Genetic Variants Associated with Gestational Hypertriglyceridemia and Pancreatitis

Severe hypertriglyceridemia is a well-known cause of pancreatitis. Usually, there is a moderate increase in plasma triglyceride level during pregnancy. Additionally, certain pre-existing genetic traits may render a pregnant woman susceptible to development of severe hypertriglyceridemia and pancreatitis, especially in the third trimester. To elucidate the underlying mechanism of gestational hypertriglyceridemic pancreatitis, we undertook DNA mutation analysis of the lipoprotein lipase (LPL), apolipoprotein C2 (APOC2), apolipoprotein A5 (APOA5), lipase maturation factor 1 (LMF1), and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) genes in five unrelated pregnant Chinese women with severe hypertriglyceridemia and pancreatitis. DNA sequencing showed that three out of five patients had the same homozygous variation, p.G185C, in APOA5 gene. One patient had a compound heterozygous mutation, p.A98T and p.L279V, in LPL gene. Another patient had a compound heterozygous mutation, p.A98T & p.C14F in LPL and GPIHBP1 gene, respectively. No mutations were seen in APOC2 or LMF1 genes. All patients were diagnosed with partial LPL deficiency in non-pregnant state. As revealed in our study, genetic variants appear to play an important role in the development of severe gestational hypertriglyceridemia, and, p.G185C mutation in APOA5 gene appears to be the most common variant implicated in the Chinese population. Antenatal screening for mutations in susceptible women, combined with subsequent interventions may be invaluable in the prevention of potentially life threatening gestational hypertriglyceridemia-induced pancreatitis.     

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.     

Comprehensive in-silico analysis of deleterious SNPs in APOC2 and APOA5 and their differential expression in cancer and cardiovascular diseases conditions

Genetic variations in APOC2 and APOA5 genes involve activating lipoprotein lipase (LPL), responsible for the hydrolysis of triglycerides (TG) in blood and whose impaired functions affect the TG metabolism and are associated with metabolic diseases. In this study, we investigate the biological significance of genetic variations at the DNA sequence and structural level using various computational tools. Subsequently, 8 (APOC2) and 17 (APOA5) non-synonymous SNPs (nsSNPs) were identified as high-confidence deleterious SNPs based on the effects of the mutations on protein conservation, stability, and solvent accessibility. Furthermore, based on our docking results, the interaction of native and mutant forms of the corresponding proteins with LPL depicts differences in root mean square deviation (RMSD), and binding affinities suggest that these mutations may affect their function. Furthermore, in vivo, and in vitro studies have shown that differential expression of these genes in disease conditions due to the influence of nsSNPs abundance may be associated with promoting the development of cancer and cardiovascular diseases. Preliminary screening using computational methods can be a helpful start in understanding the effects of mutations in APOC2 and APOA5 on lipid metabolism; however, further wet-lab experiments would further strengthen the conclusions drawn from the computational study.     

Coexistence of abnormalities of hepatic lipase and lipoprotein lipase in a large family.

A large family is reported with familial hepatic triglyceride lipase (HTGL) deficiency and with the coexistence of reduced lipoprotein lipase (LPL) similar to the heterozygote state of LPL deficiency. The proband was initially detected because of hypertriglyceridemia and chylomicronemia. He was later demonstrated to have beta-VLDL despite an apo E3/E3 phenotype and the lack of stigmata of type III hyperlipoproteinemia. The proband had no HTGL activity in postheparin plasma. Two of his half-sisters had very low HTGL activity (39 and 31 nmol free fatty acids/min/ml; normal adult female greater than 44). His son and daughters had decreased HTGL activity (normal male and preadolescent female greater than 102), which would be expected in obligate heterozygotes for HTGL deficiency. Low HTGL activity was associated with LDL particles which were larger and more buoyant. Several family members, including the proband, had reduced LPL activity and mass less than that circumscribed by the 95% confidence-interval ellipse for normal subjects and had hyperlipidemia similar to that described in heterozygote relatives of patients with LPL deficiency. All the sibs with hyperlipidemia had a reduced LPL activity and mass, while subjects with isolated reduced HTGL (with normal LPL activity) had normal lipid phenotypes. Analysis of genomic DNA from these subjects by restriction-enzyme digestion revealed no major abnormalities in the structure of either the HTGL or the LPL gene. Compound heterozygotes for HTGL and LPL deficiency show lipoprotein physiological characteristics typical for HTGL deficiency, while their variable lipid phenotype is typical for LPL deficiency.     

Gene polymorphisms in the Quebec population: a risk to develop hypertriglyceridemia

In Eastern Québec, two major lipoprotein lipase (LPL) gene mutations, P207L and G188E, lead to complete LPL deficiency in homozygote subjects and contribute to elevated predisposition to hypertriglyceridemia in heterozygotes. First, we determined the allele frequencies of LPL (D9N, G188E, P207L, D250N, N291S, and S447X), APOE (C112R and C158R), PPARalpha (L162V), and PPARgamma2 (P12A) single nucleotide polymorphisms (SNPs) in a random-based cohort of the metropolitan Québec city area. Second, we compared the LPL X447 allele frequencies observed in the random cohort and in a cohort of LPL P207L deficient patients. In the random cohort, the LPL N9 rare allele exhibited a higher prevalence than previously expected (p=0.0001). The LPL X447 allele frequency was lower in the patient cohort (Freq: 4.4%) than in the random cohort (Freq: 11.2%) (p=0.0001). These results reveal the importance of genetic screening for LPL gene mutations D9N and S447X in a population at risk to develop hypertriglyceridemia.     

Apolipoprotein CI causes hypertriglyceridemia independent of the very-low-density lipoprotein receptor and apolipoprotein CIII in mice

We have recently shown that the predominant hypertriglyceridemia in human apolipoprotein C1 (APOC1) transgenic mice is mainly explained by apoCI-mediated inhibition of the lipoprotein lipase (LPL)-dependent triglyceride (TG)-hydrolysis pathway. Since the very-low-density lipoprotein receptor (VLDLr) and apoCIII are potent modifiers of LPL activity, our current aim was to study whether the lipolysis-inhibiting action of apoCI would be dependent on the presence of the VLDLr and apoCIII in vivo. Hereto, we employed liver-specific expression of human apoCI by using a novel recombinant adenovirus (AdAPOC1). In wild-type mice, moderate apoCI expression leading to plasma human apoCI levels of 12-33 mg/dl dose-dependently and specifically increased plasma TG (up to 6.6-fold, P < 0.001), yielding the same hypertriglyceridemic phenotype as observed in human APOC1 transgenic mice. AdAPOC1 still increased plasma TG in vldlr(-/-) mice (4.1-fold, P < 0.001) and in apoc3(-/-) mice (6.8-fold, P < 0.001) that were also deficient for the low-density lipoprotein receptor (LDLr) and LDLr-related protein (LRP) or apoE, respectively. Thus, irrespective of receptor-mediated remnant clearance by the liver, liver-specific expression of human apoCI causes hypertriglyceridemia in the absence of the VLDLr and apoCIII. We conclude that apoCI is a powerful and direct inhibitor of LPL activity independent of the VLDLr and apoCIII.     

The lipoprotein lipase system: new understandings.

Hypertriglyceridemia, a risk factor for premature atherosclerosis, may result from decreased use of plasma triglycerides by tissues. The removal of triglycerides is mediated by the enzyme lipoprotein lipase (LPL). Heparin releases LPL from tissues and post-heparin plasma lipolytic activity (PHLA) has been extensively used to elucidate the mechanism of hypertriglyceridemia in various diseases. There is evidence to show that postheparin plasma contains enzymes other than LPL. Hence data on total PHLA are difficult to interpret. Availability of assays for the LPL component of PHLA has clarified equivocal findings in certain hypertriglyceridemic states. However, the LPL component is also heterogeneous. The LPL "isoenzymes" from various extrahepatic tissues behave differently under various metabolic conditions. Therefore, to understand properly the LPL system it is necessary to study the specific tissue LPL. Furthermore, the serum activator for LPL is now characterized. Its importance is evidenced by the recent discovery of a hypertriglyceridemic patient deficient in this apoprotein.     

Hypertriglyceridemia and delayed clearance of fat load in transgenic rabbits expressing human apolipoprotein CIII

Apolipoprotein CIII (apoCIII) has been implicated in hypertriglyceridemia and plasma apoCIII independently predicts risk for coronary heart disease. While hypertriglyceridemia in patients has been demonstrated to correlate with elevated plasma apoCIII levels and reduced lipoprotein lipase (LPL) activity, apoCIII transgenic mice show elevated LPL activity. In this study, we generated transgenic (Tg) rabbits expressing human apoCIII gene exclusively in liver and investigated the effect of apoCIII overexpression on lipid metabolism of rabbits. In comparison with non-Tg littermates, Tg rabbits had 3- and 3.2-fold increases in fed and fasted plasma triglycerides, respectively. In contrast, no significant differences were observed in plasma total cholesterol and high density lipoprotein cholesterol levels between Tg and non-Tg rabbits. Analysis of lipoprotein fractions revealed that elevated plasma triglyceride levels in Tg rabbits were mainly attributed to an increase in very low density lipoprotein/chylomicron-sized particles. Furthermore, Tg rabbits showed markedly delayed clearance of plasma triglycerides accompanied with significantly reduced LPL activity in post-heparin plasma compared to that in non-Tg controls. In conclusion, these results indicate apoCIII transgenic rabbits develop hypertriglyceridemia with similar mechanism in hypertriglyceridemic patients via delayed clearance of plasma triglycerides, and could be used as a valuable tool for the study of human hyperlipidemia in relation with atherosclerotic disorders.     

Studies on the mechanism of hypertriglyceridemia in the genetically obese Zucker rat

The possibility that impaired removal of lipoprotein triglyceride from the circulation may be a participating factor in the hypertriglyceridemia of the obese Zucker rat was examined. We found no significant differences in the heparin-released lipoprotein lipase (LPL) activities of the adipose tissue, skeletal muscle, and heart (expressed per gram of tissue) from the lean and obese Zucker rats. Furthermore, the kinetic properties of adipose tissue and heart LPL from the lean and obese rats were similar, indicating that the catalytic efficiency of the enzyme was unaltered in the obese animals. The postheparin plasma LPL activities of lean and obese rats were also similar. However, the postheparin plasma hepatic triglyceride lipase (H-TGL) activity in the obese rats was elevated. The higher activity of H-TGL could not alleviate the hypertriglyceridemia in these animals. Since hypertriglyceridemia in the obese rats could also be due to the hepatic production of triglyceride-rich lipoproteins which are resistant to lipolysis, we therefore isolated very low density lipoproteins (VLDL) from lean and obese rat liver perfusates and examined their degradation by highly purified human milk LPL. Although certain differences were observed in hepatic VLDL triglyceride fatty acid composition, the kinetic patterns of LPL-catalyzed triglyceride disappearance from lean and obese rat liver perfusate VLDL were similar. The isolated liver perfusate VLDL contained sufficient apolipoprotein C-II for maximum lipolysis. These results indicate that impaired lipolysis is not a contributing factor in the genesis of hypertriglyceridemia in the genetically obese Zucker rat. The hyperlipemic state may be attributed to hypersecretion of hepatic VLDL and consequent saturation of the lipolytic removal of triglyceride-rich lipoproteins from the circulation.     

Overexpression of human apolipoprotein A-II in mice induces hypertriglyceridemia due to defective very low density lipoprotein hydrolysis

Two lines of transgenic mice, hAIItg-delta and hAIItg-lambda, expressing human apolipoprotein (apo)A-II at 2 and 4 times the normal concentration, respectively, displayed on standard chow postprandial chylomicronemia, large quantities of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) but greatly reduced high density lipoprotein (HDL). Hypertriglyceridemia may result from increased VLDL production, decreased VLDL catabolism, or both. Post-Triton VLDL production was comparable in transgenic and control mice. Postheparin lipoprotein lipase (LPL) and hepatic lipase activities decreased at most by 30% in transgenic mice, whereas adipose tissue and muscle LPL activities were unaffected, indicating normal LPL synthesis. However, VLDL-triglyceride hydrolysis by exogenous LPL was considerably slower in transgenic compared with control mice, with the apparent Vmax of the reaction decreasing proportionately to human apoA-II expression. Human apoA-II was present in appreciable amounts in the VLDL of transgenic mice, which also carried apoC-II. The addition of purified apoA-II in postheparin plasma from control mice induced a dose-dependent decrease in LPL and hepatic lipase activities. In conclusion, overexpression of human apoA-II in transgenic mice induced the proatherogenic lipoprotein profile of low plasma HDL and postprandial hypertriglyceridemia because of decreased VLDL catabolism by LPL.     

A novel substitution at the translation initiator codon (ATG-->ATC) of the lipoprotein lipase gene is mainly responsible for lipoprotein lipase deficiency in a patient with severe hypertriglyceridemia and recurrent pancreatitis

A patient with severe hypertriglyceridemia and recurrent pancreatitis was found to have significantly decreased lipoprotein lipase (LPL) activity and normal apolipoprotein C-II concentration in post-heparin plasma. DNA analysis of the LPL gene revealed two mutations, one of which was a novel homozygous G-->C substitution, resulting in the conversion of a translation initiation codon methionine to isoleucine (LPL-1). The second was the previously reported heterozygous substitution of glutamic acid at residue 242 with lysine (LPL-242). In vitro expression of both mutations separately or in combination demonstrated that LPL-1 had approximately 3% protein mass and 2% activity, whereas LPL-242 had undetectable activity but normal mass. The combined mutation LPL-1-242 exhibited similar changes as for LPL-1, with markedly reduced mass, and for LPL-242, with undetectable activity. These results suggest that the homozygous initiator codon mutation rather than the heterozygous LPL-242 alteration was mainly responsible for the patient phenotypes.     

Prediction of genetic risk for dyslipidemia

The purpose of the present study was to identify genetic variants that confer susceptibility to dyslipidemia. A total of 5213 individuals from two independent populations were examined: Subject panel A comprised 3794 individuals who visited participating hospitals; subject panel B comprised 1419 community-dwelling elderly individuals. The genotypes for 100 polymorphisms of 65 candidate genes were determined. The chi(2) test and multivariable logistic regression analysis revealed that seven polymorphisms of APOA5, APOC3, APOA1, ACAT2, and LPL were significantly associated with hypertriglyceridemia, six polymorphisms of APOA5, LIPC, and CYP3A4 with low HDL-cholesterol, and three polymorphisms of APOE and CCR2 with high LDL-cholesterol in subject panel A. For validation of these associations, the same polymorphisms were examined in subject panel B. Six polymorphisms of APOA5, APOC3, APOA1, and LPL were again significantly associated with hypertriglyceridemia, three polymorphisms of APOA5 with low HDL-cholesterol, and two polymorphisms of APOE with high LDL-cholesterol. Serum triglyceride, HDL-cholesterol, and LDL-cholesterol concentrations differed significantly among genotypes of these corresponding polymorphisms in both subject panels. These results indicate that polymorphisms of APOA5, APOC3, APOA1, and LPL are determinants of hypertriglyceridemia and that those of APOA5 and APOE are determinants of low HDL-cholesterol and high LDL-cholesterol, respectively, in Japanese individuals.     

中国人群脂蛋白脂肪酶基因突变与高脂血症的相关性研究

为进行脂蛋白脂肪酶基因突变与中国人群高脂血症的相关性研究,采用单链构象多态性分析结合DNA序列测定的方法,对386例(其中108例高脂血症患者,278例正常对照)中国人群进行突变筛查。结果发现1个新的沉默突变L103L,1个错义突变P207L,3个剪接突变Int3/3′-ass/C(-6)→T和普遍存在的S447X多态性,其中发生在高脂血症组的P207L杂合子为亚洲首报,并对先证者的家系进行了研究,认为P207L是家族性高脂血症的病因之一,而在正常对照组中也有发现的Int3/3′-ass/C(-6)→T,对以往研究认为其是高脂血症易患因素的观点提出了相反的报告,对于普遍认为有益的多态性位点S447X,进一步研究认为其对于正常人群,特别是健康男性的保护作用更强。结论:脂蛋白脂肪酶基因变异与高脂血症的相关性十分复杂多样,大规模的人群筛查具有重要意义。     

Severe hypertriglyceridemia in human APOC1 transgenic mice is caused by apoC-I-induced inhibition of LPL

Studies in humans and mice have shown that increased expression of apolipoprotein C-I (apoC-I) results in combined hyperlipidemia with a more pronounced effect on triglycerides (TGs) compared with total cholesterol (TC). The aim of this study was to elucidate the main reason for this effect using human apoC-I-expressing (APOC1) mice. Moderate plasma human apoC-I levels (i.e., 4-fold higher than human levels) caused a 12-fold increase in TG, along with a 2-fold increase in TC, mainly confined to VLDL. Cross-breeding of APOC1 mice on an apoE-deficient background resulted in a marked 55-fold increase in TG, confirming that the apoC-I-induced hyperlipidemia cannot merely be attributed to blockade of apoE-recognizing hepatic lipoprotein receptors. The plasma half-life of [3H]TG-VLDL-mimicking particles was 2-fold increased in APOC1 mice, suggesting that apoC-I reduces the lipolytic conversion of VLDL. Although total postheparin plasma LPL activity was not lower in APOC1 mice compared with controls, apoC-I was able to dose-dependently inhibit the LPL-mediated lipolysis of [3H]TG-VLDL-mimicking particles in vitro with a 60% efficiency compared with the main endogenous LPL inhibitor apoC-III. Finally, purified apoC-I impaired the clearance of [3H]TG-VLDL-mimicking particles independent of apoE-mediated hepatic uptake in lactoferrin-treated mice. Therefore, we conclude that apoC-I is a potent inhibitor of LPL-mediated TG-lipolysis.     

Two novel mutations in the lipoprotein lipase gene in a family with marked hypertriglyceridemia in heterozygous carriers. Potential interaction with the polymorphic marker D1S104 on chromosome 1q21-q23

Two novel mutations in the lipoprotein lipase (LPL) gene are described in an Austrian family: a splice site mutation in intron 1 (3 bp deletion of nucleotides -2 to -4) which results in skipping of exon 2, and a missense mutation in exon 5 which causes an asparagine for histidine substitution in codon 183 and complete loss of enzyme activity. A 5-year-old boy who exhibited all the clinical features of primary hyperchylomicronemia was a compound heterozygote for these two mutations. Nine other family members were investigated: seven were heterozygotes for the splice site mutation, one was a heterozygote for the missense mutation, and one had two wild-type alleles of the LPL gene. LPL activity in the post-heparin plasma of the heterozygotes was reduced to 49;-79% of the mean observed in normal individuals. Two of the heterozygotes had extremely high plasma triglyceride levels; in three of the other heterozygotes the plasma triglycerides were also elevated. As plasma triglycerides in carriers of one defective LPL allele can be normal or elevated, the heterozygotes of this family have been studied for a possible additional cause of the expression of hypertriglyceridemia in these subjects. Body mass index, insulin resistance, mutations in other candidate genes (Asn291Ser and Asp9Asn in the LPL gene, apoE isoforms, polymorphisms in the apoA-II gene and in the apoAI-CIII-AIV gene cluster, and in the IRS-1 gene) could be ruled out as possible factors contributing to the expression of hypertriglyceridemia in this family. A linkage analysis using the allelic marker D1S104 on chromosome 1q21;-q23 suggested that a gene in this region could play a role in the expression of hypertriglyceridemia in the heterozygous carriers of this family, but the evidence was not sufficiently strong to prove this assumption. Nevertheless, this polymorphic marker seems to be a good candidate for further studies.     

Lipoprotein lipase activity and common gene variants in severely hypertriglyceridemic patients with and without diabetes

OBJECTIVES: In severe type IV hypertriglyceridemia (triglyceride levels >10 g/l), it is yet unknown whether lipoprotein lipase (LPL) differs according to the presence or not of diabetes. METHODS: We compared LPL activity and the presence of four common variants in the LPL gene (Asp 9 Asn (exon 2), Gly 188 Glu (exon 5), Asn 291 Ser (exon 6) and Ser 447 Ter (exon 9)) in a group of 34 patients of whom 17 presented diabetes mellitus. RESULTS: Maximum triglyceride, cholesterol levels and distribution of apolipoprotein E phenotypes did not differ between the two subgroups. Mean post-heparin LPL activity was lower in non-diabetic compared to diabetic patients (9.74 vs. 12.98 micromol FFA/ml/h, p=0.033). Four patients were carrying a mutation in exon 9 (1 non-diabetic), 6 patients in exon 2 (4 non-diabetic) and 1 patient in the non-diabetic subgroup in exon 5. All mutations were at the heterozygous state. CONCLUSION: We found that LPL activity was lower in type IV hyperlipidemia in the absence of diabetes. Genetic defects in the LPL gene that could lead to this lower LPL tended to be more frequently observed in patients without diabetes. These data suggest that the pathomechanisms which contribute to severe type IV hyperlipidemia are different according to the presence or not of diabetes.     

Sex- and age-related variations in the in vitro heparin-releasable lipoprotein lipase from mononuclear leukocytes in blood

An in vitro heparin release of lipoprotein lipase (LPL) from whole blood, mainly from monocytes, was demonstrated by (1) the time-course of lipolytic activity with the presence of 10 U/ml heparin at 37 degrees C, (2) the distribution of LPL activity in monocyte and lymphocyte fractions, (3) an immuno-inactivation with anti-LPL immunoglobulin (IgG) and (4) responses to various compounds such as NaCl, protamine sulfate, heparin, and serum activator. The in vitro heparin-releasable LPL activity from blood correlated well with the LPL activity of postheparin plasma obtained from both normolipidemic and hyperlipidemic rabbits. Studies in humans revealed sex- and age-related variations in the in vitro heparin-releasable LPL from monocytes in the blood of 134 normal subjects and 24 hypertriglyceridemic subjects: The mean LPL activity was significantly higher in normal females over the age of 30, than in the corresponding males. In the hypertriglyceridemic group, the LPL activity was also higher in females than in males, but it was not significant. The in vitro heparin-releasable LPL activity from monocytes in blood was comparable to the LPL activity derived from adipose tissue and postheparin plasma, and thus it reflects lipoprotein metabolism.     

Lipoprotein lipase activity determined in vivo is lower in carriers of apolipoprotein A-V gene variants 19W and -1131C

The apolipoprotein A-V (apo A-V) plays an important role in regulation of triglyceride (TG) concentration in serum. To better understand how apo A-V affects triglyceridemia and glucoregulation, the lipoprotein lipase (LPL) activity was determined using intravenous fat tolerance test (IVFTT) and oral glucose tolerance test (oGTT) was performed in carriers of apolipoprotein A-V gene (APOAV) variants known to be associated with increased triglyceridemia. Twelve carriers of 19W variant, 16 carriers of -1131C variant, 1 combined heterozygote and 16 control subjects homozygous for wild type variants (19S/-1131T) were selected from a population sample and matched with respect to body mass index and age. The APOAV variants carriers had increased TG, very low density lipoprotein-TG, and apo B concentrations (p < 0.05). The LPL activity evaluated as k(2) rate constant for clearance of Intralipid was 14 % lower in APOAV variants carriers. The depression of nonesterified fatty acids (NEFA) concentration after glucose load was delayed in APOAV variants carriers in spite of the same insulinemia and glycemia. Our results suggest that variants of APOAV combined with increased triglyceridemia are associated with lower LPL activity in vivo and with disturbances of regulation of NEFA concentration after glucose load.