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.

Lecithin:retinol acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of retinol. We have studied Lrat-deficient (Lrat ؊/؊ ) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for retinol esterification in the body. Retinoids 3 have important roles in mediating or facilitating many essential physiologic functions within the body (1). In vision, 11-cisretinal serves as the chromophore for the visual pigments present in the rod and cone photoreceptor cells (2, 3). Retinoids are also needed to maintain cell proliferation and normal differentiation, normal immune response, normal reproduction, and normal fetal development (4). It has been suggested in the literature that over 500 different genes may be transcriptionally responsive to retinoids (5). The transcriptional regulatory activities of retinoids are thought to result from the actions of all-trans-and 9-cis-retinoic acid (6 -8). These actions of retinoic acid are mediated through six distinct ligand-dependent transcription factors as follows: three retinoic acid receptors (RAR␣, 4 RAR␤, and RAR␥) and three retinoid X receptors (RXR␣, RXR␤, and RXR␥) (6 -8). Ultimately, all retinoid must be acquired from the diet either as preformed retinoid (primarily as dietary retinol or retinyl ester) or as proretinoid carotenoid (primarily as ␤-carotene) (9, 10). The different dietary retinoid forms are processed within the enterocyte and packaged along with other dietary lipids as retinyl esters in nascent chylomicrons (9, 10). Approximately 66 -75% of dietary retinoid is taken up and stored as retinyl ester in the liver (9, 10), primarily in the nonparenchymal hepatic stellate cells (also called Ito cells, lipocytes, and fat-storing cells) (11). These hepatic stores can be called upon and mobilized into the circulation as retinol bound to plasma retinol-binding protein (RBP) (12, 13). Tissues acquire retinol from the circulating retinol-RBP complex or postprandially from chylomicrons (9, 10, 12-14) and are able to oxidize enzymatically this retinoid to retinal and retinoic acid (14 -16). Most tissues also possess some capacity to esterify and thus store retinol prior to its use for the synthesis of retinal or retinoic acid (9,10,14).It is generally agreed upon in the literature that retinol is esterified primarily through the actions of the enzyme lecithin:retinol acyltransferase (LRAT) (17)(18)(19)(20). LRAT is broadly expressed in tissues and at relatively high levels in the intestine, liver, and eye where it has been proposed to catalyze the transesterification of retinol with an acyl group present in the A1 position of membrane lecithin (17-23). Recently, the gene encoding LRAT was knocked out in mice (20). It was clear from the initial studies of Lrat-deficient (Lrat Ϫ/Ϫ ) mice that LRAT is the preponderant and possibly sole enzyme responsible for retinyl ester formation in the eye and ...

Protein kinase M (PKM) is a newly described form of PKC that is necessary and sufficient for the maintenance of hippocampal long term potentiation (LTP) and the persistence of memory in Drosophila. PKM is the independent catalytic domain of the atypical PKC isoform and produces long term effects at synapses because it is persistently active, lacking autoinhibition from the regulatory domain of PKC. PKM has been thought of as a proteolytic fragment of PKC. Here we report that brain PKM is a new PKC isoform, synthesized from a PKM mRNA encoding a PKC catalytic domain without a regulatory domain. Multiple -specific antisera show that PKM is expressed in rat forebrain as the major form of in the near absence of full-length PKC. A PKC knockout mouse, in which the regulatory domain was disrupted and catalytic domain spared, still expresses brain PKM, indicating that this form of PKM is not a PKC proteolytic fragment. Furthermore, the distribution of brain PKM does not correlate with PKC mRNA but instead with an alternate RNA transcript thought incapable of producing protein. In vitro translation of this RNA, however, generates PKM of the same molecular weight as that in brain. Metabolic labeling of hippocampal slices shows increased de novo synthesis of PKM in LTP. Because PKM is a kinase synthesized in an autonomously active form and is necessary and sufficient for maintaining LTP, it serves as an example of a link coupling gene expression directly to synaptic plasticity. LTP1 is a persistent enhancement of synaptic transmission widely studied as a physiological model of memory (1). LTP can be divided into two phases: induction, which triggers the potentiation, and maintenance, which sustains it over time. Many molecules have been implicated in LTP induction, which is initiated by the activation of N-methyl-D-aspartate (NMDA) receptors and involves several protein kinases (2). In contrast, very little is known about the molecular mechanism of maintenance. Recently, however, a specific, autonomously active form of the atypical PKC isozyme (3, 4), PKM, has been found both necessary and sufficient for maintaining LTP (5-7). Overexpression of PKM also prolongs memory in Drosophila melanogaster, suggesting it is part of an evolutionarily conserved molecular mechanism for memory storage (8).The unique role of PKM in LTP maintenance is due, in part, to its unusual structural and enzymatic properties as an autonomously active kinase. PKM consists of the independent catalytic domain of a PKC isoform (5). PKC isoforms are divided into three classes: conventional, novel, and atypical (reviewed in Refs. 9 -11). Each isoform is a single polypeptide consisting of an N-terminal regulatory domain and a C-terminal catalytic domain linked by a hinge (Fig. 1A, left). The regulatory domain contains binding sites for second messengers and an autoinhibitory pseudosubstrate sequence, which interacts with and blocks the active site of the catalytic domain. Second messengers stimulate PKC by binding to the regulatory domain, translocating th...

Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region of the hippocampus. Multiple isoforms are transiently activated in the induction phase of long-term potentiation (LTP). In contrast, a single species, zeta, is persistently activated during the maintenance phase of LTP through the formation of an independent, constitutively active catalytic domain, protein kinase Mzeta (PKMzeta). In this study, we used immunoblot and immunocytochemical techniques with isoform-specific antisera to examine the distribution of the complete family of PKC isozymes and PKMzeta in rat brain. Each form of PKC showed a widespread distribution in the brain with a distinct regional pattern of high and low levels of expression. PKMzeta, the predominant form of PKM in brain, had high levels in hippocampus, frontal and occipital cortex, striatum, and hypothalamus. In the hippocampus, each isoform was expressed in a characteristic pattern, with zeta prominent in the CA1 stratum radiatum. These results suggest that the compartmentalization of PKC isoforms in neurons may contribute to their function, with the location of PKMzeta prominent in areas notable for long-term synaptic plasticity.