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.     

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.     

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.     

A missense (Asp250----Asn) mutation in the lipoprotein lipase gene in two unrelated families with familial lipoprotein lipase deficiency.

We have identified the molecular basis for familial lipoprotein lipase (LPL) deficiency in two unrelated families with the syndrome of familial hyperchylomicronemia. All 10 exons of the LPL gene were amplified from the two probands' genomic DNA by polymerase chain reaction. In family 1 of French descent, direct sequencing of the amplification products revealed that the patient was heterozygous for two missense mutations, Gly188----Glu (in exon 5) and Asp250----Asn (in exon 6). In family 2 of Italian descent, sequencing of multiple amplification products cloned in plasmids indicated that the patient was a compound heterozygote harboring two mutations, Arg243----His and Asp250----Asn, both in exon 6. Studies using polymerase chain reaction, restriction enzyme digestion (the Gly188----Glu mutation disrupts an Ava II site, the Arg243----His mutation, a Hha I site, and the Asp250----Asn mutation, a Taq I site), and allele-specific oligonucleotide hybridization confirmed that the patients were indeed compound heterozygous for the respective mutations. LPL constructs carrying the three mutations were expressed individually in Cos cells. All three mutant LPLs were synthesized and secreted efficiently; one (Asp250----Asn) had minimal (approximately 5%) catalytic activity and the other two were totally inactive. The three mutations occurred in highly conserved regions of the LPL gene. The fact that the newly identified Asp250----Asn mutation produced an almost totally inactive LPL and the location of this residue with respect to the three-dimensional structure of the highly homologous human pancreatic lipase suggest that Asp250 may be involved in a charge interaction with an alpha-helix in the amino terminal region of LPL. The occurrence of this mutation in two unrelated families of different ancestries (French and Italian) indicates either two independent mutational events affecting unrelated individuals or a common shared ancestral allele. Screening for the Asp250----Asn mutation should be included in future genetic epidemiology studies on LPL deficiency and familial combined hyperlipidemia.     

Inactive lipoprotein lipase (LPL) alone increases selective cholesterol ester uptake in vivo, whereas in the presence of active LPL it also increases triglyceride hydrolysis and whole particle lipoprotein uptake.

We have previously shown that transgenic expression of catalytically inactive lipoprotein lipase (LPL) in muscle (Mck-N-LPL) enhances triglyceride hydrolysis as well as whole particle lipoprotein and selective cholesterol ester uptake. In the current study, we have examined whether these functions can be performed by inactive LPL alone or require the presence of active LPL expressed in the same tissue. To study inactive LPL in the presence of active LPL in the same tissue, the Mck-N-LPL transgene was bred onto the heterozygous LPL-deficient (LPL1) background. At 18 h of age, Mck-N-LPL reduced triglycerides by 35% and markedly increased muscle lipid droplets. In adult mice, it reduced triglycerides by 40% and increased lipoprotein particle uptake into muscle by 60% and cholesterol ester uptake by 110%. To study inactive LPL alone, the Mck-N-LPL transgene was bred onto the LPL-deficient (LPL0) background. These mice die at approximately 24 h of age. At 18 h of age, in the absence of active LPL, inactive LPL expression did not diminish triglycerides nor did it result in the accumulation of muscle lipid droplets. To study inactive LPL in the absence of active LPL in the same tissue in adult animals, the Mck-N-LPL transgene was bred onto mice that only expressed active LPL in the heart (LPL0/He-LPL). In this case, Mck-N-LPL did not reduce triglycerides or increase the uptake of lipoprotein particles but did increase muscle uptake of chylomicron and very low density lipoprotein cholesterol ester by 40%. Thus, in the presence of active LPL in the same tissue, inactive LPL augments triglyceride hydrolysis and increases whole particle triglyceride-rich lipoprotein and selective cholesterol ester uptake. In the absence of active LPL in the same tissue, inactive LPL only mediates selective cholesterol ester uptake.     

Lipoprotein lipase (LPL) strongly links native and oxidized low density lipoprotein particles to decorin-coated collagen. Roles for both dimeric and monomeric forms of LPL

Low density lipoprotein (LDL) and oxidized LDL are associated with collagen in the arterial intima, where the collagen is coated by the small proteoglycan decorin. When incubated in physiological ionic conditions, decorin-coated collagen bound only small amounts of native and oxidized LDL, the interaction being weak. When decorin-coated collagen was first allowed to bind lipoprotein lipase (LPL), binding of native and oxidized LDL increased dramatically (23- and 7-fold, respectively). This increase depended on strong interactions between LPL that was bound to the glycosaminoglycan chains of the collagen-bound decorin and native and oxidized LDL (kDa 12 and 5.9 nM, respectively). To distinguish between binding to monomeric (inactive) and dimeric (catalytically active) forms of LPL, affinity chromatography on heparin columns was conducted, which showed that native LDL bound to the monomeric LPL, whereas oxidized LDL, irrespective of the type of modification (Cu(2+), 2, 2'-azobis(2-amidinopropane)hydrochloride, hypochlorite, or soybean 15-lipoxygenase), bound preferably to dimeric LPL. However, catalytic activity of LPL was not required for binding to oxidized LDL. Finally, immunohistochemistry of atherosclerotic lesions of human coronary arteries revealed specific areas in which LDL, LPL, decorin, and collagen type I were present. The results suggest that LPL can retain LDL in atherosclerotic lesions along decorin-coated collagen fibers.     

Insulin regulation of lipoprotein lipase (LPL) activity and expression in gilthead sea bream (Sparus aurata)

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism by virtue of its capacity to hydrolyze triglycerides circulating in the form of lipoprotein particles. Here we analyzed the fasting effects of LPL in gilthead sea bream (Sparus aurata) and also present the first study in fish of the role of insulin as a potential modulator of both LPL activity and expression. Fasting for 2 weeks provoked a clear decrease in adipose tissue LPL activity, concomitant with lower levels of plasma insulin, while no effects were observed in red muscle. To elucidate the specific role of insulin, increases of plasma insulin were experimentally induced by arginine and insulin injections. However, arginine predominantly stimulated glucagon over insulin secretion in this fish species while LPL activity did not change significantly in adipose tissue. Instead, insulin administration induced an increase in adipose tissue LPL activity 3 h after the injection, whereas LPL activity in red muscle was not affected. Changes in LPL activity were accompanied by an increase in LPL mRNA levels in the adipose tissue of insulin-injected gilthead sea bream, although changes in LPL expression were delayed in time with respect to variations in LPL activity. Finally, LPL mRNA levels in red muscle were similar between control and insulin-injected gilthead sea bream, suggesting that insulin does not play a direct role in the regulation of LPL in this tissue. The current study shows that LPL activity is regulated by nutritional condition and underscores the importance of insulin as a modulator of LPL activity and expression in the adipose tissue of gilthead sea bream.     

Identification of novel single nucleotide polymorphisms (SNPs) of the lipoprotein lipase (LPL) gene associated with fatty acid composition in Korean cattle

The lipoprotein lipase (LPL) gene can be considered a functional candidate gene that regulates fatty acid composition. In this study, genetic associations between fatty acid composition and exonic single nucleotide polymorphisms (SNPs) in the LPL gene were examined using 612 Korean cattle. We investigated the relationship between unsaturated fatty acids and five novel SNPs (c.322G>A, c.329A>T, c.527T>G, c.988C>T and c.1591G>A), and confirmed that three polymorphic SNPs (c.322G>A, c.329A>T and c.1591G>A) were associated with fatty acid composition. Korean cattle with an AA genotype of c.322G>A, c.329A>T, and GA genotype of c.1591G>A had higher levels of monounsaturated fatty acids and carcass traits (P < 0.05). Our findings confirmed that three novel SNPs we identified in the LPL gene can affect fatty acid composition and carcass traits. Therefore, selection for AA and GA genotypes should be recommended to genetically improve beef quality and flavor.