Gabriele Knipping

Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle.

Hormone-sensitive lipase (HSL) is believed to play an important role in the mobilization of fatty acids from triglycerides (TG), diglycerides, and cholesteryl esters in various tissues. Because HSL-mediated lipolysis of TG in adipose tissue (AT) directly feeds non-esterified fatty acids (NEFA) into the vascular system, the enzyme is expected to affect many metabolic processes including the metabolism of plasma lipids and lipoproteins. In the present study we examined these metabolic changes in induced mutant mouse lines that lack HSL expression (HSL-ko mice). During fasting, when HSL is normally strongly induced in AT, HSL-ko animals exhibited markedly decreased plasma concentrations of NEFA (−40%) and TG (−63%), whereas total cholesterol and HDL cholesterol levels were increased (+34%). Except for the increased HDL cholesterol concentrations, these differences were not observed in fed animals, in which HSL activity is generally low. Decreased plasma TG levels in fasted HSL-ko mice were mainly caused by decreased hepatic very low density lipid lipoprotein (VLDL) synthesis as a result of decreased NEFA transport from the periphery to the liver. Reduced NEFA transport was also indicated by a depletion of hepatic TG stores (−90%) and strongly decreased ketone body concentrations in plasma (−80%). Decreased plasma NEFA and TG levels in fasted HSL-ko mice were associated with increased fractional catabolic rates of VLDL-TG and an induction of the tissue-specific lipoprotein lipase (LPL) activity in cardiac muscle, skeletal muscle, and white AT. In brown AT, LPL activity was decreased. Both increased VLDL fractional catabolic rates and increased LPL activity in muscle were unable to provide the heart with sufficient NEFA, which led to decreased tissue TG levels in cardiac muscle. Our results demonstrate that HSL deficiency markedly affects the metabolism of TG-rich lipoproteins by the coordinate down-regulation of VLDL synthesis and up-regulation of LPL in muscle and white adipose tissue. These changes result in an “anti-atherogenic” lipoprotein profile.

Studies on the composition of pig serum lipoproteins. Isolation and characterization of different apoproteins.

1. Different lipoprotein density fractions from pig serum were isolated by phosphotungstate precipitation followed by purification in the preparative ultra-centrifuge. 2. The protein part of very low density lipoproteins was composed of approximately 52 percent lipoprotein B apoprotein and the rest of lipoprotein C II apoprotein and other as yet unidentified peptides. 3. The protein moiety of low density lipoproteins consisted primarily of lipoprotein B apoprotein (over 95 percent); the amino acid compositions of lipoprotein B apoprotein of very low and low density lipoproteins were practically identical. 4. The predominant polypeptide of pig serum high density lipoproteins exhibited an amino acid composition and a molecular weight very similar to human liprotein A I apoprotein. In contrast to human lipoprotein A I apoprotein, the apoprotein from pigs was found to release leucine first followed by alanine, threonine, and lysine upon incubation with carboxypeptidase A. 5. In pig serum the major lipoprotein C apoprotein was found to be a polypeptide similar in amino acid composition to lipoprotein C II apoprotein from human serum. The molecular weight of this polypeptide is approximately 8000. Incubation experiments with carboxypeptidase A indicate serine to be the most likely C-terminal amino acid.

Increased intrathecal production of apolipoprotein D in multiple sclerosis.

Apolipoprotein D (apoD) is a small glycoprotein responsible for the local transport of small hydrophobic ligands. Within the nervous system, apoD may be an acute phase protein that is upregulated in a variety of neuropathological conditions and is involved in the removal of lipids during nerve cell degeneration and provision of lipids during the regenerative phase. In this study, we measured cerebrospinal fluid (CSF) and serum apoD levels in patients with multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré Syndrome (GBS), infectious inflammatory neurological diseases (IND) and non-inflammatory neurological diseases (NND). We found that mean CSF apoD levels are significantly increased in patients with CIDP/GBS reflecting an acute blood-nerve barrier leakage. In contrast, MS is characterized by an increased intrathecal apoD release as measured by the apoD index. Thus, the results of our study provide the first evidence of an increased intrathecal production of apoD in MS. Moreover, we demonstrate that mean apoD indices are highest in MS patients at the time of their first clinical exacerbation. CSF apoD levels and apoD indices correlate with MS disease duration but not with disability or age. Finally, we found that corticosteroid treatment resulted in significantly elevated CSF apoD levels.

Impaired capacity of acute-phase high density lipoprotein particles to deliver cholesteryl ester to the human HUH-7 hepatoma cell line

The major role of native high density lipoprotein (HDL) is to carry cholesterol from peripheral tissues to the liver for bile excretion. As acute-phase (AP)-HDL has a decreased ability for cellular cholesterol efflux but an increased capacity for cholesteryl ester (CE) delivery to peripheral tissues, the interaction of AP-HDL with human hepatoma cells was studied. Binding studies to HUH-7 cells revealed saturable binding properties for HDL and AP-HDL at 4 degrees C. At 37 degrees C, specific cell-association of (125)I- and [1,2,6,7-(3)H]-cholesteryl palmitate ([(3)H]CE)-labeled lipoprotein particles was 2.2- and 1.6-fold higher for HDL indicating that total CE delivery was significantly (P<0.05) higher for HDL in comparison to AP-HDL. In parallel, selective CE uptake (the difference between total lipid uptake and holoparticle uptake) from AP-HDL was decreased compared with HDL. The fact that the capacity for cellular cholesterol efflux from HUH-7 cells is slightly impaired by AP-HDL (compared with HDL) is of support that scavenger receptor class B, type I (SR-BI), the only receptor so far known to mediate bi-directional lipid flux, might be involved in altered HUH-7 cholesterol hemostasis by AP-HDL. Our in vitro findings suggest that HDL and AP-HDL interact differently with cells of hepatic origin resulting in decreased hepatic cholesterol removal from the circulation during the AP reaction.

Endothelial lipase-modified high-density lipoprotein exhibits diminished ability to mediate SR-BI (scavenger receptor B type I)-dependent free-cholesterol efflux.

Endothelial lipase (EL) is a phospholipase with little triacylglycerol lipase activity. To assess structural and functional properties of EL-HDL (EL-modified high-density lipoprotein), HDL was incubated with conditioned medium from Cos-7 cells infected with adenovirus encoding human EL. After re-isolation of HDL by ultracentrifugation, TLC and HPLC analyses revealed that EL-HDL was markedly depleted in phosphatidylcholine and enriched in lyso-phosphatidylcholine compared with LacZ-HDL (control HDL) incubated with conditioned medium from Cos-7 cells infected with adenovirus encoding beta-galactosidase. The EL-HDL was enriched in non-esterified fatty acids and, as revealed by lipid electrophoresis, was more negatively charged than control HDL. The HDL particle size as well as the total cholesterol, free cholesterol and triacylglycerol content of HDL were not significantly altered after EL modification. The ability of EL-HDL to mediate 3H-cholesterol efflux from SR-BI (scavenger receptor B type I) overexpressing Chinese-hamster ovary cells was impaired and markedly lower compared with LacZ-HDL at HDL concentrations of 100 microg/ml and above. Studies with 125I-labelled HDL showed almost unaltered binding affinity (K(m) values) and a slightly but significantly decreased binding capacity (B(max) values) of EL-HDL to SR-BI, compared with LacZ-HDL. The ATP-binding-cassette transporter A1-dependent cholesterol and phospholipid effluxes were not affected by EL modification. From these results, we concluded that EL modification alters chemical composition and physical properties of HDL, resulting in its decreased binding capacity to SR-BI and a diminished ability to mediate SR-BI-dependent cholesterol efflux.

EFFECTS OF CYTOKINES, BUTYRATE AND DEXAMETHASONE ON SERUM AMYLOID A AND APOLIPOPROTEIN A-I SYNTHESIS IN HUMAN HUH-7 HEPATOMA CELLS

Serum amyloid A (SAA) and apolipoprotein A‐I (apo A‐I) are secreted by the liver. As concentrations of both apolipoproteins are inversely related under normal and acute‐phase conditions, human HUH‐7 hepatoma cells were stimulated with interleukin (IL)‐1α (100 and 200 U), IL‐6 (50 and 100 U), butyrate (2 m m) and dexamethasone (2 × 10−7 m and 1 × 10−6 m), alone or in combination. Changes in SAA and apo A‐I synthesis were monitored after metabolic labelling of the cells with [35S]‐methionine. Intracellular and secreted SAA and apo A‐I were immunoprecipitated, separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), and the radioactivity in the corresponding bands was counted. Intracellular apolipoprotein levels were increased by all stimuli, either alone or in combination, between 2.7‐ and 5.5‐fold (SAA) and between 2.8‐ and 4.1‐fold (apo A‐I), respectively. In a similar manner, apolipoprotein levels secreted by HUH‐7 cells were increased between 3.1‐ and 4.3‐fold (SAA) and between 1.9‐ and 3.3‐fold (apo A‐I). Co‐administration of cytokines, butyrate and/or dexamethasone had no pronounced synergistic effect on intracellular biosynthesis and secretion of SAA and apo A‐I. The results from the present study suggest that apo A‐I must not necessarily be considered as a negative acute‐phase reactant.

The Role of LCAT and Cholesteryl Ester Transfer Proteins for the HDL and LDL Structure and Metabolism

Since the early detection of LCAT by Glomset, a great deal of research has been conducted for establishing the role of this enzyme in the Lp metabolism. It became apparent that LCAT produces more than 3/4 of the CE found in plasma. LCAT acts primarily on Lp with high PC/FC contents and high PC/FC ratios. For expression of the full activity, apo-Lp cofactors such as apo-AI, -CI, -AIV and -E are necessary. Although it was believed for a long time that HDL are the only substrate for LCAT we could demonstrate that LDL and even apoA/C/E free LpB is utilized by LCAT. The CE formed in PC/FC rich Lp are transferred to VLDL and LDL by specific proteins, which also promote the exchange of CE against TG. TG in these particles are hydrolyzed by liver lipase providing new space in the core for further cholesterol esterification. Thus LCAT exerts its physiological role in concert with other enzymes e.g. LPL, hepatic lipase and possibly phospho-lipases as well as with exchange and transfer processes partly catalyzed by specific exchange/transfer proteins. The main function of LCAT without doubt is the reverse cholesterol transport from periphery to liver counteracting the accumulation of CE in reticulo endothelial cells by the scavenger pathway. Metabolic studies revealed that the cholesterol clearance from the circulation proceeds in the same order of magnitude as the esterification by LCAT takes place. This possibly implies that LCAT might be the rate limiting enzyme for cholesterol catabolism from blood.

Platelet membrane fluidity in type IIA, type IIB and type IV hyperlipoproteinemia

Fluorescence spectroscopy, a very sensitive index for measuring the biophysical properties of living cell systems, was used to examine the structural order of intact, resting, gel-filtered platelets from hyperlipidemic subjects (n = 48, 25-70 years) and normolipemic subjects (n = 34, 19-68 years). Fluorescence anisotropy (r[s]), which is inversely related to membrane fluidity, was estimated using 3 different fluorescent dyes, DPH, TMA-DPH, and 6-AS, known to label different regions of biological membranes. Increased membrane fluidity was observed in type IIB (n = 24, 36-62 yrs; r[s] = 0.0692 +/- 0.09) and type IV (n = 10, 33-57 yrs; r[s] = 0.058 +/- 0.006) hyperlipidemics in comparison to type IIA (n = 14, 25-70 yrs; r[s] = 0.086 +/- 0.019) and control subjects (n = 24, 28-68 yrs; r[s] = 0.079 +/- 0.012). The temperature dependency of r[s]-DPH values was significantly different (P less than 0.01) in platelets from type IIB and type IV patients compared to type IIA and control subjects of similar age. A significant positive correlation (P less than 0.005) between membrane fluidity and age was found only in healthy control subjects (n = 34, 19-68 yrs). Despite significant (P less than 0.01) differences in plasma lipid concentrations in hyperlipidemic patients and controls, significant ex vivo relations between membrane fluidity and lipoprotein concentrations, free fatty acid distribution, and increased age were found only in healthy control subjects. Plasma levels of thromboxane as well as serum selenium concentrations did not significantly differ between hypercholesterolemic, hypertriglyceridemic, and control subjects.

Factors affecting the conversion of high-density lipoproteins: experiments with pig and human plasma

The conversion of pig high-density lipoproteins (HDL) (mainly HDL3) to fractions of lower densities was studied by incubating pig plasma for 24 h at 37 degrees C in the presence and absence of lipoprotein lipase from bovine milk, lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein and triacylglycerol-rich particles (very-low-density lipoproteins (VLDL) or Intralipid). The results can be summarized as follows. In the presence of lipoprotein lipase and at a VLDL/HDL mass ratio of 2, the F-1.210 of pig HDL was shifted from 3.3 to 4.2, which is characteristic for human HDL2. This shift was caused by the excessive increase in the free fatty acid content in HDL. If 50 g/l of bovine serum albumin were added prior to incubation, the flotation rate of HDL remained in the HDL2a region. If lecithin:cholesterol acyltransferase was active in fasting pig plasma during incubation, we observed only a negligible increase of F-1.210 in HDL. If pig lipoproteins were incubated with human lipoprotein-free serum as a source of cholesteryl ester transfer activity, a slight increase in the flotation rate of HDL was observed, which was amplified in the presence of active lecithin:cholesterol acyltransferase. Pig HDL was converted to a fraction with F-1.210 of 4.2, which is typical for human HDL2, only if active lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein and triacylglycerol-rich particles were present in the incubation mixture. From our results we also concluded that apolipoprotein A-II plays no role in the HDL2 formation.

Quantification of apolipoprotein D by an immunoassay with time-resolved fluorescence spectroscopy

Apolipoprotein D (apoD), also known as gross cystic disease fluid protein-24 (GCDFP-24), is a minor protein moiety of high-density lipoproteins in human plasma. ApoD is expressed in a subset of breast carcinomas and has been proposed as a tumor marker and prognostic indicator for breast cancer progression. Here we describe a new sensitive time-resolved fluorimetric immunoassay for quantification of human apoD in biological specimens using affinity-purified polyclonal anti-human apoD rabbit antibodies and Eu3+ as a specific probe. Both purified apoD and normal human pool-serum served as reliable primary and secondary standards in the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). Plasma apoD concentrations measured by the DELFIA were 99.6 +/- 32 microg/ml. The detection limit of the DELFIA procedure was 0.5 ng/ml after sample dilution of 1/8000. The intra-assay coefficient of variation averaged 3.5%, whereas the inter-assay coefficient of variation averaged 6.9%. The concentration of apoD in breast cyst fluids ranged from 6.82 to 28.37 mg/ml. Based on the low detection limit and the high specificity of the DELFIA procedure, we have applied this technique for the measurement of apoD in breast cancer cell supernatants. In estrogen-receptor positive cells, i.e., T-47D and ZR-75-1 cells, 42.6 +/- 1.4 and 2.7 +/- 0.2 ng apoD/ml supernatant after 4 days in culture without induction of apoD synthesis were measured. A comparison of the direct sandwich DELFIA procedure with an electroimmunoassay commonly used to assay apoD revealed correlation coefficients of 0.986 (serum) and 0.975 (cyst fluids). The present findings indicate that the direct sandwich DELFIA is appropriate for apoD quantification in plasma and breast cyst fluids. Furthermore, the technique should permit studies on the induction of apoD synthesis in the low picomolar range in different carcinoma cells to gain insight into the expression of this atypical apolipoprotein.