BACKGROUND:Understanding the mechanisms of long chain fatty acid (LCFA) uptake in hepatic cells is of high medical importance to treat and to prevent fatty liver disease (FLD). ACSs (Acyl-CoA synthetases) are a family of enzymes that catalyze the esterification of fatty acids (FA) with CoA. Recent studies suggest that ACS enzymes drive the uptake of LCFA indirectly by their enzymatic activity and could promote special metabolic pathways dependent on their localization.The only protein located at the plasma membrane which has consistently been shown to enhance FA uptake is CD36.AIMS:The current study investigated whether ACSs and CD36 could regulate hepatic LCFA uptake.METHODS AND RESULTS:FATP2 and FATP4 were both localized to the ER of HuH7 and HepG2 cells as shown by double immunofluorescence in comparison to marker proteins. ACSL1 was located at mitochondria in both cell lines. Overexpression of FATP2, FATP4 and ACSL1 highly increased ACS activity as well as the uptake of [3H]-oleic acid and fluorescent Bodipy-C12 (B12) fatty acid. Quantitative FACS analysis showed a correlation between ACS expression levels and B12 uptake. FATP2 had the highest effect on B12 uptake of all proteins tested. CD36 was mainly localized at the plasma membrane. Whereas [3H]-oleic acid uptake was increased after overexpression, CD36 had no effect on B12 uptake.CONCLUSION:Uptake of LCFA into hepatoma cells can be regulated by the expression levels of intracellular enzymes. We propose that ACS enzymes drive FA uptake indirectly by esterification. Therefore these molecules are potential targets for treatment of nonalcoholic fatty liver disease (NAFLD) or steatohepatitis (NASH).

The function of membrane proteins in long-chain fatty acid transport is controversial. The acyl-CoA synthetase fatty acid transport protein-4 (FATP4) has been suggested to facilitate fatty acid uptake indirectly by its enzymatic activity, or directly by transport across the plasma membrane. Here, we investigated the function of FATP4 in basal and insulin mediated fatty acid uptake in C(2)C(12) muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl-CoA synthetase activity, and cellular uptake of oleate was enhanced similarly. Kinetic analysis demonstrated that FATP4 allowed the cells to reach apparent saturation of fatty acid uptake at a twofold higher level compared with control. Short-term treatment with insulin increased fatty acid uptake in line with previous reports. Surprisingly, insulin increased the acyl-CoA synthetase activity of C(2)C(12) cells within minutes. This effect was sensitive to inhibition of insulin signaling by wortmannin. Affinity purified FATP4 prepared from insulin-treated cells showed an enhanced enzyme activity, suggesting it constitutes a novel target of short-term metabolic regulation by insulin. This offers a new mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. FATP4 was colocalized to the endoplasmic reticulum by double immunofluorescence and subcellular fractionation, clearly distinct from the plasma membrane. Importantly, neither differentiation into myotubes nor insulin treatment changed the localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in cellular fatty acid uptake.

Silybin, the major flavonoid of Silybum marianum, is widely used to treat liver diseases such as hepatocellular carcinoma and cirrhosis-associated insulin resistance. Research so far has focused on its anti-oxidant properties. Here, we demonstrate that silybin and its derivative dehydrosilybin inhibit glucose uptake in several model systems. Both flavonoids dose-dependently reduce basal and insulin-dependent glucose uptake of 3T3-L1 adipocytes, with dehydrosilybin showing significantly stronger inhibition. However, insulin signaling was not impaired, and immunofluorescence and subcellular fractionation showed that insulin-induced translocation of GLUT4 to the plasma membrane is also unchanged. Likewise, hexokinase activity was not affected suggesting that silybin and dehydrosilybin interfere directly with glucose transport across the PM. Expression of GLUT4 in CHO cells counteracted the inhibition of glucose uptake by both flavonoids. Moreover, treatment of CHO cells with silybin and dehydrosilybin reduced cell viability which was partially rescued by GLUT4 expression. Kinetic analysis revealed that silybin and dehydrosilybin inhibit GLUT4-mediated glucose transport in a competitive manner with K(i)=60 and 116 µM, respectively. We conclude that silybin and dehydrosilybin inhibit cellular glucose uptake by directly interacting with GLUT transporters. Glucose starvation offers a novel explanation for the anti-cancer effects of silybin.

Lipid droplets emerge as dynamic organelles highly relevant for lipid homeostasis and the pathophysiology of metabolic diseases. We are using fatty acyl‐CoA synthetases (ACS), especially ACSL3 as a paradigm to investigate the formation and growth of lipid droplets. Out of eleven mammalian long chain ACS, ACSL3 is the only family member localizing specifically to lipid droplets and the ER. The distribution of ACSL3 between these two organelles depends on the cell type and whether exogenous fatty acids are being supplied. The N‐terminal region of ACSL3 is sufficient to direct reporter proteins to lipid droplets which we analysed by confocal microscopy. Mutational analysis demonstrated that only amino acids 12 to 51 are essential for targeting. ACSL3 is tightly membrane associated by an amphipathic helix contained within the N‐terminal sorting signal. During lipid droplet biogenesis, ACSL3 moves from the bilayer membrane of the ER to the phospholipid monolayer of nascent lipid droplets as we demonstrated by live microscopy. Overexpression of ACSL3 results not only in increased cellular enzyme activity but also in the enhanced uptake of exogenous fatty acids. This suggests that ACSL3 is functionally relevant for the regulation of fatty acid uptake. Moreover, we speculate that the specific localization of ACSL3 is important for the local biosynthesis of phospholipids at the surface of the lipid droplets.

Lipid droplets emerge as important intracellular organelles relevant for lipid homeostasis and the pathophysiology of metabolic diseases. Here, we present a personal view on the current knowledge about the biogenesis of mammalian cytoplasmic lipid droplets, with a focus on microscopy and especially live imaging. We also discuss difficulties related to the lipid droplet proteome, contentious views on lipid droplet growth, and last but not least the evidence for the heterogeneity of lipid droplets within a single cell. We conclude with an outline of the most important future challenges.

ABSTRACT The glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesviruses additional proteins are associated with gH/gL contributing to the cell tropism of the respective virus. Human cytomegalovirus (HCMV) gH/gL forms complexes with either gO (UL74) or proteins of the UL128-131A gene locus. While a contribution of UL128-131A to endothelial cell tropism is known, the role of gO is less clear. We studied the role of gH/gL-associated proteins in HCMV replication in human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). Deletions of UL74 alone or in combination with mutations of the UL128-131A gene region were introduced into bacterial artificial chromosome vectors derived from the endotheliotropic strain TB40/E. Deletion of UL74 caused a profound defect regarding virus release from infected HFF and HUVEC. Large numbers of capsids accumulated in the cytoplasm of infected HFF but failed to acquire an envelope. Clear cell type differences were observed in the cell-associated spread of the UL74-defective virus. In HFF, focal growth was severely impaired, whereas it was normal in HUVEC. Deletion of UL131A abolished focal growth in endothelial cells. UL74/UL128-131A dual mutants showed severely impaired reconstitution efficiency. Our data suggest that gO plays a critical role in secondary envelopment and release of cell-free virions independent of the cell type but affects cell-associated growth specifically in HFF, whereas UL128-131A contributes to cell-associated spread in HFF and HUVEC.

The molecular mechanism of fatty acid uptake and utilization is of high medical relevance for the treatment of obesity, diabetes, and cardiovascular disease. Neuronal processes, hormones, and transcription factors are master regulators of these essential processes while their fine-tuning is achieved by modulating the activity and amount of enzymes. Proteins involved in fatty acid uptake and metabolism are important pharmaceutical targets. Only basic research on these molecules will lead to new strategies for therapy. Conceptionally, the intracellular utilization of long chain fatty acids may be subdivided into three steps: uptake across the plasma membrane, activation by esterification with coenzyme A, and subsequent metabolism. Long chain acyl-CoA synthetases (ACSLs) activate fatty acids for intracellular metabolism but are also involved in the regulation of uptake. The predominant pathways for fatty acids are their storage, membrane biosynthesis, and conversion to energy. How activated fatty acids are channeled toward one particular metabolic pathway is not well understood on the molecular level. We have previously shown that ACSLs localized to either the endoplasmic reticulum or to mitochondria can regulate the extent of fatty acid uptake. Multiple different long chain ACSLs are expressed simultaneously in the same cell type but differ in their subcellular localization. The hypothesis we put forward here implies that the spatial organization of ACSL activity is a key factor in channeling fatty acids toward a particular metabolic fate.

Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the UL128-UL131A gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of UL 128 in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis; restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.

Cell-associated spread is assumed to be the predominant mode of human cytomegalovirus (HCMV) dissemination in infected patients, however the underlying mechanisms are poorly understood. We tested the hypothesis that cell-to-cell spread of HCMV may be associated with direct transfer of cytoplasmic material by analyzing focal growth of green fluorescent HCMVDeltaUL16GFP. In this recombinant virus, UL16 was partially replaced by the green fluorescent protein (EGFP). The resulting HCMVDeltaUL16GFP showed unrestricted growth and expressed EGFP from the early UL16 promoter. EGFP transmission was then investigated in relation to viral spread from productively infected cells to cocultured uninfected cells. Alternatively, microinjection of fluorescent dextrane allowed for direct visualization of inter-cell-connections. Within 5h of coculture, 8% of cells neighbouring productively infected cells had acquired EGFP. Detection of EGFP in the absence of IE antigen and during cycloheximide block excluded the possibility of de novo synthesis. Immediate distribution of microinjected fluorescent dyes from infected cells to adjacent cells proved the existence of cell-cell-fusions. These data demonstrate that focal spread of HCMV is associated with direct transfer of cytoplasmic material, most likely through cell-cell-fusions. This would withdraw the virus from the control of neutralizing antibodies and thus provide an explanation for the limited antiviral effect of the humoral immune response.