Rab2A-mediated Golgi-Lipid droplet interactions support very-low-density lipoprotein secretion in hepatocytes.
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ABSTRACT:
SUBMITTER: Mrs. Min Xu
PROVIDER: S-SCDT-10_1038-S44318-024-00288-X | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:One mechanism by which monocytes become activated postprandially is by exposure to triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDL). VLDL are hydrolyzed by lipoprotein lipase at the blood-endothelial cell interface, releasing free fatty acids. In this study, we examined postprandial monocyte activation in more detail, and found that lipolysis products generated from postprandial VLDL induce the formation of lipid-filled droplets within cultured THP-1 monocytes, characterized by coherent antistokes Raman spectroscopy. Organelle-specific stains revealed an association of lipid droplets with the endoplasmic reticulum, confirmed by electron microscopy. Lipid droplet formation was reduced when lipoprotein lipase-released fatty acids were bound by BSA, which also reduced cellular inflammation. Furthermore, saturated fatty acids induced more lipid droplet formation in monocytes compared with mono- and polyunsaturated fatty acids. Monocytes treated with postprandial VLDL lipolysis products contained lipid droplets with more intense saturated Raman spectroscopic signals than monocytes treated with fasting VLDL lipolysis products. In addition, we found that human monocytes isolated during the peak postprandial period contain more lipid droplets compared with those from the fasting state, signifying that their development is not limited to cultured cells but also occurs in vivo. In summary, circulating free fatty acids can mediate lipid droplet formation in monocytes and potentially be used as a biomarker to assess an individual's risk of developing atherosclerotic cardiovascular disease.
| S-EPMC2843797 | biostudies-literature
Project description:The very-low-density-lipoprotein secretion rate of isolated hepatocytes obtained from rats fed a high-fat diet was half that of cells from control animals. In fat-fed rats, the initial cellular uptake of [l-14C]oleate in vitro was decreased by 25%, its esterification to triacylglycerols and phospholipids by 50% and its incorporation into very-low-density-lipoprotein triacylglycerols by 70%. Exogenous oleate was not the main precursor of very-low-density lipoproteins in these animals. Lipogenesis, a minor source of very-low-density lipoproteins with the control diet in our experimental conditions, was inhibited by 84% after fat-feeding. A short-term inhibition of lipogenesis in vitro did not result in a decrease in very-low-density-lipoprotein secretion rate. The results suggest that fat-feeding decreased availability of exogenous as well as endogenous fatty acids for synthesis of very-low-density lipoproteins.
| S-EPMC1163258 | biostudies-other
Project description:The effects of different calcium-antagonists on secretion of very-low-density lipoprotein (VLDL) from cultured rat hepatocytes were examined. Verapamil (an inhibitor of voltage-dependent calcium channels) and EGTA (a calcium chelator) decreased VLDL-triacylglycerol secretion in a concentration-dependent manner, with maximum inhibition (about 90%) at 0.2 mM-verapamil and 5 mM-EGTA. Inorganic calcium-antagonists such as lanthanum, nickel, cobalt and manganese decreased secretion of VLDL-triacylglycerol by 55-95%, whereas the calcium-agonist barium did not affect secretion. Inhibition of VLDL-triacylglycerol secretion appeared within 30 min, without inhibition of triacylglycerol synthesis. Pulse-chase experiments revealed that verapamil and cobalt inhibited the secretory pathway itself. Cobalt showed a concentration-dependent inhibition of VLDL-triacylglycerol secretion, with maximal effect at 8 mM. Although inhibition by cobalt was not completely reversible, Trypan Blue exclusion and lactate dehydrogenase leakage indicated that the hepatocytes were not injured by cobalt or any of the other calcium-antagonists tested. Inhibition of protein synthesis by cycloheximide did not affect triacylglycerol secretion (up to 2 h), and the observed effects were therefore probably not due to impaired production of apolipoproteins. Taken together, these results suggest that calcium is important for secretion of VLDL particles.
| S-EPMC1148427 | biostudies-other
Project description:The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.
| S-EPMC1148864 | biostudies-other
Project description:We recently showed that the heparan sulfate proteoglycan syndecan-1 mediates hepatic clearance of triglyceride-rich lipoproteins in mice based on systemic deletion of syndecan-1 and hepatocyte-specific inactivation of sulfotransferases involved in heparan sulfate biosynthesis. Here, we show that syndecan-1 expressed on primary human hepatocytes and Hep3B human hepatoma cells can mediate binding and uptake of very low density lipoprotein (VLDL). Syndecan-1 also undergoes spontaneous shedding from primary human and murine hepatocytes and Hep3B cells. In human cells, phorbol myristic acid induces syndecan-1 shedding, resulting in accumulation of syndecan-1 ectodomains in the medium. Shedding occurs through a protein kinase C-dependent activation of ADAM17 (a disintegrin and metalloproteinase 17). Phorbol myristic acid stimulation significantly decreases DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate)-VLDL binding to cells, and shed syndecan-1 ectodomains bind to VLDL. Although mouse hepatocytes appear resistant to induced shedding in vitro, injection of lipopolysaccharide into mice results in loss of hepatic syndecan-1, accumulation of ectodomains in the plasma, impaired VLDL catabolism, and hypertriglyceridemia.These findings suggest that syndecan-1 mediates hepatic VLDL turnover in humans as well as in mice and that shedding might contribute to hypertriglyceridemia in patients with sepsis.
| S-EPMC3245353 | biostudies-literature
Project description:Hepatocytes were derived from 2-3-day streptozotocin-diabetic rats and maintained in culture for up to 3 days. Compared with similar cultures from normal animals, these hepatocytes secreted less very-low-density-lipoprotein (VLDL) triacylglycerol, but the decrease in the secretion of VLDL non-esterified and esterified cholesterol was not so pronounced. This resulted in the secretion of relatively cholesterol-rich VLDL particles by the diabetic hepatocytes. Addition of insulin for a relatively short period (24 h) further decreased the low rates of VLDL triacylglycerol secretion from the diabetic hepatocytes. The secretion of VLDL esterified and non-esterified cholesterol also declined. These changes occurred irrespective of whether or not exogenous fatty acids were present in the culture medium. Little or no inhibitory effect of insulin was observed after longer-term (24-48 h) exposure to the hormone. Both dexamethasone and a mixture of lipogenic precursors (lactate plus pyruvate) stimulated VLDL triacylglycerol and cholesterol secretion, but not to the levels observed in hepatocytes from normal animals. The low rate of hepatic VLDL secretion in diabetes contrasts with the increase in whole-body VLDL production rate. This suggests that the intestine is a major source of plasma VLDL in insulin-deficient diabetes.
| S-EPMC1133262 | biostudies-other
Project description:In contrast to water-soluble fuels such as glucose or ketone bodies, the use of lipids as an energy source for tissues has required the development of complex structures for their transport through the aqueous plasma. In the case of endogenously synthesized triacylglycerol this is achieved by the assembly and secretion of hepatic VLDL which provides the necessary stability in an aqueous medium. An essential component of this assembly process is apo B. Dietary changes which require an increase in hepatic VLDL secretion appear to be accompanied by increases in the availability of functional apo B. Interesting questions relate to: (a) the intracellular site(s) of triacylglycerol association with apo B, and (b) the mechanism(s) by which the availability of functional apo B at this site responds to metabolic and hormonal signals which reflect dietary status and, thus, the need to secrete triacylglycerol. As regards the latter, although in some cases changes in apo B synthesis occur in response to VLDL secretion hepatic apo B mRNA levels appear to be quite stable in vitro. Intracellular switching of apo B between the secretory and degradative pathways may be important in controlling VLDL assembly and post-translational modifications of the apoprotein may also play a role by influencing its ability to bind to triacylglycerol. Transport is not the only problem associated with the utilization of a concentrated energy source such as triacylglycerol and the complex problems of waste product disposal and recycling have to be dealt with. In the case of triacylglycerol, potentially toxic waste products include atherogenic remnants and LDL. The overall problem, then, in the long-term, involves the development of a 'safe' means of utilizing triacylglycerol and this requirement accounts for much of the complexity of plasma lipoprotein metabolism. In this area, the rat could teach the human a few tricks. One of these appears to be the utilization of hepatic apo B48 rather than apo B100 for VLDL assembly in response to increases in the extrahepatic utilization of hepatically synthesized triacylglycerol. Under these conditions, the remnants of hepatic triacylglycerol utilization by peripheral tissues are cleared from the plasma much more readily via a process which seems to involve the cycling of more triacylglycerol back to the liver than that which occurs in humans. The means by which this is achieved, though, are obscure and may involve a chylomicron remnant receptor, the nature of which, itself, remains controversial.(ABSTRACT TRUNCATED AT 400 WORDS)
| S-EPMC1131384 | biostudies-other
Project description:Primary cultures of rat hepatocytes were used to study secretion of very-low-density lipoproteins and metabolism of asialofetuin. The ionophore monensin inhibited both secretion of very-low-density lipoproteins and binding and degradation of asialofetuin in a concentration-dependent manner. Secretion as well as receptor binding were markedly decreased after 15 min treatment with monensin. The inhibitory effect of the ionophore was fully reversible, and no effect on protein synthesis was observed at concentrations up to 50 microM. The secretion of apoproteins (B-small, B-large and E) and that of albumin were inhibited to the same extent as was triacylglycerol secretion. Secretion of very-low-density lipoproteins was more sensitive to low concentrations of monensin than was the metabolism of asialofetuin. Maximum inhibition of very-low-density-lipoprotein secretion was obtained at 5-10 microM-monensin, whereas 25 microM was required to obtain maximum inhibition of binding and degradation of asialofetuin. The number of surface receptors for asialofetuin decreased to about half when the cells were exposed to 25 microM-monensin. It is possible that monensin inhibits endo- and exo-cytosis via a similar mechanism, e.g. by disturbing proton gradients. Since secretion of very-low-density lipoproteins was more sensitive to low concentrations of monensin, it is likely that monensin independently inhibits endocytic and secretory functions in cultured hepatocytes.
| S-EPMC1144872 | biostudies-other
Project description:RationaleSeveral reports suggest that antisense oligonucleotides against miR-33 might reduce cardiovascular risk in patients by accelerating the reverse cholesterol transport pathway. However, conflicting reports exist about the impact of anti-miR-33 therapy on the levels of very low-density lipoprotein-triglycerides (VLDL-TAG).ObjectiveWe test the hypothesis that miR-33 controls hepatic VLDL-TAG secretion.Methods and resultsUsing therapeutic silencing of miR-33 and adenoviral overexpression of miR-33, we show that miR-33 limits hepatic secretion of VLDL-TAG by targeting N-ethylmaleimide-sensitive factor (NSF), both in vivo and in primary hepatocytes. We identify conserved sequences in the 3'UTR of NSF as miR-33 responsive elements and show that Nsf is specifically recruited to the RNA-induced silencing complex following induction of miR-33. In pulse-chase experiments, either miR-33 overexpression or knock-down of Nsf lead to decreased secretion of apolipoproteins and TAG in primary hepatocytes, compared with control cells. Importantly, Nsf rescues miR-33-dependent reduced secretion. Finally, we show that overexpression of Nsf in vivo increases global hepatic secretion and raises plasma VLDL-TAG.ConclusionsTogether, our data reveal key roles for the miR-33-NSF axis during hepatic secretion and suggest that caution should be taken with anti-miR-33-based therapies because they might raise proatherogenic VLDL-TAG levels.
| S-EPMC4108429 | biostudies-literature
Project description:In this study the effects of bile acids and other organic anions on the secretion of very-low-density lipoproteins (VLDLs) were evaluated in rat hepatocytes in primary culture. Incubation of cells with portal blood concentrations (10-200 microM) of bile acids resulted in dose-dependent suppression of secretion of VLDL-associated [3H]triglyceride (TG) formed from [3H]glycerol, and also of TG mass. The degree of the inhibition was highly correlated with intracellular bile acid concentration. Prolonged incubation with 100 microM extracellular taurocholic acid (TC) decreased the secretion of [3H]TG and TG mass to 35% and 50% of the controls respectively. Cellular content of mass and of [3H]TG during prolonged incubation with TC were about 20% and 60% higher than the controls respectively. The inhibitory effect remained for at least 24 h in the presence of TC without altering VLDL-lipid and VLDL-apolipoprotein compositions or the size distribution of the particles. Secretion of apoB-100 and of apoB-48 was inhibited to a similar extent. Cells largely lost their capacity to accumulate bile acids intracellularly after 48 h in culture. In these cells TC was unable to exert its suppressive effects. Taurine and glycine conjugates of all common bile acids were capable of suppressing [3H]TG secretion. Trihydroxylated (cholic acid) and various dihydroxylated (deoxycholic, chenodeoxycholic and ursodeoxycholic acids) bile acids had similar capacities in this respect, suggesting that their common sterol-3 alpha-OH structure is required for the suppressive effect. Neither non-bile acid organic anions, e.g. bilirubin ditaurate and dibromosulphthalein, nor dianionic bile acid metabolites, e.g. sulphated taurolithocholic acid and lithocholate-3-O-glucuronide, showed any effect on [3H]TG secretion. These results indicate that bile acids might play a physiological role in regulating VLDL production by the liver, especially in the postprandial state when their enterohepatic circulation is stimulated.
| S-EPMC1217381 | biostudies-other