University of Cincinnati

UNIVERSITY OF CINCINNATI

Date:______

I, ______, hereby submit this work as part of the requirements for the degree of: in:

It is entitled:

This work and its defense approved by:

Chair: ______

Intestinal lipid uptake and

secretion of VLDL and chylomicron

By: Andromeda Nauli

August 2005

Previous degree:

Bachelor of Science in Biomedical Sciences

Degree to be conferred: Ph.D.

Department of Pathology and Laboratory Medicine

College of Medicine

University of Cincinnati

Committee chair:

Patrick Tso, Ph.D.

iii

ABSTRACT

Despite decades of research, our understanding of intestinal lipid absorption is limited. In this Ph.D. thesis, I have dealt with two main aspects of intestinal lipid absorption, namely the uptake of lipids and the formation and secretion of triacylglycerol-rich lipoproteins (very low density lipoproteins [VLDL] and chylomicrons). In terms of uptake, CD36 is one of the plasma membrane proteins implicated in mediating lipid uptake by the intestine. In order to test this hypothesis, we utilized the CD36 knockout mouse model equipped with intraduodenal and lymph cannulas. Our studies showed that the disruption of the

CD36 gene led to a significant decrease in the uptake of cholesterol but not of fatty acids. Interestingly, the role of CD36 was not limited to uptake but also appeared to affect the formation and secretion of chylomicrons, the major lipoproteins carrying the absorbed dietary fat from the gut (Chapter 2). It was first proposed by Tso et al. (202) that the small intestine secretes both VLDL and chylomicrons. Previous work by Vahouny et al. (212) suggested that female rats produced more VLDL than male rats. In addition, personal communication with

Dr. Renee LeBoeuf leads us to believe that the female C57BL/6 mice may absorb lipids less efficiently than the male mice. We therefore studied the formation and secretion of lipoproteins in male and female C57BL/6 mice. Our data agree with those of Vahouny in that the female mice had a slightly higher ratio of VLDL to chylomicron secretion relative to that of the male mice. In addition, we also found that the intestinal lymphatic lipid transport of the C57BL/6

iv female mice segregated into two groups, a phenomenon that was absent in the male mice. In summary, our work suggests that CD36 is involved not only in intestinal cholesterol uptake, but also in regulating the formation and secretion of chylomicrons. In addition to the regulation by CD36, our studies also show that the regulation of the formation and secretion of chylomicrons are potentially different between male and female animals.

Acknowledgements

I would like to express my gratitude to all of the individuals who helped me throughout this study. I am particularly thankful to Dr. Patrick Tso for all of his guidance as my mentor. I also wish to thank my committee members, Drs.

Stephen Woods, Simon Newman, Ronald Jandacek, and Sean Davidson for their continuous support. I would also like to express my appreciation to University of

Cincinnati Department of Pathology and Laboratory Medicine. Finally, I would like to thank my family, Susan, Surya, and mom.

Table of Contents

Committee Approval Form……………………………………………………… i

Title page………………………………………………………………………… ii

Abstract…………………………………………………………………………... iii

Acknowledgements……………………………………………………………... vi

Table of Contents………………………………………………………………... 1

List of Tables and Figures………………………………………………………. 4

List of Abbreviations…………………………………………………………….. 6

Chapter 1: Review of the Literature and Study Rationale………………….. 8

1.1. Introduction…………………………………………………………. 9

1.1.1. Defining intestinal lipid absorption……………………………... 9

1.1.2. The importance of intestinal lipid absorption…………………. 10

1.1.3. Limitation of the in vitro models……………………………….. 12

1.2. Anatomy of the small intestine…………………………………… 14

1.2.1. Structure supports function…………………………………….. 14

1.2.2. Understanding the histological layers…………………………. 15

1.3. Dietary lipids………………………………………………………... 17

1.3.1. The detrimental effect of saturated fat………………………… 17

1.3.2. The significance of non-dietary cholesterol…………………... 18

1.4. Digestion of dietary lipids…………………………………………. 21

1.4.1. The early digestion processes…………………………………. 21

1.4.2. The significance of pancreatic lipase…………………………. 22

1.4.3 .Hydrolysis of cholesteryl esters……………………………….. 23

1.5. Uptake of lipid digestion products by enterocytes……………... 23

1.5.1. The significance of micelles……………………………………. 23

1.5.2. The significance of bile acids…………………………………… 24

1.5.3. Fatty acid uptake……………………………………………….... 25

1.5.3.1. Fatty acid transporters…………………………………………. 25

1.5.3.2. Mode of transport across plasma membrane……………….. 27

1.5.4. Cholesterol uptake……………………………………………….. 28

1.5.4.1. The significance of bile-acid micelles………………………… 28

1.5.4.2. Cholesterol transporters………………………………………… 29

1.6. Re-esterification of lipid digestion products inside enterocytes.... 30

1.6.1. Lipid transport to the ER………………………………………….. 31

1.6.2. TG re-synthesis……………………………………………………. 32

1.6.3. Cholesterol esterification…………………………………………. 34

1.7. Formation and secretion of lipoproteins…………………………… 35

1.7.1. Intestinal lipoproteins………………………………………………. 35

1.7.2. Assembly of lipoproteins…………………………………………… 36

1.7.3. Separate pathway for VLDL and chylomicron assembly………. 37

1.7.4. ER to Golgi transport………………………………………………. 37

1.8. Regulation of lymphatic vs. portal transport………………………. 38

1.9. Study Rationale………………………………………………………. 39

Chapter 2: CD36 is important for intestinal cholesterol uptake and for the formation and secretion of chylomicrons………..……………………….. .. …... 41

Abstract……………………………………………………………………... 42

Introduction…………………………………………………………………. 43

Materials and methods…………………………………………………… 45

Results……………………………………………………………………… 50

Discussion………………………………………………………………..... 55

Chapter 3: Sex differences in intestinal lipid absorption in mice…………..... 76

Abstract…………………………………………………………………….. 77

Introduction………………………………………………………………… 78

Materials and methods…………………………………………………… 81

Results………………………………………………………………………. 85

Discussion…………………………………………………………………. 92

Chapter 4: General Conclusions and Future Directions…………………….. 115

1. Insights gained on the lipid uptake…………………………………... 116

2. Insights gained on the lipoprotein secretion………………………… 119

Literature Cited………………………………………………………………….... 124

Appendix 1………………………………………………………………………… 169

CD36 deficiency impairs intestinal lipid secretion and

clearance of chylomicrons from the blood

Appendix 2………………………………………………………………………… 170

Enterocyte fatty acid uptake and intestinal fatty acid-

binding protein

List of Tables and Figures

Table

1.1. The amount of biliary components secreted into the intestinal lumen… 20

Figures

1.1. Diagram of the small intestine……………………………………………. 16

2.1. Analysis of fatty acid uptake by the small intestines

of CD36 null and wild type mice…………………………………………. 64

2.2. Lymph [ 3H]-TG transport (A), TG mass (B), and [ 14 C]-cholesterol

transport (C) during continuous intraduodenal lipid infusion………….. 65

2.3. Total recovery of the infused TG (A) and cholesterol (B) in the

stomach, colon, intestinal lumen, intestinal mucosa, and lymph

at the end of the 6-h infusion period……………………………………… 67

2.4. Distribution of the infused TG (A) and cholesterol (B) along

4 equal-length segments of the small intestine…………………………. 68

2.5. Mucosal distribution of [ 3H]-fatty acids from infused triolein

into the major lipid classes………………………………………………… 69

2.6. Mucosal distribution of [ 14 C]-cholesterol from infusate

into the major lipid classes………………………………………………… 70

2.7. Lipoprotein particle size in the lymph of fasted mice…………………… 71

2.8. Lipoprotein particle size of lymph from lipid infused mice……………... 72

2.9. Lipid composition of chylomicrons from lipid infused mice…………….. 73

2.10. Apolipoprotein secretion into the lymph by fasted or lipid infused mice.. 74

3.1. The histograms of the total lymphatic triacylglycerol recovery

in the male (A) and the female mice (B)……………………………………101

3.2. The lymph flow rate during the continuous intraduodenal lipid infusion.. 104

3.3. The hourly lymphatic triacylglycerol output during continuous

intraduodenal lipid infusion…………………………………………………..105

3.4. The hourly lymphatic cholesterol output during the continuous

intraduodenal lipid infusion…………………………………………………..106

3.5. The total radioactive triacylglycerol recovery in the lymph

and the segments of gastrointestinal tract………………………………. . 107

3.6. The total radioactive cholesterol recovery in the lymph

and the segments of gastrointestinal tract………………………………… 108

3.7. Distribution of different classes of [ 3H]-labeled lipids

in intestinal mucosa………………………………………………………….. 109

3.8. Distribution of different classes of [ 14 C]-labeled lipids

in intestinal mucosa………………………………………………………….. 110

3.9. Lipoprotein particle size of fasting lymph………………………………….. 111

3.10. Lipoprotein particle size of lipid-infused lymph…………………………... 113

List of Abbreviations

ACAT = acyl coenzyme A:cholesterol acyltransferase

Apo = apolipoprotein

ASBT = apical sodium-dependent bile acid transporter

CE = cholesterol ester

CMC = critical micellar concentration

COP = coat protein

DG = diacylglycerol

DGAT = diacylglycerol acyltransferase

FA = fatty acid

FATP = fatty acid transport protein

G-3-P = glycerol-3-phosphate

HDL = high density lipoprotein

IBAT = ileal bile acid transporter

I-FABP = intestinal fatty acid-binding protein ilbp = ileal binding protein

KO = knock out

L-FABP = liver fatty acid-binding protein

LXR = liver x receptor

MG = monoacylglycerol

MGAT = monoacylglycerol acyltransferase

MTP = microsomal triglyceride transfer protein

NaTC = sodium taurocholate

NPC1L1 = Niemann-Pick C1-Like 1

PBS = phosphate-buffered saline

PC = phosphatidyl choline

PGC = peroxisome proliferator-activated receptors gamma

coactivator

PL = phospholipid

SPB = sucrose polybehenate

SREBP = sterol regulatory element-binding protein

TG = triacylglycerol

TLC = thin layer chromatography

VLDL = very low density lipoprotein

WT = wild type

Chapter 1

Review of the Literature and Study Rationale

1.1. Introduction

The introductory chapter (Chapter 1) discusses our current understanding of intestinal lipid absorption with the emphasis on recent findings. This chapter complements other reviews on intestinal lipid absorption that are available in the literature (156; 174; 191; 242). Chapter 1 ends with a discussion of how our studies add to the understanding of intestinal lipid absorption, particularly on lipid uptake and the formation and secretion of intestinal triacylglycerol-rich lipoproteins. Chapters 2 and 3 discuss our studies on the role of CD36 in intestinal lipid absorption and sex differences in lipoprotein secretion, respectively.

1.1.1. Defining intestinal lipid absorption

The process of intestinal lipid absorption is complex. We define “intestinal absorption” as the whole process that includes digestion, uptake, re-esterification of lipid digestion products, formation and secretion of lipoproteins, and transport to the circulation. Some investigators use the term “intestinal absorption” to refer solely to the uptake step (35), while others broaden it to the whole process as we have described (173; 190; 241). To complicate the matter further, the term

“uptake” has been replaced with “absorption” by some investigators (172).

In a simplified view, intestinal lipid absorption is an important process of transferring the hydrophobic molecules of nutrients from the aqueous environment in the intestinal lumen into the aqueous environment in the circulation. An obvious problem arises: how are these hydrophobic molecules of nutrients transferred efficiently in the hydrophilic milieu? The solution to this problem is the conversion of hydrophobic molecules of nutrients through the digestive process to forms of the molecules that are less hydrophobic; and subsequently packaging them inside the intestinal epithelial cells into large lipid particles coated with phospholipids and apolipoproteins for transport in circulation.

The problem of hydrophobicity is exemplified by the fact that medium chain fatty acids that are more water soluble are better absorbed than long chain fatty acids

(20; 70), particularly in cases of lipase insufficiency.

1.1.2. The importance of intestinal lipid absorption

The regulation of intestinal lipid absorption is not well understood. From an evolutionary viewpoint, organisms have evolved to achieve more efficient nutrient intake, storage, and utilization. A recent interest in understanding intestinal lipid absorption has arose in hopes of ultimately designing a way to reduce fat absorption. The long term goal of reducing fat absorption in Western society is obvious—preventing obesity and other cardiovascular-related diseases without significantly altering the diet. For example, the drug, orlistat, is an inhibitor of pancreatic lipase that reduces the absorption of dietary fat.

The role of intestinal lipid absorption in preventing cardiovascular diseases has been considered less important than that of the liver due to the liver’s role as the key organ regulating plasma lipid levels. According to this paradigm, the function of the intestine is simply to facilitate the absorption of nutrients. Due to the rapid metabolism of chylomicrons (produced by the gastrointestinal tract), the gut is not believed to be an important determinant of overall circulating plasma lipid levels.

Consequently, more research has focused on the liver relative to the other organs. This paradigm is challenged by recent studies that suggest that intestine also plays a significant role in regulating plasma lipid levels. Studies from mice lacking Niemann-Pick C1-Like 1 (NPC1L1), a protein implicated in intestinal cholesterol absorption, showed that these mice were resistant to hypercholesterolemia when they ate a high-cholesterol diet (55). The liver

NPC1L1 expression in these mice was negligible (5), suggesting that the intestine plays an important role in regulating lipid homeostasis. The recent studies of NPC1L1 on intestinal lipid absorption will be discussed later.

An earlier study on a unique subject designated as the “egg man” further supports the concept that intestine is an important organ in regulating plasma lipid concentration (110). The “egg man” consumed about 25 eggs a day—an unusually high cholesterol intake he followed for over 15 years. It is noteworthy that previous studies have confirmed elevated plasma cholesterols resulting from egg consumption (170). Contrary to expectation, however, the “egg man” did not

12 have a hypercholesterolemia. In fact, the “egg man” only absorbed 18% of his dietary cholesterol as compared to a normal value of about 50%, suggesting that cholesterol absorption by the intestine may be one of the important factors in determining plasma cholesterol level.

In contrast to the high cholesterol diet of the “egg man,” the Tarahumara Indians are known for their low fat and low cholesterol diet which consists mainly of corn and beans. In addition, the Tarahumara Indians are also known for their low plasma cholesterol level. When they were put on either cholesterol-free or high cholesterol diet, their cholesterol absorption index, as determined by using the dual-isotope technique, was about 28% (141). These studies, together with the studies mentioned above, suggest that intestine can be an important organ in regulating plasma lipid levels. Consequently, altering intestinal lipid absorption should be considered one of the plausible therapeutic means in fighting cardiovascular diseases.

1.1.3. Limitation of the in vitro models

Although recent findings have added significantly to our understanding of intestinal lipid absorption, many questions remained unanswered. The lack of a good cell culture model to study intestinal lipid absorption partly explains the slow progress in our understanding of the process at the cellular and molecular level.

To date, the most commonly used intestinal cells to study intestinal absorption

13 are Caco-2 cells derived from human colon carcinoma (99; 196). Although widely used, in several aspects Caco-2 cells behave rather differently from enterocytes—the epithelial cells lining the small intestine responsible for nutrient uptake. Their most important differences are in apolipoprotein B secretion and triacylglycerol (TG) synthesis. In contrast to human enterocytes that secrete only apolipoprotein B-48, Caco-2 cells secrete both apolipoprotein B-48 and B-100

(195). Furthermore, enterocytes use the monoacylglycerol (MG) pathway, while

Caco-2 cells utilize primarily the glycerol-3-phosphate (G-3-P) pathway to synthesize TG (197). The significance of these two pathways will be discussed in the succeeding section.

Other commonly used human colon carcinoma-derived cells are HT-29 cells.

These cells, however, do not differentiate without the addition of galactose in the media (248). Readers interested in comparing different intestinal cell models should refer to studies by Chantret et al. (39).

Another widely used model is the use of isolated intestinal epithelial cells

(primary cell culture). However, isolation of cells from intestine has proved to cause a significant reduction in cell viability. In addition, the intestinal epithelial cells do not seem to function well when in isolation. Shakir et al. (179) reported that the viability of isolated intestinal cells dropped to about 50% after 4 hours. A recent review of the primary culture of intestinal cells also highlighted its limitation in viability (108; 153).

All of the descriptions listed above suggest that studies using in vitro models should be interpreted cautiously. The limitation of each model should be understood. As will be discussed later, many in vitro observations could not be observed in the in vivo studies.

1.2. Anatomy of the small intestine

1.2.1. Structure supports function

In order to facilitate a better understanding of intestinal lipid absorption, a brief overview of the anatomy of the small intestine will be presented. The gastrointestinal system consists of the gastrointestinal tract and gastrointestinal glands. The gastrointestinal tract is a continuous tube consisting of mouth, esophagus, stomach, small intestine, large intestine, rectum, and anus. The major supportive glands of gastrointestinal system are the salivary glands, pancreas, and liver. The significance of these glands in intestinal lipid absorption will be mentioned later.

The whole length of an adult gastrointestinal tract is about 5 m. This extended length contributes to an enhanced area for absorption capacity. Lipid absorption mainly occurs in the small intestine, which consists of duodenum, jejunum, and ileum, with most lipids being absorbed in the duodenum and jejunum (12; 23).

There is little structural difference among the duodenum, jejunum, and ileum, only that the jejunum has more foldings (plicae circulares), which further increase the surface area for absorption. The presence of villi and microvilli of the small intestine contributes additional 10- and 20-fold increases in surface area, respectively. Altogether, these structures serve to enhance the efficiency of nutrient absorption.

1.2.2. Understanding the histological layers

The gastrointestinal tract is made out of 4 histological layers, known as the mucosa, submucosa, muscularis, and serosa. As depicted in Figure 1.1., the small intestine has many projections (villi) that are lined by enterocytes (intestinal epithelial cells, also known as absorptive cells) facing toward the lumen. This outer (luminal) layer is called the mucosal layer, which is made out of epithelial lining, lamina propria, and muscularis mucosae (layers of muscle cells). The enterocytes lining the villi (epithelial lining) are the cells responsible for taking up the nutrients from the lumen. These nutrients will then be transported to the circulation, either through blood (Figure 1, left) or lymphatic (Figure 1, center) systems. The blood capillaries and lymphatic lacteals are located in the lamina propria and extend to the outer most layer, serosa, composed of loose connective tissues. In between the epithelial lining and the lamina propria lies the basement membrane. The second layer, the submucosa, consists of connective tissues infiltrated by nerve cells, immune cells, and capillaries. The

16 third layer, the muscularis, consists of inner and outer mucularis, both of which are also infiltrated by nerve cells and capillaries. The function of the muscularis is for intestinal motility, allowing for a proper mechanical mixture and transit of the ingested nutrients in the lumen.

Figure 1.1. Diagram of the small intestine showing blood circulation (left), lymphatic circulation (center) and smooth muscle cells (right). Adapted from

Basic Histology, 9 th ed. (120)

1.3. Dietary lipids

A recent survey showed that about 30-40% of total energy of a typical Western diet was from fat (246). These studies also highlighted a significantly higher amount of fat consumed in the Western relative to the Eastern countries. About

95% of dietary fat is long-chain TG. The other 5% are phospholipids, fatty acids, and cholesterol. Most dietary cholesterol is free cholesterol. Dietary cholesteryl esters need to be hydrolyzed prior to uptake by the enterocytes (228). In this chapter, we will only discuss intestinal absorption of TG and cholesterol.

1.3.1. The detrimental effect of saturated fat

It is known that saturated fat is hyperlipidemic, and polyunsaturated fat is not.

Recent studies from Spiegelman’s laboratory (114) suggest that saturated fat, but not polyunsaturated fat, stimulates the expression of liver coactivator PGC-1β, which in turn mediates the transcription of two subsets of genes. These two subsets of genes are genes responsible for lipid synthesis in the liver (regulated by SREBP) and genes responsible for lipid transport from the liver to the circulation (regulated by LXR α). As a result of the increased synthesis and the concomitant mobilization of lipid from the liver to the circulation, plasma lipid levels increase (hyperlipidemia).

While we know that saturated fats gain entry to the body through the gastrointestinal tract, we do not know the extent of the gastrointestinal tract contribution to the hyperlipidemic effect of saturated fat. Studies comparing the absorption of saturated and unsaturated fat showed that the unsaturated fat was more efficiently absorbed (69; 152). The reason may be that the unsaturated fat is more soluble in micelles than the saturated fat. The importance of micelle solubilization will be discussed shortly.

1.3.2. The significance of non-dietary cholesterol

The source of cholesterol in the intestinal lumen is often thought to be derived mainly from diet. This view is clearly not accurate. Average cholesterol consumption is 100-500 mg/day (247). In contrast, biliary cholesterol contributes to about 800-1200 mg/day (210) (See also Table 1.1. on page 20). In addition to diet and bile, enterocyte turnover also provides a significant amount of cholesterol into the intestinal lumen. Although its value is hard to determine, we estimated from the fecal sterol studies (143) that enterocyte turnover contributes about 150-200 mg/day. The high amount of luminal cholesterol derived from enterocyte turnover is not suprising, considering that the intestine is one of the organs that produce the highest amount of cholesterol (11; 183) and that enterocyte turnover rate is about 3-4 days. An interesting recent study showed that mice infected with parasites could increase their enterocyte turnover rate as a mechanism to expel the parasites from the gut (49). It remains to be

19 determined whether intestinal parasites could indirectly affect intestinal cholesterol absorption.

Since biliary cholesterol represents about 2/3 of total luminal cholesterol, the importance of biliary cholesterol absorption becomes obvious. The difference between biliary and dietary cholesterol absorption has often been discussed

(177; 240). Several studies have concluded that biliary cholesterol is more efficiently absorbed partly due to its better solubilization in micelles (176).

However, other investigators believe that dietary cholesterol will also be eventually solubilized in micelles to the same extent as biliary cholesterol; therefore their net percent absorption should be comparable (239). The absorption of biliary cholesterol is often ignored partly due to the erroneous assumption that the source of luminal cholesterol is mainly from diet.

In fact, Breslow et al. believe that the mechanism underlying the regulation of dietary cholesterol absorption depends strongly on how much biliary cholesterol is secreted into the small intestine (178). A high cholesterol diet will supply more cholesterol to the liver, which in turn will secrete more biliary cholesterol. The more biliary cholesterol is secreted, the less dietary cholesterol is absorbed.

They proposed that both biliary and dietary cholesterol absorption compete for incorporation into micelles, and that this is the reason for reduced dietary cholesterol absorption in individuals with high biliary cholesterol secretion. Biliary cholesterol secretion may be one of the factors regulating cholesterol absorption.

However, other non-biliary factors may also be critical for its regulation (119), as will be discussed later.

Bile components Values

Bile flow 7.1 ± 0.3 µL/100 g b. wt./min

Bile salt 383 ± 18 nmol/100 g b. wt./min

Total bilirubin 0.29 ± 0.01 nmol/100 g b. wt./min

Cholesterol 5.2 ± 0.6 nmol/100 g b. wt./min

Phospholipids 47.7 ± 3.2 nmol/100 g b. wt./min

Table 1.1. The amount of biliary components secreted into the intestinal lumen.

Inc.

(http://www3.interscience.wiley.com/cgi-bin/jabout/106570044/ProductInformation.html )

1.4. Digestion of dietary lipids

1.4.1. The early digestion processes

Mastication (chewing) and antral peristalsis of the stomach provide grinding and mechanical mixing of food. In addition to grinding and mixing, the stomach also plays an important role in emptying the food into the small intestine—a complex and important process that has been reviewed recently (166).

The early digestion of TG occurs in the stomach (1) by the action of two major enzymes, namely lingual and gastric lipase. These two enzymes share many similarities; both enzymes function at low pH of about 4-6 (66; 94) and are more efficient in hydrolyzing shorter chain than longer chain TGs (113). The major products of both enzymes are diacylglycerols (DGs) and fatty acids (FAs) (65;

91; 93; 184). The fact that these enzymes hydrolyze TGs into DGs and FAs suggests that these enzymes are also critical in emulsifying the dietary lipids prior to entry into the small intestine. It has been estimated that this emulsification is responsible for reducing the size of fat droplets from 2-5 µm into about 0.5 µm (171).

The significance of these enzymes has been thought to be more pronounced in neonates since these enzymes work better on medium chain TGs, and that the major dietary fat of neonates is milk, which contains some medium chain TGs

(92). It is also believed that pancreatic lipase as well as bile acid secretion have not been fully developed in neonates (88). As will be discussed later, pancreatic lipase is the main enzyme that hydrolyzes TG. However, a study on a 5-year-old child with congenital defect reducing the activity of both pancreatic lipase and colipase showed that this child could absorb about 50% of the dietary fat (78), suggesting that other lipases, eg., gastric lipase, can compensate for TG digestion up to a certain level.

1.4.2. The significance of pancreatic lipase

Pancreatic lipase is secreted abundantly by the pancreas. However, its activity is significantly enhanced by the presence of a colipase, a coenzyme that is originally secreted in its inactive procolipase form and later activated by trypsin.

The colipase interacts with the oil-water interface allowing the lipase to bind to the interface to hydrolyze TGs. A model of how TG can get in the active site of the pancreatic lipase has been proposed. This model suggests that the pancreatic lipase has a displaceable “lid” that can expose its catalytic site and allow its nonpolar surface to stabilize its TG substrate (21; 26).

The hydrolysis of TGs by the pancreatic lipase yields 2-monoacylglycerols (2-

MGs) and FAs (133; 135; 154). However, isomerization of 2-MGs can occasionally result in a complete hydrolysis of TGs into glycerols and FAs. The digestion of TG by the pancreatic lipase is critical for its micellar solubilization.

TGs are poorly soluble in aqueous environment. In contrast, MGs and FAs are readily soluble in micellar phase.

1.4.3. Hydrolysis of cholesteryl esters

Biliary cholesterol and most dietary cholesterol are not esterified. Esterified cholesterol is not taken up by the enterocytes (227). As reported in previous studies, cholesterol esters were less efficiently absorbed than cholesterol (187).

Therefore, it can be concluded that the hydrolysis of cholesteryl esters is required for them to be taken up by the enterocytes. The main, but not the only, enzyme that mediates cholesteryl ester hydrolysis is likely to be cholesterol esterase

(cholesterol ester lipase/sterol ester hydrolase). Mice lacking cholesterol ester lipase displayed a significant decrease in cholesteryl ester absorption but had a normal absorption of free cholesterol (226).

1.5. Uptake of lipid digestion products by enterocytes

1.5.1. The significance of micelles

As mentioned earlier, the uptake of FAs, MGs, and cholesterol depends critically on the micelle formation. Micelles are lipid aggregates consisting of bile acids,

FAs, MGs, cholesterol, and phospholipids (122). The presence of a 50-500 µm thick unstirred water layer in the intestine warrants the need for the micelles (234;

238) since they greatly increase the aqueous solubility of the lipid digestion products (97; 235). Although the size of the micelles is larger than the monomeric FAs and would, therefore, reduce the diffusion rate across the unstirred water layer, the enhanced solubility by the micelles (up to about 1,000 times) overcomes this diffusion barrier.

It is noteworthy that micelles are not taken up by enterocytes as a whole (96;

112). In fact, studies showed that MGs and FAs were taken up at different rate

(95; 144). Bile acids, the principal components of intestinal micelles, are subsequently taken up in the ileum. The uptake off bile acids is mediated by ileal bile acid transporter (IBAT), also known as apical sodium-dependent bile acid transporter (ASBT).

1.5.2. The significance of bile acids

The concentration of bile acids required for micelle formation is called critical micellar concentration (CMC), which is about 2.5 - 5.0 mM depending on the bile acid species and luminal pH (14). Due to their significant role in micelle formation, bile acids are indispensable for lipid uptake. Several studies have reported that steatorrhea (fat malabsorption) could occur when bile acid concentration was below the CMC (13; 175).

In the absence of the micelle formation, lipid uptake may depend primarily on the ability of the monomeric FAs to pass through the unstirred water layer. This diffusion process is certainly not efficient, especially for the long chain FAs. It has been proposed that lipid digestion products can also be carried in vesicles

(35). However, previous studies showed that the FAs from the micelles were more efficiently taken up by the Caco-2 cells, as compared to the FAs from the vesicles (148). Nevertheless, the role of the vesicles in facilitating FA uptake may be critical in certain clinical presentations, such as in patients with cholestasis (obstruction of bile flow to the intestine).

1.5.3. Fatty acid uptake

1.5.3.1. Fatty acid transporters

The plasma membrane molecules responsible for the uptake of FAs are still largely unknown. Several of the candidate molecules have been proposed with little or no in vivo evidence supporting them. The scavenger receptor CD36 has been implicated in the uptake of FAs in many tissues, such as muscles and adipose. Chapter 2 and Appendix 1 discuss our studies on the role of CD36 in the FA uptake by the intestine. Using both a continuous lipid infusion method and a fecal fat balance method (103), we could not find any differences in the uptake of FA by the intestine between the CD36 null and wild type (WT) mice.

Consequently, our studies suggest that CD36 may not be a FA transporter in the intestine.

One of the earliest suggestions for a fatty acid transporter came from a study by

Stremmel et al. (186) which showed that a fatty acid binding protein located at the rat jejunal microvillous membrane was involved in the uptake of FAs by the small intestinal epithelial cells. It is noteworthy that this protein is different from both the intracellular fatty acid binding protein and the liver fatty acid binding protein. The authors showed that they could inhibit the binding of FAs to the brush border membrane vesicles using the antibodies that they raised against their isolated protein. Consequently, their studies generated a considerable amount of interest since inhibition of this protein may potentially inhibit the absorption of fat by the small intestine. Unfortunately, it was later reported by the same group that their plasma membrane fatty acid binding protein is closely related to the mitochondrial glutamic-oxaloacetic transaminase, therefore raising the question of whether or not the protein that they isolated and characterized was an artifact from the subcellular fractionation (19).

Another FA transporter that has been proposed is the fatty acid transport protein

4 (FATP4). FATP4 is the only FATP expressed in the small intestine. It is localized in the apical brush border of the enterocytes. Previous studies showed that the heterozygous FATP null enterocytes had a 40% reduction in FA uptake as compared to that of FATP WT enterocytes (82). However, in vivo studies did

27 not show that the heterozygous FATP null mice had an increase in percent of fecal fat relative to that of the WT mice. (Homozygous FATP4 KO mice were embryonic lethal.) The authors suggest that although FATP4 may play a role in the uptake of FAs, its absence may not be enough to cause any physiological differences in lipid absorption.

1.5.3.2. Mode of transport across plasma membrane

Although the search for FA transporters in the intestine has remained vigorous, the concept of passive transport (non carrier-mediated diffusion) has not been considered adequately. Recent studies (90; 109) briefly reviewed the significance of a flip flop mechanism on FA uptake. The flip flop process is thought to consist of adsorption, translocation, and desorption. It is believed that desorption (the process of transferring from plasma membrane to cytosol) is the rate-limiting step in the flip flop mechanism.

It is likely that both the passive diffusion mechanism and the carrier-mediated mechanism coexist in the uptake of lipids by the intestine, especially when considering that the lipid concentration in the intestinal lumen under normal absorption is relatively high. At a low FA concentration, the carrier-mediated mechanism may be critical. And at a high FA concentration, typical of that seen after a fatty meal, the passive diffusion mechanism may be more significant. This concept, though accepted and often discussed by a number of investigators in

28 the field (47; 81; 146; 209), is often missed by the other investigators who are searching for the fatty acid transporters. For further discussion on this topic, please refer to Appendix 2.

1.5.4. Cholesterol uptake

1.5.4.1. The significance of bile-acid micelles

Cholesterol uptake by the enterocytes also depends heavily on micelles. Earlier studies showed that bile-diverted rats did not transport dietary cholesterol to the lymph (181), implying that bile is obligatory for cholesterol absorption. Recent outpatient studies (243) showed that subjects given a cholic acid supplement had a higher cholesterol absorption than control subjects. Subjects with cholic acid supplements also showed increased intraluminal bile acid concentrations with a concomitant increase in micellar cholesterol. Although this study did not prove a causal effect, it is highly suggestive that bile acids can significantly increase cholesterol uptake by the intestine through the formation of micelles. These studies are a good illustration of the importance of translational research. Other investigators have also showed a similar increase in cholesterol absorption when sodium taurocholate was supplemented in rats (188).

1.5.4.2. Cholesterol transporters

Several studies have attempted to isolate cholesterol transporters from the small intestinal brush-border membrane by using the method of subcellular fractionation. One of the molecules isolated was shown to have a cholesterol transfer activity, and was subsequently proposed to be a cholesterol transporter

(nsL-TP, non-specific lipid transfer protein) (192). However, later studies showed that the protein was mainly an intracellular molecule, ruling out its possibility as a cholesterol transporter (193; 244). These studies show us that the isolation and identification of cholesterol transporters by the subcellular fractionation should be done with caution.

Recent studies have argued that NPC1L1 is one of the cholesterol transporters in the intestine (7; 54; 77). NPC1L1 is highly expressed in the enterocytes of mice

(6). In contrast, the highest NPC1L1 expression in human is in the liver with relatively low expression in the intestine (51), suggesting that NPC1L1 may have a different physiological significance among species. There have also been some conflicting results concerning the localization of NPC1L1 in the enterocytes.

Some investigators argued that the protein was localized in the plasma membrane (8); others showed that it was mainly localized in the intracellular compartment, and suggested that the function of NPC1L1 is to direct cholesterol to the Golgi aparatus (50).

As mentioned earlier, recent studies reported that NPC1L1 KO mice had a significant reduction in cholesterol absorption (9) and were resistant to hypercholesterolemia (53). These mice also showed a significant reduction in biliary cholesterol (52), arguing against the concept that biliary cholesterol level is the sole determining factor in regulating dietary cholesterol absorption. The exact role of intestinal NPC1L1 is unclear, and more studies are needed before

NPC1L1 can be considered as an intestinal cholesterol transporter.

Another plasma membrane protein that may mediate cholesterol uptake by the intestine is CD36. Studies using isolated human intestinal brush border membranes showed that antibodies against CD36 could block the uptake of cholesterol but not cholesterol esters (230). Chapter 2 further discusses our studies on the role of CD36 in intestinal cholesterol uptake. Our studies showed that mice disrupted in the CD36 gene had a significant accumulation of infused cholesterol in the intestinal lumen, suggesting that CD36 may play a role in mediating cholesterol uptake in the intestine. Further studies are needed to determine whether or not CD36 is a true intestinal cholesterol transporter.

1.6. Re-esterification of digestion products inside enterocytes

Once lipids cross the plasma membrane of the enterocyte, they will be transported to the endoplasmic reticulum (ER) for re-esterification. The process of how lipids are transported to the ER is unclear. A brief discussion on how they

31 may be transported to the ER has been presented elsewhere (136). Much of our recent understanding of the re-esterification of lipid digestion products in the intestine comes from the work of Farese et al.

1.6.1. Lipid transport to the ER

I-FABP is believed to be a protein mediating intracellular lipid transport in the intestine (15; 130; 137). It is localized in the cytosol of the small intestine.

Studies comparing a naturally occurring polymorphism of I-FABP showed that some Pima Indians of Arizona had a substitution of alanine (Ala 54 ) to threonine

(Thr 54 ) in their I-FABP (17). The Pima Indians with the less prevalent threonine

(Thr 54 ) I-FABP were associated with insulin resistance. Interestingly, Thr 54 I-

FABP had a higher affinity for long chain FAs than Ala54 I-FABP. Further studies showed that when incubated with long chain FAs Caco-2 cells expressing Thr 54 I-

FABP were able to secrete more TGs than those expressing Ala 54 I-FABP (16).

The authors suggested that I-FABP played an important role in the intracellular lipid transport in the intestine. However, in vivo studies showed that the I-FABP

KO mice had a higher body weight than the WT mice (221). Interestingly, disruption of I-FABP gene did not lead to the upregulation of the other FABPs, namely liver FABP (L-FABP) and ileal lipid binding protein (ilbp). These data suggest that FABPs may not play an important role in the intestinal lipid absorption, or that there exists such a redundancy in the system that makes it hard to be perturbed.

1.6.2. TG re-synthesis

There exist two pathways for synthesizing TG in the enterocyte, namely the monoacylglycerol (MG) pathway and the glycerol-3-phosphate (G-3-P) pathway.

The MG pathway consists of:

1) Acylation of 2-MG into diacylglycerol (DG) by monoacylglycerol

acyltransferase (MGAT).

2) Acylation of DG into TG by diacylglycerol acyltransferase (DGAT (36)).

In contrast, the G-3-P pathway, which uses a different starting substrate, consists of:

1) Acylations of glycerol-3-phosphate into phosphatidic acid by

glycerophosphate acyltransferase.

2) Hydrolysis of phosphatidic acid into DG by phosphatidate

phosphohydrolase.

3) Acylation of DG into TG by DGAT.

The relative significance of these two pathways depends on the substrate abundance. Since there are more 2-MGs present during lipid-fed stage, the MG pathway becomes the predominant pathway under these conditions. A previous study further supports this notion by showing that the presence of 2-MG could inhibit the G-3-P pathway (165).

Since MG pathway is more critical in intestinal lipid absorption, the subsequent discussion will focus on the enzymes involved in the MG pathway. So far, there are three MGATs that have been identified, namely MGAT1, MGAT2, and

MGAT3. MGAT2 but not MGAT1 is expressed in the intestine of both mice and humans (245). The importance of MGAT2 in intestinal lipid absorption has been suggested based on its high expression in the intestine and its regulation by dietary fat (34).

As depicted above, DGAT enzyme is required for both pathways. Therefore, the absence of DGAT activity would suggest a reduction in lipid absorption or perhaps even lethality. However, studies using DGAT KO mice showed that although TG synthesis was reduced in many tissues examined, these mice appeared relatively healthy (182). This data strongly suggest that there are more than one DGAT enzymes. In fact, a second DGAT enzyme, DGAT2, has been identified (38). DGAT2 KO mice were lethal shortly after birth, suggesting that its function cannot be compensated and that it is a critical enzyme (185). However, the significance of DGAT2 in intestinal lipid absorption in humans is questionable since its expression was relatively low in the human intestine (37). Although

DGAT1 may be important in intestinal lipid absorption, its absence did not cause a significant reduction in intestinal TG absorption, as determined by the fecal analysis method (30).

Interestingly, in rats the re-synthesis of the lipid digestion products into TG by the intestine turns out to be very rapid—less than 2 minutes (129). However, the TG re-synthesis does not seem to be rapid in mice since there were only about 50% of the labeled FAs incorporated into TGs by the end of the 6 h infusion (see

Chapter 2 and 3). Interestingly, the lipid transport into the lymph is also more efficient in rats than mice (unpublished observation). It remains to be determined whether this difference in lymphatic lipid transport between rats and mice is due to differences in their esterification.

1.6.3. Cholesterol esterification

About 70-80% of the total cholesterol transported to the lymph is cholesteryl esters. As discussed earlier, enterocytes take up cholesterol but not cholesteryl esters (225), implying that cholesterol must be esterified prior to its transport to the lymph. The main enzymes that are thought to be responsible for esterifying cholesterol in the enterocytes are the cholesterol esterase and acyl coenzyme

A:cholesterol acyltransferases (ACATs). It is believed that the origin of the cholesterol esterase is the pancreas, and that the enzyme is taken up by the enterocytes (71). It was also argued that cholesterol esterase was more important than ACAT in esterifying cholesterol in the intestine (72; 76). As mentioned earlier, mice lacking cholesterol esterase absorbed cholesterol normally, but had a reduced cholesterol ester absorption (224). Later studies

35 suggested that ACAT may, indeed, be more critical for cholesterol absorption

(27).

There are 2 ACATs identified so far: ACAT1 and ACAT2. ACAT1 KO mice did not display any abnormal cholesterol absorption by the intestine, as determined by dual isotope fecal analysis (142). On the other hand, ACAT2 KO mice displayed a significant reduction in intestinal cholesterol absorption only when put on a high fat high cholesterol diet, as determined by using the similar method

(28; 167). These findings suggest that ACAT2 may be an important cholesterol esterifying enzyme in the intestine (29).

1.7. Formation and secretion of lipoproteins

1.7.1. Intestinal lipoproteins

Lipoproteins are lipid particles with hydrophobic core and hydrophilic coats. By packaging the lipids with hydrophilic coats (phospholipids and apolipoproteins), lipoproteins can greatly enhance the transport of lipids to the circulation. The major lipoproteins secreted by the intestine are chylomicrons and very low density lipoproteins (VLDLs). The distinction between the two is empirical.

Chylomicrons are defined as lipoproteins with Svedberg flotation (S f) rate of more than 400, and VLDL are defined as lipoproteins with Sf rate of 20-400. Intestine secretes mostly VLDL during fasting state. In contrast, chylomicrons are the

36 major lipoproteins secreted during lipid-fed state. In addition to these two lipoproteins, the intestine has also been reported to secrete high density lipoproteins (HDLs) (18).

1.7.2. Assembly of lipoproteins

Most of our understanding of how lipoproteins are assembled in the intestine is derived from studies of the liver. Accordingly, nascent Apo B that is partially translated is brought into the ER. The nascent Apo B then moves into the lumen of ER. The addition of lipids occurs mainly in the lumen of the smooth ER.

However, if there are not enough lipids available, Apo B will be degraded. This initial process of addition of lipids is regulated by microsomal triglyceride transfer protein (MTP), suggesting that MTP may play a more important role during the initial formation of (primordial) lipoproteins (83). As the translation of Apo B is completed, it will be released to the lumen of ER, forming a lipid-poor primordial lipoprotein. More lipids will be added to this lipid-poor lipoprotein, and the mature lipoprotein will be assembled and secreted from the Golgi.

Additional discussions on the intracellular assembly of lipoproteins are available in the literature (220; 237).

1.7.3. Separate pathways for VLDL and chylomicron assembly

Tso et al. proposed that VLDL and chylomicron are assembled using different pathways (199; 203). The supporting evidences for this thesis come from the studies that showed that the hydrophobic surfactant, Pluronic L-81, could inhibit chylomicron but not VLDL secretion (200; 201). The previous data showing that the uptake of certain FAs by the intestine resulted in a unique preference towards being transported as either VLDL or chylomicron also supports the proposal

(151). This concept is worthy of further investigation because chylomicrons are thought to be more efficient in transporting lipids than VLDL. In addition, chylomicron clearance is rapid, suggesting that the intestine may potentially regulate the metabolism of absorbed lipids via its regulation of VLDL and chylomicron secretion.

1.7.4. ER to Golgi transport

The importance of ER to Golgi transport has been highlighted recently (121).

However, the mechanism of how lipids are transported from the ER to Golgi in the intestine is unclear at the moment. It seems that the transport of nascent lipoproteins (PCTV, pre-chylomicron transport vesicles) from the ER to Golgi uses the common COPII machinery (180). The COPII machinery, a common machinery used for export of nascent proteins out from the ER, involves the assembly of several COPII proteins in a stepwise fashion. In humans, mutations

38 in Sar1 GTPase, a protein critical for the initial assembly of COPII proteins, have been associated with lipid absorption disorders (107), suggesting that COPII machinery is critical in the process of intestinal lipid absorption. It has also been proposed that the rate limiting step in the intestinal lipid absorption is at the ER to

Golgi transport (127). Subsequently, the lipoproteins from Golgi are targeted to the basolateral membrane for exocytosis.

1.8. Regulation of lymphatic vs. portal transport

Once the lipoproteins are secreted by the enterocytes, they have to pass the basement membrane to get to the lamina propria. Earlier studies suggested that the integrity of basement membrane was disrupted during lipid absorption, allowing for the lipoproteins to get to the lamina propria (198). The lamina propria is filled with lymph and blood capillaries. It is commonly accepted that lipoproteins and hydrophobic molecules enter the lymphatic capillaries but small and polar molecules enter the blood capillaries. However, several studies have provided evidence that lipids could also be transported via the portal route (31;

124; 126; 140). If such regulation exists, then it may be possible that the regulation of lymphatic vs. blood (portal) transport is determined early during the packaging of the ingested materials in the enterocytes (223). This proposal is worth pursuing, especially when considering that materials transported via lymphatic and portal route may have a different metabolic fate, as will be

39 discussed in Chapter 3. Although materials transported via lymphatic system will be delivered eventually into the blood circulation, they first enter the mesenteric lymph, then the thoracic duct, and finally the blood circulation via the subclavian vein. In contrast, materials transported portally enter directly to the liver, the key metabolic organ. So although both transport systems eventually enter the blood circulation, the metabolic fate may be significantly different.

1.9. Study Rationale

The review presented above suggests that there are several concepts regarding the intestinal lipid absorption which remain unclear. First, it is not clear as to what extent uptake of lipids by the intestine is mediated by the plasma membrane proteins and the passive process. We tested a well-known candidate molecule, CD36, for its involvement in lipid uptake by using a KO mouse model.

Chapter 2 presents the results and discussions of this study. Second, it is not clear how lipoprotein formation and secretion are regulated in the intestine. In order to gain a better understanding of this regulation, we utilized two models to study: the CD36 KO mouse model and the C57BL/6 male and female mouse model. In the CD36 KO mouse model, we sought to test the hypothesis that

CD36 may also be involved in chylomicron formation and secretion (Chapter 2).

The use of C57BL/6 mouse strain for studying the mechanism of chylomicron formation and secretion was suggested to us by Dr. Renee LeBoeuf (personal

40 communication) who found that the female C57BL/6 mice absorbed lipids less efficiently than their male counterpart. The sex differences in lipoprotein secretion has also been suggested by the previous studies in rats (211). Hence, we decided to study the lipoprotein formation and secretion in male and female

C57BL/6 mice. These studies are presented in Chapter 3. Chapter 4 discusses and summarizes the overall implications of our findings and some possible future experiments.

Chapter 2

CD36 is important for intestinal cholesterol uptake and for the formation

and secretion of chylomicrons

(Submitted to Gastroenterology for consideration for publication)

Abstract

Background & Aims : The goal of our studies is to determine the role of the scavenger receptor CD36 in intestinal lipid absorption. Methods : A knock out

(KO) mouse model equipped with lymph and duodenal cannulas was used.

Results: The CD36 KO, as compared to wild type (WT) mice, exhibited significant accumulation of infused cholesterol in the intestinal lumen and significantly reduced cholesterol transport into the lymph. Triacylglycerol (TG) in the lumen of

KO mice trended higher but the effect was not significant. However, there was marked TG accumulation in the intestinal mucosa and a significant reduction in their lymphatic transport. The ratio of TG to fatty acids in the mucosa of the KO mice was slightly higher than that of the WT, arguing against impaired lipid esterification but rather for a deficiency in the formation and secretion of chylomicrons that is responsible as the cause for the reduced lymphatic TG transport. This is further supported by a marked reduction of lymphatic apolipoprotein B-48, A-IV, and A-I and by the formation of smaller lipoprotein particles in the lymph of CD36 KO mice. Conclusions : Our data suggest that

CD36 plays an important role in: 1) mediating intestinal cholesterol uptake; and

2) the formation and secretion of chylomicrons. We propose that inactivation of intestinal CD36 may lead to reduced cholesterol absorption with potential benefits in the treatment of hypercholesterolemia and the development of atherosclerosis.

Introduction

The transmembrane protein CD36 is expressed in many cell types, including platelets (158), monocytes (189), capillary endothelial cells (111), erythroblasts

(60), adipocytes (3), and intestinal (41; 117; 164), mammary, and retinal epithelial cells (89). In the rat intestinal epithelial cells, CD36 is localized in the apical brush border membranes of mainly the duodenum and jejunum (42; 163).

Using human intestinal tissues, Lobo et al. (115) also localized the CD36 expression along the proximal brush border membranes of the intestinal epithelium.

CD36 binds to a wide range of ligands, such as native and modified lipoproteins

(33; 61), anionic phospholipids (169), cholesterol (229), and fatty acids (2).

Evidence from studies conducted both in vitro (100; 159) and in vivo (63) supports an important role for CD36 in facilitating fatty acid uptake by adipose and muscle tissues. Its role in intestinal lipid uptake is less clear. In support for such a role are the high expression levels of CD36 and its expression pattern along the brush border membrane of the proximal intestine, which is typical of proteins implicated in lipid uptake would support such a role (43; 162) .

A recent study by Drover et al. (58) showed that CD36 KO mice did not exhibit reduced intestinal fatty acid uptake but had impairments in lymph TG secretion and in clearance of blood chylomicrons. Whether or not CD36 plays a

44 significant role in intestinal cholesterol uptake and their transport in TG-rich lipoproteins in vivo remains unknown. A role in uptake has been suggested by the report that the CD36 protein bound cholesterol and that CD36 antibodies inhibited cholesterol uptake by brush border membranes (233).

The goal of this study is to further our understanding of the role of CD36 in intestinal uptake and transport of cholesterol and fatty acids using the well established conscious lymph fistula model. We explored further the effect of

CD36 deficiency on formation and secretion of chylomicrons. Finally, we reexamined using a Sucrose Polybehenate (SPB) method (102) if disruption of the

CD36 gene would lead to an overall reduction in intestinal fat absorption.

Materials and Methods

Materials. Triolein, cholesterol, egg phosphatidylcholine (PC), and sodium taurocholate were purchased from Sigma (St. Louis, MO). The radioactive [9,

10-3H (N)] triolein and [4-14 C] cholesterol were purchased from New England

Nuclear (Boston, MA). Silica gel 60 plates were purchased from Fisher Scientific

(Pittsburgh, PA).

Animals. CD36 WT and KO mice were generated as described previously

(57). Animals were maintained on regular chow diet under a 12 h light / 12 h dark cycle at University of Cincinnati Laboratory Animal Medical Services. We used 4-12 month-old male animals were used in our studies.

Sucrose Polybehenate (SPB) method. The SPB method was developed and validated by Jandacek et al. (104). It is a non-invasive method for studying overall lipid absorption. The method relies on analyzing intestinal fat absorption by determining the ratio of fecal fat vs. a fecal non-absorbable fat marker.

Lymph and duodenal cannulation. Intestinal lymph ducts of anesthesized

(ketamine, 80 mg/kg and xylazine, 20 mg/kg) mice were cannulated with PVC tubing (I.D., 0.20 mm; O.D., 0.50 mm) as described by Bollman, et al. (22) with the following modifications. Suture of the lymph cannula was replaced by application of cyanoacrylate glue (Krazy Glue, Itasca, IL); in addition, a PVC tube

(I.D., 0.5 mm; O.D., 0.8 mm) was inserted into the duodenum through a fundal

46 incision of the stomach and secured by a purse-string. Following the surgery, mice were infused with 5% glucose in saline (145 mM NaCl, 4 mM KCl and 0.28

M glucose) at a rate of 0.3 ml/h. The glucose/saline solution was replaced with the prepared lipid infusate the next morning.

Lipid infusate preparation. Triolein, [ 3H] triolein, cholesterol, [ 14 C] cholesterol and PC were combined dissolved in chloroform. The chloroform content was evaporated by using nitrogen gas. The chloroform-free lipid mixture was then suspended with 19 mM NaTC and sonicated. Lipid infusate was checked for homogeneity by sampling the bottom, the middle, and the top part of the emulsion. The counts usually agreed with one another within 2 %. Aliquots of the infusate were also taken at the beginning and at the end of the infusion period to check for stability of the infusate. The counts usually agreed with one another within 5%.

The lipid emulsion was infused for 6 hours. The hourly infusate contained

4 µmol triolein labeled with [ 3H] triolein, 0.78 µmol cholesterol labeled with [ 14 C] cholesterol, 0.78 µmol egg phosphatidylcholine (PC), and 5.7 µmol sodium taurocholate (NaTC) in phosphate-buffered saline (PBS) (0.958 g Na 2HPO 4,

2.277 g NaH 2PO 4, 6.8 g NaCl, and 0.2982 g KCl per 1 L H 2O) at pH=6.4.

Collection of lymph, luminal, and mucosal samples. Lymph samples were collected hourly. At the end of the 6-h infusion period, the animals were

47 anesthetized with the ketamine-xylazine mixture, and stomach, small intestine

(cut into 4 equal segments), colon were tied off separately, and the luminal content were collected. In some experiments the lipid was administered by gavage, ([ 3H] oleate and olive oil, 100 µl/mouse, to overnight fasted mice and the mice were sacrificed 45 min later). Tissue samples were homogenized using a

Polytron homogenizer, and radioactivity of each sample was measured. For later analyses of tissue lipid content, small intestinal segments were immediately extracted using the Folch method (68). Lymphatic triacylglycerol mass was determined using a triacylglycerol kit from Randox as previously described (149).

Thin-layer chromatography (TLC) analysis of mucosal lipids. Lipids extracted from the small intestinal segments were run on silica gel 60 plates using a solvent system of petroleum ether/ethyl ether/glacial acetic acid, 25:5:1 volume ratio. After visualizing the samples and the co-migrating reference standards with iodine vapor, samples were scraped and added with 1 ml of absolute alcohol before adding the scintillation liquid (Opti Fluor for aqueous samples) for counting of radioactivity.

Lipoprotein particle size analysis. Carbon-coated formvar film on a 400 mesh copper grid (Electron Microscopy Sciences) was floated on top of a drop of the lymph sample. The grid was dried with filter paper and briefly added to 2% phosphotungstic acid (pH 6.0). For lipid-infused lymph samples, 5h and 6h lymph samples were pooled and diluted with sterile water 1:4, vol:vol. Fasting

48 lymph samples were not diluted and were added on grids as described above.

Standard beads (200 nm) were used for calibration (Duke Scientific Corp).

Electron microscopic images were taken immediately by using JEOL JEM-1230.

The size of lipoprotein particles was measured by using Adobe Photoshop and software from Reindeer Graphics. An average of 800 particles was sized per lymph sample. A previous pilot study showed that the manual and the digital counting methods agreed closely (data not shown).

Chylomicron composition. To determine the lipid composition of chylomicron in the lymph, equal aliquots of lymph samples were layered under

0.15 M NaCl and subjected to centrifugation for 30 min at 50,000 rpm in MLA-

130 rotor in a table top ultracentrifuge (Beckman instruments). Chylomicrons were removed and lipid composition was determined as described using kits from

Wako chemicals.

Western blot analysis of apolipoproteins. One minute lymph output at fasting and after 4-h infusion from CD36 WT and KO mice were run on 4-20% gradient gel. Proteins were transferred to polyvinylidene difluoride membranes, reacted with primary antibodies against Apo B (1:7500 dilution), Apo A-IV

(1:5000), and Apo A-I (1:5000), and appropriate secondary antibodies. The immunocomplexes were detected using the ECL system (Amersham

Biosciences) according to the manufacturer’s instructions. Data were quantified using Scion Image software.

Statistical analysis. The data shown are means ± SE (standard errors).

To compare groups throughout the 6-h infusion, two-way repeated measures

ANOVA with Tukey as a post-test analysis was used. A t-test was used for the rest of the data analyses. Statistical analyses were performed using Sigmastat

(SPSS Inc.), and were considered significant if P < 0.05.

Results

Fat absorption in CD36 null mice

The SPB method allows the analysis of fat absorption in ad libitum fed mice by determining in fecal samples the content of excreted fatty acids relative to the content of a non-absorbable marker. Both CD36 WT (n=4) and KO (n=4) mice showed a 91% overall fatty acid absorption (Figure 2.1.), suggesting that ablation of CD36 is not sufficient to reduce fatty acid uptake. However, this method could not determine the rate and the site at which the fat is ingested and absorbed by the gut. To acquire that information, we used the conscious lymph fistula model to study intestinal fatty acid and cholesterol absorption.

Analysis of lipid transport by the small intestine

To further determine whether CD36 plays a role in lipid transport by the small intestine, we analyzed the lymphatic lipid output of both CD36 WT and KO mice.

CD36 KO mice had a marked reduction in lymphatic output of both TG (Figure

2.2. A) and cholesterol (Figure 2.2. C). The reduction in TG output (P < 0.001) was evident as early as the first hour of infusion (Figure 2.2. A), reaching only

25% of the hourly infused TG as compared to 50% in the WT mice. The total

TG mass (Figure 2.2. B) in the lymph of the CD36 KO mice was also lower (P =

0.003) than that of CD36 WT mice throughout the entire 6 h study. About 80%

51 of the TG mass was calculated to be derived from the infused TG in both the

CD36 WT and KO mice. CD36 KO mice also showed a reduction (P = 0.002) in cholesterol transport, reaching only 10% of the hourly infused as compared to the

30% for the WT mice. The decrease was evident starting at the third hour of infusion (Figure 2.2. C).

Analysis of the luminal and the mucosal lipid of the small intestine

Most of the absorption of TG and cholesterol occurred in the small intestine since only a small amount of the infused radioactive TG and cholesterol was recovered from the colon (Figures 2.3. A and B). There was also little if any reflux of the radioactive TG and cholesterol back to the stomach (Figure 2.3., stomach).

There was a trend for luminal TG to be higher in CD36 KO than in WT mice but the difference was not statistically significant (Figure 2.3. A, lumen). This observation would support the data obtained from the SPB studies (Figure 2.1.) that showed no major difference in fatty acid absorption between the CD36 WT and KO mice. Under the same conditions, lymphatic TG transport of CD36 KO mice was significantly decreased (P = 0.0069, Figure 2.3. A, lymph) and CD36

KO mice retained more of the infused TGs in their intestinal mucosa (Figure 2.3.

A, mucosa). In a separate study, we observed a similar distribution of radioactivity when the lipid was administered by gavage instead of duodenal infusion (data not shown). Thus the fate of the infused TG was similar between the CD36 KO and the wild type animals, irrespective of whether the lipid was

52 infused as an emulsion at a constant rate (in this study) or as a gavage (data not presented).

Significantly higher luminal cholesterol counts (P = 0.0073) were obtained from the null mice (Figure 2.3. B, lumen). However, under the same conditions where

CD36 KO mice exhibited defective cholesterol output into the lymph (P = 0.0216)

(Figure 2.3. B, lymph), there was no evidence for more cholesterol counts in the

CD36-deficient mucosa (Figure 2.3. B, mucosa).

Distribution of the infused lipid along the segments of the small intestine

The infused lipid was retained more in the proximal than in the distal segments of the intestinal mucosa (M1 being the most proximal and M4 being the most distal)

(Figure 2.4, A and B). However, while more TG was retained in all segments of the small intestine of CD36 null mice (Figure 2.4. A), slightly more cholesterol was retained only in M2 and M3 relative to the WT mice (Figure 2.4. B). The low recovery of both [ 3H] and [ 14 C] in the M4 segments further imply that the lipid infused was not excreted.

Lipid distribution of the labeled fatty acids in intestinal segments

Figure 2.5. shows the distribution of the infused triolein in various enterocyte lipid fractions; cholesteryl esters (CE), triacylglycerols (TG), fatty acids (FA),

53 diacylglycerols (DG), and monoacylglycerols+phospholipids (MG+PL). In the proximal half of the mucosa of the CD36 KO mice, most of the [ 3H]-labeled lipids were in the form of TG (Figure 2.5. A and B) as compared to MG+PL in the WT mucosa; these differences were significant only in M2 (P = 0.0345 for CE and P

= 0.0019 for MG+PL). This trend was partially reversed in distal segments (M3 and M4) where CD36-deficient mucosa exhibited less accumulation of TG and more in CE.

Figure 2.6. shows the distribution of [ 14 C]-labeled cholesterol. About 80-90% of the [ 14 C]-label was recovered as free cholesterol in both CD36 WT and KO mice.

No significant differences in distribution were observed between the two genotypes, although CE trended higher in distal segments of CD36 KO mice.

Lipoprotein particle size analysis

Figure 2.7.A shows the size distribution of the lymph lipoprotein particles from the fasted CD36 WT and KO mice. Similar to CD36 WT animals, CD36 KO mice had most of the lipoproteins in the VLDL size range during fasting (Figure 2.7. B).

Figures 2.7. C and D show the fasting lipoprotein particles of CD36 WT and KO mice as analyzed by negative staining, respectively. Figure 2.8.A shows the size distribution of the lipoprotein particles during the lipid-fed stage of both the CD36

WT and KO mice. During the lipid-fed stage, the CD36 KO mice secreted appreciably less chylomicron relative to VLDL as compared to that of the CD36

WT mice (Figure 2.8. B). The lipid-fed lipoprotein particles of the CD36 WT and

KO mice are shown in Figures 2.8. C and D, respectively. The average size of the lipoprotein particles of the CD36 KO mice (841.5 ± 76.1 Å) was significantly lower (P = 0.0325) than that of the CD36 WT mice (1284.0 ± 126.1 Å) during the lipid-fed stage.

Chylomicron composition

Although less chylomicrons were secreted in the lymph of CD36 KO mice, their lipid composition was similar to that of chylomicrons from WT mice (Figure 2.9.).

The chylomicrons secreted contain mostly TG (about 88%) with small amounts of phospholipids (about 8%) and cholesterol (about 4%).

Apolipoprotein secretion by the small intestine

The CD36 KO mice secreted slightly less apolipoprotein B-48 (Figure 2.10. A), A-

IV (Figure 2.10. B), and A-I (Figure 2.10. C) than the CD36 WT mice during the fasting stage. During the lipid-fed stage, apolipoprotein B-48 (Figure 2.10. D) and A-I (Figure 2.10. F) were also secreted slightly less by the CD36 KO relative to those of the CD36 WT. The apolipoprotein A-IV secretion was comparable between both WT and KO in the lipid-fed stage (Figure 2.10. E).

Discussion

The scavenger receptor CD36 is expressed in the brush border membranes of the proximal small intestine of both rodents (40; 161) and humans (118). CD36 binds to a wide variety of ligands, including lipoproteins (32; 62), anionic phospholipids (168), and long-chain fatty acids (4). Its high expression levels and defined localization in the small intestine together with its high-affinity for lipids would suggest a role for CD36 in lipid absorption. In this study, using the

CD36 deficient mouse model equipped with lymph and intraduodenal cannulas, we directly examined the role of CD36 in uptake and lymphatic transport of both

TGs and cholesterol.

Previous work by Goudriaan et al. (86) and by Drover et al. (56) suggested that

CD36 does not function in the uptake of dietary fatty acids. We reexamined this using the SPB method, which measures the percentage of the un-absorbed

(fecal) fatty acids to the non-absorbable lipid marker. No deficiency in absorption could be detected in CD36 deficient mice. This is also supported by the similar recovery of [ 3H]-labeled fatty acids from the intestinal lumen at the end of the 6-h infusion.

Despite the fact that CD36 has been well characterized as mediating the uptake of free fatty acids in adipose tissue and muscle, in the intestine its deletion does not appear to cause an overall reduction in steady state fatty acid uptake. Our findings are consistent with those by Drover et al. (59). As a result of the high

56 fatty acid concentrations that occur in the intestine during a lipid meal, it is possible that passive transport of fatty acids may mediate most of the uptake in this tissue(208).

Although the uptake of fatty acids by the small intestine is not dependent on

CD36, the formation and secretion of chylomicrons appear to be, as supported by two lines of evidence. First, lymphatic TG transport was significantly reduced in the CD36 KO mice for both the dietary TG (infused) and the endogenous TG

(originating from the fatty acids derived from biliary phospholipids). Second, the recovery of [ 3H] TG counts in the intestinal mucosa was considerably higher in the CD36 KO than in the WT mice, thus indicating that the fatty acids were not incorporated into chylomicrons efficiently. The function of CD36 in chylomicron formation will be examined in future studies using subcellular fractionation and pulse chase experiments.

The CD36 KO mice as compared to the WT mice had higher percentages of [ 3H]- labeled lipids in the mucosal of the proximal intestine (segments M1 and M2).

This is in line with normal CD36 expression being high in proximal segments and with the concept that proximal as opposed to distal segments play the major role in fatty acid absorption and secretion (24). This interpretation is further supported by the relatively lower [ 3H] counts recovered in the distal segments

(M3 and M4). Noteworthy, fatty acid esterification into TG does not appear to be impaired in CD36 deficiency. In addition to the higher percentage of [ 3H]-labeled

TG, there were also higher [ 3H] counts. These data suggest that the reduced lymphatic TG transport reflected a defect at a step downstream of fatty acid esterification, such as packaging of the synthesized TG into chylomicrons and secretion into the lymph.

Analysis of several major apolipoproteins coating the chylomicrons, namely Apo

B-48, A-IV, and A-I also shows that CD36 KO mice secreted appreciably less chylomicron apolipoproteins than WT mice. This together with the fact that chemical composition of the secreted chylomicrons was similar for both genotypes would support the interpretation of a reduction in production rate as opposed to a defect in a specific step of chylomicron pathway. Although chylomicrons are empirically defined as TG-rich lipoprotein particles of size 800 nm in diameter or larger in contrast to less than 800 nm in diameter for VLDL,

Tso et al. (204) proposed that secretion of both particles involved two different pathways. The data in CD36 KO mice would support this concept.

However, there was a discrepancy between our studies and those of Goudriaan et al. (85). Using an inhibitor of intravascular lipolysis, Triton WR1339, they showed that CD36 KO and WT mice were comparable in the plasma recovery of infused TG and fatty acids. We cannot explain the discrepancy in the findings of their study with ours, but it should be noted that their plasma TG recovery was only about 15% of the total infused by the end of 4 hours as compared to our lymphatic recoveries of about 44% of the total infused for WT and 18% for the

KO animals. The lymph fistula model provides us with a direct measurement of the secretion of chylomicrons and VLDL by the small intestine; and this measurement is not complicated by factors such as stomach emptying. In contrast, in the WR1339 study, stomach emptying could be potentially a complicating factor. In addition, we do not know if WR1339 affects the formation and secretion of intestinal chylomicrons and VLDL, which could potentially explain the low plasma recovery in the study reported by Goudriaan et al. (84).

Lastly, the dose they infused was significantly high. Mice eat about 4 grams of chow a day. Assuming the chow is 5% fat, mice consumed about 200 mg or about 200 µl of oil in a day. In their studies, however, mice were challenged with a single bolus of 200 µl of oil.

In contrast to the uptake of fatty acids, the uptake of cholesterol was markedly reduced in the CD36 knockout animals since there was a significant accumulation of the infused cholesterol in the lumen. In addition, there was a significant reduction in lymphatic cholesterol transport which was not correlated with the accumulation of mucosal [ 14 C] cholesterol counts. Finally, the ratio of

[14 C] cholesterol to [ 14 C] cholesterol esters was similar for the KO and WT mice, implying that there was no defect in the esterification of infused cholesterol.

These data would support the interpretation that uptake of cholesterol from the lumen into the enterocyte is impaired in CD36 KO mice. However, it should be noted that infused fatty acids were less readily esterified with cholesterol in these mice. Thus the possibility that the defect in cholesterol uptake may be

59 consequent to a change in FA utilization should be considered. Another possibility is that in the absence of CD36 cholesterol and fatty acid may not be efficiently targeted to the same intracellular compartment.

Our findings that CD36 mediates cholesterol uptake were in agreement with

Werder’s studies showing that CD36 mediates the uptake of free cholesterol in the isolated human intestinal brush border membranes (231). Another plasma membrane protein that may mediate cholesterol uptake by the gut is the

Niemann-Pick C1 Like 1 (NPC1L1) protein (10). Genetic ablation of NPC1L1 gene showed that about 9% of the infused cholesterol remained in the lumen, a percentage comparable to that observed in our studies (about 11%). The exact contribution of each of these molecules in cholesterol uptake remains unclear.

Furthermore, it is not known if disruption of both genes would result in an even more marked reduction in cholesterol uptake by the gut.

Acknowledgements

This work was supported by the National Institute of Health (grants DK-56910, and DK-56863 to P Tso and DK60022 and DK33301 to N Abumrad), the pre- doctoral fellowship award from the American Heart Association, Ohio Valley

Affiliate and the National Health Research Institute scholarship for cardiovascular diseases (A. Nauli). We thank the University of Cincinnati Mouse Metabolic

Phenotype Center DK-59630 for providing many of the phenotypic tests relevant to the study.

Figure Legends

Figure 2.1. Analysis of fatty acid uptake by the small intestines of CD36 null and wild type mice . A minimum of 9 fecal samples from each group were analyzed by using the SPB method that determines the ratio of fecal fat vs. a fecal non-absorbable fat marker. Values are means ± SE.

Figure 2.2. Lymph [ 3H]-TG transport (A), TG mass (B), and [ 14 C]-cholesterol transport (C) during continuous intraduodenal lipid infusion. Mice were equipped with lymph and duodenal cannulae, and were intraduodenally infused with a lipid emulsion containing labeled triolein and cholesterol for a period of 6 hours. Lymph was collected hourly and analyzed. Values are means ± SE.

Figure 2.3. Total recovery of the infused TG (A) and cholesterol (B) in the stomach, colon, intestinal lumen, intestinal mucosa, and lymph at the end of the 6-h infusion period. Mice were equipped with lymph and duodenal cannulae, and were intraduodenally infused with a lipid emulsion containing labeled triolein and cholesterol for a period of 6 hours. The recovery of labeled lipids in intestinal mucosa, lymph, stomach, lumen, and colon were determined at the end of the 6 h by scintillation counter. Values are means ± SE.

Figure 2.4. Distribution of the infused TG (A) and cholesterol (B) along 4 equal-length segments of the small intestine . At the end of the 6hr infusion period, the intestines were harvested and divided into 4 equal length segments.

The recoveries of labeled lipids in these segments were determined by scintillation counter. From proximal to distal: M1, M2, M3, M4 (n=5). Values are means ± SE.

Figure 2.5. Mucosal distribution of [ 3H]-fatty acids from infused triolein into the major lipid classes . Mucosa was divided into 4 equal segments, from proximal to distal: M1 (A), M2 (B), M3 (C), and M4 (D). Mucosal lipids were extracted and separated by TLC into CE, TG, FA, DG, and MG+PL. CE, cholesteryl esters; TG, TGs; FA, fatty acids; DG, diacylglycerols; MG+PL, monoacylglycerols and phospholipids. Values are means ± SE.

Figure 2.6. Mucosal distribution of [ 14 C]-cholesterol from infusate into the major lipid classes . Mucosa was divided into 4 equal segments, from proximal to distal: M1 (A), M2 (B), M3 (C), and M4 (D). Mucosal lipids were extracted and separated by TLC into cholesterol and cholesterol esters. Values are means ±

SE.

Figure 2.7. Lipoprotein particle size in the lymph of fasted mice .

Distribution of particle size (A), relative VLDL/chylomicron ratio (B), and representative pictures of the fasting lipoprotein particles from CD36 WT (C) and

KO (D) mice are shown. Particles < 800 Å are considered VLDL, and 800 Å or more are considered chylomicrons. Standard bars represent 5000 Å (500 nm).

Values are means ± SE.

Figure 2.8. Lipoprotein particle size of lymph from lipid infused mice .

Distribution of particle size (A), relative VLDL/chylomicron ratio (B), and representative pictures of the lipoprotein particles of CD36 WT (C) and KO (D) mice during the lipid-fed stage are shown. Particles < 800 Å are VLDL, and 800

Å or are chylomicrons. Standard bars are 5000 Å (500 nm). Values are means ±

SE.

Figure 2.9. Lipid composition of chylomicrons from lipid infused mice .

Each chylomicron lipid, TG (white), total cholesterol (grey) and phospholipids

(black) is expressed as percentage of the total content.

Figure 2.10. Apolipoprotein secretion into the lymph by fasted or lipid infused mice . Apolipoprotein B-48 (A), A-IV (B), and A-I (C) secretions during fasting stage, and apolipoprotein B-48 (D), A-IV (E), and A-I (F) secretions during lipid-fed stages were quantified by using Scion Image software. Equal amount of samples relative to their lymph output were loaded. OD = optical density. Values are means ± SE.

Figure 2.1.

100

25 % fatty uptake acid

0 CD36 WT CD36 KO

Figure 2.2.

A) Lymph [3H]-TG transport

75 CD36 WT (n=7) CD36 KO (n=7)

* * 25 * * * % of hourly infused % of hourly * 0 0 1 2 3 4 5 6 Time (h)

B) Lymph TG (mass) transport

CD36WT (n=4) 3 CD36KO (n=7)

2 * (mg/h) * * * 1 *

Lymph triacylglycerol Lymph * * 0 0 1 2 3 4 5 6 Time (h)

C) Lymph [ 14 C]-cholesterol transport

50 CD36 WT (n=7) 40 CD36 KO (n=7)

20 * * 10 * % of hourly infused % of hourly *

0 0 1 2 3 4 5 6 Time (h)

Figure 2.3.

The recovery of [3H]-labeled FA A) 60

CD36 WT (n=5) CD36 KO (n=5) 40

20 * % % of total infused

0 Mucosa Lymph Stomach Lumen Colon

B) The recovery of [14 C]-labeled cholesterol 60 CD36 WT (n=5) CD36 KO (n=5) 40

20 % of total infused * * 0 Mucosa Lymph Stomach Lumen Colon

Figure 2.4.

20 CD36 WT CD36 KO

10 H] Count 3 % [

0 M1 M2 M3 M4 Mucosal Segment

30 CD36 WT CD36 KO 20 C] Count 14 10 % [

0 M1 M2 M3 M4 Mucosal Segment

Figure 2.5.

A) M1 75 CD36 WT (n=3) CD36 KO (n=3) 50 H] Count 3 25 % [

0 CE TG FA DG MG+PL

CD36 WT (n=3) B) M2 50 CD36 KO (n=3)

30 H] Count 3 20 * % [ 10 * 0 CE TG FA DG MG+PL

C) M3 40 CD36 WT (n=3) CD36 KO (n=3) 30

20 H] Count 3

% [ 10

0 CE TG FA DG MG+PL

40 D) M4 CD36 WT (n=3) CD36 KO (n=3) 30

20 H] Count 3

% [ 10

0 CE TG FA DG MG+PL

Figure 2.6.

A) M1 100 CD36 WT (n=3) CD36 KO (n=3) 75

C] Count 50 14

% [ 25

0 Cholesterol Cholesterol Ester

B) M2 100 CD36 WT (n=3) CD36 KO (n=3) 75

C] Count 50 14

% [ 25

0 Cholesterol Cholesterol Ester

C) M3 100 CD36 WT (n=3) CD36 KO (n=3) 75

C] Count 50 14

% [ 25

0 Cholesterol Cholesterol Ester

D) M4 100 CD36 WT (n=3) 75 CD36 KO (n=3)

C] Count 50 14

% [ 25

0 Cholesterol Cholesterol Ester

Figure 2.7. A) 50

40 CD36 WT (n=3)

30 CD36 KO (n=4)

20 % % Particles

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 Diameter (Å)

B) 100

CD36WT 75 CD36KO

50 % Particles 25

0 VLDL Chylomicron

Figure 2.8. A) 40

30 CD36 WT (n=2) CD36 KO (n=4) 20 % Particles 10

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 Diameter (Å)

100 CD36WT CD36KO 75

% % Particles 25

0 VLDL Chylomicron

Figure 2.9.

Figure 2.10.

A) B -48 (fast)

100

SE) ± ± ± ± 50

OD OD (Mean 25

0 WT (n=4) KO (n=4)

B) A-IV (fast)

40 SE) ± ± ± ± 30 20

OD OD (Mean 10

0 WT (n=4) KO (n=4)

C) A-I (fast)

40 SE) ± ± ± ± 30 20

OD (Mean 10

0 WT (n=4) KO (n=4)

D) B-48 (lipid-fed)

SE) ± ± ± ± 50

25 OD (Mean

0 WT (n=4) KO (n=4)

E) A-IV (lipid-fed)

40 SE) ± ± ± ± 30

OD OD (Mean 10

0 WT (n=4) KO (n=4)

F) A-I (lipid-fed)

30 SE) ± ± ± ±

OD OD (Mean 10

0 WT (n=4) KO (n=4)

Chapter 3

Sex differences in intestinal lipid absorption in mice

Abstract:

Intestinal lipid absorption is an essential process that governs how dietary lipid is digested and absorbed by the intestine and transported to the circulation.

Although the basic process of intestinal lipid absorption is understood, many critical aspects remain unclear. One question in particular is whether or not intestinal lipid absorption differs between sexes. In order to address potential sex differences in intestinal lipid absorption, we used the lymph fistula model to monitor uptake, re-esterification, and transport of lipids by the small intestine before they are metabolized by the peripheral tissues. Using this model, we showed that female mice, unlike male mice, segregated into either the high or the low lymphatic transport groups. The high group had similar lymphatic triacylglycerol transport to the males, and the low group had significantly less.

These differences are not due to the differences in lipid uptake or re-esterification by enterocytes, but rather may be due to the regulation of lipid transport toward lymphatic vs. portal circulation. We then tested if estrous stages regulate this lipid transport, and found that it was not. We also found that the female mice— both the high and the low transport groups—secreted more VLDL, and the male mice secreted more chylomicrons. Collectively, our data suggest the existence of sex differences in intestinal lipoprotein secretion and transport that may result in different metabolism of lipids.

Introduction

Lipids are an essential part of our diet. The main dietary lipids consist of triacylglycerols, cholesterols, and phospholipids. The absorption of dietary lipids is important for the acquisition of energy and lipid-soluble vitamins.

Dietary lipid absorption is undoubtedly a complex physiological process. It is a dynamic process that consists of digestion, uptake, re-esterification, packaging, and transport (155). The main enzyme responsible for the digestion of triacylglycerols is pancreatic lipase. Pancreatic lipase hydrolyzes triacylglycerol into 2-monoacylglycerol and two molecules of fatty acids (131; 132; 134). In the presence of bile, these lipid digestion products form mixed micelles that allow them to pass through the unstirred water layer of the small intestine (236).

These micelles consist of bile salts, cholesterol, phospholipids, and lipid digestion products.

As micelles pass through the small intestine, their lipid digestion products are taken up by enterocytes. Whether the uptake step is an active or passive process remains controversial. However, both processes may take place depending on the physiological stage (207). During fasting or a substantially low fat diet, the active process may predominate. In contrast, during normal to high lipid load, the passive process may instead be more predominant.

The lipid digestion products that are taken up by enterocytes are re-esterified into triacylglycerols by an ATP-dependent process. They are then packaged mainly as chylomicrons and transported via the lymphatic route. Although the lymphatic route is the major avenue for lipid transport, portal route has also been implicated in the transport of absorbed lipid (123; 125; 139).

Like triacylglycerol absorption, dietary cholesterol absorption also involves several steps. Dietary cholesterol enters the intestinal lumen mainly as free cholesterol. It is also taken up by enterocytes and undergoes esterification. The two enzymes responsible for cholesterol esterification are acyl-CoA cholesterol acyltransferase (ACAT) and cholesterol esterase (25; 48; 75). Once esterified, the cholesteryl esters will be packaged together with triacylglycerols into chylomicrons and transported into the lymphatic circulation. There is evidence that cholesterol esterification regulates cholesterol absorption (74).

Most of our understanding of lipid absorption has been derived from studies using male animal models. In fact, our understanding of lipid absorption in females is very limited. There have only been a few studies that directly compare the sex differences in lipid absorption. Vahouny et al. (216; 217) reported that female rats had a higher intestinal very low density lipoprotein

(VLDL) protein than male rats, and suggested that VLDL played a significant role in cholesterol transport in female rats.

Our present studies showed that the female mice could be segregated into two groups based on their lymphatic lipid absorption; one group had lipid absorption comparable to that of the males, and the other group had significantly less.

Furthermore, we showed that this segregation was not due to lipid uptake or esterification by enterocytes, but potentially lipid transport regulation. Finally, we tested whether this segregation observed in the female mice was due to the stage of their estrous cycle or not. Our data did not support that estrous cycle regulates this segregation of lipid transport. We also found that irrespective of whether the female mice were in the high or the low output group, they had a tendency to secrete more VLDL than the male mice.

Materials and Methods

Materials. Triolein, cholesterol, egg phosphatidylcholine (PC), and sodium taurocholate were purchased from Sigma (St. Louis, MO). The radioactive [9,

10-3H (N)] triolein and [4-14 C] cholesterol were obtained from New England

Nuclear (Boston, MA). Silica gel 60 plates were purchased from Fisher Scientific

(Pittsburgh, PA).

Animals. Male and female C57BL/6 mice aged 8-10 weeks were maintained at 12 h light / 12 h dark cycle with regular chow diet at University of

Cincinnati Laboratory Animal Medical Services. To initiate the cannulation procedure, an anesthetic mixture of ketamine (80 mg/kg) and xylazine (20 mg/kg) was injected intraperitoneally into the mice. Intestinal lymph ducts of mice were cannulated with PVC tubing (I.D., 0.20 mm; O.D., 0.50 mm) according to techniques described by Bollman, et al. (22) Some modifications to the methods included using cyanoacrylate glue (Krazy Glue, Itasca, IL) instead of suture to secure the lymph cannula; additional insertion of a PVC tube (I.D., 0.5 mm; O.D.,

0.8 mm) into the duodenum through a fundal incision of the stomach was secured by a purse-string. After surgery, mice were infused overnight with 5% glucose in saline (145 mM NaCl, 4 mM KCl and 0.28 M glucose) at a rate of 0.3 ml/h. The next morning the glucose/saline solution was replaced with the prepared lipid infusate. A total of 15 male and 32 female animals were studied.

We had to use more female animals for the purpose of verifying their bimodal

82 distribution (See Result). For estrous cycle determination, vaginal smear was collected at the end of the 6h infusion (see below) and determined under the light microscope. All procedures were approved by the University of Cincinnati

Institutional Animal Care and Use Committee (IACUC).

Lipid infusate preparation. Four µmol triolein with a trace amount of [ 3H] triolein, 0.78 µmol cholesterol with a trace amount of [ 14 C] cholesterol, 0.78 µmol egg phosphatidylcholine (PC), and 5.7 µmol sodium taurocholate (NaTC) in phosphate-buffered saline (PBS) (0.958 g Na 2HPO 4, 2.277 g NaH 2PO 4, 6.8 g

NaCl, and 0.2982 g KCl per 1 L H 2O) at pH=6.4 were the amounts infused every hour. To prepare the emulsion, appropriate amounts of triolein, cholesterol

(dissolved in chloroform), PC (dissolved in chloroform), [ 3H] triolein, and [ 14 C] cholesterol were pooled and evaporated under a gentle stream of nitrogen gas.

The mixture was then suspended with the corresponding volume of 19 mM NaTC and sonicated to form a homogenous lipid emulsion.

Collection of lymph, luminal, and mucosal samples . The fasting lymph was collected for one hour prior to the lipid infusion. During the 6-h lipid infusion, hourly lymph was collected. At the end of the 6-h infusion, the animals were anesthetized with ketamine-xylazine mixture. Stomach, small intestine, and colon were removed. The lumen of the small intestine was washed with 0.5 % taurodeoxycholic acid. The pre-washed small intestine was divided into four equal lengths. The four segments of the small intestine, the stomach, and the

83 colon were homogenized individually in taurodeoxycholic acid by using Polytron homogenizer. An aliquot of each sample was added to Opti-Fluor (Packard

Bioscience, Meriden, CT) and counted by liquid scintillation spectrometer (Model

TR1900 tri-carb; Packard). The total count for each sample was calculated from the total volume of the sample and the aliquot count obtained from the scintillation counter. The disintegration per minute (dpm) was converted to percent recovery by multiplying the ratio of the dpm of the sample to the dpm of the infused lipid by 100. The remaining small intestinal segments were extracted immediately according to the method described by Folch et al. (67)

Thin-layer chromatography (TLC) analysis of mucosal lipids. Lipids extracted from the segments of the small intestine were analyzed by TLC. In brief, TLC was carried out by running the samples that were dissolved in chloroform on silica gel 60 plates using a solvent system of petroleum ether/ethyl ether/glacial acetic acid with 25:5:1 volume ratio. After visualizing the plates with iodine vapor, each separated sample was scraped using co-migrating standards as reference. The scraped lipids were added to 1 ml of absolute alcohol, and radioactivity was counted as described above.

Analysis of lipoprotein particle size by electron microscopy. For fasting lymph samples, carbon-coated formvar film on a 400 mesh copper grid (Electron

Microscopy Sciences) was added with 20 µl of fasting lymph samples. After 1 min, the grid was dried gently with filter paper, and added with 2%

84 phosphotungstic acid (pH 6.0) was added. The phosphotungstic acid was also dried gently after 1 min of incubation time. For lipid-infused lymph samples, 5h and 6h lymph samples were pooled and then diluted with sterile water to 1:4.

Twenty µl of the diluted pooled samples were used as described above.

Additionally, 200 nm standard beads were used for calibration (Duke Scientific

Corp). Pictures were taken immediately by using transmission electron microscopy (JEOL JEM-1230). The lipoprotein particle size was analyzed by using Adobe Photoshop version 6.0 with an added plugin (IPTK) purchased from

Reindeer Graphics, Inc. A minimum of about 300 particles were counted per sample. Our pilot study has shown that the manual and the digital counting methods agreed closely (data not shown).

Statistical analysis. All values are expressed as means ± SE. When comparing all groups throughout the 6-h infusion (hourly data), two-way repeated measures ANOVA with Tukey as a post-test analysis was used. For comparison of total recovery of three groups, One-way ANOVA with Tukey was performed as a post-test analysis. A t-test was used for comparing two groups. The statistical analyses were performed by using Sigmastats version 2.03 (SPSS Inc.), and were considered significant if the P values were <0.05.

RESULTS

Histogram of the total lymphatic triacylglycerol output in the male and the female mice

The histograms of the lipid absorption of the male (Figure 3.1. A) and the female mice (Figure 1B) were derived from the distribution of their total lymphatic [3H]- triacylglycerol outputs. The total lymphatic [3H]-triacylglycerol outputs were obtained by summing the total [3H] counts recovered in the lymph during the six hour of infusion. Figure 3.1. A shows that the male mice had a range of total lymphatic [3H]-triacylglycerol output of 21-70% with a bell shape distribution.

However, the female mice had a distinct bimodal distribution with 12 mice falling in the range of 0-30%, and 20 mice falling in the range of 31-70%. Due to this distribution, the female mice were grouped into either high output group ( High,

31-70% of the total lymphatic triacylglycerol recovery) or low output group ( Low ,

0-30% of the total lymphatic triacylglycerol recovery) in our subsequent figures and analysis.

Lymph flow during continuous intraduodenal lipid infusion

Figure 3.2. shows that the male group had steady lymph flow rate of 0.25 ml/h.

The high-output female group had a lymph flow rate that did not differ from that of the males. The low-output female group, however, showed a decrease in the

86 lymph flow rate at the second hour of the infusion (from 0.24 ml/h to 0.14 ml/h) and remained at ~0.14 ml/h throughout the end of the infusion period. The lymph flow rate of the low-output female group was significantly different ( P < 0.001) from both the male (starting at 2h) and the high-output female groups (starting at

1h).

Hourly lymphatic triacylglycerol output

Figure 3.3.A displays hourly lymphatic [3H]-triacylglycerol recovery in the male, the low-, and the high-output female groups. As shown, the hourly lymphatic triacylglycerol output of the high-output group superimposed that of the male group, reaching a maximum of about 55% recovery of hourly infused [ 3H] at the third hour of the infusion. As expected, the low-output female group had a lower hourly lymphatic triacylglycerol recovery, reaching a maximum of only 23% recovery at the third hour. The differences in hourly lymphatic triacylglycerol output were significant ( P < 0.001) when comparing the low-output female group with both the high-output female and the male groups (from the first hour onwards). Figure 3.3.B shows hourly lymphatic [ 3H]-triacylglycerol recovery in proestrous, estrous, and diestrous mice. There was no statistical significant among them. The steady state recovery was about 35% in each group, suggesting that the low- and the the high-output females were distributed quite equally into proestrous, estrous, and diestrous.

Hourly lymphatic cholesterol output

As shown in Figure 3.4.A, the difference in hourly lymphatic [14 C]-cholesterol output between the high and the low-output female groups was more pronounced than the difference in the hourly lymphatic [3H]-triacylglycerol output (Figure

3.3.A). The output curve of the males was between those of the high- and the low-output female groups. All three groups were significantly different from one another ( P < 0.001) starting at 2h infusion. Figure 3.4.B shows that hourly lymphatic [ 14 C]-cholesterol output was not significantly different among the proestrous, estrous, and diestrous groups, and that they were all comparable to that of the males.

Total triacylglycerol recovery

Figure 3.5.A shows the percent recovery of the total [3H]-triacylglycerol in the stomach, lumen, colon, and mucosa (small intestine) at the end of the 6-h infusion period. It also includes the percent recovery of the [3H]-triacylglycerols in the lymph and those that could not be recovered at the end of the experiment

(“Others”). The percent recoveries of the [3H]-triacylglycerols in the stomach, lumen, colon, and mucosa were ~1.1%, 2.0%, 0.4%, and 13.0%, respectively, and do not show any significant difference among the three groups studied.

However, the total [3H]-lymphatic triacylglycerol recovery in the low-output female group (19.1%) was significantly lower than those in the male (46.4%, P < 0.001)

88 and the high-output female groups (51.2%, P < 0.001). The percent of the[3H]- triacylglycerols that could not be recovered in the low-output female group

(63.3%) was higher than those in the male (39.8%, P < 0.001) and the high- output female groups (31.8%, P < 0.001). In contrast, figure 3.5.B shows that the proestrous, estrous, and diestrous mice were comparable in their recoveries in lumen, stomach, colon, mucosa, and lymph. The total lymphatic [ 3H]- triacylglycerol transport was about 35% for proestrous, estrous, and diestrous mice.

Total cholesterol recovery

Figure 3.6.A shows the percent of recovery of [14 C]-cholesterol in the stomach, the lumen, colon, mucosa (small intestine), and lymph at the end of the 6h infusion period. The percent of total [14 C]-cholesterol recovery in the stomach, lumen, and colon was ~1.3%, 6.0%, and 1.2%, respectively. There was no significant difference among the three groups studied. When the hourly lymphatic cholesterol output was expressed as a cumulative recovery of the whole 6-h infusion, a similar trend as depicted in Figure 3.4. was evident. All three groups were significantly different ( P < 0.0001) from one another in their total lymphatic [14 C]-cholesterol recovery ( P < 0.01 when comparing the male group [19.7%] to the high-output female group [28.9%]; P < 0.001 when comparing the low-output female group [10.0%] with the other two groups). The total [14 C]-cholesterol recoveries in the mucosa of both female groups were

89 significantly lower (P = 0.001) than those in the male group ( P < 0.05 when comparing the male [43.5%] to the high-output female groups [31.5%]; P < 0.001 when comparing the male to the low-output female groups [26.3%]). The percent of the [14 C]-cholesterol that could not be recovered (unaccounted cholesterols) was not significantly different between the male and the high-output female groups (“Others”). The unaccounted cholesterol in low-output female group was

54.1% of the dose, significantly higher than those of the male [28.7%] ( P < 0.001) and the high-output female group [28.9%] ( P < 0.001).

Figure 3.6.B did not show any significant different among proestrous, estrous, diestrous mice in their [ 14 C] recoveries in lumen, stomach, colon, mucosa, and lymph. Their total lymphatic [ 14 C]-cholesterol transport was about 20%, comparable to that of the males.

Thin-layer chromatography analysis of mucosal lipids

Lipids extracted from intestinal mucosa were separated according to their classes by TLC. Figure 3.7. shows the distribution of [ 3H]-labeled lipids into cholesteryl esters, triacylglycerols, fatty acids, diacylglycerols, and monoacylglycerols+phospholipids. As shown, about 50% of [ 3H]-labeled lipids in the proximal half of the intestinal mucosa was in the form of triacylglycerols

(Figure 3.7. A and 7B). The [ 3H]-labeled mucosal lipids in the distal half, however, distributed more equally into triacylglycerols, fatty acids, diacylglycerols,

90 and monoacylglycerols+phospholipids (Figure 3.7. C and D). It is important to point out that the proximal half of the intestinal mucosa plays a more critical role in lipid absorption since it represented more than 80% of the total radiolabeled lipids in the mucosa (data not shown). That is, the proximal region of the small intestine, at least in our model, was the region where most triacylglycerol and cholesterol absorption occurred. There is no difference among the three groups in either one of their mucosal segments.

Figure 3.8. shows the distribution of [ 14 C]-labeled lipids into cholesterol and cholesterol esters. As portrayed, about 75% of the [ 14 C]-labeled lipids were in the form of cholesterol. Statistical analysis of the three groups also did not show any significant difference.

Lipoprotein particle size analysis.

As shown in Figure 3.9. A, the lipoprotein particles of fasting lymph of the male mice (Figure 9C) were slightly larger than those of the high-output female mice

(Figure 3.9. D), and the low-output female mice (Figure 3.9. E). When the lipoprotein particles were expressed as a ratio of VLDL (particles smaller than

800 Å) to chylomicron (particles of 800 Å or larger), the male mice were shown to have a slightly lower ratio of VLDL to chylomicron compared to those of the other two female groups (Figure 3.9. B). This slight difference in particle size was also

91 evident in the lipid-fed stage. During the lipid infusion, the male mice (Figure

3.10. C) also made slightly larger particles than both the high-output (Figure 3.10.

D) and the low output (Figure 3.10. E) female mice. The male mice showed an increase in relative chylomicron percentage from about 31% (fasting stage) to about 62% (lipid-fed stage). The female mice only reached 40% during the lipid- fed stage. Although the difference in the ratio of VLDL to chylomicron in the lipid-fed stage was not significant between the male and the other two female groups, the male mice had a tendency to produce relatively more chylomicrons while the female mice produced predominantly VLDLs during the lipid-fed stage.

DISCUSSION

Lipid absorption is a dynamic process that involves many complex steps. These steps include digestion, uptake, re-esterification, packaging, and transport (157).

Our lipid absorption studies used the lymph fistula model to allow us not only to determine any lipid uptake difference between males and females, but also to monitor the quantity of lipids transported to the lymph prior to their metabolism by the periphery.

The lymphatic triacylglycerol transport of the female mice segregated into high and low output groups. Unlike the female mice, the lymphatic transport by the male mice did not show any segregation, but rather a normal unimodal distribution. Analysis of lymphatic cholesterol transport revealed that the female mice that had low triacylglycerol transport also had low cholesterol transport to the lymph; and similarly, the female mice that had high triacylglycerol transport had high cholesterol transport. Although lymph flow in the low-output female group was significantly lower, lymph flow may not the factor responsible for these differences. The male and the high-output female groups did not show any differences in lymph flow, yet the cholesterol transport to lymph between these two groups was significantly different.

To determine the reason for the differences in lymphatic lipid transport, we investigated lipid uptake into the enterocytes and re-esterification by the small

93 intestine in these mice. Analysis of lipid uptake did not show any significant difference among the three groups studied. Although the luminal cholesterol in the male group was slightly higher than those in the two female groups, this small difference could not account for the large difference in their lymphatic transport.

The possibility that the infused lipids were excreted cannot explain the difference as shown by the low recovery in the colons and negligible radioactivity in fecal samples (data not shown). The possibility of reverse transit into the stomach was neglected by the low counts in the stomach in all three groups. Collectively these data imply that the male and the female mice were comparable in lipid uptake by the enterocytes.

To determine if there was any difference in lipid re-esterification by the enterocytes, we performed TLC analysis on the mucosal lipids. The three groups were not significantly different in the distribution of the classes of lipid from the proximal throughout the distal intestinal mucosa. Cholesterol esterification by the enterocytes has been shown to affect lipid absorption (73), however, our data did not support any difference in lipid esterification by the enterocytes.

As mentioned in the introduction, absorbed lipid can be transported via both the lymphatic and the portal routes. Cannulating both the lymph duct and the portal vein simultaneously is not a feasible approach since it will cause severe distress to the mice that could alter normal absorption processes. In the case of the lymph cannulation model, the infused lipid that did not enter the lymph and was

94 not excreted should have entered the portal blood. Cholesterol is not metabolized in the intestine, but 5-7% of the infused triacylglycerol may be metabolized by the gut (194). However, possible metabolism would represent only a small fraction of the unaccounted lipid. A study using both thoracic duct and portal cannulation in rats suggested that about 50% of the infused lipid can enter the portal circulation (138). By carefully measuring flow rate and the amount of lipid in the portal circulation, Mansbach et al. (10) also showed that there was about 39% of absorbed lipid entered the portal vein. Therefore, our derived portal transport data are in agreement with Mansbach’s report.

The low-output group had higher portal triacylglycerol transport than that of the high-output group, and vice versa. Similarly, the low-output group had higher portal cholesterol transport than that of the high-output group, and vice versa.

The differences between the two female groups in lymphatic lipid transport were not due to lipid uptake or re-esterification by the enterocytes, but were likely due to the regulation of lipid transport: lymphatic vs. portal route.

The detailed mechanism that governs this process remains elusive. Body weight was not associated with the difference in lipid transport in the female mice. The weights of the high- and the low-output groups were not different (average body weights ± SE were 22.31 ± 0.52 g and 22.22 ± 0.44 g, respectively). One factor that could be linked to these observations is the variation in the hormonal status in the females. When we tested if the estrous cycle was associated with the high

95 and the low lymphatic lipid output in the female mice, we found that it was not.

We cannot completely rule out the hormonal factor at the moment since in a particular estrous cycle, there exists a complex hormonal interaction. It remains to be determined if a particular hormone, such as estrogen, could regulate the lipid transport.

The idea of hormonal regulation of intestinal lipid absorption was indicated by

Vahouny et al. (215; 219). Their studies showed that female rats had higher

VLDL protein production compared to that of the male rats. To investigate if female mice produced more VLDLs to chylomicron than male mice, we performed an analysis on the lipoprotein particle size of lymph samples using a negative staining method. Our analysis showed that the female mice had a slightly higher VLDL to chylomicron ratio compared to that of the male mice both in fasting and lipid-fed stages. It is interesting to note that the amount of infused cholesterol in the mucosa was significantly higher in the male than in the two female groups. It remains to be determined if VLDL formation and transport is more efficient than chylomicron in transporting cholesterol, thereby clearing the cholesterol that was taken up by the enterocytes more quickly. This is possible in view that VLDL may play a more significant role in transporting dietary cholesterol than dietary triacylglycerols in females as suggested by Vahouny et al.

(214; 218). However, the ratio of VLDL to chylomicron could not explain the differences in the lymphatic lipid transport between the two female groups since the two groups were quite comparable in their VLDL to chylomicron ratios.

Our studies raise critical questions. How are the lipids transported in portal circulation (what are they bound to)? Is the proposed regulation of lipid transport mediated by a sex hormone(s)?

Despite these unanswered questions, we have contributed to the understanding that lipid absorption is not merely a process that dictates how much lipid is absorbed, but also how lipids are transported once absorbed. The amount of lipids absorbed is clearly important, however the major route for lipid absorption is also important. Lipids entering portal route will drain into the key metabolic organ, the liver. On the other hand, lipids entering the lymphatic route may have a significant time lapse before they enter the liver. Hence, it is plausible that lipid transport may affect metabolic fate.

In fact, several studies have reported such differences in dietary lipid uptake and retention by peripheral tissues between men and women. Dietary lipid uptake and retention by leg muscle tissues were reported to be greater in women than in men (98). The splanchnic uptake of dietary lipids, however, was greater in men

(150). These observations suggest that the metabolic fate of absorbed lipids can differ between male and female (105). Whether or not regulation of lipid transport plays a significant role in this process remains to be determined.

In conclusion, our studies showed that the female mice, unlike the male mice, had two distinct populations based on lymphatic triacylglycerol transport. Their transport segregated into either high or low-output groups, with the high-output group having similar lymphatic triacylglycerol transport to the males and the low- output group having significantly less. These differences in lymphatic lipid transport were not due to the difference in net lipid uptake or re-esterification by enterocytes, but most likely were due to the regulation of lipid transport. Male mice did not seem to regulate lipid transport in this manner. This observation raises the question of whether or not the proposed regulation of lipid transport was mediated by estrous cycle. Our studies, however, did not support that the estrous cycle regulates lipid transport. The mechanism underlying the regulation of lipid transport remains to be determined.

Acknowledgements:

This work was supported by the National Institutes of Diabetes and Digestive and

Kidney Diseases Grants DK-56910, DK-54504, DK-56863, and by a pre-doctoral fellowship award from the American Heart Association, Ohio Valley Affiliate. The authors would like to thank Ronald Jandacek and Cali Smith for critical reading of the manuscript.

Figure Legends:

Figure 3.1. The histograms of the total lymphatic triacylglycerol recovery in the male (A) and the female mice (B). Mice were equipped with lymph and duodenal cannulae, and were intraduodenally infused with a lipid emulsion containing labeled triolein and cholesterol for a period of 6 hours. Lymph was collected hourly and analyzed. The total lymphatic lipid recoveries were plotted in a histogram.

Figure 3.2. The lymph flow rate during the continuous intraduodenal lipid infusion. Mice were equipped with lymph and duodenal cannulae, and were intraduodenally infused with a lipid emulsion containing labeled triolein and cholesterol for a period of 6 hours. Lymph was collected hourly and analyzed. 0- h lymph represents fasting lymph. The female mice were divided into two groups, the high-output group ( High ) and the low-output group ( Low ), as discussed in

Results. Groups not having common letters are significantly different (p < 0.05).

Values are means ± SE.

Figure 3.3. The hourly lymphatic triacylglycerol output during continuous intraduodenal lipid infusion. Mice were equipped with lymph and duodenal cannulae, and were intraduodenally infused with a lipid emulsion containing labeled triolein and cholesterol for a period of 6 hours. Lymph was collected hourly and analyzed. 0-h lymph represents fasting lymph. The female mice were either divided into two groups, high-output group ( High ) and low-output

100 group ( Low ), as discussed in Results (A), or according to their estrous cycle (B).

Groups not having common letters are significantly different (p<0.05). Values are means ± SE.

Figure 3.4. The hourly lymphatic cholesterol output during the continuous intraduodenal lipid infusion. Mice were equipped with lymph and duodenal cannulae, and were intraduodenally infused with a lipid emulsion containing labeled triolein and cholesterol for a period of 6 hours. Lymph was collected hourly and analyzed. 0-h lymph represents fasting lymph. The female mice were either divided into two groups, high-output group ( High ) and low-output group ( Low ), as discussed in Results (A), or according to their estrous cycle (B).

Groups not having common letters are significantly different (p < 0.05). Values are means ± SE.

Figure 3.5. The total radioactive triacylglycerol recovery in the lymph and the segments of gastrointestinal tract. Mice were equipped with lymph and duodenal cannulae, and were intraduodenally infused with a lipid emulsion containing labeled triolein and cholesterol for a period of 6 hours. The female mice were either divided into two groups, high-output group ( High ) and low-output group

(Low ), as discussed in Results (A), or according to their estrous cycle (B).

Lymph represents the cumulative amount of radioactive triacylglycerol recovered in the lymph over the 6-h infusion. Stomach, lumen, colon, and mucosa (small intestine) were analyzed as explained in Methods. “Others” indicates portal

101 transport. Groups not having common letters are significantly different (p < 0.05).

Values are means ± SE.

Figure 3.6. The total radioactive cholesterol recovery in the lymph and the segments of gastrointestinal tract. Mice were equipped with lymph and duodenal cannulae, and were intraduodenally infused with a lipid emulsion containing labeled triolein and cholesterol for a period of 6 hours. The female mice were either divided into two groups, high-output group ( High ) and low-output group

(Low ), as discussed in Results (A), or according to their estrous cycle (B).

Lymph represents the cumulative amount of radioactive cholesterol recovered in the lymph over the 6-h infusion. Stomach, lumen, colon, and mucosa (small intestine) were analyzed as explained in Methods. “Others” represents portal transport. Groups not having common letters are significantly different (p < 0.05).

Values are means ± SE.

Figure 3.7. Distribution of different classes of [ 3H]-labeled lipids in intestinal mucosa. Mucosa was divided into 4 equal segments, from proximal to distal: M1

(A), M2 (B), M3 (C), and M4 (D). Mucosal lipids were extracted and separated by TLC into CE, TG, FA, DG, and MG+PL. CE, cholesteryl esters; TG, TGs; FA, fatty acids; DG, diacylglycerols; MG+PL, monoacylglycerols and phospholipids.

Values are means ± SE.

Figure 3.8. Distribution of different classes of [ 14 C]-labeled lipids in intestinal mucosa. Mucosa was divided into 4 equal segments, from proximal to distal: M1

102

(A), M2 (B), M3 (C), and M4 (D). Mucosal lipids were extracted and separated by TLC into cholesterol and cholesterol esters. Values are means ± SE.

Figure 3.9. Lipoprotein particle size of fasting lymph. Distribution of particle size

(A), relative VLDL/chylomicron ratio (B), and representative pictures of the fasting lymph of the male (C), the high- (D), and the low-output female (E) are shown.

Particles < 800 Å are considered VLDL, and 800 Å or more are considered chylomicrons. Standard bars represent 5000 Å (500 nm). Values are means ±

SE.

Figure 3.10. Lipoprotein particle size of lipid-infused lymph. Distribution of particle size (A), relative VLDL/chylomicron ratio (B), and representative pictures of the fasting lymph of the male (C), the high- (D), and the low-output female (E) are shown. Particles < 800 Å are considered VLDL, and 800 Å or more are considered chylomicrons. Standard bars represent 5000 Å (500 nm). Values are means ± SE.

103

Figure 3.1.

A) Male

Number of Animals of Number 2

0 0-10 11-20 21-30 31-40 41-50 51-60 61-70 Total lymph TG recovered (%)

B) Female

Number of Animals of Number 2

0 0-10 11-20 21-30 31-40 41-50 51-60 61-70 Total lymph TG recovered (%)

104

Figure 3.2.

Lymph flow: male, high- and low-output groups

Male (n=15) 0.4 Female, High (n=12) Female, Low (n=20)

0.3 a a 0.2 b 0.1 Lymph Flow (ml/h) Flow Lymph

0.0 0 1 2 3 4 5 6 Time (h)

105

Figure 3.3.

Lymphatic triacylglycerol output: male, high- and low-output groups

A) Male (n=15) Female, High (n=12) 75 Female, Low (n=20)

a 50 a

(Mean ± SE) (Mean 25 b % of Hourly Infused % of Hourly

0 0 1 2 3 4 5 6 Time (h)

Lymphatic triacylglycerol output: proestrous, estrous, and diestrous

Proestrous (n=6) B) 75 Diestrous (n=11) Estrous (n=6)

(Mean ± SE) (Mean 25

% of hourly infused % of hourly

0 0 1 2 3 4 5 6 Time (h)

106

Figure 3.4.

Lymphatic cholesterol output: male, high- and low-output groups

A) Male (n=15) 50 Female, High (n=12) Female, Low (n=20) a 40 b 30

(Mean ± SE) (Mean c 10 % of Hourly Infused % of Hourly

0 0 1 2 3 4 5 6 Time (h)

Lymphatic cholesterol output: proestrous, estrous, and diestrous

B) 50 Proestrous (n=6) Diestrous (n=11) 40 Estrous (n=6)

20 (Mean ± SE) (Mean

10 % of hourly infused % of hourly

0 0 1 2 3 4 5 6

Time (h)

107

Figure 3.5.

Total triacylglycerol recovery: male, high- and low-output groups A) 75 Male (n=15) b Female, High (n=12) a 50 Female, Low (n=20) a a a

(Mean ± SE) (Mean 25 b % of Total Infused % of Total

0 Stomach Lumen Colon Mucosa Lymph Others

Total triacylglycerol recovery: proestrous, estrous, and diestrous

B) 75 Proestrous (n=6) Diestrous (n=11) 50 Estrous (n=6)

(Mean ± SE) (Mean 25

% % of total infused

0 Stomach Lumen Colon Mucosa Lymph Others

108

Figure 3.6.

Total cholesterol recovery: male, high- and low-output groups A)

75 Male (n=15) Female, High (n=12) Female, Low (n=20) b 50 a

b a a b b

(Mean ± SE) (Mean 25 a

% of Total Infused % of Total c

0 Stomach Lumen Colon Mucosa Lymph Others

Total cholesterol recovery: proestrous, estrous, and diestrous B)

75 Proestrous (n=6) Diestrous (n=11) 50 Estrous (n=6)

(Mean ± SE) (Mean 25

% % of total infused

0 Stomach Lumen Colon Mucosa Lymph Others

109

Figure 3.7.

A) 75 Male (n=3) Female (High, n= 5) 50 Female (Low, n= 3) H] Count 3 25 % [

0 CE TG FA DG MG+PL B) 75

50 H] Count 3 25 % [

0 CE TG FA DG MG+PL C) 75

50 H] Count 3 25 % [

0 CE TG FA DG MG+PL

D) 40

20 H] Count 3

% [ 10

0 CE TG FA DG MG+PL

110

Figure 3.8.

A) 100 Male (n=3) Female (High, n= 5) 75 Female (Low, n= 3)

C] Count 50 14

% [ 25

0 Cholesterol Cholesterol Ester

B) 100

C] Count 50 14

% [ 25

0 Cholesterol Cholesterol Ester

C) 100

C] Count 50 14

% [ 25

0 Cholesterol Cholesterol Ester

D) 100

C] Count 50 14

% [ 25

0 Cholesterol Cholesteryl Ester

111

Figure 3.9.

50 Male (n=4) 40 Female (High, n=4) Female (Low, n=5) 30

20 % Particles

0 200 400 600 800 1000 1200 1400 1600 1800 2000 Diameter (Å)

100 Male (n=4) Female (High, n=4) 75 Female (Low, n=5)

50 % Particles 25

0 VLDL Chylomicron

112

113

Figure 3.10.

40 Male (n=3) Female (High, n=4) 30 Female (Low, n=3)

20 % Particles 10

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 Diameter (Å)

Male (n=3) 100 Female (High, n=4) Female (Low, n=3) 75

50 % Particles 25

0 VLDL Chylomicron

114

115

Chapter 4

General Conclusions and Future Directions

116

This chapter will discuss how our studies have provided new insights into how dietary lipids are digested, taken up by the enterocytes, and then subsequently packaged into chylomicrons for export into lymph by the small intestine. We utilized two models to help us understand the mechanism of the uptake and the packaging of chylomicrons and VLDL in the enterocytes. Briefly, we found that

CD36 plays an important role in the uptake of dietary cholesterol (Chapter 2). In addition, the presence of CD36 in the enterocytes is also important in directing the absorbed TG into chylomicrons. From our studies on the gender effect on fat absorption (Chapter 3), we found that the uptake of fatty acid and cholesterol is not different between male and female mice. However, the females can be divided into two groups: one group transported lipids efficiently into lymph and the other did not. Whether or not the female mice can transport lipids efficiently into lymph does not seem to be dependent on their estrous cycle. In the following sections, we are going to discuss the results of our studies and their implications in a greater detail.

4.1. Insights gained on the lipid uptake

From our lymph fistula studies, it does not seem that fatty acid uptake is affected to a significant extent by the lack of CD36 expression by the small intestine. This finding certainly goes hand in hand with the view of others (45; 80; 145; 206).

Fatty acid uptake by the small intestine is mostly passive rather than active when the supply of dietary fatty acids are plentiful (46; 79; 147; 205). As already

117 reviewed in Chapter 1, none of the fatty acid transporters that have been proposed to play a role in fatty acid uptake have withstood the test of time, and often the convincing evidence provided by the in vitro studies cannot be supported by the in vivo findings. We believe that the membrane transporters located in the intestinal brush border membrane are used to prevent the loss of fatty acids (especially the essential fatty acids) when the concentration of fatty acids in the lumen is low.

On the other hand, cholesterol is always abundantly present (mostly from the bile) in the intestinal lumen. Cholesterol content of the cell membrane can affect its fluidity and function. For instance, an increase in cholesterol content will increase the membrane rigidity of the membrane. Thus, the amount of cholesterol in the brush border membrane as well as other intracellular membrane is probably tightly regulated physiologically to ensure a normal function of these membranes, e.g., the normal function of transporters located in the enterocytes. For instance, there are transporters involved in the uptake of amino acids, di- and tripeptides, and water soluble ions and vitamins. There are also transporters for cholesterol uptake (CD36 and NPC1L1 are likely two of them) in addition to proteins (abcA5 and 8) involved in the active extrusion of cholesterol and similar type of molecules, such as the plant sterols (beta- sitosterol). In our CD36 studies (Chapter 2), we have demonstrated clearly that the CD36 KO animals did not take up cholesterol as efficiently as the CD36 wild type animals, thus resulting in significantly more accumulation of luminal

118 cholesterol waiting to be taken up. The fact that CD36 plays a role in the uptake of cholesterol has been suggested but has not been demonstrated physiologically until this study. The reduction of cholesterol uptake in the absence of CD36 suggests that CD36 may be an intestinal cholesterol transporter. This idea is certainly supported by its physical location in the brush border membrane of the proximal intestine (44; 116; 160) and its high lipid binding affinity. Other studies also showed that cholesterol uptake by isolated brush border membrane could be inhibited by the antibodies against CD36 (232).

Our CD36 studies raised a number of interesting questions that warrant investigation. Is there a difference in the uptake of biliary and dietary cholesterol by CD36 in the small intestine? Since CD36 may not be the only transporter involved in the uptake of cholesterol, will the deletion of CD36 up-regulate other transporters (e.g., NPC1L1)? An interesting future study would be to determine if the CD36 / NPC1L1 double KO mice exhibit an even more pronounced reduction in cholesterol uptake by the intestine.

The significance of the role of CD36 in facilitating cholesterol uptake by the intestine is clear. As discussed in Chapter 1, intestine may also be critical in regulating lipid homeostasis. Hence, being able to reduce cholesterol uptake by the intestine by CD36 inactivation may lead to reduced plasma cholesterol level.

It is unlikely, however, that the inactivation of CD36 would lead to a complete loss of cholesterol uptake by the intestine. Nevertheless, its inactivation in the

119 intestine may prove to be beneficial, such as for a therapeutic approach in combating cardiovascular diseases.

4.2. Insights gained on lipoprotein secretion

Another new concept derived from our studies is that CD36 plays an important role in the formation and secretion of chylomicrons. Despite the normal uptake of fatty acids and the subsequent re-esterification to form TGs, the TGs were less efficiently incorporated into chylomicrons for secretion in the KO relative to the

WT animals. The reduced incorporation of absorbed TGs to form chylomicrons resulted in smaller chylomicrons being secreted by the small intestinal epithelial cells during active fat absorption. This reduced TG transport, as one would expect, was also associated with decreased cholesterol transport into lymph.

The reduced ability of the CD36 KO animals to make chylomicrons resulted in more lipids being accumulated in the mucosa of the small intestinal epithelial cells. The reason why we failed to see a deficit in fat absorption in the CD36 knockout animals using the SPB method (101) is probably because the KO animals gauge its fat intake so that its small intestine can handle the ingested lipid. We hypothesize that instead of eating larger meals during the dark phase, the KO animals eat many small meals instead.

The fact that CD36 KO animals were less efficient in transporting TGs to the lymphatic circulation suggests that the handling of dietary lipids by the peripheral

120 tissues, i.e., adipose and muscles, would also be different between CD36 KO and WT animals. CD36 KO animals were slimmer (87) and were more resistant towards atherosclerosis (64). However, the readers should be reminded that the uptake of fatty acids by the peripheral tissues was also reduced in these KO animals. Therefore, the relative contribution of these two factors on the reduced body weight of the CD36 KO mice remains unclear. In addition, the macrophages in the CD36 KO mice also displayed a reduced LDL lipid uptake, which gives a potential benefit of protection against atherosclerosis. In order to study the significance of intestinal CD36 on the whole body lipid metabolism and atherosclerosis, one should generate the intestinal-specific CD36 KO mice.

Our studies that compared intestinal lipid absorption of the male and the female

C57BL/6 mice have yielded some un-expected and exciting results. Similar to the finding of Vahouny et al. (213), we demonstrated that female rodents produce more VLDL than male rodents. The notion that VLDL is the primary lipoprotein in transporting cholesterol also agrees well with our studies that showed that more of the TG-rich lipoproteins in lymph were VLDL in the female mice, and that the enterocytes of the female mice transported cholesterol more efficiently than the male animals. As mentioned, the females can be divided into two groups: one group transported lipids efficiently into lymph and the other did not. We suspected that this segregation of lipid transport was under the control of sex hormones, but we failed to observe an effect of the estrous cycle on the lymphatic transport of TG and cholesterol in the female mice. We cannot rule

121 out the involvement of a certain hormone in lipid transport because we have not directly tested the effect of individual hormones on lipid transport. It may be possible that there exists a short window of time in which lipids are less efficiently transported to the lymphatic circulation. Our studies might not be able to catch this short window of time because our studies simply correlate lipid transport with estrous stage.

In order to determine the molecular mechanism of the regulation of lipid transport, one can compare the intestinal gene expression profile in the high- and low- output mice, e.g., by utilizing the gene microarray technology. From a series of candidate genes, one could then propose the mechanism of how these genes regulate lipid transport. We propose that certain genes, which may potentially be regulated hormonally, drive the packaging of the lipids that are taken up by the enterocyte into smaller lipoproteins or carriers. As a result of the unique packaging, these lipids that are secreted into the lamina propria can enter the portal circulation instead of the lymphatic circulation. These lipids must be secreted as small size particles to allow them to pass through the endothelials lining the capillaries. Such particles include lipids bound to carriers (e.g., Apo A-

I) and high density lipoprotein (HDL). As discussed in Chapter 1, the intestine has been reported to produce a significant amount of HDL. To further test if HDL could be the potential lipoprotein carrying dietary lipids, one could isolate portal plasma (pool the samples to compensate for the smaller sample size) and determine if the labeled lipids are significantly associated with the HDL fraction.

122

To test if the HDL is of intestinal origin, one could inject labeled-HDL intravenously during the portal cannulation. If the portal HDL contains insignificant amount of labeled-HDL (intravenously administered), then this would suggest that the HDL fraction is of intestinal origin. This hypothesis is currently being investigated in our laboratory.

Another important question is: why did the TG accumulate in the mucosa of the

CD36 KO animals and not in the mucosa of the female low responders? As already discussed earlier, one possibility is that the female low responders did not have a problem in the formation of chylomicrons, but simply more of the absorbed lipids are channeled to the portal blood. The demonstration of such a possibility is not only of significance to our understanding of intestinal fat absorption but it is potentially important to the pharmaceutical industry in terms of drug delivery. Another possibility is that the TG in the CD36 knockout animals is in a pool that is not available for transport to the portal blood.

Lastly, we wondered why CD36 knockout animals have reduced formation and secretion of chylomicrons. We propose that CD36 mediates the trafficking from the ER to Golgi for a few reasons. First , studies by Mansbach et al. (128) suggested that the rate limiting step in intestinal lipid absorption is at the ER to

Golgi transport. In fact, studies in humans showed that mutations of the genes involved in ER export were associated with lipid absorption disorders (106).

Second , recent studies showed that CD36 was localized in the intracellular

123 compartment as well as plasma membrane in 3T3-L1 adipocytes. Therefore, it is conceivable that CD36 may be involved in the regulation of the trafficking of lipoproteins from the ER to Golgi transport.

124

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Appendix 1

CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons

from the blood

Due to copyright issue, only the web link to this article is provided

(http://www.jci.org/cgi/reprint/115/5/1290.pdf ).

170

Appendix 2

Enterocyte fatty acid uptake and intestinal fatty acid-binding protein

(Reproduced with permission, from Tso P, Nauli A, Lo CM, 2004, Biochem Soc

44th International Conference on the Bioscience of Lipids 75

Enterocyte fatty acid uptake and intestinal fatty acid-binding protein

P. Tso1, A. Nauli and C.-M. Lo Department of Pathology, University of Cincinnati Medical Center, 231 Albert Sabin Way, Cincinnati, OH 45267, U.S.A.

Abstract This article reviews our current understanding of the uptake of fatty acids by the enterocytes of the intestine. The micellar solubilization of fatty acids by bile salts and the factors regulating that process are discussed. The mechanism of how micellar solubilization of fatty acids promotes the uptake of fatty acids by enterocytes and their relative importance is reviewed. Additionally, discussion of the various fatty acid transporters located at the brush border membrane of the enterocytes is included. Finally, a summary of our current understanding of the function of fatty-acid-binding proteins inside enterocytes is provided.

Micellar solubilization of fatty acids acids, this does not occur by the uptake of the entire micelle by by bile salt the enterocytes, but rather when the lipophilic components Dietary fat, defined in simple terms, is that part of the diet (such as fatty acids and monoacylglycerol molecules) are that can be extracted by organic solvents. Dietary fat is taken up by the enterocytes. This concept is supported by composed of an array of compounds, from the highly non- the finding that fatty acids are taken up by the enterocytes at polar hydrocarbons to the highly polar phospholipids and a lower rate than the monoacylglycerols; this will not be the glycolipids. Fatty acids in the intestinal lumen are derived case if the whole micelle is taken up by the enterocyte [3,4]. from the digestion of triacylglycerols, phospholipids and Evidence supporting this concept further is the finding that in cholesteryl esters. Fatty acids in the intestinal lumen exist human Caco-2 cells (a model for enterocytes), the uptake of both as monomers in solution or as part of the bile salt fatty acids and monoacylglycerols compete with each other micelles. Since fatty acids are only sparingly soluble in water, [5]. the concentration of non-esterified fatty acid monomers in the aqueous medium is low, in the 10 µM range. In contrast, the concentration of fatty acids in the bile salt micellar Factors affecting the solubilization of fatty solution can reach concentrations in the range of 10 mM. acids by bile salt micelles Thus the micellar solublization of fatty acids by bile salts Since micellar solubilization plays such an important role greatly increases the concentration of fatty acids in the in the uptake of fatty acids, it is important to consider aqueous medium of the intestinal lumen. the factors that affect the incorporation of fatty acids into micelles. The pH of the medium plays a critical role, as fatty acids can exist both in the non-ionized as well as Uptake of fatty acids from bile salt micelles the ionized form. At neutral or alkaline pH, most of the Although the solubilization of lipophilic molecules by bile fatty acids exist in the ionized form. The ionized fatty acids salts has been known for a long time, the importance of are incorporated into the bile salt micelles much more effi- micellar solubization of fatty acids by bile salts for intestinal ciently than the non-ionized fatty acids [6]. Temperature lipid absorption was not fully appreciated until Hofmann also plays an important role. For instance, with a series and Borgstrom’s well-designed study on human intestinal fat of straight-chain saturated monoacylglycerols, there is a absorption was published [1]. They demonstrated that the temperature at which the solubility of each monoacylglycerol uptake of fatty acids by the intestine is dependent on the mi- for incorporation into micelles increases dramatically. When cellar concentration of the fatty acids in the aqueous phase of this particular temperature is plotted against the chain length the lumen. Work by Hoffman and Simmonds [2] advanced of the monoacylglycerol, it forms a line that runs parallel this important observation and demonstrated that while to and below the melting point of each corresponding micellar solubilization greatly facilitated the uptake of fatty monoacylglycerol [7]. Since the mono- and polyunsaturated fatty acids have lower melting points than the saturated fatty acids, their incorporation into bile salt micelles is much better Key words: brush border, cholesterol, intestinal fat absorption, lipid, passive process, transport than that of the saturated fatty acids. Saturated fatty acids are protein. not as well absorbed by the small intestine since they are not Abbreviations used: FATP4, fatty acid transporter protein 4; FABP, fatty-acid-binding protein; I-FABP, intestinal FABP; L-FABP, liver FABP; AOFA, anthroyloxy fatty acid analogue. as well solubilized in bile salt micelles as the unsaturated fatty 1 To whom correspondence should be addressed (e-mail [email protected]). acids.

C 2004 Biochemical Society 76 Biochemical Society Transactions (2004) Volume 32, part 1

Why is micellar solubilization so important specific transporters. One of the earlier studies to address for intestinal fatty acid uptake? this question was carried out by Strauss [13]. Using intestinal sacs, he demonstrated that uptake of fatty acid from This question was answered by the experiments conducted ◦ by Dietschy and his colleagues [8–10]. These investigators micellar solution occurred at 0 C, followed by resynthesis found that the brush-border membrane of enterocytes is to triacylglycerol, with the appearance of fat droplets in the apical vesicles and in the endoplasmic reticulum when the in- separated from the bulk aqueous phase in the intestinal lumen ◦ by an unstirred fluid layer. This unstirred water layer is testinal sacs were incubated at 37 C. His data implied that the uptake step occurred passively at 0◦C while the resynthesis poorly mixed with the bulk phase in the intestinal lumen. ◦ Consequently, solute molecules in the bulk phase can only progressed efficiently at 37 C. The fact that fatty acids may gain access to the brush-border membrane by first diffusing be taken up by the enterocyte via a carrier-mediated process across the unstirred water layer. For any molecule, the rate was further supported by a study conducted by Chow and of uptake by the small intestine will be dependent on the Hollander [14] in which they demonstrated that linoleate number of molecules that are in close proximity to the brush- uptake by the small intestine observes a concentration- border membrane and therefore are available for uptake by dependent dual mechanism of transport. At very low linoleate µ the brush-border membrane. Because the solubility of fatty concentration ( M), it is taken up via a carrier-dependent acids in the aqueous medium is extremely low, very few process. At higher concentrations of linoleate (mM), it is molecules will gain access to the brush-border membrane. In taken up predominantly by passive diffusion. It would contrast, micellar solubilization greatly enhances the number be useful and interesting to determine if a similar uptake of molecules that are available for uptake by the entero- mechanism also operates for other fatty acids. This is a cytes. Despite the lower diffusive rate (due to size) of the significant study but its importance does not seem to be fully micelle relative to the monomolecular fatty acid molecule, appreciated by investigators in the field. the net effect of micellar solubilization still results in a marked Work by Stremmel et al. [15] has introduced the idea enhancement of diffusion of fatty acid molecules across the that fatty acids may be taken up by enterocytes by carrier- unstirred water layer. As a result, micellar solubilization, mediated processes. Stremmel et al. [15] found that this the aqueous concentration of fatty acids next to the entero- transport protein is capable of transporting fatty acids. cytes, is increased dramatically. Treating a jejunal loop with an antibody against this protein The importance of micellar solubilization in fat absorption significantly reduced fatty acid uptake by the loop. It was was challenged by Carey and his colleagues who demon- further demonstrated [15] that this protein is present mainly strated that during fat absorption, in addition to the presence in the apical and lateral areas (in the region of the tight of fatty acid monomers in solution as well as in bile salt junction) of the villus as well as the crypt. The findings of micelles, there is also the presence of vesicles in the intestinal Stremmel and colleagues are highly suggestive of the presence lumen which contain both lipids and bile salts [11]. The of a plausible candidate transporter for fatty acids. However, vesicles are particularly abundant when the concentration the importance of this transporter in the uptake of fatty acids of bile salts in the lumen is low while the amount of lipid by the enterocytes has been questioned because (1) it was later digestion products in the lumen is high. They argued that found that this transporter is similar to the mitochondrial the vesicles could participate in the delivery of fatty acid for glutamic oxaloacetic transaminase, which is not involved in uptake by the enterocytes; this is supported by the fact that lipid absorption [16], and (2) the crypt cells express this patients with ileal disease have very low bile salt concentration protein, which are not involved in fat absorption. in the intestinal lumen [12]. However, there has not yet been Another transporter that has been suggested to play a role an experiment specially designed to determine the relative in intestinal fatty acid uptake is CD36 (also called fatty acid importance of uptake of fatty acids by the enterocytes translocase) [17]. CD36 is expressed in the intestine and is from micelles compared with the vesicles. We believe that localized mainly in the apical membrane of the intestinal while micelles are certainly the predominant vehicle used villus cells. However, a recent paper by Goudriann et al. to faciliate the uptake of fatty acids by the small intestine [18] showed that intestinal lipid absorption is not affected during normal fat absorption, the role of vesicles as vehicles in CD36-knockout mice. The knockout mice absorbed to deliver fatty acids to the enterocytes can become more triacylglycerol and fatty acid as well as the wild-type animals. important if there are fewer micelles around (e.g. in the case Although there is strong evidence that CD36 plays an of low concentrations of bile salts). With regard to other lipid- important role in the uptake of fatty acids by the adipose soluble molecules such as cholesterol, micelles seem to be the tissue, muscle and heart, its role in the modulation of the predominant vehicle delivering them to the enterocytes. uptake of fatty acids by the small intestine remains obscure [19]. Another fatty acid transporter that has attracted considerable attention in the last few years is intestinal FATP4 Uptake of fatty acids by enterocytes: (fatty acid transporter protein 4), a member of the fatty acid passive or transporter-mediated? transport protein family [20]. This protein is expressed in There has long been controversy as to whether fatty acids significant levels at the apical membrane of the enterocytes are taken up by the enterocytes passively or actively via [20]. Evidence supporting its role in fatty acid uptake is

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(1) overexpression of FATP4 in HEK-293 cells enhanced FABP [25], L-FABP [26] (where I is intestinal and L is the uptake of long-chain fatty acids, and (2) reducing the liver) and the ileal lipid-binding protein [27]. For the purpose FATP4 expression in primary enterocytes by antisense of this review, the discussion will be mostly focused on I- oligonucleotides significantly reduced fatty acid uptake. FABP and L-FABP, since fat absorption takes place mostly However, its precise role in the uptake of fatty acids by in the duodenum and the jejunum, and ileal lipid-binding the small intestine in vivo is still unclear, particularly in protein is expressed only in the ileum and colon. Using view of recent findings from the laboratory of Stremmel immunohistochemistry, Shields et al. [28] have demonstrated et al. [21,22]. First they demonstrated that FATP4 could that there is no difference in the distributions of I-FABP and function as an acyl-CoA synthetase with substrate preference L-FABP in the gastrointestinal tract. Both binding proteins for the very-long-chain fatty acids [21]. When the FATP4 are more abundant in the proximal than the distal intestine, cDNA was incorporated into COS1 cells, this resulted in more abundant in the villus cells than in the crypt cells, and a 2-fold increase in palmitoyl-CoA synthetase and a 5-fold the staining is cytoplasmic. Furthermore, consumption of a increase in lignoceroyl-CoA synthetase activity. Secondly, high-fat diet induces the expression of FABPs in the small- in a recent paper, Herrmann et al. [22] reported that the intestinal mucosa [29]. The distribution of these FABPs and deletion of the expression of FATP4 is associated with a the physiological regulation of these proteins by high-fat diet neonatally lethal restrictive dermopathy. In an independent would support the hypothesis put forward by Ockner that the study [23], Moulson and colleagues reported that FATP4 is FABPs are instrumental in the intracellular transport of actively involved in the development of skin and hair, thus the absorbed fatty acid [29,30]. However, there are differences also supporting the finding of Herrmann et al. [22]. Whether between these two FABPs. Firstly, they differ in their binding FATP4 may perform the function of both a transporter as specificity. I-FABP binds strongly to fatty acids, but L- well as an enzyme is yet to be determined. FABP will bind not only long-chain fatty acids but also Despite a number of papers published on the subject and lysophosphatidylcholine, retinoids, bilirubin, carcinogens the efforts of many laboratories, we have not come any closer and even selenium [31–33]. Secondly, using fluorescent to addressing the issue of whether there are specific fatty acid AOFA (anthroyloxy fatty acid analogues), Thumser and transporters located in the brush-border membrane to take Storch [34] demonstrated that these fluorescent fatty acid up fatty acids into enterocytes. Hamilton et al. [24] believed analogues are transferred from phospholipid vesicles to L- that the uptake of fatty acids by diffusion across plasma FABP or to I-FABP by different mechanisms. For L-FABP, membranes occurs quickly and that there appears little need they are transferred mostly by a diffusive process. In contrast, for the presence of transporters to facilitate their uptake. We the data for I-FABP suggest that a transient collisional believe that both schools of thought are probably correct, interaction of I-FABP with the phospholipid membrane depending on the concentration of fatty acids used in the occurs during AOFA extraction from the vesicles by the study. When the concentration is low, the active component protein. (involving transporters) is the major component responsible To test the importance of I-FABP for intestinal fat for the uptake of fatty acids by the enterocytes. However, absorption, I-FABP was deleted in the mouse, but the mouse when the luminal fatty acid concentration is high, the majority still absorbed lipids efficiently. However, these data have to of fatty acids are taken up by the enterocytes passively. This be interpreted carefully since it is possible that other binding argument is aetiologically reasonable as fatty acids, especially proteins may take over when I-FABP is absent. As yet, there the essential fatty acids (such as linoleic acid, arachidonic are no L-FABP-knockout animals available for physiological acid, eicosapentaenoic acid and docosahexaenoic acid), are studies. When these animals become available, it would be important for maintaining normal physiological functions in an interesting endeavour to determine the effect of a double the body, as it would be detrimental if there were insufficient knockout (I-FABP and L-FABP) on intestinal fat absorption. amounts of these essential fatty acids present in the body. Thus if the supply of fatty acids is low, then our body wants References to make sure that they are not lost in the absorptive process. 1 Hofmann, A.F. and Borgstrom, B. (1964) J. Clin. Invest. 43, 247–257 Furthermore, one might also speculate that these transporters 2 Hoffman, N.E. and Simmonds, W.J. (1971) Biochim. Biophys. Acta 241, may take up fatty acids as part of the sensing mechanism for 331–333 3 Hoffman, N.E. (1970) Biochim. Biophys. Acta 196, 193–203 fatty acids, or specific fatty acids, in the gastrointestinal tract. 4 Lee, K.Y., Hoffman, N.E. and Simmonds, W.J. (1971) Biochim. Biophys. Acta 249, 548–555 5 Ho, S.Y. and Storch, J. (2001) Am. J. Physiol. Cell Physiol. 281, C1106–C1117 Intestinal FABPs (fatty-acid-binding 6 Hofmann, A.F. (1966) Gastroenterology 50, 56–64 proteins) 7 Lawrence, A.S.C., Hume, K., Capper, C.B. and Bingham, A. (1964) To date, we do not know how the various absorbed lipids J. Phys. Chem. 68, 3470–3476 8 Dietschy, J.M., Sallee, V.L. and Wilson, F.A. (1971) Gastroenterology 61, migrate from the site of absorption to the endoplasmic 932–934 reticulum where biosynthesis of complex lipids takes place. 9 Wilson, F.A., Sallee, V.L. and Dietschy, J.M. (1971) Science 174, FABPs have been implicated in this action, but it is not 1031–1033 10 Westergaard, H. and Dietschy, J.M. (1976) J. Clin. Invest. 58, 97–108 well understood how this happens. Three FABPs have been 11 Carey, M.C., Small, D.M. and Bliss, C.M. (1983) Annu. Rev. Physiol. 45, identified in the gastrointestinal tract and these are the I- 651–677

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12 Mansbach, 2nd, C.M., Garbutt, J.T. and Tyor, M.P. (1972) Am. J. Dig. Dis. 23 Moulson, C.L., Martin, D.R., Lugus, J.J., Schaffer, J.E., Lind, A.C. and Miner, 17, 1089–1099 J.H. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5274–5279 13 Strauss, E.W. (1968) in Handbook of Physiology (Code, C.F., ed.), 24 Hamilton, J.A., Guo, W. and Kamp, F. (2002) Mol. Cell. Biochem. 139, pp. 1377–1406, American Physiological Society, Washington, DC 17–23 14 Chow, S.L. and Hollander, D. (1979) J. Lipid Res. 20, 349–356 25 Alpers, D.H., Strauss, A.W., Ockner, R.K., Bass, N.M. and Gordon, J.I. 15 Stremmel, W., Lotz, G., Strohmeyer, G. and Berk, P.D. (1985) (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 313–317 J. Clin. Invest. 75, 1068–1076 26 Gordon, J.I., Alpers, D.H., Ockner, R.K. and Strauss, A.W. (1983) 16 Berk, P.D., Potter, B.J., Sorrentino, D., Stump, D., Kiang, C.L., Zhou, S.L., J. Biol. Chem. 258, 3356–3363 Hori, Y. and Wada, H. (1990) Ann. N.Y. Acad. Sci. 585, 379–385 27 Oelkers, P. and Dawson, P.A. (1995) Biochim. Biophys. Acta 1257, 17 Abumard, N.A., Sfeir, Z., Connelly, M.A. and Coburn, C. (2000) Curr. Opin. 199–202 Clin. Nutr. Metab. Care 3, 255–262 28 Shields, H.M., Bates, M.L., Bass, N.M., Best, C.J., Alpers, D.H. and Ockner, 18 Goudriann, J.R., Dahlmans, V.E., Febbraio, M., Teusink, B., Romijn, J.A., R.K. (1986) J. Lipid Res. 27, 549–557 Havekes, L.M. and Voshol, P.J. (2002) Mol. Cell. Biochem. 239, 199–202 29 Ockner, R.K. and Manning, J.A. (1974) J. Clin. Invest. 54, 326–338 19 Ibrahimi, A. and Abumrad, N.A. (2002) Curr. Opin. Clin. Nutr. Metab. Care 30 Ockner, R.K. (1990) Mol. Cell. Biochem. 98,3–9 5, 139–145 31 Glatz, J.F., Janssen, A.M., Baerwaldt, C.C. and Veerkamp, J.H. (1985) 20 Stahl, A., Hirsch, D.J., Gimeno, R.E., Punreddy, S., Ge, P., Watson, N., Biochim. Biophys. Acta 837, 57–66 Patel, S., Kotler, M., Raimondi, A., Tartaglia, L.A. and Lodish, H.F. (1999) 32 Bass, N.M. (1988) Int. Rev. Cytol. 111, 143–184 Mol. Cell 4, 299–308 33 Bansal, M.P., Cook, R.G., Danielson, K.G. and Medina, D. (1989) 21 Herrmann, T., Buchkremer, F., Gosch, I., Hall, A.M., Bernlohr, D.A. and J. Biol. Chem. 264, 13780–13784 Stremmel, W. (2001) Gene 270, 31–40 34 Thumser, A.E. and Storch, J. (2000) J. Lipid Res. 41, 647–656 22 Herrmann, T., van der Hoeven, F., Grone, H.J., Stewart, A.F., Langbein, L., Keiser, I., Liebisch, G., Gosch, I., Buchkremer, F., Drobnik, W. et al. (2003) J. Cell Biol. 161, 1105–1115 Received 7 September 2003

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