G. M. Dallinga-Thie

Mutations in LPL, APOC2, APOA5, GPIHBP1 and LMF1 in patients with severe hypertriglyceridaemia

Abstract.  Surendran RP, Visser ME, Heemelaar S, Wang J, Peter J, Defesche JC, Kuivenhoven JA, Hosseini M, Péterfy M, Kastelein JJP, Johansen CT, Hegele RA, Stroes ESG, Dallinga‐Thie GM (Academic Medical Center, Amsterdam, the Netherlands; Robarts Research Institute, University of Western Ontario, London, Ontario, Canada; UCLA, University of California; Medical Genetics Institute, Cedars‐Sinai Medical Center, Los Angeles, CA, USA). Mutations in LPL, APOC2, APOA5, GPIHBP1 and LMF1 in patients with severe hypertriglyceridaemia. J Intern Med 2012; 272: 185–196.

Plasma apolipoprotein A5 and triglycerides in type 2 diabetes

Aims/hypothesisVariation in the human apolipoprotein (APO) A5 gene (APOA5) is associated with elevated plasma triglycerides. However, data on the exact role of plasma concentrations of APOA5 in human triglyceride homeostasis are lacking. In the present study, we estimated plasma APOA5 levels in patients with type 2 diabetes at baseline and during atorvastatin treatment, a lipid-lowering treatment that results in a reduction in plasma triglycerides and APOC3.Subjects, materials and methodsPlasma APOA5 concentration was measured by ELISA in 215 subjects with type 2 diabetes, who were taken from the Diabetes Atorvastatin Lipid-lowering Intervention (DALI) study, a 30-week randomised, double-blind, placebo-controlled study, and given atorvastatin 10 mg or 80 mg daily.ResultsAt baseline, average plasma APOA5 concentration was 25.7±15.6 μg/100 ml. Plasma APOA5 (Rs=0.40), APOC3 (Rs=0.72) and APOE (Rs=0.45) were positively correlated with plasma triglyceride levels (all p<0.001). In multiple linear regression analysis, adjusted for age and sex, the variation in plasma triglycerides was explained mostly by APOC3 (52%) and only to a small extent by APOA5 (6%) and APOE (1%). Atorvastatin treatment decreased plasma triglycerides, APOA5, APOC3 and APOE (all p<0.0001). After treatment, APOC3 remained the major determinant of plasma triglyceride levels (59%), while the contributions of APOA5 and APOE were insignificant (2 and 3%).Conclusions/interpretationOur findings reveal a positive association between plasma APOA5 and triglycerides in patients with type 2 diabetes. Treatment with atorvastatin decreased plasma APOA5, APOC3, APOE and triglycerides. In contrast to APOC3, APOA5 is not a major determinant of triglyceride metabolism in these patients.

Identification and characterization of novel loss of function mutations in ATP-binding cassette transporter A1 in patients with low plasma high-density lipoprotein cholesterol

OBJECTIVES The current literature provides little information on the frequency of mutations in the ATP-binding cassette transporter A1 (ABCA1) in patients with low high-density lipoprotein cholesterol (HDL) levels that are referred to the clinic. In 78 patients with low plasma levels of HDL cholesterol that were referred to our clinic, we routinely screened for ABCA1 gene mutations and studied the functionality of newly identified ABCA1 missense mutations. METHODS The coding regions and exon-intron boundaries of the ABCA1 gene were sequenced in 78 subjects with HDL cholesterol levels below the 10th percentile for age and gender. Novel mutations were studied by assessing cholesterol efflux capacity (using apolipoprotein A-I as acceptor) after transient expression of ABCA1 variants in BHK cells. RESULTS Sixteen out of 78 patients (21%) were found to carry 19 different ABCA1 gene variants (1 frameshift, 2 splice-site, 4 nonsense and 12 missense variation) of which 14 variations were novel. Of three patients with homozygous mutations and three patients having compound heterozygous mutations only one patient presented with the clinical characteristics of Tangier Disease (TD) in the presence of nearly complete HDL deficiency. Seven out of eight newly identified ABCA1 missense mutations were found to exhibit a statistically significant loss of cholesterol efflux capacity. CONCLUSION This study shows that one out of five patients who are referred to our hospital because of low HDL cholesterol levels have a functional ABCA1 gene mutation. It is furthermore demonstrated that in vitro studies are needed to assess functionality of ABCA1 missense mutations.

Cholesteryl ester transfer protein and hyperalphalipoproteinemia in Caucasians

It is unclear whether cholesteryl ester transfer protein (CETP) contributes to high density lipoprotein cholesterol (HDL-C) levels in hyperalphalipoproteinemia (HALP) in Caucasians. Moreover, even less is known about the effects of hereditary CETP deficiency in non-Japanese. We studied 95 unrelated Caucasian individuals with HALP. No correlations between CETP concentration or activity and HDL-C were identified. Screening for CETP gene defects led to the identification of heterozygosity for a novel splice site mutation in one individual. Twenty-five heterozygotes for this mutation showed reduced CETP concentration (−40%) and activity (−50%) and a 35% increase of HDL-C compared with family controls. The heterozygotes presented with an isolated high HDL-C, whereas the remaining subjects exhibited a typical high HDL-C/low-triglyceride phenotype. The increase of HDL-C in the CETP-deficient heterozygotes was primarily attributable to increased high density lipoprotein containing apolipoprotein A-I and A-II (LpAI:AII) levels, contrasting with an increase in both high density lipoprotein containing apolipoprotein A-I only and LpAI:AII in the other group. This study suggests the absence of a relationship between CETP and HDL-C levels in Caucasians with HALP. The data furthermore indicate that genetic CETP deficiency is rare among Caucasians and that this disorder presents with a phenotype that is different from that of subjects with HALP who have no mutation in the CETP gene.

Tissue sites of degradation of high density lipoprotein apolipoprotein A-IV in rats.

The in vivo metabolism of high density lipoprotein (HDL), labeled by incorporation of 1251-apolipoprotein (apo) A-IV, was studied in the rat and compared with the metabolism of HDL labeled with 131I-apo A-I. The 251l-apo A-IV labeled HDL was obtained by adding small amounts of radioiodinated apo A-IV to rat serum, followed by separation of the different lipoprotein fractions by chromatography on 6% agarose columns in order to avoid “stripping” of apolipoproteins by ultracentrifugation. Under both in vitro and in vivo conditions, the 1251-apo A-IV remained an integral component of HDL and was not exchanged to other lipoproteins, including the “free” apo A-IV fraction. The serum half-life, measured at between 8 and 28 hours after intravenous Injection of labeled HDL, was 8.5 f 0.5 hours for HDL apo A-IV and 10.2 & 0.7 hours for HDL apo A-I. The tissue sites of catabolism of HDL apo A-IV and HDL apo A-I were analyzed in the “leupeptin-model.” Only the kidneys and liver showed a significant leupeptindependent accumulation of radioactivity. At 4 hours after injection of 1251-apo A-IV/1311- apo A-I labeled HDL, 3.5% 21.0% and 8.4% f 2.0% of HDL apo A-IV and 4.6% * 1.3% and 2.6% f 0.6% of the HDL apo A-I were accumulated in a leupeptin-dependent process in the kidneys and liver, respectively. lmmunocytochemical studies revealed that the renal localization of apo A-IV was intraceilular and confined to the epithelial cells of the proximal tubuli. The amount of intracellular apo A-IV in rat kidneys was increased in leupeptin-treated animals. The data suggest that the leupeptin-dependent degradation of HDL apo A-IV is more active in the liver than in the kidneys, while the opposite was observed for HDL apo A-I. These results, as well as the short half-life of HDL apo A-IV as compared to HDL apo A-I, are compatible with the existence of an apo A-IV-containing HDL subtraction with a relatively fast turnover for which the liver is the major catabolic site.

Plasma pre beta-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus : Role of phospholipid transfer protein

UNLABELLED Atorvastatin lowers plasma phospholipid transfer protein (PLTP) activity, which stimulates pre-beta-HDL generation in vitro. We determined the effect of atorvastatin on pre-beta-HDL formation and its relation with PLTP activity in type 2 diabetes. METHODS Plasma pre-beta-HDL formation as well as plasma apo A-I, LpA, LpAI:AII, cholesteryl ester transfer protein (CETP) and PLTP activity were measured before and after 30 weeks treatment in 40 patients randomized to atorvastatin 80 mg daily and 41 placebo receiving patients. Pre-beta HDL formation was measured by crossed immunoelectrophoresis under conditions of lecithin:cholesterol acyltransferase (LCAT) inhibition. RESULTS Plasma pre-beta-HDL formation, triglycerides, LDL cholesterol, PLTP activity, and CETP decreased after statin treatment (all P<0.001 vs placebo), whereas HDL cholesterol increased (P<0.005). Plasma apo A-I, LpAI and LpAI:AII remained unchanged compared to placebo. In all patients combined, the changes in pre-beta-HDL formation were independently related to the decrease in plasma triglycerides (beta=0.31; P=0.006) and PLTP activity (beta=0.23; P=0.038), without a contribution of CETP. In the atorvastatin treated patients, the decrease in pre-beta-HDL formation tended to be related to the decrease in PLTP activity (beta=0.30, P=0.061) after controlling for decreases in triglycerides (beta=0.22, P=0.22). CONCLUSION High dose atorvastatin decreases the capacity of plasma to generate pre-beta-HDL particles in type 2 diabetic patients, probably via lowering of plasma PLTP activity and triglycerides. This could contribute to an improvement in the atherogenic lipoprotein profile.

Leupeptin as a tool for the detection of the sites of catabolism of rat high-density lipoprotein apolipoproteins A-I and E

Leupeptin, an inhibitor of lysosomal cathepsin activity, was injected intravenously into male rats. Tissues obtained from leupeptin-treated animals showed a depressed cathepsin activity when compared with tissues from saline-treated control animals. Leupeptin treatment did not change the hepatic activities and subcellular distribution of marker enzymes for mitochondria, microsomes and plasma membranes. Hepatic lysosomal cathepsin activity was specifically inhibited, but the subcellular distribution of all lysosomal marker enzymes tested was changed, indicating the occurrence of enlarged lysosomes in the leupeptin-treated animals. No significant differences were observed in the serum concentrations of protein, cholesterol, cholesteryl esters, phospholipids and apolipoproteins A-I, A-IV and E between leupeptin-treated rats and control animals. When radioiodinated asialofetuin was injected intravenously, the radiolabel was retained for an extended period of time in the liver of leupeptin-treated animals, indicating diminished catabolism of this protein in the liver. When rat high-density lipoprotein, labelled specifically in the apolipoprotein A-I or E moiety was injected intravenously, only the kidneys and the liver showed a leupeptin-induced accumulation of radioactivity. These studies provide evidence for an important contribution of the kidneys and the liver to the in vivo catabolism of high-density lipoprotein apolipoproteins, using a method completely different from sugar-containing labelling compounds.

Appraisal of hepatic lipase and lipoprotein lipase activities in mice.

A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required.

Role of plasma adiponectin on the HDL-cholesterol raising effect of atorvastatin in patients with type 2 diabetes

ABSTRACT Objective: Adiponectin, secreted by adipose tissue, plays an important role in lipoprotein metabolism and also affects carbohydrate and insulin pathways. We studied the effects of atorvastatin treatment on plasma adiponectin and high density cholesterol (HDL) levels in patients with type 2 diabetes. Research design and methods: In the ‘Diabetes Atorvastatin Lipid Intervention’ (DALI) study, a randomized placebo-controlled study on the effects of atorvastatin treatment in 194 patients with type 2 diabetes and mildly elevated plasma triglycerides, adiponectin levels, lipoproteins, cholesteryl ester transfer protein (CETP) mass, as well as postheparin lipoprotein lipase (LPL) and hepatic lipase (HL) activities were assessed at baseline and after 6 months of treatment (placebo, 10 mg or 80 mg atorvastatin). Results: At baseline, plasma adiponectin levels were positively associated with HDL cholesterol (r = 0.40, p < 0.001), and apoA-I (r = 0.38, p < 0.001) in both males and females. Adiponectin was negatively associated with triglycerides (r = −0.26, p < 0.001) in males as well as in females. Atorvastatin treatment had no effect on plasma adiponectin levels. However, adiponectin levels at baseline significantly predicted the effect of atorvastatin treatment on HDL-cholesterol (p = 0.007), i.e. patients with the highest baseline plasma adiponectin concentration (tertile 3) displayed the largest increase in plasma HDL cholesterol during treatment (8–10%), while the HDL-cholesterol increase in tertile 1 was almost negligible (1–3%). Conclusion: In this study, high baseline plasma adiponectin levels significantly affect the HDL-cholesterol response to atorvastatin treatment in patients with type 2 diabetes and therefore may play a role in defining future treatment strategy.

Two novel mutations in apolipoprotein C3 underlie atheroprotective lipid profiles in families.

Apolipoprotein C3 (APOC3) mutations carriers typically display high plasma high‐density lipoprotein cholesterol (HDL‐C) and low triglycerides (TGs). We set out to investigate the prevalence and clinical consequences of APOC3 mutations in individuals with hyperalphalipoproteinemia. Two novel mutations (c.‐13‐2A>G and c.55+1G>A) and one known mutation (c.127G>A;p.Ala43Thr) were found. Lipid profiles and apoCIII isoform distributions were measured. c.55+1G>A mutation carriers displayed higher HDL‐C percentiles (35.6 ± 35.8 vs 99.0 ± 0, p = 0.002) and lower TGs (0.51 (0.37–0.61) vs 1.42 (1.12–1.81) mmol/l, p = 0.007) and apoCIII levels (4.24 ± 1.57 vs 7.33 ± 3.61 mg/dl, p = 0.18). c.‐13‐2A>G mutation carriers did not display significantly different HDL‐C levels (84.0 ± 30.0 vs 63.7 ± 45.7, p = 0.50), a trend towards lower TGs [0.71 (0.54 to 0.78) vs 0.85 (0.85 to –) mmol/l, p = 0.06] and significantly lower apoCIII levels (3.09 ± 1.08 vs 11.45 ± 1.06 mg/dl, p = 0.003). p.Ala43Thr mutation carriers displayed a trend towards higher HDL‐C percentiles (91.2 ± 31.8 vs 41.0 ± 29.7 mmol/l, p = 0.06) and significantly lower TGs [0.58 (0.36–0.63) vs 0.95 (0.71–1.20) mmol/l, p = 0.02] and apoCIII levels (4.92 ± 2.33 vs 6.60 ± 1.60, p = 0.25). Heterozygosity for APOC3 mutations results in high HDL‐C and low TGs and apoCIII levels. This favourable lipid profile in patients with genetically low apoCIII levels holds promise for current studies investigating the potential of apoCIII inhibition as a novel therapeutic in cardiovascular disease prevention.