GLUTAMINE SYNTHESIS IN ADIPOCYTES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1020289
• Project No.: NJ14190
• Project Start Date: Aug 1, 2019
• Project End Date: Jul 31, 2024

Project Director
Watford, MA.

Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559

Performing Department
Nutritional Sciences

Non Technical Summary
The proposed work will provide important new data on the changes that occur in adipose tissue during obesity and the development of Type 2 Diabetes Mellitus. The goals of this project are to understand the regulation and function of glutamine synthesis in adipocytes. The results obtained will show how the amino acid glutamine produced by fat cells is important in increasing adipose tissue inflammation and insulin resistance. By specifically knocking the glutamine synthetase gene in fat cells we predict that we will reduce the size of adipocytes, lower macrophage induced inflammation and maintain insulin sensitivity.This work will develop new mouse models where the gene for glutamine synthetase has been deleted in adipocytes (fat cells) and in mammary epithelial cells (MEC) (the cells that produce milk during lactation). Using these mice models, we will determine how the role of the amino acid glutamine in both the development of obesity and the provision of the large amounts of glutamine in milk. During obesity glutamine is required for the expansion of fat cells and also to fuel the inflammatory macrophages that accumulate in fat tissue at this time.Also, by understanding the role of glutamine in lactation and neonatal development we may be able to optimize maternal diets to maintain maternal health, and also optimize the glutamine content of milk for the benefit of the neonate. As such the work not only has potential in human nutrition and development, it also has application in the domestic animal industry.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	5010	1010	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
5010 - Food;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

glutamine

amino acids

obesity

adipocyte

lactation

mammary gland

milk

gene abalation

mouse models

Goals / Objectives
We believe that both fat cells and macrophages obtain their glutamine directly from the fat cells. By deleting the ability of the fat cell to make glutamine we predict that we may prevent the development of obesity and/or possibly reverse obesity after it has developed. By removing glutamine within the fat tissue, we predict that this will prevent the accumulation of macrophages and thereby lower inflammation. Since fat tissue inflammation can lead to the development of type 2 diabetes the results may lead to novel therapies to prevent this disease. Glutamine (and glutamate) comprise 20% of the amino acids in milk and the free glutamine concentration rises as lactation progresses. It has been shown, in pigs, that supplemental glutamine is beneficial to both intestinal and immune health in suckling and post-weaning piglets. The source of the glutamine in milk is not fully known, as much as 50% is made within the mammary gland but the exact cells responsible have not been identified. We will use the adipocyte and MEC glutamine synthetase knockout mice lines to determine the role of these two cell types in providing glutamine for milk production. Using these mice, we expect lower levels of glutamine in milk. We will also use mice receiving a dietary supplement of glutamine or the branched chain amino acids, where we expect higher glutamine levels in milk. Using such mice, we will be able to determine the role of milk glutamine on neonatal growth, development and health. During lactation much of the glutamine that is exported in milk is synthesized from other amino acids, some of which arise from the breakdown of muscle protein. This means that lactation is often accompanied by a loss of lean body mass. We have shown that supplementing the diet of the lactating pig with glutamine, not only increases the glutamine content of the milk it prevents some of the loss of lean body mass in the mother. This may open novel approaches both for the prevention and treatment of obesity. Work with lactating mice will provide the first evidence of how amino acid metabolism changes during lactation and the role of fat cells in milk production. It is known that high levels of the amino acid glutamine in milk are beneficial to neonatal health and the proposed work will produce models by which we can change the glutamine content of milk. Using these procedures, we will be able to evaluate the role of glutamine on neonatal growth and health.

Project Methods
We hypothesize that adipocytes are providing glutamine that not only supports adipocyte differentiation but also maintains macrophage viability and function. During obesity such glutamine would exacerbate adipose tissue inflammation and lead to insulin resistance. We intend to determine the exact role of adipocyte glutamine synthesis in adipocyte differentiation and growth, and on the accumulation of macrophages during obesity in vivo, by developing the adipose tissue glutamine synthetase knock out (AT-GS-/-) mouse line.We are currently in the early stages developing these mice and have crossed the GS floxed mouse with the adiponectin driven Cre mouse. If the homozygote knockout strain is viable we will monitor growth, body composition and visible phenotype from birth to adulthood, including a study of up to 24 hour starvation to check for hyperammonemia. Other than the possibility of problems related to a lack of adipose tissue, we do not expect any problems with these animals. Mice for obesity experiments will be used at approximately two months of age. If the constitutive knockout strain is not viable or if homozygotes are unable to lactate, we will use heterozygotes but also immediately develop a Tamoxifen-inducible strain.We anticipate no problems with the Tamoxifen strain and mice will be at least two months old when we induce the Cre and knockout adipocyte GS. Once we have developed, and characterized, the knock out strains they will be used to study the effects of a lack of adipocyte glutamine synthesis during diet induced obesity. Adult, 6-8 weeks of age (20-25g), knockout (AT-GS-/-) mice will be provided a high fat diet (45% fat) for up to 12 weeks. In order to determine the role of adipocyte glutamine synthesis in the maintenance of obesity we will develop the Tamoxifen-induced strain. Mice (prior to treatment with Tamoxifen) will be allowed free access to the high fat (or control) diets for eight weeks (sufficient time to develop insulin resistance).and the mice continued on the diets for 2 to 8 weeks after Tamoxifen treatment.We will determine the sites of glutamine synthesis in the lactating mouse and will test the hypothesis that lactation is associated with a large increase in glutamine turnover. Work in vivo and vitro will test the hypotheses that glutamine synthesis within the mammary gland occurs in both mammary epithelial cells (MEC) and mammary adipocytes. In addition, there will be increased glutamine synthesis in skeletal muscle. There is evidence that branched chain amino acids (BCAA) are substrates for glutamine synthesis in mammary gland and other tissues. We propose that milk glutamine will be increased by long-term BCAA supplementation. To definitively determine the contribution of adipocytes and mammary gland epithelial cells to milk glutamine we will use the adipocyte glutaminase synthetase knock out mice and also develop a MEC glutamine synthetase knockout (MEC-GS-/-) model to complement. We predict that milk glutamine levels in such mouse lines will be low and such lines will be used to determine the role of milk glutamine in neonatal development. An understanding how amino acid metabolism adapts to lactation will allow novel approaches to regulate the glutamine content of milk for the benefit of both neonatal and maternal health in humans and domestic animal species.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audiences for this work are other scientists carrying out work related to the role of adipocytes in chronic diseases such as Type 2 diabetes, those interested in lactation in terms of milk quality, neonatal development and maternal health, plus those working in the general area of protein and amino acid nutrition and metabolism Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two large reviews, "Glutamine" and "Ornithine Cycle", have been published in the Encyclopedia of Biochemistry. This publication is essentially a textbook and is widely used by advanced students in all biological disciplines, including nutritional and animal sciences. How have the results been disseminated to communities of interest?Along with the publications listed in the products section of this report, a Zoom presentation "Glutamine metabolism in adipocytes" was made to the New York Adipocyte Club (September 2020) What do you plan to do during the next reporting period to accomplish the goals?We will continue to breed the glutamine synthetase knockout mice and begin experiments to detemine how they respond to feeding a high fat diet. Similarly, we will breed the homozygote females to determine if this gene ablation has any impact on lactation and neonatal development.

Impacts
What was accomplished under these goals? Working with Dr. Sheila Collins (Vanderbilt University) we have successfully established the first generation of mice where the gene for glutamine synthetase has been ablated. Currently such mice are undergoing phenotype characterization and are being used as breeding stock to produce sufficient animals for more detail experiments. Work with lactating mice has shown that glutamine synthetase is expressed at very low levels in mouse mammary epithelial cells but expression is very high in mammary adipocytes. We propose that mammary adipocytes are the source of glutamine synthesized within the mammary gland during lactation and this glutamine is exported into the milk. We have also established high expression of the enzymes of branched chain amino acid metabolism (branched chain aminotransferase and branched chain ketoacid dehydrogenase) in the mouse lactating mammary gland.This work is almost complete and will submitted for publication early in 2021. Work in pigs, with Dr. Helena Manso (Federal University of Pernambuco, Brazil) has shown that glutamine supplementation to sows, boars, and piglets improves a number of factors related to reproduction, including sperm quality, maternal health and time of return to estrus, and improvements in piglet intestinal morphology. This work is about to be submitted for publication. Work with Orangutans with Dr. Erin Vogel (Rutgers University) has shown that these animals enter ketosis when food is scarce. In addition, careful analysis of the diet has established that ketosis and endogenous protein degradation are associated iwth periods of low fruit abundance (when the diet is comprised mainly of tree bark and leaves), this work is about to be submitted for publication. Work in horses with Dr. Helio Manson (Federal University of Pernambuco, Brazil) has characerized changes in expression of the GLUT4 transporter in equine skeletal muscle during development and exercise.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Mansho Filho, H.C., Manso, H.E.C.C.C., Watford, M., McKeever, K.M. (2020) Abundance of the skeletal muscle GLUT4 glucose transporter protein in Standardbred foals during development and exercise. Comparative Exercise Physiology 16:395-402
• Type: Book Chapters Status: Published Year Published: 2020 Citation: Watford, M. (2020) Glutamine. The Encyclopedia of Biochemistry, Third Edition. Elsevier, 00028 doi.1016/B978-0-12-819460-7.00028-1
• Type: Book Chapters Status: Accepted Year Published: 2020 Citation: Watford, M.(2020) Ornithine Cycle. The Encyclopedia of Biochemistry, Third Edition. Elsevier, 00062 doi.1016/B978-0-12-819460-7.00062-1
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Naumenko, D.J., Watford, M., Erb, W.M., Utami Atmoko,S.S., Vogel, E.R. (2019) Evaluating ketosis in primate field studies: validation of urine test strips in wild Bornean organgutans Pong Pygmaeus wurmbii. Folia Primatologia 91: 159-167

Progress 08/01/19 to 09/30/19

Outputs
Target Audience:The target audiences for this work are other scientists carrying out work related to the role of adipocytes in chronic diseases such as Type 2 diabetes, those interested in lactation both in terms of milk quality and neonatal development, and maternal health. Since the project began a few months ago nothing is yet ready to report to these audiences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are continuing to cross the mice to develop the knockout line. We have just begun to determine the substrates used, and the cells involved, for glutamine synthetase in the lactating mammary gland. These studies will be completed during the next reporting period

Impacts
What was accomplished under these goals? We have begun to establish the two founder lines, Floxed GS and Adiponectin Cre, of mice that are needed to develop theadipocyte glutamine synthetase knockout line. To date we have not yet obtained a homozygote knockout but we expect to succeed within the next few months.

Publications