Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease.

SLC7A5, known as LAT1, belongs to the APC superfamily and forms a heterodimeric amino acid transporter interacting with the glycoprotein CD98 (SLC3A2) through a conserved disulfide. The complex is responsible for uptake of essential amino acids in crucial body districts such as placenta and blood brain barrier. LAT1/CD98 heterodimer has been studied over the years to unravel the transport mechanism and the role of each subunit. Studies conducted in intact cells demonstrated that LAT1/CD98 mediates a Na⁺ and pH independent antiport of amino acids. Some novel insights into the function of LAT1 derived from studies conducted in proteoliposomes reconstituted with the recombinant human LAT1. Using this experimental tool, it has been demonstrated that the preferred substrate is histidine and that CD98 is not required for transport being, plausibly, involved in routing LAT1 to the plasma membrane. Since a 3D structure of LAT1 is not available, homology models have been built on the basis of the AdiC transporter from E.coli. Crucial residues for substrate recognition and gating have been identified using a combined approach of bioinformatics and site-directed mutagenesis coupled to functional assays. Over the years, the interest around LAT1 increased because this transporter is involved in important human diseases such as neurological disorders and cancer. Therefore, LAT1 became an important pharmacological target together with other nutrient membrane transporters. Moving from knowledge on structure/function relationships, two cysteine residues, lying on the substrate binding site, have been exploited for designing thiol reacting covalent inhibitors. Some lead compounds have been characterized whose efficacy has been tested in a cancer cell line.

SLC1A5, known as ASCT2, is a neutral amino acid transporter belonging to the SLC1 family and localized in the plasma membrane of several body districts. ASCT2 is an acronym standing for Alanine, Serine, Cysteine Transporter 2 even if the preferred substrate is the conditionally essential amino acid glutamine, with cysteine being a modulator and not a substrate. The studies around amino acid transport in cells and tissues began in the ‘60s by using radiolabeled compounds and competition assays. After identification of murine and human genes, the function of the coded protein has been studied in cell system and in proteoliposomes revealing that this transporter is a Na⁺ dependent antiporter of neutral amino acids, some of which are only inwardly transported and others are bi-directionally exchanged. The functional asymmetry merged with the kinetic asymmetry in line with the physiological role of amino acid pool harmonization. An intriguing function has been described for ASCT2 that is exploited as a receptor by a group of retroviruses to infect human cells. Interactions with scaffold proteins and post-translational modifications regulate ASCT2 stability, trafficking and transport activity. Two asparagine residues, namely N163 and N212, are the sites of glycosylation that is responsible for the definitive localization into the plasma membrane. ASCT2 expression increases in highly proliferative cells such as inflammatory and stem cells to fulfill the augmented glutamine demand. Interestingly, for the same reason, the expression of ASCT2 is greatly enhanced in many human cancers. This finding has generated interest in its candidacy as a pharmacological target for new anticancer drugs. The recently solved 3D structure of ASCT2 will aid in the rational design of such therapeutic compounds.

Glutamine together with glucose is essential for body’s homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na+ and H+. Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7 and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5 and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologues. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for pharmacological intervention.