Amelioration of hypertriglyceridemia with hypo-alpha-cholesterolemia in LPL deficient mice by hematopoietic cell-derived LPL

Background

Macrophage-derived lipoprotein lipase (LPL) has been shown uniformly to promote atherosclerotic lesion formation while the extent to which it affects plasma lipid and lipoprotein levels varies in wild-type and hypercholesterolemic mice. It is known that high levels of LPL in the bulk of adipose tissue and skeletal muscle would certainly mask the contribution of macrophage LPL to metabolism of plasma lipoprotein. Therefore, we chose LPL deficient (LPL^-/-) mice with severe hypertriglyceridemia as an alternative model to assess the role of macrophage LPL in plasma lipoprotein metabolism via bone marrow transplant, through which LPL will be produced mainly by hematopoietic cell-derived macrophages.

Methods and Results

Hypertriglyceridemic LPL^-/- mice were lethally irradiated, then transplanted with bone marrow from wild-type (LPL^+/+) or LPL^-/- mice, respectively. Sixteen weeks later, LPL^+/+ →LPL^-/- mice displayed significant reduction in plasma levels of triglyceride and cholesterol (408±44.9 vs. 2.7±0.5×10³ and 82.9±7.1 vs. 229.1±30.6 mg/dl, p<0.05, respectively), while a 2.7-fold increase in plasma high density lipoprotein- cholesterol (p<0.01) was observed, compared with LPL^-/-→LPL^-/- control mice. The clearance rate for the oral fat load test in LPL^+/+ →LPL^-/- mice was faster than that in LPL^-/-→LPL^-/- mice, but slower than that in wild-type mice. Liver triglyceride content in LPL^+/+→LPL^-/- mice was also significantly increased, compared with LPL^-/-→LPL^-/- mice (6.8±0.7 vs. 4.6±0.5 mg/g wet tissue, p<0.05, n = 6). However, no significant change was observed in the expression levels of genes involved in hepatic lipid metabolism between the two groups.

Conclusions

Hematopoietic cell-derived LPL could efficiently ameliorate severe hypertriglyceridemia and hypo-alpha-cholesterolemia at the compensation of increased triglyceride content of liver in LPL^-/- mice.     

Evidence of LPL gene-exercise interaction for body fat and LPL activity: the HERITAGE Family Study.

Evidence of a gene-exercise interaction for traits related to body composition is limited. Here, the association between the lipoprotein lipase (LPL) S447X polymorphism and changes in body mass index, fat mass, percent body fat, abdominal visceral fat measured by computed tomography, and post-heparin plasma LPL activity in response to 20 wk of endurance training was investigated in 741 adult white and black subjects. Changes were compared between carriers and noncarriers of the X447 allele after adjustment for the effects of age and pretraining values. No evidence of association was observed in men. However, white women carrying the X447 allele exhibited greater reductions of body mass index (P = 0.01), fat mass (P = 0.01), and percent body fat (P = 0.03); in black women, the carriers exhibited a greater reduction of abdominal visceral fat (P = 0.05) and a greater increase in post-heparin LPL activity (P = 0.02). These results suggest that the LPL S447X polymorphism influences the training-induced changes in body fat and post-heparin LPL activity in women but not in men.     

脂蛋白脂肪酶基因突变的可能影响

脂蛋白脂肪酶（LPL）是水解富含甘油三酯脂蛋白中甘油三酯的关键酶,其基因突变常可造成严重的高甘油三酯血症和胰腺炎等疾病。近年的研究发现,LPL基因突变的影响是广泛的,不仅可能与冠心病,高血压,阿尔茨海默病,围产期疾病等相关,而且某些突变可能延缓冠状动脉疾病和阿尔茨海默病的发生和发展。     

Relative hypoglycemia and hyperinsulinemia in mice with heterozygous lipoprotein lipase (LPL) deficiency. Islet LPL regulates insulin secretion.

Lipoprotein lipase (LPL) provides tissues with fatty acids, which have complex effects on glucose utilization and insulin secretion. To determine if LPL has direct effects on glucose metabolism, we studied mice with heterozygous LPL deficiency (LPL+/-). LPL+/- mice had mean fasting glucose values that were up to 39 mg/dl lower than LPL+/+ littermates. Despite having lower glucose levels, LPL+/- mice had fasting insulin levels that were twice those of +/+ mice. Hyperinsulinemic clamp experiments showed no effect of genotype on basal or insulin-stimulated glucose utilization. LPL message was detected in mouse islets, INS-1 cells (a rat insulinoma cell line), and human islets. LPL enzyme activity was detected in the media from both mouse and human islets incubated in vitro. In mice, +/- islets expressed half the enzyme activity of +/+ islets. Islets isolated from +/+ mice secreted less insulin in vitro than +/- and -/- islets, suggesting that LPL suppresses insulin secretion. To test this notion directly, LPL enzyme activity was manipulated in INS-1 cells. INS-1 cells treated with an adeno-associated virus expressing human LPL had more LPL enzyme activity and secreted less insulin than adeno-associated virus-beta-galactosidase-treated cells. INS-1 cells transfected with an antisense LPL oligonucleotide had less LPL enzyme activity and secreted more insulin than cells transfected with a control oligonucleotide. These data suggest that islet LPL is a novel regulator of insulin secretion. They further suggest that genetically determined levels of LPL play a role in establishing glucose levels in mice.     

Equivalent binding of wild-type lipoprotein lipase (LPL) and S447X-LPL to GPIHBP1, the endothelial cell LPL transporter

The S447X polymorphism in lipoprotein lipase (LPL), which shortens LPL by two amino acids, is associated with low plasma triglyceride levels and reduced risk for coronary heart disease. S447X carriers have higher LPL levels in the pre- and post-heparin plasma, raising the possibility that the S447X polymorphism leads to higher LPL levels within capillaries. One potential explanation for increased amounts of LPL in capillaries would be more avid binding of S447X-LPL to GPIHBP1 (the protein that binds LPL dimers and shuttles them to the capillary lumen). This explanation seems plausible because sequences within the carboxyl terminus of LPL are known to mediate LPL binding to GPIHBP1. To assess the impact of the S447X polymorphism on LPL binding to GPIHBP1, we compared the ability of internally tagged versions of wild-type LPL (WT-LPL) and S447X-LPL to bind to GPIHBP1 in both cell-based and cell-free binding assays. In the cell-based assay, we compared the binding of WT-LPL and S447X-LPL to GPIHBP1 on the surface of cultured cells. This assay revealed no differences in the binding of WT-LPL and S447X-LPL to GPIHBP1. In the cell-free assay, we compared the binding of internally tagged WT-LPL and S447X-LPL to soluble GPIHBP1 immobilized on agarose beads. Again, no differences in the binding of WT-LPL and S447X-LPL to GPIHBP1 were observed. We conclude that increased binding of S447X-LPL to GPIHBP1 is unlikely to be the explanation for more efficient lipolysis and lower plasma triglyceride levels in S447X carriers.     

VLDL-induced triglyceride accumulation in human macrophages is mediated by modulation of LPL lipolytic activity in the absence of change in LPL mass

Mixed dyslipidemia of phenotype IIB is characterized by elevated levels of very low density lipoprotein (VLDL)-1 and VLDL-2 subfractions and of low density lipoprotein (LDL), which are associated with premature formation of atherosclerotic plaques, characterized by the presence of lipid-rich macrophage foam cells. Lipoprotein lipase (LPL) is a key factor in mediating macrophage lipid accumulation and foam-cell formation from native VLDL particles. The action of macrophage-derived LPL in the induction of intracellular lipid accumulation from triglyceride-rich lipoprotein (TRL) subfractions (VLDL-1, VLDL-2) is, however, indeterminate, as is the potential role of VLDL-1 and VLDL-2 in modulating macrophage LPL expression. We evaluated the role of LPL in the interaction of type IIB VLDL-1 and VLDL-2 with human macrophages. Both VLDL-1 and VLDL-2 subfractions induced significant accumulation of triglyceride (9.8-fold, P<0.0001, and 4.8-fold, P<0.0001, respectively) and of free cholesterol content (1.4-fold, P<0.001, and 1.2-fold, P=0.02, respectively). Specific inhibition (90%) of the lipolytic activity of endogenous LPL by tetrahydrolipstatin (THL) in the presence of VLDL-1 or VLDL-2 resulted in marked reduction in cellular loading of both triglycerides (-89%, P=0.008, and -89%, P=0.015, respectively) and free cholesterol (-76%, P=0.02, and -55%, P=0.06 respectively). Furthermore, VLDL-1 and VLDL-2 induced marked increase in macrophage-derived LPL enzyme activity (+81%, P=0.002, and +45%, P=0.02), but did not modulate macrophage-derived LPL mRNA and protein expression; consequently, LPL specific activity was significantly increased from 1.6 mU/microg at baseline to 4.1 mU/microg (P=0.01) and 3.1 mU/microg (P=0.05), in the presence of VLDL-1 and VLDL-2, respectively. We conclude that type IIB VLDL-1 and VLDL-2 induce triglyceride accumulation in human monocyte-macrophages primarily via the lipolytic action of LPL, which may involve stabilization and activation of the macrophage-secreted enzyme, rather than via modulation of enzyme production.     

STAT 1 binds to the LPL promoter in vitro

Interferon-gamma (IFNgamma) has been shown to decrease the expression and activity of lipoprotein lipase (LPL). Hence, we searched for IFNgamma sensitive binding sites within the murine LPL promoter. A region of the LPL promoter was identified that specifically binds nuclear, but not cytosolic, extracts isolated from IFNgamma-treated 3T3-L1 adipocytes. EMSA analysis revealed that two protein complexes bind to this site within the LPL promoter and supershift analysis demonstrated that both of these complexes contained STAT 1 proteins. In addition, we have shown that this effect is specific for IFNgamma, since LIF treatment, which also induces STAT 1, did not confer binding to this site. Interestingly, binding to this site within the LPL promoter could be effectively competed with a STAT 1 binding site that we previously identified in the PPARgamma2 promoter. Also, IFNgamma treatment resulted in decreased levels of LPL protein. In summary, we have identified a STAT 1 binding site within the murine LPL promoter which likely plays a role in the IFNgamma induced decrease of LPL expression.     

A newly identified lipoprotein lipase (LPL) gene mutation (F270L) in a Japanese patient with familial LPL deficiency

We have systematically investigated the molecular defects resulting in a primary lipoprotein lipase (LPL) deficiency in a Japanese male infant (proband SH) with fasting hyperchylomicronemia. Neither LPL activity nor immunoreactive LPL mass was detected in pre- or postheparin plasma from proband SH. DNA sequence analysis of the LPL gene of proband SH revealed homozygosity for a novel missense mutation of F270L (Phe(270)-->Leu/TTT(1065)-->TTG) in exon 6. The function of the mutant F270L LPL was determined by both biochemical and immunocytochemical studies. In vitro expression experiments on the mutant F270L LPL cDNA in COS-1 cells demonstrated that the mutant LPL protein was synthesized as a catalytically inactive form and its total amount was almost equal to that of the normal LPL. Moreover, the synthesized mutant LPL was non-releasable by heparin because the intracellular transport of the mutant LPL to the cell surface - by which normal LPL becomes heparin-releasable - was impaired due to the abnormal structure of the mutant LPL protein. These findings explain the failure to detect LPL activities and masses in pre- and postheparin plasma of the proband. The mutant F270L allele generated an XcmI restriction enzyme site in exon 6 of the LPL gene. The carrier status of F270L in the proband's family members was examined by digestion with XcmI. The proband was ascertained to be homozygous for the F270L mutation and his parents and sister were all heterozygous. The LPL activities and masses of the parents and the sister (carriers) were half or less than half of the control values. Regarding the phenotype of the carriers, the mother with a sign of hyperinsulinemia manifested hypertriglyceridemia (type IV hyperlipoproteinemia), whereas the healthy father and the sister were normolipidemic. Hyperinsulinemia may be a strong determinant of hypertriglyceridemia in subjects with heterozygous LPL deficiency.     

Effect of maternal triglycerides and free fatty acids on placental LPL in cultured primary trophoblast cells and in a case of maternal LPL deficiency

Maternal hypertriglyceridemia is a normal condition in late gestation and is an adaptation to ensure an adequate nutrient supply to the fetus. Placental lipoprotein lipase (LPL) is involved in the initial step in transplacental fatty acid transport as it hydrolyzes maternal triglycerides (TG) to release free fatty acids (FFA). We investigated LPL activity and protein (Western blot) and mRNA expression (real-time RT-PCR) in the placenta of an LPL-deficient mother with marked hypertriglyceridemia. The LPL activity was fourfold lower, LPL protein expression 50% lower, and mRNA expression threefold higher than that of normal, healthy placentas at term (n = 4-7). To further investigate the role of maternal lipids in placental LPL regulation, we isolated placental cytotrophoblasts from term placentas and studied LPL activity and protein and mRNA expression after incubation in Intralipid (as a source of TG) and oleic, linoleic, and a combination of oleic, linoleic, and arachidonic acids as well as insulin. Intralipid (40 and 400 mg/dl) decreased LPL activity by approximately 30% (n = 10-14, P < 0.05) and 400 microM linoleic and linoleic-oleic-arachidonic acid (n = 10) decreased LPL activity by 37 and 34%, respectively. No major changes were observed in LPL protein or mRNA expression. We found no effect of insulin on LPL activity or protein expression in the cultured trophoblasts. To conclude, the activity of placental LPL is reduced by high levels of maternal TG and/or FFA. This regulatory mechanism may serve to counteract an excessive delivery of FFA to the fetus in conditions where maternal TG levels are markedly increased.     

A newly identified null allelic mutation in the human lipoprotein lipase (LPL) gene of a compound heterozygote with familial LPL deficiency.

In a Japanese patient with familial LPL deficiency, a new null allelic mutation, one base pair deletion at nucleotide position 916 was identified in exon 5 of one allele. In exon 3 of the other allele, we found the same nonsense mutation as we described previously in other Japanese kindreds. For the deletional mutant allele, we developed a simple detection method and constructed the DNA haplotype.     

Support for founder effect for two lipoprotein lipase (LPL) gene mutations in French Canadians by analysis of GT microsatellites flanking the LPL gene

Mutations in the human lipoprotein lipase (LPL) gene are one of the major causes of familial chylomicronemia. We have characterized two polymorphic GT microsatellites flanking this gene. Two LPL mutations that are extremely frequent in French Canadians appear to be in complete linkage disequilibrium with specific LPL microsatellite haplotypes indicating a founder effect within this population.     

Sex differences in abdominal, gluteal, and thigh LPL activity

Lipoprotein lipase (LPL) activity is necessary for adipocytes to take up triglycerides from the circulation, and regional differences in LPL activity could help determine regional fat storage. LPL activity has been reported to increase as a function of fat cell size, but this issue has not been extensively evaluated in different depots comparing sexes. Our objective was to determine whether sex alters the relationship between LPL activity and fat cell size. Subcutaneous adipose tissue biopsies were taken from the abdomen and thigh after an overnight fast and 1 h after a meal in 65 females (BMI 25.4 +/- 0.8, means +/- SE) and 41 males (BMI 23.7 +/- 0.3); gluteal adipose samples were obtained in 47 of the females and 27 of the males. Fat cell size was greater in females than males in thigh (P < 0.005) and gluteal (P < 0.05) regions but not in the abdomen. There was a relationship between fasting LPL activity/fat cell and fat cell size in females (abdomen r2 = 0.52, P < 0.0001; gluteal r2 = 0.23, P < 0.005; thigh r2 = 0.19, P < 0.005). In males, this relationship was seen only in the abdomen (r2 = 0.51, P < 0.0001) and thigh (r2 = 0.17, P < 0.05). Males and females had a significantly different relationship in the thigh only in the fasted state. Similar results were found in the fed state, although the strength of the relationship decreased in the abdominal regions for females only. This suggests fundamental differences in the regulation of triglyceride uptake between males and females and adipose regions.