Gene environment interaction and plasma triglyceride levels: the crucial role of lipoprotein lipase.
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Chylomicrons↗
Biomedical subjects
M S Liu
Publications and source records attributed to M S Liu.
Erythrocyte Ca2+ pump is defective during sepsis.
Saturated Ca2+ extrusion rate through the Ca2+ pump of erythrocytes was determined by the cobalt-exposure method in normal subjects and septic patients. From 48 normal subjects, the value of Vmax of erythrocyte Ca2+ pump was 14.83 +/- 0.49 mmol/L cells/hr; from 29 sepsis patients, it was 9.49 +/- 0.59 mmol/L cels/hr, significantly (P < 0.001) lower than that from the erythrocytes of normal subjects. When the severity of sepsis was evaluated by the septic severity score (SSS), a significant correlation (P < 0.0001) was observed between the Vmax of Ca2+ pump and the patient's SSS, indicating that the inhibition of Ca2+ pump depended on the degree of the pathological development of sepsis. Since the ATP-dependent Ca2+ transport in rat liver plasma membrane is also reduced during the late stage of sepsis [Lau et al., Circ Shock 38:238-244, 1992], impairment of the activity of Ca2+ pump appears to have a general pathophysiological significance in the development of severe sepsis.
Adenosine Triphosphate↗
Mutagenesis in four candidate heparin binding regions (residues 279-282, 291-304, 390-393, and 439-448) and identification of residues affecting heparin binding of human lipoprotein lipase.
Lipoprotein lipase (LPL) interaction with membrane-associated polyanions is a critical component of normal catalytic function. Two strong candidate binding regions, rich in arginine and lysine residues, have been defined in the N-terminal domain (aa279-282 and aa292-304) that show homology to the heparin-binding consensus sequences -X-B-B-X-B-X- and -X-B-B-B-X-X-B-X-, respectively. Additional candidate regions appear in the C-terminal domain, (residues 390-393), which are homologous to the thrombospondin heparin-binding repeat, and the positively charged terminal decapeptide (residues 439-448). To determine residues and domains critical to heparin binding, we have generated different LPL mutants that have alanine substitutions of single arginine and lysine residues and sequence interchanges with the homologous hepatic (HL) and pancreatic (PL) lipases. The mutant cDNAs were expressed in COS-1 cells and catalytically active mutants were assessed for binding to heparin-Sepharose. All the alanine substitutions within the two regions homologous to the heparin-binding consensus sequences in the N-terminal domain either abolished activity or produced a lowering of heparin binding affinity. None of the mutants in the C-terminal domain of LPL showed a loss of activity or a reduction in heparin binding affinity. These data demonstrate that charged residues at positions 279-282 and 292-304 of LPL are important for heparin binding affinity whereas the residues 390-393 and 439-448 in the C-terminal domain are not involved in heparin binding.
Amino Acid Sequence↗
Changes of alpha 1-adrenergic receptors in human liver during intraabdominal sepsis.
We studied changes in alpha 1-adrenergic receptors in human liver plasma membranes during intraabdominal sepsis using [3H]prazosin as a radioligand. Human liver tissues were obtained from nonseptic patients undergoing elective abdominal surgery (control group) and from patients with sepsis requiring laparotomy as a therapeutic measure (septic group). Septic patients were further divided into three subgroups on the basis of septic severity scores: mild sepsis (< 21), moderate sepsis (22 to 32) and severe sepsis (> 33). Plasma membranes were prepared by means of sucrose gradient centrifugation and were purified fivefold on the basis of the enrichment of the activity of the marker enzyme, 5'-nucleotidase. [3H]prazosin-binding studies show that the maximal binding capacity was increased by 49.6% (p < 0.01) in mild sepsis, relatively unchanged in moderate sepsis and decreased by 33.4% (p < 0.05) in severe sepsis (in femtomoles per milligram: 193.7 +/- 5.7 for control [n = 6], 289.8 +/- 23.4 for mild sepsis [n = 4], 192.3 +/- 16.3 for moderate sepsis (n = 4), 129.1 +/- 18.3 for severe sepsis [n = 5]). We found a significant inverse correlation (r = 0.85, p < 0.01) between changes in the densities of alpha 1-adrenergic receptors and septic severity scores. These data indicate that alpha 1-adrenergic receptors in human liver plasma membranes undergo dynamic changes during the development of sepsis--that is, the receptor number increased in mild sepsis, returned to a normal level in moderate sepsis and finally decreased in severe sepsis.(ABSTRACT TRUNCATED AT 250 WORDS)
5'-Nucleotidase↗
High frequency of mutations in the human lipoprotein lipase gene in pregnancy-induced chylomicronemia: possible association with apolipoprotein E2 isoform.
Partial deficiency in lipolysis usually results in only mild disturbances of lipid levels. However, when this is associated with impairment of the uptake of remnant particles and increased production of triglyceride-rich lipoproteins stimulated by environmental factors such as during normal pregnancy, chylomicronemia may ensue. We have previously reported a patient who had approximately 12% of normal LPL activity and developed severe chylomicronemia during pregnancy (Ma et al. 1993. J. Clin. Invest. 91: 1953-1958). Here we report four new patients with pregnancy-induced chylomicronemia. In the nonpregnant state, these patients had mild to modest elevation of triglyceride levels ranging from 80 to 623 mg/dl (0.9-7.0 mmol/l) but during the third trimester they became severely chylomicronemic with triglyceride levels ranging from 2314 to 14,596 mg/dl (26 to 164 mmol/l). Three of these four patients had partial lipoprotein lipase (LPL) deficiency. The molecular characterization of the LPL gene in these three patients with partial LPL deficiency revealed four novel unpublished mutations. Patient #1 is a compound heterozygote for Leu252Arg and Ala261Thr mutations which are associated with 25% of normal LPL activity. In addition, she has an apoE3/2 genotype. Patient #2 is a heterozygote for a Asn291Ser substitution with 69% of LPL activity and also has an apoE3/2 genotype, while patient #3 is a heterozygote for a Trp382Stop mutation with 54% of normal LPL activity and has an apoE4/2 genotype. The fourth patient (#4) with pregnancy-induced chylomicronemia does not have LPL deficiency and has an apoE3/3 genotype. The previously reported patient (#5) who had 12% of normal LPL activity due to homozygosity for a Ser172Cys mutation also has an E3/3 genotype. Our data suggest that mutations in the LPL gene that cause partial LPL deficiency might be a frequent factor in the pathogenesis of pregnancy-induced chylomicronemia.
Adult↗
A 4 basepair deletion in exon 4 of the human lipoprotein lipase gene results in type I hyperlipoproteinemia.
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Amino Acid Sequence↗
The Whitaker database of dysarthric (cerebral palsy) speech.
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Adult↗
Biphasic intracellular redistribution of alpha 1-adrenergic receptors in rat liver during sepsis.
Changes in the distribution of alpha 1-adrenergic receptors in two subcellular fractions, the plasma membrane and the light vesicle, of rat liver during sepsis were studied using [3H]prazosin binding and photoaffinity labeling with [125I]arylazidoprazosin in combination with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hypermetabolic (hyperglycemic) phase (9 h after CLP; early sepsis) followed by a hypometabolic (hypoglycemic) phase (18 h after CLP; late sepsis). [3H]prazosin binding studies show that during early sepsis, the maximal binding capacity (Bmax) was increased by 35% in plasma membranes but was decreased by 28% in light vesicles; while during late sepsis, the Bmax was decreased by 30% in plasma membranes but was increased by 33% in light vesicles. The photoaffinity labeling studies revealed two major binding peptides with M(r) of 77,000 and 68,000 Da and one minor binding protein with M(r) of 39,000 Da. The total binding for the three labeled peptides during early sepsis was increased by 26% in plasma membranes but was decreased by 33% in light vesicles, while during late sepsis the total binding was decreased by 19% in plasma membranes but was increased by 35% in light vesicles. These data indicate that alpha 1-adrenergic receptors in the rat liver were externalized from light vesicles to plasma membranes during the hyperglycemic phase while they were internalized from surface membranes to intracellular sites during the hypoglycemic phase of sepsis.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Gene-environment interaction in the conversion of a mild-to-severe phenotype in a patient homozygous for a Ser172-->Cys mutation in the lipoprotein lipase gene.
Normal pregnancy is associated with a two- to threefold increase in plasma triglyceride levels, particularly in the third trimester, due both to the overproduction of VLDLs and to the possible suppression of lipoprotein lipase (LPL) activity. Numerous mutations in the human LPL gene causing complete LPL deficiency have been described, but naturally occurring mutations that result in defective LPL with partial activity have not yet been reported. Here we describe a 30-yr-old woman who was first diagnosed with LPL deficiency during pregnancy after she developed pancreatitis. Her plasma triglyceride levels remained mildly elevated at approximately 300 mg/dl (3.4 mmol/liter) after the first pregnancy but rose significantly after she became pregnant again (1800 to 2000 mg/dl) (20.2 to 22.5 mmol/liter). DNA sequence analysis of the LPL gene showed that the patient is homozygous for a Ser172-->Cys missense mutation in exon 5. In vitro mutagenesis revealed that the Ser172-->Cys mutation caused a mutant LPL protein that had residual activity higher than that seen in all eight other missense mutations in patients with LPL deficiency identified in our laboratory. We propose that some mutations in the LPL gene produce a defective LPL with partial activity, which usually leads to mild hypertriglyceridemia.
Adult↗
Electrophysiologic properties of isolated adult cardiomyocytes from septic rats.
In this study, we examined the effect of sepsis on the electrical properties of isolated ventricular myocytes. Sepsis was induced by cecal ligation and puncture (CLP). The control rats were sham-operated. Membrane potentials and ionic currents in isolated cardiac myocytes were measured by the tight-seal, whole-cell patch-clamp technique. The results show that the resting membrane potentials of heart cells were significantly lower in the septic group (18 hr post-CLP) than those in the control group. However, there was no significant difference in action potential duration of 50% and 90% repolarization between the two groups of cells. In voltage-clamp experiments, isoproterenol (10 nM), a beta-adrenergic agonist, caused an increase in L-type calcium current (ICa,L) in a similar magnitude in myocytes isolated from the control and septic rats. Furthermore, isoproterenol failed to modify the time constants for ICa,L inactivation and the overall shape of current-voltage relationship for both groups of cells. These results indicate that formation of a G omega seal and subsequent tight-seal whole-cell recording with patch-clamp technique can be performed in heart cells derived from CLP-induced septic rats, and that septic rat heart is capable of responding effectively to beta-adrenergic stimulation.
Animals↗
Structure-function relationships of lipoprotein lipase: mutation analysis and mutagenesis of the loop region.
The molecular models of two microbial lipases and human pancreatic lipase (PL) have suggested the existence of common structural motifs including a buried active site shielded by an amphipathic surface loop. In an effort to explore the role of residues comprising the loop of lipoprotein lipase (LPL), we have used site-directed mutagenesis to generate three new LPL variants. In variant LPLM1 we deleted 18 amino acids leaving a loop of only 4 residues which resulted in an LPL protein inactive against triolein substrates. In contrast, two other LPL variants with only partial deletions, involving the apical section of the loop [LPLM2 (-8 amino acids) and LPLM3 (-2 amino acids)] manifested normal lipolytic activity. These findings indicate a critical requirement for the maintenance of charge and periodicity in the proximal and distal segments of the LPL loop in normal catalytic function. This is further highlighted by the detection of a mutation in the proximal section of the loop in a patient with LPL deficiency at position 225 which results in a substitution of threonine for isoleucine. The intact catalytic activity of the partial deletion variants (LPLM2 and LPLM3) further suggests that the apical residues of the loop contribute minimally to the functional motifs of the active site. We support this postulate by showing that the conserved glycine in the apical turn section (G229) can be substituted by glutamine, lysine, proline, or threonine without significantly affecting catalytic activity.
Base Sequence↗
Externalization and internalization of (Na+ + K+)-ATPase in rat heart during different phases of sepsis.
Changes in the distribution of (Na+ + K+)-ATPase in two subcellular fractions, the sarcolemma and the light vesicle, of rat heart during sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The alpha-subunit of (Na+ + K+)-ATPase was photoaffinity labeled with [alpha-32P]8-N3ATP. The results show that septic rat heart exhibits hyperdynamic (hypermetabolic) phase during early (9 hr post-CLP), followed by hypodynamic (hypometabolic) phase during late (18 hr post-CLP) sepsis. Marker enzyme and beta-adrenergic receptor assays depict that the light vesicle fraction is the intracellular site of surface receptor. The incorporation of the photolabel into the alpha-subunit (M(r) = 98,000) of the (Na+ + K+)-ATPase in sarcolemmal fraction was increased by 60% (P < 0.01) during early sepsis, but was decreased by 63% (P < 0.01) during late sepsis. In contrast, the binding of 98,000-M(r) peptide in light vesicles was decreased by 40% (P < 0.01) in early sepsis, but was increased by 102% (P < 0.01) during late sepsis. The ouabain-sensitive (Na+ + K+)-ATPase activity was increased by 31% (P < 0.05) during the early sepsis, but was decreased by 32% (P < 0.01) during late sepsis in the sarcolemmal fraction; while in the light vesicle fraction, the (Na+ + K+)-ATPase activity was decreased by 21% (P < 0.01) during early sepsis, but was increased by 47% (P < 0.01) during the late phase of sepsis. The yield of membrane proteins for each specific fraction remained unchanged for control, early sepsis, and late sepsis.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
[Kinetic studies of protein kinase A in rat liver during early sepsis].
Sepsis-induced glucose dyshomeostasis has been characterized by an initial hyperglycemia followed by a progressive hypoglycemia. It is well known that the liver plays a predominant role on regulating the homeostatic level of blood glucose. Furthermore, recent studies indicate that protein kinase A, activated by c-AMP, contributes to the role of glycagon in glucogenolysis and glyconeogenesis. Kinetic studies of protein kinase were completed. During late sepsis, in order to further understand the pathophysiology of hepatic glucose disturbances during sepsis. This study investigates the role of protein kinase A in the liver regulating carbohydrate metabolism during early sepsis. The work was performed by using an animal septic model, induced by cecal ligation and puncture (CLP) operation. Through the measurement of blood sugar, a two phase change in sugar level was found. That is, blood sugar significantly increased at 4.5 hrs after CLP operation (p < 0.05) and then significantly decreased at 18 hrs (p < 0.01). In the kinetic studies of protein kinase A, the results showed that, during early sepsis, the activities of both type I (eluted at low ionic strength) and type II (eluted at high ionic strength) protein kinase A were unchanged. Moreover, the kinetic parameters, Vmax and S0.5, of protein kinase A showed no significant difference between two groups. As such, it is suggested that hyperglycemia during early sepsis is not connected to the regulation of protein kinase A.
Adenosine Triphosphate↗
Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity.
A monoclonal antibody, 5D2, which inhibits human lipoprotein lipase (hLPL) activity has been widely used for assessment of LPL immunoreactive mass in the clinical evaluation of patients [1] and for analysis of structure-function relationships of LPL [2,3]. We have mapped the epitope on LPL, recognized by the 5D2 antibody, within residues 396-405. Ala400 is the critical amino acid residue conferring epitope specificity. This knowledge confirms that the C-terminal domain of LPL plays a critical role in LPL activity and also provides important information for studies exploring the structure-function relationship of LPL using this antibody.
Amino Acid Sequence↗
Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport.
Effects of endotoxin administration on the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca2+ transport in cardiac sarcolemma was decreased by 22 to 46% (p less than 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the Vmax for ATP and for Ca2+ were decreased by 50% (p less than 0.01) and 32% (p less than 0.01), respectively, while the Km values for ATP and Ca2+ were not significantly affected after endotoxin administration. Magnesium (1-5 mM) stimulated while vanadate (0.25-3.0 microM) inhibited the ATP-dependent Ca2+ transport, but the Mg(2+)-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca2+. Additional experiments show that the Ca2+ sensitivity of the Ca(2+)-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca2+ pumping without affecting Ca(2+)-ATPase activity. Since sarcolemmal ATP-dependent Ca2+ transport plays an important role in the regulation of cytosolic Ca2+ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca2+ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.
Adenosine Triphosphate↗
Heart sarcolemmal Ca2+ transport in endotoxin shock: II. Mechanism of impairment in ATP-dependent Ca2+ transport.
The role of the phosphorylation and dephosphorylation of sarcolemma and that of the alteration of membrane lipids in the endotoxin-induced impairment of the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated. The results indicate that the ATP-dependent Ca2+ transport in canine cardiac sarcolemma was decreased by 30-35% 4 h after endotoxin administration. Phosphorylation of sarcolemma by the catalytic subunit of the cAMP-dependent protein kinase or calmodulin stimulated ATP-dependent Ca2+ transport in both groups, however, the phosphorylation-stimulated activities remained significantly lower in endotoxic animals. Dephosphorylation of sarcolemma decreased ATP-dependent Ca2+ transport in both groups, yet, the time required to reach maximal dephosphorylation was reduced from 120 to 90 min 4 h post-endotoxin. Analysis of sarcolemmal membranes reveals that phosphatidylcholine and phosphatidylethanolamine contents were decreased while their respective lysophosphatide levels were increased significantly after endotoxin injection. Digestion of control heart sarcolemma with phospholipase A2 inhibited Ca2+ transport and the inhibition was reversible by phosphatidylcholine. The inhibition caused by the in vivo administration of endotoxin was completely reversible by the addition of phosphatidylcholine. Based on these data, it is concluded that endotoxin administration impairs ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment may be due to i) a defective phosphorylation of sarcolemma; ii) a reduced number of Ca2+ pumps; iii) an accelerated dephosphorylation of sarcolemma; and iv) an alteration in membrane phospholipid profile in response to phospholipase A activation.
Animals↗
Two naturally occurring mutations at the first and second bases of codon aspartic acid 156 in the proposed catalytic triad of human lipoprotein lipase. In vivo evidence that aspartic acid 156 is essential for catalysis.
We are studying naturally occurring mutations in the gene for lipoprotein lipase (LPL) to advance our knowledge about the structure/function relationships for this enzyme. We and others have previously described 11 mutations in human LPL gene and until now none of these directly involves any of the residues in the proposed Asp156-His241-Ser132 catalytic triad. Here we report two separate probands who are deficient in LPL activity and have three different LPL gene haplotypes, suggesting three distinct mutations. Using polymerase chain reaction cloning and DNA sequencing we have identified that proband 1 is a compound heterozygote for a G----A transition at nucleotide 721, resulting in a substitution of asparagine for aspartic acid at residue 156, and a T----A transversion, resulting in a substitution of serine for cysteine at residues 216. Proband 2 is homozygous for an A----G base change at nucleotide 722, leading to a substitution of glycine for aspartic acid at residue 156. The presence of these mutations in the patients and available family members was confirmed by restriction analysis of polymerase chain reaction-amplified DNA. In vitro site-directed mutagenesis and subsequent expression in COS cells have confirmed that all three mutations result in catalytically defective LPL. The two naturally occurring mutations, which both alter the same aspartic acid residue in the proposed Asp156-His241-Ser132 catalytic triad of human LPL, indicate that Asp156 plays a significant role in LPL catalysis. The Cys216----Ser mutation destroys a conserved disulfide bridge that is apparently critical for maintaining LPL structure and function.
Amino Acid Sequence↗
Altered ryanodine receptor of canine cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock.
Effects of endotoxin administration on ryanodine receptor in canine cardiac junctional sarcoplasmic reticulum (SR) were studied. The results show that the Bmax for [3H]ryanodine binding to cardiac junctional SR was decreased by 25% (8 +/- 0.38 vs 6 +/- 0.41 pmole/mg protein for control and endotoxic, respectively; (P less than 0.01) while the kd (13.7 +/- 1 nM for control vs 13.2 +/- 2 nM for endotoxic) was unaffected 4 hr following endotoxin administration. Ca2+ activated [3H]ryanodine binding in both groups sigmoidally but the Vmax for Ca2+ activation was decreased by 24% (P less than 0.05) while the S0.5 and the Hill coefficient values remained unchanged after endotoxin injection. Caffeine, ATP, and AMP-PCP activated while calmodulin, SKF-525A, ruthenium red, and Mg2+ inhibited [3H]ryanodine binding in both groups but the A0.5 (concentration requires for half-maximum activation) and the I50 (concentration requires for half-maximum inhibition) for the above-mentioned activators and inhibitors, respectively, were unaffected during endotoxin shock. Digestion of cardiac SR isolated from control dogs with phospholipase A2 inhibited [3H]ryanodine binding and the inhibition was reversed completely by the addition of phosphatidylserine. Addition of phosphatidylserine to cardiac SR isolated from endotoxic dogs stimulated [3H]ryanodine binding and the stimulation represents a complete reversal of the inhibition caused by endotoxin administration. Based on these findings together with previous observation that phospholipase A2 activity is activated during endotoxin shock, it is concluded that endotoxin administration decreases the number of ryanodine receptor in canine cardiac junctional SR and the decrease in ryanodine receptor is associated with a mechanism involving a modification of membrane lipid microenvironment in response to phospholipase A2 activation.
Adenosine Triphosphate↗