Weight loss surgery in adolescents corrects high-density lipoprotein subspecies and their function.

BACKGROUND/OBJECTIVE: Youth with obesity have an altered high-density lipoprotein (HDL) subspecies profile characterized by depletion of large apoE-rich HDL particles and an enrichment of small HDL particles. The goal of this study was to test the hypothesis that this atherogenic HDL profile is reversible and that HDL function would improve with metabolic surgery. METHODS: Serum samples from adolescent males with severe obesity mean±s.d. age of 17.4±1.6 years were studied at baseline and 1 year following vertical sleeve gastrectomy (VSG). HDL subspecies and HDL function were evaluated pre and post VSG using paired t-tests. A lean group of adolescents was included as a reference group. RESULTS: After VSG, body mass index decreased by 32% and insulin resistance as estimated by homeostatic model assessment of insulin resistance decreased by 75% (both P<0.01). Large apoE-rich HDL subspecies increased following VSG (P<0.01) and approached that of lean adolescents despite participants with considerable residual obesity. In addition, HDL function improved compared with baseline (cholesterol efflux capacity increased by 12%, HDL lipid peroxidation potential decreased by 30% and HDL anti-oxidative capacity improved by 25%, all P<0.01). CONCLUSIONS: Metabolic surgery results in a significant improvement in the quantity of large HDL subspecies and HDL function. Our data suggest metabolic surgery may improve cardiovascular risk in adolescents and young adults.

Molecular cloning and characterization of a novel chicken gene named grni.

The full-length cDNA sequence of a novel expressed sequence tag (GenBank accession No. HQ184338) that was differentially expressed during Newcastle disease virus (NDV) infection in chickens was cloned from the chicken spleen by a rapid amplification of cDNA ends assay. This gene was further analyzed using bioinformatic methods and named grni. The full-length cDNA sequence was 1698 bp without introns, locating between 104,691,934 and 104,693,618 in galGal4 on chromosome 2. The open reading frame (ORF) contained 261 bp and encoded a deduced protein of 86 amino acid residues. Furthermore, the encoded protein contained two transmembrane regions without signal peptides, indicating that this protein is located in the mitochondrial membrane. Moreover, its homologous protein was not identified. Real-time polymerase chain reaction was used to detect the dynamic mRNA expression of this gene in the spleen, thymus, bursa of Fabricius, and trachea of NDV-infected chickens. Results suggested that the gene was involved in the transcriptional response of chicken to NDV infection. To obtain a fusion protein and prepare rabbit anti-serum, the predicted ORF of this gene was expressed in Escherichia coli. The expression of this gene at the protein level was further confirmed in the spleen, thymus, and bursa of Fabricius of NDV-infected chickens using Western blot analysis. In conclusion, a novel protein-coding gene named grni was successfully cloned and identified in chickens. Furthermore, this gene was found to be involved in the response of chickens to NDV infection.

Pancreatic acinar cell-specific overexpression of group 1B phospholipase A2 exacerbates diet-induced obesity and insulin resistance in mice.

Genome-wide association studies have identified significant association between polymorphisms of the Group 1B phospholipase A(2) (PLA2G1B) gene and central obesity in humans. Previous studies have shown that Pla2g1b inactivation decreases post-prandial lysophospholipid absorption, and as a consequence increases hepatic fatty acid oxidation and protects against diet-induced obesity and glucose intolerance in mice. The present study showed that transgenic mice with pancreatic acinar cell-specific overexpression of the human PLA2G1B gene gained significantly more weight and displayed elevated insulin resistance characteristics, such as impaired glucose tolerance, compared with wild-type (WT) mice, when challenged with a high-fat/carbohydrate diet. Pre- and post-prandial plasma ß-hydroxybutyrate levels were also lower, indicative of decreased hepatic fatty acid oxidation, in the hypercaloric diet-fed PLA2G1B transgenic mice. These, along with earlier observations of Pla2g1b-null mice, document that Pla2g1b expression level is an important determinant of susceptibility to diet-induced obesity and diabetes, suggesting that the relationship between PLA2G1B polymorphisms and obesity may be due to differences in PLA2G1B expression levels between these individuals. The ability of pancreas-specific overexpression of PLA2G1B to promote obesity and glucose intolerance suggests that target phospholipase activity in the digestive tract with non-absorbable inhibitors should be considered as a therapeutic option for metabolic disease therapy.

Two independent lipoprotein receptors on hepatic membranes of dog, swine, and man. Apo-B,E and apo-E receptors.

We have reported previously that canine livers possess two distinct lipoprotein receptors, an apoprotein (apo)-B,E receptor capable of binding the apo-B-containing low density lipoproteins (LDL) and the apo-E-containing cholesterol-induced high density lipoproteins (HDLc), and an apo-E receptor capable of binding apo-E HDLc but not LDL. Both the apo-B,E and apo-E receptors were found on the liver membranes obtained from immature growing dogs, but only the apo-E receptors were detected on th hepatic membranes of adult dogs. In this study, the expression of the apo-B,E receptors, as determined by canine LDL binding to the hepatic membranes, was found to be highly dependent on the age of the dog and decreased linearly with increasing age. Approximately 30 ng of LDL protein per milligram of membrane protein were bound via the apo-B,E receptors to the hepatic membranes of 7- to 8-wk-old immature dogs as compared with no detectable LDL binding in the hepatic membranes of adult dogs (greater than 1--1.5 yr of age). Results obtained by in vivo turnover studies of canine 125I-LDL correlated with the in vitro findings. In addition to a decrease in the expression of the hepatic apo-B,E receptors with age, these receptors were regulated, i.e., cholesterol feeding suppressed these receptors in immature dogs and prolonged fasting induced their expression in adult dogs. Previously, it was shown that the apo-B,E receptors were induced in adult livers following treatment with the hypocholesterolemic drug cholestyramine. In striking contrast, the apo-E receptors, as determined by apo-E HDLc binding, remained relatively constant for all ages of dogs studied (10--12 ng/mg). Moreover, the expression of the apo-E receptors was not strictly regulated by the metabolic perturbations that regulated the apo-B,E receptors. Similar results concerning the presence of apo-B,E and apo-E receptors were obtained in swine and in man. The hepatic membranes of adult swine bound only apo-E HDLc (apo-E receptors), whereas the membranes from fetal swine livers bound both LDL and apo-E HDLc (apo B,E and apo-E receptors). Furthermore, the membranes from adult human liver revealed the presence of the apo-E receptors as evidenced by the binding of 12--14 ng of HDLc protein per milligram of membrane protein and less than 1 ng of LDL protein per milligram. The membranes from the human liver also bound human chylomicron remnants and a subfraction of human HDL containing apo-E. These data suggest the importance of the E apoprotein and the apo-E receptors in mediating lipoprotein clearance, including chylomicron remnants, by the liver of adult dogs, swine, and man.

Accumulation of apolipoprotein E-rich high density lipoproteins in hyperalphalipoproteinemic human subjects with plasma cholesteryl ester transfer protein deficiency.

This study characterized the plasma lipoproteins of familial hyperalphalipoproteinemic patients with or without deficiency of cholesteryl ester transfer protein (CETP) activity. The subjects with CETP deficiency have increased levels of apolipoprotein (apo) E. The increased concentration of apo E in these subjects was correlated to the appearance of apo E-rich high density lipoproteins (HDL). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed that these lipoproteins contained predominantly the apo E (82%) and little amount of apo A-I (18%). These apo E-rich HDL displayed a much higher affinity than human LDL in binding to LDL receptors on human fibroblasts. Furthermore, 3.5 times fewer apo E-rich HDL than LDL were required to saturate the receptors on fibroblasts. These data indicated that the apo E-rich HDL in CETP-deficient human subjects contained multiple copies of apo E and bound to the LDL receptor through multiple interactions. The apo E-rich HDL, with similar properties as cholesterol-induced apo E HDLc, were not detectable in normal human subjects or in hyperalphalipoproteinemic subjects with normal CETP activity. The apo E-containing HDL in the latter subjects were smaller and contained only small amounts of apo E (14%). The difference in apo E-containing HDL in these subjects suggests a correlation between CETP level and the appearance of apo E-rich HDL.

A role for apolipoprotein E, apolipoprotein A-I, and low density lipoprotein receptors in cholesterol transport during regeneration and remyelination of the rat sciatic nerve.

Recent work has demonstrated that apo E secretion and accumulation increase in the regenerating peripheral nerve. The fact that apoE, in conjunction with apoA-I and LDL receptors, participates in a well-established lipid transfer system raised the possibility that apoE is also involved in lipid transport in the injured nerve. In the present study of the crushed rat sciatic nerve, a combination of techniques was used to trace the cellular associations of apoE, apoA-I, and the LDL receptor during nerve repair and to determine the distribution of lipid at each stage. After a crush injury, as axons died and Schwann cells reabsorbed myelin, resident and monocyte-derived macrophages produced large quantities of apoE distal to the injury site. As axons regenerated in the first week, their tips contained a high concentration of LDL receptors. After axon regeneration, apoE and apoA-I began to accumulate distal to the injury site and macrophages became increasingly cholesterol-loaded. As remyelination began in the second and third weeks after injury, Schwann cells exhausted their cholesterol stores, then displayed increased LDL receptors. Depletion of macrophage cholesterol stores followed over the next several weeks. During this stage of regeneration, apoE and apoA-I were present in the extracellular matrix as components of cholesterol-rich lipoproteins. Our results demonstrate that the regenerating peripheral nerve possesses the components of a cholesterol transfer mechanism, and the sequence of events suggests that this mechanism supplies the cholesterol required for rapid membrane biogenesis during axon regeneration and remyelination.

Chronic stress and Rosiglitazone increase indices of vascular stiffness in male rats.

Prolonged and/or frequent exposure to psychological stress responses may lead to deterioration of organs and tissues, predisposing to disease. In agreement with this, chronic psychosocial stress is linked to greater cardiovascular risk, including increased incidence of atherosclerosis, myocardial ischemia, coronary heart disease, and death. Thus the association between stress and cardiovascular dysfunction represents an important node for therapeutic intervention in cardiovascular disease. Here we report that 2weeks of chronic variable stress (CVS) increased indices of vascular stiffness, including increased collagen deposition in the aortic adventitia and increased resting pulse pressure, in male rats. Thus CVS may represent a useful rodent model for stress-associated CVD, especially for aging populations for which widening pulse pressure is a well-known risk factor. Additionally, we report that the thiazolidinedione Rosiglitazone (RSG) blunts chronic stress-associated increases in circulating corticosterone. Despite this, RSG was not protective against adverse cardiovascular outcomes associated with chronic stress. Rather RSG itself is associated with increased pulse pressure, and this is exacerbated by chronic stress-highlighting that chronic stress may represent an additional contributor to RSG-associated cardiovascular risk.

Inhibition by membrane-bound low-density lipoproteins of the primary inductive events of mitogen-stimulated lymphocyte activation.

Low-density lipoproteins (LDL) inhibit phytohemagglutinin (PHA)-enhanced accumulation of calcium ion and cyclic guanosine 3':5'-monophosphate, and phosphatidylinositol turnover in human lymphocytes. Inhibition by LDL of PHA-induced 32P incorporation into monophosphoinositide and cyclic guanosine 3':5'-monophosphate accumulation correlates directly with inhibition by LDL of calcium ion accumulation. These results suggest that calcium ion accumulation is required for PHA to direct turnover of monophosphoinositide and cyclic guanosine 3':5'-monophosphate synthesis in lymphocytes and that inhibition of this mitogen-induced response by LDL may be a direct consequence of the ability of these lipoproteins to prevent calcium ion accumulation by the activated cells. The ability of LDL to suppress PHA-enhanced calcium ion accumulation is directly proportional to the amount of LDL localized at the cell surface, indicating that internalization of the lipoproteins is not required for immunoregulation. This conclusion is supported by two additional lines of evidence: (a) LDL depleted of the bioregulatory constituent cholesterol are as effective as are native LDL in suppressing lymphocyte response to PHA; and (b) LDL-Sepharose complexes which cannot be endocytosed by the cells are as immunosuppressive as soluble LDL. The data clearly establish that membrane-bound LDL may have a regulatory role.

Liver-specific overexpression of LPCAT3 reduces postprandial hyperglycemia and improves lipoprotein metabolic profile in mice.

Previous studies have shown that group 1B phospholipase A2-mediated absorption of lysophospholipids inhibits hepatic fatty acid ß-oxidation and contributes directly to postprandial hyperglycemia and hyperlipidemia, leading to increased risk of cardiometabolic disease. The current study tested the possibility that increased expression of lysophosphatidylcholine acyltransferase-3 (LPCAT3), an enzyme that converts lysophosphatidylcholine to phosphatidylcholine in the liver, may alleviate the adverse effects of lysophospholipids absorbed after a lipid-glucose mixed meal. The injection of an adenovirus vector harboring the human LPCAT3 gene into C57BL/6 mice increased hepatic LPCAT3 expression fivefold compared with mice injected with a control LacZ adenovirus. Postprandial glucose tolerance tests after feeding these animals with a bolus lipid-glucose mixed meal revealed that LPCAT3 overexpression improved postprandial hyperglycemia and glucose tolerance compared with control mice with LacZ adenovirus injection. Mice with LPCAT3 overexpression also showed reduced very low density lipoprotein production and displayed elevated levels of the metabolic- and cardiovascular-protective large apoE-rich high density lipoproteins in plasma. The mechanism underlying the metabolic benefits of LPCAT3 overexpression was shown to be due to the alleviation of lysophospholipid inhibition of fatty acid ß-oxidation in hepatocytes. Taken together, these results suggest that specific LPCAT3 induction in the liver may be a viable strategy for cardiometabolic disease intervention.

Increased cholesterol esterase level by cholesterol loading of rat pancreatoma cells.

This study used the rat pancreatoma AR42J cells as a model to determine the effects of cholesterol on cholesterol esterase biosynthesis. Incubation of AR42J cells with low-density lipoproteins (LDL) or with the cholesterol-induced beta-very-low-density lipoproteins did not result in changes in cellular cholesterol levels. These cholesterol-rich lipoproteins also had no effect on cholesterol esterase biosynthesis by the AR42J cells. Cellular cholesterol level was found to increase by approx. 2-fold after incubating the AR42J cells with cationized-LDL. The increase in cellular cholesterol level resulted in a higher level of cholesterol esterase secreted into the culture medium. The increased cellular cholesterol also resulted in higher amounts of cholesterol esterase detected in the AR42J cell lysate. The increase in cholesterol esterase level corresponded to a cholesterol-induced increase in steady-state level of cholesterol esterase mRNA. The results of this study, and our previous observation of post-transcriptional activation of cholesterol esterase induced by intestinal hormones (Huang, Y. and Hui, D.Y. (1991) J. Biol. Chem. 266, 6720-6725), suggested that cholesterol esterase biosynthesis may be regulated by transcriptional and translational mechanisms in response to hormonal and nutrient stimulation of the pancreatic acinar cells. Additionally, the regulation of cholesterol esterase biosynthesis by cholesterol loading of the pancreatic cells suggested a possible role of this enzyme in cholesterol metabolism.

Interaction of plasma lipoproteins with erythrocytes. I. Alteration of erythrocyte morphology.

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL2 and HDL3, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes.

When incubated with intact erythrocytes, low density lipoproteins (LDL) decrease the phosphate content of erythrocyte spectrin allowing the cells to undergo morphological transformation. The phosphate content of spectrin depends on the balance between the activity of membrane-associated cyclic AMP-independent protein kinases and phosphoprotein phosphates. LDL do not influence the activity of membrane-associated cyclic AMP-independent protein kinases; these lipoproteins activate by 2-fold and greater membrane-associated phosphatases as determined by hydrolysis of p-nitrophenyl phosphate and by phosphate hydrolysis of phosphorylated erythrocyte membrane proteins. We conclude that LDL interact at the exterior surface of the erythrocyte to stimulate dephosphorylation of spectrin. The significance of this conclusion is augmented by the fact that spectrin, the target for LDL-induced dephosphorylation, specifies cell morphology and modulates the distribution of cell-surface receptors. LDL also render erythrocyte acetylcholinesterase less susceptible to inhition by F-. Lipoproteins in the high density class (HDL) do not stimulate dephosphorylation of spectrin, and they are consequently unable to alter erythrocyte morphology. HDL do prevent the LDL-induced activation of membrane phosphatase. The inhibitory capacity of HDL is observed over the range of LDL:HDL (w/w) which exists in the plasma of normolipemic humans.

Identity of a cytosolic neutral cholesterol esterase in rat liver with the bile salt stimulated cholesterol esterase in pancreas.

A neutral cholesterol esterase has been purified to homogeneity from the cytosolic fraction of rat liver. The 105,000 x g supernatant fraction of rat liver was applied to a DEAE-cellulose column to isolate a partially purified fraction of hepatic cholesterol esterase. Immunoblot analysis of the partially purified liver fraction with the anti-porcine pancreatic cholesterol esterase IgG demonstrated a single band with a molecular weight of 67,000. The hepatic protein was then isolated by immunoaffinity chromatography technique using a column constructed with antibodies prepared against the pancreatic cholesterol esterase. Characterization of the hepatic cholesterol esterase revealed that the hepatic enzyme shared antigenic epitopes with the pancreatic cholesterol esterase and was similarly activated by addition of bile salt such as taurocholate. Moreover, amino-terminal sequencing analysis of the hepatic cholesterol esterase showed an identical sequence with the pancreatic enzyme. Taken together, these results showed that the cholesterol esterases in the liver and the pancreas are very similar and possibly identical proteins.

Plasma cholesterol esterase level is a determinant for an atherogenic lipoprotein profile in normolipidemic human subjects.

Plasma cholesterol level is controlled by various factors. In the present study, high plasma activity of cholesterol esterase was found to correlate with plasma total cholesterol and low density lipoprotein (LDL) cholesterol levels in normolipidemic human subjects. However, the cholesterol esterase is not elevated in plasma of patients with familial hypercholesterolemia. These observations suggest that cholesterol esterase level is not determined by plasma cholesterol level, but elevated cholesterol esterase may be causative in increasing plasma cholesterol and LDL. Additional experiments further demonstrated that cholesterol esterase can convert the larger and less-atherogenic LDL to the smaller and more atherogenic LDL subspecies in vitro. These results suggest that plasma cholesterol esterase contributes to the formation and accumulation of atherogenic lipoproteins, and thus is a major risk factor for premature atherosclerosis in normal human subjects.

Binding of plasma low density lipoproteins to erythrocytes.

Low density lipoproteins (LDL) containing apolipoprotein B bind to intact, freshly isolated erythrocytes. The LDL-erythrocyte interaction is of low affinity, with a Kd of 1.1 x 10(-6) M. Binding is noncooperative. There are about 200 binding sites per cell and, within the limits of experimental uncertainty, these sites comprise a homogeneous class. Binding of LDL is a temperature-independent process. The maximum amount of LDL blood increases following proteolytic digestion of the cells with trypsin or chymotrypsin. The specificity of the binding sites for LDL is not absolute: high density lipoproteins and lipid vesicles composed of phosphatidylcholine or phosphatidylcholine/cholesterol (equimolar) complete with LDL for occupancy of 60% of the binding sites. Modification of 5--6 of the 9 apolipoprotein B arginine residues with 1,2-cyclohexanedione/borate or of 10--15 of the 20 lysine residues by reductive methylation does not alter the ability of LDL to bind to erythrocytes. Native LDL and methylated-LDL alter erythrocyte morphology. However, LDL in which the arginine residues are derivatized with 1,2-cyclohexanedione/borate do not induce the discocyte leads to echinocyte transformation. Chemically modified and native LDL exchange cholesterol with erythrocytes at equal rates and to nearly equal extents. Taken together, the data suggest that the binding sites for LDL on the erythrocyte membrane are distinct from the LDL receptors at the surface of other cells--e.g., fibroblasts and lymphocytes--which do not bind HDL and which do not recognize LDL with derivatized arginine or lysine residues. It is proposed that the biological function of the erythrocyte binding sites is to mediate the exchange of cholesterol between the cell membrane and lipoproteins.

Reduced immunoregulatory potency of low density lipoproteins with selectively modified arginine and lysine residues of apolipoprotein B.

Human plasma low density lipoproteins (LDL) suppress lymphocyte activation in vitro by inhibiting the early, membrane-associated events such as phytohemagglutinin-enhanced Ca2+ accumulation and phosphatidylinositol turnover. Chemical modification of the arginine residues of the protein constituent of LDL by 1,2-cyclohexanedione/borate or of the lysine residues by reductive methylation substantially decreases the immunosuppressive potency of LDL. The decrease in inhibitory capability of LDL correlates with a reduction in the ability of the derivatized LDL to compete with 125I-labeled LDL for lymphocyte membrane receptors. This correlation indicates that immunoregulation by LDL is the direct result of the binding of LDL to specific receptors at the cell surface. The receptor recognition site of LDL may consist of a high content of basic amino acid residues such that chemical modification of the LDL apolipoprotein reduces the LDL-lymphocyte interaction by specifically altering the change and/or steric properties of the receptor recognition site. Alternatively, chemical modification of arginine or lysine may cause a conformational change of the lipoprotein which is transmitted to the receptor recognition site. Derivatization of lysine or arginine residues does not elicit gross structural alteration of LDL, as evidenced by chemical analysis and by fluorescence quenching analysis. The intrinsic fluorescence intensity of LDL is, however, decreased by chemical modification, indicative of a minor but perhaps biologically significant structural alteration.

Molecular cloning and expression of cDNA for rat pancreatic cholesterol esterase.

A full-length cDNA complementary to the rat pancreatic cholesterol esterase mRNA was isolated by screening a rat pancreatic cDNA expression library in lambda gt11 vector with antibodies against the porcine pancreatic cholesterol esterase. The isolated cholesterol esterase cDNA is 2050 bp in length and contains an open reading frame coding for a protein of 612 amino acids. A 20-amino acid hydrophobic leader sequence is predicted, based on the position of the first ATG initiation codon upstream from the sequenced amino terminus of the isolated cholesterol esterase. The cholesterol esterase cDNA was subcloned into a mammalian expression vector, pSVL, for transfection studies. Expression of the cDNA in COS cells resulted in the production of bile salt-stimulated cholesterol esterase. Comparison of the cholesterol esterase cDNA sequence with other proteins revealed that the pancreatic cholesterol esterase is identical to rat pancreatic lysophospholipase. The primary structure of cholesterol esterase displayed no significant homology with other lipases, although the putative lipid interfacial recognition site of G-X-S-X-G is present in the cholesterol esterase sequence. However, the cholesterol esterase sequence revealed a 63-amino-acid domain which is highly homologous to the active site domain of other serine esterases. These data suggest that cholesterol esterase may be a member of the serine esterase supergene family. Analysis of the cholesterol esterase structure also revealed a repetitive sequence enriched with Pro, Asp, Glu, Ser, and Thr residues at the C-terminal end of the protein. This sequence is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that cholesterol esterase may have a short half-life in vivo. Northern blot hybridization showed that the bile salt-stimulated cholesterol esterase mRNA is present in liver suggesting that this protein may also be synthesized by liver cells.

Targeted overexpression of IGF-I in smooth muscle cells of transgenic mice enhances neointimal formation through increased proliferation and cell migration after intraarterial injury.

The response of arterial smooth muscle cells to injury is governed by a complex series of events. Significant among them is the paracrine production of peptide growth factors. To determine the impact of local IGF-I gene expression on vascular injury, the left carotid arteries of SMP8-IGF-I mice (in which IGF-I is selectively overexpressed in smooth muscle cells by means of a smooth muscle alpha-actin promoter) and wild-type controls were injured mechanically with an epon resin probe. After 7 and 14 d, a progressive increase in medial area was seen in both SMP8-IGF-I and wild-type mice, but they were not significantly different from each other. However, by 14 d there was a more than 4-fold increase in neointimal area in transgenic vs. wild-type. The intima/media ratios were also strikingly increased at 14 d in the IGF-I-overexpressing animals. The mitotic index, determined in animals injected daily with bromodeoxyuridine for 3 d before death, was markedly elevated in both the media and neointima 7 d after injury in SMP8-IGF-I mice, but the effect had subsided by 14 d. Despite a higher rate of cell division, the relative increase in medial area was less in the SMP8-IGF-I mice than in wild-type mice at both 7 and 14 d, consistent with a stimulation of cell migration to the neointima. The experiments reported here provide compelling evidence that paracrine expression of IGF-I is a powerful stimulus for smooth muscle cell proliferation and migration in vivo.

Molecular structure and tissue-specific expression of the mouse pancreatic phospholipase A(2) gene.

Pancreatic phospholipase A(2) (PLA(2)) is involved with the hydrolysis of phospholipids into lysophospholipids and unesterified fatty acids. The enzyme has been postulated to play a key role in lipid absorption by intestinal absorptive cells as well as in the regulation of secretin release from intestinal endocrine cells. This manuscript reports the genomic organization and the primary sequence of the mouse PLA(2). The results showed that the mouse PLA(2) gene contains four exons interspersed by three introns, spans over 8kb in length, and is considerably larger than the human PLA(2) gene. The mouse PLA(2) protein contains 146 amino acid residues, including the signal peptide. The mouse protein is highly homologous to the rat, dog, and human enzyme, but is two residues shorter than the human protein. Mouse PLA(2) message is synthesized predominantly in the pancreas, but the lung also contains low levels of PLA(2) mRNA.

Sequence identity between human pancreatic cholesterol esterase and bile salt-stimulated milk lipase.

Three overlapping cDNA clones covering the entire primary sequence of the bile salt stimulated lipase in human milk were isolated from a human breast lambda gt10 cDNA library by screening with the rat pancreatic cholesterol esterase cDNA. Nucleotide sequencing of the cDNA showed that the human milk lipase mRNA encodes a 748-residue protein, including a 23-residue signal peptide. The human milk lipase cDNA is highly homologous to rat pancreatic cholesterol esterase, suggesting that the milk lipase may be identical to the cholesterol esterase in human pancreas. This conclusion was confirmed by isolation and sequencing of the cDNA for human pancreatic cholesterol esterase. Analysis of the sequence for the human cholesterol esterase/milk lipase revealed similarities to other serine esterases in three distinct regions of the protein. These domains may represent the active site triads of these proteins.