Research Project: Effects of Diet and Physical Activity on Maternal/Child/Adolescent Health and Development

Location: Microbiome and Metabolism Research Unit

2020 Annual Report

Objectives
The mission of Cooperator's Arkansas Children's Nutrition Center (ACNC) is to determine the effects of diet, physical activity and nutritional status in childhood and in mothers during the pre- and post-conception periods on growth, development and health by conducting research on: 1) central nervous system and cognitive development and function of infants and children; 2) roles of maternal and child body composition, metabolism and genes (including responses to food macronutrients, phytochemicals and microbe-derived factors) on infant and child growth, development and the long-term health consequences of these factors; and 3) impact of parental and child physical activity on these parameters, singly or in combination with diet, and the biological basis for exercise-associated behaviors and health outcomes. To accomplish this mission, the ACNC has the following objectives and as appropriate, others as mutually agreed upon: A. Determine how maternal/parental diet, exercise, and metabolic health associate with psychological/behavioral, neurologic and physiological function and development in children. B. Understand how postnatal diet, childhood physical activity, and nutrition associate with psychological/behavioral, neurologic and physiological function and development in children. C. Characterize the interaction between the microbiome, host health and nutrition to modify physiology and promote health and development (skeleton, gastrointestinal and hepatic tissues, immune system, cardiovascular, and muscle, e.g.).

Approach
To accomplish these objectives, a research plan encompassing the specific research described below is authorized. Research is to be accomplished over a multi-year period as resources are available and as readiness for research and management capabilities develops. The plan shall be divided into the following areas: A. Determine how dietary components, dietary patterns, and metabolic health influence psychological-behavioral outcomes, neurodevelopment, and physiological function in children. Children will be studied longitudinally and cross-sectionally to evaluate the effects of child/parental diet, metabolic health, and body composition on psychological-behavioral, neurologic and physiological function. B. Determine how physical activity and fitness influence psychological-behavioral outcomes, neurodevelopment, and physiological function in children. Children will be studied longitudinally and cross-sectionally to evaluate the effects of child/parental physical activity patterns, fitness, metabolic health, and body composition on psychological-behavioral, neurologic and physiological function. C. Characterize the impact of host-microbe interactions on health and development. Children will be studied longitudinally and cross-sectionally to evaluate the factors linking child/parental microbiome with metabolic health, body composition, physical activity outcomes, psychological/behavioral, neurologic and physiological functions. D. Center Development. Cooperator has the following general areas of research in the Arkansas Children’s Nutrition Center (ACNC), which address the mission area of parental/child health: Brain Development and Central Nervous System Function; Dietary Factors and Physical Activity Effects on Health, Development and Function of Organ Systems (skeleton, gastrointestinal and hepatic tissues, immune system, cardiovascular, and muscle, e.g.); and Obesity and its Effects on Cellular and Physiologic Function in mothers, infants and children.

Progress Report
Examining maternal-infant cohorts over time, alongside basic research studies, provides critical information as to long-term impacts of early-life diet, maternal obesity, and factors that influence the body's development and physiology (e.g., body fat and childhood obesity risk, bone health, gut and immune functions, bioenergetics and metabolism, brain function, muscle and cardiovascular health). For the projects, "Developmental Programming Associated with Maternal Diet and Obesity" & "Antenatal factors impacting obesity and metabolism in children" we are focusing on intergenerational, mother-to-child, transmission of obesity and poor bone health. Features of potential transmission include: offspring fat utilization and storage, stem cell differentiation to fat cells, placenta inflammation, and epigenetic alterations of DNA-associated proteins. The results provide the groundwork for future childhood obesity mitigation strategies focused on mothers or women of childbearing age. Studies are determining if maternal obesity alters the fates of offspring umbilical stem cells to become white fat cells (fat storage) vs. brown fat cells ("energy-consuming" cells that may counter obesity and improve metabolic health). Results in mice indicate that "energy-consuming" brown fat is more limited in offspring born to obese dams. Other mouse studies are testing how maternal obesity impacts the offspring’s fat around blood vessels called "perivascular adipose tissue" (PVAT; thought to regulate blood pressure). Investigators are also testing if higher methylation of a chromosomal structural protein site, H3K27me3, is involved with body fat and bone dysfunction in offspring born to obese mothers. The H3K27me3 change is associated with an enzyme called Ezh2. Scientists have begun to develop new mouse models with less inflammation in placental or maternal egg (oocyte) cells. If this strategy reduces offspring obesity risk, it opens new doors for obesity prevention through prepartum anti-inflammation interventions. In humans, researchers are determining if greater maternal body fat during gestation increases infant fat and risk of childhood obesity at ages 5 and 8 yr (Glowing study). Thus far, 94 visits have been conducted at 5 yr and 10 visits conducted at 8 yr. To test if exercise during gestation can reduce maternal programming of offspring body fat, a randomized controlled trial is ongoing (Expecting study; obese mothers assigned to a fitness intervention vs. no fitness intervention). This year there were 68 (prenatal) and 125 (postnatal) visits. To determine acceptability and feasibility of the exercise protocol with pregnant women in community settings, 10 interviews and 3 focus groups were conducted. The first, second, and third focus groups, respectively, were participants with high compliance in the original clinical study (62-99%), mixed compliance (49 – 92%), and lower compliance (32 – 63%). All qualitative data analyses are complete, used to inform protocol adaptation. Community stakeholders (N = 14) (e.g., WIC, parks & recreation, trainers, churches) advise on the "real world" protocol adaptation. To investigate if even earlier, pre-conception, exercise mitigates obesity-associated metabolic dysfunction, studies were launched to test if exercise in women recruited at fertility clinics lowers inflammation indices in oocytes and follicular fluid. "Postnatal factors impacting healthy development." Early-life nutrition significantly affects colonization and metabolism of gut microbiota (the repertoire of bacteria normally found in the gut). Microbiota modulate health through metabolites (the "metabolome") and regulatory molecules (e.g., microRNAs). In one line of research, breastfeeding changed specific bacteria compared to formula (e.g., increased Bifidobacteria): Bifidobacteria produce butyric acid that promotes gut development. Bacterial tryptophan metabolites were also higher in breastfed infants; some are implicated in developing immune system tolerance and could dampen allergy pathways. Using human milk (HM) from donor banks, HM-fed piglets had higher levels of microbiota that utilize human milk oligosaccharides (HMOs). Blood microRNA profile differences were also observed in HM- vs. formula-fed piglets. Fats made by bacteria (xenolipids) were discovered, and found to be metabolized by the liver; studies are determining if xenolipids impact immune function, and how they are stored and transported in the body. Other studies tracked diabetes progression in a rat model: many gut-derived metabolites and microbiota changed with diabetes, illustrating a new host health-microbiome connection. Finally, studies are exploring how increased intakes of fruit-derived molecules ("polyphenolics") impact bone: e.g., hippuric acid or 3-(3-hydroxyphenyl) propionic acid rise in blood following blueberry (BB) intake, and regulate bone cells to promote net bone growth in rodents. Experiments are underway to test if BB feeding also promotes bone in children. Pilot studies identified BB-containing foods that have good taste and sensory properties in children, while maintaining high levels of the healthy molecules during preparation and storage. Ongoing studies are addressing the specific physiological roles for metabolites, HMOs, microRNAs, and other factors linked to early-life diet. "Pediatric Physical Activity: Mechanisms Impacting Health and Development." Regular physical activity (PA) and fitness associate with improved health and lower disease risk in children and adults. Early, frequent PA might "program" better lifelong health. Mechanisms that link PA and fitness to health, and that regulate muscle metabolism, remain largely unknown. In one line of research, small subcellular structures called mitochondria (the "powerhouses" of cells) are being studied. Mitochondrial research in children is limited to readily-accessible blood cells (e.g., platelets), which might inform on body-wide mitochondrial health. Investigators are studying how body fat, fitness, and cardiovascular health relate to platelet mitochondrial respiration in 8-10 yr old children (Mi Energy study). Preliminary results from a separate study in >100 children indicated that platelet mitochondrial respiration positively correlates with markers of poor metabolic health (central body fat, higher blood pressure). This leads to questions about the role of platelets in metabolic comorbidities. This project is also assessing the use of sugars, fats, and proteins for energy at rest and during moderate exercise in children of normal weight and with obesity, with high and low fitness levels, using specially-labeled fuels tracked in urine and breath. Other studies are testing if early life PA offers protection from obesity, and if children born to obese mothers are less active compared to normal weight mothers. Initial data suggest that 2 yr old children born to normal weight mothers tend to be more active compared to children from overweight mothers. A child's percent fat mass, father's obesity level, mother's activity level, mother's healthy eating index, and the father's education related to PA level, and associations are now being tested in kids up to 8 yr old. Identifying factors that relate to healthy PA can support effective public health strategies to promote early childhood PA. Related to the latter, a tailored, family-level intervention to increase PA and gross motor performance is being developed for preschool children of obese mothers. A "Jungle Gym" mobile app promoted moderate to vigorous PA, supporting the feasibility of increasing preschoolers' PA via mobile apps. Molecular events associated with PA, fitness, and muscle metabolism are also being explored. One line of research asks if early and frequent PA can "overcome" a genetic predisposition for low fitness and higher obesity risk. "Low capacity runner" (LCR) and "high capacity runner" (HCR) rats bred over many generations to display low and high fitness will be used to test if 8 wk of early PA blunts LCR genetic susceptibility to higher weight gain. Since a human trial in high vs. low fit women found that bile acid metabolism is fitness-related, this aspect will also be tested in LCR/HCR rats. Separate studies explored the biology of a muscle-abundant oxygen-carrying protein called myoglobin (Mb), discovered to bind specific fats. Loss of Mb in a mouse model led to modestly reduced fat oxidation and higher body weight, in females only. The results suggest that Mb influences how fat and energy are managed in the body, in a sex-specific manner. "Dietary Influences on Psychological and Neuropsychophysiological Development and Function in Children." A landmark study examining the development of functional brain circuitry in infants 2-6 months of age detailed the complex interactions between the development of the brain's functional architecture, nutritional composition and sex that fluctuate over this critical developmental period. While there were differences in brain function comparing feeding groups (soy formula fed, milk formula fed, and breast-fed infants), these differences were age-dependent and small, indicating that there was no strong or lasting effect of different feeding regimens on brain development. In a different study, in pre-adolescents who differed in body weight (obese and normal weight children, 9-10 yr), significant differences in executive function (measured as the ability to control an ongoing response) were seen in which obese children required greater cognitive effort to accomplish this important behavior. This finding suggests that the ability to inhibit impulsive responses to, e.g., high caloric value foods, could require more cognitive effort in children with obesity. Further, this study showed that all children, regardless of weight, performed the task equally well. These findings indicate that obese children may require greater cognitive effort to perform certain tasks.

Accomplishments
1. Obesity-associated disorders might be prevented with specific foods. There is a need to understand the molecular underpinnings of nonalcoholic fatty liver disease (NAFLD) in order to prevent the disease and to improve metabolic health, and the potential influence of dietary factors as a tool to prevent disease is worth considering. In particular, fibers and complex carbohydrates that can modify the normal population of bacteria in the gut ("gut microbiota") are thought to cause changes in the body's metabolism and signaling in a way that reduces fat accumulation in the liver. Scientists in Little Rock, Arkansas, collaborated with other investigators to demonstrate that in mice fed an obesity-promoting diet, the complex carbohydrate lactotrehalose prevented metabolic anomalies and increased metabolic rate, compared with mice given trehalose (a related sugar known to promote harmful bacteria). This research demonstrates that nutritional modification of the microbiota, using specific factors such as lactotrehalose (or foods rich in similar compounds) could be a useful means to promote liver health even in the face of a high fat, high sugar diet.

2. Maternal diet and body fat alter placental DNA methylation. Epigenetic changes, or changes in outcomes caused by modification of gene expression rather than alteration of the genetic code itself, provide a possible explanation for how the in utero environment "programs" health throughout the life course. Epigenetic marks can include changes in the addition or removal of natural chemicals (i.e., "methyl groups") to the DNA or the proteins (histones) onto which DNA is wrapped. Studies conducted by researchers in Little Rock, Arkansas, compared patterns of DNA methylation in placentas collected in the Glowing clinical study that is examining how maternal obesity impacts offspring growth. Researchers observed that maternal body mass index (BMI) and dietary saturated fat intake were associated with epigenetic changes in placental DNA methylation profiles, and many of these modified genes relate to fat synthesis, insulin signaling pathways, and DNA packaging. This study suggests that placental DNA methylation status is associated with maternal obesity and dietary fat, which could modify placental function and hence offspring developmental programming.

3. Phenolic acids (PAs) derived from fruits and vegetables suppresses bone-forming cell "aging". PAs such as 3-(3-hydroxyphenyl)-propionic acid (3-3-PPA) rise in blood following intake of dietary fruits such as blueberries, and 3-3-PPA has been shown to positively affect bone growth in cultured cells. To learn more about the effects of PAs in the body, investigators in Little Rock, Arkansas, administered 3-3-PPA to one-month-old female C57BL6/J mice for 30 days, finding that the PA treatment led to higher bone volume and trabecular thickness, increased bone-forming cells (osteoblast) number, decreased bone-degrading cell (osteoclast) numbers, and changes in bone formation markers in serum and bone marrow. Further studies showed that the 3-3-PPA treatment reduced senescence ("aging") signaling in bone. These results indicate that dietary factors rich in foods like blueberries promote bone health and bone growth through rebalancing bone-degrading vs. bone-forming cells, in part through a reduction in bone cell aging/senescence.

4. Neonatal diet alters gut bacteria and metabolite signals in infants. Nutrition provided in early age can significantly affect bacterial colonization and development, and there is a growing body of evidence describing the ability of the gut microbiome (the repertoire of bacteria normally found in the gut) to modulate host health through a large suite of metabolites (the "metabolome") that interact with the body. To better understand these processes, scientists in Little Rock, Arkansas, analyzed fecal samples collected from a longitudinal cohort (Beginnings), to describe the infant fecal microbiome and metabolome across the first year of life, both globally and within each age time-point. Compared to formula feeding (FF), breastfeeding (BF) was associated with increased abundances of specific bacteria that produce short chain fatty acids (SCFA), metabolites that participate in gut development. In addition, metabolites such as kynurenic acid were higher in BF relative to FF infants, and the kynurenine pathway has been implicated in developing normal tolerance of the immune system (which may optimize immune responses to, e.g., allergy-promoting factors). These results provide new information about the mechanisms by which breastfeeding promotes gut and immune health in infants.

5. Infant diet impacts liver energy metabolism. The type of neonatal diet (e.g., formula vs. breastmilk) has been shown to impact many physiological systems during infancy and beyond, with some evidence suggesting that breastfeeding can reduce childhood obesity risk. Since obesity and related outcomes involve changes in energy balance and metabolism, studies in Little Rock, Arkansas, tested if human milk (HM) or milk formula (MF) feeding impacts how fuel is burned in the small subcellular "powerhouses" of cells called mitochondria (mitochondrial bioenergetics) in piglets. The data suggest that compared to HM feeding, MF feeding elicits higher mitochondrial inefficiencies in the liver. The importance of these novel findings in terms of weight gain or overall metabolism continue to be studied, but these results highlight a very important principle that was not previously appreciated: infant diet type has the potential to "program" the body's bioenergetics systems very early in life.

6. Neonatal diet impacts unique tissue signaling factors impacting health and development. Breastfeeding is associated with positive health outcomes such as decreased rates of infections, obesity, and allergies, but the exact mechanisms are still unclear. To address this, investigators in Little Rock, Arkansas, utilized a piglet model fed either human breastmilk (HM) or dairy-based milk formula (MF) until postnatal day 21, and weaned to solid diet until postnatal day 51 (to model neonatal feeding similar to human infants). Serum levels of microRNAs (miRNAs, unique small RNAs that regulate the expression of genes in tissues) were measured on days 21, 35, and 51 using small RNA sequencing and specialized computer software to predict impacted biological pathways. miRNA differences were altered by diet, and some changes were persistent long after weaning, suggesting a persistent effect of the neonatal diet on miRNA expression. Pathway analysis suggested that many of the miRNAs are involved in immune function, suggesting that one mechanism by which breastfeeding optimizes immune health in children is through regulation by unique miRNAs transported in the blood.

7. Milk formula diet may increase inflammation and cell stress pathways in the small intestine. The mechanisms behind the positive health effects of breastfeeding, and how they associate with the immune system and gut health, are poorly understood. To address mechanisms, scientists in Little Rock, Arkansas, evaluated small intestine gene expression in piglets fed dairy milk formula (MF) compared to those fed human milk (HM). Gene expression data revealed that MF led to higher expression of genes associated with inflammation and cell death (apoptosis) pathways, whereas tight junctions (tissue structure) and pathogen detection systems were decreased relative to the HM group. The MF impacts on small intestine were maintained over the post-weaning period. These novel observations illustrate potential mechanisms driving the protective effects of breastmilk on immune function and the ability to fight pathogens.

8. Early life diet influences mammary gland gene regulating factors called microRNAs (miRNAs). Diet is a potent regulator of how the DNA code is expressed (gene expression) in the body's tissues, but the specific signals and molecular mechanisms remain to be elaborated and little is known about early-life factors that program these outcomes. One potential mechanism is through modulation of cellular molecules called miRNAs. Studying female neonatal piglets fed milk- and soy-based formulas from postnatal day 2 to 21, scientists in Little Rock, Arkansas, found that the different formulas led to disparate mammary miRNA signatures that associated with gene expression changes: e.g., miR-1, -128, -133a, -193b, -206, and -27a were negatively correlated with expression of genes associated with enhanced cell proliferation and/or increased cholesterol synthesis. The results show, for the first time, that the type of formula fed to infants can have profound and differential impacts on tissue gene regulation, in part through unique microRNAs.

9. Different infant feeding practices yield small changes in neurodevelopment from age 3 months to 6 years. To investigate the effects of infant feeding on childhood cognition and language development, scientists in Little Rock, Arkansas, characterized neurodevelopment from age 3 months to 6 years in 174 breastfed children, 169 children fed dairy milk-based formula during infancy and 161 children fed soy protein-based formula during infancy. For all groups, results were within the established norms and there were no differences in mental development between feeding groups; however, breastfed children had significantly higher motor development scores at age 3 months than children fed soy formula, significantly higher intelligence scores at 4 years, and greater language scores at ages 3 and 4 years compared with formula fed children. These results demonstrate that breastfeeding is associated with modest differences in neurodevelopment compared to formula fed children, yet formula feeding does not lead to functional impairments of brain function, and neurodevelopmental outcomes are well within age-appropriate norms.

10. Weight gain in infancy is associated with higher body mass index (BMI) and fat mass at age 5 years. To determine the relationship between weight gain and energy intake during infancy, with childhood obesity at 5 years of age, scientists in Little Rock, Arkansas, followed a group of healthy term infants from 3 months to 6 years of age, measuring body fat and weight trajectories. Higher weight gain between 3 and 12 months of age was significantly associated with higher BMI-for-age score and higher percent body fat at 5 years of age, even after adjusting for infant sex, gestational age, birth length, birth weight, maternal BMI and energy intake. Energy intake was significantly greater between birth and 12 months of age for children who were overweight at 5 years compared to their counterparts. These results demonstrate that higher weight gain during infancy is associated with higher BMI and percent body fat at 5 years of age; thus, lowering obesity risk for these children might be possible with family-focused early lifestyle interventions to modify energy intake and responses to feeding cues.

11. Identification and regulation of the unique fats produced by gut bacteria ("xenolipids"). Factors made by the natural bacteria in the gut (microbiota) are metabolized by the liver and other tissues in humans. The microbiota process molecules derived from foods and from the host, producing hundreds to thousands of unique metabolites that are thought to impact health and physiological functions in the body. Researchers in Little Rock, Arkansas, have identified unique microbiota-produced fats called long-chain cyclopropane fatty acids, but the fate of these fats in terms of absorption and metabolism have not been studied previously. Blood was analyzed from human volunteers who had specialized catheters that allowed for organ-specific measurement of cyclopropane fatty acid metabolites, and it was found that the liver appears to be a major site of metabolism: the liver has a net output of breakdown products from these fats into the bloodstream. This is the first description of gut microbe-derived fat breakdown in the body, and it is hypothesized that these metabolites serve as signals to the body in response to changes in the gut microbiome stemming from changes in diet or host health status.

12. Gut bacteria metabolite signatures change with diabetes progression in a rat model of type 2 diabetes. There are hundreds to thousands of small molecules made or modified by the natural bacteria ("microbiota") residing in the gut, and these "xenometabolites" likely beneficially or negatively influence our health. Scientists in Little Rock, Arkansas, developed a unique platform, called the XenoScan, which accurately measures >195 xenometabolites, representing one of the largest authentic standard libraries of microbe-derived molecules. In a collaborative study, this platform was used to identify intestinal and blood xenometabolites that discriminate pre-diabetes to diabetes progression over time in the UC Davis Type 2 Diabetes Mellitus Rat model: the XenoScan was able to distinguish early and late stages of diabetes, and identifies relationships between specific bacteria and levels of xenometabolites. In addition to validating a new tool for the study of the gut microbiome, these studies provide new examples of how the host’s metabolic health can influence how intestinal microbes process and metabolize foods and substrates.

13. Child-friendly foods can be healthy foods. Blueberries and some other fruits are rich in plant-derived molecules (phytonutrients), called anthocyanins and polyphenols, which have positive effects on bone, cardiovascular health, and the immune system. Despite these health benefits, very few children consume the recommended amounts of fruits and vegetables, making it imperative to identify alternative foods that contain the healthy phytonutrients in a form that kids will eat regularly. Scientists in Little Rock, Arkansas, in collaboration with University of Arkansas researchers in Fayetteville, Arkansas, tested the taste and acceptability of blueberry-enriched "kid friendly" foods (cookies, ice pops, bars, gummies) in 60 children. The studies showed that almost every test food was liked and most items retained healthful phytonutrients even after storage. Outcomes from this research highlight that it is feasible to include significant amounts of blueberries in foods familiar and well-liked by children, which could help promote a healthy diet pattern.

14. Parental body fat (adiposity) associates with body composition in newborns and 2 yr olds. The in utero and early-life factors that drive body fat regulation and childhood obesity risk remain to be fully elaborated, but may involve "programming" signals associated with parental metabolism or adiposity. To address this question, scientists in Little Rock, Arkansas, studied a large cohort of babies from infancy through 2 yr of age, measuring body composition and other metabolic factors. Maternal adiposity was positively associated with male and female 2 wk old infant fat mass, whereas paternal adiposity was negatively associated with male adiposity. Breastfeeding, female sex, gestational age and gestational weight gain were also positively associated with newborn adiposity. Maternal adiposity was associated with fat mass accretion in female but not male offspring at 2 yr of age. These results demonstrate that maternal and paternal body composition differentially associate with newborn and early childhood adiposity, supporting the hypothesis that signals associated with parental metabolism and weight status contribute to the programming of offspring body fat.

15. Too much fat in muscle can compromise the function of the blood sugar controlling hormone insulin. Research in humans, animal models, and cultured cells have pointed to the negative effects of excessive fats (and specifically, fats called saturated fatty acids) on the muscle's response to insulin. This led to the idea that a proper balance of dietary fats, along with regular physical activity, will maintain a robust and healthy muscle insulin response that in turn helps control blood sugar. To better understand the mechanisms by which saturated fatty acids reduce insulin's actions, scientists in Little Rock, Arkansas, exposed muscle cells to increasing concentrations of the fatty acid palmitate, and tested if the production of a palmitate-derived metabolite called palmitoylcarnitine triggers resistance to insulin. While this led to exacerbated insulin resistance, suggesting a causative role for palmitoylcarnitine, much of the effects of the saturated fatty acid were independent of this metabolite and must be due to accumulation of other unique fat derivatives in the cells. The studies confirmed that excessive saturated fat has a negative effect on systems controlling sugar metabolism in muscle, and highlight that as-yet unidentified fat metabolites drive insulin resistance in muscle.

16. Human milk composition differs by maternal body mass index (BMI) in the first 9 months postpartum. The components of breastmilk that promote growth and development of children remain to be catalogued, and the influence of maternal metabolic health and body composition on these factors is largely unknown. To address these knowledge gaps, scientists in Little Rock, Arkansas, analyzed human milk composition of mothers of different BMIs during the first 9 months postpartum. Human milk from overweight and obese mothers were higher in fat and protein, and lower in carbohydrate content, compared with milk from normal weight mothers. Concentrations of factors such as leptin and insulin (metabolic hormones), and C-Reactive Protein (a stress and inflammation marker), were higher in milk from overweight or obese mothers when compared with milk of normal weight mothers; leading to a 1.5– 2.5 times higher exposure of leptin and insulin in infants born to overweight mothers. The functional ramifications of these findings remains to be established, but the results point to the importance of maternal body composition and weight status on the composition of breastmilk.

17. Maternal protein intake does not negatively impact late pregnancy or offspring blood sugar control. Dietary protein and certain amino acids such as branched chain amino acids (BCAAs) have been implicated in pathways that influence actions of the blood sugar-controlling hormone insulin. Studies occurring in Little Rock, Arkansas, explored the associations between a pregnant mother's protein intake, BCAA patterns and insulin sensitivity. There was a positive association between total protein intake and plant protein intake in the last trimester of pregnancy and insulin sensitivity, and no evidence that changes in blood BCAAs throughout gestation associate with indices of insulin action or blood sugar control. Early and late pregnancy protein intake did not associate with insulin sensitivity measures in offspring at 12 and 24 months of age after adjusting for maternal body mass index, offspring sex and offspring body fat percentage. Therefore, these new results indicate that dietary protein during pregnancy does not negatively impact the hormone insulin or blood sugar control, and may actually be associated with modest increases in insulin sensitivity in mothers, at least in the last trimester.