induced Impairment of Spatial Working Memory Requires Activation of GABAergic Pathways

Background: Previous research from our laboratory implicated opioid and benzodiazepine- GABA mechanisms in other effects of N2O (antinociception and anxiolysis), so a decision was made to study these as potential mechanisms of N2O-induced dysfunction of spatial working memory. Objective: to explore potential mechanisms of N2O in reducing spatial working memory in mice. Methods: we monitored spontaneous alternation behavior (SAB) in male NIH Swiss mice exposed to N2O during a T-maze spontaneous alternation task (T-SAT). Results: mice that were exposed to 70% N2O (in O2) exhibited severely and significantly reduced spontaneous alternation behavior in the T-SAT. Mice in this environment alternated their route only 33% of the time, in comparison to the control (room air) rate of alternation at approximately 70%. Mice pretreated with the benzodiazepine antagonist, flumazenil exhibited a dose-dependent restoration of spatial working memory under 70% N2O in the T-SAT. Alternatively, pretreatment with neither the GABAA antagonist gabazine nor the opioid antagonist naloxone had any appreciable effect on the N2O-reduced SAB. Conclusion: this study verified that 70% N2O can reduce spatial working memory in mice, which appears to involve benzodiazepine mechanisms in the brain.

Impact of Exposure to Environmental Enrichment on the Anxiety-Like Behavior of Ovariectomized Mice

Objective: The aim of this study was to explore the influence of short-term (2-week) exposure to social (SE) and/or physical enrichment (PE) on the anxiety-like behavior of ovariectomized (OVX) NIH Swiss mice. Method: One week after surgery, each OVX mouse was housed under one of 4 social conditions: (1) isolated, (2) accompanied by an intact female, (3) accompanied by an intact male, or (4) in a community of 10 OVX individuals. The animals in each of these environments were divided into 2 subgroups, consisting of the presence and absence of PE. Following a 2-week exposure to the respective conditions, each OVX mouse was subjected to either the light/dark exploration test (LDT) or the elevated plus maze (EPM) to examine anxiety-like behavior. Results: The LDT and EPM showed very similar patterns. Compared to an impoverished environment, PE elicited a significant anxiolytic effect for OVX mice housed alone or in companion of an intact female (F [1, 54] = 16.11, P = 0.001). By contrast, mice living in community but without PE displayed anxiogenic-like behavior, perhaps due to crowding, compared to the animals living in isolation (F [1, 36] = 5.64, P = 0.023). Conclusion: This study emphasized the importance of taking housing conditions into account during the screening of new anxiolytic agents and the critical role of OVX in the regulation of anxiety.

A43 β-DEFENSINS AS MARKERS OF INTESTINAL DYSBIOSIS: THE NATURE OF CHANGES IN β-DEFENSINS IS DEPENDENT ON THE PROCESS UNDERLYING THE INDUCTION OF DYSBIOSIS

Background Dysbiosis may be defined as a change in the microbial composition or function that results in altered host function. Defensins are antimicrobial peptides, are part of innate immunity, and are important in host defense and maintaining homeostasis. Dysbiosis is a putative mechanism underlying the expression of many functional GI disorders like Irritable Bowel Syndrome (IBS) for which no biomarkers exist. Previous studies have revealed increased β-defensin (β-Def) levels in IBS patients, most likely due to changes in the microbiota. Aims We examined the hypotheses that: 1) Changes in β-Def are dependent on the manner in which dysbiosis is induced, and that 2) the direction of the change in β-Def depends on how dysbiosis was induced. Methods We used 4 models of experimentally induced dysbiosis to determine changes in fecal β-Def and to characterize the microbiota composition before and during the induction of dysbiosis. We used: 1) an antimicrobial cocktail (AC) in water; 2) a high-fat/ high-sugar diet (HFHSD); 3) a high salt diet (HSD) that we previously showed to induce a pro-inflammatory microbiota; and 4) mild restraint stress (MRS). All studies were performed in C57/BL6 mice except studies using MRS that were performed in NIH Swiss mice. In the AC or dietary studies, we employed a one-week intervention preceded by one-week baseline and recovery periods. In MRS studies, mice comparisons were made between a control and a stressed group. Stool samples were collected every 24 hours and were assayed for fecal β-Def levels analysis by an ELISA and microbial composition by 16S gene profiling. Results Exposure to AC or dietary change, but not MRS, resulted in significant decreases in fecal β-Def. Additionally, bacterial composition and diversity profiles were different in all mice except MRS mice (control vs. MRS males: p=0.414; control vs. MRS females: p=0.96). In contrast, mice exposed to the HSD revealed a significant increase in β-Def during treatment compared to baseline in both males (p=0.025) and females (p= 0.0019). The AC mice showed the largest changes and significant correlations between changes in β-Def levels and bacterial diversity (males: p=0.013, r=0.6; females: p=0.007, r=0.6) and richness (males: p=0.0008, r=0.70; females: p=0.003, r=0.62). However, no significant correlations were found between specific bacteria and β-Def levels in the HFHSD group. Conclusions We conclude that directional changes in fecal β-Def levels are dependent on the manner in which dysbiosis is induced. The use of β-Def as a biomarker requires comparisons with baseline levels obtained during remission in order to identify dysbiosis presence in microbiota-associated chronic GI conditions like IBS. Such an approach will identify patient subgroups that may benefit from microbiota-directed therapies. Funding Agencies CIHR

A120 INFLUENCE OF THE MICROBIOTA ON THE POSTNATAL DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM IS DEPENDENT ON MOUSE STRAIN

Background Gastrointestinal function depends on the normal formation of the enteric nervous system (ENS) during fetal and postnatal development. Prior research in an outbred strain of mice (NIH Swiss) has shown that the absence of the gut microbiome in germ-free (GF) mice results in morphological and functional abnormalities of the ENS compared to specific pathogen free (SPF) mice, including an alteration in proportion of nitrergic neurons. Increasing research has been suggesting that the genetic background of the host can impact the host response to the GF state. Aims We tested the hypothesis that the absence of the microbiome in an inbred mouse strain (C57BL/6) could influence the development of the ENS during early postnatal life. Methods C57BL/6 GF and SPF mice were sacrificed at postnatal day 3 (P3) and P28 (n=4–5 per group). Ileum and colon were collected at P3 and P28 and processed for whole mount preparations. The neuronal network in the myenteric plexus was visualized by immunohistochemistry using antibodies against the pan-neuronal marker PGP9.5. Neuronal cell bodies and nitrergic neurons were identified by immunolabeling with antibodies to the neuronal marker HuC/D and to neuronal nitric oxide (nNOS). Nerve fibre density was quantified by measuring the percentage of PGP9.5-positive pixels (μm2) compared to the whole field using an image analysis program (Volocity; reported as %). Proportions of nitrergic to myenteric neurons were determined by manually counting (blinded) the number of nNOS-positive neurons and dividing by the total number of HuC/D-positive cells per field (reported as %). Results We found a significant increase in nerve density at P3 in the GF compared to SPF mice in both ileum (43% vs. 37%; p=0.03) and colon (45% vs. 39%; p=0.03). No significant differences, however, were identified between GF and SPF mice at P28 in either ileum (27% vs. 25%; n.s.) or colon (31% vs. 32%; n.s.). At P3, no significant differences in proportion of nitrergic neurons were seen in the GF compared to SPF ileum (27% vs. 27%; n.s.). Conclusions In contrast to earlier observations in the NIH Swiss mice, in which GF mice had decreased nerve density and an increase in nitrergic neurons at P3, our findings reveal an opposite response in nerve density in the C57BL/6 mice and no change in nitrergic neurons. These results suggest that the genetic strain of the mouse model can influence the host response to changes in the microbiome. Further studies are needed to further elucidate potential underlying mechanisms. Funding Agencies NSERC

Gut microbial features can predict host phenotype response to protein deficiency

Malnutrition remains a major health problem in low and middle income countries. During low protein intake, < 0.67 g/kg/day, there is a loss of nitrogen (N2) balance, due to the unavailability of amino acid for metabolism and unbalanced protein catabolism results. However, there are individuals, who consume the same low protein intake, and preserve N2 balance for unknown reasons. A novel factor, the gut microbiota, may account for these N2 balance differences. To investigate this, we correlated gut microbial profiles with the growth of four murine strains (C57Bl6/J, CD-1, FVB, and NIH-Swiss) on protein deficient (PD) diet. Results show that a PD diet exerts a strain-dependent impact on growth and N2 balance as determined through analysis of urinary urea, ammonia and creatinine excretion. Bacterial alpha diversity was significantly (p < 0.05, FDR) lower across all strains on a PD diet compared to normal chow (NC). Multi-group analyses of the composition of microbiomes (ANCOM) revealed significantly differential microbial signatures between the four strains independent of diet. However, mice on a PD diet demonstrated differential enrichment of bacterial genera including, Allobaculum (C57Bl6/J), Parabacteroides (CD-1), Turicibacter (FVB), and Mucispirillum (NIH-Swiss) relative to NC. Additionally, statistical model fitting revealed that the relative abundance of genera such as Bifidobacterium, Ruminococcus, and Lactobacillus were significantly positively correlated with body weight, while Anaerofustis, Roseburia, and Bilophila were significantly positively correlated with ammonia excretion. Taken together, these results suggest a potential relationship between the specific gut microbiota, N2 balance and animal response to malnutrition.

Seminal fluid metabolome and epididymal changes after antibiotic treatment in mice

Paternal environment can induce detrimental developmental origins of health and disease (DOHaD) effects in resulting offspring and even future descendants. Such paternal-induced DOHaD effects might originate from alterations in a possible seminal fluid microbiome (SFM) and composite metabolome. Seminal vesicles secrete a slightly basic product enriched with fructose and other carbohydrates, providing an ideal habitat for microorganisms. Past studies confirm the existence of a SFM that is influenced by genetic and nutritional status. Herein, we sought to determine whether treatment of male mice with a combination of antibiotics designed to target SFM induces metabolic alterations in seminal vesicle gland secretions (seminal fluid) and histopathological changes in testes and epididymides. Adult (10- to 12-week-old) National Institutes of Health (NIH) Swiss males (n = 10 per group) were treated with Clindamycin 0.06 mg/kg day, Unasyn (ampicillin/sulbactam) 40 mg/kg day and Baytril (enrofloxacin) 50 mg/kg day designed to target the primary bacteria within the SFM or saline vehicle alone. Fourteen-day antibiotic treatment of males induced metabolomic changes in seminal vesicles with inosine, xanthine and l-glutamic acid decreased but d-fructose increased in glandular secretions. While spermatogenesis was not affected in treated males, increased number of epididymal tubules showed cribriform growth in this group (7 antibiotic-treated males: 3 saline control males; P = 0.01). Antibiotic-treated males showed more severe cribriform cysts. Current findings suggest antibiotic treatment of male mice results in seminal fluid metabolome and epididymal histopathological alterations. It remains to be determined whether such changes compromise male reproductive function or lead to DOHaD effects in resulting offspring.