Monitoring the partitioning of hepatic fatty acids in vivo: keeping track of control.

Noninvasive labelling of hepatic fatty acids in conscious, unrestrained rats shows how liver lipid metabolism responds acutely to physiological perturbations, such as the starved-to-refed transition. The speed with which the liver switches from fatty acid oxidation to esterification varies widely according to the requirement of the animal for continued synthesis of glucose 6-phosphate from three-carbon precursors. Ingestion of a meal also provides a strong signal for the diversion of fatty acids away from triacylglycerol synthesis and secretion, but insulin may only play an indirect role in this effect.

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.

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes.

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.

Sexual dimorphism in the preferential secretion of unsaturated lysophosphatidylcholine by rat hepatocytes but no secretion by sheep hepatocytes.

(1) Rat and ovine hepatocytes were incubated in monolayer culture with various fatty acids to determine their effects on the composition of the lysophosphatidylcholine that was secreted. (2) No lysophosphatidylcholine was detected in the medium from the ovine hepatocytes even though these cells were hormonally responsive and they secreted phosphatidylcholine and triacylglycerol in very-low-density lipoprotein. (3) Lysophosphatidylcholine was readily detected in the incubation medium of rat hepatocytes. The predominant fatty acids in this lipid were unsaturated. Stearate and arachidonate contributed 15 and 34%, and 24 and 26% of the total fatty acids when hepatocytes from male and female rats were used, respectively. The relative proportions of stearate and arachidonate in the phosphatidylcholine secreted from the hepatocytes were 20 and 14%, and 28 and 21% for the males and females, respectively. The equivalent values for stearate and arachidonate for phosphatidylcholine in the hepatocytes were 18 and 17% and 33 and 22% for male and female rats. These results provide further indications of sex differences in hepatic phospholipid metabolism and extend this to the secretion of phosphatidylcholine and lysophosphatidylcholine. (4) The addition of 1 mM stearate to the incubation medium did not significantly decrease the proportion of arachidonate in the lysophosphatidylcholine obtained from the hepatocytes of the male rats. However, the relative proportion of arachidonate was decreased in incubations that contained 1 mM oleate or linoleate. (5) The results provide evidence that the preferential secretion of unsaturated lysophosphatidylcholine by the liver may provide a system for transporting unsaturated fatty acids and choline to other organs in non-ruminant animals. However, this mechanism may not operate for ruminants.

Regulation of protein synthesis in lactating rat mammary tissue by cell volume.

The effect of changing cell volume on rat mammary protein synthesis has been examined. Cell swelling, induced by a hyposmotic challenge, markedly increased the incorporation of radiolabelled amino acids (leucine and methionine) into trichloroacetic acid (TCA)-precipitable material: reducing the osmolality by 47% increased leucine and methionine incorporation into mammary protein by 147 and 126% respectively. Conversely, cell shrinking, induced by a hyperosmotic shock, almost abolished the incorporation of radiolabelled amino acids into mammary protein: increasing the osmolality by 70% reduced leucine and methionine incorporation into mammary protein by 86 and 93% respectively. The effects of cell swelling and shrinking were fully reversible. Volume-sensitive mammary tissue protein synthesis was dependent upon the extent of the osmotic challenge. Isosmotic swelling of mammary tissue, using a buffer containing urea (160 mM), increased the incorporation of radiolabelled leucine into TCA-precipitable material by 106%. Swelling-induced mammary protein synthesis was dependent upon calcium: removing extracellular calcium together with the addition of EGTA markedly reduced volume-activated protein synthesis. Cell swelling-induced protein synthesis was inhibited by the Ca(2+) ATPase blocker thapsigargin suggesting that volume-sensitive protein synthesis is dependent upon luminal calcium.

Characteristics of L-carnitine transport by lactating rat mammary tissue.

The transport of L-carnitine by lactating rat mammary tissue has been examined. L-carnitine uptake by rat mammary tissue explants isolated from lactating rats, 3-4 days post partum, was via both Na+-dependent and Na+-independent pathways. The Na+-dependent pathway, the predominant route for L-carnitine uptake, was a saturable process: the Km and Vmax were, respectively, 132 microM and 201 pmol/2 h/mg of intracellular water. The Na+-independent pathway, which was non-saturable, had a coefficient of 0.26 microl/mg of intracellular water/2 h. The Na+-dependent component of L-carnitine uptake by mammary tissue explants was cis-inhibited by D-carnitine and acetyl-L-carnitine, but not by choline or taurine. In contrast, the Na+-independent component of L-carnitine uptake was not affected by any of these compounds. The uptake of L-carnitine by mammary tissue isolated from lactating rats, 10-12 days post partum, was qualitatively similar to that by mammary tissue taken from rats during the early stage of lactation. However, L-carnitine uptake was quantitatively lower: this was attributable to a reduction in the Na+-dependent component of L-carnitine uptake. L-Carnitine efflux from rat mammary tissue taken from animals 3-4 days post partum, consisted of at least two components; a fast extracellular component and a slow membrane-limited component. Reversing the trans-membrane Na+-gradient did not stimulate L-carnitine efflux suggesting that the Na+-dependent L-carnitine carrier operates with asymmetrical kinetics. A hyposmotic shock, hence cell-swelling, increased L-carnitine efflux from mammary tissue explants.

Submitochondrial and subcellular distributions of the carnitine-acylcarnitine carrier.

The submitochondrial and subcellular distributions of the carnitine-acylcarnitine translocase (CAC) have been studied. CAC is enriched to a much lesser extent than the carnitine palmitoyltransferases within the contact sites of mitochondria. A high-abundance protein of identical molecular size as the mitochondrial CAC that is immunoreactive with an anti-peptide antibody raised against a linear epitope of mitochondrial CAC is present in peroxisomes but not in microsomes. This suggests that CAC is targeted to at least two different locations within the liver cell and that acylcarnitine transport into peroxisomes is CAC mediated.

Mature carnitine palmitoyltransferase I retains the N-terminus of the nascent protein in rat liver.

Carnitine palmitoyltransferase I was isolated from octylglucoside extracts of rat liver mitochondrial outer membranes. This native enzyme was digested proteolytically with V8 protease. Five major peptides were obtained all of which were found in the amino acid sequence predicted from the full-length cDNA sequence of the protein. One peptide was found to correspond to the extreme N-terminus of the deduced amino acid sequence. Therefore, the mature protein retains the N-terminus of the nascent protein after import into the mitochondrial membrane. Knowledge of the identity of the N-terminus of the mature protein allows a reappraisal of the role of the two main. N-terminal hydrophobic domains of the protein and of the possible topology of the protein within the membrane.

Acute meal-induced changes in hepatic glycerolipid metabolism are unimpaired in severely diabetic rats: implications for the role of insulin.

The effect of food intake on the partitioning of diacylglycerol between phospholipid and triacylglycerol synthesis, and on the fractional rate of secretion of triacylglycerol was studied in starved-refed diabetic rats by using the technique of selective labelling of hepatic fatty acids in vivo. Acute and phasic responses in these parameters similar to those observed previously in normal animals were obtained, in spite of the absence of any insulin response to refeeding. Labelling of the major phospholipids (phosphatidylcholine and phosphatidylethanolamine) increased markedly at the expense of triacylglycerol labelling. In addition, the fractional rate of secretion of newly-labelled triacylglycerol was decreased. The data suggest that insulin is not obligatorily involved in any decrease in hepatic triacylglycerol secretion in the prandial period, but that it may act synergistically with other meal-induced signals to mediate this effect in normal animals.

A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis.

A highly purified rat liver protein kinase phosphorylates and inactivates acetyl-CoA carboxylase, and causes rapid inactivation of microsomal HMG-CoA reductase in the presence of MgATP. Both effects are stimulated in an identical manner by AMP, and are greatly reduced by prior treatment of the kinase with purified protein phosphatase. The dephosphorylated kinase can be reactivated in the presence of MgATP, apparently due to a distinct kinase kinase, and this reactivation is stimulated by nanomolar concentrations of palmitoyl-CoA. These results show that a common, bicyclic protein kinase cascade can potently inactivate the regulatory enzymes of both fatty acid and cholesterol biosynthesis.

Evidence that carnitine palmitoyltransferase I (CPT I) is expressed in microsomes and peroxisomes of rat liver. Distinct immunoreactivity of the N-terminal domain of the microsomal protein.

Mitochondria, microsomes and peroxisomes all express overt (cytosol-facing) carnitine palmitoyltransferase activity that is inhibitable by malonyl-CoA. The overt carnitine palmitoyltransferase activity (CPTo) associated with the different fractions was measured. Mitochondria accounted for 65% of total cellular CPTo activity, with the microsomal and peroxisomal contributions accounting for the remaining 25% and 10%, respectively. In parallel experiments, rat livers were perfused in situ with medium containing dinitrophenyl (DNP)-etomoxir in order to inhibit quantitatively and label covalently (with DNP-etomoxiryl-CoA) the molecular species responsible for CPTo activity in each of the membrane systems under near-physiological conditions. In all three membrane fractions, a single protein with an identical molecular mass of approximately 88,000 kDa (p88) was labelled after DNP-etomoxir perfusion of the liver. The abundance of labelled p88 was quantitatively related to the respective specific activities of CPTo in each fraction. On Western blots the same protein was immunoreactive with three anti-peptide antibodies raised against linear epitopes of the cytosolic N- and C-domains and of the inter-membrane space loop (L) domain of the mitochondrial enzyme (L-CPT I). However, the reaction of the microsomal protein with the anti-N peptide antibody (raised against epitope Val-14-Lys-29 of CPT I) was an order of magnitude stronger than expected from either microsomal CPTo activity or its DNP-etomoxiryl-CoA labelling. This suggests that the N-terminal domain of the microsomal protein differs from that in the mitochondrial or peroxisomal protein. This conclusion was confirmed using antibody back-titration experiments, in which the binding of anti-N and anti-C antibodies by mitochondria and microsomes was quantified.

Inhibition of carnitine palmitoyltransferase I by hepatocyte swelling.

Incubation of hepatocytes under conditions known to increase their volume, i.e. with amino acids (glutamine, proline) or in hypo-osmotic medium, decreased carnitine palmitoyl-transferase I (CPT-I) activity. This effect of hepatocyte swelling was antagonized by okadaic acid and dibutyryl-cAMP. Physiological concentrations of glutamate inhibited CPT-I activity in digitonin-permeabilized hepatocytes but not in isolated mitochondria. Results suggest that the amino acid-induced inhibition of CPT-I shares a common mechanism with the amino acid-induced stimulation of acetyl-CoA carboxylase and glycogen synthase [(1993) Eur. J. Biochem. 217, 1083-1089].

Volume-sensitive amino acid efflux from a pancreatic beta-cell line.

Cell swelling, induced by a hyposmotic shock, increased the fractional release of taurine from INS-1 cells. Volume-sensitive taurine release was (a) dependent upon the extent of cell swelling; (b) fully reversible; and (c) temperature dependent. Volume-sensitive taurine efflux was independent from the trans-membrane Na(+)-gradient. DIDS markedly inhibited volume-activated taurine efflux but not basal taurine release suggesting that the volume-sensitive pathway is quiescent under isosmotic conditions. Volume-activated taurine release inactivated in the continued presence of a hyposmotic shock. Cell-swelling also increased the fractional release of D-aspartate from INS-1 cells. Volume-activated D-aspartate efflux was inhibited by DIDS, albeit to a lesser extent than volume-sensitive taurine release. It is predicted that volume-sensitive amino acid efflux acts in parallel with other volume-activated transport mechanisms to regulate the volume of insulin-secreting cells.

Regulation of mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I): role of membrane-topology.

The topology of the outer membrane carnitine palmitoyltransferase (CPT I) of rat liver mitochondria was studied systematically using several experimental approaches. Studies with immobilized malonyl-CoA and octanoyl-CoA showed that functionally the active and regulatory sites of CPT I are exposed on the outer (cytosolic) surface of the mitochondrial outer membrane. Anti-peptide antibodies generated against three linear peptide sequences that occur in between and on either side of two hydrophobic, putative transmembrane domains were used to (a) ascertain which were bound by intact mitochondria and mitochondria in which the outer membrane was permeabilized to proteins; and (b) to determine the size of fragments generated by limited proteolysis (by trypsin or proteinase K) of CPT I in intact or outer membrane-ruptured mitochondria. The sizes and immunoreactivity of the proteolytic fragments generated were correlated with the effects of the proteases on CPT I activity and malonyl-CoA sensitivity. The results of all the different approaches suggested the following: (i) CPT I has two transmembrane domains; (ii) both the N- and C-termini are exposed on the cytosolic side of the membrane; (iii) the linker region between the two transmembrane domains protrudes into the intermembrane space; (iv) both the active site and the malonyl-CoA-binding site are exposed on the cytosolic side of the membrane; (v) the amino-terminus of the protein interacts with the C-terminal domain of the protein to maintain the optimal conformation required for activity of the enzyme and its sensitivity to malonyl-CoA.

Oleate acutely stimulates the secretion of triacylglycerol by cultured rat hepatocytes by accelerating the emptying of the secretory compartment.

The acute effects of addition of oleate on the rate of triacylglycerol (TAG) secretion by cultured rat hepatocytes were studied by monitoring the use of endogenous (14C-prelabeled) acyl moieties and exogenous (3H-labeled) oleate for the synthesis of secreted TAG simultaneously. Inclusion of exogenous oleate in the medium stimulated the secretion of the endogenous 14C-labeled acyl moieties by 55-100%. To find out whether the stimulation was due to increased endogenous TAG mobilization or an increased rate of processing of TAG within the endoplasmic reticulum (ER) secretory machinery, use was made of the inhibition of apolipoprotein B (apoB) synthesis (but not degradation) by Ca2+ mobilization from the ER. Inhibition of apoB synthesis stopped entry of acyl moieties (from endogenous and exogenous sources) into the secretory pathway. However, even when entry of acyl moieties into the secretory pathway was totally inhibited, exogenous oleate was still able to stimulate (twofold) the secretion [14C]TAG, indicating that oleate stimulates the emptying of prelabeled TAG from the secretory compartment at a point distal to apoB synthesis and nascent particle formation. These data indicate that exogenous oleate, besides providing additional acyl moieties for incorporation into secreted TAG, stimulates the secretion of endogenous TAG in a manner (i) that is independent of effects on apoB synthesis and/or degradation and (ii) that involves the enhanced processing of TAG resident within the ER secretory pathway.

Lipid molecular order in liver mitochondrial outer membranes, and sensitivity of carnitine palmitoyltransferase I to malonyl-CoA.

Mitochondrial outer membranes were prepared from livers of rats that were in the normal fed state, starved for 48 h, or made diabetic by injection of streptozotocin. Membranes were also prepared from starved late-pregnant rats. The latter three conditions have previously been shown to induce varying degrees of desensitization of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA inhibition. We measured the fluorescence polarization anisotropy of two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenes ulfonate (TMA-DPH) which, when incorporated into membranes, report on the hydrophobic core and on the peripheral regions of the bilayer, respectively. The corresponding polarization indices (rDPH and rTMA-DPH) were calculated. In membranes of all three conditions characterized by CPT I desensitization to malonyl-CoA, rDPH was decreased, whereas there was no change in rTMA-DPH, indicating that CPT I is sensitive to changes in membrane core, rather than peripheral, lipid order. The major lipid components of the membranes were analyzed. Although significant changes with physiological state were observed, there was no consistent pattern of changes in gross lipid composition accompanying the changes to membrane fluidity and CPT I sensitivity to malonyl-CoA. We conclude that CPT I kinetic characteristics are sensitive to changes in lipid composition that are localized to specific membrane microdomains.

The role of insulin in the control of hepatic fatty acid and glycerolipid metabolism assessed by studies in vivo.

The liver secretes triacylglycerol-rich very-low-density lipoproteins (VLDL). Teleologically, it would be expected that during the prandial/early absorptive period the rate of hepatic VLDL secretion would be inhibited, in view of the concomitant secretion of chylomicrons from the gut. Such inhibition would limit the extent and duration of post-prandial hyperlipaemia. Experiments in which the fatty acids of the liver were labelled specifically in rats in vivo show that, during a mean, triacylglycerol secretion was inhibited through a combination of diversion of flux towards phospholipid synthesis, and inhibition of the fractional rate of secretion of triacylglycerol. These adaptations occur even in diabetic rats, indicating that insulin is not obligatorily involved in mediating them. It is suggested that uptake of osmolytes (e.g. amino acids that are cotransported with Na+ ions into hepatocytes) from the portal circulation may result in increased hepatocyte volume and that this, in turn, alters fatty acid and glycerolipid metabolism independently of, but possibly in synergy with, insulin action.

Subcellular distribution of mitochondrial carnitine palmitoyltransferase I in rat liver. Evidence for a distinctive N-terminal structure of the microsomal but not the peroxisomal enzyme.

Mitochondria, microsomes and peroxisomes all express overt (cytosol-facing) carnitine palmitoyltransferase activities that are inhibitable by malonyl-CoA and are collectively termed CPTo. In order to quantify the relative contribution of the different membrane systems towards overall hepatocyte activity, all three membrane fractions and a high-speed supernatant (soluble) fraction were prepared quantitatively from rat liver homogenates. The overt (malonyl-CoA-sensitive) carnitine palmitoyltransferase activity (CPTo) associated with the different fractions were measured. In parallel experiments, rat livers were perfused in situ with oxygenated medium containing dinitrophenyl (DNP)-etomoxir in order to label covalently (with DNP-etomoxiryl-CoA) and quantitatively the molecular species responsible for CPTo activity in each of the membrane systems under near-physiological conditions. Mitochondria accounted for only 65% of total cellular overt CPT activity, with the microsomal and peroxisomal contributions accounting for the remaining 25% and 10%, respectively. A single major protein with an identical molecular size (Mr 88,000) was labelled by DNP-etomoxir perfusion in all three membrane fractions. The abundance of this 88 kDa protein in each fraction was quantitatively positively related to the respective specific activities of overt CPT. The same protein was immunoreactive with three anti-peptide antibodies raised against linear epitopes within the N- and C-terminal and loop (L) domains of the mitochondrial outer membrane CPT I of the liver mitochondrial outer membrane (L-CPT I). However, whereas reaction with anti-L and anti-C antipeptide antibodies were proportional to the respective overt CPT activities and DNP-etomoxir labelling in all three membrane fractions, reaction with anti-N peptide antibody was much stronger for microsomal CPT. We conclude that in all three membrane systems overt CPT activity is associated with the same or highly similar molecular species to mitochondrial outer membrane CPT I, but that the protein expressed in microsomes has a modified N-terminal domain, which gives the microsomal enzyme its higher malonyl-CoA sensitivity and may target the protein to its microsomal location.