Research Project: Adipose Tissue Biology

Location: Children's Nutrition Research Center

2023 Annual Report

Objectives
Objective 1: Determine if maternal obesity and high-fat diet during gestation induce adipogenic and metabolic program alterations in Wt1 expressing white adipocyte progenitor cells during development. Progenitor cell proliferation, differentiation, metabolic efficiencies will be determined, and critical transcriptional regulators will be identified. Objective 2: Assess whether carbohydrate response element binding protein alters macrophage intracellular metabolism and inflammatory response. Objective 3: Assess whether macrophage carbohydrate response element binding protein activity affects adipose tissue inflammation and the development of diet induced obesity and insulin resistance. Objective 4: Use wild type mice and an obese transgenic mouse model lacking leptin to determine organ specific metabolism of fatty acids (FA) of varying carbon chain lengths, and study their effects on the progression and/or treatment of diet induced obesity and its related metabolic disorders. Subobjective 4A: To determine the effect of FAs of varying carbon chain lengths on progression of diet-induced obesity in wild type and ob/ob mice. Subobjective 4B: To determine the effect of dietary FAs of varying carbon chain lengths on diet-induced insulin resistance and fatty liver in wild type and ob/ob mice. Subobjective 4C: To determine the effect of dietary FAs of varying carbon chain lengths on organ specific distribution and metabolism of FAs in wild type and ob/ob mice. Objective 5: Understand phenylalanine metabolism in adipogenesis Sub-objective 5.A Define the role of phenylalanine in adipocyte differentiation and adipogenesis in vitro Sub-objective 5.B Determine the dependency of phenylalanine in the development of obesity in vivo. Sub-objective 5.C Evaluate dietary phenylalanine restriction in established obese mouse models.

Approach
Our goal is to enhance the understanding of the mechanisms through which diet impacts adipose tissue during development and the understanding of the progression of obesity and related pathologies after birth. High fat-diet induced obesity is a well-recognized risk factor for a diverse array of health problems, including type II diabetes, heart diseases, and certain types of cancer. However, the mechanistic links between a high-fat diet and cellular injuries during development and after birth remain to be fully elucidated. This research will use mouse models of diet induced obesity and will focus on three general problems associated with obesity: 1) the developmental effects of maternal obesity on offspring adiposity, 2) adipose tissue inflammation that may lead to medical complications, and 3) the effects of dietary fatty acid composition on obesity. We will analyze the effects of maternal obesity on Wilms tumor 1 (Wt1) expressing white adipocyte progenitor cell development, and of the function of the intracellular glucose sensor ChREBP in macrophages and its contribution to the inflammation of fat tissues induced after long-term (months) feeding of a high fat diet. We will investigate the uptake and metabolism of dietary fatty acids of varying carbon chain lengths in different tissues, including fat tissue and their effects on progression of obesity and related disorders in wild type and obese leptin deficient mice. An expected outcome of this research is an improved understanding of the relationship between diet induced obesity and fat tissue development, inflammation, insulin resistance, and uptake and metabolism of dietary fatty acids. Researchers will also test the hypothesis that phenylalanine is essential for adipocyte differentiation and adipogenesis, and restriction of phenylalanine is anti-obesity. Outcomes from this research will provide knowledge on amino acid metabolism during adipocyte differentiation and adipogenesis and provide new avenues for targeting obesity.

Progress Report
In Objective 1, we completed analysis of body and adipose tissue weights from offspring from female mice fed a control or high-fat diet. Male offspring born to female mice fed a high-fat diet gained more body weight compared to offspring born to female mice fed the control diet. White fat cells from offspring born to female mice fed the high-fat diet were larger than white fat cells from offspring born to female mice fed the control diet. We showed that high-fat diet induced maternal obesity can impact white fat cell development in offspring. To learn how maternal high-fat diet changes the development of white fat cells in offspring, we isolated Wt1-marked white fat cells from offspring born to female mice fed control or high-fat diets and compared how these cells develop. We observed more mature lipid-containing white fat cells from the offspring of female mice fed the high-fat diet than for cells from offspring born to female mice fed the control diet. We are conducting gene expression analysis for cells isolated from offspring born to female mice fed the high-fat diet. We are generating more offspring from female mice fed the control diet and high-fat diets to isolate Wt1-labeled white fat cells for next-generation sequencing analysis to identify genes that differ between these two groups. In Objective 2, we bred macrophage-specific carbohydrate-responsive element-binding protein (ChREBP) deficient, macrophage-specific ChREBP overexpressing, and littermate control mice for the collection of bone marrow-derived macrophages. Cells were cultured to become either pro- or anti-inflammatory. We collected cell samples for analysis of ChREBP DNA binding. Previous studies showed that pro-inflammatory macrophages use more glucose, and since ChREBP regulates glucose metabolism in cells, we expect to see ChREBP binding at genes that regulate glucose metabolism in the ChREBP overexpressing cells. These will be associated with changes in macrophage inflammatory status, as observed by studying secreted factors, flow cytometry, and gene expression. We mean to uncover novel ChREBP targets that are specific to macrophages that may affect the way these cells function. Sequencing results are pending and will be integrated with gene expression datasets from similar pro- and anti-inflammatory macrophages. Integration of the datasets showing DNA binding to genes that are directly turned on or off by ChREBP with gene expression data will define new pathways through which ChREBP alters cellular nutrient utilization and inflammatory responses. In Objective 3, we placed mice that either overexpress or are deficient in ChREBP in macrophages on high- or low-fat diets to induce obesity and insulin resistance. Both obesity and insulin resistance are tied with increased pro-inflammatory macrophages in adipose tissue. Because ChREBP activity changes how these cells metabolize glucose, we expect to see altered pro- and anti-inflammatory cell numbers due to changes in macrophage metabolism, thus impacting insulin resistance in our mice. We conducted analyses of white adipose tissue and liver macrophage populations and are continuing histological studies of white adipose tissue and liver in mice lacking or overexpressing ChREBP. We indicate that ChREBP-deficient macrophage populations do not exhibit altered total macrophage numbers in white adipose tissue of high fat diet-fed mice. However, pro- but not anti-inflammatory macrophage numbers are decreased, consistent with our prior gene expression findings. We analyzed T cell populations and see a trend toward increased numbers of CD4+ and CD8+ cells. Results suggest that ChREBP activity in macrophages alters the profile of more than one type of inflammatory cell population in adipose tissue. In Objective 4, we are seeing how diets containing different types of fatty acids lead to obesity and its health-associated complications. We finished data collection on the effect of different fatty acid diets on body weights and composition, fat pads, and energy expenditure. Diets enriched in short-chain fatty acids, when compared to long or medium-chain fatty acid diet groups, reduced body fat gain and the weight of fat pads. Lean mass weight was not different among diet groups. We didn’t see a difference in food intake or energy expenditure among diet groups. We finished dietary intervention studies on glucose and insulin tolerance and liver fatty liver. Diets enriched in short-chain fatty acids improved insulin sensitivity and reduced liver weight and liver fat content. We studied how these fatty acids are stored and processed in different body tissues by adding labeled fatty acids (tracers) to the diets. These studies showed that the tracer of short-chain fatty acids was detected in very low concentrations and was mainly found in the liver, heart, skeletal muscle, and small intestine, and excreted in feces. The tracer of medium-chain fatty acids was detected in moderate concentrations and detected in various tissues and fat pads and excreted in feces. However, the tracer of long-chain fatty acid was detected the highest (approximately 50 and 5000 times higher than medium and short-chain fatty acids, respectively) and was detected in most body tissues and feces with increased storage in fat pads. We are performing RNA sequence in the liver, small intestine, and gonadal tissue, and we will correlate gene expression from these specific tissues with various measurements on body composition, tissue fatty acid content, and histological examinations. For Objective 5, obesity initiates with the expansion of white adipose tissue. Adipose tissue is composed of fat cells specialized in energy metabolism. Adipose expansion occurs through both cell hypertrophy, the expansion in size of pre-existing fat cells, and hyperplasia, the increase of cell number. Despite extensive documentation of adipose tissue in glucose and lipid homeostasis, the role of adipose tissue in protein and amino acid metabolism remains underappreciated. Previously, to study amino acid metabolism in adipocyte biology, we profiled changes of metabolism during cell growth and differentiation from pre-adipocyte to adipocyte using a widely used adipocyte model. We found that the changing pattern of phenylalanine was similar to that of three branched chain amino acids, namely leucine, isoleucine and valine. Recent studies have shown that these branched chain amino acids contribute to adipogenesis in adipocytes. An insufficient supply of phenylalanine decreased cellular lipid accumulation, which is similar as a restriction of branched chain amino acids. While more studies are emerging to understand branched chain amino acid metabolism in adipocyte biology and obesity, the role of phenylalanine in adipocyte biology and adipogenesis is unknown. We seek to understand phenylalanine metabolism in adipogenesis. We completed two feeding tests in male and female mice. Male and female mice were fed three different high fat diets: high-fat diet containing 60% energy from fat, or the high-fat diet with 85% phenylalanine restriction, or the high-fat diet with 70% phenylalanine restriction. We observed less weight gain in mice fed the phenylalanine restricted (85%) high-fat diet in comparison to the control high-fat diet. The 70% restriction of dietary phenylalanine led to no significant effect on body weight gain in either male or female mice. Mice on the 85% phenylalanine restricted high-fat diet had similar lean mass but less fat mass than the control high-fat diet group. Mice on the 85% phenylalanine restricted high-fat diet are more glucose tolerant and insulin sensitive in the glucose tolerance test and insulin tolerance test respectively. We conducted metabolic cage studies in both male and female mice. In both, those on the 85% phenylalanine restricted high fat diet showed higher physical activity and higher heat production, suggesting that the lack of weight gain is likely due to increased activity and heat production. In our other feeding study, male and female mice were fed three different low-fat diets: a diet 10% energy from fat or the low-fat diet with 85% phenylalanine restriction, or the low-fat diet with 70% phenylalanine restriction. We observed less or no weight gain in mice fed the phenylalanine restricted (85%) low-fat diet in comparison to the control low-fat diet in both male and female mice. A 70% restriction of dietary phenylalanine led to no significant effect on body weight gain. The mice on the 85% phenylalanine restricted low-fat diet had similar lean mass but less fat mass than the control group. Mice on the 85% phenylalanine restricted low-fat diet are more glucose tolerant and insulin sensitive in the glucose tolerance test and insulin tolerance test respectively. We conducted metabolic cage studies and found that those on the 85% phenylalanine restricted low-fat diet showed higher physical activity and higher heat production. We have run metabolomics on plasma and are conducting data analysis. These findings support our hypothesis that phenylalanine is essential for adipogenesis. We studied the amino acid requirement during cell differentiation using the 3T3-L1 cell, a mouse fibroblast cell line that can be induced to differentiate to fat cells. We have cultured cells under control or phenylalanine deprived conditions during the induced differentiation phase. We found that phenylalanine restriction during the cell differentiation will dampen the capability of cells to accumulate lipid, an indication of undifferentiation. We have conducted metabolomics analysis on these cells under control and phenylalanine deprived conditions before and after differentiation. The analysis revealed that phenylalanine deficiency leads to changes in vitamin B6 metabolism. This work will provide an increased knowledge of amino acid metabolism in adipocyte adipogenesis and bring new avenues for the treatment and prevention of obesity.

Accomplishments
1. Nutritional input is fundamental to the development and treatment of obesity. A diet consisting of a high intake of fat and sugar can lead to a high prevalence of obesity and its related health complications. Scientists at the Children's Nutrition Research Center in Houston, Texas, recently found that an essential amino acid (phenylalanine) commonly found in high protein foods like meat, beans, milk, and eggs, was necessary for the development of diet-induced obesity. Restriction of phenylalanine protected mice from diet-induced weight gain. These findings are important as it provides evidence that restricting nutritional phenylalanine intake could be exploited as a potential strategy to treat or prevent obesity.