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

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 st orage and metabolism of retinoids in the body has immediate implications for the overall health and metabolic homeostasis of the animal. This thesis research focus ed 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 s tored in the liver, and, of this fraction, 80-90% is stored in the hepatic stellate cell (HSC) li pid droplets as retinyl ester. These lipid droplets are a distinguishing feature of the HSC, and they have recently bee n proposed to be specialized organelles for the storage of retinoid based on thei r unique retinoid content and responsiveness to dietary retinoid status. It is also known that the bility to synthesize and store retinyl ester in HSCs is necessary for the presence of HS C lipid droplets. Interestingly, it is well established that, with the progression of liver disease in human patie ts, there is a progressive loss of total hepatic retinoid content. As hepatic disease prog resses, the HSCs transition from a quiescent to an activated phenotype, accompanied by the loss of their l ipid droplet and retinoid content. The ultimate goal of this dissertation was to further el ucidate the factors that regulate HSC retinoid storage as retinyl esters in lipid droplets and to de fine the factors that regulate HSC lipid droplet genesis and dissolution. The first aim of this research was to investigate the heter ogeneity of HSCs and their lipid droplets in healthy, uninjured liver. Our observations suggest that the H SC population in a healthy, uninjured liver is heterogeneous. One subset of the total H SC population, which expresses early markers of HSC activation, may be primed and re dy 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 le vels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthe is (LRAT); (iii) decreased triglyceride levels; (iv) increased expression of genes associated w ith 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: specific ally, we studied HSC lipid droplets in the LRAT KO mouse model, a system where HSC lipid drop lets do not form. Our findings indicate that there are not global differences in retinoi d-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 p r found changes in retinoid metabolism. Our data also shows that the LRAT KO HSCs have signi ficant differences in expression of genes related to lipid metabolism; overall, lipid biosynt hesis 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. Importantl y, we show for the first time, to our knowledge, that the lipid droplet-associated proteins may be posttran criptionally regulated. A final aim of this research was to investigate HSC lipid dr oplet dissolution in HSC activation and hepatic fibrosis, systems where HSC lipid droplets form, but are s ubs quently lost. We employed two standard models of HSC activation, the in vivo model of carbon tetrachloride (CCl4) treatment and the in vitro model, the culture of purified HSCs on plastic cell culture 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 dietor chemically-induced hepati c fibrosis. We found that, while the culture of HSCs on plastic results in the typical hallma rk events of HSC activation, including the upregulation of Col1a1, the decrease in retinyl ster 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, sphingol ipids and ceramides. Our findings allow us to hypothesize that endocannabinoids a d sphingolipids may function in activated HSCs as mediators of apoptosis. Importantly, thi s 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.