Recent developments in lipid emulsions: relevance to intensive care.

For years, intravenous lipid supply has been considered as a means of providing an efficient fuel to many tissues of the body and of preventing or correcting essential fatty acid deficiency. The potential for lipid emulsions to modulate cell function via their content of specific fatty acids and of liposoluble vitamins has not received much attention yet. Soybean [long-chain triglycerides (LCT)] emulsions provide a valuable source of energy, but they are excessively rich in omega-6 essential fatty acids (FAs). Their infusion is associated with an accumulation of linoleate and a reduction of long-chain (> or = C20) omega-6 and omega-3 fatty acids in cell membrane phospholipids, as well as with a depletion of antioxidant status, associated with a reduction of alpha-tocopherol in plasma lipoproteins. Infusions of the mixed medium-chain triglycerides (MCT)/LCT (50%:50%; wt:wt) largely bypass these disadvantages. In addition, plasma elimination of MCT/LCT is faster than that of LCT. Recent advances indicate a great potential for omega-3 FAs incorporated into membrane phospholipids to modulate cell response to various stimuli and to influence several intracellular metabolic processes. Furthermore, some of these FA directly influence the production and the action of important mediators, the eicosanoids. In practical terms, an increased intake of omega-3 FAs may reduce inflammatory and thrombotic responses while protecting tissue microperfusion and immune defenses. Such properties may find interesting applications in several types of intensive care unit patients, provided that omega-3 FA incorporation takes place promptly. We recently had the opportunity to study in vitro and in vivo the metabolism of emulsions made of a mixture of MCT, soybean LCT, and fish oil triglycerides. Plasma elimination of such preparations appeared to be very fast, and their infusion was not associated with a prolonged residence of emulsion particles. In addition, uptake of remnants enriched with omega-3 FAs and liposoluble vitamins was fairly fast and occurred in several types of cells, leading to an efficient incorporation of omega-3 FAs in cell membranes within a few hours. The understanding that remnant uptake plays a significant role in the delivery of components included in lipid emulsions opens new areas of investigation and is likely to find several conditions of applications for new types of preparations.

Isoprenylated human brain type I inositol 1,4,5-trisphosphate 5-phosphatase controls Ca2+ oscillations induced by ATP in Chinese hamster ovary cells.

D-myo-Inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase and 3-kinase are thought to be critical regulatory enzymes in the control of InsP3 and Ca2+ signaling. In brain and many other cells, type I InsP3 5-phosphatase is the major phosphatase that dephosphorylates InsP3 and D-myo-inositol 1,3,4,5-tetrakisphosphate. The type I 5-phosphatase appears to be associated with the particulate fraction of cell homogenates. Molecular cloning of the human brain enzyme identifies a C-terminal farnesylation site CVVQ. Post-translational modification of this enzyme promotes membrane interactions and changes in specific activity. We have now compared the cytosolic Ca2+ ([Ca2+]i) responses induced by ATP, thapsigargin, and ionomycin in Chinese hamster ovary (CHO-K1) cells transfected with the intact InsP3 5-phosphatase and with a mutant in which the C-terminal cysteine cannot be farnesylated. [Ca2+]i was also measured in cells transfected with an InsP3 3-kinase construct encoding the A isoform. The Ca2+ oscillations detected in the presence of 1 microM ATP in control cells were totally lost in 87.5% of intact (farnesylated) InsP3 5-phosphatase-transfected cells, while such a loss occurred in only 1.1% of the mutant InsP3 5-phosphatase-transfected cells. All cells overexpressing the InsP3 3-kinase also responded with an oscillatory pattern. However, in contrast to control cells, the [Ca2+]i returned to base-line levels in between a couple of oscillations. The [Ca2+]i responses to thapsigargin and ionomycin were identical for all cells. The four cell clones compared in this study also behaved similarly with respect to capacitative Ca2+ entry. In permeabilized cells, no differences in extent of InsP3-induced Ca2+ release nor in the threshold for InsP3 action were observed among the four clones and no differences in the expression levels of the various InsP3 receptor isoforms could be shown between the clones. Our data support the contention that the ATP-induced increase in InsP3 concentration in transfected CHO-K1 cells is essentially restricted to the site of its production near the plasma membrane, where it can be metabolized by the type I InsP3 5-phosphatase. This enzyme directly controls the [Ca2+]i response and the Ca2+ oscillations in intact cells.

Lipoprotein metabolism during and after a 6-h infusion ofMCT/LCT vs LCT emulsion in man.

We studied, in man, the intravascular metabolism of two lipid emulsions differing in their triglyceride (TG) fatty acid pattern. One emulsion was composed exclusively of soy bean long-chain triglycerides (LCT), the other of a mixture containing a (1:1, wt:wt) ratio of medium-chain triglycerides (MCT) and LCT (MCT/LCT). Both emulsions contained 10% TG and 1.2% of the same egg yolk phospholipid emulsifier. Six healthy volunteers received both emulsions, in random order, at a rate of 0.2 g TG/kg.h for 6 h. An interval of 2 weeks separated the tests. Although the MCT/LCT emulsion provided 39% more TG molecules than the pure LCT emulsion, plasma TG increased to similar levels, indicating a faster elimination of MCT/LCT. The rise of plasma non esterified fatty acids was greater with MCT/LCT (P < 0.001). LDL-TG enrichment was higher with MCT/LCT (P < 0.025) while net transfer of TG to HDL was similar with both emulsions. Cholesteryl ester (CE) enrichment in the 'VLDL' fraction (largely composed of emulsion particles) was markedly less during MCT/LCT than LCT infusions (P < 0.01). CE enrichment of the 'VLDL' fraction persisted up to 6 h after cessation of both lipid infusions. In conclusion, TG from MCT/LCT emulsion appear to be eliminated faster than LCT during an in vivo infusion in man. In accordance with our previous in vitro data, MCT/LCT infusion was associated with a higher transfer of TG to LDL and in a reverse manner, with a lesser acquisition of CE by emulsion particles as compared to LCT infusion.

D-myo-inositol 1,4,5-trisphosphate 3-kinase A is activated by receptor activation through a calcium:calmodulin-dependent protein kinase II phosphorylation mechanism.

D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase, the enzyme responsible for production of D-myo-inositol 1,3,4,5-tetrakisphosphate, was activated 3- to 5-fold in homogenates of rat brain cortical slices after incubation with carbachol. The effect was reproduced in response to UTP in Chinese hamster ovary (CHO) cells overexpressing Ins(1,4,5)P3 3-kinase A, the major isoform present in rat and human neuronal cells. In ortho-32P-labelled cells, the phosphorylated 53 kDa enzyme could be identified after receptor activation by immunoprecipitation. The time course of phosphorylation was very similar to that observed for carbachol (or UTP)-induced enzyme activation. Enzyme phosphorylation was prevented in the presence of okadaic acid. Calmodulin (CaM) kinase II inhibitors (i.e. KN-93 and KN-62) prevented phosphorylation of Ins(1,4,5)P3 3-kinase. Identification of the phosphorylation site in transfected CHO cells indicated that the phosphorylated residue was Thr311. This residue of the human brain sequence lies in an active site peptide segment corresponding to a CaM kinase II-mediated phosphorylation consensus site, i.e. Arg-Ala-Val-Thr. The same residue in Ins(1,4,5)P3 3-kinase A was also phosphorylated in vitro by CaM kinase II. Phosphorylation resulted in 8- to 10-fold enzyme activation and a 25-fold increase in sensitivity to the Ca2+:CaM complex. In this study, direct evidence is provided for a novel regulation mechanism for Ins(1,4,5)P3 3-kinase (isoform A) in vitro and in intact cells.

Molecular study and regulation of D-myo-inositol 1,4,5-trisphosphate 3-kinase.

D-myo-Inositol 1,4,5-trisphosphate (InsP3) is a critical second messenger involved in signal transduction, i.e., calcium homeostasis. InsP3-kinase directly regulates the levels of InsP3 and D-myo-inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase is a calmodulin (CaM)-dependent enzyme and is also a target for phosphorylation by protein kinase C (PKC). Molecular cloning of cDNA's encoding proteins presenting InsP3 3-kinase activity establish the existence of distinct isoenzymes (at least three: A, B and C). These isoforms are differentially expressed and regulated by calcium/CaM. Site-directed mutagenesis and chemical modification of InsP3 3-kinase A led to the identification of three charged residues involved in ATP/Mg2+ binding among the catalytic domain and a hydrophobic residue taking part of the CaM binding site.

Active site labelling of inositol 1,4,5-trisphosphate 3-kinase A by phenylglyoxal.

Chemical modification by phenylglyoxal, an arginine-specific reagent, of both native and recombinant rat brain inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase A was accompanied by irreversible inhibition of enzyme activity. This effect was prevented in the presence of the substrate ATP but not Ins(1,4,5)P3. The modification reaction obeyed pseudo-first-order rate kinetics. Complete inhibition of activity corresponded to incorporation of 1.2 mol of phenylglyoxal per mol of protein. A single [14C]phenylglyoxal-modified peptide was isolated following alpha-chymotrypsin digestion of the radiolabelled Ins(1,4,5)P3 3-kinase and reverse-phase HPLC. ATP prevented the incorporation of radioactivity to this peptide. The peptide sequence (i.e. QWREGISSSTTL) corresponded to amino acids 315 to 326 of rat brain Ins(1,4,5)P3 3-kinase A. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivative showed the modified amino acid to be Arg-317. The data directly identify a reactive arginine residue as part of the ATP-binding site. Arg-317 is located within a sequence segment which is conserved among the catalytic domain of Ins(1,4,5)P3 3-kinase isoenzymes A and B in human and rat species.

Tissue- and cell-specific expression of Ins(1,4,5)P3 3-kinase isoenzymes.

The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells.

Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase.

In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.

Identification of high molecular weight forms of inositol 1,4,5-trisphosphate 3-kinase in rat thymus and human lymphocytes.

A soluble inositol 1,4,5-trisphosphate 3-kinase (InsP3 3-kinase) has been characterized from extracts of rat thymus. The enzyme was shown to have a molecular weight within the range 98,000-114,000 M(r) as determined by regeneration of enzyme activity from sodium dodecyl sulphate polyacrylamide gels. The enzyme phosphorylates inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5-tetrakisphosphate (InsP4) with an apparent Km of 3.1 +/- 0.4 microM. The enzyme is stimulated 4-6-fold by Ca2+/calmodulin and is not recognised by polyclonal antisera raised against rat brain InsP3 3-kinase A. High levels of InsP3 3-kinase activity were also detected in soluble extracts of human lymphocyte preparations. The human lymphocyte enzyme was shown to have a molecular weight between 61,000 and 70,000 M(r) as judged by SDS-PAGE, and was stimulated approximately 10-fold in the presence of Ca2+/calmodulin. These results establish that InsP3 3-kinase from rat thymus and human lymphocyte preparations represent high molecular weight isoenzymes of the InsP3 3-kinase family.

Purification and biochemical properties of a high-molecular-mass inositol 1,4,5-trisphosphate 3-kinase isoenzyme in human platelets.

The phosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5-tetrakisphosphate (InsP4) is catalysed by InsP3 3-kinase. A method is presented for a rapid purification of the enzyme from human platelets. The purified enzyme was identified as a polypeptide of M(r) 69,000-70,000 after SDS/PAGE. It had a specific activity of 1.45 +/- 0.1 mumol/min per mg, and the degree of stimulation by Ca2+/calmodulin was 17-fold at saturating calmodulin and 10 microM free Ca2+. The Km for InsP3 and for ATP was 2.0 microM and 2.5 mM respectively. Human platelet InsP3 3-kinase was not recognized by immunodetection with anti-(InsP3 3-kinase A) or anti-(InsP3 3-kinase B) antibodies. These data provide the first biochemical evidence for the existence of a novel InsP3 3-kinase isoenzyme in human platelets, which is distinct from previously reported InsP3 3-kinase A and InsP3 3-kinase B.