Statistical methods for expression quantitative trait loci (eQTL) mapping.

Traditional genetic mapping has largely focused on the identification of loci affecting one, or at most a few, complex traits. Microarrays allow for measurement of thousands of gene expression abundances, themselves complex traits, and a number of recent investigations have considered these measurements as phenotypes in mapping studies. Combining traditional quantitative trait loci (QTL) mapping methods with microarray data is a powerful approach with demonstrated utility in a number of recent biological investigations. These expression quantitative trait loci (eQTL) studies are similar to traditional QTL studies, as a main goal is to identify the genomic locations to which the expression traits are linked. However, eQTL studies probe thousands of expression transcripts; and as a result, standard multi-trait QTL mapping methods, designed to handle at most tens of traits, do not directly apply. One possible approach is to use single-trait QTL mapping methods to analyze each transcript separately. This leads to an increased number of false discoveries, as corrections for multiple tests across transcripts are not made. Similarly, the repeated application, at each marker, of methods for identifying differentially expressed transcripts suffers from multiple tests across markers. Here, we demonstrate the deficiencies of these approaches and propose a mixture over markers (MOM) model that shares information across both markers and transcripts. The utility of all methods is evaluated using simulated data as well as data from an F(2) mouse cross in a study of diabetes. Results from simulation studies indicate that the MOM model is best at controlling false discoveries, without sacrificing power. The MOM model is also the only one capable of finding two genome regions previously shown to be involved in diabetes.

The efficiency of pooling mRNA in microarray experiments.

In a microarray experiment, messenger RNA samples are oftentimes pooled across subjects out of necessity, or in an effort to reduce the effect of biological variation. A basic problem in such experiments is to estimate the nominal expression levels of a large number of genes. Pooling samples will affect expression estimation, but the exact effects are not yet known as the approach has not been systematically studied in this context. We consider how mRNA pooling affects expression estimates by assessing the finite-sample performance of different estimators for designs with and without pooling. Conditions under which it is advantageous to pool mRNA are defined; and general properties of estimates from both pooled and non-pooled designs are derived under these conditions. A formula is given for the total number of subjects and arrays required in a pooled experiment to obtain gene expression estimates and confidence intervals comparable to those obtained from the no-pooling case. The formula demonstrates that by pooling a perhaps increased number of subjects, one can decrease the number of arrays required in an experiment without a loss of precision. The assumptions that facilitate derivation of this formula are considered using data from a quantitative real-time PCR experiment. The calculations are not specific to one particular method of quantifying gene expression as they assume only that a single, normalized, estimate of expression is obtained for each gene. As such, the results should be generally applicable to a number of technologies provided sufficient pre-processing and normalization methods are available and applied.

Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis.

The identification of mutations in ABCA1 in patients with Tangier disease and familial HDL deficiency demonstrated that inadequate transport of phospholipid and cholesterol to the extracellular space results in the hypercatabolism of lipid-poor nascent HDL particles. However, the relationship between changes in ABCA1 activity and HDL levels is not clear. To address this question directly in vivo, we have used bacterial artificial chromosome transgenic approaches, which allow for appropriate developmental and cellular localization of human ABCA1 in mouse tissues. Increased expression of ABCA1 is directly associated with an increase in HDL levels, and the relationship between the increase in efflux and HDL is completely linear (r2 = 0.87). Preliminary data have suggested that coronary artery disease (CAD) is increased in heterozygotes for ABCA1 deficiency. These results may have been biased by clinical sampling, and CAD end points are insensitive markers. We have now used surrogate end points of intima-media complex thickness (IMT) and have shown that mean IMT in ABCA1 heterozygotes is indeed increased. A strong correlation between adjusted IMT and HDL cholesterol values and apolipoprotein A-I-driven efflux has been established. These studies suggest that compromised ABCA1 activity leads to accelerated and early atherogenesis and provides a link between the cholesterol deposition in macrophages within the arterial wall and cholesterol efflux in humans.

Normal Akt/PKB with reduced PI3K activation in insulin-resistant mice.

Insulin stimulates muscle and adipose tissue to absorb glucose through a signaling cascade that is incompletely understood. Insulin resistance, the inability of insulin to appropriately stimulate glucose uptake, is a hallmark of type 2 diabetes mellitus. The development of experimental systems that model human insulin resistance is important in elucidating the defects responsible for the development of type 2 diabetes. When two strains of mice, BTBR and C57BL/6J (B6), are crossed, the resultant male offspring (BtB6) demonstrate insulin resistance in muscle tissue. Here, we report an insulin resistance phenotype in adipose tissue from lean, nondiabetic BtB6 mice similar to that observed in human muscle. Adipocytes isolated from insulin-resistant male mice display 65% less insulin-stimulated glucose uptake compared with insulin-sensitive female mice. Similarly, adipocytes from insulin-resistant mice have diminished insulin-stimulated IRS-1 phosphorylation and phosphatidylinositol 3-kinase (PI3K) activation. However, normal activation of protein kinase B (Akt/PKB) by insulin is observed. Thus BtB6 mice demonstrate the dissociation of insulin-stimulated PI3K activity and Akt/PKB activation and represent a useful model to investigate the causes of insulin resistance in humans.

Please pass the chips: genomic insights into obesity and diabetes.

Type 2 diabetes mellitus is an increasingly common disorder of carbohydrate and lipid metabolism. Approximately 16 million individuals in the United States have diabetes, and 800,000 new cases are identified each year. Two important characteristics of this disease are insulin resistance, the failure of peripheral tissues, including liver, muscle, and adipose tissue, to respond to physiologic doses of insulin, and failure of pancreatic beta-cells to properly secrete insulin in response to elevated blood glucose levels. Obesity is a significant risk factor for the development of type 2 diabetes mellitus. Recent observations of extremely lean, lipoatrophic models have revealed a similar predisposition to developing diabetes. Although it may seem paradoxical that both increased adiposity and severely reduced fat mass cause diabetes, a common pathophysiologic process in fat may be responsible for the predisposition to develop hyperglycemia in both conditions. This review will focus on the important role of adipose tissue dysfunction in the pathogenesis of diabetes, and on insights gained through the application of microarray technology to analyze adipocyte gene expression.

Genetic obesity unmasks nonlinear interactions between murine type 2 diabetes susceptibility loci.

Nonlinear interactions between obesity and genetic risk factors are thought to determine susceptibility to type 2 diabetes. We used genetic obesity as a tool to uncover latent differences in diabetes susceptibility between two mouse strains, C57BL/6J (B6) and BTBR. Although both BTBR and B6 lean mice are euglycemic and glucose tolerant, lean BTBR x B6 F1 male mice are profoundly insulin resistant. We hypothesized that the genetic determinants of the insulin resistance syndrome might also predispose genetically obese mice to severe diabetes. Introgressing the ob allele into BTBR revealed large differences in diabetes susceptibility between the strain backgrounds. In a population of F2-ob/ob mice segregating for BTBR and B6 alleles, we observed large variation in pancreatic compensation for the underlying insulin resistance. We also detected two loci that substantially modify diabetes severity, and a third locus that strongly links to fasting plasma insulin levels. Amplification of the genetic signal from these latent diabetes susceptibility alleles in F2-ob/ob mice permitted discovery of an interaction between the two loci that substantially increased the risk of severe type 2 diabetes.

The expression of adipogenic genes is decreased in obesity and diabetes mellitus.

Obesity is strongly correlated with type 2 diabetes mellitus, a common disorder of glucose and lipid metabolism. Although adipocytes are critical in obesity, their role in diabetes has only recently been appreciated. We conducted studies by using DNA microarrays to identify differences in gene expression in adipose tissue from lean, obese, and obese-diabetic mice. The expression level of over 11,000 transcripts was analyzed, and 214 transcripts showed significant differences between lean and obese mice. Surprisingly, the expression of genes normally associated with adipocyte differentiation were down-regulated in obesity. Not all obese individuals will become diabetic; many remain normoglycemic despite profound obesity. Understanding the transition to obesity with concomitant diabetes will provide important clues to the pathogenesis of type 2 diabetes. Therefore, we examined the levels of gene expression in adipose tissue from five groups of obese mice with varying degrees of hyperglycemia, and we identified 88 genes whose expression strongly correlated with diabetes severity. This group included many genes that are known to be involved in signal transduction and energy metabolism as well as genes not previously examined in the context of diabetes. Our data show that a decrease in expression of genes normally involved in adipogenesis is associated with obesity, and we further identify genes important for subsequent development of type 2 diabetes mellitus.

The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1.

Stearoyl-CoA desaturase (SCD) is a microsomal enzyme required for the biosynthesis of oleate and palmitoleate, which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides, and cholesterol esters. Two well characterized isoforms of SCD, SCD1 and SCD2, exist in the mouse. Most mouse tissues express SCD1 and 2 with the exception of the liver, which expresses mainly the SCD1 isoform. We found that asebia mice homozygous for a natural mutation of the gene for SCD1 (SCD-/-) are deficient in hepatic cholesterol esters and triglycerides despite the presence of normal activities of acyl-CoA:cholesterol acyltransferase and glycerol phosphate acyltransferase, the enzymes responsible for cholesterol ester and triglyceride synthesis, respectively, in the liver of these mice. Feeding diets supplemented with triolein or tripalmitolein to the SCD-/- mice resulted in an increase in the levels of 16:1 and 18:1 in the liver but failed to restore the 18:1 and 16:1 levels of the cholesterol ester and triglycerides to the levels found in normal mice. The SCD-/- mouse had very low levels of triglycerides in the VLDL and LDL lipoprotein fractions compared with the normal animal. Transient transfection of an SCD1 expression vector into Chinese hamster ovary cells resulted in increased SCD activity and esterification of cholesterol to cholesterol esters. Taken together, our observations demonstrate that the oleoyl-CoA and palmitoleyl-CoA produced by SCD1 are necessary to synthesize enough cholesterol esters and triglycerides in the liver and suggest that regulation of SCD1 activity plays an important role in mechanisms of cellular cholesterol homeostasis.

The role of the LDL receptor in apolipoprotein B secretion.

Familial hypercholesterolemia is caused by mutations in the LDL receptor gene (Ldlr). Elevated plasma LDL levels result from slower LDL catabolism and a paradoxical lipoprotein overproduction. We explored the relationship between the presence of the LDL receptor and lipoprotein secretion in hepatocytes from both wild-type and LDL receptor-deficient mice. Ldlr(-/-) hepatocytes secreted apoB100 at a 3.5-fold higher rate than did wild-type hepatocytes. ApoB mRNA abundance, initial apoB synthetic rate, and abundance of the microsomal triglyceride transfer protein 97-kDa subunit did not differ between wild-type and Ldlr(-/-) cells. Pulse-chase analysis and multicompartmental modeling revealed that in wild-type hepatocytes, approximately 55% of newly synthesized apoB100 was degraded. However, in Ldlr(-/-) cells, less than 20% of apoB was degraded. In wild-type hepatocytes, approximately equal amounts of LDL receptor-dependent apoB100 degradation occured via reuptake and presecretory mechanisms. Adenovirus-mediated overexpression of the LDL receptor in Ldlr(-/-) cells resulted in degradation of approximately 90% of newly synthesized apoB100. These studies show that the LDL receptor alters the proportion of apoB that escapes co- or post-translational presecretory degradation and mediates the reuptake of newly secreted apoB-containing lipoprotein particles.

Identification of a novel Arg-->Cys mutation in the LDL receptor that contributes to spontaneous hypercholesterolemia in pigs.

We previously carried out genetic and metabolic studies in a partially inbred herd of pigs carrying cholesterol-elevating mutations. Quantitative pedigree analysis indicated that apolipoprotein (apo)B and a second major gene were responsible for the hypercholesterolemia in these animals. In this study, we assessed LDL receptor function by three different methods: ligand blots of liver membranes using beta-very low density lipoprotein (VLDL) as a ligand; low density lipoprotein (LDL)-dependent proliferation of T-lymphocytes; and direct binding of 125I-labeled LDL to cultured skin fibroblasts. All three methods demonstrated that LDL receptor ligands bound with decreased affinity to the LDL receptor in these animals. In skin fibroblasts from the hypercholesterolemic pigs, the Kd of binding was about 4-fold higher than in cells from normal pigs. The cDNA of the pig LDL receptor from normal and hypercholesterolemic pigs was isolated and sequenced. We identified a missense mutation that results in an Arg'Cys substitution at the position corresponding to Arg94 of the human LDL receptor. The mutation is in the third repeat of the ligand binding domain of the receptor. By single-stranded conformational polymorphism (SSCP) analysis, we studied the relationship between LDL receptor genotype and plasma cholesterol phenotype. In contrast to humans, the hypercholesterolemia associated with the LDL receptor mutation in pigs was expressed as a recessive trait. The LDL receptor mutation made a far more significant contribution to hypercholesterolemia than did the apoB mutation, consistent with observations made in human subjects with apoB mutations. Within each genotypic group (mutated apoB or mutated receptor), there was a wide range in plasma cholesterol. As the animals were on a well-controlled low-fat diet, this suggests that there are additional genetic factors that influence the penetrance of cholesterol-elevating mutations.

Calcium induces a conformational change in the ligand binding domain of the low density lipoprotein receptor.

We previously have shown that LDL-R354, a truncated low density lipoprotein (LDL) receptor, is a calcium binding protein. LDL-R354 is composed of the ligand binding domain and repeat A of the EGF precursor homology domain of the full-length human LDL receptor. We also found that Ca2+ was required for the interaction between LDL-R354 and its ligand, LDL (Dirlam et al. 1996. Protein Expt. Purif. 8: 489-500). In the current study, calcium-induced changes in the structure and function of LDL-R354 were examined. When calcium bound to LDL-R354, its apparent size increased, as determined by native and SDS-gel electrophoresis. The calcium-saturated form of LDL-R354 was more resistant to trypsin proteolysis than the calcium-depleted form. In the presence of calcium, the disulfide bonds in the truncated receptor were stabilized, rendering them more resistant to reduction by dithiothreitol. Calcium binding affinities were measured by monitoring increased tryptophan fluorescence intensities. LDL-R354 bound Ca2+ with high affinity (EC50 = 60 nM at pH 7.4) and specificity, as 400 microM Mg2+ did not compete for calcium binding. The affinity of LDL-R354 for calcium decreased when the pH was lowered. These results suggest that calcium induces a conformational change in the ligand binding domain of the LDL receptor and that a receptor conformer capable of binding ligand should be stabilized at physiological extracellular Ca2+ concentration and pH. Drops in pH may regulate LDL receptor function by altering the amount of calcium bound to the receptor.

Interaction between BTBR and C57BL/6J genomes produces an insulin resistance syndrome in (BTBR x C57BL/6J) F1 mice.

Insulin resistance is a common syndrome that often precedes the development of noninsulin-dependent diabetes mellitus (NIDDM). Both diet and genetic factors are associated with insulin resistance. BTBR and C57BL/6J (B6) mice have normal insulin responsiveness and normal fasting plasma insulin levels. However, a cross between these two strains yielded male offspring with severe insulin resistance. Surprisingly, on a basal diet (6.5% fat), the insulin resistance was not associated with fasting hyperinsulinemia. However, a 15% fat diet produced significant hyperinsulinemia in the male mice (twofold at 10 weeks; P < .05). At 10 weeks of age, visceral fat contributed approximately 4.3% of the total body weight in the males versus 1.8% in females. In the males, levels of plasma triacylglycerol and total cholesterol increased 40% and 30%, respectively, compared to females. Plasma free fatty acid concentrations were unchanged. Oral glucose tolerance tests revealed significant levels of hyperglycemia and hyperinsulinemia 15 to 90 minutes after oral glucose administration in the male mice. This was particularly dramatic in males on a 15% fat diet. Glucose transport was examined in skeletal muscles in (BTBR x B6)F1 mice. In the nonhyperinsulinemic animals (females), insulin stimulated 2-deoxyglucose transport 3.5-fold in the soleus and 2.8-fold in the extensor digitorum longus muscles. By contrast, glucose transport was not stimulated in the hyperinsulinemic male mice. Hypoxia stimulates glucose transport through an insulin-independent mechanism. This is known to involve the translocation of GLUT4 from an intracellular pool to the plasma membrane. In the insulin-resistant male mice, hypoxia induced glucose transport as effectively as it did in the insulin-responsive mice. Thus, defective glucose transport in the (BTBR x B6)F1 mice is specific for insulin-stimulated glucose transport. This is similar to what has been observed in muscles taken from obese NIDDM patients. These animals represent an excellent genetic model for studying insulin resistance and investigating the transition from insulin resistance in the absence of hyperinsulinemia to insulin resistance with hyperinsulinemia.

The enzymatic and non-enzymatic roles of protein-disulfide isomerase in apolipoprotein B secretion.

UNLABELLED: Secretion of apolipoprotein B (apoB) from mammalian cells requires the presence of functional microsomal triglyceride transfer protein (MTP). We previously reported that co-expressing the human intestinal form of apoB, B48, with both subunits of human MTP in oleate-treated Sf21 cells led to a dramatic induction of B48 secretion. Deletion mutagenesis studies showed that the cysteine-enriched amino terminus of apoB was necessary for the MTP responsiveness (Gretch, D. G., Sturley, S. L., Wang, L., Dunning, A., Grunwald, K. A. A., Wetterau, J. R., Yao, Z., Talmud, P., and Attie, A. D. (1996) J. Biol. Chem. 271, 8682-8691). We therefore hypothesized that the small subunit of MTP, protein-disulfide isomerase (PDI), plays a role in apoB secretion by facilitating correct disulfide bond formation. To determine whether the enzymatic activities of PDI are important for MTP-stimulated apoB secretion, the wild type PDI subunit was replaced with an active site mutant, mPDI (Cys36 --> Ser/Cys380 --> Ser), lacking both disulfide shuffling and redox activities. MTP containing mPDI was fully functional in promoting apoB and triglyceride secretion. Therefore, the shufflase and redox activities of PDI are not necessary for the function of MTP. Since PDI exists in large molar excess over the other subunit of MTP, the role of free PDI (independent of the MTP complex) was investigated. PDI or mPDI was co-expressed with B48 and B17, a fragment encompassing the amino-terminal 17% of apoB. Mutant PDI significantly and specifically reduced the accumulation of the B17 and B48 both intracellularly and in the culture medium. The reduction was partially eliminated by the protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, consistent with rapid co- or post-translational degradation of apoB in the presence of mPDI. Treating the cells with oleate reversed the effect of mPDI on B48 secretion in a dose-dependent manner, but had no effect on B17. IN CONCLUSION: 1) the role of PDI in the MTP complex involves functions other than its known enzymatic activities; 2) one or both of the enzymatic activities of free PDI is/are important for the MTP-independent steps of apoB secretion; 3) oleate can affect apoB secretion at high physiological concentrations and compensate for the insufficiency of PDI activities.

Expression and characterization of a truncated, soluble, low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor mediates the clearance of apolipoprotein B- and E-containing lipoproteins from the bloodstream. In the current study, we characterized the binding properties of the amino terminus of the LDL receptor. We produced a recombinant baculovirus that encoded the first 354 amino acids, including the endogenous signal sequence, of the human LDL receptor. This truncated receptor protein (LDL-R354) was secreted from recombinant baculovirus-infected Spodoptera frugiperda (Sf-21) cells. Upon electrophoresis, LDL-R354 migrated with a mobility of 55,000. Treatment of cells with tunicamycin decreased the size of the truncated receptor, suggesting the presence of asparagine-linked carbohydrates. Nonreducing SDS-PAGE resulted in at least three discernible bands with M(r)s consistent with the truncated receptor existing as monomers and multimers, suggesting the possibility of intermolecular disulfide cross-linking. All forms of the truncated receptor bound LDL on a ligand blot in a calcium-dependent manner. The purified truncated receptor bound 125I-LDL with high affinity in a competitive binding assay (IC50 = 0.8 microgram/ml). LDL-R354 also bound calcium. This interaction was sensitive to the conformation of the ligand binding domain, as reduction of the disulfide bonds greatly decreased the affinity of the receptor for calcium. The ligand and calcium binding activities of this truncated receptor protein demonstrate that the ligand binding domain of the LDL receptor can fold into a functionally active protein.

The amino terminus of apolipoprotein B is necessary but not sufficient for microsomal triglyceride transfer protein responsiveness.

Human apolipoprotein (apo) B mediates the formation of neutral lipid-containing lipoproteins in the liver and intestine. The association of apoB with lipid is thought to be promoted by the microsomal triglyceride transfer protein complex. We have reconstituted lipoprotein assembly in an insect cell line that normally does not support this process. Expression of human microsomal triglyceride transfer protein (MTP) and apolipoprotein B48 (apoB48) together enabled Sf-21 insect cells to secrete approximately 60-fold more lipoprotein-associated triacylglycerol than control cells. This dramatic effect demonstrates that effective partitioning of triacylglycerol into the secretory pathway requires an endoplasmic reticulum-associated neutral lipid transporter (provided by MTP) and an apolipoprotein to shuttle the lipid through the pathway. Expression of the human apoB48 gene in insect cells resulted in secretion of the protein product. Including both MTP subunits with apoB48 and oleic acid specifically increased apoB48 secretion 8-fold over individual subunits alone. To assess whether specific regions of apoB are necessary for MTP responsiveness, nine apoB segments were expressed. These included NH2-terminal segments as well as internal and COOH-terminal regions of apoB fused with a heterologous signal sequence. ApoB segments containing the NH2-terminal 17% of the protein were secreted and responded to MTP activity; however, a segment containing only the NH2-terminal 17% of the protein was not significantly responsive to MTP. Segments lacking the NH2 terminus were not MTP-responsive, and five of six of these proteins were trapped intracellularly but, in certain cases, could be rescued by fusion to apoB17. These results suggest that the NH2 terminus of apoB is necessary but not sufficient for MTP responsiveness.

Lipoprotein lipase association with lipoproteins involves protein-protein interaction with apolipoprotein B.

Lipoprotein lipase (LPL) hydrolyzes chylomicron and very low density lipoprotein (VLDL) triglycerides and potentiates the cellular uptake of lipoproteins. These LPL-lipoprotein associations could involve only protein-lipid interaction, or they could be modulated by apolipoproteins (apo). ApoB is the major protein component of chylomicrons, VLDL, and low density lipoprotein (LDL). ApoB100, a large glycoprotein with a molecular mass of 550 kDa, is composed of several functional domains. A carboxyl-terminal region of the protein is the ligand for the LDL receptor. There are several hydrophobic domains that are believed to be important in lipid binding. The relatively hydrophilic amino-terminal region of apoB, however, has no known function. Using solid phase assays we quantified LPL-lipoprotein complex formation. On a molar basis, severalfold greater amounts of LPL bound to LDL and VLDL than to high density lipoprotein at all the concentrations of LPL tested (0.9-55 nM). To assess the roles of LDL protein versus lipid, we performed competition and ligand blotting experiments. LDL and an amino-terminal fragment of apoB competed better for 125I-LPL binding to LDL than did lipid emulsion particles. Delipidation of LDL-coated plates did not alter LPL binding. On ligand blots, LPL bound to amino-terminal fragments of apoB generated by thrombin digestion but not to apoA1, apoE, or carboxyl-terminal fragments of apoB. Further evidence for LPL interaction with the amino-terminal region of apoB was obtained using anti-apoB monoclonal antibodies. Antibodies directed against the amino-terminal regions of apoB blocked LPL interaction with LDL, whereas those against the carboxyl-terminal region of apoB did not inhibit LPL interaction with LDL. Thus, we conclude that a specific interaction between LPL and the amino-terminal region of apoB may facilitate LPL association with circulating lipoproteins.

Human apolipoprotein E mediates processive buoyant lipoprotein formation in insect larvae.

The expression of human apolipoprotein E in tobacco hornworm larvae causes a dramatic change in the buoyant density of the insect's endogenous lipoproteins. Larvae without apoE have lipoproteins that are found exclusively in the high-density range. Baculovirus-mediated apoE expression results in the conversion of approximately one-fourth of the endogenous lipoproteins to low-density species. This density conversion is progressive and parallels a similar change in apoE density distribution. ApoE is secreted from the lipoprotein producing fat body tissue in a lipid-poor form, but readily associates with circulating insect lipoproteins in the hemolymph where the density conversion takes place. Analysis of the buoyant lipoprotein particles indicates that they contain apoE and insect apolipophorins I and II with few or no other proteins present. Immunoprecipitation of apolipophorins I and II results in coprecipitation of apoE. This association is disrupted by detergent, consistent with the three proteins sharing the same lipoprotein particles. The ability of apoE to influence buoyant lipoprotein formation in an invertebrate system leads us to suggest that small apolipoproteins such as apoE may play a role in buoyant lipoprotein production in mammals.

Hypercatabolism of lipoprotein-free apolipoprotein A-I in HDL-deficient mutant chickens.

The Wisconsin Hypoalpha Mutant (WHAM) chicken has a sex-linked mutation associated with a 90% reduction in high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I). In the present studies, we did not detect a defect in apoA-I synthesis or secretion in liver or intestine. We tested the hypothesis that apoA-I is not binding properly to lipoprotein particles and is undergoing hypercatabolism. We therefore studied the in vivo turnover of lipid-free 125I-apoA-I. Its turnover was fourfold faster in WHAM chickens than in normal chickens. The 125I-apoA-I equilibrated more slowly with HDL in the WHAM chickens, and these animals had a much larger steady-state pool of lipid-free apoA-I than did control chickens. To determine the tissue sites of degradation of apoA- I, the tissue distribution of 125I-tyramine cellobiose apoA-I was assessed. The liver and kidneys were the major sites of apoA-I degradation, but in the WHAM chickens, the kidney made a twofold larger contribution to apoA-I degradation than in normal chickens. Total plasma phospholipid levels are reduced by 44% to 78% in the WHAM chickens. A phospholipid deficit might explain the elevated lipid-free apoA-I pool and, secondarily, the HDL deficiency of the WHAM chickens.

Human apolipoprotein B signal sequence variants confer a secretion-defective phenotype when expressed in yeast.

Hyperlipidemia arises from a disturbance in the balance between production and degradation of lipoprotein particles. Variation in the secretion of human apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, directly affects this homeostasis. Naturally occurring apoB signal peptide variants (associated with hypertriglyceridemia, altered postprandial lipid metabolism, or atherosclerosis) were investigated for their ability to direct transit through the secretion pathway. Three apoB signal peptide isoforms were fused to the secretory protein, invertase, and expressed in yeast. A deletion or insertion in the hydrophobic core of the signal peptide mediated inefficient translocation into the endoplasmic reticulum and was secretion-defective, relative to the common 27-residue isoform. Additionally, the insertion apoB isoform was observed in yeast to confer a defect in export from the endoplasmic reticulum. Secretion of the apoB signal peptide-invertase fusions responded positively to an inhibitor of calpain type I proteases. These observations suggest that the apoB signal peptide plays a role in determining the levels of apoB degradation and secretion and, thus, hyperlipidemia.