Location: Children's Nutrition Research Center
2022 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
For Objective 1, we continue to study the effects of maternal obesity on the development of white fat in children. We used a white fat cell specific gene, Wt1, to mark and identify white fat cells in mice. This year, we generated more offspring from the females treated with a high fat diet to compare with offspring from females treated with a regular diet. This allowed us to isolate white fat cells from young offspring born to the high fat diet treated female mice. Currently, we are growing these cells in culture dishes and will compare how well these cells grow and mature. To observe the effect of maternal obesity on adult offspring, we have collected fat tissues and blood from adult offspring born to the high fat and regular diet treated female mice. We have begun to analyze the body weights, white fat tissue weights, and the level of the enzymes that regulate white fat development in these tissues.
For Objective 2, we bred macrophage-specific ChREBP deficient, macrophage-specific ChREBP overexpressing, and littermate control mice for collection of bone marrow-derived macrophages. The cells were then cultured to become either pro- or anti-inflammatory. We have collected samples from these cells for metabolomic analyses. Previous studies have shown that pro-inflammatory macrophages use more glucose. Since ChREBP regulates glucose metabolism in cells, we expect to see changes in glucose-derived metabolites in the ChREBP overexpressing and deficient cells. These changes will then be associated with changes in macrophage inflammatory status, as observed by studying secreted factors, flow cytometry, and gene expression.
For 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. It is well-established that both obesity and insulin resistance are associated with increased pro-inflammatory macrophages in adipose tissue. Because we are altering the activity of ChREBP in macrophages, which changes how these cells metabolize glucose, we expect to see altered pro- and anti-inflammatory profiles due to changes in macrophage metabolism, thus impacting insulin resistance in our mice. We have conducted real-time polymerase chain reaction (PCR) for inflammatory genes in our macrophage-specific ChREBP-overexpressing and deficient mice fed high fat versus low fat diets, and have submitted liver samples for lipid analyses.
For Objective 4, we have finished the studies on the effects of different types of dietary fatty acids on body weight and composition (Sub-objective 4A). We have completed dietary intervention studies on glucose and insulin tolerance tests (insulin resistance) and liver lipid analyses (fatty liver) (Sub-objective 4B). We have also finished studies on how these fatty acids are stored and processed in different body tissues using labeled fatty acids (tracers) in the diets. We are currently analyzing the collected tissue and fecal samples for fatty acid and their labeled tracer's concentrations (Sub-objective 4C).
Additionally, a new project began this year with a new scientist that joined the center. Obesity initiates with the expansion of white adipose tissue that is a dynamic organ present throughout the human body. Adipose tissue is primarily composed of adipocytes that are specialized in energy metabolism. Adipose expansion occurs through both cell hypertrophy, the expansion in size of preexisting adipocyte, and hyperplasia, the increase of cell number. Understanding the process of adipocyte hypertrophy and hyperplasia will reveal mechanisms for the development of obesity and suggest new avenues for the treatment of obesity and its associated complications. Despite the extensive documentation of adipose tissue in glucose and lipid homeostasis, the role of adipose tissue in protein and amino acid metabolism remains largely underappreciated. Thus, we aim to extend our understanding on amino acid metabolism in adipocyte during adipogenesis.
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 like 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. Furthermore, insufficient supply of phenylamine decreased cellular lipid accumulation, which is again similar to lack 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 not known. Thus, our objective is to understand phenylalanine metabolism in adipogenesis.
Since the start of this project in April, we have continued our research in animal models. We have started two feeding tests in mice. In the first set of feeding studies, young mice at the age of 8-10 weeks old were fed a high fat control diet that contained 60% energy from fat, or phenylalanine restricted high fat diets with 85% or 70% of phenylalanine restriction compared to the control high-fat diet. With the collected data so far over the 8 weeks of feeding, we observed less or no weight gain in mice fed the phenylalanine restricted (85%) high-fat diet in comparison to the control high fat diet. A 70% restriction of dietary phenylalanine led to no significant effect on body weight gain. In the second set of feeding studies, young mice at the age of 8-10 weeks old were fed a low-fat control diet that contained 10% energy from fat or phenylalanine restricted low fat diets with 85% or 70% of phenylalanine restriction compared to the control low-fat diet. With the collected data so far over the 6 weeks of feeding, we observed similar phenotypes as in high fat diet-fed mice: less or no weight gain in mice fed the phenylalanine restricted (85%) low-fat diet in comparison to the control low fat diet, and 70% restriction of dietary phenylalanine led to no significant effect on body weight gain. Together, these exciting preliminary findings support our prior hypothesis that phenylalanine is essential for adipogenesis.
In the coming year, we will complete the ongoing dietary feeding studies. We will determine the mouse body composition of lean mass and fat mass, and metabolism of mice under the above different dietary conditions using metabolic cages. We will also determine the status of glucose metabolism and insulin sensitivity by glucose tolerance test and insulin tolerance test in the above mice. At the end of these dietary studies, we will collect tissues such as liver and adipose tissue for downstream histology, metabolic and molecular analysis, and more. These analyses will reveal the impact of phenylalanine restriction in mouse physiology and adipogenesis in the animal model. They will also reveal the details of changes in metabolism and genetic levels. While conducting the animal studies, we will also confirm the preliminary data using the adipocyte cell line. Adipocytes will be cultured with different levels of phenylalanine in the culture media, and cell differentiation and adipogenesis will be determined by the expression of cell differentiation marks and the lipid accumulation at the end of culture. We will also determine the metabolic changes during the cell differentiation and lipogenesis under the treatment of phenylalanine restriction. With the accumulated preliminary data, we are more confident that phenylalanine is required for adipogenesis. The outcome will not just provide unknown knowledge on amino acid metabolism in adipocyte adipogenesis, but potentially bring new avenues for treatment and prevention of obesity.
Accomplishments
1. Nutritional input is fundamental to the development and treatment of obesity. Consuming an unbalanced diet, particularly one containing a high intake of fat and sugar, can lead to a high prevalence of obesity and its related health complications. Scientists in Houston, Texas, recently found in mice that an essential amino acid (phenylalanine) commonly found in high protein foods like meat, beans, milk and eggs, was necessary in the development of diet-induced obesity. Restriction of phenylalanine protected mice from diet-induced weight gain. Thus, restriction of nutritional phenylalanine intake could be exploited as a potential strategy to treat or prevent obesity.
