Long-term maintenance of high rates of very-low-density-lipoprotein secretion in hepatocyte cultures. A model for studying the direct effects of insulin and insulin deficiency in vitro.

High rates of hepatic cellular triacylglycerol synthesis and very-low-density-lipoprotein (VLDL) triacylglycerol output were maintained in vitro for at least 3 days when hepatocytes were cultured in a medium lacking insulin but supplemented with 1 microM-dexamethasone, 10 mM-lactate, 1 mM-pyruvate and 0.75 mM-oleate (supplemented medium). Under these conditions VLDL output remained constant, whereas cell triacyglycerol content increased 10-fold over 3 days, suggesting that the secretory process was saturated. Insulin, present during the first 24 h period, enhanced the storage of cellular triacylglycerol by inhibiting the secretion of VLDL. This stored triacyglycerol was subsequently released into the medium as VLDL if insulin was removed. With the supplemented medium the increased rate of VLDL secretion after insulin removal exceeded that observed under 'saturating' conditions, suggesting that pre-treatment with insulin enhanced the capacity for VLDL secretion. In contrast with the short-term (24 h) effects of insulin, longer-term exposure (greater than 48 h) to insulin enhanced the secretion of VLDL compared with insulin-untreated cultures. Under these conditions, insulin increased the net rates of triacylglycerol synthesis. The results suggest that insulin affects the secretion of VLDL triacylglycerol by two distinct and opposing mechanisms: first, by direct inhibition of secretion; second by increasing triacylglycerol synthesis, which stimulates secretion. The net effect at any time depends upon the relative importance of each of these processes.     

Regulation of 3-hydroxybutyrate formation and secretion of very-low-density-lipoprotein triacylglycerol by perfused livers from fed and starved rats.

Acetoacetate was the sole ketone body formed when livers from starved rats were perfused with minimal concentrations of non-esterified fatty acid. Absence of 3-hydroxybutyrate was related to a low substrate potential, which caused a more oxidized redox state and a decreased [ATP]/[ADP] ratio. Only under conditions of comparable non-esterified fatty acid uptake was lipoprotein triacylglycerol production inversely related to ketogenesis.     

Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

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.     

Synthesis and secretion of C-reactive protein by rabbit primary hepatocyte cultures.

The synthesis and secretion of the acute-phase protein C-reactive protein by rabbit primary hepatocyte cultures was investigated. Hepatocytes prepared from animals that had received inflammatory stimuli 18-24 h before cell isolation were found to incorporate radiolabelled amino acids into C-reactive protein throughout the 48 h culture period. Intracellular C-reactive protein was found to be in steady state and there was no significant degradation of extracellular C-reactive protein, permitting direct estimation of rate of synthesis from rate of extracellular accumulation. Both C-reactive protein and total secreted protein were synthesized at constant rates for at least 24 h in culture. Mean rate of accumulation of newly synthesized total proteins in medium of cultures from six stimulated animals was 40% greater than was found in cultures from nine control (unstimulated) animals; this difference did not achieve statistical significance (0.05 less than P less than 0.10). Mean rate of C-reactive-protein synthesis represented 3.9% of total secreted-protein synthesis in cultures prepared from stimulated animals compared with 0.3% in cultures from control animals (P less than 0.001). Further, there was a correlation between C-reactive-protein synthesis by cultured hepatocytes and serum C-reactive-protein concentration at time of hepatocyte isolation (P less than 0.001). Rates of C-reactive-protein synthesis by hepatocyte cultures from stimulated animals were in good agreement with those previously measured in isolated perfused livers and those calculated from results of studies in vivo.     

Estradiol increases the secretion of hepatic lipase by rat hepatocyte cultures.

Hepatic lipase (EC 3.1.1.3) is synthesized and secreted by parenchymal hepatocytes and binds to endothelial cells of liver sinusoids. The present study shows that the activity of hepatic lipase secreted by hepatocyte cultures from male rats in increased approx. 6-fold after 10 h culture with 10 microM 17 beta-estradiol. The stimulatory effect of 17 beta-estradiol is biphasic and declines at higher concentrations. In hepatocytes from male rats: progesterone, unlike 17 beta-estradiol, had only a small stimulatory effect when present as the sole hormone and a small inhibitory effect in the presence of 17 beta-estradiol, while testosterone and dexamethasone had no effect. Hepatocyte cultures from female rats had a higher basal rate of hepatic lipase secretion than cells from male rats and showed a smaller stimulation by 17 beta-estradiol. These results suggest that 17 beta-estradiol might regulate the secretion of hepatic lipase by hepatocytes, and presumably the activity of the enzyme at either the endothelial surface of the liver sinusoids or at extrahepatic sites.     

Formation and secretion of glycolithocholate-3-sulfate in primary hepatocyte cultures

Bile acid sulfation was studied in primary hepatocyte cultures. The primary hepatocyte cultures formed glycolithocholate-3-sulfate (GLC-S) when glycolithocholate (GLC) was added to the medium. The relative percentage of GLC-S formation increased when the GLC concentration was increased from 10 microM to 100 microM. GLC-S formation was linear to 60 min. GLC-S secretion into the medium was detectable at 75 min and linear to 8 hr. In contrast to the effect of GLC concentration, there was no difference in GLC-S formation or secretion when inorganic sulfate in the medium was increased 16-fold (100 microM-1600 microM). We conclude that the rate of bile acid sulfate formation in cultured primary hepatocytes is primarily controlled by bile acid, but not inorganic sulfate, concentration.     

Polyamines in rat hepatocyte cultures.

In rat hepatocytes cultured for 120 h polyamine content was markedly modified. Putrescine concentration reached a maximum at 48 h, spermidine increased for 48 h and then remained constant, spermine after a decrease returned to its initial values. Total polyamine amount was increased by 75%. Both ornithine decarboxylase and the retroconversion pathway were responsible for these modifications. The possible correlation between polyamine metabolism and retrodifferentiation process was investigated by studying them in conditions which are known to preserve differentiated functions.     

Storage, mobilization and secretion of cytosolic triacylglycerol in hepatocyte cultures. The role of insulin.

Cytosolic triacylglycerol labelled from [3H]oleate accounted for almost 50% (57 +/- 22 nmol/mg of protein) of the total cellular triacylglycerol which was newly synthesized by cultured hepatocytes during a 24 h incubation. Insulin decreased the export of triacylglycerol as very-low-density lipoprotein (VLDL) during this period. This resulted in a sequestration of newly synthesized triacylglycerol in the cytosol, rather than in the particulate fraction of the cell. Longer periods of incubation with [3H]oleate resulted in increased concentrations of newly synthesized triacylglycerol within the cell, most of which (78 +/- 3% after 48 h; 80 +/- 3% after 72 h) was located within the cytosolic fraction. The quantity of newly synthesized triacylglycerol in the cell cytosol was further increased by insulin. During these periods there were decreases in the amounts of triacylglycerol associated with the particulate fraction of the cell, irrespective of the presence or absence of insulin. In no case was a decrease in VLDL triacylglycerol secretion in response to insulin accompanied by an increased triacylglycerol content in the particulate fraction of the cell. In some experiments, the fate of the cytosolic triacylglycerol was studied by pulse labelling with [3H]oleate. In these cases, when insulin was removed from the medium of cells to which they had previously been exposed, more newly synthesized triacylglycerol was secreted compared with cells which had not been exposed to insulin. This extra triacylglycerol was mobilized from the cytosolic rather than from the particulate fraction of the cell. Subsequent addition of insulin to the medium prevented the mobilization of cytosolic triacylglycerol. These results suggest that insulin enhances the storage of hepatocellular triacylglycerol in a cytosolic pool. Deficiency of insulin in the medium stimulates the mobilization of this pool which is channelled into the secretory pathway, entering the extracellular medium as VLDL.     

Insulin secretion from a continuously subcultivable isolate derived from foetal bovine pancreata beta cells. I. Monolayer cultures.

Monolayer cultures have been derived from bovine embryonic pancreatic cells grown in medium CMRL-1969 supplemented with foetal calf serum. The isolate has been subcultivated up to 10 population doublings. Insulin secretion from the cells into the culture medium declined with increasing passages. Of several insulin secretagogues, glucagon was found to be effective in potentiating insulin release from the cultivated cells into the medium. Insulin secretion rose to approximately 600 μU/culture/day in the presence of glucagon as compared to an average of 10 μU/culture/day in the control. This may be the first demonstration of a beta cell line developed from bovine embryonic pancreas.     

Regulation of glucose-6-P dehydrogenase activity in primary rat hepatocyte cultures

The glucose-6-P dehydrogenase specific activity in rat hepatocytes increases approximately 10-fold when the cells are placed into culture for three days. The induction requires insulin with maximum enzyme levels occurring at 10^?7 M. Pulse-labeling experiments revealed a 10-fold increase in the enzyme's relative rate of synthesis after only 8 hours in culture.     

Regulation of osteoprotegerin secretion from primary cultures of human bone marrow stromal cells

Osteoprotegerin (OPG) is a soluble receptor for receptor activator of NF kappa B-ligand, a factor required for osteoclastogenesis. OPG secreted from bone marrow stromal cells is believed to inhibit osteoclast differentiation and several agents known to influence bone resorption have been demonstrated to regulate mRNA levels of OPG. In this report we have investigated the secretion of OPG protein from primary cultures of human bone marrow stromal cells. An ELISA was developed for measuring the concentration of OPG in culture medium. OPG secretion was decreased by 50% when the human bone marrow stromal cells were treated with 1 microM of prostaglandin E(2), possibly through activation of the protein kinase A-pathway since stimulation of protein kinase A by forskolin also inhibited OPG secretion. Treatment with phorbol 12,13 di butyrate, an activator of the protein kinase C-pathway, potently stimulated the secretion of OPG from human bone marrow stromal cells. The cells were also stimulated with inflammatory mediators and glucocorticoids. Treatment with interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) stimulated OPG secretion to 500% and 400% of control whereas dexamethasone decreased OPG production by 40%. In conclusion, an ELISA measuring OPG in cell culture media was developed. Using this ELISA, the amount of OPG secreted from human bone marrow stromal cells was clearly detectable, and the secretion of OPG-protein was potently regulated by prostaglandin E(2), forskolin, phorbol 12,13 di butyrate, IL-1 alpha, TNF-alpha, and dexamethasone.     

Prostaglandins suppress VLDL secretion in primary rat hepatocyte cultures: relationships to hepatic calcium metabolism.

In primary cultures of rat hepatocytes, prostaglandin E2 and prostaglandin D2 (PGE2 and PGD2) inhibited the secretion of very low density lipoprotein (VLDL)-associated apoB, triacylglycerol, and cholesterol. These effects were concentration-dependent and remained apparent for at least 3 days of culture without an effect on the apoB/triacylglycerol ratio of the secreted VLDL. Prostaglandins had no effect on the overall synthesis of triacylglycerol but triacylglycerol accumulated within the cells, without intracellular accumulation of apoB. PGE2, when added to the medium together with glucagon, increased the inhibition of VLDL secretion, compared to that observed with glucagon alone. However, PGE2 did not increase the stimulatory effect of glucagon on ketogenesis. Unlike glucagon, the prostaglandins did not inhibit fatty acid synthesis nor did they stimulate ketogenesis or production of cAMP. Thus, of all the parameters of hepatic lipid metabolism studied, PGE2 and PGD2 selectively affected VLDL. Selective inhibition of VLDL secretion was also observed with the calcium antagonist verapamil. The divalent cation ionophore A23187 also inhibited VLDL release but, in contrast, also inhibited fatty acid and cholesterol synthesis. The results suggest that VLDL secretion is modulated at some optimal cell calcium concentration that may be mediated selectively by agents such as prostaglandins.     

Effects of insulin on lipoprotein secretion in rat hepatocyte cultures. The role of the insulin receptor

Insulin inhibits the secretion of lipoprotein components such as triglyceride, phospholipid, and apolipoproteins B and E in primary rat hepatocyte cultures. The aim of this study was to determine whether these hormonal effects are related to the interaction of insulin with its receptor on the surface of cultured hepatocytes. Half-maximal inhibition of secretion of apolipoprotein E and triglyceride occurred at 6 ng/ml porcine insulin, equivalent to a 20% receptor occupancy. When compared to porcine insulin, both guinea pig insulin and desoctapeptide insulin were 60 times less inhibitory on triglyceride and apolipoprotein secretion. These analogs were also 60 times less effective in competing with porcine 125I-insulin for receptor binding. Anti-insulin receptor IgG inhibited binding of porcine insulin to cells in a dose-dependent fashion. However, similar to the hormone itself, it reduced the secretion of triglyceride and apolipoproteins E and B. Preincubation of cells with 200 ng/ml porcine insulin for 15 h caused a 2.5-fold reduction of surface receptor number. These cells were less sensitive to the inhibitory effect of porcine insulin on secretion of triglyceride and apolipoproteins B and E. We conclude that the effects of insulin on lipoprotein processing by hepatocytes in culture are receptor-mediated, can be imitated by antibodies, to the insulin receptor, and are subject to control by receptor down-regulation.     

Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.     

Comparison of endocrine secretion by monolayer cultures derived by different procedures from neonatal hamster and rat pancreas.

Monolayer cultures derived from neonatal hamster or rat pancreas by two different epithelioid cell-enriching gravity sedimentation procedures varied in ability to maintain uniform levels of insulin secretion with increased culture age. Rat pancreatic cultures were superior in this respect to identically derived hamster preparations, depending on the preparative procedure employed. Quantitative differences in the temporal pattern of insulin secretion by different rat pancreatic culture preparations were ascribed to plating cell density and consequent terminal cell density as a function of preparative procedure such that reduced densities favored sustained secretory levels. These findings suggest the importance of tissue species and preparative procedure in deriving pancreatic monolayer cultures capable of sustained levels of insulin secretion with age.     

Regulation of ketogenesis, gluconeogenesis and the mitochondrial redox state by dexamethasone in hepatocyte monolayer cultures.

The effects of the glucocorticoid dexamethasone on fatty acid and pyruvate metabolism were studied in rat hepatocyte cultures. Parenchymal hepatocytes were cultured for 24 h with nanomolar concentrations of dexamethasone in either the absence or the presence of insulin (10 nM) or dibutyryl cyclic AMP (1 microM BcAMP). Dexamethasone (1-100 nM) increased the rate of formation of ketone bodies from 0.5 mM-palmitate in both the absence and the presence of BcAMP, but inhibited ketogenesis in the presence of insulin. Dexamethasone increased the proportion of the palmitate metabolized that was partitioned towards oxidation to ketone bodies, and decreased the cellular [glycerol 3-phosphate]. The latter suggests that the increased partitioning of palmitate to ketone bodies may be associated with decreased esterification to glycerolipid. The Vmax. of carnitine palmitoyltransferase (CPT) and the affinity of CPT for palmitoyl-CoA were not affected by dexamethasone, indicating that the increased ketogenesis was not due to an increase in enzymic capacity for long-chain acylcarnitine formation. Dexamethasone and BcAMP, separately and in combination, increased gluconeogenesis. In the presence of insulin, however, dexamethasone inhibited gluconeogenesis. Changes in gluconeogenesis thus paralleled changes in ketogenesis. Dexamethasone decreased the [3-hydroxybutyrate]/[acetoacetate] ratio, despite increasing the rate of ketogenesis and presumably the mitochondrial production of reducing equivalents. The more oxidized mitochondrial NADH/NAD+ redox couple with dexamethasone is probably due either to an increased rate of electron transport or to increased transfer of mitochondrial reducing equivalents to the cytoplasm.