Math J.H. Geelen

The AMP-activated protein kinase prevents ceramide synthesis de novo and apoptosis in astrocytes

Fatty acids induce apoptosis in primary astrocytes by enhancing ceramide synthesis de novo. The possible role of the AMP‐activated protein kinase (AMPK) in the control of apoptosis was studied in this model. Long‐term stimulation of AMPK with 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) prevented apoptosis. AICAR blunted fatty acid‐mediated induction of serine palmitoyltransferase and ceramide synthesis de novo, without affecting fatty acid synthesis and oxidation. Prevention of ceramide accumulation by AICAR led to a concomitant blockade of the Raf‐1/extracellular signal‐regulated kinase cascade, which selectively mediates fatty acid‐induced apoptosis. Data indicate that AMPK may protect cells from apoptosis induced by stress stimuli.

Short-term Hormonal Control of Hepatic Lipogenesis

Lipids are essential components of all living cells, functioning as an energy store and playing an important role in all biologic membranes. Triacylglycerols, the storage form and most abundant of the glycerolipids, represent a highly concentrated form of energy, yielding twice as many calories per gram as carbohydrate or protein. The phospholipids are amphipathic compounds which, along with cholesterol, are found in membranes where they interact to provide a polar-faced, hydrophobic continuum of critical importance for proper cell structure and function. A major function of lipogenesis is to store (as triacylglycerols) the chemical energy or foodstuffs ingested above the immediate requirements of an organism. Contrary to protein, which is not stored, and to carbohydrate, which can only be stored in limited quantities, the capacity to store triacylglycerols is almost unlimited. The ability to keep large amounts of triacylglycerols on board would not be useful, however, if it were not possible to quickly and efficiently stop diverting foodstuffs into triacylglycerol formation and to quickly and efficiently start utilizing these compounds to provide the energy necessary to synthesize ATP. For this reason, mechanisms have evolved which finely tune the synthesis and degradation of triacylglycerol to the ever changing demand for ATP. In contrast, phospholipid and cholesterol synthesis must be, at least, partially maintained (or their degradation curtailed), even in the face of limited food intake or frank starvation, to provide sufficient quantities of these lipids to fulfill their crucial role in biologic membranes. In rodents the proportion of total body fatty acids synthesized by the liver ranges from 3% (fasted animals) to 50%

Opposite Effects of Insulin and Glucagon in Acute Hormonal Control of Hepatic Lipogenesis

Conditions for the isolation of rat hepatocytes that are responsive to insulin with regard to fatty acid synthesis were explored. Cells prepared according to the procedure of Ingebretsen and Wagle require the presence of fetal calf serum for insulin expression. Cells isolated by the Seglen method are the preparation of choice, since they respond to insulin in a simple, well-defined medium and, moreover, show much higher basal rates of fatty acid synthesis. In the latter cells isolated from fed male rats, the rate of fatty acid synthesis, as determined by tritium incorporation from [3H]H2O at 37°C, is enhanced within 30 min after addition of insulin to the incubation medium; with glucagon, it is depressed. In the presence of insulin, the cellular content of malonyl coenzyme A is noticeably increased, whereas the concentrations of pyruvate, lactate, and citrate are not markedly affected. Glucagon, on the other hand, decreases the concentrations of all four intermediates. The activity of acetyl-CoA carboxylase is stimulated and depressed after addition of insulin and glucagon, respectively. In all conditions tested, the activity of acetyl-CoA carboxylase correlates with the rate of fatty acid synthesis, which in turn correlates with the cellular level of malonyl-CoA.

Immunochemical quantitation of fatty-acid-binding proteins. I. Tissue and intracellular distribution, postnatal development and influence of physiological conditions on rat heart and liver FABP

Antisera against rat heart and liver fatty acid-binding protein (FABP) were applied in Western blotting analysis and ELISA to assess their tissue and intracellular distribution, and the influence of development, physiological conditions and several agents on the FABP content of tissue cytosols. The data obtained are compared with the oleic acid-binding capacity. Heart FABP is found in high concentrations in heart, skeletal muscles, diaphragm and lung, and in lower concentrations in kidney, brain and spleen, whereas liver FABP is limited to liver and intestine. In heart and liver, FABP is only present in the cytosol. The FABP content of both heart and liver shows a progressive increase during the first weeks of postnatal development, in contrast to their constant oleic acid-binding capacity. The reciprocally declining alpha-fetoprotein content of both tissues may partially account for the complementary fraction of the fatty acid-binding capacity. The FABP content and the fatty acid-binding capacity of adult heart and liver were in good accordance under various physiological conditions. Addition of clofibrate to the diet induces an increase of liver FABP content, whereas feeding of cholesterol, cholestyramine, mevinolin or cholate caused a marked decrease. The significance of the combined determination of fatty acid-binding capacity and FABP content (by immunochemical quantitation and blotting analysis) is indicated.

Short-term effects of insulin and glucagon on lipid synthesis in isolated rat hepatocytes: Covariance of acetyl-CoA carboxylase activity and the rate of 3H2O incorporation into fatty acids

The rate of fatty acid synthesis in cultures or suspensions of isolated hepatocytes has been shown to be stimulated with added insulin [l-3] and depressed with glucagon f l-71. Insulin acting alone does not change basal CAMP levels but does partially dampen glucagon-stimulated CAMP spikes [8.9]. Although added CAMP mimicks the glucagon effect in vitro [4,10-121 it cannot be assumed that fatty acid synthesis is uniquely controlled through changes in intracellular CAMP concentrations. The activity of acetyl-CoA carboxylase (EC 6.4.2.1), long considered the limiting enzyme in fatty acid synthesis,is decreased in liver in vivo following addition of glucagon [13] or in liver slices [14] and primary cultures of hepatocytes [7] following in vitro addition of CAMP. This was substantiated by the observation [5] that in liver steady-state levels of malonyl-CoA, the product of acetyl-CoA carboxylation, were rapidly depressed to l/4 normal following glucagon injection into intact rats. On the basis of this finding acetyl-CoA carboxylase was considered to be the focal point of glucagon signalling even though the activity of acetyl-CoA carboxylase, per se, as assayed in the liver cytosol, did not seem to be affected. Attempts to correlate the insulin enhancement of fatty acid synthesis in isolated hepatocytes [l-3] with changes in the activity

Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes

An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: The hepatocytes are made permeable by digitonin. 64 micrograms of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on mitochondrial permeability. Enzyme activity is measured by coupling the carboxylase reaction to the fatty acid synthase reaction. The advantages offered by this procedure over existing assays are: rapidity, no need to prepare cell extracts, absence of product inhibition, no interference by mitochondrial enzymes, useful in systems with bicarbonate buffers, and simple separation of radioactive substrate from labelled products. Using this coupled enzyme assay a good correlation was observed between changes in the activity of acetyl-CoA carboxylase and changes in the rate of fatty acid synthesis in hepatocytes as effected by short-term modulators.

The mitochondrial citrate carrier: metabolic role and regulation of its activity and expression.

The citrate carrier (CiC), a nuclear‐encoded protein located in the mitochondrial inner membrane, is a member of the mitochondrial carrier family. CiC plays an important role in hepatic lipogenesis, which is responsible for the efflux of acetyl‐CoA from the mitochondria to the cytosol in the form of citrate, the primer for fatty acid and cholesterol synthesis. In addition, CiC is a key component of the isocitrate–oxoglutarate and the citrate–malate shuttles. CiC has been purified from various species and its reconstituted function characterized as well as its cDNA isolated and sequenced. CiC mRNA and/or CiC protein levels are high in liver, pancreas, and kidney, but are low or absent in brain, heart, skeletal muscle, placenta, and lungs. A reduction of CiC activity was found in diabetic, hypothyroid, starved rats, and in rats fed on a polyunsaturated fatty acid (PUFA)‐enriched diet. Molecular analysis suggested that the regulation of CiC activity occurs mainly through transcriptional and post‐transcriptional mechanisms. This review begins with an assessment of the current understanding of CiC structural and biochemical characteristics, underlying the structure–function relationship. Emphasis will be placed on the molecular basis of the regulation of CiC activity in coordination with fatty acid synthesis.

Regulation of triacylglycerol synthesis in the liver a decrease in diacylglycerol acyltransferase activity after treatment of isolated rat hepatocytes with glucagon

Isolated rat hepatocytes were used to investigate the possibility of a short-term effect of glucagon on the synthesis of triacylglycerols in the liver. Incubation of hepatocytes in the presence of glucagon, followed by homogenization in a buffer containing F- (50 mM) and EDTA (2.5 mM), resulted in a 53% decrease in activity of microsomal diacylglycerol acyltransferase (EC 2.3.1.20), the only enzyme that is exclusively involved in the synthesis of triacylglycerols. The activity of cholinephosphotransferase (EC 2.7.8.2), which also uses diacylglycerols as substrate, was not decreased after exposure of the hepatocytes to glucagon. This may imply that triacylglycerol synthesis can be regulated independently of phosphatidylcholine synthesis. The activity of diacylglycerol acyltransferase in microsomes isolated from a homogenate of whole liver could be reduced by preincubating the microsomes with Mg2+ (5 mM), ATP (1 mM) and 105 000 X g supernatant. The enzyme could be reactivated by incubation of the washed microsomes with a 105 000 X g supernatant in the presence of dithiothreitol (5 mM). Fluoride (50 mM) inhibited this reactivation. It is concluded that the activity of diacylglycerol acyltransferase is subject to hormonal short-term control, possibly via a phosphorylation-dephosphorylation mechanism.

Ceramide sensitizes astrocytes to oxidative stress: protective role of cannabinoids

Cannabinoids induce apoptosis on glioma cells via stimulation of ceramide synthesis de novo, whereas they do not affect viability of primary astrocytes. In the present study, we show that incubation with Delta9-tetrahydrocannabinol did not induce accumulation of ceramide on astrocytes, although incubation of these cells in a serum-free medium (with or without cannabinoids) led to stimulation of ceramide synthesis de novo and sensitization to oxidative stress. Thus treatment with H2O2 induced apoptosis of 5-day-serum-deprived astrocytes and this effect was abrogated by pharmacological blockade of ceramide synthesis de novo. The sensitizing effect of ceramide accumulation may depend on p38 mitogen-activated protein kinase activation rather than on other ceramide targets. Finally, a protective role of cannabinoids on astrocytes is shown as a long-term incubation with cannabinoids prevented H2O2-induced loss of viability in a CB1 receptor-dependent manner. In summary, our results show that whereas challenge of glioma cells with cannabinoids induces accumulation of de novo -synthesized ceramide and apoptosis, long-term treatment of astrocytes with these compounds does not stimulate this pathway and also abrogates the sensitizing effects of ceramide accumulation.

The effects of insulin and glucagon on the release of triacylglycerols by isolated rat hepatocytes are mere reflections of the hormonal effects on the rate of triacylglycerol synthesis

1. Isolated hepatocytes from meal-fed donor rats secrete newly synthesized very-low-density lipoproteins (VLDL) when incubated in a simple bicarbonate buffer. When incubated with 3H2O for 2 h, 72-81% of the 3H-labelled triacylglycerols secreted by the hepatocytes were recovered in VLDL. The secretion of newly synthesized triacylglycerols shows a lag phase of about 30 min. 2. Insulin stimulates the secretion of newly synthesized VLDL triacylglycerols, whereas glucagon has an inhibitory effect on this process. 3. When hepatocytes triacylglycerols were labelled by preincubating the cells with 3H2O or [1-14C]oleate and the cells were subsequently washed and further incubated in radioisotope-free buffer containing hormones, it was observed that the release of the pre-labelled triacylglycerols is not hormone-sensitive. This suggests that insulin and glucagon do not affect the release of triacylglycerols per se. 4. It is concluded that the effects of insulin and glucagon on the overall process of triacylglycerol secretion are reflections of the hormone-determined rate of triacylglycerol synthesis.