Project Summary/Narrative (ORIGINAL APPLICATION) This proposal utilizes cutting-edge mechanistic approaches to investigate the clinically meaningful but hard- to-study question of HOW insulin in human milk (HM) affects infant development. This research question is particularly relevant to the growing and under-studied population of infants receiving HM from mothers with obesity and/or insulin resistance (IR) - who produce milk with significantly greater insulin concentrations. Animal models show that oral insulin delivered to the infant via breast milk has far-reaching effects on normal offspring development and maturation. This proposal documents these effects in human infants and investigates how effects may be altered in the context of maternal IR (and sustained infant exposure to high concentrations of oral insulin. Specifically, we examine both systemic effects regulated by infant pancreatic function and local intestinal effects. Importantly, we will document both acute effects during the neonatal period AND effects of more “chronic” exposure. We will recruit 64 exclusively breastfed infants of both normal weight/normoglycemic (NW; n=28) and mothers with IR (n=36) and study them during the neonatal period (2-4 weeks) and after more “chronic” exposure to mother's milk (5 months). Our aims are: Aim 1 – Cross-Sectional Study – Investigate differences in metabolic development and intestinal maturation between infants exclusively breastfed by NW vs IR mothers at: 2-4 weeks and 5 months. We hypothesize that infants breastfed by IR mothers will exhibit: an altered metabolomic profile indicating differences in carbohydrate handling and insulin signaling; an altered microbial composition in the intestinal microbiome; increased relative transcript abundance of insulin-target genes in exfoliated intestinal cells indicating more active insulin signaling at the level of the enterocyte; and decreased intestinal permeability at 2-4 weeks. Aim 2 – Glycemic Study - Determine if consumption of HM from an IR mother (with high concentrations of insulin) is correlated with differences in endogenous pancreatic response at 2-4 weeks. We hypothesize that receipt of HM with high insulin will result in a lower endogenous pancreatic insulin response. Aim 3 – Glucose Tolerance Study - Determine if chronic consumption of HM from an IR mother (with high concentrations of insulin) is correlated with pancreatic response to a glucose challenge at 5 months. We hypothesize that chronic exposure to HM from an IR mother will under-stimulate pancreatic β-cells and result in a dampened endogenous insulin response to a standard glucose challenge at 5 months. Our long-term objective is to fully understand the impact of oral insulin in the development and postnatal programming of the breastfed infant. As the prevalence of insulin resistance rises among mothers – and as these women's offspring have increased risk of developing metabolic disease themselves – this work has far- rea...