Research Project: Energy Regulation and Obesity

Location: Jean Mayer Human Nutrition Research Center On Aging

2015 Annual Report

Objectives
ENERGY METABOLISM: 1: Determine the effects of specific dietary composition factors, including macronutrient type, fiber & food form on hunger, satiety & energy regulation. 2: Demonstrate effective methods for sustainable weight control & prevention of obesity in adult population groups spanning a range of ages & demographics. 3: Develop new methodology for improving the accuracy & precision of assessment of energy & nutrient intakes & energy requirements in adults. OBESITY AND METABOLISM: 1: Determine the mechanistic roles of the intestinal and hepatic proteins ACSL5 and plin2 in fatty acid metabolism, especially in delivery of dietary triacylglycerol to tissues and tissue lipogenic metabolism. 2: Determine the physiologic actions and consequences of ACSL5 in diet-induced obesity and obesity associated metabolic complications. BODY COMPOSITION: 1: Develop and validate stable isotope methodology to that can be utilized to investigate energy regulation related to sarcopenic obesity and frailty in the elderly. 2: Validate energy dispersive X-ray fluorescence methodologies for studying the prevalence of zinc deficiency and implications of zinc deficiency in the elderly.

Approach
LAB NAME: ENERGY METABOLISM The mission of the Obesity and Energetics Laboratory is to understand the effects of lifestyle factors on energy metabolism and weight regulation. Our research examines dietary and behavioral variables that influence both energy intake and metabolism throughout the adult lifecycle, and our focus is to develop and test effective lifestyle interventions for implementing sustainable, healthy weight control at all ages. Studies in our laboratory include in-depth biological examinations of the impact of different dietary factors on biochemical and neurological factors involved in energy regulation and body composition, chemical studies of food composition, and randomized controlled trials testing practical interventions that can be scaled for population-wide benefits. LAB NAME: OBESITY AND METABOLISM To address the role of acyl CoA synthetase 5 (ACSL5) and perilipin 2 (plin2) in intestinal enterocytes and hepatocytes we have generated conditional lines of knockout of ACSL5 and plin2 mice allowing us to disrupt in a tissue specific manner. We will investigate the physiological effects of ablation of ACSL5 and plin2 in mouse hepatocytes and enterocytes and the response to a high caloric diet in vivo. In these animal studies we will determine body composition, energy expenditure, insulin glucose homeostasis, fat absorption, hepatic steatosis, and in liver and intestine tissues gene expression will be determine. In isolated hepatocytes studies in our mice we will triglyceride (TG) accumulation, and TG oxidation. The studies in this project will provide novel insights that will allow researchers to direct therapeutic strategies to protect against the development of obesity and associated complications. LAB: BODY COMPOSITION Simple monitoring of isotope clearance in breath CO2 can provide detailed information on the metabolism of labeled food and help us understand the connection between food composition and energy management. Our approach includes the use of a single stable isotope administration (C-13 palmitic acid or carbohydrate) and monitoring its disappearance in breath CO2 for several days. We use both mathematical modeling and clinical validation. Repeated measurements of C-13 in breath CO2 provide us with a profile of the timing and efficiency of oxidation of the labeled fuel. The development and validation of new tools for field use include a new portable analytical device that can measure zinc content in fingernail clippings. The methodology used is non-destructive energy dispersive X-ray fluorescence (XRF). Preliminary results show that zinc measurement in protein (fingernails) is a better indicator for zinc status than zinc measurements in plasma. This is because plasma values can be easily affected by resent food intake or infection. This methodology will be tested in nursing home residents. It will become an addition to a set of portable devices that we have developed for the evaluation of nutrition status, hydration, and frailty in the field. The instruments are designed to evaluate status as well as to assess the efficacy of treatments and nutrition programs in older adults.

Progress Report
This report documents research conducted under 3 projects in a Non-Assistance Cooperative Agreement between ARS and TUFTS UNIVERSITY. Additional details for the research are associated with projects 8050-51000-097-01S, Energy Regulation During the Adult Lifespan; 8050-51000-097-02S, Regulation of Adipocyte and Adipose Tissue Metabolism in Obesity Related Inflammation and Metabolic Disorders and the final progress report for closeout of 8050-51000-097-03S, Use of Isotope Kinetics for the Assessment of Body Composition and Energy Balance in Older Adults. LAB: Energy Metabolism - The Energy Metabolism Laboratory continued its projects investigating factors determining successful weight control in adults of different ages. The laboratory has had considerable success in all projects, and, in addition, has brought in funding for a new worksite randomized controlled trial that will begin in the coming year. In our previous Objective 1, we published the first data including our primary outcomes from our human calorie restriction trial. We showed that there were no measurable harms with caloric restriction, and some potentially beneficial improvements including changes in markers of inflammation and thyroid function. Concerning psychology and mood there were no adverse effects that could be detected in symptoms of depression or sexual function. The importance of this accomplishment is major: the study is the only randomized clinical trial of human caloric restriction, and the study is implementing the largest weight loss goal of any study to date. The results from this trial will be far-reaching with respect to recommendations on caloric restriction and weight loss for long-term health. We analyzed fMRI data from our previous worksite study and that paper was published in 2014. We provided the first demonstration that brain reward system can change over time during a behavioral weight loss program such that participants come to prefer healthy food more and like unhealthy food less. This first-of-its-kind demonstration was unique not only for a behavioral intervention but also in comparison with such studies in gastric bypass patients (which have shown less liking for all foods generally without any increase in liking of healthy food). The importance of this accomplishment is that it shows brains can be retrained to enjoy healthy eating even in individuals who are obese, giving hope that American food habits can be turned around and healthy foods can become more popular with the right kinds of public health strategies. We completed a review of the role of energy density in energy regulation. A comprehensive review of the literature was conducted, and identified energy density as a significant factor influencing short-term energy regulation, while there is also a lack of research studies on the long-term impact of energy density. The significance of this work is providing a careful summary of all studies – the work on dietary composition and energy regulation tends to be very controversial, and summaries of all published work allow scientists and dietary recommendation committees to take the weight of evidence without highlighting one particular study or another unduly. LAB: Obesity and Metabolism - The family of acyl CoA synthetase proteins act within cells to add an acyl CoA group to fatty acids, and by doing this are thought to direct fatty acids to different metabolic fates. Examples of the differing metabolic fates of intracellular fatty acids include incorporation into triglyceride and phospholipids, metabolization by oxidation, and finally, in the nucleus, facilitated binding and activation of nuclear receptors. Our laboratory has hypothesized that acyl CoA synthetase 5 (ACSL5) is critical for the absorption of dietary fat. Dietary fat is hydrolyzed within the intestinal lumen to fatty acids and monoacylglycerol. The dietary fatty acids and monoacylglycerol are taken up by intestinal enterocytes, where they are activated to fatty acid acyl CoAs and reesterified into triglyceride, packaged into chylomicrons, and ultimately secreted into the blood. We hypothesize that acyl CoA synthetase (ACSL5) is the critical enzyme for activation of fatty acids into fatty acid acyl CoAs within the enterocytes and, therefore, necessary for packaging of fatty acids into triglyceride and for secretion into the blood. To investigate and prove this hypothesis, we are working to generate a line of mice in which ACSL5 is specifically ablated within intestinal enterocytes. To accomplish this, we have generated a line of floxed ACSL5 mice and mated them to mice that express the Cre enzyme (which would result in ablation of ACSL5 expression) specifically within enterocytes. During the last year, we discovered that our Cre-expressing mice were infected with fur mites. To remedy the situation, our mice were sent to Charles River Laboratories where they could be rederived and freed of fur mites. We are presently amplifying the mouse colony so we can study the effects of ablation of acyl CoA synthetase 5 expression specifically within intestinal enterocytes, and we have made progress in our studies. We predicted that we would generate mice in which the expression of acyl CoA synthetase 5 was ablated specifically in liver by year 2. We have now generated this line of mice, and will be able to start investigating the interaction between diet and liver acyl CoA synthetase 5 expression on liver triglyceride metabolism. LAB: Body Composition - We completed the data analysis of a unique experiment that uses stable isotopes to study the individual differences between subjects in the way they metabolize food. This novel method was originally designed as a way to measure energy intake. A known amount of a fatty acid (pamitic acid) fully labeled with C-13, a stable natural isotope, is administered by mouth. This fuel is absorbed and becomes available for oxidation and energy production about two hours after its administration. We can measure CO2, one of the products of oxidation in the volunteer’s breath, using simple but highly sensitive techniques. Because of the C-13 label, the oxidation product is also labeled by C-13 and is easily identified. Using only a single dose, one can monitor the metabolism of the fuel for several days just by looking for C-13 in breath CO2. Its presence slowly disappears from breath as new, non-labeled fuel enters the body with the meals that follow. As a matter of fact, the rate of this disappearance is related to the amount of incoming new fuel. Therefore, a systematic monitoring of breath samples for 7-10 days can be used to support a mathematical calculation (based on a metabolic model) of average energy intake. This is the first time that an experiment of this short has been attempted. Therefore, we had to determine all the necessary parameters (dose, timing of sampling, analytical methodology, natural C-13 interference, effect of body composition etc) with pilot experiments with human subjects and animal models. We found that, in spite of the numerous technical challenges (for example, when the isotope was administered as a pill, most of the volunteers did not absorb it fully; when given in food preparation, absorption was complete), the data supported the hypothesis that energy intake can be derived with this technique in free living adults. We also observed a significant biological variation between individuals in the timing and efficiency of metabolizing this fuel. We believe that this observation is significant because it enables this methodology to identify details of efficiencies specific to each volunteer, and therefore serve as one of our most useful tools in personalized nutrition. We developed and validated methodology that uses energy dispersive x-ray fluorescence (XRF) to study body composition, changes in hydration, and to assess frailty. The method was a significant improvement over existing techniques for analysis of bromide in biological specimens like plasma, serum, urine or saliva. It is also non-destructive, preserving 100% of the available specimen. The XRF method requires no sample preparation and avoids all the errors associated with that. In a recent study we demonstrated that, because of its simplicity and precision (better than 0.8%), repeated studies were possible with the same group of subjects in a re-feeding metabolic study. As part of our program, we expanded the capabilities of XRF to include solid samples (fingernail clippings) to study zinc deficiency and the effect of zinc supplements. This approach was recently validated against plasma zinc with a double-blind controlled intervention study in Burkina Faso (collaboration with the University of California, Davis). Based on the findings of the completed study, we initiated collaboration with a local VA hospital in Bedford, Massachusetts, which is operating a nursing home for veterans, to study zinc status in the elderly. We are also expanding the capabilities of this technology to include other nutrients such as copper, iron, and vitamin B-12.

Accomplishments
1. Retraining brains to prefer healthy food. Obesity is a serious health crisis in the United States (U.S.), and is raising health care costs, which negatively impact the competitiveness of U.S. companies. Effective, sustainable weight control programs for employees are urgently needed. ARS funded researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University in Boston, Massachusetts, completed the first demonstration that obese and overweight adults can, through a behavioral program, learn to enjoy healthy food more and unhealthy food less. This new intervention has the potential to be an important contributor to reducing the national (and worldwide) obesity epidemic.

2. Caloric restriction is healthy and may also extend lifespan. Calorie restriction (CR) is the only intervention known to extend lifespan in animal models. ARS funded researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University in Boston, Massachusetts, in collaboration with groups of scientists around the United States, published the first study of the biological effects of moderate (25%) calorie restriction in human subjects participating in a randomized controlled trial. The results indicate that CR is healthy and has some benefits that are consistent with an effect of CR on extending lifespan in humans, as it does in many animal species. This controlled study focused on biological effects of CR rather than practical application, but nevertheless the fact that the volunteers implemented it on their own and demonstrated metabolic benefits without adverse side-effects indicates that CR can now be considered for its potential role in public health.

3. Low energy density foods facilitate healthy weight management. Dietary patterns to facilitate successful weight management are a controversial topic, and progress is needed to distinguish different conflicting approaches. ARS funded researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University in Boston, Massachusetts, published a new review of the research literature on energy density demonstrating that within the limits of the available data energy density has beneficial effects on energy intake and therefore weight management. The review also highlighted areas where there is insufficient evidence and more studies are needed, providing a roadmap for the next progress needed in this controversial area.

4. Ablation of Adipocyte Acyl CoA Synthetase 4 Protects Against Diet-Induced Obesity, Fatty Liver, and Insulin Resistance. Alterations in fat cell metabolism with obesity are thought to promote increases in body fat and the risk for alterations in the regulation of glucose and insulin, which increases susceptibility to type 2 diabetes mellitus. ARS funded researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University in Boston, Massachusetts, observed that when mice, which were genetically modified do not express the protein acyl CoA synthetase 4 within fat cells, were fed a high caloric diet to induce obesity, they had reduced accumulation of body fat and fat within the liver as well as improved insulin glucose metabolism. The discovery that reduced expression of acyl CoA synthetase 4 in fat cells protects against the development of obesity and its metabolic complications provides an opportunity to investigate different diets and therapeutics that reduce ACSL5 as a potential therapy to ameliorate obesity and its complications in humans.