Gamma-aminobutyric acid-producing lactobacilli positively affect metabolism and depressive-like behaviour in a mouse model of metabolic syndrome

Metabolic and neuroactive metabolite production represents one of the mechanisms through which the gut microbiota can impact health. One such metabolite, gamma-aminobutyric acid (GABA), can modulate glucose homeostasis and alter behavioural patterns in the host. We previously demonstrated that oral administration of GABA-producing Lactobacillus brevis DPC6108 has the potential to increase levels of circulating insulin in healthy rats. Therefore, the objective of this study was to assess the efficacy of endogenous microbial GABA production in improving metabolic and behavioural outcomes in a mouse model of metabolic dysfunction. Diet-induced obese and metabolically dysfunctional mice received one of two GABA-producing strains, L. brevis DPC6108 or L. brevis DSM32386, daily for 12 weeks. After 8 and 10 weeks of intervention, the behavioural and metabolic profiles of the mice were respectively assessed. Intervention with both L. brevis strains attenuated several abnormalities associated with metabolic dysfunction, causing a reduction in the accumulation of mesenteric adipose tissue, increased insulin secretion following glucose challenge, improved plasma cholesterol clearance and reduced despair-like behaviour and basal corticosterone production during the forced swim test. Taken together, this exploratory dataset indicates that intervention with GABA-producing lactobacilli has the potential to improve metabolic and depressive- like behavioural abnormalities associated with metabolic syndrome in mice.

Ireland Ltd.) and incubating anaerobically for 48 h at 37 o C. In addition, isolated colonies were tested for GABA 36 production in vitro, as described previously 1 . Briefly, isolated colonies were grown anaerobically in MRS 37 containing 3% (w/v) and 1% (w/v) MSG at 37 o C for 55 h. Samples were then deproteinized by mixing equal 38 volumes of 24% (w/v) trichloroacetic acid (TCA) and culture, allowed to stand for 10 min and centrifuged at 39 14,000g for 10 min. Supernatants were removed and diluted with 0.2 mol/L sodium citrate buffer, pH 2.2 to 40 yield 250 nmol of each amino acid residue. Samples were then diluted with the internal standard, norleucine, to 41 yield a final concentration of 125 nm/mL. Amino acids were quantified using a Jeol JLC-500/V amino acid 42 analyser (Jeol Ltd, Garden City, Herts, UK) fitted with a Jeol Na + high-performance cation exchange column.

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To calculate the % bioconversion of 1% MSG to GABA the following calculation was used:

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Glutamate in MRS (nmol/mL) -Glutamate in sample (nmol/mL) = nmol/mL of MSG consumed 45 (GABA in sample (nmol/mL) / nmol/mL of MSG consumed) *100 46 47 Glucose and insulin tolerance tests 48 After 12 weeks of feeding, an intraperitoneal-glucose tolerance test (IP-GTT) and an intraperitoneal-insulin 49 tolerance test (IP-ITT) was performed in the LFC (n 7) and HFC (n 7) groups. After 10 weeks of intervention, 50 the IP-GTT and IP-ITT were performed on individual mice in the LFC, HFC, DPC6108 and DSM32386 groups.

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For the IP-GTT, mice were injected with a glucose load (1g/Kg body weight) directly into the peritoneal cavity, 52 following a 6 h fast. Blood glucose levels were measured before and 15, 30, 60, 90 and 120 min after glucose 53 load. For the IP-ITT, mice were injected with an insulin load (0.75IU/g bodyweight) directly into the peritoneal 54 cavity, following a 6 h fast. Blood glucose levels were measured before and 15, 30, 60, 90 and 120 min after 55 insulin load. The concentration of blood glucose during the IP-GTT and IP-ITT was determined using a glucose 56 meter (Accu-Chek Aviva, Roche Diabetes Care Ltd., West Sussex, UK) on blood samples collected from the tip 57 of the tail vein.

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The plasma insulin concentrations were measured in plasma collected from tail blood during the IP-GTT, after 61 10 weeks of intervention, using a Mouse Insulin ELISA kit (Mercodia, Uppsala, Sweden), according to the 62 manufacturer's instructions. The insulin resistance index was determined by multiplying the area under the 63 curve of both the blood glucose (0 to 120 min) and the plasma insulin (0 to 15 min) obtained from the IP-GTT.

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A mixed-meal tolerance test was performed after 10 weeks of intervention. Mice were fasted for 6 h and a 67 baseline blood sample was taken from the tail following tail incision and collected into EDTA tubes (BD

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Sequences obtained from Illumina sequencing were processed using Quantitative Insights Into Microbial 78 Ecology (QIIME) software package version 1.9 2 . The paired-end reads were associated to the corresponding 79 sample through the unique barcode and joined. Reads were further processed with the inclusion of quality 80 filtering based on a quality score of > 20 followed by subsequent removal of sequences below length threshold 2 .

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UCLUST was then used for clustering the reads left into operational taxonomic units (OTUs) at 97% identity 3 .

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PyNAST 4 was used to align OTUs with a minimum alignment of 150 bp and 80% of minimum identity, and 83 taxonomy was assigned by using Ribosomal Database Project (RDP) classifier 2.0.1 5 . QIIME was used to 84 generate alpha (Chao1, observed OTUs) and beta diversities (Bray Curtis) distance matrices, and principal 85 coordinate analysis (PCoA) plots were generated based on the beta diversity distance matrices.

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Novel object recognition 107 Following eight weeks of dietary intervention, the novel object recognition (NOR) test was used to evaluate 108 cognition (memory and learning) and was conducted as previously described 6,7 . Day 1, the habituation phase,

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± 1°C) for a single six minute trial. Water was renewed between each trial. The total time of immobility was 138 scored in the last four minutes 9 . Immobility was defined as the total absence of movement, except slight 139 motions to maintain the head above water. After the trial, mice were gently dried and single-housed for two 140 hours of recovery, before being placed back to their home cages with littermates.

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To assess stress-responsiveness, blood samples were taken in response to an acute stress (FST). First, a blood 144 sample was collected from the tail following tail incision, five minutes before the test. After the acute stress, 145 mice were singly housed following removal from the FST, and blood samples were collected at 15, 45, 90 and 146 120 minutes after the test.
Bleeding was performed in a separate room to the FST. Blood samples (50-70μl) were taken from the 148 tail and collected in heparin coated capillary tubes. The blood was then transferred to a microtainer TM collection 149 tubes containing EDTA (BD Diagnostics), thoroughly mixed in the tube and stored on ice until centrifugation 150 for 10 min at 2,000g to isolate the plasma. Isolated plasma was immediately transferred to a clean eppindorf 151 tube following centrifugation. Isolated plasma was stored at -80 °C for later corticosterone quantification.

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Corticosterone was quantified using a commercially available ELISA kit (Enzo Life Sciences (UK) Ltd., Exeter, 153 UK) according to the manufacturer's protocol.