The effects of dietary fat on pancreatic lipase gene expression.

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THE EFFECTS OF DIETARY FAT ON PANCREATIC LIPASE GENE EXPRESSION

by

Jennifer Ricketts

A Dissertation Submitted to the Faculty of the

COMMITTEE ON NUTRITIONAL SCIENCES (GRADUATE)

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1 995 UMI Number: 9604513

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read the dissertation prepared by Jennjfer Rjcketts --~~~~~~~------entitled ____T~h~e~ef~~~ec~t~s~o~f~d~ie~t~ary~t~a~t~o~n~p~a~n~cr~e~a~ti~c~h~·p~a~se~g~en~e~ex~p~r~e~ss~io~n~ ______

and recommend that it be accepted as fulfilling the dissertation

~nt for the Degree of Doctor of Philosophy M.Q~

Date I 1/9.« /16- Date .zf~,)97S Darrel E. Goll Date

Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

DisserQ~oP· ~- Date' I 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permIssIon, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED:~;;~ 4

ACKNOWLEDGEMENTS

I would like to thank Dr. Patsy Brannon for her support, effort, and time during the development of this dissertation project. I also would like to thank Dr. David K. Y. Lei, Dr. Charles W. Weber, Dr. Darrel E. Goll and Dr. Michael Wells for their participation in this project.

I am grateful to Deb Scott, Suzie Kunz and An Tsai for their help and friendship both inside and outside of the laboratory.

Special thanks is extended to Phyllis Reid for her help in final preparation of this dissertation. 5

DEDICATION

This dissertation is dedicated to the memory of Dr. B.L. Reid. He was generous in his support and encouragement and he will be missed by all. 6

TABLE OF CONTENTS

Page

LIST OF TABLES ...... 9

LIST OF FIGURES ...... 10

ABSTRACT ...... 11

1. INTRODUCTION ...... 12

2. LITERATURE REVIEW...... 15

LIPID METABOLISM ...... 15 Dietary Fats ...... 16 Physical properties ...... 16 Health and disease interactions ...... 17 Digestion ...... 20 An overview ...... 20 Preduodenal Iipases ...... 23 Duodenal Iipases ...... 23 Bile salts ...... 25 Absorption ...... 26 Enterocyte ...... 27 Transport ...... 29 Tissue Utilization ...... 30 Triglycerides (TG) ...... 30 Ketones...... 31 Lipoproteins ...... 31 PANCREAS ...... 33 Endocrine Pancreas ...... 33 Hormones ...... 33 Insulin ...... 34 Glucagon ...... 35 Somatostatin ...... 35 Exocrine Pancreas ...... 35 Ultrastructure ...... 36 Digestive enzymes ...... 37 Proteases ...... 37 Glycosidase ...... 38 Lipases ...... 38 Nucleases ...... 39 Synthesis and secretion ...... 39 Dietary and hormonal regulation ...... 42 7

TABLE OF CONTENTS--continued

page

Dietary regulation ...... 44 Hormonal regulation ...... 47 PANCREATIC LIPASE ...... 50 Gene Structure ...... 52 Function ...... 56 Dietary Regulation ...... 59 Hormonal Regulation ...... 61 Regulation of Colipase ...... 63

3. MULTIPLE MECHANISMS OF REGULATION OF PANCREATIC LIPASE IN RATS BY AMOUNT AND TYPE OF DIETARY FAT...... 65

INTRODUCTION ...... 65 METHODS...... 66 Enzyme Analysis...... 66 Isolation of Total RNA ...... 68 Quantitation of mRNA ...... 69 Statistical Analysis ...... 70 RESULTS...... 70 Enzyme Activities and Plasma Ketones ...... 70 mRNA Levels...... 74 DISCUSSION ...... 74

4. DIETARY FAT SATURATION HAS DIFFERENTIAL EFFECTS ON PANCREATIC LIPASE ACTIVITY, mRNA LEVELS, AND SYNTHESIS. . . .. 77

INTRODUCTION ...... 77 METHODS...... 78 Experimental Protocol ...... 78 Enzyme Analysis...... 79 Isolation of Total RNA ...... 79 Quantitation of mRNA ...... 80 Pancreatic Acini Isolation ...... 80 Protein Synthesis ...... 81 2-D Gels...... 81 Isolation of tRNA ...... 85 Total tRNA-phenylalanine ...... 86 Radiolabeled tRNA-phenylalanine ...... 86 Statistical Analysis ...... 86 RESULTS...... 87 Experiment 1 ...... 87 Food consumption ...... 87 8

TABLE OF CONTENTS--continued

page

Enzyme activities and plasma ketones ...... 87 Pancreatic mRNA Levels...... 91 Experiment 2 ...... 91 Body weights ...... 91 Relative synthesis ...... 91 Specific activity of tRNA-phenylalanine ...... 95 DISCUSSION ...... 95

5. THE EFFECTS OF DIETARY FAT ON THE REGULATION OF RAT PANCREATIC LIPASE FOLLOWING INHIBITION OF CHYLOMICRON TRANSPORT FROM THE ENTEROCYTE ...... 99

INTRODUCTION ...... 99 METHODS ...... 101 Experimental Protocol ...... 101 Enzyme and mRNA analysis ...... 105 Statistical Analysis ...... 105 RESULTS ...... 105 Food Consumption and Body Weight ...... 105 Enzyme Activities and mRNA Levels ...... 107 Protease activity ...... 107 Amylase activity and mRNA levels ...... 107 Lipase activity and mRNA levels ...... 107 DISCUSSION ...... 112

6. SUMMARY AND CONCLUSIONS ...... 116

APPENDIX A ...... 118

REFERENCES ...... -;. . . .. 120 9

LIST OF TABLES

Table Page

3-1. Dietary Composition 67

3-2. The Effects of Dietary Fat on Enzyme Activities and Ketone Levels ...... 73

4-1. The Effects of Dietary Fat on Body and Pancreatic Weights and Food Intake. .. 88

4-2. The Effects of Dietary Fat on Enzyme Activities and Plasma Ketone Levels ... 89

4-3. The Effects of Dietary Fat on 28s RNA and Amylase mRNA Levels...... 92

4-4. The Effects of Dietary Fat on Total tRNA Phenylalanine, tRNA-Phenylalanine Specific Activity, and Relative Synthesis of Amylase ...... 96

5-1. Dietary Composition ...... 104

5-2. The Effects of Treatment and Diet on Total Food Consumption, Weight Gain and Body Weight of Rats ...... 106

5-3. The Effects of Treatment and Diet on Amylase and Protease Activities ..... 108

5-4. The Effects of Treatment and Diet on 28s RNA and Amylase mRNA Levels.. 109 10

LIST OF FIGURES

Figure Page

2-1. Lipase Gene Family Tree 51

3-1. The Effects of Dietary Fat on Lipase Activity and mRNA Levels ...... 71

3-2. The Effects of Fat on Lipase Activity ...... 72

4-1. Linearity of 3[H] Phenylalanine Incorporation into TeA Precipitable Proteins and Lipase ...... 82

4-2. Two-Dimensional Polyacrylaminde Gel Electrophoresis of Pancreatic Proteins. . 83

4-3. The Effects of Amount and Saturation of Dietary Fat on Lipase Activity ..... 90

4-4. The Effects of Dietary Fat on Lipase and Related Protein-I mRNA Levels .... 93

4-5. The Effects of Dietary Fat on the Relative Synthesis of Lipase ...... 94

5-1. The Use of Pluronic L-81 for Determining Pre-absorptive and Post-absorptive Effects of Dietary Fat on Pancreatic Lipase Gene Expression...... 102

5-2. Experimental Design for the Use of Pluronic L-81 in Determining Pre­ absorptive and Post-absorptive Effects of Dietary Fat on Lipase Gene Expression ...... 103

5-3. Effects of Treatment and Dietary Fat on Lipase Activity...... 110

5-4. Effects of Treatment and Dietary Fat on rPL and rPLRP-I mRNA Levels ...... III 11

ABSTRACT

Pancreatic lipase is a lipolytic enzyme involved in the hydrolysis of dietary triglycerides for absorption and utilization. Both amount and type of dietary fat have been shown to regulate pancreatic lipase; however, the mediator(s) and mechanisms for this regulation have not been identified. These studies examined the effects of dietary fat ranging in polyunsaturated to saturated ratio (PIS ratio) from 0.3 to 7.9 at low fat (11 % of energy, LF) and moderate fat (40% of energy, MF) levels, including both pre-absorptive and post-absorptive effects, on lipase content

(activity), synthesis, mRNA levels and plasma ketones. Amount of fat independently increased lipase activity (p < 0.0005), synthesis (p < 0.004) and mRNA levels (p < 0.0001). Type of fat, however, affected lipase activity, but not its relative synthesis and mRNA levels. Lipase activity was significantly increased in rats fed MF diets with a high polyunsaturated fat (90% for corn oil;

172 % for safflower oil), but not significantly affected in rats fed a saturated fat (lard) or monounsaturated fat (olive oil). These results suggest that amount of fat regulates pancreatic lipase pre-translationally, whereas type of fat, specifically the degree of saturation, regulates pancreatic lipase post-translationally. Blockage of chylomicron transport out of the enterocyte with Pluronic L-81 enabled evaluation of pre-absorptive, presumably hormonally mediated, and post-absorptive, presumably peripherally mediated effects of dietary fat. Pluronic L-81 treatment decreased lipase activity 50% (p < 0.003) and mRNA levels 60% (p < 0.007) relative to control.

However, Pluronic L-81 interacted with amount and type of dietary fat resulting in a blunted effect in the MF safflower group (20% lower activity and mRNA levels). These results suggest that both pre-absorptive and post-absorptive mediators play an important role in pancreatic lipase regulation by dietary fat. 12

CHAPTER 1

INTRODUCTION

Digestion and absorption of dietary substrates requires a concerted effort from several

organs. The pancreas plays an important role in digestion by synthesizing and secreting the

digestive enzymes and hormones necessary for normal digestion and regulation of blood sugar

levels. Morphologically the pancreas can be divided into two major cell types, the endocrine cells

and exocrine cells.

The exocrine pancreas produces digestive enzymes which adapt to alterations in the diet. Several of these enzymes adapt to the amount of substrate in the diet by increasing or

decreasing mRNA levels, synthetic rates, and content of the enzyme. For example, expression of the major enzyme involved in fat hydrolysis, pancreatic lipase, increases when fat is increased

in the diet. The mechanisms involved in exocrine pancreatic adaptation to diet are not fully understood and are believed to be unique for each of the digestive enzymes.

Pancreatic lipase expression will respond to significant increases in dietary fat, but its response to moderate increases of dietary fat are less clear and depend on the type of dietary fat consumed. Polyunsaturated dietary fats at a moderate level in the diet have a more profound effect on lipase activity than do saturated fats. Saturation is likely to be only one factor influencing the response of pancreatic lipase to dietary fat. Chain length of dietary triglycerides has also been proposed as another factor influencing lipase expression, but little work has examined the effects of chain length.

The effects of dietary fat on lipase expression is likely to include several mediators.

The mediator(s) of this dietary regulation of pancreatic lipase is (are) unknown; but 13 gastrointestinal hormones, changes in cell membrane fluidity, and lipid metabolism have been proposed to mediate this regulation. Diet affects the secretion of hormones from the endocrine pancreas and from the intestine, and several of these hormones mediate selective patterns of gene expression in the exocrine pancreas in response to diet. The extent of the influence of intestinal hormones on the expression of pancreatic lipase in response to dietary fat is not known, but secretin and other members of the secretin family can regulate this lipase. Dietary fat also affects cell membrane fluidity directly and influences many physiological processes indirectly. These effects occur ov~r an extended period of time indicating that the post-absorptive metabolites of dietary fat are responsible for these changes. The mechanisms involved in the effects of dietary fat consumption and altered physiology are an active area of research. Diseases which affect energy utilization also alter the metabolism of fat post-absorptively. Diabetes and high fat feeding can result in abnormally high plasma levels of the triglyceride metabolite, ketones. Ketones have also been proposed as a mediator in pancreatic lipase expression, but the effects of ketones on lipase expression remain unclear.

This study examined the effects of the amount and the type of dietary fat on pancreatic lipase content, synthesis, and mRNA levels, at both pre-absorptive and post-absorptive stages of fat metabolism. The objectives of this study were to determine 1) the mechanism(s) whereby degree of saturation regulates pancreatic lipase; 2) the relative importance of luminal digestion and absorption and post-absorptive transport and metabolism of dietary fat to this regulation; and

3) the effects of amount and type of dietary fat on circulating levels of the ketone {3- hydroxybutyrate. In the first series of experiments designed to address objectives 1 and 3, male

Sprague-Dawley weanling rats were fed for 7 days diet with low fat (11 % of energy, LF) or moderate fat (40% of energy, MF) containing fats varying in saturation. Pancreatic enzyme 14 content, synthesis, mRNA levels, and blood ketones were determined. In the second series of

experiments to address objective 2, male Sprague-Dawley rats were fed LF and MF diets with

and without a specific inhibitor (Pluronic L-81) ofthe post-absorptive transport of digested dietary triglycerides. Pancreatic enzyme content and mRNA levels were determined. 15

CHAPTER 2

LITERATURE REVIEW

LIPID METABOLISM

Digestion, absorption, transport, and utilization of lipids is different from the other macronutrients because of their nonpolar properties. Lipids are soluble in organic solvents such as ether, chloroform, and acetone. This property allows the lipids to act as barriers in a water­ soluble environment. The body can synthesize many lipid compounds, however, essential fatty acids (EFA) exist which must be included in the diet to provide particular building blocks. The biological functions of EF A incl ude stimulation of growth, regulation of cholesterol metabolism, lipotropic activity, maintenance of growth and other physiologic and pharmacologic effects. Most importantly, on the molecular level, these fatty acids affect membrane integrity and are components of specific lipids. EFA are characterized by an unsaturated bond within the last 7 carbons of the fatty acid chain, toward the methyl end, typically n-6. The EFA are essential because the body cannot make them and they are precursors for arachidonic acid which form prostaglandins, leukotrienes, and thromboxanes (Linder, 1985). Another group offatty acids, n-

3, have been proposed to be essential along with the n-6 fatty acids because of the important role n-3 plays in structural lipids. The high amounts of n-3 found in the brain and in the retina has raised the question of n-3 essentiality during development; and although recent studies suggest this, it remains controversial in human nutrition (Sardesai, 1992). 16

Dietary Fats

Fats in the diet come from many different sources, but the two main categories are animal and vegetable sources. Each of these major sources are further characterized by their physical properties and known effects on health.

Phvsical properties. Fats and oils are chemical combinations of polyhydroxy alcohol glycerol and fatty acids (Mayes, 1988). The majority of dietary fats are made up of triglycerides

(TG) in which three fatty acids are esterified to glycerol. The chain length of fatty acids affects the digestion of triglycerides. Short chain fatty acids are less than 6 carbons long. These are mostly end products of carbohydrate fermentation by rumen organisms, however butyric acid

(C:4) is found in butter. Medium chain triglycerides (MCT) range from 6 to 14 carbons long and are found in tropical oils in small amounts (Hall, 1989). These are produced commercially because they can be absorbed more easily through a different mechanism than long chain triglycerides (LCT) and are useful in clinical nutrition therapy. The LCT include fatty acids which are 16 carbons or longer, but refer predominately to C: 18 and longer. Most TGs have a different fatty acid on each of the 3 glycerol ester positions, making them mixed TGs. The glycerol carbon atoms are labeled sn-I, 2, or 3 and various fatty acids preferentially occupy certain positions. Saturated fatty acids are usually found in the sn-l and the sn-3 position while unsaturated fatty acids usually occupy the sn-2 position. Although dietary TGs are composed of mixed TG, each source of dietary TG has a fairly constant fatty acid profile.

The level of saturation and chain length of fatty acids contribute to the TGs physical properties. The number of double bonds, or unsaturation, has a greater influence on the melting point of the fatty acid than does carbon chain length. Long chain unsaturated TGs have a much lower melting point than do long chain saturated TGs. Long chain saturated TGs have a higher 17 melting point than do medium chain saturated TGs. The location of the double bond closest to the methyl group is important in biological function. The vegetable oils contain predominately n-6 fatty acids and a diet high in n-6 decreases plasma lipids and increases membrane fluidity compared to saturated fatty acids, which predominates in animal fats.

The presence of double bonds also make fatty acids more susceptible to oxidation.

Commercial products contain preservatives to retard oxidation. Although polyunsaturated fats naturally contain antioxidants, antioxidants such as butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT) are often added to slow down free radical formation in the initiation stage of oxidation, thus improving the shelf life of the product. The naturally occurring antioxidant compounds include beta carotene and tocopherols. Polyunsaturated fatty acids (PUFA) are oxidized more rapidly than saturated or monounsaturated fatty acids, except for the long-chain

PUFA derived from EFA [dihomogamma linolenic acid (DHGL), arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA)], which are spared from oxidation

(Sardesai, 1992).

The variety and complexity in physical properties of dietary fats makes the study of the effects of dietary fat on health very challenging. Epidemiological studies have shown a strong correlation between the amount and type of fat and particular diseases, but demonstrating causal relationship and determining the mechanisms whereby dietary fat affects disease processes is complex.

Health and disease interactions. Excessive dietary fat intake has been linked to an increased risk for obesity, coronary heart disease, and certain cancers (Public Health Service,

1991). These diseases develop following abnormal tissue changes and changes in the expression of an unknown number of genes (Coleman, 1978; Bishop, 1991; MacCluer and Kammerer, 1991). 18 The correlation between a high fat diet and these diseases makes it likely that dietary fat directly

or indirectly regulates the expression of genes involved in the initiation or promotion of

pathological conditions.

There are many studies that link obesity with the excessive intake of saturated fats

(Parker et aI., 1993). Other studies report that fish oil intake is protective against obesity and

glucose intolerance (Feskens, Bowles and Kromgout, 1991). One very important and broad

influence that dietary fat has on the body is the ability to alter the composition of phospholipids

(Pan, Hulbert and Storlien, 1994), which affects membrane fluidity. Both animal and human

studies have reported that changing the membrane phospholipid profile by diet may alter metabolic

rate. Increased levels of saturated fatty acids in the diet lead to a decreased metabolic rate and

thereby increase the risk for obesity (Pan et aI., 1994). One explanation for this phenomenon is

that a membrane fatty acid profile high in polyunsaturated fatty acids (PUFA) leads to a more

fluid, or "leaky" membrane, allowing cells more access to sodium and potassium and

mitochondria more access to protons. Worthy of note, dietary arachidonic acid is preferentially

incorporated into the membrane phospholipids. Thus, membrane lipids may have a "pacemaker"

role in the overall metabolic activity of the body.

Diet has long been associated with elevated risks of heart disease. The effects of dietary substrates on plasma lipid levels have been the focus in a large amount of research.

Marine oils, which are rich in n-3 fatty acids, have the effect of lowering plasma TG more effectively than vegetable oils rich in n-6 fatty acids. The precise mechanisms by which fish oils mediate this effect is not known; however, data suggest that both the degree of saturation and the precise position of the double bonds may playa role in these effects (Coniglio, 1993). Saturated and monounsaturated fatty acids normally do not raise fasting concentrations of TG. When 19 monounsaturated fat is exchanged for carbohydrate, the fasting TG decreases. PUFA of the n-6

type may lower fasting TG levels in some people, but not others (Norum, 1992). In cholesterol

metabolism, dietary fatty acid saturation and chain length alter plasma cholesterol levels (Hayes

and Khosla, 1992). Polyunsaturated and monounsaturated dietary fats have a hypocholesterolemic

effect when compared to saturated dietary fats; however, short chain fatty acids have a

hypercholesterolemic effect when compared to long chain saturated fatty acids (Mattson and

Grundy, 1985; Denke and Grundy, 1991). Unsaturated fatty acids tend to result in larger

chylomicrons than saturated fatty acids, which may influence postprandial chylomicron clearance

because large chylomicrons appear to be cleared more rapidly than small chylomicrons (Norum,

1992). Other research on heart disease has involved the manipulation of low density lipoprotein

(LDL) receptors which are involved in atherosclerosis; however, this defect represents only 5% of people with atherosclerosis (MacCluer and Kammerer, 1991). Also, lecithin:cholesterol

acyl transferase (LCAT) has been extensively studied because of its central role in lipoprotein metabolism as a catalyst in the formation of cholesteryl esters in blood plasma (Norum, 1992).

The involvement of multiple genes in the development of atherosclerosis has been well documented, but the precise influence of diet on the regulation of these genes is still a mystery

(Kaput et aI., 1994).

Both environment, including diet and genetics, are known to be involved in r. carcinogenesis. In contrast to heart disease, various forms of cancer have been associated with increased intake of polyunsaturated dietary fats. Breast, prostate, and colon cancer are all positively correlated with a high fat diet (Cave, 1991). A suggested mechanism by which dietary fat affects colon cancer is the modification of the colonic cellular membrane lipids. Cell proliferation, which is increased in colon cancer, is influenced by membrane properties such as 20 fluidity, permeability, ion transport and enzyme activities (Bleiberg, Buyse and Galand, 1985).

Diets high in polyunsaturated fats are reflected in membrane phospholipids, and arachidonic acid is preferentially in the sn 2 position. Upon membrane or receptor stimulation, arachidonic acid is released from membrane phospholipids by phospholipase Az and is a precursor to bioactive compounds such as prostaglandins and leukotrienes, which are involved in immune function and inflammation. Release of arachidonic acid in the cell also activates protein kinase C, and this association can increase neoplastic activity (Merrill, 1989).

The role of excessive dietary fat and the etiology of so many chronic diseases underscores the power of lipids and their metabolites. Whether the effects are direct or indirect varies in different diseases. Elucidating the role of dietary fat regulated genes in the development of chronic diseases is an important focus of current research.

Digestion

An overview. Luminal digestion of dietary fats utilizes most of the GI tract. The gastrointestinal (GI) tract is a 26 foot long muscular system extending from the mouth to the anus.

Its principal organs are the stomach and intestines in which digestion of food occurs. The GI tract organs do not work autonomously, but require enzymatic, chemical and hormonal input from other ancillary organs such as the pancreas and the liver. This system allows the body to break down complex foods into absorbable nutrients. Digestion of dietary fat composed of triglycerides, phospholipids, cholesterol esters and fat-soluble vitamins and their esters yields monoglycerides, fatty acids, cholesterol and fat soluble vitamins that· can be absorbed.

Digestion begins in the mouth but is concentrated in the stomach and the duodenum where enzyme activities are greatest. Even so, luminal digestion with enzymes from other 21 sources is important to prepare chyme for the activity of pancreatic enzymes in the duodenum and to follow up the process.

In the mouth, mastication is the first step in reducing food stuff into a manageable size.

Saliva secreted by glands in the mouth contains proteins, which initiate enzymatic breakdown of particles, as well as providing a lubricating medium to aid in movement toward the stomach.

Two such enzymes are salivary amylase and, in rats, lingual lipase (Carriere et al., 1994a).

Salivary amylase can hydrolyze starch and glycogen to maltose but becomes inactive in pH less than 4.0; therefore, salivary amylase has a brief role in the initiation of digestion. Lingual lipase, however, can continue its lipolytic activity in the low pH conditions of the stomach and therefore can hydrolyze dietary triglycerides in the stomach. Milk contains short and medium chain triglycerides; and, so, lingual lipase is important for pups who are suckling. However, the majority of dietary fat in adult diets contain long chain fatty acids that are digested and absorbed in the small intestines.

In the stomach, food that has been chewed with saliva is swallowed as a bolus down the esophagus to the stomach. Entry into the stomach is through the cardiac sphincter which closes in between boluses. Once the bolus enters the stomach, gastric juice is secreted. Gastric juice is predominately water; the remainder is mucin, organic salts and digestive enzymes (Mayes,

1988). Humans and other mammals have a gastric lipolytic enzyme, gastric lipase, which functions similarly to lingual lipase in rats; however, gastric lipase activity requires a low pH environment (Carriere et al., 1994a). Hydrochloric acid (HC)) is secreted by the stomach's parietal cells with the production assisted by H,K-ATPase (Sachs, 1994). The HCl denatures proteins making them more accessible for proteolytic enzymes, such as pepsin, which is secreted as the proenzyme, pepsinogen, by the stomach's chief cells. Dietary proteins must be partially 22 broken down in the stomach so that the intestinal and pancreatic enzymes can complete their digestion. The stomach contents exit through the pyloric sphincter into the duodenum and are descriptively referred to as chyme. Chyme is intermittently released from the stomach, which can allow for a 2 to 4 h retention time for food in the stomach.

The pancreatic and bile ducts enter the duodenum very near the pyloric valve. The secretions from these ducts contain alkaline solutions which neutralize the acidic chyme and provide enzymes and emulsifiers necessary for complete digestion and absorption of the majority of nutrients. Secretion of bicarbonate by the pancreas regulates the pH of the intestinal lumen, neutralizing the acidic chyme from the stomach. The pH of the duodenum plays a role in the functioning of pancreatic enzymes and the ionization of long chain fatty acids released during hydrolysis. The partial ionization offatty acids moves them to the oil-water interface and out into the aqueous phase and into mixed bile salt micelles (Borgstrom, 1993). Bile salts are also affected by pH through ionization. The ionized bile salts solubilize into micelles. When pH is low in the intestine, the protonated bile salts have a low solubility (Zentler-Munro et aI., 1984).

In the intestine, the highly differentiated epithelium of the small intestine must orchestrate the secretions and absorption of nutrients to sustain life (Mayes, 1988). Once the food stuff has been fully digested by enzymes the components can then be transported from the brush border of the intestine through the epithelial layer to the mucosal blood and lymph vessels for distribution. The intestinal epithelium must also function in the opposite direction to provide secretions for digestion or for other regulatory functions necessary for proper digestion. Both psychological activity and chemically-induced gastrointestinal hormone release are responsible for coordinating proper digestion and absorption. 23 The small intestine is responsible for 90% of digestion and absorption of the ingested foodstuff, including dietary fat, and is divided into three major segments. The duodenum is the segment proximal to the pyloric sphincter, followed by the jejunum and then the ileum. The upper small intestine is where most intestinal secretions occur. The bile duct secretes bile acids which have been stored in the gallbladder or delivered directly from the liver. Bile acids are necessary for emulsifying dietary lipids so they can be hydrolyzed and absorbed. Once the bile acids have performed their function, bile is mostly recycled by being absorbed into the enterohepatic circulation.

Preduodenallipases. Preduodenallipases vary among species. Mice and rats secrete lingual lipase from the von Ebner's gland, whereas humans, monkeys, rabbits, horse, dogs, pigs, cats and guinea pigs secrete gastric lipase from the stomach (Carriere et aI., 1994a). In humans, the chief cells of the gastric fundus synthesize and secrete gastric lipase (Borgstrom, 1993). This lipase belongs to the acid lipase group for which low pH provides stability. Gastric lipase does not hydrolyze phospholipids, is not bile salt sensitive, and is specific to primary triglyceride ester bonds, particularly MCT ester bonds. The main products from the activity of gastric lipase are diglyceride and fatty acid. The medium chain fatty acids are partly absorbed in the stomach

(Levy et aI., 1984) and transported via the portal vein into the liver, where they appear to be extensively oxidized (Chanez et aI., 1991). Unlike LCT, significant amounts of MCT are absorbed intact as triglyceride molecules (Greenberger, Rodgers and Isselbacher, 1966). A study investigating limitations of MCT absorption found that the molecular weight of the MCT did not affect its intact absorption by the small intestine (Chow, Shaffer and Parsons, 1989).

Duodenallipases. The pancreas secretes into the duodenum several enzymes necessary for hydrolysis of lipids. Lipase and its cofactor, colipase, are primarily responsible for normal 24 digestion of dietary fats, particularly LCT; and both must be present to hydrolyze TG to 2- monoglycerides and fatty acids. Pancreatic lipase and colipase will be discussed in greater detail in later sections. Other pancreatic enzymes necessary for proper fat digestion are carboxyl ester

lipase and phospholipase A2 •

Carboxyl ester lipase has also been called cholesterol esteras~, sterol ester hydrolase, nonspecific lipase, bile-salt-stimulated lipase, and lysophospholipase. The actual role and physiological substrate for this enzyme are not exactly clear. It does not appear to be specific to either primary or secondary ester bonds, and it catalyzes hydrolysis of both water-soluble as well as water-insoluble esters. In humans, carboxyl ester lipase aggregates in the presence of bile salts to form dimers (Lombardo and Guy, 1980). Earlier work on this enzyme focused on its importance as a cholesterol esterase. Subsequent research reports that intact and poorly absorbed cholesterol esters undergo very little hydrolysis in the absence of carboxyl ester lipase, which is believed to be necessary for the release of free cholesterol that can then be absorbed (Borgstrom,

1993). Rudd and Brockman (1984) hypothesize that the actions of pancreatic lipase and carboxyl ester lipase are integrated. Initially, pancreatic lipase must generate the proper phase(s) for the hydrolysis of dietary cholesterol esters; and subsequently carboxyl ester lipase acts on the remaining esters, primary and secondary, present in the product phase generated during the first stage by pancreatic lipase.

Phospholipase A2 is secreted in a proform that is activated by trypsin in the intestine.

The activity of phospholipase A2 is much greater in the presence of emulsions, containing dietary and biliary phospholipids (Borgstrom, 1993). Human dietary phospholipids are derived mainly from ingestion of egg yolk and cell membrane fragments of animal and vegetables. The ratio of phospholipid to bile salt affects phospholipase A2 activity. Ratios between 0.2 to 2.0 increase 25 activity lO-fold, whereas increasing the ratio beyond 2 causes a decrease in catalytic activity

(Hoffman, 1979).

Intestinal phospholipase A~ has been described in the rat (Mansbach, Pieroni and

Verger, 1982). It originates in the enterocyte and is specific for phosphatidylglycerol. This phospholipase Az exhibits greater activity in the distal intestine and may play a role in gut bacterial control and in digestion of phosphatidylglycerol of vegetable origin.

Carboxyl ester lipase and the pancreatic and intestinal phospholipase Az are believed to work in concert with one another. It may be that the product of the carboxyl ester lipase is the optimal substrate for the phospholipase Az, although why one of these enzymes can affect the activity of another is not clear.

Bile snits. Bile is secreted from the liver. When the GI tract is at rest, bile is concentrated and stored in the gallbladder (Mayes, 1988). When a meal is eaten, the gallbladder secretes the bile, which contains bile salts, phospholipids, cholesterol, and an amphiphilic peptide and plays a major role in fat digestion. Bile salts are made up of conjugated forms of primary bile salts, secondary bile salts, and one tertiary bile salt. Primary bile salts are the greatest component of bile salts and are synthesized de novo in the liver from cholesterol. Secondary bile salts are formed in the intestinal lumen by the action of microflora. The tertiary bile salt is believed to be epimers of primary bile acids (Lombardo and Guy, 1980). Bile saIts are reutilized through the enterohepatic circulation, absorbed in the ileum, and maintained as a constant pool.

They are absorbed by passive diffusion (free bile acids and glycine conjugates) proximaIly and by active diffusion (taurine conjugates) distally. The composition of the circulating bile salts pool is the result of the combined effects of luminal metabolism by bacteria, absorption of the metabolites from the intestine, and their metabolism by the liver. The concentration of bile is 26 important for micelle formation. The molar relationship of bile salt-phosphatidylcholine- cholesterol is approximately 12:3:1, giving a PC/BS ratio of 0.25. At this ratio, simple bile salt micelles will coexist with small-sized mixed BS/PC micelles (Dam et a!., 1966).

The mixture of bile lipids with the lipid emulsion in chyme leads to the exchange of surface lipids, which probably exposes the oil-water interface of the chyme to the action of pancreatic lipase and colipase system. All the components of this process combine to decrease the surface tension, forming a stable emulsion that supplies a large surface area for pancreatic lipase (Borgstrom, 1993). The products of hydrolysis are then incorporated into the micelles, making mixed micelles. The function of the micelles is to solubilize the lipolytic products so that the lipids can be transported across the unstirred water layer to the intestinal brush border membrane (BBM) and absorbed (Thomas, 1989).

Through the action of mechanical, chemical and enzymatic digestion, foodstuff is prepared for absorption and utilization by the tissues. Absorption and utilization of dietary fat occurs through a different pathway than non-lipid hydrolysis products. Water-soluble products are diffused or transported into the BBM and released into intestinal capillaries which flow toward the portal vein to be removed, distributed or stored by the liver. The hydrolysis products of dietary fat must be absorbed, repackaged, and transported through the lymph before reaching the blood stream (Tso, 1994).

Absorption

Once the preparation of lipids for absorption is complete, they can proceed to the unstirred water layer. The unstirred water layer is a barrier comprising lamellae of poorly stirred water through which the lipid digestion products must pass before entering the BBM (Borgstrom,

1993). The movement through the unstirred water layer is believed to be the rate limiting step 27 of absorption because passage across the BBM is much quicker. The micelles partition at the

unstirred water layer. The lipid products detach from the bile salt micelles because of the low

pH microcompartment (Daniel et aI., 1985) and are absorbed. Monomers, or single molecules

such as fatty acids and glycerol flow into the aqueous phase from which they diffuse across the

BBM. As micelles are depleted of lipid in this aqueous phase, the micelles are shuttled back to

the intestinal lumen to incorporate more products of lipolysis for absorption. Eventually the bile

salts pass on to the ileum, where most are reabsorbed into the enterohepatic circulation. Other

factors influence movement across the unstirred water layer. Health and disease can change the

thickness and effective surface area of the unstirred water layer (Thomas, 1989).

Enteroc"te. The uptake of lipids across the BBM is thought to be a result of passive

diffusion, although Hollander and coworkers suggest there may be a mediated, as well as passive,

component to fatty acids (FA) uptake at the BBM (Ling, Lee and Hollander, 1989). Fatty acid

binding proteins (FABPs) are low molecular weight proteins found in several tissues. FABPs in

the cytosol of the enterocyte have a high affinity for long chain fatty acids. The plasma

membrane FABPs will bind free oleic, palmitic, arachidonic acid, but not cholesterol esters, bile

acids, nor phospholipids. These FABPs are not acutely regulated by hormones; however,

catecholamines may stimulate binding activity which requires increased cAMP indicating

phosphorylation regulation (Clarke and Armstrong, 1989). The purported roles of FA BPs include

cellular influx and efflux of FFA, FFA partitioning between oxidation and esterification, acting

as a "sink" to bind FFA as a cyto-protectant, and acting possibly as trans factors in the regulation of expression of lipogenic proteins (Clarke and Armstrong, 1989). There are several FABPs, two of which are similar to those found in the liver, and one which is unique to the intestine. FABP has a greater affinity for unsaturated fatty acids. More FABP is found in the jejunum than ilium, 28 and more FABP is found at the tip of the villi compared to the crypt (Ockner and Manning,

1974). Thus, FABP is most abundant in areas where fat absorption occurs. Earlier observations suggested that FABP may act as a transport vehicle in the enterocyte (Thomson, 1989). Transport of fat in the enterocyte is an area of interest because it still is not known how the major digestive products of lipids migrate from the site of absorption to the endoplasmic reticulum where biosynthesis of complex lipids takes place (Tso and Balint, 1986). The role(s) of FABPs are currently being investigated and remain controversial.

The long chain fatty acids absorbed in the enterocyte are ultimately utilized in the reformation of TG. Reformation of other lipids includes esterification of cholesterol and conversion of lysophospholipids to phospholipids. These reactions are catalyzed by their respective enzymes at the cytoplasmic surface of the smooth endoplasmic reticulum (SER) membrane (Field, 1984). The lipid products migrate through the cisternae of the SER to the

RER, where apoproteins are added and prechylomicron particles are formed (Tso and

Gollamundi, 1984). The final product comes together in the Golgi complex. The Golgi vesicle fuses with the lateral plasma membrane to extrude the chylomicron into the intercellular space

(Levy, 1991).

Lipoproteins other than chylomicrons are also synthesized in the enterocyte. Intestinal very low density lipoproteins (VLDL) form largely during a fasting state in order to carry endogenous lipids. Chylomicrons and VLDLs do not appear to use the same synthetic pathway

(Mahmood et aI., 1994). The last type of lipoprotein synthesized by the enterocyte are high density lipoproteins (HDL).

Despite intensive research on the overall scheme of digestion and absorption of dietary fat, little is known about the factors that regulate the intracellular assembly, transport, and 29 secretion of lipoproteins by enterocytes. DeSchryver-Kecskemeti and coworkers (1991) report an intracellular membrane vesicle containing intestinal alkaline phosphatase (lAP) surrounding lipid droplets in the region of the Golgi vesicles. A large bolus of fat stimulated the release of the intracellular membrane vesicle. The lAP and membrane structure resembles pulmonary surfactant and is much more abundant after fat feeding. Mahmood and coworkers (1994) suggested that these membrane vesicles playa role in intracellular movement of the droplet across the cell, with the subsequent delivery of fat and the particle to the extracellular environment.

Mahmood's work used a chylomicron blocker, Pluronic L-81, to monitor fat transport. Pluronic

L-81 is a nonionic hydrophobic surfactant, which causes a decrease in chylomicron formation and transport, but does not affect VLDL formation and transport (Mahmood et al., 1994). Significant decrease in lAP activity occurred after the blocker was administered. Administration of Pluronic

L-81 leads to the inhibition of intracellular movement of TG from the endoplasmic reticulum to the Golgi vesicles, producing large fat droplets in the cytoplasm. When Pluronic L-81 administration is stopped, the fat droplets are cleared from the enterocyte within 24 h, suggesting that the treatment is reversible and relatively nontoxic (Tso and Balint, 1986).

Factors regulating the formation of chylomicrons in the enterocyte are not completely known. The release of chylomicrons from the enterocyte requires the presence of apoprotein B

(apo B). However, it is not known if the rate of production of apo B is the physiologically rate limiting step for chylomicron formation (Tso and Balint, 1986).

Transport. The post-Golgi vesicles containing the chylomicrons and VLDL migrate toward the lateral membrane of the enterocyte, where they fuse with the membrane and release the lipoproteins into the intercellular space (Hubscher, Smith and Gurr, 1964). The basolateral membranes of adjacent enterocytes are close to each other, and separate the enterocyte from the 30 lamina propria. During active lipid transport, the intracellular space is filled with chylomicrons.

When this occurs, it is possible that there may be breaks in the basement membrane, which facilitate the movement of chylomicrons from the intercellular space to the lamina propria (Tso and Balint, 1986).

The lamina propria is connective tissue. It contains numerous cell types and is well supplied with nerves, blood and lymph vessels, and smooth muscle cells. The lacteal, which is a blind-ended lymph vessel, is located centrally at the core of the lamina propria. The mechanism through which the chylomicron migrates from the intercellular space to the lacteal is not known

(Tso and Balint, 1986). The accumulation of absorbed lipids, predominantly chylomicrons, form a milky fluid called chyle that collects in the lymph vessels of the abdominal region and passes into systemic blood through the thoracic duct. The absorbed lipids can then be transported to the tissues for utilization.

Tissue Utilization

Triglycerides (TG). During positive caloric balance, a significant proportion of dietary

TG is stored as fat in adipose tissue. However, during conditions of starvation, fatty acids are mobilized from adipose tissue and oxidized by a variety of tissues to provide energy. Oxidation of fatty acids occurs in the mitochondria of the cell (Mayes, 1988) and spares glucose for tissues

(brain and erythrocytes) that require only glucose. Regulatory mechanisms are in place to ensure a supply of suitable fuel for tissues. Several of the endocrine pancreas hormones are necessary for these mechanisms and will be discussed later. Another role of fatty acids in tissue utilization is in membrane integrity. The fatty acid arachidonic acid influences membrane fluidity and serves as an active metabolite in signal transduction (Clandinin et aI., 1991). 31 Ketones. Ketone bodies, metabolites of fatty acid oxidation, include acetoacetate, hydroxybutyrate, and acetone (Mayes, 1988). The liver produces ketone bodies under conditions associated with a high rate of fatty acid oxidation. Ketones can be oxidized in extrahepatic tissue and utilized as fuel. Higher than normal quantities present in the blood and urine are called ketonemia and ketonuria, respectively, and this condition is cdlled ketosis. Ketosis is primarily associated with diabetes mellitus, threatens normal blood pH, and can be fatal if left uncontrolled.

Lipoproteins. Lipids must be associated with water soluble molecules in plasma to reach tissues. This is accomplished by lipoprotein molecules, which contain nonpolar lipids, amphipathic lipids, and proteins (Mayes, 1988). The lipoproteins are distinguished by their density. Chylomicrons are synthesized in the intestine and have a very low density, less than

0.96, and contain 88% of total lipid as TG. Chylomicrons are associated with the protein apo

B48 and acquire apo C in the circulation. The VLDLs are synthesized predominantly in the liver and to a lesser extent in the intestine. The density of VLDL ranges from 0.96-1.006, and the

VLDL contains 56% of total lipid as TG. VLDL is associated with apo B100 and apo E.

Intermediate density lipoproteins (IDL) are partially metabolized chylomicrons and VLDL whose density ranges from 1.006-1.019. The lipid profile is now 29% TG and 34% cholesterol esters.

Low density lipoproteins (LDL) are the further metabolized product of IDL with density ranges from 1.019-1.063 containing 13% TG and 48% cholesterol esters. Finally, the liver and intestine synthesize HDLs (two fractions exist distinguished by their density) with density ranges of 1.063-

1.210. HDLs contain approximately 15% TG, 30% cholesterol esters, and 33-57% protein. The major proteins associated with HDL are apo AI and apo All.

Lipoproteins are the predominant form of lipid in circulation (Mayes, 1988).

Chylomicrons and VLDL function to distribute triglycerides to tissues, mainly adipose and 32 muscle. The apo C on the surface of the lipoprotein activates lipoprotein lipase (LPL) which is attached to the endothelial lining of the small blood vessels in the tissues (Breslow, 1994). As the VLDL gives up TG, it becomes JDL, and finally LDL with the assistance of HDL and lecithin-cholesterol acyl transferase (LCAT). The excess phospholipid, cholesterol, and protein are transferred to HDL and the chylomicron remnants are taken up via a receptor by the liver

(Shepherd and Packard, 1993). Fatty acids synthesized in the liver are packaged with cholesterol, phospholipids and apo B to form VLDL. The effects of diet on VLDL packaging and secretion are somewhat controversial. Cianflone and coworkers (1992) report that although both high carbohydrate and high fat diets increase VLDL synthesis, the high carbohydrate induced VLDL are cleared much quicker. The VLDL produced from high carbohydrate diets are larger and more likely to be catabolized without being converted to LDL than the VLDL produced from a high fat diet (Sniderman and Cianflone, 1993). Studies following LDL levels show diets high in saturated fats significantly raise plasma LDL (Nicolosi et aI., 1990). This effect appears to be the result of impaired catabolism of LDL rather than increased synthesis. High fat and high cholesterol diets in transgenic mice whose LDL receptor gene was driven by the transferrin promoter had little effect on plamsa VLDL, IDL, and LDL levels, whereas control mice showed a significant increase in all three lipoprotein fractions (Breslow, 1994). The effects of dietary fat on HDL are less clear because of the heterogeneity in HDL particles and the differences between animal models; however, diets high in PUFA tend to decrease HDL levels whereas diets high in saturated fatty acids tend to increase HDL levels (Ahn et aI., 1994).

The functions of these lipid products once they reach the tissue is varied. TGs are the form of fat most efficient for storage. The bulk of TG is found in adipose tissue where it comprises 99% of the cellular volume (Mayes, 1988). Adipocytes are found throughout the body 33 as discrete tissue and interspersed between muscle and connective tissue. TG can be converted to cholesterol, phospholipid and other lipids if required. Most importantly, adipose tissue functions to pad and insulate our bodies to protect them from injury. Phospholipids and cholesterol function primarily for the formation of cell membranes. These lipids can form a bilayer with cholesterol in the center and phospholipids at the exposed surfaces. Both phospholipids and cholesterol are substrates for other compounds. Cholesterol is a component of bile and it is a precursor to steroid hormones.

PANCREAS

The pancreas is a slender, elongated organ that runs horizontally behind the greater curvature of the stomach and is situated between the stomach and duodenum. This organ produces more protein per gram of tissue than any other organ of the body (Scheele, Bartlet and

Bieger, 1981). The main function of the pancreas is to provide secretions necessary for proper processing of ingested nutrients and to maintain blood glucose. These functions are possible because the pancreas is a mixed exocrine-endocrine gland (Gorelick and Jamieson, 1994). The exocrine cells make up 85% of the cell mass; the endocrine cells only make up 2%, ductal cells; blood vessels make up 4%; and the remaining tissue is extracellular matrix. The arterial supply comes from branches of the splenic artery which forms corridors with the gastroduodenal and superior mesenteric arteries. The autonomic innervation of the pancreas uses both the sympathetic and parasympathetic divisions.

Endocrine Pancreas

Hormones. The endocrine pancreas consists of several cell types in islets interspersed throughout the exocrine tissue, called islets of Langerhans (Granner, 1988). Although the 34 endocrine pancreas is 2% of pancreatic volume, as much as 20% of the intrapancreatic blood flow is directed toward the islets making them well vascularized. Seventy five percent of the islets are

{3 cells, 20 % are a cells and 5 % are .!l cells. In human islets, the cells are arranged so that the a cells are the outermost in the islet, the .!l cells are intermediate, and the {3 cells are innermost.

The islet cells specialize in the production of peptide hormones. The a cells produce glucagon.

The .!l cells produce somatostatin and pancreatic polypeptide. The {3 cells produce insulin. The islet cells are arranged so that they tend to release their hormones into venules which are located on the surface of the islets. These venules drain away from the centrally located {3 cells and then perfuse the exocrine acini located around the islets.

Insulin. The human {3 cells secrete 40-50 units of insulin a day, which represents about

15-20% of the hormone stored. The secretion of insulin occurs primarily in response to an increase in plasma glucose. Insulin secretion occurs rapidly and is thought to be preceded by transcriptional and post-transcriptional events (German, Moss and Rutter, 1990). Intracellular utilization of glucose is facilitated when insulin binds cell surface receptors to elicit a cellular response. Other functions of insulin include lipogenic and anabolic effects, which have serious consequences in formation and clearance of lipoproteins associated with heart disease. The lack of insulin will diminish transport of amino acids into the cell and lead to increased lipolytic activity in liver and adipose cells. Once the capacity for oxidizing fatty acids is exceeded in the liver, ketones begin to accumulate. The inhibition of lipolysis by insulin is believed to occur by activating a phosphatase which results in decreased cAMP levels (Granner, 1988). In addition, insulin stimulates the proliferation of many types of cells in culture, and plays a role in the regulation of cell growth (Scheele, 1993). 35 Glucagon. Glucagon secretion is inhibited by glucose and causes rapid mobilization

of potential energy sources by stimulating glycogenolysis and lipolysis (Granner, 1988).

Glucagon is the most potent gluconeogenic hormone and is also ketogenic. Glucagon circulates

in plasma in the free form and has a very short half life. When glucagon reaches the liver, it is

inactivated; thus the concentration of glucagon is much greater in the portal vein than in the

peripheral circulation.

Somatostatin. Somatostatin is a cyclic peptide which was first isolated from the

hypothalamus as an inhibitor of growth hormone secretion (Granner, 1988). Somatostatin is also

found in the pancreatic islets, many gastrointestinal tissues, and in the central nervous system.

In the pancreas, somatostatin inhibits other islet cell hormones through paracrine means. In large

amounts, somatostatin can reduce ketosis associated with acute insulin deficiency by inhibiting

glucagon release. Somatostatin decreases delivery of nutrients from the GI tract into the

circulation by prolonging gastric emptying which decreases gastrin secretion, exocrine pancreas

digestive enzyme secretion, splanchnic blood flow, and glucose absorption.

The endocrine pancreas is a complex system which responds to many extracellular

signals. The endocrine pancreas hormones are released into the pancreatic vein, which empties

into tht portal vein. This is an efficient arrangement, since the liver is the primary site of action

of insulin and glucagon in carbohydrate metabolism.

Exocrine Pancreas

Several cell types exist in the exocrine pancreas (Gorelick and Jamieson, 1994). The basic subunit of the exocrine pancreas is the acinus, which is mostly acini cells clustered together to utilize common ducts and bound together by connective tissue matrix. The acinar cells synthesize and secrete enzymes necessary for proper digestion of foodstuff. These cells are able 36 to synchronize secretion and conduct other cell to cell communication through gap junctions.

Other cell types are ductal cells and centroacinar cells, which mark the beginning of the ductular

system. Several types of collagen and other extracellular matrix molecules make up the

supporting matrix of the pancreas.

Ultrastructure. Although the exocrine pancreas has always been described as being

organized into true acinar units, studies using wax casts of human pancreas demonstrate that the

final ductular subdivision is an anastomosing tubular arrangement (Brockman, 1978).

Experiments using electrical coupling on mouse pancreas show the electrical subunit of the

pancreas to be approximately 500 cells in size (Petersen, 1982). The above studies indicate that

the functional portion of the pancreas is both morphologically and electrically larger than the

previously described 20 - 50 cell acinus. The individual acinar cells are coupled together by gap junctions which allow the propagation of signals throughout a population of acinar cells. These

signals are necessary to generate secretory responses as well as other cell functions. The gap junctions appear to have a small diameter allowing only molecules between 500 and 1000 daltons to pass (Bennett and Verselis, 1992). These communication molecules are ions and other small

metabolic compounds.

The majority of the pancreas is made up of acinar cells. These cells have a pyramid

shape with the apex facing the ducts for secretion while the basal portion faces the blood supply.

The basolateral plasma membrane functions as the site for uptake of nutrients from circulation

(Gorelick and Jamieson, 1994), and contains several types of hormone receptors. Binding of GI

hormones to their receptors stimulates the exocrine pancreas through second messengers systems

(Yule and Williams, 1994). The binding of the hormone cholecystokinin (CCK) and the neurotransmitters acetylcholine (Ach) and bombesin to their specific receptors activates 37 phospholipase C to generate inositol triphosphate which increases intracellular calcium.

Diacylglyceride (DAG) is also generated, which in turn increases protein kinase C (PKC). The binding of vasoactive intestinal peptide (VIP) and secretin to their specific receptors activates adenylate cyclase to increase 3'5' -cyclic monophosphate (cAMP) and protein kinase A (PKA).

These second messenger systems are thought to stimulate secretion by altering the pattern of cellular protein phosphorylation (Hootman and Williams, 1986). Both systems involve G-proteins in the initial transmembrane signal. The G proteins involved in secretin stimulation are either the

stimulatory (G,) or inhibitory (G1). A distinct G protein is involved in CCK stimulation, Gp, which in turn activates phospholipase C (Schnefel et aI., 1988). Stimulation via these messenger systems signals secretory vesicles stored in the apical portion of the acini to secrete digestive enzymes into the lumen of the duct. These secretory vesicles that store digestive enzymes and are called zymogen granules (ZG).

Digestive enzymes. There are 20 major proteins synthesized and secreted by the acinar cell. Most are necessary for efficient digestion of dietary compounds though the exact physiological role of some is stilI not known. These enzymes can be divided into 4 major categories: Proteases, glycosidase, lipases and nucleases (Lowe, 1994).

Proteases. The proteases are the greatest in number of digestive enzymes, with 12 different enzymes, several of which are isozymes. All of the known proteases are secreted in the inactive form (Light and Janska, 1989). The activation of these enzymes involves a secreted protease, trypsinogen, and enteropeptidase, a brush border intestinal enzyme. Trypsinogen accounts for 20% of the protein in pancreatic fluid (Guy et aI., 1978), and is cleaved by enteropeptidase upon entering the duodenum to produce trypsin, the active form of the enzyme.

Trypsin, in turn, can then activate other inactive enzymes present in the pancreatic fluid. 38 The exocrine pancreas contains both endo- and exopeptidases (Lowe, 1994). Trypsin

(E.C.3.4.21.4), chymotrypsin (E.C.3.4.21.1) and elastase (E.C.3.4.21.11) are endopeptidases which have specific cleavage sites. Trypsin targets peptide bonds between basic amino acids and the adjacent amino acid; chymotrypsin targets hydrophobic amino acids with aromatic side chains; and elastase, which has a broader effect, targets amino acids with aliphatic side chains. The exopeptidase is carboxypeptidase (E.C.3.4.17.1), a zinc metalloenzyme which cleaves carboxy terminal amino acids from proteins.

Glycosidase. a-Amylase (E.C.3.4.1.1) is the only glycosidase in pancreatic fluid, representing 25-30% of the total protein in pancreatic secretions (Scheele, 1986). Amylase cleaves 1,4 glycoside bonds in dietary starch to produce a-dextrins. Foodstuff encounters salivary amylase prior to pancreatic amylase; and though they have the same function, salivary amylase is present for only a short time because the low pH of the stomach terminates its activity. Once amylase has cleaved starch to produce dextrins and maltose, the intestinal enzymes a-dextrinase and maltase are present to cleave their respective substrates to produce glucose for absorption.

Lipases. Triglyceride lipase, or pancreatic lipase (E.C.3.1.1.3), predominantly hydrolyses dietary triglycerides at position sn-l and 3 to yield free fatty acids and 2- monoacylglyceride (Lowe, 1994). Pancreatic lipase must be anchored at the oil-water interface of lipid emulsions by colipase before it can exert its activity. Bile is also necessary in this process and is secreted along with pancreatic juice into the duodenum during digestion as discussed earlier. Bile lowers surface tension and enables emulsification of dietary fat, thus preparing micelles necessary for triglyceride hydrolysis. Normally, pancreatic lipase is inhibited by the presence of bile, however, the presence of colipase negates this inhibition. Pancreatic lipase will be discussed more thoroughly later in the text. 39 Carboxyl ester hydrolase (E.C.3.1.1.1), or cholesterol esterase, has several substrates

(Lowe, 1994), and catalyzes the hydrolysis of water soluble esters as well as water insoluble esters such as cholesterol oleates. Bile salts must be present for activity, hence, another name given is bile salt-stimulated lipase. Another lipase present in pancreatic fluid, as well as other tissue, is phospholipase A~ (E.C.3.1.IA). This lipase catalyzes the hydrolysis of the 2-acyl ester bond of phosphoglycerides to form Iysophospholipids. Phospholipase typically hydrolyses arachidonic acid from the number 2 position. This fatty acid has many important physiological roles.

Nilcleases. There are two nucleases present in the exocrine pancreas, deoxyribonuclease I (DNase I) and ribonuclease (RNase). DNase I degrades double-stranded

DNA to yield 5' -oligonucleotides and requires divalent metal ions for activity (Lowe, 1994).

Though DNase I has been recognized in pancreatic fluid for years, its significance is not clear.

RNase has been studied extensively. Human pancreatic fluid contains RNase, but it is much less abundant than in other species. The function of RNase in ruminants is to recover phosphorus from bacterial RNA, however, this function is not required in humans. Pancreatic RNase is specific for ribose and will cleave RNA after a pyrimidine. The presence of pancreatic RNase in animal tissues is an important issue when isolating tissue for RNA quantification. The abundance of RNase in tissues requires special attention for tissue preparation in otherwise standard protocols.

S\'nthesis and secretion. The pancreas has one of the highest rates of protein synthesis with more than 90% of newly synthesized proteins targeted to the secretory pathway (Scheele and

Kern, 1993). These proteins are synthesized as longer polypeptide chains containing N-terminal extensions of 15-30 amino acid residues (DeViIlers-Thiery et aI., 1975). The extension residues, 40 known as signal peptides, have hydrophobic segments coded at the 5' of mRNA which serve as targets for the rough endoplasmic reticulum (RER). Immediately following translation of the mRNA by free ribosomes, the signal peptide allows translocation of the nascent protein across the membrane into the cisternal space of the RER. The hydrophobic segment is recognized by a cytoplasmic signal recognition particle (SRP) consisting of 6 polypeptides and a RNA molecule which halts translation and "docks" the nascent peptide chain into the lipid bilayer of the RER membrane via a G-protein driven SRP receptor (Scheele and Kern, 1993). A key feature of RER targeting is that as soon as the specific amino-terminal signal sequence appears from the large subunit, it immediately associates with the SRP. The mRNA-SRP complex associates with a SRP receptor on the ER membrane; the SRP dissociates from the complex into the cytosol, allowing translation to continue. The SRP returns to recognize and "dock" another signal peptide (Gorelick and Jamieson, 1994).

Once the protein is fully synthesized, the signal peptide is cleaved and the complete polypeptide is released to the intravesicular space (Evans, Gilmore and Blobel, 1986). Native tertiary structure of the proteins is believed to be developed in this space. Recent findings concerning protein folding indicate specific factors, or chaperons, assist in the folding process.

Scheele and Jacoby (1982) re-analyzed their data in light of these findings and noticed that the secretory proteins which were synthesized without the presence of microsomal membranes showed conformational instability leading to little or no biological activity. When microsomal membranes contain chaperons were present, the proteins were processed into conformationally mature translocation products exhibiting biological activity, as well as solubility in an aqueous environment. Chaperons provide several types of functional assistance, including the transient interaction of nascent polypeptide chains with hydrophobic surfaces during the folding process, 41 thus preventing nonproductive (inappropriate) protein-protein interaction. Chaperon activity

occurs until the free-energy state of the folded protein excludes further interaction with chaperons

(Scheele and Kern, 1993). Other processing for protein maturation involves glycosylation and

disulfide bond formation. Both glutathione and a protein disulfide isomerase catalyze disulfide

bond formation. The RER is also where glycosyl phosphatidylinositol (GPI) anchors are attached

(Low, 1989). These folding, disulfide bond formation, and glycosylation processes render

proteins identifiable so they can be sorted and targeted to their final destination.

The steps for synthesis and secretion are as follows: first, the secretory proteins are

synthesized and processed in the cisternal space of the RER, which is interconnected to other

cisternal compartments such as the Golgi complex. The interconnection occurs through membrane

budding and fusion events (Palade, 1975). Second, the proteins go through further covalent

processing in the Golgi complex which consists of cis, mid, and trans Golgi. Third, the proteins

are shuttled into the trans-Golgi network (TGN) which serves as a central membrane compartment

where the proteins are sorted for final destination. Most secretory proteins go through a regulated

pathway by first budding into condensing vacuoles (CV) which then mature into ZG. The

maturation of ZG is believed to be a pH and ion dependent mechanism where the secretory

proteins condense into aggregates so that large quantities of protein are stored within a minimum of intracellular granule space (Orci, Ravazzola and Anderson, 1987). ZG release their contents into the ductal lumen upon cholinergic and hormonal stimulation. The regulated exocytosis of

ZG contents is ATP and calcium dependent and believed to involve low molecular weight GTP­ binding proteins (Scheele and Kern, 1993), but remains poorly understood.

Scheele (1983) developed an in-vitro system of pancreatic lobules as a model of pancreatic acinar function. Using radiolabeled amino acids, Scheele's studies provided evidence 42 that secreted proteins remain confined to membrane-bound organelles from their time of translocation into the cisternae space of the RER until their time of release by exocytosis into the ductal lumen. This work was demonstrated by the use of 2 dimensional gel electrophoresis

(Scheele, 1975). In support of Scheele's work, Bendayan and coworkers (1980) studied the tissue distribution of 9 enzymes, observing each intracellular compartment using antibodies. This work demonstrated increasing concentrations of all nine enzymes along the RER to ZG pathway. These data support the notion that post-translational degradation of pancreatic digestive enzymes is not likely.

Observations that different proportions of digestive enzymes can be present in pancreatic secretions has created the concept of parallel and nonparallel secretion in the exocrine pancreas. Dagorn (1978) proposed an existence of two populations of acinar cells, each population containing different proportions of enzymes. In this theory, all the digestive enzymes would be stored in ZG, but one population would be responsible for basal secretion and the other population would be responsible for stimulated secretion. Beaudoin and Grondin (1987) proposed two secretory pathways under resting conditions; a paragranular pathway which would bypass the classical pathway involving ZG. In this theory, the difference in enzyme ratios would reflect the contribution of the different pathways. Scheele and Kern (1993) suggest that the anticoordinate changes seen during secretagogue stimulation may result from depletion of zymogen stores of digestive proteins synthesized under one condition compared to newly synthesized proteins under new conditions, which reflect the changes observed at the level of synthesis. The mechanism(s) responsible for the differences observed in zymogen content is not fully understood.

Dietarv and hormonal regulation. The major function of exocrine pancreas secretions is to aid in the efficient digestion of foodstuff for absorption and nutrient utilization. Thus, it is 43 not surprising that diet and the hormones secreted in response to diet affect the regulation of

pancreatic secretions (Solomon, 1994). Secretion of exocrine pancreas enzymes occurs both

during, in between meals (basal or interdigestive), and after meals (postprandial). Postprandial

secretion is initiated by ingestion of a meal and is maintained throughout the digestion and

absorption period. This is thought to be the most important function of the exocrine pancreas

because pancreatic insufficiency results in maldigestion and malabsorption of key nutrients.

However, basal secretion of the exocrine pancreas may provide adequate digestive enzymes for

digestion and absorption in some species. When expressed relative to maximal secretion due to

exogenous CCK, human basal secretion is 20% (Regan, Go and DiMagno, 1980) and rat basal

secretion is 30% (Petersen and Grossman, 1977). This indicates that postprandial exocrine

pancreas secretion occurs in gross excess compared to what is necessary for efficient digestion

and absorption of foodstuff. Significant maldigestion in humans with chronic pancreatitis does

not occur until the maximal secretory capacity drops below 10% of normal (DiMagno, Go and

Summerskill, 1973).

The exocrine pancreas is under both short term and long term neural and hormonal

control. An example of short term regulation is the stimulation of secretion from food ingestion.

Long term regulation, or adaptation, consists of changes in specific digestive enzymes according to the changing needs of the organism. Changes in the concentration of hormones and

neurotransmitters exposed to the acinar cell affect gene expression of proteins in the acinar cell

(Scheele, 1993). Transcription of acinar protein genes is effected by enhancer regions either upstream or downstream of the translatable region.

The coordination of differentiated and cell-specific expression of a selective subset of genes is termed "tissue specific gene expression" and requires a combination of gene promoter 44 and enhancer regions that can confer cell specificity through transcription factors. Gene constructs in the pancreas have been studied to find tissue specific elements. Elastase I, amylase

2.2 and trypsin I have common characteristics in their gene control region. The presence of a conserved sequence of 216 base pairs (bp) termed pancreas consensus element (PCE) was identified in rats. Transcription factors PTF-l or XPF-l bind to the PCE (Swift et aI., 1984;

Boulet, Erwin and Rutter, 1986), The PTF-l is detectable at 14 day gestation, which is approximately when expression of digestive enzymes occurs (Scheele, 1993). Further studies on tissue specific gene expression in the pancreas reveal a 134 bp region (from -72 to + 205) composed of three functional segments (A,B,C), required for gene expression of digestive proteins in cultured acinar cells (Rose et aI., 1994). The A element contains the PCE. The B element is consistent with binding sites of helix-loop-helix DNA proteins. The PTF-l has two subunits, one subunit binds the A element and the other subunit binds the B element. Elements A and B can direct low levels of expression, but when coupled to the C element, transcriptional rates increase significantly (Kruse et aI., 1993).

Dietary regulation. The ability of the exocrine pancreas to adapt to dietary changes was first described in the early 1900's (Pavlov, 1902). There are 3 phases of pancreatic secretion in response to diet; cephalic, gastric, and intestinal. Pavlov (1902) was able to demonstrate the cephalic phase of pancreatic secretion using sham feedings. This phenomenon will still occur even when gastric juices are prevented from entering the duodenum. The stimulation occurs through direct vagal stimulation (Sarles et aI., 1968). The gastric phase occurs when stimuli act in the stomach. This evokes a vagovagal and gastrin response affecting pancreatic enzymes output but not water or bicarbonate secretion (Scheele, 1993). The intestinal phase is quantitatively the 45 most important phase of the pancreatic response to a meal. Digestion products are powerful stimulants in the intestine.

A number of nutritional factors can alter pancreatic function. The nutritional factors include changes in major dietary components, changes in patterns of food intake, deficiencies of trace elements, vitamins, and essential fatty acids, as well as consumption of alcohol and use of tobacco (Pitchumoni and Scheele, 1993). Many studies have been performed utilizing animal models that show altering macronutrients in the diet cause changes in enzyme content and secretion. Changes in pancreatic cellular content of enzymes occur as early as 24 h after a shift in diet (Brannon, 1990). Effects on the synthetic rate of these digestive enzymes precede changes in tissue levels of enzyme (Pitchumoni and Scheele, 1993).

The amount and type of dietary protein affects both trypsinogen and chymotrypsinogen expression (Johnson, Hurwitz and Kretchmer, 1977). Significant increases in content, synthetic rates and mRNA of these proteases occur in response to changes in amount of dietary protein from 10% to 80% of energy (Lee, Nitsan and Liener, 1984; Schick et aI., 1984a; Temler et al.,

1984). The greatest response in protease adaptation is demonstrated when the protein source is of high biological value (Johnson et al., 1977). This effect was shown by using supplementation of limiting amino acids in dietary protein to maximize pancreatic adaptation (Snook, 1965; 1969;

Johnson et al., 1977). When very low levels of protein are fed (10% and 0) with increased carbohydrate, individual enzymes and proenzymes are regulated in direct proportion to nutritional substrates (Dagorn and Lahaie, 1981), except for anionic chymotrypsin which is synthesized at near maximum levels with a 0% protein diet and synthesized at minimum levels at a 10% protein diet, followed by a gradual increase to maximum levels at a 82 % protein diet (Schick et al.,

1984a). This phenomenon may represent a survival mechanism allowing synthesis of certain 46 proteases so that in the face of diminished amino acid supplies, protein digestion can still occur

(Pitchumoni and Scheele, 1993).

Increasing dietary carbohydrate affects pancreatic amylase in a proportional fashion.

Dietary carbohydrate, specifically starch, affects enzyme content, synthesis, mRNA levels and transcription rates (Wicker and Puigserver, 1990). Changes in amylase expression occur until dietary carbohydrate reaches 58%-71 % of energy (Wicker and Puigserver, 1987; 1990). The type of dietary carbohydrate affects amylase expression. Both starch and glucose maximize amylase expression (Deschodt-Lanckman et aI., 1971). When starch digestion is inhibited (Folsch et aI.,

1981), or sucrose or lactose are the carbohydrate source (Howard and Yudkin, 1963), amylase content decreases. The use of transgenic animals has added tremendous information about the amylase gene in mice (Amy 2.2 gene). Schmid and Meisler (1992) report a region for dietary carbohydrate transcription regulation on the 5' untranslated portion of Amy 2.2. Transgenic animals with this 127 bp dietary response element fed high carbohydrate (71 % of energy) compared to low carbohydrate (11 % of energy) had nine times greater expression of amylase.

This dietary response region does not function by itself, but in conjunction with an insulin response region. This adaptation is consistent with the functional role of amylase discussed previously.

Both amount and type of dietary fat affect pancreatic lipase as will be discussed later in detail. Many studies have looked at the effects of dietary fat on lipase, however, the results have varied as much as the experimental designs used making a comparison of data difficult.

Studies using similar experimental methods report increased lipase expression from rats fed high fat diets (67 % of energy) (Wicker and Puigserver, 1987), regardless of saturation and chain length of triglyceride (Sabb, Godfrey and Brannon, 1986). When dietary fat is at moderate levels (40% 47 of energy) polyunsaturated fats stimulate lipase expression more than saturated fats do (Sabb et aI., 1986; Ricketts and Brannon, 1994).

The macronutrients and byproducts discussed influence the expression of their respective digestive enzymes whether or not the increased nutrient is at the expense of one or the other two nutrients, indicating that the response is due to the change in the nutrient in question

(Brannon, 1990).

Hormonal regulation. Hormones from the endocrine pancreas and GJ tract have been studied extensively for their effects on the exocrine pancreas. Among the many hormones released from the gut mucosa and endocrine pancreas, CCK, gastrin, secretin, gastric inhibitory peptide (GIP), peptide tyrosine tyrosine-amide (PYY), and insulin are released in response to specific nutritional substrates. These hormones mediate selective patterns of gene expression in the exocrine pancreas in response to diet (Scheele, 1993). Studies of these secretagogues have demonstrated long term regulatory effects on acinar cells; however, not all of the mechanisms have been elucidated.

The use of molecular biology tools has allowed remarkable progress in the understanding of neural and hormonal control of the exocrine pancreas in recent years. Cloning and sequencing of CCK receptors and sUbtypes and the development of synthetic antagonists have enabled the elucidation of the mechanisms and function of this one hormone (Solomon, 1994).

CCK is secreted by intestinal mucosa, acts as a potent stimulator for pancreatic secretion, and has been proposed as a regulator of several pancreatic proteins. Infusion of caerulein, a CCK analogue, causes an increase in protease synthesis and a decrease in amylase synthesis (Schick,

Kern and Scheele, 1984b). Protease synthesis varies with no change in cationic trypsinogen, a

2-fold increase of anionic trypsinogen within 4 h, and a 4-fold increase within 24 h. Neutral 48 trypsinogen showed the most dramatic change with an 8-fold increase in 4 h and IS-fold increase in 24 h. Steinhilber and coworkers (1988) report that caerulein infusion increased anionic trypsinogen synthesis 3-fold and decreased amylase synthesis 75%. These changes were not proceeded by changes in mRNA levels which suggests differential translational regulation in protein synthesis. Translational control of exocrine pancreas proteins has been demonstrated with other hormones as well. In vitro studies by Hirschi, Vasiloudes and Brannon (1994) report that caerulein increases the relative synthesis of trypsin (28 %) and tends to increase that of chymotrypsin (25%). CCK mediates dietary protein induced synthesis of proteases through interaction with monitor peptide during high protein feeding in rats (Iwai, Fukuoka and Fushiki,

1987). The monitor peptide is secreted by the pancreas and is hydrolyzed by proteases except during postprandial periods when proteases may be too occupied and monitor peptide may escape and evoke pancreatic enzyme secretion and CCK release (Chey, 1993).

Gastrin is a GI hormone produced in the gastric antrum which stimulates gastric acid and pepsin secretion (Walsh, 1994). Gastrin and CCK show homology in their amino acid sequence, particularly in the C-terminus region. The carboxyl end of gastrin is responsible for its biological activity but this hormone shows more heterogeneity in size and number of forms than any other GI hormone. Gastrin is released by gastric cells by food components and inhibited by acidification of the gastric lumen. The response to exogenous gastrin can be inhibited by other hormones such as CCK, GIP, VIP, glucagon, and somatostatin. Porcine gastrin has been shown to stimulate pancreatic secretion in humans (Cantor et aI., 1986), however, it is less clear whether endogenous gastrin is a physiologically significant stimulant of the exocrine pancreas (Konturek,

Bielanski and Solomon, 1990). It is unlikely that the amount of endogenous gastrin released by a meal plays a significant role in stimulation of pancreatic secretion (Chey, 1993). 49 Insulin is an endocrine pancreatic hormone important in glucose metabolism and known to be involved in long term regulation of a-amylase gene expression. Insulin has a stimulatory influence on enzyme secretion and has also been proposed to playa role in the mediation of pancreatic adaptation to dietary carbohydrate (Schmid and Meisler, 1992). Streptozotocin (STZ)­ induced diabetic rats, independent of diet, have decreased amylase content and mRNA, which is then restored to the respective dietary control values by insulin (Tsai et aI., 1994). Insulin binds the insulin receptor, activating tyrosine kinase for its cellular response (Feener et aI., 1992). This response results in a transcription factor, or possibly factors, binding to the insulin response element (IRE) located at the 5' region of the gene (Keller et aI., 1990). The IRE and the dietary response unit are both within a 130 bp region of the Amy 2.2 gene. Although these fragments overlap and are necessary for dietary regulation, neither fragment is capable of mediating the dietary response alone (Schmid and Meisler, 1992). Insulin affects the regulation of pancreatic lipase as well. STZ-induced diabetic rats fed high carbohydrate diets have increased lipase content and mRNA levels, while exogenous insulin administration restores lipase back to control values (Tsai et aI., 1994; Duan, Wicker and Erlanson-Albertsson, 1991). When normal rats were injected with insulin (0.5 ulloa g), lipase and co lipase content decreased 80% and 72%, respectively (Duan et aI., 1991). Amylase also showed a 25% drop in content, but this drop reflected a 21 % increase in secretion. The amylase mRNA was not changed, but lipase mRNA decreased 50%. Thus, restoration of amylase and lipase is not effected through the same mechanism, and few studies have focused on these mechanisms (Clauser, Leconte and Auzan,

1992). 50 Secretin and members of its family (GIP) regulate pancreatic lipase and have been proposed to mediate dietary fat regulation. This regulation will be discussed in detail in the following section on pancreatic lipase.

The effects of diet and hormones on exocrine pancreas protein regulation are complex and far from being completely understood. The use of transgenic animals has rapidly enhanced the knowledge of gene structure and transcription factors; however, deciphering the role of individual nutrients, nutrient metabolites, hormones, and their effect on pancreatic gene expression will require more investigation.

PANCREATIC LIPASE

The most important enzyme for hydrolysis of dietary long chain triglycerides in humans is pancreatic lipase (PL)(Figure 2-1). Pancreatic lipase is secreted as a fully active enzyme but cannot function efficiently by itself. The exocrine pancreas also secretes procolipase, which is activated by trypsin in the upper intestine. Colipase is necessary to anchor lipase in position to hydrolyze dietary triglycerides (Larsson and Erlanson-Albertsson, 1991). Without this functioning enzyme, maldigestion and malabsorption of long chain triglycerides will occur leading to steatorrhea, nutrient loss, and eventually essential fatty acid deficiency (Armand et aI., 1992).

Pancreatic lipase is a member of a gene family of proteins that includes hepatic lipase

(HL) and lipoprotein lipase (LPL)(Figure 2-1). HL hydrolyses triglycerides from very low density lipoproteins (VLDL) in the liver while LPL functions in the catabolism of triglyceride-rich lipoproteins in the circulation. These lipases have striking amino acid homology which is supported by structural analysis of the genes (Giller et aI., 1992). hLPL hHL rPL pPL hPL rPLRP1 cPLRP1 hPLRP1 mPLRP2 hPLRP2 gPL COPL

FIGURE 2-1. Lipase Gene Family Tree. Evolutionary tree of the lipase gene family based on homology of amino acid sequences. Adapted from Giller et at. (1992) and Carriere et at. (1994b). Abbreviations are as follows: hLPL, human lipoprotein lipase; hHL, human hepatic lipase; PL, pancreatic lipase; PLRP-l, pancreatic lipase related protein-I; PLRP-2, pancreatic lipase related protein-2; h, human; r, rat; p, porcine; c, canine; m, mouse; g, guinea pig; Co, coypu.

..-VI 52

Gene Structure

Several animal species have been used to study PL. The cDNAs encoding dog (c)

(Mickel et al., 1989), rat (r) (Wicker-Planquart and Puigserver, 1992; Payne et al., 1994), rabbit

(Aleman-Gomez et al., 1992), horse (Kerfelec et al., 1992), guinea pig (g) (Carriere et al.,

1994b), Coypu (Co) (Carriere et aI., 1994b), and human (h) pancreatic lipase (Lowe, Rosenblum and Strauss, 1989) have been characterized. The canine lipase gene contains two 5' sequences homologous to a 20 bp tissue specific pancreatic consensus element (PCE) (Mickel et al., 1989).

In the 5' region of canine PL, a sequence at position -81 shows a 73.3% homology to the PCE with a 15 bp overlap. Also, at position + 38 in the first intron there is a 72.2 % homology in an

18 pb overlap. Amylase, trypsinogen, and proelastase genes also contain such S'-flanking PCEs, and these sequences direct tissue specific gene transcription (Scheele, 1993); however, tissue­ specific transcription has not been demonstrated for this sequence from PL (Mickel et al., 1989).

Comparisons among PL in the different species demonstrate that some are more homologous than others. The first putative canine PL clone was isolated by Mickel and coworkers (1989). Wicker-Planquart and Puigserver (1992) screened a rat pancreatic library using this canine lipase cDNA and identified the first putative rat PL clone (rPL-l). Lowe et al.

(1989) used polyclonal antibodies to human pancreatic lipase to screen plaques from an human library and cloned human PL. Subsequently, pancreatic lipase related proteins-l and 2 were identified in both the human (Giller et al., 1992) and rat (Payne et al., 1994).

The originally mis-identified cPL and rPL-I have now been shown to be related proteins and not true PL, as will be discussed in detail. Giller et al. (1992) reported the presence of two lipase-related proteins (PLRP) in humans. These proteins are not isozymes nor alleles of lipase because they had different lipolytic activity than hPL when expressed in COS cells. When the 53 cDNAs for these lipase-related proteins (hPLRPl and hPLRP2) were compared to PL cDNAs from other species, the homology was found to be high; hPLRP-l was 85% identical to the rat clone (rPL-l) isolated by Wicker-Planquart and Puigserver (1992), but only 66% identical to the rat clone (rPL-3) isolated by Payne and coworkers (1994). To determine that the hPLRP were indeed not PL, the cDNAs were transfected into COS cells to determine lipolytic activity. The hPLRP-l was secreted into the cell media but did not show any lipolytic activity with a bile salt emulsified triolein substrate with or without colipase. In contrast to hPL, the hPLRP-2 demonstrated lipolytic activity, but was only marginally dependent on the presence of colipase.

The presence of PLRPs in the pancreas raises questions about their function and has sparked controversy regarding the validity of cDNAs (rPL-l) previously used for quantifying rPL

(Ricketts and Brannon, 1994).

Comparison of amino acid sequences with other known lipase sequences provides an abbreviated family tree (Figure 2-1). Using the pileup program within a GCG package, Giller and coworkers (1992) describe the relationship of some lipases based on homology. Three major branches are identified: hHL, hLPL, and PL. Within the PL branch exist three sub-branches which have greater homology, and within the sub-branches further branching into genes of the different species occurs. The 3 sub-branches include 2 branches of PLRPs and the third contains the bOlla fide lipases. The hPLRP2, mPLRP2, gPL and CoPL share homology; hPLRP1, cPLRPl (originally reported as cPL by Mickel et aI., 1989) and rPLRPl (originally reported as rPL by Wicker-Planquart and Puigserver, 1992) share homology; and hPL, pPL, and rPL share homology.

The rPL cDNA is 1,492 nucleotides encoding an open reading frame of 465 amino acids with a predicted 16 amino acid signal peptide. This cDNA was screened from a rat 54 pancreas library using a hPL cDNA and the similarities between this PL and hPL are strong. The rPL has an amino acid sequence with 78% identity to the hPL sequence, 66% identity to hPLRP-

1, 62 % identity to hPLRP-2, and 63 % identity to mCTLL (Giller et aI., 1992) also referred to as mPLRP2 (Payne et aI., 1994). There is only 66% identity of rPL and rPLRP-l and 65% identity between rPL and rPLRP-2 (Payne et aI., 1994).

Until recently, experiments investigating the effects of dietary fat on lipase mRNA utilized putative rPL partial cDNAs, first isolated with the canine PL cDNA (Wicker, Puigserver and Scheele, 1988). Wicker-Planquart and Puigserver (1992) first described the primary structure of a putative rPL mRNA by using a cPL clone and contirmed two clones by restriction enzyme mapping. The first, initially designated rPL-l, has 1531 nucleotides and a poly (A) tail approximately 60 nucleotides long and is a full length cDNA from a Agtlllibrary. The second, initially designated rPL-2, has 692 nucleotides and is partial length cDNA from a pUC9 cDNA library. These two clones contained the conserved residues necessary for binding and catalytic activity as well as sharing high homology, therefore they were believed to be bona fide lipases.

A third putative rPL clone, referred to as rPL-3, was reported in Genbank (M58369) by Dr.

Mark E. Lowe's laboratory. Until 1994, all effects of diet or hormones on rPL mRNA utilized the rPL-l cDNA to quantify this mRNA.

In 1994, Payne and coworkers examined the enzymatic properties ofrPL-l, rPL-3, and rPLRP2 (GP3; Wishart et aI., 1993) following the report of highly homologous related proteins without colipase dependent lipolytic activity in the human (Giller et aI., 1992). The rPL-3 clone was screened from a rat pancreas library using hPL eDNA and the rPLRP2 from a region of mPLRP-2 (also referred to as mCTLL) eDNA. Recombinant baculovirus containing these rat cDNAs were used to infect Sf9 cells and express these proteins in order to determine differences 55 in activity. Both rPL-3 and rPLRP-2 had lipolytic activity which was inhibited in the presence

of bile salts and reactivated with the addition of colipase. The rPL-3 activity was 2-fold greater

than that of rPLRP-2. The rPL-l had no lipolytic activity and was unaffected by bile salt or by

colipase. The activity of rPL-3 is similar to that of hPL (Giller et al., 1992) with which it shares

a high homology (87 %). The activity of rPLRP-2 was greater than that of hPLRP-2 with which

it shares a high homology (76%). Further, hPLRP-2 is not activated by colipase as strongly as

rPLRP-2 was. Both rPL-l and rPLRP-l failed to demonstrate lipolytic activity or colipase

dependence. The rPL-3 is thus a bona fide rat pancreatic lipase exhibiting classical colipase

dependent lipolytic activity and rPL-I is a rat pancreatic related protein (rPLRP-I) whose function

remains unknown.

The rPLRP-2 is identical to a zymogen granule membrane protein, GP3, and highly

homologous to the unusual and sole guinea pig pancreatic lipase, gPL (Carriere et al., 1994b).

Wishart and coworkers (1993) report a membrane bound rat pancreatic lipase with molecular

weight slightly greater than rPL. This is the third glycosylated protein characterized in the acinar

granule membrane and is called GP-3. Wagner et al. (J 994) further characterized GP-3 and

found its sequence was identical to rPLRP-2 reported by Payne and coworkers (1994). This study distinguished GP-3 from rPL by 2 dimensional gel electrophoresis and by using purified

antibodies synthesized to reflect nonhomologous regions of the proteins. GP-3 is not found in the ductal lumen and is more hydrophobic than rPL. It is attached to the inner ZG surface and is released with the other digestive enzymes once the ZG fuses with the apical membrane. The secretion of GP-3 also demonstrates strong regulation by CCK. The full contribution of GP-3 to duodenal lipolytic activity has not been determined; but this enzyme is highly homologous to 56 guinea pig and coypu pancreatic lipase, which have lipolytic and phospholipolytic activity, are

inhibited by bile salts, but are not dependent on co lipase (Carriere et aI., 1994b).

With the current evidence, the rat PL clone characterized by Payne and coworkers

(1994) should be referred to as rPL and not rPL-3. Also, the rPL clone characterized by Wicker­

Planquart and Puigserver (1992) should be referred to as rPLRP-l and not rPL-l. Given the

evolution of our understanding of the lipolytic nature of these two proteins, the literature prior to 1995 does not use this suggested nomenclature.

Although the function of rPLRPI remains unknown, a distinct and anticoordinate developmental expression of rPL and rPLRPI has been described (Payne et aI., 1994).

Expression of rPLRP 1 and rPLRP2 during development is greatest postnatally and declines through the weaning period and adulthood, whereas expression of rPL begins to increase during the weaning period and continues through adulthood. The rPLRPI has low activity against triolein when emulsified with bile salts only, which has led Payne and coworkers (1994) to speculate that rPLRPI might act on other substrates such as phospholipids, cholesterol esters, or vitamin esters.

Function

Pancreatic lipase functions as the most important of the digestive lipases for the hydrolysis of dietary long-chain triglycerides in the digestive tract. It is restricted to hydrophobic­ hydrophilic interfaces and dependent on colipase and the presence of emulsified substrates.

Structural evidence indicates lipase must first go through conformational changes before it can exert its activity (Winkler, D'Arcy and Hunziker, 1990). In the presence of necessary cofactors, lipase will first hydrolyze ester bonds in the sn-l and 3 positions of triglycerides, but will also act on all fatty acid esters as long as they exist as emulsions. 57 Ingestion of food stimulates the secretion of enteric hormones (among them CCK), which causes the pancreas to secrete enzymes and bicarbonate, and the gallbladder to secrete hepatic bile. The saIts of bile include glycocholic, taurocholic, and glyco- and taurodeoxycholic acids and are necessary to emulsify the dietary TG to form the interface. When a critical concentration of polar lipids is present in an aqueous medium, they form micelles. Micelles are water-soluble aggregates of dietary TG and bile salt emulsion and consists of fatty acids, monoglycerols, and bile saIts. The micelles shuttle lipids hetween the emulsion and the absorptive surface (Mead et aI., 1986). The combination of bile, TG, and TG hydrolysis provide the perfect emulsifying system at pH 6.9. This system breaks down fat particles to a size less than 0.6 J-tm and thus increases the surface area of TG by a factor of approximately 10,000 (Sickeninger,

1975). The emulsification offat is very important since the rate of lipolysis of pancreatic lipase is a function of the surface area offered to the enzyme. At physiological concentrations, bile saIts inhibit lipase activity since they displace the enzyme from the oil-water interface to the aqueous phase (Borgstrom, 1975). The inhibitory effect of hi Ie sa Its is counteracted by colipase, which forms a tight complex with lipase and restores its interfacial recognition (Patton and Carey, 1979).

Co lipase is a small protein (10 kDa) secreted as procolipase in the pancreatic juice and is activated by trypsin in the upper intestine. Colipase acts as a cofactor for pancreatic lipase by anchoring it to the interface, enahling hydrolysis of dietary TG to occur (Maylie et aI., 1971).

There is a strict interdependence of colipase and lipase activity, in which colipase deficiency has profound consequences on lipid digestion and absorption (Fukuoka et aI., 1993). Structural evidence using X-ray crystallography indicates Serine 152 is the nucleophilic residue essential for lipase catalysis and is part of an Asp-His-Ser triad, which is chemically similar to the catalytic portion found in serine proteases (Winkler et aI., 1990). CrystaIlography studies on colipase and 58 lipase binding indicate that colipase structure consists of three finger regions held together by disulfide bridges which contain the majority of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs on the other side of this site on colipase and involves polar interactions. Colipase binding occurs exclusively at the C-terminal domain of lipase. Two salt bridges and six hydrogen bonds provide the polar component of binding.

Tyrosine 403 of lipase and Arg 65 of colipase 'stack' together for the apolar binding (van

Tilbeurgh et al., 1992). Lowe (1992) demonstrated the importance of Ser 153 (which corresponds to Ser 152 in Winkler et aI., 1990) in experiments using site-specific mutagenesis of the cDNA for hPL. Amino acid substitutions were made at Ser 153 and other sites believed to be important.

These mutant cDNAs were expressed in transfected COS-I cells. The proteins with mutated Ser

153 were found to bind to interfaces but had no activity. This supports the findings regarding the role of Ser 153 in catalysis but not interfacial binding. The conformational changes of lipase involve a surface loop known as the activational flap between disulfide bridges that cover the active site with a small a-helix until the interaction of lipase and colipase. The active site is thought to be exposed at the oil-water interface after the conformational change following colipase binding and due to the physical properties at the site. Once lipase is anchored and oriented at the interface, Ser 152 is poise for catalytic activity (Winkler et aI., 1990).

The hydrolysis products of dietary fat are mainly 2-acylglycerols, some l-acylglycerol, free fatty acids, and free glycerol. The nature of the dietary fat can affect the complex and subsequent hydrolysis. The longer-chain saturated fatty acids are less completely absorbed than are the shorter-chain, or the more highly unsaturated fatty acids (Hoffman and Borgstrom, 1963).

Tristearin is poorly absorbed, however, TG containing stearate in the 2-position are more completely absorbed than those in which stearate occupies either the 1- or 3-position. Unsaturated 59 fatty acids and their monoglycerols require a lower bile acid concentration for micellar

solubilization than do saturated fatty acids and their monoglycerols (Borgstrom, 1993).

Pancreatic lipase function is affected by many variables, however, it is the Iipase­

colipase-bile salt complex that is necessary for hydrolysis of dietary fat. Type and amount of

dietary fat can influence hydrolysis further.

Dietary Regulation

Amount of dietary fat affects lipase content (Deschodt-Lanckman et aI., 1971; Gidez,

1973; Bazin and Lavau, 1979; Ouagued et aI., 1980; Saraux et aI., 1982; Sabb et aI., 1986;

Wicker and Puigserver, 1987), synthesis (Wicker and Puigserver, 1987; Ricketts and Brannon,

1994), mRNA levels (Wicker et aI., 1988; Wicker and Puigserver, 1990; Ricketts and Brannon,

1994) and transcription rate (Wicker and Puigserver, 1990). Pancreatic lipase responds to the

amount of dietary fat whether or not it replaces protein (Deschodt-Lanckman et aI., 1971) or carbohydrate (Sabb et aI., 1986), suggesting that this effect is in response to dietary fat. Studies evaluating the effect of the amount of dietary fat on lipase content in rats found increased lipase activity with 61 % of energy from lard, but not with 16% of energy from lard (Ouagued et aI.,

1980). Other studies found increased lipase activity when 72% of en'ergy came from lard compared to 9% of energy from lard (Bazin, Lavau and Herzog, 1978). Further studies found increased lipase activity with 45% of energy supplied by corn oil, but not with 27% of energy or less supplied by corn oil (Saraux et aI., 1982). Another study using corn oil reported increased activity with 54 % of energy as fat, but not 47 % of energy or less (Sabb et aI., 1986). Additional studies found increased lipase activity when 74 % of energy was derived from sunflower oil compared to 7.4 % (Wicker and Puigserver, 1987). A similar increase in lipase content in response to amount of dietary fat is also evident in lingual lipase (Armand et aI., 1990). These 60 studies demonstrate that amount of dietary fat clearly affects lipase content. Fewer studies have evaluated the regulation of the synthesis of lipase by dietary fat. Wicker and Puigserver (1987) found a 2- to 3-fold increase in synthesis when comparing 67% of energy derived from sunflower oil or more to 704%.

Prior to establishing rPL as the bOlla fide lipase, data representing effects of dietary fat on pancreatic lipase mRNA levels actually represent rPLRP I mRNA levels, except for our recent report that true rPL mRNA levels increased 1.6-fold when dietary fat increased from II % of energy to 40% of energy (Ricketts and Brannon, 1994). Safflower oil diets increased rPL mRNA 1.7-fold from LF to MF while lard diets increases rPL mRNA lA-fold. These changes in rPL mRNA level due to dietary fat were similar in rPLRP1 mRNA levels. Others also report similar regulation of rPLRP 1 mRNA levels (Wicker et ai., 1988).

Only one study has evaluated putative pancreatic lipase transcription rates, but actually used the rPLRP-l eDNA. After 24 h of consuming a high sunflower oil diet (62% of energy), transcription rates of rPLRP-l increased lA-fold (Wicker and Puigserver, 1990) and reached a maximal after 5 days. Although this study on transcription rates did not use a true rPL cDNA, these data along with the data on mRNA and synthesis support the contention that amount of fat may regulate lipase transcriptionally, but future studies need to examine the transcriptional regulation of rPL.

The effects oftype offat on pancreatic lipase are less clear. Highly unsaturated dietary fats induce greater lipase activity compared to saturated fats (Deschodt-Lanckman et aI., 1971;

Bazin et aI., 1978; Ricketts and Brannon, 1994), however; Saraux et a1. (1982) report that chain length, not saturation, affects lipase. Deschodt-Lanckman and coworkers (1971) found that when comparing effects of unsaturated dietary fats on lipase activity, corn oil (PIS ratio = 4.6) showed 61 greater activity than the more unsaturated sunflower oil (PIS ratio = 6.5). Sabb et al. (1986) found no difference in lipase activity in response to type of fat when energy levels were 67%; but when fat levels were 40-44% of energy, only the highly unsaturated safflower oil significantly increased lipase content. Ricketts and Brannon (1994) report a 3-fold increase in lipase activity from LF safflower oil to MF, but no change from LF lard to MF lard. LF lard resulted in a 2- fold greater lipase activity than LF safflower. These data indicate that there is an interaction between amount and type of fat, specifically saturation, which affects pancreatic lipase activity.

Ketones, byproducts of TG digestion, may also play a role in dietary adaptation of lipase. High fat diets are correlated with increased lipase and ketone production (Bazin et aI.,

1978). Bazin and coworkers (1978) reported that continuous infusion of the ketone, P(OH)­ butyrate, in rats results in increased lipase activity similar to that caused by a high fat diet. In vitro studies using primary acinar cells from rats fed chow report no affect of P-hydroxybutyrate

(0.01 to 2 mmol/l) on lipase activity (Hirschi, Sabb and Brannon, 1991). However, when rats were fed LF diets prior to cell isolation, the cellular lipase from the LF cells increased 264 % at

24 hand 145% at 48 h. There was no effect on lipase activity in rats when fed HF diets prior to cell isolation. Ketones may be involved in the regulation of pancreatic lipase, however their role appears to be complex and poorly understood.

Hormonal Regulation

Secretin is synthesized and secreted from the intestinal mucosa in response to duodenal acidification and the presence of fatty acids (Chey, 1993). Faichney et al. (1981) reported that administration of the dietary lipid, oleic acid, to rats increased the release of secretin from intestinal mucosa cells into the portal circulation. This hormone has been implicated in the regulation of pancreatic lipase synthesis. Rausch and coworkers (1986) infused rats with secretin 62 which increased lipase synthesis 2.8-fold after 6 hand 5.8-fold after 12 h. Proelastase 2 also increased 3.4-fold after 24 h. In contrast, secretin in vitro did not affect cellular or media lipase activity, nor did it affect lipase (Hirschi et aI., 1994). Secretin infusion does not affect lipase mRNA levels either, whereas dietary fat will increase lipase content, synthesis, mRNA levels, and transcription (Brannon, 1990). The in vitro studies do not support secretin's proposed role in regulation of pancreatic lipase; however, secretin may act in concert with other GI hormones or factors to regulate lipase. Secretin utilizes a cAMP second messenger system which has been shown to be affected by dietary fat and altered plasma membrane phospholipids (Clandinin et aI.,

1991), so there could be direct and indirect effects of dietary fat on secretin. Duan and Erlanson­

Albertsson (1992a) incubated pancreatic lobules with dibutyryl cAMP and dibutyryl cGMP to determine if these second messengers were responsible for the effects of secretin on lipase and colipase expression. Synthetic rates of lipase and colipase increased 21 % and 25%, respectively, when lobules were incubated with the cAMP; and there was no change when incubated with cGMP, indicating that cAMP may playa role in the effects of dietary fat on lipase expression.

Synthetic rates of amylase were unaffected by both cAMP and cGMP.

GIP has also been reported to stimulate synthesis of pancreatic lipase and colipase

(Duan and Erlanson-Albertsson, 1992b). GIP belongs to the secretin family. Of GIP's 42 amino acid residues, 9 residues are identical to secretin and 15 are identical to glucagon (Brown and

Dryburgh, 1971). The effects of GIP on other systems has been studied extensively. GIP has been shown to augment release of insulin during hyperglycemia, to inhibit gastric secretion

(Brown et aI., 1975), and to stimulate secretions of the small intestine (Barbezat and Grossman,

1971). Recently the effects of GIP on the exocrine pancreas have been determined. Infusions of GIP (3 ltg/h) for 24 h led to a 34% increase of lipase content with no effect on colipase or 63 amylase content or any mRNA levels. Injection of GIP three time a day for 5 days caused a dose

dependent increase of lipase and colipase content 52 and 25% and their corresponding mRNAs

60% and 160%, respectively (Duan and Erlanson-Albertsson, 1992b). It is not certain if GIP's

effects are direct or indirect since there is no report of GIP receptors on pancreatic acini,

however, synthetic GIP has been reported to increase cAMP levels in isolated pancreatic acinar

cells (Sjodin and Conlon, 1976). GIP is also involved in regulation of fatty acid metabolism

through several mechanisms such as the synthesis of lipoprotein lipase (Eckel, Fujimoto and

Brunzell, 1979) and fatty acid uptake into adipose tissue (Brown et aI., 1975). Given that

pancreatic lipase and lipoprotein lipase are part of the same gene family, the possible regulation

of both enzymes by GIP is intriguing and raises the question of a central role of GIP in regulating

lipid metabolism.

The mechanisms involved in increasing lipase content, synthesis, mRNA levels, and

transcription in response to dietary fat are unknown. Hormone levels and plasma ketone levels

are affected by fat ingestion and affect pancreatic lipase. The gastrointestinal hormones, secretin

and GIP, affecting cAMP levels, have been proposed to regulate pancreatic lipase, but whether

they mediate dietary fat regulation remains unclear.

Regulation of Colipase

Dietary fat, insulin and adrenal steroids also affect pancreatic colipase activity and

mRNA levels (Okada, York and Bray, 1993). During activation of procolipase, the N-terminal

is cleaved yielding a pentapeptide referred to as enterostatin. Studies on enterostatin demonstrate

that it has an anorectic effect on rats after administration either peripherally or centrally and that

enterostatin specifically decreases fat intake (Okada et aI., 1993). These studies determined enterostatin activity estimated from either colipase activity or procolipase mRNA expression based 64 on equimolar production of colipase and enterostatin from procolipase. Expression of pro co lipase was once believed to be exclusive to the pancreas (Lowe et aI., 1989); however, more recently low levels of expression have also been found in the stomach and duodenum ofrats (Okada et aI.,

1993). Okada and coworkers investigated the effects of both dietary fat and adrenalectomy on the expression of procolipase. Increasing dietary fat increased procolipase mRNA, but adrenalectomy effects were dependent on diet. Procolipase mRNA levels were pronounced in animals with adrenalectomies on HF diets but not LF diets. The authors suggest that both glucocorticoid hormones and dietary fat interact in the regulation of pancreatic procolipase mRNA levels. Duan and Erlanson-Albertsson (1992a) incubated pancreatic lobules with dibutyryl cAMP and cGMP to determine the effects of these important secretagogue signals. Amylase was not affected by either cAMP or cGMP, yet lipase and colipase synthesis were both increased 21 % and 25%, respectively, by cAMP, but not stimulated by cGMP. Increased levels of cAMP in acinar cells have been demonstrated when stimulated with secretin and GIP (Sjodin and Conlon,

1976).

Both lipase and colipase are clearly influenced by dietary fat. Although the mechanisms are not known at this point, evidence is accumulating that gastrointestinal hormones playa role as a mediator in dietary fat regulation of rat pancreatic lipase and colipase. 65 CHAPTER 3

MULTIPLE MECHANISMS OF REGULATION OF PANCREATIC LIPASE IN RATS BY AMOUNT AND TYPE OF DIETARY FAT

INTRODUCTION

Pancreatic lipase (E.C.3.1.1.3), the major digestive enzyme for hydrolyzing dietary

triglycerides, requires colipase and the presence of bile salts for the hydrolysis of triglycerides

to fatty acids and monoglycerides, which are subsequently absorbed. Pancreatic lipase is

regulated by dietary fat (Gidez, 1973). Different levels of dietary fat lead to adaptive responses,

beginning immediately and reaching a maximum within 5 days (Deschodt-Lanckman et ai., 1971).

Increasing dietary fat (polyunsaturated) increases lipase synthesis (Wicker and Puigserver, 1987),

rPLRPl mRNA content (Wicker et ai., 1988), and rPLRPl transcription (Wicker and Puigserver,

1990). However, the effect of type of fat - degree of saturation and chain length - on this

regulation has been controversial (Sabb et ai., 1986). Saraux et al. (1982) report that chain

length, not saturation, affects lipase; but several studies demonstrate that saturation does affect

lipase, particularly that highly unsaturated fats increase pancreatic lipase activity (Deschodt-

Lanckman et ai., 1971; Bazin et aI., 1978). Previously (Sabb et aI., 1986) looked at the effect

of type of dietary fat and found that fats differing in saturation (PIS ratio of 0.1 to 7.9) and chain

length (C12 to C18) stimulate lipase activity similarly after consumption of a high fat diet (67%

kcal), but that only highly polyunsaturated fat (safflower oil) increased lipase activity at moderate fat levels (40% of energy). Thus, type of fat appears to regulate pancreatic lipase through a

poorly understood interaction with amount of fat.

Understanding the mechanism involved in dietary adaptation of lipase requires knowledge of the effects of type and amount of fat at different regulatory steps. This study 66 investigated the independent effects of saturation and amount of dietary fat and their interactive effects on pancreatic lipase activity and mRNA levels to determine whether these two factors regulate this gene's expression pre-translationally, translationally or post-translationally.

METHODS

Male weanling Sprague-Dawley rats approximately 40 g were housed on a 12 h light/dark cycle at 24 ac. Animal protocols were approved by the University of Arizona Animal

Care and Use Committee (Appendix A). Animal facilities are ALAAC approved. Purified isoenergetic, isonitrogenous diets were fed ad libitum for 7 days (a period of maximal adaptation).

The diets differed in amount [50 g/kg (LF) and 174 g/kg (MF)] and type (safflower oil or lard) of fat (Table 3-1). The LF and MF diets also differed in content of cellulose which has been shown not to affect pancreatic adaptation (Schneeman and Gallaher, 1980). Food consumption and body weights were measured. Rats were killed by exsanguination from the abdominal aorta while anesthetized with ether in accordance with euthanasia procedures from the PHS guidelines for animal care. The pancreas was removed and divided; 200 mg of tissue was frozen in liquid nitrogen and stored at -80 aC to be later analyzed for enzyme activity; and the remaining portion was used for the isolation of total RNA to quantify mRNA.

Enzyme Analysis

Pancreatic fragments were homogenized in nine volumes of phosphate-buffered saline

(PBS) with a Polytron and centrifuged at 16,000 xg at 4 ac for 30 min. Aliquots of supernatant were taken for proteolytic enzyme analyses, and soybean trypsin inhibitor was added to remaining volume to a final concentration of 0.1 %. This supernatant was analyzed for amylase activity

(E.C.3.2.1.1) by the Phadebas blue starch method (Ceska, Burath and Brown, 1969) by which 67

TABLE 3-1

Dietary Composition'

LF MF

Components gllOO g diet

Casein 20.0 20.0

DL-Methionine 0.3 0.3

AIN Mineral Mix 3.5 3.5

AIN Vitamin Mix 1.0 1.0

Choline bitartrate 0.2 0.2

Cellulose 5.0 20.5

Fae 5.0 17.4

Cornstarch 65.0 37.1

% Energy from fat 11 40

1 Modified by Sabb et al. (1986)

2 Safflower oil was used as the polyunsaturated fat and lard as the saturated fat 68 hydrolysis of the blue starch by amylase releases a soluble dye which is measured colorimetrically. Total protdn of the homogenate was determined by the Lowry method (Lowry et aI., 1951), which is based on oxidizing copper complexed with nitrogen of the amino acid.

The oxidation of threonine, tryptophan, tyrosine and histidine turns the sample solution blue with a maximum absorbance at 750 nm. Lipase activity was determined by a titrimetric method (Sabb et aI., 1986) in which fatty acids released from a gum arabic-stabilized emulsion of neutralized triolein by lipase in the presence of excess colipase are titrated by 20 mM NaOH. Colipase was prepared from hog pancreas as described by Ouagued, Saraux and Girard-Globa (1982).

Proteolytic enzymes were activated with equal volumes of enterokinase (30% in PBS) for 1 hat room temperature (Brannon, Collins and Korc, 1987). Protease activity was determined for trypsin by the procedure of Erlanger, Kokowsky and Cohen, (1961) utilizing benzoyl D-arginine p-nitroanilide (BAPNA), which is hydrolyzed by trypsin to produce p-nitroaliline, which is a yellow pigment. Chymotrypsin was determined by the procedure of Erlanger, Edel and Cooper

(1966) utilizing n-glutaryl-L phenylalanine p-nitroanilide (GPNA) which is specific for chymotrypsin hydrolysis producing p-nitroaliline. Enzyme activities were expressed as units

(J.tmol product released/min) per mg protein and per g pancreas. The serum from the blood drawn was used to measure ketone levels «(3-hydroxybutyrate) by the method of Williamson,

Mellanby and Krebs (1962).

Isolation of Total RNA

Acinar RNA were isolated as described by Schibler et al. (1980) and modified by

Steinhilber et al. (1988). This method yields intact RNA using a cold guanidinium thiocyanate and guanidine hydrochloride procedure. All glassware and solutions were treated appropriately to remove RNA-ase contamination. Freshly isolated pancreas was homogenized in guanidinium 69 isothiocyanate - {3 mercaptoethanol buffer, and RNA was precipitated by a short (30 min) incubation at _20DC in ethanol. Samples were centrifuged; the pellet dissolved in guanidine hydrochloride-DDT buffer and reprecipitated in ethanol. The precipitate was dissolved in Tris­

EDTA (TE) buffer and extracted with chloroform:butanol (3:1). After centrifugation, the upper phase was removed and lower phase re-extracted. The upper phases were combined and precipitated with 2.5 X volume of ice-cold ethanol and incubated overnight at ~20DC. The pellet was dissolved in TE buffer followed by extraction with phenol/chloroform/isoamyl alcohol

(24124/1) 2 times. RNA was precipitated with 2.5 X volume of ice-cold ethanol for 1 hat -20 DC, centrifuged, the pellet dried in a Speed Vac for 15 min, followed by dissolving in sterile Type

I water. RNA was quantitated by UV absorption at 260 nm. To check for intact RNA, the presence of ribosomal bands following a 1% agarose gel electrophoresis and staining with ethidium bromide was assessed. Each probe yielded a single band by Northern analysis.

Quantitation of mRNA

Pancreatic tissue specific mRNA (lipase, amylase, and rPLRP-I) and a constitutive

RNA (28s) were determined by cDNA dot blot hybridization, autoradiography, and laser densitometry using area scanning and volume integration (Schibler et aI., 1980; Korc, Meltzer and Trent, 1986; Steinhilber et al., 1988; Sambrook, Fritsch and Maniatis, 1989; Chang, Brannon and Korc, 1990). Two concentrations of RNA, with triplicate samples of each, were used within a linear range of hybridization to analyze for each probe. Filters were incubated with a pre­ hybridization buffer (5 ml ultrapure formam ide, 2.5 ml 20X SSC, 1.25 ml 0.4 M sodium phosphate, 0.1 ml 10% SDS, 0.1 ml Dep C treated water, 1.0 ml Denhardts solution, 0.1 ml salmon sperm DNA) at 42 DC for 1.5 h. Radiolabeled probe was added (1.5 x 106 cpm/ml) and hybridized at 42 DC overnight. Filters were washed under increasingly stringent conditions (2X 70 sse with 1% SDS to 0.2X sse with 1% SDS) and air-dried prior to autoradiography using

Kodak XAR-5 film. The 3~P-Iabeled cDNA probes were prepared by nick translation (Sambrook et aI., 1989) for lipase and amylase and by random priming for 28s.

Statistical Analysis

Data are expressed as mean ± SEM and were analyzed by two-way analysis of variance and least significant difference and by linear regression (Steel and Torrie, 1960).

Significant differences are denoted by p < 0.05.

RESULTS

Enzyme Activities and Plasma Ketones

Pancreatic lipase activity was significantly greater (280%) in the MF safflower group compared to the LF safflower group (Figure 3-IA). There was no difference in the LF and MF lard lipase activities. Amount of fat had an independent effect (p < 0.0005), whereas amount and type showed an interactive effect (p < 0.002). When looking at actual fat consumption relative to lipase activity (Figure 3-2), there was a linear response with safflower (r=0.8322) and no difference in lard (r=0.2444). Amylase activity showed an opposite response to dietary fat levels compared to lipase (Table 3-2). There was an independent effect of amount of fat with amylase activity being greater (40%) in LF fed animals compared to MF fed animals. Type of dietary fat did not affect amylase activity. There was no effect of either amount or type of fat on protease activities (Table 3-2). Ketones ({3-hydroxybutyrate) were not significantly affected by either type or amount of dietary fat (Table 3-2). FIGURE 3-1. The Effects of Dietary Fat on Lipase Activity and mRNA Levels. Weanling rats were fed for 7 days diet containing LF (11 % of energy) or MF (40% of energy) levels of safflower or lard. Values represent mean ± SEM and those with superscript differed significantly using two way ANOV A. A. Lipase activity was determined using 6 to 7 rats. There was an independent effect of amount of fat (MF> LF, P < .0005) and an interactive effect between amount and type of fat (p < .002). B. rPL mRNA/28s RNA ratios were determined using 3 to 4 rats. There was an independent effect of amount of fat (MF> LF, P < 0.0001) and type (safflower> lard, p < 0.007) of fat, but no interactive effects between amount and type of fat. C. rPLRP-l mRNA/28s RNA ratios were determined using 3 to 4 rats. There was an independent effect of amount of fat (MF> LF, P < 0.001), but no effect of type of fat and no interactive effect between amount and type of fat. 72

150 • SAFFLOWER • LARD • 125 • >- 1-- >- .-c: 100 1--- Q) U e ~a. 75 wO> (/) E • • <' 50 e.- 0..2, • ::J • 25 • •

0 0 5 10 15 20 25 FAT INTAKE (g/rat)

FIGURE 3-2. The Effects of Fat on Lipase Activity. Fat intake was determined from total food consumption. Data analyzed by linear regression for safflower intake, r = 0.8322 (p < 0.0002), and for lard intake, r = 0.2444. The n was 6 or 7 per fat group. 73

TABLE 3-2

The Effects of Dietary Fat on Enzyme Activities and Ketone Levels'

AMYLASE TRYPSIN CHYMOTRYPSIN !3-HYDROXY- BUTYRATE U/mg protein ",moUL

SAFFLOWER LF 217 ± 34·b 1011 ± 92.8 6.5 ± 0.4 16 ± 2

MF 179 ± 19 b 1299 ± 94 6.5 ± 0.4 13 ± 3

LARD LF 296 ± 30· 1040 ± 38 6.3 ± 0.4 10 ± 1

MF 182 ± 23 b 1163 ± 96 6.5 ± 0.7 13 ± 2

I Weanling rats were fed for 7 days diet containing LF (50 g/kg diet) or MF (174 g/kg diet) levels of safflower oil or lard. Unit (U) is defined as ",mol product released/min). Values represent mean ± SEM and those with superscript differed significantly using two way ANOVA, 11 = 6 or 7. 74 mRNA Levels

Amount of fat, independent of its type, significantly affected both rPL and rPLRP-l mRNA levels, which were greater in rats fed MF diets compared with those fed LF diets

(p

DISCUSSION

The increase in lipase activity with increasing amounts of dietary safflower oil seen in this study agrees with several previous studies (Sabb et aI., 1986; Wicker and Puigserver, 1987), even though we looked at only LF and MF levels whereas the other studies included high fat (up to 67% kcal) levels. However, increasing dietary lard from LF to MF levels did not affect lipase activity. This regulation of pancreatic lipase activity by safflower oil supports our previous finding of preferential regulation by MF safflower compared to LF corn oil (Sabb et aI., 1986).

Low fat safflower oil resulted in lower lipase activity (49%) than LF lard. These findings confirm that type of fat can affect pancreatic lipase activity differently.

One hypothesis is that ketone production from fat utilization mediates the dietary regulation of lipase (Bazin and Lavau, 1979). Supporting this hypothesis is the increase in ketones and lipase in diabetes (Bazin and Lavau, 1979) and the increase in pancreatic lipase with constant infusion of ketones in vivo (Bazin et aI., 1978). However, p-hydroxybutyrate levels were not significantly different with either type or amount of dietary fat in this study. Thus, our results do not demonstrate a correlation of circulatory ketone levels and pancreatic lipase activity 75 and do not support this hypothesized role of ketones in the regulation of pancreatic lipase by

dietary fat.

Amount of dietary fat had an inverse effect on amylase activity compared to lipase

activity. Increasing dietary fat, regardless of type, resulted in decreased amylase activity. This

has been demonstrated many times and is related to the decreased amount of carbohydrate with

increased dietary fat (Brannon, 1990). Protease activities were also unaffected by type or amount

of dietary fat.

We have shown that amount of fat independent of type of fat affects lipase and its

related protein-I mRNA levels which increase 2- to 3-fold when dietary fat increases from 5% to 17 % by weight (p < 0.00 I). These results suggest that amount of fat, independent of its degree of saturation, regulates pancreatic lipase pre-translationally. A high-fat diet consisting of sunflower has been shown to increase synthesis of pancreatic lipase and mRNA levels, and transcription ofrPLRP-l using a full length cDNA (Wicker et aI., 1988; Wicker and Puigserver,

1990). These studies used large amounts of lipid in the diet (25 % by weight) to demonstrate that lipase related protein-l can be regulated transcriptionally. Most likely, the effects of lard on lipase mRNA also results from transcriptional regulation, but this has not been determined yet.

Despite increased mRNA levels with both MF lard and safflower diets, lipase activity increased only with safflower oil (4-fold) and was unchanged with lard. Type of fat, thus, interacts with amount offat to regulate pancreatic lipase translationally or post-translationally. Post-translational regulation of pancreatic digestive enzymes has not been demonstrated, but numerous examples of translational regulation by gastrointestinal hormones exists for the proteases (Steinhilber et aI.,

1988), cholesterol esterase (Huang and Hui, 1991) and lipase, itself (Brannon, 1990; Duan and

Erlanson-Albertsson 1992b; Tsai et ai., 1994). Infused secretin enhances lipase synthesis (Rausch 76 et al., 1985) but not its mRNA levels (Brannon, 1990). A secretin related hormone, gastric inhibitory polypeptide regulates lipase by acute translational and chronic pre-translational mechanisms (Ouan and Erlanson-Albertsson, 1992b). In light of the multiple mechanisms of translational and pre-translational regulation of pancreatic lipase by gastrointestinal hormones, our present findings of multiple mechanisms of regulation by amount and type of dietary fat suggest that type of fat acts through a translational mechanism whereby translational efficiency is inhibited by saturated fat or a post-translational mechanism. Repression of translational efficiency by a repressor protein or altered mRNA structure has been demonstrated for eukaryotic cells (Kozak,

1992), but this hypothesis needs to be tested in future studies of the effects of type of fat on rates of synthesis of pancreatic lipase and its translational efficiency. This study is the first to presents the effects of dietary fat on rPL mRNA levels.

In summary, the regulation of pancreatic lipase by dietary fat is complex. Therefore, we have found that amount of fat, irrespective of its saturation, appears to regulate lipase transcriptionally, whereas saturated fat at moderate fat levels appears to regulate lipase gene expression translational I y. 77

CHAPTER 4

DIETARY FAT SATURATION HAS DIFFERENTIAL EFFECTS ON PANCREATIC LIPASE ACTIVITY, mRNA LEVELS, AND SYNTHESIS

INTRODUCTION

Pancreatic lipase is an important digestive enzyme for the hydrolysis of dietary fats in adults. Adaptation of pancreatic lipase activity (Gidez, 1973; Sabb et al., 1986) mRNA levels

(Wicker and Puigserver, 1987; Ricketts and Brannon, 1994) and synthesis (Wicker and

Puigserver, 1987) to amount of fat in the diet has been clearly demonstrated in rats. Although both the amount and type of dietary fat regulate pancreatic lipase, controversy exists as to the effects associated of type of dietary fat. Deschodt-Lanckman and coworkers (1971) report that unsaturated fats stimulate lipase activity to a greater extent than do saturated fats; however, Bazin et al. (1978) report that long chain triglycerides induce greater lipase activity than medium chain triglycerides do. Sabb et al., (1986) found a similar increase in lipase activity with 67% of energy from fat, regardless of its type - saturated, polyunsaturated, long chain or medium chain.

When dietary fat levels was 40% of energy, however, only the most unsaturated fat (safflower oil) significantly increases lipase activity relative to that of the controls fed 11 % of energy as fat.

We previously investigated the effects of type and amount of dietary fat on lipase activity and mRNA levels using safflower oil and lard, both long chain triglycerides with different PIS ratios, safflower - 7.9 and lard - 0.3, at 11 % (LF) and 40% (MF) of energy (Ricketts and Brannon,

1994). Although both safflower oil and lard increased lipase mRNA levels (195%) in MF groups compared to the respective LF group, only safflower oil increased lipase activity (280%) in MF groups. These data support that amount of dietary fat regulates lipase expression pre- 78 translationally and that type of fat, specifically saturation, affects lipase expression translationally

or post-translationally.

This study investigates the response of lipase activity, synthesis, and mRNA levels to

both amount and type of fat in order to determine the mechanisms (pre-translational, translational

or post-translational) whereby type and amount of fat interact to regulate this enzyme. The effects

of LF and MF levels of four types of fats two polyunsaturated (safflower oil, PIS ratio 7.9; corn

oil, PIS ratio 4.6), one monounsaturated (olive oil, PIS ratio 0.6), and one saturated (lard, PIS

ratio 0.3) were examined in weanling rats. Lipase activity and mRNA levels were determined

for all four of the fats, but synthesis was determined only for safflower oil and lard because of

the similar responses of lipase activity and mRNA levels to safflower and corn oil and those of

lard and olive oil.

METHODS

Experimental Protocol

Male weanling Sprague-Dawley rats (35 - 60 g; Harlan, Indianapolis, IN) were housed

and fed as described in Chapter 3 (Ricketts and Brannon, 1994). Animal protocols were approved

by the University of Arizona Animal Care and Use Committee (Appendix A). The composition

of the diets is shown in Table 3-1, except that four fats were used (safflower oil, lard, corn oil

and olive oil) in experiment 1 to determine the effects of polyunsaturated, monounsaturated and

saturated fat on pancreatic lipase activity and mRNA levels. In experiment 2, only safflower oil

and lard were used to determine the effects of polyunsaturated and saturated fat on pancreatic lipase synthesis. Food consumption and body weight were measured. Animals used for determining lipase activity and mRNA levels were killed by exsanguination from the abdominal

aorta while anesthetized by ether. Pancreata were removed, and a small weighed portion was 79 frozen immediately on dry ice and stored at -80°C for enzyme analysis. The greater portion was weighed and used immediately to isolate RNA. Animals used for determining rates of synthesis were killed by carbon dioxide asphyxiation, and the pancreas was removed and used immediately for pancreatic acini isolation, protein synthesis, and tRNA isolation.

Enzyme Analysis

Pancreatic fragments were homogenized and centrifuged as described previously

(Ricketts and Brannon, 1994; Chapter 3). An aliquot of the supernatant was removed for analysis of pancreatic protease activity. Soybean trypsin inhibitor was added to the remainder for a final concentration of 0.01 %. Enzyme analysis of amylase, lipase and protease activity and total protein content were determined. Proteases were activated with equal volumes of enterokinase

(1140 U/L PBS) for 1 h at room temperature. Trypsin activity was determined by the procedure of Erlanger et al., (1961) and chymotrypsin activity was determined by the procedure of Erlanger et aI., (1966). Amylase activity was assayed by the Phadebas blue starch method (Ceska et aI.,

1969) and lipase activity was determined by titrimetric method as previously described (Ricketts and Brannon, 1994; Chapter 3). Total protein was determined by the method of Lowry et al.

(1951), using BSA as the standard. Enzyme activities were expressed as units (J!mol product release/min) per milligram of protein.

Isolation of Total RNA

Pancreatic RNA was isolated as previously described (Ricketts and Brannon, 1994;

Chapter 3). Isolated RNA was dissolved in Type I water and measured by UV absorption at 260 nm. Integrity ofthe isolated RNA was checked using 1% agarose gel electrophoresis and staining with ethidium bromide to evaluate intact ribosomal RNA bands. 80 Quantitation of mRNA

Pancreatic tissue specific mRNA levels were determined as previously described

(Ricketts and Brannon, 1994; Chapter 3). Each sample was probed using 28s cDNA, rPL cDNA, rPLRP-l cDNA, and amylase cDNA via dot blot hybridization, autoradiography and laser densitometry using area scanning and volume integration. Two concentrations of total RNA, with triplicate samples of each, were used within a linear range of hybridization to analyze mRNA levels. Filters were prehybridized for 1.5 h and hybridized overnight at 42 DC using 32p-dCTP labeled probes prepared by nick translation or random priming. Filters were washed under increasing stringent conditions and autoradiographed using Kodak XAR-5 film. The 28s RNA probe was used as a control. All mRNA levels are reported relative to 28s RNA levels.

Pancreatic Acini Isolation

Pancreatic acini were isolated according to the method of Bruzzone et al. (1985) modified by Hazlett and Brannon (1988). The pancreas was placed in a sterile petri dish containing 1 ml KRB HEPES buffer (12.5 mM Hepes, 135 mM-NaCI, 4.8 mM-KCl, 1.0 mM­

CaCI 2, 1.2 mM-KH:!PO'h 5.0 mM-NaHC03, 5 mM-dextrose, and 0.14 soybean trypsin inhibitor/ml, adjusted to pH 7.4). The tissue was minced (approximately 2 mm) and quantitatively transferred to a 25 ml Erlenmeyer flask using 3 ml of collagenase (2.0 mg/ml, 163

U. Worthington CLS 2) in KRB Hepes buffer. The tissue was vigorously shaken at 200 cycles/min at 37 DC for 6 to 10 min for digestion. Adequate digestion was determined visually when the tissue suspension appeared homogenous, generally after 8 min. Fresh KRB-HEPES with 0.1 % BSA (5 ml) was used to transfer quantitatively each sample to a 15 ml tube followed by centrifugation at 500 xg for 2 min. Tissue was washed twice using 7-8 ml of fresh KRB­

HEPES/BSA and filtered through nylon mesh (200 and 75 ILm) into a 50 ml conical tube, 81

centrifuged at 500 xg for 2 min, then layered onto 40 ml 3 % Fico II for acini to settle for 20 min.

The top 35 ml of Ficoll was discarded and the sediment was washed twice with fresh KRB­

HEPES/BSA. The acini were quantitatively transferred to a 50 ml Erlenmeyer flask using 25 ml

of oxygenated KRB-HEPES/BSA and incubated in a shaking water bath (80 cycles/min) for 30

min at 37°C. Cell viability and yield were evaluated with trypan blue and a hemocytometer.

Protein Synthesis

Acini were centrifuged at 500 xg for 2 min and resuspended in 10 ml amino acid buffer

(Arg 0.6 mM, Cys 0.1 mM, GIn 2.0 mM, His 0.2 mM, lie 0.4 mM, Leu 0.4 mM, Lys 0.4 mM,

Met 0.1 mM, Phe 0.2 mM, Thr 0.4 mM, Trp 0.05 mM, Tyr 0.2 mM, Val 0.4 mM, in KRB­

HEPES/BSA) (Korc, 1982; Hirschi et aI., 1994) and shaken at 80 cycles/min for 1 h at 37°C.

Acini were incubated with 20 /LCi/ml of 3H-phenylalanine in amino acid buffer for 0 or 15 min

[a period of linear incorporation into both total protein (TCA precipitable) and lipase, Figure 4-1],

and shaken at 80 cycles/min at 3rC. Aliquots of the radiolabeled acini were used for 2- dimensional gel electrophoresis (2-D gels, Figure 4-2) and for specific activity of the immediate precursor tRNA phenylalanine.

2-D Gels. Previous studies have been done to validate this technique for determining relative synthesis of rPL (Hirschi, 1990). The single spot identified as pancreatic lipase was the sole protein spot that exhibited colipase-dependent lipolytic activity and had a molecular weight

(MW) 49,500 and an isoelectric point (PI) of 7.0. Molecular weight and PI of rat pancreatic lipase is 50,000 and 6.8, which is in agreement with results from our lab with 2-D gel separation.

The rPLRP-l spot has not yet been identified because the PI and enzymatic activity are not yet known; however, the MW should be higher than rPL due to its additional six amino acids (Payne et aI., 1994). Although no apparent MW is reported for rPLRP-l, the rPLRP-l protein is 82

40

O~------~------~------+------~ o 15 30 45 60 Minutes

w (f) 40 a...~ ::J /e 0 ...... ~ 8 30 -o -)( ....w c ~ -. 0:: /e o '0.. a... a. 20 0:: Q o~ e· ~ E - Q. w '0 10 e/ x""'" ...... a... X ~.., / 0 0 15 30 45 60 Minutes

FIGURE 4-1. Linearity of3[H] Phenylalanine Incorporation into TeA Precipitable Proteins and Lipase. Incorporation of 3H phenylalanine was linear up to A. 30 min in TeA precipitable protein (r2 = 0.99), and B. 60 min in lipase (r:! = 0.99). 83

FI GURE 4-2. Two-Dimensional Polyacrylaminde Gel Electrophoresis of Pancreatic Proteins. Pancreatic acini were isolated from Sprague Dawley rats and sonicated in deionized water. Soluble proteins were separated in the first dimension by isoelectric focusing and in the second dimension by SDS gel electrophoresis using 10-20% polyacrylamide gradient. Protein spots were stained with Coomassie Blue dye. Lipase is identified by the arrow.

c: 80 --~...... -"- 50 • / --c.o • • • .-~ 40 .-- •• ~ ' :.. .. c: .- • --~ ~ 25 • 0 • -~ ~ 20 • - 4 5 7 9 Isoelectric Point 84 considerably larger than 50,000 kDa. Preliminary immunoprecipitation studies were attempted with a highly specific antibody to rPL that did not cross react with rPLRP-2 (Wagner et aI., 1994) in order to validate further the use of the pancreatic lipase spot on 2-D gel for these synthesis studies, but this antibody did not precipitate any radiolabeled protein. Based on current information it is highly unlikely that rPL and rPLRP-l comigrate on 2-D PAGE due to the significant difference in their MWs, but future studies need to confirm this with appropriate immunoprecipitating antibodies.

Incorporation of labeled phe was stopped by removing 1 ml labeled acini from the flask and combining it with 1 ml ice cold 40 mM phe. Acini were centrifuged 1000 xg for 2 min at

4°C; the media was removed and the pellet was washed 2 times using 2 ml of cold PBS. The acini pellets were stored at -80°C until analyzed.

Labeled acini were sonicated for 10 sec in deionized water. Aliquots of acini homogenate were analyzed for total protein using the method of Lowry et aI. (1951) and for incorporation of 3H-phe into trichloroacetic acid (TCA) precipitable protein using ice cold 10%

TCA. For TCA precipitation, acini were incubated for 20 min on ice, centrifuged 4 min at 4°C for 2000 xg, and washed 2 times with the 10% TCA. The pellets were dissolved overnight in

600 JLI 0.1 N NaOH at 4°C, and an aliquot was neutralized with 0.1 N HCI and counted in ACS liquid scintillation cocktail using a scintillation counter (Tri-Carb 460 CD, Packard, Downers

Grove, IL) (Hirschi et aL, 1994).

Relative synthesis of individual exocrine pancreas enzymes was measured using 2-D gels (Scheele, 1975; Hirschi, 1990). Proteins were separated by isoelectric point in 4% polyacrylamide in the presence of ampholites (1.0% each pH 4-6, pH 5-8, pH 9-11, and 2.5% pH 3.5-10, LKB Instruments, Inc., Gaithersburg, MD), 9.0 M urea, 0.28 M sucrose, and 0.2 85 mM PMSF. Voltage was adjusted to 100 V and run for 18 - 24 h. First dimensional gel tubes were then soaked for 5 min in 62.5 mM Tris buffer, pH 6.8, containing 2% sodium dodecyl sulfate (SDS) and 0.01 % bromophenol blue dye. The second dimension separated proteins by molecular weight in 10 to 20% polyacrylamide gradient gel containing 2% SDS. The current was adjusted to 90 rnA per gel, and the gel was run 45 min after the dye front reached the bottom of the gel. Proteins were stained using 0.06% Coomassie Blue dye and individual protein spots

(identified and unidentified) were cut out and digested overnight in 10 ml vials using 1 ml 30% hydrogen peroxide at 60°C. The radioactivity incorporated into individual protein spots was then determined by liquid scintillation counting.

The relative synthesis of individual proteins was calculated by dividing the dpm/mg incorporated into a particular enzyme spot by the total dpm/mg incorporated into all the protein spots.

Isolation of tRNA. Pancreatic tRNA was isolated by a modification (Brannon, Hirschi and Korc, 1988) of the procedure of Davey and Manchester (1969) and McKee and coworkers

(1978). Incorporation of 3H-phe into proteins was stopped by removing 1 ml of the labeled acini from the 50 ml Erlenmeyer flask and placing it in 0.5 ml ice cold bentonite/PBS (1 :4), followed by microcentrifugation. Pellets were washed one time with 1 ml bentonite/PBS then suspended

in 0.4 M sucrose - 0.1 M KC1- 6 mM MgCl 2 - 30 mM NaCI - 1 mM sodium acetate, pH 7.4), and sonicated 20 sec. Phenol (1 mt) and 0.2 ml 0.15 M NaCI - 1 mM EDTA, pH 7.4 were added, and the mixture vortexed and microcentrifuged 10 min. The supernatant was removed and re-extracted with 0.75 ml phenol, and the total supernatant (0.35 ml) was incubated with 1.25 ml ethanol and 25 JLl 20% potassium acetate at -20°C overnight. The precipitate was microcentrifuged; the supernatant discarded; and the pellet was then dissolved in 0.5 ml of 50 86

mM NaCI - 10 mM MgClz - 1 mM Na2EDTA - 10 mM sodium acetate, pH 7.4, combined with

187 ILl 5 M NaCI for a final concentration of 1.25 M NaCI, and incubated for 2 h on ice.

Samples were vortexed and microcentrifuged; the supernatant was reserved in a new

microcentrifuge tube; and the pellet was washed and incubated 0.5 h with 125 ILl 1.25 M NaCI -

10 mM MgClz - 1 mM NazEDTA, pH 7.4. The combined supernatants were precipitated with

1 ml ethanol overnight and microcentrifuged. The tRNA pellet was dissolved in 1 ml of 50 mM sodium carbonate, pH 10 for 1.5 h to release the phenylalanine. Aliquots were used to determine radiolabeled phenylalanine and total phenylalanine. Specific activity was expressed as dpm 3H phenylalanine per nmol total phenylalanine.

Total tRNA-phenvlalanine. Total phenylalanine concentrations of dissociated tRNA samples were determined using a Spectra-Physics Model 8000B high performance liquid chromotograph (HPLC) and expressed as nmollml (Lindroth and Mopper, 1979; Jones, Paabo and

Stein, 1981). Samples were run on a RP-18, 3IL spherical (Rainin Short One) column. The sample vial contained 50 ILl isolated tRNA sample, 2.5 mM internal amino acid standard and

0.085 N HC\. The total volume of the sample vial was 400 ILl and was combined with 400 ILl o-phthalaldehyde (OPA; Pierce 26010 - Fluoropa) prior to loading into the injection loop.

Radiolabeled tRNA-phenylalanine. Radiolabeled phenylalanine of dissociated tRNA samples was determined by scintillation counting of an aliquot of isolated dissociated tRNA.

Specific activity was expressed as disintegrations per minute per nmol phenylalanine (dpm/nmol phe).

Statistical Analysis

All data, expressed as mean ± SEM, were analyzed by analysis of variance (ANOVA), using least significant differences (LSD; Steel and Torrie, 1960), or Duncan's multiple range 87 (Zar, 1974) as post hoc tests. The main effects of amount of fat and type of fat and their interactions were determined by two-way ANOV A.

RESULTS

Experiment 1

Food consumption. Total body weight and pancreatic weight were not affected by either type or amount of dietary fat (Table 4-1). Food consumption was not affected by dietary treatment either; however, total fat consumption was 3-fold greater in groups fed the MF diet compared to the groups fed LF diets (p

Enzvme activities and plasma ketones. Amount offat independently affected amylase and lipase activity (Table 4-2 and Figure 4-3) and had no effect on trypsin and chymotrypsin activity (Table 4-2). Lipase activity was 58% greater in MF diet groups compared to LF diet groups (p < 0.0003), whereas amylase activity was 57% greater in LF diet groups compared to

MF diet groups (p < 0.007). Type of fat independently affected lipase activity, but had no effect on amylase activity. Lard diets had 48% greater lipase activity compared to safflower oil and corn oil diets (p < 0.03). As saturation decreased, there was progressively less lipase activity at

LF levels but similar lipase activity at MF levels. There was a 20% difference in lipase activity between LF and MF lard groups; a 30% difference between LF and MF olive oil groups; a 90% difference between LF and MF corn oil groups; and a 172 % difference between LF and MF safflower oil groups. This, however, did not result in a significant interactive effect for amount and type of fat on lipase activity in this experiment.

Amount of fat and type of fat did have an interactive effect on plasma ketone ({3- hydroxy-butyrate) levels (p < 0.03) but had no independent effect on plasma ketones (Table 4-2).

Plasma ketones were greater at LF levels compared to MF levels for the polyunsaturated 88

TABLE 4-1

The Effects of Dietary Fat on Body and Pancreatic Weights and Food Intake1

- DIET WEIGHT 7 DAY INTAKE

grams

LF BODY PANCREAS TOTAL FAT:!

SAFFLOWER 81 ± 4 0.61 ± 0.07 90 ± 6 4.49 ± 0.03 b

CORN OIL 85 ± 4 0.60 ± 0.03 86 ± 6 4.31 ± 0.03 b

OLIVE OIL 80 ± 4 0.59 ± 0.02 88 ± 9 4.41 ± 0.46 b

LARD 82 ± 2 0.58 ± 0.03 91 ± 6 4.52 ± 0.32 b

MF

SAFFLOWER 78 ± 3 0.59 ± 0.02 78 ± 7 13.57 ± 1.22 a

CORN OIL 82 ± 4 0.48 ± 0.09 78 ± 5 13.57 ± 1.02 a

OLIVE OIL 86 ± 4 0.59 ± 0.06 90 ± 9 15.60 ± 1.58 a

LARD 84 ± 4 0.54 ± 0.04 80 ± 6 13.83 ± 1.10 a

1 Values represent mean ± SEM.

2 There was an independent effect of amount of fat (MF> LF, p < 0.0001). TABLE 4-2

The Effects of Dietary Fat on Enzyme' Activities and Plasma Ketone Levels l

AMYLASE2 TRYPSIN3 CHYMOTRYPSIN3 PLASMA KETONES4 (U/mg Protein) (J.tmollL)

SAFFLOWER LF 157 ± 10 866 ± 107 8.2 ± 0.6 18 ± 2 abc

MF 133 ± 30 893 ± 48 8.9 ± 1.1 13 ± 3 bed

I CORN OIL LF 206 ± 36 1094 ± 58 11.0 ± 1.0 19 ± 2 ab

MF 150 ± 36 899 ± 61 9.4 ± 0.6 15 ± 2 abed

OLIVE OIL LF 205 ± 43 1067 ± 100 10.1 ± 0.6 12 ± 3 ed

MF 105 ± 13 819 ± 63 8.8 ± 0.6 20 ± 3 a

LARD LF 256 ± 69 1094 ± 158 10.0 ± 0.8 10 ± 1 d , , MF 135 ± 14 858 ± 59 8.4 ± 1.2 14 ± 2 abed I

I Weanling rats were fed for 7 days diet containing LF (50 g/kg diet) or MF (174 g/kg diet) levels of dietary fat. Values represent mean ± SEM (n = 4-6) and were analyzed using 1 and 2-way ANOVA and LSD.

2 There was an independent effect for amount of fat on amylase activity (LF> MF P < 0.007), but no effect of type of fat and no interactive effect.

3 There was no treatment effect for trypsin or chymotrypsin activity.

4 There was an interactive effect between amount and type of fat on plasma (j-hydroxybutyrate levels (p < 0.03), but no independent effect of either amount or type of fat.

00 \0 90

c:::J LOW FAT 60 _ MODERATE FAT ab >­ 50 I- > c 40 I-~ U e « a. 30 0> W E (f) ...... « 2, 20 (L 10

o L..-L..-..... SAFFLOWER CORN OLIVE LARD

FIGURE 4-3. The Effects of Amount and Saturation of Dietary Fat on Lipase Activity. Weanling rats were fed for 7 days diet containing LF (11 % energy) or MF (40% energy) levels of dietary fat. Values represent mean ± SEM of 6 rats. Those not sharing a superscript differed significantly using two way ANOVA and LSD. There was an independent effect of amount of fat (MF> LF. p<0.OO03) and type of fat (lard> safflower oil and com oil, p < 0.03). but no interactive effects. 91 safflower and corn oil diets, yet ketones were greater at MF levels compared to LF levels for the more saturated olive oil and lard diets.

Pancreatic mRNA Levels

Dietary treatment had no effect on 28s RNA levels (Table 4-3). Both rPL and rPLRP-

1 mRNA levels increased 1.8- and 2. I-fold from LF to MF groups (p < 0.0001 and p < 0.0002), independent of dietary fat saturation (Figure 4-4). Type of fat also had an independent effect for both rPL and rPLRP-I mRNA levels. Corn oil had a significantly greater effect than safflower, olive oil and lard (p < 0.000 I) for rPL mRNA levels; Corn oil and olive oil had significantly greater effects than safflower oil and lard (p < 0.04) for rPLRP-I mRNA levels. Amylase mRNA levels were not affected by amount of fat; however, there was an independent effect of type of fat in which corn and olive oil groups had greater mRNA levels than safflower oil and lard groups

(p<0.0004) (Table 4-3).

Experiment 2

Body weights. There was no difference in weight gain and final body weights of rats

(data not shown). The mean final body weights were 102 ± 5 g.

Relative synthesis. Amount offat independently affected lipase synthesis (Figure 4-5), but had no effect on amylase synthesis (Table 4-4). Lipase synthesis increased 2.2-fold in rats fed MF compared to LF (p < 0.004). There was no independent or interactive effect of type of fat on lipase synthesis. Type of fat independently affected amylase synthesis; safflower oil diet groups had greater synthesis of amylase than lard (p < 0.01). Although there was no independent effect of amount of fat on amylase synthesis, LF groups tended to have greater amylase synthesis than MF groups. 92

TABLE 4-3

The Effects of Dietary Fat on 28s RNA and Amylase mRNA Levels'

28s Amylase/28s2

(Relative Units)

SAFFLOWER LF 0.83 ± 0.08 1.6 ± 0.2bc

MF 0.73 ± 0.09 1.3 ± 0.3c

CORN OIL LF 0.87 ± 0.12 3.1 ± 0.43

MF 0.75 ± 0.13 2.5 ± 0.33b

OLIVE OIL LF 0.74 ± 0.16 2.8 ± 0.43

MF 0.98 ± 0.28 2.5 ± 0.63b

LARD LF 0.93 ± 0.06 1.4 ± 0.2c

MF 0.81 ± 0.18 1.3 ± 0.3c

1 Weanling rats were fed for 7 days diet containing LF (50 g/kg diet) or MF (174 g/kg diet) levels of dietary fat. Values represent mean ± SEM (n=6) and were analyzed using ANOVA and LSD. Those not sharing a superscript within a column differed significant! y.

2 There was an independent effect for type of fat (p < 0.0004), but no effect of amount and no interactive effect on amylase/28s mRNA. 93

c:J LOW FATb 1.6 _ MODERATE FAT o « Z a::: ., 1.2 en :!::c 00 ::I CI N ;:> 0 ""-« G 0.8 Z .. a::: -0 0 ;: E E -l 0.4 a.. L..

c:J LOW FATb 0.8 _ MODERATE FAT o z« 0:::: (/) 0.6 CO !! C C'I ::I "'- CD « ;:> z 0 0:::: -; 0.4 E .. -0 ..- .2 I '0 a.. .. 0.2 0:::: .....J a.. L..

FIGURE 4-4. The Effects of Dietary Fat on Lipase and Related Protein-l mRNA Levels. Weanling rats were fed for 7 days diet containing LF (11 % energy) or MF (40% energy) levels of dietary fat. Values represent mean ± SEM of 6 rats. Those not sharing a superscript differed significantly using two way ANOVA and LSD. A. rPL mRNA. There was an independent effect of amount of fat (MF> LF, P < 0.0001) and type of fat (corn oil> safflower oil=olive oil=lard, pLF,p<0.0002) and type of fat (corn oil=olive oil> safflower oil=lard, p

a 10 c::J SAFFLOWER LARD a 8 - .~ I/) Q) ..-..c: W c 6 (/) ~ I/) « Q) b 0... > b += .....J c 4 Q) L.. ~ 2

o '---"---"-- LF MF

FIGURE 4-5. The Effects of Dietary Fat on the Relative Synthesis of Lipase. Weanling rats were fed for 7 days diet containing LF (11 % energy) or MF (40% energy) levels of dietary fat. Values represent mean ± SEM of 6 rats. Those not sharing a superscript differed significantly using two way ANOVA and LSD. There was an independent effect of amount of fat (MF> LF, P < 0.004), but no independent effect of type of fat or interactive effect. 95 Specific activity oftRNA-phenyJaJanine. Total phenylalanine and the specific activity of the immediate precursor pool of tRNA were measured and were not affected by dietary treatment (Table 4-4). There was, however, high variability in the specific activity of the tRNA­ phe pool as indicated by the SEM, which ranged from 14% to 50% of the means.

DISCUSSION

The increase in lipase activity with the increase of polyunsaturated dietary fat is consistent with several previous studies (Sabb et aI., 1986; Wicker and Puigserver, 1987; Ricketts and Brannon, 1994). The effects of saturation of dietary fat has been less clear. We previously reported that a MF safflower oil diet increases lipase activity relative to LF safflower, yet MF lard diet has no effect on lipase activity relative to LF lard (Ricketts and Brannon, 1994), suggesting different mechanisms of regulation for lipase by type of dietary fat. The present study includes safflower oil, corn oil, olive oil, and lard with PIS ratios ranging between 0.3 and 7.9.

The lipase and its related protein-l mRNA levels (both rPL and rPLRP-l) increased with amount of dietary fat. Our results support previous work (Wicker and Puigserver, 1990;

Ricketts and Brannon, 1994). These results, which include the lipase mRNA levels of four different PIS ratios, further support that the amount of fat regulates pancreatic lipase pre­ translationally. Both rPL and rPLRP-I were affected similarly by the amount and type of dietary fat. Among the types of fat, corn oil demonstrated greatest mRNA levels at MF levels. The response of lipase activity to type of dietary fat at LF levels, specifically decreasing activity with increasing saturation, supports our previous findings that saturation affects lipase expression translationally or post-translationally and suggests that polyunsaturated dietary fat elicits significantly less lipase activity at LF levels compared to more saturated fats. 96

TABLE 4-4

The Effects of Dietary Fat on Total tRNA Phenylalanine, tRNA-Phenylalanine

Specific Acth'ity, and Relative Synthesis of Amylase"l

tRNA Phenylalanine Amylase Total Specific Activity % Relative Synthesis (nmol/ml) (dpm/nmol)

Safflower LF 6.3 ± 0.6 78 ± 18 55 ± 3a

MF 5.8 ± 1.1 35 ± 5 46 ± lla

Lard LF 5.4 ± 1.0 42 ± 20 38 ± 7ab

MF 5.9 ± 2.7 73 ± 29 25 ± 6b

1 Values represent mean ± SEM for 3-6 animals.

2 Values not sharing a superscript differed significantly (p < 0.01). 97 Transcriptional regulation of lipase in response to dietary fat has been studied (Wicker

and Puigserver, 1990); however, Wicker used the rPLRP-l eDNA for quantitation which has been

reported to not be the true lipase (Payne et aI., 1994). Wicker and Puigserver (1990) report that

rPLRP-l transcription rates increased 1. 7-fold after 5 days on a diet where 62 % of energy came

from highly unsaturated sunflower oil. There have been no studies that report transcription rates

using rPL eDNA, but it is likely that high amounts of dietary fat, regardless of saturation,

regulate this message transcriptionally. Our work indicates that rPL and rPLRP-I are similarly

regulated by amount and type of fat. Amount of dietary fat has been reported to increase lipase

synthesis (Wicker and Puigserver, 1987) when comparing diets containing 7% of energy from

sunflower oil to diets containing 67% of energy from sunflower oil. We report both safflower

oil and lard increase lipase synthesis when diets increase from LF to MF. These data suggest that

the effect of dietary fat saturation on lipase activity occurs after synthesis. Currently post­

translational degradation of the exocrine pancreas secretory proteins is not believed to occur. Our

results raise the possibility that post-translational degradation could occur. Other post-translation

regulation may occur in response to saturation of dietary fat, one possibility is differences in

secretion rates. The content of lipase activity is the result of the cumulative effect of transcriptional rate, synthetic rate, post-transcriptional processing, and secretion rate. Future studies need to determine whether type offat alters secretion rates and, thus, affects lipase content

Amount of dietary fat has the opposite effect on amylase activity, as has been demonstrated many times (Brannon, 1990). Type and amount of dietary fat has no effect on trypsin and chymotrypsin activity, which also has been demonstrated previously (Ricketts and

Brannon, 1994). 98 Plasma ketones have been proposed as possible mediators in the regulation of lipase

because both ketones and lipase activity are increased during diabetes (Bazin and Lavau, 1979).

However, we previously saw no relationship between lipase activity and plasma ketones ({3-

hydroxybutyrate) when feeding safflower oil and lard at LF and MF levels. In this study there

was an interactive effect between amount and type of fat on plasma {3-hydroxybutyrate levels but

no independent effect by either amount or type of fat. These results suggest plasma ketones

decrease with MF polyunsaturated fats and increase with MF saturated fats. In view of the

interactive effect between amount and type of dietary fat, the role of ketones in the regulation of

lipase is still unclear, but these results do not support the proposed mediation of dietary fat

regulation of pancreatic lipase.

The regulation of pancreatic lipase by dietary fat is complex. Safflower oil and lard have similar effects on mRNA levels and synthesis of lipase, but not on activity of lipase.

Interestingly, corn oil interacts with the amount offat to increase lipase mRNA levels beyond that of safflower oil, which suggest that the degree of polyunsaturation of dietary fat may not be the sole factor affecting interaction between amount and type of fat. This work supports the contention that there are multiple mechanisms responsible for regulation of pancreatic lipase. 99 CHAPTER 5

THE EFFECTS OF DIETARY FAT ON THE REGULATION OF RAT PANCREATIC LIPASE FOLLOWING INHIBITION OF CHYLOMICRON TRANSPORT FROM THE ENTEROCYTE

INTRODUCTION

Pancreatic lipase is the primary lipolytic enzyme for digestion of dietary fat in the adult. Type and amount of dietary fat have been shown to regulate lipase differently (Ricketts and Brannon, 1994). Amount of dietary fat appears to regulate pancreatic lipase pre- translationally because pancreatic lipase (rPL) mRNA levels increase with increasing dietary fat

(Ricketts and Brannon, 1994). Most likely this regulation is transcriptional, but no studies have examined dietary regulation of rPL transcription. Transcriptional regulation of rPLRP-l has been demonstrated for amount of dietary fat by Wicker and Puigserver (1990). The relative synthesis rates of pancreatic lipase have also been shown to be affected by amount of fat, but not type of fat (Chapter 4). Type of fat (specifically saturation of fat), however, affects pancreatic lipase post-translationally. Lipase activity increased only with increasing unsaturated dietary fat when dietary fat is increased from LF to MF levels.

The mechanisms involved in the regulation of pancreatic lipase in response the dietary fat are unknown, however, both diet and the hormones secreted in response to diet affect the regulation of pancreatic exocrine secretions (Brannon, 1990). Gut hormones such as CCK and secretin exhibit a particularly strong influence. CCK is, however, not a likely mediator of dietary fat regulation of pancreatic lipase. First, CCK, a potent stimulator for pancreatic secretion, causes an increase in protease synthesis and a decrease in amylase synthesis, but does not affect lipase synthesis (Schick et ai., 1984b). Second, consumption of high protein diets, rather than 100 high fat diets increase plamsa CCK levels (Green, Levan and Liddle, 1986). Secretin and its related peptides are more likely mediators of dietary fat regulation of pancreatic lipase. Secretin also increases pancreatic protein synthesis, but it especially increases lipase synthesis when infused into rats (Rausch et aI., 1986). GIP also increases lipase synthesis and mRNA levels (Duan and

Erlanson-Albertsson, 1992b). Second, fatty acids in the gastrointestinal lumen stimulate secretin release causing pancreatic secretion (Chey, 1993). The response of secretin to dietary fat was demonstrated when 20% TG emulsions were infused into rat duodenum leading to a 2. I-fold increased in pancreatic secretion (Guan et aI., 1991), which was significantly inhibited by antisecretin serum. The role of secretin in the regulation of pancreatic lipase is controversial because secretin had no effect on lipase expression in cultured acinar cells (Hirschi et aI., 1994).

Alternatively, the regulation of pancreatic lipase gene expression by dietary fat may involve post-absorptive metabolites of dietary fat. Ketones, metabolites of fatty acid oxidation, have been proposed as mediators of pancreatic lipase. There is an increase in ketones and lipase in diabetes (Bazin and Lavau, 1979), which also results in increased pancreatic lipase and its mRNA (Tsai et aI., 1994). Pancreatic lipase increases with constant infusion of ketones in vivo

(Bazin and Lavau, 1978). Ketones increase pancreatic lipase in acinar cells cultured from rats fed low fat diets (Hirschi et aI., 1991). However, circulating ketone levels did not differ between rats fed LF and those fed MF diets even though pancreatic lipase and its mRNA levels did

(Ricketts and Brannon, 1994). Thus the role of ketones in this regulation is controversial.

Inhibition of post-absorptive metabolites of dietary fat can be achieved by blocking transport of lipid after absorption which still allows luminal triglyceride and fatty acids to stimulate responsive gastrointestinal hormones. Pluronic L-81 (L-81) is a hydrophobic detergent made up of polyoxyethylene and polyoxypropylene copolymers, which has demonstrated to be a 101 potent inhibitor of intestinal lipid transport from the enterocyte (Halpern, Tso and Mansbach,

19S8). Triglycerides accumulate in vesiculated smooth endoplasmic reticulum of the enterocyte when rats are fed diets containing L-SI; but this effect is quickly reversed within 24 h of removing L-Sl. TGs are then rapidly mobilized and appear in the lymph. This blocker of lipid transport has been used in many studies and has proven to be effective and nontoxic to rats for up to one month of administration (Tso and Balint, 1986).

To determine if the mediator of amount or type of dietary fat on pancreatic lipase regulation occurs pre- or post-absorptively (Figure 5-1), this study examined pancreatic lipase activity and mRNA levels in rats fed LF and MF diets containing safflower oil or lard with

(Blocker) or without (Control) Pluronic L-Sl. To control for any effects of Pluronic L-Sl on food intake, a Pairfed group was fed the amount of Control diet that the Blocker group ate the previous 24 h. The Blocker diet contained by weight 0.5% L-Sl which replaced equivalent weight of cellulose.

METHODS

Experimental Protocol

Male weanling Sprague-Dawley rats (35 - 60 g; Harlan, Indianapolis, IN) were housed as described in Chapter 3. Animal protocols were approved by the University of Arizona Animal

Care and Use Committee (Appendix A). Animals were weight-matched and divided into twelve diet-treatment groups (2 x2 x3 design): LF or MF diets with safflower oil or lard, fed with

Pluronic L-Sl (Blocker) or without it (Control and Pairfed), (Figure 5-2). All diets were isonitrogenous and isocaloric. The composition of the diets is shown in Table 5-1. Control and

Pairfed groups were fed diets for 7 days, the Blocker (Pluronic L-81, a generous gift from Dr. 102

Gastrointestinal Enterocyte Circulation .& Pancreatic Lumen Tissues Acinar Cell

A. Control

MG G TG f FMA TG ?--r-..... t TG l ~ t Ketones ____

t secretin / t GIP

B. + Pluronic L-81 (Blocker)

MG MG TG FA

t secretin/ t GIP

FIGURE 5-1. The Use of Pluronic L-Sl for Determining Pre-absorptive and Post­ absorptive Effects of Dietary Fat on Pancreatic Lipase Gene Expression. The following abbreviations are: Triglycerides (TG); monoglycerides (MG); fatty acids (FA); Gastric Inhibitory Polypeptide (GIP). EXPERLMENTAL DESIGN

I weanling rats iLFi Safflower oil Lard saffeDiLard

Control Blocker Control Blocker Control Blocker Control Blocker (-LS1) (+LS1) ( -LS1) (+LS1) ( -LS1) (+LS1) ( -LS1) (+LS1)

Pairfed Pairfed Pairfed Pairfed ( -LS1) ( -LS1) ( -LS1) ( -LS1)

FIGURE 5-2. Experimental Design for the Use of Pluronic L-81 in Determining Pre-absorptive and Post-absorptive Effects of Dietary Fat on Lipase Gene Expression.

...... o w 104

TABLE 5-1

Dietary Compositionl

Diet ControIlPairfed Blocker

LF MF LF MF

Components g/100 g diet

Casein 20.0 20.0 20.0 20.0

DL-Methionine 0.3 0.3 0.3 0.3

AIN Mineral Mix 3.5 3.5 3.5 3.5

AIN Vitamin Mix 1.0 1.0 1.0 1.0

Choline bitartrate 0.2 0.2 0.2 0.2

Cellulose 5.0 20.5 4.5 20.0

Dietary fae 5.0 17.4 5.0 17.4

Pluronic L-81 -- -- 0.5 0.5

Cornstarch 65.0 37.1 65.0 37.1

1 Modified by Sabb et al. (1986)

2 Safflower oil was used as the polyunsaturated fat and lard as the saturated fat. 105 Patrick Tso, Louisiana State University) group was fed diet for 8 days, allowing one day to adjustto the Blocker diet. Control and Blocker groups were fed ad libitum whereas the Pairfed group animals were fed theamount of food consumed the previous day by the Blocker group.

Daily food consumption and body weight were recorded. Animals were killed by exsanguination from the abdominal aorta while anesthetized by ether.

Enzyme and mRNA analysis. Pancreatic tissue was used to determine digestive enzyme activity and mRNA levels. Tissue used for enzyme activity was wei'ghed and frozen immediately on dry ice and stored at -80 D C. Remaining tissue was used immediately for RNA isolation. Enzyme analysis, RNA isolation, and quantification of mRNA were preformed as described in Chapter 3 and Chapter 4.

Statistical Analysis

All data, expressed as mean ± SEM, were analyzed by three-way analysis of variance

(ANOVA) using least significant difference (LSD; Steel and Torrie, 1960) or Duncan's mUltiple range (Zar, 1974) post hoc test. Independent effects of 1) amount of fat; 2) type of fat; 3)

Blocker treatment and their interactive effects were determined.

RESULTS

Food Consumption and Body Weight

The Control groups ate 20% more food than either the Blocker or Pairfed groups

(p < 0.003, Table 5-2). Weight gain of animals differed. Amount of fat independently affected weight gain; animals in MF groups had a greater weight gain (43.6 ± 1.3) than those in LF groups [37.2 ± 1.3 (p < 0.001)]. Dietary treatment also independently affected weight gain. 106

TABLE 5-2

The Effects of Treatment and Diet on Total Food Consumption, Weight Gain and

Body Weight of Ratsl

Food Consumption:! Weight Gain3 Final Body Weight4 (g/day) (g/week) (g)

Control Safflower LF 12 ± I 52 ± 7 110 ± 9

MF 10 ± I 50 ± 6 106 ± 12

Lard LF II ± I 50 ± 4 100 ± 9

MF 11 ± I 54 ± 5 106 ± 7

Blocker Safflower LF 8 ± 1 33 ± 5 99 ± 9

MF 9 ± 1 47 ± 5 101 ± 9

Lard LF 8 ± I 31 ± 4 81 ± 9

MF 8±1 43 ± 5 96 ± 10

Pairfed Safflower LF 8±1 27 ± 2 85 ± 6

MF 9 ± I 36 ± 3 98 ± 7

Lard LF 8 ± 1 30 ± 3 86 ± 8

MF 8 ± 1 31 ± 1 92 ± 6

1 Values represent mean ± SEM for 5 rats. Animals were weight matched and divided into three treatment groups, four diet groups and fed 7 or 8 days isonitrogenous, isocaloric diets.

:! There was an independent effect of treatment (Control;:: Pairfed ~ Blocker, p<0.003).

3 There was an independent effect of amount of fat (MF> LF, P < 0.002). Treatment independently affected weight gain (Control~Blocker~Pairfed, p

4 There was an independent effect of amount of fat MF> LF, P < 0.004) and treatment (Control>Blocker=Pairfed, p

Pairfed animals (p < 0.000 I). Interaction between amount of fat and dietary treatment on weightgain was also significant (p < 0.03). Weight gain was greater for MF Blocker groups than

LF Blocker groups.

Enzyme Activities and mRNA Levels

Protease activitv. Pancreatic proteases, trypsin and chymotrypsin, were not affected by either amount of fat, type of fat, or treatment (Table 5-3).

Amylase activity and mRNA levels. Amount of fat independently affected amylase activity with a 2.3-fold greater activity in LF diets compared to MF diets (p < 0.000 I, Table 5-3).

Type of fat did not affect amylase activity. Treatment increased amylase activity in the Blocker group 1.3-fold compared to the Control (p < 0.03). Amylase activity was not different in the

Pairfed group from either the Control or the Blocker group. Amylase mRNA levels were only affected by amount of fat in which LF groups were I .8-fold greater than MF groups (p < 0.000,

Table 5-4).

Lipase activity and mRNA levels. Amount of fat independently affected lipase activity with MF groups I.8-fold greater than LF groups (p < 0.0001, Figure 5-3). There was no independent effect of type of fat on lipase activity. Treatment also affected lipase activity by decreasing activity 50% in the Blocker group compared to the Control group (p < 0.003). Lipase activity in the Pairfed group was intermediate and not different from the Control or the Blocker group. There also was an interactive effect of amount and type of fat on lipase activity (p < 0.04), which was more pronounced for MF safflower than for MF lard.

The 28s RNA levels were not affected by diet or treatment (Table 5-4). Amount of fat affected rPL mRNA levels independently by increasing mRNA levels 2-fold from LF to MF 108

TABLE 5-3

The Effects of Treatment and Diet on Amylase and Protease Activities l

Amylase2 Trypsin Chymotrypsin

U/mg protein

Safflower LF 142 ± 15 bc 729 ± 56 3.8 ± 0.7 Control MF 71 ± 9d 1009 ± 32 3.7 ± 0.2

Lard LF 141 ± 15 bc 838 ± 70 3.4 ± 0.4

MF 73 ± 5~ 1042 ± 206 3.2 ± 0.4

Safflower LF 183 ± llab 944 ± 90 3.2 ± 0.2 Blocker MF 90 ± 22cd 831 ± 86 4.4 ± 0.6

Lard LF 224 ± 3~ 629 ± 64 4.8 ± 0.8

MF 63 ± 3d 712 ± 117 3.0 ± 0.6

Safflower LF 169 ± lib 891 ± 62 4.0 ± 0.6 Pairfed MF 55 ± 9d 726 ± 36 4.4 ± 0.3

Lard LF 150 ± 32b 653 ± 107 3.9 ± 0.3

MF 89 ± l7cd 760 ± 106 3.7 ± 0.4

1 Values represent mean ± SEM.

2 There was an independent effect of amount of fat (LF> MF, p

TABLE 5-4

The Effects of Treatment and Diet on 28s RNA and Amylase

2 mRNA Levels'·

28s Amylase128s2

relative units

Safflower LF 0.79 ± 0.05 2.2 ± 0.23bed Control MF 0.74 ± 0.11 1.7 ± O.led

Lard LF 0.83 ± 0.15 2.9 ± 0.73bc

MF 0.70 ± 0.11 1.5 ± O.Sed

Safflower LF 0.83 ± 0.06 3.5 ± 0.83 Blocker MF 0.77 ± 0.16 2.1 ± O.Sbed

Lard LF 0.60 ± 0.11 3.3 ± 0.43b

MF 0.67 ± 0.08 1.5 ± O.4cd

Safflower LF 0.97 ± 0.05 2.3 ± OSbed Pairfed MF 0.81 ± 0.03 1.6 ± 0.3ed

Lard LF 0.74 ± 0.07 3.2 ± 0.23b

MF 0.60 ± 0.10 1.4 ± 0.2d

I Values represent mean ± SEM.

2 There was a significant effect of amount of fat (LF> MF, P <0.0001). 110

a 100 I'" c:::::J Control Blocker b . III (Xl ~ Pair fed ab ;I. III CII ,...~ - CIICII >- 75 - l- .e >-_ c ~ .. 1-0; 1x ~x UO >< ~ 1 ~ >< «s. 50 :a CII >< WOl :aJ tI)~ «2- n ~ I>< 0- III X X 25 r >< >< --' T >< >< >< x >< >< >< x >< >< 0 I>< safflower lard safflower lard c LF d MF

FIGURE 5-3. Effects of Treatment and Dietary Fat on Lipase Activity. Values represent mean ± SEM from 5 rats. :l-dValues for a given parameter not sharing a superscript differ significantly (p < 0.05; three-way ANOVA). c.iValues not sharing a superscript differ significantly (p < 0.0001; one-way ANOVA). There was an independent effect of treatment (Blocker < Control, p<0.003) and amount of fat (MF> LF, p <0.0001). There was also an interactive effect of amount and type of fat (p < 0.04). 111

a 0.9 Cl Control b A Cleo.. - Blocker b ~ !Xl Pair fed a c... « ~ ~ Z ~ c::: !! -'E - UJ 0.6 ~ :::II .:::- CO QQ C"J ~• =' ::.1 ...... '0 -= :"I « '! -= z 0 c::: - ~ .2 0.3 ~ E '2 =' ~ ...J ~ a.. - ~

0.0 safflower lard safflower lard

LF d MF C

1.2 r Cl Control a 8 _ Blocker b

0.6 r

0.3 T

0.0 safflower lard safflower lard LF d MF c

FIGURE 5-4. Effects of Treatment and Dietary Fat on rPL and rPLRP-I mRNA Levels. Values represent mean ± SEM from 5 rats. a-dValues for a given parameter not sharing a superscript differ significantly (p < 0.05; three-way ANOVA). c.kValues not sharing a superscript differ significantly (p < 0.0001; one way-ANOVA). A. rPL. There was an independent effect of treatment (Blocker < Control, p < 0.01) and amount of fat (MF> LF, P < 0.0001). There was also an interactive effect of amount and type of fat (p < 0.007). 13. rPLRP-l. There was an independent effect of treatment (Blocker < Control, p < 0.007) and amount of fat (MF>LF, p

Although there was no independent effect of type of fat on rPL mRNA levels, there was an interactive effect between amount and type of fat of dietary fat (p < 0.007). Similarly to effects on lipase activity, MF safflower Blocker had less of an effect on rPL mRNA levels.

The effects of amount of fat and dietary treatment on rPLRP-I mRNA levels were similar to the effects on rPL mRNA levels (Figure 5-4B). Amount of fat increased rPLRP-I mRNA levels 1.9-fold from LF to MF diets (p

Control and Blocker mRNA levels. Also, there was no interactive effect between type and amount of dietary fat on rPLRP mRNA levels.

DISCUSSION

The animals treated with Pluronic L-81 (Blocker) did not eat as much as the Control groups. The lower food intake in Blocker animals may be attributed to the bitter taste of L-81 or may possibly result from the interrupted lipid transport. The inflated Blocker animal intestines were distinguishable from non-Blocker animal intestines. Other studies using the blocker L-81 have shown no difference in overall diet consumption between Control and Blocker diets (Tso and

Balint, 1986); however, these studies lasted three to four weeks during which time full adaptation to the diet may occur resulting in equivalent food intakes. Due to the difference in food intake, a pair fed control was used to distinguish effects unique to Pluronic L-81 and those due to decreased food intake. 113 Treatment independently affected weight gain with Control group gaining 25% more than Pluronic L-81 (Blocker) group, as to be expected with animals on Blocker diets eating less and not transporting chylomicrons to the periphery. Pairfed animals also gained less weight than the Control group (40%) and the Blocker group (19%). Lower food intake of a comparable diet in a rapidly growing animal like the weanling will result in undernutrition and decreased growth as observed in the Pairfed group. Although the Pairfed and Blocker groups ate the same amount, chylomicrons would have delivered dietary triglyceride to the peripheral tissue in the Pairfed where the triglyceride would have been oxidized. In contrast, the triglyceride remained in the enterocyte in the Blocker groups and would not have been oxidized, possibly contributing to the weight difference despite comparable intakes.

The lack of influence of treatment on total protein and protease activity suggests that weight gain differences had little effect on pancreatic enzymes. Amylase activity increased in LF diets which are higher in carbohydrate compared to MF diets, and is consistent with other data.

Pluronic L-81 treatment also affected amylase activity, which was increased in Blocker groups compared to Control groups. The effects of treatment on amylase activity indicates that the lack of triglyceride transport may create a greater reliance of carbohydrate for energy utilization.

Lipase activity increased with increasing amount of fat in all dietary fat and treatment groups. Although there was a difference between the LF and MF lard groups, there was still an interaction between amount of fat and type of fat. The safflower oil diet provided greatest difference between LF and MF levels which supports our previous data. Interestingly, the

Blocker treatment resulted in a 50% decrease in lipase activity for the LF safflower group relative to Control, but only a 20% decrease for the MF safflower group. The Blocker treatment affected the two lard diets with a 35% decrease for LF and a 38% decrease for MF, relative to their 114 Control. The effects of Blocker were greater than the food restriction it induced as indicated by the generally intermediate effects !n the Pairfed. Thus food restriction can affect pancreatic lipase possibly through decreased fat intake, but the Pluronic L-81 blockage of post-absorptive metabolism of dietary trigJycerides interfere with the dietary regulation of lipase beyond the effects of food restriction. Treatment and fat had similar effects on pancreatic mRNA levels compared to enzyme activity.

These effects of treatment on lipase activity indicate that there are several factors influencing lipase gene expression. An interpretation of these data is that blocking transport of chylomicrons from the enterocyte has a peripheral inhibitory effect on lipase regulation by dietary fat. It appears that dietary adaptation of rat pancreatic lipase to fat involves pre-absorptive effects, likely through gut hormones and peripheral effects from post-absorptive dietary triglyceride metabolism.

Although these factors work together, it appears that they interact with amount and type of fat. Blocker treatment had the greatest effect on both safflower and lard at LF levels.

However, this does not explain the effect of Blocker on type of fat. Blocker treatment caused a

20% reduction in lipase activity with MF safflower, but a 38% reduction with MF lard relative to Control. Similar differences in effect are present with mRNA levels where Blocker treatment led to a 12% reduction in lipase with MF safflower, but a 35% reduction with MF lard. The regulation of lipase by type of fat may involve differential stimulatory effects by type of fat on gut hormones. These results suggest that effects of MF lard may be mediated more by post­ absorptive factors than those of MF safflower. Given that MF lard results in less pronounced induction of lipase through possible post-translational regulation (Chapter 4), these results raise 115

the possibility that post-absorptive mediators may regulate pancreatic lipase post-translationally.

Future studies need to examine this hypothesis.

Secretin and GIP are most likely the gut hormones responsible for preabsorptive effects

of dietary fat on lipase expression. Secretin uses cAMP as a second messenger and secretin, GIP

and cAMP have been shown to regulate lipase (Duan and Erlanson-Albertsson, 1992a). The

effects of amount and type of dietary fat on secretin release have not been studied, but need to

be. These effects may require a threshold level of dietary fat before differences can be

determined. The results of this study suggest that blockage of post-absorptive metabolites of dietary fat partially abolishes the dietary regulation of pancreatic lipase.

The mechanisms involved in pancreatic lipase regulation are complex. Peripheral effects of dietary fat metabolism appear to playa more important role with lard at MF levels than with safflower at MF levels. At LF levels peripheral metabolites of dietary fat appear to be more predominately involved than that at MF levels. Further studies are necessary to understand fully these differential roles of gastrointestinal hormones and post-absorptive triglyceride metabolism on the effects of amount and type of dietary fat on rat pancreatic lipase expression. 116

CHAPTER 6

SUMMARY AND CONCLUSIONS

The purpose of this dissertation research was to determine the mechanism(s) by which amount and type of dietary fat regulate the gene expression of pancreatic lipase in rats. Large

increases in amount of dietary fat have been shown to increase lipase expression within 24 h, but only polyunsaturated dietary fats have been shown to increase lipase expression with moderate increases in amount of fat. The results of this study suggest that amount of fat, independent of its degree of saturation, regulate pancreatic lipase and its related protein mRNA levels and lipase synthesis, but that the degree of saturation interacts with amount of fat to regulate pancreatic lipase content. Lipase synthesis and mRNA levels showed similar increases when diets were increased from LF to MF for both safflower oil and lard. The effects of amount of fat on lipase content, synthesis, and mRNA levels suggests that amount of fat regulates lipase at pre­ translational levels, whereas type of fat interacts with amount of fat to regulate lipase post­ translationally.

Blocking transport of dietary fat from the enterocyte to circulation did not abolish the regulation of pancreatic lipase by amount of fat, but did ·partially inhibit the stimulation of lipase activity and mRNA levels. These results suggest that both pre-absorptive mediators such as gut hormones and post-absorptive mediators such as peripheral metabolites are involved in regulation of lipase gene expression by dietary fat.

Dietary fat regulates pancreatic lipase gene expression (content, mRNA and synthesis) by multiple and interactive mechanisms which leads to complex responses to amount and type of fat. Amount of fat most likely regulates pancreatic lipase transcriptionally and type of fat, 117 specifically level of saturation, interacts with amount of fat to regulate lipase post-translationally.

Both gut hormones and peripheral metabolites contribute to this dietary regulation of lipase. 118 APPENDIX A

VERIFICATION OF REVIEW BY THE INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

PHS .Assurance No. A-3248-01 - USDA No. 86-3

IACUC Control # ~9~O-_O~1~3~3~ ______

Title: Dietary Fat Regulation of Pancreatic Lipase Gene Expression

Principal Investiqator: __P~a~t=s~y~M~. __B~r~a~n~n~o~n~ ______

DepartmeDt: ______~N~u~t=r~~~·~=·=i_o~n~&~F~o~o~d=_=S_c~i~e~n~c~e~ ______

Submission Date: ______~A~p~r~1:·=l~2~7~, __ 1~9~9~0 ______

AqeDcy: ______=U=S=D~A~ ______

The University of Arizona Institutional Animal Care and Use Committee reviews all sections of proposals relating to animal care and use. The above-named proposal:

[ ] Has been reviewed and approval withheld.

[XX] Has been reviewed and approved by IACUC on June 18, 1990 Revisions (if any), are listed below: NONE

Approval valid through June 17, 1993

Michael A. Cusanovich, Ph.D. Vice President for Research

Date: January 17, 1991 119

VERIFICATION OF REVIEW BY THE INSTITUTIONAL ANIMAL CARE AND OSE CO~TTEE PHS .Assurance No. A-3248-01 - USDA No. 86-3

IAeue Control :I ...... ::9~O:...-~O""'l!l.;3~3:::.-____

Title: Dietary Fat Regulation of Pancreatic Lipase Gene Expression

Principal Investiqator:~P~a~t~s~Y~M~._B~r~a~n~n~o~n ______

Department: ______N~u~t~=~i~~~i~o~n~& __ F~o~o~d=_~s_c:i~e~n~c~e ______

Submission Date: ______~A~p~r~~~·l=_~2_7.,~1~9~9~O~ ______=

Aqency: ______~U~S~D~A~ ______

The University of Arizona Institutional Animal Care and Use Committee reviews all sections of proposals relating to animal care and use. The above-named proposal: ( ] Has been reviewed and approval withheld. (XX] Has been reviewed and approved by IACUC on June 18, 1990 Revisions (if any), are listed below: NONE

Aporoval valid through June 17, 1993

Michael A. Cusanovich, Ph.D. Vice President for Research

Date: Janua~T 17, 1991 120

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