Regulation of lipogenesis in vivo by glucose availability and insulin secretion in maternal and foetal tissues during late gestation in the rat. Effect of glucose intubation, streptozotocin-induced diabetes and starvation.

Administration of an oral load of glucose did not change the rate of lipogenesis in maternal liver during late gestation. However, streptozotocin-induced diabetes or starvation decreased maternal liver lipogenesis at 20-22 days of gestation. Glucose intubation, on the other hand, increased foetal lipogenesis at 21-22 days. In addition, maternal starvation decreased foetal lipogenesis and plasma insulin concentration. However, chronic hyperglycaemia induced by streptozotocin administration to the mother did not change foetal liver lipogenesis.     

Insulin-like effects of dithiothreitol on isolated rat adipocytes.

The rate of 3H2O incorporation into lipid in vivo progressively decreased in liver but increased in parametrial adipose tissue during the last 3 days of gestation. These changes seem to be related to those occurring in plasma insulin and progesterone concentrations during the same period. Foetal liver showed a high rate of lipogenesis, which sharply decreased before parturition. foetal lung lipogenesis increased during days 20 and 21 of gestation.     

Changes in the fatty acid profile of lung, liver and serum of rats intratracheally given silica

The esterified (E) and nonesterified (NE) fatty acid level and profile in the lung, serum, and liver of rats are significantly altered after intratracheal administration of silica. The changes include a silica-specific increase of the total long chain (C16-C20:4) fatty acid content in the lung, and a decrease in the serum and liver of both groups of rats intratracheally given silica and/or saline. In the silicotic lung, arachidonate and palmitate accumulated at the highest rate. A heat-labile, high-molecular weight component from lung homogenates increases lipogenesis in isolated hepatocytes in vitro. These findings, taken together with evidence indicating increased lipogenesis in the liver of rats treated with silica under identical conditions, suggest a lung-liver communication mechanism which coordinates lipid uptake by the lung and lipid synthesis and release by the liver. The stimulatory factor identified in lung homogenates might play an important regulatory role-for hepatic lipogenesis in rats developing silicotic lungs.     

Endotoxin promotes preferential periportal upregulation of VLDL secretion in the rat liver

Zonation affects liver parenchymal cell function and metabolism as well as nonparenchymal cell activation, but whether VLDL production is zonated has yet to be elucidated. Infection induces enhanced VLDL secretion by the liver. Ex vivo studies were undertaken to examine the liver heterogeneity for VLDL formation and secretion and their in vivo response to endotoxin. Highly pure periportal (PP) and perivenous (PV) hepatocytes were isolated from fasted lipopolysaccharide-treated, fasted, and fed rats. They were used to assess their capacity to release VLDL-apolipoprotein B (apoB) and lipid classes in relation to de novo lipid synthesis and the expression of genes crucial to VLDL production. Despite the common superior ability of PP hepatocytes for lipid release and zonal differences in lipid synthesis, zonated secretion of VLDL particles was observed in septic but not in normal fed or fasted livers. The endotoxin-induced apoB secretion was more accentuated in PP hepatocytes; this was accompanied by a preferential PP increase in apoB and microsomal triglyceride transfer protein mRNA levels, whereas lipogenesis indicators were, if anything, similarly modified in hepatocytes of either acinar origin. We conclude that PP and PV hepatocytes exhibited similar capabilities for VLDL formation/secretion in normal conditions; however, the endotoxic pressure did zonate periportally.     

Lipid biosynthesis in liver slices of the foetal guinea pig.

Lipid synthesis as measured by the incorporation of acetate or 3H2O into slices of foetal liver, is much higher than in slices of adult liver and shows a peak at about two-thirds of gestation. At this time the synthesis from glucose was low and reached a peak 10 days later. The changes in the activity of ATP citrate lyase, which mirrored acetate incorporation, and the effect of glucose and pyruvate on acetate corporation into lipid suggests that some of the lipid synthesis occurs via intramitochondrial acetyl-CoA production from acetate. Despite this, lipid synthesis was not inhibited by (-)-hydroxycitrate. The low rate of synthesis from glucose at two-thirds of gestation is ascribed to the low activity of pyruvate carboxylase at this time and a role for a phosphoenolpyruvate carboxykinase in providing oxaloacetate for lipogenesis is proposed. The activity of fatty acid synthetase broadly agreed with the changes in lipid synthesis, whereas the activity of acetyl-CoA carboxylase was barely sufficient to account for the rates of lipid synthesis in vivo. Acetate and short-chain fatty acids are likely to be the major precursors for lipid synthesis in vivo.     

Effects of inhibition of protein synthesis by cycloheximide on lipogenesis in mammary gland and liver of lactating rats.

1. Administration of cycloheximide (an inhibitor of protein synthesis) to lactating rats raised the concentrations of amino acids, and in particular, the branched-chain amino acids (valine, leucine and isoleucine) in blood, liver and mammary gland. 2. Inhibition of protein synthesis increased the incorporation in vivo of L-[U-14C]leucine into lipids of mammary gland and liver. 3. Cycloheximide treatment caused no immediate change in the overall rate of lipogenesis in vivo (measured with 3H2O) in mammary gland but increased the rate in liver 3-fold; this latter effect also occurred in livers of virgin rats. 4. The increased rate of hepatic lipogenesis was not accompanied by significant changes in the plasma insulin concentration or the activity of acetyl-CoA carboxylase. 5. Although cycloheximide decreased the entry of total triacylglycerol into the circulation it did not alter the rate of secretion of newly synthesized saponifiable lipid. 6. Cycloheximide slightly stimulated lipogenesis from endogenous substrates in isolated hepatocytes, but this effect was abolished when lactate was the exogenous substrate. 7. Administration of cycloheximide to virgin rats decreased liver glycogen and increased the hepatic content of glucose 6-phosphate, pyruvate and lactate. 8. It is concluded that (a) there is no short-term link between the rate of protein synthesis and lipogenesis in the lactating mammary gland and (b) the increased rate of hepatic lipogenesis in cycloheximide-treated rats is mainly due to stimulation of glycogenolysis, glycolytic flux and consequent increased availability of pyruvate.     

Coupling of muscarinic receptors to the mobilization of intracellular Ca2+ stores in permeabilized SH-SY5Y human neuroblastoma cells.

1. The effects of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine; PAF) on hepatic metabolism in vivo in rats were studied. 2. PAF stimulated synthesis of hepatic lipid (saponified and non-saponified) in a dose-dependent fashion and caused hypertriglyceridaemia. There was no effect of PAF on lipogenesis in isolated hepatocytes. 3. High doses of PAF also decreased hepatic glycogen. 4. All doses of PAF decreased plasma insulin, and this was accompanied by hyperglycaemia, except at the lowest dose. 5. The selective PAF-receptor antagonist L659.989 prevented the stimulation of lipogenesis, but indomethacin did not.     

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.     

Dietary regulation of mammary lipogenesis in lactating rats.

The proportion of medium-chain fatty acids (C8:0, C10:0 and C12:0) in rat milk increased significantly between day 4 and day 8 of lactation and for the remainder of lactation these acids comprised 40-50mol% of the total fatty acids. The milk fatty acid composition from day 8 was markedly dependent on the presence of dietary fat and altered to include the major fatty acids of the fats (peanut oil, coconut oil and linseed oil). The distribution of fatty acids made within the gland, however, was independent of dietary lipid and C8:0, C10:0 and C12:0 acids accounted for over 70% of the fatty acids made. The rates of lipogenesis in both the mammary gland and liver determined in vivo after the administration of 3H2O were affected by the presence of dietary lipid. In the mammary gland the rate for rats fed a diet containing peanut oil for 7 days was only one fifth that for rats fed a fat-free diet. Coconut oil also suppressed lipogenesis. Both dietary fats also suppressed lipogenesis in the liver.     

Differences in the accessibility of the beta-adrenergic receptor in isolated hepatocytes from foetal and adult rats.

Beta-receptor number (measured by [3H]-CGP 12 177 binding) and beta-adrenergic response (measured by isoproterenol stimulated glucose liberation and isoproterenol stimulated adenylate cyclase activity) were compared in hepatocytes isolated from foetal (on day 22 of gestation), adult female and adult male rats. Beta-receptor numbers in crude membrane preparations of hepatocytes from adult female and adult male rats were found to be nearly equal (15.5 and 15.1 fmol/mg), but in crude membrane preparations of foetal rats beta-adrenergic receptor number was significantly higher (34.3 fmol/mg). Determination of number of beta-adrenergic surface receptors of intact hepatocytes showed relative high values in foetal rats (about 22,000/cell) and adult female rats (about 20,000/cell), but in male rats the number was less (about 6500/cell). Glucose liberation was stimulated by isoproterenol to the same extent in hepatocytes isolated from adult female and foetal rats (about 150% over basal), whereas no effect was found in hepatocytes isolated from adult male rats. Dose-response curves showed that in foetal rat hepatocytes glucose release was already increased by 10(-8) M isoproterenol, whereas in female rat hepatocytes at least 10(-6) M isoproterenol was required. Adenylate cyclase was stimulated by isoproterenol in lysates of hepatocytes from adult female rats by about 180% and from foetal rats by about 250%. No effects were observed using lysates of hepatocytes from adult male rats. We interpret the observed differences of beta-adrenergic responses between adult female and male rats as being primarily caused by different accessibility of the beta-receptor to the beta-agonist isoproterenol in intact hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatty acid and sterol synthesis by hepatocytes of thermally acclimated rainbow trout (Salmo gairdneri).

Incorporation of tritium from tritiated water into lipid fractions was measured in isolated hepatocytes from rainbow trout (Salmo gairdneri) acclimated to 5 degrees C and 20 degrees C. Hepatocytes from cold-acclimated trout exhibited significantly higher rates of tritium incorporation into both fatty acid and sterol fractions at assay temperatures of 15 degrees C and 20 degrees C than did hepatocytes from warm-acclimated trout. Tritium incorporation into the fatty acid fraction was nearly temperature independent in hepatocytes from warm-acclimated trout (Q10 = 1.39) but markedly temperature dependent (Q10 = 2.63) in hepatocytes from cold-acclimated trout; in contrast, rates of sterol synthesis were more temperature dependent in warm-acclimated trout. At 5 degrees C, fatty acid lipogenesis comprised a significantly greater percentage of the total tritium incorporation in hepatocytes from warm-acclimated trout and the percentage of total lipogenesis attributable to fatty acids decreased significantly in warm-acclimated trout as the assay temperature increased; the opposite trends were observed in cold-acclimated trout.     

Regulation of lipogenesis and of non-saponifiable-lipid synthesis in vivo at birth and after prolonged starvation in the newborn rat.

The rate of lipogenesis in the liver was increased by glucose injection at birth, mediated by the insulin secretion. In addition, glucagon decreased the rates of lipogenesis and non-saponifiable-lipid synthesis after birth. These rates decreased after prolonged starvation in the newborn rat. Tri-iodothyronine injection increased the rates of lipogenesis enhanced in response to glucose administration after prolonged starvation in liver and brown adipose tissue. Dexamethasone, however, increased the rates of lipogenesis enhanced in response to glucose in liver and prevented the increase in the rates of lipogenesis in brown adipose tissue.     

Hepatic FoxO1 Integrates Glucose Utilization and Lipid Synthesis through Regulation of Chrebp O-Glycosylation

In liver, glucose utilization and lipid synthesis are inextricably intertwined. When glucose availability exceeds its utilization, lipogenesis increases, leading to increased intrahepatic lipid content and lipoprotein secretion. Although the fate of three-carbon metabolites is largely determined by flux rate through the relevant enzymes, insulin plays a permissive role in this process. But the mechanism integrating insulin receptor signaling to glucose utilization with lipogenesis is unknown. Forkhead box O1 (FoxO1), a downstream effector of insulin signaling, plays a central role in hepatic glucose metabolism through the regulation of hepatic glucose production. In this study, we investigated the mechanism by which FoxO1 integrates hepatic glucose utilization with lipid synthesis. We show that FoxO1 overexpression in hepatocytes reduces activity of carbohydrate response element binding protein (Chrebp), a key regulator of lipogenesis, by suppressing O-linked glycosylation and reducing the protein stability. FoxO1 inhibits high glucose- or O-GlcNAc transferase (OGT)-induced liver-pyruvate kinase (L-PK) promoter activity by decreasing Chrebp recruitment to the L-PK promoter. Conversely, FoxO1 ablation in liver leads to the enhanced O-glycosylation and increased protein level of Chrebp owing to decreased its ubiquitination. We propose that FoxO1 regulation of Chrebp O-glycosylation is a mechanism linking hepatic glucose utilization with lipid synthesis.     

Changes in the sensitivity of lipogenesis in rat hepatocytes to hormones and precursors over the diurnal cycle and during longer-term starvation of donor animals

In rat hepatocytes freshly isolated from donor rats at different times of the day, the rates of lipogenesis (de novo fatty acid synthesis) varied with a diurnal periodicity. The maximal rate occurred approximately 5 hr after the end of the normal 8-hr feeding period and at this time was four- to fivefold higher than the minimum rate which occurred midway through the feeding period. A similar diurnal pattern of change persisted even when the supply of lipogenic substrate, present in the medium as pyruvate, was not limiting. Although insulin stimulated the basal rates of lipogenesis to different relative extents in hepatocytes isolated at different times of the day, in absolute terms the hormone had little effect on the overall pattern of change during the diurnal cycle. The presence of pyruvate protected lipogenesis against inhibition by glucagon. The degree of protection varied over the diurnal cycle. During the early stages of starvation (up to 24 hr) there was a continuous decline in the rate of hepatocyte lipogenesis, irrespective of whether insulin and/or lipogenic substrate (pyruvate) were available or not. After this time the decline in the rate of lipogenesis was much less rapid. Seventeen hr after removal of food from donor rats, a point was reached beyond which pyruvate was incapable of supporting the maximum basal rate of lipogenesis which occurred during the normal diurnal cycle of fed rats. After this time lipogenesis in the presence of pyruvate was inhibited by glucagon to a much greater relative extent than that observed during feeding. The results suggest that variations in the rate of lipogenesis over the diurnal cycle and during the first 24 hr of starvation could not be accounted for entirely by fluctuations in substrate availability. In contrast, changes which occurred subsequent to this (up to 43 hr of starvation) could be eliminated when lipogenic substrate was made more abundant. Longer periods of starvation were marked by a relative increase in the ability of glucagon to prevent the substrate-induced stimulation of lipogenesis.     

Short-term stimulation of lipogenesis by triiodothyronine in maintenance cultures of rat hepatocytes

Within 4 h following the addition of 3,3',5 triiodo-L-thyronine to monolayer cultures of hepatocytes isolated from hypothyroid rats, a very distinct stimulation of fatty acid and cholesterol synthesis, measured as incorporation of either [1-14C]acetate or [3H]H2O into these lipid fractions, is observed. A smaller but significant increase in the rate of lipogenesis occurs in hepatocytes derived from euthyroid animals. These stimulatory effects of triiodothyronine are also observed in the presence of cycloheximide, indicating that the described early and direct stimulation of lipogenesis by the thyroid hormone is, at least in part, independent of protein synthesis.     

Lipid synthesis in vivo by tissues of the maternal and foetal guinea pig.

The rate of lipid biosynthesis in vivo was determined in pregnant guinea pigs after maternal and foetal injections of 3H2O. Synthesis in the maternal tissues was low and in the foetal liver and adipose tissues relatively high. In the foetal liver it reached a peak at about two-thirds of gestation, whereas that in the foetal adipose tissue occurred later. These results were used to support the view that lipid synthesis in the foetal guinea-pig liver at two-thirds of gestation is largely from short-chain fatty acids, whereas in foetal adipose tissue glucose is probably the major substrate.     

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.     

Metabolic changes in lipids of rat plasma and hepatocytes induced by 17 alpha-ethynylestradiol treatment

Cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were used to investigate the change of lipid metabolism induced by administration of 17 alpha-ethynylestradiol. Treatment with 17 alpha-ethynylestradiol caused a decrease of rat plasma lipids (free cholesterol, cholesterol ester, triacylglycerol and phosphatidylcholine). No difference in the ability of urea nitrogen synthesis could be demonstrated between cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats and propylene glycol-treated rats (control). Total cholesterol and cholesterol ester contents of cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were increased in comparison with those of the control. Triacylglycerol content of cultured hepatocytes was not affected by 17 alpha-ethynylestradiol treatment. There was no difference in the composition of lipid content between liver tissues and cultured hepatocytes. These results suggest that hepatocytes isolated from livers maintain the character of livers treated with 17 alpha-ethynylestradiol or livers treated with propylene glycol. Free cholesterol and cholesterol ester synthesis from [14C]acetic acid by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were decreased to about 30% of the control. Triacylglycerol and polar lipid (phospholipid) synthesis from [14C]acetic acid were not affected by 17 alpha-ethynylestradiol treatment. Microsomal hydroxymethylglutaryl-CoA reductase activity of rat liver treated with 17 alpha-ethynylestradiol was decreased to about 50% of control. The secretions of free cholesterol, cholesterol ester, triacylglycerol, phosphatidylcholine, apolipoprotein BL and BS by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol treated rats were not decreased when compared with the control. Because lipid and apolipoprotein secretions from cultured hepatocytes treated with 17 alpha-ethynylestradiol were not decreased and cholesterol contents of liver tissues and cultured hepatocytes treated with 17 alpha-ethynylestradiol were increased and hepatic microsomal hydroxymethylglutaryl-CoA reductase activity was decreased by 17 alpha-ethynylestradiol treatment, it is suggested that the liver plays an important role in hypolipidemia induced by 17 alpha-ethynylestradiol by increasing the plasma lipid uptake mediated by an increased amount of lipoprotein receptors of liver membranes.