AROIVES February 2000 MASSACHUSETTS INSTITUTE OFTECHNOLOGY

IRLrF;-Y-- i--- r_ ;rr~lslu~ rr~a Hormonal Regulation of Long Chain Fatty Uptake by Adipocytes and Studies of the FATP Gene Family by David J. Hirsch B. A. Biology, Johns Hopkins University 1991 Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the Massachusetts Institute of Technology AROIVES February 2000 MASSACHUSETTS INSTITUTE OFTECHNOLOGY © 2000 Massachusetts Institute of Technology APR 0 5 LuuU All rights reserved LIBRARIES Signature of Author. ... .................................................... Department of Biology December 22, 1999 C ertified by . ........ ... ..................................................... Harvey F. Lodish Professor of Biology Thesis Supervisor Accepted by . ....... .. .. .................... ............ ......... 6 o Terry Orr-Weaver Co-Chair, Biology Graduate Committee Hormonal Regulation of Long Chain Fatty Uptake by Adipocytes and Studies of the FATP Gene Family by David J. Hirsch Submitted to the Department of Biology on December 30, 1999 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology ABSTRACT Long chain fatty acids (LCFA) are an important source of energy for most organisms. Serum fatty acid (FA) levels are dynamically regulated by hormones. We show that insulin directly stimulates adipocyte fatty acid influx suggesting that the decrease in serum FA levels seen after meals is partially mediated by an insulin-stimulated increase in FA uptake by adipocytes. We also find that TNF-ax directly inhibits FA uptake by 3T3-L1 adipocytes providing a physiologic link between the increased serum levels of TNF-oa and FA seen in Type II diabetes. Transport of LCFA across the plasma membrane is facilitated by FATP, a plasma membrane protein that increases LCFA uptake when expressed in cultured mammalian cells (Schaffer and Lodish, 1994). We report the identification of family of evolutionarily conserved FATPs. In addition to the gene first isolated by Schaffer and Lodish, humans have five additional FATP homologues (designated FATP1-5). All of the mammalian FATPs increase fatty acid uptake when over- expressed in COS cells. FATPs are also found in such diverse organisms as F. rubripes, C. elegans, D. melanogaster,S. cerevisiae, and M. tuberculosis.The function of the FATP gene family is conserved throughout evolution as the C. elegans and mycobacterial FATPs facilitate LCFA uptake when over-expressed in COS cells or E. coli, respectively. Among the mammalian FATPs, several are expressed in a highly tissue specific manner. We show that FATP4 is expressed at high levels on the apical side of mature enterocytes in the small intestine. Furthermore, reduction of FATP4 expression in primary enterocytes by anti-sense oligonucleotides inhibits FA uptake by 50% suggesting that FATP4 is the principal fatty acid transporter in enterocytes. FATP5 is expressed only in adult liver. We have isolated the FATP5 promoter and find that tissue specific expression can be recapitulated in vitro and requires a single GC box in conjunction with two novel ten nucleotide motifs. Additional study of the FATP gene family may provide a better understanding of the mechanisms whereby LCFAs traverse the lipid bilayer as well as yield insight into the control of energy homeostasis and its dysregulation in diseases such as diabetes and obesity. Thesis Supervisor: Harvey F. Lodish Title: Professor of Biology Table of Contents Apportionment of Labor Chapter 1 Introduction Chapter 2 Hormonal regulation of fatty acid uptake by adipocytes 62 Chapter 3 A family of fatty acid trasporters conserved from mycobacteria to man 89 Chapter 4 Identification of the major intestinal fatty acid transport protein 105 Chapter 5 Identification and characterization of the FATP5 promoter 132 Chapter 6 Conlcuding thoughts 164 AnDortionment of labor Chapter 1 Introduction Text David Hirsch Chapter 2 Hormonal regulation of fatty acid uptake by adipocytes Text and intellecutual contributions Andreas Stahl and David Hirsch FACS plots and data David Hirsch Radiolabeled efflux Andreas Stahl Radiolabeled uptake Andreas Stahl Chapter 3 A family of fatty acid transporters conserved from mycobacteria to man Text David Hirsch Intellectual contribution Andreas Stahl BLAST searches and alignment David Hirsch Library screening David Hirsch and Andreas Stahl FATP alignment David Hirsch FACS plots and data David Hirsch and Andreas Stahl Northern analysis David Hirsch Family tree Andreas Stahl Hirsch, D., Stahl, A., and Lodish, H. F. (1998). A family of fatty acid transporters conserved from mycobacterium to man. Proc Natl Acad Sci U S A 95, 8625-9. Chapter 4 Identification of the major intestinal fatty acid transport protein Text Andreas Stahl Intellectual contribution David Hirsch GST fusion construct and protein David Hirsch and Mariana Kotler In situ hybridization MPI Antibody purification and characterization Andreas Stahl and Mariana Kotler Immunofluorescence Andreas Stahl and Nicki Watson Electronmicroscopy Andreas Stahl and Nicki Watson Antisense and enterocyte uptake Andreas Stahl FATP4 stable cell line Andreas Stahl and MPI BODIPY competition studies MPI Stahl, A., Hirsch, D. J., Gimeno, R. E., Punreddy, S., Ge, P., Watson, N., Patel, S., Kotler, M., Raimondi, A., Tartaglia, L. A., and Lodish, H. F. (1999). Identification of the major intestinal fatty acid transport protein. Mol Cell 4, 299-308. Chapter 5 Identification and characterization of the FATP5 promoter Text David Hirsch Intellectual contribution Andreas Stahl All figures and constructs David Hirsch Chapter 6 Concluding thoughts Text David Hirsch Chapter 1 Introduction Although often neglected, obesity is arguably the single most important epidemic facing the developed world. After holding steady for about 20 years, the percentage of the United States' population classified as clinically obese almost doubled from 14.5% to 22.5% over the period from 1976 to 1994 (Taubes, 1998). The negative health consequences of obesity are significant. Severely obese people have a 40-fold greater risk of non-insulin dependent diabetes mellitus (NIDDM) than normal controls and more than 80% of NIDDM cases can be attributed to obesity (Bray, 1996). Obesity is also associated with a 160% increase in mortality from cardiovascular disease (Gillham et al., 1997). One study estimates that 34,000 of the 55,000 deaths caused by diabetes and 77,000 of the 400,000 deaths from coronary heart disease each year are attributable to obesity (Bray, 1996). Obesity is also a risk factor for cirrhosis, gall stones, and cancer. Monetarily, obesity costs the United States between $50 and $70 billion a year in direct and indirect costs (Bray, 1996; Wickelgren, 1998). Furthermore, Americans spend almost $40 billion a year on weight loss treatments (Wickelgren, 1998). The increased prevalence of obesity is generally considered to be the result of environmental factors. Essentially, people are eating more and exercising less. The average person in a developed country now receives about 40% of their daily calories from fat (Thomson et al., 1997). Almost all of this fat is in the form of triglyceride with oleate and palmitate as the major components. Although fat absorption is exclusively confined to the small intestine, digestion of fat begins in the mouth with lingual lipase, which can hydrolyze approximately one-third of the ingested fat. Gastric lipase released by the stomach can digest an additional 10-40% of the triglyceride with the remainder being digested by pancreatic lipase. The pancreas also secretes bile salts which help emulsify the fat and facilitate digestion. Fatty acids, bile salts, mono-acyl glycerols, and phospholipids form mixed micelles in the small intestine. These micelles diffuse to the brush border of the enterocytes which line the small intestine and the fatty acids are taken up by these cells. After absorption, the fatty acids are esterified to CoA, reformed into triglyceride, packaged into chylomicrons, and secreted into the lymph. Recently, a promising area of obesity therapy has been the reduction of fat intake either through inhibition of fat digestion in the small intestine or the use of non-digestible fat substitutes in food. Olestra is a fat substitute in which six to eight fatty acids are esterified to a sucrose backbone (Thomson et al., 1997). Unlike fatty acids esterified to glycerol, the fatty acids in Olestra cannot be removed by lipases and consequently are not absorbed in the small intestine. The impact of Olestra on weight reduction has been limited due to restrictions on its use in food. Xenical (orlistat) is a compound developed by Hoffman-LaRoche that inhibits gastric and pancreatic lipase. In male individuals with a daily intake of 76 grams of fat per day, 80 mg of orlistat can reduce the absorption of fatty acid uptake by 36%. However, the effectiveness of orlistat is limited by several factors including the action of lingual lipase which is unaffected by the drug. Both Olestra and Xenical can cause loose stools, vitamin malabsorption and gastrointestinal distress which further limits their usefulness. Another potential strategy for obesity therapy would be to reduce fat absorption from the mixed micelle by the enterocytes. However, for many years, absorption of fatty acids by the enterocytes was thought to be diffusion-mediated. Some investigators have challenged this notion and suggested that fatty acid uptake by enterocytes is a protein- mediated process. If so, inhibitors

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