Physiology and Pathophysiology of Retinoid and Lipid Storage in Mouse Hepatic Stellate Cell ...

245 Pages·2011·20.32 MB·English

Physiology and Pathophysiology of Retinoid and Lipid Storage
in Mouse Hepatic Stellate Cell Lipid Droplets

Diana N. D’Ambrosio

Submitted in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
under the Executive Committee
of the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY

2011
© 2011
Diana N. D’Ambrosio
All rights reserved
ABSTRACT
Physiology and Pathophysiology of Retinoid and Lipid Storage
in Mouse Hepatic Stellate Cell Lipid Droplets
Diana N. D’Ambrosio

Retinoids are important mediators of many physiological processes in the body, including
vision, reproduction, embryonic development, immunity and bone growth. Thus, the storage and
metabolism of retinoids in the body has immediate implications for the overall health and
metabolic homeostasis of the animal. This thesis research focused on two retinoid metabolites:
retinyl ester, the form in which retinoids are stored, and retinoic acid, the transcriptionally active
retinoid metabolite. Approximately 70% of retinoid in the body is stored in the liver, and, of this
fraction, 80-90% is stored in the hepatic stellate cell (HSC) lipid droplets as retinyl ester. These
lipid droplets are a distinguishing feature of the HSC, and they have recently been proposed to be
specialized organelles for the storage of retinoid based on their unique retinoid content and
responsiveness to dietary retinoid status. It is also known that the ability to synthesize and store
retinyl ester in HSCs is necessary for the presence of HSC lipid droplets. Interestingly, it is well
established that, with the progression of liver disease in human patients, there is a progressive
loss of total hepatic retinoid content. As hepatic disease progresses, the HSCs transition from a
quiescent to an activated phenotype, accompanied by the loss of their lipid droplet and retinoid
content. The ultimate goal of this dissertation was to further elucidate the factors that regulate
HSC retinoid storage as retinyl esters in lipid droplets and to define the factors that regulate HSC
lipid droplet genesis and dissolution.
The first aim of this research was to investigate the heterogeneity of HSCs and their lipid
droplets in healthy, uninjured liver. Our observations suggest that the HSC population in a
healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which
expresses early markers of HSC activation, may be primed and ready for rapid response to acute
liver injury. We show that these “pre-activated” HSCs have: (i) increased expression of typical
markers of HSC activation; (ii) decreased retinyl ester levels, accompanied by reduced
expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT); (iii) decreased
triglyceride levels; (iv) increased expression of genes associated with lipid catabolism; and (v) an
increase in expression of the retinoid-catabolizing cytochrome, CYP2S1
The second aim of this research was to investigate HSC lipid droplet formation and
maintenance in healthy, but genetically-modified liver: specifically, we studied HSC lipid
droplets in the LRAT KO mouse model, a system where HSC lipid droplets do not form. Our
findings indicate that there are not global differences in retinoid-related gene expression,
suggesting that the formation and maintenance of HSC lipid droplets is likely regulated entirely
by the synthesis and storage of retinyl ester and not by more profound changes in retinoid
metabolism. Our data also shows that the LRAT KO HSCs have significant differences in
expression of genes related to lipid metabolism; overall, lipid biosynthesis is down-regulated and
lipid catabolism is up-regulated in LRAT KO HSCs, which likely contributes to the complete
absence of lipid droplets in the HSCs of these animals. Importantly, we show for the first time,
to our knowledge, that the lipid droplet-associated proteins may be post-transcriptionally
regulated.
A final aim of this research was to investigate HSC lipid droplet dissolution in HSC
activation and hepatic fibrosis, systems where HSC lipid droplets form, but are subsequently lost.
We employed two standard models of HSC activation, the in vivo model of carbon tetrachloride
(CCl ) treatment and the in vitro model, the culture of purified HSCs on plastic cell culture
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dishes. Additionally, we studied the effects of hypervitaminosis A since there is evidence in the
literature that dietary vitamin A toxicity can cause hepatic fibrosis. Our studies suggest that,
despite being unable to synthesize and store retinyl ester in lipid droplets, LRAT KO mice are
not more susceptible than WT to the development of diet- or chemically-induced hepatic fibrosis.
We found that, while the culture of HSCs on plastic results in the typical hallmark events of HSC
activation, including the upregulation of Col1a1, the decrease in retinyl ester and the loss of lipid
droplets, it does not regulate gene expression as HSC activation does in vivo. Thus, all future
studies on HSC activation and its effects on retinoid storage should be conducted in vivo. We
also present preliminary data on the alterations in the lipidome of activated HSCs, specifically
with regard to the potent lipid signaling molecules, endocannabinoids, sphingolipids and
ceramides. Our findings allow us to hypothesize that endocannabinoids and sphingolipids may
function in activated HSCs as mediators of apoptosis. Importantly, this study demonstrates the
ability to detect these lipids in very small aliquots of in vivo-activated HSCs and provides a
strong foundation upon which all future studies may be built.
TABLE OF CONTENTS

Table of Contents………………………………………………………………………… i
List of Charts, Graphs, Illustrations……………………………………………………… v
Abbreviations…………………………………………………………………………….. ix
Acknowledgements……………………………………………………………………… xii
Chapter 1: Literature Review…………………………………………………………. 1
Adapted from: D’Ambrosio, D.N.; Clugston, R.D.; Blaner, W.S. Vitamin A Metabolism:
An Update. Nutrients 2011, 3, 63–103.
I. Introduction……………………………………………………………… 1
II. Metabolism of dietary retinoid within the gastrointestinal (GI) tract …… 2
A. Dietary forms and metabolism in the lumen of the intestine………… 2
B. Metabolism and processing within the intestinal mucosa…………… 4
1. Uptake into and efflux from the enterocyte…………………. 5
2. Enzymatic conversion of proretinoid carotenoid to retinoid… 6
3. Enterocyte esterification of retinol…………………………… 8
4. Cellular retinol-binding protein, type II (CRBPII)…………… 9
III. Chylomicrons and their metabolism in the circulation…………………… 10
IV. Hepatic retinoid metabolism……………………………………………… 11
A. Uptake and processing of chylomicron retinyl ester by the hepatocyte 12
1. Hepatic chylomicron remnant receptors……………………… 12
2. Retinyl ester hydrolysis in the hepatocyte…………………… 14
B. Transfer of retinol from hepatocytes to hepatic stellate cells (HSCs).. 17
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1. RBP-mediated transfer………………………………………. 17
2. CRBPI-mediated transfer......................................................... 18
C. Storage of retinoid in the HSC as retinyl ester in lipid droplets........... 19
1. The role of HSC lipid droplets in retinoid storage................... 19
2. Esterification of retinol in the HSC.......................................... 19
3. HSC lipid droplet content and effects of dietary retinoid status 21
4. HSC lipid droplets in hepatic disease………………………… 21
D. Mobilization of retinol from hepatic stores to peripheral tissues…….. 23
1. Hydrolysis of HSC lipid droplet retinyl ester………………… 23
2. Role of RBP in hepatic mobilization of retinol………………. 24
V. Uptake of retinoids by extrahepatic tissues………………………………. 25
A. Uptake of retinol-RBP from the blood by extrahepatic tissues………. 26
B. Extrahepatic uptake of chylomicron retinyl ester…………………….. 29
VI. HSCs and liver fibrogenesis……………………………………………… 33
A. ECM and cellular alterations in liver fibrogenesis…………………… 33
B. Stages of HSC activation…………………………………………….. 34
C. Role of cytokines and signaling pathways in HSC activation……….. 36
D. Regulation of gene transcription in activated HSCs…………………. 38
E. Interaction of activated HSCs with immune pathways………………. 39
F. Resolution of HSC activation and liver fibrogenesis………………… 40
VII. Tables and Figures………………………………………………………... 42
Chapter 2: Distinct Populations of Hepatic Stellate Cells in the Mouse Liver Have
Different Capacities for Retinoid and Lipid Storage…………………………………... 50
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Published as: D’Ambrosio, D.N.; Walewski, J.L.; Clugston, R.D.; Berk, P.D.; Rippe, R.A.;
Blaner, W.S. Distinct Populations of Hepatic Stellate Cells in the Mouse Liver Have Different
Capacities for Retinoid and Lipid Storage. PLoS ONE 2011, 6 (9): e24993.
doi:10.1371/journal.pone.0024993
I. Abstract……………………………………………………………………. 50
II. Introduction………………………………………………………………... 51
III. Materials and Methods………………………………………………….. 53
IV. Results …………………………………………………………………… 59
V. Discussion……………………………………………………………….. 65
VI. Tables and Figures………………………………………………………. 73
Chapter 3: A DNA Microarray Dissection of Key Genes and Pathways Involved in
Hepatic Stellate Cell Lipid Droplet Formation and Maintenance using the LRAT-null
Mouse Model……………………………………………………………………………. 99
I. Abstract…………………………………………………………………. 99
II. Introduction……………………………………………………………... 100
III. Materials and Methods………………………………………………….. 103
IV. Results …………………………………………………………………... 106
V. Discussion……………………………………………………………….. 110
VI. Tables and Figures………………………………………………………. 115
Chapter 4: Study of Hepatic Stellate Cell Lipid Droplet Loss in Hepatic Disease Using
Different Models of HSC Activation and Hepatic Fibrosis………………………….. 136
I. Abstract………………………………………………………………….. 136
II. Introduction……………………………………………………………… 137
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III. Materials and Methods………………………………………………….. 142
IV. Results …………………………………………………………………… 149
V. Discussion……………………………………………………………….. 154
VI. Tables and Figures………………………………………………………. 161
Chapter 5: Summary and Future Directions………………………………………… 181
I. Summary and Future Directions………………………………………… 181
II. Tables and Figures………………………………………………………. 194
References……………………………………………………………………………….. 199

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LIST OF CHARTS, GRAPHS, ILLUSTRATIONS

CHAPTER 1
Tables
Table 1-1: Retinoid-binding proteins in the adult mouse………………………………...... 42
Table 1-2: Proposed hepatic retinyl ester hydrolases (REHs)…………………………....... 43
Figures
Figure 1-1: Transcriptional regulation by the retinoid nuclear receptors………………….. 44
Figure 1-2: General scheme for the uptake and metabolism of dietary retinoids and
proretinoid carotenoids within the intestine………………………………………………... 45
Figure 1-3: Cleavage of beta-carotene…………………………………………………….. 47
Figure 1-4: Uptake of retinoids into extrahepatic tissues………………………………….. 48
Figure 1-5: Factors regulating hepatic stellate cell (HSC) activation in liver fibrogenesis... 49

CHAPTER 2
Tables
Table 2-1: ABI primers used for qRT-PCR analysis………………………………………. 73
Table 2-2: Summary of differentially expressed genes……………………………………. 74
Table 2-3: KEGG pathway enrichment……………………………………………………. 75
Table 2-4: Markers of HSC activation…………………………………………………….. 76
Table 2-5: Retinoid- and lipid-related nuclear receptors………………………………....... 77
Table 2-6: Retinol and fatty acid binding proteins………………………………………… 78
Table 2-7: Lipases………………………………………………………………………….. 79
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