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Title of document: DENOVO SYNTHESIZED FATTY ACIDS AS
REGULATORS OF MILK FAT SYNTHESIS
Diwakar Vyas, Doctor of Philosophy, 2011
Directed By: Professor Richard A. Erdman
Department of Animal and Avian Sciences
The objectives of the dissertation research were to determine the role of denovo
synthesized fatty acids (DNFA) in the regulation of milk fat synthesis. Milk fat responses to increasing amounts of short- and medium-chain fatty acids (SMCFA), added in the proportion as synthesized denovo, were studied in lactating dairy cows. The results showed a significant linear increase in milk fat concentration with SMCFA supplementation. However, milk fat yield was similar for all treatments.
A subsequent study was aimed at increasing the availability of SMCFA during trans-10, cis-12 CLA-induced milk fat depression (MFD) in lactating dairy cows to determine whether SMCFA can rescue part of CLA-induced MFD. Post-ruminal infusion of butterfat (BF) was used as a source of SMCFA. The BF treatment was compared to a mixture of fats containing only the long-chain FA (LCFA) with or without trans-10, cis-12 CLA infusion. Milk fat content and yield were significantly reduced with trans-10, cis-12 CLA. However, increased availability of SMCFA with BF infusion had no effects on milk fat yield and concentration. Trans-10, cis-12 CLA significantly reduced the mRNA expression of transcription factor SREBP-1c along with its downstream targets including ACC, FASN, LPL, SCD and AGPAT. The increased availability of SMCFA had no effect on either lipogenic gene or protein expression suggesting that nutritional manipulation was not sufficient to rescue trans-10, cis-12 CLA-induced MFD.
Finally, the effects of combination of a Rosiglitazone (ROSI), a PPAR-γ agonist, and trans-10, cis-12 CLA were examined on mammary and hepatic lipogenesis in lactating mice. Mammary lipogenesis was significantly reduced with trans-10, cis-12 CLA, reducing the milk fat content and mRNA expression of lipogenic transcription factors SREBP1-c and PPAR- γ. Trans-10, cis-12 CLA significantly increased hepatic lipid accumulation, while the mRNA expression of SREBP1-c and PPAR- γ were not altered. On the contrary, ROSI had no effects on mammary lipogenesis. However, ROSI significantly rescued trans-10, cis-12 CLA-induced hepatic steatosis.
DENOVO SYNTHESIZED FATTY ACIDS AS REGULATORS OF MILK FAT
SYNTHESISBy Diwakar Vyas Dissertation submitted to the Faculty of Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of
Professor Richard A. Erdman, Chair Associate Professor Brian J. Bequette Supervisory Research Molecular Biologist Erin E. Connor Professor Rick A. Kohn Professor Thomas W. Castonguay © Copyright by Diwakar Vyas 2011 Dedication …….. to my family
First and foremost, I would like to express my sincere gratitude to Dr. Richard Erdman for being an ideal mentor. I greatly appreciate the confidence you had in my capabilities. Thanks for teaching the basics of research, and working hard on improving my presentation, and writing skills. I am certain that guidance and training you have provided me has laid the foundation for future success.
I would like to extend special thanks to my committee members for devoting extensive time, effort and expertise towards my dissertation. I would like to recognize each member’s contribution; Dr. Brian Bequette, for all those thought provoking discussions and valuable suggestions, Dr. Erin Connor for providing inputs and relevant literature to better understand various molecular biology techniques, Dr. Rick Kohn for your valuable guidance, suggestions and career advice, and Dr. Thomas Castonguay for always being supportive and inquisitive about my research.
I will forever be thankful to Dr. Beverly Teter for helping me at each and every stage of my dissertation. Thanks for teaching me the concepts of gas chromatography, fatty acid analysis, and conducting experiments on lactating mice, and also for all those insightful discussions which were immensely beneficial in shaping up my dissertation.
I am also grateful to The Graduate School and Animal Science Graduate Student Association (ASGSA) for providing me the financial assistance to be used for dissertation research.
I extend my sincere appreciation to Dr. Liliana Piperova for helping me during initial stages of dissertation, getting acquainted with the lab atmosphere and Dr. Anil Kadegowda for being a great friend and colleague. I would like to thank Dr. Pierluigi
thank Dr. Uzi Moallem for helping me with my intensive farm studies. I wouldn’t have finished these studies successfully without your help. I also would like to acknowledge the help we received from CMREC farm crew; Michael Dwyer, Benny Erez, and Brian Spielman. I really appreciate your efforts towards my farm studies.
I am also grateful to the Porter lab (Dr. Laura Ellestad, Dr. Monica ProszkowiecWeglarz, Malini and Jyoti), Mather lab (Dr. Ian Mather, Hyunsu), Ottinger lab (Meredith), Taneyhill lab (Dr. Andrew Schiffmacher, Ranga) and Bequette lab (Umang, Qiong) for providing lab space, and valuable inputs required for conducting various molecular biology techniques. I am also thankful to Nikki Thompson for helping me with lab supplies.
I express special thanks to my friends Praveen, Li, Ratan, Shanti, Yogendra, Akshay, Lakshmi, Aashish and Kavita for constant support and motivation over the years.
All of this would not have been possible without the support from my family. The highest recognition goes to my family including my grandmother, mother, father, wife and son. Thanks to my parents, Asha Vyas and Dr. Kailash N. Vyas, for instilling in me the characters of resilience, strength, and a good work ethic and for always believing in my capabilities. I must acknowledge the support I received from my partner, Muskan Vyas and my son Tanish Vyas. It was their love, and support, without any complains or regrets despite long working hours that I have been able to finish my dissertation.
Finally, I am grateful to the almighty for providing guidance and wisdom to accomplish this opportunity.
Table of contentsDedication
Table of contents
List of tables
List of figures
List of abbreviations
Chapter 1: INTRODUCTION
Chapter 2: LITERATURE REVIEW
Milk fat synthesis
Denovo fatty acid synthesis
Uptake of preformed FA by mammary gland
Triglyceride (TG) synthesis
Transcriptional regulation of lipid synthesis
Sterol regulatory element binding protein (SREBP)
Peroxisome proliferator activated receptor- γ (PPAR-γ)
Nutritional regulation of milk fat synthesis
Conjugated linoleic acid (CLA)
Physiological effects of CLA
Effects of CLA on hepatic lipid metabolism
Hepatic FA Synthesis
Hepatic FA uptake and TG secretion
Hepatic FA Oxidation
Effect of CLA on hepatic fatty acid composition
CLA and SCD in hepatic lipid metabolism
Effect of CLA on mammary lipid metabolism
Short-and medium-chain fatty acids (SMCFA)
Absorption and metabolism of SMCFA
Milk fat responses to supplemental SMCFA
Hypothesis and study objectives
Chapter 3: EXPERIMENT 1
MATERIALS AND METHODS
Animals, Experimental Design, and Treatments
Milk Sampling and Component Analysis
Milk Fat and FA Composition
Chapter 4: EXPERIMENT 2
MATERIALS AND METHODS
Animals, Experiment Design, Treatment, and Sampling.
FA Composition Analysis
RNA Isolation and Quantitative Real-Time Reverse-Transcription PCR................ 99 Protein Isolation and Western Blotting
Chapter 5: EXPERIMENT 3
Animals, Diets, and Treatments
Lipid Extraction and FA Analysis
RNA Isolation and Quantitative Real-Time Reverse-Transcription PCR.............. 140 Statistical Analyses
Food Intake, Body and Organ Weights, and Pup Growth Rate
Milk Fat Content and FA Composition
Hepatic FA Composition
Mammary Lipogenic Gene Expression
Hepatic Lipogenic Gene Expression
Mammary and hepatic protein expression
Chapter 6: SUMMARY AND FUTURE DIRECTIONS
Studies showing trans-10, cis-12 CLA-induced changes in body, adipose and liver weights and liver lipid concentration
Studies showing trans-10, cis-12 CLA-induced changes in hepatic gene expression and circulating levels of insulin, adipokines and TNF-α
Summary of literature studies on amelioration of CLA-induced hepatic steatosis
Ingredient and chemical composition of the basal diet fed to lactating cows. 81 Table 3.2. Fatty acid composition of different feed ingredients
Amount of fatty acid supplemented, g/d
Least squares means for production responses to increasing short-and medium-chain fatty acids
Least squares means for fatty acid composition (%) of milk from cows in response to S0, S200, S400, and S600 treatments
Least squares means for yield (g/d) of fatty acids in milk from cows in response to S0, S200, S400, and S600 treatments
Ingredient and chemical composition of the basal diet
Fatty acid composition of fat supplements infused in lactating dairy cows. 115 Table 4.3. Features of primers used for qPCR analysis
Production responses from cows abomasally infused with long-chain FA (LCFA), LCFA with conjugated linoleic acid (LC-CLA), butterfat (BF) and BF with CLA (BF-CLA)
Least squares means for fatty acid composition of milk from cows infused with long-chain FA (LCFA), LCFA with conjugated linoleic acid (LC-CLA), butterfat (BF) and BF with CLA (BF-CLA)
Least squares means for fatty acid yield of C18:1 and C18:2 isomers in milk fat from cows abomasally infused with long-chain FA (LCFA), LCFA with conjugated linoleic acid (LC-CLA), butterfat (BF) and BF with CLA (BF-CLA)
Least squares means of desaturase indices in milk from cows abomasally infused with long-chain FA (LCFA), LCFA with conjugated linoleic acid (LC-CLA), butterfat (BF) and BF with CLA (BF-CLA)
Composition of experimental diets fed to lactating mice
Fatty acid composition (g/100 g FAME) of lipids added to diets of lactating mice
Primers used for RT-PCR analysis
Effects of different treatments on dam body weight, food intake, milk fat, liver weight and pup growth rate
Effects of different treatments on milk fatty acid composition in lactating mice
Effects of different treatments on hepatic fatty acid composition in lactating mice
Effects of different treatments on pup liver fatty acid composition.............. 167
The SREBP pathway (Brown and Goldstein, 1997)
Biohydrogenation pathways of unsaturated FA (Harfoot and Hazlewood, 1988)
Current concepts in the pathways of trans-10, cis-12 CLA-induced hepatic steatosis
Transport, distribution and metabolic fate of exogenous FA according to their chain length (Bach et al.,1996)
The basics of milk fat synthesis
The experimental approaches used to test the potential limitation of denovo synthesized fatty acids for milk fat synthesis
Fatty acid composition of short- and medium-chain fatty acid mixture relative to denovo synthesized fatty acids present in milk fat
Transfer efficiency of individual short-and medium-chain fatty acids into milk fat of dairy cows fed 200, 400, and 600 g/d SMCFA
Fatty acid composition of abomasally infused butterfat and long-chain fatty acids
Relative mRNA abundance of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) in response to abomasal infusion of different fat supplements........... 128 Figure 4.3.
Relative mRNA abundance of acylglycerol-3-phosphate acyl transferase (AGPAT) and diacylglycerol acyl transferase (DGAT) in response to abomasal infusion of various fat supplements