ABSTRACTTHE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME By Melissa QuinnINTRODUCTION. Growth restriction induced by undernutrition in early life increases the risk of developing chronic diseases in adulthood. We hypothesized growth restriction would alter the gut microbiome and metabolome across the lifespan, impairing vital growth signaling processes necessary for proper development, with a primary focus on muscular and hepatic Insulin-like Growth Factor (IGF-1) expression. METHODS. A cross-fostering, protein-restricted nutritive model (8% protein) was used to induce undernutrition during gestation (GUN) or lactation (PUN). At 21 days of age (PN21), all mice were weaned to a control diet (CON; 20% protein), isolating undernutrition to specific windows of early life. Fecal samples were collected weekly PN18-PN80 to determine longitudinal programming effects of growth restriction on the gut microbiome (CON N=5, GUN N=6, PUN N=6) and metabolome. Fecal sample DNA was extracted for amplification of the bacterial 16S rRNA genes using PCR, and then the amplicons were sequenced with the Illumina pipeline and analyzed using the Qiita bioinformatics software. Cecum samples were also collected at PN21 (CON N=4, GUN N=6, PUN N=5) and PN80 (CON N=5, GUN N=6, PUN N=6) for microbiome analysis. Liver samples were collected at PN21(CON N=12, GUN N=6, PUN N=7) and PN80 (CON N=13, GUN N=9, PUN N=11) and analyzed along with the cecum for metabolomics via tandem mass spectrometry (LC-MS/MS) and analyzed with the Global Natural Products Social Molecular Networking (GNPS) bioinformatics software. IGF-1 expression in the liver and gastrocnemius (CON N=15, GUN N=12, PUN N=13) was analyzed via a Total Protein NIR western blot to establish a connection between the gut microbiome, tissue metabolome and organ growth. RESULTS. The Beta-Diversity of the fecal microbiome was significantly separated by treatment group using Weighted UniFrac measures (PERMANOVA p=0.0001). Differences in the microbiome were not evident through analysis at the Phylum level (Firmicutes/Bacteroidetes ratio) but were instead driven by longitudinal alterations in the abundance of specific genera and species in PUN. Linear mixed model (LMM) analysis revealed PUN having significantly higher abundance of specific bacteria compared to GUN and CON across the lifespan including: Bacteroides uniformis, B. acidifaciens, B. ovatus, Bifidobacterium sp. and Clostridium sacchrogumia. Rikenellaceae was the only microbe that was significantly lower in abundance in the PUN group over time compared to GUN and CON. Additionally, the PUN metabolome was significantly altered compared to GUN and CON, primarily characterized by reduced: essential amino acids (EAAs: methionine, phenylalanine and tyrosine), riboflavin (B2), primary bile acids, and decreased Dehydroepiandrosterone (DHEA); and increased acylcarnitines and fecal peptides. NIR Western blot analysis revealed significantly lower IGF-1 expression in the liver at PN21 in GUN (p=0.0012) and PUN (p<0.001) as well as overall lower expression in the muscle in PUN (p=0.037) and GUN (p=0.007) compared to CON. CONCLUSION: The gut microbiome and metabolome are altered by early life growth restriction at PN21 and through adulthood. Elevated sugar-fermenting bacteria in the PUN group represent gut microbiome immaturity and delayed development. Temporary metabolic alterations of early life growth restriction are seen in decreased primary bile acids and increased synthesis of liver acylcarnitines, both of which are indicative as adaptations of the pups being calorie-restricted as a result of the low-protein fed dam. More permanent outcomes of growth restriction were evident by increased peptide excretion over the lifespan, significantly decreased methionine and riboflavin – which prevented protein synthesis to occur during early life development, and overall decreased muscle IGF-1 expression and DHEA levels in the PUN mice. Many of the metabolic pathways permanently altered by growth restriction are seen in the liver, making this organ an important site for future research on the development of treatment modalities that can limit growth restriction induced chronic disease.
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