Role of Group 1B Phospholipase A2 in Diet-induced Hyperlipidemia and Selected
Disorders of Lipid Metabolism
A dissertation submitted to the Graduate School at the University of Cincinnati
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in the Department of Pathobiology and Molecular Medicine of the College of Medicine
By
Norris Isaac Hollie, II
B.S., B.A. Oakwood University, Huntsville, Alabama
July 23, 2013
Committee Chair: David Y. Hui, Ph.D. Abstract
As obesity rates increase in developed nations, the prevalence of other obesity-related metabolic disorders such as hyperlipidemia, atherosclerosis, and non-alcoholic fatty liver disease (NAFLD) has also increased drastically. A greater understanding of the pathobiology and molecular mechanisms of these disorders becomes increasingly relevant. Recent studies demonstrated that certain alleles of the pancreatic group 1B phospholipase A2 (PLA2G1B) are associated with increased obesity and that Pla2g1b-deficient (Pla2g1b-/-) mice are protected against diet-induced obesity and diabetes. Furthermore, supplying mice with lysophosphatidylcholine (LPC), the enzymatic product of Pla2g1b, causes hyperglycemia and insulin resistance, which are risk factors for hyperlipidemia, atherosclerosis, and NAFLD. Thus, Pla2g1b may play a role in the development of these obesity-related diseases and inhibiting Pla2g1b and/or LPC may change the progression of these metabolic disorders.
The goal of the first part of this dissertation was to identify a Pla2g1b-mediated protection against hyperlipidemia. Findings from these studies indicate that Pla2g1b-/- mice are protected against high fat diet-induced hyperlipidemia and gain less weight than Pla2g1b+/+ controls due to decreased very-low-density lipoprotein (VLDL) production and increased postprandial triglyceride-rich lipoprotein clearance. Further results indicate that supplying LPC stimulates VLDL production in both Pla2g1b-/- and Pla2g1b+/+ mice and that LPC is directly used as a substrate to make VLDL-triglyceride (TG). Taken together, these findings indicate that inhibition of luminal Pla2g1b may be a viable strategy to decrease hyperlipidemia in vivo.
The goal of the second part of this dissertation was to identify a mechanism of LPC- mediated inhibition of hepatic oxidative function. Pla2g1b-/- mice have decreased plasma LPC and increased hepatic oxidation that is inhibited by intraperitoneal injection of LPC by an
Page ii unknown mechanism. Findings indicate that though low micromolar concentrations of LPC decrease the mitochondrial membrane potential, oxidation rate remains equal to controls up to incubation with 80 µM LPC. However, when isolated mitochondria or hepatocytes are supplied with 100 µM LPC, decreased substrate-stimulated oxidation and increased mitochondrial permeability are observed. These findings indicate that LPC plays a major role in the maintenance of hepatic mitochondrial integrity and function and that low micromolar changes in intra- and extracellular LPC concentration can greatly affect hepatic oxidation rate.
The goal of the third part of this dissertation was to identify a Pla2g1b-mediated protection against atherosclerosis and NAFLD. Findings indicate decreased obesity, VLDL-TG,
VLDL-cholesterol, low-density lipoprotein-cholesterol, and hepatomegaly in Pla2g1b-/-/Ldlr-/- mice compared to Pla2g1b+/+/Ldlr-/- controls. Findings also suggested decreased atherosclerotic lesion size in Pla2g1b-/-/Ldlr-/- mice.
Taken together, the findings from this dissertation have three implications in the field of metabolic research. First, these studies indicate a vital role for Pla2g1b and LPC in modulating hepatic function. Regardless of the Pla2g1b status of experimental animals, the mechanisms of
LPC action appear intact. Secondly, these studies indicate that inhibition of Pla2g1b provides protection against diet-induced obesity in two diverse murine models. Thirdly, decreased VLDL-
TG is described as a hallmark of inhibition of Pla2g1b. The development of a single drug to inhibit the action of PLA2G1B would likely lead to improvements in obesity, diabetes, hyperlipidemia, and potentially atherosclerosis.
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Copyright Notice
This dissertation is based, in part, on a published manuscript. Part of this research was originally published in the Journal of Lipid Research. Hollie NI and Hui DY. Deficiency of group 1b phospholipase A2 protects against diet-induced hyperlipidemia. J Lipid Res. 2011; 52: (11)
2005-2011. © the American Society for Biochemistry and Molecular Biology.
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Acknowledgements
This work was supported by Grant DK069967 from the National Institute of Diabetes and
Digestive and Kidney Diseases of the National Institute of Health. The author was a fellowship recipient of grants F31HL110527 from the National Heart, Lung, And Blood Institute, Grant
Number T32GM063483 from the National Institute of General Medical Sciences, and Grant
Number 11PRE7310047 from the American Heart Association during the course of this study.
The author would like to thank Dr. David Hui for allowing him to complete doctoral research in his laboratory.
The author would also like to thank Kimberly Hollie, M.Ed. (wife) and Noah Hollie (son) for their support and understanding through this busy time.
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Table of Contents
List of Figures ...... vii List of Abbreviations ...... ix Chapter 1. Introduction ...... 1 1. Significance 2. Three obesity-related disorders 3. Treatments for dyslipidemia 4. Normal lipid metabolism 5. Phospholipase A2 6. Metabolic effects of Pla2g1b Chapter 2. Materials and Methods ...... 22 1. Animals 2. Special diets 3. Hepatic VLDL production 4. Postprandial lipid clearance 5. Plasma lipid determination 6. Mitochondrial isolation 7. Mitochondrial swelling 8. Mitochondrial membrane potential 9. Mitochondrial calcium uptake 10. Mitochondrial oxygen consumption 11. Cellular oxygen consumption 12. Hepatocyte viability and mitochondrial permeability transition 13. Determination of atherosclerosis 14. Liver characterization 15. Statistical analysis Chapter 3. Results ...... 39 1. Deficiency of Pla2g1b protects against diet-induced hyperlipidemia 2. Micromolar changes in LPC concentration cause minor effects on hepatic mitochondrial permeability but major alterations in function 3. Inhibition of Pla2g1b protects against selected disorders of lipid metabolism Chapter 4. Discussion ...... 58 1. Overview of findings 2. Discussion of “Deficiency of Pla2g1b protects against diet-induced hyperlipidemia” 3. Discussion of “Micromolar changes in LPC concentration cause minor effects on hepatic mitochondrial permeability but major alterations in function” 4. Discussion of “Inhibition of Pla2g1b protects against selected disorders of lipid metabolism” 5. Clinical considerations for inhibition of Pla2g1b References ...... 74 Figures...... 87
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List of Figures
1. Schematic of lipid metabolism through the gut-circulation-liver-adipose axis
2. Responses of Pla2g1b+/+ and Pla2g1b-/- mice to hypercaloric feeding
3. Hepatic VLDL production by Pla2g1b+/+ and Pla2g1b-/- mice
4. Effect of LPC supplementation on hepatic VLDL production by Pla2g1b-/- mice
5. FPLC analysis of 3H radioactivity distribution in plasma subsequent to [3H]LPC injection
6. Postprandial plasma lipid response to a bolus lipid-rich meal
7. Effect of LPC on mitochondrial swelling
8. Effect of LPC on CaCl2-induced mitochondrial swelling
9. Effect of LPC on mitochondrial Ca2+ uptake
10. Effect of LPC on mitochondrial membrane potential
11. Effect of LPC on mitochondrial respiration
12. Effect of LPC on fatty acid-stimulated oxidation in isolated hepatocytes
13. Effect of LPC on hepatocyte mitochondrial permeability in situ
14. Effect of LPC on hepatocyte cytoplasmic activity
15. Effects of diet on body weight of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice
16. Effects of diet on glucose homeostasis in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice
17. Effects of diet on lipoprotein profile of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice
18. Total plasma lipid levels in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice after high fat
diet feeding
19. Effect of diet on liver weight of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice
20. Liver histology in high fat fed Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice.
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21. Lipid deposition in aortic arch and brachiocephalic artery of Pla2g1b+/+/Ldlr-/- and
Pla2g1b-/-/Ldlr-/- mice
22. Atherosclerotic lesion in aortic root of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice
23. Fed state plasma lipid levels in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice after chronic
Western diet feeding
24. Correlation of fed state plasma lipid to atherosclerotic lesion in Pla2g1b+/+/Ldlr-/- and
Pla2g1b-/-/Ldlr-/- mice
25. Schematic of effects of LPC and Pla2g1b inhibition on lipid metabolism the through gut-
circulation-liver-adipose axis
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List of Abbreviations
ABCA1 Adenosine triphosphate-binding cassette A1 ABCG5/G8 Adenosine triphosphate-binding cassette g5/g8 ACAT Acyl-CoA:cholesterol acyltransferase ADP Adenosine diphosphate ALT Alanine aminotransferase AM Acetoxymethylester AISF Associazione Italiana per lo studio del Fegato ApoAI Apolipoprotein AI ApoB Apolipoprotein B ApoCII Apolipoprotein CII ApoE Apolipoprotein E AUC Area under the curve BSA Bovine serum albumin CCK Cholecystokinin CsA Cyclosporin A CETP Cholesterol ester transfer protein cPLA2 Cytosolic PLA2 CPT Carnitine palmitoyltransferase CDC Center for Disease Control and Prevention CM Chylomicron CMR CM remnant CVD Cardiovascular disease DAG Diacylglycerol DKO Pla2g1b-/-/Ldlr-/- mice DMEM Dulbecco’s modified eagle medium FAO Fatty acid oxidation FCCP Carbonyl cyanide 4-(trifluoromethoxy) phenyhydrazone FFA Free fatty acid FH Familial hypercholesterolemia FI Fluorescence intensity FPLC Fast performance liquid chromatography HAART Highly active anti-retroviral therapy HDL High density lipoprotein HDL-C HDL-cholesterol HOMA-IR Homeostatic model assessment-insulin resistance HRT Hormone replacement therapy iPLA2 Calcium-independent PLA2 JNK N-terminal c-jun kinase LDL Low density lipoprotein LDL-C LDL-cholesterol LDLR LDL receptor LDLR-/- Pla2g1b+/+/Ldlr-/- mice Lp(a) Lipoprotein (a) LPA Lysophosphatidic acid
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LPC Lysophosphatidylcholine LPCAT Lysophosphatidylcholine acyltransferase LRP1 LDLR-related protein 1 LysoPL Lysophospholipid LPL Lipoprotein lipase MAG Monoacylglycerol MI Myocardial infarction MOPS 3-(N-Morpholino)propanesulfonic acid MTTP Microsomal triglyceride transfer protein NAFLD Non-alcoholic fatty liver disease NASH Non-alcoholic steatohepatitis NEFA Non-esterified fatty acid NPC1L1 Niemann-Pick C1 Like-1 OCR Oxygen consumption rate oxLDL Oxidized LDL P407 Poloxamer 407 PAF Platelet activating factor PAF-AC PAF acetylhydrolase PC Phosphatidylcholine PGC-1α PPARγ coactivator 1α PLA2 Phospholipase A2 PLA2G1B Group IB phospholipase A2 PLA2G2A Group IIA phospholipase A2 PLB Phospholipase B PLC Phospholipase C PLD Phospholipase D PNPLA3 Patatin-like phospholipase domain containing 3 PPAR Peroxisome proliferator activated receptor RCI Respiratory control index RCT Reverse cholesterol transport RMU Receptor-mediated uptake sdLDL Small dense LDL SDS Sodium dodecyl sulfate TC Total cholesterol TG Triglyceride TRL Triglyceride-rich lipoprotein UCP Uncoupling protein US United States VLDL Very-low-density lipoprotein VLDL-C VLDL-cholesterol VLDL-TG VLDL-triglyceride VSMC Vascular smooth muscle cell WHO World Health Organization WT Wild-type
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Chapter 1. Introduction
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1.1. Significance
Industrialized countries, such as the United States (US), have put great efforts into increasing the wellbeing of their citizens. As a result, life expectancy has increased over the past century from under 50 years to approximately 80 years in the US (1). Much of this increase has been due to the prevention and treatment of acute illnesses, such as smallpox, polio, pneumonia, etc. by the development of vaccines and antibiotics. In addition to this, mass production of food, government subsidies to agriculture and improvements in transportation and storage have increased the accessibility of Americans to adequate caloric intake (2). An unforeseen problem, however, that is faced as the population ages in the environment of increased caloric intake is the increasing prevalence of a number of chronic diseases. According to the Centers for Disease
Control and Prevention (CDC), obesity has increased dramatically over the past few decades in the US. In 1994, approximately one third of states had an obesity rate below 14% and about half of states had a rate between 14% and 18%. Fifteen years later, about half of states had an obesity rate between 22% and 26% and the other half of states had an obesity rate above 26% (3).
Furthermore, childhood obesity has also increased dramatically.
With the increase of obesity, the incidence and prevalence of a number of obesity-related disorders have also risen dramatically. In 1994, nearly all states had a prevalence of diabetes below 6%, with half of states below 4.5%. By 2008, however, nearly the whole country had a prevalence of above 6%, with over half of states above 7.5% (4). Cardiovascular disease (CVD) and the complications of heart attack and stroke are the top cause of death of individuals in the
US (5). Heart attacks alone affect about 715,000 Americans per year and are the cause of death
Page 2 for 385,000 individuals per year (5, 6). In addition to the immeasurable loss of human life, the cost associated with CVD is $172 billion per year and expected to increase to $276 billion by
2030 (7). In 2012, the prevalence of coronary heart disease was from 6.3-8.5% in men and 5.6-
7.6% in women, depending on race (6). Despite the presence of some non-modifiable risk factors such as age and gender, there are modifiable factors of coronary heart disease, including smoking status, weight, and dyslipidemia (8). Furthermore, between 1994 and 2008, the prevalence of non-alcoholic fatty liver disease (NAFLD) has also more than doubled to become the top cause of liver disease (9, 10). The NAFLD Expert Committee of the Associazione Italiana per lo studio del Fegato (AISF) estimates that 25% of Western countries currently lives with NAFLD and many are unaware of it since it is often silent clinically until biochemical blood analysis discovers increased liver enzymes (11, 12). A better understanding of dyslipidemia and other obesity-related diseases is therefore necessary in order to decrease the burden of these maladies.
1.2. Three obesity-related disorders
Obesity arises due to a mismatch between nutritional intake and metabolic mechanisms, in which excess fuel is stored as lipid in adipose tissue, the liver, and circulating lipoproteins.
Thus, people who are obese often suffer from a variety of metabolic disorders, including hyperlipidemia, atherosclerosis, and fatty liver disease. Individuals who have the metabolic syndrome, consisting of obesity, insulin resistance, and hypertension, also have increased risk of atherosclerosis, coronary heart disease, and stroke (13).
Dyslipidemia
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The classic Fredrickson system categorized dyslipidemia according to which lipoprotein classes were increased. These phenotypes were: Type I, in which chylomicrons are elevated due to decreased lipoprotein lipase (LPL) activity or apolipoprotein CII (apoCII) function; Type IIa, in which low-density lipoprotein (LDL) levels are elevated due to decreased LDL receptor
(LDLR) function; Type IIb, in which LDL and very-low-density lipoprotein (VLDL) are elevated due to decreased LDLR function or high rates of apoB production; Type III, in which
VLDL and chylomicron remnants are elevated due to decreased apoE function; Type IV, in which VLDL are elevated due to high rates of VLDL production; and Type V, in which chylomicrons and VLDL are elevated due to high VLDL production or decreased LPL function
(14). More recently, however, the picture of dyslipidemia has been expanded to acknowledge the roles of high-density lipoprotein (HDL), small dense LDL (sdLDL), and lipoprotein-specific cholesterol. These varied definitions may include high sdLDL, high LDL cholesterol (LDL-C), low HDL, or low HDL cholesterol (HDL-C) levels in addition to elevated lipid or apolipoprotein
B (apoB) lipoprotein levels (13, 15, 16)(15). For reference, undesirable lipid levels have been described as total cholesterol greater than 200 mg/dl, LDL-C greater than 130 mg/dl, HDL-C less than 40 mg/dl, or triglycerides greater than 150 mg/dl (13, 16).
At the heart of all hyperlipidemias is apoB, a 500 kDa protein that is specially equipped to transport triglycerides and is differentially processed to carry out this task. In the intestine, apoB translation is regulated by apoB editing complex 1 (apobec-1), resulting in a protein that is
48% of the total length and termed apoB48. Whereas apoB48 is packed with triglyceride to make large (1000 nm) chylomicron particles, in the liver, full length apoB (apoB100) is produced and filled with triglyceride to make the smaller (25-90 nm) VLDL particles. These apoB-containing
Page 4 particles (chylomicrons and VLDL) also contain small quantities of cholesterol and phospholipids in smaller quantities in order to stabilize the particles. When metabolized, these particles (VLDL more so than chylomicrons) become indirectly enriched in cholesterol and are converted to LDL and chylomicron remnants. Thus, metabolism of apoB-containing particles can influence plasma triglyceride and cholesterol levels.
Dyslipidemia can be caused by a number of genetic and environmental factors. As described in the Fredrickson phenotypes, genetic mutations in lipoprotein metabolism proteins such as LPL, LDLR, apoCII, apoE, etc. can result in increased plasma lipids (17).Furthermore, alterations in molecules important for mitochondrial function such as uncoupling proteins 1-3, carnitine palmitoyltransferase 1, peroxisome proliferator activated receptors (PPAR) α and β, and
PPAR γ coactivator 1α (PGC-1α), can also cause a buildup of plasma lipid due to decreased hepatic oxidation leading to increased lipoprotein secretion. Dyslipidemia can also occur subsequent to a combination of hypercaloric diet and sedentary lifestyle. Other factors contributing to dyslipidemia include obesity, chronic diseases of the liver or kidney, and pharmaceutical drug side effects (e.g. thiazides, highly active anti-retroviral therapy, hormone replacement therapy). The metabolic syndrome and type-2 diabetes can also lead to dyslipidemia due to hepatic insulin resistance and VLDL overproduction (18).
Atherosclerosis
In clinical terms, atherosclerosis is arteriosclerosis, or hardening of the arteries, in part due to the formation of a lipid-laden lesion (atheroma) in the vessel wall. As the atheroma increases in size, small insults may cause pieces of the atheroma to pass downstream and
Page 5 obstruct smaller vessels or expose factors that cause coagulation of blood and occlude blood flow resulting in ischemia. If plasma anticoagulant factors restore blood flow quickly, then ischemia is temporary and the episode results in angina pectoris or transient ischemic attack if the coronary or cerebral arteries, respectively, are affected. If the ischemia is not resolved, then the supplied area of tissue can die, resulting in a heart attack or ischemic stroke if the blockage is in the coronary or cerebral arteries, respectively. Atherosclerosis and cardiovascular disease risk are routinely approximated by indirect measures (e.g. plasma LDL-C and HDL-C), though more direct measures (e.g. carotid ultrasound, radioangiography) are available for patients with exceptionally high risk from prior cardiovascular events. Thus, many do not know the extent of atherosclerosis in their arteries until they suffer an ischemic event. Since half of heart attack victims die (5, 6), it is vital to understand its development in order to develop approaches to decrease and treat atherosclerosis.
Clinical studies in Framingham, Massachusettes, revealed several population-based risk factors for atherosclerosis and coronary artery disease, such as increased plasma LDL-C and decreased HDL-C, which are now known to be modifiable (8). Other risk factors include hypertension, smoking, increased VLDL, sdLDL, and the metabolic syndrome (13). A number of genetic factors can also significantly increase the risk of developing atherosclerosis. Certain defects in LDL metabolism, such as mutated LDL receptor or having certain apoE alleles, can result in elevated LDL levels and increased risk(19-23) . Furthermore, certain defects in HDL metabolism, such as reduced apoAI production (Tangier’s disease), can result in decreased HDL and increased risk for atherosclerosis.
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At the molecular level, atherosclerosis is an inflammatory disease that arises subsequent to injury of the endothelial lining and the entrance of LDL into the intimal space where it can become oxidized and start an inflammatory cascade (24). Monocytes, recruited to the site of insult by endothelial expression of adhesion molecules (VCAM, ICAM), take up the oxidized
LDL (oxLDL) via the CD36 scavenger receptor and become macrophages (24-26). As the macrophages and neighboring vascular smooth muscle cells are activated, inflammatory cytokines (e.g. MCP-1, TNF-α, etc.) are secreted (25). Recruited monocyte/macrophages and vascular smooth muscle cells continue to take up intimal lipid and become foam cells as enlarged macrophages are overwhelmed and die. This results in a necrotic, cholesterol-filled core. Other proteins, such as TGF-β, are secreted and fibroblasts are recruited, which deposit matrix to form a fibrous cap surrounding the necrotic core of cell debris and undigested lipid (26-28).
Elevated plasma HDL levels are thought to decrease risk of CVD through at least two mechanisms. The first is by mediating reverse cholesterol transport, in which lipid-poor apoAI molecules receive excess cholesterol from peripheral tissues via adenosine triphosphate-binding cassette (ABC) A1 and passive diffusion, convert to HDL particles, and transport the cholesterol to the liver and adrenal glands through scavenger receptor class B member 1 (SR-B1). In humans, but not mice, HDL also exchanges its cholesterol esters for triglycerides in the apoB-containing lipoproteins VLDL and LDL via cholesterol ester transfer protein (CETP). Subsequently, through LDLR-mediated uptake of these cholesterol-enriched apoB-containing lipoproteins, plasma cholesterol levels are reduced. The second way by which HDL particles thwart atherosclerosis is through their anti-inflammatory properties. HDL particles also contain a number of anti-inflammatory proteins such as apoAIV, apoAII, and PON1, which counter the
Page 7 effect of the inflammatory cytokines secreted by cells in an atheroma (29, 30). Thus patients with increased HDL levels have decreased risk of coronary events and CVD (8).
Increased levels of triglyceride-rich lipoproteins (TRL), such as chylomicrons and VLDL, also increase risk of atherosclerosis. After a meal, TRL are raised and contribute to endothelial dysfunction and increased expression of adhesion molecules (31). Furthermore, increased plasma glucose, such as occurs in the post-prandial state or in diabetics, can also cause damage to endothelial cells as well as a number of plasma proteins through non-enzymatic glycosylation.
Non-Alcoholic Fatty Liver Disease
The consumption of alcohol can lead to excess lipid storage in the liver and, until recently, was the most common cause of liver disease. Nonalcoholic fatty liver disease (NAFLD) is often observed concurrently in patients who have type 2 diabetes or obesity (9, 11, 32, 33), and has become the most common cause of liver disease. Free fatty acids from the action of LPL on plasma triglyceride in apoB-containing proteins and adipose tissue release are taken up by the liver, re-esterified, and stored (33). Hepatocytes store such excess triglyceride and cholesterol esters in small or large vesicles, which lead to hepatocyte damage evident by elevated liver enzymes and altered histological appearance. With time and inflammation, NAFLD can progress sequentially to nonalcoholic steatohepatitis (NASH), cirrhosis and, liver failure (12).
By definition, NAFLD excludes extreme hereditary disorders, however, the environment and genetic variability can contribute risk for fatty change (32). Since obesity and type 2 diabetes can increase the risk of NAFLD, conditions that promote these disorders, such as high fat diet
Page 8 and sedentary lifestyle, consequently increase the risk of NAFLD (34). Increased plasma triglyceride and fatty acid levels are also associated with NAFLD. Although the effects of genetic inheritance are incompletely understood, a small number of hepatic gene mutations, such as group-specific complement and lymphocyte-cytosolic protein 1, have been associated with
NAFLD (35). In Japanese subjects, NAFLD was also linked to variations in PGC-1α and patatin- like phospholipase domain containing 3 (PNPLA3) (36, 37). A recent study showed that
Mexican Americans had a higher prevalence of NAFLD than non-Hispanic whites or blacks even when controlled for lifestyle, adiposity, and metabolic factors (38)
1.3. Treatments for dyslipidemia
A first recommendation for obese patients with elevated plasma lipids is to modify their diet and add exercise to their lifestyle. Though the reduction of animal products in the diet has shown to prove effective in decreasing risk for cardiovascular disease and increasing longevity in particular populations, compliance often decreases the effectiveness of dietary intervention (39,
40). For this reason, a number alternative approaches have been developed in an effort to decrease plasma triglyceride and cholesterol levels in order to decrease atherosclerosis risk.
After diet and exercise, the next approach is inhibition of lipid-forming enzymes. The statin (e.g. simvastatin, rosuvastatin) class of drugs inhibits HMG-CoA reductase, the rate- limiting enzyme of hepatic cholesterol synthesis. This inhibition leads to deficits in intracellular cholesterol stores, increased LDLR expression, increased uptake of plasma LDL, and decreased plasma cholesterol levels. Statins can decrease plasma LDL-C by to 15-35% (41). There are also
Page 9 non-enzymatic effects of statins that decrease the risk of cardiovascular disease. However, muscle damage and pain are common adverse side effects with statin treatment. Alternatively, inhibition of microsomal triglyceride transfer protein can result in a 50% decrease in plasma triglyceride levels by inhibiting the rate-limiting enzyme of VLDL packaging. Conversely, though this approach decreases hyperlipidemia, it also causes reversible but severe hepatic steatosis.
Another approach is the alteration of gut absorption of nutrients. Bile acid sequestrants reduce bile absorption and thereby decrease caloric intake due to less efficient digestion of lipid.
Cholesterol absorption inhibitors, such as ezetimibe (Zetia, Vytorin), specifically inhibit
Niemann-Pick C1 Like-1 (NPC1L1)-mediated uptake of cholesterol and cholesterol esters (42).
By attenuating gut uptake of dietary cholesterol, plasma levels were decreased in mice; however, this approach has been less effective in reducing cardiovascular outcomes in humans. Dietary supplementation with fish oil, which is rich in ω-3 unsaturated fatty acids, has been shown to reduce inflammation and decrease plasma triglyceride levels by inhibiting hepatic VLDL production (43, 44). A study suggested that phospholipid supplementation decreases plasma and hepatic cholesterol by inhibiting lipid absorption, however this approach is not approved by the
FDA (45).High intake of nicotinic acid can increase HDL-C levels and slightly decrease LDL levels; however, flushing (hot flashes and red, irritated skin) is an adverse effect that often decreases compliance, and protective effects upon endothelial function were not seen when nicotinic acid was added to conventional statin treatment (46, 47).
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An additional method to improving lipid profile is enhancing lipid metabolism through altering fatty acid homeostasis. Thiazoladinediones and fibrates are agonists of peroxisome- proliferating activator receptor (PPAR) γ and PPARα, respectively. By increasing hepatic oxidation, there is decreased fatty acid availability for triglyceride synthesis and subsequent secretion into the plasma. Activation of PPARγ has also been used to counter insulin resistance despite the potential for increased differentiation of adipocytes (48). Since single PPAR agonists can cause hepatic dysfunction, recent efforts have been made to develop PPAR agonists with dual and multiple activities (49). LPL activators boost plasma triglyceride catabolism; however, increased plasma fatty acid concentration is associated with insulin resistance, increased adipogenesis, and increased VLDL production, so it remains to be seen if this technique will be successful in preventing obesity or dyslipidemia (33, 50, 51).
Lipoprotein homeostasis has also been targeted. ApoAI Milano is a recombinant ApoAI molecule based on a mutation in an Italian community that rapidly mobilizes cholesterol from peripheral tissues. Treatment with this protein decreased atherosclerotic lesion size, however therapy required intravenous administration and the drug has not been developed further (52, 53).
CETP inhibitors decrease the transfer of cholesterol esters from HDL to VLDL, resulting in increased plasma HDL-C levels. However, patients taking first generation CETP inhibitors did not have decreased incidence of coronary events due to a side effect of increased blood pressure, which independently increases the risk of coronary events (54). Second generation CETP inhibitors that do not increase blood pressure were designed; however, results from phase 2 clinical trials have been disappointing. Plasma levels of HDL were increased by 20%; however, there was no decrease in clinical endpoints of coronary events or death from any cause (46).
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Other modulators of lipoprotein metabolism and potential treatments are inhibitors of PCSK9, which targets LDLR for degradation; and injections of synthetic apoE to increase binding of apoB-containing lipoproteins to LDLR and LDLR-related protein 1 (LRP1) (19).
Most of the described treatments aim to combat hypertriglyceridemia and hypercholesterolemia in order to decrease the risk of atherosclerotic disease. Though procedural interventions exist to treat atherosclerosis (e.g. angioplasty, stents), these are reserved for those with the most severe disease or prior cardiovascular events. Moreover, though increasing fatty acid oxidation or decreasing cholesterol absorption were hypothesized to decrease the risk of
NAFLD, the efficacy of these approaches is minimal and does not extend to the populations most at risk, namely diabetics (32). Alas, currently, there is no definitive pharmacological treatment for NAFLD in diabetics and recent guidelines only recommend weight loss of 3-10% to improve liver histology (32). It is therefore important that these disease processes be better understood in order to develop new treatments and improve patient outcomes.
1.4. Normal lipid metabolism
When a lipid meal is consumed, cholecystokinin (CCK) released by I cells in the duodenum signal the pancreas to release many digestive enzymes into the intestinal lumen in order to digest the lumenal fat. Pancreatic lipase, colipase, cholesterol ester lipase, and phospholipases catabolize triglyceride, cholesterol ester, and phospholipid to liberate glycerol, free fatty acids, and cholesterol, as well as partially digested products monoacylglycerol (MAG), diacylglycerol (DAG), and lysophopholipid (lysoPL) (55). CCK also signals the contraction of
Page 12 the gallbladder and release of hepatic bile, which contains bile salts, cholesterol, phospholipids, into the gut lumen. Bile salts emulsify the diverse lipid molecules and promote their arrangement into mixed micelles. This process improves lipid digestion by providing a surface for lipolytic enzymes, such as phospholipase A2, to act upon (56).
Before entering the enterocyte, lumenal lipids must pass a mucosal layer which is comprised of 90% phosphatidylcholine and an unstirred water layer (57, 58). Enteroctyes absorb cholesterol from the gut lumen via NPC1L1-dependent and independent mechanisms (42, 59).
Enterocytes take up lumenal fatty acids from triglyceride and phospholipid digestion through diffusion and receptor-mediated mechanisms involving CD36 (60). Intestinal cells are also absorb dietary and bile-associated phospholipid after they are hydrolyzed to lysolipids (61). In the enterocyte, fatty acids are re-esterified to di- and triglycerides, are targeted to lipid droplets in the endoplasmic reticulum, and then bound with apoB and secreted with phospholipids as chylomicrons, which travel with the lymph until they enter the plasma circulation at the thoracic duct (Fig. 1). Some lipid molecules are also absorbed through the portal system as enterocytes transport some lipid molecules basolaterally (62).
In the plasma circulation, chylomicrons encounter LPL, which acts on triglycerides to liberate fatty acid, the excess of which is stored by the adipose tissue. When needed, the adipose tissue releases fatty acid, which is used for hepatic or peripheral tissue energy generation. As chylomicrons become chylomicron remnants, they acquire apoE and are taken up by LDLR in the liver as well as LRP1 (63). Various apoE alleles have been shown to have altered avidity to the LDLR and LRP1.The liver then uses the newly acquired lipid as well as de novo synthesis of
Page 13 fatty acids for oxidation, storage, or repackaging and secretion in VLDL particles which are released into the plasma circulation. In the circulation, triglyceride in VLDL particles is acted upon by LPL, which converts the particles to smaller LDL particles that are enriched in cholesterol and are taken up by the hepatic LDLR (20, 64). The lipid in LDL particles taken up by the liver can again be used for various purposes. Triglycerides and can again be oxidized or stored, while cholesterol is excreted in the bile or stored as cholesterol ester (Fig. 1).
The consumption of dietary fat in excess of metabolic needs results in storage and can lead to many metabolic disorders. Excess storage in adipose tissues causes obesity, while increased plasma fatty acid can result in to skeletal muscle insulin resistance, ectopic storage in the liver, increased plasma VLDL/LDL levels, and pancreatic lipotoxicity and decreased insulin production (2, 33, 65, 66).
1.5. Phospholipase A2
Phospholipids, comprised of a hydrophilic phosphate-containing head group connected to a glycerol core with a diacyl hydrophobic tail, are vitally important to human biology. The variety of head groups, including choline, serine, ethanolamine, and inositol, confer extra functionality to phospholipids based on their charge and functional groups. Phospholipids are the most prevalent lipid in the cellular plasma membrane and perform numerous functions, such as maintaining membrane integrity, mediating intra- and extracellular signaling, and interacting with various proteins. In addition, phospholipids are also major components of lipoproteins, exosomes, microparticles, apoptotic bodies, and intracellular vesicles. Phosphatidylcholine (PC),
Page 14 which was previously called lecithin, contains a phosphorylcholine head group and may be cleaved by phospholipase A1 or phospholipase A2 (PLA2) to release fatty acid at the sn-1 or sn-2 positions, respectively. Alternatively, PC can be hydrolyzed to release choline or phosphorylcholine by phospholipase D and phospholipase C, respectively. Phospholipase B
(PLB) contains both PLA1 and PLA2 activity (67).
The more than 20 members of PLA2 are grouped into five main categories: calcium- independent (iPLA2), cytosolic (cPLA2), lysosomal, platelet activating factor (PAF) acetylhydrolase (PAF-AC), and secreted (sPLA2), (68). iPLA2s are important in intracellular lipid homeostasis of adipose tissue; they are associated with mitochondrial membrane (iPLA2γ) and have triacylglycerol lipase activity (iPLA2ε,ζ,η). The activity iPLA2 is not determined by the
2+ Ca concentration, unlike some other PLA2s and iPLA2γ also plays a role in regulating mitochondrial membrane integrity (68, 69)(70). cPLA2s have C2 domains that are important for translocation to intracellular membranes and they also hydrolyze phospholipids to release arachidonic acid (68). Lysosomal PLA2s have optimal activity at pH 4.5 and are important in PC homeostasis in the macrophage and spleen (68). PAF-AH has broad lipase activity against phospholipid, DAG, and triglyceride. A secreted PAF-AH (Lp-PLA2) can modify phospholipid in oxLDL and may contribute to coronary disease risk, while intracellular PAF-AH may be involved in the oxidative stress response and in brain development (68). Secreted PLA2 enzymes contain a signal peptide sequence, are packaged into vesicles (some as inactive zymogens) and exocytosed from the cell into the lumen (e.g. group Ib) or interstitium (e.g. group IIa) (71). The
2+ 2+ activity of sPLA2 is regulated by the local concentration of Ca , and Ca is necessary for its action (68). Secreted PLA2 enzymes perform a variety of functions and have many different roles.
Page 15
For example, groups V and X are increased in mice that also display atherosclerosis (68, 71).
Group IIa PLA2 (Pla2g2a) is more active in states of inflammation, such as rheumatoid arthritis and atherosclerosis (72). In the C57Bl/6 mouse lines, Pla2g2a is inactivated by a natural mutation (68). In acute respiratory distress syndrome, the group Ib PLA2 (Pla2g1b) and Pla2g2a play a role in propagating injury (73, 74).
The human Pla2g1b was first reported in the early 1960s and was described as having similar activity to an enzyme found in snake venom (75, 76). Pla2g1b is primarily found in the pancreas, but was also found to be expressed in the lung (71, 77) and later in the brain (78). The pancreatic Pla2g1b is active in the gut lumen, functions to digest phospholipids, and is produced in excess of physiological needs (67). The pro-Pla2g1b is secreted by the pancreatic acinar cells as an inactive zymogen that can form trimers in solution. It is activated by cleavage of the first seven residues by trypsin in the duodenum (79, 80). The enzyme can also be activated by thrombin, type 1-proPLA2 activator, or by autoproteolysis (79). When Pla2g1b is aberrantly activated, acute pancreatitis or acute lung injury can occur (73, 79). With acute pancreatitis, high levels of plasma Pla2g1b correlate with the severity of disease, though when mice were treated with sPLA2 inhibitors to decrease Pla2g1b activity exhibited decreased acute lung injury (81).
PLA2G1B is located on the human chromosome 12 and on chromosome 5 in mouse and chromosome 12 in rat (82-85). The mouse enzyme is translated from 4 exons separated by introns in the DNA primary sequence (77). Despite the difference in chromosome location, the relative location of the gene is stable, as the genes flanking PLA2G1B (SIRT4 and MSI1) are identical for humans and rodents (83-85). Pla2g1b is well conserved throughout the animal
Page 16 kingdom, having homology to enzymes in chimpanzee, dog, cow, mouse, rat, chicken, pig, zebrafish, and C.elegans (71). Moreover, the amino acid sequence is greatly conserved throughout the species, with >70% sequence identity between mouse, rat, dog, human, pig, ox, and guinea-pig (77). The active Pla2g1b is 14 kDa and contains nearly 50% alpha-helix (77).
The catalytic site contains an important histidine (His48) and aspartic acid (Asp49) dyad, which polarize one or two water molecules to attack the carbonyl group of the sn-2 linked fatty acid of phospholipid (56). The activity of Pla2g1b is dependent upon binding to an anionic membrane surface such as micelle, and efficient binding cannot occur without the N-terminus, C-terminus, and the so-called “69-loop” (residues 59-72) (56). Binding of the 69-loop to phospholipid substrates is inhibited by bile salts (86).The enzyme is thought to scoot across the outer micellar surface, moving from one phospholipid to another rather than remaining stationary in order to encounter its substrate (56). In addition to releasing fatty acid from PC, the action of Pla2g1b also liberates lysophosphatidylcholine (LPC) which retains the glycerol core bound to phosphorylcholine and a single acyl tail. Both the lysophospholipid and the fatty acid may have carbon tails of various lengths, ranging from C14 to C20, though C16 and C18 are common (87).
Important R groups in this range are palmitate (16:0), oleate (18:1), and arachidonate (20:4) acid, which have important effects on oxidation and inflammation. Thus Pla2g1b may to play an important role in modulating a variety of molecular processes in addition to simple dietary digestion.
In addition to providing calories, LPC is a bioactive molecule. It has been shown to stimulate triglyceride production in hepatocytes in vitro by altering apoB metabolism, as well as affect intracellular survival pathways (88, 89). LPC causes monocytes to adhere more closely to
Page 17 endothelial cells and increases cytokine production. LPC has been shown to reproduce many of the inflammatory effects of oxidized LDL and is found in atherosclerotic lesions in high concentrations (26, 27).
Though a product of Pla2g1b, LPC can arise from a number of sources. In the plasma, other secreted PLA2 (e.g. Pla2g2a, group V PLA2, and group X PLA2) and lecithin-cholesterol acyltransferase (LCAT) can produce LPC (90). LCAT is present on HDL particles and moves fatty acids from PC to cholesterol to produce cholesterol esters and LPC. This process assists uptake of cholesterol by lipid-poor HDL by decreasing the free cholesterol concentration (90).
As mentioned earlier, in the gut lumen Pla2g1b activity selectively produces LPC from phospholipids rather than releasing both fatty acids, unlike other gut enzymes (67). Thus, in
Pla2g1b-deficient mice (Pla2g1b-/-), portal LPC transport to the liver is greatly attenuated without decreasing overall lipid absorption (62, 91). LPC can also be generated as a consequence of the Kennedy cycle, in which PLA2 acts on phosphatidic acid to generate lysophosphatidic acid
(LPA). PC was shown to be synthesized from phosphorylcholine and DAG. PC-cytidyl transferase would then use a choline group from CDP-choline to alter the head group of LPA and form LPC (92, 93)(93). The Lands cycle also was also hypothesized to generate LPC (94), though lung tissue, which is rich in Pla2g1b, was used as a source for lipid (77). LPC is also derived from the cellular plasma membrane as cPLA2 releases LPC and fatty acid from membrane phospholipid. The resulting LPC can then be converted to PAF, LPA, or endocannabinoids, or reacylated by LPC acyltransferase (LPCAT) to PC (95, 96). iPLA2γ
(Pla2g6b) is located in the mitochondrial membrane and also likely produces LPC near the mitochondrial microenvironment (70). The lipoprotein associated Lp-PLA2 (Pla2g7a) is
Page 18 associated with LDL, sdLDL, lipoprotein a (Lp(a)), and HDL in the plasma and can also generate LPC (97, 98).
1.6. Metabolic effects of PLA2G1B
In 2006, Wilson, et al. conducted a genome-wide association study to identify genes associated with obesity in a cohort of 1,200-1,400 pairs of twins. Certain single-nucleotide polymorphisms in PLA2G1B, were associated with increased central obesity (82). The mechanism for this association is currently unknown because the change in allele (GA) does not result in a change in the amino acid sequence coded (77, 82). Previously, Pla2g1b was thought to function primarily as a gut digestive enzyme that assisted cholesterol absorption by digesting phospholipids. In vitro studies had shown that increased phospholipid content inhibited cholesterol uptake by enterocytes. Thus it was thought that the digestion of phospholipid in the gut lumen removed this inhibition to enable maximal cholesterol absorption. Richmond, et al. postulated that inhibition of Pla2g1b would maintain high levels of lumenal phospholipids resulting in decreased cholesterol absorption and improved plasma lipid levels. However, the mice with Pla2g1b disrupted at the active site and decreased expression displayed slightly delayed, but equally efficient cholesterol absorption as wild type controls when fed normal chow diet (67, 91). Plasma cholesterol and triglyceride levels, as well as body weight, were similar between Pla2g1b-/- mice and Pla2g1b+/+ wild type controls. It was concluded that compensatory phospholipid digestion from other secreted phospholipases and lipolytic enzymes (PLB, carboxyl ester lipase [cholesterol esterase]) compensated for the Pla2g1b defect (67).
Page 19
However, when Pla2g1b-/- animals were challenged with hypercaloric, high-fat diet, differences were observed when compared to wild type controls. Feeding Pla2g1b-/- mice
Western diet (42% fat, 0.2% cholesterol) revealed that Pla2g1b inhibition decreased obesity and plasma leptin (99). When Pla2g1b-/- mice were fed a diabetogenic diet (60% fat, high sucrose), the protective effect against obesity was still observed despite similar food intake (91). The anti- obesity effect in the Pla2g1b-/- mice was found to be due to increased energy expenditure, as demonstrated by increased O2 consumption and CO2 output, increased body temperature, and increased postprandial hepatic fatty acid oxidation (91). The Pla2g1b-/- animals had decreased absorption of LPC and decreased fasting and postprandial plasma levels of LPC compared to
Pla2g1b+/+ animals (91). When LPC was injected to increase plasma LPC levels to those seen in
Pla2g1b+/+ mice, the rate of fatty acid oxidation in Pla2g1b-/- animals was equal to that of
Pla2g1b+/+ mice (91).
In response to Western diet feeding, plasma insulin and glucose were decreased in
Pla2g1b-/- mice compared to wild type animals. Feeding with diabetogenic diet also resulted in decreased plasma glucose in Pla2g1b-/- animals (62, 99). After exposure to both high fat diets,
Pla2g1b-/- mice had improved glucose and insulin tolerance compared to Pla2g1b+/+ mice (62).
Though pyruvate-stimulated insulin production and steady-state plasma insulin levels were similar to Pla2g1b+/+ controls fed diabetogenic diet, Pla2g1b-/- mice had decreased plasma glucose levels following challenges with oral and intraperitoneal glucose, intraperitoneal insulin, and oral administration of a mixed glucose/lipid meal (91). When injected with LPC to increase the concentration of plasma LPC to levels observed in Pla2g1b+/+ mice, glucose and insulin sensitivity in the Pla2g1b-/- animals decreased to become equal to that of Pla2g1b+/+ mice (62).
Page 20
In summary, Pla2g1b-/- mice were protected from diet-induced obesity, glucose intolerance, and insulin resistance (62, 99). Much of the protection could be removed with LPC supplementation (62). In humans, obesity often clusters with diabetes, hypertension, and dyslipidemia in the metabolic syndrome, which increases the risk of complications from atherosclerosis and the risk of the presence of occult NAFLD (13, 38). We therefore hypothesized that inhibition of Pla2g1b would protect against diet-induced hyperlipidemia, atherosclerosis, and NAFLD in mice. To test this, Pla2g1b-/- and Pla2g1b+/+ animals were fed hypercaloric diet, and lipidemia was characterized. Next, we sought to determine a mechanism for the effect of Pla2g1b-generated LPC on hepatic VLDL production and oxidation. Finally, to determine whether inhibition of Pla2g1b would decrease atherosclerosis and NAFLD, Pla2g1b-/- animals were crossed with Ldlr-/- mice in order to produce Pla2g1b-/-/Ldlr-/- and Pla2g1b+/+/Ldlr-
/- mice and challenged with high fat diet with and without cholesterol. The goal of these studies is to determine the role of Pla2g1b in the pathobiology of these obesity-related diseases.
Page 21
Chapter 2. Materials and Methods
Page 22
2.1. Animals
The Pla2g1b-/- mice were generated by homologous recombination in embryonic stem cells and backcrossed >10 times with C57BL/6J mice to obtain Pla2g1b-/- mice in homogenous
C57BL/6J background (62, 67) . Mice from Pla2g1b+/+ and Pla2g1b-/- mating colonies were used to obtain age-matched Pla2g1b+/+ and Pla2g1b-/- mice for experiments.
Wild type C57BL/6J mice were originally purchased from Jackson Laboratories and a breeding colony was established in our institutional facility. Male mice of at least 10 weeks of age were used in each experiment for isolation of primary hepatocytes and liver mitochondria.
For further studies, mice deficient in Pla2g1b (Pla2g1b-/- animals) were previously generated by our lab and backcrossed with C57BL/6J mice to obtain a stable line (62, 67). Ldlr-/- mice on the C57BL/6J genetic background were originally purchased from Jackson Laboratories and a breeding colony was established in our institutional facility. These Pla2g1b-/- and Ldlr-/- mice entered a genetic crossing program in order to produce Pla2g1b-/-/Ldlr-/- mice on the
C57BL/6J genetic background. Mice were maintained in accordance to protocols approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. Access to food and water was ad libitum, except as indicated. Mice had a 12 hour light/dark cycle.
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2.2. Special diets
Pla2g1b-/- and Pla2g1b+/+ mice were fed either basal chow diet or a hypercaloric diet
(D12331; Research Diets), which consists of 58.5% fat from coconut oil, 25% sucrose, and
16.5% protein beginning at 5-10 weeks of age for the duration as indicated for each experiment.
Ten-to-twelve week old Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice were randomly assigned into two groups to be fed either basal chow (LM485, 5% fat, 3.75 kcal/g) or Western diet (TD88137; Research Diets), which consists of 42% fat and 0.2% cholesterol. Animals were and sacrificed 1 or 10 weeks after initiation of diet.
Page 24
2.3. Hepatic VLDL production
Animals were fasted for 12 h beginning at 3-4 h into the dark cycle. Blood samples were collected to measure fasting plasma lipid levels at baseline. The mice were then given an intraperitoneal injection of Poloxamer 407 (P407) at a dosage of 1 g/kg body weight to inhibit lipolysis. For LPC supplementation studies, each mouse received 32 mg/kg of LPC, a dosage previously shown to restore LPC levels in Pla2g1b-/- mice to those observed in Pla2g1b+/+ mice
(62, 91), 1 h prior to P407 injection. Hourly blood samples were collected for plasma triglyceride measurements to determine hepatic VLDL production rates (100). Area under the curve (AUC) was calculated by trapezoidal rule and averaged. Plasma apoB levels were measured by an
ELISA kit (Uscn Life Science). In selected experiments, 1 µCi of [3H]LPC was included as tracer to assess potential use of LPC as substrate for VLDL synthesis.
Page 25
2.4. Postprandial lipid clearance
Ten-week old mice were placed on hypercaloric diet for three weeks. The animals were then fasted overnight, and blood samples were collected to measure baseline plasma lipid levels.
Each mouse received an oral gavage of olive oil (400 µl), and hourly blood samples were collected for plasma triglyceride determinations. AUC was calculated by trapezoidal rule and averaged.
Page 26
2.5. Plasma lipid determination
Blood samples were collected from mice through the tail vain or via cardiac puncture into
EDTA-containing tubes. Plasma was isolated by centrifugation and diluted in phosphate- buffered saline prior to analysis. Lipid distribution among various lipoproteins was analyzed by applying 100 µL of plasma samples to fast performance liquid chromatography (FPLC) gel filtration on two Superose 6 HR columns as described (101). Each 0.5 mL fraction was collected and analyzed. Triglyceride and cholesterol levels were measured using commercial colormetric kits (Fisher Scientific).
Page 27
2.6. Mitochondrial isolation
Hepatic mitochondria were isolated from mice using a method based on a published protocol (102). Briefly, freshly isolated mouse livers were kept at 4oC, diced into <1 mm pieces, homogenized in Isolation Media (220 mM D-mannitol (Sigma), 70 mM sucrose (Fisher), 1 mM
EDTA (Fisher), 10 mM 3-(N-Morpholino)propanesulfonic acid (MOPS, Sigma), 0.5% BSA
(Sigma), pH 7.2 with KOH (Fisher)), and centrifuged at 500 x g for 15 min. The supernatant was strained through gauze and then centrifuged again at 10,000 x g for 15 min to produce a mitochondrial pellet. This pellet was resuspended two times in Wash Media (250 mM sucrose,
10 mM MOPS, pH 7.2 with KOH) and recovered by centrifugation at 10,000 x g. The final pellet was suspended in 250-500 μL of wash medium and stored on ice until used for experiments.
Page 28
2.7. Mitochondrial swelling
Mitochondrial protein was determined by bicinchoninic acid assay (Thermo Scientific).
Isolated mitochondria (0.7 mg protein/ml) were incubated in Assay Media (250 mM sucrose, 2.5 mM MgCl2 (Fisher), 0.5 mM EDTA (Fisher), 10 mM MOPS, 0.72 mM K2HPO4 (Fisher), 0.28 mM KH2PO4 (Sigma), to pH 7.2 with KOH) supplemented with 5 mM glutamate/5 mM malate as respiratory substrates and egg LPC (Sigma) with or without 2 μM cyclosporine A (CsA,
Sigma) at room temperature (~22oC). Since LPC has detergent properties and is amphipathic, 1.7 mM SDS was used as a negative control to achieve total mitochondrial membrane solubilization
(103). Mitochondria were incubated in the indicated concentrations of LPC for 2 min prior to the addition of 75 μM or 220 μM CaCl2 (Fisher). Absorbance at 530 nm was recorded initially after exposure to LPC and after 10 min of incubation with CaCl2.
Page 29
2.8. Mitochondrial membrane potential
Mitochondria were incubated in Assay Media containing 25 μM safranin O (Sigma) and
LPC for 2 min at room temperature. Baseline absorbance at 530 nm was recorded prior to the addition of 5 mM succinate. Negative control for total mitochondrial membrane solubilization was achieved by incubation with 1.7 mM SDS. After a 3 min equilibration period, 5 mM succinate was then added by multichannel pipette to each well. Absorbance at 530 nm was measured every 30 s for 10 min after succinate addition.
Page 30
2.9. Mitochondrial calcium uptake
Mitochondria were incubated in Assay Media containing 5 mM glutamate, 5 mM malate,
1 μM Calcium Green-5N (Invitrogen), which is a fluorescent probe that binds Ca2+ and is impermeable to membranes, and varying concentrations of LPC. Fluorescence intensity was determined at 538 nm after excitation at 485 nm at baseline and every 2.5 s for 10 min after the addition of 75 μM CaCl2. In negative controls, the mitochondrial membrane was solubilized by incubation with SDS.
Page 31
2.10. Mitochondrial oxygen consumption
In order to determine the effect of intracellular LPC on mitochondrial fatty acid oxidation,
o O2 consumption was measured at 37 C with a Gilson oxymeter. Isolated mitochondria (1 mg protein/ml) were added to Assay Media containing one of the following sets of substrates: 5 mM succinate, 5 mM glutamate/5 mM malate, or 10 μM palmitoyl-carnitine/1 mM malate. After 2 min, 441 nmol ADP was added and state 3 and 4 respiration rates, phosphorylation efficiency
(ADP/O ratio), and respiratory control index (RCI) were determined from the change in chamber oxygen.
Page 32
2.11. Cellular oxygen consumption
C57BL/6J mice were anesthetized with inhaled isofluorane and primary hepatocytes were isolated as previously described by perfusion with 100 U/ml collagenase (104). Cells were then plated in Williams E media (Invitrogen) supplemented with 5% fetal bovine serum (FBS), 2 mM
L-glutamate, 100 nM insulin, 100 nM dexamethasone, 100 U/ml penicillin, and 100 µg/ml streptomycin overnight. The next day, the media was changed to glucose-free Dulbecco’s
Modified Eagle Medium (DMEM, Invitrogen) and oxygen consumption rate (OCR) was measured with an XF24 Analyzer (Seahorse Bioscience) prior to and after a dose of 100 μM sodium oleate complexed to bovine serum albumin (BSA) at a ratio of 5:1, and varying concentrations of LPC. Mitochondrial inhibitors (1 μM oligomycin, 400 nM carbonyl cyanide 4-
(trifluoromethoxy) phenyhydrazone (FCCP), and 1 μM rotenone) were then added sequentially to determine ATP turnover, respiratory capacity, and spare respiratory capacity, respectively
(105).
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2.12. Hepatocyte viability and mitochondrial permeability transition
Primary hepatocytes isolated as described above were also plated in dark-walled microtiter plates for the overnight incubation. Cells were then washed with phosphate-buffered saline (PBS), acclimated in Hepatozyme-SFM (Invitrogen) for 1 hr, treated with 100 μM oleate complexed to BSA at a ratio of 5:1 and varying concentrations of LPC for 10 min, washed three times with PBS, and then incubated in 2 μM calcein-acetoxymethylester (AM, BD Biosciences) for 30 min at 37°C in the dark (106, 107). Fluorescence intensity after excitation at 485 nm and emission at 538 nm was measured in a microplate fluorimeter to determine cytoplasmic activity.
In order to measure mitochondrial permeability, 8 mM CoCl2 (Sigma), which quenches cytosolic fluorescence of calcein for visualization of mitochondria when the mitochondrial membrane is impermeable (106, 107), was added to isolated hepatocytes prior to treatment with sodium oleate,
BSA, and LPC. Images of fluorescence intensity after excitation at 485 nm and emission at 538 nm were captured at baseline and after 10 min using Image-Pro Plus on an Olympus IX71 with a
RETIGA EXi camera (QImaging).
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2.13. Determination of atherosclerosis
Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice were sacrificed with overdose of inhaled isofluorane. Cardiac and aortic tissues were harvested and fixed in 10% formalin. The aorta, from arch and innominate arteries to femoral bifurcation, was then cut longitudinally, pinned open onto a wax surface, and then stained with Oil Red O to identify lipid-rich lesions for en face analysis. Images of equal resolution (2560 x 1920 pixels) and magnification (40x) were acquired with an Olympus SZ40 microscope fitted with a MicroPublisher 5.0 RTV camera
(QImaging) and analyzed with ImageJ. Lesion area was expressed as a percent of total area (108).
To determine area of the aortic arch for determination of atherosclerosis, the ascending aorta was cut as near as possible to the superior border of the heart. A tangent line was drawn from the proximal edge on the inferior side to the superior side of the ascending aorta to form the proximal boundary. The distal boundary was delineated by a tangent line from the distal edge of the distal innominate (left subclavian) artery. For the superior boundary, a line excluding the lumens of the innominate arteries was drawn. The areas from both halves of aortic arch were added to produce a data point. Using this protocol, consistent values for total lumen area were obtained (p=0.8 by Student’s t test).
Aortic root tissues were fixed with 10% formalin followed by 30% sucrose and embedded in Optimal Cutting Temperature media prior to obtaining frozen sections. The aortic root was obtained from 10 µm serial sections beginning at the level of the aortic valve and ending 800 µm proximally at a level distal to the left ventricle. Sections were stained with Oil
Red O or hematoxylin and eosin. Images were then acquired with an Olympus BX61 microscope fitted with a RETIGA 2000R camera (QImaging) and images were analyzed with ImageJ.
Atherosclerosis was assessed by measuring Oil Red O positive area. The total area was
Page 35 determined by outlining the elastic lamina and excluding valvular tissues. Using this approach, similar values for lumen area were obtained (p=0.4 by Student’s t test) for Pla2g1b+/+/Ldlr-/- and
Pla2g1b-/-/Ldlr-/- mice.
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2.14. Liver characterization
Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice were sacrificed with overdose of inhaled isofluorane. Liver tissue was isolated and patted dry with gauze prior to weighing. Formalin- fixed paraffin-embedded samples were then prepared from the isolated liver tissue. Sections (5
µm) were collected and stained with Masson’s trichrome or hematoxylin and eosin. Preliminary comparisons were made using a 2-point scale noting the presence or absence of lipid accumulation in 2 random low power fields from sections taken 100-200 µm deep to the superior capsular surface of the median hepatic lobe.
Page 37
2.15. Statistical analysis
Results are shown as mean ± SEM, unless otherwise noted. Unpaired Student’s t test was used for comparisons between two groups, assuming equal variance. Multiple comparisons were tested using one-way ANOVA. Differences at p<0.05 were considered significant. Microsoft
Excel was used to conduct Student’s t test, construct trend lines, and calculate R2 values where indicated. SigmaStat was used to conduct ANOVA.
In experiments with a small n (≤3), the risk of type I (α) error was reduced by performing dose-response curves. When the response had a consistent positive or negative trend, the risk of type I (α) error was considered minimized and non-contributory. With small n, type II (β) error is generally a greater concern than type I (α) error because of relatively large variance when compared to sample size. When the author considered there to be significant risk of type I (α) or
II (β) errors, results are denoted as “preliminary.”
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Chapter 3. Results
Page 39
3.1. Deficiency of Pla2g1b protects against diet-induced hyperlipidemia
Pla2g1b-/- mice have decreased fasting plasma lipids after hypercaloric diet feeding
Consistent with results reported previously (99), age-matched male Pla2g1b+/+ and
Pla2g1b-/- mice displayed similar body weights when fed basal chow diet. When the animals were challenged with a high fat/sucrose hypercaloric diet, significant body weight gain was observed in the Pla2g1b+/+ mice in a time-dependent manner (Fig. 2A). In contrast, the Pla2g1b-
/- mice were resistant to body weight gain, with 12% increase in body weight compared with the
31% increase observed in Pla2g1b+/+ mice after 10 weeks of feeding the hypercaloric diet (Fig.
2A).
Fasting plasma triglyceride levels were similar between chow-fed Pla2g1b+/+ and
Pla2g1b-/- mice. However, whereas the Pla2g1b+/+ mice showed progressive elevation of fasting plasma triglyceride levels during a 10-week hypercaloric diet (Fig. 2B), fasting plasma triglyceride levels did not change in the hypercaloric diet-fed Pla2g1b-/- mice and were significantly lower than those observed in Pla2g1b+/+ mice (Fig. 2B). Thus, after feeding the hypercaloric diet for 10 weeks, plasma triglyceride levels were 37% lower in Pla2g1b-/- mice compared with the wild-type Pla2g1b+/+ mice. The Pla2g1b-/- mice also displayed lower plasma cholesterol levels compared with Pla2g1b+/+ mice, even under basal chow dietary conditions
(Fig. 2C). Interestingly, whereas the wild-type Pla2g1b+/+ mice reached a hypercholesterolemic state after 10 weeks on the hypercaloric diet as expected (Fig. 2C), fasting plasma cholesterol levels in the Pla2g1b-/- mice were not susceptible to dietary changes, with 67 ± 2 mg/dl and 71 ±
8 mg/dl before and after feeding the hypercaloric diet (Fig. 2C). Thus, at the end of the 10-week
Page 40 hypercaloric diet, plasma cholesterol was 61% lower in Pla2g1b-/- mice compared with the
Pla2g1b+/+ mice. FPLC analysis of plasma from hypercaloric diet-fed mice showed that difference in VLDL accounted for the different plasma triglyceride levels between Pla2g1b+/+ and Pla2g1b-/- mice. Additionally, the Pla2g1b-/- mice also did not display elevated plasma HDL levels (data not shown), which are typically observed in wild-type mice after high-fat feeding
(109).
Pla2g1b-/- animals have reduced VLDL production after hypercaloric diet feeding
The mechanism underlying the differences in plasma lipid levels between Pla2g1b+/+ and
Pla2g1b-/- mice was explored by comparing hepatic VLDL production rates in these animals under both basal and hypercaloric dietary conditions. In these experiments, mice were fasted for
12 h to achieve total clearance of chylomicron-associated triglycerides and then injected with
P407 to inhibit lipolysis of nascent VLDL secreted by the liver. Plasma samples were collected at hourly intervals, and triglyceride levels were measured (100). When the animals were maintained on a basal chow diet, plasma triglyceride levels were similar between Pla2g1b+/+ and
Pla2g1b-/- mice before and every hour after P407 injection (Fig. 3A, left). Calculation of hourly changes in plasma triglyceride levels revealed no significant difference in hepatic VLDL production rates between chow-fed Pla2g1b+/+ mice (555 ± 61 mg/dl per h) and Pla2g1b+/+ mice
(538 ± 52 mg/dl per h). AUC analysis also revealed no difference in the total amount of triglyceride secreted by chow-fed Pla2g1b+/+ and Pla2g1b-/- mice after 4 h (Fig. 3A, right). In contrast, significant differences in hepatic VLDL production were observed between Pla2g1b+/+ and Pla2g1b-/- mice after feeding the hypercaloric diet (Fig. 3B, C). Feeding the hypercaloric diet for two weeks increased hepatic VLDL production rates in Pla2g1b+/+ mice to 651 ± 90 mg/dl
Page 41 per h, whereas the hepatic VLDL production rates actually decreased in Pla2g1b-/- mice to 278 ±
171 mg/dl per h (Fig. 3B, left). At the end of the 4 h experimental period, significantly more
VLDL-triglyceride was found in the plasma of Pla2g1b+/+ mice compared with that in Pla2g1b-/- mice (Fig. 3B, right). These differences in diet modulation of hepatic VLDL production rates between Pla2g1b+/+ and Pla2g1b-/- mice were observed prior to noticeable changes in their body weights (Fig. 2A). The differences in VLDL production persisted after seven weeks of hypercaloric diet feeding, when differences in body weight gains were also observed. Both the total amount of VLDL secretion over a 4-h period (Fig. 3C, right) and VLDL production rates were significantly higher in Pla2g1b+/+ mice (641 ± 43 mg/dl per h) compared with those in
Pla2g1b-/- mice (321 ± 171 mg/dl per h) (Fig. 3C, left).
LPC supplementation stimulates VLDL production in Pla2g1b-/- and Pla2g1b+/+ mice
Note that VLDL production reached a saturation level after 2 h in the Pla2g1b-/- mice.
Previous work in our laboratory has shown that Pla2g1b-/- mice on hypercaloric diet have reduced plasma levels of lysophospholipids, the enzymatic product of Pla2g1b, compared with wild-type Pla2g1b+/+ mice on similar diet (91). Moreover, restoration of plasma lysophospholipid level in Pla2g1b-/- mice to that comparable in Pla2g1b+/+ mice by intraperitoneal injection of LPC was found to ameliorate the protective effects of Pla2g1b inactivation against glucose intolerance and postprandial hyperglycemia (62, 91). Therefore, in the current study, we evaluated whether intraperitoneal injection of LPC to hypercaloric diet-fed
Pla2g1b-/- mice prior to P407 injection would also restore the linearity of the VLDL production curve. Indeed, results showed that LPC injection also increased the rate and total amount of
VLDL accumulation to levels observed in Pla2g1b+/+ mice, which were ∼2-fold greater than
Page 42 that observed in Pla2g1b-/- mice without LPC injection (Fig. 4). Injection of LPC also increased
VLDL-triglyceride levels in Pla2g1b+/+ mice to levels above those observed in mice without
LPC injection (Fig. 4C). Taken together, these results indicate that LPC promotes hepatic VLDL production and that the differences in plasma lipoprotein levels and VLDL production between
Pla2g1b+/+ and Pla2g1b-/- mice are due to reduced absorption and transport of lysophospholipids in the absence of Pla2g1b.
LPC is directly used as a substrate for VLDL-TG synthesis
In the next set of experiments, [3H]LPC was injected into mice prior to P407 administration, and plasma was collected to determine if LPC serves as substrate for VLDL synthesis. More than half of the radioactivity recovered in plasma was found to be associated with VLDL in both Pla2g1b+/+ and Pla2g1b-/- mice after 4 h, whereas the remaining radioactivity was not lipoprotein associated (Fig. 5). The amount of radioactivity from [3H]LPC incorporated into VLDL was not due to nonspecific association of the injected [3H]LPC with lipoproteins, because in vitro incubation of [3H]LPC with mouse plasma showed that most of the
[3H]LPC was associated with albumin and none of the radioactivity was associated with VLDL.
Moreover, the radioactivity found in VLDL after [3H]LPC injection was found in the triglyceride fraction after thin layer chromatography analysis, indicating that the LPC was used as substrate for VLDL lipid synthesis, with more radioactivity found in the VLDL fraction of Pla2g1b+/+ mice compared with Pla2g1b-/- mice (Fig. 5). Analysis of plasma apoB levels by ELISA found no difference between Pla2g1b+/+ and Pla2g1b-/- mice (data not shown), indicating that LPC did not change the number of particles secreted by the liver. Thus, LPC increases the production of
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VLDL particles with higher triglyceride content instead of increasing the number of apoB- containing lipoproteins secreted by the liver.
Pla2g1b-/- mice have increased postprandial TRL clearance
Plasma lipid levels are typically controlled by lipoprotein synthesis and catabolism.
Therefore, additional experiments were performed to evaluate potential differences in triglyceride-rich lipoprotein clearance between Pla2g1b+/+ and Pla2g1b-/- mice. In these experiments, the mice were fasted overnight and then fed a bolus meal of olive oil by gastric gavage. Plasma triglyceride levels were monitored at hourly intervals without lipase inhibition.
Results showed similar plasma triglyceride levels between Pla2g1b+/+ and Pla2g1b-/- mice at baseline and 60 min after oral lipid feeding. However, plasma triglyceride levels were significantly lower in Pla2g1b-/- mice compared with Pla2g1b+/+ mice 120 min after lipid feeding (Fig. 6). Importantly, plasma triglyceride levels returned to their baseline fasting levels in Pla2g1b-/- mice 180 min after lipid meal, whereas plasma triglyceride levels in the wild-type
Pla2g1b+/+ mice remained elevated compared with the fasting level throughout the 180 min study period (Fig. 6). AUC analysis of the data revealed a 2-fold decrease in postprandial lipidemia in the Pla2g1b-/- mice compared with Pla2g1b+/+ mice (Fig. 6). As Pla2g1b+/+ and
Pla2g1b-/- mice are similar in lipid absorption efficiency (67, 99), the lower postprandial triglyceride levels observed in Pla2g1b-/- mice indicated their more efficient catabolism of TRL compared with Pla2g1b+/+ mice. Thus, the significant differences in fasting plasma triglyceride and cholesterol levels observed between hypercaloric diet-fed Pla2g1b+/+ and Pla2g1b-/- mice were due to reduced hepatic production as well as increased clearance of TRL in the Pla2g1b-/- mice.
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3.2. Micromolar changes in LPC concentration cause minor effects on hepatic
mitochondrial permeability but major alterations in function
LPC induces concentration-dependent mitochondrial swelling
Exogenous LPC in the low micromolar range easily incorporates into the mitochondrial membrane and changes its permeability (110). At concentrations as low as 20 μM, LPC can form micelles (111), and concentrations from 50 to 200 μM have been used to permeabilize cell membranes (112). For initial characterization, the effect of brief egg LPC exposure on mitochondrial permeability was assessed. LPC induced permeability and swelling in a concentration-dependent manner from 40 μM to 200 μM (Fig. 7). This response was not inhibited by 2 μM CsA, confirming that LPC did not open the Ca2+-sensitive membrane transition permeability pore in the absence of Ca2+ ion.
LPC exacerbates mitochondrial response to calcium ion
Exogenous administration of Ca2+ causes membrane permeability transition (MPT) in isolated mitochondria (113, 114). Ablation of the membrane-bound iPLA2γ, which produces
LPC, has also been shown to suppress mitochondrial MPT (70). Therefore, we determined if
LPC may modify the mitochondrial response to Ca2+ (110, 115). To test this possibility, we examined the concentration-dependent influence of LPC on Ca2+-induced MPT. In the absence
2+ of LPC, addition of 220 μM CaCl2 caused rapid, CsA-responsive swelling, thus confirming Ca - induced MPT reported previously (116)(Fig. 8A). In the presence of 40 μM LPC, a similar pattern of CsA-responsive Ca2+-induced swelling protection was observed as in positive controls.
Page 45
However, in the presence of LPC at a concentration of ≥80 μM, CaCl2 caused additional swelling, but the swelling response was not rescued by CsA.
Incubation with 70 μM Ca2+ has been shown to cause minimal swelling after 10 min
(116). This study also observed minimal swelling in mitochondria after incubation with 75 μM
CaCl2. Brief exposure to 40-80 μM LPC did not result in mitochondrial swelling after incubation with 75 μM CaCl2. However, in the presence of ≥120 μM LPC, addition of 75 μM CaCl2 resulted in additional mitochondrial swelling (Fig. 8B). This LPC-exacerbated response was not CsA- sensitive. To determine whether the effect was due to massive dissolution of the mitochondrial membrane, mitochondria were solubilized using SDS as a negative control (confirmed by microscopy). Such preparations did not exhibit a decrease in absorbance after addition of either concentration of calcium, confirming that mitochondrial membrane was still maintained in the presence of 200 μM LPC.
LPC causes increased mitochondrial permeability to calcium ion
Previous studies have shown that when mitochondria were added to media containing 10
μM Ca2+ and 50-100 μM LPC, Ca2+ uptake was inhibited (115). Addition of 50 μM LPC to preparations containing mitochondria preloaded with Ca2+ resulted in temporary release of Ca2+ into the media (115). In contrast, this study examined the effect of LPC on Ca2+ homeostasis by briefly exposing mitochondria to LPC prior to addition 75 μM CaCl2 in order to determine the response of the mitochondrial membrane. To examine this, extramitochondrial Ca2+ was measured by a membrane impermeable fluorescent indicator. In the absence of LPC, Ca2+ uptake occurred gradually in a nonlinear manner within 10 min (Fig. 9A). Surprisingly, brief exposure
Page 46 to LPC resulted in more rapid Ca2+ uptake as shown by decreased equilibration times (Fig. 9A) and decreased peaks in fluorescence intensity (Fig. 9B). With 40 μM and 80 μM LPC, uptake curves were shifted to the left in a concentration-dependent manner. Moreover, with ≥120 μM
LPC, fluorescence intensity equilibrated within 20 s, suggesting freer permeability to Ca2+. As a negative control, mitochondria solubilized with SDS showed no decrease in fluorescence
2+ intensity following CaCl2 addition. Thus the order of exposure of LPC and Ca may have different effects upon membrane permeability and equilibration according to electrochemical and/or osmotic gradients.
Mitochondrial membrane potential intact with low concentrations of LPC
Maintenance of mitochondrial membrane potential is vital to maintaining the proton gradient for coupled oxidative phosphorylation. Since LPC caused increased permeability to
Ca2+, we investigated whether LPC also compromised the mitochondrial membrane potential.
After a short (2 min) exposure to LPC in the absence of respiratory substrate, a linear (R2=0.99) concentration-dependent decrease in resting membrane potential was evident (Fig. 10A). When succinate was added to activate the electron transport chain, the membrane potential remained at initial levels with LPC concentrations up to 80 μM (Fig. 10B). However, with ≥120 μM LPC, respiration caused an additional loss of membrane potential below the already depolarized baseline levels. In contrast, mitochondria solubilized with SDS showed no change in absorbance with succinate addition.
Maintenance of mitochondrial respiration in the presence of LPC
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To determine if isolated mitochondria incubated with LPC would maintain optimal oxidative function with brief (2 min) exposure to LPC, respiration was measured in the presence of 40-80 μM LPC, concentrations at which the membrane potential remained constant after substrate addition (Fig. 10) but swelling and increased permeability to Ca2+ had occurred (Figs. 7 and 9). Results showed that in the presence of up to 80 μM LPC, mitochondria incubated with succinate to activate complex II maintained maximal state 3 (with ADP) oxygen uptake similar to levels observed in controls. This occurred with no major change in state 4 (without ADP) respiration rates. After brief exposure to 100 μM LPC, state 3 respiration rate and respiratory control index (RCI) decreased dramatically (Fig. 11A).
Respiration rates were also determined in the using glutamate and malate as complex I substrates. Mitochondria briefly exposed to 20-80 μM LPC displayed state 3 respiration rates similar to controls and consistent RCI (Fig. 11B). State 3 oxygen consumption rate and RCI were decreased with brief incubation with 100 μM LPC.
Deficiencies in transport of fatty acid across the mitochondrial membrane (e.g. CPT1 deficiency) can slow the oxidation rate (117). Since LPC alters the membrane, transport of fatty acids may be affected. To test this possibility, oxidation was stimulated by addition of palmitoylcarnitine as a respiratory substrate. Again, state 3 respiration rate was similar to controls with incubations of ≤80 μM LPC. However, 100 μM LPC inhibited state 3 respiration rate without affecting state 4 respiration rate. The decrease in oxygen uptake correlated with a decrease in RCI but without a significant decrease in phosphorylation efficiency as measured by
ADP/O ratio (Fig. 11C).
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With all substrates tested, a difference of 20 μM LPC caused a great effect on respiration rates. Taken together, these results suggest that small fluctuations in LPC concentration even with brief exposure within the mitochondrial microenvironment can have a profound effect on mitochondrial function.
LPC regulates fatty acid-stimulated hepatic oxidation
The concentration of LPC in the intracellular microenvironment of mitochondria is modified in part by intracellular phospholipases and lysophospholipases, fatty acid binding proteins, and equilibration with mitochondrial and cellular membranes (69, 110, 118). The liver encounters a Pla2g1b-dependent increase in portal lysophospholipids as well as an increase in plasma non-esterified fatty acids in the postprandial state (91). Intracellular LPC concentration could possibly be modified by exogenous lysophospholipids delivered to the cell via the plasma.
In order to determine the effect of extracellular LPC on whole cell oxidative function, murine primary hepatocytes were isolated and fatty acid-stimulated OCR was measured. Albumin was added to preparations in order to better simulate plasma conditions and to decrease the membrane-permeabilizing characteristics of LPC (112). In the absence of exogenous lysophospholipids, fatty acid complexed to albumin increased OCR by 20-40%. The fatty acid- induced increase in OCR in hepatocytes was retained in the presence of 40 μM LPC. However,
100 μM LPC prevented the fatty acid-induced increase in OCR, but basal OCR was retained (Fig.
12). Incubation of hepatocytes with 200-1500 μM LPC and fatty acid resulted in OCR decreasing below baseline levels (not shown).
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Extracellular LPC induces Ca2+-independent mitochondrial permeability
In order to determine the mechanism of the LPC-mediated suppression of OCR, calcein-
AM and CoCl2 were added prior to the addition of fatty acid oxidation substrate in order to visualize mitochondrial permeability (106, 107). Co2+ quenches the fluorescence signal of calcein in the cytosol but not the mitochondria when the mitochondrial membrane is impermeable to small molecules. Mitochondrial fluorescence in hepatocytes incubated with 40
μM LPC and fatty acid was comparable to that observed in controls. However, hepatocytes incubated with and above 100 μM LPC showed decreased mitochondrial fluorescence, which is consistent with increased mitochondrial permeability (Fig. 13). Pretreatment with 2 μM CsA did not prevent the decrease in mitochondrial fluorescence observed in the presence of 100 μM or
200 μM LPC (not shown), suggesting a Ca2+-independent mechanism.
The influence of exogenous LPC on cellular metabolic activity was determined by labeling cells with calcein-AM after their incubation with LPC. A concentration-dependent decrease in fluorescence compared to control cells incubated in the absence of LPC was observed (Fig. 14). Thus while low extracellular levels of LPC caused some cytosolic dysfunction, oxidation remained intact. However, higher concentrations of LPC caused mitochondrial permeability and decreased oxidative function. Mitochondrial permeability coincided with a reduced catabolic activity in cells (Figs. 13 and 14).
3.3. Inhibition of Pla2g1b protects against selected disorders of lipid metabolism
Inhibition of Pla2g1b decreases diet-induced obesity in Ldlr-/- mice
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Consistent with results observed in mice on generic C57BL/6J background, age-matched
Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice displayed similar body weight (23 g vs. 23 g) when maintained on chow diet (Fig. 15) (99, 119). In mice challenged with high-fat, cholesterol- containing Western diet, significant weight gain was observed in Pla2g1b+/+/Ldlr-/- animals. In contrast, Pla2g1b-/-/Ldlr-/- animals were resistant to weight gain, with 30% increased body weight (to 30 g) compared to the 52% increase (to 35 g) observed in Pla2g1b+/+/Ldlr-/- mice after
10 weeks of feeding the Western diet (Fig. 15). The extent of the resistance to obesity was more pronounced in mice fed a high-fat, cholesterol-free diet during the test period. Pla2g1b+/+/Ldlr-/- animals weighed 39 g whereas Pla2g1b-/-/Ldlr-/- mice weighed 25 g (p<0.001) (Fig. 15).
Inhibition of Pla2g1b improves glucose homeostasis in Ldlr-/- mice
When maintained on basal chow diet, Pla2g1b-/-/Ldlr-/- mice had similar fasting insulin levels but decreased fasting plasma glucose levels compared to Pla2g1b+/+/Ldlr-/- mice, suggesting that inhibition of Pla2g1b improved insulin sensitivity even in the context of impaired
TRL clearance.
Previous results with mice on Ldlr+/+ background showed that age-matched Pla2g1b-/- mice fed a chronic Western diet displayed decreased, but statistically insignificant (p>0.05) fasting glucose levels compared to Pla2g1b+/+ animals (99). Current results, however, showed
Pla2g1b-/-/Ldlr-/- mice fed chronic Western diet had 32% decreased (p<0.05) fasting plasma glucose levels when compared to Pla2g1b+/+/Ldlr-/- mice (Fig. 16). In addition, Pla2g1b-/-/Ldlr-/- mice had 62% decreased fasting insulin levels compared to Pla2g1b+/+/Ldlr-/- mice (Fig. 16).
Consistent with these results, homeostatic model assessment-insulin resistance (HOMA-IR) values in Pla2g1b-/-/Ldlr-/- mice were 28% of values observed in Pla2g1b+/+/Ldlr-/- mice (Fig.
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16). Furthermore, in Pla2g1b-/-/Ldlr-/- mice challenged with a chronic high-fat, cholesterol-free diabetogenic diet, decreased plasma fasting glucose, insulin, and HOMA-IR values were also observed when compared to Pla2g1b+/+/Ldlr-/- mice (Fig. 16).
Inhibition of Pla2g1b decreases diet-induced hyperlipidemia in Ldlr-/- mice
Previous studies showed that 10-12 week old Pla2g1b-/- mice maintained on chow diet had similar hepatic VLDL production rates and lipid distribution across lipoproteins as
Pla2g1b+/+ controls (119). To determine whether this effect would persist in the Ldlr-/- background, plasma from fasting Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice maintained on chow diet was isolated, fractionated by FPLC, and lipid distribution was assessed. Results showed that, in contrast to Ldlr+/+ mice, LDL-C levels were elevated but peak values were similar between Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice (Fig. 17). In addition, VLDL-TG levels were similar in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice (Fig. 17). These findings suggest that the absence of the LDL receptor does not alter the effect of Pla2g1b on VLDL production under basal conditions.
Previous results have shown that after a high-fat diet challenge, Pla2g1b-/- animals had decreased rates of VLDL-TG secretion and increased postprandial TRL clearance which led to decreased plasma triglyceride levels (119). To assess whether this mechanism would continue to protect in the presence of inhibited TRL uptake, animals lacking LDLR were employed. Plasma from fasting Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice challenged with Western diet was isolated, fractionated by FPLC, and lipid distribution was determined. Results showed elevated
VLDL-TG, VLDL-cholesterol (VLDL-C), and LDL-C in both Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-
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/Ldlr-/- mice compared to similar mice maintained on chow diet. Pla2g1b+/+/Ldlr-/- mice exhibited a 30-fold increase in VLDL-TG, 10-fold increase in LDL-C, and 30% decrease in
HDL-C. However, Pla2g1b-/-/Ldlr-/- mice subjected to Western diet for 10 weeks displayed merely a 10-fold increase in VLDL-TG, and 6-fold increase in LDL-C (Fig. 17). Meanwhile,
HDL-C increased 2-fold (Fig. 17). Consistent with prior results in Ldlr+/+ mice, Pla2g1b-/-/Ldlr-/- had decreased VLDL-TG after Western diet challenge compared to Pla2g1b+/+/Ldlr-/- control animals (Fig. 17). In addition, Pla2g1b-/-/Ldlr-/- mice displayed peaks in VLDL-C and LDL-C that were 20% and 45%, respectively, of levels observed in Pla2g1b+/+/Ldlr-/- mice on similar diet (Fig. 17). Furthermore, when mice were challenged with a high-fat, cholesterol-free diet, decreased VLDL-TG, VLDL-C, and LDL-C were still observed in Pla2g1b-/-/Ldlr-/- mice when compared to Pla2g1b+/+/Ldlr-/- control animals (Fig. 17), further confirming decreased VLDL-
TG production with inhibition of Pla2g1b.
Total plasma triglyceride and cholesterol levels were also measured in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice after maintenance on basal diet or challenge with diabetogenic and
Western diets. Results produced similar plasma triglyceride and cholesterol levels in
Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice fed chow diet for 10 weeks (not shown). Consistent with results in Pla2g1b-/- and Pla2g1b+/+ animals on Ldlr+/+ background, Pla2g1b-/-/Ldlr-/- mice had decreased diet-induced hyperlipidemia compared to Pla2g1b+/+/Ldlr-/- animals after 10 weeks of diabetogenic diet (119) (Fig. 19). Pla2g1b-/-/Ldlr-/- mice had fasting total triglyceride and cholesterol levels that were 41% and 29% of those observed in Pla2g1b+/+/Ldlr-/- mice, respectively (Fig. 18). Previous studies by Huggins, et al. showed no difference in plasma lipids in Pla2g1b-/- and Pla2g1b+/+ mice on Ldlr+/+ background and fed Western diet for 16 weeks (99).
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However, current results in LDLR-deficient mice showed triglyceride and cholesterol levels in
Pla2g1b-/-/Ldlr-/- mice that were 44% and 38% of those observed in Pla2g1b+/+/Ldlr-/- mice, respectively (Fig. 18).
Inhibition of Pla2g1b decreases diet-induced hepatomegaly in Ldlr-/- mice
Age-matched male Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice displayed similar liver weight (1.0 g vs. 1.0 g) when maintained on chow diet (Fig. 19). Consistent with other studies in healthy mice, liver weight in both Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice was 4% of total body weight. In mice challenged with chronic cholesterol-containing Western diet, significant liver weight gain was observed in Pla2g1b+/+/Ldlr-/- animals. In contrast, Pla2g1b-/-/Ldlr-/- animals were resistant to liver weight gain, with only 60% increased liver (to 1.6 g) weight compared to the 90% increase observed in Pla2g1b+/+/Ldlr-/- mice (to 1.9 g) after 10 weeks of feeding the Western diet (Fig. 19). This finding of reduced diet-induced hepatomegaly is consistent with previous results observed in Pla2g1b-/- mice on Ldlr+/+ background (99).
Interestingly, preliminary histological examination of hepatic tissues suggested extensive lipid content in livers of both Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice (Fig. 20).
Inhibition of Pla2g1b has minimal effect on atherosclerosis in Western diet fed Ldlr-/- mice
Increased LDL-C confers risk for atherosclerosis. Since Pla2g1b-/-/Ldlr-/- mice had decreased levels of LDL-C, we sought to determine whether inhibition of Pla2g1b would decrease atherosclerosis in mice. In order to assess this, cardiac and aortic tissues from
Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice exposed to basal chow and Western diets were collected and stained with Oil Red O to visualize lipid deposits. Aortas from Pla2g1b+/+/Ldlr-/-
Page 54 and Pla2g1b-/-/Ldlr-/- mice on basal chow diet did not stain with Oil Red O in the abdominal aorta, thoracic aorta, aortic arch, or aortic root (not shown).
After feeding Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice Western diet for 10 weeks, no deposits of neutral lipid were observed in the abdominal or thoracic aortas of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice. However, Oil Red O positive staining was observed in the aortic arch of both Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice (Fig. 21). Lipid accumulation was observed primarily along the inferior border of the aortic arch and appeared to extend in a superior direction according to lesion severity. Quantification of the lesion area in the aortic arch of
Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice revealed no significant difference in size as a percentage of total area (Fig. 21).
Oil Red O positive staining was also found in the innominate arteries of Pla2g1b+/+/Ldlr-
/- and Pla2g1b-/-/Ldlr-/- mice. The most prevalent and consistent lesions were found in the proximal innominate (brachiocephalic) artery. The extent of atherosclerosis in the brachiocephalic artery was similar between Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice.
Though Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice displayed similar en face Oil Red O accumulation, the possibility remained that lesion thickness could be different in the absence of
Pla2g1b. The aortic root was, therefore, sectioned and stained with Oil Red O to quantify the thickness of the atherosclerotic lesion present. No difference in the total area of the Oil Red O positive lesion in Pla2g1b-/-/Ldlr-/- mice compared to Pla2g1b+/+/Ldlr-/- mice was observed (Fig.
22). The size distribution of the lesion area through the aortic root was also similar between
Page 55
Pla2g1b-/-/Ldlr-/- and Pla2g1b+/+/Ldlr-/- mice. Interestingly, the size of the lesion appeared to slightly increase proceeding proximally from the ascending aorta for the first 100 µm and then decrease as sections neared the left ventricle (Fig. 22)
Atherosclerosis correlates with postprandial rather than fasting lipid levels in Pla2g1b-/-/Ldlr-/- mice
Though Pla2g1b-/-/Ldlr-/- mice had decreased fasting plasma lipoproteinemia after consumption of Western diet, they did not have decreased atherosclerosis compared to
Pla2g1b+/+/Ldlr-/- mice. Despite Pla2g1b-/- mice having decreased postprandial lipemia compared to Pla2g1b+/+ on Ldlr+/+ background (119), it was considered that this mechanism may be reduced in animals on the Ldlr-/- background and contribute to the observation of similar levels of atherosclerosis in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice. Previous studies have shown that postprandial hyperlipidemia confers atherosclerosis risk (31). In order to determine whether postprandial lipemia was similar between genotypes, fed state plasma from
Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice fed Western diet for 10 weeks was collected and triglycerides and cholesterol levels were measured. Results showed that similar (p=0.4) cholesterol levels were observed between Pla2g1b-/-/Ldlr-/- and Pla2g1b+/+/Ldlr-/- mice (Fig. 23).
Furthermore, plasma triglyceride levels merely trended toward a decreased amount (p=0.09) in
Pla2g1b-/-/Ldlr-/- mice compared to Pla2g1b+/+/Ldlr-/- mice (Fig. 23).
In order to determine whether individual fed state lipid levels were an indication of the lesion severity observed, postprandial cholesterol levels in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-
/- mice were plotted against various measures of atherosclerosis. Atherosclerosis as measured by
Page 56 lesion percent area in the aortic arch and total lesion area in the aortic root had positive correlations (R2=0.67 and 0.367, respectively) with postprandial cholesterol levels in Pla2g1b-/-
/Ldlr-/- mice (Fig. 24). However no correlation was observed between these parameters in
Pla2g1b+/+/Ldlr-/- mice (R2=0.14 and 0.002, respectively) (Fig. 24). Fed state lipid levels did not correlate with severity of lesion observed in the brachiocephalic arteries of Pla2g1b+/+/Ldlr-/- or
Pla2g1b-/-/Ldlr-/- mice (R2=0.08 and 0.008, respectively) (Fig. 24).
Preliminary results of atherosclerosis determination after diabetogenic diet feeding
When Pla2g1b+/+/Ldlr-/- or Pla2g1b-/-/Ldlr-/- mice were challenged with diabetogenic diet, fasted state plasma cholesterol levels observed were less severe than similar animals fed Western diet (Fig. 18). This resulted in decreased aortic root lesion area when compared to animals fed
Western diet. However, preliminary data suggest that Pla2g1b-/-/Ldlr-/- mice have decreased lesion size when compared to Pla2g1b+/+/Ldlr-/- mice (not shown).
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Chapter 4. Discussion
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4.1. Overview of findings
Findings from this study showed that inactivation of the intestinal digestive enzyme
Pla2g1b protects mice against elevated plasma triglyceride and cholesterol levels induced by a high-fat/sucrose-supplemented (hypercaloric) diet. The protection is independent of adiposity, but it is a direct consequence of reduced hepatic VLDL production and increased TRL clearance in Pla2g1b-/- mice. This conclusion is supported by data showing ∼40% decrease in hepatic
VLDL production in Pla2g1b-/- mice compared with Pla2g1b+/+ mice upon short-term hypercaloric diet feeding, prior to any observed differences in body weight gain. Pla2g1b digests phospholipids in the intestinal lumen to facilitate lipid nutrient absorption through the gastrointestinal tract (120). The products of this enzymatic reaction are nonesterified fatty acids and LPC. The difference in hepatic VLDL production between Pla2g1b+/+ and Pla2g1b-/- mice cannot be attributed to differences in nonesterified fatty acid absorption because other phospholipases present in the digestive tract can compensate for the absence of Pla2g1b in phospholipid digestion in mediating normal fat absorption in Pla2g1b-/- mice (67), The difference in reaction products in phospholipid hydrolysis is that lysophospholipids, which are rapidly absorbed and transported to the liver through the portal vein (62, 121), are significantly reduced in the Pla2g1b-/- mice (62). Results of the current study show LPC is a direct substrate for VLDL-TG synthesis. In the absence of Pla2g1b, substrate limitation reduces VLDL-TG synthesis over time. Furthermore, VLDL production can be restored by LPC supplementation.
Thus, these results documented that absorption of lysophospholipid, in addition to fatty acids, also plays an important role in hepatic VLDL production.
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Findings from the current study also demonstrated a sensitive regulatory role of LPC on hepatic mitochondria. Isolated mitochondria sustained maximal oxidative function with differing respiratory substrates (succinate, glutamate/malate, and acyl-carnitine) in the presence of micromolar (≤80 μM) concentrations of LPC despite the presence of increased membrane permeability to water and Ca2+ and the presence of reduced membrane potential. However, slightly increasing LPC concentration to ≥100 μM caused further mitochondrial membrane permeability, increased susceptibility to Ca2+-induced damage, exacerbated impairment of mitochondrial membrane potential, and decreased in oxidative function. Our data also showed that exogenous addition of low micromolar concentrations of LPC to primary hepatocytes had minimal impact on fatty acid-stimulated oxidation. However, increasing the extracellular LPC concentration by 60 μM resulted in hepatocytes that displayed increased mitochondrial permeability and reduced oxidative function. Interestingly, the threshold concentration of LPC required to induce functional deficits in primary hepatocytes and isolated mitochondria converged at approximately 80-100 μM LPC. Thus, absorbed LPC was shown to affect oxidation in addition to VLDL production.
Finally, findings from this study showed that genetic suppression of Pla2g1b resulted in improved glucose homeostasis, decreased diet-induced obesity, and decreased diet-induced hyperlipidemia in two additional severe models of diet-induced hyperlipidemia. Decreased plasma VLDL-TG, VLDL-C, and LDL-C were observed in Pla2g1b-/-/Ldlr-/- mice after chronic high-fat feeding despite impaired TRL clearance caused by the lack of LDLR. Pla2g1b-/-/Ldlr-/- mice also had decreased fasting plasma triglyceride and cholesterol levels compared to
Pla2g1b+/+/Ldlr-/- mice after chronic feeding of high-fat, cholesterol-containing or cholesterol-
Page 60 free diet. Furthermore, inhibition of Pla2g1b resulted in decreased hepatomegaly in response to
Western diet feeding, but did not dramatically alter liver histology in in Pla2g1b-/-/Ldlr-/- mice when compared to Pla2g1b+/+/Ldlr-/- mice. Surprisingly, a decrease in atherosclerotic lesion size in the aortic arch or aortic root was not observed in Pla2g1b-/-/Ldlr-/- mice when compared to
Pla2g1b+/+/Ldlr-/- animals after feeding mice Western diet. Consistent with this result, however, was the observation that fed state plasma cholesterol levels were similar and plasma triglyceride only slightly decreased between Pla2g1b-/-/Ldlr-/- and Pla2g1b+/+/Ldlr-/- mice. Despite this similarity, atherosclerotic lesion area in Pla2g1b-/-/Ldlr-/-, but not Pla2g1b+/+/Ldlr-/-, animals correlated positively with fed state plasma cholesterol levels.
4.2. Discussion of “Deficiency of Pla2g1b protects against diet-induced hyperlipidemia”
The causative relationship of lysophospholipid absorption with VLDL synthesis is not unprecedented. Previous studies suggested that metformin decreases hepatic apoB secretion via reduction of cellular LPC (122). Although the current study showed no difference in apoB secretion between Pla2g1b+/+ and Pla2g1b-/- mice, lysophospholipids may contribute to hepatic
VLDL production through several other mechanisms. Previous in vitro cell culture experiments illustrated that LPC can be effectively reacylated to PC in hepatocytes, thereby providing the substrates necessary for VLDL synthesis under choline-deficient conditions (123).
Lysophospholipids also inhibit fatty acid β-oxidation in the liver (91), thus increasing substrate availability for VLDL-TG synthesis. Lysophospholipids may also provide additional substrates to promote both triglyceride and phospholipid synthesis in hepatocytes (124), thus decreasing intracellular degradation of apoB and facilitating VLDL secretion (88). The present in vivo data
Page 61 are consistent with the in vitro observations that reducing LPC levels limits substrate availability of VLDL synthesis and secretion. Findings showed that VLDL secretion was similar between
Pla2g1b+/+ and Pla2g1b-/- mice during the initial h of lipolysis inhibition. The differences observed were due mainly to limited VLDL production by Pla2g1b-/- mice at later time points whereas VLDL production in Pla2g1b+/+ mice continued at a linear rate (Fig. 3B, C). The provision of LPC to the Pla2g1b-/- mice restored VLDL production rate to comparable levels as that observed in Pla2g1b+/+ mice (Fig. 4). The LPC supplied to the animals was utilized directly as lipid substrates for VLDL synthesis, and additional provision of LPC further increased VLDL production in Pla2g1b+/+ mice. Interestingly, whereas previous studies showed that the cytosolic calcium-independent phospholipase A2 expressed in the liver is important for VLDL maturation and secretion (125), the current study revealed that suppression of lysophospholipid absorption via Pla2g1b gene inactivation is also effective in suppressing VLDL synthesis.
In addition to reduced VLDL secretion, hypercaloric diet-fed Pla2g1b-/- mice also displayed enhanced clearance of TRL compared with similarly fed Pla2g1b+/+ mice. Typically,
TRL catabolism occurs via LPL-mediated hydrolysis of the lipoprotein-associated triglycerides to nonesterified fatty acids, which are then taken up by extrahepatic tissues as nutrients or stored in adipose tissues as energy reserves, and via receptor-mediated uptake of remnant lipoproteins.
Although the mechanism underlying the difference in TRL clearance between Pla2g1b+/+ and
Pla2g1b-/- mice has not been determined, a difference in LPL activity between these animals is unlikely. Increased LPL activities in adipose tissues and muscle/heart to facilitate fat uptake from
TRL are known to promote obesity and insulin resistance, respectively (50, 51). However, the
Pla2g1b-/- mice are more resistant to diet-induced obesity and glucose intolerance (62, 99). In
Page 62 contrast, the increase in hepatic fatty acid oxidation (91) and reduced plasma lipid levels observed in Pla2g1b-/- mice suggests that these animals are more resistant to diet-induced suppression of Ldlr expression in the liver. Thus, the accelerated plasma clearance of TRL in hypercaloric diet-fed Pla2g1b-/- mice is likely due to increased receptor-mediated clearance by the liver. Taken together, these results indicate that Pla2g1b inactivation reduces diet-induced hyperlipidemia by decreasing VLDL production and accelerating the clearance of TRL; hence, pharmacological inhibition of PLA2 activity in the digestive tract may be sufficient to reduce diet-induced hyperlipidemia in addition to the previously reported beneficial effects on obesity and diabetes (126).
Results of the current study also showed lower plasma HDL-cholesterol levels in hypercaloric diet-fed Pla2g1b-/- mice compared with Pla2g1b+/+ mice. At first glance, these results may suggest an undesirable effect of Pla2g1b inhibition. However, note that Pla2g1b inactivation did not reduce HDL-C levels, as comparable HDL-C levels were observed between hypercaloric diet-fed Pla2g1b-/- mice and chow-fed Pla2g1b+/+ and Pla2g1b-/- mice. The
Pla2g1b-/- mice were insensitive to the increase in HDL-C levels after chronic feeding of a hypercaloric diet, suggesting that these animals were not sensing the increased supply of dietary lipids with lipid metabolism (current study) and glucose metabolism (62) similar to those observed in chow-fed animals. In fact, the dietary lipids were readily oxidized in the liver and not available to alter the metabolism in Pla2g1b-/- mice (91). The ability of Pla2g1b inactivation to reduce the synthesis of VLDL, the precursor lipoprotein of the atherogenic LDL, while sustaining normal HDL levels has direct clinical implications for targeted improvement of plasma lipoprotein profile in humans.
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4.3. Discussion of “Micromolar changes in LPC concentration cause minor effects on hepatic mitochondrial permeability but major alterations in function”
The in vitro data collected in this study has direct physiological relevance, adding mechanistic information to our previous observations that PLA2G1B-mediated phospholipid digestion in the intestinal lumen after meal consumption contributes LPC to the liver to inhibit fatty acid oxidation and promote triglyceride synthesis for storage and/or VLDL secretion (91,
99, 119). Herein we showed that meal-induced repression of fatty acid oxidation is likely caused by LPC-mediated suppression of mitochondrial function, thereby partitioning fatty acids absorbed from the meal to intracellular sites for triglyceride biosynthesis. Although the effective concentrations used in the current in vitro studies were lower than plasma LPC levels in vivo, plasma LPC binds tightly to albumin and albumin stimulates the release of hepatic LPC (119,
121, 123, 127, 128). In addition, absorbed LPC from the gut is directed to the liver by the portal vein prior to accessing the systemic circulation (62). Thus, the effective concentration of LPC that causes inhibition of hepatic mitochondrial function may be much less than LPC levels in the plasma. Alternatively, hepatocytes in situ may be acclimated to the prevailing plasma LPC concentration and the small changes in postprandial LPC are sufficient to inhibit hepatocyte fatty acid-stimulated oxidation. In either case, the current study highlighted that small changes in the effective LPC concentration could dramatically alter hepatocyte function.
The current study also showed that alterations in hepatic mitochondrial integrity and function occurred during a short incubation period with LPC. These observations are also
Page 64 consistent with previous reports showing Pla2g1b inactivation increased fatty acid oxidation in mouse liver after lipid meal consumption and that acute treatment with LPC abolished this effect
(91). Interestingly, chronic feeding of high fat diet to mice has been shown to promote hepatic steatosis, which is characterized not only by increased hepatic triglyceride content, but also increased hepatic LPC (129, 130). Diet-induced hepatomegaly can also be reduced by Pla2g1b inactivation (99). Furthermore, and consistent with our data showing the loss of mitochondrial integrity with hepatocyte toxicity at high concentrations of LPC, extended exposure of hepatocytes to LPC can cause mitochondrial release of Bax to trigger apoptosis, and LPC is an important player in palmitate-induced lipoapoptosis of hepatocytes (89, 130). Taken together, these results showed that LPC may participate in both acute and chronic responses of hepatocytes to high fat diet by modulating mitochondrial function and integrity in a concentration-dependent manner. Thus, pharmacological intervention to lower LPC levels and/or absorption in response to high fat diet may be a viable strategy to reduce high fat diet-induced hepatosteatosis and its accompanying risk of metabolic diseases. Preliminary studies showing efficacy of the generic PLA2 inhibitor methyl indoxam in improving diet-induced glucose intolerance, which can be mediated by absorbed LPC, are consistent with this possibility (62,
126).
4.4. Discussion of “Inhibition of Pla2g1b protects against selected disorders of lipid metabolism”
Previous studies showed that the decreased plasma triglyceride levels in Pla2g1b-/- mice fed high-fat diet was due to decreased VLDL-TG production and increased postprandial TRL uptake compared to Pla2g1b+/+ control animals (119). The current study adds to these results by
Page 65 demonstrating that the protective effects of Pla2g1b inhibition are not dependent on LDLR- mediated uptake. Though other receptors (e.g. LDLR related protein 1 and VLDL receptor) can remove apoB-containing lipoproteins from the plasma (104), the presence of dietary cholesterol, hepatomegaly (though reduced), and hepatic steatosis suggest the livers of Pla2g1b-/-/Ldlr-/- mice were nutrient-congested rather than nutrient-depleted and, therefore, less likely to induce these uptake pathways. Thus inhibition of Pla2g1b-mediated LPC absorption and VLDL-TG production are sufficient to alter fasting plasma lipid levels even in extreme cases of hyperlipidemia.
In the presence of decreased VLDL production and reduced plasma LDL-C, it is surprising that inhibition of Pla2g1b did not result in decreased atherosclerosis in LDLR- deficient mice. At least two possibilities may explain this finding. First, the Ldlr-/- genetic background may be too severe to display protection from atherosclerosis with Pla2g1b inhibition.
This may be due to fasting LDL-C levels that were high enough to initiate atherosclerosis (750 mg/dl) even in Pla2g1b-/-/Ldlr-/- mice. This conclusion is consistent with the observation that even in Pla2g1b-/-/Ldlr-/- mice, plasma LDL-C was increased after chronic exposure to Western diet compared to Pla2g1b-/-/Ldlr-/- mice maintained on basal chow diet. In contrast, in Pla2g1b-/-
/Ldlr+/+ mice exposed to chronic hypercaloric diet, plasma cholesterol levels remained similar to
Pla2g1b-/-/Ldlr+/+ mice and Pla2g1b+/+/Ldlr+/+ mice maintained on chow diet. Alternatively, postprandial clearance in Ldlr-/- animals may be inhibited such that plasma lipid levels remain elevated due to uninhibited chylomicron production since mice eat multiple times per day and thus stay within atherogenic range in Pla2g1b-/-/Ldlr-/- mice. This conclusion is consistent with the observation that non-fasting triglyceride and cholesterol levels were similar between
Pla2g1b-/-/Ldlr+/+ mice and Pla2g1b+/+/Ldlr+/+ mice. Prior studies have shown that postprandial
Page 66
TRL, such as chylomicrons, confer atherosclerosis risk (31). Further studies have shown that, in the postprandial state, inflammatory signals and cells are increased when compared to the fasting state. It is also possible that the observed effects may be due to both a maximum response and a postprandial hyperlipidemia effect since postprandial cholesterol levels correlated with severity of atherosclerosis in Pla2g1b-/-/Ldlr-/- mice. It was also observed that the correlated fed state lipid levels in Pla2g1b+/+/Ldlr+/+ mice (n=5) had a decreased range compared to the Pla2g1b-/-/Ldlr-/- mice (n=7).
Inhibition of Pla2g1b in Ldlr-/- mice confirmed many of the results seen in Ldlr+/+ mice, showing that many protective effects are not dependent upon postprandial lipid (62, 99).
Pla2g1b-/-/Ldlr+/+ mice have decreased diet-induced obesity and hyperlipidemia and also have improved plasma glucose and insulin levels after challenge with two high-fat diets. Though experimental mice developed atherosclerotic lesions, in humans the presence of obesity and type-2 diabetes confers increased risk of cardiovascular accidents to hyperlipidemic patients (13).
Furthermore, many post-myocardial infarction (MI) treatments (e.g aspirin, anti-platelet therapy) decrease risk of recurrent MI without directly affecting atherosclerotic plaque size. Thus developing PLA2 inhibitors for the anti-obesity, anti-diabetic, and anti-hyperlipidemic effects may still provide useful for decreasing risk of cardiovascular events in humans (126).
Furthermore, the current data suggest that applications may exist for such disorders as familial hypercholesterolemia, in which LDLR is deficient (21).
4.5. Clinical considerations for inhibition of Pla2g1b
Effects of hypercaloric diets on lipid metabolism
Page 67
When high fat diets are consumed in excess of body energy requirements, the excess fat is stored in adipocytes, leading to obesity and inflammation, which promote insulin resistance (2,
65, 66). Lipotoxicity of pancreatic islet cells due to excess fat uptake causes decreased insulin production (2). The skeletal muscle, which normally play a large role in insulin-mediated glucose uptake, are also negatively affected by high fat intake as increased plasma fatty acid levels leads to insulin resistance in these tissues as well. Insulin resistance results in hyperglycemia, leading to endothelial cell dysfunction, and increased expression of adhesion molecules, which are promote atherosclerosis (131, 132). Hepatic insulin resistance and increased plasma fatty acid levels result in improperly increased postprandial VLDL production and excess hepatic lipid storage (18, 133) (134). Excess fat also leads to the presence of small dense LDL in the plasma as LDLR is downregulated and LDL remain in the plasma longer and are metabolized by LPL (13, 21). The consumption of large amounts of saturated fat raises plasma HDL levels due to compensatory increase in reverse cholesterol transport (RCT), however, increased plasma LDL-C is also a result (135, 136).
Dietary cholesterol can also affect plasma lipoprotein metabolism and homeostasis. With ingestion, the cholesterol pool of the liver is increased, leading to decreased expression of hepatic LDLR and increased plasma LDL-C levels (20, 21). Dietary cholesterol promotes a compensatory increase RCT, which is demonstrated by increased cholesterol excretion via bile into the feces and is dependent on Abcg5/g8 expression and did not occur in Abcg5-/-/Abcg8-/- mice (135). Though there is an increase in plasma HDL-C with cholesterol ingestion, addition of cholesterol to a chow diet caused increased hepatic triglyceride storage, in addition to increased cholesterol ester storage in wild type mice without altering VLDL production rate (135, 137).
Page 68
Excessive carbohydrate intake in the diet also affects hepatic function and lipoprotein metabolism (136). In vivo, glucose stimulates insulin release, which was shown to decrease plasma apoB production (138). However, with obesity related insulin resistance, the inhibitory effect of insulin on apoB production is muted and VLDL production remains elevated in the postprandial state (18). Along with chylomicrons, this leads to excessively elevated TRL in the postprandial state. In addition to glucose, increased fructose intake due to sweeteners in processed foods and non-juice drinks has been associated with increased risk of NAFLD in humans (134). Though in vitro studies showed that glucose did not directly stimulate hepatic triglyceride production enough to stimulate apoB secretion, fructose overdose has been used with rodents to model hepatic steatosis (139) (134).
Despite the problems associated with intake of excessive fat, cholesterol, and carbohydrate, findings from this study indicate that inhibition of Pla2g1b activity decreased the severity of obesity and obesity-related disease in a variety of experimental settings that included increased intake of each of these nutrients. In the presence of increased fat and carbohydrate consumption, Pla2g1b-/- mice exhibited decreased obesity, decreased plasma lipemia, and decreased VLDL production when compared to Pla2g1b+/+ mice. These effects were seen with subacute and chronic feeding of experimental diet. When LDLR-mediated clearance was inhibited, the protective effect of inhibition of Pla2g1b remained. Pla2g1b-/-/Ldlr-/- mice showed decreased obesity, glucose, insulin, VLDL-TG, and LDL-C when compared to Pla2g1b+/+/Ldlr-/- animals on similar high-fat, sucrose-containing diet. Moreover, when cholesterol was added to a high-fat and hypercaloric diet, the protective effects still remained. Pla2g1b-/-/Ldlr-/- mice
Page 69 showed decreased obesity, glucose, insulin, VLDL-TG, and LDL-C when compared to
Pla2g1b+/+/Ldlr-/- animals. Furthermore, findings indicated that both Pla2g1b-deficient models, when maintained on basal chow, displayed no defects in metabolic activity and were similar to
Pla2g1b-competent controls in body weight and HDL-C levels. Taken together, these findings suggest that Pla2g1b may be a safe target for pharmacological inhibition since that the protective effects appeared only when needed in the context of the consumption of excessive fat, cholesterol, and/or carbohydrate.
Role of LPC in obesity-related disease
In studies with hepatoma lines, incubation with LPC resulted in increased triglyceride secretion and VLDL production. Radiolabel from LPC was found in secreted triglyceride and PC and catabolism of apoB was decreased (88). Adenovirus-mediated knockdown of Lpcat3 was shown to increase intracellular LPC and promoted microsomal triglyceride transfer protein
(Mttp) expression sufficiently to stimulate VLDL secretion (140). In vivo, intake of hypercaloric diet elevated fasting and postprandial plasma LPC levels in wild type, but not Pla2g1b-/- mice
(91). Increased LPC content in LDL decreased its binding to LDLR (141). Findings from this dissertation confirm these previous data and expand upon them in vivo. Pla2g1b-/- mice had increased TRL clearance compared to Pla2g1b+/+ animals. LPC was also found to directly stimulate VLDL production and decrease hepatic fatty acid oxidation. These effects were mitigated by inhibition of the Pla2g1b in the gut. The observation that the effect of LPC on
VLDL levels remained even in Pla2g1b+/+ and Ldlr-/- animals encourages the idea of a pharmacological approach to inhibit LPC absorption since its mechanism of action remained stable even in a variety of genetic conditions. Together, these findings illuminate a previously
Page 70 unappreciated, but modifiable, connection between gut lysophospholipid absorption and dyslipidemia.
Atherosclerosis develops due to a complex mixture of endothelial dysfunction and vascular inflammation exacerbated by oxidized LDL (oxLDL) (26, 142). In addition to increased cholesterol from oxLDL, LPC levels are also increased in the atherosclerotic lesion since lesion- associated Lp-PLA2 (Pla2g7a) can generate LPC from LDL that enters the intima (68, 97). LPC is a major component of oxLDL and has been shown to independently cause many of the effects of oxLDL in vitro, such as promote chemotaxis of mononuclear cells, promote a calcified phenotype in vascular smooth muscle cells (VSMC), increase adhesion molecule (e.g. VCAM,
ICAM) expression in endothelial cells, and promote cytokine (e.g MCP-1) production (27)(26,
27, 31, 143). Changes in LPC concentration as small as 10 nM have been shown to cause
VSMCs to have a more osteogenic phenotype (143). Though Pla2g1b-/- mice had decreased plasma LPC (91), findings from this dissertation suggest that, in the presence of defective
LDLR-mediated clearance, the effect of LPC on plasma cholesterol and triglyceride levels was more important than its effects on endothelial and VSMCs in contributing to the development of atherosclerosis. The possibility exists that the primary contribution of LPC to the progression of atherosclerosis varies with disease severity, since increased plasma LPC concentration was seen in patients with coronary atherosclerotic disease despite being only slightly dyslipidemic (144).
In this model, absorbed LPC could increase VLDL production, and then along with the secondarily increased LDL stimulate vascular dysfunction. If so, then pharmacologic inhibition of gut Pla2g1b could potentially provide a double protection by resulting in decreased hepatic
Page 71
VLDL production and subsequent LDL buildup in addition to resulting in decreased plasma LPC levels.
Prior studies showed that rats which developed NAFLD in response to high fat diet had increased hepatic LPC content compared to controls, and metformin treatment decreased adiposity and LPC content (145). LPC has been shown to function as an effector molecule of muscle insulin resistance, and hepatic insulin resistance is present in NAFLD (18, 146). LPC has also been shown to be an effector molecule of palmitate-induced lipoapoptosis of hepatocytes
(89). When NAFLD progressed to NASH, ballooned human hepatocytes were shown to exhibit decreased expression of caspase 9 (147). Huh-7 cells deficient in caspase 9 were resistant to
LPC-induced lipoapoptosis, but LPC caused increased phosphorylation of N-terminal c-jun kinase (JNK) and increased survival through increased sonic hedgehog expression (147).
Conversely, conditions of increased oxidation can also cause phosphorylation of JNK, and mice with genetically increased phosphorylation of JNK were protected against NAFLD (148).
Impaired mitochondrial beta-oxidation has been shown to lead to fatty acid excess and storage as liver triglycerides (149). Peroxisomal oxidation of surplus lipids can generate free radicals which can cause further hepatocyte damage (150). Together with previous studies, findings from this dissertation indicate that plasma LPC levels also play a role in regulating postprandial mitochondrial oxidation. Though histological differences were not appreciated between Pla2g1b-
/-/Ldlr-/-and Pla2g1b+/+/Ldlr-/- mice, reduced diet-induced hepatomegaly and reduced body weight were observed in the Pla2g1b-/-/Ldlr-/- mice and Pla2g1b-/-/Ldlr+/+ mice. Since obesity confers an increased risk of NAFLD, it thus remains possible that the beneficial effects of
Page 72 pharmacologic inhibition of Pla2g1b (e.g. decreased obesity, increased glucose tolerance) seen in mice could remain in humans and subsequently decrease the risk of NAFLD (9, 34, 126).
Taken together, the findings from this dissertation have three implications in the field of metabolic research. First, these studies indicate a vital role for Pla2g1b and gut absorption of
LPC in modulating hepatic function. In both Pla2g1b+/+ and Pla2g1b-/- animals, reduced LPC concentrations were associated with increased mitochondrial function and decreased VLDL production. Secondly, these studies confirm that inhibition of Pla2g1b provides protection against diet-induced obesity in a variety of murine models. In the presence and absence of normal postprandial lipoprotein clearance and the feeding of cholesterol-containing high-fat diet, inhibition of Pla2g1b conferred protection against obesity. Thirdly, decreased VLDL-TG was shown to be a hallmark of Pla2g1b deficiency in both a mild and a severe murine model of hyperlipidemia. Though additional interventions may be necessary to fully prevent atherosclerosis and NAFLD, the development of a gut-impermeable drug similar to methyl indoxam, which inhibits Pla2g1b, would be expected to decrease VLDL production and lead to reduced risk of these obesity-related diseases in humans (Fig. 25). These beneficial effects would be in addition to the proven improvements in obesity, diabetes, and hyperlipidemia and without the side effects of combination therapy to address each of these metabolic disorders separately.
Page 73
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Figures
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RMU
Figure 1. Schematic of lipid metabolism through the gut-circulation-liver-adipose axis. The movement of lipid molecules is symbolized by the black arrows. Dietary fats such as triglyceride
(TG), cholesterol ester (CE), and phospholipid (PL) are digested to free fatty acid (FFA), cholesterol (Ch), lysophosphatidylcholine (LPC), and monoacylglycerides (MAG) and are absorbed and secreted in chylomicrons (CM). CM are catabolized by lipoprotein lipase (LPL) to become CM remnants (CMR), which move by receptor mediated uptake (RMU) to the liver. The liver uses lipid for fatty acid oxidation (FAO), storage, or packaging into very low density lipoprotein (VLDL). VLDL are catabolized by LPL to become low density lipoprotein (LDL), and are again transported to the liver by RMU. Excess FFA in the circulation may be stored in adipose tissue and released for further metabolic needs of peripheral tissues or the liver.
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Figure 2. Responses of Pla2g1b+/+ and Pla2g1b-/- mice to hypercaloric diet feeding. The
Pla2g1b+/+ (filled bars) and Pla2g1b-/- (open bars) mice were placed on hypercaloric diets for the time indicated. Body weights (A), fasting plasma triglyceride (B), and cholesterol (C) levels were measured. Results shown are mean ± SE from 6 Pla2g1b+/+ and 5 Pla2g1b-/- mice (for 0 and 3 week time points) and 7 mice in each group at the 10 week time point. * P ≤0.05, ** P ≤
0.01, *** P ≤ 0.001 versus Pla2g1b+/+ mice; and # P ≤ 0.05, ## P ≤ 0.01, and ### P ≤ 0.001 versus week 0 data of the respective genotype.
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Figure 3. Hepatic VLDL production by Pla2g1b+/+ and Pla2g1b-/- mice. The Pla2g1b+/+ (filled symbols; WT) and Pla2g1b-/- (open symbols; KO) mice were fed basal chow diet (panel A, from five Pla2g1b+/+ and three Pla2g1b-/- mice) or a hypercaloric diet for two (panel B, from nine
Pla2g1b+/+ and eight Pla2g1b-/- mice) or seven weeks (panel C from six Pla2g1b+/+ and seven
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Pla2g1b-/- mice). The mice were fasted for 12 h, and then received an intraperitoneal injection of
Poloxamer 407 (1 g/kg body weight). Plasma samples were obtained at hourly intervals for triglyceride measurements. The left panels show time-dependent increases in plasma triglyceride levels, and the right panels show AUC analyses of the respective data. Results represent mean ±
SE, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 versus Pla2g1b+/+ mice.
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Figure 4. Effect of LPC supplementation on hepatic VLDL production by Pla2g1b−/− mice.
The Pla2g1b-/- mice (open symbols) were placed on hypercaloric diet for two weeks. (A) Fasting plasma triglyceride levels were measured after 11 h fasting (t = −1 h), and then LPC was injected intraperitoneally (32 mg/kg body weight). Poloxamer 407 was injected 1 h later at the 0 h time point, and plasma samples were obtained at hourly intervals thereafter to measure plasma triglyceride levels. (B) VLDL production rates of Pla2g1b+/+ (filled bar) and Pla2g1b-/- mice without (open bar) or with LPC injection (hatched bar). (C) Plasma triglyceride levels in
Pla2g1b+/+ (WT) and Pla2g1b-/- (KO) mice with or without LPC injection measured before
(filled bars) and 4 h after injection of Poloxamer 407 to inhibit lipolysis (open bars). Results represent mean ± SE from at least three mice in each group. Data were analyzed in comparison with Pla2g1b+/+ mice as the control group by Student t-test *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001. ns, no significant difference.
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Figure 5. FPLC analysis of 3H radioactivity distribution in plasma subsequent to [3H]LPC injection. The Pla2g1b+/+ (solid symbols, n = 3) and Pla2g1b-/- (open symbols, n = 4) mice were placed on hypercaloric diet for two weeks. LPC was injected intraperitoneally (32 mg/kg body weight) after 11 h fasting (t = −1 h). Poloxamer 407 was injected 1 h later at the 0 h time point and plasma samples were obtained 4 h later (t = 4 h). Plasma was separated by FPLC, and radiolabel in each fraction was counted by liquid scintillation. Fractions where VLDL, LDL,
HDL, and albumin were eluted from the column were identified based on comparison with standards as marked. A representative profile is shown.
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Figure 6. Postprandial plasma lipid response to a bolus lipid-rich meal. The Pla2g1b+/+ (solid symbols) and Pla2g1b-/-(open symbols) mice were placed on a hypercaloric diet for three weeks.
Fasting animals were fed 400 µl of olive oil by intragastric gavage. Plasma triglyceride levels were measured at hourly intervals as indicated. The inset shows AUC calculations. Results represent mean ± SE from six Pla2g1b+/+ and ten Pla2g1b−/− mice. *P ≤ 0.05, **P ≤ 0.01 versus
Pla2g1b+/+ mice.
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120
100 Without CsA With CsA 80
60
40 (% of control) of (%
20 Absorbancenm530 at 0 0 40 80 120 160 200 LPC (μM)
Figure 7. Effect of LPC on mitochondrial swelling. Mitochondria (0.7 mg protein/ml) were incubated with 5 mM glutamate and 5 mM malate as respiratory substrates and CsA where indicated. Mitochondrial swelling was assessed by measuring absorbance at 530 nm 1 min after exposure to LPC. The absorbance of mitochondria incubated with respiratory substrates and SDS is signified by the dotted line. Error bars signify standard error from 3 determinations.
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120 120 B 100 A 100
80 80 *
60 * 60 (% of control) of (%
40 control) of (% 40
Absorbancenm530 at Absorbancenm530 at 20 20
0 0 0 40 80 120 160 200 0 40 80 120 160 200 LPC (μM) LPC (μM)
Figure 8. Effect of LPC on Ca2+-induced mitochondrial swelling. Mitochondria (0.7 mg protein/ml) were incubated with 5 mM glutamate and 5 mM malate respiratory substrates and
LPC with or without 2 μM cyclosporin A (CsA) for 2 min prior to the addition of 220 μM CaCl2
(A) or 75 μM CaCl2 (B). Mitochondrial swelling was assessed after 10 min incubation based on absorbance at 530 nm. Open circles and dashed lines indicate incubation of mitochondria in the absence of CaCl2 and CsA (n=2-3 determinations). Filled circles and solid lines indicate incubation of mitochondria with CaCl2 (n=2-3 determinations). Filled diamonds and gray lines indicate incubation of mitochondria with CaCl2 and CsA (n=3 determinations). *p<0.05 vs. mitochondria incubated with similar LPC concentration, CaCl2, and CsA by Student’s t test.
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50 120 Add CaCl2 A B 0 μM 100 40 * 40 μM ** 80 μM SDS 80 ** *** *** 120 μM 30 160 μM 60 200 μM 20
(% of control) of (% 40
(arbitrary units) (arbitrary Fluorescenceintensity Fluorescence intensity Fluorescenceintensity 10 20
0 0 0 120 240 360 480 600 720 840 0 40 80 120 160 200 Time (s) LPC (μM)
Figure 9. Effect of LPC on mitochondrial Ca2+ uptake. Mitochondria (0.7 mg protein/ml) were incubated with 1 μM Calcium Green 5N and increasing concentrations of LPC or SDS, where indicated. Fluorescence intensity (FI) was measured at 538 nm after excitation at 485 nm prior to and after addition of 75 μM CaCl2. Shown is representative graph from 3 determinations (A).
Peak FI occurred within 35 s of addition of 75 μM CaCl2 and was measured and plotted against
LPC concentration (n=3 determinations) *p<0.05, **p<0.01, ***p<0.001 vs. control by
Student’s t test (B).
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120 5 Add succinate B A 80 μM 100 *** 0 SDS *** 0 μM *** 40 μM 80 *** *** -5
60 -10 160 μM 200 μM (% of control) of (% 40
-15 120 μM Absorbancenm530 at
20 Absorbancenm530 at (% change from baseline) change from (%
-20 0 0 40 80 120 160 200 0 5 10 15 20 LPC (μM) Time (min)
Figure 10. Effect of LPC on mitochondrial membrane potential. Mitochondria (0.7 mg protein/ml) were incubated with 25 μM safranin O and LPC (n=4 determinations). Mitochondrial membrane potential was assessed after 2 min incubation based on absorbance at 530 nm. The absorbance of mitochondria incubated with SDS is indicated by the dotted line (A). After incubation in LPC for 4 min, 5 mM succinate respiratory substrate was added and absorbance measurements at 530 nm were taken every 30 s (B). ***p<0.01 vs. control by Student’s t tests.
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200 200 200 State 3 State 3 150 A 150 B State 4 150 C State 4 State 3 * 100 State 4 100 ** 100 * 50
50 rate consumption
consumption rate rate consumption 50
consumption rate rate consumption
2
2
2
O
O
O
(nmol O/min/mg protein) O/min/mg (nmol
(nmol O/min/mg protein) O/min/mg (nmol (nmol O/min/mg protein) O/min/mg (nmol 0 0 0 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 LPC (μM) LPC (μM) LPC (μM)
5 5 5 RCI RCI RCI D 4 E ADP/O ratio 4 F ADP/O ratio 4 ADP/O ratio
3 3 3
Ratio Ratio Ratio 2 ** 2 2 ** * 1 1 * ** 1 ** ** 0 0 0 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 LPC (μM) LPC (μM) LPC (μM)
Figure 11. Effect of LPC on mitochondrial respiration. Mitochondria (1 mg protein/ml) were incubated with 5 μM succinate (A, D), 5 μM glutamate/5 μM malate (B, E), or 10 μM palmitoyl- carnitine/1 μM malate (C, F) respiratory substrates (n=4-5 determinations per concentration of
LPC). Oxygen concentration in the media was measured prior to and after addition of 441 nmol
ADP. State 3 respiration rates were determined by slope of oxygen levels over time subsequent to addition of ADP while state 4 respiration rates were determined by the slope of oxygen levels after state 3 respiration was completed (A-C). Respiratory control index (RCI) and phosphorylation efficiency (ADP/O ratio) were also calculated (D-F). *p<0.05, **p<0.01,
***p<0.001 vs. 0 μM LPC by Student’s t tests.
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300 0 μM D 250 40 μM C 100 μM 200 200 μM
150 A B
100
(% of baseline) of (%
consumptionrate 2
O 50
0 0 50 100 150 Time (min)
Figure 12. Effect of LPC on fatty acid-stimulated oxidation in isolated hepatocytes. Primary hepatocytes were isolated and incubated with DMEM. Oxygen consumption rate was measured with an XF24 Analyzer. After baseline measurements, 100 μM oleate complexed to BSA at a 5:1 ratio and LPC were added at the time indicated “A”. Subsequently, 1 μM oligomycin, 400 nM
FCCP, and 1 μM rotenone were added at times indicated “B,” “C,” and “D,” respectively. Shown is a representative graph from 3 experiments. Error bars signify standard error from 5 wells.
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Baseline 10 min
40 μM LPC μM 40 100 μM LPC μM 100
Figure 13. Effect of LPC on hepatocyte mitochondrial permeability. Primary hepatocytes were
o isolated and incubated with 2 μM calcein-AM and 8 mM CoCl2 at 37 C for 30 min in the dark.
Cells were washed and imaged using Image-Pro Plus on an Olympus IX71 microscope with a
RETIGA EXi camera after excitation at 485 nm and emission at 538 nm at baseline and 10 min after incubation with 100 μM oleate complexed to BSA at a 5:1 ratio and LPC. Mitochondria have a bright punctate appearance.
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120
100 * * 80 ** 60
40 (% of control) of (%
20 Fluorescenceintensity 0 0 40 100 200 LPC (μM)
Figure 14. Effect of LPC on hepatocyte cytoplasmic activity. Primary hepatocytes were isolated and incubated with 100 μM oleate complexed to BSA at a 5:1 ratio and LPC for 10 min, washed, and then incubated with 2 μM calcein-AM at 37oC for 30 min in the dark. Cytoplasmic activity was assessed by measurement of fluorescence intensity after excitation at 485 nm and emission at 538. *p<0.05, **p<0.01 vs. control by Student’s t tests. Error bars signify standard error from
4 experiments.
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B A C 50 50 LDLr-/- 50 LDLr-/- LDLr-/- DKO DKO DKO 40 40 * 40 30 30 30 ***
20 20 20
Body weight Body (g)
Body weight Body (g) weight Body (g) 10 10 10 0 0 0
Figure 15. Effects of diet on body weight of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice. Ten- week old Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- (DKO) mice were randomly assigned to basal chow (A), Western (B), or Diabetogenic (C) diet for 10 weeks and body weight was measured
(n=4-12 per group). *p<0.05 vs. Pla2g1b+/+/Ldlr-/- mice of similar diet by Student’s t test.
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A B C 500 500 500 LDLr-/- LDLr-/- LDLr-/- DKO DKO 400 DKO 400 400
300 300 * 300 * 200 * 200 200
100 100 100
Plasma glucose (mg/dl) Plasma glucose (mg/dl) Plasma glucose (mg/dl) 0 0 0 D E F
2.5 LDLr-/- 2.5 LDLr-/- 2.5 LDLr-/- 2.0 DKO 2.0 DKO 2.0 DKO
1.5 1.5 1.5
p=0.51 *** 1.0 1.0 1.0 ***
0.5 0.5 0.5
Plasma insulin (ng/ml) Plasma insulin (ng/ml) Plasma insulin (ng/ml) 0.0 0.0 0.0 G H I
10 LDLr-/- 40 LDLr-/- 60 LDLr-/- 8 DKO DKO 50 DKO 30 p=0.45 40 6 20 30 4 ***
20 ***
HOMA-IR (units) HOMA-IR (units) HOMA-IR HOMA-IR (units) HOMA-IR 10 2 10 0 0 0
Figure 16. Effects of diet on glucose homeostasis of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice. Ten-week old Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- (DKO) mice were randomly assigned to basal chow (A, D, G), Western (B, E, H), or Diabetogenic (C, F, I) diet for 10 weeks and plasma was isolated. Glucose (A-C), insulin (D-F), and HOMA-IR (G-I) were measured
(n=4-5 per group). *p<0.05, ***p<0.001 vs. Pla2g1b+/+/Ldlr-/- mice of similar diet by Student’s t test.
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A
25 25 VLDL LDL HDL LDLr-/- VLDL LDL HDL LDLr-/- DKO DKO 20 20
15 15
10 10 Cholesterol (μg) Cholesterol Triglyceride (μg) Triglyceride 5 5
0 0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 Fraction Fraction B
160 VLDL LDL HDL 250 LDLr-/- VLDL LDL HDL LDLr-/- 140 DKO 200 DKO 120
100 150 80
60 100 Cholesterol (μg) Cholesterol Triglyceride (μg) Triglyceride 40 50 20 0 0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 Fraction Fraction C
80 VLDL LDL HDL LDLr-/- 100 VLDL LDL HDL LDLr-/- DKO DKO 80 60
60 40
40 Cholesterol (μg) Cholesterol Triglyceride (μg) Triglyceride 20 20
0 0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 Fraction Fraction
Figure 17. Effects of diet on lipoprotein profile of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice.
Ten-week old Pla2g1b+/+/Ldlr-/- (n=5) and Pla2g1b-/-/Ldlr-/- (DKO, n=4) mice were randomly assigned to basal chow (A), Diabetogenic (B), or Western (C) diet for 10 weeks and plasma was isolated and lipoprotein fractions collected. Triglyceride and cholesterol levels in each 0.5 mL fraction were measured by commercial assay kit. Results show mean + standard deviation.
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A 300 1000 LDLr-/- LDLr-/- DKO 800 DKO 200 600 ** 400 *** 100
200
Plasma cholesterol (mg/dl) Plasma triglyceride (mg/dl) 0 0
B 600 2500 LDLr-/- LDLr-/- 500 DKO 2000 DKO 400 1500 300 ** 1000 *** 200
100 500
Plasma cholesterol (mg/dl) Plasma triglyceride (mg/dl) 0 0
Figure 18. Total plasma lipid levels in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice after high fat diet feeding. The Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- (DKO) mice were fed Diabetogenic
(A) or Western diet (B) for 10 weeks. Plasma was isolated and triglyceride and cholesterol levels were determined (n=4-5 per group). **p<0.01, ***p<0.001 vs. Pla2g1b+/+/Ldlr-/- mice of similar diet by Student’s t test.
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A B 2.5 2.5 LDLr-/- LDLr-/- DKO 2.0 2.0 DKO * 1.5 1.5
1.0 1.0
Liver Liver weight (g) Liver Liver weight (g) 0.5 0.5
0.0 0.0
Figure 19. Effect of diet on liver weight in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice. Ten- week old Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice were randomly assigned to basal chow
(A) or Western diet (B) for 10 weeks and liver weight was measured (n=7-12 per group).*p<0.05 vs. Pla2g1b+/+/Ldlr-/- mice of similar diet by Student’s t test.
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LDLR-/- DKO
LDLR-/- DKO
Figure 20. Liver histology in high fat fed Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice.
Ten-week old Pla2g1b+/+/Ldlr-/- (LDLR-/-) and Pla2g1b-/-/Ldlr-/- (DKO) mice were fed Western diet for 10 weeks. Formalin-fixed paraffin-embedded sections (5 µm) were prepared from liver tissues harvested from these animals. Preliminary comparisons were made using a 2-point scale noting the presence or absence of lipid accumulation in 2 random low power fields from sections taken 100-200 µm deep to the superior capsular surface of the median hepatic lobe. Sections stained with Masson’s trichrome revealed no fibrosis. Representative sections stained with hematoxylin and eosin are shown (n=8-10 animals per group).
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LDLR-/- DKO
A 35 B 300000 30 LDLR-/- LDLR-/- p = 0.8 DKO DKO 25 200000 20
15 100000
(% of total area) total of (% 10
Aortic arch lesion archAorticlesion Total area (pixels) Total area 5
0 0 n=5 n=7 n=5 n=7
Figure 21. Oil Red O positive staining in aortic arch of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice. After 10 weeks of Western diet feeding, the aorta from the superior border of the heart of
Pla2g1b+/+/Ldlr-/- (n=5) and Pla2g1b-/-/Ldlr-/- (DKO, n=7) mice was isolated, carefully cut longitudinally, pinned open to wax, and stained with Oil Red O. To determine area of the aortic arch for determination of atherosclerosis, the ascending aorta was cut as near as possible to the superior border of the heart. A tangent line was drawn from the proximal edge on the inferior side to the superior side of the ascending aorta to form the proximal boundary. The distal boundary was delineated by a tangent line from the distal edge of the distal innominate (left subclavian) artery. For the superior boundary, a line excluding the lumens of the innominate arteries was drawn. Representative images from Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice are
Page 109 shown. Quantification of lesion area (A) and total aortic arch area (B) are shown. The areas from both halves of aortic arch were added to produce each data point. Student’s t test was used for comparisons.
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LDLR-/- DKO
A B
400000 1000000 LDLR-/- DKO LDLR-/- 800000 300000 DKO 600000 200000 400000 100000
Lumen area Lumen(pixels) area 200000 Aortic root lesionroot Aortic (pixels) 0 0 C 50 LDLR-/- DKO 40
30
20 Aortic root lesionAortic (% of lumen area) 10
0 0 1 2 3 4 5 6 7 8 Section
Figure 22. Oil Red O positive staining in aortic root of Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice. After 10 weeks of Western diet feeding, the cardiac tissues from Pla2g1b+/+/Ldlr-/-
(LDLR-/-, n=5) and Pla2g1b-/-/Ldlr-/- (DKO, n=7) mice were fixed with 10% formalin followed by 30% sucrose and embedded in Optimal Cutting Temperature media prior to obtaining frozen sections. The aortic root was obtained from 10 µm serial sections beginning at the level of the
Page 111 aortic valve and ending 800 µm proximally at a level distal to the left ventricle. Sections were stained with Oil Red O. Images were then acquired with an Olympus BX61 microscope fitted with a RETIGA 2000R camera (QImaging) and images were analyzed with ImageJ.
Atherosclerosis was assessed by measuring Oil Red O positive area. The total area was determined by outlining the elastic lamina and excluding valvular tissues. Using this approach, similar values for lumen area were obtained from Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice
(p=0.4 by Student’s t test). Representative images from Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice are shown. Quantification of total lesion area (A), total lumen area (B), and progressive lesion area as a percentage of lumen area (C) are shown. Student’s t test was used for comparisons.
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A B 1500 LDLr-/- 2500 LDLr-/- DKO DKO p = 0.09 2000 p = 0.4 1000 1500
1000 500
500
Plasma cholesterol (mg/dl) Plasma triglyceride (mg/dl) 0 0
Figure 23. Fed state plasma lipid levels in Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice after
Western diet feeding. The Pla2g1b+/+/Ldlr-/- (LDLR-/-) and Pla2g1b-/-/Ldlr-/- (DKO) mice were fed Western diet for 10 weeks. Plasma was isolated and triglyceride (A) and cholesterol (B) levels were determined (n=8-10 per group). Student’s t test was used for comparisons.
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A B C 35 120 400000 LDLR-/- LDLR-/- LDLR-/- DKO 30 DKO 100 R2 = 0.08 DKO 300000 25 R2 = 0.14 80 2 20 R = 0.0017 60 200000 15 40 10 2
R2 = 0.67 R = 0.008 100000 (% of lumen area) Aortic arch lesionarch Aortic 20 2
5 (% of circumference)
Aorticroot lesion (pixels) R = 0.367 Brachiocephalic lesion Brachiocephalic 0 0 0 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 Fed state plasma cholesterol Fed state plasma cholesterol Fed state plasma cholesterol (mg/dl) (mg/dl) (mg/dl)
Figure 24. Correlation between measures of atherosclerosis and postprandial cholesterol in
Pla2g1b+/+/Ldlr-/- and Pla2g1b-/-/Ldlr-/- mice. Aortic arch lesion (A), brachiocephalic lesion (B), and aortic root lesion (C) were plotted against fed state plasma cholesterol levels of
Pla2g1b+/+/Ldlr-/- (LDLR-/-) and Pla2g1b-/-/Ldlr-/- (DKO) mice (n=5-8 per group). Best fit trend lines were the constructed and R2 values calculated with Microsoft Excel.
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Figure 25. Schematic of the effects of LPC and inhibition of Pla2g1b on lipid metabolism through the gut-circulation-liver-adipose axis. LPC generated by Pla2g1b activity in the gut leads to LPC-mediated increase in VLDL production and inhibition of hepatic FAO (A). In the presence of inhibition of Pla2g1b and hypercaloric diet, gut absorption of LPC is decreased. This results in decreased VLDL production and consequently decreased plasma VLDL and LDL
Page 115 levels. Inhibition of Pla2g1b also leads to increased RMU and FAO and decreased fat mass (B).
These benefits are in addition to the benefits on glucose homeostasis.
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