Nuttaporn Wongsiriroj

Hepatic stellate cell lipid droplets: A specialized lipid droplet for retinoid storage

The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease.

The Molecular Basis of Retinoid Absorption A Genetic Dissection

The intestine and other tissues are able to synthesize retinyl esters in an acyl-CoA-dependent manner involving an acyl-CoA:retinol acyltransferase (ARAT). However, the molecular identity of this ARAT has not been established. Recent studies of lecithin:retinol acyltransferase (LRAT)-deficient mice indicate that LRAT is responsible for the preponderance of retinyl ester synthesis in the body, aside from in the intestine and adipose tissue. Our present studies, employing a number of mutant mouse models, identify diacylglycerol acyltransferase 1 (DGAT1) as an important intestinal ARAT in vivo. The contribution that DGAT1 makes to intestinal retinyl ester synthesis becomes greater when a large pharmacologic dose of retinol is administered by gavage to mice. Moreover, when large retinol doses are administered another intestinal enzyme(s) with ARAT activity becomes apparent. Surprisingly, although DGAT1 is expressed in adipose tissue, DGAT1 does not catalyze retinyl ester synthesis in adipose tissue in vivo. Our data also establish that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.

Absence of hepatic stellate cell retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis

Objective Hepatic stellate cells (HSCs) contain a number of bioactive metabolites or their precursors including retinoids in their characteristic lipid droplets. The loss of lipid droplets and retinoids is a hallmark of HSC activation, but it remains unclear whether this loss promotes HSC activation, liver fibrogenesis or carcinogenesis. Design Spontaneous and experimental fibrogenesis as well as a diethylnitrosamine-induced hepatocarcinogenesis were investigated in lecithin-retinol acyltransferase (LRAT)-deficient mice which lack retinoid-containing lipids droplets in their HSCs. Results Following HSC activation, LRAT expression was rapidly lost, emphasising its importance in lipid droplet biology in HSCs. Surprisingly, there was no difference in fibrosis induced by bile duct ligation (BDL) or by eight injections of carbon tetrachloride (CCl4) between wild-type and LRAT-deficient mice. To exclude the possibility that the effects on fibrogenesis were missed due to the rapid downregulation of LRAT following HSC activation, acute as well as spontaneous liver fibrosis was investigated. However, there was no increased fibrosis in 3-, 8- and 12-month-old LRAT-deficient mice and in LRAT-deficient mice after a single injection of CCl4 compared with wild-type mice. To determine whether the absence of retinoids in HSCs affects hepatocarcinogenesis, wild-type and LRAT-deficient mice were injected with diethylnitrosamine. LRAT deficiency decreased diethylnitrosamine-induced injury and tumour load and increased the expression of the retinoic acid responsive genes Cyp26a1, RARb and p21, suggesting that the lower tumour load of LRAT-deficient mice was a result of increased retinoid signalling and subsequent p21-mediated inhibition of proliferation. Conclusions The absence of retinoid-containing HSC lipid droplets does not promote HSC activation but reduces hepatocarcinogenesis.

Apolipoprotein AII Is a Regulator of Very Low Density Lipoprotein Metabolism and Insulin Resistance

Apolipoprotein AII (apoAII) transgenic (apoAIItg) mice exhibit several traits associated with the insulin resistance (IR) syndrome, including IR, obesity, and a marked hypertriglyceridemia. Because treatment of the apoAIItg mice with rosiglitazone ameliorated the IR and hypertriglyceridemia, we hypothesized that the hypertriglyceridemia was due largely to overproduction of very low density lipoprotein (VLDL) by the liver, a normal response to chronically elevated insulin and glucose. We now report in vivo and in vitro studies that indicate that hepatic fatty acid oxidation was reduced and lipogenesis increased, resulting in a 25% increase in triglyceride secretion in the apoAIItg mice. In addition, we observed that hydrolysis of triglycerides from both chylomicrons and VLDL was significantly reduced in the apoAIItg mice, further contributing to the hypertriglyceridemia. This is a direct, acute effect, because when mouse apoAII was injected into mice, plasma triglyceride concentrations were significantly increased within 4 h. VLDL from both control and apoAIItg mice contained significant amounts of apoAII, with ∼4 times more apoAII on apoAIItg VLDL. ApoAII was shown to transfer spontaneously from high density lipoprotein (HDL) to VLDL in vitro, resulting in VLDL that was a poorer substrate for hydrolysis by lipoprotein lipase. These results indicate that one function of apoAII is to regulate the metabolism of triglyceride-rich lipoproteins, with HDL serving as a plasma reservoir of apoAII that is transferred to the triglyceride-rich lipoproteins in much the same way as VLDL and chylomicrons acquire most of their apoCs from HDL.

The multifaceted nature of retinoid transport and metabolism.

Since their discovery over a century ago, retinoids have been the most studied of the fat-soluble vitamins. Unlike most vitamins, retinoids are stored at relatively high concentrations in the body to buffer against nutritional insufficiency. Until recently, it was thought that the sole important retinoid delivery pathway to tissues involved retinol bound to retinol-binding protein (RBP4). More recent findings, however, indicate that retinoids can be delivered to tissues through multiple overlapping delivery pathways, involving chylomicrons, very low density lipoprotein (VLDL) and low density lipoprotein (LDL), retinoic acid bound to albumin, water soluble β-glucuronides of retinol and retinoic acid, and provitamin A carotenoids. This review will focus on explaining this evolving understanding of retinoid metabolism and transport within the body.

Genetic dissection of retinoid esterification and accumulation in the liver and adipose tissue

Approximately 80–90% of all retinoids in the body are stored as retinyl esters (REs) in the liver. Adipose tissue also contributes significantly to RE storage. The present studies, employing genetic and nutritional interventions, explored factors that are responsible for regulating RE accumulation in the liver and adipose tissue and how these influence levels of retinoic acid (RA) and RA-responsive gene expression. Our data establish that acyl-CoA:retinol acyltransferase (ARAT) activity is not involved in RE synthesis in the liver, even when mice are nutritionally stressed by feeding a 25-fold excess retinol diet or upon ablation of cellular retinol-binding protein type I (CRBPI), which is proposed to limit retinol availability to ARATs. Unlike the liver, where lecithin:retinol acyltransferase (LRAT) is responsible for all RE synthesis, this is not true for adipose tissue where Lrat-deficient mice display significantly elevated RE concentrations. However, when CrbpI is also absent, RE levels resemble wild-type levels, suggesting a role for CrbpI in RE accumulation in adipose tissue. Although expression of several RA-responsive genes is elevated in Lrat-deficient liver, employing a sensitive liquid chromatography tandem mass spectrometry protocol and contrary to what has been assumed for many years, we did not detect elevated concentrations of all-trans-RA. The elevated RA-responsive gene expression was associated with elevated hepatic triglyceride levels and decreased expression of Pparδ and its downstream Pdk4 target, suggesting a role for RA in these processes in vivo.

Retinoids in the pancreas

Retinoids (vitamin A and its natural and synthetic analogs) are required by most tissues for maintaining the normal health of the tissue. This is certainly true for the pancreas. The recent literature is convincing that retinoids are needed by the adult to assure normal pancreatic endocrine functions, especially those of the α- and β-cells. It is also well established that retinoids are required to insure normal pancreas development in utero, including the development of the endocrine pancreas. The actions of retinoids for maintaining normal pancreatic islet functions has drawn considerable research interest from investigators interested in understanding and treating metabolic disease. Pancreatic retinoids are also of interest to investigators studying the origins of pancreatic disease, including the development of pancreatic fibrosis and its sequelae. This research interest is focused on pancreatic stellate cells (PSCs) which store retinoids and possess the metabolic machinery needed to metabolize retinoids. The literature on pancreatic disease and retinoids suggests that there is an association between impairments in pancreatic retinoid storage and metabolism and the development of pancreatic disease. These topics will be considered in this review.

The diversity of retinoid biology.

Retinoids comprise both naturally occurring and synthetic molecules that have a structural resemblance to all- trans -retinol, which by definition is vitamin A. Thus, the term retinoid is used to refer both to vitamin A and its metabolites and to chemically-related compounds that have been specifically synthesized as potential pharmacological agents for treating disease. It has long been known that the naturally occurring retinoids are required for maintaining immunity, barrier function, male and female reproduction, embryonic development, cognitive function, and vision. Over the last decade it has become increasingly clear that aberrant actions of naturally occurring retinoids and retinoid-related proteins are associated with development of metabolic disease, including obesity, type II diabetes, liver disease and cardiovascular disease. At the cellular level, retinoids are needed for maintaining normal cell proliferation, differentiation and apoptosis. Consequently, for over 50 years there has been much research interest focused on the use of both natural and synthetic retinoids in the treatment of proliferative disorders, especially cancers and skin disease.